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Michigan Scan: University This is to certify that the thesis entitled THE BEHAVIORAL AND PHYSIOLOGICAL EFFECTS OF ZINC DEFICIENCY IN ‘ YOUNG AND AGING MALE HOLTZMAN RATS A presented by ‘ ELIZABETH FABRI GORDON has been accepted towards fulfillment of the requirements for Ph .D PSYCHOLOGY degree in 777. [Cl/U WI- __ igjor professor / Date__+lO=26.=7_9——— 0-7 639 BEHAVIORAL AND PHYSIOLOGICAL EFFECTS OF ZINC DEFICIENCY IN YOUNG AND AGING MALE HOLTZMAN RATS By Elizabeth Fabri Gordon A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Psychology '>s«» 1979 ABSTRACT BEHAVIORAL AND PHYSIOLOGICAL EFFECTS OF ZINC DEFICIENCY IN YOUNG AND AGING MALE HOLTZMAN RATS BY Elizabeth Fabri Gordon An animal model was used to study the physiological and behavioral effects of feeding a low zinc diet to young and aging male Holtzman rats. The following parameters were evaluated: (1) food intake, (2) body weight, (3) plasma zinc concentration, (4) open field behavior, including latency to leave the center circle of the field, grooming, freezing, numbers of sectors entered, rearing and defecation, and (5) discrimination learning (Study 1 only). Study 1: Three hundred day-old male Holtzman rats were randomly assigned to one of three treatment groups as follows: Group ZDA rats were fed ad libitum, a diet deficient in zinc; Group ZSP (control) rats were pair-fed a diet sup— plemented with zinc so that the daily intake for each animal was equal to the mean number of grams consumed by Group ZDA in the previous 24 hour period; Group ZSA (control) rats were fed ad libitum a diet supplemented with zinc. Animals were weighed weekly and food intake recorded daily. Plasma zinc Elizabeth Fabri Gordon levels were determined following fifteen weeks of treatment. and behavioral testing was begun. The ZDA and ZSP groups had a significantly lower intake than the ZSA group following two weeks of feeding. The ZDA group weighed significantly less than the ZSP or ZSA group following five weeks of feeding. The ZDA animals had a significantly lower plasma zinc level than the ZSP or ZSA animals. Results from the successive black—white dis— crimination learning task (T-maze) revealed slightly more errors for the ZDA group compared to the two control groups. These differences were not significant. Responses in a circular open—field demonstrated the following: (1) A sig- nificantly longer latency to leave the center circle for the ZDA group compared to the ZSP or ZSA group, (2) the ZDA group spent significantly less time grooming than the two control groups and (3) the ZDA group entered significantly fewer sectors than the controls. Study 2: Thirty-five day—old male Holtzman rats were randomly assigned to treatments and fed as described in study one. Plasma zinc concentrations were determined following seven weeks of feeding and behavioral testing was started. The ZDA and ZSP groups had a significantly lower intake than the ZSA group that was apparent following eight days of feeding. The ZDA animals weighed significantly less than the ZSP or ZSA animals and the ZSP animals weighed significantly less than the ZSA animals. These differences Elizabeth Fabri Gordon were apparent following eight days of feeding. Responses in the open—field revealed the following: (1) the ZDA animals had a significantly longer latency to leave the center circle of the field, (2) significantly fewer rearings, and (3) entered a significantly fewer number of sectors compared to the control animals. Apparent physical changes for the young ZDA animals included ruffled coats, hair loss including bald patches, inflamed paws in 50 percent of the ZDA animals and irritated conjunctiva. All of the ZDA animals had urethrititis and skin lesions. Thirty percent of the ZDA group died prior to the conclusion of the study, apparently from severe zinc deficiency. None of these physical changes were apparent in the aging ZDA animals. Results of the present studies are compared with those of previous research in the area of zinc deficiency in living organisms and comparison of differences between the two age groups are made. Study 1 is the only study, to date, that has used an animal model to assess zinc deficiency in aging orga— nisms, and, therefore previous research findings in this area have not permitted a comparison of the detrimental effects of feeding low-zinc diets for these two age groups. 5.’ v-l‘ 7&5"sz - ,1 ‘ gs ‘ L.“ . 3.7 o; .1-.~ 4 : --.-N~ s»; ‘.r (“~u,w-¢ “ u'u' 4"1. ' %"l"‘ I" '...‘ h. I) I "Al-f, \ .. ‘ .‘ .;'.kfi l .: .I‘ . .t -J‘ i ' ‘ v .,n% . , 3' . .\.. . . i a . Ksluut‘ ‘ ‘ -.b ‘14??? DEDICATION .2 -..r.;.c;q ‘ it? ‘ I ‘ t I fif To all of my family members who have been nost3_g, M responsible for my deep appreciation of the concept of life-long human development.: .' s .. “yulgrlutua ' : "-. ~7 ' (w 1. m N- .‘t: Lyin Dormant ‘75 <;' . ;‘ L: (“n LLSO~ .. VP — ‘2.. *-;v.a:ec tasks. 1111 4% k 'i J- I fin I :3 l 9’ ACKNOWLEDGMENTS I express my sincere thanks and appreciation to ’ Professor M. Ray Denny, my dissertation committee chair- person, for his continuous academic support and encourage- ment throughout these studies. I also thank the other committee members, Professors Ellen A. Strommen, John P. McKinney and Jenny T. Bond, for f their various contributions to the completion of this dis- 3 . d; sertation. it The technical assistance of Rosslyn Wofford, . “; Dwayne Moore and Paulette Valliere was very much appreciated ‘,; by me. if Special thanks from me to my daughter, Lynn Gordon, :;ED for the many summer vacation hours she spent in the base- ment of PRB assisting in a variety of research-related tasks. TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . Vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . Vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 Background . . . . . . . . . . . . . . . . . . . 1 Dietary Intake of Zinc . . . . . . . . 10 Physiological and Biochemical Aspects of Zinc Metabolism . . . . . . . . . . . . . . . . 14 Zinc Toxicity . . . . . . . . . . . . . 17 Zinc in Infection and Immunity . . . . . . . . . 19 ; Experimental Zinc Deficiency in Animals . . . . . 21 } Teratogenic Effects of Intrauterine Zinc f DEfiCienCY a o a o e o o a o o o o n a o o c 33 .“ Experimental Zinc Deficiency in Humans . . . . . . 35 Clinical Manifestations of Zinc Deficiency in Children . . . . . . . . . . . . 42 Clinical Aspects of Zinc Deficiency in Adults . . . . . . . . . . . . . 47 Zinc Deficiency and. Behavioral Impairment in Laboratory Animals . . . . . . . . . . . 51 Zinc Deficiency and Schizophrenia . . . . . . . . 56 Rationale . . . . . . . . . . . . . . . . . . . 57 STUDYl....................... 62 " MATERIALS AND METHODS . . . . . . . . . . . . . . . . 63 1, Animals and Diets . . . . . . . . . . . . . . . . 63 Apparatus . . . . . . . . . . . . . . . . . . . . 65 Procedure . . . . . . . . . . . . . . . . . . . . 66 RESULTS 0 I o a a o o o c a o n o o o o o o c o a o I 69 ' i 1:. V7 .L'J'I DISCUSSION - o c n o n o n o o o o a c o c I o o a o I 78 i h: j. 3' . .3‘:. 'é‘lfé‘h Page "I STUDY 2 . . . . . . . . . . . . . . . . . . . . . . . 83 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 84 Animals and Diets . . . . . . . . . . . . . . . . 84 Apparatus . . . . . . . . . . . . . . . . . . . . 84 Procedure . . . . . . . . . . . . . . . . . . . . 84 RESULTS . . . . . . . . . . . . . . . . . . . . . . . 86 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 96 GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . 99 REFERENCES . . . . . . . . . . . . . . . . . . . . APPENDICES A. Test Data from Study 1 and Means and Standard Deviations for Body Weight and Intake . . . . . . . . . . . . . . . B. Test Data from Study 2 and Means and Standard Deviations for Body Weight and Intake . . . . . . . . . . . . . . . xvi-A. 4:... E“! - . 'fi‘.).4.v;.:~‘1$" ‘ x a». .n g._ .1| ‘1' -\’LY L 7\ l .1 ‘ n .'K _ "C". J - R fir ,-'- v o"... Zinc Deficient Test Diet . Open—Field Data (Study 2) LIST OF TABLES LIST OF FIGURES Figure 1. Food intake of rats fed a zinc-deficient or zinc-supplemented diet for twenty weeks . . . Body weight of rats fed a zinc-deficient or zinc-supplemented diet for twenty weeks . . . . . . Mean number of errors per forty-six trial blocks on a successive black—white dis- crimination learning task . . . . Food intake of rats fed a zinc—deficient or zinc-supplemented diet for seven weeks . . . . Body weight of rats fed a zinc-deficient or zinc-supplemented diet for seven weeks g Page 7O 72 INTRODUCTION The study of zinc and its effects on the living organism has been of great interest to biologists for over one hundred years. This has gradually progressed through the disciplines of microbiology, agriculture, nutrition, medicine and more recently psychology which has begun to investigate the behavioral correlates of zinc deficiency. With the addition of psychology to these investigations we are now starting to form a comprehensive picture of the consequences of zinc deficiency for humans. w Knowledge of the biological importance of zinc dates to 1869 when Raulin showed that the element was necessary for the growth of the black mold Aspergillus niger (Raulin, 1869). Since that time extensive studies in a number of different disciplines in biology and medicine have demon- strated the importance of zinc in basic biochemical pro— cesses and in its more applied aspects in agriculture and in pathophysiology of disease. Zinc was discovered later than many of the other metals and it derives its name from the German "of unknown origin" (Vallee, 1959). It is twenty-fifth on earth in order of abundance and usually 1 occurs in combination with sulfur or oxygen. It is in group IIB in the periodic table of the elements, has the atomic number 30 and atomic weight of 65.38 (Martz, 1970; Cordas & Holland, 1977). In addition to being found on the earth it was also present in samples of moon rock but in less concentration than on earth (Halsted £3 11., 1974). Zinc was first used as an alloy with copper in the manu- facture of brass. Its medical uses began almost 2,000 years ago when the Egyptians used zinc oxide or calamine to aid in the healing of burns and wounds (Cordas and Holland, 1977). The occurrance of zinc in biological materials was first described by LeChartier and Bellamy in 1877 and was demonstrated to be present in human liver in the same year (Raoult and Breton, 1877). The influence of zinc and man- ganese on A. niger was investigated by Bertrand and Javillier in 1911 who conformed the earlier zinc work of Raulin who was a student of Pasteur. The essentiality of zinc for higher forms of plant life was demonstrated by Maze' in 1914 (Maze', 1914) and later by Somer and Lipman (Somer & Lipman, 1926). The impetus for interest in the study of the importance of the element in the biological processes of man came about through toxicological investigations of metal fume fever in the 19205 when it was shown that zinc fumes were toxic to industrial workers (Papp, 1968). These investigations aided in the development of theories relating to the role of zinc in biological functions (Vallee, 1974). During the period that the toxicologists were investigating. the untoward effects of zinc fumes on humans the agricultural chemists were finding that zinc in solution was indispensible in the growth of barley, sunflowers, buckwheat, beans and vetch in solution cultures (Somer & Lipman, 1926). As early as 1927 the application of zinc in the field was found to be beneficial for field and vegetable crops in Florida (Viets, 1966). The problem of deficiency of zinc in crops has been noted to be worldwide. The zinc content of plants averages 23 parts per million (ppm) but some plants may have a very high concentration of up to 16 percent of their ash weight (Halsted 25 al., 1974). Lutz studied the biological distribution of zinc in the rat and found it to be present in all organs (Lutz, 1926). The element was shown to be an essential in the diet of this animal by Todd and coworkers (Todd 23 31., 1934) and this work was later confirmed by Day and McCollum in 1940 (Day & McCollum, 1940). Hove, Elvehjem and Hart showed in 1937 that rats on a synthetic diet low in zinc experienced acceleration of the growth rate when zinc salts were added to the diet (Hove, Elvehjem & Hart, 1938) and were the first to describe the development of abnormalities of the coat of fur of animals on zinc deficient diets. Sub- sequent research has confirmed the significance of dietary zinc in the growth of chickens (O'Dell 95 $1., 1958) and calves (Miller & Miller, 1962). Early studies of the effect of zinc were also carried out in mice with some of the first conclusive growth studies (Day, 1942); however in recent years the rat has been most popular for investigation of the effects of zinc deficiency on rodents. At least fifteen animals including humans have now been shown to require zinc (Halsted £1 g1., 1974). Biochemical studies of the importance of zinc in physiological processes became of widespread interest follow- ing the isolation and purification of the enzyme carbonic anhydrase by Keilin and Mann in 1944 which was a compound known to contain zinc as part of its molecule (Keilin & Mann, 1944). Numerous investigations of the role of the metal have now been carried out and it is known to be an integral constituent and cofactor for more than 20 metallo- enzymes. Zinc and its sister metal copper are believed to keep the polymerases on track in winding or unwinding DNA (Cordas & Holland, 1977). Zinc deficiency has also been shown to inhibit the synthesis of both DNA and RNA (Halsted g; 31., 1974). The chemical relationships of zinc and other elements have been investigated in some detail revealing interactions between zinc and cadmium (Schroeder e; 31., 1967), zinc and calcium (Forbes, 1960), and copper and zinc (Kinnamon, 1966; Gray & Ellis, 1949; Murthy, Klevay & Petering, 1974). Zinc has in addition been shown to have a protective effect against cadmium toxicity in the testes of rats (Gunn, Gould 5 Anderson, 1961; 1963). The possible role of zinc deficiency in the diet of' the pregnant female in causing congenital malformations has been studied in rats by Hurley and Swenerton (1966). The initial work of these investigators revealed that with severe dietary restriction reproduction was impossible. Dams given a diet somewhat less restricted in zinc had fetuses were noted to be small in size, have an abnormally shaped head, clubbed feet, fused or missing digits and mal- formations of the jaw and tail. Sandstead and associates (Sandstead 31 21., 1972; McKenzie 21 11., 1975) found that the major biochemical effect of zinc restriction during pregnancy was the impairment of DNA and protein synthesis in brain, liver and the epiphyseal plate of bones. This work was carried out by Swenerton and associates using radiolabeled thymidine which showed that 12-day-old embryos had a reduced uptake of the material and indicated that the gross congenital abnormalities resulting from zinc deficiency were probably caused by a basic defect in the synthesis of DNA (1969). These studies have been extended to the effect of dietary zinc deficiency on suckling rats. Dams who received a normal diet during pregnancy were postnatally given zinc deficient diet and the pups then developed smaller forebrains and a reduced number of brain cells than con- trols (Fosmire g; 31., 1975). Studies of the effect of zinc deficiency during pregnancy on the behavior of progeny have been carried out (Halas e3 g1., 1975; Halas & Sanstead, 1975). These initial evaluations revealed that prenatally zinc deficient rats are more aggressive and tolerate elec- tric shock less well than controls. Caldwell and associates have carried out similar studies and showed long term effects in male but not female progeny (Caldwell 21 $1., 1976). The experimental pathology of zinc deficiency in the rat was first described by Todd and coworkers in 1934 (Todd 21 21., 1934) where growth failure and loss of hair was noted. The original autopsy studies of zinc deficient rats carried out in 1940 (Follis g; 11., 1941) have been recon- firmed many times (Follis, 1966). These consist of decrease in the size of the spleen and thymus and histological changes in the skin and lining of the esophagus. The testes often show reduction in tubular diameter and other changes in their structure. The influence of zinc deficiency on the functioning of the immune system has been of interest for many years. This dates to the observation of the Egyptians that zinc oxide has a healing effect on wounds. Early studies with experimental wounds in rats in 1953 and with surgical patients who had excision of pilonidal sinuses demonstrated that zinc supplementation of the diet promoted healing (Pories & Strain, 1966; 1974). Basic studies of the inter- relationships of zinc and infection were carried out at the germ warfare center by Beisel and coworkers (Pekarek & Beisel, 1971; Beisel, 1977) and the results have generally indicated a fall in serum zinc levels in a variety of infectious illnesses and in bacterial endotoxemia. Exten- sive studies have been carried out; however zinc therapy has never been shown to produce a beneficial effect in any infectious disease studied in animal models. More basic studies have involved specific limbs of the immune system and have shown that the administration of zinc in an incubation medium stimulates the DNA synthesis of lymphocytes within six to seven days. In addition, the thymus has been shown to be a zinc sensitive tissue (Prasad, 1978). More recent studies of the effects of zinc defici- ency on the immune response of mice indicate that perhaps both humoral and cellular immunity are affected and that refeeding of zinc deficient mice may restore T—cell helper function (Fraker g; g1., 1978). Interest in the possibility of zinc deficiency in humans was shown many years ago and early experiments on the absorption and excretion of zinc were carried out (Drinker 23 g1., 1927); however studies of McCance and Widdowson (1942) failed to reveal deficiencies; however, they did state that some of the individuals they identified might become deficient "if they lived long enough." They further stated that "symptoms of zinc deficiency in human beings have, however, never been described." Tribble and Scholar (1953) carried out early studies on the zinc metabolism of young college women on self-selected diets and various other nutritional investigations were continued over the years yet as late as 1962 Underwood stated that "an uncomplicated dietary deficiency of zinc has never been observed in man." The recommended daily allowance for zinc was not established until 1974 (Food and Nutrition Board, 1974). The major impetus for investigation of the impor- tance of zinc in human nutrition came about with the description by Prasad and associates of the first zinc- respcnsive syndrome in Egypt. They found that treatment of a group of patients from the farming class who were chronic- ally ill with parasitic disease and anemia and who ingested diets prepared with unleavened bread lead to dramatic increase in longitudinal growth and sexual maturation (Prasad 33 31., 1963a, 1963b). Since that time many investi- gations of the nutritional effects of zinc deficiency have been carried out including the study of phytate concentra- tions of bread as a possible mechanism for zinc deficiency through binding (Reinhold, 1971, 1972; Ismail—Beigi 33 31., 1977). Zinc responsive dwarfism caused by problems with intestinal absorption of zinc has been described and workers have shown that some children with growth problems may have impairment of taste acuity. Zinc supplementation has yielded improvement in taste ability, appetite and growth_in these children (Hambidge, 1972). The widespread use of total intravenous nutrition led to the description of a clinical syndrome consisting of dermatitis, diarrhea, hair loss and pronounced depression. Increased excretion of zinc in the urine was demonstrated and it was noted that a rapid clinical response to zinc supplementation occurred and, therefore, the illness was linked to acute zinc deficiency (Kay & Tasman-Jones, 1975; van Rij & McKenzie, 1977). Other conditions that have been shown to be related to deficiency of zinc include the rare autosomal recessive genetic disease acrodermatitis enteropathica. Symptoms include diarrhea and vomiting, skin lesions and hair loss along with growth retardation. The condition manifests itself in the period following weaning and was often fatal until the administration of zinc was found to be thera— peutic (Neldner, Hambidge & Walravens, 1978). Conditions that also involve zinc deficiency but that are less well defined include impotency in renal failure (Antoniou, Shalhoub & Sudhaker, 1977), chronic liver disease (Sullivan & Burch, 1976) and acne (Hillstrom 33 31., 1977). The other area of research involving humans is that related to the possible role of zinc in psychiatric disease. The early work with manganese administration in schizophrenics (English, 1929; Hoskins, 1934) is the basis for the speculation regarding the use of zinc in treatment of psychiatric disorders. 10 Dietary Intake of Zinc There is a wide range of values for zinc content reported in the literature for the same foods and this is thought to be due to differences in analysis, source and variety of the food (Halsted 33 31., 1974). Zinc concentra- tions in food are listed as total zinc and studies indicate that foods and diets high in protein are also high in zinc while foods and diets that are mostly carbohydrate are much lower in zinc and nitrogen content (Osis 33 31., 1972). The National Academy of Sciences has recommended the following daily dietary allowances for zinc: infants, 3-5 mg.; chil- dren, 1-10 years, 10 mg.; males, 10-51+ years, 15 mg.; pregnant women, 20 mg.; and lactating women, 25 mg. (Under- wood, 1977). Murphy and coworkers have supplied provisional tables on the zinc content of foods and showed that the major dietary sources of zinc are foods of animal origin such as meat, fish, shellfish, poultry, eggs and dairy products (1975). The zinc content of oysters is usually reported as being high but may range from 9 mg. for Pacific species to 207 mg. per 100 grams for the Atlantic variety (Murphy 33 31., 1975; Sandstead, 1973). The dietary intake of zinc is influenced by the foods consumed and their origin. Underwood (1977) reviewed the literature on dietary intake according to country of resi- dence and found that the 0.8. adult mixed diet supplied 12- 15 mg. per day while the daily intake of zinc in adults in ll nine different regions of India ranged from 14-24 mg. per day. Tribble and Schoular (1954) carried out one of the first studies of zinc metabolism using modern techniques and a sizeable number of subjects and found that self-selected diets in thirteen college women supplied from 9.8 to 14.4 mg. of zinc daily. Price and associates (1970) studied the intake of zinc in fifteen preadolescent girls and found it to range from 4.53 to 6.93 mg. per day. White and Gynne (1971) carried out a similar study in nine college women and found a mean intake of zinc of 11.5 mg. per day. A major study of dietary intake of zinc was carried out by Murphy and associates (1971) who investigated trace element intake through type A school lunches. Lunches were collected that were to be served to children in the sixth grade from 300 schools in nineteen states. Samples of food were analyzed by spectroscopy and the amount per lunch ranged between 1.77 and 7.68 mg. with a mean of 3.91 mg. Zinc content in these diets correlated well with the content of copper, manganese and strontium. Additional studies of the dietary intake of zinc have been carried out to investi- gate the zinc content of hospital diets. Osis and coworkers (1972) studied the dietary intake of zinc at a Veterans Administration hospital including nine different diets and found that the total zinc content ranged from 7.0 to 16.3 mg. per day with an average of 11.3 mg. zinc and confirmed that diets high in protein had more zinc. Zinc levels have 12 also been studied in human colostrum and range between 10 and 20 mg. per liter while the mild concentration remains at 3 mg. per liter during the first month of lactation and then declines with prolonged lactation (Hambidge, 1979). Factors that affect the concentration of zinc in foods particularly include the methods of processing and preservation. Schroeder (1971) reviewed the topic and found that when a food is divided into its components by refinement or extraction, a majority of the trace metals go with one part or the other. Zinc was found to be largely contained in the germ and bran of wheat, a marked loss of trace metals occurred when rice was polished and refined sugar was found to have a minimal amount of zinc when compared with raw sugar or sugar cane. Basic studies of the zinc available in grains and animal products were carried out to determine the growth response of chickens by O'Dell and coworkers (1972). The results indicated that in general zinc in plant seeds was less available than that in animal products. Another factor relating to the availability of zinc in foods is the amount of phytate or inositol hexaphosphate present in cereal grains. Reinhold (1971) studied the phytate con— centration of unleavened bread in Iranian villages and found it to be considerably higher than in the leavened flat breads in commercial bakeries in cities of the same region. He postulated that the excessive intakes of phytate may explain the occurrence of overt zinc deficiency in villagers 13 and its absence in the cities. Further investigation by Reinhold and associates (1973) was carried out in thirteen villages in Iran in an area where a high phytate diet is ingested. Following admission to a metabolic ward the phytate intake fell to one—fourth to one-third of the usual intake and a positive balance of zinc occurred in the major- ity of subjects. On a more basic level O'Dell and Savage (1960) investigated the possible role of phytic acid in soy protein in making zinc unavailable to laboratory chicks. They found that the zinc in isolated soy protein is less available than in casein and this is additional evidence in support of a relationship between zinc deficiency and phytic acid in the diet. Taking a somewhat different approach laboratory studies were carried out (Ismail-Beigi 33 31., 1977) to determine whether the unleavened bread ingested by Iranian villagers had any direct binding effect on zinc and iron. In these studies it was found that the removal of phytate from tanok (unleavened bread) did not reduce its binding capability. Investigators speculated that zinc or iron can form complexes with wheat fiber and that this binding can help explain why deficiencies of these metals are prevalent in the middle eastern countries and that phytate alone may ‘ not be the whole explanation. l4 Physiological and Biochemical Aspects of Zinc Metabolism Zinc is found in the biological state complexed to organic ligands rather than free in solution as metallic ion (Vallee, 1959). Important aspects of the physical chemistry of zinc include the observation that in the blood serum zinc exists in two fractions as a firmly bound and loosely bound zinc. Several proteins have been suggested as being involved in the plasma binding of zinc including transferrin and alpha—2-macroglobulin. Additionally, several amino acids have been found to bind zinc and it is possible that this amino acid bound fraction may play a significant role in biological transport (Halsted 33 31., 1974). A recent review reports that approximately 50 per- cent of plasma zinc is freely exchangeable being loosely bound to albumen, 7 percent is bound to amino acids such as histidine and cysteine and the remainder is tightly bound to the macroglobulin and other serum proteins (Walravens, 1979). Besides binding to the components of serum, zinc has been demonstrated to bind to specific sites on the hor- mone insulin. Whether this is of definite biological sig- nificance is still undecided since insulin can be produced that is free of zinc and other metals, yet is physiologically active. Cadmium is an element that is similar to zinc and has been found to replace it in several biological systems which may vary from a partial retention of zinc-dependent 15 activity to total inhibition of it (Chesters, 1978). Recent studies of physico-chemical aspects of zinc have revealed that zinc has a high affinity for sulfhydryl groups which are important determinants of protein structure and for the activity of metalloenzymes (Chesters, 1978). In addition it has been shown that zinc can alter the properties of mem- branes 13 2133. Zinc has also been found to be a trace ele- ment that is essential in vitamin A metabolism (Smith 33 31., 1973). Zinc has been shown to be involved in the metabolism of polynucleotides in RNA and DNA synthesis and in addition laboratory studies in rats have demonstrated that zinc may be involved in carbohydrate and fat metabolism. Zinc has an important role in the metalloenzymes including especially carbonic anhydrase, carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, retinene reductase and lactic—, glutamic- and d-glyceraldehyde-3- phosphate dehydrogenase (Walravens, 1979). Zinc is involved in an enzyme that also has copper, superoxide dismutase or erythrocuprein and in addition it is thought to act as a stabilizer of biological membranes. The absorption of zinc occurs mainly in the duodenum and proximal small intestine, and the mechanism probably involves active transport. Extensive studies have been carried out on the dis- tribution of zinc in the body through autopsy studies of subjects dying of accidents or of various diseases (Halsted 33 31., 1974). The findings from many studies as summarized 16 by these investigators reveal that the highest concentration of zinc in the human is in the retina (571 mg./kg. dry weight) and significant concentrations in the range of 150 to 245 mg./ kg are found in liver, kidney and muscle. The concentration in hyperplastic tissue of the prostate gland was noted to be quite elevated at 2,330 mg./kg. It is of note that zinc con- centrations in the kidney are often not decreased even when there is zinc deficiency. The concentration of zinc in serum is said to be higher than in plasma. Modern studies have revealed zinc concentrations in the range of 63 to 160 mg./ 11 ml. in adults in plasma or serum (Halsted 33 31., 1974). Another factor of importance in the study of the metabolic role of zinc is the demonstration of a circadian variation in normal volunteers (Lifschitz & Henkin, 1971). Results from the study revealed that serum zinc was above the mean from 10 AM to 10 PM and below the mean at 2 AM and 6 AM. The ceruloplasmin level tended to follow serum copper concentrations which were also being monitored and suggested that the regulation of metal binding protein may be important in the circadian pattern. Other factors that may affect serum concentration of zinc include pregnancy, oral contra- ception, myocardial infarction, extensive surgery and acute infection (Halsted 33 31., 1974). In diseases of the thyroid gland, zinc levels have been reported to be high in hyper- thyroidism and low in hypothyroidism (Hartoma 33 31., 1979). other ions that may interact with zinc include calcium l7 (Forbes, 1960) and it has been implied that interference in zinc function by calcium occurs at the cellular level. Zinc has been determined to be present in a concentration of 175 ug./gram in hair and 125 in sperm among others studied (Walravens, 1979). Hartoma and associates (1979) studied fourteen healthy male volunteers with primarily high or low serum zinc. In all subjects extensive laboratory studies were carried out showing that blood picture, carbohydrate metab— olism, fat and protein metabolism were all normal. Sig- nificant differences between those with low zinc levels, 7.50 3 21.01, and high levels, 166.12 1 24.64 were in the alpha globulin protein fraction. Serum thyroxine, effec- tive thyroxine ratio and immunoglobulin IgA showed a ten- dency to lower levels in subjects with low serum zinc. The authors suggested there is a relationship between zinc and some metabolic functions in healthy subjects without symp- toms of zinc deficiency. zinc Toxicity The extent of tolerance to ingestion of large quan- tities of zinc depends greatly on the nature of the diet, particularly its content of calcium, copper, iron and cad- mium with which it interacts in the process of absorption and utilization (Underwood, 1977). Experimental zinc intox- ication in the rat leads to anemia, high levels of zinc and reduced levels of iron, copper, cytochrome oxidase and 18 catalase. The anemia is thought to be due to the induced iron and copper deficiencies (Cox & Harris, 1960). Weanling pigs fed large amounts of zinc had depressed growth and appetite, arthritis and internal hemorrhages (Brink 33 31., 1959). Again the type of diet is important since no evidence of toxicity was seen when a diet containing 2,000 parts per million (PPM) of zinc was added to corn-soybean, corn-fish meal, or sucrose—soybean diets fed for two weeks to baby chicks. The same amount of zinc added to a sucrose-fish meal diet was more toxic. Human zinc toxicity through inhalation of metal fumes has been known for many years (Sayers, 1938) and is seen in welders. The clinical presentation in this case may be that of an upper respiratory infection, pneumonia or many other manifestations (Papp, 1968). A second type of zinc toxicity was described where oral ingestion of twelve grams of zinc sulfate over a two day period caused drowsiness, lethargy and increased serum lipase and amylase levels (Murphy, 1970). A third type of acute zinc toxicity was observed in a patient with renal failure who developed nausea, vomiting, fever and severe anemia when the water used to carry out his hemodialysis had been stored in a galvanized tank (Gallery 33 31., 1972). Callender and Gentzkow (1937) reported on zinc intoxication occurring in soldiers who drank limeade prepared in galvanized iron garbage cans and the illness consisted of gastrointestinal l9 distress and diarrhea. A complicating factor was the pres-_ ence of significant amounts of antimony and iron in the metal. A more recent report by Brown and associates (1964) described mass food poisoning where several hundred people became ill after attending a celebration where food was stored in galvanized tubs. In another episode reported by the same investigators individuals became sick after ingest— ing punch stored in a galvanized container and chemical analysis of a five ounce serving contained 525 mg. of zinc. Zinc in Infection and Immunity The first studies of zinc in infection were carried out by Vikbladh in 1951 who noted that the serum zinc con- centration was depressed in infection. The majority of studies of zinc metabolism in infection have been carried out by Beisel and associates (Pekarek & Beisel, 1971; Beisel, 1977). When infection is present zinc moves from serum to liver and in contrast copper moves from liver to the binding protein ceruloplasmin in the serum. When the zinc enters liver cells it binds to the metallothionines of the cell. The concentration of zinc in serum in humans is usually depressed in infectious diseases such as dysen- tery, typhoid fever and in tuberculosis and in experimentally induced endotoxemia (Smith 33 31., 1972). Similarly in experimental animals tularemia, rocky mountain spotted fever and other conditions cause a fall in serum zinc concentrations (Pekarek & Beisel, 1971; Powanda 33 31., 1976). It has been 20 postulated that a mediating substance for zinc depression is released by phagocytes of the host and has been shown to be produced 13 31333 by activated neutrophiles and macro- phages. This has been investigated in experimental animals where isotopic zinc tracers have been given demonstrating that there is an accelerated uptake of zinc into the liver (Beisel, 1977). The role of zinc in host defense responses were summarized by Beisel as follows: zinc contributes to plasma membrane integrity and the functioning of binding sites; in high concentrations it inhibits phagocytic functions; high concentrations cause mitogenic effects in lymphocytes and low body zinc stores are associated with dysfunction of T cells. Zinc is also important for nucleic acid and protein synthesis, has a key role in the metalloenzymes with regard to cellular function, and is also known to have negative effects on the growth of microorganisms when not in the optimal concentration. Basic studies of the effect of zinc deficiency on the immune response have been carried out by many investigators (Frost 33 31., 1977; Miller 33 31., 1968; Haas 33 31., 1976; and others). Spleen and thymus weights have been known to decrease in deficient animals and thymic atrophy has been described in zinc deficiency in the rat. Peripheral lymphocyte counts are said to be reduced, there is a deficiency in the immunologic response to experimental antigens, and there is a reduction in serum immunoglobulins 21 as well. Experimental findings recently reported by Fraker and coworkers (Fraker 33 31., 1977; Luecke 33 31., 1978) have indicated that zinc deficiency may interfere with T cell helper function; however the severe dietary deficiency caused by prolonged feeding of a zinc deficient diet will also contribute to immunologic deficiency on a nonzinc related basis. The earliest practical studies of the role of zinc in the inflammatory and healing response were surgical inves- tigations carried out by Pories and coworkers (Pories & Strain, 1974) in 1951 where it was noted that wounded and burned rats maintained on diets accidentally enriched with zinc healed more rapidly than in animals fed control diets. Since that time numerous studies of the relationship of zinc and healing have been carried out and it has been shown that the turnover of zinc is high and that it moves rapidly into healing tissues. It has been postulated that the importance of zinc in healing lies in its essentiality in the basic processes of healing such as protein synthesis and cellular growth and replication. The enhancement of wound healing by zinc may stem from a heightened metabolic demand for the element in collagen synthesis yet this is not completely documented (Underwood, 1977). Experimental Zinc Deficiency in Animals The first attempt at the study of experimental deficiency was carried out in 1922 by Bertrand and Benzon 22 who fed mice a purified diet that was believed to be free of zinc. The animals lived for an average of only twenty days because no vitamins were added to supply those destroyed during purification of the ration. Another group of animals was fed the same purified ration to which a small quantity of zinc had been added and they found that animals in the latter group lived 25 to 50 percent longer. A later study by Hubbell and Mendel (1927) in which mice received a diet supplying only 0.005 mg. zinc per animal daily and additional vitamins showed no great differences in the growth of the animals. Todd and associates (1934) were among the first workers to utilize the rat as an experimental animal in zinc research. In their early work they fed a diet contain— ing only 1.6 mg. of zinc per kg. of dry weight to weanling rats and found that growth and weight of experimental animals was considerably less than in another group of animals fed the same diet and added zinc in a concentration of 5 mg. of zinc per 100 grams of ration. Day and McCollum (1940) investigated the effect of acute zinc deficiency on the young rat using restriction of dietary intake to not more than two to four ug. of zinc per rat daily. They found that growth was greatly retarded after two to three weeks, there was marked eczema in two animals and alopecia or loss of hair as described by previous workers. Hove and associates (1937) investigated the physiology of zinc in the nutrition of the rat and made the following conclusions 23 regarding the studies at three to eight weeks of age: zinc , deficiency is prevented when 40 ug. per day is included in the diet. There was a 50 percent decrease in growth rate when 22 ug. of zinc was given per day. They were unable to detect any disorders in carbohydrate metabolism through determination of urinary sugar, blood sugar, liver glycogen or glucose tolerance. They further concluded that there is a marked delay in intestinal absorption in the zinc deficient rat and that this delay is probably greater for nitrogenous products than carbohydrates. Zinc deficient rats have been shown to have a fall in creatinine and a normal urinary nitrogen excretion per gram of food eaten in spite of the demonstrated delayed absorption, thus suggesting the pos- sibility of a lower rate of endogenous metabolism. Pitui- tary transplants carried out by injection of tissue by needle into muscle produced growth stimulation and increased efficiency in converting food to body weight. Researchers have postulated that zinc is involved in the production or utilization of some pituitary hormone which controls the motility and tone of the intestinal tract. Numerous investigations of zinc deficiency in rats have been carried out, however other investigators have used the chicken (O'Dell 33 31., 1958) and calf (Miller & Miller, 1962). The zinc requirements of chickens and swine are considerably higher than those of rats when maintained on a zinc deficient diet for a period of four to six weeks 24 beginning at one day of age. These animals developed symp—. toms ranging from mild deficiency manifestations such as slow growth, shortening and thickening of the long bones, development of frizzled feathers, abnormal respiration and an unsteady gait when kept in galvanized batteries to a very severe deficiency when kept in galvanized batteries that were lacquered. The severely deficient animals exhibited the same symptoms but to a more severe degree and also devel- oped keratosis of the skin and an increase in packed cell volume of the red cells. Microscopically there was.parakera- tosis in the esophagus and poor calcification of bone. Experimental zinc deficiency in calves was studied by Miller and Miller (1962). They used seven male Holstein calves in two experiments and fed a low-zinc purified diet with and without supplemental zinc in the two experiments. These animals developed a severe parakeratosis as originally described in swine by Tucker and Salmon (1955). Zinc deficiency symptoms in the calves included anorexia, dull and listless appearance, low weight gain, alopecia and various changes in the skin. It was also noted that the testicles were undersized and there was reduced blood zinc content and carbonic anhydrase activity. All of the con- ditions responded to zinc supplementation with the exception of the undersized testicles. The effect of zinc deficiency on food intake and feeding patterns of rats was investigated by Chesters and 25 Quarterman (1970) who found that incipient zinc deficiency . in the young rat was characterized by a cessation of growth followed by an extended period during which body weight varied very little. The voluntary food intake of zinc deficient rats fell to 70 percent of that of the controls. The day to day variation increased markedly and was associ- ated with cyclical food consumption. When food was slightly restricted at this point the troughs of the cycles dis— appeared. Concurrent with the fall in food intake the zinc deficient rats ceased to gain weight and a similar change was found in the pair-fed controls. Additional findings of note were that force feeding of rats with 140 percent of their voluntary intake rapidly induced signs of ill health. The zinc deficient animals responded to a zinc supplemented diet within one to two hours by an increased food intake. Chesters and Will (1973) studied additional factors controlling food intake in zinc deficient rats and found that when the food intake was examined for periods of two hours throughout the day the deficient rats were found to eat on fewer occasions than the control rats; however when the deficient rats did eat, the quantities eaten in two hours showed the same distribution of weights as did those for the zinc adequate diet. They also found that reduction of the environmental temperature would increase the deficient animals' intake of food. Statistical analysis of the pat- terns cf plasma zinc concentration and food intake showed 26 the inverse correlation of food intake during a day and the, plasma zinc concentration at the end of twenty-four hours was more significant than any of the other correlations tested. McConnell and Henkin (1974) investigated changes in NaCl preference and in plasma and urinary zinc in weanling rats fed ad libitum zinc deficient or zinc supplemented diets and in rats pair—fed an amount of food equal to that eaten by rats fed a zinc-deficient diet. Sodium chloride preference in the zinc deficient rats was significantly greater than in pair-fed controls or in rats fed zinc supple— mented diet ad libitum: this alternation in preference occurred within three days of initiation of the zinc deficient diet although anorexia was observed in these rats within the first two days. Both increased preference for NaCl and anorexia occur in zinc deficiency. Concentrations of plasma and urinary zinc were significantly lower in rats fed the zinc-deficient diet than in pair fed controls or those fed the zinc supplemented diet ad libitum. The concept of a physiological role for zinc in normal taste sensation together with the observation that hypogeusia (loss of taste acuity) and dysguesia (disordered taste or pica) that is correctable by zinc is now accepted (Underwood, 1977). Walker and Kelleher (1978) studied the concentra— tions of plasma, whole blood and urine zinc in young adult rats fed zinc deficient and zinc supplemented diets for a 27 period of sixty-five days. They found that within forty- eight hours the plasma zinc concentrations fell to 25 per- cent of control values and remained at this level throughout the period of the study. Whole blood values for zinc did not change during the study. Urine zinc fell to very low values ten days after the beginning of the zinc deficient diet but after thirty days rose to control values. A sub- stantial diurnal variation of zinc levels in plasma was found and the level was significantly lower in the fasting state. An investigation of possible correction of experi- mental zinc deficiency in the rat was carried out by Keen and Hurley (1977) where pregnant rats consuming a zinc deficient diet had zinc applied topically as zinc chloride. Topical zinc supplementation by application to the skin resulted in a rise in plasma values similar to the control group fed a normal diet and significantly higher than those of rats receiving no zinc application to the skin. Animals given twenty-four, four hour applications demonstrated plasma zinc values that were higher than in any other group including controls. The effect of zinc repletion late in gestation was studied by Apgar (1973) who gave zinc acetate by stomach tube to pregnant females at various times in late gestation. These experiments indicated that stressful parturition 28 could generally be avoided if the zinc was administered to deficient rats between day 19 and day 21. Extensive studies of the effects of zinc deficiency on the enzymes in tissues of rats have been carried out. Kfoury and coworkers (196 ) investigated (1) alcohol and glutamic dehydrogenase in liver and kidney, (2) alkaline phosphatase in intestinal mucosa, kidney and plasma, (3) catalase in liver, kidney and blood, (4) xanthine oxidase in liver and kidney and (5) aspartic and alanine aminotransferases in liver in rats fed diets low in zinc or protein or both and in their pair-fed controls for 45 to 180 days. The alkaline phosphatase decreased in intestinal mucosa but not in plasma or kidney. Alcohol dehydrogenase decreased marginally in the livers of rats on diets with two to four PPM of zinc but not in plasma or kidney. A correlation existed between alcohol dehydrogenase activity in liver and zinc concentration in liver. Elevated concen— trations of uric acid in plasma occurred infrequently. Prasad and Oberleas (1971) confirmed that alcohol dehydrog- enase in liver and carboxypeptidase in pancreas were sig- nificantly reduced in the deficient rats. No changes were observed when isocitric dehydrogenase (manganese dependent) and succinic dehydrogenase (iron dependent) were studied in the tissues. These results were generally con- firmed by Huber and Gershoff (1973) who also felt that decreased carbonic anhydrase in brain and red cells and the 29 decreased alkaline phosphatase in heart of zinc deficient animals seemed related in part to alterations in food intake. Many investigators have studied the effect of zinc deficiency on the synthesis of nucleic acids. Fujioka and Lieberman (1964) carried out some of the earlier work regard— ing the requirement of zinc for DNA in rat liver. They found that use of EDTA chelation largely or completely pre- vents the increase in the rate of liver DNA synthesis that normally follows experimental partial hepatectomy in the rat. Sandstead and Rinaldi (1969) confirmed the observation of these workers demonstrating that dietary zinc deficiency in the rat will impair 13 3133 zinc deficiency in the rat liver parenchymal cell. Extensive work by Sandstead and Terhune (1974) also showed that zinc deficiency in suckling rats results in a decreased activity in liver nuclear DNA dependent RNA polymerase, that the sedimentation character- istics of liver RNA are altered by zinc deficiency and that the incorporation of uridine into liver RNA is decreased by deficiency of zinc. Experimental work in pigs by Prasad and Oberleas (1974) was carried out giving a zinc deficient diet and comparing deficient animals and pair-fed controls. Results showed that growth was retarded and parakeratosis of the skin became moderately severe by the end of the second week. Extensive biochemical and histochemical studies were carried out and it was shown that the DNA content of the 30 tissues per mg. of wet weight was the same for deficient animals or pair-fed controls. Significant differences were seen, however, when the zinc dependent enzymes were expressed as activity per mg. of DNA. They concluded that glutamic dehydrogenase and carbonic anhydrase are much less suscep- tible to zinc deficiency than alcohol dehydrogenase, alka- line phosphatase and carboxypeptidase. Studies in zinc deficient rats using incorporation of radiolabeled cystine-S were carried out by Hsu (1974) who found that the incorporation of this material into skin protein was only one—seventh of that in zinc supplemented controls that had been pair-fed. He found no differences in the incorporation of the radiolabeled cystine into liver, kidney, testes or muscle but there was more incorporation of the cystine into pancreatic protein of the deficient rats which he attributed to a depletion in methionine in zinc deficient animals. Additional studies were done with multiple compounds and tritiated thymidine and he concluded also that there was slower rate of epithelial renewal as demonstrated by a decrease in thymidine incorporation into DNA and a decrease of amino acid incorporation into skin protein. Extensive studies of the concentration of ash and zinc in bones were carried out by Swenerton and Hurley (1968) and both were reduced in the femurs of rats that were zinc deficient. More recent studies have been carried out 31 to determine if the net zinc content of bone was altered during the development of zinc deficiency in the growing rat (Brown 33 31., 1978). They utilized control, zinc deficient and pair-fed groups of weanlings. They noted that the plasma zinc concentration of the zinc deficient group decreased dramatically during the first twenty-four hours of the deficient diet and continued to decline over the next two days. Body weight gain stopped after one week. Femur zinc concentration decreased 61 percent during the experi- mental period of twenty-three days while the total femur zinc decreased 37 percent. Calcium and phosphorous content con- tinued to increase and the ratio remained constant. Data indicate that zinc can be released from bone during periods of dietary zinc deficiency and furthermore the decline in zinc appears to be independent of calcium and phosphorous in the bone. The interrelationships of zinc and other elements besides calcium and phosphorous have been investigated by a number of workers. Gray and Ellis (1950) were among the first investigators to study the association between copper, molybdenum, zinc and lead in the nutrition of the rat. They noted that addition of molybdenum to diets was toxic and retarded growth while zinc produced an anemia and the addi- tion of copper corrected the anemia from excess zinc. Kinnamon (1966) studied the absorption of radiolabeled iron in the rat under the influence of dietary supplements of 32 copper or zinc. He found that dietary zinc or copper had no effect on absorption of radiolabeled iron but that diets high in zinc significantly lowered retention of iron stores. Kinnamon suggested that iron absorbed from the digestive tract apparently followed the normal metabolic pathway but later deviated under conditions of zinc toxicity while copper had no effect. A change in the ratio of zinc to copper ingested by rats was found by Klevay (1973) to cause an hypercholestero— lemia and lead to an hypothesis that such a change in the balance might be involved in the genesis of atherosclerosis Further studies led Klevay to conclude (1975) that the ratio of copper and zinc may be the most preponderant factor in heart disease; although this hypothesis is not completely accepted (Kine & Sifri, 1976). Murthy and associates (1974) carried out extensive studies of the interrelationships of zinc and c0pper in the rat and improved upon methods of use of hair analysis to evaluate zinc and copper nutriture and metabolism. They concluded that liver copper was inversely related to serum zinc, aortic copper and hair copper were directly related to serum copper and hair zinc was directly related to serum zinc. The major other area of investigation involving zinc and other metals has been in the observation (Gunn 33 31., 1960, 1963) that the toxicity of cadmium for the testes of the rat may be protected against by the b: 33 administration of zinc. The rat has long served as a useful animal for almost all aspects of experimental zinc deficiency or toxicity. Teratogenic Effects of Intrauterine Zinc Deficiency Studies with poultry were among the first attempts to look at the effect of zinc deficiency on intrauterine development. Turk 33 31. (1959) demonstrated that when hens were fed a zinc deficient diet they produced chicks that were weak and died soon after hatching. Other investi— gators (Blamberg 33 31., 1960; Keinholz 33 31., 1961) used more severe dietary restriction of zinc and noted gross mal- formations in the chick embryos such as skeletal defects, brain abnormalities and visceral herniation. Hurley and associates (Hurley 33 31., 1966, 1971) were the first to study the effect of zinc deficiency on the progeny of preg— nant rats and noted frequent embryonic death, intrauterine growth retardation, and a high incidence of congenital mal- formations of all systems. These findings have been con— firmed by numerous other workers as reviewed by Hurley (1974). More recently reported work from Hurley's labora- tory (1977) described observations that lung abnormalities were frequently found in addition to the malformations of the central nervous system so often noted. Biochemical studies of lungs indicated that fetal lung lecithin was lower in zinc deficient fetuses and found that the 34 lecithin:sphinogomyelin ratio was lower in zinc deficient fetuses indicating that zinc is important for the functional and morphological development of the lung. Hurley and investigators have shown that the uptake of tritiated thy— midine was severely depressed in zinc deficiency in twelve day old rat embryos. Autoradiography of the cerebral cortex from the embryos showed a similar effect with lower concen- trations of the silver stain in the zinc deficient fetus (Swenerton 33 31., 1969). More recent studies (Hurley, 1977) indicate that the head is more vulnerable to deficiency of zinc than the rest of the body and incorporate less tritiated thymidine into DNA and RNA relative to the control than did the body region. Bell and associates (1975) have studied the effect of zinc deficiency on nucleic acid synthesis and its effect on chromosomes. Pregnant rats given a zinc deficient diet showed bone marrow and fetal liver cell chromosomal aberrations. Additional studies of the effect of zinc deficiency on the brain of the suckling rat were carried out by Sand- stead 33 31. (1972) using radiolabeled thymidine and it was shown that the zinc deficiency impaired DNA and protein synthesis in brain tissue. More recent studies of the effect of zinc deficiency on pregnancy were reported by McKenzie 33 31. (1975) who found that deficient dams were anorexic and lost weight. The fetuses showed intrauterine growth retardation as compared to controls, the brains were 35 smaller than controls and the livers were severely affected. McKenzie noted that the total cell number was reduced and there was an increase in cell size in the brains of deficient animals. The growth failure in zinc deficient liver resulted in less total DNA, RNA, protein and lipid and there was less incorporation of radiolabeled thymidine into DNA in the liver. Postnatal zinc deficiency during the suckling period has also been investigated by Fosmire 33 31. (1975) who found that there was a depression of normal growth and that the forebrains were smaller than those of control animals. There was reduction of total RNA because of the smaller brain size and the amount of protein per brain cell was also decreased. Experimental Zinc Deficiency in Humans Human zinc deficiency with clinical manifestations was first reported from the middle east in 1963 (Prasad et a1., 1963) and in the past sixteen years the spectrum of illness related to this trace element has broadened con- siderably. The first experimental studies of zinc metab— olism in humans were carried out in the 19205 and reviewed by Lutz (1926). Investigators had shown by that time that the usual human intake of zinc was in the range of 10 to 15 mg./day and that most of this was excreted in the feces. The classical studies of the absorption and excretion of zinc were carried out in 1938-1941 by McCance and Widdowson (1942). In these studies forty-five zinc balance 36 determinations were carried out on twelve people including the intravenous injection of zinc in ten of the subjects. These studies were carried out before many studies of the response of animals to intravenous injections had been performed and it was stated that "the response of animals to zinc injections will be found to be similar to that of human beings" (McCance and Widdowson, 1942, p. 694). This is an unusual example of generalization of human results to animals rarely seen in the literature. Zinc balance studies were subsequently performed in young college women (Tribble & Secular, 1954) as described earlier in this review and more recently in thirty-six school children (Engel 33 al., 1966). These children were studied under controlled dietary conditions in a camp envi— ronment. Daily zinc intake was shown to range from 4.6 to 9.3 mg. and urinary zinc excretion was relatively constant (274 i 69 ug/24 hours) and constituted 6.5 percent of the total zinc lost through urine and feces. Enhancement of gastrointestinal absorption of zinc was encountered in twelve subjects in one of three studies during conditions of low protein intake and was more predictable in two other studies reported in the same paper. Radiolabeled zinc has been utilized for study of the metabolism of zinc in humans and it has been found that most of the zinc of the body is in a state of constant movement with accumulation and turn- over varying with the tissue being studied. It was found to 37 enter the skeleton slowly and remained firmly bound there for long periods (Prasad, 1966). The metabolism of radio— labeled zinc-65 in patients with cancers was studied by Ross and coworkers (1958) who found that the zinc rapidly appeared in leucocytes, reached a maximum in twenty-four to forty-eight hours and then decreased in concentration but persisted in the circulating leucocyte mass for several weeks. It was also found to be incorporated rapidly into red blood cells with a maximum concentration in ten days. When zinc-65 labeled erythrocytes were transfused into normal recipients the radioactivity disappeared at a rapid rate indicating that zinc-65 was not firmly bound to the red cell. The zinc was deposited into the liver in highest concentration (Ross, Ebaugh & Talbot, 1958). Prasad gt El- (1963) studied the metabolism of zinc-65 in normal subjects and patients with the syndrome of iron deficiency anemia, hepatosplenomegaly (enlargement of liver and spleen), dwarf- ism and hypogonadism. The mean plasma zinc turnover rate for normal patients was 1.00 i 0.09 mg./kg. The urinary excretion of zinc—65 in normals after thirteen days was 2.8 i 0.56 percent of the administered dose. In normal subjects the fecal excretion of zinc-65 was 0.66 i 0.19 percent of the administered dose/100 grams of stool. Spencer gt El- (1966) also studied the metabolism of zinc—65 in humans following intravenous and oral administration of the com- pound. These investigators found that the intestine was 38 the major pathway of excretion of zinc—65. The zinc was found to be deposited mainly in the liver and reticulo— endothelial system and a high uptake of zinc—65 was noted to be present in the endocrine glands. Probably the most significant findings of this investigation was that approxi- mately 50 percent of the orally administered zinc-65 was absorbed and this was not affected by the level of calcium taken in. It is of note that there was a significant dif- ference (p < 0.01) between normal subjects and the dwarfs studied by Prasad gt gt. (1963) in the turnover rate, twenty- four hour exchangeable pool, and urinary excretion of zinc— 65 while the excretion in stool in percentage of dose administered was significant at 0.05. Spencer and coworkers (Spencer gt gt., 1974) extended their earlier studies of zinc metabolism in normal individuals and in patients with neoplasms and showed that there was no correlation between excretion of zinc and urinary calcium. The data also revealed that, although a ten to 20 fold increase in urinary calcium was induced by infusing calcium, this resulted in only a very slight increase in urinary zinc excretion. Administration of ammonium chloride given orally increased the urinary calcium excretion considerably more than the excretion of zinc. The effect of starvation on urinary and plasma zinc levels was investigated by these workers using a period of total caloric restriction which resulted in marked weight loss and marked 39 and sustained increase in the excretion of zinc in the urine. These excretions reached a maximum of 6.5 mg./day and represented an approximate increase in urinary excre- tion of zinc of ten fold. The plasma zinc level did not fall in spite of this urinary loss of zinc, and in the majority of patients it increased during fasting. When other subjects were maintained on a low protein-low calorie diet containing two mg./kg. of zinc per day there was a gradual weight loss and a similar loss of zinc via the kidney. The total zinc loss in two subjects studied for sixty and sixty-four days was 270 mg. during starvation and 250 mg. on a low protein diet respectively. These same investigators also studied the effect of tetracycline admin- istration in a patient with an t. ggtt urinary tract infec- tion and found that the plasma level and urinary excretion increased during the time of administration of this anti- biotic. The studies by these workers have demonstrated that the amount of zinc necessary to attain equilibrium is approximately twelve mg. per day under normal conditions. The zinc balance in malnutrition became very positive on a normal intake and continued to be positive until the nutri- tional repletion was completed. Their studies during weight reduction have shown that the loss of zinc during weight reduction induced by a low caloric intake is as great as during total starvation. 40 Henkin gt gt. (1975) have investigated a syndrome of acute zinc loss that occurs when the amino acid histidine is administered to patients with systemic sclerosis (sclero- derma). In six patients anorexia, dysfunction in taste and smell, mental changes and cerebellar dysfunction developed on an acute basis that was associated with significant decreases in serum zinc concentration and increases in urinary excretion. Findings indicated that the adminis— tration of zinc, even in the face of continuation of the histidine, resulted in return of signs and symptoms to or toward normal within eight to twenty-four hours at the time that correction of the zinc concentration in the blood occurred. The eXplanation of the authors for these findings has been that the preferential binding of zinc to histidine alters the normal equilibrium and leads to the stripping of zinc from albumin resulting in the excretion of a sig- nificant amount of zinc in the urine since the small molec- ular weight histidine-zinc ligands can easily pass into urine. It is of interest that the administration of his- tidine leads to the lowering of zinc levels with development of anorexia and then loss of smell. This was reversible with administration of zone. This paper contributes a significant amount of information to the experimental zinc deficiency literature but the authors did not mention much about the role of the underlying disease scleroderma in the etiology of the syndrome. 41 More recently, Prasad and coworkers (Prasad gt gt., 1978) have intensively studied four patients in which they induced a marginal zinc deficiency using dietary means. They followed two subjects receiving a semi-purified diet based upon texturized soy protein for a period of fifty-two weeks and another two subjects similarly fed a special diet for sixty-four weeks. Both groups received an oral zinc supplement at some point during the experiment. The first two subjects received 2.7 mg. of zinc daily and complained of mild roughening of the skin and lethargy while the second group received 3.5 mg. of zinc daily and had no symp- toms or complaints. There was a decrease in body weight in all four subjects which was attributed to dietary zinc restriction. Urinary excretion of zinc decreased in three out of four subjects as a result of zinc restriction; how- ever in one of the four subjects there was no decrease in urinary excretion of zinc since he was also receiving a diuretic, hydrochlorothiazide, for mild hypertension during the study. One unexpected finding in the multiple chemical studies carried out was that the plasma ammonia level appeared to increase as a result of the zinc deficiency. There was also some suggestion that the zinc deficient state led to hypercatabolism of fat in the four subjects. Prasad and associates also believe that measuring the con— centration of zinc in a twenty-four hour urine may be of help in diagnosing zinc deficiency provided cirrhosis of the 42 liver, sickle cell disease and chronic renal disease are ruled out. Clinical Manifestations of Zinc Deficiency in Children The syndrome of adolescent nutritional dwarfism reported from the Middle East by Prasad gt gt. (1963) was the first documented clinical condition shown to be caused by zinc deficiency. Since that time deficiency of zinc, or in one incidence, congenital elevation of serum zinc has been demonstrated to be linked to twelve syndromes in the pediatric pOpulation. These conditions will be reviewed, beginning with the syndrome first reported, on adolescent nutritional dwarfism. Halsted (1977) described the events leading to the study of the original patient later demonstrated to be suffering from zinc deficiency and gives credit to Prasad for suspecting it. This young man of twenty-three was dwarfed, anemic and had underdevelOped gonads. Prasad soon moved to Egypt where in 1960 in Cairo he headed a U.S. Navy research unit where he found many cases similar to the ones in Iran. He found that this clinical picture had been described by Lemann (1910) who felt that this was infantilism related to hookworm disease. A number of studies have been reported describing the syndrome (Prasad gt gt., 1963a, 1963b; Sandstead gt gl., 1965; Sandstead gt gt., 1967; Eminians EE,El-r 1967; Ronaghy gt gt., 1968; Say gt gt., 43 1969) but these were not completely accepted as demonstrat- ing that zinc deficiency was the cause of the syndrome until zinc supplementation was shown to affect clinical improvement or cure (Halsted gt gt., 1972; Ronaghy gt gt., 1974). The unraveling of the puzzle was quite involved since many factors apparently enter into the clinical situation. Com- plicating factors include the presence of parasites (often multiple) in the patients, a frequent history of geOphagia (often clay or dirt eating) and the possible influence of high phytate concentration in the unleavened bread in the Middle East causing poor absorption of dietary zinc. Clinically the patients resemble patients with idiOpathic hypOpituitarism and treatment with zinc is followed by increased growth and sexual maturation which exceeds the changes resulting from adequate diet or treatment of iron deficiency. Extensive studies have shown that children with these clinical presentations do not always respond to zinc. Carter and associates (Carter gt gt., 1969) gave iron sul- phate, zinc sulphate or a placebo by use of the double blind technique and saw no differences in growth or sexual devel- Opment between the three groups after one year observation. The zinc and iron levels in the plasma returned to normal, however one criticism of this study is that the subjects actually received only five and one-half months of therapy. Coble EE.E$; (1969) carried out extensive studies of endocrine function of boys in Egypt with retarded growth, 44 delayed sexual maturation and zinc deficiency and found that aflthough the clinical appearance was similar there were two specific endocrine patterns that were involved. One group had relatively normal function and one group had poor growth hormone responses to hypoglycemia. Thus a simple relation— ship of zinc deficiency and hormonal abnormality was not apparent in all the children although they looked the same. Zinc supplementation studies carried out by Ronaghy gt gt. (1974) demonstrated significantly increased height, weight and bone age in the children receiving supplemental zinc, and the total serum protein and albumin concentration increased although the zinc levels remained abnormally low throughout most of the study. The results demonstrated a clearly defined stimulation of growth when a supplement was added to the diet that contained protein, vitamins and zinc; although a tendency toward acceleration of sexual develop- ment was noted in the boys no statistically significant changes occurred during the twenty month treatment period. Hambidge gt gt. (1972) reported their work on determination of the concentration of zinc in 338 normal adults and children whose ages ranged from birth to forty years. Ten children were found to have hair levels of zinc of less than 70 ppm. Seven of these children had a history of poor appetite and eight had weights at or below the tenth percentile; this was not explicable on a familial basis. Taste acuity was tested in six of these children 45 using four chemicals representing salt, sweet, bitter and sour and five had objective evidence of hypogeusia or diminished taste acuity. After supplementation of the diet with zinc the taste acuity was normalized in each of the children and the hair zinc levels increased. The discovery of the relationship of zinc deficiency to the etiology of the clinical manifestations of a lethal inherited disorder called acrodermatitis enteropathica is one of the most significant observations in pediatric aspects of zinc metabolism. This disease is a rare inherited disorder that is transmitted as an autosomal recessive trait vvhich appears at weaning or earlier in the baby who is not breast fed. This disease manifests as a severe skin rash, abnormal bowel functions, profound psychic changes and growth failure that responded moderately well to the admin- istration of a drug called diodoquin (di—iodohydroxyquino- line). Studies show that this drug chelates an abnormal factor (oligopeptide), that results from lack of the enzyme oligopeptidase, which binds zinc. It has now been demon- strated that the administration of zinc alone will very adequately treat this otherwise fatal condition (Moynahan, 1974). Recently it has been found that women with acro- dermatitis enterOpathica who had survived without zinc therapy may deliver infants with a high rate of congenital malformations (Hambidge gt gt., 1975). 46 Currently the possibility of zinc deficiency in relation to many other illnesses of the pediatric patient is being investigated. Impairment in cellular immunity is a good example and clinical research has shown that zinc deficient children will respond to zinc therapy with improvement in immunocompetance. This indicates that zinc deficiency may be a factor in the increased susceptibility to infections in conditions such as protein-energy mal- nutrition (Walravens, 1979). Sickle cell disease and the syndrome of adolescent nutritional dwarfism described from the Middle East have many clinical characteristics in common. This partially relates to poor growth and delayed sexual deve10pment and led Prasad gt gt. (1977) to study the effect of treatment with zinc on the development of the picture and it is important to note that the plasma, red blood cell and hair levels of zinc were significantly decreased in sickle cell disease patients as compared to controls. A limited and uncontrolled trial of zinc therapy revealed beneficial effects of zinc on growth and development of secondary sexual characteristics in teenagers and young adults. Zinc has been described as being of importance in the clinical picture of intestinal malabsorption (Sandstead, 1973) in the form of the adolescent dwarfism syndrome and similarly more recently with cystic fibrosis (Dodge & Yassa, 1978). In addition zinc may have a role in treatment of 47 rheumatoid arthritis since it has been found that serum zinc levels are low in that condition (Simkin, 1976). Two controlled studies have also indicated that oral zinc sul- phate is useful in treatment of acne, a common pediatric problem (Michaelsson gt gt., 1977; Hillstrom gt gt., 1977). Hereditary hyperzincemia was described in 1976 (Smith gt gt., 1976) in a twenty-eight year old black man, his siblings, his son, and in one of two nieces. Although the only mani- festation of the syndrome noted so far is abnormally high blood levels of zinc it is currently being looked at for pathological significance. Clinical Aspects of Zinc Deficiency in Adults Zinc deficiency in adults is more generally dis- tributed throughout the various organ systems and is less specific than in children where it usually is directly related in some way to decreased intake in the diet. It is important to note that an extensive study of the molec— ular basis of taste and its diseases has been carried out (Henkin gt gt., 1969) and these workers have reported a decrease in taste acuity that accompanies many disease processes. It may well be that the zinc deficiency seen in some adult conditions (e.g., lung cancer) is due to decreased dietary intake of the element because of some toxicity on the taste mechanism rather than a more direct effect by the disease process on the metabolism of zinc itself. 48 Zinc levels may be decreased in a variety of con- ditions that are either acute or chronic in nature and these disorders associated with zinc deficiency are listed by Cordas and Holland (1977) as follows: Nutritional Alcohol and Cirrhosis Renal Disease with Proteinuria Malabsorption Syndrome Burns Iatrogenic (a) COpper Administration (b) Penicillamine Therapy (c) EDTA or other Chelating Compound (d) Hemodialysis (e) Total Parenteral Hyperalimentation (f) Oral Contraception Acrodermatitis Enteropathica Sickle Cell Disease Porphyria Malignancies, Especially Lung and Leukemia Chronic Leg Ulcer PostOperative Situation Schizophrenia (Some Instances) Postmyocardial Infarction Post Cardiac Surgery During Infection Down's Syndrome Atherosclerosis--Moderate to Advanced In addition to this long list we can add several topics as follows: Diminished sexual function in men on hemodialysis for renal failure. When zinc chloride was added to the dialysis solution there was a striking increase in the testosterone and follicle stimulating hormone levels in plasma to normal values (Antoniou gt gt., 1977). In addition in another study of male subjects with oligospermia (decrease in sperm) zinc administration was accompanied by increased levels of plasma testosterone and increased sperm count (Hartoma gt gt., 1977). 49 Diabetes mellitus has been shown by Pidduck and coworkers (Pidduck gt gt., 1970) to have some abnormality in zinc metabolism. Diabetics were shown to have a sig- nificant elevation of zinc excretion in urine but investi- gations of the leucocyte contents did not demonstrate any difference in leucocyte zinc levels between patients and controls (Pidduck gt gt., 1971). Plasma zinc and COpper in obese patients before and after intestinal bypass surgery were investigated by Atkinson and coworkers (Atkinson gt gt., 1978) who found that obese patients showed significantly lower zinc levels than lean control subjects. After the intestinal bypass surgery their plasma zinc and c0pper levels were significantly lower than pre-operatively. One of the patients deve10ped leucopenia (fall in white blood count) associated with a very low copper level but responded well to oral c0pper sulfate treatment. They recommended that careful obser- vation and replacement therapy be carried out in all patients after intestinal bypass surgery. Extensive studies of zinc metabolism in alcoholic and postalcoholic cirrhosis have been carried out by Sullivan and Lankford (1962) and by Sullivan and Heaney (1970). These workers found that there is an increased urinary excretion of zinc in most patients with post- alcoholic cirrhosis. Approximately 50 percent of chronic alcoholics without cirrhosis also show excess excretion of 50 zinc in the urine. Halsted and coworkers (Halsted gt gt., 1968) investigated plasma zinc levels in patients with various lever diseases and noted that in all types of liver disease the plasma zinc is consistently less than in con- trols. They were unable to differentiate between the dif- ferent types of liver disease, i.e., infection or cirrhosis due to alcoholism based upon the zinc levels. Relatively few studies of the zinc status of the elderly have been carried out. Gregor (1977) investigated the dietary intake of sixty-five institutionalized aged subjects and found that the dietary intake was generally adequate compared to the recommended daily allowances except for zinc and magnesium. She found that 5 percent of the subjects had hair zinc levels indicative of defici- ency. The levels were significantly correlated to protein intake in men and were significantly lower in women taking medication for coronary heart disease. Approximately one- fifth of the subjects had decreased taste acuity but neither dietary zinc intake, hair zinc levels nor smoking correlated to taste acuity. Weismann and associates (Weissman gt gt., 1978) investigated the geriatric pOpulation of an institution with 585 inhabitants and found that twenty-six peOple with a mean age of eighty-two years had skin lesions suggestive of zinc deficiency. Ten of the patients had a subnormal plasma zinc concentration. This group was treated for four 51 weeks with oral zinc with no improvement of the skin con- dition and it was concluded that the skin changes were not caused by zinc deficiency. It was noted that the plasma albumin was subnormal in all patients and lower than the levels in patients with normal zinc concentrations. They concluded that the plasma zinc concentration may be low in the elderly because of low plasma albumin. Plasma zinc concentrations did rise however after oral administration of the zinc. A recent study by Vir and Love (1979) determined the concentration of zinc in plasma and hair of 146 subjects aged 65 to 95 years. They found that the plasma zinc was less than 50 ug./100 ml. in 2.1 percent of subjects and hair zinc levels were less than 100 ug. in 2.7 percent of subjects. They found no correlation between zinc levels in hair and plasma. The administration of multivitamin supple- ments appears to increase hair zinc levels but this was significant only for females. Zinc Deficiency and Behavioral Impairment in Laboratory Animals The importance of zinc as it relates to behavior has not been extensively studied. This observation is interesting in that many studies make brief reference to the marked lethargy, slowing of behavior, apathy and decreased sexual activity in both males and females as part of the overall scenario of zinc deficiency (Apgar, 52 1968a; Ronaghy gt gt., 1969; Caldwell gt gt., 1970; Witenack gt gt., 1970; Miller & Stake, 1974). Animal models have typically been employed when research in this area has been carried out primarily because of ethical con— siderations but also because certain questions are not readily answerable from studies involving known zinc deficient humans. The rat has been the most popular but not the exclusive animal for carrying out investigations of the influence of zinc deficiency on behavior. Eichelman and coworkers (1973) studied aggression in animals resulting from prenatal zinc deficiency; pregnant Long-Evans rats were given either a zinc supplemented or zinc deficient diet starting on day 14 and continuing until day 20 of pregnancy. The pups of the low zinc dams were markedly smaller for the first forty days of life; however by day 75 there were no differences between zinc deficient and control groups. Females were tested for aggressive behavior at seventy-five days. The zinc deficient animals were paired with those that were zinc supplemented. All rats received two levels of shock for 0.5 seconds and spaced 5.0 seconds apart. Aggressive attacks were defined as directed movement toward the opponent resulting in contact that included at least one of the following responses: biting, sparring, upright attack posturing or supine sub- missive posturing adopted by the attacked rat. The zinc deficient rats displayed a significantly higher percent of 53 aggressive attacks. The level of shock (1.3 mA or 1.6 mA) made no difference in number of aggressive attacks in either group of animals. Peters (1977) studied the long term effects on aggression and affiliation behavior of male rats that had experienced either prenatal zinc deficiency or adequate prenatal nutrition. The animals were placed in a T-maze and their interactions observed. Zinc deficient animals were more aggressive than the well nourished animals and pre- natally well nourished animals preferred to affiliate with similar animals. Caldwell, Oberleas, Clancy and Prasad (1970) inves- tigated the effects of severe, chronic zinc deficiency on postweaning age rats. Learning was measured by performance in a Lashley III water maze. Latency scores to traverse the maze, number of cul-de-sac entries, and number of alley re-traces were recorded. Emotionality was assessed by counting the number of squares traversed in a square, Open- field. Zinc deficient animals had significantly greater latencies in coursing the water maze and made a greater number of combined cul-de-sac and re-trace errors between the first and final maze trials compared to the zinc supple- mented controls. Zinc supplemented animals were observed to be less emotional (i.e., had greater activity scores in the Open field) than the zinc deficient animals. These 54 same workers next investigated the effects of chronic, mild. zinc deficiency on gravid rats and in the behavior of their offspring (Caldwell, 1976). There was a high incidence of mortality in progeny of zinc deficient dams thus greatly reducing the number available for behavior testing. The results from those tested revealed similar behavioral impair- ment, although not of the magnitude reported in the first study. The evidence from these two studies suggests behav- ioral deficits occur whether the zinc deficiency occurs prenatally or postnatally. The following criticisms can be made of these studies: (1) plasma zinc concentrations of the animals were not determined in these studies thus making it necessary to define zinc deficiency based on intake alone; (2) the only control group employed was pair-fed zinc supplemented diet in the amounts consumed by the zinc deficient animals. Zinc deficient diets are known to cause anorexia thus decreasing intake in both groups. A second ad libitum control group would improve the design of this study since this would allow the zinc deficient group to be compared to an optimally nourished group. (3) it is impossible to state when testing physically debilitated animals whether the zinc deficiency actually resulted in decreased ability to learn or simply reflected animals less motivated to perform. Lokken and coworkers (1973) sought to address this third issue by renourishing rats that had been deprived of 55 zinc from birth through twenty-one days of age and then adequately fed for twenty-three days prior to testing. They found that even after improving the zinc status of these animals acquisition of an elevated Tolman-Honzik maze was impaired compared to control animals, suggesting that zinc deficiency occurring during the critical period of brain development results in permanent learning deficits. Halas and Sandstead (1975) employed an additional control group thus dealing with the second issue mentioned previously. Their study included a zinc deficient group, a zinc supplemented pair-fed group and a zinc supplemented ad libitum group. The zinc deficiency occurred prenatally and male animals were tested using a shock avoidance task. Performance was best for the ad libitum zinc supplemented group, somewhat less good for the pair-fed control and poorest for the zinc deficient group. The effects of zinc deficiency on the brains and behavior of rats and Rhesus monkeys were recently reported by Sandstead and coworkers (1977). These investigators found that the brains of viable rat pups from zinc deficient mothers contained less DNA than those of pair-fed dams that were also given a controlled amount of zinc. Behavioral studies were carried out in deficient pups and it was found that active shock avoidance was impaired in the deficient males. Female pups did not display this impaired active shock avoidance. This study was unique in that sex 56 differences were demonstrated. Zinc deprivation in pregnant monkeys resulted in a rash and loss of hair and a decrease of plasma zinc to very low levels. Abortion was frequent and the pair-fed mothers tolerated the procedure poorly suggesting that the monkey is not a completely satisfactory animal for this type of study. Additional work with Rhesus monkeys by the same group (Sandstead gt gt., 1978) showed that the infants of the zinc deprived mothers played and explored less than the control infants and they associated with the mother a greater percentage of time and were less active. Zinc Deficiency and Schizophrenia There is a possible role of zinc in the etiology of psychiatric disease. The early work with administration of manganese in schizophrenia (English, 1929; Hoskins, 1934) is the basis for speculation regarding the potential use of zinc in treatment of psychiatric disorders. Blood histamine levels were noted by Pfeiffer and Cott (1974) to vary in schizophrenics and these authors postulated that abnormal- ities of trace metals might be related to these differences. Previous workers had found that brain autOpsy specimens of schizophrenics contained approximately half of the amount of zinc as noted in brains of patients dying of other causes (Kimura & Kumura, 1965). Pfeiffer and Cott (1974) demonstrated that approximately 11 percent of schiZOphrenics studied had a low serum zinc level and that there were, in 57 addition, interrelationships between copper, manganese and . zinc. Additional evidence suggesting that zinc deficiency might be related to schiZOphrenia has been provided in the use of penicillamine in the treatment of this psychiatric illness (Pfeiffer & Iliev, 1972). The reported improvement of these patients may be explained by the previously dis- cussed relationships of zinc and copper. Presumably the penicillamine bound the c0pper and increased its excretion thus causing an elevation of zinc in body tissues possibly also including the brain. Rationale The importance of the role of zinc for living organisms is evident from the review of the research literature. Evident, also, is the paucity of research on 'the behavioral correlates of zinc deficiency in humans. The frequent mention of lethargy, apathy and decreased sexual activity appears in reports from investigations where the behavioral aspects of the syndrome were not of primary interest. Apgar (1973) investigated the effects of zinc repletion on parturition and included a brief state- ment about behavior. Henkin and coworkers (1975) reported mental changes as part of the overall scenario of acute zinc loss upon administration of the amino acid histidine, used in experimental treatment of scleroderma. Diminished sexual function was noted in men on hemodialysis for renal 58 failure (Antoniou gt gt., 1977) and this symptom was reversed when zinc chloride was added to the dialysis solution. Pronounced depression was described as one of the characteristics found following the widespread use of total intravenous nutrition (Hambidge, 1972). Zinc supplementa- tion resulted in rapid reversal of symptoms. Marginal zinc deficiency was induced eXperimentally in humans by Prasad and coworkers (1978) for the purpose of evaluating dietary requirements. Two levels of zinc were utilized; the two subjects on the lower level reported, among their many complaints, feeling lethargic. Human zinc deficiency was first described in the Middle East (Prasad gt gt., 1963) but recent evidence indi- cates that zinc deficiency may be common in the United States and not limited to any specific age group. No fewer than eleven syndromes related to zinc metabolism have been demonstrated in the pediatric pOpula- tion and most of these are related to dietary intake. Poor growth related to zinc has been described in infants and older children and low zinc levels are common in emotionally disturbed children (Cordas & Holland, 1977). A few studies have evaluated the zinc status of youngsters from low income families and found them to be more frequently affected (Hambidge gt gt., 1976). Zinc deficiency has been reported in adults where the condition is often unrelated to intake but rather 59 occurs through various disease states including cirrhosis from alcoholism, atherosclerosis and malignancies, including lung cancer and leukemia. Decreased zinc levels have been noted in females taking contraceptive drugs, known to be in wide use (Cordas & Holland, 1977). Obese adults were found to have significantly lower zinc levels than lean controls and the levels were even lower following intestinal bypass surgery. Low plasma zinc concentrations in aging individuals have been reported by Weismann and associates (1978) who found that levels rose with the oral administration of zinc. Vir and Love (1979) found low zinc levels in 2.7 percent of subjects aged sixty-five to ninety-five years, while Gregor (1977) found 5 percent of institutionalized aged subjects to be zinc deficient. This brief summary demonstrates that although poorly defined there are behavioral consequences related to zinc deficiency and that this syndrome is more common than previously believed, affecting peOple of all ages. Only a few studies to date have specifically inves- tigated the behavioral aspects of zinc deficiency (Caldwell gt gt., 1970; Lokken EE.El-v 1973; Halas & Sandstead, 1975; Sandstead gt gt., 1977). Animal models have typically been employed primarily because of ethical considerations; but also confounding present in human studies particularly in the elderly makes it difficult to attribute effects to the 60 treatment under investigation. The experimental designs of’ all of these studies involved at least one of these criti- cisms: (1) Plasma zinc levels were not always determined and zinc deficiency was inferred from low intake; (2) Typically a well-nourished control group was not used. This is extremely important since the zinc deficiency is known to cause a dramatic decrease in intake and the pair— fed control is thus possibly debilitated by low intake. Adding the free-fed control allows a comparison of the pair- fed groups to a normally nourished group; (3) All studies have failed to evaluate the Open-field response other than number of squares entered; (4) Studies have assessed blatantly sick animals on learning tasks, rendering the results impossible to interpret since any difference could easily be attributed to physical disability rather than reduced learning capacity; (5) Most studies have assessed behavioral effects of prenatal and postnatal zinc defici— ency and in fact no animals over three months old have been evaluated. In the present studies: (1) Young and aging rats were used. The strain, sex, laboratory conditions, diet composition and experi- menter were the same to allow for comparisons including body weight and food intake changes over time. 61 (2) A free-fed control group was used in addition to (3) (4) (5) (6) the zinc deficient and zinc supplemented pair-fed groups. Body weight and intake data were collected through- out the studies so that the effects of feeding low zinc diets could be assessed. Plasma zinc concentrations were determined so that zinc status was not inferred from low-zinc intake. A learning task was used to evaluate learning in the older animals since these animals did not appear sick; however, learning was not assessed in the second study since the young zinc deficient animals did appear sick. Assessment of behavior in an Open-field was used for both age groups. Latency, grooming, freezing, number of sectors entered, rearing and defecation responses were measured in the present studies so that comparisons on many specific behaviors could be made between zinc deficient and zinc supplemented animals. STUDY 1 MATERIALS AND METHODS Animals and Diets The subjects were 150 day old male rats obtained from the Holtzman Company, Madison, Wisconsin, and housed in the animal quarters of the Psychology Research Building for the purpose of aging. Animals were maintained on a standard laboratory diet regimen until they were 300 days old. They were, then, randomly assigned to one of three treatment groups as follows: Group ZDA rats were fed, gg libitum, a diet deficient in zinc. Group ZSP (control) rats were pair-fed a diet supplemented with zinc so that the daily intake for each animal was equal to the mean number of grams consumed by rats in Group ZDA in the pre- vious 24-hour period; Group ZSA (control) rats were fed, gg libitum, a diet supplemented with zinc. The diets were commercially prepared by Teklad Test Diets, Madison, Wisconsin (Table l). Diets were isocaloric, differing only in that the supplemented diet contained zinc carbonate (ZnCOB) at a level of 0.089 g./kg. of diet whereas the zinc-deficient diet contained less than one ppm zinc and 636.49 g./kg. of dextrose. 63 64 Table l Zinc Deficient Test Diet* Ingredient g/Kg Egg White Solids, Spray Dried 200.000 Dextrose 636.498 Corn Oil 100.000 Non-Nutritive Fiber (Cellulose) 30.000 Sodium Chloride 5.551 Potassium Phosphate 10.687 Calcium Phosphate 2.489 Magnesium Sulfate 1.651 Calcium Carbonate 9.944 Ferric Citrate 0.911 Potassium Iodide 0.026 Manganese Sulfate 0.008 Cupric Sulfate 0.009 Cobalt Chloride 0.001 Biotin 0.004 Vitamin B 0.020 Calcium Pantothenate 0.016 Choline Chloride 1.500 Folic Acid 0.001 Niacin 0.025 Pyridoxine HCl 0.004 Riboflavin 0.006 Thiamin HCl 0.010 Vitamin A Palmitate 0.020 Vitamin D 0.002 Vitamin E Acetate 0.220 Menadione 0.001 Chlortetracycline HCl 0.390 *In the Control Diet, Zinc Carbonate (ZnCO ) is added at a level of 0.089 g/Kg of diet and the Dex rose is reduced to 636.409 g/Kg of diet (adds 50 ppm zinc). 65 The living environment of the animals was made as metal free as possible prior to the start of the study to control for contamination. All rats were transferred to stainless steel cages held on stainless steel racks. They were given free access to deionized water contained in glass bottles with vinyl tops and stainless steel water spouts. The diets were contained in glass food cups. All water bottles and food cups were soaked in 4N hydrochloric acid for 24 hours and rinsed with deionized water. The laboratory was kept on a 12-hour photoperiod daily. Each animal was weighed and the weight recorded weekly and food intake weighed and recorded daily. Notes were kept regarding changes in physical appearance. Apparatus Testing equipment included a T-maze with black or white removeable interiors and a circular Open-field similar to the one described by Price and Huck (1976), six feet in diameter, enclosed by a sixteen inch high steel wall. The floor had concentric circles equidistant apart and a circle with a diameter of fifteen inches was located in the center of the circle. A total of 47 sectors each had approximately the same dimensions. An incandescent light was used during field testing. 66 Procedure Determination of Zinc Status All animals had been under the dietary conditions for fifteen weeks prior to the determination of zinc status. Each rat was etherized and a 2 ml. blood sample obtained using the orbital sinus bleeding technique. The plasma zinc content was determined as described by Prasad EE.§l° (1967) using atomic absorption spectrophotometry. This was a double—blind procedure in that the samples were coded and recoded prior to being sent to the animal nutrition labora- tory so that there was no way for the testers to know which samples were from which animals. Successive Black-White Discrimi- nation Learning Task Animals were approximately 415 days old when tested. The task was to learn to turn consistently to the right (left) alley when the interior of the maze was white and to the left (right) when the interior was black. The procedure was similar to that described by Davis (1957). Rats were habituated to the T-maze following 22 hours of water depri- vation (drinking was permitted between the hours of 4:00 PM and 6:00 PM each day). Each rat was placed in the start box and allowed to explore freely for a period of five minutes on each of four successive days. The interior was white on days one and three and black on days two and four. On day five each animal was placed in one of the two goal 67 boxes where a deionized water reinforcer was available. This procedure was continued until all animals were drinking within one minute of being placed in the goal box. The animals were then placed in the start box and allowed free access to all parts of the maze. Water reinforcement was available in each goal box. This phase of the habituation procedure was continued until animals were drinking water within one minute of being placed in the start box. Habitu- ation was completed on day 16. Animals were randomly assigned to be reinforced to either left-black, right-white or left-white, right-black to control for possible direction preference. Each rat received twelve trials per day for a total of 156 trials. The first eighteen trials were forced correct in that the incorrect alley was not accessible. A variable intertrial interval (ITI) was employed. The acqui- sition criterion was ten correct responses in twelve trials. The rate of habituation and number of errors were recorded. Activity and Exploration Animals were 435 days old at the time of testing. Each animal was placed in the center circle of the field and observed for five minutes. Six responses were measured and recorded as follows: (1) Latency to leave the center circle (seconds), (2) Grooming (seconds), (3) Freezing (seconds), (4) Number of sectors entered, (5) Number of rearings and (6) Defecation (number of boluses). Following 68 this test animals were saved to be used in an immunology study. Statistical Analysis A trend analysis was performed on the intake and weight data followed by univariate linear F tests. Means and standard deviations were determined for the weight intake data. A multivariate analysis of variance was used to analyze the plasma zinc and open-field data followed by univariate linear F tests. A minimum .05 level of significance was specified prior to the start of the study and nondirectional tabled values were used. RESULTS Food Intake A multivariate test of significance revealed an overall significant treatment effect = 78.26; p i (F2,17 .00001) (Figure 1). The amount of variance accounted for by the treatment was approximately 90 percent. The linear univariate F for all groups was signifi- cant (F = 76.99; p 1 .00001). The ZDA and ZSP groups 1,18 had a significantly lower intake than the ZSA group. Significant differences in food intake between groups were apparent following two weeks of feeding, and continued throughout the fifteen week study (Fl,18 = 8.78; p : .00832). Means and standard deviations for intake are reported in Appendix A. Body Weight A multivariate test of significance revealed an overall significant treatment effect (F4,52 = 8.89; p 3 .00002) (Figure 2). The amount of variance accounted for by the treatment was approximately 65 percent. The linear univariate F for all groups was signifi- cant (F = 15.36; p E .00004). 2,27 69 70 Fig. l.--Food intake of rats fed a zinc-deficient or zinc— supplemented diet for twenty weeks. 71 ZSA DA ZSP 36-? 32-1 84h 4m. Weeks 72 Fig. 2.--Body weight of rats fed a zinc-deficient or zinc- supplemented diet for twenty weeks. 73 ZSA ZSP ZDA 21 I I 19 Water cDeprivation 1 1 17 15 I I z I I I I l o 13 11 / I... 68%? 640... P b p P . ply/II nfl, mu nu 0. 0. Oj,, 0 6 2 8 4 0 6 5 5 4 4 4 mamuo mo prE52 and: Weeks 74 The ZDA group weighed significantly less than the ZSP group (T18 = 4.35; p i .002) and the ZSA group (Tl8 = 5.21; p g .002). Significant differences in body weights were appar- ent following five weeks of feeding, at which time the ZDA group weighed significantly less than the ZSP group (t18 = 2.75; p i .05) and the ZSA group (t18 = 7.19; p i .002). Weights between groups ZSP and ZSA were not significantly different; however following seven weeks of feeding the ZSP group weighed significantly less than the ZSA group (t18 = 2.94; p i .01). Means and standard deviations for body weight are reported in Appendix A. Plasma Zinc Concentrations and Field Data A multivariate test of significance was performed on the zinc and field data. The overall F was significant (F14,30 = 6.56; p E .00002). The amount of variance accounted for by the treat— ment was approximately 94 percent. Univariate F tests revealed the following: (1) Sig- nificant differences for zinc levels (F2,21 = 67.55; p E .00001); (2) Significant differences for latency to leave the center circle of the field (F = 18.56; p E .00002); 2,21 (3) Significant differences for time spent grooming (F2 21 = I 6.04; p E .00846); (4) Nonsignificant differences for freez— ing behavior (F2 21 = 2.31; p 3 .12416); (5) Significant 75 differences for number of sectors entered (F = 3.12; 2,21 p : .06479); (6) Nonsignificant differences for number of rearings (F = 2.065; p : .15174); (7) Nonsignificant 2,21 differences for number of boluses (F .363; p E .70023). 2,21 = Plasma Zinc - The ZDA group had significantly lower plasma zinc levels than the ZSP group (t18 = 18.567; p i .002) and the ZSA group (t18 = 16.026; p i .002). Differ- ences between the ZSP and ZSA groups were not significant. Latency — The ZDA group took significantly longer to leave the inner circle of the field than the ZSP group ( = 9.423; p f_.002). Differences between the ZSP and t14 ZSA groups were not significant. Grooming - The ZDA group Spent significantly less time grooming than the ZSP (tl4 = 3.29; p §_.01) and ZSA = 6.012; p < 002). The ZSA group spent sig— groups (t15 nificantly more time grooming than the ZSP group (tl4 = 2.72; p fi_.02). Sectors - The ZDA group entered significantly fewer sectors than the ZSP group (t14 = 4.33; p f_.002) or the ZSA group (tl4 = 2.15; p fi_.05). Differences between the ZSP and ZSA groups were not significant. Successive Black-White Discrimination Learning Time The ZDA group made slightly more errors than the ZSP or ZSA groups, however differences were not significant (Figure 3). 76 Fig. 3.--Mean number of errors per forty-six trial blocks on a successive black-white discrimination learn- ing task. 77 60-, A PA D SS ZA 22 I4 Av. It nw .0 4 2 muouum mo umnasz cows 93-138 47-92 1-46 TRIALS DISCUSSION The purpose of Study 1 was to induce zinc deficiency in aging rats and to evaluate the consequences on several physiological and behavioral parameters. No studies have reported behavioral data from aging zinc deficient labora- tory animals. Complete intake and body weight data were collected for each subject for twenty—one weeks: The intake of the animals free-fed a zinc deficient diet became significantly lower following two weeks of feeding. These data are in agreement with those of other investigators who have reported anorexia occurring as one of the symptoms of zinc deficiency (Day & McCollum, 1940; Swenerton & Hurley, 1968; Chesters & Quarterman, 1970); however the amount of feeding tme prior to this effect was greater than that in other studies. This difference can probably be attributed to the fact that animals from the present study were older and zinc depletion would be expected to be slower in aging organisms because of increased bone concentration of zinc over time. Body weights of the zinc deficient animals were reduced significantly by the fifth week of treatment com- pared to both control groups. Retarded growth has been a 78 79 typical finding in studies with younger organisms (Hurley, 1974). A study on experimental zinc deficiency in 55 to 65 year old humans resulted in weight loss (Prasad gt gt., 1978) in the subjects. The differences found between the zinc deficient animals and the pair-fed controls is of interest in that intake differed only in amount of zinc and not calories or other nutrients. This strongly suggests that zinc plays a key role in metabolic processes. Plasma zinc concentrations were significantly less for the animals on the low zinc diet. Low hair zinc levels have been previously reported in aging humans (Gregor, 1978); however they were not correlated with dietary zinc intake. This could possibly be attributed to cardiovascular drug therapy since the medications may inter- act with zinc metabolism. Prasad's group (1978) also got reduced levels for the four subjects in their study; how- ever intake changes were not reported. Results from Gregor's work demonstrate that low zinc levels can occur in the absence of reduced zinc intake while the data from the present study clearly show that reduction in zinc intake also results in low zinc levels in aging animals. The zinc deficient animals made slightly fewer errors in the learning task, however these differences resulted from this group having a greater number of trials where no direction choice was made. None of the animals 8O approached the learning criterion. At least two explana- tions are possible. A deionized water reinforcer was used for correct responses. Zinc deficient animals are known to have decreased taste and olfactory acuity (Henkin gt gt., 1975); therefore to insure that the reinforcement had equal incentive value for all subjects, tasteless and odorless water was used. Deionized water may not be reinforcing to animals in any experimental condition. The animals were on a strenuous water deprivation schedule, drinking only two hours each day following testing, and thirst was evident as demonstrated by immediate drinking by rats in all groups upon being returned to the home cage. A second interpretation is that successive discrimination is too difficult a task for aging rats. Bitterman and Wodinsky (1953) compared single simultaneous, multiple simultaneous, and successive dis- crimination learning tasks and found the first to be least complex, the second to be most complex and the third at a level of complexity between the two. When simple simultaneous discrimination tasks were used to compare young and old rats no age related differ- ences were found; however, when multiple simultaneous learning tasks were presented the young rats made signifi- cantly fewer errors than the old ones (Field, 1953). Suc- cessive discrimination has not previously been tested in aging rats and is therefore difficult to state if the level of difficulty was too great for the animals in the study. 81 Behavioral responses in an open-field revealed a longer latency for the zinc deficient subjects prior to leaving the center circle, significantly less grooming and significantly fewer sectors entered. The highly significant longer latency suggests an initial tendency not to readily explore novel situations. The shorter grooming time and fewer number of sectors entered could be interpreted as decreased activity level in the zinc deficient animals. Caldwell and coworkers (1970) attributed the fewer number of squares entered by the zinc deficient group to increased emotionality. Use of the Open—field to assess emotionality has been reviewed and criticized by Archer (1973). He reported that several types of evidence failed to support the validity of this measure for the assessment of emotion— ality and suggested that a variety of field behaviors be measured and interpreted without making the assumption that they represent a unitary major motivational construct. The present study attempted t9 use the field data responses in this way. The results from the present study confirm the pos- sibility of inducing zinc deficiency in aging lab animals, so that behavioral consequences can be evaluated. Both weights and intakes were lower and zinc deficiency was defined by lower plasma zinc levels in the rats fed a low zinc diet. Subsequent testing in an open-field demonstrated behavioral deficits on several parameters. Further 82 assessment using a less complex learning task with appro- priate reinforcement would be of value in future studies as would rehabilitation and retesting for possible reversal of behavioral effects in the open field. STUDY 2 MATERIALS AND METHODS This section is the same as in Study 1 with the following exceptions. Animals and Diets The subjects were 28 day-old male weanlings obtained from the Holtzman Company, Madison, Wisconsin and housed in the animal quarters of the Psychology Research Building. Animals were maintained on a standard laboratory diet regi- men for one week, and then randomly assigned to one of three treatment groups described in Study 1. Apparatus The T-maze was not used in Study 2. Procedure Determination of Zinc Status All animals had been under the dietary conditions for seven weeks prior to the determination of zinc status. Successive Black-White Discrimi- nation Learning Task This learning task was omitted in Study 2. 84 85 Activity and Exploration Two judges were trained to measure the field behav- ior and the data collected by Judge 1 and Judge 2 were compared. RESULTS Food Intake A multivariate test of significance revealed an overall significant treatment effect (F2,l7 = 65.39; p : .00001) (Figure 4). The amount of variance accounted for by the treatment was approximately 88 percent. The linear univariate F for all groups was signifi- cant (F = 133.59; p E .00001). The ZDA and ZSD groups 1,18 had a significantly lower intake than the ZSA group. Significant differences in food intake were apparent following eight days of feeding and continued throughout the seven week study (Fl,18 = 88.00; p E .00001). Means and standard deviations for intake are reported in Appendix B. Body Weight A multivariate test of significance revealed an overall significant treatment effect (F4,52 = 138.95; p E .00001) (Figure 5). The amount of variance accounted for by the treatment group was approximately 99 percent. The linear univariate F for all groups was signifi- cant (F = 1456.05; p E .00001). 2,27 86 87 Fig. 4.--Food intake of rats fed a zinc-deficient or zinc- supplemented diet for seven weeks. 88 ZSA ZDA ZSP n- 2 5 air .11! “H 1' 6 3 l' S y a .18 D 2 3 AT : 0 412 A ., :12 l 11. IV 4 r) i? _ p b — b p _ . . . a . _ . 2 8 4 0 6 2 8 4 3 2 2 2 l l mEmuw mo HmQESZ cmmz 89 Fig. 5.-—Body weight of rats fed a zinc-deficient or zinc- supplemented diet for seven weeks. 90 400. F 360“ b — nu m 4 O 2 2 mamuw mo Monacz cmmz 320-, 280., 160-» 120J- 80w 404 52 44 36 28 20 12 Days 91 The ZDA group weighed significantly less than the ZSP group (t19 = 20.99; p i .002) and the ZSA group (tl8 = 89.37; p g .002). The ZSP grOUp weighed significantly less than the ZSA group (t18 = 68.38; p i .002). Significant differences in body weight were apparent following eight days of feeding, at which time the ZDA group weighed significantly less than the ZSP group (t18 = 2.32; p i .05) and the ZSA group (t18 = 13.75; p g .002). The ZSP group weighed significantly less than the ZSA group (t18 = 11.43; p i .002). Means and standard deviations for body weight are reported in Appendix B. Plasma Zinc Concentrations and Field Data A multivariate test of significance was performed on the field data. The overall F was significant (F10,42 = 6.00; p : .00002). The amount of variance accounted for by the treatment was approximately 84 percent. Univariate F tests revealed the following: (1) Sig- nificant differences for latency to leave the center circle of the field (F2,24 = 14.76; p 1 .00007); (2) Nonsignificant differences for time spent grooming (F2,24 = 2.31; p g .1211); (3) Nonsignificant differences for freezing behavior (no F's reported since no animals displayed this response); (4) Significant differences for number of sectors entered 92 (F2,24 = 20.07; p : .00001); (5) Significant differences for number of rearings (F2,24 = 11.01; p i .0004); and (6) Nonsignificant differences for number of boluses (F2!24 = 2.29; p 3 .12341). Latency - The ZDA group took significantly more time to leave the inner circle of the field than the ZSP group (tl6 = 6.82; p i .002). Differences between the ZSP and ZSA groups were not significant (t16 = .2178). Number of sectors entered - The ZDA group entered significantly fewer sectors than the ZSP group (tl6 = 31.83; p i .002) or the ZSA group (tl6 = 19.64; p g .002). The ZSP group entered significantly more sectors than the ZSA group (t18 = 12.19; p j .002). Rearing - The ZDA group reared significantly fewer times than the ZSP group (t = 10.51; p i .002) or the ZSA 16 group (tl6 = 5.10; p i .01). The differences between the ZSP and ZSA groups were significant. The ZSP group reared significantly more than the ZSA group (t18 = 7.41; p i .002). The ratings of the two judges for the Open field test were approximately the same (see Table 2). Physical Changes Apparent physical changes for the ZDA animals included ruffled coats (following fifteen days of treatment), hair loss and bald patches (following twenty-eight days of treatment), inflamed paws in 50 percent of the ZDA animals (following thirty days of treatment), a irritated conjunctiva 93 0 mm hmH o o N 0 mm ¢0N o m m 0H 0 mH omH o o H 0 0H NmH o o H m o m HVH o o v o m HVH o o m m o mN 00H 0 o m o mN 00H 0 o m h o mN va o o N o mN va o o N o o m NvH o N H o m ovH o N H m o mH mVN o m m o vH vMN o m m g 0 0m HmH o o n o mN mnH o o h m o NN moH o v m o mN NhH o v o N o VH mmH o o o o vH mmH o o o H mmN o m on o w HH 0 m on o m 0H m o v mm o o h o v mm o o w h o h mHH o o m o n VHH o o m o v n mm o 0 NH v n Hm o 0 NH m m v mm o 0 MN m v om o o vN v o v mm 0 0H HH 0 v ms 0 mH NH m o mH mm o v mH o vH on o g mH N H OH mm o VH v H OH mm o NH v H «ON .Hom .ummm .uomm .Nwoum .Eoouw mHouHU .Hom .Hmmm .uomm .Nomum .Eoouw wHouHO nonesz A.owmv mEHB umnaoz A.owmv mEHB 039 mmpzh mco mmGSN |||.|l ' -.|IA I .III‘ 0‘ ’..I.- lulttl'l'l AN sesame mama meHmucmmo m magma 94 N oH ONH o o v N CH mHH o o v OH 0 H MhH o w H 0 NH NhH o m H m o m mOH o o m o m 00H 0 00 N m o @H an o m m 0 SH mmH o m m h 0 MH OHN o N m 0 MH HHN o N m o o vH 05H 0 mH N H MH vnH o mH N m H m ovH o v n H m mmH o v n v o oH vNH o m m o OH MNH o m m m o vH NMH o v v o «H va o v v N o v mOH o m m o v 00H 0 0H m H mmN .Hom .ummm .uowm .Nmmum .Eoouu mHouHO .Hom .ummm .uowm .Nmmum .Eoouw mHoHHU nonfioz A.ommv mEHB nonfisz A.ommv mEHB 039 mop:h mco mopso pmscHucou m anms 95 in all ZDA animals (onset from fourteen to thirty-six days). All of the ZDA animals had urethritis by six weeks of treat- ment and skin lesions were apparent on various parts of the body. Thirty percent of this group died prior to the con— clusion of this study. DISCUSSION The goal of Study 2 was to produce zinc deficiency in young rats and assess resulting physiological and behav- ioral deficits and to employ an improved design compared to those used in previous behavioral research on zinc defic- iency (Caldwell gt gt., 1976; Halas & Sandstead, 1975). Significantly lower intakes and body weights for the zinc deficient animals were apparent following eight days of feeding a low zinc diet. Decreased weights and intakes have been previously reported by Chesters and Quarterman (1970) who found that incipient zinc deficiency in young rats pro- duced cessation of growth with food intake falling to 70 percent of free-fed controls. The differences were greater in their investigation compared to the present study probably because their rats were put on zinc-deficient diets at an earlier age. The differences in weight between the pair-fed groups suggest metabolic deficiency in low zinc animals. Tucker and Salmon (1955) reported weight loss and anorexia in calves fed low-zinc purified diets and Hambidge and coworkers (1972) reported that zinc deficient children and adults had weight loss and poor appetites. Comparison across these studies using rats, calves and humans indicates 96 97 that, at least for body weight and intake, there are similar findings across species. Plasma levels of zinc in zinc deficient rats (if lower) are in agreement with find- ings of Sandstead and coworkers (1977) and Kelleher (1978). Behavioral responses in an Open-field revealed a significantly longer latency to leave the center circle of the field, significantly fewer number of sectors entered and fewer rearings for the zinc deficient animals compared to the two control groups. The longer latency suggests an initial tendency not to readily explore novel environments. The zinc deficient animals were clearly less active as indicated by entering a fewer number of sectors. The differences for sectors entered were significant for the ZSP and ZSA groups in that the ZSP group was more active than the ZSA group. One explanation is that the ZSP was the starved group and were extremely hyperactive to the point of nearly jumping from the experimenter's hand when being placed in the field. The significantly lower number of rearings may have been related to physical weakness. These animals appeared sick and displayed "Kangaroo" like posture described by Swenerton and Hurley (1968) who studied severe zinc deficiency in male and female rats. The time spent grooming in the zinc deficient animals was not significantly different, however it should be noted that their grooming was characterized by licking the front paws which were inflamed. This symptom of zinc 98 deficiency was previously reported (Follis, 1966). The control animals engaged in a variety of grooming behaviors. Learning was not assessed in these young zinc deficient animals. Previous studies have used various learning tasks (Caldwell gt gt., 1970; Caldwell gt gt., 1976; Halas & Sandstead, 1975); however results from such tests are difficult to interpret since young zinc deficient animals are quite ill. Lokkens and coworkers (1977) renourished animals deprived of zinc from birth to twenty-one days of age and then evaluated learning ability. The rehabilitated animals showed impaired performance compared to controls on an elevated Tolman-Honzik maze. These animals were immedi- ately put on low zinc diets following birth. The present study utilized low zinc diets starting at thirty-five days of age. Future work in this area might look at the effect of rehabilitation on rats that were zinc deprived somewhat later than in previous studies to determine if learning deficits are greater when animals are deprived during the early, most critical period of brain develOpment. Also of interest would be the comparison of performance of zinc deficient animals on tests varying in complexity. It may be that, given a simple task, no deficits will be evident and that only as the tasks become more complex will zinc deficient animals display reduced learning capacity. GENERAL DISCUSSION Comparisons of results of both studies indicate that, although physiological and behavioral deficits were present for each age group the aging animals were less affected than the young ones and zinc deficiency was induced in the young ones in a much shorter period of time. Significant differences between the ZDA and ZSA groups in food intake and body weight were apparent follow- ing two weeks and five weeks respectively for the aging animals, while the differences for both of these parameters were significant by eight days for the younger rats. One important consideration in evaluating these age-related differences is that the young animals were put on the low zinc diets during a period of rapid growth rate, and daily intake was not at a maximum. These effects are easily demonstrated by comparing weight and intake trends of the young zinc deficient animals to their free-fed zinc supplemented controls. Many physical changes were apparent for the young zinc deficient animals that were not evident in the aging group. Fifteen days into the treatment the coats of the ZDA rats were ruffled and hair loss including bald patches was evidenced by twenty-eight days of treatment. The paws 99 100 of 50 percent of the young zinc deficient animals were inflamed and the conjunctiva were irritated. All of the zinc deficient animals had urethritis by six weeks of feeding and skin lesions appeared on various parts of the body. Two animals from this group died prior to testing and three more died the week following testing so that the overall mortality was 50 percent. No animals in the two control groups were lost and all animals in the aging study survived. The field data revealed longer latency for both age groups of zinc deficient rats compared to controls. Groom- ing differences were significant in the first but not the second study; however, the aging zinc deficient animals did not have inflamed, irritated paws and grooming in the zinc deficient young animals consisted entirely of licking the sore paws. Both zinc deficient age groups were less active in the field as measured by the number of sectors entered. Rearings were not significantly different for aging zinc deficient animals compared to their controls but were for the young zinc deficient animals, who displayed "kangaroo" like posture possibly indicating neurological damage. A comparison of data from studies of young and aging rats indicates that zinc deficiency results in physio- logical and behavioral deficits for both ages, but zinc deficiency induced in early life has greater detrimental effects for all parameters measured and has a high morbidity 101 and mortality rate, compared to the zinc deficient aging animals. The physiological and behavioral differences were caused by the decreased zinc intake in the ZDA animals as indicated by the very high amount of variance accounted for in each study. REFERENCES REFERENCES Antoniou, L. D.; Shalhoub, R. J.; & Sudhaker, T. EE.E£- Reversal of uremic impotence by zinc. Lancet 2 (1977):897. Apgar, J. Effect of zinc repletion late in gestation on parturition in the zinc-deficient rat. J. Nutr. 103 (1973):973. Apgar, J. Effect of zinc deficiency on parturition in the rat. Amer. J. Physiol. 215 (1968):160. Arakawa, T.; Tamura, T.; Igarashi, Y.; Suzuki, H.; & Sandstead, H. H. Zinc deficiency in two infants during "total parenteral hyperalimentation for diarrhea." Am. J. Clin. Nutr. 29 (l976):l97. Archer, J. Tests for emotionality in rats and mice; a review. Animal Behavior 21 (1973):205. Atkinson, R. L.; Dahms, W. T.; Bray, G. A.; Jacob, R.; & Sandstead, H. H. Plasma zinc and cepper in obesity and after intestinal bypass. Ann. Int. Med. 89 (l978):49l. Barnes, R. H.; Robertson, 8.; Zimmerman, R.; Simmons, H.; McLeod, R. B.; & Krook, L. Influence of nutritional deprivations in early life on learning behavior of rats as measured by performance in a maze. J. Nutr. 89 (l966):399-4ll. Beisel, W. R. Zinc metabolism in infection. In: Zinc Metabolism, Current Aspects in Health and Disease, edited by G. J. Brewer and A. S. Prasad. New York: Alan R. Liss, Inc., 1977, p. 155. Bell, L. T.; Branstrator, M.; Roux, C.; & Hurley, L. 8. Chromosomal abnormalities in maternal and fetal tissues in magnesium or zinc-deficient rats. Teratol. 12 (l975):221. 102 103 Bertrand, G., & Javillier, M. Influence du zinc du mangan- ese sur la composition minerale de l'aspergillus niger. C. R. Acad. Sci. (Paris) 152 (1911):1337. Bertrand, G., & Benson, B. Recherches sun l'importance du zinc dans l'alimentation des animaux. Experiences sur la souris. Compt. Rend. Acad. Sci. 175 (1922): 289. Bitterman, M. E., & Wodinsky, J. Simultaneous and successive discrimination. Psychol. Rev. 60 (l953):371. Blamberg, D. L.; Blackwood, U. B.; & Supplee, W. C. Effect of zinc deficiency in hens on hatchability and embryonic development. Proc. Soc. Exp. Biol. Med. 104 (1960):217. Brown, M. A.; Thom, J. V.; Cova, P.; & Juarez, J. Food poisoning involving zinc contamination. Environ. Health 8 (l964):657. Brown, E. D.; Chan, W.; & Smith, J. C. Bone mineralization during a developing zinc deficiency. Proc. Soc. Exp. Biol. Med. 157 (1978):211. Caldwell, D. F.; Oberleas, D.; Clancy, J. J.; & Prasad, A. S. Behavioral impairment in adult rats following acute zinc deficiency. Proc. Soc. Exp. Biol. Med. 133 (l970):l4l7. Caldwell, D. F.; Oberleas, D.; & Prasad, A. S. Psychobio- logical changes in zinc deficiency. In: Trace Elements in Human Health and Disease, edited by A. S. Prasad and D. Oberleas. New York: Academic Press, 1976, p. 311. Callender, G. R., & Gentzko, C. J. Acute poisoning by the zinc and antimony content of limeade prepared in a galvanized iron can. Milit. Surg. 80 (l937):67. Carter, J. P.; Grivetti, L. B.; Davis, J. T.; Nasiff, S.; Mansour, A.; Mousa, W. A.; Atta, Alla-El-Din; Patwardhan, V. N.; Moneim, M. A.; Abdou, I. A.; & Darby, W. J. Growth and sexual development of adolescent Egyptian village boys. Am. J. Clin. Nutr. 22 (1969):59. Chesters, J. K. Biochemical functions of zinc in animals. Wld. Rev. Nutr. Diet. 32 (1978):135. 104 Chesters, J. K., & Quarterman, J. Effects of zinc deficiency on food intake and feeding patterns of rats. Brit. J. Nutr. 24 (1970):106l. Chesters, J. K., & Will, M. Some factors controlling food intake by zinc—deficient rats. Brit. J. Nutr. 30 (l973):555. Coble, Y. D.; Bardin, C. W.; Ross, G. T.; & Darby, W. T. Studies of endocrine function in boys with retarded growth, delayed sexual maturation and zinc defici- ency. J. Clin. Endocr. 32 (l971):36l. Cordas, S., & Holland, C. Zinc: clinical considerations. Osteopathic Medicine, August, 1977, p. 44. Cox, D. H., & Harris, D. L. Effect of excess dietary zinc on iron and copper in the rat. J. Nutr. 7 (1960): 514. Davis, R. H. The effect of spacing units on simultaneous vs. successive discrimination. J. Comp. Physio. Psychol. 50 (1957):207. Dawson, J. R., & Walker, B. E. Direct determination of zinc in whole blood, plasma and urine by atomic absorp— tion spectroscopy. Clin. Chim. Acta 26 (l969):465. Day, H. G., & McCollum, E. V. Effects of acute dietary deficiency in the rat. Proc. Soc. Exp. Biol. Med. 45 (1940):282. Day, H. G. Effects of zinc deficiency in the mouse. Fed. Proc. 1 (1942):188. Dodge, J. A., & Yassa, J. G. Zinc deficiency in a British youth with cystic fibrosis. Brit. Med. J. l (1978): 411. Drinker, K. R.; Thompson, P. K.; & Marsh, M. Effects of long continued ingestion of zinc in the form of zinc oxide by cats and dogs. Am. J. Physiol. 30 (1927):31. Eichelman, B.; DeJong, W.; & Williams, R. B. Aggressive behavior in hypertensive and normotensive rat strains. Physiol. Behav. 10 (l973):301. Eminians, J.; Reinhold, J. G.; Kfoury, G. A.; Amirhakimi, G. H.; Sharif, H.; & Ziai, M. Zinc nutrition of children in Fars province of Iran. Am. J. Clin. Nutr. 20 (l967):734. 105 Engel, R. W.; Miller, R. F.; & Price, N. 0. Metabolic patterns in preadolescent children. In: Zinc Metab- olism, edited by A. S. Prasad. Springfield: C. C. Thomas, 1966. English, W. M. Report of the treatment with manganese chloride of 181 cases of schiZOphrenia, 33 of manic depression, and 16 of other defects or psychoses at the Ontario Hospital, Brockville, Ontario. Amer. J. Psych. 9 (l929):569. Evans, G. W.; Grace, C. I.; & Votava, H. J. A prOposed mechanism for zinc absorption in the rat. Amer. J. of Physiol. 228 (1975):501. Follis, R. H., Jr.; Day, H. G.; & McCollum, E. V. Histo- logical studies of the tissues of rats fed a diet extremely low in zinc. J. Nutr. 22 (l94l):223. Follis, R. H., Jr. The pathology of zinc deficiency in zinc metabolism. In: Zinc Metabolism, edited by A. S. Prasad. Springfield: C. C. Thomas, 1966. Food and Nutrition Board. Recommendations of National Academy of Sciences. In: Underwood, E. J. Trace Elements in Human and Animal Nutrition, edition 4. New York: Academic Press, 1977, p. 228. Forbes, R. M. Nutritional interactions of zinc and calcium. Fed. Proc. 19 (1960):643. Fosmire, G. J.; Al-Ubaidi, Y. Y.; & Sandstead, H. H. Some effects of postnatal zinc deficiency on developing rat brain. Pediat. Res. 9 (l975):89. Fraker, P. J.; Haas, S. M.; and Juecke, R. W. Effect of zinc deficiency on the immune response of the young adult A/J mouse. J. Nutr. 107 (1977):1889. Fraker, P. J.; DePasquale-Jardieu; Zwickl, C. M.; & Luecke, R. W. Regeneration of T-cell helper function in zinc-deficient adult mice. Proc. Natl. Acad. Sci. 75 (l978):5660. Frost, P.; Chen, J. C.; & Rabbani, I. The effect of zinc deficiency on the immune response. In: Zinc Metabolism, Current Aspects in Health and Disease, edited by G. J. Brewer and A. S. Prasad. New York: Alan R. Liss, Inc., 1977, p. 143. 106 Fujioka, M., & Lieberman, 1. A zinc requirement for syn- thesis of deoxyribonucleic acid by rat liver. J. of Biol. Chem. 239 (l964):1l64. Gallery, E. D. M.; Blomfield, J.; & Dixon, S. R. Acute zinc toxicity in hemodialysis. Brit. Med. J. 4 (1972): 331. Gray, L. F., & Ellis, G. H. Some interrelationships of c0pper, molybdenum, zinc and lead in the nutrition of the rat. J. Nutr. 40 (l950):44l. Gregor, J. L. Dietary intake and nutritional status in regard to zinc of institutionalized aged. J. of Geront. 32 (l977):549. Gunn, S. A.; Gould, T. C.; & Anderson, W. A. Zinc protec- tion against cadmium injury to rat testis. Archiv. of Path. 71 (l961):274. Gunn, S. A.; Gould, T. C.; & Anderson, W. A. D. The selec- tive injurious response of testicular and epididymal blood vessels to cadmium and its prevention by zinc. Amer. J. Path. 46 (1963):685. Haas, S.; Fraker, P.,; & Luecke, R. W. The effect of zinc deficiency on the immunoresponses of A/J mice. Fed. Proc. 35 (l976):659. Halas, E. S.; Hanlon, M. J.; & Standstead, H. H. Intra- uterine nutrition and aggression. Nature 257 (1975): 221. Halas, E. S., & Sandstead, H. H. Some effects of prenatal zinc deficiency on behavior of the adult rat. Pediat. Res. 9 (1975):94. Halsted, J. A.; Smith, J. C.; & Irwin, M. I. A conspectus of research on zinc requirements of man. J. Nutr. 104 (1974):345. Halsted, J. A. GeOphagia in man: its nature and nutri- tional effects. Am. J. of Clin. Nutr. 21 (1968):1364. Halsted, J. A.; Ronaghy, H. A.; & Abadi, P. gt gt, Zinc deficiency in man: the Shiraz experiment. Amer. J. Med. 53 (l972):277. Halsted, J. A. Events surrounding the original demonstra- tion of human zinc deficiency. In: Zinc Metabolism: Current Aspects in Health and Disease, edited by G. J. Brewer and A. S. Prasad. New York: Alan R. Liss, Inc., 1977. 107 Halsted, J. A.; Hackley, B.; Rudzki, C.; & Smith, J. C., Jr. Plasma zinc concentrations in liver diseases. Com- parison with normal controls and certain other chronic diseases. Gastroent. 54 (1968):1098. Hambidge, K. M.; Hambidge, C.; Jacobs, M.; & Baum, J. D. Low levels of zinc in hair, anorexia, poor growth and hypogeusia in children. Pediat. Res. 6 (1972): 868. Hambidge, K. M.; Neldner, P. A.; & Walravens, P. A. Zinc, acrodermatitis enterOpathica, and congenital mal— formations. Lancet 1 (l975):577. Hambidge, K. M. Trace elements. In: Pediatric Nutrition Handbook. Evanston: American Academy of Pediatrics, 1979, p. 41. Hartoma, T. R.; Nahoul, K.; & Netter, A. Zinc, plasma androgens and male sterility. Lancet 2 (1977):1125. Hartoma, T. R.; Sotaniemi, E. A.; & J. Maattanen. Effect of zinc on some biochemical indices of metabolism. Nutr. Metab. 23 (1979):294. Henkin, R. I.; Patten, B. M.; Re, P. K.; & Bronzert, D. A. A syndrome of acute zinc loss. Cerebellar dys- function, mental changes, anorexia, and taste and smell dysfunction. Arch. Neurol. 32 (l975):745. Henkin, R. I.; Graziadei, P. P. G.; & Bradley, D. F. The molecular basis of taste and its disorders. Ann. Int. Med. 71 (l969):791. Hesse, G. W. Chronic zinc deficiency alters neuronal func- tion of hippocampal nerve fibers. Science 205 (1979):1005. Hillstrom, L.; Pettersson, L.; Hellbe, L. gt gt. Comparison of oral treatment with zinc sulphate and placebo in acne vulgaris. Brit. J. of Derm. 97 (l977):679. Hoskins, R. G. The manganese treatment of "schizophrenic disorders." J. Nerv. Mental. Dis. 79 (1934):59. Hove, B.; Elvehjem, C. A.; & Hart, E. B. The physiology of zinc in the nutrition of the rat. Amer. J. of Physiol. 119 (l937):768. 108 Hsu, J. M. Metabolic studies in rat skin during zinc deficiency. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories gt gt. Spring- field: C. C. Thomas, 1974, p. 43. Huber, A. M., & Gershoff, S. N. Effects of dietary zinc on zinc enzymes in the rat. J. Nutr. 103 (1973): 1175. Hurley, L. S., & Swenerton, H. Congenital malformations resulting from zinc deficiency in rats. gtgc. Soc. Exp. Biol. Med. 123 (l966):692. Hurley, L. S.; Gowan, J.; & Swenerton, H. Teratogenic effects of short-term and transitory zinc deficiency in rats. Teratol. 4 (1971):199. Hurley, L. S. Zinc and its influence on develOpment in the rat. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories gt a1. Springfield: C. C. Thomas, 1974, p. 57. —_ Hurley, L. S. Zinc deficiency in prenatal and neonatal develOpment. In: Zinc Metabolism: Current Aspects in Health and Disease, edited by G. J. Brewer and A. S. Prasad. New York: Alan R. Liss, Inc., 1977. Ismail-Beigi, F.; Faraji, B.; & Reinhold, J. G. Binding of zinc and iron to wheat bread, wheat bran, and their components. Am. J. of Clin. Nutr. 30 (1977):1721. Kay, R. G., & Tasman-Jones, C. Acute zinc deficiency in man during intravenous hyperalimentation. Aust. NZ J. Surg. 45 (1975):325. Keen, C. L., & Hurley, L. S. Zinc absorption through skin: correction of zinc deficiency in the rat. Am. J. of Clin. Nutr. 30 (l977):528. Keilin, D., & Mann, T. Activity of purified carbonic anhydrase. Nature 153 (1944):107. Kfoury, G. A.; Reinhold, J. G.; & Simonian, S. J. Enzyme activities in tissues of zinc-deficient rats. g. Nutr. 95 (1968):102. Kienholz, E. W.; Turk, D. E.; Sunde, M. L. gt gt. Effects of zinc deficiency in the diets of hens. J. Nutr. 75 (l961):211. Kime, Z. R., & Sifri, M. Objections to the Klevay hypoth- esis. Amer. J. Clin. Nutr. 29 (1976):1065. 109 Kimura, K., & Kumura, J. Preliminary reports on the metab- olism of trace elements in neurOpsychiatric disease. I. Zinc in schiZOphrenia. Proc. Jap, Acad. 41 (1965):943. Kinnamon, K. E. The role of iron in the c0pper-zinc inter— relationship in the rat. J. Nutr. 90 (l966):315. Klevay, L. M. Hypercholesterolemia in rats produced by an increase in the ratio of zinc to copper ingested. Am. J. of Clin. Nutr. 26 (1973):1060. Klevay, L. M. Coronary heart disease: the zinc/copper hypothesis. Amer. J. of Clin. Nutr. 28 (l975):764. Lemann, I. T. A study of the type of infantilism in hook- worm disease. Arch. Int. Med. 6 (l910):l39. Lifschitz, M. D., & Henkin, R. I. Circadian variation in c0pper and zinc in man. J. of Appl. Physiol. 31 (1971):88. Lokken, P. M.; Halas, E. 8.; & Sandstead, H. H. Influence of zinc deficiency on behavior. Proc. Soc. Exp. Biol. Med. 144 (1973):680. Luecke, R. W.; Simonel, C. B.; & Fraker, P. J. The effect of restricted dietary intake on the antibody medi- ated response of the zinc deficient A/J mouse. J. of Nutr. 103 (l978):88l. Lutz, R. E. The normal occurrence of zinc in biological materials. J. Ind. Hyg. 8 (1926):177. MacCaslin, E. F. Successive and simultaneous discrimination as a function of stimulus similarity. Amer. J. of Psychol. 67 (l954):308. Maleki, M. Food consumption and nutritional status of 13 year old village and city schoolboys in Fars pro- vince, Iran. Ecol. of Food and Nutr. 2 (l973):39. Maze, P. Influences respectives des elements de la solution minerale sur le development du mais. Ann. Inst. Pasteur 28 (l914):21. McCance, R. A., & Widdowson, E. M. The absorption and excretion of zinc. Biochem. J. 36 (l942):692. 110 McConnell, S. D., & Henkin, R. I. Altered preference for sodium chloride, anorexia, and changes in plasma and urinary zinc in rats fed a zinc-deficient diet. 9. Nutr. 104 (1974):1108. McKenzie, J. M.; Fosmire, G. J.; & Sandstead, H. H. Zinc deficiency during the latter third of pregnancy: effects on fetal rat brain, liver and placenta. J. Nutr. 105 (1975):l466. Mertz, W. Some aspects of nutritional trace element research. Fed. Proc. 29 (l970):l482. Michaelsson, G.; Juhlin, L.; & Ljunghall, K. A double~blind study of the effect of zinc and oxytetracycline in acne vulgaris. Brit. J. Derm. 97 (l977):561. Miller, J. K., & Miller, W. J. Experimental zinc deficiency and recovery of calves. J. Nutr. 76 (1962):467. Miller, E. R.; Luecke, R. W.; Ullrey, D. E. gt gt. Bio- chemical, skeletal, and allometric changes due to zinc deficiency in the baby pig. J. Nutr. 95 (1968) :278. ’— Miller, W. J., & Stake, P. E. Zinc metabolism and homeo— stasis--some new concepts. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories gt gt. Springfield: C. C. Thomas, 1974, p. 130. Mills, C. F.; Quarterman, J.; Chesters, J. K.; Williams, R. B.; & Dalgarno, A. C. Metabolic role of zinc. Am. J. of Clin. Nutr. 22 (1969):1240. Moynahan, E. J. Acrodermatitis enterOpathica: a lethal inherited human zinc-deficiency disorder. Lancet 2 (1975):399. Murphy, E. W.; Page, L.; & Watt, B. K. Trace minerals in type A school lunches. J. Am. Diet Assoc. 58 (1971): 115. Murphy, E. W.; Willis, B. W.; & Watt, B. K. Provisional tables on the zinc content of foods. J. Am. Diet Assoc. 66 (1975):345. Murphy, J. V. Intoxication following ingestion of elemental zinc. J.A.M.A. 212 (l970):2119. Murthy, L.; Klevay, L. M.; & Petering, H. G. Interrelation- ships of zinc and cepper nutriture in the rat. J. Nutr. 104 (1974):1458. 111 Neldner, K. H.; Hambidge, K. M.; & Walravens, P. A. Acro- dermatitis enteropathica. Int. J. Dermatol. 17 (1978):380. O'Dell, B. L.; Newberne, P. M.; & Savage, J. E. Significance of dietary zinc for the growing chicken. J. Nutr. 65 (1958):503. O'Dell, B. L., & Savage, J. B. Effect of phytic acid on zinc availability. Proc. Soc. Exp. Biol. Med. 103 (1960):304. O'Dell, B. L.; Burpo, C. E.; & Savage, J. E. Evaluation of zinc availability in foodstuffs of plant and animal origin. J. Nutr. 102 (1972):653. Osis, D.; Kramer, L.; Wiatrowski, E.; & Spencer, H. Dietary zinc intake in man. Amer. J. of Clin. Nutr. 25 (l972):582. Papp, J. P. Metal fume fever. Postgraduate Medicine, March, 1968, p. 160. Pekarek, R. S., & Beisel, W. R. Characterization of the endogenous mediator(s) of serum zinc and iron depression during infection and other stresses. Proc. Soc. Exp. Biol. Med. 138 (l97l):728. Peters, D. Effects of prenatal nutritional deficiency on affiliation and aggression in rats. Physiol. and Behav. 20 (1978):359. Pfeiffer, C. C., & Iliev, V. A study of zinc deficiency and c0pper excess in the schiZOphrenias. In: Neurobiology of the Trace Metals Zinc and COpper. International Review of Neurobiology, Supplement 1, edited by C. C. Pfeiffer. New York: Academic Press, 1972. Pfeiffer, C. C., & Cott, A. A study of zinc and manganese dietary supplements in the copper loaded schizo- phrenic. In: Clinical Applications of Zinc Metab- olism, edited by W. J. Pories et a1. Springfield: C. C. Thomas, 1974, p. 260. __H—_ Pidduck, H. G.; Keenan, J. P.; & Price-Evans, D. A. Leuco- cyte zinc in diabetes mellitus. Diabetes 20 (1971): 206. Pidduck, H. G.; Wren, P. J. J.; & Price-Evans, D. A. Hyper- zincuria of diabetes mellitus and possible genetical implications of this observation. Diabetes 19 (1970): 240. 112 Pories, W. J., & Strain, W. H. Zinc and wound healing. In: Zinc Metabolism, edited by A. S. Prasad. Spring- field: C. C. Thomas, 1966, p. 378. Pories, W. J., and Strain, W. H. Zinc sulfate therapy in surgical patients. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories gt gt. Springfield: C. C. Thomas, 1974, p. 139. Powanda, M. C.; Kenyon, R. H.; & Moe, J. B. Alterations in plasma c0pper, zinc, amino acids and seromucoid during rocky mountain spotted fever in guinea pigs. Proc. Soc. Exp. Biol. Med. 151 (l976):804. Prasad, A. S.; Miale, A.; Farid, Z.; Sandstead, H. H.; Schulert, A. R.; & Darby, W. J. Biochemical studies on dwarfism, hypogonadism and anemia. Arch. Int. Med. 111 (1963):65. Prasad, A. S.; Miale, A.; Fariz, Z.; Sandstead, H. H.; & Schulert, A. R. Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepato- splenomegaly, dwarfism and hypogonadism. J. of Lab. and Clin. Med. 61 (1963):537. Price, E. O., & Huck, U. W. Open-field behavior of wild and domestic Norway rats. Animal Learning and Behavior 4 (l976):125. Ronaghy, H. A.; Reinhold, J. G.; Mahloudji, M.; Ghavami, P.; Spivey-Fox, M. R.; & Halsted, J. A. Zinc supple- mentation of malnourished schoolboys in Iran: increased growth and other effects. Amer. J. of Clin. Nutr. 27 (1974):112. Ronaghy, H. A.; Moe, P. G.; & Halsted, J. A. A six-year follow—up of Iranian patients with dwarfism, hypo- gonadism, and iron-deficiency anemia. Amer. J. of Clin. Nutr. 21 (1968):709. Ross, J. F.; Ebaugh, R. G., Jr.; & Talbot, T. R., Jr. Radioisotopic studies of zinc metabolism in human subjects. Trans. Assoc. Amer. Phys. 71 (1958):322. Sandstead, H. H.; Shukry, A. S.; Prasad, A. S.; Gabr, M. K.; El Hifney, A.; Mokhiar, N.; & Darby, W. J. Kwashiorkor in Egypt. 1. Clinical and biochemical studies, with special reference to plasma zinc and serum lactic dehydrogenase. Amer. J. of Clin. Nutr. 17 (1965):15. 113 Sandstead, H. H.; Prasad, A. S.; Schulert, A. R.; Farid, Z.; Miale, A.; Bassilly, S.; & Darby, W. J. Human zinc deficiency, endocrine manifestations and response to treatment. Amer. J. of Clin. Nutr. 20 (l967):422. Sandstead, H. H.; Fosmire, G. J.; Halas, E. 8.; Jacob, R. A.; Strobel, D. A.; & Marks, E. O. Zinc deficiency: effects on brain and behavior of rats and rhesus monkeys. Teratol. l6 (l977):229. Sandstead, H. H.; Strobel, D. A.; Logan, G. M., Jr. gt gt. Zinc deficiency in pregnant rhesus monkeys: effects on behavior of infants. Amer. J. Clin. Nutr. 31 (1978):844. Sandstead, H. H., & Terhune, M. W. Zinc deficiency: effect on the activity of liver RNA polymerase, sucrose density gradients and tg vivo uridine incorporation. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories gt gt. Springfield: C. C. Thomas, 1974, p. 9. Sandstead, H. H., & Rinaldi, R. A. Impairment of deoxy- ribonucleic acid synthesis by dietary zinc deficiency in the rat. J. Cell. Physiol. 73 (1969):81. Sandstead, H. H. Zinc nutrition in the United States. Amer. J. of Clin. Nutr. 26 (l973):1251. Sandstead, H. H.; Gillespie, D. D.; & Brady, R. N. Zinc deficiency: effect on brain of the suckling rat. Pediat. Res. 6 (1972):1l9. Say, B.; Ozsoylu, S.; & Berkel, I. Geophagia associated with iron-deficiency anemia, hepatosplenomegaly, hypogonadism and dwarfism. A syndrome probably associated with zinc deficiency. Clin. Peds., November, 1969, p. 661. Sayers, R. R. Metal fume fever. Pub. Health Rept. 53 (1938):1080. Schroeder, H. A.; Nason, A. P.; Tipton, I. H.; and Balassa, J. J. Essential trace metals in man: zinc, relation to environmental cadmium. J. Chr. Dis. 23 (1970): 123. Schroeder, H. A. Losses of vitamins and trace minerals resulting from processing and preservation of foods. Amer. J. of Clin. Nutr. 241 (l97l):562. 114 Simkin, P. A. Oral zinc sulphate in rheumatoid arthritis. Lancet 2 (l976):539. Smith, J. C., Jr.; McDaniel, E. G.; McBean, L. D.; Doft, F. S.; & Halsted, J. A. J. Nutr. 102 (1972):7ll. Smith, J. C., Jr.; McDaniel, E. G.; Fan, F. F.; & Halsted, J. A. Zinc: a trace element essential in vitamin A metabolism. Science 181 (l973):954. Smith, J. C., Jr.; Zeller, J. A.; Brown, E. D. gt gt. Elevated plasma zinc: a heritable anomaly. Science 193 (l976):496. Somer, A. L., & Lipman, C. B. Evidence of the indispensible nature of zinc and boron for higher green plants. Plant Physiol. 1 (1926):231. Spencer, H.; Rosoff, B.; Lewin, I.; & Samachson, J. Studies of zinc—65 metabolism in man. In: Zinc Metabolism, edited by A. S. Prasad. Springfield: C. C. Thomas, 1966, p. 339. Spencer, H.; Osis, D.; Kramer, L.; & Wiatrowski, E. Studies of zinc metabolism in normal man and in patients with neoplasia. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories et a1. Spring- field: C. C. Thomas, 1974, p. 101. _—'—_ Sullivan, J. F., & Lankford, H. G. Urinary excretion of zinc in alcoholism and postalcoholic cirrhosis. Amer. J. of Clin. Nutr. 10 (1962):153. Sullivan, J. F., & Heaney, R. P. Zinc metabolism in alcoholic liver disease. Amer. J. of Clin. Nutr. 23 (l970):l70. Sullivan, J. F., & Burch, R. E. Potential role of zinc in liver disease. In: Trace Elements in Human Health and Disease, edited by A. S. Prasad. New York: Academic Press, 1976, p. 67. Swenerton, H.; Schrader, R.; & Hurley, L. S. Zinc—deficient embryos: reduced thymidine incorporation. Science 166 (1969):1014. Swenerton, H., & Hurley, L. S. Severe zinc deficiency in male and female rats. J. Nutr. 95 (l968):8. 115 Terhune, M. W., & Sandstead, H. H. Decreased RNA polymerase activity in mammalian zinc deficiency. Sciengg 177 (1972):68. Todd, W. R.; Elvehjem, C. A.; & Hart, C. B. Zinc in the nutrition of the rat. Amer. J. Physiol. 107 (1934):l46. Tribble, H. M., & Scoular, F. I. Zinc metabolism of young college women on self-selected diets. J. Nutr. 52 (1954):209. Tucker, H. F., & Salmon, W. D. Parakeratosis or zinc deficiency disease in the pig. Proc. Soc. Exp. Biol. Med. 88 (l955):613. Turk, D. B.; Sunde, M. L.; & Hoekstra, W. G. Zinc deficiency experiments with poultry. Poultrnycience 38 (1959): 1256. Underwood, E. J. Trace Elements in Human and Animal Nutri- tion. 4th Edition. New York: Academic Press, 1977. Vallee, B. L. Foreward. In: Clinical Applications of Zinc Metabolism, edited by W. J. Pories gt gt. Spring- field: C. C. Thomas, 1974. Vallee, B. L. Biochemistry, physiology and pathology of zinc. Physiol. Rev. 39 (1959):443. Van Rij, A. M., & McKenzie, J. M. Hyperzincuria and zinc deficiency in total parenteral nutrition. In: M. Trace Element Metabolism in Man and Animals-3, Proc. of 3rd International Symposium, edited by M. Kirchgessner. Fed. Rep. Germany: Freising, 1977, p. 288. Viets, F. R., Jr. Zinc deficiency in the soil-plant system. In: Zinc Metabolism, edited by A. S. Prasad. Springfield: C. C. Thomas, 1966, p. 90. Vir, S. C., & Love, A. H. G. Zinc and c0pper status of the elderly. Amer. J. of Clin. Nutr. 32 (l979):l472. Walker, B. E., & Kelleher, J. Plasma whole blood and urine zinc in the assessment of zinc deficiency in the rat. J. Nutr. 108 (1978):1702. Walravens, P. A. Zinc metabolism and its implications in clinical medicine. West. J. of Med. 130 (l979):l33. 116 Weismann, K.; Wanscher, B.; & Krakauer, R. Oral zinc therapy in geriatric patients with selected skin manifesta- tions and a low plasma zinc level. Acta. Dermatoven 58 (1978):157. White, H. S., & Gynne, T. N. Utilization of inorganic ele- ments by young women eating iron-fortified foods. J. of Amer. Diet. Assoc. 59 (1971):27. Whitenack, D. L.; Luecke, R. W.; & Whitehair, C. K. Fed. Proc. 29 (l970):297. Abstract 293. APPENDICES APPENDIX A TEST DATA FROM STUDY 1 AND MEANS AND STANDARD DEVIATIONS FOR BODY WEIGHT AND INTAKE OH O m h o m v m N O.mH m.mH N.ON m.ON m.ON NN m.NN MN m.VN mN 5.5N H mmN m.mH NH m.mH m.wH m.mH m.ON HN HN m.ON m.¢N m.hN OH m.HN m.mH m.MN HN ON m.NN VN m.MN m.mN m.hN ON a m.mH m.ON MN m.NN m.HN m.NN mN ON m.hN v.0N m.mN m m.bH m.hH OH ¢.mH mH m.HN MN m.MN m.vN VN mN h m.mH m.mH m.mH m.wH OH m.HN v.mH m.OH HN m.MN m.hN o m.mH m.HN m.ON m.ON m.NN m.NN v.mN v.VN m.MN m.VN ON m m.mH HN m.ON m.HN m.MN m.NN m.NN m.MN mN M.VN m.Om v m.ON m.HN OH m.ON m.ON vN mN m.vN m.vN mN ON m m.mH m.mH ON m.HN m.mH ON m.HN ON m.©N ON Om N m.mH m.mH OH m.HN m.HN. m.HN m.NN m.MN m.VN VN m.ON H mo deoz<8m Qz< mzmo omsozmnmm OHmHmnuN scsum 136 N OH ONH O O s N OH OHH O O s OH O H meH O O H O eH NeH O m H O O O OOH O O H O O OOH O O N O O OH eOH O O m O eH mOH O O m e O NH OHN O N H O mH HHN O N m O O sH OeH O OH N H HH seH O OH N m H O OsH O s e H O OmH O s e s O OH sNH O O O O OH mNH O O m O O sH NMH O s s O sH sMH O s s N O s OOH O O H O s OOH O OH H H «mm .Hom .Hmmm .Uuwm .NwthH .EOOHU OHUHHU .Hom .Hmmm .Hme .NOQHW .EOOHU QHOHHU nmnesz H.OOOO meHe umnssz H.OOOO msHe 03H. mOOoO mco OOOHHO 137 Om. O.NN Os.OmO sN.OH OO.NsO OO. os.NH Os.NH ON.HOs HN os. OO.ON OO.OOO O0.0H OH.mmO HO. OO.OH OO.NH OO.NOs ON os. O0.0N Om.OsO Os.OH OO.NmO Om. OO.OH HO.NH ON.an OH sm. OH.ON Om.OOO HN.OH OO.msO HO. Om.OH OO.NH OO.sOs OH Hm. O0.0N ON.OOO HH.OH O0.0sO Os. Os.OH OH.HH ON.OOs OH Os. OH.NN O0.000 Om.mH O0.0sO sm. Os.OH Om.HH O0.00s OH NO. OH.NN Om.OOO ss.mH O0.0sO Om. O0.0H Os.NH OO.mOs OH OO. OH.ON ON.mOO OO.mH Om.OsO Nm. O0.0H O0.0H O0.00s sH OO. OO.ON OH.OOO OO.mH ON.NOO Os. O0.0H m0.0H os.NOs mH sm. OO.ON Om.mOO Hm.mH OO.sOO Om. O0.0H N0.0H O0.00s NH Om. OH.NN ON.OOO mO.NH OH.OOO Ns. O0.0H Os.mH OO.sOO HH OO. O0.0N OH.OOO Ns.HH O0.000 Os. os.OH ms.NH os.OHO OH mO. ON.NN ON.OOO OO.HH ON.OOO sO. OH.ON ON.O O0.0HO O Om. O0.0N O0.000 HH.NH O0.000 Ns. Os.ON H0.0 OO.mNO O Hs. ON.ON O0.000 OH.NH ON.OOO NO. O0.0N ON.O OH.ONO O HO. OO.NN OO.HOO Hm.HH O0.0NO os. OO.HN O0.0 OO.smO O OO. O0.0N Om.HOO O0.0 Os.NOO OO. OO.NN NO.mH O0.0mO O NO. OO.NN Om.mOO Om.O OO.ONO OO. OH.mN OO.mH O0.0sO s HO. N.ON os.OOO OH.O OO.HOO HO. ON.sN OO.NH ON.HOO m OO. N.ON OH.OOO. OO.O ON.mOO Os. O0.0N O0.0 O0.000 N mO. OO.NN OO.sOO N0.0 OO.NNO OO. O0.0N m0.0 O0.0NO H 2mm cmwz cam: 5mm cam: 2mm cmmz 2mm cum: OxmucH uOOsz usOHmz mxmucH uanmg «ON Osouw OON Odouo OON msouo mxmucH coon cam unwflm3 xvom Mom 2mm can mammzlla >USUm 138 I I I I Hs.O OO.mOm I I I I mO.N OO.sOH I I I OO.s OO.HmH sH OO. os.NN ON.O os.OOm I I I I NO.H 0.00H HN. Om.O OO.s Om.NmH mH mm. OO.mN ON.O OH.ONm I I I I OH.N ON.OOH ON. OO.O OH.O O0.0mH NH sm. OO.mN Hm.O OO.sOm I I I I sO.N ON.OOH Nm. O0.0 OH.s O0.0sH HH Os. os.sN OO.s O0.0sm I I I I ON.N OO.sOH sm. O0.0 mO.m OO.ssH OH Os. O0.0N OO.s ON.Omm I I I I Om.N NO.OOH sm. O0.0 OH.s OH.ssH O sm. OH.mN mH.O ON.ONm I I I I mO.H ON.HOH sO. OH.HH sO.m ON.HsH O mO. ON.sN sO.s OO.sOm I I I I HO.H OO.OOH OO. O0.0H ON.m O0.0sH N Om. mO.mN Om.O OO.OON I I I I mO.H OO.sOH OO. OH.NH Os.m- O0.0sH O HO. ON.ON Om.O os.smN I I I I NO.H OO.OOH OO. ON.OH OO.m ON.NmH O HO. O0.0H sN.O OO.NON I I I I HH.N ON.NOH hO. O0.0H OO.m ON.smH s NO. ON.OH mH.s OO.HNH I I I I OO.N ON.OsH mO. O0.0 Os.m os.smH m ON. N.OH OO.N OH.OmH I I I I OO.N os.OmH sm. os.O OO.m OO.NNH N NH. OO.sH OO.H OO.sOH I I I I ON.m os.NHH OO. OO.NH ms.N ON.HHH H 2mm OOO: 2mm OOO: Omm “HOO EOO OOO: 2mm cams 2mm cam: mxmucH usomm3 mxmucH unmflm3 wxmucH unmflmz OON msouw OON msouu dam muouu OxmucH Ooom OOO unOHmz OOom now 2mm Omm OOOOzIIN mwsum