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Studies on the biochemistry of lead, iron and copper

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Studies on the Biochemistry of Lead, Iron and
Copper
H f H w it t H W iM m a t
A Thesis presented by,
Sidney Lionel Tompsett B«Sc* (Lond*), Ph.D* (Glas*),
t
r «1
* r*
•n t
for the Degree of Doctor of Science of the University
of Glasgow
ProQuest Number: 10662628
All rights reserved
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Preface
The work that has been described in this thesis has
been, done during the period 1934 until the present time*
All
of the estimations have been done by the writer himself.
The
animals were under the sole charge of the writer who possesses
an Animal License* Certificates A and B.
Much of the work
has been published in the form of papers* a list of which
follows.
Heprints of those which are available are enclosed
under separate cover.
1. fThiolacetic acid as a reagent for the determination of the
inorganic iron content of certain biological materials*
Biochemical Journal (1934)
28,
1536 - 1543
2. *The copper content of blood*
Biochemical Journal
(1934) 28,
1544
- 1549
3. *Studies of the complexes of iron with various biological
materials*
Biochemical Journal
(1934) 28,
1802 - 1804
4. *The excretion of copper in urine and faeces and its relati
to the copper content of the diet*
Biochemical Journal
(1934) 28,
2088 - 2091
5. *The copper and *inorganic* iron contents of human tissues*
Biochemical Journal
(1935) 29,
480 -486
6. *The copper content of the blood in pregnancy*
British Journal of Experimental Pathology
(1935)
(with D. P. Anderson)
7# *The lead content of human tissues and excreta*
Biochemical Journal
(1935) 29,
(with A. B. Anderson)
2-*
1851 - 1864
16,
67
8* 'The distribution of lead in human bones'
Biochemical Journal
(1936)
30,
345 - 346
9# 'Lead poisoning*
Lancet
(1939)
1,
559
(with A. B* Anderson)
10, 'The determination of lead in biological materials'
Biochemical Journal . (1939)
33,
1331 - 1236
11, 'The influence of certain constituents of the diet upon
the absorption of lead from the alimentary tract*
Biochemical Journal
(1939)
33,
1237 - 1240
12, 'Studies in lead mobilisation*
British Journal of Experimental Pathology
(1939)
30,
408 - 416
(with J. IT, M, Chalmers)
13, 'Further studies on the absorption, mobilisation and
excretion of lead*
British Journal of Experimental Pathology
(1939)
20,
512 - 516
14, *A study of factors influencing the absorption of iron
and copper from the alimentary tract'
Biochemical Journal
(1940)
in preparation
Index
page
Lead
Introduction
7
The estimation of lead in animal tissues
and excreta
19
The distribution
of lead in human tissues
40
The lead content
foetuses
of the tissues
46
of
human
Lead in milk
48
The lead content of human excreta
50
Origin of the lead present in human tissues
and excreta
53
Factors influencing the absorption of lead
56
The lead content of the blood and excreta in
suspected plumbism
64
The mobilisation
of lead
68
The lead content
lead absorption
of the tissues
in
increased 83
The excretion of lead
86
Discussion
88
References
94
Iron and Copper
Introduction
100
Determination of iron in biological materials
108
Fon-haematin iron
112
The dialysis of iron salts
121
Factors influencing the absorption of iron
from the alimentary tract
127
The reduction of ferric iron in the alimentary
tract and its significance in absorption
142
Bggg.
The non-haeraatin iron of human serumand plasma
148
The non-haematin iron of whole blood
155
The iron content of urine
161
The 'inorganic1 iron content of human tissues
164
The determination of copper in biological
materi al s
168
The copper content of blood
183
The copper content of urine and faeces
191
The copper content of human tissues
194
Factors influencing the absorption of copper
from the alimentary tract
200
The copper content of the blood in certain
conditions
207
Discussion
216
References
225
Lead, Iron and Copper
Contrasts and comparisons
231
LEAD
Introduction
Lead is one of the commonest elements in the earth*!
crust*
Lead and its salts are of biological importance
because of their toxicity*
Lead salts precipitate proteins and act as local
astringents*
Acute toxicity is generally low but cumulativ<
effects tend to be produced*
Acute lead poisoning is rare
and is usually produced by the acetate*
Chronic lead
poisoning is commoner and possesses characteristic
synptons.
These are as follows:
1*
lead line at the margin of the gums*
2.
anaemia,
3*
paroxysmal colic and constipation,
4*
*dropl wrist and other peripheral motor paralyses,
5*
tremors and convulsions,
6*
psychic changes (encephalopathia etc.)
There are three possible modes of entrance of lead
into the body;
3. skin.
1. alimentary tract, 2* respiratory tract,
Absorption of lead from the alimentary tract
has been recognised from time immemorial*
Absorption of
lead from the respiratory tract would result from the
inhalation of dust and vapour containing lead or its
compounds.
This mode of absorption has been held in disput«
but Aub et alia (1926) have shown that this does occur*
It is debatable whether or not lead or its compounds are
absorbed through the intact skin, but it is agreed that
absorption can occur through the broken skin.
Agreement
also exists that, lead tetra-ethyl, a constituent of
5anti-knock* petrols, can be absorbed through the intact skii
but this is due to its fat soluble properties.
Phe toxic effects of lead have been recognised
from time immemorial.
Hippocrates, about 370 B. C.,
recognised lead as being responsible for attacks of colic
in a man who was described as an extractor of metals.
Nicardo, who lived in the 2nd century B. 0., noted the
relationship between lead and constipation, abdominal pain
and pallor and mentioned lead palsy for the first time.
Discoides is quoted by Alderson (1852) as saying that fthe
drinking of litharge causes oppression to the stomach, belly
and intestines., with intense wringing pains, , sometimes it
even wounds the intestines by its pressure, it suppresses
the urine, while the body swells and acquires an unsightedly
blue hue1.
He also mentioned paralysis and delirium as
being produced by lead.
About 1000 A. I)., Avicenna
recommended the use of violent purgatives to relieve a form
of colic which was probably due to lead.
Oreato, who lived
about 1600 A. D., attributed the colic which was prevalent
in Moravia at that time and was known to end in paralysis
to the use.of ‘falsified wine5.
A similar conclusion was
reached by Gitois, a physician of Poitiers, regarding the
colic which was prevalent in his district*
It appears to
have been the practice on the Continent at this period to
treat sour wine with litharge or red lead in order to remove
the acidity and so make it palatable.
In 1656 Stockhusen
published a treatise in which he recognised that lead
poisoning could result from the inhalation of dust containing
lead*
In 1745 Hexham described a condition appearing in
Devonshire which had symptons similar to those of lead colic,
Later this was shown by Sir John Baker (1767) to be lead cola
and was due to the lead utensils used in the preparation
and the storage of cider.
A similar type of colic of simile
aetiology was described by Luauricaga in Spain in 1796.
At
the beginning of the 19th century several brilliant clinical
descriptions of lead poisoning appeared.
Grisolle (1836, 16
described lead colic, line and encephalopathy very
accurately.
Three years later, Tanquerel des Planches
published his epoch making book in which he described the
clinical aspects of the disease*
He laid particular emphasj
on the lead line, this becoming known as the Burtonian line,
because of the description published by Burton in 1840.
Sine
the time of Tanquerel, little appears to have been added to 1
clinical knowledge of the disease except perhaps by
Dejerine-Kliampfe (1889) who wrote an excellent treatise on
lead palsies.
Experimental studies on lead poisoning appeal
to have been started by Qrfila in 1814.
An important clinical sign in lead poisoning is the
lead line.
Typically it occurs in the gingiva near the
border of the teeth, usually near decaying teeth, either in
the gum or in the mucosa of the lip or cheek opposite these.
Rapid caries seems to develop whenever a lead line appears
near teeth apparently good.
The true lead line is situated
within the tissues and is peculiar to lead poisoning except
possibly in bismuth poisoning.
Microscopical examination
shows that the line is composed of irregular amorphous granul
which are entirely sub-epithelial.
It is presumed that it i
produced by the interaction of dissolved lead and hydrogen
sulphide to form insoluble lead sulphide.
In experimental
work on animals, Aub et alia (1926) found that lead line coul
be produced in carnivorous animals e.g. cat, but not in
herbivorous animals. e*:g* rabbit.
They believed that the
lead line is, produced from hydrogen sulphide derived from
the putrefaction of food debris left in the mouth.
In suppo
of this theory it has been shown that the incidence of lead
line is related to dental hygeine.
Of cases of suspected
lead poisoning admitted to the Glasgow Royal Infirmary within
recent years, often no lead line could be found even when
the diagnosis was confirmed.
This appears to be due to the
fact that on examination, the patients were found to be
edentulous.
The extraction of remaining teeth appears to
be often advised as part of the treatment of early symptons.
Extensive clinical and pathological studies have
been made of lead palsy, colic and encephalopathy, and of the
relationship of lead to constipation.
These have been
reviewed by Aub et alia (1926) in their monograph.
Lead
colic is generally believed to be produced by the action of
lead on the smooth muscle of the intestine.
Encephalopathy, the most severe of the toxic episodes
of lead poisoning is. now fortunately rare owing to more
10
stringent public health measures.
The pathology of these
conditions, in spite of the extensive work done, is still
in a debatable state.
Chronic lead poisoning is always accompanied by
an anaemia.
A marked feature of the blood picture is the
large number of stippled cells present*
Although stippled
cells are not specific to lead poisoning but appear in
other conditions, they are most constant in lead poisoning.
Blood examinations for haemoglobin content and for the
presence of stippled cells form part of the usual
routine clinical examination of lead workers.
In the view
of a large number of clinicians, these examinations give
the most exact picture of the patients' condition.
An
enormous amount of work has been done on the effect of
lead upon blood end its formation.
This has been reviewed
by Aub et alia (1926).
It has been shown that lead has a marked effect on
the germ cell.
Lead poisoning in the mother and even in the
father has been shown to result often in still-births•
The mortality rate of the offspring of such parents is
also high, particularly during the first three years of
life ^Paul (1860), Porak (1894), Lewin (1904), Oui (1907),
Legge and Goadby (1912) "j .
Numerous cases have been
reported of the use of lead, chiefly as lead diachylon,
to produce abortion
£ Ransom (1900), Scott (1902), Hall
(1905), Hall and Ransom (1906)
J . The reaction appears
to be due to the effect of lead on the smooth muscle of
/A
the uterus in the same way as it acts on the smooth muscle
of the intestine during colic.
In severe lead poisoning the liYer is often affecte
The effects of lead on the liver have occupied much of the
attention of German and Italian workers within recent years
[ Viglianl (19,5), L.geaer (1954), Vlgllanl
Angsl.rl
(1935, 1936), Fellinger (1936), Carrie (1936), Vigliani
(3.936), Mertens (1937), Vigliani and Waldenstrom (1937),
Vigliani and Libowitaky (1937)
Exposure to unusual amounts of lead has resulted
from,
1. the contamination of foodstuffs and drinking water
with lead,
2. the extensive use of lead and its compounds in industr
Contamination of foodstuffs with lead must hare bee
extremely common at one time*
With more stringent
public health measures this has now been much reduced,
but cases do still occur.
In modern times contamination
of foodstuffs with lead occurs usually during processing.
The most notable exception has been in the case of fruit.
It has become prevalent, especially in the U. S. A., for
lead arsenate to be used as an insectide, resulting in
fruit and vegetables becoming contaminated.
A large
amount of work has been done in the XI. S. A., on the
toxicity of lead arsenate
£ Talbert and Taylor (1933),
Greene (1936, 1937), Shields, Mitchell and Huth (1939)
j .
Contamination is often due to the use of utensils made of
lead, containing lead solder or glazed with lead compounds•
This is particularly true when the material has an acid
reaction e.g. fruit juices and fermented liquids.
Numerous
cases of lead poisoning due to this have been reported;
wines
Campbell (1886), Alden (1889), Arnaud (1936)*
Yang ( 1 9 3 6 ) * Yang, Chang and Liu ( 1 9 3 7 )
(1 9 3 8 ) J
| fruit juices
Duy ( 1 9 3 5 ) "j .
f beer j~ Sutton
Tinned sardines hav
recently been found to be heavily contaminated with lead*
due to preparation over a lead containing grill
and Rooke ( 1 9 3 3 )
.
£ Lampitt
An unusual case, resulting in an
epidemic of lead poisoning, has been reported from Austria
£ Berger, Studeney and Rosigger (1 9 3 S )
jj . Lead was used
to counterbalance a grinding wheel in a flour mill with the
result that the flour was impregnated with fine particles
of metallic lead*
Drinking water, contaminated with lead, has been
held responsible for outbreaks of lead poisoning on many
occasions.
free of lead.
Natural waters are usually almost completely
The contaminations have always been caused
by the use of lead pipes.
Lead pipes for the transport of
water are still used extensively in some localities.
The
plumbosolvency of natural waters is extremely variable.
Soft waters, especially those containing much dissolved
oxygen and carbon dioxide are particularly plumbosolvent.
Hard waters are considered to have a low plumbosolvency
and it is considered safe to transport, such waters through
lead pipes as they become coated with a layer of insoluble
calcium salts and are thus protected from corrosion.
Waters from peaty areas, containing large amounts of organic
acids are very plumbo so1vent.
In this country no legal
limit exists as to the maximum amount of lead permitted in
drinking water*
Various authorities have on occasions
expressed their opinion*
Within recent years outbreaks
of lead poisoning due to contaminated water supplies have
r
Poisoning produced by the consumption of foodstuffs
and drinking water contaminated with lead is due entirely
to alimentary absorption*
A large amount of lead poisoning exists in industry
With the rise of industry and the more widespread use of
lead, this has assumed great importance*
In industry
absorption can take place from the respiratory tract as well
as from the alimentary tract.
Many authorities consider
that the former is the more important entrance.
The number of industrial processes using lead or
its derivatives is legion*
The following represents a
short summary!
1.
lead mining
S.
lead smelting
3.
handling and fabrication of lead
a.
manufacture of lead articles
b*
handling metallic lead in hot processes e.g.
lead burning, soldering, tempering and plumbing
4.
c.
brass and other founding in which lead is used
d.
buffing and polishing metallic surfaces
manufacture of lead salts and compounds, lead pigments
and organic lead compounds
5*
manufacturing processes in which lead compounds are used
storage battery, paint, glass, rubber and chemical
industries
6.
application and removal of lead containing paints,
enamels and glares
painting, spray painting, vitreous enamelling, pottery
dipping
sandpapering, scraping and chipping painted surfaces
flame cutting of painted metal
7.
tree spraying of lead containing insectides
8*
typographic trades
type founding, electro-typing, stereotyping
In lead mining, the incidence of poisoning appears
to be much higher in the new countries e.g. U. S. A., than
in the older countries e. g. those in Europe.
This is due
apparently to differences in the type of ore mined.
In the
older countries the mines are deep and the ore consists
chiefly of sulphide which is poorly soluble.
In the newer
mines, which are shallow^ the ore consists chiefly of
oxidised forms of lead e.g. carbonate, sulphate and oxides,
which are much more soluble.
It is to be expected that
as these mines become deeper, the yield of sulphide will
IS.
increase, with a consequent decrease in the incidence of
lead poisoning.
The manufacture and handling of lead tetra-ethyl
is
particularly dangerous owing to the ease with which it is
ahsorted through the skin*
At times unusual cases of lead poisoning are reporOne of the strangest cases was the occurrence in a large
number of families in Baltimore and Nashville, Tenessee,
produced by the use of discarded storage battery cases as
fuel*
Several cases of encephalopathy were reported
£ Williams et alia (1933), Crutcher (1933) J * Cases of
lead poisoning have been reported from the use of lead opiui
pipes £ Tang, Chang and Liu (1937)
, theatrical grease
paint containing lead pigments |" Bartleman and Dukes (1936)
and from shrapnel and bullets left in wounds £ Londres (193'
Leschke (1934), Haenisch (1936)
* The use of cosmetics
containing lead compounds has resulted in a large number of
cases of lead poisoning amongst women and suckling children
in Japurr
Kato (1932)
* Many children have been shown
to have suffered from lead poisoning in Queensland, Austral:
£ Nye (1933)
. It was concluded that the source of
lead was from the paint used on the verandas and railings
\
where the children played*
Under the action of the tropic*
sun, this paint powdered readily and as a result was in
a form readily assimilated by the children*
The
Queensland Government have prohibited the use of lead paints
containing more than
dfo
soluble lead*
Lead poisoning, due to eating and sucking toys etc.
containing lead, have been reported in children
Aub et al
(1926), Taylor and Schram (1936)J .
Extensive lead poisoning has been reported amongst
wild ducks in certain well hunted districts in the U. S. A.
[ Torr.y, Tfccrpe
Or.ham (1934) ] . Thls „ „ oused Py
the birds swallowing lead shot which was retained in the
bulbo-ventricuius•
Dowdell and Green (1937) have recommend
the use of shot made of lead magnesium alloy as this,
disintegrates rapidly on contact with water.
The incidence of lead poisoning as a result of
ingestion of contaminated foodstuffs and drinking water is 1
The occurrence of lead contamination in sardines in this
country was met effectively by a meeting of Port Medical
Officers of Health at the Ministry of Health where it was
decided to condemn any consignment which contained more than
20 parts of lead per million.
At a later meeting it was
decided to reduce the limit to 6 parts per million.
Limits of lead content are not applied to foodstuff
in general in this country, but only in particular cases
when evidence of gross contamination has been brought to the
notice of the Ministry of Health as in the case cited above.
The incidence of lead poisoning in industry has
decreased very much*
The measures adopted to prevent lead
poisoning in industry are chiefly concerned with personal
hygeine and the prevention of fumes and dust being inhaled.
A great deal of work in this field has been done by
Sir Thomas Oliver (1913 - 1921} and Legge and Goadby (1912)
in Britain, Meilliere (1902, 1903) in France, TeXeky
(1909 - 1921) in Germany and Hamilton (1911 - 1921) in the
TJ* S. A*
Lanza (1935) in a recent review considers that in
spite of the preventative measures taken, a considerable
amount of mild lead poisoning exists, much of which escapee
diagnosis.
This view warrants a much more extensive
investigation into the biochemistry of lead than hitherto
has taken place in an attempt to discover more delicate
methods of diagnosis.
The Estimation of Lead in Animal Tissues and Excreta,
The major problem which had to he solved before
any investigations could be contemplated, was to devise
a method for the estimation of the small amounts of lead
that occur in animal tissues and excreta.
The requirements
of such a method were accuracy, speed and sensitivity
in order that large quantities of material were not
necessary as the estimation of the lead content of the
blood of living persons was contemplated.
Biological materials of the nature of tissues, and
excreta consist of a heterogeneous mixture of organic
compounds and mineral salts of which lead, if present,
forms a very small part*
As a result, any method resolves
itself into three essential stages; 1. destruction of the
organic matter, 2. separation of the lead, 3. estimation
of the lead.
Almost all methods that have been devised,
consist of these three stages.
Up to 1934, almost all
methods have employed reactions which are used in
macro analytical chemistry and as a result the data obtained
have been very unsatisfactory.
To destroy organic matter, two methods have been
employed;
1. ignition, 2. digestion with sulphuric acid
and some oxidant e.g. nitric acid.
To separate lead, the following reactions, singly
or in combination have been used;
1. precipitation as the sulphide,
2. precipitation as the sulphate,
tn
3. precipitation as the chromate,
4* deposition in the metallic form or as the peroxide by
electrolysis*
As a general rule* the first reaction has been used as a
preliminary to the others*
The objection to precipitation
methods for separating small amounts of a substance is
obvious*
In the first place, the precipitate is usually
colloidal in nature and difficult to filter.
In the second
place,-all ?insoluble1 substances have definite solubilities
which although small become appreciable when small
quantities are involved.
Electrolytic methods usually fail
In the presence of large amounts of iron
I Francis et alia
(1 9 2 9 )
A large number of methods have been devised to
estimate lead after separation*
These are as followsj
1* nephelometrically as the sulphide,
2. nephelometrically as the sulphite
Cooksey and Walton
3* colorimetrically by the blue colour produced when
lead peroxide and tetramethyldiaminophenylmethane interact,
4. colorimetrically by the colour produced when lead Ghromate
and semicarbaside interact j^Fairhall ( 1 9 £ 4 ) ^ J ,
5. by titrating the iodine liberated when potassium iodide
is added to lead chromate in the presence of sulphuric acid
Fairhall (1924) J
None of the above reactions are specific for lead*
Reactior
3 to 5 ai*e to be condemned because they are not concerned
with lead but with the ion to which it is attached*
The sulphide test is probably the oldest of the above
but it lacks sensitivity.
A real objection may be laid
against the tetramethyldiaminodiphenylmethane test as it
involves the electrolytic deposition of lead as the peroxide
and manganese, a constant constituent of tissues and
excreta, tends to be deposited as the dioxide at the same
time and this reacts with the reagent.
Fairhall (1924) described a method in which the
lead was precipitated as sulphide and then as chromate.
The lead chromate was then determined either 1*
colorimetrically with semicarbazide or 2. by titration
with sodium thiosulphate after the addition of potassium
iodide and sulphuric acid.
This method was used by Aub
et alia (1926) in their investigations.
Kehoe et alia
(1926, 1933} used a modification of this method.
Later
they (1935) acknowledged a loss of 0.0? mg* Pb per sample
by this method.
In an examination of the lead content of
urine, Oooksey and Walton (1929) made a, preliminary
separation of the lead by electrolysis, and estimated it
nephelometrically as sulphite.
Francis, Harvey and Buchan
(1929) described a process involving the precipitation
of lead as sulphide, followed by electrolysis and
precipitation as sulphate.
The lead was estimated
nephelometrically as the sulphide.
Weyrauch and Muller
(1933) and Litzner and Weyrauch (1932, 1933} investigating
the distribution of lead in man, separated the lead as
ZJ.
sulphide and then as the peroxide by electrolysis.
They
estimated the lead colorimetrically with
tetramethyldiaminodiphenylmethane*
An estimation by
any of these methods would take about 3 days to complete*
The spectrograph has been used to identify and
estimate lead in biological materials.
The results obtains*
have been very conflicting*
The Author1s Method
The method consists of 3 stagesj 1* destruction
of the organic matter, 2* separation of the lead, 3.
estimation of the lead.
It will be considered "under these
headings in order for convenience, although this was not
the order of development.
Destruction of organic matter
Two methods are available $ 1* digestion with
sulphuric acid and an oxidant e.g. nitric acid, perchloric
acid, 2. ignition*
The objection to the first is that often large
quantities of sulphuric acid and oxidant are required and
consequently a large quantity of alkali is necessary for
neutralisation.
This leads to a high blank even when the
purest reagents are used*
Destruction by ignition, which avoids this high
blank, has been criticised on the grounds that lead may
be lost by volatilisation.
The writer has found that materj
containing a large amount of ash, consisting chiefly of
phosphate, may be ignited in a silica dish over a bunsen
aa.
burner without loss of lead.
When materials of low ash
content are treated similarly, a loss of lead may occur.
For
this reason, the ash content of such materials has been
increased by the addition of sodium phosphate (HagHPO^.) .
Biological materials have therefore been divided into two
classes;
1. high ash content - ignited without the addition of
sodium phosphate e.g. urine, faeces, milk and bone,
2. low ash content — sodium phosphate added prior to
ignition e.g. blood and soft tissues*
This ignition
satisfactory under the
method has been found to be quite
above conditions*
Towards the end,
ignition may be assisted by allowing the ash to cool,
adding a little concentrated nitric acid and re-heating.
Separation of the lead
Separation of
was not
lead by precipitation-or by electrolysj
considered for reasons stated already.
A method
suggested by Allport and Skrimshire (1932) for separating
lead from solutions of the ash of dyestuffs appeared at
first to have possibilities*
An alkaline solution of the
ash was shaken with a chloroform solution of
diphenylthiocarbazone (dithizone).
Lead was extracted by
the chloroform as a leaxUdithizone complex.
Iron was
not extracted and other metals were not extracted if
cyanide was present.
difficulties.
The writers recognised certain
The aqueous solutions must be perfectly
clear, the slightest turbidity due to iron or phosphates
interfering with the separation.
As the extractions must
be made in alkaline solution, this is difficult, for even
when citrates have been added, a solution may be perfectly
clear and yet iron or phosphates, may be precipitated in
colloidal form and so prevent complete extraction of lead*
The pH of the solution needs careful adjustment, which is
difficult when some classes of material are used.
If the
organic matter has been destroyed by wet oxidation, the
nature of the oxidant used has a marked influence.
Allport
and Skrimshire (1932) found that if nitric acid had been
used, extraction of the lead was generally incomplete.
The writer has found the method to give erratic results
and so was abandoned.
Lynch, Slater and Osier (1934) used
this method of separation in an examination of the lead
content of some human tissues.
The lead was estimated by
the sulphide reaction.
In a search for a more satisfactory method, the
writer found that sodium diethyIdithiocarbamate offered
,possibilities.
This substance was described by Gallon and
Henderson (1929) as being suitable for the estimation of
small amounts of copper and since then it has been used
extensively for this purpose.
As a preliminary, the writer
carried out an investigation of the general properties of
this substance.
Sodium diethyldithiocarbamate, a white substance, is
easily soluble in water.
It reacts with metals to form
organic complexes, many of which are soluble in organic
solvents e.g. ether.
The nature of these complexes was not
24.
studied#
Of the metals that occur in biological materials
and react thus are iron, copper, lead, zinc, manganese and
cohalt*
These complexes have low solubilities in water
but are easily soluble in ether#
The lead complex is. white and dissolves in ether
to form a colourless solution.
With the exception of
zinc, the complexes of the other metals are coloured and
form coloured solutions in ether#
The lead complex
is formed in acid and alkaline solution and in alkaline
solution its formation is not inhibited by the presence
of citrate, pyrophosphate or cyanide.
The complexes of
the other metals are also formed in both acid and
alkaline solution but in alkaline solution they are not
formed if cyanide is present*
In alkaline solution-the
iron complex is not formed if citrate or pyrophosphate
is present.
The effects of other organic solvents was not
investigated as ether Y/as found to be quite satisfactory
as an extractant*
Before lead can be estimated by the process to be
described in the next stage, it is necessary to obtain
it free from iron and copper#
This may be done by the use
of sodium diethyldithiocarbamate.
The separation may be
carried out by adding to a solution of the salts of the
metals, sodium citrate, ammonia to make alkaline and
cyanide*
On the addition of sodium diethyldithiocarbamate,
the lead complex alone is formed, and may be extracted with
zs:
ether.
The presence of citrate is essential as it
prevents the precipitation of phosphates and metallic
hydroxides in alkaline solution*
This technique has been found to be quite
satisfactory for urine, soft tissues and blood.
In the
case of milk, faeces and bone, certain modifications
have been found to be advantageous.
The ash of this class
of material contains a considerable amount of calcium
phosphate, which in spite of the presence of citrate,
tends to precipitate when the solution is made alkaline.
This is liable to prevent a quantitative separation of
lead.
For this reason, a twofold extraction has been employ
Soldium diethyldithiocarbamate is added to an acid
solution of the ash.
All the metallic complexes are
formed and all are extracted by ether.
The extracted metals
are converted into the inorganic state and the process
of extraction repeated but in alkaline solution in the
presence of citrate and cyanide.
Under these conditions
the lead complex alone is formed and this alone is
extracted with ether*
Estimation of lead
Fischer and Leopoldi (1934) described a very
sensitive colori&etrie method for the estimation of lead.
An alkaline solution of the lead salt was shaken with a
carbon tetrachloride solution of dithizone.
The lead
formed a complex with dithizone which was extracted
by the carbon tetrachloride to produce a pink coloured
ab
solution.
It was decided that this reaction merited
investigation.
Dithizone, an organic substance, dissolves in
carbon tetrachloride and chloroform to produce green
coloured solutions.
It is soluble in water only if alkalin
in reaction, the solution being coloured brown.
When an
alkaline solution of a lead salt is shaken with carbon
y
tetrachloride and dithizone, pink lead-dithizone is
formed which is extracted by the carbon tetrachloride.
If excess dithizone has been used, the colour of the lead
complex will be masked by the green colour of unchanged
dithizone.
A pure extract of the lead complex may be
obtained by separating the carbon tetrachloride and
extracting it repeatedly with dilute potassium cyanide
solution.
Unchanged dithizone but not the lead-dithizone
is removed by this process.
The reaction was studied quantitatively. . It is
necessary to have the lead present in a suitable medium.
As a result of separation, the lead will be present as
an organic complex.
To convert this into an inorganic
form, digestion with sulphuric acid and an oxidant is
most suitable.
This leaves a residue containing lead
sulphate which is insoluble in water.
To ensure solution,
water, acetic acid and sufficient ammonia was added to
make the final reaction alkaline*
In such a solution, lead
sulphate is easily soluble and by using a suitable amount
of ammonia, the correct reaction at which lead and
dithizone react may he obtained*
A series of lead solution
concentrations ranging from 0*005 to 0*8 mg* Pb, were
prepared in a medium of the above composition.
Using 10 ml
carbon tetrachloride, the lead complex was prepared.
These
were compared one against another in a colorimeter and it
was found that the depth of colour was proportional to the
concentration of lead.
Pischer and Leopold!, prior to colorimetric
comparison, treated carbon tetrachloride extracts of the
lead complex with mineral acid, the colour changing from
pink to green.
The writer has found that no advantages are
derived from this.
The writer has found that for colorimet
comparison, the best depth of colour is that containing
about 0.01 - 0.02 mg. Pb.
Excess dithizone must be used to produce a
quantitative result but too great an excess must be
avoided as under such conditions the formation of the
lead complex is inhibited.
Dithizone is very susceptible to oxidation to
produce a substance which dissolves in carbon tetrachloride
to produce a yellow coloured solution, and cannot be
removed by cyanide extraction.
This substance is produced
from dithizone in the presence of iron and copper salts
and by bright sunlight.
The absence of iron and copper
salts is effected by the separation described above.
In dealing with biological materials, the effect of copper
is practically negligable owing to the low concentrations
2.8
present.
Bright sunlight, but not diffuse light such as
present in a laboratory, produces this oxidation product
rapidly.
The writer believes that it is the ultra-violet
component of bright sunlight which is responsible for
this reaction.
In diffuse light, a carbon tetrachloride
solution of the lead complex will remain unchanged for a
very long period.
The specificity of the reaction was then
investigated.
A large number of metals were examined and
none of these interfered with the estimation of lead with
dithizone under the conditions stated above.
apparent exception was bismuth.
The only
Bismuth, even in the
presence of cyanide, was found to produce a complex with
dithizone which was extracted by carbon tetrachloride to
form an orange coloured solution.
The bismuth complex is
unstable, for when a carbon tetrachloride solution is
extracted repeatedly with dilute cyanide solution it is
removed.
More extractions with cyanide are usually
required to remove bismuth-dithizone complex than free
dithizone.
Although bismuth does not occur naturally
in human tissues and excreta, it is liable to be present as
bismuth is used in therapeutics.
The amounts of bismuth
that would occur in tissues and urine under such conditions
would not interfere with the estimation of lead.
If present
bismuth will be separated along with lead as its complex
with sodium diethyldithiocarbamate has the same properties.
Its presence will be detected at the commencement of the
colorimetric estimation of lead*
In actual experiment it
was found that 0.01 mg. Pb could be accurately estimated in
the presence of 0.1. mg. bismuth.
As a result of medication
with 1stomach powders1, containing bismuth salts, faeces
can at times contain considerable amounts of bismuth.
Under
such conditions, an estimation of lead is impracticable
and it is advisable to stop the medication and to collect
the specimen when the alimentary tract is free of bismuth.
During the course of this work, bismuth was found
in three samples of faeces, none in any other types of
material.
Apparatus and reagents
All glassware was Pyrex, ignitions were carried out
in silica dishes and glass distilled water was used.
Filter papers were washed with dilute acid and then with
distilled water.
All chemicals, even the purest, contain traces of
lead and these if used in any quantity produce quite a
large blank.
It is possible to reduce this by further
purification but it was wished to avoid this as it was
hoped to produce a method which would be suitable for
routine purposes.
For this reason, the amounts of reagents
have been kept as low as possible except in a few cases.
Fairly large quantities of sodium phosphate and citrate
are required but simple methods of purification have been
devised.
Commercial dithizone contains a yellow oxidation
product which dissolves in carbon tetrachloride to form a
30
yellow solution and it cannot be removed by cyanide
extraction.
Purification is thus essential.
It has not
been found practicable to keep purified solutions, hence it
is purified just before use.
1.
Concentrated hydrpchloric acid - analar reagent
2.
Concentrated sulphuric acid - analar reagent.
3.
Concentrated nitric acid - analair reagent
4.
Glacial acetic acid - analar reagent
5.
Perchloric acid (SO$) - analar reagent
6.
Ammonia (sp. gr. 0.88) - analar reagent
7. Potassium cyanide (10$) - Pbl (B. D.
H.)
8. Potassium cyanide (1$) Reagent 7 diluted 1 in 10
9. Ether - analar reagent
10. Sodium diethyldithioearbamate - 2$ in water
11. Carbon tetrachloride - analar reagent
12. Sodium citrate - 20$ (Lead free)
lo 1 litre of a 20$ solution of sodium citrate are added
100 ml. of 0.1$ dithizone in chloroform and the mixture
shaken well.
As required, a portion is separated and
filtered to remove suspended particles of chloroform.
13. Sodium phosphate (HagHP04 .12HgO) - io$ (lead free)
This solution is added to soft tissues andblood
prior
to ignition.
As required, the necessary volume of a stock solution
is placed in a separating funnel, ether andsodium
diethyldithioearbamate added and the mixture shaken.
31.
After allowing to settle, the lead free aqueous layer
is run off,
14*
Standard solution of lead acetate
0.1831 g. of lead acetate PbCCgHgOgJg^SHgO, is
dissolved in distilled water containing 5 ml. of
glacial acetic acid*
with water*
mg. Pb*
The volume is made up to 1 litre
1 ml, of this solution is equivalent to 0*1
This solution is diluted as required so that
1 ml, is equivalent to 0.01 mg. Pb*
15*
Dithizone reagent
A stock solution of 0*1% commercial dithizone in
carbon tetrachloride is kept.
When required, an aliquot
is shaken with an equal volume of 0*5% ammonia*
After
allowing to settle, the aqueous layer containing pure
dithizone is separated and used*
In the following, details of the method as applied
to various types of materials are described.
The final
colorimetric estimation is the same in each case.
Urin©
500 ml. of urine are evaporated to dryness in a
silica dish and then ashed by ignition.
The ash is dissolved in 100 ml. of water containing
5 ml. of concentrated hydrochloric acid.
The solution is
transferred to a separating funnel, 50 ml. of 20% sodium
citrate added and the mixture made alkaline by the
addition of ammonia, sp. gr. 0.88.
5 ml. of 10% potassium
cyanide are added*
5 ml. of 2% sodium
diethyldithioearbamate are added and the mixture extracted
three times with ether, 25 ml* being used on each occasion*
The ether extracts which are washed separately, are
transferred to a hard glass round bottomed flask.
The ether is evaporated off and the residue
digested with 1 ml. concentrated sulphuric acid and 1 ml.
perchloric acid to destroy organic matter.
The residue is diluted with water, 1 ml. glacial
acetic acid and 5 ml. ammonia, sp« gr. 0.88, added and
the mixture diluted to 25 ml. with water.'
Soft Tissues
To 100 ml. of lead free 1C% sodium phosphate in a
silica dish are added 100 g. fresh tissue.
After drying,
the mixture is ashed by ignition.
The ash is dissolved in 100 ml. of water containing
10 ml# of concentrated hydrochloric acid.
The procedure
is then as for urine.
Blood
To 100 ml. of lead free 10% sodium phosphate in
a silica dish are added 20 ml* blood.
After drying the
mixture is ashed by ignition*
The ash is dissolved in about 50 ml. of water
containing 5 ml. of concentrated hydrochloric acid*
The
solution is transferred to a separating funnel, 5 ml. of
20% sodium citrate added and the mixture made alkaline by
the addition of ammonia, sp. gr. 0.88.
5 ml. of 10% potassi
cyanide are added.
2 ml. of 2J& sodium
diethyldithioearbamate are added and the mixture extracted
twice with ether, 20 ml. being used on each occasion.
The
ether extracts which are washed separately are collected
in a hard glass round bottomed flask.
The ether is evaporated off and organic matter
destroyed by digestion with 0.2 ml. concentrated sulphuric
acid and 0.5 ml. perchloric acid.
To the digest are added, 3.5 ml. water, 0.2 ml.
glacial acetic acid and 1*5 ml. ammonia, sp. gr. 0.88.
Faeces
10 g. of dried faeces are ashed by ignition in a
silica dish.
The ash is dissolved in 100 ml. of water
containing 10 ml. concentrated hydrochloric acid.
The
solution is diluted to 200 ml. with water.
50 ml. of the ash solution are introduced into
>
,
a separating funnel and 10 ml. of 2% sodium*
diethyldithioearbamate added.
The mixture is extracted
three, times with ether, 25 ml. being used on each
occasion..
The ether extracts are collected in a hard glass
round bottomed flask and the ether evaporated off.
The
residue is digested with 1 ml. concentrated sulphuric
acid and 1 ml. perchloric acid.
The residue is diluted with water, 1 ml. concentrate<
hydrochloric acid added and the mixture heated.
The
solution is transferred to a separating funnel and diluted
to about 50 ml. with water, 5 ml. of
ZQPjo
sodium citrate
added and the mixture made alkaline by the addition of
ammonia, sp. gr. 0.88.
5 ml. of
1 0 fo
potassium cyanide
are then added, followed by 5 ml. of 2f 0
diethyldithioearbamate.
sodium
The mixture is then extracted
three times with ether, 25 ml. being used on each
occasion.
The ether extracts are collected in a hard glass
round bottomed flask.
The ether is evaporated off and the residue digested
with 1 ml. concentrated sulphuric acid and 1 ml. perchloric
acid.
To the residue are added, water, 1 ml. glacial
acetic acid, and 5 ml. ammonia, sp. gr. 0.88.
The mixture
is diluted to 25 ml. with water.
Bone
20 g. of bone are ashed by ignition in a silica
dish.
The ash is dissolved in water containing hydrochloric
acid and the solution diluted to 200 ml. with water. 50 ml. of this solution are taken and proceeded with as in
the case of faeces.
Milk
500 ml. of milk are evaporated to dryness in a
silica dish and then ashed.
The ash is dissolved in water
containing hydrochloric acid and proceeded with as in the
case of faeces.
The colorimetric estimation of lead
Preparation of the standard
;
The following mixture is prepared.
To 1 ml. of
concentrated sulphuric are added water, 1 ml. glacial acetic
35T -
and 5 ml. ammonia, sp. gr. 0.88.
The mixture is diluted
to 25 ml. with water.
A known amount of lead is added to 5 ml. of this
solution.
To this are added 5 ml. of 1fo potassium cyanide
and 10 ml. carbon tetrachloride.
An ammoniacal solution
of dithizone is added drop by drop, with constant shaking
until excess has been added.
Too great ah excess must be
avoided. 'Sufficient excess is indicated when the carbon
tetrachloride layer has reached its maximum intensity of
redness and the aqueous layer in tinged brown.
layer is then separated and discarded.
The aqueous
The carbon
tetrachloride layer, containing the red coloured lead
complex, is shaken repeatedly with aliquots of 5 ml.
of 1fo potassium cyanide until excess dithizone has been
removed as shown by the aqueous layer being no longer
coloured.
The carbon tetrachloride extract is then passed
through a filter paper to remove droplets of water and is
then ready for comparison.
A range of standards may be prepared but the
writer prefers to use a standard containing 0.02 mg. Pb
and using a volume of unknown to conform to this.
This
standard may be prepared by using 2 ml. of a standard
solution of lead acetate containing 0.01 mg. Pb per ml.
Preparation of the unknown ;
Urine, faeces, soft tissues, bone and milk ;
The lead is contained as lead sulphate in a
solution of ammoniacal ammonium acetate with a volume of
25 ml.
To 5 ml. of this solution are added 5 ml. of 1^
potassium cyanide and 10 ml. carbon tetrachloride.
The
colour is developed in the same way as that of the standard.
In the event of the lead content of the unknown
being low, 10 ml. of the solution is used, the amounts
of the other reagents being the same.
If the lead content
of the unknown is high, a smaller volume than 5 ml. is used.
In this case the solution is diluted to 5 ml. by the
addition of an ammoniacal ammonium acetate solution
having the same composition as that used to prepare the
standard.
The amounts of the other reagents are the same.
Blood ;
In the case of blood, the whole of the lead
containing solution is used.
To the mixture, containing
the lead in the flask used for digestion, are added 5
ml. of
l< f0
potassium cyanide and 10 ml. of carbon
tetrachloride and the colour developed as above.
Blank
;
A blank should always be done on a new set of
reagents.
In estimating the blank, the complete process
is carried out.
The blank is small and is thus difficult
to estimate accurately.
has been adopted.
As a result, the following method
Before the estimation, 0.02 mg. Pb
is added to the blank.
This after development of the
colour with dithizone is compared with a standard
containing 0.02 mg. Pb.
The blank is then calculated from
the difference.
o
3 * f.
With the exception of “
blood, the development of
the colour is “best carried out in glass stoppered tubes.
A aeries of recovery experiments were carried out
and the results are shown in Table 1.
It will be
seen that added lead could be estimated accurately.
The method that has been described is specific
for lead and gives accurate results.
An estimation can
be completed in a comparatively short time.
drying and ignition, an
Excluding
estimation may be completed in
1 hour.
Since this method has been published, new methods
for estimating lead in biological materials have been
published by other workers
;
spectrographic J^Blumberg
and Scott (1935), Gholak (1935) ^ ; polarigraphic
£ Teisinger (1936)
;
colorimetric dithizone methods
j^Hubbard (1937), Willoughby and Wilkins (1938)J .
3 8.
Table I. The recovery o f added Pb
0-042
0-042
0-042
0-155
0-155
Pb added
mg.
0-050
0-100
0-200
0-250
0-500
0-020
0-050
0-100
0-200
0*400
Total Pb
found
mg.
0*163
0-215
0-308
0-362
0-625
0-079
0-096
0-140
0-360
0-565
Pb
recovered
mg.
0-043
0-095
0-188
0-242
0-505
0-037
0-054
0-098
0-205
0-410
Faeces (10 g.)
0-030
0-030
0-030
0-042
0-042
0-042
0-031
0031
0-031
0-200
0-500
1-000
0-030
0-050
0-100
0-040
0-080
0-120
0-235
0-549
1-036
0-079
0*096
0-140
0-072
0-115
0-154
0-205
0-519
1-006
0-037
0-054
0-098
0-041
0-084
0-123
Milk (500 ml.)
0-050
0-050
0-050
0-050
0-050
0-100
0-200
0-500
0-105
0-145
0-248
0-560
0-055
0-095
0-198
0-510
Liver (100 g. fresli)
0-243
0-243
0-243
0-243
0-050
0-100
0-200
0-400
0-299
0-350
0-440
0-640
0-056
0-107
0-197
0-397
Pg-
^g10
20
40
10
20
40
10
20
40
Pg-
P g-
27
37
58
28
39
58
22
34
53
11
21
42
9
20
39
10
22
41
Urine (500 ml.)
Initial Pb
content
mg.
0-120
0-120
0-120
0-120
0-.120
Blood (20 ml.)
16
16
16
19
19
19
12
12
12
3<J.
The Distribution of Lead in Human Tissues
f.
The available data concerning the presence or
otherwise of lead in 'normal* human tissues has been in
a very conflicting state.
Meilliere (1903) stated that he could detect lead
in the organs of nearly all the subjects examined by him.
Aub et alia (1926) state that the lead retained by an
apparently normal individual is held almost exclusively
by the skeleton.
Later work illustrated the presence of
lead in 'normal* bones but there was considerable
variation.
Barth (1931) found 0.01 - 0.06 mg. Pb per g. ash
or approximately 5 - 30 mg. Pb per kg. fresh bone, while
Lynch et alia (1934) found 14 - 146 mg. Pb per kg. fresh
bone.
Yfeyrauch and Muller (1933) found no appreciable amoun
of lead in liver, spleen or brain.
Sheldon and Ramage (1931
using a spectrographic method, found lead occurring
spasmodically in 'normal! human tissues while Boyd and De
(1933), also using a spectrographic method, found lead
well marked in the liver and present in all organs examined
except the brain.
Lynch et alia (1934), in an examination
of a few organs, obtained values of the order of 1.5 mg.
Pb per kg. fresh tissues in some livers and none in others.
Kehoe et alia (1933) found appreciable amounts of lead in
the tissues of two cases apparently normal shortly before
death.
The conflicting results reported are undoubtedly
due to inaccurate methods.
Tissues were obtained post-mortem from persons with
no history of exposure to lead other.; than the 1normal®
hazard, and the lead content determined,
The results are
shown in fable 2a.
In a later investigation, the lead contents of
different types of bone viz. rib, vertebra, femur and
tibia were examined,
The results are shown in fable 2b.
The lead content of normal blood was then
determined*
This was taken from living persons and was
undertaken as it was considered that such an estimation
might prove of value in the diagnosis of lead poisoning.
The blood was collected in an all glass syringe fitted
with a stainless steel needle, transferred to a Byrex
teat tube and measured as quickly as possible.
20 ml. were used.
About
The results are shown in Table 2c.
In an examination of the tissues of persons with
no history of exposure to lead other than the ’normal®
hazard, lead was found in every one examined viz. bone,
liver, spleen, kidney, lung and brain.
concentrations were present in bone.
The highest
Bones such as
femur and tibia were found to contain much higher
concentrations of lead than rib or vertebra.
Of the soft
tissues, the highest concentrations were found in the liver
It would appear that the lead content of such bones as
femur and tibia increases with age.
The lead contents of r
vertebra and soft tissues are unaffected by age.
Lead was found to be invariably present in
circulating blood.
In 40 cases, the figures ranged from
40 - 70y**g. Pb per 100 ml., with an average of 55y^g.
Pb per 100 ml. blood.
Since this work was completed and published,
investigators in various parts of the world have recorded
the lead content of 1normal1 human blood.
These confirm
the figures reported here ^Blumberg and Scott (1935)
- spectrographic method ; Kehoe, Thamann and Cholak (1935)
- spectrographic method ; Teisinger (1936) - micropolarigrapi
method ; Taeger and Schmitt (1937) - dithizone method ;
Willoughby and Wilkins (1938) - dithizone method
<
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Table 2q
The lead content of the blood of ’normal1
men and women
yM-g. Pb per 100 ml*
No* of cases
Blood lead
Average
40
5
45
7
50
10
55
5
60
8
65
4
70
1
55
Total
■
40
The Lead Content of the Tissues of
Human Foetuses
The tissues of four stillborn human foetuses were
obtained and their lead content estimated*
The results
are shown in Table 3.
Appreciable amounts of lead were found in these
tissues but the concentrations were much lower than in
tha adult*
It must be assumed that lead is transferred by*
the mother to the foetus through the placental circulation.
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Lead in Milk
As lead could "be detected and estimated in all
tissues and excreta, examined, one would naturally expect
it to be secreted in milk and an excellent opportunity
arose to investigate this.
On a farm in a neighbouring county, water was
supplied through a lead pipe.
'This water was very
plumbosolvent and was found to contain 6 mg. Pb per litre.
This water was consumed by the cows.
Samples of milk
were obtained from various cows and the lead content
estimated.
The results are shown in Table 4.
Lead in appreciable amounts was present in all
of the samples, values of 0.069 - Q.&12 mg. Pb per litre
being obtained.
Table 4
The Lead Content of Milk
mg. Pb per litre
1.
0.094
2.
0.069
3.
0.093
4.
0.083
5*
0.170
6
.
0.212
7.
0.143
4*1-
The Lead Content of Homan Excreta.
Originally it was the practice for a sample
of urine or faeces to he sent to the laboratory to he
tested for the presence of lead if plumbism was sus­
pected.
A positive result was taken to support the
diagnosis.
Later it was realised that lead occurred
in urine and faeces under normal conditions, although
the amounts were small.
Several, investigators then
attempted to determine the normal range, especially
that in urine
^Cooksey and Walton (1929), Francis et
alia (1929), Kehoe et alia (1933) J *
These
investigations were undertaken in order to determine
a standard upon which a diagnosis of plumbism might
he made in a suspected case.
were taken as to diet etc.
No particular precautions
The results were expressed
in mg. Pb per litre in the case of urine and in mg.Ph.
per 100 g. dried faeces or in mg. Ph per g. faecal
ash.
The mean values for urinary lead were much the
same hut the ranges showed great variation.
It has been the experience of the writer that
often in plumb ism, especially in the mild type, the
excretion of lead does not greatly exceed that in the
normal.
According to Kehoe et alia (1933)# removal
from exposure to large amounts of lead, results in a
rapid fall in excreted lead until values just above the
normal/
SO
*
normal are reached, these levels being then maintained
for a long period.
It is the mild type that is liable
to defeat the clinician and it is in this type of case
that the biochemist's assistance is most needed.
The writer considers that results should be
expressed in terms of mg. Pb per day, as this would
eliminate many variable factors.
In addition the results
should have reference to some standard diet, the lead
content of which is known, in fact one should attempt
to do a lead balance.
Por a number of years it has been our practice
in the Glasgow Royal Infirmary to do this.
The patient
is placed on a standard diet, the lead content of which
is known, and the urine and faeces collected over a
period of 3 days.
The lead content is then estimated.
A number of normal patients were examined
thus.
The diet contained 0.22 mg. Pb per diem.
The
results are shown in Table 5 &&& indicate that for all
practical purposes they were in balance.
At the head
of the Table are shown the lead contents of the urine
and faeces of 3 persons whose diets were uncontrolled.
These are much higher.
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a
Origin of the Lead present in Normal Tissues and Excreta.
The lead present in the tissues and excreta of
’normal1 human "beings appears to he of twofold origin;
1. lead in foodstuffs and drinking water, 2. lead present
in the atmosphere.
A large amount of the food that we consume
contains traces of lead.
by Monier-Williams (1938)•
This subject has been reviewed
This lead is not due entirely
to the manufacturing processes through which much of the
food of a civilised community passes, for lead is distri­
buted very widely throughout this planet, even normal soil
containing traces.
Kehoe et alia (1933) found lead present
in the urine, faeces and foodstuffs of a community of
Indians living under primitive conditions.
The figures
were lower than those of a civilised community.
Drinking
water is responsible for a certain amount of lead for
although natural waters do not as a rule contain
appreciable amounts of lead, the use of lead pipes and
storage tanks is still common.
The writer has estimated
the lead content of the drinking water used in various part
of Glasgow.
The results are shown in Table 6 indicating
that appreciable amounts are present.
Another source of lead is that present in dust,
especially in industrial regions.
This lead would be
absorbed from the respiratory tract.
gations/
A number of investi­
investigations of the lead content of both the air
and dust in industrial towns has been made.
In
Bradford, Leeds and Huddersfield, Manley (1937) found
that the dust contained 0.053 - 1.234^ lead.
5^
Table
6.
The Lead Content of the Drinking Water in the
City of Glasgow.
The results are expressed
in mg. per litre.
Remarks.
1.
O.OJ
iron pipes.
2.
0.09
lead pipes only.
3.
0.06
«
4.
0.08
"
5*
0.43
lead pipes and (
storage tank.
6.
0.57
M
7.
0.55
8.
0.34
n
9.
0.27
«
Factors Influencing the Absorption of Lead.
Lead may be absorbed from the respiratory tract,
alimentary tract and skin.
In industrial lead poisoning
absorption from the respiratory tract is considered to
be the important factor.
In the ’normal* hazard and in
lead poisoning due to contamination of foodstuffs and
drinking water, absorption is from the alimentary tract.
Absorption from the alimentary tract is undoubtedly
influenced by a number of factors.
Aub et alia (1926) compared the degree of
absorption of lead in two groups of cats, one group being
on a milk diet and the other on a milk free diet. They
could detect no marked difference and concluded that the
amount of calcium in the diet had no influence upon the
amount of lead absorbed from the alimentary tract.
Shelling (1932) produced evidence which seemed to suggest
that diets high in calcium render lead more toxic than
diets high in phosphorus.
Weyrauch and Necke (1 9 3 3 )
found that the simultaneous oral administration of milk
or mucilage and white lead to rabbits did not decrease to
any appreciable extent the amount of lead absorbed.
On
the other hand they found that oil and margarine increased
the amount of lead absorbed by more than 10 times. Sobel
et alia (1 9 3 8 )
a study of experimental lead poisoning
in/
s b
in rats, found that about twice as much lead was
absorbed on a diet containing viosterol as without it.
Adult male mice were the animals used throughout
this work.
Two basic diets were used, namely a high
calcium diet and a low calcium diet of the following
compositions;
Low calcium diet
Shelling (19J2)
Whole wheat flour
t>*
400
Casein.
100
Corn starch.
325
Wheat gluten
50
Olive Oil.
40
Sodium chloride.
20
Potassium chloride.
15
Butter
omitted.
High calcium diet.
Whole wheat flour
£•
700
Whole milk powder.
J00
Marmite
50
In the first experiments the effect of high
and low calcium diet upon the absorption of lead was studied.
The mice were fed with the specified diets (2.5
per/
5*j
&•
per mouse per day) to which were added supplements of
lead in the form of lead acetate. Bach mouse was housed
in a separate glass jar, floored with sawdust.
Ho
restriction was made upon the amount of water drunk.
The amount of diet supplied was constant per mouse per
day and was made up into a thick paste with water and
placed in a porcelain dish, this minimising loss by
spillage.
Ho difficulty was experienced in obtaining
complete consumption of the daily rations.
experimental period was 14 days.
The
At the end of this
period the jars were cleaned out and all the animals
placed on the high calcium diet for 4 days to remove
unabsorbed lead from their alimentary tracts.
animals were then killed.
The
The lead content of each
whole animal was then determined.
The lead content of
control animals and also of unsupplemented diets were
also estimated.
The results are shown in Table 7«
It will be seen that absorption of lead was
high on a low calcium diet and low on a high calcium
diet.
In view of this, experiments were carried out
in which mice were fed on the low calcium diet containing
added supplements of lead to which had been added calcium
glycerophosphate (0.5 £• P©** mouse per day).
This in
effect converted the low calcium diet into a high calcium
diet/
S
*
diet. Here again the effect of the high calcium diet
was to produce only a small absorption of lead (Table 7)*
The effect of fat and vitamin D upon the
absorption of lead was then stxxdied.
It was considered
that fat might hinder the absorption of lead by the
formation of insoluble lead soaps.
In this series of
experiments the mice were placed upon the low calcium
diet, containing supplements of added lead, to which
was added olive oil at the rate of
day.
1
ml. per mouse per
Ho specific influence of fat upon the absorption
of lead could be detected (Table 7)-
It was considered
that vitamin D might aid the absorption of lead in the
same way that it aids the absorption of calcium.
In this
series of experiments mice were placed on a) the high
calcium diet, b) the low ealcium diet, containing
supplements of added lead to which was added cod liver oil
at the rate of 3 drops per mouse per day.
The experimental
results obtained did not indicate any marked influence by
vitamin D upon the absorption of lead (Table 7)*
It is interesting to speculate as to the reasons
for the influence of calcium upon the absorption of lead
from the alimentary tract. According to Shields et alia
(1 9 3 9 ) absorption of lead takes place not in the stomach
but rapidly from the small intestine.
be/
5?*
Lead in diets1 will
be in insoluble forms but will pass into solution rapidly
in the stomach owing to the action of the acid of the gastric
juice.
Upon entering the small intestine, the stomach
contents will meet the alkaline pancreatic fluid and bile,, as
a result the now intestinal contents will tend to become
neutral and possibly alkaline in reaction.
will tend to precipitate the lead.
This process
It is generally agreed
that lead to be absorbed must be in solution.
As a result
the degree of lead absorption will depend upon the rate at
which the stomach contents are neutralised in the small
intestine.
It is suggested that upon a low calcium diet,
owing to the low base content, the stomach contents are more
acid than usual and as a result neutralisation in the small
intestine will take longer while on high calcium diet, owing
to the high base content, the opposite takes place.
Although large amounts of lead were absorbed on a
low calcium diet, the absorption by animals receiving the
same quantities of lead in different experiments showed
marked variation.
Other important factors must therefore be
involved such as the amount of water consumed etc.
Under
comparable conditions however, the amount of lead absorbed
is related to the amount of lead consumed.
Telfer (1939) has shown that the absorption of
calcium and magnesium from the alimentary tract is increased
by the oral administration of hydrochloric acid.
probable/
fo'O
It is
probable that lead would behave similarly.
The following
experiment was carried out. A number of mice were fed with
high calcium diet containing 1 mg. Pb (as lead acetate)
and
0.5
ml. N. hydrochloric acid per mouse per day for a
period of 14 days.
At the end of this period they were
placed on the high calcium diet alone for 4 days to remove
unabsorbed lead from their alimentary tracts and then
killed.
The total lead content of each animal was then
estimated.
The results are shown in Table
8
. It will be
seen that lead is more easily absorbed under these
conditions.
The results of this experiment lend support
to the theory advanced above, and indicate the importance
of the degree of gastric acidity on the absorption of lead.
are expressed as (A) mg. total Pb, (B) mg. Pb per 100 g. body wt.
Supplement of Pb
(mg. Pb per mouse
per day)
A
B
Exp. 1
2
3
4
Average
0-046
0-143
0-057
0-133
0-224
0-681
0-259
0-665
0-457
0-10
A
B
Low Ca diet
0-159
0-795
0-211
1-055
0-149
0-709
0-182
0-867
0-856
Exp. 5
0
Average
0-033
0-042
0-173
0-221
0-197
High Ca diet
0-042
0-210
0-054
0-284
0-247
Exp. 7
8
Average
0 ■05
0-50
1-00
A
B
A
B
0-254
0-510
0-200
0-450
1-270
2-55
0-87
2-37
1-76
0-445
0-909
0-385
0-666
2-42
5-05
1-68
1-33
2-63
0-055
0-042
0-282
0-200
0-241
Low Ca diet + Ca glycerophosphate (high Ca diet)
0-027
0-142
0-028
0-155
0-029
0-129
0-026
0-108
0-027
0-117
0-030___ 0-115
0-125
0-136
0-122
Exp. 9
10
Average
0-133
0-125
Exp. 11
12
Average
0-128
0-133
Exp. 13
0-029
0-057
0-317
0-050___ 0-2_17
0-267
0-028
0-03G
0-143
0-180
0-162
1-25
3-10
2-18
0-366
0-833
1-74
3-97
2-86
Low Ca diet -food liver oil
0*222
0-609
0-925
0-714
0-665
0-159
0-757
0-360
0-841
0-637
3-10
2-00
2-55
0-800
0-500
4*21
2-63
3-42
High Ca diet + cod liver oil
0-132
0-167
0-040
0-046
0*230
0-054
0-13E
0-633
0-571
0-602
Low Ca diet + olive oil
0-182
0-860
0-250
0-252
1-200
0-714
1-030
Pb content of control animals = 0-020 ±0-003 mg. Pb.
Pb content of the high Ca diet = 0-002 mg. Pb per mouse per day.
Pb content of the low Ca diet = 0-002 mg. Pb per mouse per day.
62
IAB»l E 8-
■Adult M ale M ice. High Calcium D iet -\- 1 mg. P b (lead acetate)
0-5 ml. N H C l per mouse per day. Period, 14 days.
1
2
3
4
5
6
7
8
Total lead.
(mg.)
1*429
0*909
1*111
1* 176
1*000
0*667
0*909
1*111
Weight.
(g.)
28
19
21
21
23
17-5
21-5
31-5
£>3
Lead.
(mg. Pb per 100 g.)
5*10
4*78
5*29
5*60
4*35
3*81
4*23
3*53
Average 4*58
The Lead Content of the Blood and Excreta in Suspected
Plumb ism.
The lead content of the blood and excreta in a
number
of cases of
suspected plumbism has been examined.
The excreta were collected over periods of J days
while the
patients
were on
the standard diet (see pageS
i
In three cases the excreta were collected when the patients
were on other diets ; Case No*19 was on low calcium diet +ammonium chloride during one period and on high calcium
diet during another period; Case No.29 was on the standard
diet during one period and on high calcium diet during
another period;
Case No.JO was on the standard diet during
one period and on the standard diet and ammonium chloride
during another period.
In every case, the blood lead was
estimated once or several times but the excreta were not
examined in every case. The results are shown in Tables
9a and 91>-
Table 9s- are included those cases in which
the blood lead did not exceed 100 jfig. Pb per 100 ml.
9b
Table
includes those cases in which the blood lead at some
period exceeded 100^ug. Pb per 100 ml.
in Table
9b
The cases included
were all diagnosed as plumbism while of the
cases included in Table 9&* only Case No.19 was confirmed
as such.
In the cases of lead poisoning diagnosed as such
(Table
some/
9^>)»
"the blood lead was at some period increased.
In
some of these oases, the excretion of lead was increased
greatly hut in others it did not greatly exceed the normal.
In Case No. 24 it will he noted that the amount of lead
excreted decreased to a level not greatly exceeding the
normal as the time of removal from exposure to lead
increased.
This is in agreement with the findings of
Kehoe et alia (1933)Of the cases shown in Tahle 9a, only No,19
was finally diagnosed as plumbism as although the hlood
lead was normal, the excretion of lead was high.
In the
other cases, the hlood lead and lead content of the
excreta were normal or raised only slightly.
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The Mobilisation of Lead.
Aub and his co-workers (1926) believed that lead
is stored in the bones and as such exerts no toxic action.
They believed that the lead present in the soft tissues
is solely responsible for the toxic episodes that occur in
plumbism.
As a consequence, the condition of a person who
has absorbed large quantities of lead will depend upon
the distribution of lead between the soft tissues and the
skeleton.
Aub et alia (1926) have shown clinically that
the toxic episodes of plumbism are relieved quickly by
high calcium therapy and that the clinical improvement is
accompanied by a decrease in the amount of lead excreted.
This would suggest that lead is being laid down in the
skeleton.
The clinical side of this work has been confirmed
by Belknap (I929).
Two different views are held regarding the
treatment of plumbism ; in one a high calcium intake is
recommended in order to keep lead stored in the bones;
the other favours a process of de-leading as it is
considered that a large store of lead in the bones cons­
titutes' a potential danger as in metabolic disturbances
it is liable to be mobilised and pass into the soft tissues.
Aub et alia (1926) support the second view.
Aub et alia (1926) carried out an investigation
of/
■
of agents likely to produce mobilisation of lead.
They
found that acidosis producing substances e.g. ammonium
chloride and mineral acids, potassium iodide, sodium
bicarbonate and low calcium intake have this property.
Similar results were obtained with parathormone in con­
junction with a low calcium intake by Hunter and Aub (1937)*
In this work the efficiency of the agent was assessed by
its ability to increase the total excretion of lead.
Litzner, Weyrauch and Barth (1931) could confirm the
efficiency of sodium bicarbonate but not of acidosis pro­
ducing substances, their conclusions being based upon the
effect of the agent upon the urinary excretion of lead.
Apart from effects upon excretion, no worker has
shown experimentally that these agents do actually produce
a liberation of lead from the skeleton and their effect
upon the blood lead has not been studied.
The following
investigations were concerned with these.
A
These investigations were carried out on animals,
adult male mice being used.
The lead content of a number of mice was
increased by feeding them with a low calcium diet containing
lead (as lead acetate) at the rate of 1 mg. Pb per mouse
per day for a period of 14 days.
In the first experiment, the animals were fed
on/
on the low calcium diet and lead as described above. At
the end of the period, one third of the animals were kept
on low calcium diet, but without lead for 4 days and
then killed (a).
The remainder were placed on a high
calcium diet for 7 days and then half of their number
were killed (b).
The remainder were placed on the low
calcium diet, and then killed at the end of 7 days (c)*
In each mouse, the soft tissues were separated from the
skeleton and their lead contents estimated.
The percentage
of the total lead present in the soft tissues and skeleton
was calculated.
The results are shown in Table 10.
It will be seen that following absorption of
lead on a low calcium diet, lead predominated in the soft
tissues.
The effect of high calcium diet was to cause
lead to predominate in the skeleton but it should be noted
that the amount of lead in the soft tissues is not
negligible and is probably quite capable of producing
damage.
The transference of the mice from high calcium to
low calcium diet resulted in an increased percentage.of
lead in the soft tissues.
These results indicate that
the distribution of lead between the soft tissues and
skeleton is influenced by the calcium content of the diet.
In the following experiments the effect of other
agents were studied.
1.
These were,
Potassium iodide with low calcium intake,
2/
7a
2.
Sodium bicarbonate with low oalcium intake.
3.
Ammonium chloride with low calcium intake.
4.
Potassium iodide with high calcium intake.
5.
Sodium "bicarbonate with high calcium intake.
6.
Ammonium chloride with high calcium intake.
The lead content of adult male mice was increased
as described above.
They were then placed on a high ♦
calcium diet for 7 days in order to produce a predominance
of lead in the skeleton.
The animals were then placed
on treatment for 7 days, after which they were killed and
the lead content of their soft tissues and skeletons
estimated.
The percentage of the total lead present in
soft tissues and skeleton was then calculated.
The
results are shown in Table 11.
It will be seen that these substances produced
mobilisation of lead on both high and low calcium intake.
That the substance was producing an effect on low calcium
intake, which itself has a mobilising effect, is indicated
by the higher percentage of lead in the soft tissues.
Several important facts emerge from this
investigation.
Acidosis producing substances, which are
usually used in conjunction with a low calcium intake to
produce mobilisation, are effective on a high calcium
intake.
Acidosis from metabolic causes should therefore
be avoided in plumbism as a high calcium intake cannot
neutralise/
7/.
neutralise its effect. Potassium iodide and sodium
bicarbonate should not be administered to persons with an
excess
of lead unless mobilisation of lead is desired.
’Stomach1 powders, containing sodium bicarbonate, are
common offenders in this respect.
of the soft tissues
and bones of mice.
The animal was skinned and the carcase heated with a
small quantity of distilled water in a silica dish on a
steam bath.
At the end of JO minutes, the bones could be
separated quite easily from the soft tissues.
It was
considered that under such conditions, possible solution
of lead present in the bones would be a minimum.
skin was included with the soft tissues.
The
IO
The results are expressed in m g. Pb.
a
.
On loiv calcium diet alone.
Mouse No.
Lead content of soft tissues
Lead content of skeleton
T otal lead .
.
.
.
.
Percentage of lead in skeleton
Percentage o f lead in soft tissues
b
.
l.
0-154
0-105
0-259
40-6
59-4
2.
0-168
0-125
0-293
42-8
57-2
3.
0-150
0-100
0-250
40
60
Low calcium diet followed by high calcium diet.
Mouse No.
4 .
Lead content of soft tissues
Lead content of skeleton
T otal lead .
.
.
.
.
Percentage o f lead in skeleton
Percentage of lead in soft tissues
0-077
0-250
0*327
76-4
23-6
5 .
0-089
0-312
0-401
77-8
22-2
6.
0-080
0-282
0-362
77-9
22-1
c. Low calcium diet followed by high calcium d iet, followed by
low calcium diet.
Mouse No.
7.
Lead content of soft tissues
Lead content of skeleton
Total lead .
.
.
.
.
Percentage of lead in skeleton
Percentage of lead in soft tissues
0-077
0-143
0-220
65
35
8 .
0-087
0-151
0-238
64
36
9 .
0-090
0-178
0-268
62
38
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B.
In this investigation, the effect of certain
agents upon the lead content of the "blood in plumb ism was
investigated.
In three of the cases, the lead content
of the excreta was determined.
The results are shown in
Charts 1 - 11.
The effect of variation in the calcium content of
the diet upon lead mobilisation as shown by the amount of
lead in the blood is illustrated by Cases 4 - 1 1 , a high
calcium intake being followed by a marked fall in blood
lead.
As the treatment in some cases lasted over a period
of weeks, this may have been due in part to loss of lead
by excretion.
That the calcium content of the diet does
exert a powerful influence is shown by Case 7> who was
placed alternately on high and low calcium intake.
high calcium intake, the lead content
On
ofthe blood fell
while on low calcium intake it rose.
The effect of an acidosis producing substance via.
ammonium chloride in conjunction with a low calcium diet
was studied in Cases 1 - 3 *
ln Cases 1 and 3> "the effect
was to produce marked increases in blood lead,
the levels
being very much higher than with low calcium diet alone.
In Case 1, in which the rate of lead exception was
investigated, the total amount of lead excreted was
increased but not to anything like the same level as the
increase/
increase in blood lead.
In Case 3# high calcium intake
and acidosis therapy were alternated several times.
On
each occasion responses were obtained as evidenced by
changes in the blood lead.
In Case 2, the blood lead
was hardly affected by ammonium chloride treatment although
the rate of lead excretion was increased.
This may be
explained by the fact that this subject had been exposed
to lead for only a short period and consequently his store <
lead would be comparatively low.
Case 11 illustrates the
effect of potassium iodide upon the blood lead.
In this
case also, the increase in blood lead was not accompanied
by a similar increase in lead excreted.
Changes in the excretion of lead as the result
of mobilisation were most marked in the faeces, the
urine being hardly affected.
Case
I.
Lead "burner (battery works)
Male - 2 9 years.
Lead poisoning.
The investigations were carried out over an
extended period.
Following high calcium intake for 14
days, the patient was placed on low calcium diet and
ammonium chloride for 9 days.
Following this, high
calcium diet was maintained.
During the beginning of the
last period, injections of calcium gluconate were given.
During the period of medication with ammonium chloride
days before and after it, the excreta were
6
collected in periods of
estimated.
days and the lead content
The lead content of the blood was estimated
at intervals.
346
3
IS39
The results are shown in Charts la and lb.
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Case
2.
Red lead worker.
Male -
years.
32
lead poisoning.
This patient was treated similarly to Gase
/
1.
The results are shown in Charts 2a and 2b.
387
267 252 267
CHART 2 A
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White lead worker.
Male -
66
years.
Lead poisoning.
This patient was placed on alternate periods
of high calcium intake and low calcium intake and
ammonium chloride,
finally he was maintained on
high calcium intake. The lead content of the "blood
was estimated at intervals.
Chart 3.
The results are shown in
Lead workers with vague symptoms.
Males - 2 3 , 33 and 56 years.
Increased lead absorption
The lead content of the blood of these
patients was determined initially and then after a
period of high calcium intake.
The results are shown
in Charts 4, 5# an(i 6 .
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4NO TREATMENT
COL DIET-
CASE
7
Lead poisoning due to a lead contaminated drinking
water.
Female - 27 years.
The effect of high and low calcium intake upon
the lead content of the blood was investigated.
The
results are shown in Chart 7*
Cases 8. 9 and 1 0 .
Increased lead absorption due to a lead contaminated
drinking water.
Males - 34 and 7 years.
Female - 9 years.
The lead content of the blood was determined
initially and then after a period of high calcium intake.
The results are shown in Charts 8, 9 &&d 10.
m
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Case 11
Solder maker
Male
Lead poisoning
The effect of potassium iodide upon the blood lead
and the amount of lead excreted was investigated*
are shown in
n
G>H R f t T
The result
H i©
Chart No . 11#
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The Lead Content of the Tissues in Increased
Lead Absorption.
Tissues were obtained from 4 cases with
histories of abnormal exposure to lead and their lead
contents estimated*
The results are shown in Table 12.
The lead content of the tissues of the second
case are within normal limits except rib which shows a
slight increase. The exposure was probably only slight.
In the other eases, all the soft tissues show
marked Increases.
In bone, the most marked increases
*
are in rib and vertebra .
From an examination of *normal* tissues, one
concludes that the long bones have the greatest affinity
for lead.
During life there appears to be an accumulation
of small quantities of lead, moat of which is laid down
in such bones as femur and tibia but not in bones of
the structure of rib and vertebra.
The probability is
that such lead is difficult to mobilise.
When com­
paratively large quantities of lead are being absorbed
over a short period, it appears to be laid down in such
bones.as rib and vertebra rather than in tibia and femur.
This is probably due to their more vascular nature, and
it would appear that such lead is comparatively easy to
mobilise.
If it were possible to compare the total lead
content of an old person with no history of abnormal
exposure/
^3
exposure to lead with that of a young person with a history
of abnormal exposure to lead and with active symptons of
plumbism, one might find little difference, the difference
between the two conditions being due to differences in
distribution, chiefly amongst the bones.
In a chemical examination of the tissues of a case of
suspected plumbism we have in the past been inclined to lay
stress on the results obtained from the long bones.
The
results obtained here indicate that the data obtained from
rib and vertebra are more important#
The results are expressed in mg. Pb per
kg. fresh tissues.
Sex.
Age.
1.
2.
3-
4.
M.
M.
M.
P.
60
41
60
26 .
Liver
4.50
2.40
5.40
7 *14
Kidney
1.00
2.88
2.00
4.62
Brain
1.00
-
1.36
J.08
119.4
22.0
50.6
52.1
18.8
8.5
13.3
Rib
Vertebra
Femur
-
-
50.6
52.1
Tibia
mm
—
78.9
55-3
Metal
worker.
Lead eontaminat
drinking water.
Remarks
Painter
Printer
The Excretion of Lead.
Kehoe et alia (1933) have shown that after removaj
from exposure to lead, excretion falls rapidly until levels
just exceeding the noiimal are reached.
The writer
considered that it would he of interest to determine the
rate at which recently absorbed lead could be eliminated.
The lead content of adult male mice was increased
by the method described already.
The animals were then
placed on high calcium diet for 7 days to remove unabsorbed
lead from their alimentary tracts and to produce a
transference of lead from soft tissues to skeleton.
They
were then placed in separate glass jars, floored with
filter paper.
Half of the mice were placed on high calcium
diet and the remainder on low calcium diet for a period
of 14 days.
At the end of this period the animals were
killed and their total lead contents estimated.
The lead
content of the material in the jars was then estimated.
After allowing for the lead content of the filter paper and
the diet consumed dux^ing the experimental period, the amounl
of lead excreted was calculated.
Fx^om these figures, the
percentage of the original total lead that had been
eliminated, was calculated.
The results are shown in
Table 1 }.
It will be seen that recently absorbed lead may
be eliminated very rapidly.
On low calcium diet, about 60fo
was excreted while on high calcium diet about 251° was excrel
-L e a d E xcreted in 14 days on L o iv a n d H ig h C a lc iu m D ie ls .
L o w C a lc iu m D ie t.
2.
0-150
0-225
0-375
Mouse No. 1.
Total lead (mg.)
. 0-105
. 0-175
Excreted lead (nrg.)
Original total lead (mg.) .
. 0-280
Per cent, original total lead
. 62-5
excreted
3.
0-141
0-220
0-361
4.
0-099
0-198
0-297
60-9
66-7
6.
7.
8.
0-184
0-049
0*231
0-289
0-068
0-357
0-320
0-082
0-402
60
H ig h C a lc iu m D ie t.
Mouse No. 5.
. 0-193
Total lead (mg.)
. 0-060
Excreted lead (mg.)
. 0*253
Original total lead (mg.) .
Per cent, original total lead
. 23-7
excreted
21-2
19
20-4
Discussion.
A method has been described for the estimation
of lead in biological materials.
It is specific for
lead and gives accurate results.
An estimation may be
carried out quickly and as no complex apparatus is
required, it is very suitable for routine use.
In the
hands of other workers it has proved successful.
It
cannot be over emphasised that in work of this nature,
reliable data cannot be obtained unless the method of
analysis is satisfactory.
Much of the earlier work on
the biochemistry of lead suffered from this defect.
It has been shown quite conclusively that lead
occurs in the tissues and excreta of persons with no
history of exposure to lead other than the ’normal*
hazard.
The origin of this lead has been discussed viz.
food and lead containing dust.
The distribution of lead
throughout the body is however uneven.
The highest
concentrations appear to be situated in the skeleton but
even here, the distribution is uneven, for such bones
as femur and tibia contain higher concentrations than
rib and vertebra.
Of the soft tissues examined, liver
was found to have the highest concentration.
The-
concentration of lead in the long bones appears to
increase with age whereas that of the other tissues is
unaffected.
of/
There appears to be a very slight retentioi
of lead throughout life, most of which is laid down or
stored in the long hones.
All lead absorbed from the
alimentary tract must pass through the liver, where much of
it is trapped and excreted, thus protecting the other tissue)
Lead absorbed from the respiratory tract does not pass
directly through the liver and hence absorption from this
site is more dangerous.
The results indicate that as for
other metals e.g. iron and copper, liver tissue has a
marked affinity for lead.
Lead is a constant constituent of blood*
In the
normal, the range is narrow and similar ranges have been
obtained by workers in various parts of the World.
Lead occurs in the organs of foetuses, being
derived from the mother, presumably through the placental
circulation.
This indicates that at no time is man free of
lead.
Lead has been found to occur in the excreta.
This
is to be expected as it has been shown that foodstuffs,
drinking water and even the atmosphere contain traces of
lead.
d
It has been stated that the excretion of metals such
as lead can occur through the intestine as well as the
kidney.
This statement is sometimes difficult to prove
owing to the difficulty in distinguishing betvireen unabsorbed
and excreted metal in the faeces.
The writer, however,
has produced evidence to shovf that lead can be excreted
through the intestine and that the excretion by this route
may exceed that by the kidney.
The kidney appears to
have no power of concentrating lead, as the lead content
of urine is much
smaller than that of blood.
Similar
re stilts have been obtained with iron and copper (later
sections).
Evidence has been presented to show that the
absorption of lead from the alimentary tract is influenced
by the composition of the diet.
Absorption is high on a
low calcium diet and low on a high calcium diet.
have been put forward to account for this.
D appeared to have no influence.
Reasons
Pat and vitamin
Absorption of lead was
increased very much when mineral acid was added to the
diet.
This indicates the importance of the gastric acidity
in lead absorption.
The effect of various agents upon the distributio]
of lead between the soft tissues and skeleton has been
studied.
Low calcium intake, ammonium chloride, sodium
bicarbonate and potassium iodide produce mobilisation of
lead, as evidenced by the high percentage of the total lead
present in the soft tissues of mice after treatment with
these.
High calcium intake has the reverse effect.
The
effect of sodium bicarbonate, ammonium chloride and
potassium iodide was shown to be independent of the calcium
content of the diet.
On high calcium intake, the quantity
of lead present in the soft tissues could not be regarded
as being negligable and probably was quite capable of
producing/
producing damage.
The effect of the above agents upon the
lead content of the blood in men with histories of
increased exposure to lead was studied.
Mobilising agents
produced marked increases in blood lead while high calcium
intake produced a marked fall.
In man it was found that mobilising agents
produced much more marked changes in the blood than in the
excreta.
The question of the wisdom of de-leading arises t
mobilisation may result in an influx of lead into the soft
tissues without a compensatory increase in excretion.
On
the other hand, high calcium therapy may leave nonnegligable amounts of lead in the soft tissues.
The
obvious answer, is more stringent preventative measures.
It is suggested that de-leading should never be attempted
without adequate laboratory control.
This should include
frequent estimations of the lead content of the blood and
excreta.
It has been shown that in mice, after removal fr<
exposure to lead, absorbed lead may be eliminated very
rapidly.
Frequently persons with suspected lead poisoning
are ilot admitted to hospital for examination until some
time has elapsed since the last exposure and as a result
the amount of lead excreted may not greatly exceed the
normal.
Under such conditions, it cannot be assumed that
a comparatively low lead excretion suggests a mild exposur
References/
9/.
References to individual susceptibility to lead
are often made.
The writer believes that much can be
explained by the biochemical knowledge that has accumulated.
Absorption of lead from the alimentary tract is influenced
by the composition of the diet and by the degree of gastric
acidity, both very variable factors.
The clinical condition
is influenced not by the total amount of absorbed lead but
by its distribution between the soft tissues and skeleton.
Low calcium intake, acidosis from metabolic disorders and
the indiscriminate use of such drugs as sodium bicarbonate
and potassium iodide will produce mobilisation.
Such
factors must be taken into consideration when the question
of susceptibility arises.
Various procedures have been used in the diagnosis
of plumbism ; 1. clinical, 2. haematological, 3 .
radiological, 4. chemical.
Of these, the chemical examinati
produces the only evidence of increased lead absorption.
Chemical examinations have usually been carried out on
isolated samples of urine and/or faeces.
The writer conside
that chemical examinations should be much fuller.
The
examination of the lead content of the excreta often gives
important Information, but the collection should be made
while the patient is on a diet of known lead content.
This
and the interpretations to be derived therefrom have been
discussed already.
Of particular value, is the estimation
of the lead content of the blood.
This gives information
as to whether the lead is confined mainly to the skeleton
or is present in high concentration in the soft tissues.
In a case of plumbism the blood lead could be normal due
to conditions of diet etc*
It is suggested that in a
case of suspected plumbism, in the event of the blood
lead being normal, the estimation should be repeated after
the patient has been under one of the lead mobilisation
procedures.
An examination of the lead content of the
excreta at the same time would give confirmatory evidence.
Lanza (1935) has suggested that a considerable amount of
mild lead poisoning occurs, much of which escapes diagnosis.
In view of this, the above technique appears to be justified
There is no justification in making a diagnosis
of lead poisoning on evidence of increased lead absorption
alone, in fact the final diagnosis must remain with the
clinician.
Clinical symptoms are often obscure.
Satisfactory conclusions can only be made through the
closest co-operation between clinician and biochemist.
Under natural conditions, lead occurs in food­
stuffs and animal tissues.
The question must remain open
as to whether these minute amounts of lead are accidental
or have some physiological function as in the case of
copper and cobalt.
93
References.
Alden (1889) Lancet 1, 728.
Alderson (1852) Lancet, 2, 73, 95
Arnaud (1936) Analyst 61, 219
Aub, Pairhall, Minotfc and Reznikoff (1926)
’Lead poisoning*, Baltimore.
Baker (1767) quoted by Tanquerel des Planches (1839)
Barth (1931) Virchow’s Arch. 281, 148
Bartleman and Dukes (1936 ) Brit. Med. J. 1, 528.
Belknap (1929) Wisconsin Med. J. 28, 346.
Berger, Studeney and Rosegger (I923) Wien, Klin. Wschr.
4£,
586.
Blumberg and Scott (1935) Bull. Johns Hopkins Hosp.
56 , 276, 311
Boyd and De (1933) Ind. J. Med. Research 20, 789*
Burton (1840) G-az. med. de Paris, 2me serie, 8, 470.
Callan and Henderson (I929) Analyst %4, 550*
Campbell (1886) Practioner 2Z, 477*
Carrie (1 9 3 6 ) Med. Welt. 10, 104.
Chi-Shih Yang (1936 ) Chinese Med. J* ^0, 165 .
Chi-Shih Yang, Shi-Lu Chang and Kusum Liu (1937)
Chinese Med. J.
945*
Cholak (1935) Ind. Eng. Chem. 2, 287
Cooksey and Walton (I929) Analyst j?4, 97*
Dejerine-Klumpfe (I889) *Des polynevritis en general et
des paralysees et atrophies saturnines en
particuliere *, Paris.
Dowdell and Green (1937) Mining and Met. 3.8, 463 .
Duy (1935) Wien. Klin. 48, 1413.
Fairhall (1924) J. Biol. Chem. 60, 485.
Fellinger (1936) Arch. Gewerbepath. Gewerbehyg. £, 414.
f
Fischer and Leopold! (1934) Z. angew. Chem. 47. 90.
Francis, Harvey and Buchan, (1929) Analyst 54, 725•
Greene (1936) Aria. Pub. Health Hews (Aug.) 126, 1.
(1937) U.S. Pub. Health Eng. Abstr. 1£, Mi, 6 .
Grisolle (1SJ6) Au Bureau du Journal Hebdomadaire
(1837) Oaz. hebd., Ho.40, 50, 51* &nd 52.
Haenisch (1936) Chem. Zentr. 1, 5362.
Hall (1905) Brit. Med. J. 1, 584.
Hall and Hansom (1906) Brit. Med. J.l, 428.
(1906) Lancet 1, 510.
Hamilton (1911) U.S. Bureau Labour Bull. Ho.95*
(1912) J. Amer Med Assoc. ££, 777 .
(1912) Bull, of U.S. Bureau of Labour Ho.104
Ind. Accid. and Byg. Series Ho.4.
(1913) J. Amer. Med. Assoc. 60, 772.
(1914) U.S. Bureau of Labour Statistics Ho.141.
Ind. Accid. Byg..Series Ho.4.
(1914) Amer. J. Pub, Health 4, 477*
(1914) Bull, of U.S. Bureau of Labour Statistics.
Ind. Accid. and Byg. Series Ho.6.
(1920) J. Ind. Hygeine 1, 177*
Hubbard (1937) lud. Eng. Chem. 9, 493*
Hunter and Aub (1927) Quart. J. Med, 20, 123.
Ingleson (1938) Analyst 63,, 546.
Kato (1932) Amer, J. Dis. Child, 44, 569 *
Kehoe, Thamann and Cholak (1926) J. Amer. Med. Assoc.87,2081
(1933) J. Ind. Byg. 15,306 - 339*
(1935) J* Amer. Med. Assoc. 104,9C
95“^
Kruse and Fischer (1931) Bull. Hyg . 6, 74.
Lampitt and Rooke (1933) Analyst j>8, 736.
Lanza (1935) J* Amer. Med. Assoc. 104. 85.
Legeder (1934) Arch. Verdauuskrankh. 56, 237 .
Legge and Goadhy (1912) ’Lead poisoning and lead absorption’
London and N.Y.
Lesche (1934) Chem. Zentr. 1, 3880.
Lev/in (1904) Klin. Yfschr. 41 - 42, 1298.
Litzner, Weyrauch and Barth (1931)
Arch. Gewerbepath. Gewerbehyg. 2, 330.
Litzner and Weyrauch (1932)
Arch. Gewerbepath. Gewerbehyg. 4, 74.
(1933) Med. Klin. 2£, 381.
Londres (1934) Presse med. 42, 465*
Luzuriaga (1796) ’Dissertio medica sabro el colico’ Madrid.
Lynch, Slater and Osier (1934) Analyst gg, 787*
Manley (1937) Analyst 62, 544.
Meilliere (1902) Compt. Rend. Soc. Biol. ^4, 1134.
(1903) Compt. Rend. Soc. Biol.
517.
(1903) ®Le saturnism©’, Paris.
Mertens (1937) Klin. Wschr. 16, 6l.
Monier-Williams (1938) Rep. Pub. Health Med. Sub j . No .88
London.
Nye (1933) Med. J. Australia 2, 235-
Oliver (1913) Lancet 2, 527.
(1914) ’Lead poisoning’, London and N.Y.
(1921) Brit. Med. J. 2, 108.
Oui (1907) Echo med. du Nord, Lille, 11, 44
Paul (I860) Arch. gen. de Med., 1st series, 5* 15.* 513 •
Porak (1894) Arch.de Med. exp. et d ’Anat. pax.h0"™6, 192.
96.
Bansom (1900)
Brit. Med. J. 1, 1590*
Beport of the University of Aberdeen(1935)
Lancet 2,374.
Scott (1902) Quart. J. Med. 10, 148.
Sheldon and Bamage (1931) Biochem. J. 23. 1608.
Shelling (1932) Proc. Soc. Exp. Biol. Med. £0, 248.
(1932) J. Biol. Chem. 2£, 197.
Shields, Mitchell and Ruth (1939) J. Ind. Byg. 21, 7 .
Sobel, Gawron and Kramer (1938) Proc* Soc. E x p . Biol. Med.
Stockhusen (1656) quoted by Aub et alia (1926)
Sutton (1938)
Analyst 63. 637 •
Taeger and Schmitt (1937) 2. ges* exp.
Med. 100.717.
Talbort and Taylor (1936) Mo. Agri. E x p . Stn. Eesearch Bull
m s
3.
Tanquerel des Planches (1809) ?Traites des maladies de
plomb en saturnines ’, Paris.
Teisinger (1936) Z. ges. exp. Med. 100, 320.
Teleky (1909) Deutsch, z . f . lervenk. 37* 234.
(1909) Schrft * a. d. G-esamtgebiet der Gewerbehyg.
I* 1 (1921)
Zentralb. f. Gewerbehyg. £, 3'
Telfer (1939) Glas. Med. J. 131* 257 .
Thouvenet (1931) quoted by Lanza (1935)
Torrey, Thorpe and Graham (1934) Cornell V et. 24, 289.
Vigliani (1933) Minerva med* 11, 674.
Vigliani and Angeleri (1935) Clin. med. ital 66, 5*
(1936) Klin. Wschr. 1^, 700.
Vigliani (1936) Bass* med. applicata lavoro ind* 2* 355Vigliani and Waldenstrom (1937) Deut« Arch. Klin. Med.
180, 182 *
77
Vigliani and Libowitzky (1937) Klin. Wschr. 16, 1243.
Water Pollution Research Board, Technical Paper Ho *4.
H. M. Stat o Off, (1934)
Weyrauch and Muller (1933) 2* Byg. Infectionskrankh, 113*2
Williams, Schulze, Rothschild, Brown and Smith (1933)
J. Amer* Med. Assoc. 100, 1485.
Willoughby and Wilkins (1938) J. Biol. Chem. 124, 639 .
Iron and Copper
INTRODUCTION
(a.) Iron.
The amount of iron in the body Is small but its
functions are of the highest importance. It is estimated that
the iron content of an adult man or woman is about 3
of which is in the form of haemoglobin.
most
There is no consideral:
reserve of relatively inactive iron in the body, hence if the
intake fails to equal the output, anaemia soon develops.
Investigations of iron metabolism have as a result been largely
concerned with the study of haemoglobin formation and anaemia.
In addition to respiratory function, there is evidence that
iron compounds are concerned with a large number of other
biological processes.
It has long been known that iron is essential to the
nutrition of both animals and plants and that It is present
in the ash of such. A few decaxles ago it was assumed that iron
existed in the food as oxide or phosphate and that haemoglobin
was formed by the combination of Iron and protein.
Results
obtained during the latter half of the 1 9 th century threw
doubt upon the utilisation of Inorganic iron for the productioj
of haemoglobin.
It was believed that iron salts when Injected
acted.as poisons and when given by mouth were almost quantit:
:atively eliminated in the faeces.
In spite of this, inorganic
iron salts were used and found efficient in the treatment of
anaemia.
To harmonise these views with clinical experience,
it was suggested that inorganic iron might act by absorbing
/do
the hydrogen sulphide of the intestine and thus protecting the
food iron.
Another view was that inorganic iron salts might
aot b, stimulation of tn. abstain* tls.uo. [bu„s. (1884), von
Hosslin (1882), G-ottlieb (1891), Socin (1891), Woltering (1895;
Tartakowsky (1903, I904)
Bunge examined the iron present in a number of
foodstuffs and could detect hut traces of inorganic iron.
Wroi
egg yolk he isolated a compound containing iron which he callec
1haematogen* and regarded it as the precursor of haemoglobin.
Since the World War the question of iron absorption
has become very much clearer.
It has been shown that inorganic
iron salts are absorbed and converted into haemoglobin whereas
iron compounds of the nature of haematin are not
f Hart
et alia
(1928), Elvehjem (1932)
Elvehjem and his co-workers found that the efficiency
of a foodstuff to cure nutritional anaemia in rats was dependen
on its inorganic Iron content.
They estimated the inorganic
iron content of various foodstuffs directly, by the use of
-dipyridyl jjfilvehjem et alia (1933) ? Elvehjem et alia (1 9 3 4 )
Sherman et alia (I9 3 4 ) "j .
They found that as a rule, a large
part of the total iron of foodstuffs was in the inorganic form*
Following this work a large amount of research has
been concerned with the absorption of iron from the alimentary
tract, humans being used as experimental subjects.
These have
indicated that upon a large intake of inorganic iron, the body
was capable of absorbing large amounts and that it was not
subsequently excreted |* Hutchison (1937)? Brock (1937)?
/©/
Widdowson and MeGance (1937)
]
In anaemia it has been
suggested that only a small amount of this absorbed iron'is
converted into haemoglobin.
It is generally agreed that inorganic iron to be
absorbed must be in a dialysable form and that absorption takes
place in the upper part of the small intestine. Lintzel (1931)
and Heubner (1926) state that ferric but not ferrous iron forms
non-ionisable complexes with proteins.
As a result they belies
that ferrous Iron is absorbed easily but that ferric iron must
be reduced in the intestine prior to absorption.
Whipple and
Robcheit-Robbins (1936)* Hahn (1937) a-nd Brock and Hunter
believe that ferric iron can be absorbed as such.
The gastric acidity has been recognised as being a
very important factor in the assimilation of iron, especially
the food iron and it has been recognised that a lack of
secretion of hydrochloric acid in the stomach is responsible fo
certain forms of hypochromic anaemia £ Lintzel (I93I),
Starkenstein (1934)* Strauss (1934)* ICellog and Met tier (1936),
Barer and Fowler (1937)
The question of the Iron content of the human diet
has received attention.
Studies have failed to shox«7 any
correlation between the total iron content of the diet, the
concentration of other constituents and the incidence of anaerni
(b) Copper.
Although copper as a metal has been known for
centuries, the importance of its compounds in biological
lo z
activities in other than certain invertebrates has been known
only recently.
It has been known for a long* time that copper is a
constituent of plants, Meissner (18 1 "/) being; probably the
first worker to note this.
Sarzeau (183O) noted the presence
of copper in plants and made quantitative estimations.
Deschamps (1848) showed that a relationship existed between
the copper In plants and that in soil and Ohevreul (1868)
suggested that copper was distributed widely in organic matter
Later work showed the universal distribution of copper through
:out the plant world and the suggestion was made that it was
essential in plant metabolism as the highest concentrations
were found present in the more active parts e.g. young shoots,
leaves etc. J^Maquenne and Demoussy (I92O), Guerithault (1 9 2 0 ),
Fleurant and Levi (192O), Bertrand (l920)^j.
It has been known for a long period that certain
marine animals contain considerable amounts of copper.
Harles
(1 8 4 7 ) detected copper in Eleclone and Helix pomatia and showed
that it did not exist as a salt.
Frederica (1878) obtained a
copper containing protein from the blood of Octupus vulgaris
and although he was the first to give this compound a definite
physiological function, earlier workers had suggested the
importance of a compound of this nature in the blood of
invertebx^ates j^Blasius (1866), Bert (1 8 6 7 )~| •
Since this
period a considerable amount of investigation has been carried
out into the chemical nature and physiological properties of
the ha emocyan ins J”Parsons and Parsons (1923), Begemann (1924),
to %
Redfeld et alia (1926), Btedman and Btedman (1926), Hogben and
Pinhey (1926, 1927) J •
has "been established.
The occurrence of different haemocyanin
The chemistry of. the haemocyanins has
received attention but no copper compound of the nature of
haematin has been isolated
J
Dhere* and Burdell (1919), Dhere*
(1 9 2 0 ), Conant, Dersch and Mydam (1 9 3 4 )
j.
An interesting copper compound is turacin, a pigment
found in the feathers of the South African bird, Turaco, and
was first described by Ohurcli (1869).
It is believed to be a,
copper porphyrin. La&dlow (1904) showed that haematoporphyrin
would combine with iron and copper, the compound with the latte
resembling turacin.
Fischer and Hilger (1923) have shown that
turacin porphyrin is probably uroporphyrin, a pigment excreted
in the urine of man in conditions known as congenital
porphyrinuri a ♦
The distribution of copper in the tissues of higher
animals has only received serious attention within recent years
t
Bodansky (1 9 2 1 ), McHargue (1 9 2 5 ; 1 9 2 6 ), Cunningham (1931)
J.
The first definite evidence that copper is essential
in the metabolism of the higher animals was obtained by the
Wisconsin workers who showed that it is necessary for the
formation of haemogloblin. They fed young animals (rabbits,
rats etc.) with cows1 milk, a diet which as well as being
deficient in iron has a low copper content, and eventually an
anaemia developed.
The administration of purified iron salts
produced no response until a oopper salt was included also
j" Hart
et alia (1 9 2 5 , 1927, 1 9 2 8 ), Waddell et alia (1929)
tof-
This work was confirmed by McHargue et alia (1 9 2 8 ), Krauss
(I9 2 9 )i Titus et alia (1 9 2 9 ) an^ Cunningham (1 9 3 1 )*
Some workers have attempted to show that purified
iron salts were effective in curing this type of nutritional
anaemia
£ Beard and Myers (193^*) > Keil and Nelson (1 9 3 1 ) J >
while others have suggested that elements other than copper
have a similar effect £ Titus et alia (1929), Myers and Beard
(1929, 193^-) J •
All of this work has since been discounted.
A considerable amount of woik has been done on the
relation of copper in the treatment bf certain types of anaemia
in man.
A number of workers have presented evidence to show
that iron in combination with copper is more effective than
iron alone in the treatment of nutritional anaemia in children
£ Lewis (1 9 3 1 ); Josephs (1 9 3 1 ); Bloxsom (1932), Oaldwell and
Dennett (1932), Cason (1 9 3 4 )> Hutchison (1 9 3 7 ) J *
These
results are not surprising as it has been shown that the copper
content of ordinary infant dietaries does not greatly exceed
the supposed requirement.
Ram age, Sheldon and Sheldon (1 9 3 3 )
found reduced copper contents in the livers of infants during
the nursing period and increased values as soon as they were
placed on a mixed diet.
(I933)
Parsons and Hawks ley (1933^ an^- Maokay
are inclined to believe that copper deficiency does not
play any important part in common nutritional anaemias in
babies in England.
It has been reported that better results were
obtained when certain types of anaemia in adults were treated
/OS'
with iron in combination with copper than with iron alone
£
Dwyer (I93O)', Mills (I93O, I93I), Waugh (193I), Adamson and
Smith (1 9 3 1 )> G**oss (1932), Dameshek (1933)> Wintrobe and
Biebe (1933 )» Machold (1934) J*
Other workers have concluded
that in the treatment of the usual cases of anaemia in adults,
no advantage is gained by the inclusion of copper in therapeuti
preparations of iron Jj-Ieath (1933)> Bethell et alia (1934);
Davidson and Leitch (1934) J •
It is generally accepted that
iron and copper are ineffective in pernicious anaemia.
Workers have found that iron in combination with
copper is effective in combating nutritional anaemia, in sucklin
pigs £ Hart et alia (1930), Hunt and Carroll (1933) ^ •
In higher animals it has been shown that copper has
no influence upon the absorption of iron but influences its
o n ™ ,.!™
I n t o t a - o g l o M n J 0 « n » i n Bh .
J o ..* .
(1932 ),
Elvehjem and Sherman (1 9 3 2 )
The quantity of copper in foodstuffs has received
considerable attention.
Ho foodstuff has been found free of
copper but great variations have been found, the figures
ranging from 0.1 mg. per k.g, fresh celery to 44*1 mg. per kg.
fresh calx* liver.
Copper catalyses the oxidation of sulphydril
compounds.
Factors affecting this reaction have been studied
and their relation to biological activities discussed J^Matthews
and Walker (1 9 °9 ), Harrison (1927), Warburg (I927), Elvehjem
(I93.O), Elliott (I93O), Bummer and Poland (1933) > Hellerman et
alia (1933)? Bersin and Legemann (3-933); Michaelis and Kunstrom
/06
(1934)1 .
The value of copper In the growth of higher plants
has been reviewed by Hoagland (1932). Felix (1927) and Allison
(1927) have improved the production of a large vai’iety of plants
on peat soils by the addition of copper.
It has been shown
that the production of cytochrome in yeast necessitates the
presence of copper as well as iron I Elvehjem (1931), Parsons
and Hickmans (1933) 1 •
/o
y
The Determination of Iron In Biological M a t e r i a l s .
For the estimation of small amounts of iron such as
are present in biological materials, several reagents and
methods are available.
These may be enumerated as follows:
1.
Potassium thiocyanate - colorimetric.
2.
Thiolacetic acid - colorimetric £ Lyons (1927)"J .
3. <=*&*1dipyridyl - colorimetric [Hill (I9 3 I)
Of these methods, the reaction between ferric iron
and potassium thiocyanate is probably the oldest,
occurs with ferrous iron.
ho reaction
Various methods employing this
1
reaction are in existence.
Some workers prefer to compare' the
colours in aqueous solution while others extract the ferric
thiocyanate with amyl alcohol prior to colorimetric comparison,
Certain difficulties may be experienced.
Pyrophosphates
depress the reaction partially or completely.
When it is
necessary to ash organic materials containing much phosphorus,
a wet digestion method in which an excess of sulphuric acid
is present at the end of the reaction is essential.
Elvehjem
(I9 3 O) lias employed ignition to destroy organic matter but
heated the ash with alkali to convert pyrophosphates into
orthophosphates.
Kennedy (1 9 2 7 ) and Elvehjem and Hart (1 9 2 6 )
t
state that pyrophosphates are not formed during sulphuricpercholorio acid digestion, but the writer has noted that
unless excess of sulphuric acid is present at the end of the
reaction, they are liable to be formed.
McFarlane (1 9 3 2 ) has
shown that the depth of colour produced, ie influenced by the
/o8
concentrations of thiocyanate and sulphuric acid present.
The reaction between iron and thiolacetic acid was
first described by Andreasch (1879)*
Lyons (1 9 2 7 ) showed that
the reaction could be employed to estimate small amounts of
iron.
When thiolacetic acid is added to a solution of an iron
salt, ferric or ferrous, and alkali added, a purple colour is
produced.
Lyons believed that when thiolacetic acid is added
to a solution of a ferric salt, a substance Fe
.CH2 .COOIiJ-j
is formed which is immediately reduced to Fe £ S.CHg .GOOH*j 2 &nd
this -
in alkaline solution gives an intensely coloured ion -
Fe £ S.GHg,GOo"J . The presence of oxidising agents prevents the
formation of the colour due to the formation of dithioglycollic
acid which does not give the reaction.
Lyons found that strong
bases produced rapid colour fading and that ammonia was the
most satisfactory to use.
Lyons showed that the reaction was uninfluenced by
the presence of a large number of substances, both inorganic
and organic.
Hanzal (1933) an& Burmester (1 9 3 4 ) have applied
the reaction to the estimation of iron in biological materials.
Hill (I9 3 I) Las u s e d d i p y r i d y l to estimate iron
in biological materials.
This substance reacts with ferrous
iron but not ferric iron to produce a red colour.
Ferric salts
will react if a deducing agent e.g. sodium hydrosulphite is
added.
The writer wished to make direct determinations, if
possible, of non-haematin iron in biological materials and it
was considered that thiolacetic acid might serjve this purpose.
The range of the reaction was studied.
iron was prepared containing 0 .0 8 5 5
A standard solution of
°£ iron alum in 1 litre
of ifo sulphuric acid (l ml. - 0 .0 1 mg. Fe).
Into a series of
tubes were measured, 0 .0 0 1 , 0 ,0 0 2 , O.OO3, 0 .0 0 4 , 0 .0 0 5 , 0 .0 1 ,
0 .0 2 , 0.03, 0 .0 4 , and 0,05 mg. Fe.
These were diluted to 5 ml*
with water, 2 drops thiolacetic acid added and then 1 ml.
ammonia, sp. gr. 0 .8 8 .
It was found that in the tubes
containing 0.005 - 0.03 mg. Fe, the depths of colour were
strong enough to permit comparison in a colorimeter.
On
comparison it was found that the depth of colour was proportion!
to the concentration.
Although distinct differences in depth
of colour could be distinguished in the tubes containing 0.001 .
0,005
Be, they were too weak to compare in a colorimeter.
To estimate amounts of iron of this order, it has been found
best to use direct comparison with a series of standards in
standard tubes.
,
The reaction has been found to be uninfluenced
by the presence of such substances as amino acids, pyrophosphat
:es, citrates etc.
A number of estimations of total iron have been made
in biological materials.
The majority of these have been made
on mice and rat tissues.
In these, thiolacetic acid has been
used.
Since the colour is developed in a medium with an
alkaline reaction Interference from calcium phosphate, if
present, appeared probable.
It has been found that precipitate
of calcium phosphate may be removed by centrigulisation and
accurate results may be obtained.
na
In genera]., organic matter was desti^oyed by ignition
in a silica dish over a bunsen burner under the same conditions
as that used in lead estimations,
A few estimations of the iron content of urine and
egg have been made.
In these, the thiocyanate reaction was
used, as certain technical difficulties made the use of
thiolacetic acid impracticable,
ofct*-dipjfridyl has been used as a test for ferrous
iron in certain sections of this thesis.
It has been stated
that it is effective over a range pH 3 . 5 - 8 .5 , this has been
confirmed.
Non-haematln Iron.
It Is generally recognised that iron exists in
biological materials In at le-asA two forms (a) Maematin iron,
(b) non-haematin iron.
Non-haematin iron has received
considerable attention within recent years.
In the hens' egg, nearly all the iron is located in
the yolk and many believe that it exists as an organic complex
with protein like properties.
This complex was first studied
by Bunge (1 8 8 4 ) who named it 'haematogen1, and regarded it as
the precursor of haemoglobin.
Hugounenq and Morel (1905) found
that decomposition of 'haematogen' by acids produced a black
pigment, containing 2.6$ iron, which they called thaematovin'.
Hill (I93I) has submitted evidence to show that all
the iron in egg yolk Is present as ferric iron, probably as
colloidal ferric hydroxide and that no organic complex exists.
He found that when
©fe/-d±pyri&yl was added to a suspension of
egg yolk in an acid acetate buffer, no colour developed, but on
the addition of sodium hydrosulphite a red colour was produced
and determined the amount of Iron present by comparison with a
standard.
He found that the iron content of egg yolk as
determined by this simple procedure agreed with the iron contenl
estimated after ashing.
McFarlane (1932 ) £°und that lecitho-vitellin prepared
from egg yolk contained a fair percentage of iron which appearec
to be present in very stable combination.
I
He concluded that
part at least of the iron of the egg is present as an organic
c omple x .
/ / 2L
Additional evidence appeared to be necessary to
decide this point.
The contents of one egg x^ere well mixed, treated
with an equal volume of 2 0 trichloroacetic acid and filtered.
To 5 rcl. 0£ ^ ie filtrate were added 2 drops of thiolacetic
acid followed "by 1 ml, ammonia, sp, gr. 0 .8 8 .
produced.
No colour was
A known volume of standard iron solution was added
to another portion of the filtrate and the test repeated.
The depth of colour produced corresponded to the amount of
iron added, indicating that none of the constituents of the
filtrate interfered with the reaction.
To the contents of another egg some iron alum
solution was added and the proteins precipitated with
trichloroacetic acid as before.
The filtrate gave a negative
reaction for iron.
The following experiment was then carried out.
5 ml. of thiolacetic acid were added to the contents of one
egg and the proteins precipitated by the addition of an equal
volume of 20fa trichloroacetic acid. 1 ml. of ammonia, sp.gr.
0.88, was added to 5
*he filtrate;
a positive reaction
for iron was obtained.
A series of experiments was then made in which the
iron content as estimated directly on the protein free filtrate
was compared with the total iron content estimated after ashing
The contents of an egg were diluted to 100 ml. with
water, 10 ml. of the mixture were taken, 2 ml. of thiolacetic,
// 3
acid added followed by 15 ml. of 20$ tr ichi or0acet>ic acid and
the volume made up to 3 °
filtered.
with water.
The mixture was then
filtrate was added 1 ml. of ammonia,
To 5
sp.gr. 0 . 8 8 and then compared with a standard, prepared as
dollows.
To 2 ml. of standard iron solution ( 2 ml. = 0 . 0 2 mg.
Fe) were added O.33 ml. thiolacetic
acid and 2.5
trichloroacetic acid and the volume made up to 5
1 ml. of ammonia, sp. gr. 0 .8 8 , was then added.
ml. of 20$
with water.
The results
obtained by this method were compared with those determined
after ashing.
The results are shown in Table 14.
the
At the same time
recovery of added iron was studied, using
determinations in a protein free extract.
shown in Table 15,
direct
The results are
It will be seen that the iron content of
the egg as estimated by the two methods is the same within the
limits of experimental error and that added iron could be
estimated quantitatively.
From the above investigations it would appear that
the iron of the hens1 egg is the part of some organic molecule.
Added ferric iron behaves in exactly the same way therefore
one must conclude that the iron of the hens’ egg is in a
similar state to ferric iron added to an organic mixture of
the
nature of egg yolk.
Thiolacetic acid has a reducing action
and
one would assume that ferrous iron does not form such
strong combinations as ferric iron with organic compounds such
as are present in egg yolk.
There seems to be little evidence
to suggest that the iron of the hens’ egg exists as the
hydroxide.
Investigations were then carried out to determine
whether substances other than thio3.acetic acid were capable
of liberating iron from egg yolk.
The yolk of one egg was diluted to 100 ml. with
water.
Four 10 ml. samples were treated as follows:
1 . control - diluted to 15 ml. with water.
2 . 2 ml. thiolacetic acid added and the volume made up to
15 ml. with water.
3. 5 rol.of 4$ sodium pyrophosphate added and the volumemade
up to 15 ml. with water.
4. 2.5 ml. of 5i° sodium hydrosulphite added and the volume
made up to 15 nil. with water.
After standing for 5 minutes, 15 ml. of 20$
trichloroacetic acid were added and the mixture filtered.
To
5 ml. of the filtrate were added 2 drops of thiolacetic acid
followed by 1 ml. ammonia, sp. gr. 0 ,8 8 .
The depths of colour
produced were compared with a standard (0 , 0 1 5 ffig. Fe), prepared
similarly.
The results are shown in Table 1 6 .
Sodium pyrophosphate and hydrosulphite were both
capable of liberating the iron of egg yolk quantitatively.
The latter is effective because of its reducing action while
the former has the property of forming non-ionised compounds
with iron*.
Experiments were carried out in which sodium
glycerophosphate and citrate and dithioglycollic acid were
used.
The filtrates gave negative reactions for iron.
Investigations were then carried out to determine
its
thetnature of the substances with which ferric iron forms
such stable complexes.
Iron alum solutions were added to milk, solutions
of sodium caseinate, gelatin and edestin and suspensions of
egg white and lecithin and samples treated in the same way
as the egg yolk suspensions described above.
Within the limits of the amounts used, milk, sodium
caseinate and egg white gave negative reactions for iron.
Lecithin, gelatin and edestin contained appreciable amounts
of iron which were allowed for.
The lecithin suspension was
prepared by pouring an ethereal solution into boiling water.
The results are shown in Table 1 7 .
Iron was not
present in the control filtrates of milk, lecithin or sodium
caseinate but was recovered quantitatively in the control
filtrates of gelatin and edestin.
Iron was present in
control filtrates of egg white but recovery was incomplete,
this being probably due to traces of lecithin. Milk, sodium,
caseinate and lecithin reacted similar to egg yolk. Although
ferric iron forms complexes with simple proteins, it must be
concluded from these results that compounds with phosphatidejs
and phosphoproteins are much more stable.
Table 14
Tbe iron content of the hen!s egg
mg. Fe per whole
egg
tftloeyanate
Direct determination
method after
W H i n . i H i a m in * ! m
in trichloroacetic
m i
m .i > —
fc te g w hm . i d
acid extract
Whole egg
0.92
0.90
.
1.26
1.25
3.
1.00
0.96
4.
1.27
1.21
5.
1.25
1.17
6#
1.11
1.18
I'm
1.27
1.19
2
'77-
mam
Table 15
The recovery of* iron added to egg and
determined by the thiolacetic acid
method in trichloroacetic acid extracts
Initial iron
content
mg.
Iron added
mg.
Total iron
content
mg.
Iron
recovere<
mg.
1.
0.067
0.088
0.153
0.086
2.
0.097
0.070
0.159
0.062
3«
0.072
0.070
0.136
0.064
4«
0.087
0.088
0.174
0.087
5.
0.088
0.088
0.172
0.084
6.
0.079
0.088
0.160
0.081
IFU*>L£
The
tttort
Content
lg
of
r“»G-. Pe PER
1
2
3
4
5
Thiolacetic
acid
1*28
1*24
0-9G
1*10
MG
Sodium
pyrophosphate
1*24
1*18
0-99
1*12
M2
M9*
E
&Gr "Vomv.
EfrCV-
Sodium
hydrosulphitc
1*22
1*20
1*00
1*17
1*10
Control
Nil
Nil
Nil
Nil
Nil
R ccovprv
op
GslOUOfc-fC-Fll-
nro
iison
v ar io u s
fiT^R 1ft k S .
Ferric iron.
Iron recovered (mg.)
Iron added
mg.
1 % lecithin
suspension
2 % sodium
caseinogenate
Milk
Egg-white
2 % edestin
2% gelatin
0-10
0-05
0-10
005
0-10
0-05
0-10
0-10
0-15
0-10
0-10
0-15
0-20
0-10
0-15
0-20
Control
Nil
Nil
Nil
Nil
Nil
Nil
0-091
0-081
0-124
0-078
0-099
0-145
0-193
0-094
0-144
0-198
rao
Thiolacetic
acid
0-099
0-052
0-09G
0-052
0-10G
0-053
Sodium
pyro­
phosphate
0-097
0-054
0*102
0-053
0-105
0-047
Sodium
hydro sulphite
0-09G
0-051
0-105
0-051
0-107
0-048
_
___
0-102
0-10G
0-098
0-107
0-103
—
0-106
_
---
_
__
---.
---
-__
_
___ .
_
__ _
---
_
_
_
,
The Dialysis of Iron Salts
The object of the present experiments was to determin
some of the factors which might influence the dialysis of
inorganic iron from organic mixtures and their possible
relationship to alimentary absorption*
In a previous section, the writer has shown that ferr
iron forms very stable complexes with substances of the natur
of phosphatides and phosphoproteins*
are not produced with protein*
Such stable complexes
It was considered that the
formation of such complexes might influence the rate at which
inorganic iron dialyses.
Parchment thimbles (Whatman) were used as dialysing
membranes*
In each case 5 ml# of mixture, containing an
added iron salt (1 mg* Fe) was dialysed against 15 ml. of
distilled water for 4 hours.
At the end of this period, the
iron content of the external fluid was estimated#
Egg yolk and egg. white suspensions, containing added
iron (ferrous sulphate, iron ammonium citrate, iron alum)
were dialysed#
Ferrous iron dialysed readily whereas the
dialysis of ferric iron was nil or negligable*
Under normal conditions, absorption of iron probably
takes place from the small intestine from a mixture of digest
protein#
The reaction of such contents is probably acid*
Dialysis was investigated under similar conditions.
Suspensit
of egg white and egg yolk were digested with pepsin and
hydrochloric acid.
These peptic digests, to which iron
(ferrous sulphate, iron ammonium citrate, iron alum) had been
added, were dialysed against distilled water.
I ZS s
Ferrous sulpha*
dialysed readily*
Ferric iron dialysed readily from peptic
digests of egg white but not from peptic digests of egg yolk.
Acid peptie digests of casein had the same effect as egg yolh
As these digests were acid in reaction, inability of ferric
iron to dialyse could not be due to precipitation as phosphat
The inability of ferric iron to dialyse is undoubtedly due
to the formation of strongly combined undissociated compounds
between ferric iron and such substances as phosphatides and
phosphoproteins or their degradation products*
Peptic digests of egg white and egg yolk were
neutralised by the addition of sodium bicarbonate and the
dialysis experiments, described above, repeated*
Dialysis ot
ferric and ferrous iron from egg white digests were little
affected by neutralisation#
The dialysis of ferrous iron
from egg yolk digests was reduced very markedly by
neutralisation*
In a previous section it has been shown that
pyrophosphates are capable of liberating ferric iron from its
complexes with phosphatides etc.
As a result, experiments
were carried out in order to determine whether pyrophosphates
were capable of Increasing the rate of dialysis of ferric ire
The pyrophosphate was added as the sodium salt*
In acid
peptic digests of egg yolk, pyrophosphate had little effect*
This could be due to the rapidity with which it is hydrolysed
to orthophosphate by mineral acids.
lyrophosphates increased
to a marked extent the dialysis of ferric iron from
neutralised digests of egg yolk and from undigested egg yolk
and egg white.
/2 2 .
Dialysis of ferric iron from suspensions of undigestet
egg white was increased by the addition of acid.
Examples of the results obtained from these dialysis
experiments are shown in Table 18a.
Similar dialysis experiments were carried out, using
lead acetate and copper sulphate (1 mg# Pb,
1 mg# Cu). The
results indicated that these metals did not form strongly
combined complexes with phosphatides and phosphoproteins or
their degradation products.
As a result one must conclude th«
these substances should have no specific inhibiting effect
upon the absorption of these metals from the alimentary tract<
Typical results are shown in Table 18b.
Methods of Analysis
Iron - directly with thiolacetic acid and ammonia
Copper - directly with sodium diethyldithiocarbamate (see late
section)
Lead - with dithizone after digestion with sulphuric and
perchloric acids
Table 18 a .
Dialysis of I r o n ,
Quantity of iron subjected to dialysis - lmg.
The figures refer to mg. of Fe that have dialysed in 4 hours
Ferrous sulphate
O.56
Iron alum
0,00
Iron ammonium citrate
0,01
Iron alum
* sodium
4
piyro phosphate
0.28
Egg* white digested with Pepsin
pH -
4 .0
Ferrous sulphate
0.41
Iron alum
O.41
Iron ammonium citrate
0 .3&
Peptic digest of Egg White
neutralised with sodium
bicarbonate
Ferrous sulphate
O.36
Iron alum
0.20
Iron ammonium citrate
0.19
Iron alum + sodium
pyrophosphate
6.19
Undigested Egg Yolk
Ferrous sulphate
O.52
Iron alum
0.00
Iron ammonium citrate
0.00
Iron alum + sodium
pyrophosphate
0,26
sIn
pH - 4.1
F’errous sulphate
O .56
Iron alum
0.03
Iron ammonium citrate
O .03
Iron alum + sodium
pyropho sph ate
0.03
Peptic digest of Egg Yolk
neutralised with sod i m
'bicaivbonate
Ferrous sulphate
0.03
Iron alum
0.00
Iron alum + sodium
pyropho sphat e
0.20
digested with Pepsin
pH - 4.2
Ferrous sulphate
Iron alum
Iron ammonium citrate
Acidified undigested Egg
White
pH - 4.0
Iron alum
Table 18 b .
Dialysis of Copper.
Quantity of copper subjected to dialysis
- Img.
The figures refer to mg. of Gu that have dialysed in 4 hour
Undigested egg white
0.00
Peptic digest of egg white (pH - 4 .2 )
°.3 8
Peptic digest of egg white
neutralised with sodium bicarbonate
0.05
Undigested egg yolk
0.0 4
Peptic digest of egg yolk (pH - 4 *5 )
O.38
Peptic digest of egg yolk
neutralised with sodium bicarbonate
0.04
Acidified undigested egg white (pH - 4 .6 )
0.22
Dialysis of Lead
Quantity of lead subjected to dialysis - Img.
The figures refer to mg. of Pb that have dialysed in 4 hour
.
0.00
Peptic digest of egg white (pH - 4 .2 )
0.45
Peptic digest of egg white neutralised
with sodium bicarbonate
O.O3
Undigested egg yolk
O.O3
Peptic digest of egg yolk (pH - 4 *5 )
O.48
Peptic digest of egg yolk neutralised
with sodium bicarbonate
0.02
Acidified undigested egg white (pH - 4 -8 )
0.26
Undigested egg white
Factors influencing; the Absorption of Iron from
the Alimentary Tract
Little work appears to have been done regarding this
important problem*
Results obtained from dialysis and other
experiments have indicated the probability that materials
containing phosphatides and phosphoproteins are likely to
interfere with the absorption of iron from the alimentary trac
This is of great importance as certain foodstuffs, ranking
high nutritionally, are rich in these e.g. egg, milk.
The
writer has shown that lead absorption is dependent on the
calcium content of the diet.
The probability is that the
absorption of iron is influenced in a similar manner*
In the first instant, the effect of phosphatides
and phosphoproteins upon the absorption of iron was studied.
In these experiments, young female mice (aged 3 weeks) were
used*
In general, the experiments were carried out under
similar conditions to those with lead.
The diets, which were
of low calcium content, had the following compositions
5
Diet A
&♦
Whole wheat flour
300
Casein
200
Corn starch
325
Wheat gluten
50
Olive oil
40
Sodium chloride
20
Potassium chloride
15
Butter
15
Diet B
•
Whole wheat flour
400
Corn starch
325
Wheat gluten
50
Olive oil
40
Sodium chloride
20
Potassium chloride
15
Butter
15
The diets were fed at the rate of 2.5 g. per mouse per day
and contained supplements of iron alum (0.8 mg. Fe per mouse p
day).
The duration of the experiments was 14 days.
At the
end of this period the animals were placed on the diets less
added iron for 4 days to remove
unabsorbed iron from their
alimentary tracts, killed and their total iron contents
estimated.
The animals were placed on the following diets,
, .tz%
i)
1. Diet A,
2* Diet B
+
1 ml. egg white per mouse per day,
3* Diet B
1 ml# egg yolk per mouse per day*
Control animals were killed at the commencement of the
e:xperiment and their total iron contents estimated#
The results are shown in Table 19a and it will be see:
that the presence of phosphatides and phosphoproteins in the
diet does inhibit the absorption of ferric iron from the
alimentary tract#
The experiments were repeated using iron pyrophosphat
The results, which are not reported in detail, agreed with th<
obtained with iron alum#
This was due probably to the hydrol^
of the pyrophosphate by the acid of the gastric juice#
The experiments were repeated using ferrous sulphate
as the source of iron#
These results are shown in Table 19b
and agree with those obtained with iron alum#
The influence of the calcium content of ‘the diet upon
the absorption of iron has been examined*
The diets used had
the following compositions ;
High calcium diet
Whole wheat flour
700
Whole milk powder
300
Marmite
50
Low calcium diet
&•
Whole wheat flour
400
Casein
100
Corn starch
325
Wheat gluten
50
Olive oil
40
Sodium chloride
20
Potassium chloride
15
Butter
15
The diets were fed at the rate of 2.5 g. per mouse per day*
Adult male mice were used in the following experiment
The animals were placed on a) high calcium diet, b) low caleii
diet, to which was added iron (iron ammonium citrate) at the :
of 2 mg, Fe per mouse per day for a period of 21 days*
The
animals were then placed on the diets less added iron for
4 days to remove unabsorbed iron from their alimentary tracts
killed and their total iron contents estimated.
A number of
animals of the same group were killed before the commencement
of the experiment and their total iron contents estimated.
The results are shown in Table 19c.
In this experiment, female mice, aged 3 weeks, were
used.
These animals were placed on a) high calcium diet,
T&) low calcium diet, to which had been added iron as iron
ammonium citrate at the rate of 0,8 mg. Fe per mouse per day.
At the end of 14 days, the animals were placed on the diets 1<
iron for 4 days to remove unabsorbed iron from their
alimentary tracts, killed and their total iron contents
;z ®
estimated.
A number of* animals of* the same group were killed
before the commencement of the experiment and their total iroi
contents estimated.
The results are shown in Table 19c.
The results obtained indicate that the absorption of
iron from the alimentary tract is influenced by the calcium
content of the diet in the same way as lead.
Absorption of
iron is high on a low calcium diet and low on a high calcium
diet.
The writer has shown that the addition of hydrochloric
acid to the diet increases the absorption of lead#
probable that iron would behave in a similar manner.
It seemed
The
following e:xperiment was therefore carried out.
Female mice, aged 3 weeks, were placed on the high
calcium diet, containing 0.8 mg. Fe (as iron ammonium citrate)
and 0.5 ml. of N. hydrochloric acid per mouse per day for a
period of 14 days.
The animals were then placed on the high
calcium diet alone for 4 days to remove unabsorbed iron from
their alimentary tracts, killed and their total iron contents
estimated.
The results are shown in Table 19c and indicate
that iron is absorbed more easily when mineral acid is added
to the diet.
This experiment indicates the Importance of the
gastric acidity in the absorption of iron.
Xn animal experiments, the subject usually consumes a
diet, every mouthful of wnicn has the same composition.
man, this does not occur.
workers in nutrition.
In
This point is often neglected by
In man the composition of the diet
should vary througnout the day.
In view of the fact that the
absorption of essential constituents of the diet may be
subjected to mutual inhibition it seems apparent that the dail;
/3 A
diet should be so arranged into meals that mutual inhibition
is reduced to a minimum.
To attempt to simulate this in
animals, young female mice were fed on the high and the low
calcium diets on alternate days together with added iron for
14 days.
At the end of this'period, they were placed on high
calcium diet alone for 4 days to remove unabsorbed iron from ,
their alimentary tracts, killed and their total iron contents
estimated.
The results are shown in Table 19d.
It will be
seen that under sucn conditions, iron absorption was good
and in addition, their growth was much better than in the otb
experiments.
Method of Analysis
The animal was ignited in a silica dish over a bunsen
burner.
The ash was dissolved in water containing 5 ml.
concentrated hydrochloric acid and the volume made up to
50 ml.
The iron content was estimated in an aliquot with
thio lacetic acid and ammonia.
Table 19a
Young female mice (aged 3 weeks)
Added iron - 0*8 mg* Fe (iron alum)
Time - 14 days
Weigfot
g.
Total iron
.
mg.
Iron
mgY Fe p e r
100 g. mouse
Controls
!•
9*5
0.59
©.02
2*
11.0
0.67
6.09
3.
9*0
0.59
6.55
4.
10.5
0.71
6.76
5.
10.5
0.71
6.76
6.
10.5
0.71
6.76
7.
9.5
0.53
5.57
3*
9.0
0.45
5.00
Average
0.62
6.25
9.
14.0
0.63
4.50
H
O
•
Diet A
18.0
0.70
3.90
11.
19.5
0.71
3.64
12.,
T7
c:
JLfi miJ
0.78
4 #47
13.
16.0
0.78
4.38
14.
12*0
0.64
5.23
Average
0.71
4.36
Weight
g*
Diet B _±_
Total Iran
mg*
li*on
ragT^Pe pet*
100 g. mouse
y o ik
15.
19.0
0.68
3.58
16.
19.0
0.67
3.52
17.
IB. 5
0.79
4.27
18.
20.5
0.82
4.00
19.
20.0
0.78
3.90
20.
15.5
0.73
4.71
21.
17.5
0.65
3.72
22.
21.5
0.73
3.39
23.
18.5
0.70
3.79
24.
17.5
0.59
3.37
25.
17.0
0.71
4.18
26.
20.5
0.78
3.80
A veva& e
0.71
3.85
Diet „B... *4“ Egg White
27.
17.0
0.90
5.88
28.
17.5
1.06
6.06
29.
14. 5
1.00
6.96
30.
14.5
1.06
7.31
.
f4
CO
13.5
0.91
6.70
32.
14.5
0.89
6.10
33.
14.5
0.93
6.41
34.
15.5
1.06
6.84
35.
12.5
0.91
7.24
36.
14.0
0.98
7.00
/ 3^.
Total Iron
mg.
Iron
ingT"Pc per
100 g< mouse
37.
16.5
1.14
7.00
38;.
12.0
1.10
6.92
1.00
6.80
/ 35\
'"ifytij.. • tySJfr
" ^'■y»;'/-
Table 19 b
Female mice (aged 3 weeks)
Added iron - 0.8 mg. Fe (ferrous sulphate) per day
Time - 14 days.
Iron
mg. per 100
g . mous e
Weight
Total iron
mg ;
1•
8.5
O .44
5.17
2.
7.0
0.53
7.57
3.
9.0
0.43
4.78
4-
9 .5
0.53
*yO
5-
9.0
0.45
5.00
6.
10.0
0.62
6.20
Controls
B + Egg white
Average
0.30
7-
16.0
1.20
7-5°
8.
17.5
1.15
6 .5^
9.
15.5
1.03
6 .64
10.
13.0
O .98
7.54
11.
11.5
0.80
6 .94
12.
12.5
0.86
6.88
1.00
7.01
Average
I 56
gf
Weight
S.
Diet B 4.
Tot al iro n
Iron
mg. per 1 0 0
a;, mouse
Egg Yolk
13.
17.5
O.73
4.17
14. .
I3 .O
O.65
5.00
15.
15.5
O.65
4-19
16.
16.0
O.63
3.94
17.
13.5
0.73
5*41
18 .
15.5
O174
4. 77
O .69
4.58
13 7 .
Table 19 o
Adult made mice
Added Iron - 2 mg. Fe (Iron Ammonium Citrate) per mouse per day
Time 21 days.
Weight
§•
Total iron
mg.
Iron
mg. per 100
g. mouse
Gontrols
1.
30
1.80
6.00
2.
24.5
1.67
6 .5 4
3*
21
1.26
6.00
4.
24.5
1.56
6.54
5*
27
1.80
6.67
Average
m wW BjSrfi
.
6,.3.5,
High oaloium diet
6.
22
1 .4 5
6.6 0
7.
1 9 .5
1.14
§.90
8.
23
1 .3 4
5.83
9*
18
1.54
6.56
6.22
Low calcium diet
23
2.03
8.83
23.5
1.96
8.34
24.5
2.03
8.29
23
2.35
10 22
*
Average
im*
m.MWiw m uni1*' 11fji
8.92
Female mice (aged 3 weeks)
Added iron - 0*8 mg. Fe (Iron Ammonium Citrate) per mouse per
day.
Time - 14 days
Total iron
mg.
g.
Iron
mg. per 100 g.
mouse
Controls
1.
IO.5
O.53
5.05
2.
10.5
0.54
5.14
3.
10.0
0.68
6.80
4.
10.5
0.54
5.14
5.
11.5
0.58
5.04
6.
9
0.51
5.67
7-
8
0.50
6 .2 5
8*
8
0.41
5.12
0.51
3 *°I
11,0
0 .7 5
6.82
10.
14.0
0.81
5.78
11.
9 -0
O.70
7-77
12.
8 .5
0.63
7.53
13.
9.5
0.71
7.47
14.
11.0
0.83
7 .5 5
15.
11.0
Q.83
7 .5 5
16 •
9.0
0.69
7.66
Average
High, calcium diet
9.
Average
03%
O.74
7.27
HW ItiAfM W iM P
Weight
&’*
Total iron
mg
Iron
mg. per 100
g. mou se
Low calcium diet
17.
13
1.11
8 .5 4
18.
12
1.09
9.08
19 .
10.5
1.09
10.38
20.
8
1.38
17.25
21.
8.5
0.97
11.41
22.
8 .5
1.17
13.76
23.
9
1.09
12.11
24.
7-5
0 .9 0
12.00
1.10
12.04
Average
calcium filet; and hydrochloric acid
25-
11.0
I.09
9.91
26.
1 1 .5
1.01
8 .7-5
27.
10.0
1.10
11.00
28.
10.5
1.00
9 .5 2
29.
9
I.05
11.66
3 °.
8 .5
1.08
12.71
31.
8 .5
0.98
11.53
32.
8
1.06
13 .25
1.04
11.04
Average
Table 19 d *
B'emale Mice (aged
3
weeks)
Added iron - 0*8 rag.Fe (iron Ammonium Citrate)
Time -
14
days
Total iron
mg.
Iron
mg, per 100
mouse
High and low calcium di et on
alternate days
33.
22
1*10
5.00
34.
16
0*70
4.38
35.
19
1.03
5.42
36.
22
1.15
5.23
37.
17
1.00
5-99
38.
19
0.88
4*62
39.
17.5
0.88
5.00
40.
14
0.92
5.67
Average
0.96
iin .i i. f
The Reduction of Ferric Iron in the Alimentary Tract and its
Significance in Absorption
Many workers believe that ferric iron cannot be
absorbed as such but must be reduced to the ferrous state pri
to absorption*
Tnis reduction is said to occur in tne small
intestine 1 Lintsel (1931), Starkenstein and We den (1930),
Reimann and Fritsch* (1930) , Moore et alia (1939) 1 • Other
workers believe that ferric iron can be absorbed as such
Whipple and Robscheit^Robbins (1936), McGance and Widdowsoi
(1937, 193S), Brock and Hunter (1937)
presented by both sides is conflicting*
views on the following
5 1
•
The evidence
The former base theia
. ferric but not ferrous iron forms
non-dissociated compounds with proteins, 2* in the treatment
of anaemia, ferrous salts often produce a better response
than ferric salts*
following ;
1
The latter base their views on the
* in iron balance experiments, ferric iron
has been found to be absorbed as easily as ferrous iron,
#
2
in the treatment of anaemia, ferric iron often produces as
good a response as ferrous iron*
Those who hold the first vie
have in no case indicated that reduction of ferric iron does
take place in any part of the alimentary tract*
To examine th
the following eixperiments were carried out*
The effect of various foodstuffs, used in animal diets
upon ferric iron was examined.
added to about
1
1
mg. Fe++*’ (iron alum) was
g* of the foodstuff suspended in about
of water and a little oCC<,~dipyridyl added*
5
ml.
- dipyridyl reac
with ferrous iron to produce a red colour but not with ferric
iron*
In neutral solution, no reaction occurred but in acid
solution (pH 4 - 5) a number of substances produced a marked
reduction of ferric iron as evidenced by the production of a
red colour with
dipyridyl*
The substances capable of
reducing ferric iron in acid solution were,
whole wheat flour,
whole milk powder,
egg white (boiled and unboiled),
wheat gluten,
cows1 milk (boiled and unboiled),
commercial egg albumin •
Casein, glucose, lactose, egg yolk (boiled and unboiled) were
without effect*
then examined*
Peptic digests of egg white and egg yolk wer
An acid solution of egg white (pH - 4*2)
produced rapid reduction, of ferric iron whereas after
neutralisation with sodium bicarbonate it had no effect*
Acid and neutralised peptic digests of egg yolk had no effect
The effect of those substances which had no reducing
action on ferric iron, upon ferrous iron was then examined.
With the exception of egg yolk no effect was observed*
Ferroi
iron allowed to stand in contact with egg yolk became oxidiset
to the ferric state.
This was shown by the fact that when
1 mg. Fe4'* (as ferrous sulphate) was added to 5 ml* of egg yol
the mixture would give no reaction for ferrous iron with
oW- dipyridyl at the end of 1. hour*
This had been suspected
since ferrous iron added to egg yolk suspensions could not be
recovered quantitatively in trichloroacetic acid extracts as
would have been expected.
The writer considers that the
phenomenon is due to autoxidation, the phosphatides being
143
;responsible#
The reducing effect of certain foodstuffs should now
be considered#
The effect was most marked with egg white,
egg albumin and gluten, proteins which contain a high
percentage of cysteine#
The writer considers that the
reducing action is in all probability due to the cysteine
molecule.
In view of this, the reducing action of whole whea
flour should be due to gluten and that of milk to lactalbumin
The Inability of casein to reduce ferric iron is probably due
to its low cysteine content#
Ferric iron was found to dialyse readily from acid an
neutralised peptic digests of egg white and from acidified
but not neutral egg white#
The ready dialysis of ferric iron
from such acid mixtures is undoubtedly due to reduction i.e#
ferrous iron passed through the membrane#
Dialysis of ferric
iron from neutralised egg white digests was found to be due
to the development of an acid reaction during dialysis#
Ferr
iron was observed to dialyse from egg yolk and from acid pept;
digests of egg yolk*
That this was possible is due to the fa<
that autoxidation takes time*
The writer believes that the
inability of ferrous iron to dialyse from neutralised peptic
digests of egg yolk was due primarily to its precipitation as
phosphate*
Similar factors may be responsible for the fact that
added ferric iron may be recovered quantitatively in
trichloroacetic acid extracts of egg white etc but not from
egg yolk etc.
The absorption of ferric iron as such would be hinders
in fact one might say completely prevented by the presence of
phosphatides etc in the diet*
It is probable that all protein;
act in a similar manner unless
reduction takes place*
As to
the fate of the phosphatides in the alimentary tract, very
little is known, they probably have a long survival*
In addit:
bile will have a similar action owing tds its lecithin content*
As a result the writer considers that under normal conditions
of diet, absorption of ferric iron as such is either nil or
negligable*
Ferrous iron should be absorbed easily#
The writer
hen
produced evidence to show that ferric iron can be reduced in tl
alimentary tract, not in the small intestine as others have
suggested, but in the stomach. This has been confirmed in viv<
Two rats were fed on a) boiled egg white
boiled egg yolk
+
iron alum, b)
iron alum, respectively and at the end of
two hours killed and their stomach and intestinal contents
examined for the presence of ferrous iron with
dipyridyl*
The animal that had been fed on egg white, showed the presence
of ferrous iron in both stomach and intestine very markedly
whereas in the animal that had been fed on egg yolk, no trace
of ferrous iron could be found in either stomach or intestine*
Ferric iron must be reduced to the ferrous state prior
to absorption* and as indicated, this can occur in the stomach*
This is influenced by two factors, 1) the gastric acidity,
2) the composition of the diet*
The diet should have a rectucii
effect towards iron i.e. it should contain substances of the
nature of albumin* gluten etc#
The gastric acidity is of
importance since the reducing power of the diet only becomes
t* fS
effective at acid reactions*
When iron is administered along with food its fate
depends to a large extent on the diet*
If the diet is
predominately reducing then it will be immaterial whether th<
iron is administered in the ferric or ferrous state since in
both cases an equilibrium mixture, predominating in ferrous
iron, of the same composition, will result in both cases*
la
autoxidisable properties predominate tnen wnether the iron
is administered in the ferric or ferrous state, the final st<
of the iron will be ferric.
If the diet is 1inert1, then
ferrous and ferric iron will remain unchanged, the former
being absorbed and the latter not#
One is now able to obtain a clearer insight into the
results obtained from the animal experiments reported in the
previous section*.
The low absorption of iron by mice on a diet contains
egg yolk was no doubt due to the predominating autoxidation
properties of the diet#
Oft. egg white, iron absorption was
much higher, this would be due to the predominating reducing
properties of the diet*
Iron absorption was poor on a diet
containing a large amount of casein, this probably being due
to the comparative inertness of the diet.
It also becomes
apparent why similar results were obtained when ferrous iron
and ferric iron were fed on diets containing added egg yolk
and egg white.
Hose et alia (1934) and Sherman, Elvehjem and Hart
(1934) found that egg yolk produced very little regeneration
in nutritional anaemia in rats.
They believed that copper
deficiency was the primary factor although the former workers
considered tnat there were other factors e#g# the form inwhici
the iron was present in the egg yolk#
The question naturally arises as to whether the iron
of the egg is available at all, that is wnetner the effect of
white predominates over the yolk or vice-versa#
Experiments
were carried out to determine this.
Peptic digests of unboil*
and boiled whole egg were prepared#
At pH 4 - 5# a reducing
effect was observed#
as described earlier#
This was confirmed in vivo on a rat 5
These results indicate that the effect
the white does predominate over that of the yolk#
The Non-Haematln Iron of Human Serum
and Plasma.
Many writers, have estimated what they describe as the
non-haematin iron content of serum and plasma#
The following
results have been reported.
mg# Fe per 100 c
Starkenstein and Weden (1928)
0.57
- 0.79
Rieeker and Winters (1930)
0.9
- I#4
Locke et alia (1932)
0.06
- 0.12
Marlow and Taylor (1934)
0.4
- 0.7
Moore (1937)
0.06
- 0.17
In these investigations the serum or plasma was ashed prior
to the estimation of iron.
The writer has found that, as in the case of egg yolk,
trichloroacetic acid extracts of serum or plasma gave negative
reactions for iron and added inorganic iron could not be
recovered in such extracts.
As a result the following technic
was adopted#
TO' 5 ml. of serum or plasma were added 8 drops of
thiolacetic acid and 5 ml. of 20% trichloroacetic acid and
the mixture centrifuged*
1 ml# of ammonia, sp. gr. 0.88, was
added to 5*ml. of the supernatant fluid.
Under such condition
a definite reaction for iron was obtained and added inorganic
iron could be recovered quantitatively.
As the depth of colou
was too faint to compare with a standard in a colorimeter,
the iron content was estimated by direct comparison with a
series of standards.
From the results shown in Table 20, it will be seen
that added inorganic iron could be estimated quantitatively
by this method*
The iron content of 10 normal sera was
estimated and the results are shown in Table 21, the range
being 0*12 - 0*20 mg. Fe per 100 ml*
It would appear convenient to refer to the serum iron
estimated above as ’inorganic* iron*
After this work had bee
published, Fowweather (1934) published a paper in which he
reported the total iron content of human plasma, shown to be
free of haemoglobin.
His range for total iron agrees with tb
writer’s range for ’inorganic’ iron.
One is forced to concln
that the high figures reported by some writers must have been
due to the use of serum and plasma in which there had been
considerable haemolysis*
The effect of plasma upon the dialysis of iron salts
(iron alum, iron ammonium citrate, ferrous sulphate) has beer
studied.
These were carried out in a similar manner to those
described in an earlier section#
The results are shown in
Table 22 and indicate that whereas ferrous iron dialysed
easily, ferric iron was almost non-dialysable*
To a sample of plasma,
dipyridyl was added#
A rec
colour did not develop until after the addition of sodium
hydro sulphite, this indicating that in the sample, the iron
was in the ferric state#
A sample of serum (5 ml#) to which
had been added 1 rag. ferric iron, as iron alum, and a little
dipyridyl was acidified to give pH 4 - 5*
A red colour
developed, indicating that on the acid side, the serum prote:
are capable of reducing ferric iron to. the ferrous state*
T1
was rather to be expected since the plasma proteins contain
a high content of sulphur containing amino acids*
Barkan
(192?) showed that plasma iron was not dialysable but became
so after incubation with 0*4% hydrochloric acid at 37 ° C for
24 hours*
He used this as the basis of a method for the
estimation of plasma iron*
He estimated the iron content of
an ultrafiltrate prepared from plasma that had been subjected
to the above procedure*
He obtained values of 0.08 - 0*17
mg. Fe per 100 ml*, which are in agreement with those obtaine<
by the writer.
This phenomenon observed by Barkan can be
explained by tne results obtained by the writer.
The 1inorganic1 iron content of sera from cases of
pernicious anaemia and anaemia following haemorrhage has been
estimated.
The results are shown in Table 23*
It will be
seen that the values are increased in the former and decrease!
in the latter condition.
Table 20
Recovery of ferric iron added to
serum
mg* Fe
Initial
Iron added
Total
iron
1.
0.004
0.01
0.013
0.009
2*
0.004
0.01
0.013
0.009
3.
0.005
0.015
0.020
0.015
4.
0.004
0.01
0.014
0.01
5.
0.005
0.015
0.019
0.014
>$7
Iron
recovered
Table 21
The 'Inorganic1 iron content of normal sera
mg. Pe per 100 ml.
1.
0.20
6.
0.20
2.
0.18
7.
0.14
3.
0.14
8.
0.16
4*
O.14
9.
0.20
5.
0.18
10 .
0.12
/5~Z
Table
22
Dialysis of iron from S e r u m .
Quantity of Iron subjected to dialysis - 1 mg.
TJae figures refer to mg. Fe that have dialysed in 4 hour
1 mg Fe
(ferrous sulphate)
0,30
1 mg Fe
(iron alum)
0.01
1 mg.Fe
(Iron ammonium citrate)
0.01
1 mg.Fe
(iron alum)
and sodium pyrophosphate
0.30
The *inorganic * iron content of
human serum,
mg. Fe per 100 ml.
Pernicious Anaemia.
Hb.
$
Serum Iron
1.
36
0.26
2.
60
0.21
3-
3°
O.23
4.
51
O.32
following Haemorrhage
Kb. jo
Serum Iron
1.
30
O.O5
2.
73
0.09
3-
45
0.06
4.
43
0.06
5-
33
O.O5
6.
40
0.06
7-
38
0.04
8.
35
0.04
9•
41
0 .0 6
The Uon-Haematin Iron of Whole Blood
a)
*Inorganic* Iron
Trichloroacetic acid extracts of whole blood Were
prepared and it was found that they gave positive reactions
for inorganic iron but added inorganic iron could not be
recovered quantitatively#
To estimate the finorganic1 iron
content of whole blood, the following technique was used#
To 5 ml* of whole blood were added a) 5 ml# of wate;
and 8 drops of thiolacetic acid or b) 5 ml# of 4% sodium
pyrophosphate*
5 ml. of 20% trichloroacetic acid were then
added and the mixture filtered.
Iron was estimated directl;
in the filtrate by the addition of thiolacetic acid and
ammonia#
Inorganic iron, added as iron alum, could be
recovered quantitatively as shown by the results in Table
2*
It has been, found that thiolacetic acid attacks
haemoglobin slowly to produce 1inorganic1 iron#
The proces
Is slow and the filtrations in. the above method are
exceedingly rapid*
Comparison was made between the results
obtained by the use of a) thiolacetic acid, b) sodium
pyrophosphate, to liberate finorganicT iron In whole blood.
The results as shorn in Table 25, indicate that no
appreciable differences exist#
The finorganic* iron content of whole blood from a
number of men has been estimated#
The results are shown in
Table 26, and range from 0.93 to 1.50 mg. Fe per 100 ml.
It will be obvious that these amounts of 1inorganic* iron
are present mainly in the red corpuscles.
Using the above method, Shorlands and Wall (1936)
tS T
obtained similar results to those of the writer.
To a trichloroacetic acid extract of whole blood
in which sodium pyrophosphate had been used to liberate
*inorganic1 iron, o(o< 'dipyridyl was added and an immediate
red colour was produced.
This could indicate that at least
part of the 1inorganic1 iron of the red blood corpuscles is
in the ferrous state or it could have been the result of the
reducing action of the plasma when the trichloroacetic acid
was added.
This result must be held in doubt although one
might expect that part at least of the iron in the H. B. C.
is in the ferrous state since they contain such reducing
substances as glutathione etc.
b) 'Easily split Iron
Barkan (1927 - 1938) has estimated what he terms
the 'easily split iron* in blood.
He regards 'easily split
iron1 as the precursor of bilirubin.
'Easily split iron'
has been estimated by a number of writers using various
methods ;
1, treatment of blood with hydrochloric acid at 37° C follow
by ultrafiltration and estimation of the total iron content
of the ultrafiltrate (Barkan),
2, heating blood with
5K
sulphuric acid and estimation of
the total iron content of a trichloroacetic acid extract
j" Starkenstein and Weden (1928)
,
3, heating blood with normal acid and estimating the total
iron content of a trichloroacetic acid extract ^Moore (1937
'Easily split iron' values are considerably higher
than the 'Inorganic* iron values obtained by the writer.
'Easily split iron' values appear to1 depend upon the method
/516
used. e.g. lower values are obtained when hydrochloric acid
is used than when sulphuric or nitric acid is used
Moore
(1937)
The writer is of the opinion that much of the iron
that is included under the heading of Easily split iron*
is derived by decomposition processes from haemoglobin
and as such is of no physiological or pathological
significance.
More work is necessary before our ideas on
this subject are clarified.
Table 24
The
recovery of iron added to
whole blood,
mg.
Initial
Iron added
Total iron
0.020
0,015
0.033
0.013
0.020
0.020
0.042
0.022
0.020
0.010
0.030
0.010
0.026
0.010
0.037
0.011
0.026
0.015
0.043
0.017
0.026
0.020
0.045
0.019
Recovery.
The results are expressed as mg.
Pe per 100 ml.
blood.
A,
-
B.
thiolacetic acid
sodium pyrophosphate
A.
B.
1.
1.30
1.29
2.
1*34
1-33
3.
1.14
1.11
4.
1.40
1.38
Table 26
The 1inorganic 1 iron content
of whole hlood.
The results are expressed in
mg. Fe per 100 ml. blood.
1.
1.14
5.
O .96
2.
1.06
6,
1.08
3*
0.93
7.
1.40
4.
1*36
8.
1.56
/60
The Iron content of U r i n e .
Earlier writers have reported the presence of iron
in normal urine but more recent work has thrown doubt on this.
Lintzel (1929 ) and Lanyar et alia (1 933 ) state that
the iron content of normal urine does not exceed 0.01 mg. per
litre.
Henriques and Roland (1928) obtained values of 0.08
to O.32 mg. Fe per litre while Coons (1932 ) reported values
of 0.06 ho 0.48 mg. Fe . per^litre in the urine of pregnant
women.
Later Marlow and Taylor (1934 ) have obtained values
of O.O3 to 0 .8 mg. Fe per litre in the urine of normal men.
To estimate the small amounts of iron that may be
present in urine, special precautions must be taken.
In the
present study, urine from normal males was used and was
collected in paraffin wax coated bottles. It was found that
a thiocyanate colorimetric method was the most suitable to
use and was carried out as follows.
A mixture of $0
urine and 5 mil. concentrated
nitric acid was evaporated almost to dryness In a pyrex flask.
5 ml. of concentrated sulphuric acid were added and the mixture
heated to remove nitric acid.
5
perchloric acid were
then added and the mixture heated until all the organic matter
was destroyed.
A blank was carried out at the same time.
After cooling, the residue was diluted to 20 ml. with water,
4 ml. of 4
potassium thiocyanate added and the red ferric
thiocyanate extracted with 5
of amyl alcohol.
The amyl
alcohol layer was separated and compared with a standard
i (ot-
prepared as follows.
The standard containing O.OO5 mg. Fe
and 1 ml. concentrated sulphuric acid was diluted to 10 ml.
with water.
2 ml. of 4 0?° potassium thiocyanate were added
and the red ferric thiocyanate extracted with 5
of amyl
alcohol.
Although the purest chemicals that could be obtained,
were used, the blank was appreciable.
males was examined.
Urine from 6 normal
The colorimeter readings of the blanks
and the urines were almost the same, the iron contents of
these urines in no case exceeding 0 .0 1 mg. per litre.
The
results of Lintzel and Lanyar et alia were thus confirmed.
The probability is that the other Investigators neglected to
take blanks into account.
Lanyar et alia found that no increase in the iron
content of urine resulted from the oral administration of
iron salts.
This was repeated, Iron ammonium citrate being
administered in three cases.
The results are shown in Table 27
and it will be seen that the iron content of the urine was
increased.
/62
The iron content of urine before and after the
administration of iron ammonium citrate (90 gr. per day).
The results are expressed in mg. Fe per litre.
Case 1
Before iron treatment
<
0.01
(average of 3 days)
Luring iron treatment
Q.52
(average of 5 days)
Gase 2
< 0 ,0 1
Before iron treatment
(average of 3 days)
During iron treatment
O.52
(average of 2 d^y.s)
/
L®.
< 0 .0 1
Before iron treatment
(average of 3 days)
During iron treatment.
Q.42
(average of 3 days)
/63*
The 1Inorganic1 Iron Content
of
Human
Tie sues.
The following method was adopted:
Borne broken glass and 10 g. of finely out up
tissue
were ground up in a porcelain mortar.
20 ml. of
4$ sodium pyrophosphate were added and the grinding continued.
The mixture was allowed to stand for 3-5 minutes after which
20 ml. of 20$ trichloroacetic acid were added and the mixture
ground up.
After a further 15 minutes the mixture was
filtered and the residue washed with 10$ trichloroacetic
acid until the volume of the extract was either 100 ml.
(liver and spleen) or $0 ml. (other tissues).
An aliquot
portion of the filtrate, containing about 0,03 mg. Fe, was
diluted to 5 ml. with water, 2 drops of thiolacetic acid
added and then 1 ml, of ammonia, sp. gr. 0 .6 8 .
This was
compared with a standard prepared similarly.
At the same time, the total iron content of rib
and vertebra was estimated.
This was carried out on a
solution of the ash in hydrochloric acid.
The results are shown in Table 2 8 .
Of the soft
tissues, liver and spleen had the highest concentrations of
’inorganic1 iron, which is to be expected.
The 'inorganic1
iron content of the spleen was fairly constant but that of
the liver showed1great variations.
This is to be expected
as the liver is regarded as being the chief store for iron.
I
The ‘inorganic1 iron contents of the other soft tissues were
low hut comparatively constant.
The iron content of such
hones as rib and vertebra was high.
These bones
a re
very
vascular and much of the iron could have been present in
other than bone tissue e.g. bone marrow.
The distribution of iron throughout a number of
rats was determined.
The results are shown in Table 29, and
indicate that less than
in the skeleton.
14$
of the total iron was present
These figures are probably on the high
side as it was impossible to separate bone marrow.
The
skeleton appears to have little affinity for iron and
therefore one must conclude that the reserve iron must be
held mainly in the soft tissues.
~K,a
E
\
1
Hor*mw
JftoN
6
8
10
11
12
10-1
1-6
1-6
1-6
6-2
10-6
8-6 11-2
11-1
1-6
10-8
11-2
6-6
/(?&>+
op
tissues
The figures are expressed in mg. Fe per kg. fresh
Liver
Kidney
Brain
-*•------ ^ r 1
r
(2)
(1),
(2)
(1)
(2)
(1)
—
162-4
178-6
—
2-4
36-6
51-2
9-6
3-4
8-7
3
69-2
166-0
3-3
14-8
20-7
4
50-6
75-9
9-2
3-0
7-6
5
80-7
144-3
5-1
4-0
5-2
—
27-7
38-8
7-4
—
7
64-6
71-1
7-9
1-5
71-4
128-5
4-6
1-4
15-5
9
45-6
68-4
5-4
14-6
19-7
84-2
138-9
8-4
2-7
8-4
91-6
124-6
7-6
3-0
13-4
39-4
76-4
6-9
1-9
7-3
1
2
CONTE1NT
tissue (1) and in mg. Fe per organ (2).
Spleen
(1)
93-6
.—.
—
.
—
169-4
100-3
96-4
164-6
84-6
126-3
116-4
84-6
~~{2)
9-4
_ _
• ____
—
29-6
10-0
14-5
23-0
8-9
13-9
15-1
10-2
Pan­
creas
(1)
18-2
10*8
7-0
4-2
13-4
8-4
6-7
8-4
9-3
8-4
7-1
Verte­
bra
(1)
128-6
123-1
142-9
134-0
133-2
167*4
146-1
126-0
111-6
136-4
154-3
121-4
Rib
(1)
103-4
147-6
119-5
111-8
114-6
161-4
151-8
111-0
109-2
146-1
138-4
109-6
Table 29
The distribution of iron
in the rat (mg#)
1#
2.
3.
4.
Skin
1 •05
1.16
1.08
1.00
Liver
0*87
0.92
1.17
1.06
Kidneys
0*21
0.18
0.15
0.17
Spleen
0.41
0.71
0.35
0.53
Bones
0.93
0.82
0.80
0.87
Remainder
3*25
3.68
2.88
3.60
Total
6.72
7.47
6.38
7.23
% Total iron in
the skeleton
13.8
10.9
12.6
12. 0
THE DETERMINATION OP COPPER IN BIOLOGICAL MATERIALS,
A large number of methods have been devised and
used for the estimation of the small amounts of copper
present in biological materials.
Some writers have used
macro-chemical methods but owing to the large quantities
of material required, are generally unsatisfactory.
A
number of colorimetric methods have been devised and are
as follows
1. potassium.ethyl xanthate method,
2. Biazzo method (1926),,
3. dlethyldithlocarbamate method I Gallon and Henderson
(19)
Potassium ethyl xanthate reacts with copper to
produce a yellow colour.
In the Biazzo method, copper is
allowed to react with pyridine and potassium thiocyanate, a
green colour being produced.
In the third method, copper
is allowed to react with sodium dlethyldithlocarbamate, a
golden brown colour being produced.
This is a very sensi­
tive reaction, it being possible to detect one part of
copper in ten million, parts of solution.
The accuracy of the Biazzo method has been question
ed by a number of writers I Ansbacher et alia (1931), Walker
l
(1930) j Drabkin and Waggoner (1930) J . A serious disadvantag
of the method is interference by iron
5
a preliminary separation of the copper.
this necessitates
This method
has been used by the Wisconsin workers in their investigations
into the metabolism of copper.
McFarlane (1932) appears to have been the first
to use sodium diethyidithiocarbamate for the estimation of
copper in biological materials.
He showed that the
sensitivity of the reaction was increased by extracting the
pigment with amyl alcohql*
pH but iron interferes*
The reaction is independent of
McFarlane (1932) showed that
interference from iron could be prevented by carrying out the
reaction in an alkaline solution containing sodium
pyropho spn ate•
The writer has applied the reaction to the
estimation of copper in various types of biological materials
but in some cases modifications were found necessary or
advantageous.
Blood
As a preliminary a slight modification of
McFarlane1 s method for blood was used*
5 ml. of blood was digested with
5
ml. of
concentrated nitric acid in a pyrex teat tube in a boiling
water bath..
When the contents were almost dry, 1 ml * of
concentrated sulphuric acid was added and the mixture heated.
Perchloric acid was added and the heating continued until
all the organic matter was destroyed.
The residue was
diluted to 10 ml. with water, 5 ml* of 4$ sodium pyrophosphat
added and the mixture made alkaline to litmus by the addition
of ammonia.
0.5 ml. of
5 ml. of amyl alcohol were added followed by
2%
.sodium dlethyldithlocarbamate and the mixture
shaken vigorously * The amyl alcohol layer, containing the
golden brown copper complex, was separated and compared in
a colorimeter with a standard containing 0.01 mg.
Cu,
prepared similarly.
The amyl alcohol extracts contained suspended
particles of water which were removed by filtration or by
centrifuging.
Satisfactory results were obtained.
Locke et alia (1932) attempted to estimate copper
in human serum by applying sodium diethyldithiocarbamate
directly to trichloroacetic acid extracts.
The possibility
of estimating copper directly in trichloroacetic acid extracts
of blood was investigated.
As a preliminary, the effect of various substances,
many of which occur in protein free extracts of blood was
7
Investigated,
These substances, the nature and amounts
used being shown below, were added to 0.01 mg. Cu (as
copper sulphate) and the reaction developed as described
above.
Substance
nc use
Glucose
Tyrosine
Alanine
Glutathione
Thiolacetic acid
Urea
Glycine
Potassium cyanide.
With the exception of potassium cyanide, none of the above
substances prevented the quantitative development of the
reaction.
No colour developed in the presence of cyanide.
In the case of glutathione and thiolacetic acid, on the
addition of the reagent no colour developed until the
contents of the tubes were shaken vigorously after which
the amyl alcohol extracts were found to match the control
quantitatively.
This was due probably to the reduction of
the copper to the cuprous state, which does not react with
the reagent, this being reversed on shaking with air.
1 volume of blood, plasma or serum was diluted
with 3 volumes of water and 1 volume of SO% trichloroacetic
acid added.
The mixture was filtered.
To 25 ml. of the
filtrate were added 2 ml. of 4$ sodium pyrophosphate and
the mixture made alkaline to litmus by the addition of
ammonia.
0.5 ml. of
5 ml. of amyl alcohol were added, followed by
2%
sodium diethyldithiocarbamate and the mixture
shaken vigorously.
The amyl alcohol layer was separated
and compared with a standard, containing 0.01 mg.
Cu,
prepared similarly.
Results obtained by this method were compared
with those obtained after destruction of the organic matter..
The results are shown in Table 30.
It will be seen that copper present in the
trichloroacetic acid filtrates reacts directly with sodium
diethyldithiocarbamate.
The results obtained by the two
methods are the same within the limits of experimental error
This indicates that the copper present In the blood is in a
comparatively simple form.
There were Indications that the
copper present in the corpuscles Is present, at least in
part, In the cuprous state.
The results shown In Table 31 indicate that added
copper could be estimated quantitatively by the second
Urine.
Urine contains a large amount of calcium phosphate
which tends to precipitate in alkaline solution.
this, sodium citrate was added.
To prevent
It was found that Inter­
ference from iron was prevented in alkaline solution.
50 - 100 ml. of urine were evaporated almost to
dryness with 5 ml. of concentrated nitric acid in a Kjeldahl
flask.
2
ml. of concentrated sulphuric acid were added and
the mixture heated until the nitric acid was driven off.
The remainder of the organic matter was destroyed by the
addition of perchloric acid and further heating.
was diluted to a known volume with water.
The digest
Tp ah aliquot,
containing about 0.01 mg. Cu, were added 5 ml. of
20%
sodium
citrate and the mixture made alkaline by the addition of
ammonia.
5 ml. of amyl alcohol were added, followed by 0,5
ml. of
sodium diethyldithiocarbamate and the mixture
2%
shaken vigorously.
The amyl alcohol layer was separated
and compared In a colorimeter with a standard, prepared
similarly.
Faeces.
For similar reasons, the technique used for faeces
was similar to that used for urine*
1 g. of dried faeces was (a) ignited in a silica
basin over a bunsen burner, or (b) digested with 5 ml. of
concentrated sulphuric acid and 15 - 25 ml. of perchloric
acid to destroy organic matter.
The residue was dissolved
in distilled water, containing hydrochloric acid, and diluted
to a known volume.
AnaLiquot, containing about 0.01 mg. Gu,
was taken for analysis which was carried out in the same way
as urine.
Diets
To estimate the copper content of diets the follow­
ing method was employed.
-,
A half day sample of the diet was dried and ground
up.
An aliquot of this was transferred to a silica basin and
100 ml. of 10^ sodium phosphate added.
and ignited over a bunsen burner.
The mixture was dried
The ash was dissolved in
water containing hydrochloric acid and made up to a known
volume.
An aliquot, containing about 0.01 mg. Gu, was taken
for analysis which was carried out as for urine and faeces.
To destroy organic matter, an ignition method was
adopted because a large amount of material had to be used
as ;
ash content was low.
When samples of diet were
ashed in silica dishes, low results and poor recoveries of
/
added copper were obtained.
Urine and faeces could be ignite
under similar conditions to give accurate results.
These
materials usually gave a large ash residue whereas in the
case of diets it was small.
When diets were ashed, it was
found that invariably the silica of the basin was attacked.
These difficulties were overcome by mixing the diet with
sodium phosphate prior to ignition.
Under such conditions
consistent results and quantitative recoveries of added coppe
were obtained.
From the results shown in Table 32, it will be
seen that copper added to urine, faeces and diets could be
estimated quantitatively.
Soft Tissues.
Owing tothe low ash content of soft tissues,
the
method adopted was similar to that for diets.
It was decided to see whether copper could be
estimated directly in trichloroacetic acid extracts of soft
tissues as in the case of blood.
The following method was
us ed.
10 g. offinely cut tissue was ground up with some
broken glass:in a mortar. 40 ml. of 10$ trichloroacetic
acid were added and the grinding continued.
The supernatant
fluid was filtered and the residue washed with 10$
trichloroacetic acid until the requisite volume of filtrate
was obtained.
Extracts of liver tissue were made up to
100 ml. while
extracts of other tissues were made up to 50
ml.
The final estimation wa3 made directly, using 20 ml.
of extract, as described for blood.
The results obtained by this method were compared
with those obtained after ashing.
Table 33.
The results are shown in
It will be seen that copper may be estimated
directly in trichloroacetic acid extracts of soft tissues.
The results obtained by the two methods are the same within
the limits of experimental error.
Bone.
Bone offers special difficulties owing to its very
high calcium phosphate content.
the copper is necessary.
A preliminary separation of
Many writers have attempted to
separate copper as the sulphide but this is time consuming
and liable to lead to difficulties.
The following method
has been found to give rapid and accurate separations of
copper.
A sample of bone (30 - 40 g.) was asfhed in a silic«
basin over a bunsen burner.
The ash was dissolved in water
containing hydrochloric acid and the volume made up to 250
ml.
50 ml. of the solution were introduced into a separating
funnel, sodium dlethyldithiocarbamate added and the mixture
extracted 3 times with ether, 25 ml. being used on each
occasion.
The combined ether extracts were collected in a
digestion flask and the ether evaporated off.
The residue
was digested with 1 ml. of concentrated sulphuric acid and
1 ml. of perchloric acid to destroy organic matter.
The
residue was diluted to 25 ml. with water and the copper
determined in an aliquot.
Milk. ' -
5
For similar reasons, the copper content of milk was
estimated by the same method as that used for bone.
Prom the results shown in Table 34, it will be
seen that copper added to milk or solutions of bone ash were
estimated quantitatively.
The method, as described, is specific for copper,
in so much that none of the metals that occur normally in
animal tissues and excreta and plant tissues interfere.
Bismuth does interfere.
Bismuth does not occur in animal
tissues and excreta undier normal conditions but the metal
and its salts are used therapeutically.
Bismuth forms a
lemon yellow complex which is soluble in amyl alcohol under
the same conditions as copper.
To he certain that the polour
in the amyl alcohol layer is due to copper, after colori­
metric comparison, the extract should be shaken with a solu­
tion of potassium cyanide.
If due to copper, the colour
will disappear, if due to bismuth, it will remain.
It is
obvious that an estimation of the copper content of faeces
should never be made after oral administration of bismuth
carbonate.
Throughout this work, the writer has not been
troubled with bismuth.
It has been found that the depths of colour pro­
duced by amounts of copper of the order of 0.01 mg. Ou,
proved very satisfactory for colorimetric analysis.
As a
result, throughout this work, a standard containing 0.01 mg.
Cu and amounts of unknown to conform to this have been used.
T A B L E
50
Th© copper content of blood
and serum
(mg. Cu per 100 ml.)
Complete destruction of the organic matter.
Direct determination on trichloroacetic acid filtrate.
Whole biood
Serum
A
S
0.174
0.186
0.169
0.174
0.157
0.171
0.165
0.156
0.200
0.182
0.193
0.199
A
0.256
. B
0.263
0.204
0.205
0.238
0.235
0.220
0.229
0.196
0.204
r
/y d
rpATvnr
x jix jx jx lb
O JL
«
Recovery of copper added to
whole blood or serum
(mg. Cu per 100 ml.)
A
Initial copper content
B
Copper added
C
Total copper estimated
D
Copper recovered
Whole blood
B
0
D
0.156
0.143
0.289
0.133
0.156
0.286
0.435
0.279
0.156
0.429
0.683
0.427
0.223
0.143
0.378
0.155
0.223
0.286
0.500
0.277
A
B
C
D
0.245
0.143
0.378
0.133
0.245
0.286
0.526
0.281
0.217
0.143
0.351
0.134
0.217
0.143
0.356
0.139
0.217
0.286
0,490
0.273
A :
Serum
'79,
3 2.
R c c o v c r y
Faeces
Urine
1
© «=
c o p p e r
t o
U
b
8F\J ^
^m d
Initial copper
content
mg.
0-216
(a)
(b)
(c)
Diet
r i j d e c j
2
0-160
1
2
3
4
5
6
0-104
0-109
0-083
0-081
0-091
0-088
1
2
0-086
0-102
3
0-096
(a)
(b)
(c)
(a)
(b)
(c)
(a)
(6)
/ 90
Copper
added
mg.
0-1
0*2
0-3
0-1
0-2
0-3
0-2
0-1
0-1
0-2
0-2
0-2
0-10
0-10
0-20
0-40
0-20
0-50
Total
copper
mg.
0*301
0-400
0-630
0-262
0-368
0-454
Copper
recovered
mg.
0-085
0-184
0-314
0-102
0-208
0-294
0-300
0-211
0-179
0*296
0-310
0-292
0-180
0-210
0-300
0-506
0-28S
0-595
0-196
0*102
0-096
0-215
0-219
0-204
0-094
0-108
0-198
0-404
0-192
0-499
I ME
<L>OE»r»ER
C/OMTENT
OF
T
i S-SUES.
(mg. Cu per 1000 g.)
A
Determined directly
in trichloroacetic
acid extract
22-24
6-20
5-69
4-94
6-26
3-33
3-63
3-01
6*96
2-27
4-67
1-40
1-16
2-27
4-00
1-96
2-22
Tissue
1
Liver
2
3
4
5
6
7
8
9
Brain
10
11
12
13
14
16
16
17
/®/
B
Determined after
ashing tissue
22-80
6-16
6-74
6-06
6-10
3-52
3-46
2-84
7-14
2-20
4-61
1-38
1-16
2-18
3-79
2-06
2-20
TABLE 54.
Recovery of added, copper
A
Initial copper content
B
Copper added
C
Total copper content
D
Copper recovered
Bone (mg.)
A
B
C
0.051
0.050
0.106
0.055
0.051
0.075
0.127
0.076
0 .051
0.100
0.159
0.108
0.051
0.200
0.259
0.208
0.051
0.300
0.353
0.302
M M
D
Milk (mg. per litre)
A
B
0.140
0.100
0.246
0.106
0.140
0.200
0.342
0.242
0.140
0.300
0.448
0.308
0.140
0.400
0.541
0.401
0.140
0.500
0.648
0.508
C
M M
/82
D
M M
Content of Blood.
I—M « « ■■
HI.J I MlLI'f H *
The copper content of the blood has been estimated
on many occasions.
The following results have been reported.
Human whole blood.
0.113 - 0.114 mg.per 100 ml. - Schonheimer and Oshima (1929)
0.185 - 0.210 mg.per 100 ml. - McFarlane (1932)
Human serum or plasma.
0.124 mg. per 100 ml* - Warburg and Krebs (1927)
0.056 - 0.075 mg.per 100 ml. - Guillemet (1931)
0.08
- 0.095 mg. per 100 ml. « Locke et alia (1932)
Horse whole blood.
,0.058 mg. per 100 ml. - Elvehjem et alia (1929)
Horse serum.
0.19 mg. per 100 ml. - Abderhalden and Miller (1928)
0.09 mg. per 100 ml. - Locke et alia (1932)
Ox whole blood
0.14 mg. per 100 ml. - McHargue (1925)
0.185 - 0.226 mg. per 100 ml. - McFarlane (1932)
Ox serum.
0.058 - 0.082 mg. per 100 ml. - Guillemet (1931)
Pig aerum.
0.194 mg. per 100 ml. - Warburg and Krebs (1927)
0.067 - 0.118 mg. per 100 ml. * Guillemet (1931)
Rabbit serum.
0.104 mg. per 100 ml. - Warburg and Krebs (1927).
.
•
0.05 - 0.072 mg. per 100 ml. - Locke ©t alia (1932)
. • /93 . " ■ .
It would appear that the copper content of the
blood of different species and even of individuals within
the same species must vary widely or most of the methods
are inaccurate.
Guillemet used a method In which the blood was
ashed and the copper precipitated first as the hydroxide
r ‘ ..
and then as the metal by electrolysis,
^hon jthe copper was
dissolved in phosphomolybdic acid, a blue solution result­
ing and the copper estimated'by titrating this solution
with potassium permanganate until the blue colour disappear­
ed.
This writer estimated amounts of copper of the order of
0.1 mg. by this method.
Warburg (1927) utilised the fact
that copper catalyses the oxidation of cysteine to cystine
to estimate the copper content of serum.
Using large
quantities of blood, McHargue (1925) and Abderhalden and
Miller (1929) employed macro chemical methods.
Elvehjem et
alia (1929) used the Blaazo method while Locke et alia (1932)
and McEarlane (1932) used sodium diethyldithiocarbamate to
estimate copper.
The present writer has estimated the copper
content of the blood of 21 normal males and 10 normal
females.
The estimations were made directly on
trichloroacetic acid filtrates as described earlier.
The
results are shown in Table 35.
The range, 0.185 - 0.229 mg.
Cu per 100 ml., is very narrow.
those obtained by McFarlane.
These results agree with
Since this work-was published,
Sachs et alia (1935) have determined the copper content of
normal human blood but obtained a slightly lower range.
The question arises as to the distribution of
copper between the corpuscles and the plasma. .Guillemet
(1932) found much higher concentrations of copper in the
plasma than in the whole blood in the ox, pig and dog.
Schlndel (1931) obtained higher values for the copper
content of the plasma than the cells in man.
Elvehjem et
alia (1929) found more copper in the corpuscles than in the
plasma in defribinated horse blood.
McHargue et alia (1928)
found more copper in the plasma than the corpuscles in the
ox.
The values obtained by Elvehjem et alia and Guillemet
were much lower than those obtained by the writer.
The
figures obtained by Schlndel were of the same order as those
obtained by the writer.
Serum from clotted blood, plasma from citrated
blood and whole blood were examined.
in Table 36.
The results are shown
It will be seen that plasma contains slightly
higher concentrations of copper than corpuscles.
The copper content of whole blood and plasma from
a number of animals of different species has been determined
Prom the results shown in Table 37, it will be seen that
the ranges are of the same order as that in man.
t$4>
TABLE 55.
The- copper content of normal
Human blood.
mg. Cu per 100 ml.
Males
'
1.
0.187
8.
0.194
15.
0.207
2.
0.197
9.
0.198
16.
0.194
3.
0.220
10.
0.222
17.
0.186
4.
0.229
11.
0.189
18.
0 *218
5.
0.220
12.
0.219
19.
0.208
6 • 0.202
13.
0.194
20.
0.194
7.
0.185
14.
0.192
21.
0.216
1.
0.208
.5.
0.196
2.
0.228
6 • 0.188
3.
0.198
7.
0.196
4.
0.216
8.
0.214
Females
0.196
10.
0.216
Table 56
Distribution of copper between corpuscles
and plasma (human)
mg. Cu per 100 ml.
Whole blood
Serum
1.
0.184
0.197
2.
0.225
0.245
3.
0.174
0.183
4.
0.192
0.209
.5.
0.195
0.197
6.
0.210
0.217
7.
0.238
0.205
8.
0.202
0.220
Plasma
;
9.
0.225
0.238
10.
0.227
0.227
11.
0.208
0.227
12.
0.238 :
0.227
13.
0.220
0.236
14.
0.207
0.220
/
TABLE 37
The copper content of the whole blood and
plasma of various species
mg. Cu per 100 ml.
Whole blood
Sheep 1 .
Ox
Plasma
. 0.163
0.172
2.
0.167
0.156
3.
0.172
0.172
4.
0.180
0.183
5.
0.156
0.161
0.223
0.208
2.
0.200
0.208
3,.
0.190
0.180
4.
0.180
5.
0.192
0.192
1.
:
0.190
Pig
1.
0.181
0.191
.
2.
0.165
0.161
Horse
3.
0.168
4.
0.185
0.200
5.
0.167
0.185
1.
0.179
0.187
2.
0.187
0.199
3.
4.
0.208
0.205
I 93.
'
0.208
0.217
Whole blood
Rabbit 1.
0,143
2.
0.147
3.
0,144
4.
0 .155
5.
0.139
Guinea
""Pig. 1.
0.179
2.
0.192
3.
0,186
4.
0..184
i<jO~
The Copper Content of Ui»ine and Faeces .
So far only one Investigation into the copper
content of urine apjjears to have been made.
Rabinowitch
(1933) obtained values of a trace to 0.41 mg. Cu per litre
of urine.
He found that the addition of copper to the diet
produced an increase in the copper content of the urine.
In the present investigation the copper content
of the faeces and daily diet was estimated as well as that
of the urine.
The results are shown in Table 38.
With the exception of Case Ho.17, there was a
balance between the intake and output of copper within the
limits of experimental error.
In Case No.17 the intake.of
copper was low and there was a negative' balance• From this
case one might assume that the minimum amount of copper
necessary to preserve equilibrium is of the order 0.7 mg.
Cu per day.
The copper content of a number of urines is shown
in Table 39,
These figures agree, closely with those obtained
by Rabinowitch.
/9/
1.
2,
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Case
Normal
Fracture
Fracture
Fracture
Fracture
Fracture
Normal
Normal
Normal
Normal
Fracture
Fracture
Normal
Amputation of leg
Nephritis
Nephritis
Carcinoma of stomach
No. of
days of
collection
6
15
13
21
21
20
7
10
9
14
16
20
12
18
8
8
8
Av. daily
excretion
of copper
in the
urine
mg.
0-29
0-36
0-52
0-45
0-50
0-36
0-38
0-33
0-29
0-30
0-44
0-22
0-21
0-18
0-32
0-16
0-11
Av. daily
excretion
o f copper
in the
faeces
mg.
2-60
1-98
1-88
2-08
2-15
2-08
2-18
2-07
2-00
1-93
1-70
1-83
1-67
1-56
1-24
100
0-52
Total
daily
excretion
of copper
mg.
2-89
2-34
2-40
2*53
2*65
2-44
2-56
2*40
2*29
2*23
2*14
2*05
1*88
1*70
1*56
1*16
0*63
Av. daily
content
of copper
in diet
mg.
2*72
2*44
2*44
2*42
2*42
2*42
2*41
2*41
2*41
2*34
2*29
2*22
2*12
1*89
1*43
1*21
0*21
T
h s
:
o © p P t£ P i
Copper excretion in mg. per litre
Copper excretion in mg. per diem
& o n t t E fr^’T -
No. of
cases
16
24
o
f
U (R In E“.
Minimum
0-08
0-12
Maximum
0-48
0-52
Average
0-18
0-28
The Copper Content of Human Tissues.
Several writers have determined the distribution
of copper inhuman tissues.
Herkel (1930) obtained values
of 3.88 - 12.9 mg. Cu per kg* fresh liver and ranged in
descending order of concentration his results may be
summarised as follows:liver, kidney, spleen, pancreas and
bone.
Cunningham (1931) found that the average concentration
of copper in three human livers was 24.9 mg. Cu per kg. dry
tissue and ranged in descending order of concentration his
results may be summarised as follows:
pancreas.and spleen.
results
liver, kidney, brain,
Other writers have obtained similar
£schonheimer and Oshima (1929), Kleinmann and Klinke
(1930), Cherbuliez and Ansbacher (1930), Cordon and
Rabinowitch (1933) J .
The majority of distribution studies such as
these must almost necessarily be done on pathological
subjects.
In certain pathological conditions it has been
shown that the copper content of the liver is very much
increased — pigmented and non-pigmented cirrhosis of the
liver, yellow atrophy of the liver, and haemochromatosis
j^Schonheimer and Oshima (1929), Cherbuliez and Ansbacher
(1930), Kleinmann and Klinke (1930), Herkel (1930)1 . It
lias been shown that the copper content of foetal organs
are very much higher than In the adult £ Kleinmann and
Klinke (1930), Cunningham (1931), Sheldon and Ramage (1931)
The question of bone seems to have been neglected.
Of the above writers, Herkel and Sheldon and Ramage alone
have examined this tissue.
The former, as a result of two
analyses, obtained values of 3.7 and 4.03 mg. per kg.,
while Sheldon and Ramage state that bone contains only a
trace of copper.
Tissues were obtained from a number of cases post
mortem, and their copper contents estimated.
The results
are shown in Table 40 and agree with those obtained by
other workers. Rib and vertebra contained comparatively
high concentrations of copper but these results are probably
high due to their vascular nature and the inability to
separate bone marrow.
The distribution of copper between the soft tissues
and skeleton of 4 adult mice was determined.
The results are
shown in Table 41 and indicate that no more than 15$ of the
total copper was present in the skeleton*
These results are
probably high due to the inability to separate bone marrow.
The skeleton must have little affinity for copper and
reserves of copper must be stored in the soft tissues.
Tissues were obtained from 4 foetuses and their
copper contents estimated.
The results are shown in Table
42 and indicate that the copper contents of the livers were
much increased.
IABtE 4 0
e
COPPER
G O N T p N T OF
TISSUES
The figures are expressed in mg. Cu per lrg. fresh tissue (1) and in mg. Cu per organ (2).
Kidney
Liver
■"1
1
2
3
4
5
6
7
8
9
10
11
12
(1)
3-09
9-09
6-59
5-43
4-86
3-74
2-96
5-46
7-94
3-16
4-44
6-12
(2)
3-40
12-99
15-82
8-14
8-75
5-24
3-26
9-83
11-81
5-21
6-04
11-97
(
(1)
2-27
2-36
3-82
3-57
2-5G
2-12
—
2-91
3-42
2-16
2-84
3-01
Spleen
Brain
t----- A_
A
(2)
0-54
0-83
1-91
1-25
0-82
0-44
—
1-02
1-02
0-69
1-11
0-84
o7~
—
2-27
4-57
3-16
—
—
2-16
3-96
4-84
2-22
3-04
3-24
(2)
(1)
(2)
—
--
—
—
3-18
6-40
4-42
—
—
2-81
5-44
6-54
2-84
3-65
3-56
----
1-16
2-27
1-96
2-04
1-96
1-84
2-41
1-92
—
—
0-20
0-36
0-28
0-28
0-20
0-20
0-28
0-23
Pan­
creas
(1)
2-10
1-96
2-20
—
2-46
2-36
2-06
2-22
2-86
2-54
2-16
2-04
Verte­
bra
(1)
2-83
1-63
2-84
2-13
4-88
3-40
1-81
2-96
3-04
2-91
1-84
4-16
Rib
(1)
6-40
32-06
8-95
47-70
10-21
3-71
4-02
9-81
21-62
14-61
9-45
8-61
Table 41
Distribution of copper in the mouse
2
1
3
4
Weight (g.)
32
33.5
29-5-
35
Soft tissues (mg*)
0.085
0.078
0.068
0.073
Skeleton (mg.)
0.015
0.012
0.008
0.010
Total copper (mg.)
0.100
0.090
0.076
0.083
15.0
12.5
10.5
11.9
%
Total copper in
Skeleton
*9‘Q
Table 42
The copper content of the tissues
of human foetuses
mg. Cu per kg. fresh tissue
1
Brain
1.25
Liver
55.6
Kidney
7.6
Femur
1.06
2
1.06
48.6
3
4
0.90
1.16
47.4
40.3
3.26
-
-
1.26
1.96
2.06
Factors influencing the Absorption of Copper ,
from the Alimentary Tract
The writer ha3 shown that the absorption of both
iron arid lead from the alimentary tract is influenced by
the calcium content of the diet.
It has been shown also
that the addition of acid to the diet increases the
absorption of these elements.
It seemed highly probable
that the absorption of copper from the alimentary tract is
governed by similar factors.
The purpose of the following
experiments was to confirm this.
Dialysis experiments
have indicated that the absorption of copper should not
b© specially inhibited by the presence of phosphoproteins
or phosphatides as in the case of ferric iron.
The following basic diets, fed at the rate of
2.5 g, per mouse per day, were used throughxrut these
experiments and had the following compositions:
Dow calcium diet
Whole wheat flour
Casein
Corn starch
g*
400
100
325
Wheat gluten
Olive oil
Sodium chloride
Potassium chloride
Butter
50
40
15
15
15
High calcium diet.
S*
Whole wheat flour
700
Whole milk powder
300
Marmite
50
In the first experiment, adult male mice were
placed on (a) low calcium diet, (b) high calcium diet,
-
(c) high calcium diet containing 0.5 ml. of N . hydrochloric
acid per mouse per day, to which had been added copper (as*
copper sulphate at the rate of
for a period of 21 days.
2
mg. Cu per mouse per day
At the end of this period, the
mice were placed on the high calcium diet alone for 4 days
to remove; unabsorbed copper from their"alimentary tracts,
killed and their total copper contents estimated.
. In the second experiment, young female mice (aged
3 weeks) were placed on (a) low calcium diet, (b) high
calcium diet, (c) high calcium diet containing 0.5 ml. of
N. hydrochloric acid per mouse per day, to which had been
added copper (as copper sulphate) at the rate of 0.8 mg. Cu
per mouse per day for a period of 14 days.
At the end of
this period the animals were placed on high calcium diet
alone to remove unabkorbed copper from their alimentary
tracts, killed and their total copper contents estimated.
The results are shown In Table 43, and it will be
seen that they are very similar to those obtained with lead.
They indicate that absorption of copper is highest on a low
calcium diet and is influenced by the gastric acidity.
Even
under.apparently optimum conditions, one must conclude that
either the absorption of copper Is low or else the retaining
power of the tissues is low.
Under similar conditions, lead
appears to be much easier absorbed and retained.
2oZ*
Table 43
A
Adult male mice -
2
rag. Cu per mouse per day
Period - 21 days.
Weight
g
Total: copper
mg
Copper
mg.per 100
g . mouse.
1.
19
0.031
0.16
2.
25
0.031
0.12
3.
20
0.033
0,17
4.
21
0.029
0,14
5.
19
0.031
0.16
6.
24
0.023
0,10
7.
16
0.025
0.16
8.
25
0.025
0.10
Average
0.14
0.062
0.29
Controls
Low calcium diet
9.
21
10.
19
0.040
0.21
11,
21
0.052
0.25
19
0.048
0.25
13.
15
0.046
0.31
14.
20.5
0.050
0.24
15.
21
0.054
0.26
16.
18
0.062
0.33
Average
0.27
, 1 2 .
Weight
E*
High calcium diet
17 .
19.5
Total copper
mg.
Copper
mg.per 100
g. mouse
21
0,038
0.18
19.
18.5
0.028
0,15
20.
23
0.039
0.17
21.
22.5
0.033
0.14
22.
18.5
0.029
0.14
23.
20
0.034
0.17
Average
0.16
•
0.16
00
H
0.032
High calcium diet and hydrochloric acid.
24,
24
0.065
0.28
25.
•23
0.062
0.27
26 .
23.5
0.071
0.30
27.
22,5
0.050
0.22
28.
.25.5
0.081
0.32
29.
23
0.048
0.21
30.
23
0.057
0.24
31.
21.5
0.081
0.37
Average
0.28
Female mice (3 weeks) - 0*8 mg. Cu per mouse per day
Period - 14 days.
Weight
g.
Total copper
mg.
Copper
rag.per 100
g. mouse
Controls
1.
9.5
0.027
0.28
2.
9
0.025
0.26
3*
8.5
0.025
0.29
4.
8.5
0.020
0.24
5.
7.5
0.016
0.21
0.023
0.25
0.063
0.39
Average
Low calcium diet
• .'■ 6. 16
7.
14 .
0.043
0.31
•8 ♦
10.5
0.036
0.34
9.
13
0.042
0.32
10.
14
0.042
0.30
11,
14.5
0.100
0.69
IS.
13.5
0.046
0.34
13.
13.5
0.048
0.36
Average
0.053
0.38
Weight
g.
*Total copper
mg.
High calcium diet
14.5
14.
mg.per 100
g. mouse
0.028
0.19
15.
14.5
0,031
0.21
16.
13.5
0.029
0.22
17 .
16
0.030
0,19
18.
17
0.031
0.18
19.
12.5
0.025
0.20
Average
0.029
0.22
High calcium diet and hydrochloric acid.
:
20.
14
0,051
0.36
21.
12,5
0.053
0,42
22.
.14,.5
0.048
0.33
23.
12.5
0.052
0.42
24.
13.5
0.040
0.30
25.
13.5
0.048
0.36
26.
13
0.042
0.32
0.048
0.36
Average
i?0 6 .
•
The Copper Content of the Blood In Certain
Conditions.
Increased copper content of the hlood has been
reported dp p r e t t y [ w.rPprg end KreP. (1987), KreP. (1988)
Locke et alia (1932) J . The normal values obtained by these
workers were very much lower than those obtained by the
writer*
The copper content of blood from women in various
stages of pregnancy was estimated and the results are shown
in Table 44.
It will be seen that the copper content of the
blood is increased in pregnancy, the increase occurring
chiefly in the last three months.
Dleckmann and Wegrier
(1936) have shown that in pregnancy, although there is a
slight decrease in the haemoglobin
increase in blood volume.
fo>
there is a large
The copper content of the organs,
especially the liver is much increased, in the foetus. Either
or both of these phenomena are probably connected with the
increased blood copper In pregnancy.
Warburg and Krebs (1927) have reported increases
in the copper content of the sera of pigeons with anaemia
due to haemorrhage.
Sheldon and Ramage (1931), using,a .
spectrographic method, noted In an examination of 28 human
bloods that the strongest lines due to copper were in a
case of carcinoma and a case of anaemia due to haemorrhage.
Locke et alia (1932) reported increases in the copper
content of the serum in carcinoma but decreases in anaemias.
The copper content of blood from cases of anaemia
has been estimated and the results shown in Table 4-5.
A marked increase in blood copper was observed in
many of those conditions associated with an anaemia;
obstructive jaundice, acholuric jaundice, hyperthyroidism,
leukaemia, after haemorrhage.
pernicious anaemia.
There were no increases in
Where anaemia was associated with
carcinoma, there were increases with the exception of one
case which showed a sub-normal value.. This case showed by
balance, experiment that there was a daily loss of copper
(Case 17 - p a g e ).
In a series of cases of anaemia
associated with nephritis, the blood copper was increased,
normal or decreased,
^hese increases’ of blood copper are
undoubtedly associated with increased regeneration of blood.
This large influx of copper into the blood stream could be
derived from other tissues or by an increased retention,
probably the former.
Sub-normal values are probably due
to a negative copper balance and would suggest that the
administration of copper salts would be beneficial.
At the conclusion of this work, a paper by Sachs
et alia (1935) appeared which confirmed all the results
reported here.
These writers could not offer any explana­
tion for the low blood copper values in anaemia associated
with nephritis.
The copper content of the blood was estimated
during treatment in 3 cases of post haemorrhagic anaemia..
From the results shown in Table 46, it will be seen that
there was a fall towards normality.
In obstructive jaundice, cirrhosis of the liver,
hyperthyroidism, myxbedema, pneumonia, diphtheria, diabetes
mellitus, carcinoma and nephritis, when there was no anaemia,
the blood copper was normal.
Table 44-.
The copper content of the blood,
in pregnancy
mg. Cu per 100 ml.
1.
0.200
2.
0.257
5 - 6 months1 pregnancy
3.
0.216
4.
0.228
5.
0.228
6.
0.232
7.
0.246
8.
0.255
9.
0.284
10.
0.363
7 months1 pregnancy.
11.
12.
0.204
.
0.255
13.
0.286
14.
0.286
Z i ’ e.
8 months* pregnancy.
15.
0.250
16.
0.367
17.
0,229
18
0.244
19.
0.250
20.
0.250
21.
0.250
.
0.258
at term.
22
0.259
24.
V 0*259
25.
0.280
26.
0.286
27 *
0.290
28.
0.294
29.
0,321
30.
0.333
31.
0.359
to
•
23.
0.359
33.
0.365
34.
0.366
35.
0.380
Zf/.
Table 4 5 .
The copper content of blood
In anaemias
mg# Cu per 100 ml#
Hb
$
Acholuric jaundice
1.
0,301
48
#
0.311
42
3.
0.305
32
. 0.273
23
Myeloid leukaemia
2
Lymphatic leukaemia
4•
Carcinoma of the Breast
'r A
5#
0.245
75
.
0.294
70
7,
0.290
40
.
0.290
38
0.290
55
6
8
Carcinoma of the Colon
9#
Carcinoma of the Prostate
10
.
0.289
52
Carcinoma of the Stomach
11
0.158
.
£/e.
22
Hb
°/
jaundice
.
0.333
48
13.
0.253
45
14.
0.267
73
15.
0.330
59
16.
0.288
44
17.
0.320
43
18«
0.385
42
19.
0.400
42
.
0.294
40
21,
0.400
38
0.420
38
23.
0.340,
38
24»
0.360
35
25.
* 0.370
33
26.
0.320
30
27,
0.246
68
28.
0.177
68
29 .
0.177
65
30.
0.200
65
31.
0.240
65
12
^rhage
20
22.
:
Nephritis
Nephritis
Hb
%
32..
0.275
65
33.
0.196
64
34.
0;176
64
35.
0.147
60
36.
0.275
55
37.
0.227
4-5
38.
0.167
45
39.
0.215
45
40.
0.218
45
41.
0.325
45
42.
0.152
42
43.
0,309
35
44.
0.136
28
45.
0.133
28
46.
0.212
45
47.
0.215
45
48.
0.223
44
49.
0.208
54
50.
0.223
50
51.
0.227
48
Pernicious Anaemia
Table 46
Cage 1
33
40
42
45
50
78
0.36
0.29
0.21
0.22
0.21
0.19
Hb %
67
67
68
75
Blood copper
0.29
0.22
0.20
0.18
Hb $
40
57
61
74
Blood copper
0.39
0.31
0.18
0.19
Hb
%
Blood copper
Case 2
f ... ..................................
Case 5
Discussion
The writer has shown that ferric iron but not ferrous
iron forms very stable compounds with substances of the nature
of phosphatides and phosphoproteins.
Whether such stable
compounds are formed with simple proteins is debatable.
Failure to realise this in the past, has resulted, in the
isolation of iron containing compounds, many of which have
been designated as haemoglobin precursors.
Iron which can
be extracted with trichloroacetic acid with or without the
aid of thiolacetic acid, sodium hydro sulphite or sodium
pyrophosphate and capable of direct.estimation with .thiolaceti
acid in such extracts has been designated as ’inorganic1 iron
to distinguish it from haematin iron.
It has been considered
that this is appropriate since inorganic iron salts added to
biological materials react in a similar manner.
A simple
method for estimating the ’inorganic* iron content of biologic
materials has been described.
Evidence has been produced to show that ferric iron
as such is not absorbed from the alimentary tract and that all
iron that is absorbed is in the ferrous state.
It has been
shown that reduction of ferric iron can occur in the stomach.
The following factors have been shown to influence the
absorption of iron
5
1
* gastric acidity,
2
. the calcium content of the diet,
3* the oxidation - reduction potential of the stomach
contents as determined by the diet.
The gastric acidity is important in two respects,
2/^
1) it aids solution of the iron, 2) it is necessary for the
reducing action of the diet, if any, to take effect.
Lack of
gastric acidity has been held responsible for the hypochromic
anaemia which often occurs in achlorhydria.
Ferric iron woulc
not be absorbed under such conditions but ferrous iron should
be absorbed if the diet does not contain substances that
produce autoxidation and substances e.g. phosphates, that are
liable tp precipitate iron.
The calcium content of the diet has a marked effect.
The effect is similar to that produced with lead
probably of similar cause.
and is
During the first few months of
man’s separate existence, his diet consists solely of milk.
Milk is deficient in iron and as a result, a store of iron is
laid down in the fioetus, chiefly in the liver, to suffice for
this period.
Milk is not the best medium for iron to be
absorbed from, owing to its high calcium content.
The writer
suggests that Nature In her wisdom realises this and instead
of secreting milk of a high iron content to the detriment of
the mother and much of which would be wasted, has laid down a
store in the foetal liver.
It has been shown that babies fed on cows* milk,
develop an anaemia which is more severe and develops earlier
than those on human milk T Mackay (1931), Fullarton (1937)
.
It has been suggested that human milk contains a higher iron
content than cows’ milk and that it is more easily absorbed.
The writer considers that this may be explained*
Human milk
differs from cows* milk in two respects, 1) lower calcium
content, 2) higher 1act albumin content*
Both of these factors
should produce a better absorption of iron, the former by
producing a lower buffering action and the latter a more
reducing medium*
Attempts have been made to determine the minimum
amount of iron necessary in the daily diet
£ Sherman (1 9 3 3 ) j
and the relationship between diet and the incidence of
Davidson et alia (1 9 3 3 ) J # All have failed.
anaemia
The writer considers that it is impossible to state any
particular quantity of iron
as being a minimum as the degree
of iron absorption depends on the other constituents of tne
diet*
The same applies to tne other investigation*
Workers in nutrition usually consider human
requirements in terms of the daily intake*
The writer has
shown that absorption of essential substances may result in
mutual inhibition.
As a result, a daily diet should be plann
into meals in such a way that mutual inhibition is reduced to
a minimum*
into courses
One could even go a stage further and plan a meal
since it is well known that food tends to layer
in the stomach in such a way that digestion and absorption of
one course is independent of another.
This is of particular
importance in the case of growing children in which the
requirements are large.
A large number of iron salts, both ferric and ferrous
have been used in the treatment of hypochromic anaemias.
The;
may be administered on an empty stomach or after a meal.
The
advantage of the latter is that their passage through the
intestine will be delayed and absorption should be greater.
When administered after a meal, it should be immaterial
Z/&.
whether ferric or ferrous salts are used, provided that the
diet contains iron reducing substances and hydrochloric acid
secretion is normal.
The latter can be remedied if defective
by the administration of hydrochloric acid.
It is an obvious
advantage that the diet should have predominating reducing
properties.
Although ferric iron appears to be capable of dialysii
readily in the presence of pyrophosphates, this does not appei
to be of any physiological importance.
’Inorganic' iron has been shown to be distributed
throughout the tissues.
The iron content of the skeleton is
low and therefore its capacity as a reserve for iron is
negligable.
and liver.
The highest concentrations were found in spleen
In the former, the iron is derived probably from
haemoglobin of broken down R. B. G. while in the latter the
iron will be derived from this source and from absorbed iron.
The ’inorganic1 iron content of other tissues were found
to be low but fairly constant.
The liver is usually regarded
as holding the greater part of the iron reserves but the fact
should not be neglected that no inconsiderable amount may be
held in the soft tissues at low concentrations ; the importan
factor is quantity, a point often neglected.
The ’inorganic’
iron content of plasma is low and is considered to represent
transport iron.
is higher.
The ’inorganic’ iron
content of the R. B. C
Its function is obscure.
The tissues, especially the liver, are said to have
a great affinity and retaining power for iron in that iron
from the haemoglobin of broken down R. B. C. is not excreted
or only excreted with difficulty.
MeCance and Wi&dowson
(19;
have stated that recently absorbed iron is only excreted with
difficulty and then in the urine and not by the intestine*
Thet go so far as to suggest that other metals are not excret<
by the intestine.
With regard to iron, the writer has no
results to contradict this but considers that their statement
is probably incorrect since it has been irrefutably shown
that both lead and copper are excreted by the intestine, in
fact it appears to be the more important path of excretion.
The great affinity of the tissues for iron is due probably
to the fact that much of the iron is in the ferric state
and -that pliosphatides are distributed widely throughout the
body*
A simple method for the estimation of copper in
biological' materials has been described*
It has been shown
that copper is extracted quantitatively from tissues by
trichloroacetic acid and in such extracts reacts directly witl
sodium diethyldithiocarbamate.
including blood.
Copper occurs in all tissues
The quantity of copper in the skeleton
is small and therefore the capacity of this tissue to act as
a reserve of copper is negligable*
The highest concentrations
of copper were found in the liver ; this organ is usually
regarded as holding the greater part of the copper reserves
oi
the body.
It has been shown that the absorption of copper from
the alimentary tract is influenced by 1) the degree of gastric
acidity, 2) the calcium content of the diet*
During the firsl
few months of man’s separate existence, his diet consists
solely of milk*
Milk is deficient in copper and as a result,
a store of copper is laid down in the foetus, chiefly in the
liver, to suffice for this period*
Milk is not the best medit
for copper to be absorbed from, owing to its high calcium
content*
The writer suggests that Mature in her wisdom
realises this and instead of secreting milk of high copper
content, to the detriment of the mother and much of which
would be wasted, has laid down a store in the foetal. liver*
After weaning, the diet should contain a much higher copper
content.
Undoubtedly during this period positive copper
balances are necessary*
composition of the diet.
Absorption of copper depends on the
Remarks have been made as to the
importance of arranging the daily diet into meals in such a
way as to obtain maximum absorption and minimal mutual
inhibition.
Similar remarks may be applied to copper.
During periods of increased haemoglobin formation
there will be increased demands for ’inorganic* iron.
This
will result in a flow of iron from the reserves to the site
of haemoglobin formation.
Such iron must be carried by the
blood stream, the plasma probably being tne chief carrying
agent.
The plasma iron appears to be in the ferric state and
as such non-dialysable.
It seems probable that at some period
it becomes dialysable i.e. It becomes redxiced to the ferrous
state, before it can be utilised.
It is however useless to
speculate on such problems without experimental evidence.
Thi
aspect of iron metabolism has been sadly neglected and the
writer considers that research along these lines may produce
very fruitful results.
The iron content of urine under normal conditions is
low and is probably connected with the low plasma iron and the
fact tnat it appears to be in the ferric state*
The iron
content of the urine is increased after oral ingestion of
iron salts*
The copper content of urine is comparatively high
This is probably connected with a comparatively high plasma
copper and the fact that copper is more easily dialysable thar
iron.
It has been proved definitely that copper catalyses
tne production of haemoglobin from inorganic1 iron.
exact role of copper is however unknown.
The
It has been suggeste
tnat an intermediate compound between copper and a porphyrin
like substance is formed but there is no definite evidence.
At the present time there is no experimental evidence to
indicate how copper acts.
We have however the fact that in
hypochromic anaemias and in the later stages of pregnancy,
periods of increased haemoglobin formation, the blood copper
is in general increased very much.
As a result, the writer
is o3S the opinion that the copper effect probably takes place
in the blood itself.
There nas been much controversy as to whether the
administration of copper is essential in anaemias in man.
Hone of these studies appear to have included any estimations
of blood copper.
The writer considers that such estimations
might throw some light on the problem.
The prevalence of
low blood coppers in anaemias associated with nephritis
requires explanation.
The writer considers that this indicat€
a copper Insufficiency, this being due probably to the nature
222.
of the diets used in tne treatment of* nephritis.
Very little is known as to the fate of haemoglobin
except that the iron is held tenaciously by tne tissues and
tne porphyrin part of the molecule is excreted in the bile
as bilirubin.
Numerous workers
^Barkan (1925 - 1937),
Starken stein and Weden (1930), Moore et alia (1939) J have
concerned tnemselves with what they describe as the 1easily
split1 iron of tne blood.
It is located in the corpuscles.
Barkan and Senales (1937) believe that this is derived from
so called pseudo-haemoglobins which they regard as
intermediates in the decomposition of haemoglobin.
is inclined to the view that it is an artifact.
The write:
Estimations
of ‘easily split1 iron involve drastic treatment of blood,
a most complex mixture, with acid at high temperature.
No
worker has shown that haemoglobin is not attacked under such
circumstances.
The writer has been working on the problem
recently and although the work has not been completed, has
obtained sufficient evidence to show that haemoglobin does
decompose under such conditions*
Haemochromatosis, although a rare condition, has
received considerable attention.
Of particular interest, is *
origin of the large deposits of iron in the soft tissues,
especially the liver.
A considerable amount of work has been
done on the subject and has been reviewed by Sheldon (1934).
It is now considered that these large deposits of iron are du<
to a positive iron balance over a long period.
The tissues,
especially the liver, generally show an increased copper
content.
Mallory and his co-workers (1925, 1931) have
attempted to show a relationship between copper poisoning
and haemochromatosis#
This view is not accepted#
The many riddles with which we are confronted are due
to our very incomplete knowledge of the fundamentals of iron
metabolism#
It is strange that although the importance of
iron in respiration has been known for a long period* so littl
is known about the physiological processes in which this
element is involved#
This lack of knowledge is probably
responsible for our inability to understand the rble of copper
Methods of approach to these subjects have been very similar#
It appears to the writer that different and more variable
methods of approach are required#
References
Abderhalden and Waller (192B) Z, physiol. Chem.
Adamson and Smith (1931)
Can ad* Med. Ass. J..
Allison, Bryan and Hunter (1927)
Andreasch (1879)
Ansbacher, Remington and Culp (1931)
Barkan (1925)
(1937)
(1927)
Klin, wschr.
Barkan and Berger
(1928)
6,
t
mm“
1391.
J • Ind. Eng. Chem.
148,
59,
Biochem. Z.
(1933)
Z. physiol. Chem.
785.
124.
1615.
224.
53.
216,
1, 17.
(1933)
221.
241.
(1935)
236.
97.
(1936)
239,
97.
(1936)
244,
81, 257
(1937)
Klin, wschr.
16. 1265.
Beard and Myers (1931) J. Biol. Chem.
Begemann (1924)
(1933)
Z. vergl. Physiol.
2, 381.
Z. physiol. Chem. 220,
Compt. rend. Acad.
Bertrand (1920)
94, 71.
loc. cit, Jordan (1925)
Bersin and Legemann
65,
Bull. Soc. Hyg. Aliment.
Biazzo (1926) Ann. Chim. Appli.
16,
209.
300.
Bethell, Goldhamer, Isaacs and Sturgis
Blasius (1866)
3,
Arch. f. exp. Path. u. Pharmakol.
136. 278.
(1930)
(1867)
793.
171, 179, 194.
(1927)
Bert
12,
Arch. Int. Med.
Z. physiol. Chem.
24 ,
95
Flo. Agr. Exp. Stn. Bull.
Ber. deut. chim. Ges.
Barer and Fowler
1V6,
8,
49.
(1934) J. Amer. Med
103. 797
2.
Z. ration. Med., 3 reihe,
26, 250.
Bloxsom
(1932)
Bodansky
Brock
South. Med* J.
(1921)
J. Biol. Chem.
(1937) Brit. Med. J.
Brock and Hunter
Bunge
25 , 401.
(1937)
48, 361.
1,
314.
Quart. J. Med.
(1884) Z. physiol. Chem.
9,
6,
5.
49.
Caldwell and Dennett
(1932)
Med. J. Rwcord. 135,
Callan and Henderson
(1929)
Analyst 54,
Cason
(1934) J. Ped.
4,
Church
(1868)
(1869)
650.
614.
Cherbulies and Ansbacher
Chevreul
286.
(1930)
Compt. rend.
Arch. klin. Med.
Acad.
66,
567*
Proc. Hoy. Soc. Lond.
17,
436.
278,
365<
Conant, Chow and Shoenbach
(1933)
J. Biol. Chem.
101, 463.
Conant, Dersch and Myrdans
(1934)
J. Biol. Chem.
105, 755,
Coons (1932)
Cunningham
Dameshek
J. Biol. Chem.
(1931)
(1933)
Biochem, J.
(1933)
Davidson and Leitch
Dhere
(1848)
(1920)
215.
25,
(1934)
(1930)
685.
Hutr, Abstr. Rev.
J. Physiol. Path.
Drab kin and Waggoner
Elliott
1,
540.
3,
J. pharm. Chim., 3 ser., 13,
Dieekmann and Waggoner
(1930)
100,
Brit. Med. J.
Gen.
Dhere" and Burdell (1919)
Dwyer
1267.
J-. Amer. Med. Assoc.
Davidson et alia
Deschamps
97,
(1936)
(1930)
1081*
IS,
685.
Arch. Int. Med.
J. Michigan State Med. Soc.
Bio chem.
Elvehjem and Hart
(1926)
J.
Elvehjem and Lindow
(1929)
24,
53,
89,
29,
71.
51.
420.
310.
J. Biol. Chem.
Elvehjem, Steenbock and Hart
91.
18,
J. Biol. Chem.
901.
67,
43.
(1929) J. Biol.Chem.
J. Biol. Chem.
81,
435.
83,
21.
Elvehjem
(1930)
J* Biol#
(1930)
Biochem. J.
(1931)
J. Biol#
Elvehjem and Sherman
Elvehjem
(1932)
Chem#
(1927)
(1932)
90, 111.
J. Biol. Chem.
(1933)
309#
J,Biol.
(1923)
(1920) Bull, Soc.
(1934)
Biochem, J.
28,
Fullarton
(1937)
Arch. Dis. Child.
Gordonand Rabinowitch
Guillemet
(1891)
(1933)
Z. physiol. Chem.
(1920)
39,
(1937)
Harless
(1847)
Harrison
7, 535.
Med.
51,
171,
196.
108,
32.
109,
652.
249.
Arch. Anat. Physiol. 148.
(1927)Biochem, J.
21,
335.
Hart, Steenbock, Elvehjem and Waddell
Hart, Elvehjem, Waddell and Herrin
(1925)
(1927)
Hart, Steenbock, Waddell and Elvehjem
J. Biol. Chem
65, 67,
<T. Biol. Chem.
72, 299.
(1928)
J. Biol. Chem
77, 797.
Hart, Elvehjem, Steenbock, Berhstedt and Fargo (1930)
«J. Biol. Chem. 86,
Heath
143.
567.
Compt. rend. Soc. Biol.
16,
27, 440,
15, 371.
Compt. rend. Acad.
Medicine
167.
91.
Arch. Int.
Clin. Med. Surg,
(1931)
12,
(1932)
Hahn
61
1160.
Arch. zool. exp. et gen.
Guerithault
103,
128,
Chim.
(1878)
(1932)
Chem.
Z* physiol, Chem.
Fredericq
Gros
309.
Phytopath. 17, 49#
Fleur ant and Levi
Gottlieb
98,
J. Amer, Med. Assoc. 98,
Fischer and Hilger
Foweather
24, 415.
Chem.
Elvehjem, Hart and Sherman
Felix
86, 466#
(1933) .Arch. Int. Med.
51,
459.
277.
He Herman, Perkins and Clark
Henriques and Poland
Herkel
(1930)
Heubner
Hill
Hoagland
(1928) Biochem. Z.
Beitr. Path. Anat.
(1926)
(1931)
(1933) Proc. Hat* Acad# Sci*,
Washington, D.C., 19, 855*
Klin, wschr.
85,
5,
513.
B107,
Ann. Review Biochem.
Hogben and Pinhey
(1926)
479*
588.
Proc. Roy. Soc. Lond.
(1932)
201,
205.
1,
618.
Brit. J. Exp. Path.
4,
203.
5,
55.
(1927)
von Hosslin (1882)
loc. cit. Sherman (1924)
fChemistry of Food and Nutrition1, London
Hugounencq and Morel
(190E) Compt. rend. Acad. Sci.
140,
141.
Hunt and Carroll
Hutchison
Josephs
(1933)
(1937)
(1931)
loc. cit. Elvehjem (1935)
Physiol. Rev. 15, 471.
Arch. Dis. Child.
Bull. Johns Hopkins
(1932) J.
Keil and Nelson
Biol. Chem.
(1931)
(1936)
Kennedy (1927)
Biol. Chem.
J.
Kleinmann and Klinke
Laidlaw
(1929)
(1904)
(1931)
Lintzel (1929)
(1931)
96,
246.
559.
93,
Arch. Int. Med.
J. Dairy Sci.
J. Physiol.
74,
12,
49.
58,
278.
385.
242.
31,
(1933)
464.
Z. physiol. Chem.
J. Amer. Med. Assoc,
Z.
49,
(1930) Arch. Path. Anat. Physiol.
275, 422.
Lanyar, Lieb and Verdino
Lewis
305.
Hosp.
J. Biol. Chem.
Kellog and Mettier
Krauss
12,
Biol. 89 ,
Erg. Physiol.
Locke, Main and Rosbash
(1932)
2ZB'
96,
217,
16
1135.
89.
31,
844.
J. Clin. Investig.
12,
967
Lyons
(1927)
Macho Id
J. Amer. Chem# Soc.
(1934)
Mackay
Mallory
Z. klin. Med..
(1933)
Arch. Dis. Child.
Amer. J. Patn.
(1931)
Maquenne and Dempussy
Marlow and Taylor
(1920)
McFarlane / (1932)
1,
117.
7,
Lancet
2,
(1938)
J. Physiol.
551.
6,
299.
94,
J. Biol. Chem.
107,
583.
(1926)
£7,
245.
(1928)
J, Biol. Chem.
Ann. chim. phys.
Michaelis and Runstrbm
(1934)
4,
Can. Med. Assoc. J.
(1931)
J.
(1939)
154.
(1937)
J. Clin. Investj
16, 613.
J. Clin. Investg.
(1929)
J. Amer. Med. Assoc.
(1931)
J. Biol. Chem.
(1923)
637.
175.
Amer. Med. Assoc. 182,
Parsons and Parsons
78,
106.
22,
Moore, Arrowsmith, Quilligan and Read
% e r s and Beard
309.
Proc. Soc. Exp. Biol. Med*
32, 343*
(1930)
Moore, Minnich and Welch
148.
26, 1034, 1061.
Amer. J. Physiol. 72,
(1817)
87.
680.
(1925)
McHargue, Healy and Hill
170,
53,
J. Biol. Chem.
(1937)
(1934)
351.
Compt. rend. Acad.
Biochem. <J.
McFarlane and Milne
145.
Arch. Int. Med.
(1909)
Me Cane e and Widdowson
8,
Amer. J. Path.
(1934)
Matthews and Walker
Mills
27.
Med. Res. Council, Spec. Rep. Ser., London.
Mallory and Parker
Meissner
127,
1919.
(1931)
(1925)
McHargue
49,
94,
93,
18 ,
1210.
71.
J, Gen. Physiol.
6,
153.
Parsons and Hawksley
(1933)
Arch. Dis. Child.
8,
117.
Parsons and Hickmans
(1933)
Arch. Dis. Child.
8,
95.
543*
Rabinowitch
(1933)
J. Biol. Chem.
Redfeld, Coolidge and Hurd
100,
479.
(1926) J. Biol. Chem.
Reimann and Fritsch
(1930)
Z* f. klin. Med.
Riecker and Winters
(1930)
Amer. J. Physiol.
Rose, Vahlteich, McCollum and Macleod
Sachs, Levine and Fabian
Sarzeau
(1830)
Schindel
(1931)
Klin, wschr.
Lancet
J. Biol* Chem.
104,, 217.
Z.
58,
523.
505.
physiol. Chem.
25,
180, 249.
1608.
1031.
(1933) Chemistry of Food and Nutrition, London.
Shorlands and Wall
Socin
2,
92,196.
743.
and Ram age (1931) Biochem. J.
Sheldon(1934)
Sherman
10,
(1929)
(1934)
16,
475.
115,13.
Arch. Int. Med.
J. pharm. et chim.
Schonheimer and Oshima
Sheldon
(1935)
69,
(1891)
(1936) Biochem. J. 30,
Z. physiol. Chem.
Starkenstein and Weden
Starkenstein
(1930)
(1928)
15,
1049.
93.
Arch. exp. Path. u. Pharmak.
134, 278.
Arch. exp. Path. u. Pharmak.
150,
324
(1934)Handhuch. der allgemeinen Haematologie.
Stedman
Strauss
and Stedman (1926) Biochem. J.
(1934)
J. Amer. Med. Assoc.
Sumner and Poland
Tartakowsky
(1933)
(1903)
(1904)
Warburg
(1930)
(1927)
103, 1.
30,
5.
100, 581.
101, 423.
(1929)
Waddell, Steenbock and Hart
Walker
938.
Proc. Soc. Exp. Biol. Med.
Pflugers Arcliiv.
Titus, Cave and Hughes
20,
J. Biol. Chem.
(1929)
J. Biol. Chem. 83,
243
(1929)
84,
115
J. Assoc. Offic.-Agr. Chem. 13,
Biochem.Z.
80, 565.
187,255.
246.
Warburg and Krebs
Waugh
(1931)
(1927)
Biochem* Z#
Arch.# Int. Med.
47,
190,
71.
Whipple and Hobscheit-Robbins
(1936)
Widdowson and McCance
(1936)
J. Hyg.
(1937)
Biochem. J.
Wintrobe and Biebe
Weltering
(1895)
(1933)
143#
Amer. J. Med. Sci.
36,
13#
31,
Medicine
12,
187#
Z. physiol. Chem.
21,
186.
2029.
Lead, Iron and Copper
Contrasts and Comparisons
Iron, copper and lead are representatives of the
heavy metals and although the interest in the two former
is chiefly physiological and in the latter chiefly
toxicological, it is of interest to compare and contrast
them biochemically in so far as our knowledge permits*
All three exist in two forms of different valencies
iron in the divalent and trivalent states, copper in the
monovalent and divalent states and lead in the divalent and
tetravalent states#
We know that the two forms of iron may
be found in animal tissues and diets and in the case of
copper we have evidence that although the divalent form is
more general, the monovalent form may be found in certain
situations e.g. the R. B* C.
There is no evidence to suggef
that lead exists in biological materials in anything but the
divalent state*
The possible differences in physiological
action of two forms of a metal with different valencies
should not be overlooked.
Under normal conditions, the human diet contains
considerably more iron than copper and considerably less
lead than copper*
Marked differences may be observed in the
capacity of the body to absorb and retain these.
Lead
appears to be most easily absorbed and retained and because
of its toxic properties, it is just as well that it exists
in normal diets in small quantities.
The absorption of thee
three metals appears to be dependent upon the calcium
content of the diet and the gastric acidity in the same way.
The absorption of iron is complicated by the fact that
ferrous iron is absorbed but not the ferric form.
This
necessitates a reducing mechanism in the alimentary tract.
Lead appears to he most easily retained.
This is probably
connected with the fact that whereas the skeleton has a
great affinity for lead, it has little affinity for iron
or copper.
The liver has a marked affinity for all three
metals.
Lead and copper probably exist in animal tissues
in comparatively simple forms but iron exists in various
forms, some simple and others complex e.g. haemoglobin,
ferric complexes.
Lead and copper appear to be fairly easily excretec
the intestine being important in this respect.
Iron on
the other hand does not appear to be as easily excreted.
This is probably due to two reasons, 1. much of the body
iron is present as haemoglobin, 2. the distinctive propert:
of ferric organic complexes.
The kidney is usually regarded as a concentrating
organ.
Although iron, copper and lead are present in urine
their concentrations are much lower than in plasma.
causes probably contribute to this.
Two
In the first place,
these metals are probably circulating as metallic protein
complexes which are poorly ionised and as a result the
concentration of metal in the glomerular filtrate will be
representative of the ionised part only and in the second
place no concentration occurs during passage through the
tubules.
The mobilisation of lead under the influence of
various agents is unique and is connected with its deposit]
in the skeleton.
In this respect it resembles calcium*
Reprinted from
The B ritish Journal of Experim ental Pathology)
1939, Vol. X X, p. 408.
.STUDIES IN LEAD MOBILIZATION.
S. L; TOMPSETT
and
J. N. M. CHALMERS.
F r o m the Biochemical Laboratory) the Institute of Pathology of the University
and Royal Infirmary, Glasgow.
L ondon:
H. K.
L e w is &
Co.
L td .,
130
C o w e d S 'H ie e t ,
W.C. 1.
M ade and printed in Great B rita in .
Reprinted from
The British Journal of Experimental Pathology,
1939, Vol. X X , p. 408.
STUDIES IN LEAD MOBILIZATION.
S. L. TOMPSETT
and
J. N. M. CHALMERS.*
From the Biochemical Laboratory, the Institute of Pathology of the University
and Royal Infirmary, Glasgow.
Received for publication August L8th, LiKiU.
A r e v ie w o f th e su b ject o f lead poisoning up to 1925 w as m ade by Aub
et. alia (1926) in their m onograph, and further in v estig a tio n s in th e decade
follow ing w ere review ed by A ub (1935). A lthough th e a n a ly tic a l m eth od s
described in th e m onograph h ave been show n to be som ew h at inaccurate,
considerable in terest w as aroused in th e biological im portance o f lead.
U sin g a new sen sitiv e and accurate m eth od for th e estim a tio n o f lead in
hum an tissu es and excreta (T om p sett and A nderson. 1935), lead can be d etected
in th e tissu es o f persons w ith no h istory of exposure to lead other th an th e
“ norm al ” hazard. T he “ norm al ” con ten t o f lead in hum an tissu es is derived
p resu m ably from th e in h a la tio n of d u st containing lead, and from th e con­
sum ption o f food and w ater containing traces o f lead. T he q u estion o f the
* McCJunn Scholar.
409
STUDIES IN' LEAD MOBILIZATION.
occurrence o f lead in foodstuffs has been review ed b y M onier-W illiam s (1938),
and th a t o f lead in drinking-w ater b y In g leso n (1934).
T he con cen tration o f lead varies in th e different tissu es, th e h igh est con cen ­
trations being found in th e bones, esp ecially th e long bones, such as th e fem ur
and tibia (L ynch, Slater and Osier, 1934: T o m p sett and A nderson, 1935;
T om p sett. 1936). A ub and his co-w orkers b eliev e th a t lead is stored in the
sk eleton exertin g no to x ic action , w hile th e lead present in th e so ft tissu es and
circulation is so lely responsible for th e sy m p to m s and to x ic episodes in plum bism .
C onsequently the con d ition o f a person w ho has absorbed large q u a n tities
o f lead depends en tirely on th e relative d istrib u tion o f lead b etw een th e soft
tissues and th e sk eleton . A ub and his colleagues h ave sh ow n clin ically th a t
the to x ic episodes in plum bism are relieved q u ick ly b y high calcium th erap y,
and th a t th e clinical im p rovem en t is accom p an ied b y a decrease in th e am ou n t
o f lead excreted. T his su ggests th a t lead is being laid dow n in th e bones.
The clinical side o f th is work has b een confirm ed b y B elkn ap (1929).
Two different view s are held regarding the treatm ent o f plumbism ; in
one a high calcium intake is necessary in order to keep the lead locked in the
bones ; the other favours a process of de-leading, as it is considered th a t a large
store of lead in the bones is a potential danger, and in m etabolic disturbances is
liable to be mobilized and pass into the soft tissues and circulation. Aub
(1935) supports the second view.
From a stu d y o f agents lik ely to produce m ob ilization and increased ex cre­
tion o f lead, it w as found th a t sodium bicarbonate, p otassium iodide and a
low calcium in tak e Avith or w ith o u t acidosis-producing su b stan ces were effective
(Aub, Fairhall, M inot and R etznikoff, 1926). Sim ilar results w ere ob tain ed
w ith parathorm one in con ju n ction w ith a I o a v calcium d iet (H unter and A ub,
1927). Litzner. W eyrauch and B arth (1931) confirm ed th e efficiency o f sodium
bicarbonate, but n ot o f acidosis-producing su bstan ces, th e conclusions being
based upon th e effect o f th e agen t upon th e urinary excretion o f lead w hereas
th ose o f Aub were based upon th e to ta l excretion.
L ead is presen t in norm al hum an b lood in am ou n ts ranging betw een
40-60 pg. Pb per 100 ml. (T om p sett and A nderson, 1935). F ifty in d ividuals
in th e G lasgow area w ith no h istory o f lead exp osu re other th a n th e norm al
hazard were fou n d to con tain 30-80 pg. Pb per 100 ml. blood, w ith a m ean o f
54 pg. Pb per 100 m l. blood (Chalmers, 1939). S om ew hat sim ilar results
have been reported b v other w orkers (B lum berg and S co tt, 1935 ; Teisinger,
1936 ; T eager and S ch m itt, 1937 ; W illou gh by and W ilkins, 1938 ; K ehoe,
Tham ann and Cholak. 1935).
In 1938 w e published th e results o f in v estig a tio n s carried ou t on a case
o f lead poisoning produced b y th e oral ad m in istration o f lead a ceta te. I t w as
found th a t th e level o f blood lead w as affected v ery m ark ed ly b y certain
changes in d iet and by m ed ication . A high calcium in ta k e p roduced a fa ll in
blood lead, w hereas a low calcium d iet w ith or w ith o u t parathorm one produced
a rise in blood lead. In a case o f lead poisoning o f in d u strial origin, it w as
n oted th a t p otassium iodide produced a m arked rise o f blood lead, w hich at
th e sam e tim e w as accom panied b y a return o f th e to x ic sy m p to m s o f plum bism
(T om p sett and A nderson, 1935 ; Chalm ers an d T o m sett, 1938).
T hese in vestigation s h ave b een ex ten d ed to further cases o f clinical plum bism
410
S. L. TOMPSETT AND J. N. M. CHALMERS.
and increased lead absorption, w ith special reference to the effects of changes
of diet and of m edication upon the level of the blood lead. In tw o cases the
rate of excretion of lead was studied over a period. The m ethods o f analysis
were those of Tom psett and Anderson (1935) and of T om psett (1939a).
The low calcium diets em ployed contained 0-13 g. Ca per day, and the
high calcium diets contained 2-7 g. Ca per day. In some cases th e high calcium
diet was supplem ented by daily intramuscular injections of 10 ml. of a 5 per
cent, solution of calcium gluconate. To produce acidosis, amm onium chloride
was given by m outh in doses of 1 g. four tim es per day.
Clinical and liEematological investigations were carried out at the same
time, but the results of these are being recorded elsewhere.
346
/S39 3 6 3 0
II
CHART
3630
/A
200
1000
30
£*
100
500
40 0
50
WEEK5
300
200
/
2
3
k
4
n>
S
6
»
to / /
/Z /3
22
00
DAYS
3
6
9
12
/S
18
TOTAL EXCRETION OVER PERIODS OF3 DA
URINARY ~
■
* *
■
ft
§
C a s e 1.— L ead burner (b a tte iy w orks). Male, 29 years. L ead poisoning.
T he in v estig a tio n s w ere carried out over an ex ten d ed period. F ollow in g high
calcium in ta k e for 14 days, th e p a tien t w as placed on Ioav calcium d iet and
am m onium chloride for 9 days. FolloAving th is, high calcium d iet Avas m a in ­
tained. D uring th e b eginning o f th e la st period injections o f calcium glu con ate
were given. D uring th e period o f m ed ication Avith am m onium chloride, and
for 6 d ays before and after it, th e excreta w ere collected in periods o f 3 d a y s and
their lead co n ten t estim ated . T he high calcium d iet Avas m a in ta in ed for a
long period.
The results obtained are shown in Charts l a and lb.
C a s e 2. — R ed -lead worker. M ale, 32 years. L ead poisoning. T his
p a tien t w as trea ted sim ilarly to Case 1. T he results ob tain ed are shoAvn in
Charts 2a and 2b.
C a s e 3.— W h ite-lea d A v o r k e r . M ale, 66 years. L ead poisoning. T his
p a tien t Avas placed on altern ate periods o f high calcium in ta k e and I o a v calcium
§
2,
iHIGH CaDlETKLOWCa DIETa-NH}CTXWGH CciDILO
lead
1250
150
/up Po per zoo m.z
(500
B lood
BLOOD
L £/}£) pp Pi) p<sr/00m . Z.
I
• I ll
STUDIES IN LEAD MOBILIZATION.
in tak e w ith am m onium chloride. F in a lly he w as m ain tain ed on high calcium
diet.
T he resu lts are show n in Chart 3.
267
252
267
I I I
CHART 2 A
200
c h a r t
2 B
600
it iso
500
5 too
§
§
? 2-0
4 00
300
50
'o
*0
/.55kS
200
100
0 0-5
/
X--
J
4-
*-
1
/3
S
*
ts $
*55 v5
v3^§
v<+
20
HUGH CaDIETXLOW Ca D/ET+NH*CIH H/CH Ca DIET
TOTAL E X C R E T IO N O VER P E R IO D S O r 3 D A T S •
U R IN A R Y
§ Sk
■■
-
-
-
'
*
1
SOD
248 307
444
f-o
CHARTS
5 200
|
6 150
<
100
§
*<1
50
14EEK 5
2
3
5 Si
SIS vS klk
£ 1
4 - 5
■Hv-KS
k
6
L. .
7
~K-
.J_
/o
//
/2
k
I
g
I
I
/J
/4
29
%
3
vS
1
C a s e s 4, 5 and G.— L ead w orkers w ith v a g u e sy m p to m s.
M ales, 25, 33
and 56 years. In creased lead absorption. T h e b lood lead o f th ese p a tien ts
w as determ ined in itia lly , and th en later after a period o f h igh calcium in tak e.
T he results ob tain ed are show n in Charts 4, 5 and 6 resp ectiv ely .
BLOOD L E A D P b p e r L O O m l
38 7
412
S. L. TOMPSETT AND J. N. M, CHALMERS.
Case 7.— L ead poisoning due to a lead con tam in ated drinking-w ater.
F em ale, 27 years. T he effect o f high and low calcium in tak e u p on th e blood
lead w as in v estig a ted . T he results ob tain ed are show n in Chart 7.
Cases 8, 9 and 10.— In creased lead absorption due to a lead -con tam in ated
drinking-w ater. M ales, 34 and 7 years ; fem ale, 9 years. T he blood lead w as
'S2 200
CHART
^
4-
200
charts
Kj
|
^ 150
^ 150
too
SA
i
100
I
5
50
50
100
^ 50
§
§
/
WEEKS
CHARTS
Z
3
4-
S
W EEKS
NO TREATMENT*HIGH Ccl D/EE-
N> 200
CHART
m
WEEKS
/
TREATM ENriH/CrHCa
1 2
3
4-
DIET- -
7
J
k
150
^
100
§
Vi
50
^
50
50
1
WEEKS
100
4-
/
k
S
6
fei
7
WEEKS
/
NO TREATMENT*-
HIGH Ca DIET-
v2
I
§
vi
I
d eterm ined in itia lly , and th en th e p a tien ts were m ain tain ed on h igh calcium
in take. T h e b lood lead w as determ ined a t in tervals. T he resu lts ob tained
are sh ow n in Charts 8, 9 and 10 resp ectively.
T he effect o f va ria tio n in th e calcium con ten t o f th e d iet u p on lead m o b ili­
zation as sh ow n b y th e am ou n t o f lea d in th e circulation is illu stra ted by
Cases 4 -1 0 , a high calcium in ta k e being follow ed q u ick ly b y a fa ll in b lood
lead. A s th e trea tm en t in som e o f th e cases lasted over a period o f w eek s, th is
could b e due in p a rt to loss o f lead b y excretion. T h a t th e calcium con ten t
o f th e d iet does exert a pow erful influence is b est illu strated b y Case 7, w ho
STUDIES IN LEAD MOBILIZATION.
413
was placed altern a tely on high and low calcium diet. On high calcium diet
the b lood lead fell, w hile on low calcium d iet it rose.
T he effect o f acidosis-producing su bstan ces, v iz. am m onium chloride, in
conjunction w ith a low calcium d iet w as stu d ied in Cases 1 -3 . I n Cases 1 and
3 th e effect o f trea tm en t w as to produce m arked rises in blood lead, th e levels
being m uch higher th a n th ose ob tain ed w ith low calcium d iet alone. In Case 1,
in w hich th e rate o f ex cretio n w as stud ied , th e to ta l a m ou n t o f lead excreted
was increased, but n ot to a n y th in g like th e sam e le v el as th e increase in blood
lead. In Case 3 high calcium th erap y and acidosis th era p y w ere alternated
several tim es. On each occasion responses w ere ob tain ed as ev id en ced b y
the changes in blood lead. In Case 2 th e b lood lead w as hardly affected by
am m onium chloride treatm en t, alth ou gh th e rate o f ex cretio n w as increased.
CHtf&T/ O
’iQ
^ 100
T ioo
50
WEEKS
50
/
N O T R E im iK n
2
-
3
4-
S
6
.
HIGH C a D/ET~
WEEKS
/
A'0 TREATMENTS
-HIGH C a D /E T -
This m ay be exp lain ed b y th e fa ct th a t th e su b ject w as ex p o sed to lead for a
com p aratively short period and con seq u en tly his store o f absorbed lead w ould
be sm all. P atien t 7 died ; th e lead con ten ts o f certain tissu es are show n in
'Fable J. W hen com pared w ith th e norm al figures for h um an tissu es as
ob tain ed by T o m p sett and A nderson (1935). it w ill be seen th a t all th e soft
tissues show an increased lead content. T he lead co n ten t o f rib is increased,
but th a t o f tib ia and fem ur falls w ith in norm al lim its.
T a b l e I . — L e a d Content of the Tissues of Case 7.
The results are expressed in mg. Pb per kg. fresh tissue.
Brain
.
.
.
.
.
3 • 08
Liver
.
.
.
.
.
7*14
K idney
.
.
.
.
.
4*62
R
i b .............................................................52*1
T i b i a .............................................................53*3
Femur
.
.
.
.
.
52 • 1
T he changes described hi this paper up to n ow h ave n o t b een show n ex p eri­
m en tally to be due to differences o f d istrib u tion o f lea d b etw een so ft tissues
and skeleton. T he follow ing anim al ex p erim en ts w ere therefore carried out.
Mice were fed w ith abnorm al am ou n ts o f lead, and th e d istrib u tion o f lead
betw een soft tissu es and sk eleto n d eterm ined under different conditions. I t
has been show n (T om psett, 19396) th a t lead is absorbed from th e alim entary
414-
S. L. TOMPSETT ANT) J. N. M. CHALMERS.
tract, rapidly on a lo w calcium d iet an d slow ly oil a high calcium d iet. A series
o f m ice w ere fed w ith a lo w calcium d iet o f th e follow ing com p osition , togeth er
w ith an added su p p lem en t o f lead in th e form o f lead a ceta te a t th e rate of
1 mg. P b. per m ouse per d a y for a period o f 14 days.
L o w C a l c i u m Diet (Shelling, 1932).
W hole w heat flour
.
.
.
.
Corn starcli
.
.
.
.
.
Casein
.
.
.
.
.
.
W h eat gluten
.
.
.
.
.
B u tte r
.
.
.
.
.
.
Olive oil
.
.
.
.
.
Sodium chloride .
.
.
.
.
P otassium chloride
.
.
.
.
(2-5 g. per m ouse per day.)
100 g.
g.
g.
g.
g.
g.
20 g.
15 g.
325
100
50
50
40
A t th e en d o f th is period th e y were placed on th e diet, less lead, for a period
o f 3 days to rem ove unabsorbed lead from their alim en tary tracts. A third
o f their num ber were k illed (a).
T he rest were placed on a h igh calcium diet
for 7 days. T his w as affected b y th e ad d ition o f 0-5 g. o f calcium g ly cero ­
p h osp h ate to th e low calcium diet.
A t th e end o f th is period one h a lf of th e rem ainder were k illed a t once (b),
and th e others w ere placed on th e low calcium d iet for a further period of
7 days before b ein g k illed (c). In each anim al th e sk eleton w as sep arated
from th e so ft tissu es and th e lead co n ten t o f each determ ined. T h e results
are show n in T able II.
T a b le II.
The results are expressed in mg. Pb.
A. O n low calcium diet alone .
Mouse No.
L ead content of soft tissues
L ead content of skeleton
T otal lead .
.
.
.
Percentage of lead in skeleton
Percentage of lead in soft tissues
l.
2.
3.
0*154
.0 * 1 0 5
0*259
40*6
59*4
0 *168
0*125
0*293
42*8
57*2
0 *150
0*100
0 *250
40
60
B. L o w calcium diet followed by high calcium diet.
Mouse No.
Lead content of soft tissues
Lead content of skeleton
T otal lead .
.
.
.
Percentage of lead in skeleton
Percentage of lead in soft tissues
4.
5.
G.
0*077
0*250
0*327
76*4
23*6
0 *089
0 * 312
0*401
77*8
22*2
0*080
0*282
0*362
77*9
22*1
415
.STUDIES IN LEAD MOBILIZATION.
c. L o w calcium diet followed by high calcium diet, followed by
loio calcium diet.
Mouse N o.
7.
Lead content of soft tissues
Lead content of skeleton
Total lead .
.
.
.
.
Percentage of lead in skeleton
Percentage of lead in soft tissues
0 -0 7 7
0 -1 4 3
0 -2 2 0
65
35
8.
0 -0 8 7
0-151
0 -2 3 8
64
36
9.
0 -0 9 0
0 -1 7 8
0 -2 6 8
62
38
D IS C U S S IO N .
T he in v estig a tio n o f hum an su b jects w hose tissu es con tain a large am ou n t
o f lead lias show n th e extrem e m o b ility o f lead. T he low calcium d iets w ith
or w ith ou t am m onium chloride produce alm ost w ith o u t ex cep tio n an increase
in blood lead, w hereas high calcium d iets produce a fall o f blood lead, in som e
cases to alm ost norm al levels. T he m o b ility o f lead is sh ow n further b y th e
fact th a t the processes could be rep eated several tim es in succession. I t has
been show n th a t am m onium chloride, as a rep resen ta tiv e o f acidosis-producing
su bstances, is an a ctiv e m obilizer o f lead. T his supports th e w ork o f A ub,
Fairhall, M inot and RetznikofE (1926), b u t con trad icts th e results o f th e Germ an
workers.
Increase in blood lead does n o t necessarily parallel increase in lead excretion.
Similar results have been obtained by T om psett and Anderson (1935) in a case
of lead poisoning which was tre a te d w ith potassium iodide. This raises the
question of the wisdom of de-leading a p atien t w ithout adequate laboratory
control. The question of the wisdom of de-leading a t all m ust arise, as the
process m ay result in a large influx of lead into th e soft tissues and circulation
w ithout com pensatory increase in excretion.
The technique for the study of agents likely to produce m obilization of lead
appears also to require revision. In the p ast interpretations have been based
solely upon changes in p a rt of or in the to ta l excretion of lead. In our view
it is necessary in such studies to exam ine th e blood lead in addition to th e to tal
lead excretion.
In the diagnosis of plum bism , owing to th e influence of diet, a single
estim ation of blood lead m ay lead to erroneous results. We would suggest th a t,
in a suspected case, in th e event of the blood lead being norm al, th e estim ation
should be repeated after a course of low calcium therapy, preferably in con­
junction w ith am m onium chloride. I t is also im p o rtan t if conditions perm it
to exam ine th e lead excretion a t th e same tim e.
From an ex am in ation o f norm al tissu es lead is p resen t in th e h igh est con cen ­
trations in th e sk eleton , b u t th e d istrib u tion is u n ev en , bones such as th e fem ur
and tib ia con tain in g m uch higher con cen trations th an ribs and vertebrae.
In the case described in th is paper hi w hich an abnorm al a m ou n t o f lead had
been absorbed over a period o f tw o years, th e ribs show ed a m arked increase, b u t
n ot th e fem ur or tib ia. I t is probable th a t under con d ition s in w hich abnorm al
am ou n ts o f lead are being absorbed, th e m ore vascu lar bones such as ribs and
vertebras ta k e th e lead in preference to bones o f th e structure o f th e fem ur
S. L. TOM PSETT A N D J . N . M. CHALMERS.
416
and tibia. Sim ilar resu lts were ob tain ed in a case reported b y T o m p sett and
A nderson (1939). T h e p rob ab ility is th a t lead is laid dow n slow ly in th e long
bones and is m ore stable, b u t m a y be laid dow n q u ickly in th e vascu lar bones
and is more easily m obilized. I t is im p ortan t, therefore, th a t in a chem ical
an alysis o f tissu es in a su sp ected case o f lead poisoning, th e vascular bones
such as ribs and vertebra? should be exam in ed , since th e results o b tain ed from
th ese appear to be as im p ortan t as th o se ob tain ed from other tissues.
The anim al experim ents illustrate particu larly well the effect of low and
high calcium intake upon th e distribution of lead between soft tissues and
skeleton. On a low calcium diet lead predom inates in th e soft tissues, although
considerable am ounts were found in th e skeleton. On high calcium diet lead
predom inated in th e skeleton, b u t the am ount of lead in th e soft tissues was not
negligible, and was probably capable of producing damage. This point of
view would su pport de-leading.
In all probability im perfections exist in both courses of th erap y suggested
for the tre atm e n t of plum bism . The safest course is naturally preventive.
SU M M A R Y .
1. T he effect o f high calcium d iet an d o f low calcium d iet w ith and w ith o u t
am m onium chloride has been stud ied in cases o f lead poisoning and in creased
lead absorption. H igh calcium d iets produced a fall in blood lead, w hile low
calcium d iets w ith and w ith o u t am m onium chloride produced a rise in blood
lead.
2. There w as no parallelism b etw een blood lead lev el and lead ex cretio n in
the tw o cases exam in ed th u s.
3. T he effect o f high and low calcium d iets upon th e d istrib u tion of lead
b etw een so ft tissu es and sk eleto n in m ice has b een studied.
4. T he q u estion o f trea tm en t in lead p oisoning and th e aid o f th e b io ­
chem ical lab oratory in th e diagnosis o f lead poisoning has b een discussed.
In conclusion w e w ish to record our th an k s to Dr. J o h n Gracie, Prof. J. W.
M cN ee, D r. G. A. A llan, D r. W . R . Snodgrass and D r. A. E . Struthers for givin g
us facilities to stu d y and ex a m in e cases in their w ards, to Sister R ose for her
co-operation w ith diets, and to Dr. A. B . A nderson for helpful criticism and
advice.
REFERENCES.
Aub, J. C.— (1935) J. Amer. med. Ass., 1 0 4 , 87.
Idem, F a i r h a l l , L. T., M i n o t , A. S., a n d R e t z n i k o e p , P.—-(1926) ' Lead Poisoning
Baltimore.
B e l k n a p , E. L.— (1929) Wis. med. J ., 2 8 , 346.
B l u m b e r g , H ., and S c o t t , T. F. M.— (1935) Johns Hopk. Hosp. Bull., 5 6 , 276, 311.
C h a l m e r s , J. N. M.— (1939) Unpublished.
Idem a n d T o m p s e t t , S. L.— (1938) Lancet, i, 994.
H u n t e r , D., and A u b , J. C.— (1927) Quart. J. M ed., 2 0 , 123.
I n g l e s o n , H .— (1934) Dept. Scient. Ind. Res., Technic. Paper No. 4, H.M. Stationery
Office.
417
S T U D IE S IN LEA D M OBILIZATION.
I v e h o e , R. A., T h a m a n n , F., a n d C h o l a k , J.— (1935) J . Amer.
L i t z n e r , S. T., W e y r a u c h , F,, a n d B a r t h , E.— (1931) Arch.
med. Ass., 1 0 4 , 90.
Oewerbepath. Gewer-
behyg., 2, 330.
L y n c h , G. R., S l a t e r , R. H., a n d O s l e r , T.
M o n i e r - W i l l i a m s , G. W . — (1938) ‘ Rep. Pub.
G.— (1934) A nalyst, 59, 787.
Health and Med. Subj.’, Ministry of
Health, H.M. Stationery Office.
T e a g e r , H., a n d S c h m i t t . F .— (1937) Z. ges. exper. M ed., 1 0 0 , 717.
T e i s i n g e r , J. — (1936) Ibid., 9 8 , 520.
T o m p s e t t , S. L.— (1936) Biochem. J., 3 0 , 345.— (1939a) Ibid., 3 3 ,
Ibid., 3 3 , 1237.
Idem a n d A n d e r s o n , A. B.— (1935) Ibid., 2 9 , 1851.— (1939) Lancet,
W i l l o u g h b y , C. E., a n d W i l k t n s , E. S.— (1938) J. hiol. Chem., 1 2 4 ,
M ad e a n d p rin te d in Great B r it a in f o r
I i . K . L e w is <0 Co. L td ., by
A d la rd tt- Bon, L td ., 21 B lo o m sb u ry W ay, L o n d o n , W .C. 1.
1231.— (19396)
i, 559.
693.
Reprinted from
The British Journal of Experimental Pathology,
1939, Vol. X X , p. 512.
FURTHER STUDIES ON THE ABSORPTION, MOBILIZATION
AND EXCRETION OF LEAD.
S. L. TOMPSETT.
Infirm ary, Glasgow.
L ondon :
H. K.
L e w is
& Co.
L t d . , 136 G o w e r S t r e e t ,
W.C. 1.
Reprinted from
The British Journal of Experimental Pathology.
1939, Vol. X X , p. 512.
FURTHER STUDIES ON THE ABSORPTION, MOBILIZATION
AND EXCRETION OF LEAD.
S. L. TOMPSETT.
From the Biochemical Laboratory, Department of Pathology, The Royal
Infirm ary, Glasgow.
Received for publication Novem ber 18th, 1980.
THE ABSORPTION OF LEAD.
I n previous com m unications th e m ethods o f analysis and th e com position
o f th e anim al d iets u sed h ave b een described (T om psett, 1939a, 6), and it
has been show n th a t lead is absorbed readily on a low calcium d iet b u t w ith
difficulty on a high calcium d iet and su ggestions were p u t forw ard to accou n t
for th is (T om p sett, 19395).
T elfer (1939) has show n th a t th e ad d ition o f
hydrochloric acid to th e d iet increased th e am ount o f calcium and m agnesium
absorbed from th e alim entary tract.
In v iew o f th is it w as considered th a t
acidification o f th e d iet w ould aid th e absorption o f lead.
A d u lt m ale m ice were placed on a high calcium diet containing lead aceta te
at th e rate o f 1 m g. Pb per m ouse per day, and 0-5 ml. o f N hydrochloric acid
per m ouse per d ay for a period o f 14 days.
A t th e end o f th is period th e
anim als were placed upon th e high calcium diet alone for 4 d ays to rem ove
unabsorbed lead from their alim entary tracts and killed.
Their to ta l lead
con ten t w as estim a ted and th e results are show n in Table I.
T a b le
I .— A d u lt M a le M ic e . H igh C alcium D iet -(- 1 mg. P b (lead acetate)
0-5 ml. N IIC l p er mouse p er day. P erio d , 14 days.
Weight.
1
2
3
4
5
0
7
8
(g.)
28
19
21
21
23
17*5
21*5
31 - 5
Total lead.
(mg-)
1*429
0- 909
1-111
1* 176
1-000
0-667
0- 909
1*111
Lead.
(mg. Pb per 100 g
A verage
5*10
4*78
5- 29
5*60
4- 35
3-81
4 - 23
3-53
4-58
It was previously found th a t m ice receiving high and low calcium d iet and
lead aceta te a t th e rate o f 1 mg. per m ouse per day for a period o f 14 days,
contained on an average 0-215 and 2-03 mg. Pb per 100-g. m ouse resp ectively
513
ABSORPTION, MOBILIZATION AND EXCRETION OF LEAD.
(Tom psett, 19395). In th e experim ent described in this paper the mice
contained on an average 4-58 mg. P b per 100-g. mouse. These results show
th a t the addition of hydrochloric acid to the diet favours th e absorption of
lead from the alim entary tra c t.
T H E M O B IL IZ A T IO N O F L E A D .
In a previous com m unication th e effect o f certain agen ts on th e m obilization
o f lead w as exam in ed (T om p sett and Chalmers, 1939). In m en w ith histories
o f abnorm al exposure to lead it w as fou n d th a t lo w calcium in tak e, w ith or
w ith ou t am m onium chloride, produced a rise in b lood lead. H igh calcium
intake w as fou n d to produce a fall in blood lead. In m ice a high calcium
intake resulted in lead being transferred from th e so ft tissu es to th e skeleton,
whereas a low calcium in tak e had th e reverse effect.
To produce m ob ilization o f lead in m an th e follow ing are th e com m onest
substances used : am m onium chloride, p otassiu m iodide, sodium bicarbonate.
I t is u sual to use am m onium chloride in con ju n ction w ith a low calcium diet,
and th e other tw o in conjunction w ith w h a t is term ed an ordinary diet.
A du lt m ale m ice were placed on low calcium d iet containing lead a cetate
at th e rate o f 1 mg. Pb per m ouse per d a y for a period o f 14 days. T h ey were
placed on high calcium d iet for 7 d a y s to produce a transference o f lead from
th e so ft tissu es to th e sk eleton. T h ey were th en placed in groups on various
d iets for 7 d a y s and killed. T he lead co n ten t o f th eir so ft tissu es and skeletons
w as estim a ted separately. T he p ercentage o f th e to ta l lead p resent in th e
so ft tissu es and sk eleton was th en calculated. T he results are show n in
T able I I. I t w ill be seen th a t am m onium chloride, p o tassiu m iodide and
sodium bicarbonate produced m ob ilization o f lead on b o th high and low
calcium intake.
THE
E X C R E T IO N
OF L E A D .
Kehoe el al. (1933) have shown th a t, in man, the q u an tity of lead excreted
decreases rapidly after rem oval from exposure until it does not greatly exceed
the norm al, a t 'which level it rem ains for a long period. I t was considered
of interest to estim ate th e q u an tity of absorbed lead th a t could be excreted
during a period after rem oval from exposure.
A d u lt m ale m ice were placed upon low calcium d iet con tain in g lead a cetate
at th e rate o f 1 mg. Pb per m ouse per day for a period o f 14 days. T h ey were
then placed upon high calcium d iet for 7 d a y s to rem ove unabsorbed lead from
their alim entary tracts and to produce a transference o f lead from so ft tissu es
to skeleton. The anim als were th en placed in separate glass jars floored w ith
several layers o f filter-paper for 14 days. H a lf o f their num ber w ere placed on
a high calcium d iet and th e rem ainder on lo w calcium d iet. T he anim als
were then killed and their to ta l lead co n ten t w as estim ated . T he lead co n ten t
o f th e con ten ts o f th e jars w as also estim ated . T he q u a n tity o f lead excreted
during th e period w as th en calculated after m aking allow ances for th e lead
con ten t o f th e filter-paper and d iet consum ed during th e period. The per­
centage o f th e to ta l original lead excreted w as th en calculated. The resu lts,
S. L. TOMPSETT.
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ABSORPTION, MOBILIZATION ANT) EXCRETION OF LEAD.
show n in Table III, indicate th e rap id ity w ith w hich recen tly absorbed lead
m ay be elim inated. On low calcium intake, a p p roxim ately tw o-th ird s o f th e
to ta l lead w as excreted in 14 d ays, and on high calcium in tak e ap p roxim ately
one-quarter o f th e to ta l lead w as ex creted during th e sam e period.
Table
I I I . — Lead, Excreted in 14 d a ys on L ow and H igh C alcium D iels.
L o w C a lc iu m D ie t .
2.
Mouse
N o. 1.
Mou
T otal lead (mg.)
E xcreted lead (m g.)
Original to ta l lead (m g.)
Per cent, original to t
excreted
. 0*105
. 0*175
. 0*280
lead
. 6 2 -5
0*150
0*225
0*375
3.
0*141
0*220
0*361
60*9
60
4.
0*099
0*198
0*297
66*7
H ig h C a lc iu m D ie t.
Mouse No. 5.
T otal lead (mg.)
E xcreted lead (m g.)
Original to ta l lead (nig.)
Per cent, original to t
excreted
. 0*193
. 0*060
. 0*253
lead
.23*7
G.
7.
0*184
0*049
0*231
0*289
0*068
0*357
2 1 -2
19
s.
0*320
0*082
0*402
20*4
D IS C U S S IO N .
I t has been show n th a t th e ad d ition o f hydrochloric acid to th e d iet increases
th e absorption o f lead from th e alim en tary tract. G astric a cid ity, w hich can
show great variation s, is an im p ortan t factor in th e absorption o f lead.
A m m onium chloride, p otassiu m iodide and sodium bicarbonate h a v e been
show n to effect m ob ilization o f lead on b oth high and low calcium intake.
This su ggests th a t such substan ces as potassium iodide and sodium bicarbonate
should n ot be adm inistered to persons w ho h a v e absorbed large am ounts
o f lead unless m ob ilization o f lead is desired, otherw ise to x ic sym p tom s are
liable to develop. H igh calcium in tak e under such conditions w ould be
ineffective. Com m on offenders in th is respect are “ stom ach pow ders ” , th e
m ajority o f w hich con tain sodium bicarbonate.
T h at m a n y lead-w orkers
ad m itted to th e G lasgow R o y a l Infirm ary w ith sy m p to m s o f lead-poisoning
had been treated w ith such pow ders w as show n b y th e presence o f large am ounts
o f b ism u th in their faeces. T he irresponsible use o f “ stom ach pow ders ” b y
lead-workers is a p o ten tia l danger.
A fter rem oval from exposure to lead, absorbed lead m ay be elim in ated
very rapidly. It freq u en tly happens th a t p atien ts, w ith histories o f abnorm al
exposure to lead, are n ot a d m itted for ex am in ation u n til som e tim e has elapsed
since th e la st exposure, and as a resu lt th e q u a n tity o f lead excreted m ay n ot
greatly exceed the norm al. I t cannot be assum ed th a t, under such conditions,
an alm ost norm al lead excretion su ggests a m ild exposure.
s. h. tompsett .
516
The question o f su scep tib ility o ften arises in lead-poisoning. T he w riter
considers th a t v a riation in diet, gastric a cid ity ,' adm inistration o f drugs,
m etabolic disorders, etc., p la y an im p ortan t part.
In all th e exp erim en ts in w hich m ice w ere used, th e ad m inistration o f
relatively large am ou n ts o f lead appeared to h ave little effect upon th e general
bodily h ealth o f th e anim als.
SUM M ARY.
The ad d ition o f hydrochloric acid to th e diet increases th e absorption o f
lead from th e alim en tary tract.
A m m onium chloride, sodium bicarbonate and p otassium iodide produce
m obilization o f lead on b o th a low and a high calcium intake.
R ecen tly absorbed lead m ay be excreted very rapidly after rem oval from
exposure.
The q u estion o f su scep tib ility is discussed.
I w ish to th an k Dr. A. B . A nderson for his helpful criticism and advice.
REFERENCES.
R ehoe, R. A., Tiiamann, F., and Cholak, J.— (1933) J . industr. Hyg., 15, 306.
Telfer, S.— (1939) Glasgow Med. J ., 13, 257.
ToMrsETT, S. L.— (1939a) Biochem. J ., 33, 1231.— (19395) Ibid., 33, 1237.
Idem, and Chalmers, J. N. M.— (1939) Brit. J . exp. Path., 20, 408.
M a d e a n d p r in te d in G reat B r it a in f o r
H . I I . L e w is ifc Co. L td ., by
A d la rd <£ Son, L td ., at th e ir w orks, B arth olo m eiv Press, D o rk in g .
LEAD-POISONING
LEAD CONTENT OF BLOOD AND OF EXCRETA
BY
SIDNEY LIONEL TOMPSETT, B.Sc. L ond., P h .D., F.I.C.
A SSIST A N T BIO C H EM IST, ROYAL IN FIR M A R Y , GLASGOW
AND
ALAN BRUCE ANDERSON, B.Sc. A del ., P h.D., M.R.C.S.
CLINICAL BIO C H EM IST, ROYAL IN FIR M A R Y , A N D LEC TU R E R IN
PATHOLOGICAL BIOCHEM ISTRY IN THE U N IV E R S IT Y ,
GLASGOW
R eprin ted fro m T he L ancet , M arch n , 1939 , P . 559
LEAD-POISONING
LEAD C O N T E N T OF B L O O D A N D OF
EXCRETA
T h e im p ro v ed a n a ly tica l m eth o d s w h ich h a v e
b ecom e a v a ila b le in th e la st few y ea rs for th e e s tim a ­
tio n of sm a ll q u a n tities of lead in b io lo g ica l m aterials,
an d th e gen eral recognition th a t lea d is n orm ally
ex cr eted b y th e hu m an su b ject, h a v e en a b led a
closer approach to b e m ad e to th e p ro b lem of th e
ch em ica l d iagn osis of lead-p oison in g. R ecen t w ork
on th is p roblem h a s been d iscu ssed b y M inot (1938),
W illo u g h b y and W ilk in s (1938), and M on ier-W illiam s
(1938). In a p reviou s paper (T o m p sett a n d A nd erson
1935) w e rep orted figures for th e ex cretio n of lea d b y
p a tie n ts in h o sp ita l w ho h ad n o t b een ex p o sed to lead
an d for th e le v e l of lead in th e b lood of th e s e p a tien ts.
T h e av era g e ex cretio n on h o sp ita l d iet w as 0-05 m g.
lea d d a ily in th e u rin e an d 0-22 m g. d a ily in th e
faeces, and th e b lood -lead varied from 40 to 70 jxg.
per 100 m l., w ith a m ean of 65 (xg.
O nly o n e p a tie n t
w ith lead -p oison in g h a d been ex a m in ed a t th a t tim e,
an d h e sh ow ed as m uch as 380 p,g. lead p er 100 m l.
of b lood.
T h e p resen t pap er is a report of fche resu lts of th e
a n a ly sis of b lo o d an d ex creta from cases o f su sp ected
lea d -p o iso n in g a d m itted to th e R o y a l Infirm ary
during th e la st th ree years.
METHODS
T h e m eth o d s u sed w ere essen tia lly th o se d escrib ed
in th e p reviou s paper. In v iew of certain im p ro v e­
m en ts w h ich h a v e b een m ad e in th e m eth o d for th e
ex tra ctio n an d estim a tio n of lea d , th e m eth o d as n ow
ad a p ted for th e a n a ly sis of b lood is g iv en in d eta il.
A p p ro x im a tely 20 m l. of v en o u s b lo o d w a s w ith ­
draw n w ith a n a ll-glass syringe a n d a sta in lessste e l n eed le. T h e b lood w as im m ed ia te ly poured
in to a tu b e, an d before co a g u la tio n to o k p lace a
m easu red q u a n tity w as p ip etted in to 100 m l. of leadfree 10 per cen t, s o lu tio n of so d iu m p h o sp h a te in a
silic a d ish . T he sod iu m p h osp h ate, w h ich w a s k ep t
as a 10 per cen t, sto c k so lu tio n , w as a lw a y s d e-lead ed
ju s t before u se in th e fo llo w in g m an n er. T o every
100 m l. so lu tio n in a sep aratin g fu n nel 5 m l. of a
2 per cen t, so lu tio n of sod iu m d ieth y ld ith io ca rb a m a te
w a s ad d ed , an d th e w hole w as sh ak en v ig o ro u sly
w ith eth er. T he lead-free aq u eou s so lu tio n w a s th e n
4
run in to th e d ish . T he co n ten ts of th e d ish w ere th e n
ev a p o ra ted to d ryn ess on a s te a m b a th a n d a sh ed
as described in our origin al paper, w ith o n ly 1 m l.
of n itric a cid . T he ash w a s d isso lv ed in 50 m l,
o f w a ter c o n ta in in g 2 m l. of co n cen tra ted HC1,
a n d th e so lu tio n w a s transferred t o a sep a ra tin g
fu n n el. T he so lu tio n , w h ich w ith w a sh in g s a m o u n ted
t o 1 00-150 m l., w a s m a d e fa in tly a lk a lin e t o litm u s
b y th e a d d itio n of a m m o n ia (specific g r a v ity 0*880)
a n d cooled . T o th e cool so lu tio n w a s a d d ed 5 m l.
of a 10 per cen t, so lu tio n o f p o ta ssiu m cyan id e,
fo llo w ed b y 2 m l, of a 2 x>er cen t, so lu tio n of so d iu m
d ieth y ld ith io ca rb a m a te an d b y 25 m l. of eth er ;
th e m ix tu re w as sh a k en v igorou sly an d th e aq ueous
la y er a llow ed t o run off. T he eth er e x tr a c t w a s
w ash ed w ith 25 m l. of w ater a n d th e n run in to a
150 m l. ro u n d -b o tto m ed P y r e x flask, a fu rth er 10 m l.
of eth er b ein g u sed to w a sh th e fu n n el. T he aq u eou s
so lu tio n w a s re-ex tra cted w ith 10 m l. of ether, w h ich
w as a d d ed to th e first e x tr a c t, to g eth er w ith a secon d
w a sh in g of 10 m l. of eth er. T he ether w as th e n
ev a p o ra ted off on a stea m b a th a n d th e organic
m aterial in th e residue d estro y ed b y d ig estio n w ith
0*2 m l. of sulp hu ric acid a n d 0*2 m l. of perchloric acid .
To th e flask w ere a d d ed 3*5 m l. of w ater, 0*2 m l.
of g la cia l a cetic acid, 1 m l. of a m m o n ia (specific
g r a v ity 0*S80), a n d 6 drox)S of a 5 per cen t, so lu tio n of
sulxfhurous acid, in t h a t order, a n d th e n th e w h ole
co n ten ts of th e flask w ere transferred t o a 50 m l.
g lass-stop p ered x)yrex v o lu m etric flask, a n d th e
d ig estio n flask w a s w ash ed o u t w ith 5 m l. of a 1 per
cen t, so lu tio n of p o ta ssiu m cyan id e in to th e v o lu ­
m etric flask. T o th e m ix tu re 10 m l. of carbon te tr a ­
chloride an d ex cess of a fresh ly prepared aq u eou s
so lu tio n of d ip h en ylth iocarb azon e w ere ad d ed . (The
d ip h en yltliiocarb azon e so lu tio n differs from t h a t
described in our p reviou s paper a n d w a s prepared
in th e follow in g m anner : a k n ife-p o in t of d ip h en y l­
th iocarb azon e w a s d issolved in 1 m l. of CC14 an d
sh a k en w ith 5 m l. of a £ per cen t, so lu tio n of N H 4O H .
T he aq u eou s so lu tio n w as th e n p ip etted off an d u sed .)
A fter vigorous sh a k in g , th e co n ten ts of th e flask were
poured in to a te st-tu b e , th e aq u eou s la y er w as rem oved
w ith a te a t p ip e tte , an d th e carbon tetrach lorid e
la y er w as retu rn ed to th e flask, w here ex cess d ip h en y l­
th iocarb azon e w as rem o v ed b y rep ea ted ex tr a c tio n
w ith 10-m l. lo ts of a 1 per cen t, so lu tio n of K C N .
U su a lly four to s ix e x tr a c tio n s were n ecessary.
T he p in k e x tr a c ts w ere th e n w a sh ed w ith w ater,
filtered th rou gh lead-free filter-paper, a n d com pared
w ith a stan d ard in a colorim eter. A b lan k estim a tio n
w as carried th rou gh on th e rea g en ts w ith o u t blood, an d
to th e final so lu tio n o b ta in ed a k n ow n a m o u n t of
lead w a s ad d ed to prepare th e sta n d a rd so lu tio n , th u s
a llo w in g for th e sm all a m o u n ts of lea d p resen t in th e
rea g en ts. F or n orm al blood, a sta n d a rd co n ta in in g
0-01 m g. of lea d w as fo u n d to be su ita b le w hen
5
20 m l, of b lo o d w a s u sed , F or p articu lars of th e
rea g en ts reference sh ou ld he m ad e t o our origin al
d escrip tion of th e m eth o d ,
R ESULTS
In accordance w ith our p reviou s su g g estio n th a t
“ th e estim a tio n of b lood -lead w o u ld ap p ear to
b e a m ore sa tisfa cto ry m eth o d of in v e stig a tio n th a n
th e d eterm in ation of th e excretion of le a d ,” th e bloodlead w a s d eterm in ed in all cases. T h e ex cretio n of
lead w a s also d eterm in ed in a m ajority. T h e e x cr e­
tio n w as u su a lly m easured w h ile th e p a tie n t w a s
h a v in g th e ordinary h o sp ita l d iet, b u t in so m e cases,
w h ich w ere a d m itted to th e m eta b o lic w ard s, th e
excretio n on a h igh -calciu m or low -calciu m d iet w a s
also in v estig a ted .
T h e u p per lim it of n o rm a lity for b lo o d -lea d w as
ta k en as 100 pg. o f lea d per 100 m l., for th is v a lu e
h a s been su g g ested b y several au th ors (T aegcr and
S ch m itt 1937, T eisinger 1936, B lu m b erg an d S co tt
1935a), an d our resu lts h a v e been classified accord ­
in g ly . In ta b le I are g iven all th o se cases w ith a
b lo o d -lea d b elow 100 p g., and in ta b le II all th o se
w ith on e or m ore readings a b o v e 100 pg. T h e
ex cretio n in th e urine and fasces is g iv en in m g.
d a ily from th e average of th ree d a y s’ ex cretio n .
W e em p h asise th e im p ortan ce of g iv in g th e a m o u n t
ex creted d a ily an d n o t per litre of urine or per w eig h t
of dried faeces, for th e se la tte r figures are u seless for
comxmrison. F o r th e d eta ils o f th e p resen ce of
p u n c ta te b a so p h ilia and a blue lino in th e g u m s w e
are in d eb ted to th e clin ical n otes. In so m e cases
b lood -film s w ere n o t exam in ed .
T a b le I sh ow s th a t in m o st p a tie n ts th e b lo o d lea d an d th e excretio n of lea d w ere w ith in our
p rev io u s n orm al lim its, and th ere w as no q u estion
of p lu m b ism . Case 19 is an ex cep tio n , for, th o u g h
h is b lood -lead w as o n ly 75 pg. per 100 m l., h e ex creted
1*27 m g. o f le a d per d a y on an ordinary d iet. T h is
m an h ad b een w ork in g in an ex p lo siv es fa c to r y in
on e of th e D o m in io n s an d h ad been ex p o sed to lea d in
p ow d er form for six m o n th s before th e sy m p to m s
ap peared a b o u t a yea r before adm ission. H e h a d
b een off w ork for eig h t m on th s, during w h ich tim e
a d iagn osis of lead -p oison in g w a s m ade. H o w a s
seen soon after h is return to G lasgow , w h en h e w a s
com p lain in g of colic, co n stip a tio n , and w ea k n ess
o f th e han ds. In v ie w of h is large ex cretio n of lea d ,
h e w a s classified as a case of chronic p lu m b ism .
H is ex cretio n o f le a d w a s in creased slig h tly b y a low calcium d iet, com b in ed w ith th e ad m in istration of
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8
am m on iu m ch lorid e, an d d ecreased w h en
011 a h igh -calciu m d iet (ta h le I).
T h is
th a t th e d eterm in a tio n of th e ex cretio n
w ell as th e b lo o d -lea d , m a y b e n ecessa ry
cases.
he w as p u t
case sh ow s
of lea d , as
in d o u b tfu l
Case 7, a m etal-w ork er, a d m itted w ith drop -w rist,
appeared a t first as a p o ssib le ca se of p lu m b ism ,
an d b lo o d -lea d of 91 pg. w a s on th e bord er-lin e, b u t
h is co n d itio n d eterio ra ted , w ith th e d ev elo p m en t of
fa cia l p a ra ly sis, an d h e d ied , th e clin ica l d iagn osis
of cerebral so ften in g b ein g confirm ed a t a u to p sy .
A n a n a ly sis of som e of th e tissu e s w a s carried o u t
p o st m ortem w ith th e fo llo w in g resu lts.
Lead in nag. per kg. of w et tissue
Liver
..
ITemur
..
. . 43*3
Tibia
..
. . 78-9
K idney
..
B i b .................................... 19-6
Brain
..
Vertebra
..
. . 50 *G
..
..
5*4
2*0
1-36
T h e figures for fem u r an d tib ia fa ll w ith in th e lim its
g iv en for th e se b o n es b y T o m p se tt (1936) an d L y n ch ,
S later, an d Osier (1934), T h e rem aind er are com p ar­
ab le w ith th o se g iv en in our origin al p ap er (T o m p se tt
an d A nd erson 1935) for a p a tie n t w ith o ccu p a tio n a l
exp osu re to lea d — n a m ely , a p a in ter— ex ce p t th a t th e
lea d co n ten t of th e v erteb ra is higher.
In case 12, w hoso b lo o d -lea d w a s also n ear th e
border-line, th e ex cretio n of lea d w a s n o t d eterm in ed .
In n o n e of th e s e p a tie n ts w a s p u n c ta te b a so p h ilia
or a b lu e lin e in th e g u m s n oted .
A ll th e ca ses w ith a b lo o d -lea d of m ore th a n 100 pg.
d eta iled in ta h lo I I w ere d iagn osed as lead -p oison in g.
T h e ta b le sh ow s th a t th e ra te o f ex cretio n o f lea d w a s
n o t p rop ortion al to th e le v e l of lead in th e b lo o d —
e.g ., ca se 24, w h o se b lo o d co n ta in ed 200 pg. per
100 m l., ex creted 2-82 m g. d a ily , w h erea s ca se 25,
on read m ission w ith a b lo o d -lea d o f 322 pg., o n ly
ex creted 0-49 m g. d a ily . Case 25 on first ad m ission
h a d a b lo o d -lea d of o n ly 80 pg. an d no p u n c ta te
b a so p h ilia an d w a s disch arged w ith an in co m p lete
d iagn osis. F iv e m o n th s la ter h e w a s rea d m itte d
w ith sev ere colic an d m ek en a, w h en h is b lo o d -lea d
w a s 322 p g., an d th e b lood sh o w ed d efin ite p u n c ta te
b a sop h ilia. F rom ta b le I I it w ill b e seen t h a t th e
le v e l of th e lead in th e b lo o d fell slo w ly during tr e a t­
m en t an d in so m e p a tie n ts rea ch ed a n o rm a l figure
in six or sev en w eek s ; th is fa ll w a s accom p an ied
or p reced ed b y clin ical im p ro v em en t and d isap pearan ce
of sy m p to m s. In th is sm a ll series co lick y ab d om in al
p ain w a s th e co m m o n est sy m p to m .
9
D ISC U SSIO N
F rom tlie s e resu lts w o con clu d e t h a t , w h en th e
le v e l of lea d in t lie h lo o d is a b o v e 100 ^xg, p er 100 m l.,
som e degree of p o iso n in g w ith sy m p to m s w ill b e
p resen t. T h u s far w e agree w ith B lu m b erg and
S c o tt (1935a), T eisinger (1936), an d T aeger and
S ch m id t (1937), all of w h o m p u t th e critica l lim it
for b lo o d -lea d a t 100 \ i g , ; b u t w e su g g est th a t in a ll
q u estio n a b le cases th e to ta l ex cretio n of lea d sh o u ld
b e d eterm in ed , an d th a t a d a ily ex cretio n of 1 m g.
or m ore w o u ld in d ica te p oison in g, ev en if th e b lo o d lead w ere b elo w th e critical le v e l o f 100 [xg. K eh o e,
T h am an n , an d C liolak (1933) con sid er a d a ily faecal
ex cretio n of 1*1 m g. as a sso cia ted w ith p oison in g.
T h ere is on e fa lla c y to b e gu ard ed a g a in st in u sin g
th e ex cretio n of lead for d ia g n o stic p u rp o ses, an d
th a t is an a ccid en ta lly h ig h in ta k e of lead in th e d iet
during th e p eriod o f te s t. I t is preferable to d eterm in e
th e ex cretio n on a d iet of k now n lea d co n te n t, an d
u rin e an d faeces sh ould b e co llected over a p eriod of
a t le a st th ree d ays.
W e fo u n d , as rep orted p rev io u sly , th a t th e ord in ary
d iet in th e R o y a l Infirm ary h a d a d aily lea d c o n te n t
o f 0-22 m g. T h e w a ter con ta in ed 0*03 m g. of lea d
per litre ; h en ce th e average d a ily in ta k e w a s a b o u t
0*25 m g. T h e m ean to ta l d a ily o u tp u t for p a tie n ts
w a s 0*27 m g. T h e p a tie n t in h is ow n h o m e, h o w ev er,
m a y be in g estin g m u ch larger a m o u n ts of lea d from
varied sources. To q u o te M onier-W illiam s (1 9 3 8 ):
“ T h e t o ta l d a ily in ta k e of lea d from a ll sou rces,
in clu d in g w a ter, is n orm ally a b o u t 0*5 m g. A n
in ta k e of 1 m g ., or p o ssib ly even less, m u st b e regarded
w ith su sp icio n .”
T h e large difference b etw een th e co n cen tra tio n of
lea d in th e b lo o d an d th a t in th e u rin e ob serv ed in th e
p resen t cases is w o rth y of n ote. In th e p a tie n ts w ith
lead -p o iso n in g th e co n cen tra tio n in th e b lo o d w a s
te n to tw e n ty tim e s th a t in th e u rin e, a n d ev en in
th e m an w ith a n orm al b lo o d -lea d (case 19) it w a s
s ix tim e s t h a t in th e urine. T h is rela tio n sh ip m ig h t
b e ex p la in ed if th e lea d w as p red o m in a n tly in th e
b lood -cells. B lu m b erg and S c o tt (1935b ) fo u n d t h a t
in clin ical lea d -p o iso n in g th e greater p a rt of th e lea d
w a s carried b y t h e cells. F or n orm al b lo o d W illo u g h b y
and W ilk in s (1938) report th a t in 90 per cen t, of th e
b loo d s a n a ly sed th e serum fraction s co n ta in ed no d e t e c t ­
ab le a m o u n t of lead. B y co n tra st, T eisinger (1936) fo u n d
a m ore eq u a l d istrib u tio n b etw een cells a n d p la sm a .
Su ch clin ica l sign s of p lu m b ism as th e b lu e lin e on
th e g u m s an d p u n c ta te b a so p h ilia are b y no m ea n s
»
10
a lw a y s x^resent. Cases of su sp ecte d p o iso n in g are
o n ly to o o ften fo u n d to b e ed en tu lo u s w h en a b lu e
lino is so u g h t. T h e ex tra c tio n of a n y rem ain in g
te e th m a y h a v e b een a d v ised as p a r t of th e trea tm en t
of ea rly sy m p to m s.
T h e d ia g n o sis of lea d -p o iso n in g in th e ea rly sta g es
is su fficien tly difficult to w arran t th e clin ician callin g
th e la b o ra to ry to liis aid , an d th e u su a l b rief “ m ed ical
in sp ectio n ” w h ich m a n y w ork m en e x p o sed to lea d
h azard s undergo a t in terv a ls w o u ld appear to b e
in su fficient to d e te c t ea rly p oison in g.
In th e p resen t s ta te of k n o w led g e, it ca n n o t b e
claim ed th a t th e ch em ica l d iagn osis of lea d -p o iso n in g
is in fallib le, an d a larger n u m ber of a n a ly ses b y
accu ra te m eth o d s of th e b lo o d an d of t h e ex cr eta
of norm al an d p oiso n ed p erson s w ill b e required
before th e p resen t u n certa in ty is rem oved .
SUMMAET
(1) F ig u res for lea d c o n te n t of b lo o d an d ex cr eta
of 29 ca ses of su sp ecte d lea d -p o iso n in g , of w h ich
11 w ere fin ally d ia g n o sed as p lu m b ism , are g iv en .
Som e im p ro v em en ts in th e m eth o d of estim a tio n of
lead are described.
(2) In 10 of th e cases o f p o iso n in g th e b lo o d -lea d
w a s greater th a n 100 pg. per 100 m l. o f b lo o d and
va ried from 100 to 400 pg. T o ta l d a ily ex cretio n
in u rin e an d faeces v a ried from 0-22 to 2-82 m g.
and did n o t run p arallel to th e le v e l o f lea d in th e b lood .
T h e rem ain in g p a tie n t sh o w ed a b lood -lead of o n ly
75 pg. b u t a d a ily ex cretio n of 1*27 m g.
(3) I t is su g g este d th a t m ore th a n 100 pg. of lead
I>er 100 m l. o f b lood or a t o t a l d a ily ex cretio n of 1 m g.
or m ore of lead in d ic a te s lead -p oison in g.
Our th a n k s are due t o sev era l m em b ers of th e sta ff
of th e R o y a l Infirm ary for p erm issio n t o q u o te from
th e clin ica l n o te s on th ese cases.
REFERENCES
Blumberg, H ., and Scott, T. F. M. (1935a) Johns Hopk. IIosp.
Bull. 56, 27G.
— — (1935b) Ibid, p. 311.
K ehoe, H. A., Thamann, F ., and Cholak, J. (1933) J . industr.
Hyo. 15, 30G.
Lynch, G. R ., Slater, R , H ., Osier. T. G. (1934) Analyst, 59, 787.
Minot, A. S. (1938) Physiol. Rev. 18, 554.
Monier-Willittms, G. W . (1938) Rep. publ. H lthm ed. Subj., Lond.
No. 88.
Taeger, H ., and Schm itt, F. (1937) Z . ges. exp. Med. 100, 717,
Teisinger, J. (1936) I b id , 98, 520.
T om psett, S. L. (193G) Biockcm. J. 30 345.
— and Anderson, A. B. (1935) Ibid, 29, 1851.
W illoughby, O. E ., and W ilkins, E. S., jun. (1938) J . Mol. Chem.
124, 639.
___________________________
The Lancet Office,
7, Adam Street, Adelphi, London, W.C.2.
[F rom THE BIOCHEMICAL JOURNAL, Vol. XXVIII, No. 6, pp. 2088-2091,1934]
[A l l Mights reserved)
P R IN T E D I N G R E A T B R IT A IN
CCLXXIV. TH E EXCRETION OF COPPER IN
URINE AND FAECES AND ITS RELATION TO
TH E COPPER CONTENT OF TH E DIET.
B y SIDNEY LIONEL TOMPSETT.
From the Biochemical Laboratory of the Institute of Pathology of the
Royal Infirmary and University of Glasgow.
{Received October 29th, 1934.)
In a previous paper the present writer [1934] described a method for the de­
termination of copper in blood. The reaction employed was the formation of a
yellow-coloured complex of copper with sodium diethyldithiocarbamate which
could be extracted with amyl alcohol. The amyl alcohol layer was removed and
compared in a colorimeter with a standard similarly prepared. This reaction is
extremely sensitive, it being possible to detect 1 part of copper in 10 million
parts of solution. Using this technique human blood was found to contain
0*185-0-230 mg. Cu per 100 ml.
So far only one investigation of the copper content of urine appears to have
been made. Rabinowitch [1933] obtained values of a trace—0*41 mg. Cu per
litre for the copper content of normal urine. This writer stated that addition of
copper to the diet definitely increased the amount excreted in the urine, but did
not publish any analyses of the copper content of the faeces or the daily diet. In
the present investigation the copper contents of the daily diet and faeces were
determined as well as that of the urine.
Methods.
1. Collection of urine and faeces. The urine and faeces were collected during
periods of 6-21 days. The daily urines were made up to a constant volume and
a composite sample prepared. The faeces were dried on a steam-bath and weighed,
ground up and stored in sealed bottles.
2. Diet. An average half-daily sample of the diet (with the exception of
butter) was weighed. After drying on a steam-bath, it was weighed, ground up
and stored in a sealed bottle.
Technique .
In the determination of copper in blood, the proteins were precipitated with
trichloroacetic acid and the copper determined directly in the filtrate. Naturally
such a procedure would be impossible with urine, faeces or a food. Ashing of
some form is necessary. These materials contain large amounts of calcium phos­
phate which tends to precipitate during the estimation since the reaction is
carried out in alkaline solution. In the past investigators have carried out a
preliminary separation of the copper as sulphide in acid solution. This procedure
is time-consuming and needs very careful attention to ensure accurate results.
The present writer has found that the addition of sodium citrate prevents the
precipitation of calcium phosphate without interfering with the reaction. A
copper-free sample of sodium citrate was used throughout the investigation.
2089
EXCRETION OF COPPER
Throughout the investigation 0-01 mg. Cu was used as the standard since the
colour value obtained in this region presents sharp comparison in the colorimeter.
After each determination the unknown extract was shaken with a solution of
sodium cyanide, and in each case it was noted that the colour disappeared, i.e.
the colour was due to copper only. This precaution is necessary since bismuth
gives a yellow colour with sodium diethyldithiocarbamate and is soluble in
organic solvents. The reaction with bismuth is far less sensitive than that with
copper— 1 mg. of bismuth gives less colour than 0-01 mg. of copper. Bismuth
is not likely to be present often in material derived from experiments such as
these. Manganese gives a pink to dirty brown colour soluble in organic solvents.
Manganese occurs in foodstuffs and faeces in higher concentration than copper.
The present writer has noted that just as in the case of iron, the presence of
pyrophosphate in an alkaline medium prevents any reaction between manganese
and sodium diethyldithiocarbamate so that the metal is not extracted. Man­
ganese therefore must form un-ionised complexes with pyrophosphate in the
same way as iron.
Urine. 50-100 ml. of urine were measured into a 300 ml. Kjeldahl flask and
boiled almost to dryness with 5 ml. concentrated nitric acid. 2 ml. of concen­
trated sulphuric acid were then added, and the heating was continued until the
nitric acid was driven ofE. The remainder of the organic matter was finally
destroyed by the addition of perchloric acid and further heating. The digest was
made up to a known volume with distilled water and a volume containing about
0-01 mg. Cu taken for analysis (usually equivalent to 50 ml. urine). 5 ml. of
20 % sodium citrate were added, followed by 2 ml. of 4 % sodium pyrophosphate,
and the solution was made alkaline to litmus by the addition of ammonia.
5 ml. of amyl alcohol were added, followed by 0-5 ml. 2 % sodium diethyl­
dithiocarbamate, and the whole was vigorously shaken. The amyl alcohol layer
was removed, passed through a filter-paper to remove any suspended particles
of water and compared in a colorimeter with a standard containing 0*01 mg. Cu
prepared similarly.
Faeces. 1 g. of dried faeces was heated in a Kjeldahl flask with 5 ml. of con­
centrated sulphuric acid and 15-25 ml. perchloric acid until all the organic
matter was destroyed. When the excess of perchloric acid had been removed, the
mixture was cooled and diluted with distilled water to 100 ml. If the digest
contained any white insoluble matter it was diluted to about 20 ml. with distilled
water, 1 ml. concentrated hydrochloric acid was added and the whole heated
until solution was complete, after which it was diluted to 100 ml. with distilled
water. A volume containing about 0-01 mg. Cu (usually about 10 ml.) was taken
for analysis, which was carried out similarly to that described under urine. In
the case of faeces 5 ml. of 4 % sodium pyrophosphate were added during the
estimation instead of 2 ml. as used in the case of urine.
Diet. Owing to their low concentration of copper, samples of diet were ashed
in silica basins by a dry method. It was found that when samples of such diets
were ashed in silica basins, low results and poor recoveries of added copper were
obtained. On the other hand it was found that urine and faeces could be ashed
by this method to give satisfactory results. These materials usually gave a large
amount of ash, consisting mainly of phosphates, and the silica of the basin did
not appear to be attacked. When diets are ignited, the ash is usually very much
smaller in comparison and the silica of the basin is invariably attacked even when
the temperature is kept as low as possible. When the sample of diet was mixed
with sodium phosphate the silica of the basin did not appear to be attacked and
consistent results and accurate recoveries of added copper could be obtained.
2090
S. L. T O M P S E T T
The following was the method adopted for the determination of the copper
contents of the diets. A sample of 10 g. of the dried diet plus the appropriate
amount of butter was placed together with 5 g. of copper-free sodium phosphate
in a silica basin and ignited at as low a temperature as possible. Final traces of
carbon were destroyed by the addition of concentrated nitric acid and further
heating. The ash was dissolved in distilled water containing 5 ml. concentrated
hydrochloric acid and the solution diluted to 50 ml. A sample containing about
0-01 mg. Cu was taken for analysis which was carried out as described under
urine. It was usual to add 5 ml. 4 % sodium phosphate as in the case of faeces.
Copper-free reagents were used throughout this investigation and all filter papers were washed first with dilute acid and then with distilled water.
From the results shown in Table I, it will be seen that copper added to urine,
faeces or samples of diet could be estimated quantitatively by the technique just
outlined.
Table I. Recovery of copper added to urine, faeces or diet.
Urine
1
Initial copper
content
mg.
(a)
0-216
F)
F)
2
0-160
(a)
F)
F)
!Faeces 1
2
3
4
5
6
0-104
0-109
0-083
0-081
0-091
0-088
Diet
1
2
0-086
0-102
(a)
F)
F)
3
0-096
(a)
F)
Copper
added
mg.
0-1
0-2
0-3
0-1
0-2
0-3
0-2
0-1
0*1
0-2
0-2
0-2
Total
copper
mg.
0-301
0-400
0*630
0-262
0*368
0*464
0-300
0-211
0-179
0-296
0-310
0-292
Copper
recovered
mg.
0-085
0-184
0-314
0-102
0-208
0-294
0-196
0-102
0-096
0-215
0-219
0-204
0-10
0-10
0-20
0-40
0-20
0-50
0-180
0-2.10
0-300
0-606
0-288
0-595
0-094
0-108
0-198
0-404
0-192
0-499
In Table II are shown the average daily excretions of copper in the urine and
faeces during the periods stated of 17 cases. The average daily copper contents
of the diets are also shown. Several preparations of the same half-day diet were
made and the average copper content taken. It will be noted that several of the
cases show the same copper content of the diet—these were on the same diet.
Cases 1-14 had no organic disease while cases 15 and 16 had subacute nephritis
of the nephrotic type. Case 17 had carcinoma of the stomach and was only able
to take fluids. The diets consumed by Cases 1-14 were well varied and were
normal mixed diets.
It will be seen that, with the exception of No. 17, within the limits of experi­
mental error total excretion balances the intake. The copper contents of the diets
were found to be slightly higher than when calculated from the analyses of food­
stuffs by Lindow el al. [1929]. The normal intake of copper would appear to vary
from 2 to 2-5 mg. per diem on an average. As would be expected No. 17 shows a
marked loss of copper—the loss being chiefly in the faeces. The fact that the total
amount of copper excreted was 0-63 mg. per diem suggests that the minimum
amount required per diem to preserve copper equilibrium may be in the region
of this figure.
EXCRETION OF COPPER
2091
Table II.
Case
1. Normal
2.
3!
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Fracture
Fracture
Fracture
Fracture
Fracture
Normal
Normal
Normal
Normal
Fracture
Fracture
Normal
Amputation of leg
Nephritis
Nephritis
Carcinoma of stomach
No. of
days of
collection
6
15
13
21
21
20
7
10
9
14
16
20
12
18
8
8
8
Av. daily
excretion
of copper
in the
urine
mg.
0-29
0-36
0-52
0-45
0-50
0-36
0-38
0-33
0-29
0-30
0-44
0-22
0-21
0-18
0-32
0-16
0-11
Av. daily
excretion
of copper
in the
faeces
mg.
2-60
1-98
1-88
2-08
2-15
2-08
2-18
2-07
2-00
1-93
1-70
1-83
1*67
1*56
1-24
1-00
0-52
Total
daily
excretion
of copper
mg.
2-89
2-34
2-40
2*53
2*65
2*44
2*56
2*40
2*29
2*23
2-14
2*05
1*88
1*70
1*56
1*16
0*63
Av. daily
content
o f copper
in diet
mg.
2*72
2*44
2*44
2*42
2*42
2*42
2*41
2*41
2*41
2*34
2-29
2-22
2*12
1-89
1*43
1*21
0*21
In Table III are shown the copper contents of human urines as obtained b
Lie present writer.
Table III.
1. Copper excretion in mg. per litre
2. Copper excretion in mg. £>er diem
No. of
cases
16
24
Minimum
0-08
0-12
Maximum
0*48
0*52
Average
0*18
0-28
Those agree very closely with the figures obtained by Rabino witch [1933].
Sum m ary.
1. The copper contents of the urine and faeces of 17 persons have been de­
termined and compared with the copper contents of the diets.
2. Human urine contains 0-08-0-48 mg. of copper per litre.
3. An average daily diet would appear to contain 2-2-5 mg. of copper.
In conclusion I wish to thank Dr D. P. Cuthbertson for the use of material
from his cases and for his advice and criticism. I also wish to thank the Medical
Research Council for a grant towards expenses.
REFERENCES.
Lindow, Elvehjem and Peterson (1929). J . B io l. Chem. 82, 465.
Rabinowitch (1933). J . B io l. Chem. 100, 479.
Tompsett (1934). Biochem. J . 28, 1544.
[From THE BIOCHEMICAL JOURNAL, Vol. XXX, No. 3, pp. 345, 346, 19363
[All Bights reserved]
PRINTED IN GREAT BRITAIN
L IV . T H E D I S T R I B U T I O N O F L E A D
IN H U M A N B O N E S .
B y SIDNEY LIONEL TOMPSETT.
From the Biochemical Laboratoi'y, Department of Pathology of the
University and Royal Infirmary, Glasgow.
(.Received January 7th} 1936.)
I t lias been shown [Tompsett and Anderson, 1935] that lead occurs in appreciable
amounts in the tissues of persons with no history of exceptional exposure to
lead. The highest concentrations of lead were found in bones (rib and vertebrae).
The concentration of lead in the rib varied from 5*2 to 12-9 mg. Pb per kg. (wet
tissue) and in the vertebrae from 4-2 to 14*7 mg. Pb per kg. (wet tissue). Lynch
et al. [1934], in an examination of human tibiae and femora, found 14-146 mg.
Pb per kg. (wet tissue). It was decided therefore to compare the concentrations
of lead in the rib, the vertebrae, the shaft of the femur and the shaft of the tibia.
These bones were obtained from cases of widely different ages having no history
o f special exposure to lead.
Experimental.
A weighed portion of the bone (tibia and femur 1-2 g .; rib and vertebrae
10-20 g.) was dried and incinerated in a silica basin containing 100 ml. of leadfree sodium phosphate. Lead was then determined as described previously
[Tompsett and Anderson, 1935].
Table I.
The results are expressed in mg. Pb per kg. fresh bone.
Age
Occupation
1
2
3
25
28
29
Quarryman
Newsagent
Clerk
4
5
6
7
8
35
50
58
59
66
Boiler fireman
Hairdresser
Labourer
Engineer
Iron moulder
9
67
Retired
10
11
12
67
68
74
Retired
Blacksmith
Restaurant proprietor
13
14
12
19
Shop assistant
15
16
17
22
30
39
Clerk
Housowife
Housewife
18
19
49
61
Housewife
Housewife
Diagnosis
Males.
Nephritis
Aspirin poisoning
Ulcers of small in­
testine
Care, o f stomach
Care, of stomach
Nephritis
Uraemia
Care, of pelvic
colon
Cerebral
haemorrhage
Fractured skull
Care, of bronchus
Coronary
Females.
Malignant thymoma
Subacute bacterial
endocarditis
Pernicious anaemia
Burns
Subacute bacterial
endocarditis
Toxic goitre
Abscesses of the
kidneys
( 345 )
Rib
Vertebrae
Femur
Tibia
5-9
9-8
4-2
4-6
6-4
4-6
33*9
25*6
21-1
27*8
27*0
20*6
13-3
7-9
8-9
12-8
8-6
7-3
4-6
8-6
11-6
6-5
48-2
48-1
29-0
70-0
42*3
28*6
42*1
26*5
62*7
48*6
9-1
8-2
55*9
43*7
11-1
11-3
134
12-4
7-6
12-6
108*3
74*1
82-2
96*5
43*4
81*6
4*0
16*5
3*4
10*6
19*1
74*8
17*9
60*0
7*2
9*5
9*8
6*2
8*4
10*6
18*2
34*1
44*5
15*3
29*8
46*8
8*7
17*5
9*0
16*5
33*3
61*7
27*8
61*5
346
S. L. T O M P S E T T
The pink colours developed with diphenylthiocarbazone are quite stable
unless exposed to bright sunlight when rapid fading will take place.
The results are shown hi Table I.
From the results it will be seen that femora and tibiae contain much higher
concentrations of lead than ribs and yertebrae. The concentrations of lead in
tibiae and femora appear to be of the same order and agree with those reported
by Lynch et al. [1934]. It seems that in cases of suspected lead poisoning,
different types of bone should be examined.
Summary.
1. Femora and tibiae contain much higher concentrations of lead than ribs
and vertebrae.
2 . In a series of 19 cases, the concentrations of lead were, rib 4*0-17*5,
vertebrae 3*4-16*5, femur 18*2-108*3, tibia 15*3-96*5 mg. Pb per kg. fresh bone.
In conclusion I wish to thank Dr A. B. Anderson for his helpful criticism
and advice.
REFERENCES.
Lynch, Slater and Osier (1934). A n a lyst, 59, 787.
Tompsett and Anderson (1935). Biochcm. J . 29, 1851.
[From THE BIOCHEMICAL JOURNAL, Vol. XXX III, No. 8, pp. 1231-1236, 1939]
\A ll rights reserved ]
PR IN TED IN GREAT BRITAIN
CL. T H E D E T E R M I N A T I O N O F L E A D
IN B I O L O G I C A L M A T E R I A L S
By
SIDN EY LIONEL TOMPSETT
From the Biochemical Laboratory, Department of Pathology,
Royal Infirmary, Glasgow
{Received 22 June 1939)
F urther experience of the method of Tompsett & Anderson [1935] for the
estimation of lead in human tissues and excreta has shown that in the analysis
of different types of material modifications are an advantage in special cases. Also
certain general improvements make it desirable to describe the method in more
detail. A survey of the various methods for estimating minute amounts of lead
in biological materials is not included as this has been done by us [1935] and
more recently by Minot [1938].
The method consists essentially of three stages: (1) destruction of organic
material, (2) extraction of Pb with ether as a complex with Na diethyldithio­
carbamate, (3) colorimetric estimation of lead with ditliizone.
Destruction of organic material
Two methods are available, viz. digestion with H 2S 0 4 and some oxidant, e.g.
H N 0 3 or HCIO4, and ignition.
The objection to the first method is that often large quantities of H 2S 0 4
and oxidant are required and consequently large quantities of alkali are necessary
for neutralization. This leads to a large blank even when the purest reagents are
used.
Destruction by ignition, which avoids this high blank has been criticised 011
the grounds that Pb may be lost by volatilization. The writer has found that
materials containing a large amount of ash, consisting in the main of phosphates,
may be ignited in a silica dish over a bunsen burner without loss of Pb. When
materials containing a low ash content arc treated in this manner, a loss of Pb
may occur. For this reason, the ash content of such materials is increased by
the addition of Na phosphate. Biological materials are therefore divided into
two classes:
(1) High mineral content—ignited without the addition of Na phosphate,
e.g. urine, faeces, milk and bone.
(2) Low mineral content—Na phosphate added before ignition, e.g. blood,
soft tissues.
The ignition method has been found to be'quite satisfactory under these
conditions and has been used throughout. Towards the end, ignition may be
assisted by allowing the ash to cool, adding a little cone. H N 0 3 and re-heating.
Separation of Pb with ether and N a diethyldithiocarbamate
Na diethyldithiocarbamate reacts with various metals to form complexes,
many of which are soluble in organic solvents. Amongst the metals which occur
in biological materials and react thus are Fe, Cu and Pb. Pb forms a complex
which is soluble in ether. This complex is produced in acid and in alkaline
solution and its formation is not inhibited by the presence of such substances as
1232
S. L. T O M P S E T T
citrate, pyrophosphate or cyanide. The formation of the Cu complex is inhibited
if the solution is alkaline and contains cyanide in addition. The Ee complex is
not formed if the solution is alkaline and contains in addition pyrophosphate,
citrate or cyanide.
Before Pb may be estimated with the dithizone reagent it is essential to
separate it from Fe and recent experience has shown that it is advantageous to
separate it from Cu. Use of the above is made to achieve this. The separation
is carried out by adding to a solution of the metals, Na citrate, N H 3 to make
alkaline and then Na diethyldithiocarbamate. The Pb complex, which is the
only one formed under these conditions, may then be extracted with ether. The
presence of the Na citrate is essential so as to prevent the precipitation of
hydroxides anti phosphates in the alkaline medium.
This technique has been found to be quite satisfactory for urine, soft tissues
and blood, but in the case of mill?:, bone and faeces, certain modifications have
been found to be advantageous. The ash of the second class of material contains
a considerable amount of Ca phosphate. As a result, sometimes in spite of the
presence of citrate, troublesome precipitates of Ca phosphate appear when the
mixture is made alkaline. Such precipitates may prevent the quantitative
separation of Pb. For this reason, a twofold extraction is employed in the
examination of these materials.
Na diethyldithiocarbamate is added to an acid solution of the ash. All the
metallic complexes arc formed and all are extracted with ether. The extracted
metals are converted into the inorganic state and the process of extraction
repeated but in an alkaline solution containing citrate and cyanide. Under these
conditions, the Pb complex alone is formed, and this alone extracted by the
ether.
The colorimetric estimation of Pb with dithizone
The extracted Pb is converted into the sulphate by digestion with H 2S 0 4.
After digestion, water is added and then acetic acid. The mixture is then made
alkaline by the addition of NH3. An alkaline reaction is essential for the oc­
currence of the reaction between Pb and dithizone and the presence of ammonium
acetate ensures that the PbS0 4 is in solution.
The reaction is as follows. KCN and CC14 are added to the alkaline acetate
solution of Pb, Dithizone solution is then added and the whole well shaken.
The Pb forms a complex which dissolves in the CC14 to produce a red solution.
The solution of dithizone in ammoniacal solutions is brown and in CC14 green.
After the shaking process, unchanged dithizone will be distributed between the
aqueous and CC14phases. The next stage is to separate the CC14 layer and to free
it from unchanged dithizone by shaking it with KCN solution, when uncombined
dithizone is removed and an extract containing the red coloured Pb complex
alone is obtained. This reaction is quantitative and the Pb present may be
estimated by colorimetric comparison with a standard.
Certain precautions are however necessary. Excess of dithizone must be
used to ensure that all the Pb present has been converted into dithizone complex,
but too great an excess must be avoided.
Dithizone is very susceptible to oxidation and a substance is produced which
is soluble in CC14to produce a yellow coloured solution. This substance cannot be
removed from CC14 by cyanide solutions. It is produced when Fe or Cu salts are
present and by bright sunlight. In the earlier work, the influence of Fe salts was
realized and they were carefully eliminated. The effect of Cu was not then
known but owing to its generally low concentration in biological materials, the
LEAD DETERMINATION
1233
effect was almost negligible. Occasionally trouble was experienced and this was
found to occur in materials having a high Cu content. Upon removal of the Cu,
no trouble was experienced. It has been decided, therefore, to eliminate Cu as
well as Fe.
Bright sunlight causes a rapid production of the yellow oxidation product.
This does not take place in ordinary diffuse light. It is believed that it is the
ultraviolet component of bright sunlight which is responsible for this reaction
[Tompsett, 1936]. In ordinary diffuse light the CC14 solution of the red coloured
Pb complex will remain stable for a very long period.
Apparatus and reagents
Pyrex glassware was used throughout. Silica dishes were washed with hot
dilute HC1 before use. Glass-distilled water was used and filter papers were
washed with dilute HC1 followed by water.
The reagents used were the same as those described by Tompsett & Anderson
[1935] with the following exceptions:
Dithizone reagent. Commercial dithizone contains an oxidation product
which must be removed. In earlier work a mass purification was done but this
has now been abandoned. A 0-1 % solution of 0-1% crude dithizone in CC14
is prepared. Just before use, a small volume of this is shaken with an equal
volume of 0*5% N H 3. Dithizone but not the oxidation product passes into the
aqueous layer. After allowing the mixture to settle, the ammoniacal extract
is separated and used directly.
N a 2B P O i — 10% in water—lead-free. Before use the requisite volume of a
stock solution is introduced into a separating funnel, sodium diethyldithio­
carbamate added and the mixture shaken vigorously with ether. After settling,
the lead-free aqueous layer is run off.
The method
In the following, details of the method as applied to various materials are
described. The final colorimetric estimation is the same in each case.
A. Extraction of lead
Urine. 500 ml. of urine are evaporated to dryness in a silica dish in a hot
air oven. The residue is then ashed by ignition over a bunsen burner in a fume
cupboard.
The ash is dissolved in 100 ml. of water containing 5 ml. of cone. H.C1. The
solution is transferred to a separating funnel, 50 ml. of 20% Na citrate added
and the mixture made alkaline by the addition of ammonia. 5 ml, of 10%
KCN are added and the mixture cooled thoroughly. 5 ml. of 2 % Na diethyl­
dithiocarbamate arc added and the mixture extracted 3 times with ether, 25 ml.
on each occasion. The ether extracts which are separate^ washed with water
are transferred to a hard glass round-bottomed flask.
The ether is evaporated off and the residue digested witli 1 ml. conc. H 2S 0 4
and 1 ml. HC104 to destroy organic matter.
The residue is diluted with water, 1 ml. glacial acetic acid added, followed by
5 ml. ammonia sp.gr. 0-88, and the mixture diluted to 25 ml. by the addition of
water.
Soft tissues. To 100 ml. of lead-free 10 % Na 2H P 0 4 in a silica basin are added
100 g. fresh tissue. After drying in a hot air oven, the material is ashed.
The ash is dissolved in 100 ml. water containing 10 ml. conc. IlCl. The
procedure is then as for urine.
1234
S. L. T O M P S E T T
Blood. To 100 ml. of lead-free 10 % Na 2H P 0 4in a silica dish are added 20 ml.
blood. After drying in a hot air oven, the material is ashed.
The ash is dissolved in about 50 ml. water containing 5 ml. conc. HC1. After
transference to a separating funnel, it is cooled, 5 ml. of 20% Na citrate added
and the mixture made alkaline to litmus by the addition of ammonia. 5 ml. of
10 % KCN are added, followed by 2 ml. of 2 % Na diethyldithiocarbamate. The
mixture is extracted twice with ether, 20 ml. being used on each occasion. The
ether extracts, which on each occasion are washed with water, are transferred to
a 100 ml. hard glass round-bottomed flask.
The ether is evajjorated off and the residue digested with 0*2 ml. conc. H 2S 0 4
and 0-5 ml. HC104. To the digest are added 3-5 ml. water, 0-2 ml. glacial acetic
acid and 1*5 ml. ammonia, sp.gr. 0-88.
Faeces. 10 g. of dried faeces are ashed in a silica basin. The ash is dissolved
in 100 ml. water containing 10 ml. conc. HC1. The solution is then made up to
a volume of 200 ml. by the addition of water.
50 ml. of the ash solution are introduced into a separating funnel and 10 ml.
of 2 % Na diethyldithiocarbamate added. The mixture is extracted 3 times with
ether, 25 ml. on each occasion. The ether extracts are collected in a hard glass
flask and the ether evaporated off. The residue is digested with 1 ml. conc. H 2S 0 4
and 1 ml. HC104.
•
i
The residue is diluted with water, 1 ml. conc. HC1 added and the mixture
heated to dissolve the digest. The solution is transferred to a separating funnel
to make a volume of about 50 ml., 5 ml. of 20% Na citrate are added and the
mixture made alkaline to litmus by the addition of N H 3. 5 ml. of 10% KCN
are added, followed by 5 ml. of 2 % Na diethyldithiocarbamate. The mixture is
then extracted 3 times with ether, 25 ml. being used on each occasion. The
combined ether extracts are transferred to a hard glass round-bottomed flask.
The ether is evaporated off and the residue digested with 1 ml. conc.
sulphuric acid and 1 ml. perchloric acid. To the residue are added, water, 1 ml.
glacial acetic acid and 5 ml. ammonia, sp.gr. 0-88, and the mixture is diluted
to 25 ml. with water.
Bone. 20 g. of bone are ashed in a silica dish. The ash is dissolved in water
containing HC1 and the solution diluted to 200 ml., 50 ml. of this are taken and
treated as in the case of faeces.
M ilk. 500 ml. of milk are evaporated to dryness in a silica dish in a hot air
oven and then ashed. The ash is dissolved in water containing HC1 and treated
as in the case of faeces.
B. The colorimetric estimation of lead
1.
Preparation of standard. The following mixture is prepared. To 1 ml. conc.
H 2S 0 4 are added a little water, 1 ml. glacial acetic acid and 5 ml. ammonia,
sp.gr. 0-88 and the whole diluted with water to a volume of 25 ml. giving
ammonium acetate.
A known amount of Pb is added to 5 ml. of this mixture. To this are added
5 ml. of 1 % KCN and 10 ml. of CC14. To this mixture is added drop by drop an
ammoniacal solution of dithizone reagent, with constant shaking until excess
has been added. Too great an excess must be avoided. Sufficient excess is
indicated when the CC14 layer has reached its maximum intensity of redness and
the aqueous layer is tinged brown. At this stage, the aqueous layer is removed
and discarded. The CC14 layer, containing the red coloured lead complex, is
shaken repeatedly with aliquots of 5 ml. of 1 % KCN until the aqueous layers
1235
LEAD DETERMINATION
are 110 longer coloured. The CCI4 layer may be passed through a filter to remove
droplets of water and is then ready for comparison.
A range of standards may be prepared but the writer prefers to use a standard
containing 0*02 mg. Pb, and to take the requisite amount of the unknown to
conform to this. This standard may be obtained by using 2 ml. of a standard
solution of lead acetate, containing 0*01 mg. Pb per ml.
2.
Preparation of unknown urine, faeces, soft tissues, bone and milk. The
extracted Pb is contained as PbS0 4 in a solution of ammoniacal ammonium
acetate having a volume of 25 ml. For the estimation, 5 ml. of the solution are
taken and to this are added 5 ml. of 1 % KCN and 10 ml. of CC14. The colour is
developed as described above.
In the event of the Pb content of the unknown being low, 10 ml. of the
solution are used, the amounts of the other reagents being the same. If the Pb
content of the unknown is high, a smaller volume than 5 ml. is used. In this
case the solution is diluted to 5 ml. by the addition of an ammoniacal ammonium
Table I. The recovery of added Pb
Initial Pb
content
mg.
0*120
0*120
0*120
0*120
0*120
0*042
0*042
0*042
0*155
0*155
Pb added
mg.
0*050
0*100
0*200
0*250
0*500
0*020
0*050
0*100
0*200
0*400
Total Pb
found
mg.
0*163
0*215
0*308
0*362
0*625
0*079
0*090
0*140
0*360
0*565
Faeces (10 g.)
0*030
0*030
0*030
0*042
0*042
0*042
0*031
0*031
0*031
0*200
0*500
1*000
0*030
0*050
0*100
0*040
0*080
0*120
0*235
0*549
1*036
0*079
0*096
0*140
0*072
0*115
0*154
Milk (500 ml.)
0*050
0*050
0*050
0*050
0*050
0*100
0*200
0*500
0*105
0*145
0*248
0*560
0*055
0*095
0*198
0*510
Liver (100 g. fresli)
0*243
0*243
0*243
0*243
0*050
0*100
0*200
0*400
0*299
0*350
0*440
0*640
0*056
0*107
0*197
0*397
Mg*
16
16
16
19
19
19
12
12
12
Mg*
10
20
40
10
20
40
10
20
40
Mg27
37
58
28
39
58
22
34
53
Mg*
11
21
42
9
20
39
10
22
41
Urine (1500 ml.)
Blood (20 ml.)
Pb
recovered
mg.
0*043
0*095
0*188
0*242
0*505
0*037
0*054
0*098
0*205
0*410
.
0*205
0*519
1*006
0*037
0*054
0*098
0*041
0*084
0*123
1236
S. L. T O M P S E T T
acetate solution, having the same composition as that used to prepare the
standard. The amounts of the other reagents are the same. The development of
the colour is best carried out in glass-stoppered tubes.
Blood . In the case of blood, the whole of the Pb-containing solution is used.
To the mixture containing the Pb in the flask used for the digestion, add 5 ml.
1 % KCN and 10 ml. CCl4. The colour is developed as above.
C. Blank
A blank should always be done on a new set of reagents. In estimating the
blank, the complete process is carried out. The blank is small and is thus difficult
to estimate accurately so that the following method has been adopted. Before
colorimetric estimation, 0*02 mg. Pb is added to the blank. This, after the
development of the colour with dithizone, is compared with a standard con­
taining 0*02 mg. Pb. The blank is then calculated from the difference.
A scries of recovery experiments is shown in Table I.
Discussion
It will be seen from the Table that added Pb could be estimated accurately.
The method is fairly quickly performed. It is designed so that little apparatus
is required and so that large scale purification of reagents is avoided.
The accurate estimation of Pb is of some importance as Pb is a “ normal”
hazard which at times may become unduly large, and it is also an important
industrial hazard. In a recent publication Toinpsett & Anderson [1939] have
shown the value of Pb estimations, especially in the blood, in the diagnosis of
plumbism.
Summary
The estimation of lead in biological materials is described. The method
comprises an earlier one in which are incorporated certain modifications.
In conclusion I wish to thank Dr A. B. Anderson for his helpful criticism and
advice.
REFERENCES
Minot (1938). P hysiol. Rev. 18, 554.
Tompsett (193(i). Biochem. J . 30, 345.
& Anderson (1935). Biochem. J . 29, 1851.
-------------- (1939). Lancet , p. 559.
[From THE BIOCHEMICAL JOURNAL, Vol. XXXIII, No. 8, pp. 1237-1240, 1939]
[A ll rights reserved ]
PRIN TED IN GREAT BRITAIN
CLI. T H E I N F L U E N C E O F C E R T A I N
C O N S T I T U E N T S O F T H E DIET U P O N
T H E ABSORPTION OF LEAD F R O M
THE ALIMENTARY TRACT
By
SIDNEY LIONEL TOMPSETT
From Ihe Biochem.ical Laboratory, Department of Pathology,
Royal Infirmary, Glasgow
{Received 22 June 1939)
Lead may be absorbed from the respiratory tract, from the alimentary tract
and from the skin. In industrial Pb poisoning, absorption from the respiratory
tract is usually the important factor. In the “ normal” hazard, absorption is
chiefly from the alimentary tract. This at times may be largely due to con­
tamination of foodstuffs and drinking water with undue amounts of Pb. The
object of the experiments to be described was to see whether variation in the
composition of the diet had any influence upon the absorption of Pb from the
alimentary tract.
Aub et al. [1926] compared the degrees of absorption of Pb in two groups of
cats, one group being on a milk diet and the other on a milk-free diet. They
could detect no marked difference in the amounts of Pb absorbed by the two
groups ancl concluded that the amounts of Ca in the diet had no influence upon
the amount of lead absorbed from the alimentary tract. Sobcl el al. [1938], in a
study of experimental Pb poisoning in rats, found that about twice as much Pb
was absorbed on a diet containing viosterol as without it.
E xperimental
Adult male mice were the animals used throughout this work. Two basic
diets were used, namely a low Ca diet and a high Ca diet, of the following comi.
High Ca diet
Low Ga diet
Shelling [1932]
ggWhole wheat flour 700
Whole wheat flour 100
Whole milk powder 300
Casein
100
Corn starch
Marmite
50
325
Wheat gluten
50
40
Olive oil
20
NaCl
15
KC1
Omitted
Butter
In the first series of experiments, the effects of high and low Ca diets upon the
absorption of Pb were studied.
S. L. TOMPSETT
1238
The mice were fed with the specified diets (2-5 g. per mouse per day), to
which were added supplements of Pb in the form of lead acetate solution. Each
mouse was housed in a separate glass jar, which was floored with sawdust. No
restriction upon the amount of water drunk was made. The amount of diet
supplied was constant per mouse per day. The diet together with the Pb supple­
ments was made up into a thick paste with water and supplied in a porcelain
dish, thus minimizing loss due to spillage. No difficulty was experienced in
obtaining complete consumption of the daily diets by the mice. The experi­
mental period was 14 days. At the end of this period, the jars were cleaned out
and the animals placed upon the high Ca diet for 4 daj^s to remove unabsorbed
Pb from their alimentary tracts. The animals were then killed and the Pb
content of the whole animal determined by the method published previously
[Tompsett & Anderson, 1935; Tompsett, 1939]. The Pb content of control animals
and also that of unsupplemented diets were determined.
It Avas found that absorption of Pb was not very marked with the high Ca
diet but Avas high upon the loAAr Ca diet. In the latter case, absorption increased
with increase in the amount of added Pb.
Table I. Experim ental period, 14 days
The results are expressed as (A) mg. total Pb, (B) mg. Pb per 100 g. body Avt.
Supplement of Pb
(mg. Pb per mouse
per day)
A
B
Exp. 1
2
3
4
Average
0-046
0-143
0-057
0-133
0-224
0-681
0-259
0-605
0-457
0-10
B
A
Low Ca diet
0-795
0-159
0-211
1-055
• 0-149
0-709
0-182
0-867
0-856
Exp. 5
6
Average
0-033
0-042
0-173
0-221
0-197
High Ca diet
0-042
0-210
0-054
0-284
0-247
Exp. 7
8
Average
0 05
0-50
B
A
B
0-254
0-510
0-200
0-450
1-270
2-55
0-87
2-37
1-76
0-445
0-909
0-385
0-666
2-42
5-05
1-68
1-33
2-63
0-055
0-042
0-282
0-200
0-241
0057
0-050
0-317
0-217
0-267
0-028
0-036
0-143
0-180
0-162
Low Ca diet + Ca glycei'ophospliate (high Ca
0-142
0-155
0-027
0-028
0-029
0-026
0-108
0-027
0-117
0-030
0-125
0-136
Exp. 9
10
Average
0-133
0-125
Exp. 11
12
Average
0-128
0-133
Exp. 13
0-029
1-00
A
diet)
0-129
0-115
0-122
Low Ca diet + olive oil
0-182
0-860
0-250
0-252
0-714
1-200
1-030
1-25
3-10
2-18
0-366
0-833
1-74
3-97
2-86
Low Ca diet -t- cod liver oil
0-222
0-609
0-925
0-714
0-665
0-159
0-757
0-360
0-841
0-637
3-10
2-00
2-55
0-800
0-500
4-21
2-63
3-42
High Ca diet + cod liver oil
0-167
0-046
0-132
0-040
0-230
0-054
0-135
0-633
0-571
0-602
Pb content of control animals =0-020 ±0-003 mg. Pb.
Pb content of the high Ca diet = 0-002 mg. Pb per mouse per day.
Pb content of the low Ca diet = 0-002 mg. Pb per mouse per day.
ALIMENTARY Pb ABSORPTION
1239
In view of this, experiments were then carried out in which mice were fed
with low Ca diet containing added supplements of Pb, to which had been added
Ca glycerophosphate (0-5 g. per mouse per day). This in effect converted the
low Ca diet into a high Ca diet. Here again the effect of the high Ca diet was to
produce only a small absorption of Pb.
The effects of fat and vitamin D upon the absorption of Pb were then studied.
It was considered that fat might hinder the absorption of Pb by the formation
of insoluble Pb soaps. In this series of experiments, the mice were placed upon
the low Ca diet, containing supplements of added Pb to which was added olive
oil at the rate of 1 ml. per mouse per day. No specific influence of fat upon the
absorption of Pb could be detected. It was considered that vitamin D might aid
the absorption of Pb in the same way as it assists the absorption of Ca. In this
series of experiments the mice were placed on (a ) the high Ca diet, (b) the low
Ca diet containing supplements of added Pb, to which was added cod liver oil
at the rate of 3 drops per mouse per day. The experimental results obtained
did not indicate any marked influence by vitamin D upon the absorption
of Pb.
The results obtained are shown in Table I.
Discussion
Examination of the influence of variation in the concentration of certain
constituents of the diet upon the absorption of Pb from the alimentary tract in
mice has shown that from a high Ca diet the absorption of Pb was small and was
not influenced to any great extent by the amount of Pb administered. Upon a
low Ca diet, the amount of Pb absorbed was large and depended to a large extent
upon the amount of Pb administered.
It is interesting to speculate as to the reasons for this. According to Shields
et al. [1938], absorption does not take place from the stomach but takes place
rapidly from the small intestine. The phosphate content of both diets is high
and as a result the Pb will be chiefly present as Pb phosphate. The solubility
of this Pb phosphate in the intestinal contents, and hence its capacity to be
absorbed, will depend to a great extent upon the reaction of the intestinal
contents. It is suggested that upon the low Ca diet, the reaction of the intestinal
contents tend to become more acid whereas on the high Ca diet the contents
tend to become more alkaline.
Although large amounts of Pb were absorbed on a low Ca diet, the absorption
by animals receiving the same quantities of Pb in different experiments showed
marked variation. Other important factors must be involved, e.g. the speed
with which contents pass through the intestine, the amount of water consumed,
the capacity to excrete absorbed Pb etc.
No specific action of vitamin D upon the absorption of Pb could be observed.
Upon a high Ca diet the absorption of Pb was low and upon a low Ca diet the
absorption of Pb was of the same order as that on a diet which did not contain
additional vitamin D.
Large quantities of fat in the diet did not appear to influence the absorption
of Pb.
Summary
The effect of variation in the concentration of certain constituents of the
diet upon the absorption of Pb from the alimentary tract of mice has been
studied.
1240
S. L. T O M P S E T T
On high Ca diets, the absorption of Pb was low while upon low Ca diets it
was high. Eat and vitamin D were without influence.
I wish to thank Dr A. B. Anderson for his helpful criticism and advice.
REFERENCES
Aub, Fuirhall, Minot & Reznikoff (1926). Lead poisoning. Baltimore: Williams and Wilkins.
Shelling (1932). J . biol. Chem. 96, 197.
Shields, Mitchell & Ruth (1938). J . In d . H yg . T o xic. 21, 7.
Sobel, Gawron & Kramer (1938). Proc. Soc. exp. B io l. N .Y . 38, 433.
Tompsett (1939). Biochem. J . 33, 1231.
& Anderson (1935). Biochem. J . 29, 1851.
[P ro m
THE BIOCHEMICAL JOURNAL,
V o l.
XXIX, No. 8, pp. 1851-1804, 1935]
[A 11 R ights reserved]
PR IN TED IN GREAT BRITAIN
CCXVI. T H E L E A D C O N T E N T O F
H U M A N TISSUES A N D EXCRETA.
By SIDN EY LIONEL TOMPSETT and ALAN BRUCE ANDERSON.
From the Biochemical Laboratory, Department of Pathology of the
University and Royal Infirmary, Glasgow.
{Received M ay 31st, 1935.)
That lead is normally excreted in human urine and faeces is now well established.
The literature has been reviewed by Kelioe et al. [1933, 1, 2], who have carried
out an exhaustive study of the excretion of lead by normal American adults
and children.
The occurrence of appreciable amounts of lead in “ normal” human bones
is also generally agreed upon, although there is considerable variation in the
figures published, e.g. Bartli [1931] lincls 0-01-0-06 mg. Pb per g. ash or approxi­
mately 5-30 mg. Pb per kg. fresh bone, whilst Lynch et al. [1934] find 14-146 mg.
Pb per kg. fresh bone. The position as regards the soft tissues is very unsatis­
factory; few analyses are available and the evidence for the occurrence of
“ normal” lead in tissues is conflicting. Mcillerc [1903] states that small amounts
of lead were present in the organs of nearly all the subjects examined by him
(1-2 mg. per kg. on the average in the liver and spleen). Aub et al. [1926] state
that the lead retained by an apparently normal individual is hold almost ex­
clusively by the skeleton. Weyrauch and Muller [1933] found no appreciable
amount of lead in the liver, kidney, spleen or brain. Sheldon and Ramage [1931],
using a spectrograpliic method, found lead occurring spasmodically in normal
organs, whilst Boyd and De [1933], also using a spectrograpliic method, found
lead well marked in the liver and present in all the other organs examined except
the brain. Lynch et al. [1934] in an analysis of a few organs found 1-5 mg. per kg.
in some livers and kidneys and none in others. Kehoe et al. [1933, 1] found
appreciable amounts of lead in most of the tissues from two cases apparently
normal shortly before death.
Whilst the estimation of lead in bones is comparatively easy, the soft tissues,
having a high iron content and yielding only a small amount of asli, present
considerable difficulties. It is probable that the differences in the published
results may be attributed to the methods of analysis used, which are open to
several criticisms. A number of methods have been used. Fairhall [1924]
described a method in which the lead was precipitated as sulphide and then as
the chromate. The lead chromate was determined either (1) colorimetrically with
diphenylcarbazide or (2) by titration with thiosulphatc after the addition of potas­
sium iodide. Kehoe et al, [1926; 1933, 1, 2] used modifications of this method.
Cooksey and Walton [1929], in an examination of urine, made a preliminary
separation of lead by an electrolytic method. The lead was subsequently esti­
mated nephelometrically as the sulphite. Francis et al. [1929] described a process
involving the precipitation of lead as sulphide followed by electrolysis and pre­
cipitation as the sulphate. Finally the lead was estimated colorimetrically as the
sulphide. Weyrauch and Muller [1933] and Litzner and Weyrauch [1932; 1933],
investigating the distribution of lead in man, separated lead as the sulphide and
then as the peroxide by electrolysis. They estimated the lead colorimetrically by
( 1851 )
1852
S. L. T O M P S E T T A N D
A. B. A N D E R S O N
the blue colour formed by the interaction of the peroxide and tetramethyldiaminodiphenyl methane.
One of the major problems in the determination of lead is to separate it from
substances that would interfere in the final stage of the estimation. The chief
of these is iron. None of the above methods can claim to perform this process
satisfactorily. Electrolytic methods usually fail in the presence of large amounts
of iron [Francis et al., 1929], while precipitation as lead sulphate or chromate is
unsuitable as these substances have solubilities which are appreciable when
fractions of a mg. of lead are being dealt with. In a recent paper [1935] Kehoe
et al. acknowledge a loss of 0-07 mg. Pb per sample in the earlier method they
employed.
A method suggested by Allport and Skrimsliire [1932, 1] for separating lead
from solutions o f the ash of dycstuffs appeared to solve such difficulties. An
alkaline solution of the ash was shaken up with a chloroform solution of diphenylthiocarbazone (dithizonc). Lead was extracted by the chloroform as a leaddiphcnylthiocarbazone complex. Iron was not extracted and other metals, with
the exception of bismuth, were not extracted if cyanide were present. Under
the latter conditions then, only two metals, lead and bismuth, were extracted.
With this method it is recognised that certain difficulties are encountered. The
aqueous solutions must be perfectly clear, the slightest turbidity due to iffiosphatcs, iron etc., preventing a complete extraction of lead. As the extractions
must be carried out on alkaline solutions, this is difficult, even when citrates
have been added, for a solution may appear perfectly clear and yet iron, phos­
phates etc. may be precipitated in colloidal form and so prevent a complete
extraction. The p n of the solutions needs careful adjustment which is not always
easy when certain classes of materials are being examined. I f the organic matter
lias been destroyed by a Avet oxidation method, the nature of the oxidant appears
to exert a marked influence. Allport and Skrimsliire [1932, 2] found that if
nitric acid had been used as the oxidant, extraction of the lead was generally
incomplete. This appears to bo due to traces of oxidant remaining in the digest.
We have found that in practice, Avlien used to separate lead from solutions of
the ash of urine, liver etc., the method gave erratic results. In every case the
solutions appeared perfectly clear.
In the final stage of the estimation of lead, tlie sulphide reaction appears to
have been most commonly used. Unfortunately this is not specific for lead,
bismuth giinng a similar reaction. The sulphide reaction also lacks sensitivity.
A more sensitiAre reaction is required for the determination of lead in blood, as
the amount of blood that can be taken from a patient under routine conditions
is limited. The objection to the tetrametliyldiaminodiplienylmethane reaction
is that although it is A7ery sensitive, it is not specific for lead peroxide, substances
such as manganese dioxide reacting similarly. Apart from the objections to
electrolytic methods in general already referred to, manganese tends to be de­
posited as the dioxide along with lead. This is an especial failure of the method
as manganese occurs in human tissues and excreta in appreciable amounts.
The present paper is diAdded into tAvo parts. In the first a method for the
estimation of lead, in which the difficulties outlined above have been overcome,
is described. The second part deals with the lead content of human tissues
obtained post mortem, and also the lead content of blood and excreta from
hospital patients and normal individuals.
L E A D C O N T E N T O F TISSUES A N D E X C R E T A
1853
I. THE METHOD FOR THE ESTIMATION OF LEAD.
B y S. L. Tompsett.
The separation of lead. Wlien an aqueous solution of sodium dietliyldithio carbamate is added to a solution of a copper salt a yellow organic copper complex
is formed which may be extracted with ether. The extraction is complete in
acid, neutral or alkaline solution, i.e. is independent of p n , but is preferably
carried out in alkaline solution in the presence of pyrophosphate to prevent the
extraction of iron. The exact adjustment of^»n is uninqiortant [Tompsett, 1935].
Lead also was found to form an organic complex with sodium dietliyldithio carbamate, which could be extracted with ether. The lead complex is white and
therefore ethereal extracts are colourless. Amounts of lead varying from 0*01
to 0*2 mg. could be extracted quantitatively by such a technique. The complex
of lead and sodium dietliyldithiocarbamate is very insoluble in water, turbidities
appearing when the reagent is added to 0*05 mg. Pb or more in 100 ml. water.
Sodium dietliyldithio carbamate itself is insoluble in ether, so that the amount
of organic material extracted is minimum.
The estimation of lead. Fischer and Leopoldi [1934] have published a colori­
metric method using diplicnylthiocarbazone for the estimation of small amounts
of lead. When an alkaline solution of a lead salt was shaken with a carbon
tetrachloride solution of diphenylthiocarbazone, a pink complex with lead was
formed, which was extracted by the organic solvent. After the shaking process,
the carbon tetrachloride layer contained pink lead complex and also unchanged
green diplicnylthiocarbazone. Unchanged diphenylthiocarbazone was removed
by repeatedly shaking the carbon tetrachloride with 1 % KCN solution. Finally
the pink extract was shaken with dilute acid, which changed the colour to green
and then compared in a colorimeter with a standard. They stated that the method
was quantitative and that amounts of lead of the order 6 to 120y could be
estimated, also that the reaction was specific for lead.
The writer has found that the pink colour is just as sensitive to colorimetric
comparison as the green colour developed after shaking with acid. Using 10 ml.
CCld to extract the complex it was found that the depth of colour was pro­
portional to the Pb concentration within the range 5 to 70y. The best depth of
colour for colorimetric comparison appears to be in the region of 0*01 and
0*02 mg. Pb. W ith amounts of lead above 0*03 mg. the colour was too strong
for colorimetric comparison.
Reagents.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
determination of lead in urine and faeces.
Concentrated hydrochloric acid—analar reagent.
Concentrated nitric acid—analar reagent.
Perchloric acid—analar reagent.
Glacial acetic acid—analar reagent.
Ammonia (sp. gr. 0*88)—analar reagent.
Ether—analar reagent.
10% potassium cyanide—PbT (B.D.H.). This was diluted 1 in 10 as
required.
(8) Carbon tetrachloride— analar reagent.
(9) 5 % sulphurous acid—lead-free.
(10) 20 % sodium citrate—lead-free.
A lead-free solution was prepared as follows. To 1 litre of a 20 % solution
in water, 100 ml. of 0*1 % diphenylthiocarbazone in chloroform were added and
1854
S. L. T O M P S E T T A N D
A. B. A N D E R S O N
the mixture was shaken vigorously. As required, a small portion was passed
through a filter-paper to remove suspended particles of chloroform.
( 11) 0-1 % diphenylthiocarbazone in carbon tetrachloride.
Commercial diphenylthiocarbazone contains a yellow oxidation product
which is soluble in carbon tetrachloride but is not extracted by alkali cyanide
solutions. The commercial product was purified as follows. 100 ml. of 0-1%
diphenylthiocarbazone in carbon tetrachloride were extracted with several
100 ml. portions of 0-5% ammonia. Diphenylthiocarbazone passes into the
aqueous phase, leaving the oxidation product in the carbon tetrachloride. The
ammoniacal extracts were passed through filter-paper to remove suspended
particles of chloroform and then acidified with sulphurous acid. The green pre­
cipitated diphenylthiocarbazone was then extracted with 100 ml. of carbon
tetrachloride. This solution if preserved under a layer of sulphurous acid (5%)
will keep indefinitely.
( 12) 2 % sodium diethyldithiocarbamate.
Before use a small volume was shaken up with ether to remove traces of lead.
(13) Standard solution of lead acetate.
0-1831 g. of lead acetate Pb(C2H 30 2)2, 3H20 was dissolved in distilled water
containing 5 ml. of glacial acetic acid. The volume was then made up to 1 litre
with distilled water. 1 ml. of this solution is equivalent to 0-1 mg. Pb. This
solution was diluted as required so that 1 ml. was equivalent to 0*01 mg. Pb.
The water used was glass-distilled. Pdter-papers were washed with dilute
acid, followed by distilled water. Pyrex glassware was used. Silica dishes were
always cleaned out with hot dilute acid prior to use.
Method. 500 ml. of urine were evaporated to dryness in a silica dish on a
steam-bath and then ignited over a Bunsen burner in a fume cupboard. Final
traces of carbon were removed by adding 5 ml. of concentrated nitric acid to
the cooled ash and heating further.
The ash was dissolved in 100 ml. of water containing 5 ml. of concentrated
IlCl, and the solution transferred to a 750 ml. separating funnel. 100 ml. of 20 %
sodium citrate solution were added and the mixture was made slightly a litaline
to litmus by the addition of ammonia (sp. gr. 0*88). The volume of the solution
at this stage should be about 400 to 500 ml. 10 ml. of sodium diethyldithio­
carbamate solution were added followed by 25 ml. of ether. The mixture was
shaken vigorously. The aqueous layer was run off and the ether extract washed
twice with 25 ml. of water. Tlie ether extract was run into a 300 ml. Kjeldahl
flask, the separating funnel being rinsed with 10 ml. of ether. The aqueous
solution and washings were extracted a second time with ether. The combined
extracts were evaporated to dryness on a steam-bath and the organic matter
was destroyed by heating with 1 ml. concentrated sulphuric acid and 1 ml.
perchloric acid. After digestion fumes were removed by a water pump. The
following were added to the digest in order: 10 ml. water, 1 ml. glacial acetic
acid, and 5 ml. ammonia (sp. gr. 0*88) and the volume was made up to 25 ml.
with water.
The final stage of the estimation was carried out as follows. Three glassstoppered 50 ml. volumetric flasks were taken. 5-10 ml. of the diluted digest
were measured into one of the flasks. Similar amounts of blank solution were
measured into the other two flasks. The blank solution was prepared in exactly
tlie same way as the unknown. For every 5 ml. solution in the flasks 6 drops
of sulphurous acid were added. Into one of the blank flasks 1 or 2 ml. of standard
lead acetate solution (equivalent to 0*01 or 0*02 mg. Pb), depending on the lead
L E A D C O N T E N T O F TISSUES A N D E X C R E T A
185S
content of the unknown, were measured. To each flask were now added 5 ml.
1 % KCN solution, 10 ml. carbon tetrachloride and 0*5 ml. 0*1 % diphcnylthiocarbazone. After vigorous shaking the contents of the flasks were poured into
test tubes and the aqueous layers removed with a teat pipette. After being
returned to the flasks, excess diphenylthiocarbazone was removed from tlie
carbon tetrachloride layers by repeated extraction with 10 ml. lots of 1 % KCN
solution. Usually 4-6 extractions were necessary. The pink extracts were then
washed with water and compared with a standard in a colorimeter.
After washing with distilled water the extracts were quite clear and did not
require filtration. Filtration should be avoided as yellow tints tend to develop
after the extracts have passed through filter-paper. After the diluted digest
has been shaken with diphenylthiocarbazone, the aqueous layer should bo
coloured brown, indicating that excess of the reagent is present, otherwise more
must bo added. In the event of 5 ml. of diluted digest containing more than
0*03 mg. Pb, a smaller volume should be used which should be diluted to 5 ml.
with blank solution. A blank was always done to control contaminations from
outside sources. The blanks showed perceptible pink tinges but were too small
to be measured. That the blanks showed a reaction at all is due to the extreme
sensitivity of the test.
In the case of faeces, 5-10 g. of dried material were ignited in a silica dish
and the estimation proceeded with as above.
From the results shown in Table I it will be seen that lead added to urine
or faeces may be estimated quantitatively by the above method. The urine and
faeces used in these recovery experiments were collected with no special pre­
cautions to exclude contamination. The figures therefore cannot justifiably bo
taken as representative of the lead content of urine and faeces in the normal
subject.
Table I. The recovery of lead added to urine and faeces.
Urine.
Volume used—500 ml.
Faeces.
Weight of dried faeces used— 10 g.
Initial lead
Lead
Lead
Initial lead
Lead
Lead
content
content
added
recovered
added
recovered
mg.
mg.
mg.
mg.
mg.
S
0-143
0-106
0-500
0-482
0-063
0-100
1
0-104
0-053
9
0-500
0-5.L2
0-053
0-050
2
0-025
0-096
0-500
0-100
0-100
0-525
3
10
0-048
0-210
1.1
0-400
0-200
4
0-063
0-423
0-167
0-099
0-250
12
0-254
0-062
0-100
5
0-100
0-095
6
0-031
0-100
0-105
0'077
7
The specimens of urine and faeces were collected with no special precautions to exclude con­
tamination from outside sources. The results must not bo taken as representative of the normal.
By the use of an ignition method for destroying organic matter, blanks have
been reduced to a minimum. There were no losses observed when this method
was applied in the estimation of lead in urine and faeces. This is probably due
to the high ash content of these materials, much of which is in the form of
phosphates. It was not necessary to add pyrophosphates to prevent extraction
of iron in the preliminary separation of lead as they are formed in sufficient
quantity during the ignition process.
In preliminary experiments the carbon tetrachloride extracts containing the
lead diphenylthiocarbazone complex often had yellow tints which made colori­
metry difficult. It was considered that this was due to traces of perchloric acid
in the digests. The addition of 6 drops of sulphurous acid to every 5 ml. of
1856
S. L. T O M P S E T T A N D
A. B. A N D E R S O N
diluted digest used prevented the formation of this yellow colour and perfect
matching colours could be obtained.
Although sodium dietliyldithio carbamate is not a specific reagent for the
separation of lead, those metals that are extracted, e.g. copper, zinc, bismuth,
do not interfere, as the complexes that they form with diphenylthiocarbazone
arc unstable in the presence of cyanide. The bismuth complex is orange-coloured
and if present will be seen in the carbon tetrachloride layer after the first
shaking. It is removed however during the subsequent extractions with cyanide.
In actual experiment it was found that 0-01 mg. Pb could be estimated in the
presence of 0-1 mg. Bi, tlie bismuth complex being completely removed at the
fourth extraction. The complexes of the other metals are so unstable in the
presence of cyanide that they arc not formed at all.
The separation of lead as the complex with sodium diethyldithiocarbamate
serves the very useful purpose of removing those substances such as iron and
the phosphates of the alkaline earths which interfere with its reaction with
diphenylthiocarbazone.
After extraction of the dietliyldithio carbamate complexes with ether, a few
ml. of dilute copper sulphate solution should be added to the residual aqueous
solution. The formation of a golden brown colour will indicate that excess of
the diethyldithiocarbamate reagent has been adclccl. In the majority of cases
tlie amount of reagent indicated in this paper is in marked excess but in a few
cases more may be necessary.
II. THE LEAD CONTENT OF HUMAN TISSUES AND EXCRETA.
By S. L. Tompsett and A. B. Anderson.
E xperimental.
Urine and faeces. The urine was collected in paraffined bottles and 500 ml.
were taken for each estimation. The faeces were collected directly into large
pyrex glass dishes in which they were dried. For each estimation 5-10 g. of
dried and powdered material were used.
Soft tissues. The organs were first weighed and, except in the case of lung,
100 g. of tissue were chopped into small slices and placed in 100 ml. of lead-free
10% sodium phosphate solution in a silica dish. The whole right lung was first
dried at 110° in a p 3U’ex dish, ground to a powder and an aliquot portion of
powder added to the phosphate solution in a silica dish. The mixture of tissue
and phosphate solution was then evaporated to dryness on a steam-bath, and
the subsequent procedure was the same as that described for urine and faeces.
In the final colorimetric estimation 5-10 ml, of diluted digest were taken and
compared with standards containing 0 -01- 0-02 mg. of lead.
Bone. About 5 g . of bone in small pieces were put into a silica dish without
phosphate solution and ashed directly. The ash was treated in the same manner
as that from other tissues.
Blood. Some modification of the method was necessary when dealing with
blood owing to the smaller quantity of material used. The method is therefore
given here in some detail. Approximately 20 ml. of blood were drawn from a
vein with an all-glass syringe and stainless steel needle. Syringe and needles
were sterilised by boiling in distilled water. The blood was immediately poured
into a pyrex tube and rapidly pipetted into 100 ml. of lead-free 10 % sodium
phosphate solution in a silica dish. The exact volume of blood obtained was
L E A D C O N T E N T O F TISSUES A N D
EXCRETA
1857
noted. The contents of the dish were then evaporated to dryness on a steam
bath and ashed in the same manner as the tissues, using only 1 ml. of nitric
acid. The ash was dissolved in 50 ml. of water containing 2 ml. of concentrated
HC1 and the solution transferred to a separating funnel. The solution, which
with the washings amounted to 100-150 ml., was made faintly alkaline to litmus
by the addition of ammonia (sp. gr. 0-880), and cooled. After the addition of
2 ml. of 2 % sodium diethyldithiocarbamate and 25 ml. of ether the mixture
was shaken vigorously and allowed to separate and the aqueous layer run off.
The ether extract was washed with 25 ml. of water and then run into a 150 ml.
round-bottomed pyrex flask, a further 10 ml. of ether being used to wash the
funnel. The aqueous solution was re-extracted with 10 ml. ether, which were
added to the first extract, together with a second washing of 10 ml. of ether.
The ether was then evaporated off on a steam-bath and the organic material
in tlie residue destroyed by digestion with 0-2 ml. of sulphuric acid and 0-2 ml.
of perchloric acid. The fumes were then removed from the flask by suction with
a water pump, and 3-5 ml. water, 0-2 ml. glacial acetic acid and 1-0 ml. ammonia
(sp. gr. 0-88) added in that order. After the addition of G drops of 5% sul­
phurous acid the contents of the digestion flask were transferred to a 50 ml.
glass-stoppered pyrex volumetric flask and the digestion flask was washed out
with 5 ml. of 1 % KCN into the volumetric flask. To the mixture 10 mi. of carbon
tetrachloride and 0-2 ml. diphenylthiocarbazone solution were added. The colour
was then developed in the usual way and compared with a standard prepared
similarly. For normal blood a standard containing 0-01 mg. Pb was found to
be suitable when 20 ml. of blood were used. Sodium citrate was not added in
the analysis of blood as any slight haze of calcium or magnesium phosphates did
not interfere with the extraction.
Sodium phosphate was added to the blood and soft tissues before ashing for
two reasons; firstly to increase the amount of ash and to prevent the formation
of insoluble ferric oxide; secondly to form an excess of pyrophosphate which
prevents the extraction of iron in the separation process. The sodium phosphate
which was kept as a 10 % stock solution was always de-leaded just before use
in the following manner. To every 100 ml. of solution in a separating funnel
5 ml. of 2 % sodium diethyldithiocarbamate solution were added and the whole
was shaken vigorously with ether. The lead-free aqueous solution was then run
off. The phosphate solution was not added to urine, faeces or bone, as these
substances contain enough phosphate.
The results of analysis of organs from 22 post mortems are given in Table II,
which is divided into 20 cases with no known occupational lead exposure and
2 cases with occupational exposure to lead, namely a painter and a printer.
The figures are given for concentration in mg. Pb per kg. of fresh tissue and
also for the total organ where possible. In the cases of rib and vertebra, concen­
trations alone are given. In order to obtain a representative sample of lung the
whole organ was first dried and powdered as described above. For this reason
only the total lead content of the lung is given. The cases in the first division
range in age from G9 years to 6 weeks and consist of 11 males and 9 females.
Lead in appreciable amounts was found in all the tissues examined, and with
the exception of the spleen, the amounts present were remarkably constant
considering the widely different pathological states involved. The figures for
bone: rib, mean concentration 8-55 mg. per kg., and vertebra, mean concen­
tration 7-09 mg. per kg., are the most constant, the greatest deviation from tlie
mean being in the vertebra of No. 7 with a concentration of 14-7 m gv, or just
more than twice the mean value. The child of 2 years of age, No. G, shows the
1858
S
.L. TOMPSETT AND A. B. ANDERSON
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LEAD CONTENT OF TISSUES AND EXCRETA
1050
same concentrations of lead as the adults in liver, kidney, brain and bones.
The spleen was not analysed. The figures for the 6-weeks old infant are re­
markably high for some of the organs. The concentrations in the kidney and
spleen of 3-55 and 3-08 mg. respectively are approximately twice the mean
values for adults. The concentrations in the bones, 1-57 mg. for rib, and 2-6 for
vertebra, are considerably less than the adult figures, as would bo expected.
The post mortem on this child showed a normal well-nourished baby and the
organs, with the exception of the brain, were normal. Of the two cases with
occupational exposure to lead, the painter shows an excessive deposition of lead
in the bones; the concentration in the rib of 119 mg. is fourteen times the mean
value, and that in the vertebra, 18-8, approximately two and a half times the
mean. Tlie printer shows a slight excess in the rib only. There is no suggestion
of lead poisoning in the clinical notes on these two cases.
Table III. Lead content of tissues from hum,an foetuses.
Liver
Months
gesta­
Sex
tion
fM
8
2 f twin
IB
M
8-5
3
M
4
7
t
Weight mg, per
kilo
g2400
0-33
0*83
2100
.—
0-63
0-95
1600
Mean
0-68
A
Kidney
(
Total
mg.
0-04
0-07
0-0G
0-06
0-06
mg. per
kilo
0-66
0-63
0-63
0-67
0-65
,
Brain
-------- V .
Total
mg.
0-01
0-01
0-01
0-01
0-01
mg. per
kilo
0-12
0-21
0-18
0-16
0-17
Total
mg.
0-03
0-04
0-07
0-04
0-046
Femur
mg. per
kilo
1-49
2-66
1-47
1-30
1-73
The tissues of four stillborn foetuses, two of which were twins, wore also
analysed, using the same method except that, in the case of the kidney, the
modifications described for blood wore used. For the analysis of bone, both
femurs were used. The results are given in Table III. Lead was present in
appreciable concentrations in all the tissues examined. The mean values for
liver, kidney, and brain are from one-lialf to one-third of the adult values. The
concentration in the femurs approximates to that found in the rib of the infant
of 6 weeks. Of the twins, the female shows higher values than the male. For
comparison, the copper in these tissues was determined at the same time; the
mean values obtained were, in mg. per kg., liver—44-6, brain— 1*08, and
femur— I '86. These results show the usual selective absorption of copper by
the foetal liver. The amount in the livers was at least five times that reported
for the adult [Tompsett, 1935].
Urine and faeces were collected from three laboratory workers, as normals,
and from ten hospital patients, in periods of two or three consecutive days. The
normals were working and eating their ordinary diet. The patients, with the
exception of No. 5 who was a case of pernicious anaemia, wore in the metabolic
wards for determination of basal metabolic rate, which in every case was within
the normal limits. The diagnosis in most cases was tachycardia of unknown
origin or “ neurosis” . The figures for the daily excretion of lead in the urine
and faeces are given in Table IV. It will be seen that the normals excreted daily
0-16to0-03 mg. in the urine and 0*40 mg. Pb in the faeces. The patients’ excretion
in the urine varied from 0-07 to 0-025 mg., with a mean of 0-05 mg., and in the
faeces from 0*26 to 0-20 mg., with a mean of 0-22. A half-day’s diet as given
in the metabolic ward was dried down in a pyrex dish, and on analysis by the
method used for tissues gave 0-22 mg. Pb per diem . The hospital water was
found to contain 0-03 mg. Pb per litre. Patients on this diet would appear to
excrete a fairly constant amount of lead, and this amount is less than that
1860
S
.L. TOMPSETT AND A. B. ANDEBSON
excreted by the normal laboratory workers. This difference can probably be
accounted for by a greater intake of lead in the diet and drinking water of the
normals. The occurrence of large amounts of bismuth in the faeces during
bismuth medication will interfere with the lead estimation. The only remedy is
to discontinue the bismuth, and analyse the faeces in a few days’ time, when the
small amounts of bismuth that may be present will not interfere, because the
bismuth diphenylthiocarbazone complex is unstable when shaken with cyanide.
Table IV. The excretion of lead in urine and faeces.
Sex
Days
collection
1
2
3
M
M
M
3
3
1
<1
5
(j
7
8
9
10
11
12
12
M
F
M
M
F
F
M
M
M
M
3
3
3
2
3
3
2
2
3
2
Occupation
(a) Normals.
Laboratory worker
Do.
Do.
Urine
mg. pe r diem
Faeces
mg. per diem
0-16
0-03
0-085
0-40
0-40
0*39
0-04
0-025
0-06
0-055
0-07
0-05
0-06
0-05
0-04
0-06
0-05
0-20
0-23
0-26
0*23
0*24
0*20
0*22
0-24
0*20
0*20
0*22
(b) Hospital patients.
Warehou s cm an
Housewife
Miner
Packer
Housewife
Do.
Miner
Engineer
Brass moulder
Packer
Mean for patients
Before estimating the lead in the blood some recoveries of lead added to
blood and serum were undertaken to test the method as modified for the smaller
quantities. As will be seen from the figures given in Table V, a good recovery
of lead added to blood was obtained. I t should be emphasised here that the
figures for the lead content of blood in Table V have no significance because
the blood was mixed and obtained from contaminated sources.
Blood was then collected from the same normals as before and from 18 hos­
pital patients. Twenty-five samples in all were taken. The figures are given in
Table VI. Tlie patients were chosen from among those not acutely ill, and where
no diagnosis is given it is to be understood that no physical signs of organic
disease were present. The blood lead varied from 70 to 40 y per 100 ml., with a
mean value of 55y. The figures arc given to the nearest 5 y . In the case of
normal No. 3 several figures are given; these represent determinations done on
blood collected on different days both before and after meals.
Of several cases of suspected lead poisoning investigated only one showed
definite plumbism and this case is discussed here. The clinical history is briefly
as follows. A young man of 30 years of age was admitted to the medical wards
Table V. Recovery of lead added to serum and blood.
Initial lead
Lead
Recovered
content
added
lead
r
7
7
(a) Scrum. 10 ml. samples.
5-5
5
6-0
5*5
10
9*0
5-5
5
4-5
5-5
10-5
10
Initial lead
content
7
Lead
added
Recovered
lead
7
7
(b) Blood., 10 ml. samples.
12-9
12-9
12-9
14-0
14*0
5
10
100
5
10
5-6
10*5
102*1
5*5
9*0
LEAD CONTENT OP TISSUES AND EXCIIETA
1861
Table VI. Lead content of blood.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Age
32
19
29
30
26
—
53
32
—
62
—
25
65
.—
50
45
52
26
—
72
15
Sox
M
M
M
M
F
M
M
F
M
F
M
M
M
M
M
M
M
F
M
F
M
Occupation
Laboratory worker
Do.
Do.
Motor driver
Housewife
—
Brass moulder
Housewife
.—
Housewife
—
.
Engineer
Steelworker
—
Packer
Colliery repairer
Park labourer
Housewife
Miner
Teacher
Unemployed
Blood load
y per
Diagnosis
100 ml.
Normal
50
40
Do.
Do.
70,40,60,50
50
Duodenal ulcer
60
Hysteria
—
55
Debility
50
Epilepsy
55
.—
45
Hypertension
60
— .
45
60
Duodenal ulcer
Hemiplegia
65
— .
55
Neurasthenia
60, 50
Osteoarthritis
60
60
Gastric ulcer
Vasovagal attacks
60
—
50
65
Neurosis
Abdominal pain
65
Mean
55
complaining of severe pains in the stomach with vomiting of a week’s duration.
The pain had no relation to food and was not affected by food. His occupation
was that of a solder-maker. In this trade, which he had followed all his adult
life, he mixed molten lead, tin and other metals. The clinical findings were a
moderate anaemia, blood count showed 3,000,000 red colls, and 40 % haemo­
globin, with some punctate basophilia, no wrist drop or other signs in the
nervous system. There was a marked blue line round the gums. The stools con­
tained blood. An X-ray of the stomach did not reveal any lesion.
Table VII. Case J . C. adm itted to medical toard 27. i. 35.
Date
28. i. 35
29. i. 35
19. ii. 35
21. ii. 35
5. iii. 35
18. iii. 35
15. iv. 35
16. iv. 35
19. iv. 35
24. iv. 35
Urine
Blood lead
,---------------- A---------------- v
Faeces
D iet le
y per 100 ml. mg. per litre
mg. per diem , mg. per diem■
mg.
—
0-27
0-12
—
—
135
—
—
—
—
125
0-073
0*135
0-27
0-13
Started medication with potassium iodide 15 gr. pe r diem
135
0-107
0-17
0-19
—
Discharged on potassium iodide 15 gr. per diem
Readmission to metabolic ward complaining of return of symptoms
380
—
'—
—
—
—
0-036
0-041
0-44
0-22
240
—
—
—
—
The chemical investigation is summarised in Table VII. It will be seen that
soon after admission the urinary and faecal excretion of lead was not higher
than would be expected in a normal person. In this connection tlie intake of
lead must also be considered. Tor the first few days he was on a milk diet and
later was given a peptic ulcer diet. A sample of the latter diet was analysed
and found to contain 0*13 mg. of lead per diem. This alkaline milky diet was
probably increasing the storage of lead in his bones. The figures for urine show
the importance of calculating tlie excretion per day and not relying on the
amount per litre. In contrast to his excretion of lead the blood lead was high,
1862
S
.L
. TOMPSETT AND A. B. ANDERSON
being at least twice the normal. On 21. ii. 35 medication with potassium iodide,
15 grains per diem , was started. Ten days afterwards blood lead and the excre­
tion of load were the same as before. He was now free from pain and was dis­
charged and given the potassium iodide, which he continued to take until he
was readmitted 4 weeks later to the metabolic ward complaining of sharp pains
in the stomach. The potassium iodide was discontinued on admission, and blood
taken the next day gave the surprisingly high figure of 380y Pb. He was on
tlie ordinary ward diet, and a 3-clay collection of excreta showed a normal
urinary excretion but a faecal excretion about twice that of patients on the
wrard diet. The pain became less and less severe, and 10 days after admission
his blood lead had fallen to 240 y. The lead line had almost disappeared when
he was readmitted.
This man had been subjected to a prolonged occupational exposure to leacl,
both as solid and vaporised, and must have had large deposits in his bones.
His symptoms were of the gastro-intestinal typo probably with intestinal ulcera­
tion giving blood in the stools. Though showing clinical signs of lead poisoning
and having a high blood lead, he showed little increase in lead excretion. The
results of administration of potassium iodide for a period of weeks were a return
of the abdominal pain and an increase in the blood leacl to nearly treble the
original figure.
D iscussion.
The salient features of the analysis of “ norm al” soft tissues arc the presence
of lead in all the tissues examined, and the comparatively constant concentra­
tion for the adult irrespective of age, sox or cause of death. The presence of
small quantities of lead in the soft tissues might be deduced from the fact that
leacl is a constant constituent of blood. It is not suggested, however, that lead
is necessarily present in these concentrations in the normal during life. The
changes in metabolism preceding death may bring about a mobilisation of lead
from the deposits in the bones. Lead was found in the bones in amounts of the
same order as those reported by Kehoe et al. [1933] and Barth [1931]. The
figures show less variation from the mean than those for the soft tissues. Analysis
of bones other than the ribs and vertebrae taken in this investigation would
probably give different results. The higher figures reported by Lynch et al. [1934]
were obtained in the analysis of the shaft of the femur principally.
Whilst our results for tissues may be taken as “ norm al” for cases in the
Glasgow Royal Infirmary, it is possible that a similar investigation in another
part of the country would reveal different “ normals” . This “ normal” figure is
of importance in the evaluation of results undertaken for toxicological purposes.
Whilst lead was present in all the foetal tissues examined, the amounts present,
which were of the same order as that in normal blood, clo not indicate any
selective absorption of this clement in the foetus. T$y contrast, copper was
concentrated in the foetal livers.
In any investigation of the excretion of lead, the important figures are those
for the amount excreted over a given period of time. For this reason we have
given all results for urine and faeces as mg. per day. In the case of urine the
concentration of lead varies with the volume secreted, and results expressed in
mg. per litre, which is the usual practice, may bo misleading.
The estimation of blood lead described above has many advantages. It can
be completed in 24 hours, and, as only 20 ml. of blood are required, it can be
repeated several times on the same subject, if necessary. The lead content of
the normal bloods examined showed only comparatively small variations and the
LEAD CONTENT OF TISSUES AND EXCRETA
1863
mean value of 55-y/lOO ml. agrees with that of 60-yreported by Kehoe el al. [1935]
for a group of medical students. On the other hand Litzner and Weyrauch
[1933] consider that a figure above 40 y indicates an increased lead absorption.
The method used by Litzner and Weyrauch is open to several criticisms which
have been mentioned already. The estimation of blood lead would appear to
be a more satisfactory method of investigation than the determination of the
excretion of lead, the significance of which is unavoidably obscured by the
presence of unabsorbcd lead in the faeces, where the greater excretion is to be
expected. This is illustrated by the case of plumbism reported here.
Lead is normally considered to be an accidental body constituent, and the
term “ normal” has been decried on these grounds. When the general occurrence
of lead in foodstuff's and its presence in the tissues generally are taken into
account it seems more reasonable to describe lead as a normal constituent of
the human body.
With the information at present available the results of analyses of tissues,
blood and excreta only justify a statement as to whether normal or abnormal
amounts of lead are present. The final diagnosis of lead poisoning is in the
province of the clinician, who can correlate the clinical, haematologieal and
chemical findings.
Sum m ary.
1. Air accurate method has been described for the estimation of lead in
human tissues, blood and excreta. After ashing, the lead was extracted with
ether as a complex with sodium diethyldithiocarbamate. The lead in the other
extract, after destruction of the organic material, was determined colorimetri­
cally with diphenylthiocarbazone.
2. Lead was found in all the tissues examined. The mean concentrations in
mg. Pb per kg. for adults wore: liver— 1-73, kidney— 1-34, spleen— 1-68, brain—
0-5, rib—8-55, vertebra— 7-09. Tissues from a case of known exposure to lead
showed higher figures, more especially the rib, with 119 mg. per kg.
3. Analysis of four foetuses of 7-8 months’ gestation gave mean concentra­
tions in mg. per kg. of: liver—0*68, kidney—0*63, brain—0-17, femur— 1*73.
Lead was found in all those tissues in each case.
4. Figures for tlie excretion of lead in urine and faeces by normal laboratory
workers and hospital patients arc given. The mean daily excretion of lead by
10 patients was 0*05 mg. for urine and 0*22 mg. for faeces.
5. The analysis of 25 samples of blood obtained from 3 normals, and 18
patients, none of whom were acutely ill, gave values of 40-70y per 100 ml.,
with a mean value of 55 y.
6 . A case of plumbism in a solder-maker showing very high concentrations
of blood lead is reported.
Our thanks are due to Dr Muir Crawford for permission to use the clinical
notes on one of his cases, to Dr J. C. Middleton and other members of the staff
of the Royal Infirmary for specimens from the wards and also to Dr Dugald
Baird for arranging the supply of foetal material.
Part of the cost of this investigation was defrayed from a grant from the
Rankin Fund of tlie University of Glasgow.
1864
S. L. T O M P S E T T A N D A. B. A N D E R S O N
REFERENCES.
Allport and Skrimsliire (1932, I). A n a ly s t, 57, 440.
-------------- (1932, 2). Q uart. J . I 3harm . Pharm acol. 5, 461.
Aub, Fairhall, Minot and Rcznikofl: (1926). Lead poisoning, p. 56.
Barth (1931). Virchoto’s A rc h . 281, 146.
Boyd and De (1933). In d . J . M ed. Research, 20, 789.
Cooksey and Walton (1929). A n a ly s t , 54, 97.
Fairlmll (1924). J . B io l. C hon. 60, 485.
Fischer and Lcopoldi (1934). Z . angew. Chem, 47, 90.
Francis, Harvey and Buchan (1929). A n a ly s t , 54, 725.
Kehoe, Edgar, Tliamann and Saunders (1926). J . A m c r. M ed. Assoc. 87, 2081.
Tliamann and Cholak (1933, 1). J . In d u s t. Ily g . 15, 273.
---------------------- (1933, 2). J . In d u s l. Ily g . 15, 301.
---------------------- (1935). J . A m e r. M ed. Assoc. 104, 90.
Litzner and Weyrauch (1932). A rc h . Qewerbepath. Gewerbehyg. 4, 74.
■
-------------- (1933). M ed. K lin . 29, 381.
Lynch, Slater and Osier (1934). A n a ly s t, 59, 7S7.
Mcillere (1903). Compt. Rend. Soc. B io l. 55, 517.
Sheldon and Ramage (1931). Biochem. J . 25, 1608.
Tompsett (1935). Biochem. J . 29, 480.
Weyrauch and Muller (1933). Z . Ily g . In jc c tio n s k ra n k . 115, 216.
[F
rom
THE BIOCHEMICAL JOURNAL, V o l. XXIX, No. 2, pp. 480-480, 1935]
[ A l l R ights reserved )
P R I N T E D I N G R E A T B R IT A IN
LV. THE COPPER AND “INORGANIC” IRON
CONTENTS OF HUMAN TISSU ES.
B y
SID N E Y LIONEL TOMPSETT.
From the Biochemical Laboratory of the Institute of Pathology of the
R oyal Infirm ary and U niversity of Glasgow.
{Received December 27th, 1934.)
The present paper is concerned with the concentration of copper and “ inorganic ”
iron in various tissues and bones of the human body and also with suitable
methods for their determination.
Copper.
Several writers have determined the distribution of copper in human tissues.
Herkel [1930] obtained values of 2*88-12*9 mg, Cu per kg. fresh liver and ranged
in descending order of concentration his results may be summarised as follow s:
liver, kidney, spleen, pancreas and bone. Cunningham [1931] found that the
average concentration of copper in three human livers was 24*9 mg. per kg. of
dry tissue and ranged in descending order of concentration his results m ay be
summarised as follows: liver, kidney, brain, pancreas and spleen. Others have
published analyses of single organs, especially the liver, which agree more or
less with the analyses of the above two writers [Schonheimer and Oshima, 1929;
Kleinmann and Klinke, 1930; Cherbuliez and Ansbacher, 1930; Gordon and
Rabinowitch, 1933].
The majority of distribution studies such as these m ust almost necessarily
be done with pathological subjects. In certain pathological conditions of the liver
in the adult, there appears to be a very marked mcrease in the concentration
of copper. This occurs often in both pigmented and non-pigmented cirrhosis of
the liver [Schonheimer and Oshima, 1929; Cherbuliez and Ansbacher, 1930;
Kleinmami and Klinke, 1930; Herkel, 1930]. Gordon and Rabinowitch [1933]
in one ease of yellow^ atrophy of the liver obtained the extraordinarily high value
of 179*3 mg. copper per kg. fresh tissue. Herkel [1930] states that in haemochromatosis the concentration of copper is increased hr all the organs, with the
exception of the kidney and bone. The copper content of foetal organs appears
also to be higher than in adults [Kleinmann and Klinke, 1930; Cunningham, 1931;
Sheldon and Ramage, 1931], Sheldon and Ramage [1931] report that five foetal
livers examined by them contained 5-7 times the concentration of copper present
in adult organs. In a survey such as is intended here, it will be necessary then to
exclude foetal organs and organs from cases of liver disease.
The question of bone seems to have been neglected. Of the above-men­
tioned writers only Herkel [1930] and Sheldon and Ramage [1931] appear to
have examined this material. The former, as a result of two analyses, obtained
values of 3*7 and 4*03 mg. per kg. fresh material. Sheldon and Ramage state
that bone contains only a trace of copper.
The various colorimetric methods for the determination of copper in bio­
logical material have been discussed elsewhere [Tompsett, 1934, 2]. W ith the
exception of Sheldon and Ramage, who used a spectrographic method, the abovementioned analyses were obtained by modifications of the Biazzo method. The
writer has found the method of Callan and Henderson [1929] as modified by
( 480 )
Cu A N D
“I N O R G A N I C ” Fe C O N T E N T S O F T I S S U E S
481
McFarlane [1932] to be the most sensitive and accurate. In this method sodium
diethyldithiocarbamate is added to the solution under test, a yellow complex
with copper is formed and this is extracted by shaking with amyl alcohol. The
extract is compared in a colorimeter with a standard prepared similarly. This
reaction, although independent of p u> is carried out in alkaline solution in the
presence of pyrophosphate to prevent interference by iron.
Methods.
Tissues. The following method is based on that used for blood and reported
previously [Tompsett, 1934, 2].
A porcelain pestle and mortar and some broken glass were treated several
times with hot dilute hydrochloric acid to render them copper-free. The tissue
to be examined was cut up into small pieces and 10 g. ground up with the
broken glass. About 40 ml. of 10 % trichloroacetic acid were added and the
grinding continued. The supernatant fluid was filtered through an acid-washed
filter-paper and the residue washed with 10 % trichloroacetic acid until the
requisite volume of filtrate was obtained. Extracts of liver tissue were made
up to 100 ml., while extracts of other organs were made up to 50 ml. The final
estimation was made directly, using 20 ml. of extract with a standard con­
taining 0*01 mg. copper as described for blood.
The results obtained by this method were compared with those obtained
after ashing either with sulphuric and perchloric acids or by ignition in a silica
basin. For the ignition method the tissues (10-20 g.) were mixed with 5 g. of
copper-free sodium phosphate for reasons that have been stated in a previous
paper in connection with the analyses o f samples of diet [Tompsett, 1934, 4].
From the figures shown in Table I it will be seen that the copper contents
of tissues as determined by the two methods are the same within the lim its of
experimental error, that is copper may be extracted completely from tissues by
trichloi’oacetic acid and reacts directly with sodium diethyldithiocarbamate.
Table I. Copper content of fresh tissue.
{mg. Cu per 1000 g.)
Tissue
Liver
1
2
3
4
5
Kidney
6
7
8
Brain
9
10
11
12
Spleen
13
14
Pancreas 15
16
17
A
Determined directly
in trichloroacetic
acid extract
22-24
6-20
5-69
4-94
5-26
3-33
3-63
3-01
6-96
2-27
4-57
1-40
1-16
2-27
4-00
1-96
2-22
B
Determined after
ashing tissue
22-80
6-16
5-74
5-06
5-10
3-62
3-45
2-84
7-14
2-20
4-61
1-38
M6
2*18
3-79
2-06
2-20
482
S
.L
. TOMPSETT
Bona. Bone offers special difficulties in the determination of copper, owing
to its high content of calcium phosphate. Since the final determination is carried
out at alkaline reaction, precipitation of calcium phosphate would be so great
as to interfere with the result. The same difficulties are m et with in milk, owing
to its relatively high concentration of calcium phosphate and low concentration
of copper. In the case of faeces, urine and samples of diet, materials containing
relatively much lower concentrations of calcium phosphate, copper m ay be de­
termined directly in solutions of their ash by addition of sodium citrate to
prevent precipitation of the phosphates [Tompsett, 1934, 4], With hone or milk,
however, there is no alternative but to make a prehminary separation of copper.
Many authors have separated copper as the sulphide but this is time-con­
suming and liable to lead to difficulties. The following method has been found
to give accurate and rapid separations of copper.
The sample of bone (30-40 g.) was ashed in a silica basin. The final traces
of carbon were destroyed by the addition of 10 ml. concentrated nitric acid and
further heating. The ash was dissolved in distilled water containing 15 ml. of
concentrated hydrochloric acid filtered through an acid-washed filter-paper
and diluted to 250 ml. A sample of 50 ml. was measured into a 750 ml. sepa­
rating funnel and diluted to about 400 ml. with distilled water. To the solution
100 ml. of 20 % sodium citrate were added and the whole made slightly alkaline
to litmus by the addition of ammonia. After the addition of 5 ml. of 2 %
aqueous sodium diethyldithiocarbamate, the yellow copper complex which was
formed was extracted with ether. 25 ml. of ether were added and the whole
vigorously shaken. A further 25 ml. of ether were added and the vigorous
shaking repeated. The ether layer containing the copper complex separated
very quickly. The aqueous layer was run off and the ether washed twice with
about 50 ml. of distilled water. The ether extract was then run off into a 300 ml.
Kjeldahl flask and the separating funnel rinsed with 25 ml. of ether which was
transferred to the Kjeldahl flask. The aqueous layer and washings were re­
extracted with 25 ml. ether which was washed and added to the other ether
extracts. The ether was evaporated off on a steam-bath and the organic matter
destroyed by heating with 1 ml. concentrated sulphuric acid and 1 ml. of per­
chloric acid. The contents of the flask were diluted to 25 ml. with distilled water
and the copper content determined in an aliquot (about 5 ml.) using 0-01 mg. Cu
as standard.
Erom the results shown in Table II it will be seen that copper added to
solutions of bone ash could be recovered quantitatively by this technique.
Table II. The recovery of copper added to an acid solution of bone ash.
1
2
3
4
5
6
Initial copper
content
mg.
0-046
0-046
0*046
0-064
0*064
0-064
Copper
added
mg.
0-050
■ 0-075
0-100
0-050
0-075
0-100
Total copper
content
mg.
0-095
0*118
0*152
0*111
0-144
0-157
Copper
recovered
mg.
0-049
0*072
0-106
0-047
0-080
0*093
Table III gives the copper content of cows’ milk as determined by the above
procedure. No special precautions were taken in the collection of this miHr
which was unpasteurised and delivered in iron cans. These figures agree very
closely with those obtained in America by Lindow et al. [1929] who obtained
an average value of 0*15 mg. Gu per litre.
Cu A N D
“I N O R G A N I C ” Fe C O N T E N T S O F T I S S U E S
483
Table III. The copper content of cows’ milk.
(mg. Cu per litre.)
1
2
0-11
0*13
3
4
0-14
0-12
5
6
0-11
0*16
7
8
0*10
0*14
Average— 0*13.
“ I norganic” iron.
No figures for the “ inorganic” iron content of human tissues appear to be
available. Copper may be extracted from biological materials with trichloro­
acetic acid but “ inorganic” iron is extracted either partially or not at all
[Tompsett, 1934, 1, 3].
It has been shown that the whole of the iron of egg yolk is in the “ inorganic ”
form, which confirms the earlier work of Hill [1931]. When a suspension of egg
yolk was treated with trichloroacetic acid, no iron could be detected in the
filtrate, but if, prior to the precipitation of the proteins, thiolacetic acid, sodium
hydrosulphite or sodium pyrophosphate were added, the whole of the iron was
present in the filtrate. This work was extended further when it was shown that
this phenomenon observed with egg yolk was due to the presence of indiffusible
phosphorus compounds, e.g. phospholipins and phosphoprotems. When solu­
tions of iron salts were added to a suspension of lecithin or solutions of caseinogen
or to milk and the protein or phospholipin precipitated with trichloroacetic
acid, no iron could be detected in the filtrate, but if thiolacetic acid, sodium
hydrosulphite or sodium pyrophosphate were added prior to the addition of
trichloroacetic acid, the iron could be estimated quantitatively in the filtrates.
On the other hand, when mixtures of ferric salts and either egg white or solutions
of edestin were treated with trichloroacetic acid, the iron could be estimated
quantitatively in the filtrates. It has been suggested that ferric but not ferrous
iron forms complexes with these indiffusible phosphorus compounds, and that
upon reduction of the ferric iron by sodium hydrosulphite or thiolacetic acid
these complexes are destroyed. The complex also appears to be unstable in the
presence of sodium pyrophosphate which is probably due to the property of
iron of forming noil-ionised compounds with pyrophosphates.
The iron was detected and determined with thiolacetic acid, which gives a
purple colour with iron salts on the addition of ammonia. The reaction is very
delicate, is quantitative and is not affected by the presence of organic substances
such as may be present in filtrates like the above. Roth ferric and ferrous
salts give the reaction since the former are reduced to the ferrous state by
thiolacetic acid.
In his investigations Hill [1931] used aa'-dipyridyl. This substance gives a
red colour with ferrous salts and no reaction with ferric salts. He found that
when egg yolk was suspended in an acid acetate buffer and aa'-dipyridyl added,
no colour developed, whereas when a reducing substance such as sodium hydro­
sulphite was added, a red colour developed. When this colour was compared
with standards, he noted that it accounted for all the iron of the yolk.
McFarlane [1934] has studied the iron of the rat’s liver. He found that
trichloroacetic acid extracts gave only faint reactions for iron with the thiocyanate reagent, whereas the “ inorganic” iron as determined by H ill’s aa'dipyridyl method was very much higher.
484
S. L. T O M P S E T T
Method,
For the complete extraction of “ inorganic ’5iron by trichloroacetic acid, the
complexes must be broken down first. For this type of extraction it was con­
sidered that sodium pyrophosphate would answer this purpose better than a
reducing agent such as thiolacetic acid or sodium hydrosulphite. The extraction
and estimation were carried out as follow s:
Some broken glass and 10 g. of finely cut tissue were ground up in a porcelain
mortar. 20 ml. of 4 % sodium pyrophosphate were added and the grinding
continued. The mixture was allowed to stand 15 minutes after which 20 ml. of
20 % trichloroacetic acid were added and the mixture ground. After a further
15 minutes the mixture was filtered through an acid-washed filter-paper and
the residue washed with 10 % trichloroacetic acid until the volume of the
extract was either 100 ml. (liver and spleen) or 50 ml. (other tissues). An aliquot
portion of the filtrate, containing no more than 0*03 mg. Fe, was diluted to 5 ml.
with distilled water and 6 drops of thiolacetic acid were added, followed by 1 ml.
of ammonia (sp. gr. 0*88). This was compared in a colorimeter with a standard
similarly prepared and containing 0*005, 0*010 or 0*020 mg. Fe.
If copper is being estimated in the same tissue as “ inorganic” iron, it may
be determined in the extract prepared for the determination of “ inorganic”
iron, so that only one extraction is necessary.
Table IV.
A. Copper.
1'he figures aro expressed in mg. Cu per kg , fresh tissue (1) and in mg. Cu per organ (2).
Liver
A
r
1
2
3
4
5
6
7
8
9
10
11
12
(i)
3-09
9-09
6-59
5-43
4-86
3-74
2-96
5-46
7-94
3-16
4-44
612
■
(2)
3-40
12-99
15-82
8-14
8-75
5-24
3-26
9-83
11-81
5-21
6-04
11-97
Kidney
^
^
(2)
(i)
2-27
0-54
2-36
0-83
3-82
1-91
3-57
1-25
0-82
2-56
2-12
0-44
2-91
3-42
2-16
2-84
3-01
A
t
(2)
—
3-18
6-40
4-42
—
—
(2)
—
(1)
—
—
—
—
—
—
—
2-81
5-44
6-54
2-84
3*65
3-56
'I
—
0-20
0*36
0*28
0-28
0*20
0*20
0*28
0*23
1-16
2-27
1-96
2-04
1-96
1-84
2-41
1*92
—
2-16
3-96
4-84
2-22
3-04
3-24
1-02
1-02
0-69
1-11
0-84
A
(
(1)
—
2-27
4-57
3-16
—
—
Spleen
Brain
r
Pan­
creas
(1)
2*10
1*96
2*20
—
2*46
2-36
2-06
2*22
2-86
2-54
2*16
2-04
Verte­
bra
(1)
2-83
1*63
2-84
2*13
4*88
3-40
1*81
2*96
3*04
2*91
1-84
4-16
Bib
(1)
6*40
32*06
8*95
47*70
10*21
3*71
4*02
9*81
21*62
14*61
9*45
8*61
B. “ Inorganic' ” iron.
The figures are expressed in mg. Fe per kg . fresh tissue (1) and in mg. Fe per organ (2).
Liver
K
<---------------
(1)
1
2
3
45
6
7
8
9
10
11
12
162-4
36-6
69-2
50-6
80-7
27-7
64*6
71-4
45-6
84*2
91-6
39*4
Kidney
\
(2)
178-6
51-2
166-0
75*9
144-3
38-8
71-1
128-5
68-4
138-9
124-6
76-4
Brain
^
t
uT
10-1
9-6
3-3
9-2
5-1
7-4
7-9
4-6
5-4
8-4
7-6
6-9
(2)
2-4
3-4
1-6
3-0
1-6
1-6
1-5
1-4
1-6
2-7
3-0
1-9
(1)
(2)
—
—
6-2
14-8
7-6
4-0
—
8-6
11-1
14-6
8-4
11-2
6-6
Spleen
A
(
8-7
20-7
10-6
5-2
—
11-2
15-5
19-7
10-8
13-4
7*3
(1)
93-6
(2)
9*4
—
—
—
—
—
169-4
100*3
96*4
164*6
84*5
126*3
116*4
84*6
—
29-6
10-0
14-5
23-0
8-9
13*9
15*1
10*2
Pan­
creas
(1)
18*2
10*8
7-0
—
4-2
13*4
8-4
6-7
8-4
9-3
8-4
7*1
Verte­
bra
(1)
128-6
123*1
142*9
134*0
133-2
167-4
146*1
126-0
111-6
136*4
154*3
121*4
Rib
(1)
103*4
147*6
119*5
111*8
114*6
161*4
151*8
111*0
109*2
146*1
138*4
109*6
Cu AND “INORGANIC”Fe CONTENTS OF TISSUES
The
copper and
“ inorganic”
485
iron contents of tissues.
In Table IV are shown the concentrations of copper and “ inorganic” iron
in the tissues of 12 cases. These cases had no organic disease of the liver. The
bones examined were the rib and the vertebra. Figures are given for the iron
content of these bones. These were determined in the solutions of their ashes
by the thiolacetic acid method as used for the tissue extracts. Although these
figures cannot be classed as “ inorganic” iron yet it is more than likely that the
greater part of the iron in bones is in the inorganic form. A precipitate of
calcium phosphate always appears during the estimation of iron in bone but
this m ay be removed by centrifuging without interfering with the results.
Discussion.
In a previous paper [1934, 2] the writer has reported that when the proteins
of blood are precipitated with trichloroacetic acid, the whole of the copper is
present in the filtrate and reacts directly with sodium diethyldithiocarbamate.
It has been shown in this paper that the copper of tissues such as liver, kidney,
brain, pancreas and spleen m ay be completely extracted with trichloroacetic
acid and the copper may be determined directly in the extracts with sodium
diethyldithiocarbamate in the same way as blood. During the progress of this
work McFarlane [1934] published a paper on similar lines. He extracted rat
livers with trichloroacetic acid, found that these extracts gave a direct reaction
for copper with sodium diethyldithiocarbamate and that direct estimations
using these extracts agreed with those obtained after ashing.
A method has been described for the extraction of copper from solutions of
bone and milk ash by means of sodium diethyldithiocarbamate and ether. This
extraction is independent of <pB so that it is as efficient in acid as in alkaline
solution. I t is carried out in alkaline solution in the presence of pyrophosphate
to prevent the extraction of iron. I f the extraction be carried out as soon as
the solution of the ash is obtained it is not necessary to add pyrophosphate
shice sufficient of this substance is formed during the ignition process. Since
the reagent itself is insoluble in ether, the ethereal extracts contain only minimum
amounts of organic material. Chloroform was tried as a solvent but it was found
that the solubility of the copper complex in this solvent is very poor, nor does
chloroform separate very easily.
The values obtained for the copper content of various tissues with the excep­
tion of bone agree with those obtained by other workers. Vertebra contains a
very low concentration of copper which is fairly constant throughout the series.
Rib on the other hand appears to contain very varied amounts of copper,
exceeding that in the fiver in many cases even when differences of water con­
centration are taken into consideration. It appears possible that the bones may
act as stores for copper as well as the fiver.
As would be expected, liver and spleen contain the highest concentrations of
“ inorganic” iron. Tissues such as brain, kidney or pancreas contain on an
average less than a tenth of the concentration of “ inorganic” iron of the liver
or spleen. The values obtained for the spleen are more constant than those for
the liver but from the size of the latter organ it must be the principal store of
“ inorganic” iron. Vertebra and rib contain fairly high concentrations of iron
and, in contrast to copper, the concentrations are almost identical in the two
types of bone.
The term “ inorganic” iron is probably incorrect since this iron will be in
some form of organic combination. It may be justified since ferric salts added
*186
S
.L
. TOMPSETT
to egg yolk etc. react in the same way and require the same methods for their
separation.
All tissues such as have been examined contain considerable amounts of
haematin iron. It would appear, however, that the whole or almost the whole
of the iron present in plasma is in the ‘’'inorganic” form. The writer [1934, 1]
has estimated the “ inorganic” iron content of normal sera and obtained values
of 0*12-0*22 mg. per 100 ml. Fowweather [1934] in a recent paper has estimated
the total iron of plasma, obtaining values of 0*06-0*18 mg. per 100 ml. He noted
that precipitation of the proteins with trichloroacetic acid removed a con­
siderable portion of the iron.
Sum m ary.
1. Copper may be determined directly with sodium diethyldithiocarbamate
in trichloroacetic extracts of liver, kidney, spleen, brain and pancreas.
2. A method has been described for the determination of copper in bone
and milk.
3. The copper and “ inorganic” iron contents of a series of tissues including
bone have been determined.
4. The values obtained for the copper contents of liver, spleen, kidney,
pancreas and brain agree with those obtained by other writers. Vertebra contains
very low concentrations of copper while the concentrations in rib are extremely
variable.
6.
Liver and spleen contain high concentrations of “ inorganic” iron whereas
kidney, brain and pancreas contain very low concentrations.
In conclusion I wish to thank Dr A. B. Anderson for his helpful criticism
and advice and the Medical Research Council for a grant towards expenses.
R EFERENCES.
Callan and Henderson (1929). A n a ly s t, 54, 650.
Cherbuliez and Ansbacher (1930). A rch . M in . M ed, 278, 365.
Cunningham (1931). Biochem. J . 25, 1267.
Fowweather (1934). Biochem. J . 28, 1160.
Gordon and Rabinowitcli (1933). A rc h . I n t . M ed. 51, 143.
Herlcel (1930). B e itr. P ath . A n a t. 85, 513.
Hill (1931). Proc, Roy. Soc. Bond, B 107, 205.
Kleinmann and IClinke (1930). A rc h . P ath . A n a t. P h ysio l. 275, 422.
Lindow, Elvehjem and Petersen (1929). J . B io l. Ghem. 82, 465.
McFnrlane (1932). Biochem. J . 26, 1022.
(1934). J . B io l. Chem. 106, 245.
Sheldon and Ramage (1931). Biochem. J . 25, 1608.
Schonheimer and Oskima (1929). Z . jd iy s io l. Chem. 180, 249.
Tompsett (1934, 1). Biochem. J . 28, 1536.
(1934, 2). Biochem. J . 28, 1544.
(1934, 3). Biochem. J . 28, 1802.
(1934, 4). Biochem. J . 28, 2088.
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