Copper deficiency and arachidonic acid enhance insulin secretion in isolated pancreatic islets from lean (FaFa) Zucker rats.

Based on reports that copper deficiency can result in altered arachidonic acid (AA) metabolism and the knowledge that AA can modulate insulin secretion, we investigated the interaction of copper deficiency and AA on glucose-stimulated insulin secretion by using isolated islets. Lean male (Fa/Fa) Zucker rats were fed either a copper-adequate (Cont) or low-Cu (Cu Defic) diet (8.7 vs. 0.5 micrograms copper/g diet) for 5 weeks. Pancreatic islets were isolated and incubated in medium containing 3.3, 8.3, or 16.7 mM glucose with or without 100 microM AA. Insulin secretion was measured after either 5 (phase I) or 45 (phase II) min of incubation. In the absence of AA, islets from Cu Defic rats secreted more insulin during phase I than those from Cont rats; phase II insulin secretion was similar between the groups. Exogenous AA treatment increased insulin secretion from islets during both phases of insulin secretion, regardless of dietary copper treatment. In the presence of AA, islets from Cu Defic rats secreted more insulin in phase I than did islets from Cont rats at the 3.3 and 8.3 mM glucose levels. Similar findings were made for phase II insulin secretion. Consistent with this, Cu Defic rats tended to have higher plasma insulin levels than Cont rats (472.6 vs. 199.6 pM). In summary, Cu deficiency increases insulin release in isolated islets; this response is particularly pronounced when AA is present in the incubation medium.

Rat embryos cultured under copper-deficient conditions develop abnormally and are characterized by an impaired oxidant defense system.

Rat embryos (gestation days 9.0 and 10.0) obtained from dams that were fed a Cu-adequate (8 micrograms Cu/g) or Cu-deficient (< 0.5 micrograms Cu/g diet were cultured for 48 hr in Cu-adequate (16.2 microM) or Cu-deficient (1.0 microM) rat serum. Control embryos cultured in control serum were morphologically normal. Embryos from Cu-deficient dams developed abnormally when cultured in Cu-deficient serum; the abnormalities included distended hindbrains, blisters, blood pooling, and cardiac defects. Control embryos cultured in Cu-deficient serum and Cu-deficient embryos cultured in control serum also showed abnormal development, but to a lesser degree than that of the Cu-deficient embryos cultured in Cu-deficient serum. To test the idea that the above abnormalities were due in part to free radical induced damage occurring secondary to an impaired oxidant defense system, a chemiluminescence assay was used to detect superoxide dismutase (SOD) activity in the cultured embryos. SOD activity was lowest in embryos cultured in Cu-deficient serum. When the Cu-deficient serum was supplemented with antioxidants (CuZnSOD or glutathione peroxidase), its teratogenicity was reduced. These data support the idea that an impaired oxidant defense system contributes to the dysmorphology associated with Cu deficiency. However, the Cu-deficient embryos also had low cytochrome c oxidase activity compared to control embryos--thus, multiple factors are likely contributing to Cu deficiency-induced abnormalities.

Marked and rapid decreases of circulating leptin in streptozotocin diabetic rats: reversal by insulin.

Evidence for regulation of circulating leptin by insulin is conflicting. Diabetes was induced in rats with streptozotocin (STZ; 40 mg.kg(-1).day(-1) x 2 days) to examine the effect of insulin-deficient diabetes and insulin treatment on circulating leptin. After 12 wk, plasma leptin concentrations in untreated rats were all < 0.4 ng/ml versus 4.9 +/- 0.9 ng/ml in control animals (P < 0.005). In rats treated with subcutaneous insulin implants for 12 wk, which reduced hyperglycemia by approximately 50%, plasma leptin was 2.1 +/- 0.6 ng/ml, whereas leptin concentrations were 6.0 +/- 1.6 ng/ml in insulin-implanted rats receiving supplemental injections of insulin for 4 days to normalize plasma glucose (P < 0.005 vs. STZ untreated). In a second experiment, plasma leptin was monitored at biweekly intervals during 12 wk of diabetes. In rats treated with insulin implants, plasma leptin concentrations were inversely proportional to glycemia (r = -0.64; P < 0.0001) and unrelated to body weight (P = 0.40). In a third experiment, plasma leptin concentrations were examined very early after the induction of diabetes. Within 24 h after STZ injection, plasma insulin decreased from 480 +/- 30 to 130 +/- 10 pM (P < 0.0001), plasma glucose increased from 7.0 +/- 0.2 to 24.8 +/- 0.5 mM, and plasma leptin decreased from 3.2 +/- 0.2 to 1.2 +/- 0.1 ng/ml (delta = -63 +/- 3%, P < 0.0001). In a subset of diabetic rats treated with insulin for 2 days, glucose decreased to 11.7 +/- 3.9 mM and leptin increased from 0.5 +/- 0.1 to 2.9 +/- 0.6 ng/ml (P < 0.01) without an effect on epididymal fat weight. The change of leptin was correlated with the degree of glucose lowering (r = 0.75, P < 0.05). Thus insulin-deficient diabetes produces rapid and sustained decreases of leptin that are not solely dependent on weight loss, whereas insulin treatment reverses the hypoleptinemia. We hypothesize that decreased glucose transport into adipose tissue may contribute to decreased leptin production in insulin-deficient diabetes.

Altered zinc metabolism contributes to the developmental toxicity of 2-ethylhexanoic acid, 2-ethylhexanol and valproic acid.

It has been hypothesized that the developmental toxicity of certain compounds is, in part, due to maternal toxicity resulting in alterations in zinc (Zn) metabolism that affects the developing conceptus. In the present work the effects of developmentally toxic doses of 2-ethylhexanoic acid (EHXA), 2-ethylhexanol (EHXO), and valproic acid (VPA) on Zn metabolism were investigated in the pregnant rat. In experiment 1, dams were intubated with EHXA (3.13, 6.25, 9.38 or 12.5 mmol/kg), EHXO (6.25, 9.38 or 12.5 mmol/kg), VPA (1.56, 3.13, 6.25 or 9.38 mmol/kg), or corn oil (control; 1.0 ml/kg) at 14:00 h on gestation day (GD) 11.5, intubated with 32 microCi 65Zn at 22:00 h, and then killed at 08:00 h on GD 12.5. At the higher dose levels of EHXA and EHXO, and at all dosages of VPA, the percentage of 65Zn retained in maternal liver was higher, while that in the embryos was lower, than in controls. Chemical-associated changes in 65Zn distribution were associated with increased maternal liver metallothionein (MT) concentrations. In experiment 2, dams were fed diets containing 1, 25 or 97 microg Zn/g from GD 0-16 and intubated with 3.5 mmol EHXA or 1.0 ml corn oil/kg/d from GD 8-15. Dams were killed on GD 16 or 19. High incidences of encephalocele and tail defects were noted in the GD 16 fetuses of EHXA-treated dams fed either the low or adequate Zn diet, the highest incidences being in the low Zn group. On GD 19 the incidence of tail defects tended to be higher in the EHXA groups than in oil-treated controls, the highest incidence occurring in the low Zn EHXA group. Encephalocele was only observed in the low Zn EHXA-treated group. Fetal weight and crown-rump lengths were decreased by EHXA treatment and low dietary Zn. The incidence of rib anomalies was higher in the EHXA-exposed groups than in their respective oil controls. In experiment 3, GD 10.5 embryos collected from control dams were cultured for 48 h in serum from control or EHXA-treated male rats fed 4.5 or 25.0 microg Zn/g diets. Embryos cultured in either EHXA or low Zn sera exhibited delayed development; the addition of Zn to these sera eliminated their developmental toxicity. These results support the hypothesis that certain chemicals which induce maternal toxicity act, in part, to influence embryonic Zn metabolism and trigger abnormal development. Importantly, the teratogenic effects of these chemicals can be modulated by dietary Zn intake.

Effect of copper deficiency on prenatal development and pregnancy outcome.

Copper deficiency during embryonic and fetal development can result in numerous gross structural and biochemical abnormalities. Such a deficiency can arise through a variety of mechanisms, including low maternal dietary copper intake, disease-induced or drug-induced changes in maternal and conceptus copper metabolism, or both. These issues are discussed in this article along with the use of in vitro embryo culture models to study the mechanisms underlying copper deficiency-induced teratogenesis. Current data suggest that changes in free radical defense mechanisms, connective tissue metabolism, and energy production can all contribute to the dysmorphogenesis associated with developmental copper deficiency.

Copper, lysyl oxidase, and extracellular matrix protein cross-linking.

Protein-lysine 6-oxidase (lysyl oxidase) is a cuproenzyme that is essential for stabilization of extracellular matrixes, specifically the enzymatic cross-linking of collagen and elastin. A hypothesis is proposed that links dietary copper levels to dynamic and proportional changes in lysyl oxidase activity in connective tissue. Although nutritional copper status does not influence the accumulation of lysyl oxidase as protein or lysyl oxidase steady state messenger RNA concentrations, the direct influence of dietary copper on the functional activity of lysyl oxidase is clear. The hypothesis is based on the possibility that copper efflux and lysyl oxidase secretion from cells may share a common pathway. The change in functional activity is most likely the result of posttranslational processing of lysyl oxidase. Copper is essential for organic cofactor formation in amine oxidases such as lysyl oxidase. Copper-containing amine oxidases have peptidyl 2,4,5 tri(oxo)phenylalanine (TOPA) at their active centers. TOPA is formed by copper-catalyzed oxidation of tyrosine, which takes place as part of Golgi or trans-Golgi processing. For lysyl oxidase, recent evidence (Science 1996;273:1078-84) indicates that as an additional step, a lysyl group at the active center of lysyl oxidase reacts with TOPA or its precursor to form lysyl tyrosylquinone.

Incorporation of copper into lysyl oxidase.

Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation.

Modulation of lysyl oxidase by dietary copper in rats.

Lysyl oxidase levels were estimated in rat tissues using an enzyme-linked immunosorption assay (ELISA) and a functional assay standardized against known amounts of purified lysyl oxidase. High concentrations of lysyl oxidase (> or = 150 micrograms/g of tissue or packed cells) were detected in connective tissues, such as tendon and skin. Values for aorta, kidney, lung and liver ranged from 30 to 150 micrograms/g of tissue; values for skeletal muscle and diaphragm were < 30 micrograms/g tissue. Purified rat skin lysyl oxidase catalyzed the release of 50-100 Bq of tritium per micrograms enzyme in assays that used 3H-elastin-rich substrates. In dense connective tissues, good agreement was obtained for the values from ELISA and those derived from measurements of functional activity in aorta, lung, skin and tendon (r2 > 0.9). When egg white-based experimental diets containing 2 or 10 micrograms/g added copper were fed to weanling rats, values for skin lysyl oxidase functional activity in the group fed 2 micrograms/g added copper were one-third to one-half the values for skin lysyl oxidase functional activity in rats fed 10 micrograms/g copper. This reduction in lysyl oxidase activity, however, had minimal effect on indices of collagen maturation in rat skin, e.g., collagen solubility in neutral salt and dilute acid or the levels of acid stable cross-links. Moreover, copper deficiency did not influence the steady-state levels of lysyl oxidase specific mRNA in rat skin or the apparent amounts of lysyl oxidase in rat skin as determined by ELISA. These observations underscore that the concentration of lysyl oxidase is relatively high in dense corrective tissues, and although decreasing dietary copper influences functional activity, there is little apparent effect on the production of lysyl oxidase protein.

Copper absorption, endogenous excretion, and distribution in Sprague-Dawley and lean (Fa/Fa) Zucker rats.

We previously observed a rapid reduction in plasma ceruloplasmin activity in lean Zucker (Fa/Fa) rats fed a marginal copper (Cu)-deficient diet compared to similarly fed obese Zucker (fa/fa) and lean Sprague-Dawley rats. In an effort to understand the mechanisms underlying this response, we utilized the isotope dilution method to investigate the absorption and excretion of Cu in lean Zucker rats fed control and marginal Cu diet. Sprague-Dawley (SD) and homozygous lean Zucker rats were fed either a Cu-adequate (Cont; 7.5 micrograms Cu/g diet) or a low Cu (Low; 1.1 micrograms Cu/g diet) casein-based diet for 23 d. Two weeks following initiation of the dietary treatment, each rat was injected intramuscularly (im) with 11.2 microCi of 67Cu. Urine and feces were collected daily. On the 9th d following isotope injection, rats were killed and tissues collected. Significant dietary effects were observed in the relative absorption and endogenous fecal excretion of 67Cu. The tissue distributions of nonisotopic Cu and 67Cu activity were also different between dietary treatments. Tissues from rats fed the low-Cu diet typically had high concentrations of 67Cu and low concentrations of nonisotopic Cu compared to controls. An increase in relative 67Cu absorption was evident for rats fed the low-Cu diet (57.2 and 39.3%, for SD Low, Zucker Low, respectively, and 17.9, and 28.5% SD Cont and Zucker Cont, respectively). Rats fed the low-Cu diet also had reductions in endogenous fecal excretion of 67Cu compared to their respective controls. Although strain effects were not evident for either percent Cu absorption or endogenous fecal Cu excretion, the relative adaptive changes appeared more marked for the Sprague-Dawley rats compared to the lean Zucker rats.

Drinking water source and reproductive outcomes in Sprague-Dawley rats.

As part of our work on the influence of water source on reproductive outcome, Sprague-Dawley rats were randomized to tap water, bottled water, or deionized water treatment groups, utilizing 160 animals per treatment; animals received the water prior to and during pregnancy. Rats were shipped in four batches (A-D). Batch effects were seen for several reproductive parameters. Because the tap water supply was interrupted by an earthquake resulting in an unbalanced design, primary analyses utilized only batches C and D, which included most of the tap water-treated rats. A treatment effect with respect to resorption frequency was seen that was marginally significant using a fixed-effects analysis of variance (P = 0.053), but not when batch was entered as a random effect (P = 0.36). The data were modeled by logistic regression, controlling for batch, litter size, and batch-treatment interaction. The odds ratio comparing tap to bottled water was 1.8 (95% CI 1.0 to 3.3, P = 0.05), which was similar to the epidemiologic result that prompted this study. The magnitude of this association varied by batch, and the difference in resorption frequency was within the range of variation seen for control animals. Although these findings do not justify public health action at this time, further investigation is warranted.

Maternal zinc deficiency, but not copper deficiency or diabetes, results in increased embryonic cell death in the rat: implications for mechanisms underlying abnormal development.

The mechanisms underlying the teratogenicity of maternal copper deficiency, zinc deficiency, and diabetes are largely unknown. Here we investigated whether these insults are associated with altered patterns of cell death in gestation day (GD) 11.0 rat embryos. Four weeks prior to mating, rats in the copper-deficient group (CuD) were fed a copper-deficient diet supplemented with the chelator, triethylenetetramine, to facilitate the depletion of tissue copper stores. Rats in this group were switched to a triethylenetetramine-free copper-deficient diet 1 week prior to mating. Dams in the diabetic and control groups were fed a control (8 micrograms copper, 25 micrograms zinc/g) diet throughout the study. On GD 3.0, one subset of the control dams was assigned to the zinc-deficient group (ZnD) and fed a zinc-deficient diet. A second subset of control dams was assigned to a restricted fed group and fed the control diet in quantities consumed by the zinc-deficient dams. Litters were taken by cesarean section on GD 11.0. Embryos were examined for gross morphology and assessed for patterns of cell death using Nile blue sulfate. Embryos from the CuD dams were characterized by edematous hindbrain. Embryos from the diabetic group were characterized by delayed development. Altered patterns of cell death were only detected in embryos from the ZnD dams. Within the ZnD group, embryos were either characterized by small size, edematous head region, and control patterns of cell death, or normal size, normal morphology, and increased cell death. These different patterns of morphology and cell death in the embryos of ZnD dams were associated with different patterns of maternal food intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary macronutrient composition influences tissue trace element accumulation in diabetic Sprague-Dawley rats.

The purpose of the present investigation was to study the role of two potential contributory factors, hyperphagia and alterations in fuel metabolism, on the development of tissue trace element accumulation in the experimentally induced diabetic rat. The role of increased mineral intake associated with diabetic hyperphagia on tissue trace element accumulation was evaluated by feeding control and diabetic rats high-carbohydrate (HC) diets which varied in Zn, Cu, Mn, and Mg concentrations. Diabetic rats were hyperphagic and had lower plasma Mg, and higher liver Zn, Cu, and Mn concentrations than control rats, regardless of dietary mineral intake. In a second study, diabetic hyperphagia was reduced by feeding control and diabetic rats a HC, high-fat (HF), or high-protein (HP) diet; the effects of altering diabetic metabolism on trace element status was studied. Liver Mn and Zn concentrations of diabetic rats fed the HF diet were lower than diabetic rats fed the HC diet and HP diet, and were similar to control rats. Liver Cu concentrations of diabetic rats fed the HF and HP diets were lower than diabetic rats fed the HC diet and were similar to control rats. While diabetic rats, in general, had higher plasma glucagon concentrations and lower percent body fat than control rats, diabetic rats fed the HF diet had similar plasma glucagon and percent body fat to control rats. These data suggest that tissue-specific biochemical needs, such as the need for metals as cofactors for enzymes, rather than hyperphagia per se, may drive the accumulation of trace elements in the diabetic animal.

Effect of maternal diabetes and dietary copper on fetal development in rats.

To test whether diabetes associated alterations in copper metabolism contribute to diabetes-induced teratogenicity in rats, pregnancy outcome was compared between diabetic and nondiabetic rats fed either a copper adequate (12 micrograms/g diet) or low copper diet (1 microgram/g diet). The dietary regimen was begun two weeks prior to mating and continued throughout pregnancy. To facilitate the reduction of maternal copper stores in the low copper groups, the low copper diet was supplemented with a copper chelator, triethylenetetraamine, at 1% for one week; the chelator was removed from the diet one week prior to mating. Pregnancy was terminated on gestation day 20. Maternal and fetal tissues were assessed for copper concentrations, the activities of the cuproenzymes copper, zinc superoxide dismutase and ceruloplasmin, and the copper binding protein metallothionein. Dams fed the low copper diet had low tissue copper concentrations, and low plasma ceruloplasmin and erythrocyte superoxide dismutase activities compared to copper-adequate dams. Fetuses in the low copper groups were characterized by low liver copper concentrations. Gross structural and skeletal anomalies were only observed in the diabetic groups; maternal copper intake did not influence the frequency of these anomalies. However, fetuses in the low-copper nondiabetic group, and both diabetic groups, were characterized by low liver copper, zinc superoxide dismutase activity suggesting that fetal copper metabolism was influenced by both copper intake and diabetes.

Effect of the severity of maternal zinc deficiency on pregnancy outcome and infant zinc status in rhesus monkeys.

To investigate the effects of the severity of maternal zinc deficiency on early development, rhesus monkeys were fed diets that were either moderately zinc-deficient (MZD) (2 micrograms Zn/g) or marginal in zinc (M) (4 micrograms Zn/g) throughout pregnancy and lactation. Dams in the MZD group developed overt signs of zinc deficiency. Compared with control dams fed diets adequate in zinc (C) (50 or 100 micrograms Zn/g), both M and MZD dams showed low mitogen response. Pregnancy outcome was similar in all groups, and infants were considered healthy at delivery. From birth until d 30, infants were closely monitored for signs of zinc deficiency. On d 30, infants were killed and tissues were analyzed for several parameters reported to be affected by zinc status. MZD infants tended to have lower plasma zinc concentrations than C infants, although the difference was only significant at d 14. M infants tended to have lower plasma zinc concentrations than C infants. Mitogen response was lower in MZD and M infants than in C infants. However, mitogen responses were similar in MZD and M infants. Liver zinc concentrations were similar among the three groups of infants; however, zinc and metallothionein concentrations in (10,000 x g) liver supernatant fractions were lower in the MZD and M groups than in the C group. 65Zn absorption/retention was higher in MZD and M mothers and infants than in C mothers and infants; there were no marked differences between MZD and M mothers or infants. In contrast to whole-body absorption, 65Zn uptake/retention by isolated hepatocytes was similar among the three infant groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and accumulation of lysyl oxidase, elastin, and type I procollagen in human Menkes and mottled mouse fibroblasts.

Menkes syndrome in humans is an X-linked disorder characterized in part by abnormal copper transport, cellular copper sequestration, and defective crosslinking of collagen and elastin. A decrease in the functional activity of lysyl oxidase, a cuproenzyme, is thought in part to be responsible for the decreased crosslinking of collagen and elastin. It has also been suggested that low levels of lysyl oxidase activity may occur secondarily to disturbances in intracellular copper translocation and consequently impaired incorporation of copper into lysyl oxidase. Herein, we examine the expression and accumulation of selected extracellular matrix proteins in fibroblasts from a Menkes patient, as well as fibroblasts from the tortoiseshell (MoTo/y) mouse. The MoTo mutation is an allele of the mottled (Mo) locus, which is considered to be a murine analog of the human Menkes locus. In both Menkes and tortoiseshell fibroblasts, levels of lysyl oxidase mRNA transcripts were less than 15% of levels for corresponding controls. The level of elastin mRNA transcripts was also markedly lower in both cell lines in comparison to controls. In contrast, the levels of procollagen Type I mRNA were similar or enhanced in Menkes and MoTo/y fibroblasts compared to their respective controls. Consequently, we conclude that the connective tissue defects associated with Menkes syndrome and those occurring in mottled mouse mutants involve more than abnormal copper utilization in the formation of lysyl oxidase holoenzyme. Based on the present studies in cell culture, the production of essential enzymes and matrix proteins, such as lysyl oxidase and elastin, appear to be altered at the level of transcription or mRNA turnover.

65zinc metabolism is altered during diabetic pregnancy in rats.

To assess further diabetes-induced alterations in gestational Zn metabolism we examined the uptake/retention and distribution of 65Zn during pregnancy. Control and streptozotocin-induced diabetic rats, with and without insulin treatment, were gavaged with 65Zn on gestational d 12 or 18. Six hours post-intubation, untreated diabetic dams at d 12 of gestation had a lower percentage of retained 65Zn in bone, plasma, erythrocytes, pancreas and spleen than did controls. Retention of 65Zn in amniotic fluid and sacs and in embryos was similar among groups. Untreated diabetic dams at d 18 of gestation had a higher percentage of retained 65Zn in liver, intestine and urine, and most notably a lower percentage in amniotic fluid, placenta, fetus and fetal liver. Lower percentages of 65Zn were found in bone, muscle, plasma, erythrocytes, lung, spleen, heart, pancreas and uterus of diabetic dams compared with controls. HPLC fractionation of samples from maternal livers showed a higher percentage of Zn, Cu and 65Zn associated with the metallothionein peak in samples from untreated diabetic dams compared with other groups, whereas the percentages of Zn, Cu and 65Zn per fraction in fetal liver were similar. The marked decrease in retained 65Zn in products of conception at d 18 suggests either decreased placental transport or altered maternal and/or fetal Zn-binding ligands. Insulin treatment significantly reversed the streptozotocin diabetes-induced derangements in maternal and fetal Zn metabolism.

The effects of water source on reproductive outcome in Fischer-344 rats.

To investigate the relation between maternal water consumption and pregnancy loss, we conducted a reproductive toxicology study using Fischer rats. On day 0 of gestation, dams were assigned to tapwater, commercial bottled water, or purchased high pressure liquid chromatography grade water. On day 20 of gestation, we killed the dams and removed their litters for examination. There were no marked effects of dietary water source on maternal body weight, number of implantation sites, number of live fetuses, sex of offspring or number of resorptions per litter. Fetal length and weight, placenta weight, and soft tissue and skeletal malformations were also similar among groups. The tapwater group had somewhat higher resorption frequency (5.3%) and frequency of affected fetuses (6.5%) than those fed bottled water (3.8% and 4.5%, respectively).

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.

Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin.

Lysyl oxidase (protein-lysine 6-oxidase; EC 1.4.3.13) is a copper-containing enzyme that functions extracellularly and catalyses the oxidative deamination of peptidyl lysine. Lysyl oxidase was purified 150-175-fold from urea extracts of rat skin and uteri. Features of the enzyme were similar to those reported previously for lysyl oxidase obtained from rat aorta and bovine ligamenture. However, both approximately 40 and approximately 32 kDa polypeptide chains could be isolated from rat skin with apparent lysyl oxidase activity. Antibodies raised in chickens against the approximately 40 kDa form of lysyl oxidase detected the approximately 32 kDa form in immunoblots. Consequently it is inferred that the approximately 32 kDa form of lysyl oxidase is processed from the approximately 40 kDa form of the enzyme. The antibodies were also used to prepare anti(rat lysyl oxidase) affinity columns to facilitate the separation of lysyl oxidase from other proteins in studies to assess the extent to which lysyl oxidase serves as a reservoir for skin copper. At 16 h after an oral dose of copper, as 67Cu, about 6-8% of the total 67Cu incorporated into rat skin was found in association with lysyl oxidase. The lysyl oxidase concentration in rat skin was 2.5-7.5 nmol/g (determined by e.l.i.s.a.). Changing the copper status of rats by feeding a diet deficient in copper did not appear to influence lysyl oxidase accumulation in skin nor the percentage of incorporation of 67Cu in skin as lysyl oxidase. However, when rats were deprived of copper, the functional activity of lysyl oxidase in skin was one-third to one-half the normal values.

Influence of marginal maternal zinc deficiency on pregnancy outcome and infant zinc status in rhesus monkeys.

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)