Intracellular Ca2+ and cytotoxicity.

Following injury or activation in some immune cell lines, elevation of intracellular Ca2+ concentration (Cai2+) is an early and major event that precedes cell death. Agents shown to elevate Cai2+ and to result subsequently in the death of some cells include human immunodeficiency virus (HIV) (in T4+ cells), 25-hydroxy cholesterol, tumor necrosis factor (TNF), cyclosporine, dexamethasone, alpha-interferon, and Ca2+ ionophores. The effects of these agents, both on Cai2+ and on cytotoxicity, are additive. This type of Ca2+-related cytotoxicity may be associated with either accelerated synthesis of triglycerides (TNF), accelerated synthesis of cholesterol ester (25-hydroxy cholesterol), or cholesterol (HIV) and terminally with declining synthesis of structural phospholipid. Agents that can lower Cai2+ (e.g., phorbol esters, diglycerides, lipoproteins [LDL], oleic acid, or serum) under appropriate conditions ameliorate the Ca2+-induced cytotoxicity. Metabolism of other divalent metals, i.e., Zn2+ and Cd2+, also become altered with cell injury, e.g., glucocorticoids elevate Cai2+, but block uptake of Zn2+. These observations support the idea that chronic elevation of Cai2+ by many chemically unrelated agents leads to cell death by creating imbalance both in cell biosynthetic mechanisms--especially in those controlling lipid metabolism--as well as creating imbalances in metabolism of other trace metals, especially Zn2+.

Parakeratosis of the labial mucosa in zinc-deficient rats.

Epithelial parakeratosis has been observed in zinc-deficient animals of several species. In the rat, it has been reported on the epidermis, footpad, esophagus, soft palate, buccal mucosa, and dorsal tongue. This study reports that the lower lip also shows a striking hyperparakeratosis, as well as epithelial hyperplasia and an increase in mitoses. The appearance of these features was rapid with changes noted at Day 7 and persisting through Day 14 and Day 28. On Day 32, zinc was restored to the diet at control levels and by 42 days the zinc-deficient group showed dramatic increases in food intake, body weight, femur zinc and, in addition, mucosa of the lower lip resembled zinc-adequate controls. These differences are statistically significant. Morphometric comparison of spinous cells from zinc-deficients with those of the controls did not show significant differences.

Effect of dietary zinc deficiency on protein synthesis in cell-free systems isolated from rat liver.

This study presents evidence for the direct involvement of zinc in the translation of polypeptide chains in rat liver. Cell-free systems consisting of polyribosomes and fractions, enriched with aminoacyl-tRNA synthetase for incorporation of amino acids into trichloroacetic acid-insoluble proteins, were prepared from livers of three populations of rats: 1) rats fed ad libitum a diet containing 25 ppm zinc; 2) rats fed a diet containing less than 1 ppm zinc; and 3) rats pair fed to the zinc-deficient group a diet containing 25 ppm zinc. Group 2 showed typical signs of zinc deficiency, including decreased bone zinc. The protein synthetic ability of systems isolated from zinc-deficient rats was considerably depressed, resulting in incorporation of 59-69% of the amino acids incorporated by systems from pair-fed rats and 54-59% of those incorporated by ad libitum-fed control animals. This effect was significant after only 14 d of the dietary regimen. Experiments performed by mixing polyribosomes and synthetase fractions from the different groups indicated that a defect is located in the synthetase fraction. Acylation of tRNA by the synthetase fraction also was assayed and found to be 69-90% of that in zinc-sufficient preparations. It is suggested that one or more of the aminoacyl-tRNA synthetase enzymes from the rat liver may be zinc dependent.

Zinc and the central nervous system.

The effect of zinc nutriture and metabolism on brain function has been reviewed. Zinc nutriture and its effect on the concentration and metabolism of essential elements (e.g. zinc, copper, manganese, magnesium, sodium, potassium and calcium) and on the concentration and metabolism of toxic elements (e.g. aluminum and lead) are discussed in relationship to brain function. In addition, possible interrelationships between zinc nutriture and metabolism and its effect on a number of diseases including acrodermatitis enteropathica, Pick's disease, Alzheimer's disease, schizophrenia, fifth day fits, and epilepsy are discussed. Descriptions and comparisons of methods to measure brain zinc are presented. Behavioral changes and the altered brain morphology which have been associated with zinc deficiency are reviewed. Some possible mechanisms for the association of anorexia with zinc deficiency are outlined. Perinatal brain damage produced by early zinc deficiency followed by rehabilitation with adequate zinc appears to be long term, maybe permanent. Interrelationships between zinc nutriture and aspects of neurochemistry are outlined. Some of the neurochemistries discussed include nucleic acid and protein synthesis, cytoskeletal proteins, neurotransmitters (e.g. catecholamines, indoleamines, glutamate, gamma-aminobutyric acid, and neuropeptides), neurotransmitter receptors, 7S nerve growth factor and zinc-binding proteins. Recent evidence linking zinc and neurotransmission is discussed.

Appraisal of the methodology and applications for measurement of the zinc content of blood components as indicators of zinc status.

Methods for the measurement of the zinc content of blood components (plasma, erythrocytes, platelets, mononuclear leukocytes, and polymorphonuclear neutrophils) as indicators of the zinc status of animals have been reviewed and evaluated. The values of plasma or serum zinc concentrations as indicators of zinc status in experimental animals or humans is questionable. Consequently, the zinc content of blood cellular components has been suggested as better indicators of zinc status. Methodological problems, such as incomplete cellular separation or zinc contamination, occur in some of the procedures applied to the quantitation of blood cellular component zinc. Specialized microprobe procedures involving, for example, proton-induced X-ray fluorescence, scanning ion technology, mass spectrometery, or laser microprobe mass analysis might be used to measure the absolute zinc level in individual cells subjected to minimal handling. Then, more conventional methods can be tested against the true standard. Nevertheless, there appears to be species differences in regard to the response of blood cellular zinc concentrations to dietary zinc deficiency. Blood cellular component zinc is conserved during severe zinc deficiency in the rat. In contrast, the zinc content of blood cellular components in humans may reflect the whole body zinc status.

Methionine metabolism in isolated perfused livers from rats fed on zinc-deficient and restricted diets.

1. Sulphur amino acid metabolism in livers from rats which had been fed ad lib. on a restricted diet (malnourished) or a Zn-deficient diet was investigated. Perfusion of normal livers with different amounts of L-methionine revealed that homocysteine was recycled four to five times before being eliminated via the transsulphuration pathway. 2. From the perfusion experiments, we found no evidence that any of the enzymes involved in recycling homocysteine back to methionine (methylation pathway) were adversely effected by Zn deficiency or malnutrition. 3. The intracellular concentration of cystathionine and S-adenosylmethionine increased in all livers in response to increased concentrations of L-methionine (L-Met) in the perfusate. 4. The intracellular concentration of S-adenosylhomocysteine remained the same in all livers regardless of the concentration of perfusate methionine. 5. Although homocysteine did not accumulate in the cell, it was excreted into the perfusate. The amount of homocysteine in the perfusate of livers from Zn-deficient rats was higher than either ad lib. or pair-fed rats. 6. The breakdown of homocysteine, via the transsulphuration pathway, was augmented by Zn deficiency. This was apparent from the greater amount of alpha-ketobutyrate excreted by livers from Zn-deficient rats compared with pair-fed or ad lib.-fed controls. 7. The increase in metabolism of L-Met, via the transsulphuration pathway, in the livers from Zn-deficient rats appears to reflect the lack of demand for this compound in protein synthesis and methylation reactions (Wallwork & Duerre, 1985).

Zinc content of blood cellular components and lymph node and spleen lymphocytes in severely zinc-deficient rats.

Several investigators have suggested that the measurement of leukocyte zinc may be useful for the assessment of zinc nutriture. However, in an earlier study, we found that the methods used did not adequately separate platelets from the different leukocyte populations. We subsequently developed a method which does adequately separate platelets, mononucleated cells (MNC), polymorphonucleated cells (PMN), and erythrocytes (RBC) from a single blood sample. This method was applied to a study of severe zinc deficiency in adult male rats. Significant reductions in plasma and femur zinc indicated severe zinc deficiency in the zinc-deficient animals compared with pair-fed or ad libitum-fed control rats in two separate experiments. However, we noted no differences in the zinc content of the MNC, PMN, RBC or lymph node lymphocytes, on a per cell basis, between the zinc-deficient and ad libitum- or pair-fed control groups of rats. A minor, but not significant (P greater than 0.05) reduction of platelet zinc was noted in zinc-deficient rats in two separate studies. These data indicate that the zinc contents of blood cellular components are not sensitive indicators of zinc deficiency in rats.

Effect of zinc deficiency on methionine metabolism, methylation reactions and protein synthesis in isolated perfused rat liver.

A perfusion technique was utilized to assess the rate of absorption and metabolism of L-methionine by livers isolated from rats fed a diet deficient in zinc. The endogenous concentration of S-adenosyl-L-methionine in the livers from the zinc-deficient rats was near normal, while the concentration in pair-fed controls was approximately 50% of that found in normal livers (ad libitum fed). The rate of uptake of methionine by the livers isolated from zinc-deficient rats was significantly less than in the pair-fed or ad libitum-fed controls. The synthesis of L-methionine, S-adenosyl-L-homocysteine and S-adenosylmethionine was not impaired in the livers from the zinc-deficient rats. However, the methyl group of the S-adenosylmethionine turned over much more slowly in the livers from zinc-deficient rats than in either control group. This was reflected in the depressed rates of methylation of various macromolecules, particularly DNA and histones. The synthesis of nuclear proteins (histones and nonhistone chromosomal proteins) was depressed in the livers from zinc-deficient rats. The reduced synthesis of chromosomal proteins and marked reduction in DNA methylation would be consistent with the finding that DNA biosynthesis and cellular proliferation are markedly depressed in zinc-deficient animals.

Zinc content of cellular components of blood: methods for cell separation and analysis evaluated.

Platelets, mononucleated cells, polymorphonucleated cells, and erythrocytes were separated from whole blood by use of discontinuous gradients of colloidal polyvinylpyrrolidone-coated silica ("Percoll"). We measured the zinc content of these cells by flame atomic absorption spectrophotometry, using a modified technique for micro-samples that obviated matrix interferences. Thus, results obtained by conventional flame atomic absorption and by the micro-method were identical. Inter-comparisons of separation methods indicated that separation of platelets and mononucleated cells by a two-gradient system of "Ficoll-Hypaque" (a synthetic polymer of sucrose) or Percoll was relatively poor, whereas there was a good separation when a tertiary gradient system of Percoll was used. The apparent zinc content of mononucleated cells depended on the degree of separation from the platelets, with contamination by platelets resulting in artificially high values for mononucleated cells.

Zinc-binding proteins (ligands) in brains of severely zinc-deficient rats.

Previous studies from this laboratory reported the presence of a metallothionein-like protein in brain with an apparent estimated molecular weight of 13,000-15,000 daltons. The synthesis of this protein, which incorporates large quantity of cysteine, is stimulated following administration of zinc and copper and is blocked by actinomycin D. In this study, we report that the synthesis of this metallothionein-like protein is considerably lower in brains of severely zinc-deficient rats in comparison with pair-fed orad libitum fed groups. Furthermore, incubation of partially purified metallothionein-like protein with(65)Zn and chromatography on DEAE A-25 Sephadex produced similar elution patterns in the three experimental groups. However, the extent of binding of(65)Zn to the metallothionein-like protein from the zinc-deficient rats was significantly (p<0.05) lower than the control groups. On the other hand, the total concentration of zinc in brains of zinc deficient rats did not vary from control groups. Since the synthesis of this metallothionein-like protein is reduced by zinc deficiency and is stimulated following administration of zinc, we postulate that the free pool of zinc may regulate the synthesis of its binding protein in the brain.

Effect of zinc deficiency on the biosynthesis of phosphatidylcholine in rat microsomes.

Phosphatidylcholine is the major lipid of all cellular membranes. Phosphatidylcholine biosynthesis in microsomes involves two enzyme pathways, choline phosphotransferase and phosphatidyl-ethanolamine methyltransferase. The present study was designed to examine the effect of zinc deficiency on these two enzymes. Male, weanling Long-Evans rats were fed a biotin-enriched 20% egg white diet deficient in zinc for 15-45 d. The specific activity (pmol phosphatidylcholine formed/min/mg microsomal protein) of choline phosphotransferase, phsophatidylethanolamine methyltransferase, and phos-phatidyldimethylethanolamine methyltransferase was determined. The latter assay measures the third methylation of phosphatidyl-ethanolamine to phosphatidylcholine. Zinc deficiency resulted in a significant increase over controls in the specific activity of phospha-tidylethanolamine methyltransferase and phosphatidyldimethyl-ethanolamine methyltransferase in liver and spleen microsomes. A significant increase in the picomoles of phosphatidylcholine formed by the choline phosphotransferase pathway occurred in liver microsomes of zinc-deficient animals. In the brain microsomes a significant decrease in specific activity of phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase, and choline phosphotransferase occurred among zinc-deficient ani-mals. These data suggest that zinc deficiency alters the biosynthesis of phosphatidylcholine, the major lipid of cellular membranes.

Severe zinc deficiency: effects on the distribution of nine elements (potassium, phosphorus, sodium, magnesium, calcium, iron, zinc, copper and manganese) in regions of the rat brain.

The effect of severe zinc deficiency on the distribution of nine elements (potassium, phosphorus, sodium, magnesium, calcium, iron, zinc, copper and manganese) in brain regions (olfactory lobes, right and left hippocampi, cerebellum and the rest of the brain) has been studied. After male rats (30 days old) were fed a zinc-deficient diet for 28 days, the zinc concentration of most brain parts was similar to zinc-adequate control values. Olfactory lobe zinc, on the other hand, was slightly depressed. However, the levels of other metals were dependent on zinc nutriture. Zinc deficiency caused an elevation in copper concentrations in most brain parts. Restriction of food intake caused a similar increase in brain copper but generally the effect was less than with zinc deficiency. Levels of calcium, manganese, sodium and potassium, in certain brain regions, also appeared to be altered by the zinc status of an animal. Of the minerals examined, only zinc and copper displayed asymmetrical distribution between the right and left hippocampus, and severe zinc deficiency did not affect lateral distribution of these trace metals in the hippocampus. The data suggest the hypothesis that changes in brain metal content, associated with zinc deficiency, contribute to the behavioral abnormalities that occur.

The effect of interactions between dietary egg white protein and zinc on body weight, bone growth and tissue trace metals in the 30-day-old rat.

Nine groups of 30-day-old rats were fed different diets, which contained 8, 15 or 20% egg white (equivalent to 6.0, 11.3 and 15.0% protein, respectively) each at 3 levels of zinc (6, 12 and 18 ppm zinc) for 30 days. The rats consuming the 6.0% protein, regardless of zinc intake, gained less weight than the groups fed 11.3 or 15.0% protein. Plasma zinc was not influenced by dietary egg white protein content, whereas, plasma zinc levels were lower in the rats fed 6 ppm dietary zinc compared to those fed 12 or 18 ppm zinc. Of the liver trace metals measured, only iron appeared to be influenced by an egg white protein-zinc interaction. Increasing dietary egg white protein led to increased concentrations of liver zinc and magnesium. Dietary zinc levels had no effect on the concentration of these metals in the liver. Liver calcium and copper concentrations were not significantly influenced by either dietary zinc or egg white protein. Femur weights and lengths were lower in the rats fed 6.0% than in those fed 11.3 or 15.0% protein diet but were unaffected by dietary zinc. Femur width was related to dietary protein and zinc. Dietary protein and zinc were interrelated in their affect on femur zinc levels. Femur zinc concentrations increased with increasing dietary zinc levels. Also femur zinc levels decreased with increasing dietary protein content. Other femur metal (copper, iron and calcium) concentrations were higher in rats fed the low level versus the higher levels of protein, but were not influenced by dietary zinc. These results show that dietary zinc and egg white protein are interrelated with regard to their effect on body and femur growth and tissue trace metal content.

Effect of zinc deficiency on appetite and free amino acid concentrations in rat brain.

Brain amino acids were measured in 30-day-old male Long-Evans rats subsequent to feeding a 20% egg white biotin-enriched zinc-deficient diet for 9 days. The zinc-deficient (ZD) group was given distilled deionized water. Zinc-supplemented control groups included pair-fed (PF), ad libitum-fed (AL) and ad libitum-fed, overnight fasted (OF) animals. Brain tyrosine concentrations and related amino acid ratios tended to be higher when food was consumed in all groups. Brain tryptophan concentrations and a brain amino acid ratio (glycine + serine + glutamine + taurine:leucine + isoleucine + valine + methionine) were not related to food intake in ZD rats in contrast to zinc-adequate controls. Also the brain ratio of tryptophan to the sum of large neutral amino acids minus tryptophan was not related to food intake in the ZD and AL-OF groups in contrast to the PF group. There were some differences in brain amino acid concentrations between ZD rats and the control groups; however, the pattern of the brain amino acids in ZD rats did not suggest that food intake was directly influenced by them.

Epiphyseal plate development in the zinc-deficient rat.

The effect of zinc deficiency on shearing strength, histological changes and proline utilization of the epiphyseal plate of the tibia of the weanling male rat was studied. A diet was fed based on sprayed egg white and containing less than 2 mg of zinc per kilogram. Over 27 day, the force required to displace the epiphysis of the zinc-deficient (ZD) rats was always less than that required for pair-fed (PF) controls. After 18 days, approximately 15% more force was required to displace the epiphysis of the PF rats than was required in the ZD rats. The thickness of the outside compact bone next to the epiphyseal plate region as determined by scanning electron microscopy was thicker in the ZD rats than in the PF controls of comparable age. The epiphyseal plates narrowed as the rats aged, and were clearly discernible in PF controls but not in ZD rats. The incorporation of L-[U-14C]proline into the epiphysis was significantly less in rats deprived of zinc for 16-22 days than in PF controls.

Influence of dietary zinc on rat brain catecholamines.

Weanling rats were fed a 20% sprayed egg white, zinc-deficient diet for 9 or 10 days. One group (zinc-deficient) was fed ad libitum and given distilled deionized water; a second group was individually pair-fed to rats in the zinc-deficient group; a third group was fed ad libitum; a fourth group was also fed ad libitum, but was fasted overnight prior to slaughter. The latter three groups were given 25 ppm zinc (as zinc acetate) in the water. The brain was excised and the catecholamines were extracted with 0.1 M perchloric acid separated by reverse phase HPLC and measured electrochemically. There did not appear to be a correlation between food intake and brain catecholamine concentrations in any of the groups examined. Brain norepinephrine concentrations in the zinc-deficient rats, however, were significantly higher than in the pair-fed or ad libitum-fed rats.

Mild zinc deficiency and undernutrition during the prenatal and postnatal periods in rats: effects on weight, food consumption, and brain catecholamine concentrations.

Rat dams were fed a 20% sprayed egg white diet, containing 10 ppm zinc, during gestation and lactation (ZD) and compared to zinc-adequate pair-fed (PF) and ad libitum fed (AL) control animals. AL dams consumed more food than the ZD group. Throughout gestation AL dams were heavier than PF and ZD dams. During lactation AL dams were heavier than the ZD dams which in turn were heavier than PF dams. Birth weights of ZD pups were less than the PF and AL pups. Postnatal daily weight gains of ZD pups were less than the PF pups which were less than AL pups. After weaning ZD pups fed commercial rat food gained less weight than PF and AL pups, and weight gain of PF and AL pups was similar. ZD dams had lower plasma zinc than control dams at parturition and at the end of lactation. Femur zinc of ZD, PF and AL dams was similar at parturition. ZD dams had lower femur zinc than PF and AL dams after lactation. Femur and liver zinc of ZD pups was lower than in PF and AL pups at birth. Regional brain catecholamine levels were not measured, but whole brain concentrations of catecholamines were not different among the groups. Thus, while mild zinc deficiency adversely affected growth of pups, effects on brain remain to be identified.