The response of copper metabolism to dietary copper challenge was investigated in jaundiced rats with elevated plasma concentrations of conjugated bilirubin as a result of impaired canicular transport of bilirubin glucuronides. Control and jaundiced rats were fed purified diets with either normal (64 µmol Cu/kg) or high (640 µmol Cu/kg) concentration of added copper. Copper loading produced a greater increase in hepatic copper concentrations in the jaundiced than in control rats. The greater dietary-copper-induced increase in hepatic copper in the jaundiced rats can be explained by the observed smaller rise in biliary copper excretion and a greater efficiency of dietary copper absorption. In individual rats, there was a positive relationship between hepatic copper concentrations and biliary copper concentrations. It is suggested that not the transport of copper from liver cells to bile but that from plasma to bile is diminished in the jaundiced rats. The elevated plasma copper concentrations in the jaundiced rats may support this suggestion.

The effect of high arsenic intake on copper metabolism was investigated. Male rats aged 6 wk had free access to purified diets containing either 0 or 100 mg As/kg diet and demineralized water for a period of 2 wk. Arsenic was added to the diet in the form of NaAsO2. The high-arsenic diet decreased feed and water intake and body weight gain, but significantly increased liver weight. Kidney weight was not affected. Arsenic feeding drastically elevated kidney copper concentration, but significantly reduced copper concentration in plasma. Both true absorption and biliary excretion of copper were decreased significantly in rats fed the high-arsenic diet. True copper absorption was lowered essentially through the lower copper intake in the rats fed arsenic. It is speculated that arsenic feeding primarily leads to copper accumulation in the kidney, followed by a decrease in feed intake and thus in true, absolute copper absorption, a decrease in plasma copper concentration, and a decrease in biliary copper excretion.

Thyroid lesions develop in most NOD.H-2h4 mice 6 weeks after they are given 0.05% NaI in drinking water. B cells are required for spontaneous autoimmune thyroiditis (SAT) development, and anti-thyroglobulin autoantibody levels correlate with SAT severity. Immunohistochemical staining of thyroids obtained 2-10 weeks after administration of NaI water suggested that CD4+ T cells initially infiltrated the thryoid, followed by CD8+ T cells and B cells. Intrathyroidal CD4+ T cells are more numerous than CD8+ T cells. CD4+ T cells and B cells form aggregates in the thyroid, while CD8+ T cells are scattered throughout the thyroid. Intrathyroidal germinal centre-like structures could be observed in thyroid lesions with 2-3+ SAT and intrathyroidal B cells co-expressed OX40L. By RT-PCR, intrathyroidal expression of OX40L, OX40, CD40L, IL-2R, CTLA-4 and Igbeta mRNA correlated closely with the SAT severity score. These molecules were not expressed in normal thyroids. In the spleen, OX40L-positive cells were detected at 2 weeks and increased 4-6 weeks after NaI water. OX40, OX40L, CD40L, IL-2R and B7-1 as well as IFN-gamma and IL-4 mRNA were minimally expressed in normal spleens, usually began to be expressed at 2 weeks and increased to maximal level 4-8 weeks after NaI water. These results suggest that in NOD.H-2h4 mice, the OX40L, OX40, CD40L and B7 molecules, which increase in the spleen and thyroid of these mice after receiving NaI water, may play a role in SAT development, implying that one or more of these molecules might be good targets for the prevention or treatment of SAT.

The sodium requirement of adult cats for maintenance was determined using a randomized block design of eight dietary sodium treatments (0.1, 0.4, 0.5, 0.66, 0.8, 1.2, 1.6 or 2.0 g Na/kg in a casein-lactalbumin-based purified diet) administered for periods of 4 wk. A total of 35 adult specific-pathogen-free domestic shorthaired cats (26 males and 9 females, 1.5-3 y of age) was given an equilibration diet (2 g Na/kg) for 14 d before assignment (or reassignment) to the treatments. A total of 12 cats (8 males, 4 females) was randomly assigned to the lowest six levels of sodium, and four cats to the highest two sodium levels. Cats consuming the diet containing 0.1 g Na/kg had significantly elevated aldosterone concentration in plasma, and packed cell volume. In addition, these cats exhibited anorexia, body weight loss, reduced urinary specific gravity and sodium excretion, and had a negative sodium balance. However, adult cats did not develop polydypsia and polyuria reported in sodium-deficient kittens. Cats given the diet containing 0.66 g Na/kg did not have an increased packed cell volume, but aldosterone concentration in the plasma was significantly elevated. However, cats given diets containing >/=0.8 g Na/kg had plasma aldosterone concentrations </=0.7 nmol/L (reference value for sodium-replete cats) and normal packed-cell volumes. A minimal sodium requirement of adult cats for maintenance of 0.8 g Na/kg diet (energy density = 22 kJ/g diet) or 0.4 mmol Na. kg body weight-1. d-1 is proposed.

Folate is a generic descriptor referring to a group of related compounds of which folic acid is the simplest form. Folic acid is readily assimilated and converted to the active cofactors necessary for single-carbon metabolism and DNA synthesis. Folate has been shown to be an essential dietary component for growing kittens (Thenen and Rasmussen 1978). Because the folate requirement of growing kittens had not been experimentally determined, the National Research Council (1986) suggested a tentative requirement of 0.8 mg/kg diet based on data from swine. In this study, we determined the minimal folate requirement of growing kittens based on hematological parameters and formiminoglutamic acid excretion after a histidine load. Materials and methods. The experimental protocols adhered NIH guidelines (NRC 1985) and were approved by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis. Animals and housing. Specific-pathogen–free domestic short-haired kittens (initial mean body weight 1.33 6 0.03 kg) from the Feline Nutrition and Pet Care Center of the University of California, Davis were used. Kittens were housed individually in stainless steel metabolism cages (60 3 60 3 60 cm) in rooms with a controlled temperature (21 6 2°C) and light cycle (lights on: 0600–2000 h). They had free access to the experimental diets and tap water. Diets. A gelatin-based purified diet supplemented with amino acids was used as a basal diet, the diet contained all nutrients in sufficient amounts to meet the requirements of growing kittens except for folate. Experimental diets were prepared by adding various amounts of folic acid (pteroylglutamic acid, Sigma Chemical, St. Louis, MO) to the basal diet at the expense of cornstarch. The folic acid concentrations of the experimental diets were 0.1, 0.15, 0.2, 0.3, 0.6, 0.9 and 1.2 mg/kg diet, which were confirmed by a Lactobacillus casei microbiological assay (Martin et al. 1990). Experimental design. Kittens were weaned at 8 wk of age and given the basal diet and tap water until 10 wk of age (wk 0 of the experiment) when they were randomly allocated into seven groups of eight kittens (4 males and 4 females) in each group. Each group was randomized to one of the seven experimental diets and maintained on the diet for 20 wk. Food intake was measured daily and body weight weekly. Blood samples were taken at wk 0 and 20 from the jugular vein of unanesthetized kittens with potassium EDTA as an anticoagulant. Plasma was prepared from the blood samples by centrifugation at 1100 3 g for 20 min. For the measurement of formiminoglutamic acid (Figlu), urine was collected for a period of 48 h from kittens given a histidine load (0.22 g L-histidine/kg body weight) administered by gastric tube at wk 0 and 20. Urine was collected into a plastic bottle containing 1 mL of 18 mol/L sulfuric acid (Fisher Scientific, Fair Lawn, NJ). Food was withheld overnight before the oral histidine load. A broken-line method (Robbins 1986, Robbins et al. 1979) was employed to estimate the minimum folate requirement of growing kittens. Sample analysis. Hematological parameters were measured using a blood cell counter (Mascot, CDC Technologies, Oxford, CT). A microbiological assay described by Tamura (1990) with L. casei (7469, American Type Culture Collection, Rockville, MD) and the Bio-Rad Model 2550 EIA 1 Presented as part of the Waltham International Symposium on Pet Nutrition and Health in the 21st Century, Orlando, FL, May 26–29, 1997. Guest editors for the symposium publication were Ivan Burger, Waltham Centre for Pet Nutrition, Leicestershire, UK and D’Ann Finley, University of California, Davis. 2 Supported by Waltham Centre for Pet Nutrition, Leicestershire, U.K. and a grant from the Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis. 3 To whom correspondence should be addressed. 4 Composition of the basal diet (g/kg): gelatin (150 Bloom Type A, Grayslake Gelatin, Grayslake, IL), 380; amino acid mixture, 40; sucrose, 100; animal fat (Florin Tallow, Dixon, CA), 300; starch 93 (Melojel, Bridgewater, NJ); cellulose, 20; mineral mixture (Williams et al. 1987), 50; vitamin mixture, 10; choline chloride, 4.7 and taurine, 2.5. The amino acid mixture (Ajinomoto U.S.A., Raleigh, NC) was composed of (g/kg diet): L-isoleucine, 4.64; L-leucine, 7.32; L-methionine, 7.04; L-phenylalanine, 3.12; L-threonine, 3.72; L-tryptophan, 2.32; L-valine, 4.24; L-histidine, 3.20 and L-lysine z HCl, 4.40. The vitamin mixture contained (mg/kg diet) retinyl palmitate, 40; cholecalciferol, 5; dl-a-tocopheryl acetate, 320; menadione, 15; thiamin mononitrate, 25; riboflavin, 10; pyridoxine z HCl, 10; nicotinic acid, 100; calcium pantothenate, 20; myo-inositol, 200; cyanocobalamin in mannitol, 0.05; D-biotin, 1; ascorbic acid, 400 (as a preservative). 5 Abbreviations used: Figlu, formiminoglutamic acid; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.

Aldosterone concentrations in the plasma of specific-pathogen-free, domestic, short-hair cats given sodium replete diets were measured by a radio-immunoassay. Plasma samples assayed were taken from male and female cats of 8, 12, 17, 20, 26, 29, 40 and > 52 weeks of age, from adult non-pregnant queens, and from queens in the ninth weeks of pregnancy and in the second and fifth week of lactation. Plasma aldosterone concentration was also measured in samples taken four times over a 24 h period (06:00-06:30, 11:00-11:30, 17:00-17:30 and 23:00-23:30) in a group of seven adult male cats. neither age nor sex had a significant effect on the plasma aldosterone concentration. Also, queens had similar aldosterone concentrations in plasma whether they were non-pregnant, pregnant, or lactating. No circadian rhythm was observed in plasma aldosterone concentrations in adult male cats. The plasma aldosterone concentrations of 148 cats (87 males and 61 females, ranging in ages from 8 to > 52 weeks) had a positive skew distribution with a median of 161 pmol l-1. The upper value for plasma aldosterone concentration in healthy domestic cats as defined by the 95 percentile was 700 pmol l-1.

Dysfunction of T lymphocytes in aging has been causally related to a gradual loss of the thymic microenvironmental function. However, in view of the fact that T cells are generated from bone marrow-derived stem cells that settle in the thymus, we have investigated the possibility that aging effects on the bone marrow have an impact on T cell development. Our approach was based on seeding of bone marrow cells, from young and old mice, onto lymphoid-depleted fetal thymus explants, and examining the patterns of T lymphocyte development under organ culture conditions. The results indicate multifactorial effects of aging, on pre-thymic and intra-thymic developmental processes, as well as on feedback regulation by mature T cells.

The mechanism underlying the reduced Cu status in rats fed on a high-sulphide diet was investigated. Male rats aged 6 weeks were fed ad libitum on purified diets containing either 0 or 500 mg S 2- /kg and demineralized water for a period of 2 weeks. The high-sulphide diet had no effect on feed intake, body-weight gain or weight of liver and kidney bat significantly reduced Cu concentrations in plasma and kidney. Biliary Cu excretion was decreased significantly in rats fed on the high-sulphide diet. Apparent Cu absorption (Cu intake -faecal Cu) and true Cu absorption (Cu intake - (faecal Cu - biliary Cu)) were significantly lowered after sulphide feeding for 2 weeks. Rats fed on the high-sulphide diet excreted less Cu in urine than did the controls. We conclude that high sulphide intake reduces Cu status in rats through inhibition of Cu absorption which is reflected by a decrease in biliary Cu excretion as a secondary feature

The mechanism underlying the reduced Cu status in rats fed on a high-Sn diet was investigated. Male rats aged 4 weeks were fed ad lib. on purified diets containing either 1 or 100 mg Sn/kg and demineralized water for a period of 4 weeks. The high-Sn diet had no effect on feed intake, body-weight gain or weight of liver and kidney but significantly reduced Cu concentrations in plasma, liver and kidney. Biliary Cu excretion was decreased significantly in rats fed on the high-Sn diet. Apparent Cu absorption (Cu intake−faecal Cu) was not affected by the high-Sn diet, but the estimate of true Cu absorption (Cu intake−(faecal Cu−biliary Cu)) was significantly reduced. We conclude that high Sn intake reduces Cu status in rats through inhibition of Cu absorption. The decreased biliary Cu excretion observed on the high-Sn diet is a result of the reduced Cu absorption.

The metabolism of iron and copper in male Nagase analbuminaemic (NA) and Sprague Dawley (SD) rats was compared. Relative liver weight was higher and spleen weight significantly lower in NA than SD rats. In NA rats, red blood cell count, haemoglobin and haematocrit were lower, whereas plasma transferrin, total iron-binding capacity and mean corpuscular haemoglobin were higher when compared with SD rats. Iron concentrations in plasma, liver, kidneys and heart were higher, and those in the spleen and tibia were lower, in NA rats. The iron concentrations in liver and spleen were positively correlated with the amount of brown pigment as observed histopathologically. Bile flow as well as biliary iron and copper excretion were higher in NA than SD rats. Copper concentrations in liver, kidneys and plasma were higher in NA rats. Plasma levels of ceruloplasmin were about two-fold higher in NA rats. The feeding of a high-iron diet reduced kidney copper concentrations in both strains of rats, which was associated with a decrease in the absorption and biliary excretion of copper.

The feeding of diets enriched with ascorbic acid (10 g/kg) to rats has previously been shown to lower plasma and liver copper concentrations. The present studies corroborate this. We hypothesized that ascorbic acid initially reduces copper absorption, this effect being masked later by the stimulatory effect on copper absorption of the impaired copper status. We also hypothesized that the impaired copper status as induced by ascorbic acid feeding is followed by a diminished biliary excretion of copper in an attempt to preserve copper homeostasis. Our hypotheses are supported by the present studies. Ascorbic acid feeding initially reduced apparent copper absorption, and in the course of the experiment this effect tended to turn over into a stimulatory effect. Copper deficiency, as induced by feeding a diet containing 1 mg Cu/kg instead of 5 mg Cu/kg, systematically increased copper absorption. Biliary excretion of copper in rats given ascorbic acid was unaffected initially but became depressed after prolonged ascorbic acid feeding. A similar time course was seen for fecal endogenous copper excretion that was calculated as the difference between true and apparent copper absorption. Copper deficiency systematically reduced biliary copper excretion and fecal endogenous copper loss.

High intakes of Fe may impair Cu status, but the underlying mechanism is not known. Male rats, aged 7 weeks, were given purified diets adequate in Cu (8 mg Cu/kg) and containing either 7, 40 or 389 mg Fe/kg. After 6 weeks the concentrations of Fe in liver and spleen were positively related with dietary Fe level and those of Cu were negatively related with dietary Fe level. Increasing Fe intakes reduced apparent absorption and biliary excretion of Cu in a dose-dependent fashion. In individual rats, biliary Cu excretion showed a significant, positive correlation with Liver Cu concentration. It is concluded that increased Fe intakes depress Cu absorption which produces a decrease in plasma and organ Cu concentrations. As a result, biliary Cu excretion is lowered which contributes to achieving Cu balance at high Fe intakes. Because the concentrations of Cu in plasma and bile, and also plasma ceruloplasmin (EC 1.16.3.1) activities, showed much greater percentage reductions with increasing Fe intake than did the concentrations of Cu in organs, it is possible that increased Fe status interferes with the mobilization of Cu stores.

The interactions between copper, zinc, and iron intake in rats were investigated with regard to copper status. Weanling male rats were fed purified diets containing two levels of each of the three elements in a 23 factorial design. The added amounts of copper, zinc, and iron in the diets were 5, 12, and 35 mg/kg feed or were 10 times as high. After feeding on the experimental diets for 4 wk, the rats were killed and copper concentrations in plasma and organs measured. Plasma copper concentration was lowered by high zinc and iron intakes but this was seen only in the rats fed the normal-copper instead of the high-copper diets. In essence, the effects of zinc and iron were additive. Neither in rats fed the normal-copper diets nor in those fed the high-copper diets did extra iron or zinc intake alter copper concentrations in liver, spleen, kidney, and tibia.

To compare the changes in Fe metabolism during the development of vitamin A and Fe deficiencies, rats were given either a control diet with sufficient Fe (35 mg added Fe/kg feed) and retinol (1200 retinol equivalents/kg feed), a diet without added vitamin A or a diet with sufficient vitamin A but only 3.5 mg added Fe/kg feed. During a period of 10 weeks, indicators of vitamin A and Fe status were monitored. Neither vitamin A nor Fe deficiency produced clinical signs. Fe deficiency induced an immediate fall in blood haemoglobin concentration. Vitamin A deficiency produced a mild anaemia as the first change in Fe metabolism, pointing to unpaired erythropoiesis. This effect was followed by a rise in Fe absorption and an increased amount of Fe in the spleen. By the end of the study, blood haemoglobin, packed cell volume, plasma Fe and Fe content in kidney and femur had increased above control levels, while total Fe-binding capacity had decreased. We speculate that the initial anaemia was masked later by haemoconcentration. The decrease in Fe mobilization, shown by lower total Fe-binding capacity, and the increase in Fe absorption may have caused the observed continuous rise in tissue Fe concentration in rats with vitamin A deficiency. In the rats with Fe deficiency, low tissue Fe levels coincided with high Fe absorption and high total Fe-binding capacity. Thus, changes in Fe metabolism with vitamin A deficiency differed from those with Fe deficiency.

The hypothesis was tested that dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats, which in turn causes a decreased copper absorption. Male rats were fed adequate-copper (5 mg Cu/kg) diets containing either fructose or glucose (709.4 g monosaccharide/kg) for a period of 5 wk. Fructose vs glucose significantly lowered copper concentrations in plasma and the liver, but did not alter hepatic copper mass. Fructose feeding resulted in a significantly lesser intestinal solubility of copper as based on either a smaller soluble fraction of copper in the liquid phase of small intestinal contents or a lower copper concentration in the liquid phase. The latter fructose effect can be explained by the observed fructose-induced increase in volume of liquid phase of intestinal digesta. After administration of a restricted amount of diet extrinsically labeled with 64Cu, rats fed fructose also had significantly lower soluble 64Cu fraction in the digesta of the small intestine. Although this study shows that fructose lowered intestinal copper solubility, only a slight reduction of apparent copper absorption was observed. It is suggested that the fructose-induced lowering of copper status in part counteracted the fructose effect on copper absorption at the level of the intestinal lumen.

The effect of dietary iron loading on biliary iron excretion was investigated with male Wistar rats aged 6 wk. The rats were fed purified diets with either 174 or 1740 mg FeSO4. 7H2O/kg diet and demineralized water for 6 wk. Blood haemoglobin, hematocrit, and iron concentrations in kidney and heart were not affected and iron concentrations in liver, spleen, and tibia were significantly raised after feeding the high-iron diet. The high-iron diet did not raise biliary iron excretion, suggesting that biliary iron excretion does not play an important role in regulating iron metabolism in rat after dietary iron loading.