Copper-deficient Rats Research Articles

Copper deficiency increases total protein and apolipoprotein A-I synthesis in the rat small intestine.

This study was designed to determine whether an enhanced intestinal synthesis of apolipoprotein (apo) A-I is associated with the hyperapolipoproteinemia observed in copper-deficient rats. Male weanling Sprague-Dawley rats were assigned to two dietary treatments, Cu deficient (0.6 ppm Cu) and Cu adequate (6.0 ppm Cu) for 6 weeks. In vivo studies were then performed after rats were injected with a flooding dose of 150 microM [3H]phenylalanine (PHE, 50 microCi/ml/100 g body wt). Three rats from each treatment were sacrificed at 5, 10, 15, 30, and 60 min postinjection. The small intestine was rapidly rinsed and frozen in liquid N2. In vitro studies were performed by labeling freshly isolated 6-cm segments from duodenum, jejunum and ileum with [3H]PHE (33 microCi/ml, 49.7 Cl/mmol) in PHE-free minimum essential medium for 7 and 14 min. In vivo and in vitro intestinal samples were sonicated, solubilized in 1% Triton X-100, and centrifuged to provide the detergent soluble fraction for the isolation of nascent apo A-I and total protein. Radioactivities associated with nascent apo A-I isolated by immunoprecipitation and SDS-PAGE, and with total protein precipitated by trichloroacetic acid, were measured to determine the influence of Cu deficiency on nascent apo A-I and total protein synthesis. In the Cu-deficient small intestine, the synthesis of total protein was measured only in the duodenum and was enhanced after 1 hr for the in vivo studies. Moreover, total protein synthesis was enhanced at both 7 and 14 min of the in vitro studies for all three small intestinal segments of the Cu-deficient rats. Apo A-I synthesis was measured only at the jejunum and was also enhanced by Cu deficiency in the in vitro studies. Thus, an increase in intestinal apo A-I synthesis may contribute to the elevated plasma apo A-I level in Cu-deficient rats.

Copper deficiency increases hepatic apolipoprotein B secretion and mRNA editing in rats

Male weanling Sprague-Dawley rats were assigned to copper-deficient (9.0 mumol Cu/kg diet) or copper-adequate (102 mumol Cu/kg diet) dietary treatments for 6 wk. Pulse-chase studies using freshly isolated rat liver parenchymal cells demonstrated that apolipoprotein B (apoB)-48 and apoB-100 syntheses were not altered, but secretion was increased twofold in hepatocytes derived from copper-deficient rats. Both plasma apoB-48 and apoB-100 levels were increased by copper deficiency, but only the apoB-48 increase was significant. Hepatic apoB mRNA editing, expressed as a ratio of apoB-48 mRNA to apoB-48 plus apoB-100 mRNA, was significantly increased from 60.8% in copper-adequate to 70.2% in copper-deficient rats. Moreover, hepatic apoB mRNA abundance was not significantly altered by copper deficiency. Thus the increased amount of nascent apoB-48 secreted into the medium as well as the enhanced apoB mRNA editing may have contributed to the differential increase in plasma apoB-48 over apoB-100 level in copper-deficient rats.

Lysyl oxidase activity, collagen cross-links and connective tissue ultrastructure in the heart of copper-deficient male rats

Copper deficiency is characterized by multiple connective tissue manifestations, such as skeletal and joint abnormalities, and vascular lesions that lead to aneurysms and aortic rupture. However, the rupture of the heart observed in cooper-deficient male rats is not fully understood. We demonstrated the effect of copper deficiency on cardiac collagen content and solubility, regional differences in cardiac lysyl oxidase activity, collagen cross-links, and on the ultrastructural morphology of collagen in the myocardium. Weaned male rats fed copper-deficient or copper-adequate diets were examined. Copper-deficient rats died prematurely of heart rupture at the apex, and those who survived exhibited heart enlargement, decreased cardiac lysyl oxidase activity with increasing heart soluble collagen content. Mature collagen cross-links, as assessed by the concentrations of pyridinoline and deoxypyridinoline, were lower both in the apex and in the right and left ventricles of copperdeficient rats as compared with copper-adequate controls. However, in copper-deficient rats, cross-links levels were significantly lower at the apex than at the left and right ventricles. In addition, severe ultrastructural abnormalities in the size and shape of endo-and epimysium collagen fibers were observed in the apex of copper-deficient rats. Transmission electron microscopy showed giant, spiralled or frayed, and spiny collagen fibers. These findings allow us to postulate that the reduced cardiac lysyl oxidase activity accompanied by altered collagen cross-links and an abnormal connective tissue ultrastructure play a significant role in the manifestation of copper deficiency in the male rat.

Spontaneously transformed rat pancreatic epithelial oval cells give rise to ductal type adenocarcinomas

Oval epithelial cells that proliferate in the pancreas of the rats maintained on a copper-deficient diet are considered as stem cells with a potential to differentiate into hepatocytes. We isolated these oval cells from a copper-deficient rat and maintained these as a cell line in our laboratory. During 13th passage, oval cells showed increased growth and cellular pleomorphism and were analyzed for spontaneous transformation by anchorage-independent growth in soft agar and tumorigenicity in nude mice. Oval cells formed large colonies in soft agar and developed tumors in nude mice after subcutaneous transplantation. The tumors, by light and electron microscopy, showed features of well differentiated ductal-type adenocarcinomas. The phenotypic properties of these tumors included expression of neutral mucins, keratin filaments, carcinoembryonic antigen and glutathione S-transferase-pi and absence of gamma-glutamyltranspeptidase. The results of this study demonstrate that spontaneously transformed oval cells can form typical ductal-type adenocarcinomas. These observations are of particular interest, since bonafide ductal adenocarcinomas have not been described in the rat pancreas before.

Responses of insulin to oral glucose and fructose loads in marginally copper-deficient rats fed starch or fructose

The purpose of this study was to assess the effects of dietary fructose either alone or in combination with marginal copper deficiency in weanling male rats exposed to their respective diets for only 2 wk. This short duration of exposure to inadequate copper intake prevents progressive morbidity brought about by increasing periods of exposure to dietary copper deprivation. Weanling male rats were fed a copper-deficient (0.6 μg Cu/g) or a copper-adequate (6.0 μg Cu/g) diet containing 62% fructose or 62% starch for 2 wk. Either an oral glucose or an oral fructose tolerance test was conducted after an overnight fast. Insulin levels were elevated by either oral glucose or oral fructose at fasting and at 30 min postload in rats fed fructose compared with those fed starch. Despite high levels of plasma, insulin blood glucose was not reduced. Marginal copper deficiency had no effect on either plasma insulin or blood glucose. Data identify fructose as the sole agent responsible for inducing adverse changes in glucose metabolism. Two weeks of fructose consumption was sufficient to produce these changes.

Lipid fluidity of triacylglycerol-rich lipoproteins isolated from copper-deficient rats

Triacylglycerol-rich lipoproteins (TGRLP) were isolated from Cu-deficient and control rats. TGRLP from Cu-deficient rats appeared more fluid than those from controls as sensed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). This high fluidity was related to a low cholesterol:phospholipid ratio and high triacylglycerol content in these lipoproteins. TGRLP from Cu-deficient rats were more susceptible to in vitro peroxidation than lipoproteins from control rats as shown by the rate of diene conjugation. The damage induced by the peroxidation resulted in a more ordered state of the lipid fraction especially in lipoproteins from Cu-deficient rats. Thus, after in vitro peroxidation, TGRLP from Cu-deficient rats were more rigid than those from controls. These results suggest that Cu deficiency induces modifications in physicochemical properties of TGRLP which could affect their metabolism.

Increased hepatic synthesis and accumulation of plasma apolipoprotein B100 in copper-deficient rats does not result from modification in apolipoprotein B mRNA editing.

Experimentally induced copper deficiency in the rat results in increased plasma apolipoprotein B100 (apo B100) concentration in association with increased hepatic apo B100 synthesis. This enhancement of apo B100 synthesis and plasma accumulation accounts for the rise of plasma low density lipoprotein in these animals. In the present study, we have investigated if the selective increase in hepatic apo B100 synthesis is accounted for by changes in apo B mRNA editing. Reverse transcription coupled with polymerase chain reaction amplification and primer extension analysis of apo B cDNA revealed no differences in apo B mRNA editing in either the liver or small intestine between control and copper-deficient rats. We speculate that the increase in apo B100 synthesis in the liver of copper-deficient rats reflects posttranslational alterations in gene expression accompanying changes in very low density lipoprotein assembly and secretion.

Aspects of Cardiomyopathy Are Exacerbated by Elevated Dietary Fat in Copper-Restricted Rats

The objective of this study was to determine if a high fat diet having a 2:1 saturated:polyunsaturated fatty acid ratio exacerbates signs of copper deficiency. Male weanling Long-Evans rats were randomly placed into one of the following treatment groups: adequate copper low fat, adequate copper high fat, deficient copper low fat or deficient copper high fat. The levels of fat used were 31 or 12% of daily energy, and copper concentrations were 94.5 µmol/kg and <15.8 µmol/kg in the copper-adequate and copper-deficient diets, respectively. Cardiac hypertrophy as well as lower liver copper levels and superoxide dismutase activity were observed in both groups of copper-deficient rats. Irrespective of copper level, consumption of the high fat diet resulted in thickening of the interventricular septum and left ventricular free wall. Electrocardiograms revealed that the copper-deficient high fat diet led to a significantly smaller QT interval compared with all other groups. Significantly greater S-wave voltage due to copper deficiency was observed. Significantly lower heart cytochrome c oxidase (CCO) activity was found in the copper-deficient groups with the copper-deficient high fat group showing the lowest activity. Western blots of the cardiac non-myofibrillar fraction demonstrated lower amounts of CCO nuclear encoded peptides in the copper-deficient groups, with the least amount seen in the copper-deficient high fat treatment. These data suggest that a high level of dietary fat exacerbates some of the signs of copper deficiency.

Auditory Startle Response is Diminished in Rats after Recovery from Perinatal Copper Deficiency

Recovery from perinatal copper deficiency was studied in female and male Sprague Dawley rats for 6 mo. Month-old offspring reared by dams on copper-deficient treatment starting d 7 of pregnancy had up to 80% reductions in regional brain copper concentrations compared with offspring from copper-supplemented dams. Liver copper concentrations and plasma ceruloplasmin diamine oxidase activities of copper-deficient rats were restored to control levels within 1 mo of nutritional repletion with dietary copper. However, brain copper concentrations, with the exception of the hypothalamus and medulla, remained lower than in controls even after 5 mo of treatment. Rats were screened for startle responses and foot splay after 1, 3 and 5 mo of repletion. Diminished auditory startle was evident in rats of both sexes at all repletion times tested, whereas tactile startle and preimpulse inhibition of tactile startle were not influenced by prior copper deficiency, suggesting auditory sensory perception abnormalities. In a separate study, postweanling male rats deprived of dietary copper for 5 wk exhibited clear signs of copper deficiency but normal acoustic startle responses and foot splay. Long-term neurochemical and behavioral abnormalities persist in rats after perinatal copper deficiency.

Blood glucose and insulin are modified by levels of dietary iron in copper-deficient rats

The present study was designed to test the hypothesis that lowering of hepatic iron in copper-deficient rats fed fructose will modify plasma insulin and glucose. One hundred ninety-two weanling male rats were randomly assigned to eight dietary groups. These diets contained either starch or fructose, adequate or inadequate copper and adequate or low iron. Rats were fed their respective diets for 2 weeks. Following an overnight fast rats were given oral glucose loads (250 mg glucose/100 g body weight). Blood was collected at fasting and at 30, 60 and 120 min post-load. Rats that had been fed the low-iron diet tolerated oral glucose load better than those fed the adequate-iron diet. These responses, however, were not due solely to decreased iron intake, but to a combination of low-iron and low copper intake and/or fructose feeding with low iron intake. The reduced levels of hepatic and pancreatic iron were accompanied by elevations of copper levels. Fructose-fed, copper-deficient rats benefited from these changes by improving insulin efficiency. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Allopurinol an inhibitor of xanthine oxidase reduces uric acid levels and modifies the signs associated with copper deficiency in rats fed fructose

This study was designed to focus on the potential stress that xanthine oxidase could produce in copper-deficient rats fed fructose. Fructose consumption results in an excess production of uric acid due to an increased degradation of nucleotides. The enzyme xanthine oxidase catalyzes the oxidation of both hypoxanthine and xanthine. During the oxidation process free radicals are generated, which in turn, induce lipid peroxidation and premature death. Allopurinol-a competitive inhibitor of xanthine oxidase-could alleviate the combined effects of fructose feeding and copper deficiency. Twenty-five male rats were fed for 4 weeks from weaning a copper-deficient or adequate diet containing fructose. Twelve rats were given a daily oral dose of 5 mg allopurinol/100 g b.wt. Two copper-deficient rats that were not treated with allopurinol died prematurely during the fourth week of the study. No mortality occurred in the group of copper-deficient rats that had been treated with allopurinol. Anemia was alleviated by allopurinol, which in turn, could be responsible for improved growth rate. Allopurinol was effective in inhibiting xanthine oxidase activity in vivo as measured by the dramatic reduction of uric acid production. Lipid peroxidation, however, was not affected by allopurinol. It is concluded that the beneficial effects of allopurinol in copper deficiency do not appear to be related to prevention of oxygen radicals, but rather, to the protection against the catabolic destruction of purines, which in turn, increases nucleotide pool.

Microvascular control mechanisms in copper deficiency

Dietary copper deficiency has long been associated with exaggerated inflammatory responses in both humans and experimental animals. However, the mechanisms for this increased response are not understood. By using the rat cremaster muscle microcirculatory model as a window to the in vivo microcirculation, we have identified and studied three functional changes occurring in the microcirculation that operate during the inflammatory response and appear to be copper-dependent. First, we documented an increased postcapillary venule leakage of protein in response to histamine released by mass cells in copper-deficient rats. This response appears to be the result of increased numbers of mast cells and thereby increased available histamine. The second microvascular change we noted in copper deficiency is that there is decreased in vivo thrombogenesis with a resulting prolonged bleeding time. Additionally, in in vitro studies platelet-endothelial cell adhesion is diminished and the platelet concentration of the adhesion molecule von Willebrand Factor (vWF) is reduced. We propose that reduced vWF-mediated platelet adhesion accounts for the depressed thrombogenesis in copper deficiency. The third difference we found is that nitric oxide-mediated arteriole vasodilation is compromised, which may lead to increased peripheral vascular resistance. This functional deficit to NO can be reversed by the addition of Cu, Zn-SOD. This suggests that it may result from increased scavenging of NO by O2− during copper deprivation. These observations demonstrate that dietary copper has several important functional roles in microvascular control mechanisms affecting inflammation, microhemostasis, and resistance to blood flow. © 1996 Wiley-Liss, Inc.

Antioxidant enzyme gene transcription in copper-deficient rat liver

Antioxidant enzymes, Cu/Zn- and Mn-superoxide dismutase, catalase, and glutathione peroxidase, constitute an important defense mechanism against cytotoxicity of reactive oxygen species. Copper is essential for the activity of Cu/Zn-superoxide dismutase. Oxidative stress, therefore, is expected in organs of rats fed copper-deficient diet due to reduced Cu/Zn-superoxide dismutase activity. Our previous studies have shown that the expression of antioxidant enzymes was altered in copper-deficient rat liver. The present report was undertaken to study further the transcription of these enzymes in liver nuclei of rats made copper-deficient for 4 weeks. While copper deficiency decreased the copper in liver by about 80%, it did not alter the copper content in liver nuclei. In spite of a 100% elevation in nuclear iron concentration, liver nuclei from copper-deficient rats showed normal appearance. The transcriptional rates for Cu/Zn-superoxide dismutase, glutathione peroxidase, and glyceraldehyde-3-phosphate dehydrogenase were not altered by dietary copper deprivation. In contrast, transcriptional rates for Mn-superoxide dismutase and β-actin were increased but that for catalase was reduced in the nuclei isolated from the copperdeficient rat liver. These results suggest that oxidative stress, resulting from copper deficiency, differentially modulates the gene transcription for the antioxidant enzymes in rat liver.

Microvascular control mechanisms in copper deficiency

Dietary copper deficiency has long been associated with exaggerated inflammatory responses in both humans and experimental animals. However, the mechanisms for this increased response are not understood. By using the rat cremaster muscle microcirculatory model as a window to the in vivo microcirculation, we have identified and studied three functional changes occurring in the microcirculation that operate during the inflammatory response and appear to be copper-dependent. First, we documented an increased postcapillary venule leakage of protein in response to histamine released by mass cells in copper-deficient rats. This response appears to be the result of increased numbers of mast cells and thereby increased available histamine. The second microvascular change we noted in copper deficiency is that there is decreased in vivo thrombogenesis with a resulting prolonged bleeding time. Additionally, in in vitro studies platelet-endothelial cell adhesion is diminished and the platelet concentration of the adhesion molecule von Willebrand Factor (vWF) is reduced. We propose that reduced vWF-mediated platelet adhesion accounts for the depressed thrombogenesis in copper deficiency. The third difference we found is that nitric oxide-mediated arteriole vasodilation is compromised, which may lead to increased peripheral vascular resistance. This functional deficit to NO can be reversed by the addition of Cu, Zn-SOD. This suggests that it may result from increased scavenging of NO by O2− during copper deprivation. These observations demonstrate that dietary copper has several important functional roles in microvascular control mechanisms affecting inflammation, microhemostasis, and resistance to blood flow.

Blood glucose and insulin are modified by levels of dietary iron in copper‐deficient rats

The present study was designed to test the hypothesis that lowering of hepatic iron in copper-deficient rats fed fructose will modify plasma insulin and glucose. One hundred ninety-two weanling male rats were randomly assigned to eight dietary groups. These diets contained either starch or fructose, adequate or inadequate copper and adequate or low iron. Rats were fed their respective diets for 2 weeks. Following an overnight fast rats were given oral glucose loads (250 mg glucose/100 g body weight). Blood was collected at fasting and at 30, 60 and 120 min post-load. Rats that had been fed the low-iron diet tolerated oral glucose load better than those fed the adequate-iron diet. These responses, however, were not due solely to decreased iron intake, but to a combination of low-iron and low copper intake and/or fructose feeding with low iron intake. The reduced levels of hepatic and pancreatic iron were accompanied by elevations of copper levels. Fructose-fed, copper-deficient rats benefited from these changes by improving insulin efficiency. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Copper deficiency increases hepatic apolipoprotein A-I synthesis and secretion but does not alter hepatic total cellular apolipoprotein A-I mRNA abundance in rats.

This study was designed to determine whether an increase in hepatic apolipoprotein A-I (apo A-I) synthesis and mRNA abundance is responsible for the enlarged plasma apo A-I pool observed in copper-deficient rats. Weanling male Sprague-Dawley rats were divided into two dietary treatments: copper-adequate (102.2 mumol Cu/kg diet) and copper-deficient (9.0 mumol Cu/kg diet). Copper deficiency resulted in a significant increase (124%) in intravascular apo A-I pool size after 6 wk of treatment. Following intraportal injection of a flooding dose of [3H]phenylalanine, in vivo hepatic apo A-I synthesis and secretion were significantly greater in the copper-deficient animals as detected by [3H]phenylalanine incorporation into immunoprecipitable apo A-I isolated from liver homogenates and plasma using anti-rat apo A-I antibodies. Pulse-chase experiments using freshly isolated hepatocytes demonstrated that a significant increase (148%) in apo A-I secretion by hepatocytes derived from copper-deficient rats may have resulted from increased hepatic synthesis rather than altered intracellular degradation of apo A-I. Hepatic total cellular apo A-I mRNA abundance was not altered by copper deficiency when expressed per microgram of RNA. Thus, the enhanced hepatic apo A-I synthesis, observed in copper-deficient cells, may have resulted from alterations in post-transcriptional and translational processes.

Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.

Anemia in copper-deficient rats: role of alterations in erythrocyte membrane fluidity and oxidative damage.

This study was designed to make precise the nature and the mechanism of the anemia induced by dietary copper (Cu) deficiency. Male Wistar rats were pair fed from weanling for 6 wk either a Cu-deficient or a control diet. The reduced red blood cell (RBC) 51Cr survival indicates an increased destruction of RBC during Cu deficiency. 1,6-Diphenyl-1,3,5-hexatriene fluorescence polarization studies revealed an increase in the fluidity of erythrocyte membranes from deficient rats. The reduced cholesterol-to-phospholipid ratio was consistent with the increased fluidity. Other results indicate an increased vulnerability of RBC to hemolysis in dilute hydrogen peroxide and an increased formation of lipid peroxidation products. Before exposure to free radical stress, electron spin resonance studies in intact RBC revealed decreased correlation time of 16-doxyl-stearic acid, confirming a more fluid membrane in RBC from Cu-deficient rats. After in vitro peroxidation, RBC from Cu-deficient rats showed a more ordered state of membrane lipids compared with controls. Together, these studies demonstrate the hemolytic nature of the anemia. The shortened survival of erythrocytes apparently results from changes in membrane fluidity and enhanced susceptibility to peroxidation.

Copper deficiency increases metallothionein-I mRNA content selectively in rat liver

A variety of inducers of metallothionein (MT) synthesis have been identified. Copper loading, in particular, has been shown to increase MT expression in the liver and other organs of rodents. In the present study, the effect of severe dietary copper deficiency on MT expression was determined. Weanling rats were fed a purified diet deficient in copper (0.4 μg/g of diet) or containing adequate copper (5.7 μg/g of diet) for 4 weeks. Mineral analysis revealed that copper concentrations were severely depressed in the liver, heart, and kidney of the animals fed the copper-deficient diet. The mRNA content for MT-I was markedly increased in the copper-deficient liver but not in the heart or kidney. The metallothionein protein concentration, however, was not elevated in either liver or heart and was decreased in the kidney of copper-deficient rats. The results demonstrate that copper deficiency enhances MT-I transcription selectively in the liver of rats. It also suggests that copper is not essential for MT transcription in rats.

Body temperature and thyroid hormone metabolism of copper-deficient rats

The hypothesis that the effects of copper deficiency on body temperature are mediated via the thyroid hormone system was examined in 24 male Sprague-Dawley rats, weighing 240 to 250 g, matched by weight and fed ad libitum diets containing adequate (78.7 μmol/kg), marginal (31.5 μmol/kg), or deficient (<7.9 μmol/kg) amounts of copper for 35 days. Anemia was not found in the copper-restricted groups. Plasma cholesterol concentrations increased (P < 0.05) and plasma copper, ceruloplasmin, and liver copper concentrations decreased (P < 0.05) in the rats fed the copper-restricted diet as compared with the copper-adequate diet. Cytochrome c oxidase and glutathione peroxidase activities in liver and brown adipose tissue were decreased (P < 0.05) in the copper-deprived rats. Body temperatures at 24 °C were less (P < 0.05) in the severely copperdeficient as compared with the moderately copper-deficient and copper-adequate animals. Plasma thyroxine concentrations were decreased (P < 0.05) in severely copper-deficient as compared with moderately copperdeficient and copper-adequate rats. Plasma tri-iodothyronine concentrations were reduced (P < 0.05) and plasma thyroid-stimulating hormone concentrations were elevated (P < 0.05) in both groups of copper-restricted rats relative to the copper-adequate rats. Thyroxine 5′-monodeiodinase activities in liver and brown adipose tissue were decreased (P < 0.05) in the copper-restricted animals. Hepatic triiodothyronine receptors were increased (P < 0.05) by dietary copper restriction. Body fatness was greater (P < 0.05) in copper-restricted as compared with copper-adequate rats. These findings indicate that copper deficiency without anemia decreases tissue copper and selenium status and is associated with impaired thyroid hormone metabolism and mild hypothermia in rats at maintained at 24 °C. (J. Nutr. Biochem. 6:445-451, 1995.)