Longitudinal changes in the mineral composition of mouse milk and the relationship to zinc metabolism of the suckling neonate.

We studied changes in the mineral composition of milk of lactating Swiss-Webster mice and the relationship of those changes to mineral metabolism of suckling mouse pups. Concentrations of Zn, Cu, Mg and Ca were analyzed in maternal and neonatal tissues and in milk; Zn metabolism was studied using 65Zn. Although Cu, Ca and Mg concentrations in milk declined during the first 2 d of lactation, only the concentration of Zn decreased progressively throughout 30 d. Various pup tissues were characterized by developmental changes in concentrations of some elements. Turnover of Zn in neonatal tissues was studied by radiolabeling litters in utero and fostering them at birth to nonradiolabeled lactating dams. The turnover of whole-body 65Zn in suckling mice decreased during development, and, at 20 d of age, the biological half-life of 65Zn in the neonate was approximately the same as for a nonpregnant, nonlactating adult female (20 d). The decreased turnover of 65Zn in lactating dams that characterized progressive lactation and was reflected in the Zn concentration of milk is in agreement with changes in whole-body 65Zn turnover observed for the suckling mouse. These findings demonstrate that the metabolism of the suckling neonate is directly related to longitudinal changes in the composition of maternal milk.

Mineral status of mice suckling early-, mid- and late-lactating foster dams.

We have demonstrated that the zinc (Zn) concentration of mouse milk declines significantly over the lactation period. Pups radiolabeled in utero with 65Zn were forward-fostered (FF) to nonradiolabeled dams at a later stage of lactation to study the effects of early milk deprivation. Other groups of radiolabeled pups were back-fostered at 5, 10 and 15 days of age to a nonradiolabeled dam who had just given birth; this provided additional colostrum during the suckling period. Litters fostered at birth to an unlabeled dam at d 0 of lactation were used as controls. Weight gain of FF litters decreased and signs of Zn deficiency increased as the foster dam's days of lactation increased. The Zn concentration of kidney, brain and plasma tended to decrease with increasing lactation days of the foster dam. Tibia Zn concentration declined progressively as the lactation days of the foster dam increased, and the concentration of calcium also was lower in all three groups of FF litters than in controls, indicating that bone calcification may have been impaired. Kinetic data proved to be a more sensitive index of Zn status than tissue Zn concentration. The biological half-life of whole-body 65Zn for FF suckling mice increased in a linear fashion with increasing lactation days of the foster dam; whole-body retention of 65Zn for back-fostered litters did not differ from that for controls. Brain, small intestine, kidney muscle, plasma and tibia of pups FF to late-lactating dams seemed to have greater retention of Zn than did controls. Thus, deprivation of early milk impaired growth and development of the mouse neonate despite some ability to conserve Zn.

Influence of maternal dietary zinc intake on in vitro tubulin polymerization in fetal rat brain.

The hypothesis that one of the biochemical lesions underlying zinc deficiency-induced teratogenicity is altered microtubule formation was tested. Day 19 fetuses from zinc-deficient Sprague-Dawley dams were characterized by low brain supernate zinc concentrations and slow brain tubulin polymerization rates compared to controls. Brain supernate tubulin and protein concentrations were similar in zinc-deficient and control fetuses. In vitro brain tubulin polymerization rates were increased following addition of zinc to either control or zinc-deficient brain supernates; however, the stimulatory effect of added zinc on polymerization was significantly higher in brain supernates obtained from zinc-deficient fetuses compared to controls. These results support the idea that one effect of fetal zinc deficiency is a reduction in tubulin polymerization, which in turn may result in altered microtubule function.

Effects of marginal zinc deficiency on microtubule polymerization in the developing rat brain.

One of the possible mechanisms that has been proposed to underlie the deleterious effects of zinc deficiency on brain development is an impairment in the normal formation of the cytoskeletal network. In the current study, in vivo microtubule polymerization was characterized in brain supernatant fluids, from 20-d-old pups whose dams were fed diets containing control (50 micrograms zinc/g) or marginal levels of zinc (10 micrograms zinc/g) throughout pregnancy and lactation. Pup brain and body weights were similar between the groups; however, plasma zinc concentrations were lower (27%) in pups fed the marginal zinc diet than in controls. Tubulin concentrations in 100,000 g brain supernates were similar between the groups; however, tubulin polymerization in the brain supernates was significantly lower in pups fed the marginal zinc diet compared to controls. Primarily, the early events of polymerization were affected; the lag period of the reaction was doubled, and the initial velocity was slower (26%) in supernates from pups fed the marginal zinc diet than in controls. These findings support the idea that some of the negative effects of marginal zinc deficiency on brain development and function may be mediated by an alteration in microtubule formation.

The effect of valproic acid on 65Zn distribution in the pregnant rat.

The effect of valproic acid on the distribution of gavaged 65Zn in maternal and embryonic tissue of Sprague-Dawley rats was examined 24 h after gavaging of the drug on d 13 of pregnancy. Valproic acid treatment resulted in a significantly higher retention of 65Zn in maternal liver and lower amounts in uterus, placenta and embryos than in controls. Compared to controls, gel chromatography of maternal liver from valproic acid-treated dams showed higher 65Zn counts associated with a protein peak of molecular weight of 6,500, the approximate molecular weight of the Zn-binding protein metallothionein. These results support the idea that the teratogenicity of valproic acid is in part due to an induction of embryonic Zn deficiency secondary to a drug-induced sequestering of Zn into maternal liver that results in a decrease in maternal plasma Zn and subsequent reduction in embryonic Zn uptake.

Effect of 6-mercaptopurine on 65Zn distribution in the pregnant rat.

The effect of 6-mercaptopurine (6-MP) on the distribution of gavaged 65Zn in maternal and embryonic tissues of Sprague-Dawley rats was examined 24 hr after injection of the drug on day 13 of pregnancy. 6-MP injection resulted in a significantly higher retention of counts of 65Zn in maternal liver and lower counts in maternal plasma, uterus, placenta, and embryo than in controls. Compared to controls, gel chromatography of maternal liver from 6-MP injected dams showed higher counts associated with a protein peak of molecular weight 6,000-8,000, the approximate molecular weight of the zinc-binding protein metallothionein. These results support the idea that the zinc deficiency, which is observed in day 21 fetuses from dams injected with 6-MP during midgestation, may be the result of a drug-induced sequestering of zinc into maternal liver followed by a decrease in maternal plasma zinc and subsequent reduction in fetal zinc uptake. We suggest that this 6-MP-associated redistribution of zinc into maternal liver may be due to induction of maternal metallothionein synthesis by the drug.

Effect of zinc or copper deficiency on erythrocyte purine nucleoside phosphorylase and pyruvate kinase activity in the rat.

The effect of deficiencies of zinc or copper in the rat on the activity of purine nucleoside phosphorylase and pyruvate kinase in erythrocytes, liver and muscle was investigated. The data showed no effect of either zinc or copper deficiencies on the activity of these enzymes in the tissues tested. It is concluded that these enzymes are not useful markers for either zinc or copper deficiency.

Effect of kainate-induced seizures on tissue trace element concentrations in the rat.

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.

Studies of marginal zinc deprivation in rhesus monkeys. III. Use of liver biopsy in the assessment of zinc status.

Studies of marginal zinc deficiency in rhesus monkeys have demonstrated that plasma Zn levels are often a poor indication of Zn status. To better assess the Zn status of these animals, we examined their liver concentration of Zn as well as of other minerals, metallothionein (MT), and superoxide dismutase (SOD). Liver-wedge biopsies were obtained from adult rhesus monkeys fed for 15 mo, either a control (100 micrograms Zn/g) or a marginally Zn deficient diet (4 micrograms/g; ZD). Liver Zn and MT concentrations were lower in ZD monkeys than in controls whereas iron concentration was higher in ZD monkeys than in controls. Liver copper, manganese, and magnesium concentrations and activities of CuZnSOD and MnSOD were similar in the two groups. Data from the groups were pooled for regression analysis. Measurement of liver Zn and MT concentrations are useful in the assessment of the effects of long-term Zn deprivation in primates.

Studies of marginal zinc deprivation in rhesus monkeys. VIII. Effects in early adolescence.

Arrested adolescent growth and sexual maturation are striking symptoms of severe dietary zinc deprivation. More general implications of mild or marginal Zn deficiency during adolescence are not known. Five marginally Zn-deprived (ZD) male monkeys (4 mg Zn/kg diet) were compared with five controls pair fed a diet containing 100 mg Zn/kg during early adolescence. Mean plasma Zn levels were 38% lower in ZD group than in controls when evaluations began. During rapid growth plasma Zn decreased in controls but not ZD animals. ZD animals had delayed onset of accelerated weight gain and linear growth; loss of subcutaneous fat typical of early adolescence did not occur. ZD monkeys required two to three times more trials for both learning and reversal a visual discrimination task. Immune function was depressed 20-30% as reflected in early reduced proliferative response of peripheral lymphocytes and later lower immunoglobulin levels. Marginal dietary Zn deprivation affects growth and function in adolescence without producing frank developmental retardation.

Marginal zinc deficiency affects maternal brain microtubule assembly in rats.

The possible physiological role of Zn in tubulin polymerization was studied. Tubulin assembly in vitro was characterized in brain supernatants from rats fed a marginally Zn-deficient diet (10 micrograms Zn/g) during pregnancy and lactation. The initial rate of tubulin polymerization was significantly lower (27%) in brain supernatants from the marginally Zn-deficient animals than from controls. This was associated with a lower Zn concentration in the brain supernatants from the marginally deficient rats. Total protein and tubulin concentrations were the same in the supernatants from both groups. These data show that Zn is necessary for normal tubulin assembly and indicate that one biochemical defect underlying brain alterations in Zn deficiency could be abnormal microtubule function.

The effect of variable magnesium intake on potential factors influencing endurance capacity.

Rats fed a magnesium (Mg) deficient diet have a lower endurance capacity than rats fed Mg adequate diets. The current study evaluates the effects of marginal, moderate, and severe Mg deficiencies on physiological and biochemical changes that may contribute to the reduced endurance capacity of Mg deficient rats. Variable levels of dietary Mg (400, 200, 100, 50 micrograms/g) were fed for 23 d to 5-wk-old male Osborne-Mendel rats. Indirect blood pressure and heart rate were measured during dietary treatment. Forty-eight hours after an endurance test, rats were killed and sampled for plasma glucose, insulin, and triglyceride levels. Organ weights, mineral and trace element concentrations, and carcass composition were determined. Blood pressure was lower in rats fed 50 and 100 ppm Mg during the first half of the study than in controls (400 ppm Mg). There were no significant differences in blood pressure among groups at the end of the study. Heart rate was not affected by dietary Mg intake. Plasma insulin was lowered by decreasing dietary Mg; however, plasma glucose and triglyceride concentrations were not affected by dietary Mg intake. Rats fed 100 and 50 ppm Mg diets had significantly higher calcium concentrations in plasma and gastrocnemius muscle than controls. Dietary Mg variably affected tissue trace element (iron, zinc, copper, and manganese) concentrations but did not affect Mg concentrations in any organ studied. Body composition was significantly altered by dietary Mg intake. In conclusion, variable Mg intake differentially affects the parameters evaluated. Thus, the decreased endurance capacity of the Mg deficient rat is apparently not the result of a single biochemical lesion but is likely to be multifactorial.

Gestational zinc deficiency amplifies the regulation of metallothionein induction in adult mice.

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.

Long-term marginal zinc deprivation in rhesus monkeys. IV. Effects on skeletal growth and mineralization.

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.

Mineral composition and zinc metabolism in female mice of varying age and reproductive status.

Zinc metabolism of adult female C57BL/6J mice varying in age and reproductive status was studied using 65Zn. Animals were injected intraperitoneally with isotope, and whole-body and tissue turnover of 65Zn was measured. Biological half-life of whole-body 65Zn for pregnant females was 129% of that for nonpregnant, nonlactating females of similar age (25.7 and 20.0 d, respectively). Conversely, the half-life of zinc was less for lactating (11.1 d) and aged (15.3 d) females than for either pregnant or nonpregnant, nonlactating young adult females. Retention of 65Zn was generally lower for all tissues of pregnant and lactating dams than for nonpregnant, nonlactating females except for brain and tibia. In aged females, specific activity at 20 d postinjection did not differ from that of nonpregnant, nonlactating young adult females for any tissue except bone. While specific activity of tibia increased during pregnancy and lactation, it was lower in aged females than in nonpregnant, nonlactating young adult females. Increased organ content of zinc resulted from changes in zinc concentration or in organ mass or both, and was supported by increased food intake. Thus, greater food intake for these groups than for young adult females contributed to the higher turnover of tissue 65Zn. Differences in concentrations of Cu, Ca and Mg were also observed among groups for some tissues; the significance of these differences and their relationship to zinc metabolism are not clear.

Altered mineral metabolism: a mechanism underlying the fetal alcohol syndrome in rats.

Excessive ethanol intake during pregnancy can cause birth defects in humans and is referred to as fetal alcohol syndrome (FAS). Because of the characteristic changes that are similar in FAS and zinc (Zn) deficiency, we have examined the role of Zn nutriture in the teratogenicity of ethanol in Sprague-Dawley rats. Female Sprague-Dawley rats were adapted to liquid diets containing Zn at 2 micrograms/ml (LZn), 30 micrograms/ml (AZn), or 300 micrograms/ml (HZn); ethanol contributed either 0% or 36% of kilocalories. Ethanol consumption resulted in reduced fetal growth and retarded skeletal development. Ethanol had no effect on whole body fetal Zn concentrations; however, copper (Cu) deficiency was induced in the HZn fetuses. Ethanol consumption resulted in higher than normal fetal liver CuZnSOD activity in the LZn and AZn groups. Fetuses from HZn dams showed no ethanol effect on CuZnSOD activity, suggesting that the low availability of Cu to the fetus prevented the increase in CuZnSOD activity in response to ethanol. The increase in the activity of fetal CuZnSOD in LZn and AZn groups is consistent with the concept that the metabolism of ethanol results in free radical generation in fetal tissue. Because excessive free radical levels may result in tissue damage, this may be one mechanism contributing to the expression of FAS.

Iron and zinc concentrations and 59Fe retention in developing fetuses of zinc-deficient rats.

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)

Zinc deficiency in anorexia nervosa.

Adolescents with anorexia nervosa were evaluated for clinical and biochemical evidence of zinc deficiency. To assess whether these patients would benefit from zinc supplementation, a double-blind, randomized, controlled trial was conducted. The mean zinc intake of the anorexic group calculated on the basis of three-day dietary records was 7.7 +/- 5.2 mg/day, which is significantly below the recommended daily allowance of 15 mg for adolescents (p less than 0.001). The mean urinary zinc excretion in the anorexic group was 257.1 +/- 212.7 micrograms/24 hours compared to 749.9 +/- 897.8 micrograms/24 hours in the control group (p less than 0.005). This result suggests that the zinc status of anorexia nervosa patients may be compromised due to an inadequate zinc intake. Zinc supplementation (50 mg elemental zinc/day) was followed by a decrease in the level of depression and anxiety as assessed by the Zung Depression Scale (p less than 0.05) and the State-Trait Anxiety Inventory (p less than 0.05), respectively. Our data suggest that individuals with anorexia nervosa may be at risk for zinc deficiency and may respond favorably after zinc supplementation.

Dietary magnesium intake influences exercise capacity and hematologic parameters in rats.

The effects of feeding varying concentrations of dietary Mg (50, 100, 200, or 400 ppm) for 22 days on exercise capacity and hematologic parameters were investigated in male rats. Five-week-old male Osborne-Mendel rats fed diets containing 50 or 100 ppm Mg developed transitory priapism and hyperemia, signs of Mg deficiency. Based on a treadmill test, these rats showed a markedly lower exercise endurance capacity (four hours) than rats fed the higher levels of dietary Mg (six hours). They also showed a mild macrocytic anemia. Consumption of a mineral water containing 85 ppm Mg prevented these signs of Mg deficiency. These results show that a reduction in exercise capacity can be an early effect of Mg deficiency. Hematologic changes that occurred with Mg deficiency such as macrocytic anemia may be responsible, at least in part, for the depression of exercise observed in these rats. These data are important because they illustrate the potential significance of even a marginal deprivation of dietary magnesium.

Increased methionine synthetase activity in zinc-deficient rat liver.

To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.