1 Experimental Zinc Deficiency in Humans An Overview of Original Studies
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
ANANDA S. PRASAD Wayne State University School of Medicine, Harper-Grace Hospitals, Department of Internal Medicine, Detroit, MI 48201, and Veterans Administration Medical Center, Allen Park, MI 48101
During the past two decades, essentiality of zinc for man has been established. Deficiency of zinc in man due to nutritional factors and several disseased states, has been recognized. A marginal deficiency of zinc appears to be prevalent in many segments of population in developed countries and more severe deficiencies are widespread in many parts of the world. In our experimental human model, a marginal deficiency of zinc was induced by dietary means. Loss of body weight (less than 10% in six months on zinc restricted diet), testicular hypofunction, hyperammonemia and a decrease in plasma, urinary and neutrophil zinc concentration were observed. Changes in zinc dependent enzymes such as deoxythymidine kinase in newly synthesized connective tissue and plasma alkaline phosphatase were also observed as a result of zinc restriction and repletion in our model. Although the r o l e of z i n c i n human subjects has been now defined and i t s d e f i c i e n c y recognized i n s e v e r a l c l i n i c a l cond i t i o n s , these examples are not r e p r e s e n t a t i v e of a pure z i n c d e f i c i e n t s t a t e i n man. I t was, t h e r e f o r e , considered d e s i r a b l e to develop a human model which would a l l o w a study of the e f f e c t s of a m i l d z i n c d e f i c i e n t s t a t e i n man. Recently such a model has been e s t a b l i s h e d s u c c e s s f u l l y i n human volunteers w i t h the use of a s e m i - p u r i f i e d d i e t based on t e x t u r i z e d soy p r o t e i n . Long-standing n u t r i t i o n a l z i n c d e f i c i e n c y has been reported to cause primary hypogonadism i n human subjects ( 1 , 2). D e f i ciency of z i n c o c c u r r i n g i n a s s o c i a t i o n w i t h c e r t a i n diseases has a l s o been reported t o a f f e c t adversely t e s t i c u l a r f u n c t i o n ( 3 ) . In experimental animals, z i n c - r e s t r i c t e d d i e t i s known t o produce primary hypogonadism ( 4 ) . In both animals and human s u b j e c t s , t e s t i c u l a r hypofunction 0097-6156/83/0210-0001$06.00/0 © 1983 American Chemical Society In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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due t o z i n c d e f i c i e n c y was c h a r a c t e r i z e d by decreased f u n c t i o n of the Leydig c e l l s and oligospermia ( 3 ) . A decrease i n serum androgens, an increase i n serum gonadotropins, and a r r e s t of spermatogenesis were observed i n t e s t i c u l a r b i o p s i e s obtained from some p a t i e n t s who were z i n c - d e f i c i e n t . A l s o , z i n c supplementation has been used t o increase plasma t e s t o s t e r o n e l e v e l and sperm count i n i n f e r t i l e men ( 5 ) . In previous r e p o r t s , a d e f i c i e n c y of z i n c was b e l i e v e d t o cause male hypogonadism, but because of the complicated nature of the c l i n i c a l problems i n a l l such cases, other f a c t o r s respons i b l e f o r hypogonadism, n u t r i t i o n a l or otherwise, could not be r u l e d out. However, i n a l l such cases reported so f a r , mode r a t e l y severe d e f i c i e n c y of z i n c was present f o r s e v e r a l years. In t h i s study we have shown that even a m i l d d e f i c i e n c y of z i n c can adversely i n f l u e n c e t e s t i c u l a r f u n c t i o n i n a d u l t s u b j e c t s , which was r e v e r s i b l e w i t h z i n c supplementation. In t h i s paper, I w i l l summarize our experience w i t h t h i s model. Methods and R e s u l t s Four male volunteers p a r t i c i p a t e d i n the f i r s t experiment. The f i r s t p a i r of two s u b j e c t s ( P a t i e n t 1, P a t i e n t 2) were 57 and 55-year-old white men, r e s p e c t i v e l y . P a t i e n t 1 had degenerat i v e o s t e o a r t h r i t i s and a l l e r g i c r h i n i t i s . R e s u l t s of p h y s i c a l examination, r o u t i n e blood t e s t s , and z i n c c o n c e n t r a t i o n of plasma e r y t h r o c y t e s and h a i r were w i t h i n normal l i m i t s before the study. P a t i e n t 2 was diagnosed as having m i l d diabetes m e l l i t u s and m i l d hypertension. At the time the p a t i e n t s entered the study, there were no abnormal p h y s i c a l f i n d i n g s , and z i n c s t a t u s was w i t h i n normal l i m i t s . The second p a i r of subjects were P a t i e n t 3, a 56-year-old b l a ^ k man who was being followed up f o r e s s e n t i a l hypertension and gouty a r t h r i t i s , and P a t i e n t 4, a 65-year-old white man who had c h r o n i c s i n u s i t i s and m i l d diabetes i n s i p i d u s . At the time of the study P a t i e n t 3 and 4 were asymptomatic, w i t h no abnormal p h y s i c a l or l a b o r a t o r y f i n d i n g s . Zinc s t a t u s as assessed by z i n c c o n c e n t r a t i o n i n plasma, e r y t h r o c y t e s , and h a i r was w i t h i n normal l i m i t s i n both s u b j e c t s . A f t e r a thorough p h y s i c a l examination, r o u t i n e l a b o r a t o r y t e s t s were done, i n c l u d i n g r o u t i n e hematologic t e s t s , serum e l e c t r o l y t e s , blood urea n i t r o g e n , serum c r e a t i n i n e , and f a s t i n g blood sugar. Serum l e v e l s of calcium, i n o r g a n i c phosphate, t o t a l cholesterol, triglyceride, total protein, uric acid, total b i l i r u b i n , serum g l u t a m i c - o x a l a c e t i c transaminase, v i t a m i n A, and carotene were a l s o measured. These r o u t i n e blood t e s t s were r e peated once a month. Routine chest roentgenograms and u r i n a l y s i s were c a r r i e d out i n the beginning and p e r i o d i c a l l y as needed. The subjects were kept on the metabolic ward, and two p h y s i c i a n s f o l l o w e d them up r e g u l a r l y . Psychologic t e s t i n g was done
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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by a c l i n i c a l p s y c h o l o g i s t t h r i c e , i n i t i a l l y , once at the end of the r e s t r i c t e d z i n c i n t a k e p e r i o d and again a f t e r z i n c supplementation. A s e m i - p u r i f i e d d i e t based on t e x t u r i z e d soy p r o t e i n (purchased from General M i l l s Company, M i n n e a p o l i s , Minnesota (Bontrae Products) and Worthington Foods Company, D i v i s i o n of M i l e s Laboratory, E l k h a r t , Indiana) was developed f o r t h i s study. Soy p r o t e i n i s o l a t e was used as soy f l o u r i n the baked goods (purchased from General B i o c h e m i c a l s , Teklad M i l l s , Chagrin F a l l s , Ohio). The t e x t u r i z e d soy meals used were hamburger granules, chicken s l i c e s , turkey s l i c e s , and chicken chunks. The t e x t u r i z e d soy p r o t e i n and soy p r o t e i n i s o l a t e were washed twice w i t h e t h y l e n e d i a m i n e t e t r a a c e t a t e , then r i n s e d three times w i t h d e i o n i z e d water, b o i l e d f o r 30 min. and kept f r o z e n u n t i l ready to be used. Most r e c i p e s used i n t h i s study were adapted from Soy P r o t e i n Recipe Ideas, published by I n s t i t u t i o n Volume Feeding Management Magazine, Chicago, I l l i n o i s . The foods were cooked i n l a r g e q u a n t i t i e s and s t o r e d i n a f r e e z e r f o r 1 to 3 months. As needed, the food items were def r o s t e d i n the r e f r i g e r a t o r , heated, weighed, and then served to the v o l u n t e e r s . Every 4 weeks f o r 7 days, b r e a k f a s t , l u n c h , d i n n e r , and snacks served each day were homogenized i n a blender. A l i q u o t s of the composite homogenized meals were weighed, f r o z e n , l y o p h i l i z e d , and then analyzed f o r f a t , n i t r o g e n , and z i n c content of the meals. For the f i r s t two s u b j e c t s ( P a t i e n t 1 and 2 ) , the d i e t s u p p l i e d 1865 k c a l , 53 g of p r o t e i n , and a l l e s s e n t i a l v i t a m i n s and minerals except z i n c , according to recommended d i e t a r y allowances. The second set of p a t i e n t s ( P a t i e n t s 3 and 4) r e c e i v e d a v i t a m i n t a b l e t ( P o l y - V i - S o l , Mead Johnson Laboratori e s , E v a n s v i l l e , Indiana) and a m i n e r a l mixture. The second p a i r of s u b j e c t s a l s o r e c e i v e d p r o t e i n supplement ( S t u a r t Amino Acids Powder, S t u a r t Pharmacy, Wilmington, Delaware). The d a i l y i n t a k e of c a l o r i e s was 2352 k c a l and p r o t e i n , 58 g. The f i r s t p a i r of s u b j e c t s ( P a t i e n t s 1 and 2) r e c e i v e d hosp i t a l d i e t f o r 2 weeks; then they r e c e i v e d the experimental d i e t w i t h 10 mg of supplemental z i n c (as z i n c acetate) d a i l y o r a l l y for 6 weeks. A f t e r t h i s , they were given only experimental d i e t ( d a i l y z i n c i n t a k e of 2.7 mg) f o r 24 weeks. At the end of t h i s phase, w h i l e c o n t i n u i n g the experimental d i e t , the two s u b j e c t s r e c e i v e d 30 mg of z i n c supplement (as z i n c acetate) d a i l y o r a l l y f o r 12 weeks. F i n a l l y , these s u b j e c t s were maintained on h o s p i t a l d i e t w i t h t o t a l d a i l y i n t a k e of 10 mg z i n c p l u s 30 mg of o r a l z i n c supplement (as z i n c acetate) f o r 8 weeks. The h o s p i t a l d i e t provided the same amount of c a l o r i e s and p r o t e i n as the e x p e r i mental d i e t . Thus, these two s u b j e c t s were observed f o r a t o t a l p e r i o d of 52 weeks. The second two s u b j e c t s ( P a t i e n t s 3 and 4) r e c e i v e d h o s p i t a l d i e t (10 mg of z i n c i n t a k e d a i l y ) f o r 3 weeks, f o l l o w e d by the experimental d i e t w i t h 30 mg of o r a l z i n c supplement (as z i n c acetate) f o r 5 weeks. A f t e r t h i s , they were given only e x p e r i -
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
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mental d i e t (3.5 mg of z i n c i n t a k e d a i l y ) f o r 50 weeks. The r e p l e t i o n phase was begun w i t h the a d m i n i s t r a t i o n of 30 mg of z i n c (as z i n c acetate) o r a l l y w h i l e the same experimental d i e t was maintained and continued f o r a t o t a l p e r i o d of 8 weeks, at the end of which the h o s p i t a l d i e t replaced the experimental d i e t . The c a l o r i e and p r o t e i n i n t a k e from the h o s p i t a l and the experimental d i e t s were the same. O r a l z i n c supplement (30 mg as z i n c acetate) was continued along w i t h the h o s p i t a l d i e t f o r a p e r i o d of 8 weeks. A l t o g e t h e r these two s u b j e c t s were observed f o r 64 weeks. Deionized water was given f o r d r i n k i n g purposes throughout the study p e r i o d . Care was taken to ensure that the d i e t provided the same number of c a l o r i e s and p r o t e i n q u a n t i t y throughout the study p e r i o d , i n c l u d i n g the p e r i o d i n which the s u b j e c t s r e c e i v e d the h o s p i t a l d i e t . Whenever a p p r o p r i a t e , e x t r a t r a y s of the h o s p i t a l d i e t (composite of 7 days) were homogenized and analyzed f o r f a t , p r o t e i n , and z i n c content. The p a t i e n t s were weighed three times a week, and s k i n f o l d measurements were taken p e r i o d i c a l l y of the abdomen, t r i c e p s , subscapular areas, u s i n g s k i n f o l d c a l i p e r s according to the procedure o u t l i n e d by the manufacturer (Cambridge S c i e n t i f i c I n d u s t r i e s , Inc., Cambridge, Maryland). Urine and f e c a l samples were c o l l e c t e d f o r 7 consecutive days every 4 weeks. F e c a l samples were analyzed f o r n i t r o g e n , f a t , and z i n c content. F e c a l samples were weighed, l y o p h i l i z e d , d i g e s t e d w i t h n i t r i c a c i d , d i l u t e d to volume w i t h d e i o n i z e d water, and analyzed by the atomic a b s o r p t i o n spectrophotometer, model 303 or 306 ( P e r k i n Elmer, Norwalk, C o n n e c t i c u t ) . Food samples were weighed, wet d i g e s t e d w i t h n i t r i c a c i d , and d i l u t e d to volume w i t h d e i o n i z e d water, then analyzed f o r z i n c l e v e l ( 6 ) . N i t r o g e n l e v e l s of d r i e d samples (food or feces) were determined by K j e l d a h l procedure ( 7 ) . The f a t content of d r i e d samples (food or feces) was a s c e r t a i n e d by ether e x t r a c t i o n of the l i p i d s (8). Blood samples were drawn every other week, u s i n g p l a s t i c s y r i n g e s and tubes. The whole blood was c e n t r i f u g e d and plasma p i p e t t e d i n t o p l a s t i c tubes, then p r e c i p i t a t e d w i t h 10% t r i c h l o r o a c e t i c a c i d , d i l u t e d 1:4 w i t h d e i o n i z e d water, and analyzed f o r z i n c by the atomic a b s o r p t i o n spectrophotometer, model 303 or 306. The e r y t h r o c y t e s were washed three times w i t h normal s a l i n e . A f t e r the l a s t c e n t r i f u g a t i o n 1 ml of packed e r y t h r o cytes were p i p e t t e d i n t o p l a s t i c tubes, and 2 ml d e i o n i z e d water was added. Hemoglobin l e v e l was measured using cyanide technique on Beckman DK-2 spectrophotometer ( F u l l e r t o n , C a l i f o r n i a ) . E r y t h r o c y t e s were digested w i t h n i t r i c a c i d and d i l u t e d to volume w i t h d e i o n i z e d water. Zinc was then assayed by atomic absorption spectrophotometer and values expressed i n termes of micrograms of z i n c per gram of hemoglobin. The leukocytes were separated by a technique reported by R o t h s t e i n , Bishop, and Ashenbrucker ( 9 ) , and z i n c content was measured by atomic absorpt i o n spectrophotometer.
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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Zinc Deficiency in Humans
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For plasma ammonia l e v e l s , whole blood was c e n t r i f u g e d and plasma p i p e t t e d i n t o ammonia-free tubes. The plasma was then f r o z e n immediately and kept f r o z e n u n t i l ammonia was measured by an autoanalyzer. Urea n i t r o g e n i n the plasma and c r e a t i n i n e e x c r e t i o n i n the u r i n e were a l s o measured by the autoanalyzer. L a c t i c dehydrogenase a c t i v i t y i n the plasma was measured by a method published by Bergmeyer, Bernt, and Hess (10). Ribonuclease a c t i v i t y was a s c e r t a i n e d by a modified Sekine method (11, 12). The a c t i v i t y of a l k a l i n e phosphatase i n the plasma was measured by a c o l o r i m e t r i c procedure. In each of the f i r s t two s u b j e c t s ( P a t i e n t s 1 and 2 ) , a f t e r l o c a l a n e s t h e s i a , one p o l y v i n y l sponge measuring approximately 4 cm χ 3 cm χ 3 mm was implanted subcutaneously on the l a t e r a l aspect of the chest. This was done i n order to o b t a i n newly s y n t h e s i z i n g c o l l a g e n connective t i s s u e f o r assay of deoxythymidine kinase a c t i v i t y . Twenty-one days a f t e r i m p l a n t a t i o n the sponges were i s o l a t e d by b l u n t d i s s e c t i o n , and the capsules surrounding the sponges were c o l l e c t e d f o r study. T o t a l p r o t e i n , t o t a l c o l l a g e n , r i b o n u c l e i c a c i d (RNA) to d e o x y r i b o n u c l e i c a c i d (DNA) r a t i o , and the a c t i v i t y of deoxythymidine kinase were assayed by techniques reported p r e v i o u s l y (13). Sponge implanta t i o n was done t w i c e , once at the end of the z i n c r e s t r i c t i o n period and again a f t e r z i n c supplementation f o r 12 weeks, w h i l e the s u b j e c t s r e c e i v e d the same experimental d i e t otherwise. C l i n i c a l and psychologic e v a l u a t i o n s during the study p e r i o d remained e s s e n t i a l l y unchanged. The f i r s t p a i r of s u b j e c t s ( P a t i e n t s 1 and 2 ) , on 2.7 mg d a i l y z i n c i n t a k e , complained of m i l d roughening of s k i n and l e t h a r g y ; but these were not observed i n the second p a i r ( P a t i e n t s 3 and 4 ) , on 3.5 mg of d a i l y z i n c intake. R e s u l t s of r o u t i n e l a b o r a t o r y t e s t s remained e s s e n t i a l l y the same i n a l l four subjects throughout the study except f o r blood urea n i t r o g e n , which decreased s i g n i f i c a n t l y soon a f t e r the subjects s t a r t e d r e c e i v i n g the experimental d i e t . This change occurred even before the z i n c i n t a k e was r e s t r i c t e d , suggesting that the decrease i n the blood urea n i t r o g e n was due to the change from animal p r o t e i n to c e r e a l p r o t e i n i n the d i e t . In the f i r s t two subjects ( P a t i e n t s 1 and 2 ) , who r e c e i v e d 2.7 mg of z i n c d a i l y , the weight l o s s was more pronounced i n comparison w i t h the second p a i r of s u b j e c t s ( P a t i e n t s 3 and 4 ) , who r e c e i v e d 3.5 mg of z i n c d a i l y . A f t e r r e p l e t i o n w i t h z i n c , the weight s t a b i l i z e d i n three out of four s u b j e c t s ; and i n one subject ( P a t i e n t 1 ) , although the weight l o s s continued during the zinc-supplemented p e r i o d , the r a t e of weight l o s s as d e t e r mined by the slope of the curve was decreased. The changes i n body weight c o r r e l a t e d h i g h l y w i t h the subscapular t h i c k n e s s i n the two s u b j e c t s i n whom these data were obtained ( r = 0.839, Ρ < 0.001; r = 0.938, Ρ < 0.001). Further c a l c u l a t i o n (14), showed t h a t the weight l o s s could be
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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accounted f o r as f o l l o w s : 50% f a t , 30% water, and 20% other (approximately). I n two subjects the intake of f a t was 108.3 g/day, and f e c a l f a t (mean ± SD) was 5.6 ± 1.33 g/day i n one subj e c t ( P a t i e n t 4) and i n the other 4.11 ± 0.94 g/day throughout the study p e r i o d . Thus no e f f e c t of z i n c d e p l e t i o n - r e p l e t i o n was observed on f a t balance. The n i t r o g e n e x c r e t i o n i n the f e c e s , u r i n e , and the balance throughout the experimental study period i n the second p a i r of subjects ( P a t i e n t s 3 and 4) showed no remarkable changes. The balance data were apparent balances, inasmuch as the n i t r o g e n e x c r e t i o n i n the sweat was not considered i n c a l c u l a t i n g these balances. U r i n a r y e x c r e t i o n of z i n c decreased i n three out of four subjects as a r e s u l t of z i n c r e s t r i c t i o n . I n one case ( P a t i e n t 4) there was no decrease i n u r i n a r y z i n c e x c r e t i o n . This was due t o the d i u r e t i c therapy (hydrochlorothiazide) that he r e ceived f o r m i l d hypertension during the study. In the f i r s t two s u b j e c t s , during the z i n c r e s t r i c t i o n phase (2.7 mg z i n c d a i l y i n t a k e ) , the apparent negative balance f o r z i n c ranged from 1 t o 4 mg/day, whereas i n the second group of subjects the apparent negative balance f o r z i n c was 1 to 2 mg/ day. A f t e r supplementation w i t h 30 mg of z i n c , the p o s i t i v e z i n c balance ranged from 11 t o 22 mg/day i n the f i r s t p a i r of subj e c t s , suggesting a r e t e n t i o n of approximately 33% to 70% of z i n c i n t a k e . I n the second p a i r of subjects ( P a t i e n t s 3 and 4 ) , during the b a s e - l i n e period when the d a i l y z i n c i n t a k e was 33.5 mg, the p o s i t i v e balance f o r z i n c was 3 t o 4 mg d a i l y . On the other hand, these s u b j e c t s , on the same l e v e l of z i n c intake (33.5 mg d a i l y ) a f t e r z i n c d e p l e t i o n phase, showed a p o s i t i v e z i n c balance of 14 t o 16 mg d a i l y . The plasma z i n c l e v e l decreased s i g n i f i c a n t l y i n a l l 4 subj e c t s as a r e s u l t of z i n c r e s t r i c t i o n and increased a f t e r supplementation w i t h z i n c . The changes were more marked from p a t i e n t s on 2.7 mg d a i l y z i n c i n t a k e compared w i t h those on 3.5 mg d a i l y z i n c i n t a k e . The erythrocyte z i n c l e v e l decreased s i g n i f i c a n t l y i n the f i r s t group of two subjects ( P a t i e n t s 1 and 2) although the decrease was not evident u n t i l 12 weeks on the r e s t r i c t e d z i n c i n t a k e . I n the second group of s u b j e c t s , although the erythrocyte z i n c l e v e l d i d not decrease s i g n i f i c a n t l y during the z i n c - r e s t r i c t e d p e r i o d , i t showed a marked increase a f t e r z i n c supplementation. Leukocyte z i n c decreased s i g n i f i c a n t l y due t o z i n c r e s t r i c t i o n i n the second group of subjects i n whom t h i s v a r i a b l e was measured. Plasma a l k a l i n e phosphatase was monitored c a r e f u l l y i n the second group of s u b j e c t s . I n both cases, the a c t i v i t y s l o w l y d e c l i n e d as a r e s u l t of z i n c r e s t r i c t i o n , and a f t e r supplementat i o n w i t h z i n c , the a c t i v i t y n e a r l y doubled i n 8 weeks. I n a l l four s u b j e c t s , the a c t i v i t y of plasma ribonuclease was almost twice as great during the z i n c - r e s t r i c t e d period as i n the z i n c supplemented phase. Plasma l a c t i c dehydrogenase a c t i v i t y de-
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
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creased w i t h z i n c r e s t r i c t i o n and increased during supplementat i o n phases i n two s u b j e c t s . Plasma ammonia l e v e l s were higher during z i n c r e s t r i c t i o n and decreased a f t e r z i n c supplementation i n two s u b j e c t s ( P a t i e n t s 3 and 4 ) . In the sponge connective t i s s u e of the f i r s t p a i r of subj e c t s , t o t a l p r o t e i n and t o t a l c o l l a g e n increased s i g n i f i c a n t l y during the z i n c supplementation phase i n comparison w i t h the z i n c r e s t r i c t i o n phase ( P a t i e n t 1, r e s t r i c t i o n phase: t o t a l prot e i n , 108 mg; t o t a l c o l l a g e n , 29.3 mg; versus supplementation phase: t o t a l p r o t e i n , 226 mg; and t o t a l c o l l a g e n , 58.4 mg; P a t i e n t 2, r e s t r i c t i o n phase: t o t a l p r o t e i n , 94 mg; t o t a l c o l l a g e n 0.013 mg; versus supplementation phase: t o t a l p r o t e i n , 240 mg; t o t a l c o l l a g e n , 121.2 mg). The RNA-DNA r a t i o i n the sponge connective t i s s u e a l s o increased a f t e r z i n c supplementat i o n ( P a t i e n t 1, r e s t r i c t i o n phase: 0.69 versus supplementation phase: 0.82; P a t i e n t 2, r e s t r i c t i o n phase: 0.71 versus supplemental phase: 0.95). There was no d e t e c t a b l e a c t i v i t y of deoxythymidine kinase i n the sponge connective t i s s u e during the z i n c r e s t r i c t i o n phase, but i t was 0.385 u n i t s i n P a t i e n t 1 and 0.321 u n i t s i n P a t i e n t 2 a f t e r supplementation w i t h z i n c . The a c t i v i t i e s a f t e r supplementation became 70% of the mean normal values (14). The e f f e c t of marginal z i n c d e f i c i e n c y on gonadal f u n c t i o n s were s t u d i e d i n 5 male v o l u n t e e r s (15). T h e i r ages ranged from 51 t o 65 years. These s u b j e c t s had normal gonadal f u n c t i o n . They had no medical d i s o r d e r , and they had taken no drugs p r i o r t o the s t u d i e s known t o a f f e c t t e s t i c u l a r f u n c t i o n . P h y s i c a l examination, r o u t i n e l a b o r a t o r y t e s t s , and chest roentgenogram were unremarkable. T e s t i c u l a r f u n c t i o n as e v a l u ated c l i n i c a l l y and by the measurement of serum androgens, FSH, LH, and sperm count were normal i n a l l p a t i e n t s . Zinc s t a t u s as assessed by the determination of z i n c c o n c e n t r a t i o n i n plasma, e r y t h r o c y t e s , and h a i r was w i t h i n normal l i m i t s . During s t a b i l i z a t i o n p e r i o d , a l l s u b j e c t s r e c e i v e d h o s p i t a l d i e t (10 mg of z i n c i n t a k e d a i l y ) . A f t e r s t a b i l i z a t i o n , Subject 1 r e c e i v e d the experimental d i e t p r o v i d i n g 2.7 mg of d a i l y z i n c , w i t h 10 mg of supplemental z i n c (as z i n c acetate) o r a l l y f o r 6 weeks. Subjects 2 and 3 r e c e i v e d the experimentl d i e t p r o v i d i n g 3.5 mg of d a i l y z i n c , w i t h 30 mg of supplemental z i n c (as z i n c acetate) o r a l l y f o r 5 weeks. Subjects 4 and 5 were switched d i r e c t l y from the h o s p i t a l d i e t to the experimental z i n c r e s t r i c t e d d i e t . The experimental z i n c - r e s t r i c t e d d i e t provided 2.7 mg of d a i l y z i n c i n Subject 1 f o r a p e r i o d of 24 weeks; 3.5 mg of d a i l y z i n c i n Subjects 2 and 3 f o r a p e r i o d of 40 weeks; and 5 mg of d a i l y z i n c i n Subjects 4 and 5 f o r a p e r i o d of 40 and 32 weeks, r e s p e c t i v e l y . In Subjects 1, 2, and 3, the r e p l e t i o n phase was begun w i t h the a d m i n i s t r a t i o n of 30 mg of z i n c (as z i n c acetate) o r a l l y w h i l e the same experimental d i e t was maintained f o r a p e r i o d of
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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12 weeks i n Subject 1 and 8 weeks i n Subjects 2 and 3. In Subject 4, 10 mg of z i n c (as z i n c acetate) was mixed w i t h the experimental d i e t , f o r a p e r i o d of 12 weeks. A f t e r the e x p e r i mental d i e t , the h o s p i t a l d i e t (10 mg of d a i l y z i n c ) p l u s the z i n c supplementation were given f o r a p e r i o d of at l e a s t 8 weeks i n these four s u b j e c t s . Subject 5 was not given a d d i t i o n a l z i n c a f t e r h i s experimental d i e t was terminated but i n s t e a d r e c e i v e d r e g u l a r d i e t at home. The c a l o r i e s and p r o t e i n i n t a k e of the h o s p i t a l and the experimental d i e t remained the same. T h e r e a f t e r , a l l p a t i e n t s r e c e i v e d home d i e t s i m i l a r to the h o s p i t a l d i e t p r o v i d i n g 10 mg of d a i l y z i n c . Blood samples were drawn f o r the determination of LH, FSH, and t e s t o s t e r o n e before and a f t e r s t i m u l a t i o n w i t h GnRH. These determinations were performed twice during s t a b i l i z a t i o n p e r i o d , twice at the end of z i n c - d e p l e t i o n p e r i o d , twice during the f i r s t 6 months of z i n c - r e p l e t i o n p e r i o d , and twice a f t e r 6 to 12 months of z i n c r e p l e t i o n . Thus, the z i n c r e p l e t i o n p e r i o d was d i v i d e d i n t o two phases: the e a r l y phase which r e f l e c t e d the f i r s t 6 months of r e p l e t i o n and the l a t e phase which was extended beyond 6 months and up to 12 months of z i n c r e p l e t i o n . Blood samples were drawn from an i n d w e l l i n g intravenous c a t h e t e r between 9:00 and 9:30 a.m. At l e a s t two b a s e l i n e samples 30 min apart were drawn f o r the determination of serum LH, FSH, and t e s t o s t e r o n e , and then 200 μg of GnRH were i n j e c t e d i n t r a v e n o u s l y . Blood samples were drawn f o r the measurement of serum LH and FSH 15, 30, 60, 120, 180, and 240 min l a t e r . Blood samples were drawn f o r the determination of serum t e s t o s t e r o n e 60, 120, 180 and 240 min a f t e r GnRH i n j e c t i o n . The b a s e l i n e value of each hormone was d e r i v e d by c a l c u l a t i n g a mean of two to four b a s e l i n e determina t i o n s . Means of two d i f f e r e n t determinations of GnRH-stimulation t e s t s were c a l c u l a t e d f o r the s t a b i l i z a t i o n , z i n c - r e s t r i c t i o n , and e a r l y and l a t e phases of z i n c - r e p l e t i o n p e r i o d s . Thus f l u c t u a t i o n of these hormones from hour to hour, as w e l l as from day to day, was avoided. Serum t e s t o s t e r o n e , LH, and FSH were measured according to radioimmunoassay techniques. Semen a n a l y s i s was done every 4 to 12 weeks and a f t e r a b s t i nence from sexual a c t i v i t y f o r 3 to 7 days before e j a c u l a t i o n . A n a l y s i s was done w i t h i n 30 min of e j a c u l a t i o n . R e s u l t s of sperm count were expressed i n c o n c e n t r a t i o n of sperm per m i l l i l i t e r as w e l l as t o t a l number of sperm per e j a c u l a t e . A mean of two to f i v e sperm counts during each phase of the study were c a l c u l a t e d . Oligospermia was d e f i n e d as a t o t a l sperm count l e s s than 40 m i l l i o n per e j a c u l a t e . Zinc concentrations i n the e r y t h r o c y t e and plasma decreased s i g n i f i c a n t l y (p < 0.01) during z i n c r e s t r i c t i o n i n comparison to the s t a b i l i z a t i o n l e v e l s . During the e a r l y phase of z i n c r e p l e t i o n a s l i g h t i n c r e a s e i n e r y t h r o c y t e and plasma z i n c l e v e l s was noted, but these values were not s t a t i s t i c a l l y s i g n i f i c a n t i n comparison to the z i n c - r e s t r i c t i o n l e v e l s . A marked increase i n e r y t h r o c y t e and plasma z i n c c o n c e n t r a t i o n was observed during the
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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l a t e phase of z i n c r e p l e t i o n , and these l e v e l s were s t a t i s t i c a l l y s i g n i f i c a n t (p < 0.005) when compared w i t h the values of z i n c r e s t r i c t i o n and e a r l y phase of z i n c r e p l e t i o n . Sexual h a i r and t e s t i c u l a r s i z e showed no change throughout the study p e r i o d . However, sexual d r i v e was diminished during z i n c - r e s t r i c t e d p e r i o d as compared t o the s t a b i l i z a t i o n and z i n c r e p l e t i o n p e r i o d s , but an o b j e c t i v e assessment was unobtainable. The b a s e l i n e determinations of serum androgens and p i t u i t a r y gonadotropins during s t a b i l i z a t i o n p e r i o d were normal. The mean ± S.E.M. of b a s a l serum testosterone i n a l l f i v e s u b j e c t s was 4.81 ± 0.78 ng/ml (normal 5.4 ± 0.3); serum LH, 14 ± 3.7 mIU/ml (normal 12.4 ± 2 ) ; serum FSH, 12.2 ± 2 mIU/ml (normal 10 ± 1 ) . The range of sperm c o n c e n t r a t i o n i n a l l f i v e s u b j e c t s was 131 t o 825 m i l l i o n s / m l , w i t h a mean ± S.E.M. of 305.9 ± 76.4. The range of t o t a l sperm count per e j a c u l a t e was 87 t o 3925 m i l l i o n s , w i t h a mean ± S.E.M. of 1114.7 ± 411.6 m i l l i o n s . The sperm count d e c l i n e d s l i g h t l y during z i n c r e s t r i c t i o n and continued t o d e c l i n e i n the e a r l y phase of z i n c r e p l e t i o n . Oligospermia ( t o t a l sperm count per e j a c u l a t e l e s s than 40 m i l l i o n ) was observed i n four out of f i v e subjects as a r e s u l t of d i e t a r y z i n c r e s t r i c t i o n . The mean ± S.E.M. of sperm concen t r a t i o n f o r the e n t i r e group was 59.2 ± 17.4 m i l l i o n s / m l , and the t o t a l sperm count per e j a c u l a t e was 53.6 ± 12.5 m i l l i o n s during the e a r l y phase of z i n c r e p l e t i o n ; the d i f f e r e n c e between these two values and those of the s t a b i l i z a t i o n p e r i o d were s i g n i f i c a n t . During the l a t e phase of z i n c r e p l e t i o n , the sperm c o n c e n t r a t i o n increased t o 207 ± 47.9 m i l l i o n s / m l , and the t o t a l sperm count per e j a c u l a t e increased t o 416.3 ± 102.9 m i l l i o n s (mean ± S.E.M.). These values a r e considered t o be w i t h i n the normal range. There was a s i g n i f i c a n t c o r r e l a t i o n between the sperm c o n c e n t r a t i o n and the t o t a l sperm count (4 = 0.90, ρ < 0.001). Changes i n sperm m o t i l i t y and morphology throughout the study p e r i o d were unremarkable. The b a s e l i n e serum testosterone decreased s i g n i f i c a n t l y during the e a r l y phase of z i n c r e p l e t i o n and returned t o normal l e v e l s during the l a t e phase of z i n c r e p l e t i o n . There was a s l i g h t d e c l i n e i n the maximal r i s e of serum testosterone a f t e r GnRH s t i m u l a t i o n during z i n c r e s t r i c t i o n and a more s i g n i f i c a n t d e c l i n e during the e a r l y phase of z i n c r e p l e t i o n , w i t h recovery to normal l e v e l during the l a t e phase of z i n c r e p l e t i o n . The changes of serum dihydrotestosterone were s i m i l a r t o those of serum t e s t o s t e r o n e , but s t a t i s t i c a l l y not s i g n i f i c a n t . Although the mean maximal r i s e i n LH a f t e r GnRH s t i m u l a t i o n was highest during e a r l y phase of z i n c r e p l e t i o n , the values were not s t a t i s t i c a l l y s i g n i f i c a n t as compared t o the other p e r i o d s . B a s e l i n e mean serum FSH was h i g h e s t during z i n c r e s t r i c t i o n p e r i o d , but again the values were not s t a t i s t i c a l l y s i g n i f i c a n t as compared t o the other p e r i o d s .
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
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Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
Discussion The purpose of our study was to produce only a m i l d z i n c d e f i c i e n t s t a t e i n human v o l u n t e e r s , inasmuch as severe d e f i ciency of z i n c may be l i f e - t h r e a t e n i n g , as seen i n a c r o d e r m a t i t i s enteropathica. Furthermore, i t i s the marginal d e f i c i e n c y of z i n c that appears to be prevalent and l i k e l y to be missed c l i n i c a l l y . Our data show that we were s u c c e s s f u l i n producing a m i l d z i n c - d e f i c i e n t s t a t e i n human v o l u n t e e r s by d i e t a r y means. Zinc c o n c e n t r a t i o n of plasma, e r y t h r o c y t e s , leukocytes, and u r i n e decreased when the d a i l y i n t a k e of z i n c was r e s t r i c t e d to 2.7 and 3. 5 mg i n human v o l u n t e e r s . Changes i n the a c t i v i t i e s of zinc-dependent enzymes such as a l k a l i n e phosphatase and ribonuclease i n the plasma and thymidine kinase i n the sponge connective t i s s u e during the z i n c r e s t r i c t i o n phase were a l s o supportive of the c o n c l u s i o n that a z i n c - d e f i c i e n t s t a t e was induced i n the vonunteers. A f t e r supplementation w i t h z i n c , a l l the above mentioned v a r i a b l e s returned toward normal l e v e l s . Inasmuch as a l l the other d i e t a r y c o n s t i t u e n t s remained the same except f o r z i n c i n t a k e , i t i s safe to presume that the above biochemical changes were due to z i n c d e f i c i e n c y . Our z i n c balance data show that we slowly depleted the body s t o r e of z i n c by approximately 500 mg during the study p e r i o d . Presuming the t o t a l body s t o r e of z i n c i n a 70-kg man to be around 1.5-2.5 g, 500 mg of z i n c d e p l e t i o n represents approximately 30% or l e s s of the body s t o r e . Thus the changes described here are those of a mild zinc-deficient state. One unexpected f i n d i n g was that the plasma ammonia l e v e l appeared to increase as a r e s u l t of z i n c d e f i c i e n c y . We have reported s i m i l a r f i n d i n g s i n z i n c - d e f i c i e n t r a t s (16). This may have important h e a l t h i m p l i c a t i o n s concerning z i n c d e f i c i e n c y i n man, because i n l i v e r disease hyperammonemia i s b e l i e v e d to a f f e c t the c e n t r a l nervous system adversely. In the f i r s t two subjects ( P a t i e n t s 1 and 2 ) , the weight l o s s may have been p a r t l y due to a m i l d r e s t r i c t i o n i n c a l o r i c i n t a k e i n a d d i t i o n to a d i e t a r y z i n c r e s t r i c t i o n . A l s o , that the s y n t h e t i c d i e t based on soy p r o t e i n may have been l i m i t i n g i n c e r t a i n amino a c i d s was considered. Because of these c o n s i d e r a t i o n s , i n the second group of subjects we increased the c a l o r i c i n t a k e , and i n the b a s e - l i n e period of 5 weeks before z i n c r e s t r i c t i o n , we supplemented these two s u b j e c t s w i t h 30 mg of z i n c d a i l y . The weight remained s t a b l e during the b a s e - l i n e p e r i o d but decreased when the z i n c was r e s t r i c t e d subsequently. A s l i g h t l y delayed and l e s s marked response to z i n c r e s t r i c t i o n i n the second group of subjects was r e l a t e d to the f a c t that they r e c e i v e d z i n c supplement (30 mg d a i l y ) f o r 5 weeks before r e s t r i c t i o n , which may have b u i l t up the body s t o r e of z i n c ; furthermore, they received 3.5 mg of d i e t a r y z i n c d a i l y i n s t e a d of 2.7 mg as was the case w i t h the f i r s t two s u b j e c t s . The amino a c i d content of the experimental d i e t was adequate according to
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Downloaded by UMEA UNIV on October 6, 2014 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0210.ch001
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the recommended d i e t a r y allowances, Food and N u t r i t i o n Board, N a t i o n a l Research C o u n c i l , N a t i o n a l Academy of Sciences. Thus, the changes observed w i t h respect to weight i n our experiments were r e l a t e d to a d i e t a r y z i n c r e s t r i c t i o n . Our data suggest that the z i n c - d e f i c i e n t s t a t e may have l e d to hypercatabolism of f a t i n our s u b j e c t s . This i s suggested by an increased f a t l o s s and normal a b s o r p t i o n of f a t during the z i n c r e s t r i c t i o n phase. In experimental animals an increase i n f r e e f a t t y acids has been observed as a r e s u l t of z i n c d e f i c i e n c y (17). Indeed, more s t u d i e s are required i n human subjects to document increased f a t catabolism due to z i n c r e s t r i c t i o n . D i r e c t measurement of DNA and p r o t e i n synthesis i n e x p e r i m e n t a l animals suggest that i n z i n c d e f i c i e n c y p r o t e i n synthesis i s adversely a f f e c t e d ( 1 , 2). Our own data on human subjects i n these experiments show that the t o t a l p r o t e i n , t o t a l c o l l a g e n , and RNA-DNA r a t i o increased as a r e s u l t of z i n c supplementation. The a c t i v i t y of deoxythymidine kinase was not measurable during the z i n c r e s t r i c t i o n phase but became 70% of normal l e v e l a f t e r supplementation w i t h z i n c f o r 3 months. S i m i l a r data have been published f o r experimental animals. Thus, our data show that deoxythymidine kinase i n human subjects a l s o i s a zinc-dependent enzyme, and an adverse e f f e c t of z i n c d e f i c i e n c y on t h i s enzyme may be r e s p o n s i b l e f o r decreased p r o t e i n s y n t h e s i s . Our s t u d i e s do not r u l e out an adverse e f f e c t of z i n c d e f i c i e n c y on p r o t e i n c a t a b o l i s m . Further studies are r e q u i r e d to e s t a b l i s h the e f f e c t of z i n c r e s t r i c t i o n on p r o t e i n catabolism. Our data i n d i c a t e that plasma a l k a l i n e phosphatase, plasma l a c t i c dehydrogenase, and deoxythymidine kinase i n sponge connect i v e t i s s u e i n human subjects are zinc-dependent enzymes, i n a s much as changes i n t h e i r a c t i v i t i e s were r e l a t e d to only one d i e t a r y m a n i p u l a t i o n , namely, z i n c i n t a k e . Changes i n the a c t i v i t i e s of plasma r i b o n u c l e a s e a l s o appear to be r e l a t e d to z i n c i n t a k e under the c o n d i t i o n s of our experiments. Other t r a c e elements may a l s o i n h i b i t i t s a c t i v i t y i n v i t r o , but the e f f e c t of z i n c i s more pronounced. Previous i n v e s t i g a t i o n s have r e l a t e d changes i n the a c t i v i t i e s of plasma r i b o n u c l e a s e to prot e i n intake (18); i n these s t u d i e s , changes i n p r o t e i n i n t a k e may have been associated w i t h changes i n plasma trace elements, thus accounting f o r a l t e r a t i o n s i n r i b o n u c l e a s e a c t i v i t y . The measurement of the a c t i v i t i e s of these plasma enzymes may be h e l p f u l i n c o r r e l a t i n g uncomplicated z i n c status i n humans, p a r t i c u l a r l y i f the changes are observed a f t e r z i n c supplementation f o r a short period of time. Changes i n the plasma z i n c c o n c e n t r a t i o n were observed w i t h i n 4 to 6 weeks and c o r r e l a t e d w i t h the s e v e r i t y of d i e t a r y z i n c r e s t r i c t i o n . Thus, plasma z i n c may be very u s e f u l i n assessment of z i n c status i n humans provided i n f e c t i o n s , myocard i a l i n f a r c t i o n , i n t r a v a s c u l a r hemolysis, and acute s t r e s s are r u l e d out (2). As a r e s u l t of i n f e c t i o n s , myocardial i n f a r c t i o n , and acute s t r e s s , z i n c from the plasma compartment may
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
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NUTRITIONAL BIOAVAILABILITY OF
ZINC
r e d i s t r i b u t e to other t i s s u e s , thus making an assessment of z i n c s t a t u s i n the body a d i f f i c u l t task. I n t r a v a s c u l a r hemolysis would a l s o s p u r i o u s l y increase the plasma z i n c l e v e l inasmuch as the c o n c e n t r a t i o n of z i n c i n the erythrocytes i s much higher than i n the plasma. Changes i n the e r y t h r o c y t e z i n c c o n c e n t r a t i o n were slow to appear as expected; on the other hand, changes i n the leukocyte z i n c c o n c e n t r a t i o n appeared more s e n s i t i v e to changes i n z i n c i n t a k e . U r i n a r y e x c r e t i o n of z i n c decreased as a r e s u l t of d i e t a r y z i n c r e s t r i c t i o n , suggesting that r e n a l c o n s e r v a t i o n of z i n c may be important f o r the homeostatic c o n t r o l mechanism i n man. Thus, measuring z i n c c o n c e n t r a t i o n i n a 24-h u r i n e t e s t may be of a d d i t i o n a l h e l p i n diagnosing z i n c d e f i c i e n c y provided c i r r h o s i s of the l i v e r , s i c k l e c e l l d i s e a s e , and chronic r e n a l diseases are r u l e d out. These c o n d i t i o n s are known to have h y p e r z i n c u r i a and a s s o c i a t e d z i n c d e f i c i e n c y . Our data a l s o i n d i c a t e that d u r i n g the z i n c - d e f i c i e n t s t a t e the subjects were i n a more p o s i t i v e balance f o r z i n c . This would suggest that perhaps a t e s t based on o r a l challenge of z i n c and subsequent plasma z i n c measurement may be able to d i s t i n g u i s h between the z i n c - s u f f i c i e n t and the z i n c - d e f i c i e n t s t a t e i n human s u b j e c t s . A study to t e s t t h i s p o s s i b i l i t y i s c u r r e n t l y underway i n our l a b o r a t o r y . In our study, m i l d z i n c d e f i c i e n c y as induced by d i e t a r y z i n c r e s t r i c t i o n has shown d e f i n i t e e f f e c t on gonadal f u n c t i o n . The sperm count was s u s c e p t i b l e to d i e t a r y r e s t r i c t i o n of z i n c . Although there was only a s l i g h t decrease i n the sperm count d u r i n g z i n c r e s t r i c t i o n , o l i g o s p e r m i a became s i g n i f i c a n t during the e a r l y phase of z i n c r e p l e t i o n . The nature of the delayed e f f e c t of z i n c d e f i c i e n c y on sperm count i s not w e l l understood. However, an explanatory hypothesis can be drawn on the b a s i s of other s t u d i e s . The developmental progression of spermatogenesis, from the o r i g i n of spermatozoa i n the germinal e p i t h e l i u m to mature spermatozoa, i s a prolonged process. The d u r a t i o n of human spermatogenesis i s 74 ± 4.5 days (19). Therefore, an i n s u l t a f f e c t i n g the germinal c e l l s may not be evident u n t i l s e v e r a l months l a t e r . The e f f e c t of c e r t a i n therapeutic drugs on spermatogenesis i s an example. In p a t i e n t s t r e a t e d w i t h cyclophosphamide, oligospermia or azoospermia occurred s e v e r a l months a f t e r the treatment was s t a r t e d (20-23). S i m i l a r l y , spermatogenesis returned to normal 15 to 49 months a f t e r cyclophosphamide therapy was stopped. I t i s not s u r p r i s i n g t h e r e f o r e f o r an i n s i d i o u s i n s u l t to germinal c e l l s , such as z i n c d e f i c i e n c y , to be manifested a f t e r such a long period of time. I f the temporal r e l a t i o n s h i p between the onset of i n j u r y to germinal e p i t h e l i u m and the sperm count i s t r u e , one would expect an equal period of time to be required f o r the recovery of spermatogenesis, once the ensuing d i s o r d e r has been c o r r e c t e d . In our s u b j e c t s , the recovery of o l i g o spermia occurred i n s i m i l a r f a s h i o n . The sperm count returned
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
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1.
PRASAD
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to the b a s e l i n e l e v e l a f t e r s e v e r a l months of z i n c supplementa tion. The long d u r a t i o n of human spermatogenesis, however, pro v i d e s only a p a r t i a l explanation f o r the e f f e c t s of z i n c on gonadal f u n c t i o n , inasmuch as we a l s o observed a s i m i l a r delayed e f f e c t of d i e t a r y z i n c r e s t r i c t i o n on plasma testosterone l e v e l s i n our s u b j e c t s . Another c o n t r i b u t o r y f a c t o r f o r the l a c k of synchronism i n t e s t i c u l a r hypofunction w i t h the periods of z i n c r e s t r i c t i o n and z i n c r e p l e t i o n i s perhaps the design of our study. The slow i n d u c t i o n of z i n c d e f i c i e n c y by d i e t a r y means, w i t h i t s n u t r i t i o n a l and metabolic consequences, r e q u i r e d a r e l a t i v e l y longer period of time t o be r e f l e c t e d c l i n i c a l l y on the t e s t i c u l a r f u n c t i o n . Body s t o r e s of z i n c were very slowly de p l e t e d and l a t e r were slowly r e p l e t e d w i t h z i n c supplementation. The dosage of supplemental z i n c i n these subjects was w i t h i n a p h y s i o l o g i c r a t h e r than a t h e r a p e u t i c range. I t i s evident from our data that r e p l e t i o n of body z i n c s t o r e was not accomplished u n t i l the l a t e phase of z i n c r e p l e t i o n which extended up t o 12 months, and t h i s c o r r e l a t e d w e l l w i t h the observed e f f e c t s on t e s t i c u l a r f u n c t i o n i n our study. Acknowledgement Supported i n part by S i c k l e C e l l Center Grant from N a t i o n a l Heart, Lung and Blood I n s t i t u t e , NIH, and USDA Competitive Re search Grant. L i t e r a t u r e Cited
1. Prasad, A.S. "Trace Elements in Human Health and Disease"; Academic Press, New York, New York, 1976, Chapter 1. 2. Prasad, A.S. "Trace Elements and Iron in Human Metabolism"; Plenum Publishing, New York, New York, 1978, 251. 3. Abassi, A.A.; Prasad, A.S.; Ortega, J.; Congco, E.; Oberleas, D. Ann. Intern. Med. 1976, 85, 601. 4. Lei, K.Y.; Abbasi, Α.; Prasad, A.S. Am. J. Physiol. 1976, 230, 1730. 5. Hartoma, T.R.; Nahoul, K.; Netter, A. Lancet 1977, 2, 1125. 6. Prasad, A.S.; Oberleas, D.; Halsted, J.A. J. Lab. Clin. Med. 1965, 66, 508. 7. Concon, J.M.; Soltess, D. Anal. Biochem. 1973, 53, 35. 8. Bauer, J.D.; Toro, G.; Ackerman, P.G. "Bray's Clinical Laboratory Methods," 6th ed., C.V. Mosby Co., St. Louis, Missouri, 1962, 333. 9. Rothstein, G.; Bishop, C.R.; Ashenbrucker, H.E. Blood, 1971, 38, 302. 10. Bergmeyer, H.; Bernt, E.; Hess, B. "Methods of Enzymatic Analysis"; Bergmeyer, H., Ed.; Academic Press, New York, New York, 1963, 736.
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
14 11. 12. 13. 14. 15. 16.
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17. 18. 19. 20. 21. 22. 23.
NUTRITIONAL BIOAVAILABILITY OF ZINC
Sekine, H.; Nakano, E,; Sakaguchi, K. Biochim. Biophy. Acta 1969, 174, 202. Prasad, A.S.; Oberleas, D. J. Lab. Clin. Med. 1973, 82, 461. Fernandez-Madrid, F.; Prasad, A.S.; Oberleas, D. J. Lab. Clin. Med. 1973, 82, 951. Prasad, A.S.; Rabbani, P.; Abbasi, Α.; Bowersox, E.; Fox, M.R.S. Ann. Int. Med., 1978, 89, 483. Abbasi, A.A.; Prasad, A.S.; Rabbani, P.; DuMouchelle, E. J. Lab. Clin. Med. 1980, 96(3), 544. Rabbani, P.; Prasad, A.S. Am. J. Physiol. 1978, 235(2), E203. Underwood, E.J. "Trace Elements in Human and Animal Nutri tion"; Underwood, E.J., Ed., Academic Press, New York, New York, 1977, 196. Albanese, A.A.; Orto, L.A. "Modern Nutrition in Health and Disease," 5th ed.; Goodhart, R.S.; Shils, M.E. Eds.; Lea and Febiger, Philadelphia, Penn., 1973, 28. Heller, C.H.; Clermont, Y. Rec. Prog. Horm. Res. 1964, 20, 545. Kumar, R.; Biggard, J.D.; McEnvoy, J.; McGrown, M.G. Lancet 1972, 1, 1212. Qureshi, M.S.; Goldsmith, H.J.; Pennington, J.H.; Cox, P.E. Lancet 1972, 2, 1290. Fairley, K.F.; Barnie, J.U.; Johnson, W. Lancet 1972, 1, 568. Buchanan, J.D.; Fairley, K.F.; Barnie, J.U. Lancet 1975, 2, 156.
RECEIVED September 29,
1982
In Nutritional Bioavailability of Zinc; Inglett, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.