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The hypothesis that a biochemical lesion underlying the development of fetal alcohol syndrome is an ethanol-induced reduction in placental zinc transfer was tested. Placental zinc transfer was assessed in control and ethanol-fed dams on d 14-15 of pregnancy. Litters from ethanol-fed dams were characterized by a high resorption frequency and fewer live fetuses per litter than litters from control dams. In addition, fetuses from the ethanol-fed dams weighed less than control fetuses. However, despite the negative effects of ethanol intake on litter outcome, placental and fetal retention of 65Zn was similar in the two groups. Therefore, an ethanol-induced fetal zinc deficiency does not seem to have a role in the production of the gross structural malformations associated with fetal alcohol syndrome when adequate zinc is provided in the diet.

Dry ashing and wet ashing are two commonly used methods for the preparation of biological materials for trace element analysis by atomic absorption spectrophotometry. In this paper, National Bureau of Standards (NBS) bovine liver was dry ashed at 450°C for 24 h in silica glass (Vycor) or procelain crucibles; the resulting ash was dissolved in either concentrated nitric or hydrochloric acid. Dry ashing efficiency was evaluated by comparing iron, copper, zinc, and manganese concentrations of the samples with the values certified by NBS. Highest recoveries were obtained by dry ashing in silica glass (Vycor) crucibles. Dissolving the resultant ash in either hydrochloric or nitric acids did not significantly alter the results. A comparison between dry and wet ashing shows the latter method to be superior for the preparation of biological tissues for analysis of iron, copper, zinc, and manganese.

The molecular localization of zinc in rat milk and pup intestine was determined by gel chromatography. Commerical gels were found to give low recoveries of zinc and also caused anomalous elution patterns. Treatment of the gel with sodium borohydride corrected these problems. With treated gels, all of the zinc in rat milk eluted with high molecular weight proteins (MW greater than or equal to 30,000). In neonatal intestine, zinc eluted in two major peaks with molecular weights 30,000 and 6,000-8,000, respectively. However, incubating intestinal homogenates at 37 degrees for 30 minutes produced a redistribution of zinc with a new major low molecular weight (less than or equal to 1,500) peak. These results demonstrate the importance of sample preparation and chromatographic conditions when studying trace element ligands and also suggest that rat milk is not analogous to human milk in regard to zinc binding.

Skeletal maturation was evaluated from ages 1 to 3 y in rhesus monkeys that had been subjected to a diet marginally deficient in zinc (4 micrograms/g Zn) from conception through age 3 y. Skeletal development was assessed at 18, 24, 30, and 36 mo of age and compared with that of controls fed ad libitum. Skeletal maturation was determined by the presence of epiphyseal ossification centers. To evaluate endochondral bone mineralization the appearance of the zone of provisional calcification on the metaphyseal side of the growth plate and the width of the growth plate were observed. Marginal Zn deprivation was associated with delayed skeletal maturation in monkeys up to age 3 y. Defective mineralization of bone was evident in these monkeys up to age 6 mo. Between ages 6 mo and 3 y bone mineralization increased in some of the marginal-Zn monkeys to values that were only slightly below those for control monkeys.

Earlier studies have demonstrated that zinc deprivation, when begun in young NZB mice, can retard the development of autoimmunity and result in an increased life span. The present study evaluated the possible benefits of zinc deprivation in NZB mice with established disease, as this model is more relevant to the human patient. Female NZB mice aged 6-8 mo were fed diets containing either 80 micrograms Zn/g and 10 micrograms Cu/g (control) or 1 microgram Zn/g and 10 micrograms Cu/g (zinc-deficient) for 4 mo. In addition, another group was fed a diet containing 1 microgram Zn/g and 100 micrograms Cu/g to determine whether zinc deficiency could be exacerbated by high dietary copper through a competition of copper with zinc at the intestinal level (zinc deficient + high copper). A fourth group of mice was fed the control diet at the same intake as that of the zinc-deficient group in order to control for the inanition associated with zinc deficiency (restricted intake). Regardless of dietary treatment, all mice developed antierythrocyte antibodies at the same rate. At the end of 4 mo, 82% of the control and the restricted-intake groups had survived, whereas the zinc-deficient group had a 38% survival rate and the zinc-deficient + high copper group had a 50% survival rate. These observations show that, in contrast to findings with younger NZB mice, zinc deprivation of adult NZB mice with established autoimmunity will not improve survival. Indeed, severe zinc deficiency increased the mortality rate, demonstrating the need to consider the potential hazards of dietary extremes.

In separate experiments, domestic short hair kittens were fed diets based on soy or egg white protein containing from 1.98 to 9.64 or 0.58 to 9.8 micrograms Cu/g diet, respectively. Moreover, kittens were also nursed from queens fed diets containing either 0.58 or 12 micrograms Cu/g of diet. In each experiment, copper was analyzed in liver, spleen, lung, heart, muscle and brain samples. The liver copper concentration was the most responsive to changes in dietary copper intake. For example, in weanling kittens fed 0.58 or 9.8 micrograms Cu/g diet, the liver copper concentration was 24 or 45 micrograms/g wet weight, respectively. Likewise, the levels of hepatic Cu,Zn-superoxide dismutase were reduced in kittens fed the low copper diet. However, the concentrations of zinc, iron and manganese in liver, lung, spleen, brain, muscle and kidney were not consistently changed in response to low copper intakes. Anemia was also not a consistent feature in the copper-deficient cat or kitten, but signs of connective tissue lesions were observed in kittens born to and weaned from queens fed diets containing less than 1 micrograms Cu/g diet. Based on these observations and data for changes in growth in response to the differing levels of dietary copper, the copper requirement for the kitten was estimated to be 4-5 micrograms/g of diet (energy content approximately 5 kcal or 21 kJ per gram).

An abnormality in copper metabolism during both the prenatal and postnatal (preweaning) periods was found to be associated with the autosomal recessive gene "crinkled" (cr) in mice. Liver copper concentration was significantly lower in crinkled mice (cr/cr) than in littermate controls (+/?) from 18 days of gestation to 20 days after birth. Crinkled mice older than 20 days of age and liver copper concentrations similar to those of littermate controls. Liver zinc and brain copper and were similar in crinkled and noncrinkled mice at all times tested. In both crinkled and noncrinkled mice, brain copper concentration increased during the suckling period, and liver copper concentration decreased.

Earlier studies have shown that diets that increase tissue catabolism reduce the teratogenic effects of zinc deficiency. The hypothesis that zinc may be released from body tissues when the metabolic state is altered was further tested. Nonpregnant Sprague-Dawley females were injected with 65Zn; after equilibration, the two major pools of zinc, bone and muscle had different specific activities, muscle being much higher. Females were mated and fed diets adequate in zinc and calcium, deficient in zinc alone or deficient in both zinc and calcium. Calculations using weight loss, zinc content of maternal bone and muscle and total zinc content of the fetus at term indicated that most of the zinc in the fetus at term in both the zinc-deficient and zinc-calcium-deficient groups came from breakdown of maternal muscle in the last 3 d of pregnancy. The relatively small amount of additional zinc released from bone in the zinc-calcium-deficient rats early in pregnancy was sufficient to prevent abnormal organogenesis. Specific activity of zinc in the zinc-deficient and zinc-calcium-deficient fetuses was equal and high, indicating that most zinc in these fetuses came from maternal tissues and from the same maternal sources in both groups. In contrast, specific activity of zinc in the fetuses from rats fed adequate zinc and calcium was less than 30% of that in either of the deficient groups; this is consistent with the hypothesis that most zinc accrued by these fetuses came directly from the diet.

Age-related changes in serum copper and zinc concentrations were evaluated in approximately 800 Beagle dogs. A 20% increase with age was established for both elements. Seasonal variation was also found to have a marked effect on serum zinc with the values positively correlated to ambient temperature. In contrast, serum copper was not affected by temperature. Hemolysis, but not lipemia, was found to affect analytical results.

Iron supplements are commonly administered to infants in order to prevent iron deficiency. We wished to determine whether iron administration could compromise zinc nutrition as might be suspected from previous studies. Measures of iron nutrition, serum zinc, and serum copper were measured before and after randomization of 291 healthy 1-yr-old infants to a 3 mo course of placebo or iron treatment (30 mg iron as ferrous sulfate given before a meal). There was no significant difference in serum zinc or copper in the two groups before or after treatment; thus iron administration did not result in any evidence of zinc deficiency in a healthy, well-nourished group of T-yr-old infants.

Adriamycin (ADR) administration can result in cardiac toxicity. One suggested mechanism of damage is through increased lipid peroxidation. We have evaluated the biochemical response of mice to ADR treatment when fed Mn-sufficient, vitamin E-sufficient (+Mn, +E); Mn-sufficient, low vitamin-E (+Mn, -E); Mn-deficient, vitamin E-sufficient (-Mn, +E); or Mn-deficient, low vitamin-E (-Mn, -E) diets. Mice were injected i.p. with ADR (10 mg/kg bw) or saline (0.9% w/v). ADR injection resulted in a reduction of food intake by 2 days postinjection; by 5 days postinjection ADR-treated mice lost an average of 6.0 g. Heart Mn superoxide dismutase (SOD) activity of -Mn mice was 50% that of +Mn mice; ADR did not affect MnSOD activity. Lipid peroxidation was highest in the -Mn, -E mice, being 2-fold higher than that observed in +Mn, +E mice. ADR injection did not affect lipid peroxidation. An interaction between Fe and ADR treatment was apparent; ADR injected -Mn, -E mice had liver Fe concentrations which were twice that of the saline injected mice fed -Mn, -E diets. These data show that the antioxidant status of an animal may influence ADR toxicity.

Because disturbances in iron metabolism might contribute to the teratogenicity of zinc deficiency, we examined the effect of zinc deficiency on fetal iron accumulation and maternal and fetal retention of 59Fe. Pregnant rats were fed from mating a purified diet containing 0.5, 4.5 or 100 micrograms Zn/g. Laparotomies were performed on d 12, 16, 19 and 21 of gestation. Maternal blood and concepti were analyzed for zinc and iron. Additional groups of dams fed 0.5 or 100 micrograms Zn/g diet were gavaged on d 19 with a diet containing 59Fe. Six hours later maternal blood and tissues, fetuses and placentas were counted for 59Fe. Maternal plasma zinc, but not iron, concentration was affected by zinc deficiency on d 12. Embryo zinc concentration on d 12 increased with increasing maternal dietary zinc, whereas iron concentration was not different among groups. On d 16-21 plasma iron was higher in dams fed 0.5 micrograms Zn/g diet than in those fed 4.5 or 100 micrograms/g, whereas plasma zinc was lower in dams fed 0.5 or 4.5 micrograms Zn/g than in those fed 100 micrograms Zn/g diet. On d 19 zinc concentration in fetuses from dams fed 0.5 micrograms/g zinc was not different from that of those fed 4.5 micrograms/g zinc, and iron concentration was higher in the 0.5 microgram Zn/g diet group. The increase in iron concentration in zinc-deficient fetuses thus occurs too late to be involved in major structural teratogenesis. Although whole blood concentration of 59Fe was not different in zinc-deficient and control dams, zinc-deficient dams had more 59Fe in the plasma fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

We proposed that the circadian rhythm of corticosterone in diabetic rats would have a different pattern than that in non-diabetic control rats. To test this hypothesis, 20 male Sprague-Dawley rats were given ad libitum access to a stock diet and housed individually in a light and temperature controlled room. Ten rats were made diabetic by two subcutaneous injections of streptozotocin. Ten rats which were not injected served as controls. Thirteen days after induction of the diabetes, tail blood samples were taken every 4 h for 24 h. Plasma corticosterone levels were significantly higher in diabetic rats than in control rats at 3 time points during the light cycle; however, concentrations were similar during the dark cycle. We speculate that diabetes may cause alterations in the steroid feedback mechanism to the hypothalamus and/or pituitary, resulting in an abnormal circadian rhythm of plasma corticosterone.

Copper nitrilotriacetate (NTA) was evaluated for its ability to ameliorate effects of the recessive mutant gene crinkled (cr) in mice. Copper-NTA was superior to copper sulfate in increasing postnatal survival and body copper content of offspring of dams supplemented during pregnancy and lactation. Feeding of NTA alone during these periods had no effect on survival. Postnatal supplementation with copper did not increase survival of the mutants. The therapeutic use of copper-NTA, and the necessity for prenatal intervention for successful treatment of the mutant, are discussed in relation to Menkes' syndrome.

Indicators of copper and iron metabolism were studied in pregnant rats and their 90-d-old offspring fed copper-sufficient or copper-deficient diets containing marginal or adequate levels of iron from the beginning of pregnancy until the offspring were 90 d of age. Offspring had more severe signs of copper deficiency (including anemia, hypertrophy of the heart, decreased activity of ferroxidase I and II, depression of growth and death) than the dams. In both dams and offspring, copper deficiency resulted in anemia when dietary iron was marginal but not when it was adequate. Liver iron was elevated in copper-deficient male offspring, but not in female offspring. Anemia and growth retardation were more pronounced in copper-deficient males than in females, despite similarly low levels of ferroxidase I and II. Iron absorption was reduced by copper deficiency only in female offspring. Activity of 59Fe in various tissues 6 or 48 h after gavage did not reveal any other effect of copper deficiency on iron metabolism. Thus age at the time copper-deficient diets were introduced, sex and dietary iron strongly influence the effect of copper deficiency.

The utilization and distribution of copper during dietary copper deficiency was studied in the pregnant rat, and the effects of maternal copper deficiency on fetal development were compared with those of maternal zinc deficiency. Sprague-Dawley rats were fed from mating to term (per gram diet): 1) a control diet (10 micrograms copper, 100 micrograms zinc) or 2) a copper-deficient diet (0.7 micrograms copper, 100 micrograms zinc) or 3) a zinc-deficient diet (10 micrograms copper; 0.7 micrograms zinc). Dams fed the copper-deficient diet deposited only 15.5% of the dietary copper consumed during pregnancy into the products of conception (fetuses, uterus and placentas); in comparison dams fed the zinc-deficient diet deposited more zinc into their litters than was consumed (240%). Copper concentration in the fetuses of copper-deficient dams was 30% of that of controls, but the size and number of live fetuses was unaffected. The zinc concentration of the zinc-deficient fetuses was 78% of that of the controls, and both the size and number of live fetuses were considerably lower than normal. Accumulation of copper in the products of conception may be accounted for by dietary copper intake, whereas accumulation of zinc in fetuses of zinc-deficient females is dependent in part on catabolism of maternal tissues.

Catch-up growth during recovery from undernutrition is characterized by rapid body weight gain often marked by disproportionately high fat gain. In this study dietary zinc intake of mouse pups during recovery from undernutrition affected composition of gain as well as growth. Mouse pups were undernourished during the suckling period and then fed 25% casein recovery diets containing 5, 10, 40 or 110 micrograms Zn/g diet. Pups given dietary zinc levels of 40 micrograms Zn/g ad libitum achieved recovery body weights and had normal body composition. However, previously undernourished pups given marginal levels of dietary zinc (10 micrograms Zn/g diet) had similar food intakes, similar final body weights, lower lean body mass gains and greater fat gains (males only) than controls. Pups fed low zinc diets (5 micrograms Zn/g diet) were unable to attain recovery body weight and had less protein gain than previously undernourished animals fed higher zinc-containing diets or healthy pups fed the same zinc levels. The results were consistently more pronounced in males. Thus dietary zinc deficiency during catch-up growth after undernutrition limited both recovery and protein gain, whereas marginal dietary zinc intake limited protein gain in mice.

The teratogenicity of copper deficiency is well known, but underlying mechanisms have not been delineated. One method of studying the biochemical lesions of copper deficiency is the use of chelating drugs with different chemical characteristics. The teratogenicity of a copper deficient diet and of diets containing either D-penicillamine or triethylenetetramine is quite different, although all three diets result in decreased fetal liver copper levels. Feeding D-penicillamine can result in decreased fetal liver zinc, while feeding triethylenetetramine can result in increased fetal liver zinc. The effect of these three diets on fetal liver copper and zinc molecular localization was determined. Gel filtration showed that fetal liver copper and zinc in controls was localized in 3 fractions with MWs of greater than 50,000 (H), 30,000 (I) and 8-10,000 (L). Independent of dietary treatment, as liver copper diminished, copper was missing first from the L peak, then the I peak and with severe deficiency, from the H peak. Drug induced increases and decreases in fetal liver zinc were reflected in the L peak. These data suggest that the absolute levels of copper in the liver of the term fetus determines the distribution of the element among its binding ligands.

The concentrations of iron, copper, zinc, manganese, calcium, magnesium, protein, carbohydrate and fat were analyzed in cats' milk during the course of lactation. Cats' milk is different from most species in that the concentrations of iron, copper, zinc and manganese are lower during the first 2 days of lactation than on days 3-7. After the initial rise in the concentration of these elements, the concentration of iron decreased from 5.9 micrograms/ml to 3.0 micrograms/ml, with most of the decrease occurring between days 8 and 21. Copper concentration declined from 1.6 micrograms/ml to 0.8 microgram/ml, with most of the change occurring between days 8 and 28. Concentration of manganese increased during lactation, from 0.14 microgram/ml of 0.39 microgram/ml. Calcium concentration increased rapidly during the first 3 weeks from 550 micrograms/ml to 1500 micrograms/ml, with little change thereafter. The magnesium concentration (congruent to 100 micrograms/ml) and zinc concentration (congruent 6 micrograms/ml) were not affected by stage of lactation. Protein increased during lactation from 4% to 7%, and fat from 3% to 5%, whereas carbohydrate concentration (congruent 4%) did not change significantly. These data demonstrate that the nutrient intake of the kitten changes markedly during the early neonatal period and that these changes should be taken into account in evaluating studies of suckling cats.

Creatine phosphokinase (CPK) concentrations in CSF and in serum of dogs with a variety of neurologic diseases were evaluated. Concentrations of CPK in CSF and serum were diagnostically nonspecific. Although a diagnostic advantage was not found in measurement of CPK concentrations in CSF and serum, it appeared that increased CPK concentration in CSF was associated more frequently with a guarded-to-poor prognosis. The concentration of CPK in CSF was statistically independent of CPK concentration in serum. Cerebrospinal fluid CPK concentration was significantly (P = 0.01) correlated to the number of RBC in CSF. However, CSF concentration of CPK was not significantly correlated to serum, blood, or CSF variables evaluated.

The molecular localization of maternal and fetal zinc and copper metalloproteins in diabetic and control rats was studied. Compared to controls, liver and kidneys of diabetic dams showed an increased concentration of zinc and copper that was associated with metallothionein. In contrast, fetuses of diabetic dams had lower zinc and metallothionein levels than fetuses from controls. The abnormal maternal trace element metabolism seen with diabetes resulted in alterations of zinc uptake and/or retention of their fetuses.

Food intake and body weight gain in rats fed a zinc-deficient diet are reported. The severity of zinc deficiency-induced anorexia was observed to be dependent upon the age, sex and reproductive status of the animal. Rapidly growing rats such as weanlings, adult males, or ovariectomized females demonstrated more rapid induction of anorexia and more severe effects on cumulative food intake and body weight gain when fed zinc-deficient diets than did slowly growing (nonpregnant female adult) rats given the same diet. These results suggest that feeding in zinc-deficient animals is not regulated by dietary zinc concentration alone, but rather is mediated by one or several as yet undefined factors.

Rats with diets containing 0.2% propylthiouracil (PTU) throughout gestation had progeny with persistent cyanosis and high neonatal mortality. Histological and histochemical studies failed to reveal lung abnormalities in these pups. Studies of the blood of PTU-fed dams demonstrated that hemoglobin, packed cell volume, and numbers of erythrocytes were significantly reduced. In their 21-day fetal young, erythrocytopenia was accompanied by an elevated mean corpuscular volume and a reduced mean corpuscular hemoglobin concentration. Imprints of marrow from dams and of liver and spleens of the young showed normoblastic erythropoiesis. A granulocytic leucocytosis was present in the blood of the PTU-fed dams, whereas their progeny had a granulocytopenic leucopenia. Tissue concentrations of copper, zinc, manganese, magnesium and iron were determined. The most striking changes observed were the significant elevations of copper in the dams' brain, liver and kidneys. No changes in the concentration of any of the trace minerals were found in the livers of the pups. Food restriction to the dam failed to significantly alter maternal or fetal hematologic or trace element concentrations as compared with controls. It is evident that PTU, when fed to pregnant rats, has demonstrable effects on erythropoiesis, granulocytopoiesis, and maternal trace element distribution. It is not presently known whether these phenomena are interrelated.

Graphite furnace atomic absorption spectrophotometry is a method used for the measurement of low concentrations of manganese (ppb range). Despite the widespread use of this technique, there is considerable inconsistency concerning sample preparation and choice of instrumental parameters. In this paper, we determined manganese concentrations of National Bureau of Standards (NBS) bovine liver by both graphite furnace (Instrumentation Laboratory IL 555B) and flame atomic absorption following wet digestion of the sample with nitric acid. The following instrumental parameters for the graphite furnace were found optimal for the measurement of manganese in digested NBS bovine liver: inert gas flow=14 SCFH, drying temperature 100°C/15 s (step 1), 125°C/15 s (step 2), pyrolysis temperature 500°C/15 s (step 3), and 1000°C/15 s (step 4); atomization temperature 2250°C/10 s (step 5). For optimal results, the nitric acid concentration of the sample should be between 2 and 4M. There were no significant differences found for manganese concentrations determined by either peak height or peak area measurement. Additionally, no significant differences were found in manganese concentrations determined by flame or furnace methods. Assuming proper sample preparation and choice of instrumental parameters, values obtained for manganese concentration by graphite furnace and flame atomic absorption spectrophotometry are similar. Therefore, data obtained by these two methods can be compared directly.

The effect of superoxide dismutase (SOD) activity and isoenzyme pattern of detergents, incubation time, and sonication in the preparation of rat liver samples was investigated. The activity of the manganese form of the enzyme (Mn-SOD) was found to decrease significantly after 4 hr of incubation at room temperature, and activity of the copper, zinc form of the enzyme (Cu, Zn-SOD) was not changed significantly even after 24 hr, although levels were somewhat decreased. Sonication of the sample did not affect Cu, Zn-SOD activity, but total Mn-SOD activity was increased. Addition of detergents did not increase Mn-SOD activity when homogenates were sonicated, indicating that Mn-SOD is not membrane bound. Detergents also had no effect on Cu, Zn-SOD activity. None of the treatments investigated altered the isoenzyme patterns, providing evidence that these isoenzymes are not degradation products.

The effects of manganese (Mn) toxicity on carbohydrate homeostasis was examined in Sprague-Dawley and Osborne-Mendel rats. Mn injection was followed by increases in Mn concentration in both liver and pancreas. Concentrations of Mn in the pancreas increased more rapidly than in the liver. Plasma insulin levels decreased, plasma glucose levels increased, and a spike in glucagon concentration was observed following Mn injection. Increases in blood glucose in response to Mn injection were also observed in 24- and 48-h fasted rats, although the magnitude of the increase was less than that observed in fed rats. Both strains of rats appeared to respond similarly to Mn injection. The present studies demonstrate that acute Mn injection can affect glucose homeostasis. These effects may be mediated through altered endocrine pancreatic function.

Metal-chelating drugs were employed to investigate the role of copper (Cu) in elastin metabolism during the period of alveolarization in rat lung. Six groups of pregnant Sprague-Dawley rats were fed one of six semipurified diets, i.e., sufficient or deficient in copper, or the same basal diet containing 0.2% or 0.4% D-penicillamine (DPA), or the same basal diet containing 0.2% or 0.4% triethylenetetramine (TETA). The dams were fed throughout gestation, parturition, and lactation. The pups were then killed postnatally at day 10 and day 21 for the various analyses. At day 21, liver copper in the Cu-deficient pups was 3-5% control levels. In drug-treated groups, liver copper was 16-30% control levels. In the 21-day-old Cu-deficient rats, the concentration of lung elastin was only 75% of its concentration in control. Lung lysyl oxidase activity was lower in Cu-deficient rats, and data for the relative distribution of lung elastin cross-linking amino acids indicated impaired cross-linking in the pups from both the Cu-deficient and the 0.4% DPA groups. Morphologic examination of the lung also indicated dilation of airways in these two groups. Data on the distribution of cross-linking amino acids in elastin samples were also consistent with previous suggestions that impaired cross-linking observed in copper deficiency or from DPA treatment results from different mechanisms. Since the intakes of DPA and TETA were chosen to be in the therapeutic range of intakes used to treat diseases of abnormal copper metabolism, an important feature of these studies is that DPA and perhaps TETA have the potential of impairing normal lung development.

To test the hypothesis that the high absorption of manganese (Mn) in suckling rats compared to weanling rats is due in part to maturational differences in mucosal cell uptake of Mn, uptake kinetics of Mn were examined in isolated brush-border membrane vesicles prepared from the small intestine of rats at various ages (d 14, d 18, d 21). Initial uptake of Mn was rapid by vesicles from all age groups and reached an equilibrium plateau by 5 min in vesicles from suckling rats (d 14) and 10 min in vesicles from weanling rats (d 18, d 21). Uptake of Mn was associated with an osmotically active space. Uptake velocity was similar in all age groups and was nonsaturable at Mn concentration of 1-90 microM. The data were representative of a diffusional transport process. Mn uptake did not appear to be influenced by a putative ligand, L-histidine. However, incubations that included ascorbate did result in increased uptake of Mn by membrane vesicles. The results do not support the hypothesis that age-related differences in Mn retention in rats are due to maturational differences in the transport of Mn across small-intestinal brush-border membranes.

The concentrations of iron, copper, zinc, manganese, calcium, magnesium, protein, carbohydrate, and fat were analyzed in dog milk during the first 45 days of lactation. The concentration of iron decreased significantly from 13 microgram/ml on day 0 to 6 microgram/ml. Zinc concentration decreased only slightly from 9.6 microgram/ml to 8.7 microgram/ml. Calcium concentration increased significantly during early lactation, from 1,366 microgram/ml to 1,757 microgram/ml on day 10, but changed little thereafter. Protein concentration increased throughout lactation, from 4.3% early in lactation to 6.3% late in lactation. Fat concentration increased from 2.4% to 4.5% in early lactation, and then decreased to 2.7% in late lactation. The concentrations of copper, manganese, magnesium, and carbohydrate did not show strong developmental patterns; average values were 1.8 microgram/ml, 0.14 microgram/ml, 59 microgram/ml, and 4.5%, respectively, throughout lactation. These data indicated that the nutrient intake of the pup changed markedly during the early neonatal period and that these changes should be taken into account in evaluating studies of suckling dogs.

The objective of this study was to determine whether alterations in HDL composition observed previously were due specifically to zinc deficiency or to reduction in food intake. Rats were placed into three groups and fed for 21 d a zinc-adequate diet (100 ppm) ad libitum, or a zinc-deficient diet (less than 1 ppm) ad libitum, or the zinc-adequate diet at an intake equal to that of the deficient group. Blood plasma was fractionated into VLDL, LDL and HDL fractions, and plasma and the isolated fractions were analyzed for lipids and apolipoprotein composition. Analysis of plasma and liver zinc indicated that animals consuming the low zinc diet were deficient. Zinc deficiency and restricted feeding were associated with lower plasma and VLDL triglyceride levels and lower HDL cholesterol levels. The similarities in overall pattern of lipoprotein composition between the zinc-deficient and restricted-intake groups indicate that reduction in food intake may account for the differences in lipoprotein composition which were observed.

The effects of manganese (Mn) deficiency on carbohydrate metabolism in the Sprague-Dawley rat were investigated. Oral glucose tolerance tests were performed on offspring from Mn-sufficient female rats fed 45 micrograms Mn per gram diet (C-C), offspring from Mn-deficient female rats fed 1 microgram Mn per gram diet (D1-D1) and rats not exposed to Mn deficiency in utero but fed D1 diet from weaning to maturity (D1). Mn-deficient rats, D1 and D1-D1, had significantly lower concentrations of Mn in liver, kidney, heart and pancreas than controls. D1-D1 rats responded with a diabetic type of glucose tolerance curve when given oral glucose. Insulin levels of D1-D1 rats were not commensurate with their high glucose levels. Measurements made by using an isolated perfused-pancreas preparation indicate that insulin output due to release of stored hormone was 76% of control levels in D1-D1 rats. This suggests either lower insulin stores or impaired release of insulin in D1-D1 rats. Second phase insulin release was also significantly lower in D1-D1 rats than in controls. Pancreatic insulin content was 63% of control levels in D1-D1 rats, further supporting the idea of lower insulin stores in Mn-deficient rats. No differences in plasma glucose levels were observed between D1 rats and controls after an oral-glucose load. These findings indicate that dietary Mn deficiency can result in impaired insulin secretion producing impaired carbohydrate metabolism; however, the timing of the deficiency may be a critical factor in the expression of this abnormality.

The teratogenicity of D-penicillamine (DP) (dimethyl cysteine) was studied using the Sprague-Dawley rat. D-Penicillamine was fed during pregnancy at a level of 0 (control), 0.17, 0.83, or 1.66% of the diet. The frequency of resorptions, and the frequency and severity of malformations, increased with increasing levels of the drug. Maternal and fetal tissue copper levels were significantly lower in the DP groups than in controls, with the levels decreasing in a dose-related manner. Maternal and fetal zinc levels were lower in the 1.6% DP group than in controls. Maternal liver iron concentration was higher in the drug-fed rats than in controls, increasing in a dose-related manner. Fetal iron concentration was not consistently affected by the drug. Maternal and fetal manganese, calcium, and magnesium concentrations were similar among all groups. These results suggest that the teratogenicity of D-penicillamine may be due in part to induction of copper deficiency and, at high dose levels, zinc deficiency, caused by its chelating properties.

The control of Zn and Cu homeostasis in the gut is not well understood; a role for bile and pancreatic secretions has been suggested. In the present study, the changes in biliary and pancreatic Zn and Cu after surgery were followed. Rats were prepared surgically with pancreatic, biliary, and intestinal cannulae, placed in restraint cages, and allowed to recover from anesthesia. Fluids were collected for up to 120 hours. Aliquots of the collected samples were analyzed for Zn and Cu. Zn in pancreatic juice did not follow a consistent pattern; however, the concentration of Zn was well correlated with pancreatic protein concentration. Cu in pancreatic juice was very low and was apparently not affected by surgery. In bile, Zn concentration increased markedly at about 40 hours postsurgery but declined to its initial level by days 4-5. Cu concentration in bile was similar to that of Zn initially, then increased 10-fold after 20-30 hours postsurgery, and returned to the initial level by days 4-5. These results demonstrate that the surgical procedure of cannulating bile and pancreatic ducts has a pronounced effect on the concentrations of Zn and Cu in bile and pancreatic fluid. This effect must be considered when designing and evaluating experiments concerned with the role of these secretions in trace element homeostasis.

Manganese nutrition in the neonatal period is poorly understood, due in part to a lack of information on the amount of manganese in infant foods and its bioavailability. Since the molecular localization of an element in foods is one determinant of its subsequent bioavailability, we have studied the binding of manganese in human and cow's milk. An extrinsic label of 54Mn was shown to equilibrate isotopically with native manganese in milks and formulas. Milk samples were separated into fat, casein and whey by ultracentrifugation. In human milk, the major part (71%) of manganese was found in whey, 11% in casein and 18% in the lipid fraction. In contrast, in cow's milk, 32% of total manganese was in whey, 67% in casein and 1% in lipid. Within the human whey fraction, most of the manganese was bound to lactoferrin, while in cow's whey, manganese was mostly complexed to ligands with molecular weights less than 200. The distribution of manganese in formulas was closer to that of human milk than of cow's milk. The bioavailability of manganese associated with lactoferrin, casein and low molecular weight complexes needs to be assessed.

The influence of a higher-than-normal intake of vitamin A on the detrimental effects of zinc deficiency on vitamin A metabolism was investigated in pregnant Sprague-Dawley rats. At mating, rats were fed diets containing 100 (control), 4.5, or 0.5 micrograms/g zinc combined with 4 (control) or 8 micrograms retinyl acetate/g. Low intake of zinc, but not of vitamin A, caused food intake, total body weight change, fetal weight and placental weight to be low. Incidence of teratogenic effects was more pronounced in low zinc groups than in controls. Concentrations of vitamin A in maternal plasma and liver were affected by the amount of zinc in the diet. Dietary vitamin A, however, did not affect either of these parameters. Maternal plasma zinc concentration was affected only by low dietary zinc, whereas plasma copper and iron were unaffected by the dietary treatments. Maternal liver iron was higher in zinc-deficient rats than in controls; however, maternal liver zinc and copper concentrations were not altered by dietary treatments. No significant differences in vitamin A concentration of fetal liver, fetal plasma, or placenta were seen among the groups. Fetuses from zinc-deficient dams had significantly lower levels of liver vitamin A and liver zinc than did controls. Fetal liver iron was higher in zinc-deficient fetuses than in controls, whereas fetal liver copper was not affected by dietary treatment. These data suggest that supplemental dietary vitamin A does not ameliorate the effect of zinc deficiency on vitamin A metabolism during pregnancy.

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.

It has been shown that epileptics have lower mean blood concentration of manganese than do controls but the cause of this abnormality has not been determined. In order to investigate the effects of seizures on manganese distribution in the body, rats were treated with kainic acid to produce spontaneous seizures which were quantitated for number and severity. Manganese, zinc, copper and iron concentrations were determined in blood, brain, liver, heart and kidney. Kainate-treated animals ate more food but gained less weight than controls. Liver and kidney manganese concentrations were significantly higher in kainate-treated animals than in controls. Blood manganese concentration showed a significant negative correlation with seizure index while heart manganese concentration showed a significant positive correlation with seizure index. None of the other trace elements showed a significant correlation between trace element concentration and seizure index in any of the tissues, although iron concentration was lower in brain and copper concentration was lower in kidney of kainate-treated animals than in their appropriate controls. These data show that manganese concentrations are generally elevated in tissues of kainate-treated animals. This increased manganese concentration may be related to the increased energy demand of these animals.

To investigate the use of whole blood manganese (Mn) as an indicator of total body Mn, we measured Mn in whole blood and liver of rats fed purified diets containing adequate (45 micrograms Mn/g diet) or deficient (1 microgram Mn/g diet) Mn. The mean hepatic Mn concentration was significantly lower (P less than 0.001) in the Mn-deficient group compared to the control group, 0.36, microgram Mn/g and 1.73 micrograms Mn/g, respectively. Furthermore, whole blood Mn was significantly reduced (P less than 0.001) in the deficient group when compared to the control group, 4.0 ng Mn/ml and 8.6 ng Mn/ml, respectively. Hepatic Mn linearly regressed against whole blood Mn yielded a statistically significant (P less than 0.001) correlation coefficient of 0.775. These data suggest that whole blood Mn is a valid indicator of body Mn status and thus may be useful, in addition to the measurements of serum copper and zinc, for the diagnosis and prognosis of diseases in which the metabolism of trace elements is affected. In addition, this paper describes and delineates operational parameters for the measurement of whole blood Mn using the IL 551 atomic absorption spectrophotometer and the IL 555 B flameless atomizer.

The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 μg/g) and five levels of zinc (1, 4.5, 10, 100, 1000 μg/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 μg Cu, 100 μg Zn/g diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily when dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.

To determine if prenatal zinc deficiency has a persistent effect on metallothionein (MT) regulation, Swiss-Webster mice were mated and fed a diet containing either control (100 micrograms Zn/g) or low levels of zinc (5 micrograms Zn/g) from Day 7 of gestation to parturition. After birth all mice were given the control diet. Liver zinc and MT levels were 50% lower in newborn pups from dams fed the low zinc diets than in control pups. In control pups, liver zinc and MT concentrations were relatively stable during the first week of postnatal life. In contrast, in pups prenatally deprived of zinc, liver levels of zinc and MT increased such that by Day 3 of postnatal life, the levels were not significantly different from controls. At Day 56, serum IgM concentrations were significantly lower in the low zinc offspring. Liver zinc concentrations in the two groups of mice were similar at Day 70 postnatal, and in both groups liver MT levels were below detection limits. However, when Day 70 mice were given zinc injections to stimulate MT synthesis, the prenatally zinc deprived offspring showed markedly higher liver MT levels than did control mice given similar injections, despite similar liver zinc concentrations in the two groups. These results show that prenatal zinc deficiency has pronounced effects on postnatal MT metabolism which can persist into adulthood.

Zinc and copper absorption from five infant cereal products mixed with water, human milk, or cow's milk was measured using an in vivo absorption model (rat pup) involving gastric intubation of extrinsically radiolabeled diets. Whole-body copper 64 uptake, nine hours after intubation, ranged from 14% to 31% of the dose given for the different cereal combinations. The resultant bioavailability of copper from human milk-cereal combinations (23% to 26%) was significantly lower than that from human milk alone (38%). Whole-body zinc 65 uptake, nine hours after intubation, ranged from 13% to 54% of the dose given for the different cereal combinations. These values were significantly lower than the whole-body zinc 65 uptake from milk alone (61%). Zinc availability was lower (13% to 25%) from dry cereal combinations that contained phytic acid (oatmeal and high-protein varieties) compared with the ready-to-serve cereal-fruit combinations (24% to 54%). The highest zinc uptake (37% to 54%) was from rice-fruit combinations that do not contain phytic acid. We estimated the amounts of zinc and copper that would be absorbed from these cereal products and speculated on the potential impact of these foods on the weaning infant's zinc and copper nutriture. Depending on the feeding practices employed during the weaning period, it is apparent that infant cereals may compromise utilization of zinc and copper from milk diets during weaning.

The effects of hydralazine (HZ) on mineral metabolism was investigated in rats. Sprague-Dawley rats were fed HZ (0.1-4%, w/w) in three separate experiments: (1) a 2-week dose-response study; (2) a 2-month long-term feeding study; and (3) a pregnancy study. At the end of each experiment, tissues were removed and concentrations of Mn, Cu, Zn and Fe were measured. Results from the three experiments were similar. HZ had no effect on tissue Mn and Zn concentrations, while tissue Fe concentrations were lower and kidney Cu concentrations were higher in HZ-treated groups compared to controls. The effect of HZ on mineral metabolism may be, in part, responsible for some of its negative side effects.

To investigate the effects of marginal zinc deficiency on early development, rhesus monkeys were fed a diet marginally deficient in zinc (M; 4 micrograms/g) throughout pregnancy and during the first month of lactation. Despite the low concentration of zinc in the diet. M dams did not develop overt signs of zinc deficiency. However, compared to control dams fed diets adequate in zinc (C; 100 micrograms Zn/g), M dams showed a low response to the mitogens concanavalin A and phytohemagglutinin. Pregnancy outcome was similar in the two groups and all of the neonates were judged to be healthy at delivery. From birth until d 30 of age, the infants were closely monitored for signs of zinc deficiency, and at d 30, they were killed and tissues were removed and analyzed for a number of parameters reported to be affected by zinc status. At birth, M infants had low plasma zinc concentrations compared to controls; however, this difference was not observed at d 30. D 30 M infants showed a normal response to the mitogens concanavalin A and phytohemagglutinin, but showed a low response to pokeweed mitogen. Tissue (liver, brain, spleen, kidney, and heart) trace element concentrations were similar in the two groups of infants, as were liver metallothionein concentrations and 65Zn uptake/retention by isolated hepatocytes. Infant wt gain was inversely correlated with plasma zinc, liver zinc, and liver metallothionein concentrations in both the M and C groups.(ABSTRACT TRUNCATED AT 250 WORDS)