EXPERIMENTAL NUTRITION
COPPER TOXICITY, RATS AND WILSON'S DISEASE
The ability to store ionic copper in tissues such as the liver and the brain may be one of the main causes of copper accumulation in these organs of patients with Wilson's disease. In rats drinking water high in copper, the organs which can take up ionic copper store it and they are not protected from copper toxicity. On the other hand, those organs which require copper bound in ceruloplasmin for storage, do not show excess copper and thus, are protected from copper toxicity.
Key Words: copper concentration, ceruloplasmin, liver, iron
The etiology of copper deposition in tissues of patients with Wilson's disease has not been clearly established. It may be due to excessive absorption of copper from the intestine, an inability to excrete copper, or inability to convert copper to ceruloplasmin. Hypotheses remain hypotheses although each has some data to support it A search for an experimental animal which mimics Wilson's disease has led, as usual, to the rat. There is dispute, however, whether copper loading in rats is similar to Wilson's disease because consumption of great quantities of copper is not a feature of Wilson's disease. In Wilson's disease, copper levels are ten times normal or more in the liver, kidney, brain, and cornea despite normal dietary levels in food. However, only slight increases are found in spleen, lung, erythrocytes and skin, and normal or decreased concentrations are often found in other organs' In long term studies of copper loading in rats extending for two and a half years, a general parallel was found between artificial copper toxicity in the rat and the tissue copper levels demonstrated in
patients with Wilson's disease. After two years, the rats' liver contained copper a t levels 65 times normal, kidney and small bowel five times normal, large bowel three times normal, and brain two times normal. Copper content in lung, heart, bone marrow, testes, and erythrocytes remained almost normal. Other organs demonstrated only small increases in copper. The increase in copper in the small intestine occurred immediately after copper was placed in the drinking water and persisted for the next two years. The high concentration of copper in the small intestine of rats compared with normal or decreased levels in human beings with Wilson's disease, was most likely due to the increased copper content of drinking water given to the rats. The human gut is not exposed to high levels of dietary copper. Another difference noted was that copper concentration in hepatic bile of rats was extremely high compared with no increase in the bile of subjects with Wilson's disease. Rats were also different in that the copper concentration was increased in the skin and nails and found to be very high in the hair while in Wilson's disease these areas are usually normal with hair copper content decreased. NUTRITION REVIEWSIVOL 33, NO. Z/FEBRUARY 1975
51
I t was concluded that despite a normal intake o f copper by patients with Wilson's disease, that the mishandling of copper by them was similar to that noted when normal rats were fed copper in excess. The data, however, did not clearly indicate which hypothesis was correct concerning the abnormality of copper metabolism in Wilson's disease. In rats, when tracer copper in ionic form was injected intravenously, specific activity in organs was less than expected. (The expected activity was derived from data using normal rats not drinking excess copper in their water rations.) This was true even for organs that had increased copper content in the toxic rats. The highest specific activities, although s t i l l below those expected, were found in bone marrow, heart, kidney, erythrocytes, and muscle. The response was between one-half and one-third of normal. Lung, spleen, and testes were approximately one-fifth normal while brain, skin, liver, and small bowel were one-tenth of normal. Large intestine and bone were lower than one-tenth o f normal. When ceruloplasmin labeled with Cu was injected, completely different specific activities were noted. Values were normal (again, normal judged from ceruloplasmin studies in normal rats) in the brain, heart, bone marrow, spleen, and testes and about half of normal in the kidney, lung, and muscle. Liver and other organs showed low specific activities o f ceruloplasmin. The radioactive copper remained almost entirely within the ceruloplasmin molecule during the 48-hour test period. When the specific activities of ceruloplasmin were compared with ionic copper in the copperladen rats, only the liver showed less specific activity after ceruloplasmin Cu than after ionic Cu. The specific activity of ceruloplasmin was greater than ionic copper in the lung, testes, skin, bone, muscle, heart, bone marrow, brain, and spleen. It was equal t o ionic copper in erythrocytes and in kidney, small, and large intestine. Because of the well-known influence o f copper deficiency on mobilization of iron 52
NUTRITION REVIEWS/VOL. 33, NO. PIFEBROARY 1975
stores, the iron concentrations were studied in the organs of five copper-laden rats. Tissue iron levels were significantly increased in the kidney, lung, muscle, spleen, and testes and were modestly increased in all other organs except the large and small intestine. There was no obvious parallel between changes in concentration of iron and copper. Because injection of ionic copper failed to induce a normal specific activity, a defensive mechanism by which massive copper deposition is prevented may have been operative. If an organ of a r a t accepted ionic copper, then it was that organ which accumulated a significant amount of copper during copper loading. If, however, the organ ordinarily took up ceruloplasmin preferentially, then l i t t l e stable copper was accumulated. The preferred form of copper in the brain appeared t o be ionic while the liver failed to differentiate between forms of metal, ionic, or as ceruloplasmin. This failure t o differentiate the form of copper o r t h e preference f o r ionic copper appeared, therefore, t o explain the organ accumulation of copper. Copper toxicity induced in rats resulted in increased copper in those organs known t o handle ionic copper directly; the liver, kidney, small intestine, and brain (to a lesser degree). The liver removes copper from blood to synthesize ceruloplasmin and also excretes copper into bile and stores excess. The kidney excretes copper into urine and absorption of copper occurs primarily in the upper small intestine. The defense mechanism of some organs (identified as the requirement of ceruloplasmin formation before copper storage resu I ted) was imperfect because these organs did accumulate copper slowly. Organs such as lung, testes, and erythrocytes, on the other hand, which require ceruloplasmin for storage of copper, when normal levels of copper are fed, store l i t t l e if any stable copper when fed toxic levels. Treatment of Wilson's disease consists of both D-penicillamine and low copper diet therapy. Speech will deteriorate to a per-
ceptibly significant degree if a low copper diet is not followed, even though penicillamine is taken by these patients.* This correlation between a specific defect in speech and normal copper of the diet needs further study t o determine i f the copper is taken up in ionic form in speech areas of the brain. 0
1. C. A. Owen, Jr.: Similarity of Chronic Copper Toxicity in R a t s t o Copper Deposition of Wilson's Disease. Mayo Clin. Proc. 49: 368-376, 1974 2. W. R. Berry, A. E. Aronson, F. L. Darley, and N. P. Goldstein: Effects of Penicillamine Therapy and Low-Copper Diet on Dysarthia in Wilson's Disease (Hepatolenticular Degeneration). Mayo Clin. Proc. 49: 405-408, 1974
EFFECT OF (-)-HYDROXYCITRATE ON LIPOGENESIS, APPETITE AND BODY WEIGHT IN RATS A dministra tion of (-)-H ydrox ycitra te (H Cl, (a competitive inhibitor of the enzyme A TP-citrate lyase) to rats causes marked reduction in in vivo lipogenesis, food intake, body weight gain, and total body lipid. Chronic administration of HC elevates in vitro rates of lipogenesis.
Key Words: lipid, lipogenesis, hepatic, cholesterol, enzyme
(- 1- Hy dr ox yci tra t e ( HC ) the principal acid of the fruit rinds of Garcinia cambogia has been shown t o be a competitive inhibitor of ATP-citrate lyase (EC 4.1.3.81, the enzyme catalyzing the extramitochondrial cleavage of citrate t o oxaloacetate and acetyl C0A.l Inhibition of this enzymatic reaction should limit the availability of 2-carbon u n i t s f o r f a t t y acid and c hot esteroI synthesis. Indeed A. C. Sullivan and her co-workers2 using a meal-fed r a t as an animal model geared to elevated lipid synthesis, demonstrated that acute administration of HC inhibits, in a dose dependent manner, in vitro rates of lipogenesis in hepatic cell-free and slice systems, and also i n vivo rates of hepatic fatty acid and cholesterol synthesis. In two recent papers the same group of workers (A. C. Sullivan, J. G. Triscari, J. G. Hamilton, 0. N. M i l l e r , and V. R. Wheat Ie y ) i nvestigated the effects of chronic administration of HC on lipo-
genesis, weight gain, and appetite in rat^.^'^ The characteristics of inhibition of lipogenesis after the acute administration of HC were also investigated further. In vivo rates of lipogenesis were determined by the intravenous administration of Galanine and H-water as precursors of lipids The latter revealed total fatty acid and cholesterol synthesis independent of the source of carbon precursors of the acetyl groups. Earlier experiments had indicated that 14Galanine was equivalent to either l4Gpyruvate or I4C-lactate as a carbon precursor for lipogenesis. Female rats weighing 120 to 160 g were housed under controlled conditions of temperature and duration of light. They were fed a commercial diet (G-70) containing 23 percent casein and 1 percent corn oil, with glucose, cellulose, vitamins, and minerals. Feeding was done once a day between 8 to 11 A.M. (meal feeding). When HC or citrate was added, the equivalent weight of sucrose was deleted from the diet. NUTRITION REVIEWS/VOL. 33, NO. Z/FEBRUARY 1975
53
COPPER TOXICITY, RATS AND WILSON'S DISEASE
The ability to store ionic copper in tissues such as the liver and the brain may be one of the main causes of copper accumulation in these organs of patients with Wilson's disease. In rats drinking water high in copper, the organs which can take up ionic copper store it and they are not protected from copper toxicity. On the other hand, those organs which require copper bound in ceruloplasmin for storage, do not show excess copper and thus, are protected from copper toxicity.
Key Words: copper concentration, ceruloplasmin, liver, iron
The etiology of copper deposition in tissues of patients with Wilson's disease has not been clearly established. It may be due to excessive absorption of copper from the intestine, an inability to excrete copper, or inability to convert copper to ceruloplasmin. Hypotheses remain hypotheses although each has some data to support it A search for an experimental animal which mimics Wilson's disease has led, as usual, to the rat. There is dispute, however, whether copper loading in rats is similar to Wilson's disease because consumption of great quantities of copper is not a feature of Wilson's disease. In Wilson's disease, copper levels are ten times normal or more in the liver, kidney, brain, and cornea despite normal dietary levels in food. However, only slight increases are found in spleen, lung, erythrocytes and skin, and normal or decreased concentrations are often found in other organs' In long term studies of copper loading in rats extending for two and a half years, a general parallel was found between artificial copper toxicity in the rat and the tissue copper levels demonstrated in
patients with Wilson's disease. After two years, the rats' liver contained copper a t levels 65 times normal, kidney and small bowel five times normal, large bowel three times normal, and brain two times normal. Copper content in lung, heart, bone marrow, testes, and erythrocytes remained almost normal. Other organs demonstrated only small increases in copper. The increase in copper in the small intestine occurred immediately after copper was placed in the drinking water and persisted for the next two years. The high concentration of copper in the small intestine of rats compared with normal or decreased levels in human beings with Wilson's disease, was most likely due to the increased copper content of drinking water given to the rats. The human gut is not exposed to high levels of dietary copper. Another difference noted was that copper concentration in hepatic bile of rats was extremely high compared with no increase in the bile of subjects with Wilson's disease. Rats were also different in that the copper concentration was increased in the skin and nails and found to be very high in the hair while in Wilson's disease these areas are usually normal with hair copper content decreased. NUTRITION REVIEWSIVOL 33, NO. Z/FEBRUARY 1975
51
I t was concluded that despite a normal intake o f copper by patients with Wilson's disease, that the mishandling of copper by them was similar to that noted when normal rats were fed copper in excess. The data, however, did not clearly indicate which hypothesis was correct concerning the abnormality of copper metabolism in Wilson's disease. In rats, when tracer copper in ionic form was injected intravenously, specific activity in organs was less than expected. (The expected activity was derived from data using normal rats not drinking excess copper in their water rations.) This was true even for organs that had increased copper content in the toxic rats. The highest specific activities, although s t i l l below those expected, were found in bone marrow, heart, kidney, erythrocytes, and muscle. The response was between one-half and one-third of normal. Lung, spleen, and testes were approximately one-fifth normal while brain, skin, liver, and small bowel were one-tenth of normal. Large intestine and bone were lower than one-tenth o f normal. When ceruloplasmin labeled with Cu was injected, completely different specific activities were noted. Values were normal (again, normal judged from ceruloplasmin studies in normal rats) in the brain, heart, bone marrow, spleen, and testes and about half of normal in the kidney, lung, and muscle. Liver and other organs showed low specific activities o f ceruloplasmin. The radioactive copper remained almost entirely within the ceruloplasmin molecule during the 48-hour test period. When the specific activities of ceruloplasmin were compared with ionic copper in the copperladen rats, only the liver showed less specific activity after ceruloplasmin Cu than after ionic Cu. The specific activity of ceruloplasmin was greater than ionic copper in the lung, testes, skin, bone, muscle, heart, bone marrow, brain, and spleen. It was equal t o ionic copper in erythrocytes and in kidney, small, and large intestine. Because of the well-known influence o f copper deficiency on mobilization of iron 52
NUTRITION REVIEWS/VOL. 33, NO. PIFEBROARY 1975
stores, the iron concentrations were studied in the organs of five copper-laden rats. Tissue iron levels were significantly increased in the kidney, lung, muscle, spleen, and testes and were modestly increased in all other organs except the large and small intestine. There was no obvious parallel between changes in concentration of iron and copper. Because injection of ionic copper failed to induce a normal specific activity, a defensive mechanism by which massive copper deposition is prevented may have been operative. If an organ of a r a t accepted ionic copper, then it was that organ which accumulated a significant amount of copper during copper loading. If, however, the organ ordinarily took up ceruloplasmin preferentially, then l i t t l e stable copper was accumulated. The preferred form of copper in the brain appeared t o be ionic while the liver failed to differentiate between forms of metal, ionic, or as ceruloplasmin. This failure t o differentiate the form of copper o r t h e preference f o r ionic copper appeared, therefore, t o explain the organ accumulation of copper. Copper toxicity induced in rats resulted in increased copper in those organs known t o handle ionic copper directly; the liver, kidney, small intestine, and brain (to a lesser degree). The liver removes copper from blood to synthesize ceruloplasmin and also excretes copper into bile and stores excess. The kidney excretes copper into urine and absorption of copper occurs primarily in the upper small intestine. The defense mechanism of some organs (identified as the requirement of ceruloplasmin formation before copper storage resu I ted) was imperfect because these organs did accumulate copper slowly. Organs such as lung, testes, and erythrocytes, on the other hand, which require ceruloplasmin for storage of copper, when normal levels of copper are fed, store l i t t l e if any stable copper when fed toxic levels. Treatment of Wilson's disease consists of both D-penicillamine and low copper diet therapy. Speech will deteriorate to a per-
ceptibly significant degree if a low copper diet is not followed, even though penicillamine is taken by these patients.* This correlation between a specific defect in speech and normal copper of the diet needs further study t o determine i f the copper is taken up in ionic form in speech areas of the brain. 0
1. C. A. Owen, Jr.: Similarity of Chronic Copper Toxicity in R a t s t o Copper Deposition of Wilson's Disease. Mayo Clin. Proc. 49: 368-376, 1974 2. W. R. Berry, A. E. Aronson, F. L. Darley, and N. P. Goldstein: Effects of Penicillamine Therapy and Low-Copper Diet on Dysarthia in Wilson's Disease (Hepatolenticular Degeneration). Mayo Clin. Proc. 49: 405-408, 1974
EFFECT OF (-)-HYDROXYCITRATE ON LIPOGENESIS, APPETITE AND BODY WEIGHT IN RATS A dministra tion of (-)-H ydrox ycitra te (H Cl, (a competitive inhibitor of the enzyme A TP-citrate lyase) to rats causes marked reduction in in vivo lipogenesis, food intake, body weight gain, and total body lipid. Chronic administration of HC elevates in vitro rates of lipogenesis.
Key Words: lipid, lipogenesis, hepatic, cholesterol, enzyme
(- 1- Hy dr ox yci tra t e ( HC ) the principal acid of the fruit rinds of Garcinia cambogia has been shown t o be a competitive inhibitor of ATP-citrate lyase (EC 4.1.3.81, the enzyme catalyzing the extramitochondrial cleavage of citrate t o oxaloacetate and acetyl C0A.l Inhibition of this enzymatic reaction should limit the availability of 2-carbon u n i t s f o r f a t t y acid and c hot esteroI synthesis. Indeed A. C. Sullivan and her co-workers2 using a meal-fed r a t as an animal model geared to elevated lipid synthesis, demonstrated that acute administration of HC inhibits, in a dose dependent manner, in vitro rates of lipogenesis in hepatic cell-free and slice systems, and also i n vivo rates of hepatic fatty acid and cholesterol synthesis. In two recent papers the same group of workers (A. C. Sullivan, J. G. Triscari, J. G. Hamilton, 0. N. M i l l e r , and V. R. Wheat Ie y ) i nvestigated the effects of chronic administration of HC on lipo-
genesis, weight gain, and appetite in rat^.^'^ The characteristics of inhibition of lipogenesis after the acute administration of HC were also investigated further. In vivo rates of lipogenesis were determined by the intravenous administration of Galanine and H-water as precursors of lipids The latter revealed total fatty acid and cholesterol synthesis independent of the source of carbon precursors of the acetyl groups. Earlier experiments had indicated that 14Galanine was equivalent to either l4Gpyruvate or I4C-lactate as a carbon precursor for lipogenesis. Female rats weighing 120 to 160 g were housed under controlled conditions of temperature and duration of light. They were fed a commercial diet (G-70) containing 23 percent casein and 1 percent corn oil, with glucose, cellulose, vitamins, and minerals. Feeding was done once a day between 8 to 11 A.M. (meal feeding). When HC or citrate was added, the equivalent weight of sucrose was deleted from the diet. NUTRITION REVIEWS/VOL. 33, NO. Z/FEBRUARY 1975
53
