Relative availability of zinc in ground beef and soybean protein for young swine compared with zinc carbonate as the standard.

The experimental objective was to determine the relative availability of endogenous Zn in soybean protein (SP) and cooked ground beef (GB) compared with the availability of Zn from ZnCO(3) for young swine. Crossbred pigs (4 trials with total n = 104) were weaned at an average age of 11 d, placed in metabolism cages, and allotted to 13 treatments. The basal semipurified negative control (NC) diet contained 2 mg of Zn/kg and wet-autoclaved spray-dried egg albumen (AEA) as the protein source. Reference diets were made by adding 3, 6, or 9 mg of Zn/kg from ZnCO(3) to the NC diet. The AEA in the NC diet was replaced with SP or GB to make diets with 3, 6, and 9 mg of Zn/kg from SP and GB. Positive control (PC) diets were made by adding 101 mg of Zn/kg from ZnCO(3) to the diets with 9 mg of Zn/kg from ZnCO(3), SP, or GB. Diets were mixed with deionized water and fed to appetite in gruel form every 3 h from 0800 to 2300 h. Pigs were weighed and blood samples were taken on d 7, 14, 21, and 28. Plasma concentrations of Cu, Fe, Zn, glucose, and urea N were determined. Total urine and fecal grab samples (Cr(2)O(3) in diets) were collected from d 7 to 14 and d 21 to 28 to determine Cu, Fe, Zn, and N apparent balance and energy utilization. On d 29, pigs were scored visually for parakeratosis and dosed orally with (65)Zn from ZnCl(2). Pigs were counted for whole body gamma radioactivity on d 29 and d 34 to 36, with Zn retention estimated by extrapolation to zero time. Pigs were killed on d 36 (NC on d 29). Samples of skin, tongue, and esophagus were taken for histological evaluation, and a tibia bone was excised for measurements and breaking strength. Pigs fed the diets with 9 mg Zn/kg from GB or ZnCO(3) were not different in Zn availability and retained about 70% Zn (apparent balance and (65)Zn) compared with 30% (P ≤ 0.05) for pigs fed the diet with 9 mg Zn/kg from SP. Also, pigs fed the diets with 9 mg Zn/kg from GB or ZnCO(3) had greater (P ≤ 0.05) growth performance, bone weight and breaking strength, % N retention, and % ME utilization, and smaller plasma urea N concentrations and parakeratosis scores (visual and histological) than pigs fed the diet with 9 mg Zn/kg from SP. Pigs fed the PC diets were not different in any response criteria. In conclusion, apparent Zn availability was not different from cooked GB and ZnCO(3), and Zn availability from cooked GB and ZnCO(3) was markedly greater than Zn availability from SP for young swine.

Neuropeptide Y fails to normalize food intake in zinc-deficient rats.

Zinc deprivation results in decreased and cyclic food intake in rats. We determined the response of zinc-deprived rats to neuropeptide Y (NPY). In a preliminary experiment, rats were fed a low (-Zn; <1 mg/kg) or adequate zinc diet (+Zn; 100 mg/kg) for 4 days. NPY (5 or 10 microg) was then administered via an intracerebroventricular (ICV) cannula and food intake measured for 4 h. NPY stimulated food intake in all rats, but the difference in food intake due to zinc deprivation persisted. In a subsequent experiment, rats were fed the low zinc and adequate zinc diets for 4, 5 or 6 days. Food intake was suppressed in rats fed the low zinc compared to the adequate zinc diet on all of these days. When NPY (10 microg) was administered at the onset of the light cycle, the food intake was approximately 2.5-fold greater regardless of dietary zinc status, but the amount of food consumed by rats fed low zinc was approximately one-half the quantity consumed by NPY-stimulated zinc-adequate rats. NPY administered at the onset of dark failed to stimulate food intake in either dietary group although the total intake difference due to zinc status persisted. ICV administration of 5 nmol of zinc prior to NPY injection failed to correct the food intake response of the zinc-deficient rats. We conclude that the basis of the reduced food intake of zinc-deficient rats does not relate to NPY quantity or release, or to impairment of its signal transduction. There appears to be another undefined factor that limits food intake in zinc deficiency.

Characterization of the region containing the jcpk PKD gene on mouse Chromosome 10.

The jcpk gene on mouse Chromosome 10 causes a severe, early onset form of polycystic kidney disease (PKD) when inherited in an autosomal recessive manner. In order to positionally clone this gene, high resolution genetic and radiation hybrid maps were generated along with a detailed physical map of the approximately 500-kb region containing the jcpk gene. Additionally, sixty-nine kidney-specific ESTs were evaluated as candidates for jcpk and subsequently localized throughout the mouse genome by radiation hybrid mapping analysis. Previous studies indicating non-complementation of the jcpk mutation and 67Gso, a new PKD translocation mutant had suggested that 67Gso represents a new allele of jcpk. Fluorescence in situ hybridization (FISH) analysis using key bacterial artificial chromosome clones from the jcpk critical region, refined the 67Gso breakpoint and provided support for the allelism of jcpk and 67Gso.

Role of zinc in plasma membrane function.

The concentration of plasma zinc is the generally accepted index of zinc status. Although low plasma zinc is an essential criterion of deficiency, alone it is inadequate. To supplement this index, we sought to determine the first limiting biochemical defect in animals fed zinc-deficient diets and concluded that the limiting function is associated with a posttranslational change in plasma membrane proteins. Among the signs of zinc deficiency in rats is a bleeding tendency associated with failure of platelet aggregation, a phenomenon that correlates with impaired uptake of Ca(2+) when stimulated. Zinc-deficient guinea pigs exhibit signs of peripheral neuropathy, and their brain synaptic vesicles exhibit impaired Ca(2+) uptake when they are stimulated with glutamate. Red cells from zinc-deficient rats show increased osmotic fragility associated with decreased plasma membrane sulfhydryl concentration. Both phenomena are readily reversed (2 d) by dietary zinc repletion. Volume recovery is dependent on Ca-dependent K channels and the sulfhydryl redox state. Both the impaired aggregation and calcium uptake of zinc-deficient platelets are corrected by in vitro incubation of blood with glutathione. Considering the fact that plasma membranes from several cell types show impaired function that is associated with a decreased rate of calcium uptake, it is postulated that a defect in calcium channels is the first limiting biochemical defect in zinc deficiency. The calcium uptake defect and consequent impaired second-messenger function likely results from an abnormal sulfhydryl redox state in the membrane channel protein.

Zinc Deprivation and the Nervous System.

This review is concerned with the role of zinc in the function of the nervous system with emphasis on the effects of dietary zinc deprivation. Zinc deficiency leads to several pathological signs, some of which occur within a few days in growing animals deprived of zinc. Depletion eventually leads progressively to more severe behavioral changes, to abnormal stance, and to peripheral neuropathy. The nervous system contains many essential zinc metalloproteins that are highly stable, i.e. have high zinc association constants. These proteins, which include enzymes, transcription factors and storage proteins, do not become limiting during incipient zinc deficiency. It is likely that other, yet unidentified, zinc dependent proteins become limiting within a few days after animals are deprived of dietary zinc and lead to behavioral changes such as decreased and cyclic feeding. One candidate for the first limiting zinc component of nerve tissue is the "chelatable" zinc pool. Another is the plasma membrane zinc pool; the latter pool is decreased in zinc deficient rat erythrocytes, leading to oxidation of protein thiol groups and malfunction of that membrane. While the defective biochemical mechanism(s) that leads to the signs of deficiency is not entirely clear, there is evidence that synaptosomes from zinc deficient guinea pigs do not take up calcium normally when stimulated. It is postulated that the most vulnerable zinc pool serves a protective role against oxidative damage to plasma membrane proteins including the proteins involved with calcium channels. If so, the first limiting role of zinc is protective rather than catalytic or that of a neurotransmitter.

Decreased plasma membrane thiol concentration is associated with increased osmotic fragility of erythrocytes in zinc-deficient rats.

Zinc deficiency leads to pathological signs that are related to impaired function of plasma membrane proteins. The purpose of this study was to assess the effect of dietary zinc status on the sulfhydryl (SH) content of erythrocyte plasma membranes and erythrocyte function. Three experiments were performed. In the first, immature male rats were fed for 21 d either a low-zinc (<1.0 mg/kg) diet free choice (-ZnAL), an adequate-zinc (100 mg/kg) diet free choice (+ZnAL), or the adequate-zinc diet limited to the intake of -ZnAL pair-mates (+ZnPF). Tail blood was sampled to measure osmotic fragility and SH concentration of erythrocyte membrane proteins. The zinc-deficient rats were then repleted for 2 d and erythrocytes assayed for fragility and SH content. In the second experiment blood was sampled at 3-d intervals to determine the time course of change in fragility and SH concentration. In the third experiment the SH concentration of erythrocyte band 3 protein and the binding of zinc to isolated plasma membranes were measured. SH concentration decreased from approximately 75 nmol/mg protein to 68 nmol/mg protein during 21 d of depletion and returned to control level within 2 d of repletion. There was an inverse relationship between osmotic fragility and SH concentration of erythrocyte membrane proteins. Maximal decrease in SH occurred within 6 d of consuming the low-zinc diet. The SH content of band 3 protein isolated from deficient rats was also significantly lower than that of pair-fed controls (45 vs. 51 nmol/mg protein). The zinc-binding affinity of plasma membrane proteins tended to be decreased by zinc deficiency. In summary, low-zinc status lowers the plasma membrane SH concentration, and the decreased reducing potential is inversely related to osmotic fragility, and presumably, with impaired volume recovery of erythrocytes.

Zinc deprivation of murine 3T3 cells by use of diethylenetrinitrilopentaacetate impairs DNA synthesis upon stimulation with insulin-like growth factor-1 (IGF-1).

Growth failure in zinc-deficient animals is associated with decreased DNA synthesis; zinc deprivation of 3T3 cells, by use of diethylenetrinitrilopentaacetate (DTPA), impairs thymidine incorporation when the cells are stimulated with fetal bovine serum (FBS). The purpose of this study was to determine the step of cell cycle progression that is affected by zinc deprivation. Swiss murine 3T3 cells were cultured for 3 d in complete media and then for 2 d in low serum media. Cells were then placed in serum-free media and stimulated in sequence with platelet-derived growth factor (PDGF; 3 h), epidermal growth factor (EGF; 0.5 h) and insulin-like growth factor-I (IGF-I; 16 h). The combination of growth factors stimulated thymidine incorporation to the same extent as 10% FBS, and DTPA or EDTA (0.6 mmol/L) inhibited thymidine incorporation. Inhibition was prevented by addition of zinc, but not calcium, iron or cadmium (0.4 mmol/L). When DTPA was present during all stages with no addition of zinc, or zinc added during the competency-priming (PDGF and EGF) step, the IGF-I step, or both steps, the zinc effect occurred at the IGF-I step. Zinc addition 4 h before the measurement of thymidine incorporation had no ameliorative effect, but the presence of zinc during the prior 12 h increased incorporation. Thus zinc exerts its major effect on DNA synthesis during the IGF-I stimulatory phase of the cell cycle. The total zinc concentration of 3T3 cells treated with DTPA for 16 h was not different from that of untreated cells; hence only a small compartment of the cell is affected by DTPA.

Chelation of extracellular zinc inhibits proliferation in 3T3 cells independent of insulin-like growth factor-I receptor expression.

Depletion of zinc inhibits growth in animals and proliferation of cultured cells. Additionally, zinc can serve as an antioxidant protecting many compounds, including proteins, from oxidation. Regulation of cell division also involves insulin-like growth factor type I (IGF-I) and its receptor, especially during late G1 phase, allowing progression of the cell to S phase with subsequent DNA synthesis. We examined the effects of zinc depletion from the culture media of Swiss 3T3 cells on the cell cycle and IGF-I receptor expression. Cells were exposed to reduced fetal bovine serum concentrations to induce growth arrest, then returned to normal fetal bovine serum concentrations with the divalent cation chelator diethylenetriamine pentaacetic acid. Reducing the fetal bovine serum concentration did not induce quiescence in the cells as previously suggested. Zinc depletion reduced the proliferative fraction (S and G2/M phases) of the cell cycle. The addition of glutathione to the zinc-depleted media partially returned the proliferative fraction to the control level. Fetal bovine serum deprivation reduced IGF-I receptor expression whereas the absence of zinc had little effect on receptor expression. We conclude that depletion of zinc from culture media inhibits 3T3 cell proliferation independent of insulin-like growth factor-I receptor expression, and part of this inhibition is due to the antioxidant capacity of this divalent cation.

Personal reflections on a galvanizing trail.

This article encompasses my perception of, and experience in, an exciting segment of the trace element era in nutrition research: the role of zinc in the nutrition of animals and humans. Zinc has been a major player on the stage of trace element research, and it has left a trail that galvanized the attention of many researchers, including myself. It is ubiquitous in biological systems, and it plays a multitude of physiologic and biochemical functions. A brief historical overview is followed by a discussion of the contributions the work done in my laboratory has made toward understanding the physiological and biochemical functions of zinc. The effort of 40 years has led to the belief that one of zinc's major roles, and perhaps its first limiting role, is to preserve plasma-membrane function as regards ion channels and signal transduction. Although substantial knowledge has been gained relating to the importance of zinc in nutrition, much remains to be discovered.

Reduced food intake in zinc deficient rats is normalized by megestrol acetate but not by insulin-like growth factor-I.

Zinc deficiency in rats results in impaired growth accompanied by decreased and cyclic food intake. These signs are associated with decreased plasma insulin-like growth factor-I (IGF-I), a major mediator of growth. The purpose of this study was to determine the relationship between decreased plasma IGF-I and the impairment of appetite and growth in zinc deficiency. Immature male rats were fed free choice a low zinc (<1 mg/kg) diet (-Zn) or a zinc adequate (100 mg/kg) control diet (+Zn). Plasma IGF-I concentrations were normalized in zinc-deficient rats by the following two methods: osmotic pump infusion of IGF-I (2.4 mg/kg body weight daily) and oral administration (50 mg/kg body weight twice daily) of the synthetic progestin, megestrol acetate (MA). Infusion of IGF-I for 8 d sustained plasma IGF-I concentrations in zinc-deficient rats at control levels but had no effect on either food intake or growth rate. MA administration for 8 d maintained the plasma IGF-I of deficient rats and significantly increased food intake. The early aspects of cyclic food intake were eliminated, and, after a few days, food intake of deficient rats given MA was not different than that of controls. MA increased food intake and fat deposition regardless of zinc status, but it had no effect on the growth rate of deficient rats. MA significantly decreased body weight of controls, uncoupling energy intake and gain. The results suggest that reduced food intake precedes the decreased plasma IGF-I concentration and that IGF-I is not responsible for the decreased growth and food intake of zinc-deficient rats. The appetite and growth impairment of zinc-deficient rats may arise from disrupted function of IGF-I receptors in the brain and peripheral tissues, but not from low circulating levels of IGF-I.

Morphophysiologic characterization of peripheral neuropathy in zinc-deficient guinea pigs.

Zinc-deficient guinea pigs develop a peripheral neuropathy characterized by abnormal posture and gait, hyperesthesia, slowed motor nerve conduction velocity (MNCV), and decreased sciatic nerve Na,K-ATPase activity. This study was designed to further investigate longitudinally the morphophysiologic features of the neuropathy. Weanling guinea pigs were fed a low-zinc (<1 mg/kg) diet ad libitum (-ZnAL), an adequate-zinc (100 mg/kg) diet ad libitum (+ZnAL), or the adequate diet restricted in intake ((+ZnRF). Electrophysiologic, morphologic, and biochemical parameters of peripheral nerves were examined at 2.5, 4.0, and 5.5 weeks. Serum zinc was significantly lower by 2.5 weeks and growth rate reduced by 4 weeks in -ZnAL animals. Postural abnormalities were first obvious at 4 weeks, although MNCVs were significantly slower in zinc-deficient animals at all time intervals. The conduction of sensory impulses, as measured by spinal cord somatosensory evoked potentials (sSSEP), was significantly slower in the -ZnAL animals at 5.5 weeks. Examination of teased preparations and histologic sections of sciatic nerves at 5.5 weeks revealed no degenerative lesions or differences in density of myelinated fibers (MF). The size frequency distribution of MF in all groups was unimodal, with a trend toward smaller myelinated nerve fibers in -ZnAL and +ZnRF animals. Sciatic nerve Na,K-ATPase activity in the -ZnAL animals was significantly reduced after 4 weeks of zinc deprivation. At 5.5 weeks, nerve concentrations of myo-inositol, glucose, fructose, and sorbitol were significantly decreased in -ZnAL animals compared with the +ZnRF and +ZnAL controls. The peripheral neuropathy associated with acute zinc deficiency is a parenchymatous axonal disorder characterized by slowed motor and sensory nerve impulse conduction and reduction in nerve Na,K-ATPase activity and nerve concentrations of simple sugars and their metabolites.

Endpoints for determining mineral element requirements; an introduction.

There are numerous measures of essential mineral element status, including growth rate, tissue and physiological fluid concentrations, enzyme concentrations and activities, chemical balance and mobilizable stores. For the adult human, blood and its specific nutrient concentrations provide a useful but frequently inadequate index. The first limiting biochemical system should provide the most valid index, but in many cases it is unknown or not readily measured. Chemical balance and mobilizable stores provide valid measures but are difficult to determine. Two indices are infinitely more valuable than one and should be determined if possible. More research is needed to establish valid indicators of nutritional status for mineral elements.

Design of a physiologic pulsatile flow cardiopulmonary bypass system for neonates and infants.

Cardiopulmonary bypass surgical techniques that allow a surgeon to operate on the infant's heart use an extracorporeal circuit consisting of a pump, oxygenator, arterial and venous reservoirs, cannulae, an arterial filter, and tubing. The extracorporeal technique currently used in infants and neonates is sometimes associated with neurologic damage. We are developing a modified cardiopulmonary bypass system for neonates that has been tested in vitro and in one animal in vivo. Unlike other extracorporeal circuits which use steady flow, this system utilizes pulsatile flow, a low prime volume (500 ml) and a closed circuit. During in vitro experiments, the pseudo patient's mean arterial pressure was kept constant at 40 mmHg and the extracorporeal circuit pressure did not exceed a mean pressure of 200 mmHg. In our single in vivo experiment, the primary objective was to determine whether physiologic pulsatility with a 10 F (3.3 mm) aortic cannula could be achieved. The results suggest that this is possible.

Zinc deficiency decreases the concentration of N-methyl-D-aspartate receptors in guinea pig cortical synaptic membranes.

Zinc deficiency in guinea pigs decreases glutamate-stimulated calcium uptake in cortical synaptosomes. Glutamate not only stimulates calcium uptake but also potentiates the binding of the drug dizocilpine (MK-801) to an internal site of the N-methyl-D-aspartate receptor/calcium channel, a subtype of the glutamate receptor. The purpose of this study was to determine whether the effect of zinc deficiency on calcium uptake by glutamate-stimulated synaptosomes is related to N-methyl-D-aspartate receptor number or function, as measured by MK-801 binding. Immature guinea pigs consumed a low zinc (< 1 mg/kg) diet ad libitum or an adequate zinc (100 mg/kg) diet, either ad libitum or restricted to maintain weight similar to that of the low zinc animals. Binding of MK-801 to cortical membranes was measured first in the presence of saturating concentrations of glutamate or N-methyl-D-aspartate in combination with glycine. Zinc deficiency significantly reduced the concentration of MK-801 binding sites (20%) regardless of the potentiating agonist used, but had no effect on binding affinity. The binding of MK-801 in response to 1, 10 and 100 mumol/L glycine, in the presence of 100 nmol/L glutamate, was then measured and found to be significantly reduced (12%). The results suggest that zinc deficiency decreases the number of functional N-methyl-D-aspartate receptor/channels in cortical membranes, probably because of impaired channel opening.

Chronological loss of bone zinc during dietary zinc deprivation in neonatal pigs.

This research was conducted to measure the chronological changes in zinc concentrations of biopsied bone, hair, and plasma samples collected weekly during dietary zinc deprivation. Pigs 1-2 wk of age were fed a basal diet (< 1 microgram Zn/g) during a 1-wk depletion period and then assigned to one of three dietary regimens for 4 wk: a low-zinc diet (4 micrograms Zn/g) fed ad libitum, an adequate-zinc diet (100 micrograms/g) fed ad libitum, and an adequate-zinc diet restricted in intake to allow eight gain comparable with that of the low-zinc group. Bone zinc remained at approximately 120 micrograms/g dry wt for the control groups fed adequate zinc but steadily declined in pigs fed the low-zinc diet, leveling off at approximately 25% of the control values. Plasma and hair zinc concentrations also decreased but at a more rapid rate. Bone zinc is mobilizable in neonatal pigs, and biopsied bone zinc concentration is a reliable index of zinc status.

Low zinc status in guinea pigs impairs calcium uptake by brain synaptosomes.

Zinc deficiency results in defective central nervous system function and in peripheral neuropathy. Calcium serves as second messenger in both pre- and postsynaptic membranes. Presynaptic uptake of calcium occurs via voltage-gated channels, whereas postsynaptic uptake occurs by way of a glutamate-activated channel, the N-methyl-D-aspartate (NMDA) receptor-channel. This study was designed to determine the effect of zinc status on calcium uptake by synaptic membranes prepared from guinea pigs deprived of zinc. Within each group of three guinea pigs, one animal was allowed to consume a low zinc (< 1 mg/kg) diet ad libitum (-ZN), one an adequate zinc (100 mg/kg) diet ad libitum (+AL), and one the adequate zinc diet restricted (+RF). When the -ZN guinea pig within a group developed clinical signs of deficiency, synaptosomes were prepared from brain cortices and calcium uptake measured by use of 45Ca. Both high potassium- and glutamate-stimulated calcium uptakes by synaptosomes from zinc-deficient guinea pigs were significantly lower than those of controls, with the glutamate-stimulated uptake 40% lower. In vitro addition of either magnesium or zinc resulted in lower uptake in synaptosomes from all dietary groups. Regardless of in vitro conditions, calcium uptake was impaired by zinc deficiency. The impaired function of calcium channels may explain the neurological disturbances observed in zinc-deficient animals.

Fructose and mineral metabolism.

Fructose affects to some extent the bioavailability of iron, zinc, and copper. Its effect on copper has been studied most intensively. Fructose forms stable complexes with iron and promotes its absorption and also that of zinc. Compared with starch, fructose and sucrose decrease copper bioavailability in rats fed diets based on egg white and containing 60% carbohydrate. There is no apparent fructose effect on copper bioavailability in pigs. In humans fructose consumed as 20% of energy has no effect on copper balance and minimal effects on the criteria of copper status.

Interleukin-2 production is altered by copper deficiency.

Copper is an essential nutrient for optimal function of the immune system; deficiency results in impairment of both humoral and cell-mediated components. Copper deficiency in rodents results in decreased numbers of CD4+ (helper) and total T cells. This defect has been traced to impaired production of interleukin-2, a cytokine essential for T-cell division and differentiation. Impairment of quiescent cell proliferation is reversed by both in vivo and in vitro copper supplementation.

Low zinc status in rats impairs calcium uptake and aggregation of platelets stimulated by fluoride.

Platelets from rats of low zinc status exhibit impaired aggregation in response to ADP stimulation. The abnormality has been traced to defective uptake of calcium from the external medium. This study was designed to determine the location of the molecular defect and whether or not the ADP receptor is involved. Washed platelets were collected from rats fed a low zinc diet (< 1 mg/kg) and control groups that consumed a zinc-adequate diet (100 mg/kg), ad libitum- and pair-fed. Fluoride, a G-protein stimulant, was used to bypass the ADP receptor. F- stimulated platelet aggregation and calcium uptake; both of these functions were impaired by zinc deficiency. At 10 mM F-, the time to half maximal aggregation was increased from 1.8 min in platelets from control to 2.8 min in zinc deficient rats. At 8 mM F-, the uptake of calcium was decreased from 170 to 85 nM cytosolic free calcium. At this concentration of F- there was no release of internal calcium. The results show that the molecular defect in the zinc-deficient platelet is located in the aggregation pathway beyond the ADP receptor and suggest a point between, or including, a G-protein and the plasma membrane calcium channel.