The effects of oral zinc supplementation in the mouse

.J. COMP.

THE

PATH.

1

1977. VOL. 87.

EFFECTS

OF

ORAL ZINC SUPPLEMENTATION IN THE MOUSE BY

ELIZABETH University

AUGHEY

Department of Veterinary Histology and Embyology, of Glasgow Veterinary School, Bearsden, Glasgow G6I

IQH,

Scotland

and

LIONEL

GRANT

Glasgow Institute of Radiotherapeutics and Oncology, Western Injrmary, Glasgow Gll S.NT, Scotland and

BRIAN

L. FURMAN

and WILLIAM

F. DRYDEN

Department of Plysialogy and PharmacoloQ, University of Strathclyde, Glasgow GI IXW, Scotland

INTRODUCTION

Since the discovery by Strain, Dutton, Heyer, Pories and Ramsay (1953) that a zincsupplemented diet led to more rapid healing of thermal burns and excised wounds in rats, oral zinc therapy has been applied to a variety of conditions in experimental animals and in man. The results have not been free from controversy. Oberleas, Seymour, Lenaghan, Hovanesian, Wilson and Prasad ( 197 1) and Lavy ( 1972) suggested that healing was promoted in incised wounds of zinc-fed rats when compared with controls, but Kim and Rosenthal (1970) in similar experiments were unable to find any difference. Similar controversy has arisen from clinical investigations. Pories, Henzel, Rob and Strain (1967) reported, from a controlled trial, that healing and granulation of wounds resulting from the excision of pilonidal sinus tracts in young men was accelerated by daily administration of oral zinc. Husain (1969) reported a controlled trial in which he found that orally administered zinc accelerated the healing of leg ulcers, while from an uncontrolled trial, Greaves and Skillen (1970) also claimed that oral zinc administered to patients with chronic venous leg ulceration promoted healing. On the other hand, Clayton (1972), in a double-blind trial on patients with similar conditions, was not able to confirm these results. Hallbook and Lanner (1972) p er f ormed a double-blind trial on a group of 50 patients, and found that significant healing of leg ulcers occurred only in patients who had initially low serum zinc values. Much of the evidence has been reviewed by Carruthers (1973). The indiscriminate use of zinc in therapy may not be without potential hazards. Little evidence is available on the metabolic and physiological effects of subtoxic zinc supplementation as opposed to zinc depletion in the mammal. Excess dietary zinc has been reported to cause histological changes in the adrenal glands of rats (Roventa, Pora, Sahleanu and Vaduva, 1968) and Quarterman (1973) confirmed that adrenal weight was increased in zinc-supplemented animals. This work was undertaken to investigate zinc on endocrine glands and to attempt change in body zinc levels produced.

further the effects of supplemental to correlate these effects with any

2

E. AUCHEY et al. MATERIALS

AND

METHODS

Animals. One hundred and fifty mice of the C3H strain were used from an inbred stock maintained in the Glasgow Institute of Radiotherapeutics and Oncology and kept in polypropylene cages with stainless steel tops. Population densities were kept low, and not more than 15 animals were held in a cage at any time. The animals were received when aged between 6 weeks and 2 months, and the sexes were kept separate. Oxoid breeding diet was supplied ad libitum and 0.5 g/l zinc in the form of zinc sulphate was provided in distilled water for drinking ad libitum. Untreated distilled water was available for the control animals. The mice were maintained for up to 14 months before killing. Control and test animals were removed from the colonies in groups of five, usually at monthly intervals, for the following estimations. Plasma zinc. Control and zinc-fed animals were removed from holding cages and approximately 1 ml blood was removed from the femoral vein under ether anaesthesia. The blood was immediately centrifuged and the plasma removed and diluted to 1 ml using 10 per cent propanol in deionized water. Zinc estimations were carried out by atomic fluorescence spectrometry (Hussein and Ottaway-to be published). Plasma glucose and insulin. Blood samples were removed as before and the plasma glucose in undiluted plasma was estimated by the method of Haslewood and Strookman (1939). Plasma insulin was measured by the radioimmunoassay method of Hales and Randle (1963) with materials supplied by The Radiochemical Centre, Amersham. The animals were not starved prior to these estimations. Tissue rinc. Samples of skin, liver and spleen were removed and desiccated in a vacuum oven at 60 “C for 24 to 48 h. The dried tissue was weighed and digested in a heated nitric perchloric acid mixture by the method of Mills, Dalgarno, Williams and Quarterman ( 1967). Z inc analysis on the diluted digest was performed by atomic absorption spectrophotometry. Histology. Small blocks of adrenal, pancreas and hypophysis cerebri were fixed in Balanced Neutral Formalin (BNF) (Lillie, 1965), embedded in paraffin wax and 5 urn sections were stained with haematoxylin and eosin (HE). Histochemistry. Blocks of adrenal were fixed in BNF and the frozen cryostat sections were stained with Sudan Black and the Schultz method for cholesterol (Lillie, 1965). Electron microscopy. Sections from the adrenal, pancreas and adenohypophysis were examined. The adenohypophysis was obtained after perfusing the head region of mice anaesthetized with barbiturate with paraformaldehyde via a common carotid artery. One mm cubes of tissue were fixed in cold buffered isotonic paraformaldehyde (Peters and Palay, 1966) post-fixed in 1 per cent cold buffered isotonic osmium tetroxide, dehydrated in acetone and embedded in epon (Luft, 1961). Thick sections (1 to 2 pm) were cut and stained with toluidine blue-pyronin (Ito and Winchester, 1963) and the block suitably trimmed. Thin sections cut on an LKB ultramicrotome were stained with uranyl acetate and lead citrate and viewed in a Hitachi HS8 electron microscope at 50 kV. RESULTS

The visible. weight

zinc-treated The weight of zinc-fed

animals remained healthy and no outward effect was of the control mice ranged from 21 to 30 g and the mean mice was consistently higher (about 1 g) in each group

examined. Appetite and activity Plasma

zinc

remained at a high level throughout.

Levels

An estimate of the amount of zinc swallowed by the experimental animals was difficult due to spillage during drinking and uncertain intestinal absorption. Accordingly, plasma zinc levels were monitored to ensure that the supplemental

ZINC

SUPPLEMENTATION

IN

:5

lMICE

zinc was made available to the tissues. The mean [and standard error of the mean (s.e.m.)] plasma zinc level of a population of adult mice was 15-C 1.35 PM (1.02 kO.09 pg/ml). Th is value was constant in control mice at all ages during the experiment. The change in plasma zinc of zinc-supplemented mice was followed over a period of 34 days, and the results are depicted in Fig. 1. The rise in plasma zinc after supplementation was rapid, rising to a peak of 2-l pg/ml (31 PM) in 3 days. Thereafter a plateau was evident, with fluctuations reflecting the wide variation within the relatively small experimental groups. It was concluded that the method of administration was efrective in elevating plasma zinc levels sufficiently to correspond to levels experienced in the treatment of man without incurring acute toxic manifestations. 2.5r

0

IO Duralion

Fig.

20 of zinc

supplementation

30 (days)

1. Change in plasma zinc concentration during oral zinc supplementation. The stippled band indicates the range of the standard error of the mean (s.e.m.) of the population before zinc supplementation. The points represent the mean + s.e.m. of groups of 5 mice.

Tissue zinc Concentmtions The liver, spleen and skin were chosen for study. Liver and spleen are sites for storage and for protein synthesis and might respond to increased circulating zinc by increasing their intracellular zinc. Skin was chosen because the suggested effectiveness of zinc therapy in wound healing (Carruthers, 1973) might indicate that it was a target organ. In no case did the tissue zinc concentration of zinc-fed animals differ significantly from controls over a. 6-month period, thus confirming the observations of Arachi (1972) in zincsupplemented rats. No difference in the zinc content of tissues from either sex was evident. The mean vaIues for the tissues after 5 months of supplementation are shown in Table 1.

4

E. AUGHEY

et a/.

TABLE ZINC

CONTENT

----____-___ Controls (mean f standard demation)

Tissue

86.8k

Liver

OF TISSUES

Skin

5

MONTHS

(&g

DRY

WEIGHT)

M&S

15.3

202.8k21.3

Spleen

AFTER

1

46.2 + 18.7

Females
supplemented

95.6 + 5.8 195.7k21.0 58.9 f 5.0

Controls (mean $ standard devaation)

80.7 + 3.8 224.6k41.2 55.8 k 16.2

-----.+zc supplemented

79.0&

13.3

193.9k40.7 53.8 + 18.0

Plasma Insulin and Glucose The plasma insulin and glucose in mice supplemented by zinc for 6 months were not significantly different from control values of 12$1*4 pU/ml and 1.42 &O-O6 mg/ml respectively. Histology of the Pancreas The histological appearance of the control mouse pancreatic islet is typical of the species. In the 9 to 12 month zinc-supplemented mice the pancreatic islet was often larger, even visible macroscopically and the individual islet cells had a vacuolated appearance (Fig. 2). The cells were separated by wide vascular channels which are not typical of the control islet tissue. The microscopic changes in this and other tissues were particularly obvious in the

Fig. 2. Pancreas cells (I) separate

of 14-month-old are lightly stained the islet cells. HE.

for 12 months. The pancreatic islet mouse on zinc supplement and slightly larger than in control mice. Wide vascular channels x 227.

ZINC

12-month 3 months. liltrastructure

zinc-supplemented Prior to 3 months

SUPPLEMENTATION

IN

MICE

D

mouse, but can be seen to a lesser degree from no changes in histology were obvious.

of the Pancreas

The typical insulin-secreting beta cells of the pancreatic islet contain osmiophilic granules retracted from the enclosing membrane. The Golgi apparatus was not prominent, and small mitochondria were present throughout the cytosome. Small dilated cisternae of the endoplasmic reticulum were present, there were few ribosomes and the membranes were mainly smooth surfirced. In the zinc-supplemented mouse, the individual beta cells appeared to be larger, and some crystalline forms of the beta cells were present (Fig. 3). The

Fig. 3. Pancreas of 14-month-old mouse on zinc supplement for 12 months. There appears to be an increase in the number of granules in the p cell illustrated here. Many of the secretory granules are close to the plasma membrane (arrowed). The mitochondria (M) are larger and apparently more numerous. A prominent Golgi apparatus (G) is present in one cell. Uranyl acetale. 6000.

granules were arranged along the plasma membrane, and occasionally fused to the membrane. Individual granules showed varying degrees of* fusion. The mitochondria were large and the internal arrangement of the cristae more complex compared with the control. The Golgi apparatus was a prominent cell organelle (Fig. 4). Alpha cells were confined to the periphery of the islrt and as in control animals no obvious ultrastructural changes were seen in these cells. Histology of the Adrenal Cortex In the zinc-supplemented mouse the cortex was thicker than in the control animals. The majority of the fasciculata cells were hypertrophied and vacuolated, the distension causing compression of the vascular channels (Figs 5 and 6).

6

E. AUGHEY

Fig. 4. Pancreas of 14-month-old mouse on Golgi apparatus (G) in many of the p granules could be an artefact, but A number of granules are seen along (asterisks). Uranyl acetate. X 12 150.

et

al.

zinc supplement for 12 months. The complexity of the cells is illustrated here. The apparent fusion ofjuxtaposed is not present in similarly treated control specimens. the plasma membrane, and points of fusion are evident

Fig. 5. Adrenal gland of 14-month-old mouse on zinc supplement for 12 months. The adrenal cortex is thicker than normal, the zona fasciculata (ZF) cells are larger, and both outer (OF) and inner (IF) layers are distended with lipid. The zona reticularis (ZR) forms a deep layer of small dark cells separated by vascular channels between the zona fasciculata and the medulla (M). HE. x260.

ZINC

SUPPLEMENTATION

IN MICE

Fig. 6. Adrenal cortex of 14-month-old mouse on zinc supplement for 12 months. Most of the zona fasciculata (ZF) cells show a vacuolated appearance, and the vascular channels appear compressed. The extensive zona reticularis (ZR) consists of a network of small dark cells sepa rated by wide vascular channels between the fasciculata and the medulla (M). HE. :I 600.

Fig. 7. Adrenal gland of 5-month-old control mouse. lipid reaction; the paler zone at the periphery is the zona reticularis. Sudan Black B. x 450.

The majority of the cortical cells show a strong is the zona glomerulosa and next to the medulla

Fig. 8. Adrenal gland of 5-month-old mouse on zinc supplement for 3 months. The cortex is markedlv thicker, mainly due to the increase in the zona fasciculata. There appears to be an increase in the depth of the zona reticularis. Sudan Black B. x 450.

8

E. AUGHEY

Differences in lipid content of The zona reticularis consisted wide vascular channels. There apparently due to an increase Histochemistry

f?! a/.

outer and inner fasciculata cells were not obvious. of anastomosing cords of cells separated by very was a marked increase in the depth of this zone, in the cell content.

of the Adrenal Cortex

In the control mouse, the Sudan black B reaction was confined mainly to the zona fasciculata cells, the zona glomerulosa was lightly stained and the zona reticularis almost unstained (Fig. 7). In the zinc-supplemented mouse, however, the hypertrophied zona fasciculata reacted strongly with Sudan black B (Fig. 8). The glomerulosa and reticularis form relatively unstained thin bands of cells on either side of the fasciculata. These changes were evident in the adrenal cortex of mice after 3 months zinc supplementation. The cholesterol reaction was strongly positive throughout the three zones after zinc supplementation for 6 months and over. Prior to this in the 3 to 6 months mice the reaction was weaker, often confined to the zona fasciculata and present to a lesser degree in the control mice. This reaction was particularly obvious in short term experimental female mice, though clear cut results were not obvious in the male mice on short term feeding. Ultrastructure

of the Adrenal Cortex

In the control mouse adrenal cortex the outer zona fasciculata cell secretion droplets were separated by a cytosome containing mitochondria with complex tubular cristae, and smooth membranes of the endoplasmic reticulum. The vascular channels were lined by a fenestrated epithelium. The cells of the zona reticularis were smaller than the fasciculata cells and the lipid droplets fewer and densely osmiophilic. In the zinc-supplemented mouse the ultrastructural characteristics of the fasciculata cells are shown in Fig. 9. The cells had many lipid droplets in the cytosome and there was a granular deposit in the sub-endothelial space. The blood vessels also contained this granular deposit and were much contracted in appearance. In the zona reticularis cells of the zinc-supplemented mouse, the cells were still small with the characteristics seen in the control mouse, but arranged in anastomosing cords with wide vascular channels (Fig. 10). This formed an extensive zone between the fasciculata and the medullary capsule. Histology of the Pars Distalis The adreno-corticotrophic hormone (ACTH) secreting cell in the control mouse was agranular, irregular in shape and often sent cytoplasmic projections between adjacent cells. In the pars distalis of the zinc-supplemented mouse, the cytoplasm of the ACTH cell appeared more granular, presented an irregular appearance and was surrounded by somatotrophin (STH) cells (Fig. 11). cells contained many more In the zinc-supplemented mouse, the ACTH ranging in size from 80 to 240 pm (Fig. 12). The granules than controls,

Fig. 9. Adrenal cortex (zona fasciculata) of 14-month-old mouse on zinc supplement for 12 months. The p&capillary space (PC) and the blood vessel (BV) contain a granular material. The secretory vesicles (V) are more numerous than in the normal adrenal fasciculata cells, and many have coalesced to form irregular large “spaces”. The mitochondria (M) are closel) associated with these vesicles. Uranyl acetate. x 7000.

Fig.

10. Adrenal gland (zona reticularis) of 14-month-old mouse on zinc supplement for 12 months. Increased number of typical reticularis cells separated by wide blood channels (BV). Some collagen fibres are present, but in markedly smaller amounts than in the control mouse. Uranyl acetate. x 3500.

Fig.

11. Pars distalis of 14-month-old mouse on zinc supplement for 12 months. The irregular secreting cell (A) is surrounded by somatotrophin secreting cells (S). Toluidine Blue.

ACTH x 1200.

Fig.

12. Pars distalis of 14-month-old mouse on zinc supplement for 12 months. The ACTH cell (A) contains a large number of granules compared to controls, and the mitochondria, Golgi (G) and the rough endoplasmic reticulum (RER) are more prominent organelles. A small lipid droplet (1) is present. The ACTH cell is surrounded by somatotrophic hormone cells (S) and sends long processes between the cells. The adjoining capillary is labelled (CAP). Uranyl acetate. x 6000.

ZINC

SUP.PLEbXENTAtCION

IN

MICE

11

density of the granules also varied from relatively low to a density comparable with the granules of the STH cell. Small areas of smooth membrane comthese were surrounded 1)~. parable to the Golgi complex were also present; granules. Rough endoplasmic reticulum and free ribosomes were also prcscnt in the cytosome. Elongated mitochondria with numerous cristnc were also more A small droplet of osmiophilic material frequently seen than in controls. similar to that seen in control mouse ACTH cells is prcscnt and an assoc;a(c(l dcnrc body. DISCUSSION

Zinc is known to be a nutrient essential for many metabolic proccsscs including DIVA synthesis and cell replication (Mills, Quarterman, Chcstcrs, ‘CYilliams and Dalgarno, 1969; Rubin, 1972, 1973; Fell, 1975). While the erects of zinc deficiency in man and animals have been studied in some’ depth, the effects of supplementing dietary zinc have recei\,ed less attention. Studies with ruminants (Ott, Smith, Harrington, Stob, Parker and BC~SOII~ 1966; Ott, Smith, Harrington, Parker and Beeson, 1966; Stake, Miller. Gentry and Neathery, 1975) have indicated that extremely high plasma concentrations were rapidly attained, and that zinc was equally rapidly taken up, principally by the liver, pancreas and kidney. High plasma levels of zinc inhibited further intestinal absorption of the element. At the highest dosage levels, toxic effects were noted which included a reduction in haemoglobin and packed cell volume, and the replacement of pancreatic acinar tissue by connective tissue. In rats, however, no change in liver zinc during supplementation at our dosage levels was observed by Arachi (1972), although he did observe a small but not statistically significant rise in the zinc content of the spleen. Our observation of little change in skin, liver or spleen zinc content would confirm his report and may indicate a difference between rodents and ruminants. Histological changes had been noted in the adrenal glands of zincsupplemented rats (Roventa et nl., 1968) together with increases in weight (Quarterman, 1973). It is evident from the results reported here that the prolonged administration of additional but subtoxic quantities of zinc to the laboratory mouse affected the adrenal cortex, the pancreatic islets and also the cells of the anterior 101~ of the pituitary gland. ;\ll the cell types in our pituitary specimens showed increases in size with evidence suggesting increases in both synthetic and secretory activity. If this is so, then confirmation should be obtained from an examination of target tissues. Of the tissues examined, the pancreatic islets and the zona fasciculata both show hypertrophy. The islets are known to respond in this manner to raised plasma levels of somatotrophin and corticotrophin (Abrams, Baker, Inglc and Li, 1953; Kinash, McDougall, Evans, Bryans and Hoist, 1953)) while the zona fasciculata is similarly sensitive to ACTH and gonadotrophin (Christian, 1964; Jones, 1949). Thus the suggestion that anterior pituitary activity in these animals was elevated by prolonged zinc administration would appear to be reasonable.

12

E. AUGHEY

et

d.

Although the light microscopic appearance of the adrenal cortex and the pancreatic islets is suggestive of pituitary hyperactivity, the electron micrographs reveal confljcting evidence. The accumulation of secretory vesicles in the zona fasciculata cells, together with the evidence of large quantities of cholesterol is not to be expected from a postulated increase in the circulat’ng ACTH. Miller (1950) has demonstrated that ACTH-induced hypertrophy of the adrenal cortex is associated with a decrease in intracellular lipid and cholesterol. In contrast to our observation, Quarterman (1972) reported that the adrenal glands of zinc-deficient rats always contained more cholesterol. He also showed that the secretory response of the adrenal gland to injected ACTH was greater in these animals, and suggested that the zinc-deficient adrenal was hypersensitive to ACTH. There is the possible corollary, therefore, that while synthetic activity in peripheral endocrine glands of zinc-supplemented animals is either unaffected or increased, the release of the synthesized products may be prevented, causing the observed intracellular accumulation of secretory vesicles and lipids. The depression of plasma corticosteroid levels would in turn reflexly stimulate the pituitary to secrete ACTH. Whether this postulated inability of the zona fasciculata cells to release their secretory products is attributable to the occupation by zinc of plasma carrier ligands, or to the known stabilization by zinc of lysosomal membranes (Chvapil, Ryan and Zukoski, 1972) and subsequent inhibition of secretory product release (Baczy and Rappay, 1974) is not at present known. Clearly information on plasma corticosteroid levels in such animals must be obtained. Despite the differing involvement of the adrenal cortex in sexual maturation in mice, no differential effect of zinc on the sexes was noted. The beta-cell hypertrophy in the pancreatic islets is not reflected in raised levels of plasma insulin thus supporting the theory of impaired hormone release. The mechanism of action of zinc in these animals is obscure. Since zinc supplementation in these animals cannot be correcting any dietary deficiency, the effects observed may be regarded as a pharmacological action of zinc, with profound implications for the endocrinological balance, and the response of the individual to trauma. SUMMARY

C3H mice were given zinc sulphate in their drinking water for periods of up to one year. Histological confirmation of hypertrophy of the adrenal cortex and the pancreatic islets after three months was obtained and changes consistent with hyperactivity were noted in the pituitary. Further work is suggested to correlate structural changes with possible functional changes in the islets, the adrenal cortex and the pars distalis. Zinc content of the liver, spleen and skin was not altered. Plasma insulin and glucose concentrations in zincsupplemented animals were not significantly different from control values.

ZINC

SUPPLEMENTATION

IN

MICE

13

ACKNOWLEDGMENTS

We are indebted to Dr A. H. W. Nias, Glasgow Institute of Radiotherapeutics and Oncology for the supply of animals, to Drs G. S. Fell, Department of Pathological Biochemistry, University of Glasgow and J. M. Ottaway, and to Mr F. E. R. Hussein, Department of Pure and Applied Chemistry, University of Strathclyde, for the zinc determinations. REFERENCES

Abrams, G. D., Baker, B. L., Ingle, D. J., and Li, C. H. (1953). The influence of somatotropin and corticotropin on the Islets of Langerhans of the rat. Endocn’n-

ology, 53, 253-260. Arachi, H. (1972). Studies on the relation between zinc and malignant tumour. Journal of the Sara Medical Association, 23, 177-186. Baczy, E., and Rappay, G. (1974). Fine structural localization of aryl sulphatases in rat adrenal cortex. In Electron Microscopy and Cytochemistry, E. Wisse, W. Th. Daems, I. Molenaar and P. Van Duijn, Eds, p. 63. North-Holland Pub. Co., Amsterdam and London. Carruthers, R. (1973). Oral zinc in cutaneous healing. Drugs, 6, 161-164. Chvapil, M., Ryan, J. N., and Zukoski, C. F. (1972). The effect of zinc and other metals on the stability of lysosomes. Proceedings of the Society for Experimental Biology and Medicine, 140, 642-650. Christian, J. J. (1964). Effect of chronic ACTH treatment on the maturation of intact female mice. Endocrinology, 74, 669-679. Clayton, R. J. (1972). Double-blind trial of oral zinc sulphate in patients with leg ulcers. British Journal of Clinical Practice, 26, 368-370. Fell, G. S. (1975). Zinc in clinical nutrition. Scottish Medical Journal, 20, 101-102. Greaves, M. W., and Skillen, A. W. (1970). Effects of long-continued ingestion of zinc sulphate in patients with venous leg ulceration. Lancet, i, 889-891. Hales, C. N., and Randle, P. J. (1963). I mmunoassay of insulin with insulin-antibody precipitate. Biochemical Journal, 88, 137-146. Hallbrook, T., and Lanner, E. (1972). Serum-zinc and healing of venous leg ulcers. Lance& ii, 780. Haslewood, G. A. D., and Strookman, T. A. (1939). A method for the estimation of “true” sugar in 0.05 ml blood. Biochemical Journal, 33, 920-923. Husain, S. L. (1969). Oral zinc in leg ulcers. Lancet, ii, 1069-107 1. Ito, S., and Winchester, R. J. (1963). The fine structure of the gastric mucosa in the bat. Journal of Cell Biology, 16, 541-577. *Jones, C. I. (1949). The relationship of the mouse adrenal cortex to the pituitary. Endocrinolopv, 45, 514-536. Kim, Z. W., and Rosenthal, S. P. (1970). The effect of orally administered zinc sulphate on healing incised wounds. Journal of Surgical Research, 10, 597-599. Kinash, B., McDougall, I. Evans, M. A., Bryans, S. E., and Hoist, R. E. ( 1953). preparations on Effects of anterior pituitary extracts and of growth hormone the Islets of Langerhans and the pancreas. Diabetes, 2, 112-121. Lavy, U. I. (1972). The effect of oral supplementation of zinc sulphate on primary wound healing in rats. British Journal of Surgery, 59, 194-196. Lillie, R. D. (1965). Histopathologic Technique and Practical Histochemistry, 3rd edit. McGraw-Hill, New York. Luft, J. G. (1961). Improvements in epoxy resin embedding methods. Biophysical and Biochemical Cytology, 9, 409-414. Miller, R. A. (1950). Cytological phenomena associated with experimental alteration in the adrenal cortex of mice. American Journal of Anatomy, 86, 405-437. Mills, C. F., Dalgarno, A. C., Williams, R. B., and Quarterman, J. (1967). Zinc deficiency and the zinc requirements of calves and lambs. British Journal qf .Nutrition, 21, 75 l-768.

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C. F., Quarterman, J., Chesters, J. K., Williams! R. B., and Dalgarno, A. C. (1969). Metabolic role of zinc. AmericanJournal ofCltnzcat Nutrition, 22, 1240-1249. Oberleas, D., Seymour, J. K., Lenaghan, R., Hovanesian, J., Wilson, R. F., and Prasad, A. S. (1971). Effect of zinc deficiency on wound healing in rats. American Journal of Surgery, 121, 566-568. Ott, E. A., Smith, W. H., Harrington, R. B., Parker, H. E., and Beeson, W. M. ( 1966). Zinc toxicity in ruminants. IV. Physiological changes in tissues of beef cattle. Journal of Animal Science, 25, 432-438. Ott, E. A., Smith, W. H., Harrington, R. B., Stob, M., Parker, H. E., and Beeson, W. M. (1966). Zinc toxicity in ruminants. III. Physiological changes in tissues and alterations in rumen metabolism in lambs. Journal of Animal Science, 25, 424-43 1. Peters, A., and Palay, S. L. (1966). The morphology of the lamina A and Al of the dorsal nucleus of the lateral geniculate body in the cat. Journal of Anatomy,

100, 45 l-468. Pories, W. J., Henzel, J. H., Rob, C. G., and Strain, W. H. (1967). Acceleration of wound healing in man with zinc sulphate given by mouth. Lancet, ii, 121-124. Quarterman, J. (1972). The effect of zinc deficiency on the activity of the adrenal glands. Proceedings of the .Nutrition Society, 31, 74A. Quarterman, J. (1973). The effects of zinc deficiency or excess on the adrenals and the thymus in the rat. In Trace Element metabolism in Animals, 2, W. G. Hoekstra, J. W. Sutty, H. E. Dauther and W. Mertz, Eds, p. 742. University Park Press, Baltimore. Roventa, E., Pora, E. A., Sahleanu, V., and Vaduva, E. (1968). Corelatii functionale dintre suprarenala si cupru sau zinc. Studia Universitatis BabepBolyai, Seria Biologica, 13, 107-l 14. Rubin, H. (1972). Inhibition of D.N.A. synthesis in animal cells by ethylene diamine tetraacetate and its reversal by zinc. Proceedings of the National Academy of Sciences, U.S.A., 69, 712-716. Rubin, H. (1973). pH, serum and zinc in the regulation of D.N.A. synthesis in cultures of chick embryo cells. Journal of Cellular Physiology, 82, 231-237. Stake, P. E., Miller, W. J., Gentry, R. P., and Neathery, M. W. (1975). Zinc metabolic adaptations in calves fed a high but non-toxic zinc level for varying time periods. Journal of Animal Science, 40, 132-l 37. Strain: W. H., Dutton, A. M., Heyer, H. B., Pories, W. J., and Ramsey, G. H. (1953). Unibrersity of Rochester Report. [Received for publication,

February 2nd, 19761