Cornelis J. A. Van den Hamer

In Vitro Exchangeable Erythrocytic Zinc

Exchangeable erythrocytic zinc is measured by65Zn uptake in and release from erythrocytes under standarized and near, physiological conditions: 7.6 μM zinc and 580 μM albumin in the medium. The intracellular exchangeable erythrocytic zinc pool in healthy volunteers amounts to 5 μmol zinc/L packed cells. The half-time of the exchange is 7 h, its activation energy 84 kJ/mol. The effects of the variation in temperature and the concentrations of albumin, as well as the effects of some zinc carriers, cell transport inhibitors, and stress hormones on the65Zn uptake are measured.

Zinc uptake by blood cells of rats in zinc deficiency and inflammation

In zinc deficiency, the function of leukocytes is impaired. However, the results of studies on the zinc concentration of blood cells in zinc deficiency are conflicting, probably in part because of technical and analytical problems. The aim of this study was to investigate, under standard conditions, the uptake of65Zn-labeled zinc by blood cells, taken from zinc-deficient rats and from rats in which an inflammation is induced. In both conditions, the serum zinc concentration is reduced. In clinical practice, this makes it difficult to determine whether the decrease in serum zinc is the result of a real or an apparent zinc deficiency. In stress, like an inflammatory disease, the decrease of zinc reflects an apparent zinc deficiency because of redistribution of serum zinc into the liver and because of decrease in serum albumin concentration. Over 70% of the serum zinc is bound to albumin. Blood cells from zinc-deficient and control rats were isolated using a discontinuous Percoll gradient and incubated under nearly physiological conditions in a65Zn-containing medium. A significant increase in the in vitro uptake of65Zn-labeled zinc by the blood cells of zinc-deficient rats was seen: erythrocytes 1.3, mononuclear cells 2.0, and polymorphonuclear cells 2.6 times the control values. During inflammation, no change in65Zn-labeled zinc uptake by erythrocytes and mononuclear cells was demonstrated after 2 d, although the serum zinc and albumin concentrations were decreased, but a small but significant increase in zinc uptake by polymorphonuclear cells was observed. This study of65Zn uptake in vitro under standard conditions may prove of value for distinguishing in patients real zinc deficiency from apparent zinc deficiency owing to, e.g., stress, although additional experiments should be performed.

Laboratory Assessment of Early Dietary, Subclinical Zinc Deficiency: A Model Study on Weaning Rats

ABSTRACT: Male weaning rats were pair-fed a low-zinc diet or a controt diet. After 10 d, the animals fed the low-zinc diet showed physiologic signs of deficiency; however, they showed no clinical symptoms. Their estimated whole body zinc was 25 μtmol versus 39 μmol for the controls. The 65Zn absorption increased 2-fold and the tissue distribution altered: muscle and erythrocytes contained more, small intestine and liver less activity at 0.5 h postdose. in vitro, the erythrocyte 65Zn uptake rate increased also. The 65Zn uptake experiments required small quantities of erythrocytes. The difference observed between the deficient and control cells was significant and showed little overlap. The increase of the 65Zn uptake from a medium was not affected when the animals underwent endotoxn exposure 24 h before, as was reported to occur in whole blood 65Zn uptake. Therefore, we suggest the in vitro erythrocyte 65Zn uptake, performed in a standardized, near physiologic medium, to detect early, subclinical zinc deficiency.

Zinc exchange by blood cells in nearly physiologic standard conditions

Determination of zinc concentrations in white blood cells has been used to establish zinc deficiency. During pathological conditions changes in zinc concentrations in these blood cells were observed. However, these investigations were hampered by the low amount of zinc in this form per mL blood. Earlier we demonstrated that, in the case of zinc deficiency, the uptake of zinc was increased, using the in vitro exchange of zinc by the various blood cells with extracellular zinc labeled with65Zn in fairly physiologic conditions. In case of inflammation, no increase in zinc uptake by erythrocytes was seen, indicating that this method probably can be used to differentiate real from apparent zinc deficiency. Only during the first days of the inflammatory process, probably representing the redistribution phase during which zinc moves from the serum to the liver, a small increase in in vitro zinc uptake was seen in mononuclear cells (MNC) and polymorphonuclear cells (PMNC).Earlier papers raised some questions; e.g., is the uptake part of an exchange process and can the efflux of zinc by the cells be measured by the same method; what is the influence of time on the process of zinc uptake; what is the magnitude of the uptake of zinc by the cells compared to the zinc concentration in the cells; and, what is the influence of temperature on the uptake of zinc?In the present study, the influence of incubation time and temperature on the uptake of zinc by human and rat blood cells and on the release of zinc by rat blood cells was studied. At least three phases of uptake of zinc in the various cells were found by varying the incubation time—a fast phase during the first half hour, probably caused by an aspecific binding of zinc on or in the cell membrane; a second fast uptake between 60–330 min, probably caused by an influx of zinc in the cell as part of the exchange process of zinc; and a slow third phase after 5.5 h, in which probably the in- and efflux of the rapidly exchangeable intracellular pool is more or less equilibrated. For mononuclear cells, polymorphonuclear cells, and erythrocytes of rats, the rapidly exchangeable intracellular pool is 40%, 53%, and 10%, respectively, of the total zinc content of the cells. This study is also performed in human cells; in human cells the exchangeable pool of mononuclear cells and erythrocytes is 17 and 3.5% of the total zinc content of the cells, respectively. The efflux of zinc by blood cells can be measured by the same method. Both the uptake and the loss of zinc by blood cells of rats were compared and are of the same magnitude, indicating that the in vitro uptake of zinc described elsewhere is part of an exchange process. Increasing temperature during incubation procedures results in an increase of zinc uptake by human blood cells, even at high temperatures of 41°C, although there are gradual differences between the various blood cells. Both the in- and efflux of zinc by blood cells are very small at 4°C.

Uptake and turnover of65Zn in subcellular fractions of brain of rat under normal and zinc-deficient conditions

After a single injection,65Zn is slowly taken up by the brain of the rat to a maximum after 7 d, followed by a turnover phase, with a half-time of about 3 wk. In the brain of rats on a zinc-deficient diet, the65Zn content in the brain continued to increase up to 30 d after the injection.The uptake and turnover phases in six different subcellular fractions of the brain showed a pattern similar to that of the whole brain in both the control and zinc-deficient rats. There was no internal redistribution of65Zn in the brain under conditions of progressive zinc deficiency.The results are discussed in a model for zinc homeostasis in the brain.

Histidine supplement and Zn status in Swiss random mice.

The influence of either histidine supplement or nutritional Zn deficiency on growth and the organ and tissue Zn content of mice during a 21-d period was compared with a control group. When the histidine intake was increased from 5 to 9 μmol/g body wt/d we noted increased body weight and higher Zn concentrations in liver, pancreas, spleen, and muscle. As a result, the estimated whole body Zn mass increased. This was explained by enhanced utilization of dietary Zn. These results differed from those seen in Zn deficient animals (fed 5 nmol Zn/g body wt/d instead of 29).Dietary Zn deficiency was characterized by anorexia and growth retardation, lower Zn concentrations in pancreas, muscle, bone, tail, and plasma, plus higher Zn concentrations in spleen and fur. As a result, the estimated total body Zn mass was 20% lower than in the control animals, despite a two-to threefold increase in utilization of dietary Zn.These results are discussed in view of the available literature. It is concluded that in humans and in animals both the absorption and the excretion of Zn may be increased by histidine. Below a certain dose the former will prevail, viz., a situation of increased utilization exists, preventing the development of Zn deficiency.

Zinc and copper of fetal organs during the second trimester of pregnancy

In fetus with a mean gestational age of 18 weeks (range 15–25,n=14), zinc and copper concentrations in liver, femur, rib, and skeletal muscle were measured. Zinc and copper concentrations are highest in liver. A trend of decreasing liver zinc concentrations during gestational age is suggested. Zinc concentrations are significantly correlated with copper concentrations in liver and in femur, suggesting steady growth in both organs. Femur zinc values rank ca. 30% of those in liver, femur copper, ca. 2%. Zinc or copper concentrations in rib are of the same levels as in skeletal muscle. Their concentration for zinc ranks ca. 20%, for copper, ca. 5% of the values in liver. All zinc and copper values are lower than reported in third trimester fetal organs.Calculated zinc/copper molar ratios are distinctive for the various organs: in liver, 6±1, in femur, 73±8, and in soft tissues, 26±3. Calculated ratios from published values obtained from the third tri|mester of pregnancy show that the ratios in liver and skeletal muscle maintain these levels. The zinc/copper molar ratio can serve as an internal reference in zinc and/or copper measurements.

A histidine supplement and regulation of the zinc status in Swiss random mice

The effects of histidine on the zinc status are controversial. In mice, we studied the effects of a moderate histidine supplement on the regulation of the zinc status using subcutaneously administered65Zn. In animals fed a zinc-adequate diet, histidine supplement did not cause changes in the zinc status (zinc concentrations,65Zn tissue distribution, and tissue specific activities). Neither effects on the regulation of the zinc status (65Zn retention, excretion and biological half-life) could be demonstrated. However, the combination of a low zinc diet and moderate histidine supplementation caused changes in the regulation of the zinc status (lower65Zn retention, associated with increased fecal excretion and a shorter biological half-life), aggravating the dietary deficiency (lower bone zinc, a shift in the65Zn tissue distribution). Reviewing the literature, it seems that only a molar histidine/zinc ration of 2,000 or higher will cause zinc deficiency.

Dietary zinc deficiency has no effect on auditory brainstem responses in the rat

Zinc has been shown to effect—in vitro—a number of processes associated with neurotransmission. We have tested whether the rate of impulse conduction—in vivo—as measured from the latencies of auditory brainstem responses (ABR), is influenced by dietary zinc deficiency in the rat.Dietary zinc deficiency for up to 26 wk had no effect on the wave I–IV interval compared to zinc-adequate fed animals. The results are discussed in relation to the observed constancy of brain overall and extracellular fluid zinc concentrations under conditions of dietary zinc deficiency.

Effect of excess dietary histidine on rate of turnover of65Zn in brain of rat

The effect of the chronic administration of histidine on the brain zinc level was examined in growing, male Wistar rats. Using a purified diet, the minimum zinc requirement for normal growth and normal plasma and tissue zinc levels was found to be around 10 ppm. Given this zinc content; the diet was supplemented with 5% and 8% histidine, respectively, or with 10% glycine (as control). Brain zinc was analyzed by measuring the rate of turnover of65Zn from 2–4 weeks after a single injection of the tracer. Feeding the diet supplemented with 5% histidine caused a small decrease in the plasma zinc concentration and a slight increase in the rate of turnover of65Zn in the cerebrum and the cerebellum as compared to the control group. The animals fed the diet supplemented with 8% histidine became severely zinc deficient (as evidenced by a 50% reduction in the plasma zinc content), however, the rate of turnover of65Zn in all brain regions examined was significantly decreased as compared to the control group. The results indicate that histidine has no specific complexing action on the brain zinc.