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The pig as a biomedical model to study human protein calorie malnutrition Thacker, Philip Alfred 1978

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THE PIG AS A BIOMEDICAL MODEL TO STUDY HUMAN PROTEIN CALORIE MALNUTRITION by PHILIP ALFRED THACKER B.Sc. (Agr.), U n i v e r s i t y of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1977 O ) P h i l i p A l f r e d Thacker, 1977 MASTER OF SCIENCE i n In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, T agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my department or by his re p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permi ss ion. Department of Animal Science, The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, B.C., CANADA. Date: 5.Wit ABSTRACT Two experiments were.undertaken to eya1uate the:baby pig as a biomedical model with which to study P r o t e i n - C a l o r i e m a l n u t r i t i o n . In the f i r s t experiment, 32'. Y'orksh i re . and Yorksh i re X Landrace pigs weaned at 21 days were fed e i t h e r an 18% o r k % p r o t e i n r a t i o n . Blood samples were taken biweekly from the a n t e r i o r vena cava and the serum samples analyzed f o r calcium, phosphorus, glucose, c h o l e s t e r o l , l a c t i c dehydrogenase, glutamic o x a l o a c e t i c transaminase, amylase, a 1ka1ine phosphatase, t o t a l p r o t e i n , albumin and blood urea nitrogen. S i g n i f i c a n t (p — .01) treatment e f f e c t s were observed f o r t o t a l p r o t e i n , albumin, amylase, a l k a l i n e phosphatase, l a c t i c dehydrogenase, c h o l e s t e r o l , calcium and phosphorus. In the second experiment, kO Y o r k s h i r e and Yor k s h i r e X Landrace pigs weaned at 28 days were fed r a t i o n s c o n t a i n i n g 18%, 10%, 8%, 6%, and k% p r o t e i n . Blood samples were again taken biweekly and serum samples were analyzed f o r the same parameters as in t r i a l one. In a d d i t i o n , serum copper, i r o n , magnesium, and z i n c were measured. The l i v e r s of any animals which died on the low p r o t e i n d i e t s , were f a t e x t r a c t e d , and the l e v e l of f a t compared to that obtained from l i v e r s of animals k i l l e d as s u c k l i n g pigs at a slaughter p l a n t . Total body water was determined on three animals on the 18% r a t i o n and three on the h% r a t i o n u t i l i z i n g t r i t i a t e d water as a t r a c e r . Total p r o t e i n , albumin, amylase, l a c t i c dehydrogenase, calcium, phosphorus, copper, iron and magnesium c o r r e l a t e d well with d i e t a r y p r o t e i n intake. S i g n i f i c a n t treatment . e f f e c t s were observed f o r t o t a l body water and f a t content of the l i v e r . An attempt was made to f i n d a biochemical - i i parameter which might be used in diagnosing developing protein c a l o r i e malnutrition. The results of the study would indicate.that serum phosphate and amylase are the most sensitive parameters to dietary protein intake. Not every lesion or biochemical serum change occurring in man was reproduced in the present study. Nevertheless, cha r a c t e r i s t i c symptoms such as the development of fat t y l i v e r , growth retardation, abnormal hair texture, hypoalbuminemia, and apathy were reproduced in'the protein deficient swine. The baby pig would therefore appear to be a good model for the study of protein-calorie ma 1 n u t r i t i o n . TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v i i LIST OF APPENDIX TABLES i x ACKNOWLEDGEMENTS . x i INTRODUCTION 1 LITERATURE REVIEW '. > A. BIOCHEMICAL ASPECTS OF PCM IN MALNOURISHED INFANTS h 1. P r o t e i n Metabolism in PCM ^ a. Total Proteins b. Albumin ' 5 c. Blood Urea Nitrogen 7 d. Amy 1 ase 8 e. A l k a l i n e Phosphatase 9 f. Serum Glutamic Oxaloacetic Transaminase 10 g. L a c t i c Dehydrogenase 11 2. Fat Metabolism in PCM. . . 12 a. General 12 b. Serum Cho l e s t e r o l 13 c. Fatty L i v e r 15 3. Carbohydrate Metabolism | N P C M 17 a. Blood Glucose. 17 h. Water and E l e c t r o l y t e Metabolism in P C M . . 19 a. Edema 19 i v -b. Serum Calcium 2h c. Serum Copper 25 d. Serum Iron. 27 e. Serum Magnesium: 29 f. Serum Phosphorus; 33 g. Serum Zinc . . . . 33 B. BIOCHEMICAL ASPECTS OF PCM IN SWINE . . 36 1. P r o t e i n Metabolism. 36 a. Serum Proteins 36 b. Serum Amino Acids. 36 c. Serum Enzymes. 37 d. Antibodies . 37 2. Water and Elec.trolyte Metabol ism in PCM. 37 a. Edema . .37 b. E l e c t r o l y t e s . 38 3. Fat Metabolism in PCH . 38 a. Fatty L i v e r 38 b. Serum Cholesterol .39 k. Hormones in PCM 39 a. General 39 5. Miscellaneous Aspects of PCM i n Swine kO a. Organ Weights . hO b. Behavior .40 c. Hematology. kO ' d. R e h a b i l i t a t i o n 40 - v -EXPERIMENT I kl A. MATERIALS AND METHODS. kl a. Obj e c t i v e s kl b. -Exper i m e n t a l P r o c e d u r e s kl c. Feed A n a l y s i s k3 d. B i o c h e m i c a l A n a l y s i s kS e. S t a t i s t i c a l A n a l y s i s o f D a t a . kS B. RESULTS • kS a. G e n e r a l kS b. P r o t e i n M e t a b o l i s m . 50 c. Fat M e t a b o l i s m 50 d. C a r b o h y d r a t e M e t a b o l i s m • • .50 e. M i n e r a l M e t a b o l i s m 65 C. DISCUSSION 65 a. P r o t e i n M e t a b o l i s m 65 b. F a t Metabol ism 70 c. C a r b o h y d r a t e M e t a b o l i s m 71 d. M i n e r a l M e t a b o l i s m 71 EXPERIMENT I I . . . Ik A. MATERIALS AND METHODS . . Jk a. O b j e c t i ves Ik b. E x p e r i m e n t a l P r o c e d u r e s Ik c. A n a l y t i c a l Methods. 75 d. B i o c h e m i c a l A n a l y s i s 75 e. S t a t i s t i c a l . A n a l y s i s •. • - 7 7 - v i -B. RESULTS . . . • 81 a. General 81 b. P r o t e i n Metabolism 81 c. Fat Metabol ism. 83 d. Carbohydrate Metabolism . 83 e. Water and E l e c t r o l y t e Metabolism 84 C. DISCUSSION 103 a. P r o t e i n Metabolism 103 b. Fat Metabol ism 105 c. Carbohydrate Metabolism 106 d. Water and E l e c t r o l y t e Metabolism 106 CONCLUSIONS 112 BIBLIOGRAPHY. 115 APPENDIX TABLES 131 - v i i -LIST OF TABLES Table Page 1 Composition of Experimental Diets ( T r i a l 1). 46 2 Proximate A n a l y s i s of Experimental Rations ( T r i a l 1) kl 3 Mineral Ana 1 ys is of Rat ions ( T r i a l 1). . . 48 4 Body Weights (kg) of Swine Fed Varying Levels of P r o t e i n ( T r i a l 1). . . . . . 52 5 Weekly Feed Consumption (kg) in T r i a l 1 53 6 Serum Total P r o t e i n (g/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1). . . , 54 7 Serum Albumin Levels (g/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 55 8 Blood Urea Nitrogen Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 56 9 Serum Amylase Levels (Somogyi Units/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 57 10 Serum A l k a l i n e Phosphatase Levels (m I.U./ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 58 11 Serum Glutamic Oxaloacetic Transaminase Levels (Karmen Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1). .59 12 Serum L a c t i c Dehydrogenase Levels (Wroblewski Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 60 13 Serum Cholesterol Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 61 14 Serum Glucose Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 62 15 Serum Calcium Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) • 63 16 Serum Phosphate Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1) 64 17 Composition of Experimental Diets ( T r i a l 2) 78 18 Proximate A n a l y s i s of Experimental Rations ( T r i a l 2) 79 19 Mineral A n a l y s i s of Rations ( T r i a l 2) 80 - v i i i -20 Weekly Feed Consumption (kg) in T r i a l 2 , 86 21 Body Weights (kg) of Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). . 87 22 Serum Total P r o t e i n (g/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) . . . . 88 23 Serum Albumin Levels (g/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2 ) . . . . . . . 89 24 Blood Urea Nitrogen Levels (mg/100 ml) in Swine Fed Varying P r o t e i n Levels ( T r i a l 2) . . 90 25 Serum Amylase Levels (Somogyi Units /100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2)' 91 26 Serum Aika 1i ne Phosphatase (m I.U./ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). . . 92 27 Serum Glutamic Oxaloacetic Transaminase (Karmen Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). 93 28 Serum L a c t i c Dehydrogenase (Wroblewski Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) 94 29 Serum Cholesterol Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) . . .95 30 Serum Glucose Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) • 96 31 Serum Calcium Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) 97 32 Serum Copper Levels (ug/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) . . 98 33 Serum Iron Levels (ug/100 ml) i n Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) • • • 99 34 Serum Magnesium Levels (mg/100 ml) i n Swine Fed Varying Levels of P r o t e i n ( T r i a l 2) . 100 35 Serum Phosphate Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2), . . . . .101 36 Serum Zinc Levels (ug/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). 102 - ix -LIST OF APPENDIX TABLES Tab!e Page 1A Analys is of Var iance f o r Body Weight in T r i a l 1 132 2A A n a l y s i s of Var iance f o r Serum Total P r o t e i n in T r i a l 1. . . • -133 3A A n a l y s i s of Va r iance f o r Serum Albumin in T r i a l 1. . .134 4A , Analys i s of Var iance f o r Blood Urea Nitrogen in T r i a l 1 . . . . 135 5A A n a l y s i s of Var iance f o r Serum Amylase in T r i a l 1 . .136 6A A n a l y s i s i n Tra i 1 of 1 Var iance f o r Serum A l k a l i n e Phosphatase . • 137 7A A n a l y s i s Transam i i of Var iance nase in T r i a l f o r 1 Serum Glutamic Oxaloacetic 8A Analys i s of Var iance f o r L a c t i c Dehydrogenase in T r i a l 1. . • • 139 9A Ana 1ys i s of Variance fo r Serum Cho l e s t e r o l in T r i a l 1. . . . . .140 10A A n a l y s i s of Variance f o r Serum Glucose in T r i a l 1 141 11A A n a l y s i s of Var iance fo r Serum Calcium in T r i a l 1 142 12A Analys i s of Variance f o r Serum Phosphate in T r i a l 1 . . . . . . 143 13A Ana 1ys i s of Va r i ance fo r Body Weight in T r i a l 2 . . . . . . . . 144 14A Analys i s of Va r iance f o r Serum Total Proteins in T r i a l 2 . . . .145 15A Ana l y s i s of Va r iance fo r Serum Albumin in T r i a l 2 , .146 16A Ana 1ys i s of Var iance fo r Blood Urea Nitrogen in T r i a l 2 . . . . 147 17A Ana 1ys i s of Variance f o r Serum Amylase in T r i a l 2 148 18A A n a l y s i s T r i a l 2 of Variance fo r Serum A l k a l i n e Phosphatase in 19A A n a l y s i s of Variance Transaminase in T r i a l f o r 2 Serum Glutamic Oxaloacetic . . 150 2 OA Analys i s in T r i a l of 2 Var iance f o r Serum L a c t i c Dehydrogenase . .151 21A Analys i s of Variance fo r Serum Cholesterol in T r i a l 2 , . . . . 152 22A Ana 1ys i s of Var iance f o r Serum Glucose in T r i a l 2 . . . . . • . 153 23A Ana 1ys i s of Var iance f o r . . .154 24A An a l y s i s of Var iance fo r . . .155 25A Ana l y s i s of Var iance f o r . . 156 26A. A n a l y s i s of Var iance fo r . . 157 27A Analys i s of Var iance f o r . . 158 28A Analys i s of Variance fo r . . 159 29A Analys is of Var iance f o r Total Body Water in T r i a l 2 . . . 160 3 OA Ana 1ys i s of Var iance f o r 161 - x i -ACKNOWLEDGEMENTS The author wishes to express h i s g r a t i t u d e to Dr. H.S. Saben, who proposed t h i s study and provided the f i n a n c i a l a s s i s t a n c e necessary to complete i t . Thanks are a l s o extended to the members of my committee and e s p e c i a l l y Dr.'J.A. S h e l f o r d a n d Dr. D.B. Bragg f o r t h e i r help in the preparation of t h i s manuscript. The author a l s o wishes to thank Mrs. Mabel S t r i k e r who spent considerable time in a s s i s t i n g with the s t a t i s t i c a l a n a l y s i s . F i n a l l y , the author wishes to thank the graduate students and support s t a f f of the Department of Animal Science f o r t h e i r many suggestions and encouragement supplied at c o f f e e time. - 1 -INTRODUCTION P r o t e i n - C a l o r i e m a l n u t r i t i o n (PCM) is one of the most important p u b l i c health problems in underdeveloped c o u n t r i e s . PCM i s l a r g e l y responsible f o r the f a c t that in many areas of the world up to one-half the c h i l d r e n born do not s u r v i v e to the age of f i v e years. Death rates in these c h i l d r e n may be 20-50 times higher than the rate in r i c h and prosperous communities in North America and Europe. P r o t e i n - C a l o r i e m a l n u t r i t i o n , describes a spectrum of c l i n i c a l d i s o r d e r s . At one end, marasmus is due to a continued r e s t r i c t i o n of both d i e t a r y energy and p r o t e i n . At the other end, i s kwashiorkor, due to a q u a n t i t a t i v e and q u a l i t a t i v e d e f i c i e n c y of p r o t e i n , but in which energy may be adequate. These two syndromes are the extremes. Between them are forms in which the c l i n i c a l features are due to varying combinations of pro t e i n and energy d e f i c i e n c y together with d e f i c i e n c i e s of vitamins and minerals and with associated i n f e c t i o n s . The study of disease in the human has fr e q u e n t l y been advanced by the development of an experimental model system in animals which mimicks the c o n d i t i o n in the human. Attempts at reproducing PCM in research animals are numerous. To-date, the m a j o r i t y of research p r o j e c t s have u t i l i z e d the monkey (Coward and Whitehead, 1972; Deo et a_L, 1965), the dog (Heard, 1968; Stewart, 1968) and the rat (Ki rsch _et a],. , 1968a; Anthony and Edozien, 1975)- Considerable d i f f e r e n c e s of opinion have been voiced concerning the adequacy of these animal models and e x t r a p o l a t i o n of r e s u l t s from these sources to man. The most common complaints against these models are that they f a i l to develop severe edema, extensive s k i n and h a i r changes and - 2 -s p r u e - l i k e bowel changes as seen in man (Madi et a K , 1970; Blackburn and V i n i j c h a i k u l , I969). During the l a s t decade, many workers have elaborated on the p o t e n t i a l of swine as an ideal experimental animal. Glauser (1-966) has reviewed some of the advantages of pigs as experimental animals. Most importantly, the pig has many p h y s i o l o g i c a l analogies with man. K i r s c h £ 1 a_L ,(1968b); Bustad (1966) and Douglas (1972) have emphasized the s i m i l a r i t i e s between swine and man. P i g l e t l i t t e r s i z e may run between 12-16 in number enabling the use of s t a t i s t i c a l l y s i g n i f i c a n t numbers of p i g l e t s from the same l i t t e r . Considering that the same sow has two l i t t e r s per year and sometimes three, a large number of subjects of the same parentage are a v a i l a b l e f o r study during a year. It i s therefore p o s s i b l e to run e n t i r e experiments from the progeny of one sow. This p r a c t i c e leads to less v a r i a b i l i t y in the environmental, g e s t a t i o n a l and other genetic f a c t o r s a f f e c t i n g experimental parameters than occurs with other species. Swine r e a d i l y accept experimental d i e t s at an e a r l y age which reduces the time spent in feeding and handling animals. Other large animals such as the dog or monkey g e n e r a l l y refuse experimental d i e t s and must be force fed. In animal experimentation, the cost of buying experimental animals is s i g n i f i c a n t . In comparison with dogs or monkeys, p i g l e t s are r e l a t i v e l y inexpensive. One t h e o r e t i c a l l i m i t a t i o n to the use of the pig is that growth is r e l a t i v e l y more rapid than in man who has a very long j u v e n i l e period between weaning and puberty (Evans and M i l l e r , I968). Despite t h i s - 3 -c o n s i d e r a t i o n , i t is believed that the pig i s a more ideal animal model than others f o r research in PCM. In human PCM, various biochemical parameters are a l t e r e d as a consequence of the i n s u f f i c i e n t supply of energy and amino acids to the t i s s u e s . In order to evaluate the pig as a model to study PCM, the bi o -chemical changes o c c u r r i n g i n the p r o t e i n d e f i c i e n t p i g w i l l be compared to those o c c u r r i n g in malnourished c h i l d r e n as reported in the 1 i t e r a t u r e . If the pig proves to be a s u i t a b l e model, r e s u l t s might be obtained u t i l i z i n g t h i s model to f u r t h e r the knowledge of the a e t i o l o g y of PCM and u l t i m a t e l y enable s u i t a b l y pretreated animals to be used f o r t r i a l s on the examination of t h e r a p u t i c agents. The prognosis of PCM i s o f t e n d i f f i c u l t to assess on c l i n i c a l grounds alone, and various attempts have been made to f i n d an accurate biochemical index of s e v e r i t y or response to treatment (Barclay, 1 9 7 3 ; Stephens, 1974; MacFarlane, 1969; B r a d f i e l d , 1973; Whitehead and Dean, 1964). In a d d i t i o n to e v a l u a t i n g the pig as a model to study PCM, an attempt w i l l be made to i n v e s t i g a t e a number of biochemical v a r i a b l e s which may r e f l e c t s e v e r i t y and response to treatment of subjects with PCM. - 4 -LITERATURE REVIEW A. BIOCHEMICAL ASPECTS OF PCM IN MALNOURISHED INFANTS 1. P r o t e i n Metabolism in PCM a. Tota1 Prote i ns One of the most c o n s i s t e n t serum biochemical a l t e r a t i o n s observed in PCM i s lowered t o t a l p r o t e i n s . Ismadi et aj.. (1971) reported values of 6.73 + .17 g/100 ml in well nourished c h i l d r e n , compared with a mean value of 3-43 + .50 g/100 ml in malnourished c h i l d r e n . Several workers have considered t o t a l p r oteins to be a r e l i a b l e i n d i c a t o r of developing PCM (Baertl et aj.. , 1974; Haddad and Harfouche, 1971) but t h i s i s not a commonly accepted view. The lowering of t o t a l p r o t e i n s i s due to a great extent to the reduction of the albumin f r a c t i o n (Scrimshaw and Behar, 1961). This reduction in albumin i s p a r t i a l l y obscured by changes in the g l o b u l i n f r a c t i o n (Scrimshaw and Behar, 1961). During the e a r l y phases of m a l n u t r i t i o n , there is a r e c i p r o c a l r e l a t i o n s h i p between the albumin and g l o b u l i n con-c e n t r a t i o n s , w i t h decreasing albumin and incre a s i n g g l o b u l i n s (Coward et a l . , 1972). A n a l y s i s of the i n d i v i d u a l g l o b u l i n components show that the r i s e in g l o b u l i n concentration is due mainly to increases in gamma g l o b u l i n , and to a less e r extent alpha g l o b u l i n s . The r i s e in alpha g l o b u l i n i s thought to be r e l a t e d to c e l l u l a r d e s t r u c t i o n , w h i l e the high l e v e l s of gamma g l o b u l i n are believed to be re l a t e d to i n f e c t i o n ( V i t e r i _et.aj.., 1964). - 5 -During the l a t e r p a t h o l o g i c a l stages of PCM, t o t a l g l o b u l i n concentrations f a l l mainly because of a reduction in the alpha and beta g l o b u l i n f r a c t i o n (Coward et aj ., 1972). It i s i n t e r e s t i n g to note that the immuno g.lobuj'ins are the only group of proteins in plasma normally synthesized o u t s i d e the l i v e r . Therefore, a serious derangement in the f u n c t i o n a l c a p a c i t y of the l i v e r might e x p l a i n the f i n a l f a l l s in albumin and alpha and beta g l o b u l i n s (Coward et aj_. , 1972). The increase in gamma g l o b u l i n s suggests the p r e f e r e n t i a l use of amino acids f o r the synthesis of these proteins when i n f e c t i o n i s present and t h i s demand f o r amino acids may a c t u a l l y aggrevate the p r o t e i n d e f i c i e n c y (Cohen and Hansen, 1962). b. Albumi n Hypoalbuminemia is a c o n s i s t e n t f i n d i n g in PCM. Ismadi et a l . (1971) observed albumin l e v e l s of 1.54 + .30 g/100 ml in 14 malnourished c h i l d r e n which was considerably lower than the mean 3-89 + .13 g/100 ml of the c o n t r o l group. Several workers have suggested that serum albumin l e v e l s are a good index with which to estimate the s e v e r i t y and prognosis of recovery from PCM (Wh i tehead et a k , 1973; Baertl e_t aj.., 1974). However, McFarlane (1970), showed that serum albumin l e v e l s were not a guide to the subsequent progress of the malnourished c h i l d . He observed no d i f f e r e n c e s in the serum albumin l e v e l s of c h i l d r e n who survived and those who died. Reeds and Laditan (1976) concluded that serum albumin concentrations are poor i n d i c a t o r s of a marginal reduction in n u t r i t i o n a l s t a t u s . - 6 -Wh i tehead _e_t aJL (1973) proposed that the concentrations of serum albumin could be used as an i n d i c a t o r of the development of edema. Their p r e l i m i n a r y observat ions indicated that 3 -0 g/100 ml was the c r i t i c a l serum albumin concentration as f a r as the appearance of e a r l y edematous signs was concerned. Approximately 75% of the t o t a l c o l l o i d osmotic pressure of plasma is normally derived from albumin, as a r e s u l t of i t s r e l a t i v e l y low molecular weight and high net charge (Searcy, 1969) . Albumin plays an important r o l e in the t r a n s p o r t a t i o n of metabolic products. Albumin is e s s e n t i a l f o r the transport of u n e s t e r i f i e d f a t t y a c i d s , hormones such as estrogens., testosterone and catecholamines, and anions such as calcium. As a r e s u l t of hypoalbuminemia in PCM, the transport of these products would be reduced, which could r e s u l t in metabolic disturbances throughout the body. T h e o r e t i c a l l y , the reduction in albumin l e v e l s in PCM could r e s u l t e i t h e r from a lower rate of synthesis or from an increased rate of catabolism. However, experimental evidence suggests that the c a t a b o l i c rate i s a c t u a l l y reduced in PCM. Nine c h i l d r e n with PCM and nine who had 131 recovered from m a l n u t r i t i o n were i n j e c t e d with albumin I and were then studied during consecutive periods in which the amount of d i e t a r y p r o t e i n was changed (James and Hay, 1968). Malnourished c h i l d r e n had s i g n i f i c a n t l y lower c a t a b o l i c rates of albumin than d i d the recovered c h i l d r e n on the same p r o t e i n intake. Growth hormone has been shown to produce a decrease in the c a t a b o l i c r a t e of albumin in p a t i e n t s with c i r r h o s i s (Gabuzda et a l . , 1963 ) . However, i t i s not presently known i f the elevated growth hormone l e v e l s o c c u r r i n g in malnourished c h i l d r e n are the cause of the lower c a t a b o l i c rate of albumin (Pimstone et a 1., 1966 ) . - 7 -James and Hay (1968), measured the rate of synthesis of albumin in malnourished c h i l d r e n . Their malnourished group had a s y n t h e t i c rate of 101 mg/kg/day which was s i g n i f i c a n t l y lower than the 148 mg/kg/day observed in t h e i r c o n t r o l group. It has been concluded that the diminution in the rate of synthesis of albumin i s caused by a reduction in the a v a i l a b i l i t y of amino acids (Rothschild et a j . , 1969). L i v e r s obtained from fasted r a b b i t s synthesized 18 + 1 mg of albumin. The a d d i t i o n of methionine, l y s i n e , l e u c i n e , v a l i n e , or threonine, at 10 umoles/ml f a i l e d to a l t e r albumin s y n t h e s i s . Tryptophan increased albumin synthesis 38~75%. The a d d i t i o n of is o l e u c i n e r e s u l t e d in an 89% increase in albumin s y n t h e s i s . When the donor r a b b i t s were fed, albumin synthesis averaged 33 mg and no increases were observed with the a d d i t i o n of tryptophan or i s o l e u c i n e . B a e r t l et a l . (1974) noted high c o r r e l a t i o n c o e f f i c i e n t s between tryptophan and the branched chain amino a c i d s , and serum albumin l e v e l s . c. Blood Urea Nitrogen Blood urea nitrogen has been reported to be low in PCM. Arroyave _et _aj. (1962) reported l e v e l s of 7.8 mg/100 ml in malnourished c h i l d r e n compared with a mean of 14.9 mg/100 ml in recovered c h i l d r e n . S i m i l a r reductions have been reported by Bjornesjo et, aj.. (1965), Kelman S i a J . (1972) and Edozien _e_£ .aj.. (i960). Van der Westhuysen ej; _a±. (1975) reported no d i f f e r e n c e s in blood urea nitrogen l e v e l s between c o n t r o l s and malnourished c h i l d r e n . Ammonia, a hi g h l y t o x i c agent, i s continuously generated in a l l t i s s u e s as an end product of p r o t e i n catabolism. Its accumulation i s prevented by the conversion of any ammonia not required f o r amino a c i d - 8 -synthesis i n t o urea. The q u a n t i t y and q u a l i t y of d i e t a r y p r o t e i n are important determinants of c i r c u l a t i n g l e v e l s of urea. Ingestion of large amounts of r e a d i l y absorbable p r o t e i n produce s u b s t a n t i a l increases in blood urea nitrogen. Low values of blood urea nitrogen are a r e f l e c t i o n of diminished deamination of amino a c i d s . An e l e v a t i o n in blood urea nitrogen i s most of t e n i n t e r p r e t e d as i n d i c a t i v e of p o s s i b l e renal d i s f u n c t i o n (Searcy, 1969)- In PCM, glomerular f i l t r a t i o n rate and renal plasma flow are reduced and there is evidence of impaired tubular f u n c t i o n as shown by an amino-aciduria, occasional renal phosphaturia and an i n a b i l i t y to excrete an a c i d load (Mann et a_L , 1972). Since the l i v e r i s one of the major s i t e s of urea s y n t h e s i s , the c e l l u l a r damage o c c u r r i n g as a r e s u l t of f a t t y i n f i l t r a t i o n could r e s u l t in lower c i r c u l a t i n g urea l e v e l s . However, the l i v e r has a great f u n c t i o n a l reserve and urea synthesis i s not l i k e l y to be c u r t a i l e d unless hepatic t i s s u e i s a c u t e l y damaged (Searcy, 1969)- In a d d i t i o n , urea formation may take place In other t i s s u e s than the l i v e r . Such mechanisms could t h e r e f o r e augment c i r c u l a t i n g urea l e v e l s . Urea i s h i g h l y water s o l u b l e and d i f f u s e s f r e e l y through a m a j o r i t y of c e l l membranes. As a r e s u l t of the increases in e x t r a c e l l u l a r body water due to the presence of edema, i t i s p o s s i b l e that c i r c u l a t i n g urea l e v e l s would be reduced. d. Amylase Serum amylase l e v e l s are reduced in PCM. Edozien (1961) measured the serum amylase l e v e l s in malnourished and healthy Nigerian - 9 -c h i l d r e n . Serum amylase values of malnourished c h i l d r e n were reduced to an average of 72.6% of the normal. The healthy group had a mean serum l e v e l of 157 somogyi units / 100 ml whi1e the ma 1nourished group had a mean of 114 somogyi units / 100 ml. S i m i l a r reductions have been reported by S r i k a n t i a et aj.. (1964) , Mukherjee and Werner (1954), and Dean and Schwartz (1953). In the body, amylase i s present in a number of organs and t i s s u e s . The greatest concentration i s present in the pancreas where the enzyme is synthesized by the a c i n a r c e l l s and then secreted into the i n t e s t i n a l t r a c t f o r the d i g e s t i o n of starches. The s a l i v a r y glands secrete a potent amylase to i n i t i a t e h y d r o l y s i s of starches while the food i s s t i l l in the mouth or esophagus. The decrease in serum amylase in PCM i s believed to i n d i c a t e pancreatic d i s f u n c t i o n ( V i t e r r et a]_. , 1964), since the major source of serum amylase i s the pancreas (Wiberg and Tuba, 1952). In a d d i t i o n to lowered serum amylase l e v e l s , Thompson and Trowel 1 (1952) showed lower concentrations of amylase in duodenal contents of malnourished c h i l d r e n . e. A l k a l i n e Phosphatase A l k a l i n e phosphatase values are low in PCM. Edozien (1961), noted that serum l e v e l s were reduced to 59-2% of the normal. Malnourished c h i l d r e n showed a mean value of 16 King-Armstrong units / 100 ml compared with a normal value of 27 King-Armstrong units / 100 ml. S i m i l a r reductions have been reported by Sandstead et .aj.• (1965), Schwartz (1956), and Scrimshaw e£ .al . (1955). A good c o r r e l a t i o n has been shown to e x i s t between the l e v e l s of a l k a l i n e phosphatase and albumin (Edozien, I96I). - 10 -A l k a l i n e phosphatase may be derived from a number of d i f f e r e n t organs such as l i v e r , bone, kidney, and i n t e s t i n e , but that found in the serum of young c h i l d r e n is mostly of bone o r i g i n (Waterlow and Stephen, 1969). A l k a l i n e phosphatase i s believed to be involved in the d i f f e r e n t i a t i o n of both o s t e o b l a s t s and chondroblasts, as well as in the synthesis of f i b r o u s p r o t e i n and the formation of preosseous bone matrix (Searcy, 1969). The e x c r e t i o n of hydroxyproline peptides i s lower in PCM (Waterlow and Stephen, 1969) and t h i s combined with the low serum l e v e l s of a l k a l i n e phosphatase would seem to r e f l e c t a decreased rate of formation and remodelling of the bone matrix in PCM. Rosenthal et; _§_].. (1952), observed higher concentrations of a l k a l i n e phosphatase in the l i v e r s of ad u l t rats fed d i e t s d e f i c i e n t in pr o t e i n . It has been suggested that the changes in l i v e r concentrations might be explained by the f a c t that the l i v e r normally disposes of a l k a l i n e phosphatase, and that t h i s f u n c t i o n i s impaired during p r o t e i n d e p l e t i o n (Schwartz, 1956). f. Serum Glutamic Oxaloacetic Transaminase Serum glutamic o x a l o a c e t i c transaminase (SGOT) l e v e l s have been shown to be moderately increased in PCM. Sandstead .et a j . (I965), obtained 36 sigma u n i t s in malnourished c h i l d r e n compared to a co n t r o l value of 23 sigma u n i t s . A s l i g h t e l e v a t i o n was a l s o reported by Zaki et a l . (1970), Baron (i960) and Edozien (196I) . Contrary to the above r e p o r t s , Smith (1962) estimated SGOT a c t i v i t y in 60 malnourished c h i l d r e n and showed - l i -no a l t e r a t i o n s which could be a t t r i b u t e d to the s t a t e of m a l n u t r i t i o n . The transaminases c o n s t i t u t e a group of enzymes which c a t a l y z e the i n t e r c o n v e r s i o n of amino acids and a 1pha-ketoacids by the tr a n s f e r of amino groups. SGOT c a t a l y z e s the t r a n s f e r of the amino group from glutamic a c i d to o x a l o a c e t i c a c i d . This provides a mechanism f o r r e d i s t r i b u t i n g nitrogen according to the p a r t i c u l a r needs of the organism. The greatest amount of GOT i s present in the l i v e r , followed by l e s s e r amounts in the heart muscle and s k e l e t a l muscle. A small amount is present in the kidney, pancreas, red blood c e l l s , b r a i n and s k i n . Under normal c o n d i t i o n s , only minute amounts of the enzyme are present in the serum (Searcy, 1969)- However, in c o n d i t i o n s in which there i s t i s s u e d e s t r u c t i o n , serum l e v e l s are increased. It i s thought, t h e r e f o r e , that elevated l e v e l s in malnourished chi1dren i n d i c a t e l i v e r damage (Waterlow, 1959)- Edozien (1961), suggested that small increases were due to muscle breakdown and la r g e r increases were due to l i v e r n e c r o s i s . g. Lact i c Dehyd rogenase L a c t i c dehydrogenase (LDH) l e v e l s are high in PCM and tend to decrease during p r o t e i n r e p l e t i o n . Zaki et aj_. (1970), showed LDH l e v e l s of 326.5 + 28.1 units/ml serum in malnourished i n f a n t s , compared with a l e v e l of 205.5 + 14.5 i n c o n t r o l s . Elevated l e v e l s were a l s o observed by Weimer et .al.. (1959) in p r o t e i n d e f i c i e n t rats and by Sandstead et a 1 . (1965) in Egyptian malnourished c h i l d r e n . LDH i s a hydrogen t r a n s f e r enzyme which c a t a l y z e s the ox i d a t i o n of L - l a c t a t e to pyruvate with the mediation of NAD as hydrogen - 12 -acceptor. Enzyme l e v e l s present in various t i s s u e s (Wroblewski-LaDue u n i t s / ml) are very high as compared to serum: l i v e r 260,000 units/gm; heart, 160,000 t o 240,000 u n i t s ; kidney, 250,000 to 300,000 u n i t s ; and s k e l e t a l muscle, 133,000 u n i t s . Thus, t i s s u e l e v e l s are about 1000-fold higher than those normally found in serum, and leakage of the enzyme from even a small mass of damaged t i s s u e can increasethe observed serum l e v e l (Kachmar, 1970). The major f a c t o r c o n t r i b u t i n g to the increase LDH l e v e l s in PCM is believed to be degeneration of somatic t i ssue with release of the enzyme in t o the c i r c u l a t i o n (Weimer et aj.. , 1959). It i s well e s t a b l i s h e d that s k e l e t a l muscle undergoes the g r e a t e s t loss in weight during d e p l e t i o n and contains large amounts of LDH. 2. Fat Metabolism in PCM a. Genera 1 Fat malabsorption i s o f t e n present in PCM. Gomez et a l . (1956), showed that the average absorption of f a t was 48% in malnourished inf a n t s on admission to h o s p i t a l . A f t e r s i x weeks of treatment, absorption rose to 79%. There are several p o s s i b l e reasons f o r t h i s decrease in f a t absorption. F i r s t l y , i t has been shown that pancreatic l i p a s e s e c r e t i o n i s diminished in malnourished c h i l d r e n (Thompson and Trowel 1, 1952). Secondly, i t has been reported that j e j u n a l b i l e s a l t s were deconjugated in eight out of twenty South A f r i c a n cases of PCM (Redmond et a±., 1972). This deconjugation would decrease fragmentation of the f a t g l o b u l e s . - 13 -F i n a l l y , mucosal atrophy could r e s u l t in decreased f a t absorption. A serious r e s u l t of t h i s f a t malabsorption would seem to be an impairment of absorption of f a t s o l u b l e vitamins such as vitamin A. (Konno e£ _aj_. , 1968) - In a d d i t i o n , there i s the p o s s i b i 1 i t y . o f an e s s e n t i a l f a t t y a c i d d e f i c i e n c y . Naismith (1964) measured the amounts of l i n o l e i c , a r a c h i d o n i c , and e i c o s a t r i e n o i c a c i d in the serum of Yoruba c h i l d r e n w i t h PCM. The r a t i o of e i c o s a t r i e n o i c a c i d to arach i d o n i c a c i d was 1.08 which i s in excess of the .4 considered to be i n d i c a t i v e of an e s s e n t i a l f a t t y a c i d d e f i c i e n c y (Holman, I960). The resemblance between the s k i n l e s i o n s in cases of PCM and those produced experimentally by d i e t s l a c k i n g e s s e n t i a l f a t t y acids i s s t r i k i n g (Hansen ei. . a l . , 1958; Dean, 1965). A d e f i c i e n c y of e s s e n t i a l f a t t y acids has been shown to lead to an impairment in the u t i l i z a t i o n of p r o t e i n in the rat (Naismith, 1962) and there f o r e could aggravate the p r o t e i n d e f i c i e n c y . b. Serum Cholesterol The f a c t that t o t a l serum c h o l e s t e r o l i s low in PCM is a gen e r a l l y agreed upon f i n d i n g . Schendel and Hansen (1958) measured the serum c h o l e s t e r o l in 48 cases of PCM and observed a mean of 93 mg/100 ml. This c h o l e s t e r o l l e v e l was s i g n i f i c a n t l y lower than the 173 mg/100 ml observed in the c o n t r o l group. Following treatment, there was a s i g n i f i c a n t increase in serum c h o l e s t e r o l l e v e l s . They noted a c l o s e c o r r e l a t i o n between c h o l e s t e r o l r i s e and c l i n i c a l improvement and suggested that the measurement of serum c h o l e s t e r o l might provide a s e n s i t i v e biochemical index of success or f a i l u r e of treatment. Low c i r c u l a t i n g l e v e l s of ch o l e s t e r o l have a l s o been reported by Matthew and Dean (i960), Jaya Rao - 14 -and Prasad (1966) and Cravioto et a l . (1959). Cholesterol serves as a precursor f o r many of the s t e r o i d hormones as wel l as the b i l e s a l t s . C h o l e s t e r o l e s t e r s may a l s o play a r o l e in the transport of f a t t y a c i d s , i n a d d i t i o n , i t i s believed that c h o l e s t e r o l may f u n c t i o n in the production and conduction of e l e c t r i c a l impulses (Searcy, 1969)-Several f a c t o r s may account f o r the reduction in serum c h o l e s t e r o l in PCM. Exogenous sources of preformed c h o l e s t e r o l are l i m i t e d since the ma j o r i t y of the pre-PCM d i e t i s almost t o t a l l y of vegetable o r i g i n . One of the s t e r o l s in such a d i e t , s i t o s t e r o l , has a l s o been shown to be associated w i t h reduced c h o l e s t e r o l absorption and hypocholesterolemia (Friedman et a j . 1956). Diarrhea, which is common in PCM, may a l s o play a part in producing the hypocholesterolemia, e i t h e r through general metabolic disturbances and s t r e s s or through increased e x c r e t i o n of endogenous c h o l e s t e r o l (Schendel and Hansen, 1958). The l i v e r i s the main s i t e of c h o l e s t e r o l s y n t h e s i s , but the s k i n , adrenals, gonads, i n t e s t i n e , and aorta can car r y out the b i o s y n t h e s i s . Acetate r a d i c a l s , c h i e f l y in the form of acetyl-coenzyme A, are a l l that the body requires as s t a r t i n g m a t e r i a l . Consequently many amino a c i d s , carbohydrates, and f a t t y a c i d s , when supplied in excess of other metabolic needs, can c o n t r i b u t e to the c h o l e s t e r o l pool. Since the d i e t may be d e f i c i e n t i n p r o t e i n and energy, endogenous synthesis of c h o l e s t e r o l may be reduced. Under normal c o n d i t i o n s c h o l e s t e r o l is released from the l i v e r in the form of l i p o p r o t e i n s . Serum l i p o p r o t e i n s have been estimated to be reduced in PCM (Cravioto et a i - , 1959). - 15 -c. Fatty L i v e r An extremely f a t t y l i v e r i s one of the most s t r i k i n g c h a r a c t e r i s t i c s of PCM. However, de s p i t e the c l i n i c a l importance of t h i s f a t t y c o n d i t i o n , i t s pathogenesis is not yet resolved. There are several p o s s i b l e mechanisms which may r e s u l t in a f a t t y l i v e r . F i r s t l y , i t i s p o s s i b l e that decreased o x i d a t i o n of f a t t y 14 acids leads to the b u i l d up. However, using very small doses of C-palmitate, Lewis et a 1. (1967), discovered that plasma f r e e f a t t y a c i d s were o x i d i z e d to r e s p i r a t o r y CO^ more r a p i d l y than normal in PCM. This i s the opposite of what would be expected i f the f a t t y l i v e r r e s u l t e d from decreased o x i d a t i o n of the f a t t y a c i d s . The r o l e of l i p o t r o p h i c f a c t o r s on f a t accumulation has a l s o been i n v e s t i g a t e d . The d i e t s of c h i l d r e n who develop PCM may be la c k i n g in ch o l i n e and methionine (Truswel1, 1975). If l i p o t r o p h i c f a c t o r d e f i c i e n c y c o n t r i b u t e s to f a t t y l i v e r , t h i s presumably comes about by reduced synthesis of p h o s p h a t i d y l c h o l i n e in the l i v e r . P h o s p h a t i d y l c h o l i n e i s the p r i n c i p a l transport phospholipid in plasma. If f a t t y l i v e r i s caused by a d e f i c i e n c y of l i p o t r o p h i c f a c t o r s , cases with gross f a t t y 1iver would be expected to have low serum ph o s p h a t i d y l c h o l i n e , with t o t a l phospholipids reduced more than other l i p i d c l a s s e s . However, serum phospholipids are not as reduced as c h o l e s t e r o l or t r i g l y c e r i d e s in untreated PCM, and phospholipids are not lower in cases with severe f a t t y l i v e r (Truswel 1 ej; aj.., 1969). It is therefore u n l i k e l y that l i p o t r o p h i c substances have a r o l e in the formation of f a t t y 1iver. - 16 -Another poss i b i 1 i ty Is that there i s increased f a t t y a c i d synthesis in the l i v e r . F l e t c h e r (1966) observed a s t r i k i n g reduction in glucose-6-phosphatase a c t i v i t y in the l i v e r of malnourished c h i l d r e n and considered i t a key f a c t o r in the pathog enesis of f a t t y 1 i v e r . He postulated that with the reduction in t h i s enzyme, there i s an impaired mechanism f o r s e c r e t i n g glucose. With a continued a d d i t i o n of carbohydrate from the d i e t , the l i v e r in malnourished subjects receives a greater load of carbohydrate than i t can handle. Some of t h i s i s deposited as glycogen while the remainder i s converted by normal metabolic processes into f a t . However, Lewis gtaj. (1964), discovered that 10% of the f a t in the l i v e r of malnourished subjects was l i n o l e i c a c i d . This would not occur i f the f a t were synthesized in the l i v e r because l i n o l e i c a c i d i s an e s s e n t i a l f a t t y a c i d u l t i m a t e l y derived from the d i e t . In a d d i t i o n , F l e t c h e r (1966) 14 measured f a t t y a c i d s y n t h e s i s from C-acetate i n v i t r o i n l i v e r biopsy samples from c h i l d r e n w i t h PCM. The s y n t h e t i c rate was reduced. Another p o s s i b l e mechanism f o r the accumulation of l i v e r f a t i s increased m o b i l i z a t i o n of f r e e f a t t y a c i d s from adipose t i s s u e . Lewis et a j . ( 1 9 6 4 ) , showed that the concentration of n o n e s t e r i f i e d f a t t y acids i s increased in the plasma of c h i l d r e n with PCM. They suggested that in the acute phase of the disease, the c h i l d becomes deprived of c a l o r i e s because of anorexia, vomiting and d i a r r h e a . As a r e s u l t , the plasma glucose concentrations decrease. This reduction in plasma glucose leads to an increased output of NEFA from f a t depots which in turn causes d e p o s i t i o n of excess f a t in the l i v e r . Lewis et ,aj_. ( 1 9 6 6 ) , went on to show that the f l u x of f r e e f a t t y acids through plasma was increased in i n f a n t s with PCM. However, m o b i l i z a t i o n of NEFA cannot be the t o t a l mechanism f o r f a t t y l i v e r , because in marasmus, where the plasma f r e e f a t t y a c i d s are as high as - 17 -in kwashiorkor, the l i v e r i s able to cope with an excessive input of f a t t y acids and no f a t t y l i v e r develops. Fat accumulated or produced in the l i v e r i s secreted i n t o the plasma in combination with p r o t e i n in the form of l i p o p r o t e i n s . It now appears that the l i p i d s cannot be released from the l i v e r because of low concentrations of beta l i p o p r o t e i n s and that t h i s i s probably the r e s u l t of reduced hepatic synthesis of the p r o t e i n moiety of the l i p o p r o t e i n (Truswell and Hansen, 1969)-3. Carbohydrate Metabolism in PCM a. Blood Glucose There is some disagreement in the l i t e r a t u r e on the question of blood glucose l e v e l s in PCM. Slone et a_l_. (196T) , i n v e s t i g a t e d blood sugar l e v e l s in malnourished South A f r i c a n Bantu and observed a mean glucose l e v e l of 51 mg/100 ml, which was s i g n i f i c a n t l y lower than the 76 mg/100 ml of the c o n t r o l group. Hypoglycemia has a l s o been reported by Kassem et a l . (1975), Baig and Edozien (1965), and Wharton (1970). Bowie (1964) however, reported normal l e v e l s in malnourished South A f r i c a n c h i l d r e n as did Jaya Rao (1965) working w i t h malnourished Indian c h i l d r e n . There i s co n s i d e r a b l e disagreement as to the cause of the hypoglycemia when i t does occur. One p o s s i b l e explanation f o r the hypoglycemia may be i n t e s t i n a l malabsorption of carbohydrates. Sugar absorption was studied by j e j u n a l perfusion in f i v e malnourished .chiIdren both on admission and a f t e r at l e a s t two months high p r o t e i n feeding - 18 -(James, 1968). Al1 chiIdren i n i t i a l l y had poor glucose absorption, four had d e f e c t i v e l a c t o s e h y d r o l y s i s and two had a defect in sucrose h y d r o l y s i s . The h y d r o l y t i c defects were r e l a t e d to low d i s a c c h a r i d a s e a c t i v i t i e s in j e j u n a l mucosal t i s s u e as a r e s u l t of mucosal damage. Digestion of st a r c h i s presumably impaired by the severe reduction of pancreatic amylase s e c r e t i o n reported in PCM (Thompson and Trowel 1, 1952). Kerpel-Fronius and Kaiser (1967), concluded that malabsorption of carbohydrate d i d not play a leading r o l e in e l i c i t i n g hypoglycemia since severe hypoglycemia was a l s o observed in cases in which the a b i l i t y to s p l i t d i s a c c h a r i d e s was preserved. Slone et a l . (1961), postulated that a decrease in g1uconeogenesis may be a f a c t o r leading to hypoglycemia. They c i t e d high amino ac i d l e v e l s and low blood urea l e v e l s as evidence of impaired gluconeogenesis and suggested that the block may be in the deamination of amino a c i d s . However, Arroyave et _al_. (1962) showed that the t o t a l amino a c i d l e v e l in c h i l d r e n with PCM was approximately one-half of those in healthy c h i l d r e n . Whitehead and Dean (1964) a l s o reported s i g n i f i c a n t l y reduced l e v e l s of amino a c i d s . Whitehead and Harland (1966) measured the blood sugar, l a c t a t e and pyruvate l e v e l s of 69 Ugandan c h i l d r e n during treatment of PCM. The ma j o r i t y of untreated cases had low l e v e l s of blood glucose but high l e v e l s of l a c t a t e and pyruvate. It i s th e r e f o r e p o s s i b l e that there i s a metabolic block in the gluconeogenic pathway from pyruvate v i a the c i t r i c a c i d c y c l e . A d e f i c i e n c y of thiamine may cause t h i s , or of pantothenic a c i d , the precursor of coenzyme A. The r e l a t i o n s h i p between hypoglycemia and the glycogen content of the l i v e r has a l s o been i n v e s t i g a t e d . A l l e y n e and S c u l l a r d (1968) showed that l i v e r glycogen i s depleted in PCM. Wayburne (1963) reported that needle biop s i e s taken w i t h i n a few minutes of death in hypoglycemic p a t i e n t s have - 19 -shown a d i r e c t c o r r e l a t i o n between the presence of glycogen and blood sugar l e v e l s . U s u a l l y , when blood sugar was below 30 mg/100 m l , glycogen was absent from the 1iver. In a d d i t i o n , i t is p o s s i b l e that there i s a defect in the m o b i l i z a t i o n mechanism of the glycogen s t o r e s . Some i n v e s t i g a t o r s a t t r i b u t e the presence of hypoglycemia to a d e f i c i e n c y of the enzymes glucose - 6 -phosphatase and phosphoglucomutase (Durbin et aj_., 1959; F l e t c h e r , 1966). These f i n d i n g s however, are not unanimously accepted. A l l e y n e and S c u l l a r d (1968) reported that g1ucose-6-phosphatase l e v e l s were elevated in PCM. Low glucagon l e v e l s would a l s o r e s u l t in hypoglycemia. However, Kabadi et a 1. (1976), showed that glucagon s e c r e t i o n i s normal in pro t e i n d e f i c i e n t r a t s . Kassem e_k .§_].. (1975), i n j e c t e d glucagon i n t r a m u s c u l a r l y , but observed l i t t l e response in PCM p a t i e n t s . They postulated that the hepatic enzyme systems involved in g l y c o g e n o l y s i s are impaired in PCM. The l i t e r a t u r e on the incidence, c l i n i c a l s i g n i f i c a n c e and pathogenesis of hypoglycemia in PCM contains many p h y s i o l o g i c a l c o n t r a d i c t i o n s . It i s d i f f i c u l t to r e c o n c i l e the hypoglycemia with the low i n s u l i n (Becker et aj_., 1972) and high C o r t i s o l l e v e l s (Alleyne and Young, 1967) r e p o r t e d in PCM. Further research would seem to be needed in t h i s area. k. Water and E l e c t r o l y t e Metabolism in PCM a. Edema One of the most s t r i k i n g features of PCM i s the formation of - 20 -edema. Total body water, expressed as a percentage of body weight i s c o n s i s t e n t l y increased in PCM. Smith (I960), measured t o t a l body water in Zk Jamaican c h i l d r e n s u f f e r i n g from severe chronic m a l n u t r i t i o n and observed a mean t o t a l body water (TBW) of 8k.5% of body weight in the presence of edema and a mean of 62.6% upon recovery. S i m i l a r elevated l e v e l s have been reported by Brinkman et aj_. (1965), Flynn ejt a_L. (1967), and Schneiden et al.. (1958). There appears to be s i g n i f i c a n t a l t e r a t i o n s in body water d i s t r i b u t i o n in PCM. When expressed in absolute terms or as a percentage of body weight, the e x t r a c e l l u l a r body water i s increased. Muscle b i o p s i e s obtained from f i v e c h i l d r e n on admission and before treatment was s t a r t e d , gave a mean value f o r ECW of 226 + 26 ml and f o r i n t r a c e l l u l a r body water of 225 + 38 ml per 100 grams of f a t f r e e s o l i d s , compared with c o n t r o l values of 131 + 5 and 236 + 5 ml r e s p e c t i v e l y (Metcof f. et aj . . ,1960). The main cause of t h i s r e l a t i v e expansion of the e x t r a c e l l u l a r space appears to be the r e s u l t of d i s p r o p o r t i o n a t e losses in the body s t r u c t u r e . The s k i n , nervous t i s s u e , and skeleton of the body do not change much with loss of body weight. However, these s t r u c t u r e s contain a considerable proportion of the ECW. The major losses of body weight in m a l n u t r i t i o n r e s u l t from losses of f a t and protoplasmic mass which cont a i n r e l a t i v e l y l i t t l e ECW (Kerpel-Fronius, i960). The basic cause of the development of edema in PCM i s s t i l l not c l e a r . A hypothesis that found wide acceptance f o r a long time was the c l a s s i c a l one of S t a r l i n g . This concept was that physiochemical a l t e r a t i o n s brought about by low p r o t e i n l e v e l s in plasma could e x p l a i n the development of edema in p r o t e i n d e f i c i e n t s t a t e s . This explanation i s now recognized as f a i l i n g to e x p l a i n several known f a c t s about the edema - 21 -of PCM, one of them being that c h i l d r e n s u f f e r i n g from PCM shed t h e i r edema during treatment long before there i s any s i g n i f i c a n t increase in t h e i r serum p r o t e i n s . Hypoprotei netnia is probably a modifying f a c t o r , but not the basic cause of the water r e t e n t i o n (Water low et , i960). Garrow (1965) goes so f a r as to say that the hypoproteinemia may be the r e s u l t rather than the cause of the edema. There is evidence that renal f u n c t i o n may be impaired in PCM, and that these defects may be important in the production of the edema of PCM. The glomerular f i l t r a t i o n rate has been shown to be reduced in PCM (Alleyne, 1967). This reduction in GFR was a t t r i b u t e d mainly to a s u b s t a n t i a l f a l l in c a r d i a c output and renal blood flow. A l l e y n e (1967), considered the p o s s i b i l i t y that glomerular membrane or c a p i l l a r y c e l l d i s f u n c t i o n might be the cause of the f a l l in GFR. In the presence of a diminished GFR, with r e s t r i c t i o n in the amount of f l u i d presented to the d i s t a l segment of the kidney, the body would be unable to c l e a r water maximally. Potassium d e p l e t i o n may r e s u l t in r e t e n t i o n of water and s a l t (Black and Milne , 1952), and consequently potassium d e f i c i e n c y i s very l i k e l y a f a c t o r in the development of the edema of PCM. Garrow (I965), observed that the :edematous type of c h i l d w i t h PCM was even more depleted in potassium than in p r o t e i n . Marasmic c h i l d r e n , c h a r a c t e r i z e d by a lack of edema, were not potassium d e f i c i e n t . It has been reported that aldosterone, the most potent sodium r e t a i n i n g s t e r o i d known, might be responsible f o r the edema in PCM. (Hansen, 1956)• Aldosterone acts on the kidney to increase tubular reabsorption of sodium and water while u r i n a r y e x c r e t i o n of potassium is enhanced. However, L u r i e and Jackson (1962) observed no r e l a t i o n s h i p - 22 -between u r i n a r y aldosterone l e v e l s and sodium r e t e n t i o n or edema in PCM. They suggested that mechanisms other than aldosterone s e c r e t i o n were responsible f o r the water and e l e c t r o l y t e disturbances in PCM. However, i t is p o s s i b l e that the s e c r e t i o n rate of aldosterone i s not r e f l e c t e d in the u r i n a r y e x c r e t i o n . Leonard and MacWilliam (1965) measured the percentage of bound aldosterone in the serum of s i x malnourished c h i l d r e n and observed i t to be almost one-half the c o n t r o l value. Aldosterone i s bound almost e x c l u s i v e l y to albumin in the serum. In PCM, the serum albumin l e v e l i s g r e a t l y reduced, which could cause a decrease in the amount of bound aldosterone, and an increase in the p h y s i o l o g i c a l l y a c t i v e f r e e form (Leonard and MacWilliam, 1965). Be i t ? ns et a l . (1974) measured the plasma aldosterone l e v e l s in malnourished c h i l d r e n and observed them to be higher when compared with those of a c o n t r o l group. They reported that the aldosterone s e c r e t i o n rate remained normal and suggested that the increased plasma concentrations might be the r e s u l t of an a l t e r a t i o n in the metabolic clearance rate of the s t e r o i d . Unfortunately, they d i d not c o r r e l a t e the r a i s e d aldosterone l e v e l s with edema formation and th e r e f o r e the r o l e of aldosterone in the formation of edema is s t i l l to be determined. In most mama 1ian species the major c o n t r o l over aldosterone s e c r e t i o n i s exerted by a n g i o t e n s i n , which is derived from a plasma p r o t e i n when the kidney releases the enzyme r e n i n . Plasma renin a c t i v i t y was measured by bio-assay in 100 c h i l d r e n with PCM and in 20: healthy c h i l d r e n ( K r i t z i n g e r et a i . , 1974). Renin a c t i v i t y was s i g n i f i c a n t l y increased in c h i l d r e n with PCM. This increased plasma renin a c t i v i t y must be the r e s u l t - 23 -of e i t h e r an increase in renin s e c r e t i o n by the kidney or a decrease in renin clearance by the l i v e r w i t h the l a t t e r more l i k e l y ( K r i t z i n g e r et a l . , 1974). The renin-angiotensin system opposes s a l t and water e l i m i n a t i o n by d i r e c t a c t i o n on the kidney (DeBono et a_L , 1963) and a l s o by r e l e a s i n g vasopressin (Bonjour and Malvin, 1970), and aldosterone (Mulrow et a_j_. , 1962). The r e n i n - a n g i o t e n s i n system a l s o increases water consumption by inducing t h i r s t (Fitzsimmons and Simons, 1969)• In a f u r t h e r study, increased plasma renin a c t i v i t y was found in PCM, but there was no s t r i c t r e l a t i o n s h i p to the degree of edema (Van der Westhuysen et aj_. , 1 9 7 5 ) . Many c h i l d r e n with high renin a c t i v i t y d i d not develop edema and v i c e versa. In a d d i t i o n , work on malnourished pigs has shown that the increase in renin a c t i v i t y occurs a f t e r the edema develops (Van der Westhuysen et a_]_. , 1977). These f i n d i n g s suggest that the r e n i n -angiotensin system i s not responsible f o r the i n i t i a l f l u i d r e t e n t i o n and the development of edema in PCM. It has re c e n t l y been observed that during PCM, there are elevated l e v e l s of a n t i d i u r e t i c hormone (ADH) in the c i r c u l a t i o n ( S r i k a n t i a and Mohanran, 1970). ADH i s secreted from the neurohypophysis and acts on the d i s t a l and c o l l e c t i n g tubules of the kidney, thereby increasing the reabsorption of water. There are two p o s s i b l e explanation f o r the increased ADH l e v e l s in the c i r c u l a t i o n . F i r s t l y , i t has been shown that in PCM, the a b i l i t y of the l i v e r to i n a c t i v a t e ADH i s consi d e r a b l y impaired ( S r i k a n t i a and Gopalan, 1958). Secondly f e r r i t i n , the i r o n - p r o t e i n complex, has been shown to exert considerable a n t i d i u r e t i c a c t i v i t y (Baez et aj.., 1952). F e r r i t i n i s mainly present in the 1iver, spleen, and bone marrow, and i s not normally observed in the c i r c u l a t i o n . However, probably as a r e s u l t of - 2h -l i v e r damage, a c t i v e f e r r i t i n has been observed in the c i r c u l a t i o n of c h i l d r e n with PCM ( S r i k a n t i a , 1958). The elevated l e v e l s of-plasma ADH may therefore be explained on the basis of d e f e c t i v e i n a c t i v a t i o n of the hormone as a r e s u l t of l i v e r damage, and an increased s e c r e t i o n of the hormone as a r e s u l t of s t i m u l a t i o n of the neurohypophysis by f e r r i t i n . It has been shown that c h i l d r e n with marasmus do not have elevated ADH. In a d d i t i o n , f o l l o w i n g therapy, and disappearance of edema, l e v e l s of the hormone return to normal ( S r i k a n t i a and Mohanram, 1970). These r e s u l t s suggest that the high l e v e l s of ADH are the cause of the edema in PCM. b. Serum Calcium Studies on calcium metabolism in PCM have shown v a r i a b l e r e s u l t s . Jayalakshmi et aj,. (1957), reported that serum calcium concentrations were subnormal in malnourished c h i l d r e n . They observed a mean of 8.25 mg/100 ml in ]h malbourished c h i l d r e n compared with a mean of 10.50 mg/100 ml a f t e r s i x weeks of treatment. Hypocalcaemia has a l s o been reported by Sandstead et a l . (1965), and Senecal (1958). They a t t r i b u t e the lower serum l e v e l s to a lowering of the p r o t e i n bound f r a c t i o n secondary to hypoproteinemia. Khali 1 et a_l_. (197*0, reported that serum calcium values in c h i l d r e n with PCM did not d i f f e r s i g n i f i c a n t l y from c o n t r o l s . Erythrocyte l e v e l s however, were s i g n i f i c a n t l y reduced. They thought that the maintenance of t h i s normal concentration in s p i t e of d e f i c i e n t intake and inadequate a b s o r p t i o n , was a r e f l e c t i o n of increased f r e e l y d i f f u s i b l e calcium through bone mobi1izat ion. - 25 -Shenolikar and Narasinga Rao (1968) demonstrated reduced calcium a c c r e t i o n rates in the bones of p r o t e i n deprived rats using 45 Ca as a t r a c e r . This reduction of calcium a c c r e t i o n was associated with a reduced i n c o r p o r a t i o n of p r o l i n e i n t o hydroxyproline and suggests an impairment in bone co l l a g e n s y n t h e s i s . Le Roith and Pimstone (1973), demonstrated a s i g n i f i c a n t reduction in i n t e s t i n a l calcium absorption in p r o t e i n d e f i c i e n t r a t s . Kalk and Pimstone (1974), observed that in the p r o t e i n d e f i c i e n t r a t , there was a s i g n i f i c a n t reduction in i n t e s t i n a l calcium binding p r o t e i n a c t i v i t y . They f e l t that as a consequence of d e f i c i e n t amino a c i d s u b s t r a t e , synthesis of calcium binding p r o t e i n was reduced. Shenolikar and Narasinga Rao (1968) observed a higher f e c a l calcium e x c r e t i o n in rats on a low p r o t e i n d i e t . They observed that the major component of t h i s f e c a l calcium was of endogenous o r i g i n . Endogenous calcium i s co n t r i b u t e d by b i l e , pancreatic j u i c e and the s e c r e t i o n of e p i t h e l i a l c e l l s along the i n t e s t i n e s . It is not known which f a c t o r s c o n t r i b u t e to the increased f e c a l e x c r e t i o n . Leonard et aj_. (1968), reported s i g n i f i c a n t l y higher calcium l e v e l s in n a i l c l i p p i n g s from ma 1nourished chi1dren. They suggested that the a n a l y s i s of n a i l e l e c t r o l y t e s may be of considerable value as a guide to a l t e r a t i o n s in t i s s u e e l e c t r o l y t e s . c. Serum Copper Serum copper l e v e l s have been shown to be reduced in PCM. Lahey et a). (1958), studied 10 Guatemalan c h i l d r e n with PCM and indi c a t e d - 26 -that the concentration of copper in serum was s u b s t a n t i a l l y reduced. Gopalan et a l . (1963), observed low serum copper in kwashiorkor but reported normal l e v e l s in marasmic i n f a n t s . Edozien and Udeozo (i960), reported a reduction in serum copper concentration from 180 T k~] ug/100 ml in normal chi1dren to 86 + 23 ug/100 ml in ch i I d r e n wi th PCM. The p o s s i b i l i t y e x i s t s that a d i e t a r y d e f i c i e n c y of copper occurs concomitantly w i t h a d e f i c i e n c y of p r o t e i n . The d a i l y requirement f o r copper i s approximately 50 ug/kg body weight. Dried cassava, which i s a s t a p l e food of most developing c o u n t r i e s , contains approximately 1 AO ug/ 100 gm. However, i t has been e s t a b l i s h e d that only about 5% of the copper in an ordinary d i e t i s r e t a i n e d . If such i s the case in c h i l d r e n with PCM, i t would appear l i k e l y that the copper intake would be below normal. However, a study of the copper content o f the home d i e t of c h i l d r e n with PCM revealed that the d i e t a r y intake was not inadequate (Gopalan etaj., 1963)-I t i s known that copper i s excreted in the b i l e (Cartwright and Wintrobe, 1964), and i t i s therefore p o s s i b l e that diarrhea r e s u l t i n g in an excessive loss of b i l a r y contents may be a f a c t o r r e s u l t i n g in low c i r c u l a t i n g copper l e v e l s . Lahey et a_L. (1958), and Sanstead et a j . (1965) a t t r i b u t e the reduced serum copper l e v e l s to a reduction in serum p r o t e i n s . Over 95% of the copper in serum i s bound to the ceruloplasmih component of the alpha -2-globu1 in f r a c t i o n of serum p r o t e i n . Gopalan et a_[. (1963) observed a h i g h l y s i g n i f i c a n t c o r r e l a t i o n between serum copper l e v e l s and ceruloplasmin. Warren et .a] . (1969), determined the concentration of copper in the l i v e r s of c h i l d r e n s u f f e r i n g from PCM. This study showed that there - 27 -was a d e c r e a s e i n the c o n c e n t r a t i o n o f c o p p e r i n t he l i v e r s o f PCM p a t i e n t s . The p i g m e n t a r y changes i n t h e h a i r commonly noted i n PCM may be due t o a d e f i c i e n c y o f c oppe r a t t h e t i s s u e l e v e l (Lahey e_t a_k, 1958) . A coppe r c o n t a i n i n g enzyme, t y r o s i n a s e , i s known t o be i n v o l v e d i n t h e p r o d u c t i o n o f m e l a n i n f r om t y r o s i n e ( F l e s c h , 1949). Gopa l an ej: a_[. (1963), measured t he coppe r c o n t e n t i n t he h a i r o f m a l n o u r i s h e d and normal c h i l d r e n . The coppe r c o n t e n t o f the h a i r i n PCM was low and ave raged o n l y 9-1 gamma/gm o f h a i r compared t o 19-3 gamma/gm o f h a i r i n t h e normal g r o u p . However, among c a s e s o f PCM, t he coppe r c o n t e n t o f h a i r was u n i f o r m l y low, i r r e s p e c t i v e o f whethe r t h e h a i r was normal o r n o t . Copper i s known t o p l a y a r o l e i n e r y t h r o p o i e s i s but whe the r t he anemia i n PCM i s i n any way r e l a t e d t o t he a b n o r m a l i t i e s i n t he m e t a b o l i s m o f t h i s e l ement i s not known. However, i t s h o u l d be no ted t h a t t h e anemia i n c o p p e r d e f i c i e n t a n i m a l s i s c h a r a c t e r i s t i c a l l y m i c r o c y t i c and h y p o c h r o m i c , w h i l e t he anemia o f PCM i s r a r e l y t h i s t y p e ( E d o z i e n and Rah im-Khan, 1968) . d . Serum I ron Serum i r o n and i r o n b i n d i n g c a p a c i t y a r e low i n PCM. E d o z i e n and Udeozo ( i 9 60 ) showed t h a t 35 N i g e r i a n c h i l d r e n w i t h PCM had a serum i r o n c o n c e n t r a t i o n o f 40.6 + 21.5 ug/100 ml compared w i t h 62.7 + 17-0 ug/100 ml i n 37 c o n t r o l s . The i r o n b i n d i n g c a p a c i t y was f u r t h e r reduced f r om a c o n t r o l v a l u e o f 274.0 + 59-0 ug/100 ml t o 119-1 + 54.4 ug/100 ml i n m a l n o u r i s h e d c h i l d r e n . These f i n d i n g s have been c o n f i r m e d by Lahey e t a 1. ( 1958 ) , i n m a l n o u r i s h e d Mex i c an c h i l d r e n and by E1 -Sho l my et. a j . . (1962) i n m a l n o u r i s h e d E g y p t i a n c h i l d r e n . - 28 -Sood _§_[• (J965) studied the absorption of iron using 59 Fe in nine p r o t e i n d e f i c i e n t and four c o n t r o l monkeys. At 8-10 weeks of p r o t e i n d e f i c i e n c y , there was a f a l l in iron absorption ranging from 9-23% over the basal value in seven out of nine animals. The average value f o r iron absorption in the d e f i c i e n t group was 39-9% compared with the basal value of 50.8%. A p o s s i b l e reason f o r malabsorption of iron in PCM r e l a t e s to the changes that occur in the i n t e s t i n a l mucosa of the small bowel. Extensive atrophy, as well as f u n c t i o n a l a b n o r m a l i t i e s have been demonstrated (Barbezat et a_l_. , 1967) -It i s g e n e r a l l y accepted that ferrous i r o n only i s transported across the i n t e s t i n a l w a l l . The reduction of f e r r i c to ferrous iron i s f a c i l i t a t e d by the reducing powers of as c o r b i c a c i d . Andersson et a 1. (1956) reported a low d i e t a r y intake of as c o r b i c a c i d in malnourished South A f r i c a n Bantu. In a d d i t i o n , Edozien and Rahim-Khan (1968) reported low serum a s c o r b i c a c i d in over 75% of malnourished cases examined. Amino acids such as h i s t i d i n e and l y s i n e a s s i s t in iron absorption (Van Campen and Gross, 1969). It is suggested that a d i r e c t r e a c t i o n between iro n and h i s t i d i n e occurs and that an amino a c i d - i r o n chelate may be formed and absorbed. In PCM the le v e l of h i s t i d i n e in the g a s t r o i n t e s t i n a l t r a c t would be low and there f o r e i r o n absorption may be impa i red. Losses of iron from the bodies of normal i n f a n t s occur from the g a s t r o i n t e s t i n a l t r a c t , p r i m a r i l y in the form of desquamated i n t e s t i n a l e p i t h e l i a l . eel I s , from the s k i n and in u r i n e . It i s p o s s i b l e that f a t losses in stearorrhea could be accompanied by increased desquamation of g a s t r o i n t e s t i n a l mucosal c e l l s . - 2 9 -Malnourished c h i l d r e n are a l s o subject to p a r a s i t i c i n f e s t a t i o n s , which can act as f u r t h e r d r a i n s on the very meager s u p p l i e s of d i e t a r y i r o n . The iron binding p r o t e i n resides in the beta g l o b u l i n , t r a n s f e r r i n . El-Sholmy et a ] , ( 1 9 6 2 ) , reported a c l o s e c o r r e l a t i o n between the iron binding c a p a c i t y and beta g l o b u l i n . It is th e r e f o r e probable that the reduction in serum ir o n and iron binding c a p a c i t y are due to profound metabolic a l t e r a t i o n s produced by p r o t e i n d e p l e t i o n . El Shobaki et j_L. (1972) observed that the l i v e r non-heme iron concentration was higher in malnourished c h i l d r e n than in c o n t r o l s . Chattopadhyay and Banerjee ( 1 9 7 5 ) reported that the synthesis of hemoglobin was impaired in PCM. Since e x c r e t i o n of iron i s very l i m i t e d , the only way to get r i d of the n o n - u t i l i z e d part i s to store i t in the l i v e r . e. Serum Magnesiurn Magnesium d e f i c i e n c y appears to be a r e l a t i v e l y frequent occurrence in PCM. Evidence of magnesium d e p l e t i o n has been derived from a n a l y s i s of muscle biopsy m a t e r i a l , balance s t u d i e s , and estimations of u r i n a r y , plasma and serum magnesium. A gross t i s s u e d e p l e t i o n of magnesium was f i r s t reported in PCM by Montgomery (1960)- He performed muscle b i o p s i e s on 12 Jamaican c h i l d r e n s u f f e r i n g from PCM and observed s i g n i f i c a n t l y lower l e v e l s of magnesium in muscle. Caddell and Goddard (1967) a l s o reported low magnesium values in muscle samples of Nigerian p a t i e n t s . Linder et a j . (1963) showed that cumulative magnesium r e t e n t i o n in the f i r s t 21 days of treatment was - 30 -2.1 meq/g N with and .Sk meq/g N without magnesium supplementation. Increased magnesium r e t e n t i o n p e r s i s t e d f o r as long as s i x weeks, showing that r e p l e t i o n takes a long time. There appears to be" a discrepancy regarding serum magnesium l e v e l s in PCM. Linder et a l . (1963) reported low magnesium values in over two-thirds of t h e i r o b s ervations, while Montgomery (1960 ) reported unaltered l e v e l s . A large proportion of body magnesium i s in the skeleton and muscle, and therefore blood l e v e l s may be normal. Urinary magnesium i s extremely low and i s probably a b e t t e r index of magnesium d e f i c i e n c y than i s the serum l e v e l (Linder .et aj_., 1963) -Leonard et a_l_. (1968) reported that normal plasma e l e c t r o l y t e l e v e l s are f r e q u e n t l y observed in a s s o c i a t i o n with decreased t i s s u e l e v e l s rendering measurement of the former of l i t t l e value in assessing the c e l l u l a r e l e c t r o l y t e s t a t u s . They reported s i g n i f i c a n t l y reduced magnesium l e v e l s in n a i l c l i p p i n g s from PCM p a t i e n t s and suggested that the a n a l y s i s of n a i l c l i p p i n g s may be of considerable value as a guide to a l t e r a t i o n s in t i s s u e e l e c t r o l y t e s . According to Caddell and Goddard (1967)', the magnesium d e f i c i e n c y in m a l n u t r i t i o n r e s u l t s from prolonged losses of magnesium through the Gl t r a c t during diarrhea and vomiting coupled with a low magnesium intake. Ch i l d r e n on high m i l k p r o t e i n therapy o f t e n d i e suddenly and unexpectedly j u s t as they are beginning to recover and feed themselves. It has been shown that p r o t e i n , calcium, phosphorus and potassium increase the metabolic demand f o r magnesium and t h i s therapy may a c t u a l l y increase the magnesium d e f i c i e n c y syndrome. In a d d i t i o n , c h i l d r e n recovering from PCM grow at a f a n t a s t i c rate and t h i s must e n t a i l a need f o r large amounts - 31 -of magnesium. For these reasons, i t has been suggested that supplemental magnesium be given r o u t i n e l y to a l l severely malnourished c h i l d r e n during treatment (Caddell, 1966). Caddell (1967) conducted a double-blind paired sequential t r i a l in malnourished Nigerfan .chi1dren'to assess the e f f i c a c y of parenteral magnesium therapy. Her p r e l i m i n a r y f i n d i n g s i n d i c a t e d that t h i s form of therapy was of such s i g n i f i c a n t value that the t r i a l was stopped before completion in order to give the c o n t r o l p a t i e n t s the b e n e f i t of the therapy. The c l i n i c a l symptoms a t t r i b u t e d to magnesium d e p l e t i o n included weakness, anorexia, tremors, s l e e p l e s s n e s s , h y p e r i r r i t a b i 1 i t y hypotension, and hypothermia, a l l of which improved r a p i d l y a f t e r magnesium a d m i n i s t r a t i o n . Another t r i a l of magnesium therapy was undertaken in South A f r i c a (Rosen .et 1970). Although i n i t i a l plasma magnesium values were commonly lower than normal and tended to f a l l t r a n s i e n t l y in untreated cases, the t r i a l f a i l e d to demonstrate any t h e r a p u t i c b e n e f i t of magnesium supplementation. In no instance was i t p o s s i b l e to recognize c l i n i c a l l y the c h i l d r e n who had the lowest plasma magnesium values; nor were there any s p e c i f i c symptoms i d e n t i f i a b l e w i t h magnesium d e p l e t i o n . P o s s i b l y the South A f r i c a n c h i l d r e n were less severely depleted than are those described by Caddell (1967)', t h e i r s t a p l e d i e t being maize which has a higher magnesium content than does cassava, the main substance of d i e t in N i g e r i a . A magnesium d e f i c i e n c y would help to e x p l a i n many of the biochemical l e s i o n s associated w i t h PCM. A d e f i c i e n c y of magnesium would help to e x p l a i n the f a t t y l i v e r commonly observed in PCM. Severe f a t t y i n f i l t r a t i o n , w i t h o b l i t e r a t i o n of c e l l s by f a t t y changes and marked f i b r o s i s , has been described i n p a t i e n t s with d e p l e t i o n of serum magnesium (Waterlow, 1962). Oxidative phosphorylation, which i s dependant on - 32 -magnesium a c t i v a t e d enzyme systems, i s severely decreased in f a t t y l i v e r s ( G r i f f i t h s and Rees, 1957). Magnesium i s a l s o an a c t i v a t o r in alpha keto f a t t y a c i d metabolism ( V a l l e e , i960). The human bone i s thought to be a h i g h l y l a b i l e reserve f o r magnesium. The marked bone changes in PCM (Higginson, 1954) might be re l a t e d to a very s u b s t a n t i a l bone d e f i c i t (Montgomery and C h i r , 1961). Absorption of carbohydrate has been shown to be depressed in PCM (James, 1968). The a c t i v e absorption of glucose, g a l a c t o s e , and fr u c t o s e involves the hexokinase r e a c t i o n . The hexokinases f o r glucose and f r u c t o s e are a c t i v a t e d by magnesiurn : (Cori and S l e i n , 19^7)-Magnesium i s an a c t i v a t o r f o r a l l the enzymes that r e q u i r e thiamine pyrophosphate as a c o f a c t o r (Brown, 1962). This coenzyme i s responsible f o r alpha keto f a t t y a c i d metabolism and f o r o x i d a t i v e d e c a r b o x y l a t i o n of pyruvate i n muscle and b r a i n . Pyruvic a c i d i s a key substance in intermediary metabolism, being a stage reached by g l y c e r o l , many amino a c i d s , and a l l carbohydrates. PCM pa t i e n t s manifest d i s -turbances in pyruvate metabolism; the blood pyruvate i s ra i s e d and pyruvate decarboxylation i s reduced (Whitehead and Harland, 1966). Pyridoxine phosphate requires magnesium f o r optimal a c t i v i t y . In a d d i t i o n , magnesium i s needed f o r the phosphorylation of r i b o f l a v i n to form i t s coenzyme. Since magnesium i s required f o r coenzyme formation of r i b o f l a v i n and f o r a c t i v a t i o n of the coenzyme of py r i d o x i n e , i t might be that the s k i n l e s i o n s of PCM, so s i m i l a r to those of r i b o f l a v i n and pyridoxine d e f i c i e n c i e s , are r e l a t e d to disturbed metabolism of these vitamin B coenzymes in magnesium d e f i c i e n t subjects ( C a d d e l l , 1965)-- 33 -f. Serum Phosphorus Hypophosphataemia has been observed in PCM. Sandstead e t a l . (1965), measured the serum phosphorus l e v e l s in 39 malnourished Egyptian c h i l d r e n . Upon admission, they had a mean serum l e v e l of 2.8 + 1.1 mg/100 ml compared with a l e v e l of 4.7 +1.1 mg/100 ml upon c l i n i c a l cure. This i s in agreement with the f i n d i n g s of Bjornesjo et aj_. (V965). Smith (i960) reported that serum phosphorus l e v e l s tended to be lower in those i n f a n t s who died w i t h i n seven days of admission compared with those who surv i v e d . In a l l f a t a l cases, the mean serum phosphorus l e v e l was 3-7 + T.6 mg/100 ml, but among 51 s u r v i v o r s the mean was 4.39 + 1.43 mg/100 ml. In PCM, inorganic and organic phosphate are decreased in muscle (Waterlow and Mendes, 1957). Upon recovery, both kinds of phosphate increase. In cases ending in death, inorganic phosphate increase at the expense of organic phosphate. g. Serum Zinc Zinc d e f i c i e n c y i s manifested by the occurrence of s k i n l e s i o n s , retarded growth, d i a r r h e a , vomiting, a l o p e c i a , d i s t u r b e d p r o t e i n metabolism, and a decrease in blood a l k a l i n e phosphatase and pancreatic amylase. H i s t o l o g i c a l s tudies have revealed h y p e r k e r a t i r i i z a t i o n , t h i c k e n i n g of the epidermis and i n t r a and i n t e r c e l l u l a r edema. It becomes apparent that a s t r i k i n g s i m i l a r i t y e x i s t s between the signs and symptoms observed in animals in a z i n c d e f i c i e n t s t a t e and the corresponding f i n d i n g s reported by various workers i n v e s t i g a t i n g PCM. - 34 -Kumar and Jaya Rao (1973), estimated the plasma and ery t h r o c y t e z i n c l e v e l s in c h i l d r e n s u f f e r i n g from PCM. Plasma z i n c l e v e l s were low on admission and returned to normal l e v e l s a f t e r n u t r i t i o n a l r e h a b i l i t a t i o n . Erythrocyte z i n c concentrations were unalt e r e d . These observations are in l i n e with those reported e a r l i e r by Smit and P r e t o r i u s (1964), and by Sandstead et a±. (I965). The low l e v e l s of serum z i n c during the acute phase of PCM may be due to several f a c t o r s . Plant products p a r t i c u l a r l y c e r e a l s , are not s a t i s f a c t o r y sources of z i n c because of t h e i r p h y t i c a c i d content and i t s binding e f f e c t s on the z i n c ion (O'Dell and Savage, i960). The pre-PCM d i e t of malnourished c h i l d r e n i s high in c e r e a l s and other vegetable products and very low in animal p r o t e i n . Therefore, because of the presence of phytate and other b i o l o g i c a l c h e l a t o r s , the a v a i l a b i l i t y of z i n c f o r absorption is probably low. The p r o t e i n l e v e l of the d i e t has been shown to a f f e c t z i n c absorption. To assess some of the e f f e c t s of p r o t e i n m a l n u t r i t i o n on zi n c metabolism, rats were fed d i e t s w i t h 5 or 15% c a s e i n and 9 or 33 mg/kg z i n c (Van Campen and House, 1974). Rats on the 5% p r o t e i n d i e t retained less of a s i n g l e o r a l dose of In3^ than those on the 15% p r o t e i n d i e t with e i t h e r amount of p r o t e i n . Sandstead _et a j . (1965) a s c r i b e the z i n c d e f i c i e n c y to the presence of i n f e c t i o n s or to i n t e s t i n a l loss of z i n c due to di a r r h e a . Ninety percent of c i r c u l a t i n g z i n c i s bound to serum p r o t e i n s . It i s th e r e f o r e not s u r p r i s i n g that in PCM where serum proteins are low, that the t o t a l z i n c l e v e l s should be low. - 35 -The loss of z i n c i n t o the i n t e r s t i t i a l compartment i s yet another f a c t o r which might c o n t r i b u t e to the low l e v e l s of serum z i n c in PCM. Edema f l u i d was found to contain s u b s t a n t i a l amounts of z i n c (Kumar and Jaya Rao, 1973). In PCM cases the z i n c content of the l i v e r has been observed to be markedly reduced (Warren e_t aj.,. , 1969) • It i s conceivable that some of the changes in sk i n and h a i r observed in PCM are r e l a t e d to z i n c d e f i c i e n c y . A l o p e c i a , coarseness and dispigmentation of the h a i r occurs in z i n c d e f i c i e n t r a t s . Zinc was estimated in the h a i r of 43 Peruvean Andean Indian c h i l d r e n on admission to h o s p i t a l and again a f t e r recovery ( B r a d f i e l d et a_f., 1969). There was no s i g n i f i c a n t d i f f e r e n c e between admission and recovery. Impaired i n s u l i n s e c r e t i o n i s a c h a r a c t e r i s t i c f i n d i n g in PCM (Becker .et. aj.., 1972). Recent f i n d i n g s i n d i c a t e that z i n c d e f i c i e n c y r e s u l t s in the impairment of i n s u l i n s e c r e t i o n from the pancreas ( S u l l i v a n eJt.a_l-> 1974). Huber and Gershoff (1973) fed rats d i e t s c o n t a i n i n g 1, 20, or 1200 mg/kg z i n c . Feeding of high or low z i n c d i e t s d i d not a l t e r the i n s u l i n content of the pancreas, but immunoreactive serum i n s u l i n and t o t a l serum i n s u l i n l i k e a c t i v i t y were s i g n i f i c a n t l y reduced in the z i n c d e f i c i e n t group. - 36 -B. BIOCHEMICAL ASPECTS OF PCM IN SWINE 1. P r o t e i n Metabolism a . Serum Prote i hs Tumbleson e_t a_l_. (1972b) measured serum proteins in malnourished swine. Mean serum t o t a l p r o teins and albumin concentrations were lower f o r the undernourished pigs than f o r the c o n t r o l s . From 4-32 weeks of t e s t , the undernourished pigs had higher serum t o t a l g l o b u l i n s as a percentage of t o t a l p r o t e i n t h a n d i d c o n t r o l p i g s . Mean serum beta g l o b u l i n c o n c e n t r a t i o n f o r the pigs fed the low p r o t e i n d i e t was lower than f o r the c o n t r o l s . b. Serum Am i no Ac ids Badger and Tumbleson (1974), reported that normal r e l a t i o n s h i p s among concentrations of amino acids were a l t e r e d in young miniature swine fed 5% p r o t e i n d i e t s . Mean concentrations of 11 f r e e amino acids were a l t e r e d s i g n i f i c a n t l y in the serum of malnourished p i g l e t s . Taurine, threonine, glutamic a c i d , v a l i n e , i s o l e u c i n e , l e u c i n e , t y r o s i n e , and phenylalanine were reduced, while a l a n i n e , alpha amino-butyric a c i d and methionine were increased. S i m i l a r f i n d i n g s were reported by Grimble and Whitehead (1970). - 37 -c. Serum Enzymes Tumbleson (1972), reported l i t t l e d i f f e r e n c e in the a l k a l i n e phosphatase, glutamic o x a l o a c e t i c transaminase and l a c t i c dehydrogenase l e v e l s of c o n t r o l and malnourished pigs. A l k a l i n e phosphatase and l a c t i c dehydrogenase were s l i g h t l y reduced. S u r p r i s i n g l y , SGOT l e v e l s were a l s o s l i g h t l y lower. Heard . e t _ a j . (1957) observed lower plasma amylase l e v e l s in pigs on a 5% p r o t e i n d i e t . d. Ant i bod ies Hook et _al.. (1972) measured antibody responses in undernourished S i n c l a i r miniature swine. There were no d i f f e r e n c e s in the antibody responses of the two d i e t a r y groups tested a f t e r 0, 4, or 8 weeks. A f t e r 12 or 16 weeks on t e s t , the appearance of maximumtitres of serum antibody was delayed in undernourished swine. Serum antibody not only appeared l a t e r .1 in undernourished swine tested a f t e r 20-24 weeks, but the a n t i b o d i e s a l s o f a i l e d to a t t a i n the l e v e l s reached in corresponding c o n t r o l swine. In a d d i t i o n , there was a decrease in the number of antibody producing c e l l s obtained from the lymph nodes of undernourished swine. 2. Water and E l e c t r o l y t e Metabolism in PCM a. Edema Tumbleson et _§J. (1969) u t i l i z e d deuterium oxide to measure - 38 -t o t a l body water and sodium thiocyanate to measure e x t r a c e l l u l a r body water in malnourished pigs. There was l i t t l e d i f f e r e n c e in t o t a l body water but e x t r a c e l l u l a r body water was s i i g h t l y e l evated. Payne and Done (1959) however, reported s i g n i f i c a n t l y higher t o t a l body water in a pig fed a low pro t e i n d i e t with carbohydrate supplement. b. E l e c t r o l y t e s Tumbleson .et_ a l . (1972a) measured serum e l e c t r o l y t e s in undernourished S i n c l a i r miniature swine. Mean serum calcium and phosphorus concentrations were lower from two through twenty-eight weeks on t e s t f o r the undernourished pigs compared with the c o n t r o l s . The undernourished group had a lower mean sodium l e v e l from 14 through 24 weeks on t e s t . Mean serum concentrations of potassium, urea n i t r o g e n , and c r e a t i n i n e were not a l t e r e d . The undernourished pigs had s i g n i f i c a n t l y higher mean serum c h l o r i d e l e v e l s compared with the c o n t r o l s . P i a t t and Frankul (1962) reported lower serum l e v e l s of iron and z i n c in t h e i r malnourished group of pig s . 3. Fat Metabolism in PCM a. Fatty L i v e r Gupta (1973a) conducted a study on the q u a n t i t a t i v e changes in the l i v e r l i p i d s of Indian pigs s u f f e r i n g from severe p r o t e i n m a l n u t r i t i o n . The l i v e r s of a l l pigs fed low p r o t e i n showed p e r i p o r t a l to d i f f u s e f a t t y changes. There was a two f o l d increase in the t o t a l l i p i d content and an - 39 -eleven f o l d increase in the t r i g l y c e r i d e content of the l i v e r s of pigs fed the low p r o t e i n d i e t . The low p r o t e i n d i e t a l s o increased the c h o l e s t e r o l content in the l i v e r . P r o t e i n d e f i c i e n c y r e s u l t e d in marked reduction of hepatic phospholipids in malnourished p i g s . b. Serum Cholesterol Tumbleson jet a±. (1969) measured the serum c h o l e s t e r o l values in undernourished Hormel miniature swine. They found no s i g n i f i c a n t d i f f e r e n c e due to d i e t a r y treatment a f t e r 12 weeks on t e s t . k. Hormones in PCM a. General P i a t t and Stewart (1967), reported that the endocrine glands of PCM pigs were smaller than those of pigs fed normally. However, r e l a t i v e to body weight, the adrenals were l a r g e , the hypophysis w i t h i n the normal range, the thymus small and the pancreas and t h y r o i d showed wide v a r i a t i o n s a t t r i b u t a b l e to d i f f e r e n t degrees of edema. B a l d i j a o et . a l . (1976) measured c o r t i c o s t e r o i d s in malnourished and c o n t r o l p i g s . No s i g n i f i c a n t d i f f e r e n c e s were observed f o r t o t a l plasma c o r t i c o s t e r o i d s . Free c o r t i s o L however, was s i g n i f i c a n t l y higher in the p r o t e i n depleted group compared to c o n t r o l s . - 40 -Atinmo et a l . (1976b) measured immunoreactive growth hormone l e v e l s in p r o t e i n depleted p i g s . Post weaning p r o t e i n d e p r i v a t i o n r e s u l t e d in higher growth hormone l e v e l s during the r e s t r i c t i o n period compared with c o n t r o l s . At i nmo _et _a_l. (1976a) conducted an experiment to i n v e s t i g a t e changes in plasma i n s u l i n l e v e l s during PCM i n swine. P r o t e i n r e s t r i c t i o n a f t e r weaning r e s u l t e d in p e r s i s t e n t l y low i n s u l i n l e v e l s during the d e p l e t i o n and r e h a b i l i t a t i o n p e r i o d . 5. Miscellaneous Aspects of PCM i n Swine a. Organ Weights Badger et aj_. (1972) conducted a study to determine the e f f e c t s of PCM on body and organ weights. Malnourished pigs weighed s i g n i f i c a n t l y l e s s at 63 days than d i d c o n t r o l s and consumed s i g n i f i c a n t l y less feed. Organ weights were lower in the malnourished group. The order or v u l n e r a b i l i t y from the l e a s t to the most a f f e c t e d was as f o l l o w s : cerebrum, cerebellum, eye, heart, kidney and l i v e r . Tumbleson e_t aj.. (I969) reported s i g n i f i c a n t l y lower kidney, gastroenemius muscle, spleen, l i v e r , lungs, adrenals, heart, t i b i a , t h y r o i d , and b r a i n weights in t h e i r malnourished group. Gupta (1973b) reported s i m i l a r changes i n malnourished Indian p i g s . b. Behavior Barnes et aj_. (1970) studied behavioral changes in baby pigs malnourished for a period of e i g h t weeks by r e s t r i c t i n g p r o t e i n or energy - 41 -intake. An apparatus was designed f o r the measurement of changes in the l e v e l of excitement or emotiona1ity under c o n d i t i o n s of s t r e s s , as wel l as changes in le a r n i n g performance in a conditioned avoidance s i t u a t i o n . The most s t r i k i n g behavioral change" due to e a r l y m a l n u t r i t i o n was the heightened excitement of the pigs when exposed to adverse s t i m u 1 i , a 1 though there was a l s o an i n d i c a t i o n of decreased le a r n i n g a b i l i t y . c. Hematology Burks .et „aj.. ( 1974 ) performed a hematological study in malnourished swine. Mean e r y t h r o c y t e count was lower from 12 through 28 weeks on tes t f o r malnourished p i g s . From 4 through 2 8 weeks on t e s t , mean packed c e l l volumes and hemoglobin concentrations were lower f o r pigs fed the k% p r o t e i n d i e t . Malnourished pigs had lower mean corpuscular volumes and lower mean corpuscular hemoglobin from 4 through 26 weeks on t e s t . Mean percent of ne u t r o p h i l s was greater f o r malnourished pigs from 8 through 16 weeks; mean percent lymphocytes was lower from 8 through 18 weeks on t e s t . d. Rehab?1i t a t i o n Pigs have a l s o been used in studies on r e h a b i l i t a t i o n from PCM. Pond et a l - ( 1 9 7 0 conducted a study to compare the adequacy of c a s e i n , i s o l a t e d soy p r o t e i n , or f i s h concentrate f o r r e h a b i l i t a t i o n from PCM. A s i m i l a r study was performed by Barnes et J I K ( 1 9 6 6 ) . They concluded that f i s h concentrate and casei n were super i o r to i s o l a t e d soy p r o t e i n in promoting growth, feed intake, and regeneration of serum pr o t e i n s and pancreatic enzymes. - 42 -EXPERIMENT 1 A. MATERIALS AND METHODS a. Object lyes T r i a l 1 was undertaken to determine the s u i t a b i l i t y of the pig as a biomedical model with which to study p r o t e i n - c a l o r i e m a l n u t r i t i o n . Various biochemical parameters are a l t e r e d in the malnourished human and the o b j e c t of t h i s t r i a l was to determine i f s i m i l a r changes occur in the malnourished p i g . b. Experimental Procedures Thirty-two Y o r k s h i r e and Yorkshire X Landrace pigs were selected at approximately the same age and weight f o r the study. The experimental animals were weaned at twenty-one days of age and assigned to a d i e t a r y treatment on the basis of weight, l i t t e r , and sex. Each treatment was f u r t h e r d i v i d e d into two pens with eight pigs in each (four barrows and four g i l t s ) . The duration of the t r i a l was ten weeks. The experiment was conducted at the Swine Research Unit at the U n i v e r s i t y of B r i t i s h Columbia. The b u i l d i n g was i n s u l a t e d and supplemental i n f r a r e d heat lamps were used to maintain the a i r temperature at approximately 25°C. The b u i l d i n g was v e n t i l a t e d w i t h t h e r m o s t a t i c a l l y c o n t r o l l e d a i r exhaust fans. The.pens had concrete f l o o r s , p a r t i a l l y s l a t t e d and a t o t a l area of 4.8 square meters. - 43 -Pigs we re fed ad 1i b j turn f rom a portable wooden feed trough. Feed consumption records were kept d a i l y and weekly.tota1s were recorded. The composition of the experimental r a t i o n s i s shown in Table 1. The experimental r a t i o n s were formulated to meet NRC n u t r i e n t requirements f o r growing pigs (National Academy of Sciences, 1973). The only exception was the p r o t e i n l e v e l of the PCM r a t i o n . The r a t i o n s were formulated to be s i m i l a r in energy l e v e l and u t i l i z e d adjustments in the l e v e l s of cassava and soybean in c o n t r i b u t i n g to the p r o t e i n d e f i c i e n c y . Water was supplied ad l i b i t u m by d r i n k i n g n i p p l e . The experimental animals were weighed biweekly and weight gains were recorded. Following weighing, blood samples were c o l l e c t e d from the a n t e r i o r vena cava, u t i l i z i n g the method of Mackenzie (1961). A twenty mi 1 1 i 1 i t r e • blood sample was obtained through a twenty gauge heparinized needle. The pigs were fasted f o r a period of twelve hours p r i o r to sampling, and the samples were taken at the same time of day to'minimize, d i u r n a l v a r i a t i o n s . Samples were allowed to c l o t , c e n t r i f u g e d and the serum obtained. The samples were then frozen f o r l a t e r a n a l y s i s . c. Feed Analys is Dry matter determinations were c a r r i e d out on t r i p l i c a t e samples of feed, by drying to a constant weight in a forced draught oven at 100°C. Nitrogen was determined in t r i p l i c a t e according to the macrokjeldahl method (A.OiA.C., 1975). Nitrogen content of the feed and feces was converted to crude p r o t e i n using the f a c t o r of 6.25 and r e s u l t s were expressed as a percentage of the i n i t i a l sample dry matter. - 44 -A c i d D e t e r g e n t F i b r e was d e t e r m i n e d i n t r i p l i c a t e , u t i l i z i n g t h e m i c r o - d i g e s t i o n p r o c e d u r e o f Waldern (1971). E t h e r e x t r a c t was d e t e r m i n e d on t r i p l i c a t e samples o f f e e d a c c o r d i n g t o t h e methods o f the A.O.A.C. (1975), u t i l i z i n g a g o l d f i s c h f a t e x t r a c t o r . Ash was d e t e r m i n e d on t r i p l i c a t e samples o f fee d by h e a t i n g a known w e i g h t o f sample i n a m u f f l e f u r n a c e a t 600°C f o r s i x h o u r s . Gross energy was d e t e r m i n e d on f e e d and f e c e s u s i n g a Gallenkamp A d i a b a t i c bomb c a l o r i m e t e r and t h e r e s u l t e x p r e s s e d as k i l o c a l o r i e s per k i l o g r a m o f sample. The a p p a r e n t d i g e s t i b i 1 i t y o f the f e e d s was d e t e r m i n e d u s i n g a m o d i f i e d method o f McCarthy et _aj_. (1974). F e c a l samples were c o l l e c t e d f o r a p e r i o d o f f o u r d a y s , t h r e e t i m e s d u r i n g t h e t r i a l and a f e e d sample was a l s o o b t a i n e d . F e c a l samples were d r i e d f o r 48 hours a t 60°C and then ground i n a hammer m i l l . F i v e grams o f f i n e l y ground f e e d o r f e c e s were b o i l e d i n 100 ml o f 4 N HC1 f o r t h i r t y m i n utes i n a 250 ml e r l e n m e y e r f l a s k . They were then f i l t e r e d t h r o u g h s i n t e r e d g l a s s f i l t e r i n g c r u c i b l e s which had been oven d r i e d , d e s i c c a t e d and weighed. They were then washed f r e e o f a c i d w i t h b o i l i n g w a t e r and ashed a t 600° C f o r s i x h o u r s . The samples were then reweighed and a c i d i n s o l u b l e ash was e x p r e s s e d as a p e r c e n t a g e o f the o r i g i n a l sample d r y m a t t e r . D i g e s t i b i l i t y was c a l c u l a t e d by d e t e r m i n i n g t h e r a t i o o f t h e c o n c e n t r a t i o n o f t h e r e f e r e n c e s u b s t a n c e t o t h a t o f a g i v e n n u t r i e n t i n t he f e e d and the same r a t i o i n the f e c e s r e s u l t i n g from t h a t f e e d . The r e s u l t s o f t h e p r o x i m a t e a n a l y s i s and d e t e r m i n a t i o n o f d i g e s t i b l e energy a r e p r e s e n t e d i n T a b l e 2. - h5 -Mineral content of the experimental rations was determined u t i l i z i n g a new block digestion procedure developed in the U.B.C. Animal Science Laboratory by Mr. Edward B.Cathcart. The procedure involved mixing .15 g of dried ground sample w i t h 6 mis of reagent grade H^ SO^  and pre-digesting with 1 ml 30% H 2°2 ' After the solution cleared, 3 grams of catalyst consisting of reagent grade K^SO^ and reagent grade HgO in a r a t i o of 22.kg:lg were added. The solution was then allowed to digest on a block heater pre-set to a range between 4l0°C and 425°C. Following digestion, the samples were allowed to cool and made up to volume with.deminera1ized water. Calcium, magnesium, iron, zinc and copper were determined by atomic absorption spectrophotometry while phosphorus was determined u t i l i z i n g the Technicon Autoanalysis method number 327-74W. Results of the mineral analysis are presented in Table 3-d. B i ochem i ca1 Aha l y s i s Serum calcium, phosphate, total proteins, albumin, cholesterol, glucose, l a c t i c dehydrogenase, a l k a l i n e phosphatase, glutamic-oxaloacetic transaminase, and blood urea nitrogen were determined using a survey model sequential multiple auto analyzer (S'MA-.l 2-90) . Amylase was determined on a single channel auto analyzer at the same commercial laboratory as the multi-channel analysis (B.C. Biomedical Lab). e. S t a t i s t i c a l Analysis of Data The data were subjected to analysis of variance using the computer program UBC BMD:10V (Bjerr i ng .et .aj.. , 1975). Sex, l i t t e r , and T a b l e 1. C o m p o s i t i o n o f E x p e r i m e n t a l D i e t s ( T r i a l 1 ) . D i e t a r y P r o t e i n L e v e l I n g r e d i e n t {%) 18% 4% Corn 21.70 21.70 Cassava 35.00 72.25 Soybean 32.30 .85 Prem i x * 4.00 4.00 Corn O i l 7.00 .1.20 V i t a m i n - M i n e r a l P r em ix s u p p l i e d / k g : V i t a m i n A, 4545 I .U.; V i t a m i n D, 363 I .U.; V i t a m i n E, 5-5 I .U.; C a l c i u m , 9-0 gm.; Pho spho ru s , 4 .5 gm.; S a l t , 7-5 gm.; I r o n , 120 mg.; I o d i n e , 160 mg. ; Z i n c , 120 mg. Table 2. Proximate A n a l y s i s of Experimental Rations ( T r i a l 1). Dietary P r o t e i n Level Component 18% k% Moisture 10.96 11.86 Crude P r o t e i n 20.74 6.36 Acid Detergent F i b r e 5.90 8.37 Ether E x t r a c t 6.60 1.61 Ash 7.91 7.94 Nitrogen Free E x t r a c t 47.89 63.86 Acid Insoluble Ash 0.86 2.10 Gross Energy (kcal/kg) 4481.0 3989-7 D i g e s t i b l e Energy (kcal/kg) 3955-2 3359-0 Table 3- Mineral A n a l y s i s of Rations ( T r i a l 1). Dietary P r o t e i n Level Mineral 18% 4% Calcium (%) 1.23 1.07 Phosphorus {%) .54 .42 Magnesium (%) .18 .13 Iron (%) .01 .01 1 Copper (mg/kg) 27.10 27.80 ^° Zinc (mg/kg) 121.80 104.20 - kS -treatment were the f a c t o r s taken i n t o account in the a n a l y s i s of variance t a b l e . Since there were empty eel 1s, no i n t e r a c t i o n terms were included. B. RESULTS a. Genera 1 Data f o r body weight gains are presented in Table 4. Pigs fed the 18% p r o t e i n r a t i o n gained an average of 24.1 kg during the du r a t i o n of the t r i a l compared with only 2.1 kg f o r the k% group. There was a s i g n i f i c a n t treatment e f f e c t f o u r weeks on t r i a l s (p< .01). There was no s i g n i f i c a n t sex or l i t t e r e f f e c t during the t r i a l . Weekly feed consumption i s presented in Table 5. The animals on the low p r o t e i n d i e t consumed considerably less feed than animals on the co n t r o l d i e t . However, the d i f f e r e n c e i s not as great when feed consumption is c a l c u l a t e d per kilogram of body weight. The low p r o t e i n group were very apa t h e t i c in t h e i r feeding habits and consumed less feed as the t r i a l progressed. Animals in the low p r o t e i n group had sparse h a i r , which was b r i t t l e and thinner than normal and seemed to lack the l u s t r e of the c o n t r o l group. Some animals showed abnormal g a i t , t h e i r legs became s t i f f , the hind legs being more severely a f f e c t e d than the fore legs. In a d d i t i o n , as the t r i a l progressed, there were frequent outbreaks of d i a r r h e a in the low p r o t e i n group. - 50 -b. P r o t e i n Metaboli sm Values f o r t o t a l serum pr o t e i n s are given in Table 6. Total p r o t e i n s were s i g n i f i c a n t l y lower ( p ^ . 0 1 ) in pigs a f t e r four weeks on the pr o t e i n d e f i c i e n t d i e t . Values f e l l from a mean of 5.16 g/100 ml at the beginning of the t r i a l to 4.03 g/100 ml a f t e r ten weeks. There was a s i g n i f i c a n t l i t t e r e f f e c t at the beginning of the t r i a l ( p ^ - O l ) , while sex e f f e c t was not s i g n i f i c a n t during the e n t i r e t r i a l . Serum albumin l e v e l s are given in Table 7. A f t e r four weeks on t r i a l , values f o r serum albumin were s i g n i f i c a n t l y lower (p±= .01) f o r pigs on the low p r o t e i n d i e t . The l e v e l s f e l l from a mean of 2.85 g/100 ml at the beginning of the t r i a l to a l e v e l of 1.76 g/100 ml a f t e r e i g h t weeks. Sex and l i t t e r showed no e f f e c t on serum albumin l e v e l s . Blood urea nitrogen values are given in Table 8. There were no s i g n i f i c a n t d i f f e r e n c e s f o r sex, l i t t e r or treatment. Values f o r serum amylase are presented in Table 3. Serum amylase l e v e l s were s i g n i f i c a n t l y lower ( p — . 0 1 ) in the p r o t e i n d e f i c i e n t group a f t e r four weeks on t r i a l . There was a s i g n i f i c a n t l i t t e r e f f e c t (p H£.0\) at the beginning of the t r i a l and a f t e r four weeks. Sex had no e f f e c t on l e v e l s of serum amylase through out the feeding t r i a l . Serum a l k a l i n e phosphatase values are given in Table 10. Serum a l k a l i n e phosphatase l e v e l s were s i g n i f i c a n t l y lower ( p ^ .01) in the p r o t e i n d e f i c i e n t pigs a f t e r four weeks on t r i a l . At the beginning of the t r i a l , a l k a l i n e phosphatase values averaged 135-5 m I.U./ml and t h i s value f e l l to 61.2 m I.U./ml on the four percent r a t i o n . Sex and l i t t e r had no influence on a l k a l i n e phosphatase l e v e l s . - 51 -Serum glutaminc o x a l o a c e t i c transaminase l e v e l s are given in Table 11. A s i g n i f i c a n t d i f f e r e n c e (p — . 01) was obtained a f t e r s i x weeks on t r i a l but t h i s d i f f e r e n c e was not maintained during the balance of the t r i a l . L i t t e r had a s i g n i f i c a n t i n f l u e n c e at the beginning of the t r i a l and a f t e r s i x weeks ( p ^ . 0 1 ) . However, no d i f f e r e n c e s were observed f o r sex. L a c t i c dehydrogenase values are given in Table 12. L a c t i c dehydrogenase l e v e l s were s i g n i f i c a n t l y higher (p— .01) in the p r o t e i n d e f i c i e n t pigs a f t e r ten weeks on t r i a l . Values rose from a mean of 321.6 Wroblewski units/ml at the beginning of the t r i a l to a mean of 519-7 Wroblewski units/ml a f t e r ten weeks. Sex and l i t t e r had no s i g n i f i c a n t e f f e c t on l a c t i c dehydrogenase l e v e l s during the t r i a l . c. Fat Metabolism Values f o r serum c h o l e s t e r o l are given in Table 13. P r o t e i n d e f i c i e n t pigs had s i g n i f i c a n t l y lower ( p ^ . 0 1 ) serum c h o l e s t e r o l values a f t e r e i g h t weeks on t r i a l . Sex and l i t t e r e f f e c t s were not s i g n i f i c a n t during the e n t i r e t r i a l . d. Carbohydrate Metaboli sm Values f o r serum glucose are given in Table 14. No s i g n i f i c a n t d i f f e r e n c e s were observed f or sex, l i t t e r , or treatment. Table h. Body Weights (kg) of Swine Fed Varying Levels of Pro t e i n ( T r i a l 1) Dietary P r o t e i n Level Weeks 18% k% 0 5.18 + .25 a 5.45 + .23 a 2 6-93 + .hSa 5.46 + -.29a k 9-56 + .77 a 5.45 + ' . 3 7 b 6 15.11+1.19* 5.73 + .hkb 8 2 , - 9 6 +1.5! a 6.10 + .54 b 1 0 29.26 +2.00a 7.57+I.36 b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (p-^-.Ol) Table 5. Weekly Feed Consumption (kg) in T r i a l 1. Dietary P r o t e i n Level Weeks 18% 4% 1 26.40 (16) 24.50 (16) 2 36.30 (16) 20.90 (13) 3 54.00 (16) 19.80 (13) 4 59-50 (16) 16.70 (13) 5 77.20 (16) 20.50 (12) 6 99-50 06) 16.00 (12) 7 117,80 (16) 16.30 (11) 8 137.50 (16) 18.90 (11) 9 152.60 (16) 14.80 (8) 10 171.50 (16) 6.20 (6) Values in parenthesis denote the average number of pigs on t r i a l during a week. ] 2 Table 6. Serum Total P r o t e i n (g/100 ml) in Swine Fed Varying Levels of Pr o t e i n ( T r i a l 1). ' Dietary P r o t e i n Level Weeks 18% 4% 0 5.31 + .2ka 5-16 + .15 a 2 4.96 + .10 a 4.76 + .12 a 4 4.76 + .19 a 4.18 + .09 b 6 4.99 + .10 a 3.91 + -10 b 8 5.83 + -10 a 3.89 + -15 b 10 5.87 + .I6 a 4.03 + .20 b 1 2 Values are Means + Standard Error of the Mean. Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). Table 7- Serum Albumin Levels (g/100 ml) in Swine Fed Varying Levels of Protein ( T r i a l 1) Dietary P r o t e i n Level Weeks 18% k% 0 2 . 9 5 + AkB 2 .85 + -12 a 2 2.96 + ,06 a 2.91 + .Ok3 k 2.31 + . 0 8 A l.g8 + .06 b 6 2 .98 + .06 a 2.55 + -03 b 8 3.18 + .10 a ]. 76 + .07 b 1 0 3.41 + .12 a 2.05 + - I 6 b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) 1 2 Table 8. Blood Urea Nitrogen Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1). ' Dietary P r o t e i n Level Weeks 18% 4% ° , 6-31 T 16.25 + . 8 5 a 2 17.20 r l . l 4 a 13.46 t . 9 I a 4 16.94 + 0.97 a 13.31 + ] . , 3 a 6 16.56 +0.79 a 16.83 +1.10a 8 15.44 + 0.86 a , 6.2,5 + .g 2a , 0 13.69 + 0.53 3 16.75 +3.6l a ON 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). Table 9- Serum Amylase Levels (Somogyi Units /100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1). ' Dietary P r o t e i n Level Weeks 18% 4% ° 320.2 + 2 1 . l a 335., + 2 g . g a 2 305.9 + 23.2 a 3,4.8 + 28.9 a k 310.8 + 15.9 a 234 .3 + 19.9 b 6 255.2 + 11.2a ,86.7 + 13.6b 8 245 .9 + 13.8a ,84 .0 + 18.5 a 1 0 307.4 + 17.2 a ,66.3 + 4 l . l b 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). Table 10. Serum A l k a l i n e Phosphatase Levels (m I.U./ml) in Swine Fed Varying Levels of Prote ( T r i a l 1). 1» 2 Dietary P r o t e i n Level Weeks 18% k% 0 125.6 + 9.0 A 135.5 + 7.2 A 2 125.5 + 6.5 A 122.8 + 6.8 A 4 136.8 + 8.9 A 87.8 + 9-9 B 6 143-9 + 9.3 A 77.5 + 6.4 B 8 " 130.6 + 6.6 A 64.6 + 9.8 B 10 122.9 + 8.8 A 61.2 +17.5 B 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (p — .01). Table 11. Serum Glutamic Oxaloacetic Transaminase Levels (Karmen Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l l ) . l ' 2 Dietary P r o t e i n Level Weeks 18% h% 0 39.47 + 2.60 a 41.50 + 2.71 a 2 41.13 + 3.50 a 43.00 + 2.56 a 4 57.75 + 4.53 a 43.62 + 3.71 a 6 58.00 + 2.05 a 49.08 + 1.65b 8 47.25 + 1.50a 41.20 + 2 .13 a 10 47.56 + 2.17 a 43.50 + 6.25 a 1 2 Values are Means + Standard Error of the Mean. Values w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). Table 12. Serum L a c t i c Dehydrogenase Levels (Wroblewski Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1)J>2 Dietary P r o t e i n Level Weeks 18% 4% 0 397.3 + 44.7 a 321.6 + 19.9 a 2 550.4 + 65.0 a 698.2 + 51.6 a h 552.9 + 74.7 a 488.0 j 38.5 a 6 320.6 + 15. l a 324.9 + 25.7 a 8 • 372.1 + 19.7 a 387.9 + 21.4 a 1 0 401.6 + 15.2a 519.7 + 28.3 b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). Table 1 3 - Serum Cholesterol Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a Dietary P r o t e i n Level Weeks . 1 8 % k% 0 82.87 + 5.74a 81 .75 + 8.17 a 2 95-53 + 2.70 a 98.62 + 3.82 a k 85.37 + 3.25 a 86.00 + h.27a 6 108.30 + 3.90 a 98.67 + 3.68 a 8 103.10 + 2.10 a 87.82 + 6 . 9 2 b 1 0 115-30 + 3 . 4 2 a 91.75 + 6.15 b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (p — .01) Table 14. Serum Glucose Levels (mg/100 ml) in Swine Fed Varying Levels of Pr o t e i n ( T r i a l 1) Dietary P r o t e i n Level Weeks 18* k% 0 132.5 + 7.7 a 113-1 + 4.4 a 2 85.3 + 2.7 a 79.1 + 5-0 a 4 80.6 + 4.0 a 91.2 + 3.5 a 6 87.5 + 1.8a 89.5 +-5-9a 8 93.2 + 2.9 a 84.6 + 5.2 a 10 94.6 + 3.0 a 100.0 +12.l a 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) Table 15- Serum Calcium Levels (mg/100 ml) in Swine Fed Varying Levels of P r t e i n ( T r i a l 1). Dietary P r o t e i n Level Weeks ]8% k% 0 9.16 + .22 a 8.95 + .24 a 2 9.90 + .12 a 9.84 + .15 a 4 9.26 + .22 a 8.86 + . I 8 a 6 8.97 + -13 a 7.82 + .17 b 8 10.03 + . I 3 a 8.16 + .31 b !0 9-85 + .10 a 8.13 + -25 b 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p ~ . 0 l ) Table 16. Serum Phosphate Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 1 Dietary P r o t e i n Level Weeks 1 8 % 4 % 0 6.87 + . 4 8 a 6 .73 + .24 a 2 8.19 + .25 a 7.28 + .I4 b 7.53 + -23 a 5.85 + . 1 9 b 6 8 . 0 4 + .17 a 6 .34 + . 3 1 b 8 8 . 6 4 f . l 3 a . 6 .25 + -I8 b 10 8 . 6 4 + .25 a 6 .70 + . 4 l b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) - 65 -e. Mineral Metabolism Serum calcium l e v e l s are l i s t e d in Table 15. Values f o r serum calcium were s i g n i f i c a n t l y lower ( p i z.Ol) a f t e r s i x weeks on the low p r o t e i n d i e t . There was a s i g n i f i c a n t l i t t e r e f f e c t a f t e r four weeks ( p ~ .01), w h i l e sex e f f e c t was not s i g n i f i c a n t during the e n t i r e t r i a l . Serum phosphate l e v e l s are given in Table 16. P r o t e i n d e f i c i e n t pigs had s i g n i f i c a n t l y lower (pi=..Ql) serum phosphate values a f t e r two weeks on t r i a l . Sex and l i t t e r had no s i g n i f i c a n t e f f e c t on serum phosphate l e v e l s during the e n t i r e t r i a l . C. DISCUSSION a. P r o t e i n MetabOli sm One of the most c o n s i s t e n t biochemical a l t e r a t i o n s found in PCM is lowered t o t a l serum p r o t e i n s . Several workers have considered t o t a l p r o teins to be a r e l i a b l e i n d i c a t o r of developing PCM (Baertl e_t_a_K, 1974; Haddad and Harfouche, 1971). In the present study, serum t o t a l p r o t e i n was s i g n i f i c a n t l y lower ( p — .01) in the malnourished group. This is in agreement with e a r l i e r work by Tumbleson et _a_L (1972b), in malnourished S i n c l a i r miniature swine. The c o n t r o l values are s i m i l a r to those obtained by Mi 1 ler\e_t j a j . (1961) f o r swine of a s i m i l a r age. - 66 -There are.several explanations f o r the reduced l e v e l of serum p r o t e i n in the malnourished p i g . Owing to the p r o t e i n d e f i c i e n c y , not only is the t o t a l concentration of amino acids low, but in a d d i t i o n , the pattern of ami no acids i s d i s t o f t e d (Whitehead and Dean, 1964). A l s o , as a r e s u l t of f a t t y i n f i l t r a t i o n , the f u n c t i o n a l c a p a c i t y of the l i v e r i s reduced, r e s u l t i n g in lower p r o t e i n s y n t h e s i s . Hypoalbuminemia i s another constant f i n d i n g in human PCM. In the present study, albumin l e v e l s in the p r o t e i n d e f i c i e n t group were reduced to 60% of those of the c o n t r o l s . S i m i l a r r e s u l t s have been obtained in swine by Tumbleson e_t .§_[. (1972b) . The reason albumin l e v e l s drop i s not c l e a r . T h e o r e t i c a l l y , the reduction in albumin l e v e l s could a r i s e e i t h e r from a lower rate of synthesis or an increased rate of catabolism of serum albumin. Research has indi c a t e d that c o r t i s o n e enhances albumin d e s t r u c t i o n (Searcy, 1969)- A l l e y n e and Young (I967), observed high l e v e l s of c i r c u l a t i n g c o r t i s o n e in malnourished c h i l d r e n . However, work by James and Hay (I968), i n d i c a t e that the c a t a b o l i c rate of albumin is reduced in PCM. It there f o r e seems more l i k e l y that a reduction in albumin synthesis is the cause of the reduction of c i r c u l a t i n g albumin. James and Hay (1968), observed that the rate of albumin synthesis was s i g n i f i c a n t l y lower in malnourished c h i l d r e n . This diminution in the rate of albumin synthesis is believed to be caused by a reduction in the a v a i l a b i l i t y of amino acids (Rothsch i 1 d ej: .aj.. , 1 969) -Blood urea nitrogen l e v e l s have been reported to be low in human PCM (Ar royave et _al_. , I962). However, in t h i s study, d i e t a r y treatment d i d not a f f e c t blood urea l e v e l s . Tumbleson (1972), a l s o f a i l e d to note any changes in blood urea l e v e l s in h i s malnourished pigs. - 67 -The q u a n t i t y and q u a l i t y of d i e t a r y p r o t e i n are important determinants of circu1 a t i n g 1evels of urea. Addis et a l - (1947), showed that the l e v e l of serum urea is p r o p o r t i o n a l to the p r o t e i n intake. As a r e s u l t of t h e i r observations,' i t was expected that serum urea l e v e l s would be low in the present t r i a 1 . The higher values observed may be explained by increased catabolism of body proteins due to a very small supply of proteins and c a l o r i e s . Serum amylase l e v e l s have been reported to be reduced to 72.6% of normal in Nigerian c h i l d r e n with PCM (Edozien, 1961)- The r e s u l t s of the present study i n d i c a t e that swine are more severely a f f e c t e d . Serum amylase in the malnourished group l e v e l s>"we re reduced to almost 50% of the c o n t r o l . Heard e t _ a l . (1957) reported s i m i l a r reductions in t h e i r malnourished group of pi g s . The reduction in serum amylase in PCM i s be l i e v e d to r e f l e c t pancreatic d i s f u n c t i o n ( V i t e r i et aj.. , 1964). The pathophysiologic a l t e r a t i o n s of the pancreas in PCM have been studied e x t e n s i v e l y (Pitchumoni, 1973; Barbezat and Hansen, 1968). The turnover of p r o t e i n in the pancreas is among the highest of any organ (Wheeler e_t a l . , 1949), and there f o r e i t is not s u r p r i s i n g that the pancreas i s r a p i d l y a f f e c t e d in sta t e s of p r o t e i n d e p r i v a t i o n . An e a r l y paper on the e s s e n t i a l pathology of PCM goes so f a r as to suggest that i t i s p r i m a r i l y a pancreatic d i s o r d e r secondary to m a l n u t r i t i o n , r e s u l t i n g in c i r r h o s i s of the l i v e r , p ancreatic c i r r h o s i s and a form of n e p h r i t i s (Davis, 1948). H i s t o l o g i c a l s t u d i e s in PCM pati e n t s have revealed atrophy o f ' a c i n a r . e e l 1s, with a diminution in the number of secretory granules (Pitchumoni, 1973)-- 68 -Reductions in serum a 1ka1ine phosphatase have been reported in human PCM. Edozien -'(.1 '961") noted that serum l e v e l s were reduced to 59.2% of norma.l . In the present t r i a l , a f t e r ten weeks on t e s t , a l kal ine phosphatase values were reduced :to49 .8% of normal. Although the a l k a l i n e phosphatases are found in most t i s s u e s , the source of the a c t i v i t y in serum i s s t i l l underfined. The l o c a t i o n of a l k a l i n e phosphatase in the growing individua 1 i s in the o s t e o b l a s t s and the condroblasts of the skeleton. Thus, most of the a l k a l i n e phosphatase is present in the bones of t h i s age group. In the a d u l t , the gas t o i n t e s t i n a 1 mucosa and l i v e r c o n t a i n the l a r g e s t amount of enzyme, .whi1e lung, spleen, t h y r o i d and placenta, a l s o contain the enzyme. It is believed that the low serum l e v e l s of a l k a l i n e phosphatase in PCM r e f l e c t a decreased r a t e of formation and remodelling of the bone matrix (Waterlow and Stephen, 1969)• It is p o s s i b l e that the disturbances of g a i t e x h i b i t e d by the malnourished pigs in the present study were a r e s u l t of t h i s a l t e r e d bone metabolism. Diminished serum a l k a l i n e phosphatase values have been noted in c h i l d r e n with underactive t h y r o i d glands (Cassar and Joseph, T 969)-Experimental work with pigs has shown that the a c t i v i t y of the procine t h y r o i d gland i s reduced by PCM ( P i a t t and Stewart, 1962). It was suggested that PCM a f f e c t s the t h y r o i d gland in two ways; by reducing the supply of thyro t r o p h i n and by reducing the supply of p r o t e o l y t i c enzymes required f o r the breakdown and release of the stored product ( P i a t t and Stewart, 1967). Another p o s s i b l e explanation f o r the low serum l e v e l s of a l k a l i n e phosphatase could be increased u r i n a r y e x c r e t i o n . It i s p o s s i b l e that the u r i n a r y output of t h i s enzyme i s influenced by adrenal c o r t i c a l - 69 -hormones, since the u r i n a r y a c t i v i t y of t h i s enzyme i s high in.Cushing 1s disease (Searcy, 1 9 6 9 ) . A l l e y n e and Young (I967) r e p o r t e d high plasma C o r t i s o l l e v e l s in chi1dren with PCM. Magnesium i s one of the ions necessary f o r the f u n c t i o n of a l k a l i n e phosphatase, and a magnesiurn d e f i c i e n c y r e s u l t s in lowered serum a l k a l i n e phosphatase (Wolf and W i l l i a m s , 1973). Linder e t - a l . (1963), reported lower serum magnesium l e v e l s in PCM p a t i e n t s . L a c t i c dehydrogenase values are elevated in human PCM (Zaki et a_L, I97O). Tn the present study, malnourished pigs had a mean LDH a c t i v i t y 20% higher than the c o n t r o l s a f t e r ten weeks on t r i a l . L a c t i c dehydrogenase acts in the g l y c o l y t i c c y c l e to c a t a l y z e the conversion of l a c t i c a c i d to pyruvic a c i d . It is widely d i s t r i b u t e d throughout the body with l e v e l s in t i s s u e s about 1000 f o l d higher than those normally observed in serum. Therefore leakage of the enzyme from even a small mass of damaged t i s s u e can increase the observed serum l e v e l . In man, elevated serum l e v e l s of LDH occur in myocardial i n f a r c t i o n , p ernicious anemia, leukemia, hepatic n e c r o s i s , renal disease and t r o p i c a l sprue (Wolf and W i l l i a m s , 1973). The major f a c t o r c o n t r i b u t i n g to the increase in c i r c u l a t i n g LDH l e v e l s in PCM i s b e l i e v e d ' t o be degeneration of somatic t i s s u e (Weimer et . a j . , 1959). There was no apparent d i e t a r y e f f e c t on serum l e v e l s of glutamic o x a l o a c e t i c transaminase in the present t r i a l . The s i g n i f i c a n t d i f f e r e n c e obtained a f t e r week s i x is d i f f i c u l t to e x p l a i n . In l i g h t of data obtained in experiment two, i t is believed that a real d i f f e r e n c e did not occur. Tumbleson et .a_l_. (1969) a 1 so did not observe any d i f f e r e n c e s in SGOT l e v e l s between c o n t r o l s and ma 1nourished pigs. - 70 -There i s considerable controversy in the 1 i t e r a t u r e regarding SGOT l e v e l s in ma 1nourished c h i I d r e n . Elevated l e v e l s have been reported by Sandstead et _§J_. (I965) , whi le others (Edozien, 1961 ; Smith, 1962).have reported no e l e v a t i o n of SGOT in PCM. SGOT ca t a l y z e s the t r a n s f e r of thealpha amino group from glutamic a c i d to o x a l o a c e t i c a c i d . The carbon skeleton so formed, can be used f o r energy s y n t h e s i s . The greate s t amounts of SGOT are present i n the 1iver, followed by l e s s e r amounts in the heart, and s k e l e t a l muscle. A small amount is present in the kidney, pancreas, red blood c e l l s , b r a i n and s k i n . Normally, almost a l l the transaminases are located w i t h i n the c e l l and r e l a t i v e l y small amounts of the enzyme c i r c u l a t e in the serum. Therefore, hypertransaminasemia i s an expression of a release of the enzyme through c e l l u l a r d e s t r u c t i o n . It i s thought that in PCM, the c i r c u l a t i n g enzymes a r i s e from the f o c i of nec r o s i s in l i v e r or muscle. It is a l s o p o s s i b l e that enzyme might leak out from i n t a c t c e l l s whose c e l l membranes may have suffered s t r u c t u r a l damage induced by p r o t e i n d e f i c i e n c y (Edozien and Rahim-Khan, 1'9'68). The coenzyme pyridoxal phosphate is necessary f o r the a c t i o n of transaminase enzymes. Pyridoxine d i s o r d e r s have been reported in PCM (Theron et. a±., 1961). b. Fat Metabolism Child r e n with PCM havebeen reported to have low serum c h o l e s t e r o l l e v e l s (Schendel and Hansen, 1958). S i m i l a r r e s u l t s were discovered in the present t r i a l . This however, is in cont r a s t to the animal - 71 -studies of Tumbleson (1969) who found no d i f f e r e n c e s between c o n t r o l and malnourished p i g s , and Tumbleson (1972) who reported elevated serum c h o l e s t e r o l in h i s malnourished group; The i n i t i a l step in the synthesis of c h o l e s t e r o l involves the combination of acetate with coenzyme A. Consequently, many amino a c i d s , carbohydrates and f a t t y a c i d s , when supplied in excess of other metabolic needs can c o n t r i b u t e to the c h o l e s t e r o l pool. Whitehead and Harland (1966), on the basis of elevated blood pyruvate l e v e l s , postulated that there was a depression of pyruvate o x i d a t i o n and impaired entry of pyruvate i n t o the Krebs c y c l e of PCM p a t i e n t s . Therefore, i t would appear that acetate l e v e l s may be lower in PCM, which may be r e f l e c t e d in reduced synthesis of c h o l e s t e r o l . c. Carbohydrate Metabolism D i e t a r y treatment d i d not appear to have any e f f e c t on serum glucose l e v e l s in the present study.. This is in agreement with work done by Bowie (1964) and Jaya Rao (I965) in human studies and Tumbleson (1972) in p igs. d. Mineral Metabolism Although studies on calcium metabolism in human PCM have shown v a r i a b l e r e s u l t s , work with swine appears to.be more c o n c l u s i v e . Tumbleson et .a]. (1972) and Tumbleson _et a]_. (1969) reported reduced serum calcium in malnourished swine. This has been confirmed in the present study. - 72 -There are many f a c t o r s which could c o n t r i b u t e . t o the diminished calcium l e v e l s . Prolonged a d m i n i s t r a t i o n of adrena1 c o r t i c a 1 hormones or ad renal hyperfunction i s o f t e n followed by s k e l e t a l r a r e f a c t i o n (Sissons, 1971). A l l e y n e and Young (1967) have shown elevated l e v e l s of a d r e n a l c o r t i c a l hormones in PCM. Severe inflammatory attacks of pancreatic disease are o f t e n accompanied by an impressive d e c l i n e in serum calcium l e v e l s (Searcy, 1969). It has been suggested that PCM i s p r i m a r i l y a pancreatic disease secondary to m a l n u t r i t i o n (Davis, 1948). Large q u a n t i t i e s of calcium can be l o s t in cases of d i a r r h e a , p a r t i c u l a r l y those i n v o l v i n g excessive f e c a l e x c r e t i o n of f a t (Thomas and Howard, 1964). Shenolikar and Narasinga Rao (1968) observed higher f e c a l calcium e x c r e t i o n in rats on a low p r o t e i n intake. Serum calcium is r a p i d l y reduced to a low l e v e l in c o n d i t i o n s of hypoparathyroidism (Searcy, 1969). The e f f e c t s caused by PCM upon endocrine fun c t i o n s are not well known. There are no published reports concerning the a c t i v i t y of the parathyroids in PCM. However, PCM has been shown to adversely a f f e c t other endocrine glands (Heard and Stewart, 1971; Godard, 1974) and i t i s p o s s i b l e that the parathyroids might a l s o be a f f e c t e d . It is p o s s i b l e that the reduced a l k a l i n e phosphatase l e v e l s observed in the present t r i a l r e s u l t in less bone calcium being m o b i l i z e d . In a d d i t i o n , i t has been shown that calcium binding p r o t e i n i s reduced in the i n t e s t i n e of p r o t e i n d e f i c i e n t rats (LeRoith and Pimstonej 1973). If the same i s true in p i g s , t h i s reduction could help to e x p l a i n the lower serum calcium l e v e l s observed. - 73 -Hypophosphataemia was observed in the p r o t e i n d e f i c i e n t p i g s . This is in accord w i t h . the : human studies of Sandstead _§_t _a_l_. (T965) , and Bjornesjo et_ aj_. (I965) as well as the animal studies of Tumbleson (1972). Circulating.phosphorus l e v e l s could be reduced by an impair-ment in i n t e s t i n a l absorption or through some impairment in the renal excretory mechanism. Phosphorus absorption c o r r e l a t e s p o s i t i v e l y with the concentration of calcium present and i t i s reduced in c o n d i t i o n s i n v o l v i n g an impairment in the i n t e s t i n a l uptake of calcium . (Searcy, 1969)- Thus the lowered calcium binding p r o t e i n in PCM (LeRoith and Pimstone (1973) may be r e f l e c t e d in lower absorption of phosphorus. The renal tubular mechanism f o r phosphate reabsorption, operates to maintain c i r c u l a t i n g l e v e l s of the mineral w i t h i n normal l i m i t s . The reabsorption process i s v a r i a b l e and i s influenced by the s t a t e of the body reserves of the m i n e r a l , parathyroid hormone, i n s u l i n , vitamin D, adr e n o c o r t i c a l hormones as well as p r o t e i n intake (Searcy, T9&9)• The lower serum a 1kaline phosphatase l e v e l s observed in the present t r i a l could r e s u l t in less bone phosphorus being m o b i l i z e d . - Ik -EXPERIMENT 11 A. MATERIALS AND METHODS a. Object ives The r e s u l t s of t r i a l one would seem to i n d i c a t e that the pig is a good model r e l a t i v e to the human with which to study p r o t e i n - c a l o r i e m a l n u t r i t i o n . However, i t is not known at what l e v e l of p r o t e i n intake PCM develops. The object of t h i s t r i a l was to study the e f f e c t s of pr o t e i n intake on the development of the symptoms of PCM. In a d d i t i o n , i t was intended that t h i s experiment would provide some i n s i g h t i n t o which parameter i s the most s e n s i t i v e index of the n u t r i t i o n a l s t a t e of the animal b. Experimental Procedures Forty Yorkshire and Yo r k s h i r e X Landrace pigs chosen as c l o s e as p o s s i b l e to be of the same age and weight were used in t h i s study. The experimental animals were weaned at 28 days of age and assigned to a d i e t a r y treatment on the basis o f sex, l i t t e r and weight. Five treatments were used with one pen per treatment. Each pen contained four barrows and four g i 1 t s . D e t a i l s of research f a c i l i t i e s , feeding methods, bleeding methods, and weighing procedure are as given f o r experiment one. The composition of the experimental r a t i o n s i s shown in Table 17- The experimental r a t i o n s were formulated to meet NRC requirements f o r growing pigs with the exception of the p r o t e i n l e v e l . In t h i s t r i a l , the d i f f e r e n t p r o t e i n l e v e l s were obtained by d i l u t i n g the basal r a t i o n (18%) - 75 -with corn s t a r c h . These d i e t s allowed the amino ac i d r a t i o s presented to the experimental animals to.be constant among treatments. D i f f e r e n t l e v e l s of animal t a l l o w were added to keep the d i e t s i s o c a l o r i c . Rations were formulated to contain 18%, 1 0%','.8%,:$%, and k% p r o t e i n . Actual l e v e l s of p r o t e i n are presented in Table 18. c. A n a l y t i c a l Methods A n a l y t i c a l methods for. proximate a n a l y s i s , d i g e s t i b l e energy, and mineral a n a l y s i s are given in t r i a l one. The r e s u l t s of the proximate a n a l y s i s are given in Table 18 while mineral a n a l y s i s is presented in Table 19-d. Biochemical A n a l y s i s Biochemical a n a l y s i s f o r parameters repeated in experiment two are as given f o r experiment one. Serum copper, i r o n , magnesium, and z i n c , were determined by atomic absorption, u t i l i z i n g a Unicam SP 90 spectrophotometer. For the determination of magnesium, samples were d i l u t e d f i f t y times and f o r serum z i n c the samples were d i l u t e d f i v e times. Copper and iron were determined on undiluted serum samples. The 1ivers of any animals which died on t r i a l were removed and frozen f o r l a t e r a n a l y s i s . Control l i v e r s were obtained from pigs of approximately the same:weight, ki1 led as s u c k l i n g pigs at a slaughter p l a n t . The l i v e r s were d r i e d at60°C f o r 48 hours and ground in a hammer - 76 -m i l l . Fat was extracted on t r i p l i c a t e one gram samples u t i l i z i n g a g o l d f i s c h f a t e x t r a c t o r . The f i n a l r e s u l t s were expressed as a percentage of the dr i e d sample weight. Total body water was determined using the t r i t i a t e d body water technique of B r i n kma n e_t _aj_. (1965). Three animals s e l e c t e d at random from the \8% and k% treatments were used in t h i s study. T r i t i a t e d water was purchased from New England Nuclear w i t h a s p e c i f i c a c t i v i t y of one m i l l i c u r i e per m i l l N i t r e . It was considered d e s i r a b l e to maintain the t r a c e r in a concentrated form. Therefore, 2.5 m i l l N i t r e s of the t r a c e r was d i l u t e d with p h y s i o l o g i c a l s a l i n e to make an i n j e c t a b l e dose of 100 uc/ml. Ten micr o c u r i e s per kilogram body weight were then i n j e c t e d intraperitonea11y. Approximately f i v e and s i x hours post i n j e c t i o n , f i v e m i l l N i t r e s of blood was obtained by vena cava puncture. The samples were then allowed to c l o t , c e n t r i f u g e d and the serum obtained. In the past, i t has been deemed necessary to obt a i n water f r e e of pigment and p r o t e i n in order to achieve adequate counting e f f i c i e n c y . Several methods have been used i n c l u d i n g benzene d i s t i l l a t i o n (Werbin, 1959), d e p r o t e i n i z a t i o n with TCA (Langhan et a j . 1956) and vaccum sublimation (Vaughan and B o l i n g , 1961). Recently, several new s c i n t i l l a t i o n c o c k t a i l s have come on the market which promise much higher counting e f f i c i e n c i e s at much higher water contents ( i . e . Handifluor or Aquasol). A p r e l i m i n a r y t r i a l was run to compare counting water obtained by l y p h i 1 i z a t i o n to d i r e c t counting of serum. The d i f f e r e n c e was not s i g n i f i c a n t and ther e f o r e f o r t h i s t r i a l vaccum sublimation was not used. T r i p l i c a t e one-tenth m i l l N i t r e serum samples were added to ten mi 11i1i t r e s of the s c i n t i 1 1 a t i o n c o c k t a i l Handifluor (Ma 11inckrodt). - 77 -An a l i q u o t of the i n j e c t e d dose was d i l u t e d 1/200 with disti1 1ed water and 1/10 mi 1 1i1itre of t h i s sample was counted in the same manner as the samples. A quench c o r r e c t i o n curve was obtained by counting quenched standards from Amershan/Searle and p l o t t i n g counting e f f i c i e n c y vs channels net count rate r a t i o . Background was determined by counting a reference background v i a l from Amershan/Searle. Mean serum a c t i v i t y was used to c a l c u l a t e body water according to the equation of Kay, Jones and Smart (1966). ... Dose i njected (dpm) Body Water (ml) = — : — ^ — _ E q u i l i b r i u m a c t i v i t y / m l serum e. Stat i s t ica1 Analys i s The data were subjected to a n a l y s i s of variance using the computer program UBC BMD: 1 0V ( B j e r r i ng _et_ aj_., 1975). Sex, l i t t e r , and treatment were the f a c t o r s taken i n t o account in the a n a l y s i s of variance t a b l e . Since there were empty c e l l s , no i n t e r a c t i o n terms were included. Means from comparisons showing a s i g n i f i c a n t "F" were tested using Tukey's te s t (1953). Table 17'. Composition of Experimental Diets ( T r i a l 2). Ingredients (%) 18% D i eta ry 10% P r o t e i n Level 8% 6% 4% Corn 21.70 12.05 9.64 7.23 4.82 Cassava 35.00 19.44 15.55 11.66 7-77 Soybean 32.30 17.94 14.35 10.76 7.17 Corn Starch 0.00 40.57 50.71 60.85 70.74 Corn Oi1 7.00 0.00 0.00 0.00 0.00 Tallow 0.00 6.00 5.75 5-50 5.50 Premix* 4.00 4.00 4.00 4.00 4.00 Vitamin-Mineral Premix supplied/kg: Vitamin A, 4545 I.U.; Vitamin D, 363 I.U.; Vitamin E, 5.5 I.U.; Calcium, 9-0 gm; Phosphorus, 4.5 gm; S a l t , 7-5 gm; Iron, 120 mg; Iodine, 160 mg; Zin c , 120 mg. Table 18. Proximate A n a l y s i s of Experimental Rations ( T r i a l 2). Dietary P r o t e i n Level Component 18% 10% 8% 6% 4% Moi sture 10.96 12.76 12.57 14.04 13.65 Crude P r o t e i n 20.74 11.84 10.70 8.07 5.80 A.D.F. 5.90 3-35 2.77 2.08 1.28 Ether Extract 6.60 5-50 5.19 6.21 5.76 Ash 7.91 6.33 6.19 5.33 4.71 N.F.E. 47.89 60.22 62.58 64.27 68.80 A.I.A. 0.86 0.42 0.39 0.41 0.15 G.E. (kcal/kg) 4481.6 4 324.9 4274.9 4309-9 4288.3 D.E. (kcal/kg) 3955.2 4035.1 3940.9 4052.0 4147.3 Table 19- Mineral A n a l y s i s of Rations ( T r i a l 2). Dietary Protein Level Mineral 18% 10% 8% 6% 4% Calcium (%) 1.23 1.03 1.10 0.96 0.96 Phosphorus (%) 0.54 0.46 0.47 0.44 0.42 Magnesium (%) 0.18 0.11 0.10 0.07 0.07 Iron (%) 0.01 0.01 0.01 0.01 0.01 Copper (mg/kg) 27-10 29.60 27.10 31.40 27.80 Zinc (mg/kg) 121.80 50.50 94.70 112.40 103-50 - 81 -B. RESULTS a. Genera 1 Weekly feed consumption data i s recorded in Table 20. There was a l i n e a r r e l a t i o n s h i p between feed consumption and percent d i e t a r y p r o t e i n ( r 2 =/98 at week 10). Body weights are recorded in Table 21. There was a s i g n i f i c a n t (p .01) treatment e f f e c t a f t e r two weeks on t r i a l . In a d d i t i o n , there was a s i g n i f i c a n t ( p ~ .01) l i t t e r e f f e c t up to s i x weeks on t r i a l . Sex had no i n f l u e n c e on body weight gain during the t r i a l . As in t r i a l one, the animals on the low p r o t e i n d i e t s e x h i b i t e d disturbances of g a i t , apathy, d i a r r h e a , and sparse h a i r growth. There were no v i s u a l symptoms of edema. b. P r o t e i ri Metabolism The r e s u l t s of the a n a l y s i s f o r serum t o t a l p r oteins are presented in Table 22. There was a s i g n i f i c a n t treatment e f f e c t a f t e r four weeks on t r i a l {o~ .01). Serum t o t a l p r o t e i n s d e c l i n e d with decreasing d i e t a r y p r o t e i n . There was no s i g n i f i c a n t sex or l i t t e r e f f e c t during the t r i a l . Mean values f o r serum albumin are presented in Table 23. There was a s i g n i f i c a n t {p— .01) treatment e f f e c t a f t e r four weeks on t r i a l . Serum albumin declined with decreasing d i e t a r y p r o t e i n . There was a s i g n i f i c a n t ( p _ ^ . 0 l ) l i t t e r e f f e c t a f t e r four weeks and at the beginning of the t r i a l . There was no s i g n i f i c a n t sex e f f e c t during the e n t i r e t r i a l . - 82 -Blood urea nitrogen (BUN) values are presented in Table 24. There was a s i g n i f i c a n t (pz=. .01) treatment e f f e c t a f t e r s i x a n d e i g h t weeks on t r i a l , but t h i s disappeared at week ten. The eighteen percent r a t i o n had s i g n i f i c a n t l y higher BUN l e v e l s than d i d the other treatments. There was a s i g n i f i c a n t (p^r.OT) l i t t e r e f f e c t a f t e r "four weeks on t r i a l , w h i l e sex had no i n f l u e n c e on BUN values. Mean values f o r serum amylase are presented in Table 25. There was a s i g n i f i c a n t treatment e f f e c t a f t e r four weeks on t r i a l ( p — .01). Serum amylase values d e c l i n e d w i t h decreasing d i e t a r y p r o t e i n . There was a s i g n i f i c a n t (p.£~.0l) l i t t e r e f f e c t at the beginning of the t r i a l and a f t e r four and ten weeks on experiment. Sex had no e f f e c t . The r e s u l t s of the a n a l y s i s f o r a l k a l i n e phosphatase are presented in Table 26. There was a s i g n i f i c a n t (p.fz.01) treatment e f f e c t a f t e r four weeks on t r i a l . At four and s i x weeks, a l k a l i n e phosphatase values d e c l i n e d with decreasing d i e t a r y p r o t e i n . However, at e i g h t and ten weeks, there was a quadratic e f f e c t , with the eight percent p r o t e i n r a t i o n being higher than the other groups. L i t t e r e f f e c t was s i g n i f i c a n t (p zz. .01) at the beginning of the t r i a l and a f t e r four weeks. Sex had no influe n c e on a l k a l i n e phosphatase l e v e l s during the t r i a l . Values f o r serum glutamic o x a l o a c e t i c transaminase are presented in Table 21. There was no s i g n i f i c a n t e f f e c t f o r sex, l i t t e r , or treatment. The r e s u l t s of serum l a c t i c dehydrogenase a n a l y s i s are presented in Table 28. There was a s i g n i f i c a n t (p — . 0 1 ) treatment e f f e c t a f t e r ten weeks on t r i a l . The four percent p r o t e i n r a t i o n had s i g n i f i c a n t l y higher LDH l e v e l s than d i d the other treatments. Sex and l i t t e r had no i n f l u e n c e on LDH l e v e l s during the t r i a l . - 83 -c. Fat Metabolism The r e s u l t s of the a n a l y s i s f o r serum c h o l e s t e r o l are presented in Table 29. There was a s i g n i f i c a n t (p ^ . 0 1 ) treatment e f f e c t at two weeks and at ten weeks. At two weeks, the T8% r a t i o n was lower than the other treatments. At ten weeks the four percent r a t i o n was s i g n i f i c a n t l y lower than.the other treatments. There was no s i g n i f i c a n t e f f e c t f o r sex or l i t t e r . L i v e r s obtained from those animals on the low p r o t e i n r a t i o n which died during the experiment were smaller and contained s i g n i f i c a n t l y more f a t ( p i ~ . 0 l ) than l i v e r s obtained from animals k i l l e d at the slaughter p l a n t . Malnourished animals had a mean of 3l'.2k% l i v e r f a t compared with a mean of 11.83% f o r the c o n t r o l s . d. Carbohydrate Metabolism Serum glucose l e v e l s are presented in Table 30. There was a s i g n i f i c a n t (p£ = . 0 1 ) treatment e f f e c t a f t e r f o u r , s i x , and e i g h t weeks, but t h i s disappeared at week ten. A f t e r weeks four and s i x , serum glucose l e v e l s appeared to increase with decreasing d i e t a r y p r o t e i n . A f t e r week e i g h t , glucose l e v e l s were lowest on the 8% r a t i o n and highest on the 6% r a t i o n . There was a s i g n i f i c a n t ( p ^ . 0 1 ) sex e f f e c t a f t e r two weeks on t r i a l , w h i l e l i t t e r was not s i g n i f i c a n t during the t r i a l . - 84 -e. Water and E l e c t r o l y t e Metabolism Mean values f o r serum calcium are presented in Table 31-There was aLsignificant treatment e f f e c t a f t e r s i x weeks on t r i a l . Serum calcium declined with d i e t a r y p r o t e i n l e v e l . Sex and l i t t e r were not s i g n i f i c a n t . The r e s u l t s of serum copper a n a l y s i s are presented in Table 32. No d i f f e r e n c e s in serum copper were picked up u n t i l a f t e r ten weeks on the t r i a l . A f t e r ten weeks, there was a s i g n i f i c a n t treatment e f f e c t (p — .01). Serum copper l e v e l s d e c l i n e d with decreasing d i e t a r y p r o t e i n . There was a steady d e c l i n e u n t i l the 8% l e v e l was reached, t h e r e a f t e r , serum copper values l e v e l l e d o f f . There was a s i g n i f i c a n t ( p — .01) l i t t e r e f f e c t a f t e r ten weeks on t r i a l , w h i l e sex e f f e c t was not s i g n i f i c i a n t during the e n t i r e t r i a l . Values f o r serum ir o n are presented in Table 33. No s i g n i f i c a n t d i f f e r e n c e s were observed f o r sex, l i t t e r , or treatment. The r e s u l t s of serum magnesium a n a l y s i s are presented in Table 34- There was a s i g n i f i c a n t treatment e f f e c t a f t e r s i x weeks on t r i a l (p — .01). Serum magnesium values d e c l i n e d with decreasing p r o t e i n l e v e l . There was no s i g n i f i c a n t sex or l i t t e r e f f e c t . Mean values f o r serum phosphate are presented in Table 35. There was a s i g n i f i c a n t treatment e f f e c t a f t e r two weeks on t r i a l (p~ .01). Serum phosphate values d e c l i n e d w i t h decreasing d i e t a r y p r o t e i n . There was a s i g n i f i c a n t l i t t e r e f f e c t at the beginning of the t r i a l , w h i l e sex e f f e c t was not s i g n i f i c a n t during the e n t i r e t r i a l . - 85 -Mean values f o r serum z i n c are presented in Table 36- There was a s i g n i f i c a n t treatment e f f e c t a f t e r ten weeks on t r i a l (pfE:.01). The eight percent p r o t e i n ration,had s i g n i f i c a n t l y higher serum z i n c l e v e l s than d i d the other treatments. No sex or T i t t e r e f f e c t was observed. The animals on the four percent r a t i o n had s i g n i f i c a n t l y greater t o t a l body water (p — . 0 1 ) than d i d those pigs fed the 18% r a t i o n . The three animals on the 4% r a t i o n had a mean t o t a l body water of 78.71% compared with a mean of 67-84% f o r the three animals fed the 18% r a t i o n . Table 20. Weekly Feed Consumption (kg) in T r i a l 2. feeks 18% 10% Dietary P r o t e i n 8% Level 6% 4% 1 14.80(8) 12.00(8) 10.60(8) 11.30(8) 12.10(8)* 2 23.20(8) 17.30(8) 14.60(8) 14.50(8) 10.50(8) 3 29.90(8) 19.90(8) 16.30(8) 13.40(8) 11.20(8) 4 37-20(8) 24.70(8) 19.70(8) 16.50(8) 13.30(8) 5 59.80(8) 34.10(8) 24.40(8) 21.40(8) 16.70(8) 6 64.70(8) 37.30(8) 25.60(8) 18.80(8) 11 .20(8) 7 73.40(8) 46.10(8) 35.40(8) 22.00(8) 11.40(7) 8 87.90(8) 47.80(8) 35.80(8) 21.00(8) 10.20(7) 9 115.10(8) 58.20(8) 47.10(8) 26.40(8) 9.10(6) 10 89.30(8) 48.70(8) 29.80(8) 17.20(7) 3-60(5) Values in parenthesis are average number of animals per pen during the week. 1 2 Table 21. Body Weights (kg) of Swine Fed Varying Levels of Pr o t e i n ( T r i a l 2). ' Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 2 4 6 8 10 7.38+ .22 a 7.27 + -24 a 6.74 + -39 a 8 . 7 9 ± . l 8 a 8.06 + . 23 a b l . k h + M a b 12.80 + .45 a 9.80 + .36 b 8.50 + .51 b 19.26 + .85 a 11.90 + .51 b 9.45 + -50 c 28.53 +1.22a 16.25 +1.01b 12.12 + .hSC 34.37 +1.583 20.72 +1.57b 14.45 + -46 c 6.90 + .49 a 7.00 + .50 a 7.41 + .51 a b7-04 +. .45 b 7.64 + .51° 7.30 + .75° 8.05 + .56° 7.03 + .59° 8.75 + -65 d 7.43 + .45 d 8.80 + .76 c d6.78 + .55 d 00 1 2 Values are Means + Standard E r r o r of the Mean. Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) . Table 22. Serum Total P r o t e i n (g/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). Dietary P r o t e i n Level Weeks 18* 10% 8% 6% 4% 0 4.94 + • 05a 4.95 + . l l a 4.86 + .04 a 4.91 + • 07 a 5.01 + . l l a 2 4.44 + .08 a 4.23 + .09 a 4.24 + .10 a 4.28 + • I3 a 4.15 + . l l a 4 4.73 + .10 a 4.18 + . 12 b c 4.15 + . 08 b c 4.23 + .12 b 3.71 + • 07C 6 5.21 + .13 a 4.25 + .I6 b 4.21 + . l l b 4.20 + • 13 b 3-67 + . l l b 8 5.64 + . l l a 4.35 + • 15 b 4.25 + . l l b 4.08 + . 20 b c 3.40 + .08 c 10 5-31 + • 17a 4.26 + .I6 b 4.31 + .I6 b 3.93 + • 33 b 3.28 + • 13b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (p ~.01) . Table 23. Serum Albumin Levels (g/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 3.18 + .08 a 3.18 + .13 a 3.04 + .12 a 3.15 + . i o a 3.21 + . i o a 2 2.46 + • 0 9 a 2.29 + • 07a 2.30 + • 093 2.31 + .08 a 2.28 + • 09 3 4 2.26 + . l l a 1.86 + . l l b 1.77 + .08 b 1.76 + .08 b 1.67 + .08 b 6 2.41 + . l l a 1.51 + • 09b 1.44 + V 1.28 + .06 b 1 .24 + .08 b 8 3.17 + .10 a 1.83 + .08 b 1.61 + . 08 b c 1.40 + .07° 1.23 + .06 c 10 2.70 + .19 a 1.86 + . l l b 1.45 + .08 b 1.26 + . l l b 1.12 + . l l b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p - ^ . 0 1 ) . Table 24. Blood Urea Nitrogen Levels (mg/100 ml) in Swine Fed Varying Protein Levels ( T r i a l 2). Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 11.75 + 1.21 a 11.87 + 1.04a 11.62 + 1.22a 12.25 + 1 -32 a 12.75 + 1.28' 2 12.62 + 1.31 a 9.25 + 0.78 c 11.12 + 0.96a 9.63 + 0.78 a 10.00 + 0.73' h 12.00 + 0.5ka 13.62 + 0.97 3 13-71 + 0.77 3 13.00 + 0.95 3 13-43 + 1.36' 6 12.87 + 0.58 a 10.62 + 1.00 a b 9-86 + 0.85 a b 8.63 + 0.6 3 b 8.86+0 .80 ' 8 16.14 + 0.85 a 12.00 + 1.24 a b 10.37 + 0.78 b 14.37 + "l . '17 a b 9-8 3 + 0.65 10 11.25 +1 . 2 7 3 13-00 +1.87 3 9-75 + 0.92 a 11.83 + l . l l a 9.60+1.16' 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p ^ . 0 1 ) . Table 25. Serum Amylase Levels ( T r i a l 2 ) . 1 ' 2 (Somogyi Units/100 ml) in Swine Fed Varying Levels of P r o t e i n Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 228.6+17.l a 242.1+21.6a 236.9+25.7a 276.4+29.l a 271.2+29.l a 2 348.8+18.6a 367.6+31.63 356.8+20.2a 325.8+31.5a 330.7+26.9a 4 379.0+12.7a 336.4+16.l a b 347.9+31.6ab 292.8+26.4b 283.1+33.6b 6 446.7+16.8a 406.9+26.3 a b 377.8+25.7ab 325.7+22.8b 285.0+28.5b 8 374.0+19.3a 333.4+26.0 a b c 342.3+15.6ab 257.4+22.0bc 217.4+24.3C 10 444.1+34.5a 438.5+22.0ab 448.9+26.6a 309-4+29.9b c 228.6+21.9° 1 2 Values are Means + Standard E r r o r of the Mean. Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) . Table 26. Serum A l k a l i n e Phosphatase (m I.U./ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). ' Dietary P r o t e i n Level Weeks 18% . 10% 8% 6% 4% 0 183.1+16.6a 190.6+20.3a 179-4+17.4a 184.9+19.8a 184.4+15.4a 2 130.6+ 7.2 a 152.7+13-5a 125-9+ 9-2 a 125.0+ 5 . l a 115.9+ 7.9 a 4 134.7+ 9.6 a 124.2+ 9 .9 a b 119.6+ 9 -5 a b 91.6+ 9 . 7 b c 76.9+ 5.2 C 6 138.5+ 9.0 a 134.1+ 9-9 a 123.9+13.4ab 79-6+ 9 . 1 b c 49.1+ 6.1 c 8 119.3+ 5 .9 a b 145.1+ 9.4 a 149.9+I3.6a 95.0+11.7b c 49.67+4.3° 10 97.9+ 9 -7 a C 177.9+13.8a 272.9+26.73b 112.8+21.9 a° 43.8+ 5.1° 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) . Table 27. Serum Glutamic Oxaloacetic Transaminase (Karmen Units/ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2).1,2 Dietary P r o t e i n Level Weeks. 18% 10% 8% 6% 4% 0 71.3 + 0.1 a 68.6 + 5.0 a 68.0 + 5.9 a 66.1 + 3.2 a 82.9 + 5.8 a 2 41.6 + 2.9 a 50.3 + 2.6 a 45.1 + 2.6 a 42.3 + 3.9 a 46.4 + 4.4 a 4 57.1 +10.2a 63.5 +10.5a 49.3 + 4.7 a 45.5 + 2.5 a 60.7 + 6.0 a 6 51.1 + 4 .1 3 52.2 + 5.8a 48.3 + 4.8 a 48.1 + 7.0 a 55.7 +13.8a 8 52.6 + 4.0 a 44.9 + 3.8 a 44.1 + 2.9 a 43.4 + 2.5 a 40.17 + 5.41 10 48.1 + 4 .1 a 50.4 + 2.8 a 49.8 + 6.1 a 38.7 + 3.6 a 60.0 + 16.85 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (p — . 0 1 ) . Table 28. Serum L a c t i c Dehydrogenase (Wroblewski Units/ml) in Swine Fed Varying Levels of Pr o t e i n ( T r i a l 2 ) . 1 ' 2 Dietary P r o t e i n Level Weeks ' 18% 10% 8% 6% 4% 0 727.5+86.0a 600.2+46.3* 711.9+56.2a 810.7+100.9* 740.2+33-3* 2 386.4+21.6a 445.5+18.9* 464.5+16.l a 450.7+23.0* 402.1+9-5* 4 393-4+25.2a 661.6+126.9* 426.9+22.8a 430.0+21;8* 518.9+60.5* 6 393-0+17.3a 429.1+21.7a 404.3+26.9* 454.6+48.4* 462.0+74.2a 8 376.9+23.2a 395.6+15.7* 4 31.7+17.6 a 408.6+23.7* 410.2+33.2a 10 415.2+25.3* 473.5+16.7* 666.7+81.4a 598.7+63-2a 736.4+124.3b 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) . T a b l e 29. S e r u m C h o l e s t e r o l L e v e l s (mg/100 m l ) i n S w i n e F e d V a r y i n g L e v e l s o f P r o t e i n ( T r i a l 2). ' D i e t a r y P r o t e i n L e v e l W e e k s 18% 10% 8% 6% 4% 0 94.00 + 6.62a 95.62 + 5.49a 104.70 +10.68a 87.50 +4.56a 101.70+ 11.38a 2 81.50 + 3-88a 87.75 + 2.96 a b 91-37 + 5.03 a b 105.50+ 5.03b 98.25+ 4.05 a b 4 95.62 + 4.78a 105-00+ 6.32a 105-30+ 4.88a 113-00+ 7-53a 109.10+ 4.40a 6 101.00+ 3-52a 117.10+ 5.24a 112.30+ 6.66a 118.70+ 6.21a 109.10+ 6.80a 8 117-10+ 6.01a 133-4 + 6.67a 129-90+ 8.7 3 a 117.60+7.84 103-2 + 7-45a 10 105-70+ 2.94a 115.10+ 6.68a 123.40+ 3.27* 103-80+ 5.90a 65-20 + 6.47b 1 2 V a l u e s a r e M e a n s + S t a n d a r d E r r o r o f t h e M e a n . V a l u e s w i t h i n r o w s w i t h t h e s a m e s u p e r s c r i p t s a r e n o t s i g n i f i c a n t l y d i f f e r e n t ( p - ^ . 0 1 ) . Table 30. Serum Glucose Levels (mg/100 ml) in Swine Fed Varying Levels of Pr o t e i n ( T r i a l 2). ' Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 109.5+3.7a 102.4+5.8a 106.1+5.9a 99-8+4.2a 108.0+3•9a 2 86.5 +5-3a 75-5+2.6a 88.3+6.5a 88.4+5.4a 89.9+4.9a 4 66.8 +2.9a 63.6+2.8a 76.6+3-7ab 94.0+8.4b 85.l±5-3a 6 75.5 +2.0ab 71.5±2.0ab 66.3+2.la 73-9+3.0ab 83.1+4.6b 8 79.4 +3-9ab 70.9+2.4ab 62.3+2.2a 83.2+5.5b 72.0+6.83' 10 77.8 +2.7a 70.4+4.la 68.4+7.6a 67.2+3.8a 70.8+9.63 1 2 Values are Means + Standard Error of the Mean. Values w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p ~ . 0 l ) . Table 31. Serum Calcium Levels (mg/100 ml) in Swine Fed Varying Levels of Pro t e i n ( T r i a l 2). Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 9.54+.07a 9.48+.113 9.41+.173 9.34+.10a 9. 51 ± - T 0 a 2 9.46+.083 9.03+.093 9.14+.173 8.98+.203 9.21+.163 4 9.72+.15a 9.34+.15a 9-33+.I4a 9.46+.I6a 9.11+.163 6 9-95+.12a 8.95+-12b 8.77t.06 b c 8.25+.17° 8 . 3 9 ± - 2 0 b c 8 10.69+.12a 9.25+.1 9.00+.15b 8.77+.I4b c 8.14+.14° 10 10.04+.273 9.10+.06b 8.64+.05b c 8.52+.22b 7.72+.28c 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) . Table 32. Serum Copper Levels (ug/100 ml) in Swine Fed Varying Levels of Pr o t e i n ( T r i a l 2). ' Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 * 162.0+8.8a 146.1+8.6a 157.0+13 -1 3 161.9+10.2a 166.7+16.2a 6 154.9+5.8a 107.9+4.2a 130.4+18.3a 119-5+10.6a 133 .0+24 .9 3 10 220.4+9.l a 158.0+12.0b 93.3+13-5° 121.0+14.6bc 136.8+15-3b 1 2 Values are Means + Standard E r r o r of the Mean. Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — . 0 1 ) . Table 33. Serum Iron Levels (ug/100 ml) in Swine Fed Varying Levels of Protein ( T r i a l 2). ' Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 217.7+15.2a 215.6+21.9a 177.0+17.4a 215.4+16.3a 239-8+28.l a 6 170.6+11.l a 124.5+8.5a 146.0+13.13 169-4+26.5a 106.5+4.0a 10 181.0+19-4a 148.7+7.3a 151.6+24.l a 142.3+20.7a 141.4+31.4a 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). Table 3k. Serum Magnesium Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). ' Dietary P r o t e i n Level Weeks 18% 10% 8% 6% k% 0 2.37+-183 2.96+.I6 a 2.81+.113 2.85+.13 a 2.56+.17a 6 2.51±.12a 2.28+.15ab 1.79+.20 a b c 1 . 7 4 + . l l b c 1.39+.H C 10 2.75+-17a 2.68+.043 2.27+.093 2.15+.25 a b 1.33+.08b 1 Values are Means + Standard Error of the Mean. 2 Values w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (p — . 0 1 ) . Table 35- Serum Phosphate Levels (mg/100 ml) in Swine Fed Varying Levels of P r o t e i n ( T r i a l 2). Dietary P r o t e i n Level Weeks 18% 10% 8% 6% 4% 0 7.49+.I6a 7.74+.13a 7.64+.17a 7.54+.13a 7.76+.22a 2 8.03+.273 7.85+.17ab 7.30+.17a b 7.l6+.19 a b 7.03+.l6 b 4 9.05+.283 8.13+.l8 b 8.10+.10b 7-53±.26 b c 6.90+.16C 6 8.74+.I4a 7.75+.H b 7.10+.l6b c 6.71+.27C 6.40+.l6 c 8 8.81+.259 8.19+.12 a b 7.66+.21 b c 7.06+.26c 5.78+.17d 10 8.74+.203 8.51+.123 8.13+.223 6.52+.48b 5.90+.l l b 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p ^ .01). Table 36. Serum Zinc Levels (ug/100 ml) in Swine Fed Varying Levels of Pr o t e i n ( T r i a l 2 ) . ' ' * Dietary P r o t e i n Level Weeks 18% 10% 8% 6% . 4% 0 186.6+20.7a 198.9+32.7a 193 - 7+21.8a 214.0+23.6a 214.0+20.4a 6 213-0+11.0a 200.3+22.9a 176.0+21.6a 141.4+15.03 149.7+J8.9 3 10 188.5+27.4 a b 176.9±l4.3a 260.0+21.5b 179.3+14.l a 106.0+6.6a 1 Values are Means + Standard E r r o r of the Mean. 2 Values w i t h i n rows with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t ( p — .01). - 103 -C. DISCUSSION This t r i a l had two o b j e c t i v e s . The f i r s t was :to t r y and determine at what l e v e l of p r o t e i n : i n t a k e PCM develops. U t i l i z i n g the c l i n i c a l c r i t e r i a of growth r e t a r d a t i o n , abnormal h a i r t e x t u r e , emaciation, and apathy i t was p o s s i b l e to c l a s s animals on the k% and 6% r a t i o n s as having developed PCM. The second o b j e c t i v e was to f i n d the most s e n s i t i v e biochemical index of the n u t r i t i o n a l s t a t e of the animal. To be of value as an index of the n u t r i t i o n a l s t a t e of the animal, biochemical parameters must f u l f i l l three c r i t e r i a . F i r s t , i t must have a high c o r r e l a t i o n w i t h d i e t a r y p r o t e i n intake. Second, i t must be s e n s i t i v e enough to p r o t e i n d e f i c i e n c y to a l l o w s i g n i f i c a n t treatment e f f e c t s to be observed r e l a t i v e l y e a r l y . F i n a l l y , i t must be able to d i s c r i m i n a t e between those animals on a marginal p r o t e i n intake (8% and 10%) and those animals.with PCM (4% and 6%). In t h i s t r i a l , many of the same parameters were measured as in t r i a l one, and f o r the most pa r t , s i m i l a r r e s u l t s were obtained. Discussion of the p h y s i o l o g i c a l s i g n i f i c a n c e of these parameters and the changes that occur in PCM were presented f o l l o w i n g data f o r t r i a l one and w i l l not be repeated here. Discussion of these parameters w i l l be l i m i t e d to the e f f e c t of p r o t e i n intake on the development of symptoms of PCM. However, several new parameters were measured in t h i s t r i a l , and the s i g n i f i c a n c e of these r e s u l t s w i l l be discussed in the f o l l o w i n g pages. a . PrOtein Metabolism • Total serum proteins declined with decreasing d i e t a r y p r o t e i n .32 at week ten). S i g n i f i c a n t d i f f e r e n c e s between treatments intake (r = - 104 -fo r serum t o t a l p r o teins occurred e a r l y in the t r i a 1. However, the measurement of serum t o t a l p r o teins was not s e n s i t i v e enough to a 1 low one to d i f f e r e n t i a t e between animals on the 10%, 8%, 6% or 4% r a t i o n s . Therefore, t o t a l serum pr o t e i n s wouid not appar to be a good i n d i c a t o r of the n u t r i t i o n a l s t a tus of the animal. There was a s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between d i e t a r y 2 pro t e i n intake and serum albumin l e v e l s (r = .'98 at week t e n ) . However, as with t o t a l p r o t e i n s , serum albumin d i d not appear to be s e n s i t i v e enough to a l l o w one to d i f f e r e n t i a t e between PCM animals and those on marginal p r o t e i n intake. There was l i t t l e c o r r e l a t i o n between l e v e l s of BUN and d i e t a r y 2 p r o t e i n intake (r = .09 at week ten). This is rather s u r p r i s i n g as i t is believed that the l e v e l of serum urea i s pr o p o r t i o n a l to the p r o t e i n intake (Addis et a l . , 1947). However, s i m i l a r r e s u l t s were obtained in t r i a l one, and i t i s p o s s i b l e that increased catabolism of body pr o t e i n s could e x p l a i n the observed values. Serum amylase l e v e l s d e c l i n e d with decreasing d i e t a r y p r o t e i n 2 intake (r = .50 at week ten ) . S i g n i f i c a n t treatment d i f f e r e n c e s appeared as e a r l y as four weeks. A f t e r ten weeks, i t was p o s s i b l e to d i f f e r e n t i a t e between animals on the 8%, 6% and k% r a t i o n s . It is f e l t that the measurement of serum amylase i s a good i n d i c a t o r of the n u t r i t i o n a l status of the an imal. The r e s u l t s of the a n a l y s i s f o r a l k a l i n e phosphatase are very d i f f i c u l t to e x p l a i n . There was no c o r r e l a t i o n between a 1ka1ine 2 phosphatase l e v e l s and d i e t a r y p r o t e i n intake (r =.001 at week t e n ) . However, as in t r i a l one, the four percent r a t i o n had s i g n i f i c a n t l y lower - 105 -(p—.01) alkaline phosphatase levels than did the eighteen.percent group. Why the eight percent ration had s i g n i f i c a n t l y higher.alkaline phosphatase a c t i v i t y after ten weeks is not known. ' However, serum zinc levels were also elevated in animals on the eight percent ration. Li (1966) reported that zinc was required for the a c t i v i t y of a 1ka1ine phosphatase and i t is possible that the elevated zinc levels are responsible for the elevation in serum a 1ka1ine phosphatase. 2 SGOT did not correlate with dietary protein intake (r = -.03 at week ten). No si g n i f i c a n t differences were picked up between treatments and therefore, SGOT would be of l i t t l e value as an indicator of the nutritional status of the animal. Lactic dehydrogenase levels correlated f a i r l y well with 2 dietary protein intake (r. = -.75 at week ten). However, no si g n i f i c a n t treatment effects were observed unti1 after ten weeks on t r i a l . It was f e l t that LDH was not sensitive enough to be of value as an indicator of protein status. b. Fat Metaboli sm Serum cholesterol levels did not correlate well with dietary 2 protein intake (r = .17 at week ten). No si g n i f i c a n t treatment effects were observed u n t i l after ten weeks on t r i a l . It was therefore f e l t that serum cholesterol was not a good indicator of the protein status of the animal. F a t t y i n f i l t r a t i o n of the l i v e r is an important feature of human PCM. The pattern of fat d i s t r i b u t i o n is quite.characteristic of the syndrome (Davis, 1948). Quantitative l i p i d analysis in the present study - 106 -showed s i g n i f i c a n t l y higher f a t concentrations in the ma 1nourished pigs. This is in agreement with the work of Gupta (1973a) in malnourished I'nd ian pigs. The mechanism of t h i s f a t t y c o n d i t i o n i s not c l e a r l y understood. One of the f a c t o r s associated with the increase in the l i v e r 1ipid i s an increase in the serum f r e e f a t t y acids (Jaya Rao and Prasad, 1966). Synthesis of t r i g l y c e r i d e s from carbohydrates may be another f a c t o r ( F l e t c h e r , 1966). However, i t now appears that the l i p i d s cannot be released from the l i v e r because of low concentrations of beta l i p o p r o t e i n s , and that t h i s is probably the r e s u l t of reduced hepatic synthesis of the pro t e i n moiety of the l i p o p r o t e i n (Truswel1 and Hansen, 1969). c. Carbohydrate Metabolism Serum glucose l e v e l s d i d not c o r r e l a t e with d i e t a r y p r o t e i n 2 intake (r = .048 at week e i g h t ) . In a d d i t i o n , no s i g n i f i c a n t d i f f e r e n c e s f o r treatments were picked up a f t e r ten weeks. Some treatment e f f e c t s were observed at weeks 4, 6 and 8 but they would appear to be the r e s u l t of something other than p r o t e i n intake. The measurement of serum glucose i s of l i t t l e value as an i n d i c a t o r of the p r o t e i n status of the animal. d. Water and E l e c t r o l y t e Metabolism Total body water, expressed as a percentage of body weight, i s c o n s i s t e n t l y increased in PCM (Smith, 1960; Brinkman jet a]_., 1965; Flynn et aj_., 1967). In the present study, pigs on the 4% p r o t e i n r a t i o n - 107 -had s i g n i f i c a n t l y higher t o t a l body water than d id those animals on the 18% r a t i o n . The ac tua l s i g n i f i c a n c e of t h i s increase is d i f f i c u l t to a s c e r t a i n because of d i f f e r e n c e s in body f a t between the.two t reatments . It was unfortunate that determinat ions on ; ext race l1u1ar water were not performed. Serum ca lc ium l e v e l s c o r r e l a t e d extremely wel1 w i th d i e t a r y 2 p ro te i n intake (r = .96 a t week t e n ) . S i g n i f i c a n t treatment e f f e c t s were observed a f t e r s i x weeks on t r i a l . However, i t was not f e l t that the measurement of serum calc ium'was s e n s i t i v e enough to-= a l l ow one to d i f f e r e n t i a t e between PCM animals and those on marginal p r o t e i n in take. Serum copper l e v e l s have been shown to be reduced in human PCM (Lahey et _§_[. , 1958; Gopa 1 an et , 1963). In the present study there was a moderate c o r r e l a t i o n between the l e v e l s of serum copper and d i e t a r y 2 p ro te i n intake (r = .67 at week t en ) . It i s p o s s i b l e that copper abso rp t ion on "the lower p r o t e i n d i e t s was poor. Copper i s absorbed mainly from the small i n t e s t i n e and co lon in pigs (Bowland et^aj_. , 1961), and there i s evidence of morpholog ica l a l t e r a t i o n s in the i n t e s t i n a l mucosa of expe r imenta l l y malnourished pigs ( P i a t t .e_t_a±., 1964). A copper b ind ing p r o t e i n has been demonstrated by Starcher (I969) in the mucosal c e l l s of the duodenum of the ch i ck which plays a r o l e in copper ab so rp t i on . If a s imi1ar mechanism e x i s t s in swine, reduced synthes i s is p o s s i b l e in much the same manner as the ca lc ium b ind ing p ro te i n in p r o t e i n d e f i c i e n t r a t s (Kalk and Pimstone, 1974)'. It i s known that copper i s excreted in the b i l e (Cartwr ight and Wintrobe, 1964) and there was a p o s s i b i 1 i t y of exces s i ve loss of b i l a r y - 108 -contents in the presence of diarrhea,.resu 1ting in lower c i r c u l a t i n g copper l e v e l s . It i s i n t e r e s t i n g to note the low serum copper l e v e l s in the 8% p r o t e i n group in combination with the high serum z i n c l e v e l s . Zinc i s believed to have an a n t a g o n i s t i c e f f e c t on copper absorption (Van Campen and S c a i f e , 1967) -Although serum copper c o r r e l a t e d r e l a t i v e l y w e l l with d i e t a r y p r o t e i n intake, i t was not p o s s i b l e to d i f f e r e n t i a t e between those animals e x h i b i t i n g c l i n i c a l signs of PCM and those on a marginal p r o t e i n intake. Therefore, serum copper i s not a r e l i a b l e i n d i c a t o r of the n u t r i t i o n a l status of the anima1. Many workers have reported lower iron and iron binding capacity in the serum of human PCM p a t i e n t s (Edozien and Udeozo, I960; Lahey et _a_L , 1958; El Sholmy j s j _a_L., 1962). In the present study, serum iron • • • 2 c o r r e l a t e d extremely w e l l with the d i e t a r y intake of p r o t e i n (r = .92). However, although there was a good c o r r e l a t i o n , there were no s i g n i f i c a n t treatment e f f e c t s . The lack of s i g n i f i c a n c e would seem to be explained by the r e l a t i v e l y high standard e r r o r s associated with the treatment means. In a d d i t i o n there are several other explanations f o r t h i s apparent discrepancy between human and animal s t u d i e s . There i s evidence that some of the human pre-PCM d i e t s are d e f i c i e n t in iron (Metz and S t e i n , 1959)- In the present study, NRC recommendations f o r d i e t a r y i r o n . i n growing swine were followed and therefore a true iron d e f i c i e n t d i e t d i d not e x i s t . Iron is transported in serum completedly bound to t r a n s f e r r i n (Holmberg and Laurel 1, 1947). In normal individua 1s, only 30-40% of the - 109 -t r a n s f e r r i n c a r r i e s i r o n , the remainder of the t r a n s f e r r i n being known as the l a t e n t iron binding c a p a c i t y (Underwood, 1 9 7 1 ) . In l i g h t of the reduced serum pr o t e i n s observed, i t would appear as though the p r o t e i n d e f i c i e n t pigs had a higher percentage iron s a t u r a t i o n . The animal body has a r e l a t i v e l y large storage c a p a c i t y f o r iron in the form of e i t h e r f e r r i t i n or hemosiderin. It i s p o s s i b l e that due to the short d u r a t i o n of the t r i a l , serum iron l e v e l s were s t i 1 1 being maintained at the expense of body s t o r e s . There i s evidence that vitamin C increases the e f f i c i e n c y of iron absorption in man ( P i r z i o - b i r o l i et _§_[., 1 9 5 8 ) . It i s believed that a s c o r b i c acid aids in the reduction of f e r r i c to ferrous i r o n . Andersson et a j . . (I956) reported a low d i e t a r y intake of a s c o r b i c a c i d in malnourished South A f r i c a n Bantu c h i l d r e n and i t was suggested that t h i s low intake might e x p l a i n the lower serum ir o n l e v e l s in PCM. It i s i n t e r e s t i n g to note that pigs do not have a d i e t a r y requirement f o r vitamin C as i t is believed that they can synthesize adequate amounts of vitamin C f o r t h e i r needs (National Academy of Science, 1 973 ) -Malnourished c h i l d r e n are subject to p a r a s i t i c i n f e s t a t i o n s which act as a d d i t i o n a l drains on the bodies supply of i r o n . As a r e s u l t of the concrete f l o o r in the swine u n i t and p e r i o d i c deworming as a management technique, the existence of p a r a s i t e s in the t e s t animals is not 1 i ke 1 y. Magnesium d e f i c i e n c y appears to occur r e l a t i v e l y f r e q u e n t l y in human PCM. Linder et a}. ( 1 9 6 3 ) reported low serum magnesium values in p a t i e n t s w i t h PCM. In the present study, magnesium values dec 1ined with decreasing d i e t a r y p r o t e i n intake (r = . 6 4 at week ten ) . However, i t was - no -not f e l t that serum magnesium was s e n s i t i v e enough to p r o t e i n intake to be of much value-as an i n d i c a t o r of n u t r i t i o n a l s t a t u s . Accord i ng to Caddel1 and Goddard (1967) the magnes ium d e f i c i e n c y in m a l n u t r i t i o n r e s u l t s from prolonged losses of magnesium through the Gl t r a c t during d i a r r h e a and vomiting coupled with a low magnesium intake. In serum, magnesium i s bound to albumin and a 1pha g1obu1in with a ma j o r i t y associated with the former. In a d d i t i o n , a small part of the serum magnesium i s a l s o combined with phospholipids in the form of a c o l l o i d a l phosphate complex. In l i g h t of the lower serum proteins observed in the present t r i a l , i t i s p o s s i b l e that t h i s could account f o r the lowering of serum magnesium observed. An over production of aldosterone is accompanied by hypomagnesemia and a negative magnesium balance (Milne e t a j . , 1957). It i s believed that the negative balance i s due to renal losses that cause a reduction in c i r c u l a t i n g magnesium l e v e l s . Be i t i ns et a 1. (1974) measured the plasma aldosterone l e v e l s in malnourished c h i l d r e n and reported that they were higher than those of a c o n t r o l group. Lower serum z i n c l e v e l s have been observed in human p r o t e i n c a l o r i e m a l n u t r i t i o n (Kumar and Jaya Rao, 1973; Sandstead et j J . , 1965). In a d d i t i o n , P i a t t and Frankel •(• 1-962). observed lower serum z i n c l e v e l s in malnourished pigs. The r e s u l t s . o f the a n a l y s i s f o r serum z i n c in the present t r i a l are d i f f i c u l t to e x p l a i n . Van Campen and House (1974) reported that the p r o t e i n l e v e l of the d i e t a f f e c t e d z i n c absorption. For t h i s reason, i t was believed that serum'zinc l e v e l s would d e c l i n e with decreasing p r o t e i n intake. Contrary to expec t a t i o n s , there was very l i t t l e c o r r e l a t i o n - I l l -between serum z i n c and d i e t a r y p r o t e i n intake ( r z = .09. at.week.ten). The 8% group had s i g n i f i c a n t l y higher serum z i n c than d i d the other treatments, though whi1e not s i g n i f i c a n t , the four percent r a t i o n appeared to have lower serum zi n e 1evels.. The loss of z i n c i n t o the i n t e r s i t i a l compartment i s a f a c t o r which may have co n t r i b u t e d to the lower serum z i n c values on the k% r a t i o n (Kumar and Jaya Rao, 1973)- Animals on the k% d i e t had s i g n i f i c a n t l y higher t o t a l body water than d i d those animals on the \8% r a t i o n . Unfortunately, e x t r a c e l l u l a r body water was not determined in the present study. However, i t i s probable that at l e a s t a po r t i o n of the observed increase in t o t a l body water was e x t r a c e l l u l a r . S k e l e t a l a b n o r m a l i t i e s are a regular and conspicuous feature of zinc d e f i c i e n c y (Mi 1ler e£ , 1968). It i s p o s s i b l e that the disturbances of g a i t observed in the low p r o t e i n d i e t s are r e l a t e d to the lower serum z i n c l e v e l s observed. - 112 -SUMMARY AND CONCLUSIONS There i s general agreement by workers engaged in the study of p r o t e i n c a l o r i e m a l n u t r i t i o n that two d i s t i n c t c l i n i c a l forms can be recognized. Kwashiorkor is p r i m a r i l y a c o n d i t i o n of e a r l y l i f e (1-3 years) but older c h i l d r e n and a d u l t s may be affected:whenever there i s prolonged consumption of a d i e t low in p r o t e i n and high in carbohydrate. Kwashiorkor is c h a r a c t e r i z e d c l i n i c a l l y by r e t a r d a t i o n of growth and development, loss of weight with muscular wasting, edema, f a t t y l i v e r , abnormal h a i r t e x t u r e , and apathy. Marasmus i s p r i m a r i l y a disease of infancy ( l e s s than one year) and i s associated w i t h d i e t s that are low in p r o t e i n and energy. Marasmus i s c h a r a c t e r i z e d by r e t a r d a t i o n of growth and development, loss of weight with severe muscular wasting, and a minimum of changes in blood compos i t ion. An attempt was made in t h i s study to reproduce the symptoms of the "kwashiorkor" type of PCM in the baby p i g . Not every l e s i o n o c c u r r i n g in man was reproduced in the present study. However, because of the mu l t i f a c e t e d nature of the disease, i t i s doubtful i f the e n t i r e gamut of le s i o n s could ever be reproduced. It i s d i f f i c u l t to expect a research animal to develop a l l of the known l e s i o n s , when man from one geographical area often does not e x h i b i t the same l e s i o n s which may be c h a r a c t e r i s t i c of the disease in another region or country. PCM i s a disease compounded by d e f i c i e n c i e s of vitamins and m i n e r a l s , which in combination with concurrent i n f e c t i o n s , can a l t e r the c l i n i c a l and metabolic pattern of PCM in an endless array of patterns. - 113 -Nevertheless, c h a r a c t e r i s t i c symptoms such as the development of f a t t y l i v e r , growth r e t a r d a t i o n , abnormal h a i r t e x t ure and apathy were reproduced in p r o t e i n d e f i c i e n t swine. In a d d i t i o n , most of the biochemical parameters which are a l t e r e d in the human c o n d i t i o n were a l t e r e d in the pr o t e i n d e f i c i e n t swine. It i s th e r e f o r e concluded that the baby pig i s a good model with which to study p r o t e i n - c a l o r i e m a l n u t r i t i o n . The prognosis of PCM is oft e n d i f f i c u l t to assess on c l i n i c a l grounds alone and various attempts have been made to f i n d an accurate biochemical index of developing PCM. It was hoped that t h i s experiment would provide some i n s i g h t into the most s e n s i t i v e index of the n u t r i t i o n a l s t a t e of the animal. Such parameters as z i n c , a l k a l i n e phosphatase, serum glutamic o x a l o a c e t i c transaminase, c h o l e s t e r o l , glucose, and blood urea nitrogen can be discarded as i n d i c a t o r s s i n c e no c o r r e l a t i o n was observed between these parameters and d i e t a r y p r o t e i n intake. L a c t i c dehydrogenase and iron did not appear to be s e n s i t i v e enough to p r o t e i n d e f i c i e n c y since s i g n i f i c a n t treatment e f f e c t s occurred only a f t e r ten weeks of t r i a l . Copper, t o t a l p r o t e i n , albumin, calcium and magnesium a l l c o r r e l a t e d w e l l with d i e t a r y p r o t e i n intake but f a i l e d to d i f f e r e n t i a t e between those animals on marginal p r o t e i n intake and those developing PCM. This leaves amylase and serum phosphate as being the only parameters which met the three c r i t e r i a f o r a d i a g n o s t i c index. Serum phosphate was the f i r s t parameter to be a f f e c t e d by di m i n i s h i n g d i e t a r y p r o t e i n intake and as such these r e s u l t s were rather unexpected. Unfortunately, hypophosphatemia occurs in other c o n d i t i o n s such as Addison's disease, Fanconi's syndrome and m u l t i p l e myeloma. This holds true f o r other parameters as well and i t is doubtful i f any one index can be considered as being c h a r a c t e r i s t i c of a c e r t a i n disease. - 114 -Under the condit ions of the current t r i a l , serum phosphate and serum amylase would appear to be the best biochemical indices of the nu t r i t i ona l status of the prote in deprived p ig . However, because of the many factors a f f ec t i n g the serum levels of these parameters, i t is important that they be used in conjunction with c l i n i c a l c r i t e r i a in diagnosing develop PCM. It is concluded that the young pig could be e f f e c t i v e l y used for research on the PCM snydrome as i t occurs in humans. For example, studies of therapeutic d ietary regimes for t reat ing the condi t ion might well be undertaken with th i s species. - 115 -BIBLIOGRAPHY Addis, T., E. B a r r e t t , L.J. Poo and D.W. Yeun. 1947. 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Van Der Westhuysen, J.M., F. Kanengoni, J . J . Jones and CH. Van Niekerk. 1975-Plasma renin a c t i v i t y in edematous and marasmic c h i l d r e n with p r o t e i n energy m a l n u t r i t i o n . S. A f r . Med. J . 49: 1729"1731. Vaughan, B.E. and E.A. Bo l i n g . 1961. Rapid assay f o r t r i t i u m l a b e l l e d water in water f l u i d s . J . Lab. C l i n . Med. 57: 159-164. V i t e r i , F., M. Behar, G. Arroyave and N.S. Scrimshaw. 1964. C l i n i c a l aspects of p r o t e i n m a l n u t r i t i o n . P. 523-568. jjn: H.N. Munroe (ed) Mammalian P r o t e i n Metabolism. V o l . 11. Academic Press, New York. Waldern, D.E. 1971. A rapid m i c r o - d i g e s t i o n procedure f o r neutral and a c i d detergent f i b r e . Can. J . Anim. S c i . 51: 67-69-- 129 -Warren, P.J., J.D. Hansen and B.Hi Lehmana*. 1969- The concentrations of copper, z i n c , and manganese in the l i v e r o f . A f r i c a n . c h i l d r e n w i t h marasmus'and kwashiorkor. Proc. Nutr. Soc. 28: 6A. Warton, B. 1970. Hypoglycemia in c h i l d r e n with kwashiorkor. Lancet 1: 171-173. Waterlow, J.C. 1959- P r o t e i n n u t r i t i o n and enzyme changes in man. Fed. Proc. 18: 1143-1155-Waterlow, J.C. 1962- O x i d a t i v e phosphorylation in human i n f a n t s . Nutr. Rev. 20: 150-152. Waterlow, J.C. and C.B. Mendes. 1957. Composition of muscle in malnourished human i n f a n t s . Nature, 180:1361-1362. Waterlow, J . C , J . Cravioto and J.M. Stephen. 1960. P r o t e i n m a l n u t r i t i o n in man. Advan. P r o t e i n Chem. 15: 131-238. Waterlow, J.C and J.M. Stephen. 1969- Enzymes and the assessment of p r o t e i n n u t r i t i o n . Proc. Nutr. Soc. 28: 234-242. Wayburne, S. 1963- Hepatic f a i l u r e in m a l n u t r i t i o n . Lancet 1: 447 -447. Weimer, H.P., A.W. Carpenter, A.W. Nayor-Foote, R.W. McKee and H. N i s h i h a r i . 1959. E f f e c t s of i n a n i t i o n , p r o t e i n d e p l e t i o n and r e p l e t i o n on serum l a c t i c a c i d dehydrogenase l e v e l s in r a t s . P i r o c . Soc. Exp. B i o l . Med. 101: 344-345. Werbin, H.B., M.R. Chaikoff and'M.R. Imada. 1959- Rapid s e n s i t i v e method f o r determining t r i t i a t e d water in body f l u i d s by l i q u i d s c i n t i l l a t i o n spectrometry. Proc. Soc. Exp. B i o l . Med. 102: 8-12. Wharton, B. 1970. Hypoglycemia in c h i l d r e n with kwashiorkor. Lancet 1: 171-173. Wheeler, J.E., F.D. Lukens and P. G.yory. 1949- Studies on the l o c a l i z a t i o n of tagged methionine w i t h i n the pancreas. Proc. Soc. Exp. B i o l . Med. 70: 187-I89. Whitehead, R.G. and R.F. Dean. 1964. Serum amino acids in kwashiorkor. 1. R e l a t i o n s h i p to c l i n i c a l c o n d i t i o n . Amer. J . C l i n . Nutr. 14: 3I3-3I9. Whitehead, R.G. and P.S. Harland. 1966. Blood glucose, l a c t a t e , and pyruvate in kwashiorkor. B r i t . J . Nutr. 20; 825-83T. Whitehead, R.G., W.A. Coward and P.G. Lunn. 1973- Serum albumin concentrations and the onset of kwashiorkor. Lancet 1; 63-66. Wiberg, G.S. and J . Tuba. 1955. On rat serum amylase. I l l The c o n t r i b u t i o n by v a rious t i s s u e s to serum amylase a c t i v i t y . Can. J . Biochem. 33: 817-825. - 130 -Wolf, P.L. and W i l l i a m s . 1973- P r a c t i c a l C l i n i c a l Enzymology. John Wi1ey and Sons, New York. Z a k i , A.H., B. El-Kammah, I. Fayad, A.H. Shehata and S. Mahmoud. 1970. Serum enzymes in p r o t e i n m a l n u t r i t i o n . Acta. B i o l . Med. Germ. 2k: 137-140. - 131 -A P P E N D I X T A B L E S Table 1A. An a l y s i s of Variance fo r Body Weight in T r i a l 1. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 0.4637 1 0.3686 1 2.4009 1 0.7341 1 3.0496 1 3.0157 1 L i t t e r 1.4099 3 1.9450 3 4.8739 3 18.8840 3 . 44.2650 3 98.8950 3 Treatment 0.2430 1 15.3520 1 118.1000* 1 546.0300* 1 1391.5000* 1 1016.0000* 1 1 Error - 0.8187 21 2.6693 22 5.9732 23 13.8620 22 20.7330 21 48.4780 14 p ~ .01 Table 2A. An a l y s i s of Variance f o r Serum Total P r o t e i n in T r i a l 1 Source 0 Weeks 2 Weeks Mean Square + Degree of Freedom 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment E r r o r 0.0156 1 2.0642 3 0.1616 1 0.4286 21. 0.3957 1 0.2651 3 0.2540 1 0.1469 22 0.0001 1 0.6487 3 2.6311* 1 0.3419 23 0.0105 1 0.0796 3 7.2164'' 1 0.1816 22 0.0049 1 0.1646 3 22.7770* 1 0.2308 21 0.4408 1 0.3982 3 6.1917* 1 0.3949 « 14 P — .01 Table 3A. Ana l y s i s of Variance f o r Serum Albumin in T r i a l 1. Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment Error 0.2861 1 0.7412 3 0.1760 1 0.1714 21 0.1776 1 0.0329 3 0.0185 1 0.0420 22 0.2512 1 0.0051 3 0.8498* 1 O.0836 23 0.0022 1 0.0139 3 1.1537* 1 0.0529 21 0.0005 1 0.0938 3 11.2380* 1 0.1507 21 0.1046 1 0.1933 3 3.6143* 1 1 _. 0.2442 14 p — .01 Table 4A. An a l y s i s of Variance f o r Blood Urea Ni trogen in T r i a l 1 . Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 54.457 1 10.808 1 1.737 1 0.0204 1 29.308 1 12.783 1 L i t t e r 33.639 3 8.598 3 5.130 3 13.782 3 0.458 3 13.564 3 1 Treatment 8.594 1 100.170 1 86.392 1 0.031 1 8.278 1 39.603 CJ 1 ^ 1 Error 10.793 19 16.881 22 17.345 23 12.375 22 11.454 21 12.033 14 p — .01 Table 5A. A n a l y s i s of Variance f or Serum Amylase in T r i a l 1 Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment Error 584130.0 1 3513300.0* 3 65248.0 1 450830.0 20 2451700.0 1 732010.0 3 5654.3 1 883140.0 22 7728.7 1 2084100.0* 3 4480600.0* 1 244800.0 23 1601.5 1 510870.0 3 3919200.0* 1 171600.0 22 1157.6 1 646290.0 3 2055900.0 1 301920.0 20 1173000.0 1 758440.0 3 5213300.0* ' 1 vo ON 384070.0 14 Table 6A. An a l y s i s of Variance f o r Serum A l k a l i n e Phosphatase in T r i a l 1 Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li t t e r Treatment Err o r 6132.7 1 2473.4 3 492.8 1 523.3 21 452.4 1 1541.9 3 184.7 1 501 .2 22 308.9 1 2124.1 3 16605.0* 1 1122.8 23 619.2 1 1773-6 3 30957.0'' 1 928.0 22 1011.2 1 1272.8 3 32189.0* 1 764.4 21 2020.2 1 1323.4 3 10089.0* 1 1118.8 14 V/J P — .01 Table 7A. An a l y s i s of Variance fo Serum Glutamic Oxaloacetic Transaminase in T r i a l 1 Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li t t e r Treatment Error 26.02 1 402.38* 3 10.01 1 56.21 21 401.88 1 7.40 3 34.20 1 144.43 22 581.07 1 57.64 3 1421.20 1 261.16 23 9.34 1 229.89* 3 639.06* 1 31.09 22 0.19 1 83.48 3 201.35 1 36.67 20 393.40 1 92.56 3 0.50 1 70.31 14 CO p ^ .01 Table 8A. Source Analysis of Variance for Lactic Dehydrogenase in Trial 1. Mean Square + Degree of Freedom 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li tter Treatment Error 13810.0 1 54465.0 3 46697.0 1 14389.0 21 113330.0 1 24987.0 3 179140.0 1 51048.0 21 3.4 1 10514.0 3 34769.0 1 64397.0 21 187.8 5997-7 3 455.3 1 5638.4 22 235.6 1 5082.8 3 3036.8 1 6058.2 21 73.9 1 5693.1 3 44488.0* 1 3437.7 14 00 U3 p — .01 Table 9A. A n a l y s i s of Variance f o r Serum Cholesterol in T r i a l 1 Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment Error 916.19 1 347.04 3 25.41 1 666.83 -21 61.48 1 134.65 3 29-70 1 153.03 22 841.84 1 201.34 3 • 72 1 170.28 23 305.42 1 519.31 3 591.54 1 152.25 22 341.95 1 235.90 3 1989.70* 1 248.95 21 62.67 1 71.26 3 2091.90* 1 131.34 14 4 T -O p .01 Table 10A. A n a l y s i s of Variance f o r Serum Glucose in T r i a l 1. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li t t e r Treatment Error 7.45 1 998.25 3 2678.50 1 576.42 21 95.32 1 160.15 3 357.09 1 224.60 22 6.59 1 218.94 3 633.47 1 206.39 23 32.80 1 411.99 3 158.84 1 190.57 22 324.40 1 347.57 3 512.26 1 175.42 21 190.17 1 89.80 3 0.35 -1 i 243.44 14 Table 11A. A n a l y s i s of Variance f o r Serum Calcium in T r i a l 1. Sou rce 0 Weeks 2 Weeks Mean Square + Degree of Freedom 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment 1.3481 1 1.6332 3 0.3662 1 0.0492 1 0.7750 3 0.1334 1 0.0531 1 2.8038'' 3 1.2437 1 0.1914 1 0.0109 3 8.3812>' 1 0.4605 1 0.2751 3 23.1400* 1 0.1739 1 0.0994 3 6.5797* 1 Error 0.5704 21 0.2150 22 0.3101 22 0.3834 22 0.6662 21 0.1913 14 Table 12A. A n a l y s i s of Variance f o r Serum Phosphate in T r i a l 1 Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment Error 8.2598 1 6.5122 3 0.6522 1 1.5307 21 1.9846 1 0.9511 3 5.0232* 1 0.5508 22 0.1416 1 2.0520 3 19.6620* 1 0.5485 23 0.4590 1 1.4786 3 17.0860* 1 0.6901 22 1.0451 1 0.2188 3 35.3770' 1 0.2822 20 0.2876 1 1.9648 3 10.6320* 1 0.8382 14 p — .01 Table 13A. Analysis of Variance for Body Weight in Trial 2. Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li tter Treatment Error 0.8027 1 4.7546* 5 0.5733 4 0.5905 29 0.4267 1 4.1672* 5 4.1250* 4 0.7197 29 0.0659 1 5.1212* 5 40.0040* 4 1.1946 27 0.0000 1 8.2966* 5 189.2400'' 4 2.0803 27 6.9606 1 7.4171 5 497•2900* 4 4.6085 26 13.9750 1 16.9540 5 822.3900* 4 9.0858 24 Table 14A. An a l y s i s of Variance f o r Serum Total P r o t e i n in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li t t e r Treatment Error 0.0239 1 0.0428 5 0.0240 4 0.0614 29 0.0395 1 0.0676 5 0.0889 4 0.0856 29 0.0049 1 0.1450 5 0.9673* 4 0.0664 27 0.2588 1 0.2303 5 2.3235* 4 0.1061 27 0.2125 1 0.2436 5 4.3502' 4 0.1286 26 0.1908 1 0.2521 5 3.1681* 4 0.2636 24 -C-p — .01 Table 15A. A n a l y s i s of Variance f o r Serum Albumin in T r i a l 2. Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li t t e r Treatment Error 0.0474 1 0.4555-5 0.0385 4 0.0434 29 0.0402 1 0.1224 5 0.0471 4 0.0431 29 0.1021 1 0.1901* 5 0.3961* 4 0.0455 27 0.0310 1 0.1211 5 1.7928* 4 0.0516 27 0.0167 1 0.1271 5 4.0972* 4 0.0332 26 0.0095 1 0.1283 5 2.6533* 4 0.1281 24 o\ p — .01 Table 16A. An a l y s i s of Variance f o r Blood Urea Nitrogen in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex Li t t e r Treatment Error 3.2111 1 27.3820 5 1.6625 4 9.4288 29 0.4812 1 3.4022 5 13.8700 4 7.7616 29 0.4247 1 21.3910' 5 4.4935 4 4.2228 27 2.0876. 1 2.7848 5 22.9830* 4 5.191.7 27 9.0137 1 6.9594 5 50.2470* 4 7.7239 26 38.7620 1 6.6551 5 17.1630 4 13.0940 24 -c-p — .01 Table 17A. A n a l y s i s of Variance f o r Serum Amylase in T r i a l 2. Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex L i t t e r Treatment Error 1 2 4 2 0 0 . 0 1 2 0 0 6 1 0 0 . 0 * 5 3 6 6 2 8 0 . 0 4 2 4 7 1 5 0 . 0 29 77910.0 1 1416600.0 5 252690.0 4 416360.0 29 883770.0 1 1825200.0* 5 1077200.0* 4 221840.0 27 31426.0 1 902830.0 5 2799200.0'' 4 370430.0 27 1 4 2 6 4 0 . 0 1 3 8 6 4 8 0 . 0 5 2811800.0* 4 3 5 4 4 0 0 . 0 26 491290.0 1 1695500.0* 5 5515400.0* 4 386600.0 24 Table 18A. An a l y s i s of Variance f o r Serum A l k a l i n e Phosphatase in T r i a l 2. Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 504.10 1 25.24 1 1.61 1 59.71 1 65.69 1 47.08 1 L i t t e r 13386.00' 5 1170.80 5 2096.10' 5 1878.70 5 1897.80 5 4394.40 5 Treatment 131.65 4 1497.10 4 4238.10* 4 10748.00* 4 10928.00* 4 51447.00* 4 Error 686.22 29 574.76 29 384.25 27 571.31 27 577.89 26 2906.70 24 p — .01 Table 19A. A n a l y s i s of Variance f o r Serum Glutamic Oxaloacetic Transaminase in T r i a l 2. Mean Square + Degree of Freedom Sou rce 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 1 0 Weeks Sex 220.90 1 13.22 1 2.65 1 1995.40 1 278.96 1 37.14 1 L i t t e r 341.03 5 220.45 5 718.33 5 385.06 5 53.37 5 185.97 5 Treatment Error 357.56 4 2 0 8 . 6 6 2 9 70.73 4 70.86 2 9 446.76 4 396.85 27 123.40 4 433.73 27 143.84 4 100.04 26 301.21 4 o 355.05 24 p — . 0 1 Table 20A. A n a l y s i s of Variance f o r Serum L a c t i c Dehydrogenase in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 6024.3 1 1676.1 1 97016.0 1 21625.0 1 10293.0 1 24.6 1 Li t t e r 80464.0 5 3187.1 5 31063.0 5 I6966.O 5 6405.5 5 34114.0 5 Treatment 46181.0 4 8838.3 4 82326.0 4 7864.0 4 3339.0 4 123560.0* 4 Error 32181.0 29 2660.2 29 29932.0 27 12740.0 27 3039.0 26 281.40.0 24 p — .01 Table 21A. An a l y s i s of Variance f o r Serum Cholesterol in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 1207.60 1 10.94 1 10.21 1 4.10 1 165.27 1 185.59 1 Li t t e r 1307.60 5 303.52 5 310.60 5 4 5 1 . 8 7 5 702.68 5 102.57 5 Treatment 366.78 4 7 0 6 . 4 8 * 4 339.17 4 395.06 4 821.00 4 2 5 2 2 . 4 0 * 4 M Error 367.63 29 121.44 29 255.89 27 217.07 27 376.56 26 188.07 2 4 Table 22A. A n a l y s i s of Variance f o r Serum Glucose in T r i a l 2. .01 Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 529.66 1 1167.20* 1 237.33 1 74.69 1 1.16 1 648.97 1 L i t t e r Treatment 109.95 5 129.71 4 421.87 5 383.90 4 127.17 5 1329.60* 4 58.27 5 282.47* 4 212.60 5 550.51* 4 122.91 115.08 4 Ul UJ Error 159.57 29 130.25 29 209.17 27 65.95 27 123.03 26 233.73 24 Table 23A. A n a l y s i s of Variance f o r Serum Calcium in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 0.1493 0.0027 0.1412 0.3002' 0.01899 0.2236 1 1 1 1 1 1 L i t t e r 0.2589 0.2660 0.0427 0.1142 0.0692 0.4832 5 5 5 5 5 5 Treatment 0.0535 0.2630 0.3682 3-5738* 5.8099* 4.7033* 4 4 4 4 4 4 Error O.0787 0.1752 0.2171 0.1622 0.1573 0.2134 29 29 27 27 26 24 4^ p — .01 Table 24A. Ana l y s i s of Variance f o r Serum Copper in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 6 Weeks 10 Weeks Sex 757-54 1 2508.00 1 1792.80 1 Li t t e r 3940.10 5 1485.10 5 4251.30* 5 Treatment Error 173.02 4 604.58 27 1981.80 4 1463.00 27 I8565.OO* 4 612.91 25 p — .01 Table 25A. An a l y s i s of Variance f o r Serum Iron in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 6 Weeks 10 Weeks Sex 3956.60 916.93 3178.10 1 1 1 L i t t e r 3978.60 3410.90 2330.50 5 5 5 Treatment 4739-80 5265.90 2330.50 4 4 4 E r r o r 2339-20 1566.30 2915-40 22 25 24 Table 26A. A n a l y s i s of Variance f o r Serum Magnesium in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 6 Weeks 10 Weeks Sex 0.2171 1 0.0463 1 0.2223 1 Li t t e r Treatment Error 0.2712 5 0.4631 4 0.1894 28 0.3592 5 0.3558' 4 0.1132 26 0.0678 5 1.6924* 4 0.1804 25 Table 27A. An a l y s i s of Variance f o r Serum Phosphate in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks Sex 0.5377 1 0.0278 1 0.0716 1 0.6287 1 0.0127 1 0.2253 1 Li t t e r 0.6682* 5 0.7204 5 0.9026 5 0.5255 5 O.669I 5 0.6327 5 Treatment 0.1160 4 1.4684* 4 4.5256' 4 6.5366* 4 8.0350* 4 8.9269' 4 Error 0.1582 29 0.2416 29 0.2546 27 0.1879 27 0.2767 26 0.4174 24 Table 28A. A n a l y s i s of Variance f o r Serum Zinc in T r i a l 2. Mean Square + Degree of Freedom Source 0 Weeks 6 Weeks 10 Weeks Sex 256.87 1078.70 2304.40 1 1 1 Li t t e r Treatment Error 7093-40 1573.30 8056.60 5 5 5 1 1116.10 7176.50 20541.00* -4 4 4 g 1 4013.00 2346.80 1954.00 27 24 25 p — .01 Table 29A. A n a l y s i s of-Variance f o r Total Body Water, Source Mean Square + Degree of Freedom Treatment 177.1 2-' 1 Error 9 - 7 7 k .01 o Table 30A. A n a l y s i s of Variance f o r Fatty L i v e r s . Source Mean Square + Degree of Freedom Treatment 2097-21* 1 Error 182.13 Zk 

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