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Absorption and excretion of organic compounds of copper by sheep
J. COMP.
PATH.
1983. VOL.
93.
551
ABSORPTION AND COMPOUNDS
EXCRETION OF COPPER
OF ORGANIC BY SHEEP
BY
0. M. University
of
MAHMOUD
and E. J. H.
FORD
Department of Veterinary Clinical Studks, Liverpool, Leahurst, Neston, South Wirral Lti4 7TE,
U.K.
INTRODUCTION
Our interest in the uptake and transport of organic compounds of copper arose from reports of the toxic effect of a copper containing preparation when used to prevent the occurrence of copper deficiency and its clinical consequences in sheep. Ishmael, Howell and Treeby (1969) reported deaths after the injection of sheep with the copper calcium complex of ethylene diamine tetra-acetic acid (Cu Ca EDTA) and Ishmael, Treeby and Howell (1970) described the lesions found at necropsy. The main finding was acute liver damage in the form of centrilobular cell necrosis accompanied by haemorrhage. There was also degeneration of kidney tubules, oedema of the lungs, excess fluid in the serous cavities and epicardial and sub-endocardial haemorrhages. When housed sheep were injected with Cu Ca EDTA in amounts which contained between 3 and 5 mg copper per kg body weight, 7 out of 9 sheep died and characteristic liver lesions w&e present. Smaller doses of copper in this form caused transient liver damage which was detected by the release of liver specific enzymes into plasma. Ishmael and Gopinath ( 1972) gave intravenous injections of copper sulphate to sheep and death was accompanied by lesions indistinguishable from those caused by Cu Ca EDTA. They concluded that there was a strong indication that Cu Ca EDTA produces a form of acute copper poisoning due to the copper liberated from the complex. However, there are no reports of deaths due to the use of 2 other organic compounds of copper, the methionate complex and diethylamine copper oxyquinoline sulphate, which are also used for the prophylaxis of copper deficiency in sheep. It therefore seemed useful to make a comparison of the toxic effects of the 3 organic compounds of copper and this was done by Mahmoud and Ford ( 1981). We confirmed previous findings (Ishmael et al., 1969, 1970; Ishmael and Gopinath, 1972) on the toxicity of Cu Ca EDTA and showed that the oxyquinoline sulphonate complex killed sheep when 2 mg of copper per kg body weight was given. One mg of copper per kg body weight caused non-fatal centrilobular hepatic necrosis accompanied by the release of liver specific enzymes into plasma. On the other hand, up to 6 mg of copper per kg body weight had no deleterious effect when given as the methionine complex. In fact, Ishmael, Howell, Treeby and Bramley (1977) had injected sheep with copper methionate containing 13.5 mg of copper per kg body weight without signs of toxicity. These results suggested that although the copper part of the 002 l-9975/83/04055
I+08
$03.00/O
0
1983 Academic
Press Inc.
(London)
Limited
552
0.
M.
MAHMOUD
AND
E. J. H.
FORD
molecule was likely to be the toxic agent, the organic part of the molecule could affect the toxicity of the copper. Mahmoud and Ford (1981) reported that subcutaneous swellings due to the injection of copper methionate were larger and persisted longer than those which followed the injection of Cu Ca EDTA or of the oxyquinoline sulphonate complex. They also described a different pattern of changes in the concentration of copper in the serum of sheep after the injection of each of the three compounds. After the injection of copper methionate, serum copper concentration rose gradually during the first 5 to 10 days, maintained the increase for up to 25 days and then fell slowly to pre-injection values. The injection of the other 2 compounds produced a more rapid effect. A peak concentration was reached within a few hours, then there was a rapid fall over 12 to 24 hours, followed by a more gradual fall until pre-injection values were reached at 30 days. These results suggested that the copper in the 3 compounds was absorbed at different rates and the present paper gives information on the absorption, transport, and excretion of the copper component of each of the three compounds. MATERIALS
AND
METHODS
Six adult female Welsh Mountain sheep were placed in metabolism cages with facilities for collecting separately the total output of faeces and urine. They had free access to water and chopped hay. Two animals (Group 1) were each given a subcutaneous injection of copper methionate containing 4 mg of copper per kg body weight, 2 animals (Group 2) were each given a subcutaneous injection of Cu Ca edetate containing 2 mg of copper per kg body weight and 2 animals (Group 3) were each given a subcutaneous injection of copper oxyquinoline sulphonate containing 1 mg of copper per kg body weight. Blood samples were collected from all animals before injection, at 1, 4, 8, 12 and 24 h after injection, then every second day for 4 weeks and weekly for a further 4 weeks after which the animals were killed for the examination of copper-containing proteins in the liver. Urine, faeces or liver samples were digested with an acid mixture as described by Thompson and Blanchflower (1971) and the copper content of the digest was measured by atomic absorption spectrophotometry. Caeruloplasmin activity was measured by the method of Smith and Wright (1974) and results were expressed as change in optical density in 10 min. Electrophoresis of protein in serum collected before and at 9 and 60 days after injection was carried out on cellulose acetate strips as described by Neil (1963). After staining with amidoblack, the strips were cleared and scanned in a Chromoscan 200 densitometer (Joyce Loebl Ltd). For the separation of liver proteins, liver collected at necropsy was homogenized 0.05 N KCl. The homogenate with 2 parts of 0.01 N tris buffer (pH 8.0) containing was centrifuged at 105,000 g at 4 “C for 1 h, passed through a 3.0 pm millipore filter and 4 ml was applied to a 2.6 x 40 cm column of Sephadex G200 superfine (Pharmacia Sweden) at 4 “C. Ten millilitre fractions were extracted by passing tris buffer through the column at the rate of 9.5 ml per h and the copper content of the fractions was measured by atomic absorption spectrophotometry. The approximate molecular weight of the protein in the fractions was obtained by applying haemoglobin and cytochrome C to the column as markers. RESULTS
Clinical Findings One
sheep
of Group
3 died
24 h after
it had
been
injected
and
showed
the
ORGANIC
COPPER
IN
553
SHEEP
acute liver and kidney damage described by Mahmoud and Ford ( 198 1). Both sheep of Group 2 were dull for 2 to 3 days but neither of Group 1 sheep nor the survivor of Group 3 showed any untoward effects of the injection. Copper Concentrations Group 1. Both sheep showed the same pattern of changes. Figure 1 shows that the injection was followed by a gradual rise in the concentration of copper in whole blood and in serum and in caeruloplasmin activity in serum for about 10 days. Values remained high for a further 10 days after which there was a gradual decline to pre-injection values at between 30 and 40 days. No increase in the concentration of copper in urine or faeces was detected. -z L
0.6 (0)
-
Weeks -Hours+-
DW
*
Fig. 1. Changes in (a) the caeruloplasmin activity in serum, and (b) the concentration of copper in serum (t-0) and whole blood ( X __ X ), of one of the sheep of Group 1. The sheep was injected with copper methionate at the time indicated by the arrow.
Group 2. Both sheep of Group 2 (Fig. 2) sh owed a rapid increase in the concentration of copper in whole blood and in serum to reach a peak by 4 h. Figure 2 also shows that there was a sharp fall in the next few hours but, thereafter, the concentration remained greater than before injection and fell gradually to reach the pre-injection value between 30 and 40 days. The copper concentration in urine rose rapidly for 12 h and then fell equally rapidly to an undetectable value at 24 h. No increase in the copper content of the faeces was detected. Caeruloplasmin activity in serum followed a similar pattern to that of Group 1 sheep, i.e. it rose gradually to a plateau which was maintained up to 15 days, after which it fell gradually to pre-injection values at about 40 days. Group 3. Figure 3 shows that in the surviving Group 3 sheep the time of
554
0. -z b
2 E 0
=
M.
MAHMOUD
AND
E. J.
H.
FORD
0.6 (a)
50-
(b)
40-
& LI
8 ::
zo-
Ok+T++--f/
0
24 = =
-Weeks-Hours Fig. 2. Changes in (a) the caeruloplasmin of copper in serum (O--O) and whole was injected with copper calcium edetate
I II
I 21
I 31
I 41
I 51
61
Days --
activity (O---O) in the serum and (b) the blood ( x ~ x ), of one of the sheep of Group at the time indicated by the arrow.
concentration 2. The sheep
.C0 b s” 9
0.6-
5g -E
04-
(a)
--
-10
-5
+--Weeks-ours+ Fig. 3. Changes in (a) the caeruloplasmin copper in serum (O---O) and whole was injected with copper oxyquinoline
0
/,I ’ ’ ’ ’ ’
24
II
z
2-1
31
Days
activity (O---O) in the serum blood ( x ~ x ) of the surviving sulphonate at the time indicated
41
51
t and (b) the concentration sheep of Group 3. The by the arrow.
of sheep
ORGANIC
COPPER
IN
555
SHEEP
commencement and duration of the change in the activity of caeruloplasmin in serum was similar to that of Group 2. Figure 3 also shows that the peak which occurred in serum and whole blood copper concentration was greater than in Group 2 and the fall was not so rapid although the dose of copper was only half as great. The copper concentration in urine rose rapidly in the first 12 h after injection and then fell and became undetectable after 24 h. There was no increase in the copper content of the faeces. Serum proteins. Electrophoresis of serum collected before injection and 9 and 60 days later showed that, in the serum of Group 1 sheep, there was an increase in the amount of P-globulin at 9 days but this increase was not found at 60 days. No increase in this fraction was found in sheep of Groups 2 or 3 at any time. Liuer protein containing copper. In liver homogenates of a sheep which had not been injected with copper (liver copper content 40 ppm) there were small amounts of at least 2 copper containing proteins with molecular weights (mw) of approximately 180 x IO3 and 60 x 103. Figure 4 showed that the amount of Molecular
weight
(x IO31
i0 Volume Fig. 4. The liver (copper methionate
copper content (0-a)
content and molecular 350 ppm) of a Group and of an uninjected
of eluate
(ml)
weight of protein fractions &ted 1 sheep 60 days after the subcutaneous sheep (O-0) (liver copper content
from
homogenates of injection of copper 40 ppm).
the higher molecular weight protein was increased in the liver (350 ppm Cu) of a Group 1 sheep and the lower molecular weight protein was considerably increased. The pattern was similar but the increases were less in a Group 2 sheep and the Group 3 sheep showed a small increase in both proteins.
556
0.
M.
MAHMOUD
AND
E. J. H.
FORD
DISCUSSION
The absorption of ingested dietary copper and its distribution to the tissues has been studied by many investigators and reviewed by Evans (1973). It is known that the presence of other trace elements affects copper absorption by competing for binding sites on intestinal metallothionein. After absorption, copper is transported in portal plasma as a Cu-albumin complex. Ingested copper rapidly disappears from plasma (Jensen and Kanin, 1957) and there is an increase in the liver copper content (Osborne, Roberts and Walshe, 1963). Caeruloplasmin, a protein of molecular weight 160 x 103 which accounts for 60 to 90 per cent of plasma copper, is synthesized in the liver and is a copper donor for the tissues (Owen, 1965). Erythrocyte copper content remains constant in spite of changes in the copper status of the animal and this fraction is not involved in the transport of copper to and from the tissues (Gubler, Lahey, Cartwright and Wintrobe, 1953). Faecal copper includes that excreted in the bile and unabsorbed dietary copper. A small amount is excreted in the urine. In the present experiments the similarity between the changes in the concentration of copper in whole blood and in serum indicates that the amount of copper in the erythrocytes was unchanged. This indicates that none of the injected copper was transported in this way irrespective of differences in the rate of absorption. The time scale of the changes in the concentration of copper in serum and urine indicates that absorption from the subcutaneous site was more rapid in the sheep of Groups 2 and 3 than it was in Group 1. Further evidence of differences in the rates of transport to the liver of injected organic complexes of copper was obtained in experiments reported by Mahmoud and Ford (1981) which described the histological changes in the liver and kidneys and changes in copper and other constituents of blood and serum after the injection of organic copper compounds. In these experiments it was also found that there was no increase in the liver copper content of liver biopsies taken from sheep 48 h after the injection of copper methionate whereas there was a marked increase in the liver copper content of sheep which died 48 h after or survived the injection of either copper calcium edetate or oxyquinoline sulphonate. A more recent report (Mahmoud and Ford, 1982) showed that, in spite of this difference at 48 h after injection, the injection of all three compounds produces a marked rise in liver copper content at 1 month and that this persists for at least 6 months. The increase in circulating caeruloplasmin which occurred in all 3 groups of sheep during the period between 10 and 20 days after injection suggests that the subcutaneous injection of organic copper stimulates the synthesis of caeruloplasmin by the liver in a similar manner to that which follows the absorption of copper from the alimentary tract and that the onset and duration of the synthesis is not affected by the different rates of absorption of the injections. The increased amount of P-globulin found in the serum 9 days after injection of Group 1 sheep with copper methionate but not with the other 2 compounds suggests that the synthesis of this protein in the liver was stimulated by the slower absorption of copper from the methionate. The excess ,&globulin had
ORGANIC
COPPER
IN
SHEEP
557
disappeared at 60 days by which time absorption of copper from the injection site was likely to have ceased. It is also likely that the much greater increase in the amount of the two copper containing proteins in the liver of sheep injected with copper methionate than in those injected with the other 2 compounds was a consequence of increased synthesis stimulated by the slower absorption of the copper injected as methionate. On the basis of its molecular weight one of the 2 protein fractions may have had a high caeruloplasmin content. Our results support the view that slow absorption of copper from methionate is the reason for the low toxicity of the copper in this form and that rapid absorption followed by rapid transport to the liver and kidney is the reason why copper in the other two forms, i.e. copper calcium edetate or copper oxyquinoline sulphonate, will cause liver and kidney lesions and death within 48 h of administration. SUMMARY
When sheep are injected subcutaneously with copper calcium edetate or copper oxyquinoline sulphonate there is a rapid increase in the concentration of copper in whole blood, serum and urine within the first 24 h. When sheep are injected with copper methionate the concentration of copper in whole blood and serum rises slowly over a period of about 10 days and there is no detectable increase in urinary copper. After the injection of each of the three compounds, there was a steady increase in the caeruloplasmin activity in serum over a period of 10 to 20 days, followed by a slow fall to pre-injection activity by 40 days. There was a marked increase in the p-globulin fraction of serum 9 days after the injection of copper methionate but not after the other 2 compounds and the amounts of 2 copper containing proteins in liver were greater 60 days after the injection of copper methionate than after the injection of the other two compounds. The copper content of the 3 organic compounds is absorbed and excreted at different rates by sheep. The amounts of copper-containing protein produced in the liver also differ according to the organic component of the compound injected. The results suggest that the lower toxicity of copper injected as methionate compared with that injected as copper calcium edetate or copper oxyquinoline sulphonate is due to the slower absorption and transport of the copper to the liver and kidney. ACKNOWLEDGMENTS
We are grateful to several firms for gifts of the copper injections and to the Overseas Development Administration for a Technical Training Award to 0. M. Mahmoud whilst on study leave from the University of Khartoum. REFERENCES
Evans, G. W. (1973). Copper homeostasis
in the mammalian
system. Physiological
Rezn’ews, 53, 535470.
Gubler, C. J., Lahey, M. E., Cartwright,
G. E., and Wintrobe, M. M. (1953).
558
0.
M.
MAHMOUD
AND
E. J.
H.
FORD
Studies on copper metabolism. IX. The transportation of copper in blood. Journal of Clinical Investigation, 32, 405-414. Ishmael, J., Howell, J. McC., and Treeby, P. J. (1969). Death in ewes following the administration of copper calcium EDTA for the prevention of swayback. Veterinary Record, 85, 205-206. Ishmael, J., Treeby, P. J., and Howell, J. McC. (1970). Lesions found in ewes which have died following the administration of copper calcium EDTA for prevention of swayback. In Trace Element Metabolism in Animals, C. F. Mills, Ed., E. and S. Livingstone, Edinburgh, pp. 268270. Ishmael, J., and Gopinath, C. (1972). The effect of a single small dose of inorganic copper on the liver of sheep. Journal of Comparative Pathology, 82, 47-57. Ishmael, J., Howell, J. McC., Treeby, P. J., and Bramley, P. S. (1977). Copper methionate for parenteral copper therapy in sheep. Veterinary Record, 101, 410. Jensen, W. N., and Kanin, H. (1957). Copper transport and excretion in norma subjects and in patients with Laennec’s cirrhosis and Wilson’s disease. A study with (W4. Journal of Laboratory and Clinical Medicine, 49, 200-210. Mahmoud, 0. M., and Ford, E. J. H. (1981). Injection of sheep with organic compounds of copper. Veterinary Record, 108, 114-I 17. Mahmoud, 0. M., and Ford, E. J. H. (1982). Changes in the liver copper content of Welsh Mountain sheep after the injection of organic compounds of copper. Veterinary Record, 111, 534-535. Neil, D. W. (1963). Joyce-Loebel Review. Team Valley, Gateshead. Osborne, S. B., Roberts, C. N., and Walshe, J. M. (1963). Uptake of radioactive copper by the liver. A study of patients with Wilson’s disease and various control groups. Clinical Science, 24, 13-22. Owen, C. A. (1965). Metabolism of radioactive copper (CUDS) in the rat. American Journal of Physiology, 209, 900-904. Smith, B. S. W., and Wright, H. (1974). Improved manual and automated procedures for estimation of caeruloplasmin oxidase activity. Clinica Chimica Acta, 50, 359-366. Thompson, R. H., and Blanchflower, W. J. (1971). Wet ashing apparatus to prepare biological materials for atomic absorption spectrophotometry. Laboratory Practice, 20, 859-861. [Received for publication,
August 18th, 19821
PATH.
1983. VOL.
93.
551
ABSORPTION AND COMPOUNDS
EXCRETION OF COPPER
OF ORGANIC BY SHEEP
BY
0. M. University
of
MAHMOUD
and E. J. H.
FORD
Department of Veterinary Clinical Studks, Liverpool, Leahurst, Neston, South Wirral Lti4 7TE,
U.K.
INTRODUCTION
Our interest in the uptake and transport of organic compounds of copper arose from reports of the toxic effect of a copper containing preparation when used to prevent the occurrence of copper deficiency and its clinical consequences in sheep. Ishmael, Howell and Treeby (1969) reported deaths after the injection of sheep with the copper calcium complex of ethylene diamine tetra-acetic acid (Cu Ca EDTA) and Ishmael, Treeby and Howell (1970) described the lesions found at necropsy. The main finding was acute liver damage in the form of centrilobular cell necrosis accompanied by haemorrhage. There was also degeneration of kidney tubules, oedema of the lungs, excess fluid in the serous cavities and epicardial and sub-endocardial haemorrhages. When housed sheep were injected with Cu Ca EDTA in amounts which contained between 3 and 5 mg copper per kg body weight, 7 out of 9 sheep died and characteristic liver lesions w&e present. Smaller doses of copper in this form caused transient liver damage which was detected by the release of liver specific enzymes into plasma. Ishmael and Gopinath ( 1972) gave intravenous injections of copper sulphate to sheep and death was accompanied by lesions indistinguishable from those caused by Cu Ca EDTA. They concluded that there was a strong indication that Cu Ca EDTA produces a form of acute copper poisoning due to the copper liberated from the complex. However, there are no reports of deaths due to the use of 2 other organic compounds of copper, the methionate complex and diethylamine copper oxyquinoline sulphate, which are also used for the prophylaxis of copper deficiency in sheep. It therefore seemed useful to make a comparison of the toxic effects of the 3 organic compounds of copper and this was done by Mahmoud and Ford ( 1981). We confirmed previous findings (Ishmael et al., 1969, 1970; Ishmael and Gopinath, 1972) on the toxicity of Cu Ca EDTA and showed that the oxyquinoline sulphonate complex killed sheep when 2 mg of copper per kg body weight was given. One mg of copper per kg body weight caused non-fatal centrilobular hepatic necrosis accompanied by the release of liver specific enzymes into plasma. On the other hand, up to 6 mg of copper per kg body weight had no deleterious effect when given as the methionine complex. In fact, Ishmael, Howell, Treeby and Bramley (1977) had injected sheep with copper methionate containing 13.5 mg of copper per kg body weight without signs of toxicity. These results suggested that although the copper part of the 002 l-9975/83/04055
I+08
$03.00/O
0
1983 Academic
Press Inc.
(London)
Limited
552
0.
M.
MAHMOUD
AND
E. J. H.
FORD
molecule was likely to be the toxic agent, the organic part of the molecule could affect the toxicity of the copper. Mahmoud and Ford (1981) reported that subcutaneous swellings due to the injection of copper methionate were larger and persisted longer than those which followed the injection of Cu Ca EDTA or of the oxyquinoline sulphonate complex. They also described a different pattern of changes in the concentration of copper in the serum of sheep after the injection of each of the three compounds. After the injection of copper methionate, serum copper concentration rose gradually during the first 5 to 10 days, maintained the increase for up to 25 days and then fell slowly to pre-injection values. The injection of the other 2 compounds produced a more rapid effect. A peak concentration was reached within a few hours, then there was a rapid fall over 12 to 24 hours, followed by a more gradual fall until pre-injection values were reached at 30 days. These results suggested that the copper in the 3 compounds was absorbed at different rates and the present paper gives information on the absorption, transport, and excretion of the copper component of each of the three compounds. MATERIALS
AND
METHODS
Six adult female Welsh Mountain sheep were placed in metabolism cages with facilities for collecting separately the total output of faeces and urine. They had free access to water and chopped hay. Two animals (Group 1) were each given a subcutaneous injection of copper methionate containing 4 mg of copper per kg body weight, 2 animals (Group 2) were each given a subcutaneous injection of Cu Ca edetate containing 2 mg of copper per kg body weight and 2 animals (Group 3) were each given a subcutaneous injection of copper oxyquinoline sulphonate containing 1 mg of copper per kg body weight. Blood samples were collected from all animals before injection, at 1, 4, 8, 12 and 24 h after injection, then every second day for 4 weeks and weekly for a further 4 weeks after which the animals were killed for the examination of copper-containing proteins in the liver. Urine, faeces or liver samples were digested with an acid mixture as described by Thompson and Blanchflower (1971) and the copper content of the digest was measured by atomic absorption spectrophotometry. Caeruloplasmin activity was measured by the method of Smith and Wright (1974) and results were expressed as change in optical density in 10 min. Electrophoresis of protein in serum collected before and at 9 and 60 days after injection was carried out on cellulose acetate strips as described by Neil (1963). After staining with amidoblack, the strips were cleared and scanned in a Chromoscan 200 densitometer (Joyce Loebl Ltd). For the separation of liver proteins, liver collected at necropsy was homogenized 0.05 N KCl. The homogenate with 2 parts of 0.01 N tris buffer (pH 8.0) containing was centrifuged at 105,000 g at 4 “C for 1 h, passed through a 3.0 pm millipore filter and 4 ml was applied to a 2.6 x 40 cm column of Sephadex G200 superfine (Pharmacia Sweden) at 4 “C. Ten millilitre fractions were extracted by passing tris buffer through the column at the rate of 9.5 ml per h and the copper content of the fractions was measured by atomic absorption spectrophotometry. The approximate molecular weight of the protein in the fractions was obtained by applying haemoglobin and cytochrome C to the column as markers. RESULTS
Clinical Findings One
sheep
of Group
3 died
24 h after
it had
been
injected
and
showed
the
ORGANIC
COPPER
IN
553
SHEEP
acute liver and kidney damage described by Mahmoud and Ford ( 198 1). Both sheep of Group 2 were dull for 2 to 3 days but neither of Group 1 sheep nor the survivor of Group 3 showed any untoward effects of the injection. Copper Concentrations Group 1. Both sheep showed the same pattern of changes. Figure 1 shows that the injection was followed by a gradual rise in the concentration of copper in whole blood and in serum and in caeruloplasmin activity in serum for about 10 days. Values remained high for a further 10 days after which there was a gradual decline to pre-injection values at between 30 and 40 days. No increase in the concentration of copper in urine or faeces was detected. -z L
0.6 (0)
-
Weeks -Hours+-
DW
*
Fig. 1. Changes in (a) the caeruloplasmin activity in serum, and (b) the concentration of copper in serum (t-0) and whole blood ( X __ X ), of one of the sheep of Group 1. The sheep was injected with copper methionate at the time indicated by the arrow.
Group 2. Both sheep of Group 2 (Fig. 2) sh owed a rapid increase in the concentration of copper in whole blood and in serum to reach a peak by 4 h. Figure 2 also shows that there was a sharp fall in the next few hours but, thereafter, the concentration remained greater than before injection and fell gradually to reach the pre-injection value between 30 and 40 days. The copper concentration in urine rose rapidly for 12 h and then fell equally rapidly to an undetectable value at 24 h. No increase in the copper content of the faeces was detected. Caeruloplasmin activity in serum followed a similar pattern to that of Group 1 sheep, i.e. it rose gradually to a plateau which was maintained up to 15 days, after which it fell gradually to pre-injection values at about 40 days. Group 3. Figure 3 shows that in the surviving Group 3 sheep the time of
554
0. -z b
2 E 0
=
M.
MAHMOUD
AND
E. J.
H.
FORD
0.6 (a)
50-
(b)
40-
& LI
8 ::
zo-
Ok+T++--f/
0
24 = =
-Weeks-Hours Fig. 2. Changes in (a) the caeruloplasmin of copper in serum (O--O) and whole was injected with copper calcium edetate
I II
I 21
I 31
I 41
I 51
61
Days --
activity (O---O) in the serum and (b) the blood ( x ~ x ), of one of the sheep of Group at the time indicated by the arrow.
concentration 2. The sheep
.C0 b s” 9
0.6-
5g -E
04-
(a)
--
-10
-5
+--Weeks-ours+ Fig. 3. Changes in (a) the caeruloplasmin copper in serum (O---O) and whole was injected with copper oxyquinoline
0
/,I ’ ’ ’ ’ ’
24
II
z
2-1
31
Days
activity (O---O) in the serum blood ( x ~ x ) of the surviving sulphonate at the time indicated
41
51
t and (b) the concentration sheep of Group 3. The by the arrow.
of sheep
ORGANIC
COPPER
IN
555
SHEEP
commencement and duration of the change in the activity of caeruloplasmin in serum was similar to that of Group 2. Figure 3 also shows that the peak which occurred in serum and whole blood copper concentration was greater than in Group 2 and the fall was not so rapid although the dose of copper was only half as great. The copper concentration in urine rose rapidly in the first 12 h after injection and then fell and became undetectable after 24 h. There was no increase in the copper content of the faeces. Serum proteins. Electrophoresis of serum collected before injection and 9 and 60 days later showed that, in the serum of Group 1 sheep, there was an increase in the amount of P-globulin at 9 days but this increase was not found at 60 days. No increase in this fraction was found in sheep of Groups 2 or 3 at any time. Liuer protein containing copper. In liver homogenates of a sheep which had not been injected with copper (liver copper content 40 ppm) there were small amounts of at least 2 copper containing proteins with molecular weights (mw) of approximately 180 x IO3 and 60 x 103. Figure 4 showed that the amount of Molecular
weight
(x IO31
i0 Volume Fig. 4. The liver (copper methionate
copper content (0-a)
content and molecular 350 ppm) of a Group and of an uninjected
of eluate
(ml)
weight of protein fractions &ted 1 sheep 60 days after the subcutaneous sheep (O-0) (liver copper content
from
homogenates of injection of copper 40 ppm).
the higher molecular weight protein was increased in the liver (350 ppm Cu) of a Group 1 sheep and the lower molecular weight protein was considerably increased. The pattern was similar but the increases were less in a Group 2 sheep and the Group 3 sheep showed a small increase in both proteins.
556
0.
M.
MAHMOUD
AND
E. J. H.
FORD
DISCUSSION
The absorption of ingested dietary copper and its distribution to the tissues has been studied by many investigators and reviewed by Evans (1973). It is known that the presence of other trace elements affects copper absorption by competing for binding sites on intestinal metallothionein. After absorption, copper is transported in portal plasma as a Cu-albumin complex. Ingested copper rapidly disappears from plasma (Jensen and Kanin, 1957) and there is an increase in the liver copper content (Osborne, Roberts and Walshe, 1963). Caeruloplasmin, a protein of molecular weight 160 x 103 which accounts for 60 to 90 per cent of plasma copper, is synthesized in the liver and is a copper donor for the tissues (Owen, 1965). Erythrocyte copper content remains constant in spite of changes in the copper status of the animal and this fraction is not involved in the transport of copper to and from the tissues (Gubler, Lahey, Cartwright and Wintrobe, 1953). Faecal copper includes that excreted in the bile and unabsorbed dietary copper. A small amount is excreted in the urine. In the present experiments the similarity between the changes in the concentration of copper in whole blood and in serum indicates that the amount of copper in the erythrocytes was unchanged. This indicates that none of the injected copper was transported in this way irrespective of differences in the rate of absorption. The time scale of the changes in the concentration of copper in serum and urine indicates that absorption from the subcutaneous site was more rapid in the sheep of Groups 2 and 3 than it was in Group 1. Further evidence of differences in the rates of transport to the liver of injected organic complexes of copper was obtained in experiments reported by Mahmoud and Ford (1981) which described the histological changes in the liver and kidneys and changes in copper and other constituents of blood and serum after the injection of organic copper compounds. In these experiments it was also found that there was no increase in the liver copper content of liver biopsies taken from sheep 48 h after the injection of copper methionate whereas there was a marked increase in the liver copper content of sheep which died 48 h after or survived the injection of either copper calcium edetate or oxyquinoline sulphonate. A more recent report (Mahmoud and Ford, 1982) showed that, in spite of this difference at 48 h after injection, the injection of all three compounds produces a marked rise in liver copper content at 1 month and that this persists for at least 6 months. The increase in circulating caeruloplasmin which occurred in all 3 groups of sheep during the period between 10 and 20 days after injection suggests that the subcutaneous injection of organic copper stimulates the synthesis of caeruloplasmin by the liver in a similar manner to that which follows the absorption of copper from the alimentary tract and that the onset and duration of the synthesis is not affected by the different rates of absorption of the injections. The increased amount of P-globulin found in the serum 9 days after injection of Group 1 sheep with copper methionate but not with the other 2 compounds suggests that the synthesis of this protein in the liver was stimulated by the slower absorption of copper from the methionate. The excess ,&globulin had
ORGANIC
COPPER
IN
SHEEP
557
disappeared at 60 days by which time absorption of copper from the injection site was likely to have ceased. It is also likely that the much greater increase in the amount of the two copper containing proteins in the liver of sheep injected with copper methionate than in those injected with the other 2 compounds was a consequence of increased synthesis stimulated by the slower absorption of the copper injected as methionate. On the basis of its molecular weight one of the 2 protein fractions may have had a high caeruloplasmin content. Our results support the view that slow absorption of copper from methionate is the reason for the low toxicity of the copper in this form and that rapid absorption followed by rapid transport to the liver and kidney is the reason why copper in the other two forms, i.e. copper calcium edetate or copper oxyquinoline sulphonate, will cause liver and kidney lesions and death within 48 h of administration. SUMMARY
When sheep are injected subcutaneously with copper calcium edetate or copper oxyquinoline sulphonate there is a rapid increase in the concentration of copper in whole blood, serum and urine within the first 24 h. When sheep are injected with copper methionate the concentration of copper in whole blood and serum rises slowly over a period of about 10 days and there is no detectable increase in urinary copper. After the injection of each of the three compounds, there was a steady increase in the caeruloplasmin activity in serum over a period of 10 to 20 days, followed by a slow fall to pre-injection activity by 40 days. There was a marked increase in the p-globulin fraction of serum 9 days after the injection of copper methionate but not after the other 2 compounds and the amounts of 2 copper containing proteins in liver were greater 60 days after the injection of copper methionate than after the injection of the other two compounds. The copper content of the 3 organic compounds is absorbed and excreted at different rates by sheep. The amounts of copper-containing protein produced in the liver also differ according to the organic component of the compound injected. The results suggest that the lower toxicity of copper injected as methionate compared with that injected as copper calcium edetate or copper oxyquinoline sulphonate is due to the slower absorption and transport of the copper to the liver and kidney. ACKNOWLEDGMENTS
We are grateful to several firms for gifts of the copper injections and to the Overseas Development Administration for a Technical Training Award to 0. M. Mahmoud whilst on study leave from the University of Khartoum. REFERENCES
Evans, G. W. (1973). Copper homeostasis
in the mammalian
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Gubler, C. J., Lahey, M. E., Cartwright,
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August 18th, 19821