Zinc Blood and Tissue Cells
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The objective of this research was to investigate the distribution of zinc in the various leukocyte forms and to make comparisons of its concentration therein with acid and alkaline phosphatases in the same tissues. It was also expected to correlate zinc and enzyme levels in rabbit leukocytes under such stress conditions as induced leukocytosis and after Versene (EDTA) administration. It was desired to determine whether the zinc levels parallel the known effects of stress on the enzymes, whether the altered activities result from changes in the pH maxima, and whether zinc functions as an activator in vivo. Histochemical detection of zinc A histochemical technique for the detection of zinc in blood cells, which had been previously partially developed, was further developed and extended to permit a survey of other tissues. Specificity for zinc was achieved by means of a dithizone complex-forming solution. Zinc in prostate and stomach sections could be clearly differentiated from other metallic constituents. In all prostate sections studied, the cytoplasm of the epithelial cells showed a distinct red-orange color with red-purple granules. In all areas the apical portions of these secretory cells were often most deeply stained. Connective tissue and smooth muscle did not stain. The anterior and posterior lobes of dog, human, and monkey prostates exhibited no essential difference in dithizone-staining properties. In all sections of stomach from dog, man, monkey, and rabbit the parietal cells were clearly demarcated when stained for zinc and exhibited dark red cytoplasm in addition to red granules. Epithelial cells displayed variable shades of color, from light pink to an intense orange-red, while connective tissue was unstained. Dithizone in aqueous acetone solution demonstrated metallic non-specific staining in stomach, prostate, pancreas, and erythrocytes of dog, man, rabbit, and rat. Metallic staining as contrasted with concentration of the dye by fat was shown by loss of stain in tissues previously chelated with ethyl-enediaminetetraacetic acid (EDTA). staining of phagocytized zinc oxide in macrophages indicated penetration of the cellular membranes by dithizone. Zinc acetate given intravenously to rabbits showed a marked concentration of zinc in the epithelial cells of small and large intestines. This experiment indicated that the intestines, particularly the small intestine, were a possible excretory route for zinc. Many attempts to utilize the staining technique on leukocytes produced such infrequent success it is not as yet possible to employ routinely the procedure to detect the zinc in such cells. Zinc distribution in rabbit blood Zinc was determined quantitatively in polymorphonuclear leukocytes obtained from rabbit intraperitoneal exudates, in lymphocytes obtained by cannulation of the thoracic duct, in leukocytes obtained from peripheral blood by fibrinogen sedimentation, in normal erythrocytes and plasma, and in peripheral leukocytes, erythrocytes, and plasma obtained from rabbits treated with Versene and DNA. Cell suspensions and plasma were analyzed for zinc by extracting with dithizone in carbon tetrachloride and measuring the optical densities at two wave lengths (525 and 625 millimicron). The absorption maxima were verified and the required experimental constants evaluated. The average distribution values for zinc in millimicrograms per million cells and the standard error were mononuclear leukocytes 346.6 (calculated), polymorphonuclear leukocytes (from peritoneal exudates) 11.8 ± 1.5, immature lymphocytes (from thoracic duct) 1.81 ± 0.3, erythrocytes 4.32 ± 0.47, and plasma 268 (micrograms per 100 ml) ± 39. No statistically significant difference was found betvreen zinc levels of peripheral leukocytes from normal rabbits and those treated with Versene. The zinc content of mixed leukocytes from untreated rabbits was 1.7 times greater than in DNA-treated rabbits, and this dif ference was significant at the 0.05 level of probability. The zinc in normal plasma was 2.3 times greater than in the DNA animals, which corresponded to a P value of 0.02. Erythrocytes were statistically significantly depleted of zinc by Versene treatment. Correlation analysis of zinc per million cells vs. per cent mononuclear leukocytes gave a correlation coefficient (r) of 0.629 and a P value of 0.01. Thus, a high correlation exists between per cent non-granular leukocytes and total zinc content. Since the per cent of polys present is equal to 100 minus the per cent of mononuclear cells, r is equal to -0.629 for zinc levels vs. per cent polys. Thus, a negative correlation exists for this latter system. The regression equation was found to be: Y(zinc) = -.0054 + 0.0035 X(mononucl.) These data indicate that unit zinc content of rabbit peripheral leukocytes is confined to the mononuclear cells. The polymorphonuclear leukocytes have essentially no zinc. The exudate studies showed zinc levels of 11.8 millimicrograms per million cells; however, this level is probably a reflection of the 5 per cent or less mononuclear cells present in most exudate samples. It was calculated that 5 per cent non-granular leukocytes corresponds to 15.0 millimicrograrns. Distribution of acid and alkaline phosphatases in rabbit blood Enzyme levels, in mgm phosphorus liberated per hour per 10^10 cells, were determined on the same samples obtained for zinc analysis. The pH optima for acid and alkaline phosphatases, using sodium beta-glycerophosphate as substrate, in exudate leukocytes and lymphocytes was established at 5.0 and 9.9. This is an essential agreement with reported values for other substrates. The average values for the acid and alkaline phosphatases of rabbit blood were polymorphonuclear leukocytes 12.8 and 155, immature lymphocytes 1.9 and 1.6, and per 100 ml plasma 0.29 and 1.38. The low values obtained for the lymphocyte samples were unaltered by the addition of glycine, zinc ion, change of substrate, or by varied incubation conditions. The same pH optima, pH 5.0 and 9.9, were observed in the lew~ocytes of leukemia patients. The phosphatase activities of these cells remained unchanged when treated as the lymphocyte samples. Fibrinogen sedimented leukocytes from peripheral blood of either Versene or DNA-treated rabbits showed no significant difference with samples from normal animals. However, a trend toward higher values of alkaline phosphatase in DNA-treated animals was apparent. The acid phosphatase activity of peripheral leukocytes showed a positive correlation which approached significance with the mononuclear leukocytes, while no correlation could be established for alkaline phosphatase. A correlation coefficient of 0.625 was obtained for the concentration of zinc per million white cells and its acid phosphatase level. This corresponded to a P value between 0.01 and 0.02. No statistically significant correlation existed between alkaline phosphatase and zinc concentration. The composite data aside from that given in the histochemical section warrants the following conclusions: The zinc in peripheral leukocytes is mainly in the mature mononuclear leukocytes, there being nruch less in the young lymphocyte and granular leukocytes. The metal is firmly bound, as noted in the similarity in zinc levels of both normal cells and those subject to chelating agents. This may be contrasted with the more labile zinc of the erythrocyte, which is removed by chelating agents. The weight of evidence from the literature and from these experiments, both analytical and histochemical, showed the acid phosphatase and zinc to be non-nuclear constituents.
Thesis (Ph.D.)--Boston University
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