J. Cecil Smith

Determination of Individual Carotenoids in Human Plasma by High Performance Liquid Chromatography

Abstract A high performance liquid chromatography procedure using an internal carotenoid standard, echinenone, is described. The method uses a C-18, reverse-phase column, an isocratic solvent (acetonitrile:methylene chloride:methanol, 70:20:10) and requires only thirteen minutes. Most human plasmas gave seven distinct peaks; six carotenoids are identified. The sum of seven peaks is 90 percent or more of the total carotenoids in a total lipid extract.

The vitamin A-zinc connection: a review.

A general summary of typical results involving animal studies is shown in TABLE 7. A consistent and established finding is a reduction of plasma vitamin A concentration in zinc-deficient animals despite diets adequate in vitamin A. However, it appears that the depressed plasma vitamin A is not a result of zinc deficiency per se but rather is nonspecific, resulting from food- and growth-restriction factors associated with zinc deficiency. Food restriction apparently is the critical factor, since both immature and mature nongrowing zinc-deficient animals have exhibited the decreased plasma vitamin A concentration. However, this point needs clarification. Liver vitamin A concentration is usually unaltered by the zinc deficiency, suggesting no defect in absorption or transport to the liver. In regard to retinol-binding protein, it appears from the animal studies that zinc deficiency per se has an effect on both the plasma and liver RBP concentrations. We have hypothesized that zinc deficiency impairs RBP synthesis. It is speculated that only severe zinc deficiency results in a deficit of a sufficient magnitude for impairment of vitamin A metabolism at the cellular level. For example, retinene reductase, an apparent zinc-metallo alcohol dehydrogenase of the retina, appears to be sensitive to a severe zinc deficiency in animal studies. In humans, impaired dark adaptation may be a result of inadequate supplies of the metabolizable zinc necessary to maintain the activity of the enzyme system. Thus, the conversion (dehydrogenation) of vitamin A alcohol to vitamin A aldehyde is impaired, with a resulting abnormality in dark adaptation, i.e., night blindness. Indeed, the limited number of human studies suggest that zinc supplementation may be beneficial to vitamin A metabolism only in conditions where zinc deficiency is prevalent as indicated by low (less than 70 micrograms/100 ml) plasma zinc. Conversely, zinc supplementation is of little benefit in conditions where vitamin A metabolism is altered but zinc status is normal. Therefore, definitive clinical studies involving primary zinc deficiency must be conducted before final conclusions can be made regarding the interrelationships of zinc and vitamin A in health and disease.

Dietary fructose exacerbates the cardiac abnormalities of copper deficiency in rats.

Copper deficiency has been shown to result in severe cardiovascular lesions in several species of animals. The principal carbohydrate in the copper-deficient diet most often used with rats is sucrose, which is known to have adverse effects on carbohydrate and lipid metabolism and thus may contribute to cardiovascular disorders. These observations prompted experiments in which starch and fructose were substituted for sucrose in a copper-deficient diet, to see if the effects of the copper deficiency might be modified. In the hearts from rats fed copper-deficient diets with fructose or sucrose, there was marked, mostly ventricular hypertrophy, and mild to severe myocardial inflammation, degeneration, and fibrosis. Aneurysm of the left ventricle and pericarditis also were common. Hearts from the starch, copper-deficient groups were much less hypertrophic, and very few were affected by myocardial inflammation, degeneration, or fibrosis. Defects of elastin or other structures were not observed in the aortas or pulmonary or coronary arteries of any specimens.

Effect of Copper or Insulin in Diabetic Copper-Deficient Rats

Abstract The effects of copper and insulin on lipogenesis and glucose tolerance were studied using diabetic, copper-deficient rats. Diabetes was induced by intraperitoneal injection of 50 mg streptozotocin/kg body weight to rats fed a sucrose-copper deficient diet for 7 weeks. Five days later the rats were injected intraperitoneally with [14C]glucose with either saline, insulin, copper, or copper plus insulin. The disappearance of serum [14C]glucose at 30, 60, and 120 min postin-jection and the incorporation of [14C]glucose into lipid of epididymal fat 2 hr after administration were determined. The combined effect of copper and insulin significantly decreased peak blood glucose at 30 min and increased the incorporation of [14C]glucose into lipid in the epididymal fat pad when compared to either copper or insulin alone. The enhancement of glucose utilization may be due to a formation of a more stable complex which will increase insulin binding and/ or decrease its degradation.

Transferrin-iron and proinflammatory cytokines influence iron status and apical iron transport efficiency of Caco-2 intestinal cell line

Abstract Endogenous factors that regulate the absorption of dietary iron remain unknown. Differentiated cultures of Caco-2 human intestinal cells grown on membrane inserts were used to study the characteristics of transferrin-iron uptake across the basolateral surface, the effects of transferrin-iron uptake on cellular ferritin content, the transport of apical iron across the monolayer, and the influence of proinflammatory cytokines on these processes. Caco-2 cells accumulated transferrin-iron from the basolateral chamber by a temperature-dependent, saturable process that was enhanced in less differentiated cultures and attenuated by pre-exposure to high-iron medium. Exposure of Caco-2 cells to 10 μmol/L diferric transferrin for 36 hr increased cellular ferritin protein 3.4-fold and decreased the transport of apical 59 Fe to the basolateral compartment by 45%. Pretreatment of cells with a combination of interleukin-1β, interleukin-6, and tumor necrosis factor-α increased transferrin-iron uptake by 70% and cellular ferritin content by 54%. Also, cytokine treatment decreased apical iron transport across the monolayer by 40% without altering paracellular transport of mannitol. These results suggest that transferrin-iron and proinflammatory cytokines are capable of modulating the iron status and iron transport activity of intestinal epithelial cells.

Copper deficiency fails to affect vitamin A status

Abstract The effect of dietary copper (Cu) deficiency on vitamin A status as measured by serum and liver vitamin A was examined. Weanling male Sprague Dawley rats were randomly assigned by weight to one of four groups of 11–12 animals. Group 1 was fed a Cu deficient diet; Group 2, Cu supplemented, pair fed; Group 3 Cu supplemented, weight-matched to Group 1; Group 4 Cu supplemented, ad lib. All diets were supplemented with vitamin A (1,200 retinol equivalents/Kg). After six weeks, the animals were sacrificed and indices of Cu and vitamin A status measured. There was no significant difference for body weight gain or food intake among Groups 1, 2 and 3, eliminating these as variables. Copper deficiency was verified in Group 1 by significantly lowered hematocrit as well as lower serum, heart, and liver Cu concentrations. In addition, liver Cu−Zn superoxide dismutase (SOD) activity was significantly lowered, and cardiac hypertrophy was evident in the Cu deficient group. Serum and liver vitamin A concentrations did not differ significantly among all four groups. Total liver vitamin A content was not significantly different among the livers of rats fed the Cu deficient diet (Group 1), the Cu supplemented diet (pair-fed, Group 2) or Cu supplemented diet (weight-matched, Group 3). The results demonstrate that Cu deficiency does not affect vitamin A metabolism as assessed by serum and liver vitamin A concentrations, using rats as the model.

Indices for Assessing Copper Nutriture in Animals and Humans

The importance of copper in animal nutrition was first demonstrated by feeding a diet (milk) low in copper (1). Copper deficiency also developed in human infants fed diets similar to those employed for inducing copper deficiency states in experimental animals (2,3,4). Although the importance of adequate copper content of animal diets has long been recognized, only recently has the importance of adequate dietary copper for humans received renewed interest. Indeed, the recommendation of a “safe and adequate” daily dietary intake of 2–3 mg by the Food and Nutrition Board of the U.S. National Academy of Sciences in 1980 (5) has resulted in increased attention to methods for assessing copper status in both animals and humans.

Effect of prednisone on growth and zinc metabolism in rats

Abstract Growth failure and increased susceptibility to infection are common complications of prednisone administration, which may be associated with reduced zinc nutriture. To test the hypothesis that prednisone administration interferes with zinc metabolism and impairs growth, 41 male, weanling Charles River rats weighing 43–60 g were randomly assigned to four groups. Three groups of 12 rats received prednisone daily for five weeks at dosages of 0.5, 2.0 and 8.0 mg/kg body weight per day, respectively. A fourth group receiving the maximum dose of prednisone. In the fifth week, all the animals received 65 Zn at 0.5 uCi/100 g body weight by stomach tube. The retention of radioactivity was measured in a small animal whole body gamma counter. The results show that the rate of weight gain decreased in the prednisone-treated animals. In addition, while there were no significant differences in food intake between the three groups of prednisone-treated rats, the food efficiency ratio of 0.10±0.03 of the maximally-treated group was significantly lower (P 65 Zn retention in the pair-fed control animals at 24 and 216 hours was greater than 90% and 60%, respectively, as compared with the retention of 60% and 10% at 24 and 216 hours in the rats administered the highest dose (8 mg/kg/day). Prednisone treatment depressed the capacity of the liver, kidney, muscle, bone and testes to accumulate 65 Zn. Urinary excretion of zinc and nitrogen increased in proportion to the doses of prednisone used. We conclude that using growing rats, prednisone treatment impaired body weight gain and reduced food efficiency. At a high dosage the steroid decreased whole body 65 Zn retention and increased the relative dry liver weight.

EFFECT OF PREDNISONE ON GROWTH AND ZINC METABOLISM IN RATS

To test the hypothesis that prednisone administration interferes with zinc metabolism and impairs growth, 41 male, weanling. Charles River rats weighing 43-60 g were randomly assigned to four groups. Three groups of 12 rats received prednisone daily for five weeks at dosages of 0.5, 2.0 and 8.0 mg/kg per day, respectively. The control group were pair-fed with the third treatment group receiving the maximum dose of prednisone. In the fifth week, all animals received 65Zn at 0.5 uCi/100 g body weight by stomach tube. Retention was measured in whole body gamma counters. The rate of weight gain decreased in the prednisone-treated animals. While food intakes between the three groups of treated rats were no different, the food efficiency ratio of 0.10 ± 0.03 of the third group was significantly lower (P <.001) compared to the pair-fed controls (0.36 ± 0.03) as well as the other treatment groups (0.25 to 0.29). The whole body 65Zn retention in the pair-fed control animals at 24 and 216 hrs was 90% and 60%, respectively, compared to retention of 60% and 10% in the maximally treated rats. Prednisone treatment depressed the capacity of the liver, kidney, muscle, bone and testes to accumulate 65Zn. Urinary excretion of zinc and nitrogen increased in proportion to the doses of prednisone. We conclude that in growing rats, prednisone treatment impaired weight gain, reduced food efficiency and decreased 65Zn retention.