The impact of micronutrient supplementation in alcohol-exposed pregnancies on reaction time responses of preschoolers in Ukraine.

The potential of micronutrients to ameliorate the impact of prenatal alcohol exposure (PAE) on attentional regulation skills was explored in a randomized clinical trial conducted in Ukraine. Women who differed in prenatal alcohol use were recruited during pregnancy and assigned to one of three groups [No study-provided supplements, Multivitamin/Mineral Supplement (MVM), or MVM plus Choline]. Their offspring were seen in the preschool period and a reaction time task was administered. Participants were asked to press a response button as quickly as possible as 30 stimuli from the same category (animals) were presented consecutively and then followed by six stimuli from a novel category (vehicles). Number correct, mean latency of the response over trials, and variability in the latency were analyzed separately by sex. During the initial animal trials, boys whose mothers received MVM during pregnancy had more correct responses and reduced response latency compared to boys whose mothers had no MVM treatment. During vehicle trials, maternal choline supplementation was associated with increased response speed in males without a PAE history. Females receiving supplements did not show the same benefits from micronutrient supplementation and were more adversely impacted by prenatal alcohol exposure. Relationships between maternal levels of choline, betaine, and dimethylglycine (DMG) and task performance were also assessed. Although no effects were found for choline after adjusting for multiple comparisons, lower baseline DMG level was associated with greater accuracy and shorter latency of responses in the initial animal trials and shorter latency in the vehicle trials in female preschoolers. Level of betaine in Trimester 3 was associated with reduced variability in the latency of male responses during the animal trials. Maternal micronutrient supplementation in pregnancy appears to improve preschool reaction time performance, but the effects varied as a function of sex and PAE exposure status.

Copper supplementation in quaking mutant mice: reduced tremors and increased brain copper.

Mice homozygous for the mutant gene quaking (qk) with a high frequency of axial tremors had a low concentration of copper in the brain. Supplementation during pregnancy and lactation with a high level of dietary copper greatly reduced the frequency of tremors and brought brain copper level to normal in the off-spring. It is suggested that qk affects copper metabolism.

Implications of altered maternal cytokine concentrations on infant outcomes in children with prenatal alcohol exposure.

Excessive alcohol consumption has been shown to increase serum plasma levels of numerous immune cytokines. Maternal immune activation and elevated cytokines have been implicated in certain neurological disorders (e.g., autism and schizophrenia) in the offspring. We investigated the hypothesis that elevated cytokines during pregnancy are a risk factor in women who gave birth to a child with Fetal Alcohol Spectrum Disorder (FASD) or a child with neurobehavioral impairment, regardless of prenatal alcohol exposure. Moderate to heavy alcohol-exposed (AE) (N = 149) and low or no alcohol-exposed (LNA) (N = 92) women were recruited into the study during mid pregnancy (mean of 19.8 ± 5.8 weeks' gestation) in two regions of Ukraine: Khmelnytsky and Rivne. Maternal blood samples were obtained at enrollment into the study at early to mid-pregnancy and during a third-trimester follow-up visit and analyzed for plasma cytokines. Children were examined at 6 and/or 12 months of age and were classified as having FASD if their mothers reported alcohol use and if they had at least one standardized score (Bayley Scales of Infant Development II Mental Development Index [MDI], or Psychomotor Development Index [PDI]) below 85 with the presence or absence of physical features of FASD. In multivariate analyses of maternal cytokine levels in relation to infant MDI and PDI scores in the entire sample, increases in the ratio of TNF-α/IL-10 and IL-6/IL-10 were negatively associated with PDI scores at 6 months (p = 0.020 and p = 0.036, respectively) and 12 months (p = 0.043 and p = 0.029, respectively), and with MDI scores at 12 months (p = 0.013 and p = 0.050, respectively). A reduction in the odds ratio of having an FASD child was observed with increasing levels of IL-1ß, IL-2, IL-4, IL-6, and IL-10 in early to mid-pregnancy and IL-1ß and IL-10 during late pregnancy. However, women that failed to increase IL-10 levels in the third trimester in order to maintain the balance of pro- and anti-inflammatory cytokines had an elevated risk of having an FASD child, specifically a significant increase in the odds ratio of FASD with every one-unit log increase in late pregnancy TNF-α/IL-10 levels (aOR: 1.654, CI: 1.096-2.495, p = 0.017). These data support the concept that disruptions in the balance between pro- and anti-inflammatory cytokines may contribute to neurobehavioral impairment and alter the risk of FASD.

Evaluation of negative staining technique for determination of CN--insensitive superoxide dismutase activity.

The different forms of superoxide dismutase (superoxide-superoxide oxidoreductase, EC 1.15.11) have been studied, in tissues of rat, mouse and chicken, by the ectrophoresis-nitro blue tetrazolium technique proposed by Beauchamp. Similar enzyme patterns were evident in every tissue. A fast migrating CN--sensitive form of dismutase activity was present in isolated liver mitochondria of each species. Chicken and mouse liver mitochondria, as well as whole homogenate of every tissue of these two species, showed two additional slow-migrating bands of CN--insensitive activity. In contrast, such bands were not detectable in mitochondria isolated from rat liver or in any of the rat tissues analyzed by this technique. Prior to their electrophoretic separation, the samples were analyzed for CN--insensitive superoxide dismutase activity by a spectrophotometric assay; by this assay it was possible to demonstrate and quantitate a CN--insensitive superoxide dismutase activity in every preparation. Two units of CN--insensitive activity were applied to the gels for each sample. These results indicate that the electrophoresis-nitro blue tetrazolium technique is unsuitable for the detection of the rat CN--insensitive form of superoxide dismutase in crude preparations such as whole tissue homogenates or isolated mitochondria.

Folate and homocysteine metabolism in copper-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.

Influence of maternal dietary Zn intake on expression of diabetes-induced teratogenicity in rats.

Diabetic rat pregnancies are characterized by altered maternal and fetal Zn metabolism and a higher frequency of fetal malformations. In this study, the effect of varying maternal dietary Zn on pregnancy and fetal outcome and on maternal and fetal trace element status were investigated. Starting on day 0 of gestation, streptozocin-induced diabetic and nondiabetic control rats were fed a low-Zn diet (4.5 micrograms/g diet), an adequate-Zn diet (24.5 micrograms/g diet), or a high-Zn diet (500 micrograms/g diet) throughout gestation. Fetuses were taken by cesarean section on gestation day 20. Fetuses from diabetic dams were smaller, weighed less, and had less calcified skeletons and more malformations than fetuses from control dams. In the controls, maternal dietary Zn had a minor effect on fetal malformation frequency. In contrast, in the diabetic animals, the low-Zn diet had a strong teratogenic effect. In diabetic dams, the adequate- and high-Zn diets improved fetal length and weight more than it did in fetuses from nondiabetic dams. However, supplemental dietary Zn during diabetic pregnancy did not further improve malformation frequencies. Liver and kidney Zn, Cu, and metallothionein concentrations were higher in diabetic dams than in control dams. In contrast, liver Zn, Cu, and metallothionein concentrations in fetuses of diabetic dams were lower than in fetuses from control dams, regardless of maternal dietary Zn intake. These results show that diabetes during pregnancy can amplify the teratogenic effects of a mild maternal Zn deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of maternal diabetes on trace element status and fetal development in the rat.

Despite improvements in prenatal care, there is a high incidence of congenital malformations in diabetic pregnancies. Not only is the diabetic patient characterized by a disorder of total fuel metabolism, but abnormal trace element metabolism occurs as well. In the present study, maternal and fetal zinc (Zn), copper (Cu), manganese (Mn), iron (Fe), magnesium (Mg), and calcium (Ca) status has been studied in Sprague-Dawley (SD) and Wistar rats. In addition, the effect of maternal diabetes on fetal development was also investigated. Rats were injected 27 days before mating with streptozocin (STZ, 45 mg/kg) in citrate buffer. On day 20 of gestation, litters were taken by cesarean section. Fetuses from diabetic dams weighed less, and had shorter crown-rump lengths and larger placentas than fetuses from controls. Evaluation of fetal skeletal development revealed fewer calcified sternal sites, anterior phalanges and caudal vertebrae, and an increased frequency of malformations in fetuses of diabetic dams. In dams, diabetics had larger adrenals, kidneys, and liver, and smaller thymus. Abnormal trace element metabolism was evident in diabetic dams and their fetuses. Mn was elevated in maternal liver, kidney and placenta of diabetic animals as well as in fetal liver of pups from diabetic dams. Maternal Cu and Zn levels were also higher in the liver and kidney of diabetic rats. In contrast, fetal liver Zn from fetuses of diabetic mothers was significantly decreased when compared with controls. These results suggest that diabetes may have induced fetal Zn deficiency. If this deficiency is present during embryogenesis/organogenesis, this could be one of the mechanisms of the teratogenicity of the diabetic state.

Copper, zinc, manganese, and magnesium status and complications of diabetes mellitus.

OBJECTIVE: To evaluate copper, zinc, manganese, magnesium, and other indices of peroxidative status in diabetic and nondiabetic human subjects. RESEARCH DESIGN AND METHODS: Convenience sample of 57 insulin-dependent or non-insulin-dependent diabetic subjects recruited from the diabetes clinic of the University of California, Davis, Medical Center and 28 nondiabetic subjects recruited from the staffs of the Departments of Internal Medicine and Nutrition. Individuals conducting laboratory analyses were blind to subject group. A fasting blood sample was collected from all subjects and appropriately processed for future analyses. A 24-h urine collection was obtained in a subset of subjects. RESULTS: Hyperzincuria and hypermagnesuria were evident in diabetic subjects compared with control subjects. There were no differences in plasma magnesium or whole-blood manganese between groups. Plasma copper was higher and plasma zinc was lower in diabetic than in control subjects. When data were viewed with respect to specific diabetes-associated complications, diabetic subjects with retinopathy, hypertension, or microvascular disease had higher plasma copper concentrations compared with both diabetic subjects without complications and with control subjects. There were no significant differences between control and diabetic subjects in erythrocyte copper-zinc superoxide dismutase activity or whole-blood glutathione peroxidase or glutathione reductase activities. Plasma peroxide concentrations were higher in diabetic than control subjects. CONCLUSIONS: Diabetes can alter copper, zinc, magnesium, and lipid peroxidation status. Perturbations in mineral metabolism are more pronounced in diabetic populations with specific complications. It is not known whether differences in trace element status are a consequence of diabetes, or alternatively, whether they contribute to the expression of the disease.

Hematologic and trace element alterations following chronic maternal ingestion of propylthiourea.

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.

The impact of micronutrient supplementation in alcohol-exposed pregnancies on information processing skills in Ukrainian infants.

The potential of micronutrients to ameliorate the impact of prenatal alcohol exposure (PAE) was explored in a clinical trial conducted in Ukraine. Cardiac orienting responses (ORs) during a habituation/dishabituation learning paradigm were obtained from 6 to 12 month-olds to assess neurophysiological encoding and memory. Women who differed in prenatal alcohol use were recruited during pregnancy and assigned to a group (No study-provided supplements, multivitamin/mineral supplement, or multivitamin/mineral supplement plus choline supplement). Heart rate was collected for 30 s prior to stimulus onset and 12 s post-stimulus onset. Difference values (∆HR) for the first 3 trials of each condition were aggregated for analysis. Gestational blood samples were collected to assess maternal nutritional status and changes as a function of the intervention. Choline supplementation resulted in a greater ∆HR on the visual habituation trials for all infants and for the infants with no PAE on the dishabituation trials. The latency of the response was reduced in both conditions for all infants whose mothers received choline supplementation. Change in gestational choline level was positively related to ∆HR during habituation trials and levels of one choline metabolite, dimethylglycine (DMG), predicted ∆HR during habituation trials and latency of responses. A trend was found between DMG and ∆HR on the dishabituation trials and latency of the response. Supplementation did not affect ORs to auditory stimuli. Choline supplementation when administered together with routinely recommended multivitamin/mineral prenatal supplements during pregnancy may provide a beneficial impact to basic learning mechanisms involved in encoding and memory of environmental events in alcohol-exposed pregnancies as well as non- or low alcohol-exposed pregnancies. Changes in maternal nutrient status suggested that one mechanism by which choline supplementation may positively impact brain development is through prevention of fetal alcohol-related depletion of DMG, a metabolic nutrient that can protect against overproduction of glycine, during critical periods of neurogenesis.

Dynamics of insulin and glucagon release in rats: influence of dietary manganese.

The effect of manganese on endocrine pancreatic function was examined in manganese-sufficient (control) and manganese-deficient (Mn-) Sprague-Dawley rats. Pancreatic insulin release was lower (P less than 0.05) in Mn- rats than in controls in response to both a 300 mg/dl and a 100 mg/dl glucose stimulus. The 300 mg/dl glucose stimulus induced the synthesis of 19.4 micrograms insulin/g pancreas in control rats. Additionally, no appreciable intracellular degradation of insulin occurred over an 80-min perfusion period. By contrast, in Mn- rats, there occurred an intracellular insulin degradation amounting to 7.8 micrograms/g pancreas. This enhanced degradation was partially compensated by a net insulin synthesis of only 3.4 micrograms insulin/g pancreas. Initial (min 1-3) insulin release by Mn- rats in response to 10 mM arginine was lower (P less than 0.05) than that observed in controls. Pancreatic glucagon release in response to 10 mM arginine was not affected by manganese deficiency. These findings demonstrate that manganese deficiency results in depressed pancreatic insulin synthesis and enhanced degradation. These factors may be responsible for the abnormal carbohydrate metabolism observed in Mn- animals.

Hypertension-induced alterations in copper and zinc metabolism in Dahl rats.

It has been suggested that one risk factor in the development of hypertension and vascular disease may be abnormal copper and zinc metabolism. In the current study we tested the hypothesis that hypertension itself may result in alterations in the metabolism of these essential elements. Dahl salt-sensitive rats were fed diets containing 0.4 or 8.0% NaCl for 32 days. At the conclusion of the study, blood pressure was significantly higher in the rats fed a high NaCl diet than in controls. Liver, kidney, and heart copper concentrations were significantly lower in the rats fed a high NaCl diet compared with controls, while plasma copper levels were higher. In contrast, tissue zinc levels were higher in the rats fed a high NaCl diet than in controls, while plasma zinc levels were lower. It is hypothesized that alterations in copper and zinc metabolism may be one factor underlying tissue damage in these animals.

Influence of hypertension and dietary copper on indexes of copper status in rats.

The Dahl salt-sensitive rat was used to investigate the effect of hypertension on indexes of copper status and to determine the extent to which dietary manipulation of copper attenuated, or exacerbated, the rate of sodium chloride-induced hypertension. Weanling salt-sensitive rats were fed, in a 2 x 3 factorial design, one of six diets that contained one of three levels of copper (2.0 micrograms/g marginal, 12 micrograms/g adequate, or 50 micrograms/g supplemental) and either control (0.4%) or high (4%) levels of sodium. Diets were fed to the rats for 11 weeks. Rats fed the high sodium diets were characterized by high plasma copper concentrations and ceruloplasmin activities compared with their respective control sodium rats. The magnitude of the sodium-induced rise in plasma copper and ceruloplasmin was affected by dietary copper intake; however, dietary copper intake had no effect on the development of hypertension in the high sodium groups. These results suggest that altered copper metabolism is secondary, rather than primary, to the development of sodium chloride-induced hypertension in the salt-sensitive rat. Red blood cell superoxide dismutase activity was reduced in rats fed the low copper diets compared with the adequate and supplemented copper groups. At the lower levels of copper intake, sodium chloride-induced hypertension increased red blood cell superoxide dismutase activity in a manner consistent with the plasma copper and ceruloplasmin changes observed. However, at adequate or supplemental levels of dietary copper, red blood cell superoxide dismutase activity plateaued, suggesting possible saturation of copper at sites of hematopoeisis.

Zinc deficiency induces oxidative stress and AP-1 activation in 3T3 cells.

It has been postulated that one mechanism underlying zinc deficiency-induced tissue alterations is excessive cellular oxidative damage. In the present study we investigated if zinc deficiency can induce oxidative stress in 3T3 cells and trigger select intracellular responses that have been associated to oxidative stress. Cells were exposed to control media or to chelated media containing 0.5, 5, or 50 microM zinc for 24 or 48 h. The oxidative status of the cells was evaluated as an increase in the fluorescence of the probe 5(or 6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate (DCDCDHF). After 24 and 48 h of exposure, the fluorescence intensity was significantly higher (4- to 15-fold) in the 0.5 and 5 microM Zn groups compared to the 50 microM Zn and control groups. The activity of the antioxidant enzymes CuZn (CuZnSOD) and Mn (MnSOD) superoxide dismutases was significantly higher in the 0.5 and 5 microM Zn cells compared to the 50 microM Zn and control groups at both the 24 and 48 h time points. These higher activities were associated with higher levels of MnSOD mRNA. After 24 h in culture, the level of activated AP-1 was markedly higher in the 0.5 and 5 microM Zn cells than in the control (72 and 58%, respectively) and 50 microM Zn cells (73 and 60%, respectively). NF-kappaB binding activity was lower in the 0.5 and 5 microM Zn cells than in controls. Thus, oxidative stress is induced by zinc deficiency in 3T3 cells. This oxidative stress results in an upregulation of oxidant defense mechanisms.

Retinol binding protein expression is induced in HepG2 cells by zinc deficiency.

Zinc (Zn) deficiency is often associated with low plasma vitamin A (retinol) concentrations. It has been suggested that the reduction in plasma retinol is secondary to reduced liver retinol binding protein (RBP) synthesis. In the present study, RBP expression was determined in HepG2 cells cultured in either Zn adequate media or chelated media containing varying concentrations of Zn. Levels of RBP mRNA increased in a time- and Zn concentration-dependent manner such that 0.5 microM Zn-treated cells exhibited a >7.5-fold increase while cells treated with 15 microM Zn were increased 2.9-fold at 72 h compared to controls. RBP protein also progressively increased by 72 h to levels >8-fold and 3-fold higher than controls, in 0.5 microM and 15 microM Zn-treated cells, respectively. The increase in RBP occurred without any change in DNA concentration between groups through 72 h. The Zn deficiency-induced elevations in RBP transcript levels could be reversed within 24-48 h of repletion in Zn adequate media. Thus, the reductions in plasma retinol observed in Zn deficiency are in part a direct consequence of the deficiency.

Zinc absorption through skin: correction of zinc deficiency in the rat.

The therapeutic effect of topical administration of zinc was tested in pregnant rats consuming a diet deficient in the element. Four groups of rats were fed a zinc-deficient diet for 24 hr. Half of the animals were treated during this period with a topical application of oil saturated with zinc chloride, for the full 24 hr in one group, and for the last 8 hr in the other. In the two remaining groups, oil without zinc chloride was applied under the same conditions as described above, and in all cases oral ingestion of the supplement was prevented. At the end of the 24-hr period, the animals were killed and plasma zinc was determined. An additional group of animals consuming a diet adequate in zinc was killed without any treatment to provide control values of normal plasma zinc. Rats consuming the deficient diet and without topical zinc supplementation had plasma zinc values significantly lower than all other groups after 24 hr. Animals receiving zinc supplementation for 8 hr had plasma levels similar to those of the control group fed an adequate zinc diet and significantly higher than those of rats that received no zinc application to the skin. In animals in which zinc was applied for 24 hr, plasma zinc values were significantly higher than in any other group, including normal controls. The results indicate that percutaneous transport of zinc may be of sufficient magnitude to be clinically significant and that topical application of this element may be useful in cases of dietary zinc deficiency or diseases producing a zinc deficiency state.

Should vitamin-mineral supplements be recommended for all women with childbearing potential?

Interest in maternal nutritional status as a critical factor for prenatal development has increased. When supplemented before conception, iodine prevents cretinism and folic acid reduces neural tube defect risks. Other nutrient supplements may also reduce pregnancy complications. Thus, should supplements be advocated for all women with childbearing potential? Potential supplementation benefits include 1) improved nutritional status, 2) reduced risk of some developmental defects, 3) improved antioxidant and immune defense systems, 4) lower incidence and/or slower progression of some diseases, and 5) harmonization of government and health care professionals' dietary recommendations for optimal health. Potential questions are, will the supplement reduce a woman's motivation to maintain and/or improve dietary quality? Will the supplement result in excessive nutrient intakes and/or adverse nutrient-nutrient interactions? Will supplement use encourage the perception that all women are, by definition, well nourished? These issues should be clarified before widespread supplementation programs are implemented.

An experimental model for studies of zinc bioavailability from milk and infant formulas using extrinsic labeling.

The ability to assess zinc bioavailability from various diets is essential as the zinc content on many foods can be low or marginal. We have investigated the absorption of zinc from human milk, cows' milk, cows' milk formula (whey-adjusted) and soy protein formula as these fluids can comprise the majority of an infant's diet. Radiozinc was added to the diets in tracer amounts. The extrinsic 65Zn was shown by ultracentrifugation, ultrafiltration, and gel filtration to add to milk fractions and individual binding compounds in a manner analogous to the distribution of native zinc, validating the use of extrinsically labeled milk diets. Labeled diets were fed by intubation to 16-day-old suckling rats. Animals were killed after 4 h and tissues removed and counted. Zinc bioavailability was 28% from human milk, 24% from whey-adjusted cows' milk formula, 15% from cows' milk, and 10% from soy formula. Intubation studies using adult rats showed that zinc absorption was lower from all the diets; however, it was still highest from human milk and cows' milk formula. These results show that the rat pup model may provide a rapid, inexpensive, and sensitive method to assay bioavailability of zinc from infant foods.

Copper nitrilotriacetate: a potent therapeutic agent in the treatment of a genetic disorder of copper metabolism.

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.

Manganese binding proteins in human and cow's milk.

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.