UBC Theses and Dissertations

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UBC Theses and Dissertations

Effect of protein-calorie malnutrition on intestinal disaccharidase activities and disaccharide absorption… Wilson, Judith Leslie 1973

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•cl  EFFECT OF PROTEIN-CALORIE MALNUTRITION ON INTESTINAL DISACCHARIDASE ACTIVITIES AND DISACCHARIDE ABSORPTION IN THE RAT by JUDITH LESLIE WILSON B.H.E., U n i v e r s i t y o f B r i t i s h Columbia, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the D i v i s i o n of Human N u t r i t i o n School o f Home Economics  We a c c e p t t h i s t h e s i s as conforming t o the required  standard  The U n i v e r s i t y o f B r i t i s h A p r i l , 1973  Columbia  In p r e s e n t i n g  t h i s t h e s i s i n 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, I agree t h a t 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 r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be granted by  permission.  Department The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada  Department or  I t i s understood t h a t copying or  of t h i s t h e s i s f o r f i n a n c i a l gain written  the Head of my  Columbia  s h a l l not be  publication  allowed without  my  i  ABSTRACT  The purpose o f the p r e s e n t e f f e c t o f prolonged  i n v e s t i g a t i o n was t o study the  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 on i n t e s t i n a l  d i s a c c h a r i d a s e a c t i v i t i e s and on d i s a c c h a r i d e a b s o r p t i o n , as carbohydrate ing  i n t o l e r a n c e i s a major problem i n c h i l d r e n s u f f e r -  from 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 . Four groups of r a t s (90 t o 120 grams) were fed the f o l -  lowing d i e t s f o r 8 t o 9 weeks: 66% c a r b o h y d r a t e ) ; talbumin,  c o n t r o l (18% l a c t a l b u m i n ,  low p r o t e i n low carbohydrate  66% c a r b o h y d r a t e ) ;  low p r o t e i n h i g h  (0.5% l a c t a l b u m i n , 83.5% c a r b o h y d r a t e ) ; restricted  (0.5% l a c carbohydrate  and low p r o t e i n  ( 1 % l a c t a l b u m i n , r e s t r i c t e d t o 4 grams per d a y ) .  A f t e r 8.5 weeks, p a r t o f the group on the 0.5% l a c t a l b u m i n low carbohydrate  d i e t was f e d the c o n t r o l d i e t  (18% l a c t a l -  bumin, 66% carbohydrate) f o r 8 weeks. At the end o f the f e e d i n g p e r i o d , the f o l l o w i n g assays were performed:  1) iri v i v o a b s o r p t i o n of r a d i o a c t i v e ("*"C) 4  l a c t o s e , sucrose, and maltose; 2) a c t i v i t i e s o f i n t e s t i n a l l a c t a s e , sucrase,  and maltase; 3) plasma albumin  concentra-  t i o n s ; and 4) mucosal p r o t e i n c o n c e n t r a t i o n s . In the three p r o t e i n d e f i c i e n t groups (0.5% l a c t a l b u m i n low carbohydrate,  0.5% l a c t a l b u m i n high carbohydrate,  l a c t a l b u m i n ) , the a c t i v i t y of both the j e j u n a l and  and 1%  ileal  d i s a c c h a r i d a s e s and the a b s o r p t i o n of l a c t o s e , sucrose and maltose were s i g n i f i c a n t l y higher when compared w i t h the controls.  The j e j u n a l sucrase and maltase a c t i v i t i e s were  s i g n i f i c a n t l y higher  i n the 0.5% l a c t a l b u m i n h i g h  carbo-  ii hydrate  group than i n the 0.5% l a c t a l b u m i n low  carbohydrate  group, b u t the a b s o r p t i o n o f l a c t o s e , sucrose and maltose were alike.  When the 1% l a c t a l b u m i n  ( r e s t r i c t e d t o 4 grams per day)  and the 0.5% l a c t a l b u m i n low carbohydrate  groups were compared,  there were no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the s p e c i f i c a c t i v i t i e s o f the i n t e s t i n a l d i s a c c h a r i d a s e s or the a b s o r p t i o n of the d i s a c c h a r i d e s . The  a b s o r p t i o n of l a c t o s e , sucrose,  or maltose were s i m i l a r  i n the c o n t r o l s and the p r o t e i n r e p l e t e d group. c h a r i d a s e a c t i v i t i e s were a l s o s i m i l a r except f o r a s i g n i f i c a n t d e p r e s s i o n i l e a l sucrase and maltase a c t i v i t i e s  i n these  The d i s a c two groups  o f j e j u n a l maltase and i n the p r o t e i n r e p l e t e d  group. Therefore,  these  results indicate that protein depriva-  t i o n i n r a t s f o r 8.5 weeks causes an i n c r e a s e i n s p e c i f i c a c t i v i t i e s o f the i n t e s t i n a l d i s a c c h a r i d a s e s  i n both the  jejunum and ileum, and t h a t these changes caused by p r o t e i n d e p l e t i o n may be r e v e r s e d by f e e d i n g a d i e t h i g h i n p r o t e i n . A l s o , an i n c r e a s e i n the carbohydrate deficient diet results maltase a c t i v i t i e s . disaccharidases  content  o f the p r o t e i n  i n an i n d u c t i o n of j e j u n a l sucrase and  The h i g h s p e c i f i c a c t i v i t y o f the i n t e s t i n a l  f o l l o w i n g 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 may be  i n p a r t due t o a p r e f e r e n t i a l l o s s of s t r u c t u r a l p r o t e i n s r a t h e r than t o an increase i n enzymatic p r o t e i n i n the i n t e s t i n a l mucosa. The  i n c r e a s e i n the d i s a c c h a r i d a s e a c t i v i t i e s  i n the  p r o t e i n d e f i c i e n t r a t s i s accompanied by an i n c r e a s e i n d i s a c c h a r i d e a b s o r p t i o n which c o u l d be due t o the higher  l e v e l s of  d i s a c c h a r i d a s e s o r t o an i n c r e a s e i n the t r a n s p o r t o f the  c o n s t i t u e n t monosaccharides.  The demonstration 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 d i f f e r e n c e s i n sucrase and maltase a c t i v i t i e s between the 0.5% l a c t a l b u m i n h i g h carbohydrate the 0.5% l a c t a l b u m i n low carbohydrate comitant  i n c r e a s e i n sucrose  groups w i t h o u t  and maltose a b s o r p t i o n ,  and  a consupports  the view t h a t the h i g h e r a b s o r p t i o n of sucrose and maltose i n the p r o t e i n d e f i c i e n t r a t s i s a r e s u l t of i n c r e a s e d  monosac-  charide transport. The r e s u l t s o f t h i s study are not c o n s i s t e n t w i t h the suggestion  that p r o t e i n - c a l o r i e malnutrition i s responsible  for disaccharide intolerance i n c h i l d r e n .  ACKNOWLEDGEMENT  The author would l i k e t o s i n c e r e l y thank h e r a d v i s o r , Dr. J . L e i c h t e r , f o r h i s d i r e c t i o n and h e l p throughout the study; Dr. M. Lee and Dr. J.C. Brown f o r t h e i r advice i n the p r e p a r a t i o n  o f the manuscript; and Miss U s e Borgen  f o r her kind a s s i s t a n c e  i n the l a b o r a t o r y .  T h i s study was supported by Grant No. A6249 from the N a t i o n a l Research C o u n c i l o f Canada.  V  TABLE OF CONTENTS CHAPTER I  PAGE LITERATURE REVIEW  1  A. I n t r o d u c t i o n  1  B. D i g e s t i o n and Absorption o f Carbohydrates  2  v  1. Review o f d i g e s t i o n and absorpt i o n o f carbohydrates  2  2. L o c a t i o n o f the i n t e s t i n a l disaccharidases  3  3. Changes i n i n t e s t i n a l d i s a c c h a r i d a s e a c t i v i t i e s w i t h age....  5  4.  E f f e c t o f d i e t a r y carbohydrate on a c t i v i t y levels of intestinal disaccharidases  5  C. 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 and Carbohydrate Absorption i n Man........  6  1. The e f f e c t o f 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 on the i n t e s t i n a l mucosa i n man  6  2. The e f f e c t o f 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 on the i n t e s t i n a l d i s a c c h a r i d a s e s i n man  7  3. The e f f e c t o f 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 on d i s a c c h a r i d e a b s o r p t i o n i n man  7  4.  The r e l a t i o n s h i p between l a c t o s e intolerance in protein-calorie m a l n u t r i t i o n and the i n c i d e n c e o f primary l a c t a s e d e f i c i e n c y  5. The e f f e c t o f i n f e c t i o n s and n u t r i t i o n a l d e f i c i e n c i e s on the a c t i v i t y o f the i n t e s t i n a l disaccharidases  9  10  vi  D. 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 and Carbohydrate Absorption i n Laboratory Animals 1. The e f f e c t o f 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 on the i n t e s t i n a l mucosa i n l a b o r a t o r y animals  12  2. The e f f e c t o f 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 on i n t e s t i n a l d i s a c char idases i n l a b o r a t o r y animals.  14  3. The e f f e c t o f 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 on a b s o r p t i o n o f disaccharides i n laboratory animals.....  18  4. The e f f e c t o f n o n - p r o t e i n f a c t o r s on the a c t i v i t y o f the i n t e s t i n a l disaccharidases i n laboratory animals....  19  E. O b j e c t i v e s o f the Present Study II  MATERIALS AND METHODS  21 23  A. Animals and D i e t s  23  B. A b s o r p t i o n o f D i s a c c h a r i d e s  25  C. D i s a c c h a r i d a s e A c t i v i t i e s  27  D. Other Assays  29  1. Plasma albumin c o n c e n t r a t i o n  29  2. Mucosal p r o t e i n d e t e r m i n a t i o n  29  E. Materials III  12  RESULTS  29 31  A. Appearance, Body weight, and Plasma Albumin c o n c e n t r a t i o n  31  B. I n t e s t i n a l D i s a c c h a r i d a s e A c t i v i t i e s . . . .  39  C. I n t e s t i n a l A b s o r p t i o n o f D i s a c c h a r i d e s in Vivo  46  vii  IV V  VI  DISCUSSION  51  SUMMARY AND RECOMMENDATIONS  62  A. Summary  62  B. Recommendations  65  BIBLIOGRAPHY  67  viii  LIST OP TABLES TABLE I II III IV V VI  VII  VIII  IX  X  PAGE D i g e s t i o n and A b s o r p t i o n o f Carbohydrates.... Composition of D i e t s  24  I n i t i a l and F i n a l Body Weights o f the S u r v i v o r s i n Each D i e t a r y Group Percentage of S u r v i v o r s D i e t a r y Regimens...  4  3  4  on the D i f f e r e n t 38  Plasma Albumin C o n c e n t r a t i o n of C o n t r o l , P r o t e i n Depleted, and P r o t e i n Repleted R a t s . .  40  L a c t a s e A c t i v i t i e s i n Jejunum and Ileum o f C o n t r o l , P r o t e i n Depleted and P r o t e i n Repleted Rats  41  Sucrase A c t i v i t i e s i n Jejunum and Ileum of C o n t r o l , P r o t e i n Depleted, and P r o t e i n Repleted Rats  42  Maltase A c t i v i t i e s i n Jejunum and Ileum of C o n t r o l , P r o t e i n Depleted, and P r o t e i n Repleted Rats  43  Mucosal P r o t e i n C o n c e n t r a t i o n i n Jejunum and Ileum o f C o n t r o l , P r o t e i n Depleted, and P r o t e i n Repleted Rats  47  L a c t o s e , Sucrose, and Maltose A b s o r p t i o n i n C o n t r o l , P r o t e i n Depleted, and P r o t e i n Repleted Rats  48  ix  LIST OP FIGURES FIGURE 1.  2.  3.  PAGE Changes i n Body Weight o f C o n t r o l , P r o t e i n Depleted, and P r o t e i n R e p l e t e d Rats  33  T y p i c a l Appearance o f Rats on the Following Three d i e t s : 1% Lactalbumin, R e s t r i c t e d t o 4 g per Day ( L e f t ) ; 18% Lactalbumin (Center); and 0.5% L a c t a l bumin High Carbohydrate (Right)  36  A Close-up o f the Rat on the 0.5% L a c t a l bumin High Carbohydrate D i e t Shown i n Figure 2  37  1  CHAPTER I LITERATURE REVIEW A.  Introduction Protein-calorie malnutrition  problems i n d e v e l o p i n g c o u n t r i e s . c l e a r l y shown t o be a s s o c i a t e d i n t e s t i n a l disaccharidases  is s t i l l  suffered not  been  (2).  Bowie e t a l .  of c h i l d r e n w i t h p r o t e i n - c a l o r i e  a c t i v i t i e s are p r i m a r i l y due  malnutrition  and  diminished  to protein  depriv-  deficiencies  of  other than p r o t e i n .  In order to c l a r i f y t h i s problem o f carbohydrate erance i n p r o t e i n - c a l o r i e d e f i c i e n c y , t o r s have s t u d i e d  a number of  intol-  investiga-  the e f f e c t o f p r o t e i n d e f i c i e n t d i e t s  i n t e s t i n a l disaccharidase  activities  i n young r a t s  on  (4,5).  r e s u l t s obtained i n those s t u d i e s were s u r p r i s i n g because  protein deprivation  i n r a t s produced an  increase  s p e c i f i c a c t i v i t i e s of d i s a c c h a r i d a s e s and maltase and this  (3)  However, i t i s  intolerance  a t i o n , or t o g a s t r o i n t e s t i n a l i n f e c t i o n , and  The  of  (1) which i s p r o b a b l y a consequence  c l e a r whether the d i s a c c h a r i d e  nutrients  i t has  w i t h depressed a c t i v i t i e s  from malabsorption of c a r b o h y d r a t e s .  disaccharidase  of the major  In c h i l d r e n ,  of a l t e r a t i o n s of i n t e s t i n a l s t r u c t u r e noted t h a t 60%  one  sucrase.  increase  I t has  in  the  p a r t i c u l a r l y of  been suggested, t h e r e f o r e ,  i n disaccharidase  activities  a d a p t a t i o n t o the h i g h p r o p o r t i o n  i n r a t s was  of carbohydrate i n  p r o t e i n d e f i c i e n t d i e t s s i n c e most of the  that an  the  investigators  2  r e p l a c e d the p r o t e i n p o r t i o n o f the c o n t r o l d i e t w i t h hydrate. designed  carbo-  I t should a l s o be p o i n t e d out t h a t i n the experiments t o show the e f f e c t of p r o t e i n d e f i c i e n t d i e t s on  i n t e s t i n a l d i s a c c h a r i d a s e a c t i v i t e s , the experimental  animals  have n o t been kept on the p r o t e i n d e f i c i e n t d i e t s long enough t o develop edema which i s so c h a r a c t e r i s t i c o f the f u l l kwashiorkor syndrome i n c h i l d r e n  (4,5,6).  Almost a l l o f the s t u d i e s on the e f f e c t o f p r o t e i n maln u t r i t i o n on carbohydrate  absorption reported  i n the l i t e r a -  t u r e determined changes i n i n t e s t i n a l d i s a c c h a r i d a s e without  measuring the a b s o r p t i o n o f d i s a c c h a r i d e s .  activities Depressed  l e v e l s o f d i s a c c h a r i d a s e s can be i m p l i c a t e d as b e i n g t i o n a l l y s i g n i f i c a n t only levels are r e f l e c t e d as w e l l  (7).  i f variations i n disaccharidase  i n changes i n d i s a c c h a r i d e  A l s o , i t i s important  absorption  t o study the a b s o r p t i o n  o f the d i s a c c h a r i d e s by an in_ v i v o technique which separate  func-  as procedures  the i n t e s t i n e from the animal c r e a t e a  c o n d i t i o n i n which the b l o o d v e s s e l s a r e no l o n g e r  present  t o c a r r y away the absorbed m a t e r i a l . With the p r e c e d i n g  ideas i n mind, i t seemed  necessary  to f u r t h e r study the e f f e c t o f long-term p r o t e i n - c a l o r i e d e p r i v a t i o n on i n t e s t i n a l d i s a c c h a r i d a s e a c t i v i t i e s and on in vivo absorption of disaccharides. B.  D i g e s t i o n and Absorption 1.  o f Carbohydrates  Review o f d i g e s t i o n and a b s o r p t i o n o f carbohydrates.  Carbohydrates a r e i n g e s t e d i n complex forms which must be broken down t o t h e i r simpler components b e f o r e  they can be  3  absorbed.  The  carbohydrates hydrates  process  o f d i g e s t i o n and a b s o r p t i o n of d i e t a r y  i s shown i n Table I .  occurs  The d i g e s t i o n o f  carbo-  i n the lumen o f the s m a l l i n t e s t i n e w i t h  a c t i o n o f amylase on the p o l y s a c c h a r i d e s , amylose and p e c t i n , and  the  amylo-  a t the s u r f a c e of the mucosal c e l l s of the  small  i n t e s t i n e w i t h the a c t i o n o f the d i s a c c h a r i d a s e s ( l a c t a s e , sucrase, and  and  maltase) on the d i s a c c h a r i d e s  maltose).  The  f i n a l products  are the monosaccharides: The  glucose,  ( l a c t o s e , sucrose,  of disaccharide h y d r o l y s i s f r u c t o s e , and  galactose.  monosaccharides are then t r a n s p o r t e d a c r o s s the mucosal  membrane by e i t h e r a c t i v e t r a n s p o r t , as i n the case o f  glucose  and g a l a c t o s e , or f a c i l i t a t e d d i f f u s i o n , as i n the case o f fructose  (8).  h y d r o l y s i s and  When the complete process  of disaccharide  monosaccharide a b s o r p t i o n were s t u d i e d i n  the a c t i o n of l a c t a s e was  found to be  the r a t e - l i m i t i n g  i n the a b s o r p t i o n of l a c t o s e (9), whereas  step  the mucosal t r a n s -  f e r o f g l u c o s e was  the r a t e - l i m i t i n g step i n sucrose  maltose a b s o r p t i o n  (10,11).  2.  man,  and  L o c a t i o n o f the i n t e s t i n a l d i s a c c h a r i d a s e s .  The  a c t i v i t i e s o f i n t e s t i n a l d i s a c c h a r i d a s e s , l a c t a s e , sucrase, and  maltase, are not u n i f o r m l y d i s t r i b u t e d along  the  length  o f the s m a l l i n t e s t i n e w i t h the h i g h e s t a c t i v i t y b e i n g i n the j e j u n a l s e c t i o n and i n the  the lowest a c t i v i t y b e i n g  i l e a l s e c t i o n i n r a t s (12), p i g s  The  (13), and man  found  found (14).  l a c t a s e , sucrase and maltase a c t i v i t i e s are l o c a t e d  i n the b r u s h b o r d e r of the mucosa (15) w i t h most of the a c t i v i t y being i n the c r y p t s  found along (16).  The  the s i d e s of the v i l l i  a c t i v i t i e s o f sucrase  and  and  not  maltase  TABLE I * DIGESTION AND ABSORPTION OF CARBOHYDRATES CARBOHYDRATE  SITES OF DIGESTION Intraluminal  Starch  amylase  (MODE OF TRANSPORT)  Muscosal  maltose  amylose  maltotriose maltase  amylopectin  ABSORPTION  amylase  glucose  active  a l p h a d e x t r inase -> g l u c o s e  active  glucose  active  maltose ^  maltotriose alpha d e x t r ins  Sucrose  sucrase  Lactose  lactase  *Taken from: Ward A. Olsen, "Carbohydrate Post Graduate Medicine, 51:149-152, 1972.  -> f r u c t o s e glucose 'galactose  facilitated active active  D i g e s t i o n and A b s o r p t i o n , "  diffusion  5  have been a s s o c i a t e d w i t h s m a l l knobs on the o u t e r edge o f the  microvilli 3.  age.  (17,18).  Changes i n i n t e s t i n a l d i s a c c h a r i d a s e a c t i v i t i e s w i t h  At the time o f b i r t h i n the r a t , the l e v e l s o f l a c t a s e  a c t i v i t y a r e h i g h and decrease d u r i n g the immediate  post  weaning p e r i o d t o the low l e v e l s c h a r a c t e r i s t i c o f the a d u l t animal.  Sucrase and maltase a c t i v i t i e s a r e low a t b i r t h , and  by 4 t o 5 weeks o f age, i n c r e a s e t o l e v e l s found i n a d u l t rats  (19,20). In  man, sucrase and maltase a c t i v i t i e s  I) a t b i r t h a r e the same as i n a d u l t l i f e of  (except f o r maltase  (14).  I n the case  l a c t a s e , the l e v e l o f a c t i v i t y found a t b i r t h may e i t h e r  remain the same throughout the l i f e o f the i n d i v i d u a l or decrease w i t h i n the f i r s t  decade of l i f e  a c t i v i t y which would then be permanent.  t o a lower l e v e l o f The p o p u l a t i o n s i n  which a decrease i n l a c t a s e a c t i v i t y a f t e r e a r l y c h i l d h o o d has been shown t o be v e r y p r e v a l e n t a r e l i s t e d by Herber ( 1 4 ) . 4. of  E f f e c t o f d i e t a r y carbohydrate on a c t i v i t y  i n t e s t i n a l disaccharidases.  levels  The a c t i v i t y o f the i n t e s t i n a l  d i s a c c h a r i d a s e s can be i n f l u e n c e d by the carbohydrate c o n t e n t of  the d i e t .  I t has been demonstrated  t h a t a f t e r a 3 day  f a s t , a 70% sucrose o r maltose d i e t causes i n c r e a s e d  jejunal  sucrase and maltase a c t i v i t i e s i n the r a t when compared t o animals f e d a c a r b o h y d r a t e - f r e e d i e t a f t e r the f a s t In  (7,21).  the case o f l a c t a s e , some r e s e a r c h e r s have found an  i n c r e a s e i n i n t e s t i n a l l a c t a s e a c t i v i t y i n r a t s i n response to  h i g h l e v e l s o f l a c t o s e i n the d i e t a f t e r weaning, (20,  6  22,23) w h i l e o t h e r s were not able t o c o n f i r m  these f i n d i n g s  (12,19,24). Studies done on the e f f e c t o f d i e t a r y carbohydrate the a c t i v i t i e s o f i n t e s t i n a l d i s a c c h a r i d a s e s have shown t h a t an  increase  i n d i e t a r y sucrose and  r e s u l t s , a f t e r 3 t o 5 days, i n s i g n i f i c a n t sucrase and  maltase a c t i v i t i e s .  d i e t s containing and  i n man  increased  (25,26) fructose  increases  in  When s u b j e c t s were g i v e n  0 t o 80% sucrose o r g l u c o s e ,  maltase a c t i v i t i e s  j e j u n a l sucrase  i n the s u b j e c t s  receiving  the sucrose or g l u c o s e i n the d i e t as compared t o the hydrate-free  group (27).  The  increases  i n sucrase,  maltase a c t i v i t i e s were p r o p o r t i o n a l to the and  g l u c o s e i n the d i e t .  C.  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 and in 1.  The  and  l e v e l s of sucrose  Carbohydrate  Absorption  e f f e c t o f 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 on  gastrointestinal tract  i n the mucosal t i s s u e of  i n c l u d e t h i n n i n g of the  mucosa; broadening and  h e i g h t o f the e p i t h e l i a l c e l l s .  the s u r f a c e  the  intestinal  atrophy o f the  i n c r e a s e d c e l l u l a r i t y o f the lamina p r o p r i a ; and  of lymphocytic  the  In 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 changes t h a t take p l a c e  in  carbo-  Man  i n t e s t i n a l mucosa i n man.  w a l l and  on  villi; decreased  There i s a l s o the presence  i n f i l t r a t i o n between the e p i t h e l i a l c e l l s  o f the v i l l i  (28).  on  These changes have been noted  c h i l d r e n s u f f e r i n g from 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  S t a n f i e l d e t a l . (2), Burman (29), Barbezat e t a l . (30),  by  7  Brunser e t a l . (31), and B e r k e l e t a l . (32) and  i n mal-  n o u r i s h e d a d u l t s by Tandon e t a l . (33), and Mayoral e t a l . (34,35) . The renewal o f the i n t e s t i n a l mucosa as expressed by the mitotic  index (which i s the percentage o f the e p i t h e l i a l  c e l l s undergoing m i t o s i s i n the c r y p t of Lieberkuhn  (36) has  been shown t o be lower f o r marasmic p a t i e n t s than i n kwashi o r k o r p a t i e n t s and both are lower than i n h e a l t h y i n d i v i d uals  (31,36). 2.  The e f f e c t o f 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 on the  i n t e s t i n a l d i s a c c h a r i d a s e s i n man.  The r e s u l t s o f s t u d i e s on  the a c t i v i t y o f i n t e s t i n a l d i s a c c h a r i d a s e s i n c h i l d r e n  suffer-  i n g from 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 i n v a r i o u s areas of t h e w o r l d i n c l u d i n g Uganda (2), South A f r i c a Turkey  (32) have demonstrated  significantly  (30), and lower l e v e l s o f  the t h r e e d i s a c c h a r i d a s e s , l a c t a s e , s u c r a s e , and maltase, when compared t o the l e v e l s o f a c t i v i t y i n h e a l t h y c h i l d r e n o f s i m i l a r age.  f o r these enzymes  James (37)  studied  Jamaican c h i l d r e n w i t h kwashiorkor and marasmus and  found  t h a t 8 o f the 19 c h i l d r e n had decreased l a c t a s e , s u c r a s e , and maltase a c t i v i t i e s  i n the j e j u n a l b i o p s y  specimens.  The c h i l d r e n diagnosed as marasmic had h i g h e r l e v e l s o f l a c t a s e , s u c r a s e , and maltase a c t i v i t i e s than the c h i l d r e n diagnosed as marasmic-kwashiorkor 3.  or kwashiorkor.  The e f f e c t o f 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 on d i s -  acchar ide a b s o r p t i o n i n man.  Bowie e t a l . (38) proposed  t h a t the d i a r r h e a , which i s o f t e n a s e r i o u s c o m p l i c a t i o n o f  8  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 , i s caused by i n t o l e r a n c e t o disaccharides.  To t e s t t h i s h y p o t h e s i s , they removed c a r b o -  hydrates from the d i e t o f 13 malnourished A f r i c a n c h i l d r e n s u f f e r i n g from d i a r r h e a . f r e e d i e t , there was  A f t e r 3 days on the c a r b o h y d r a t e -  a s i g n i f i c a n t decrease i n s t o o l weight  and a decrease i n f e c a l l a c t i c a c i d  i n 9 of the 13  children.  L a c t o s e , g l u c o s e / g a l a c t o s e , or g l u c o s e t o l e r a n c e t e s t s performed on 3 o f the c h i l d r e n showed an i n t o l e r a n c e t o l a c t o s e b u t not t o the g l u c o s e / g a l a c t o s e mixture o r g l u c o s e alone. F u r t h e r s t u d i e s by Bowie e t a l . (3,39) of malnourished c h i l d r e n w i t h d i a r r h e a showed t h a t the use o f a c a r b o h y d r a t e f r e e formula c o r r e c t e d the d i a r r h e a i n 60 t o 65% o f the children.  The c h i l d r e n who  d i e t demonstrated  improved  on the c a r b o h y d r a t e - f r e e  an i n t o l e r a n c e t o a l a c t o s e l o a d but not t o  an e q u i v a l e n t load o f e i t h e r sucrose, maltose or g l u c o s e p l u s galactose. was  Lactase a c t i v i t y  below normal  i n the j e j u n a l b i o p s y samples  i n 8 out of 20 malnourished c h i l d r e n ,  rase a c t i v i t y was below normal was below normal  i n 3, and maltase  suc-  activity  i n one.  P r i n s l o o e t a l . (40) s t u d i e d the e f f e c t s o f v a r i o u s d i e t a r y monosaccharides  and d i s a c c h a r i d e s i n the c o n t r o l of  diarrhea i n children with protein-calorie  malnutrition.  They d i v i d e d 120 malnourished Bantu c h i l d r e n i n t o 6 groups and gave each group a d i f f e r e n t source o f d i e t a r y carbohydrate i n t h e i r formula.  F i v e o f the groups were g i v e n the same  b a s i c formula except t h a t the carbohydrate source was  either  9  l a c t o s e , g l u c o s e , s u c r o s e , d e x t r i n - m a l t o s e , or c a r b o h y d r a t e free.  The s i x t h group o f c h i l d r e n was  powdered m i l k formula.  fed a  full-cream  The c h i l d r e n on the formula w i t h  l a c t o s e or the f u l l - c r e a m powdered m i l k s u f f e r e d lactic acid  the most  severe diarrhea  and had the h i g h e s t  excretion  i n the s t o o l s .  The s e v e r i t y o f the d i a r r h e a was about the  same i n the groups of c h i l d r e n w i t h glucose, sucrose, or d e x t r i n - m a l t o s e i n t h e i r d i e t s and the c a r b o h y d r a t e - f r e e group.  The a c t i v i t i e s o f i n t e s t i n a l l a c t a s e , sucrase, and  m a l t a s e f o r the malnourished c h i l d r e n i n these s t u d i e s by P r i n s l o o e t a l . (40) were lower than those f o r normal c h i l d r e n w i t h l a c t a s e a c t i v i t y b e i n g the most a f f e c t e d . James  (41,42,43) s t u d i e d malnourished Jamaican  u s i n g an i n t e s t i n a l p e r f u s i o n generalized tion.  defect  infants  technique, and demonstrated a  i n g l u c o s e , sucrose, and l a c t o s e  absorp-  There was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between  the r a t e o f h y d r o l y s i s o f the d i s a c c h a r i d e s of disaccharidase  activity  and the l e v e l  i n the j e j u n a l mucosa.  f o r 2 t o 4 months w i t h a h i g h p r o t e i n d i e t r e s u l t e d increase 4.  i n the a b s o r p t i o n  i n an  o f g l u c o s e , s u c r o s e , and l a c t o s e .  The r e l a t i o n s h i p between l a c t o s e  protein-calorie malnutrition lactase deficiency.  Treatment  intolerance i n  and the incidence  Lactose i n t o l e r a n c e  o f primary  i n a d u l t s due t o  primary l a c t a s e d e f i c i e n c y e x i s t s w i t h v a r y i n g  frequency i n  d i f f i c u l t e t h n i c groups as reviewed by Herber  (14), and many  of the p o p u l a t i o n  groups w i t h h i g h prevalence o f primary  l a c t a s e d e f i c i e n c y are p o p u l a t i o n s i n which p r o t e i n - c a l o r i e  10  malnutrition e t a l . (45)  i s also prevalent. studied  the  Wharton e t a l . (44)  incidence  of d i s a c c h a r i d e  i n the Ugandan Bantu p o p u l a t i o n  and  erance t o l a c t o s e which d i d not  improve w i t h the  d e f i c i e n c y of l a c t a s e  The  f a m i l y h i s t o r y was  e t a l . (46). (<pH  6.0)  Of the  stools.  100  intol-  treatment  t o the  underlying  activity.  presence o f h e r e d i t a r y  v e r i f i e d by  be due  Cook  intolerance  suggested t h a t the  f o r 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 may genetic  and  l a c t a s e d e f i c i e n c y which reported  from I n d i a by  infants studied,  50% had  When l a c t o s e , sucrose, and  was  Chandra  low  pH  maltose  toler-  ance t e s t s were performed on those 50 c h i l d r e n , 39 were i n t o l e r a n t t o l a c t o s e , 25 t o s u c r o s e , and  16 t o maltose.  A f t e r treatment w i t h a h i g h p r o t e i n d i e t f o r 3 months, 4 of the  50 c h i l d r e n were s t i l l  considered  i n t o l e r a n t t o l a c t o s e and  t o have primary l a c t a s e d e f i c i e n c y .  Therefore, i n t o l e r a n c e and  i t has been w e l l documented t h a t depression  of d i s a c c h a r i d a s e  problem f o r man^f c h i l d r e n who calorie malnutrition.  The  disaccharide  activities  is a  a r e s u f f e r i n g from p r o t e i n -  However, i t i s not c l e a r whether  protein-calorie malnutrition 5.  were  i s the cause o f these problems.  e f f e c t o f i n f e c t i o n s and  i e s on the a c t i v i t y o f the  intestinal  nutritional deficiencdisaccharidases.  P r o t e i n d e f i c i e n c y u s u a l l y does not occur w i t h o u t  other  accompanying n u t r i t i o n a l d e f i c i e n c i e s or w i t h o u t the t i o n o f an accompanying i n f e c t i o n .  complica-  In c h i l d r e n w i t h 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 , admission t o the h o s p i t a l may r e s u l t of an acute episode o f d i a r r h e a  be  the  of unknown cause.  11  James (43) concluded from h i s survey on the e f f e c t s o f 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 on i n t e s t i n a l a b s o r p t i o n the r e l e v a n t tion  that  importance o f acute i n f e c t i o n s or o f m a l n u t r i -  i t s e l f on the changes which occur i n the i n t e s t i n a l  f u n c t i o n and s t r u c t u r e have n o t been determined.  In the  study by Barbezat e t a l . (30), there was some c o r r e l a t i o n between the presence o f G i a r d i a Iambiia i n f e s t a t i o n and the depression  o f l a c t a s e and sucrase a c t i v i t i e s .  I n the s t u d i e s  by P r i n s l o o e t a l . (40) and Bowie e t a l . (3,39), however, there was no c o r r e l a t i o n between the presence o f pathogens and the s e v e r i t y o f the d i a r r h e a .  Mata e t a l . (47) r e p o r t e d  t h a t the s t u d i e s performed a t the c l i n i c a l u n i t a t INCAP ( I n s t i t u t e o f N u t r i t i o n o f C e n t r a l America and Panama) on 13 c h i l d r e n w i t h kwashiorkor or marasmus showed t h a t  there  was more b a c t e r i a i n the stomach and jejunum o f malnourished c h i l d r e n w i t h d i a r r h e a than i n malnourished c h i l d r e n who were n o t s u f f e r i n g from d i a r r h e a . A decrease i n i n t e s t i n a l d i s a c c h a r i d a s e  a c t i v i t i e s may  occur due t o damage o f the i n t e s t i n a l mucosa caused by infestation.  Hookworms a r e an example o f t h i s  T r i p a t h y e t a l . (50) noted abnormal absorption  (48,49).  r e s u l t s o f D-xylose  t e s t s and mucosal damage i n c h i l d r e n w i t h heavy  Ascaris i n f e c t i o n s . Therefore, been d i s c u s s e d of disaccharide  the r e s u l t s o f these s t u d i e s which have j u s t show t h a t the r o l e o f i n f e c t i o n i n the e t i o l o g y intolerance i n protein-calorie malnutrition  i s not completely understood and needs t o be s t u d i e d  further.  12  Certain n u t r i t i o n a l d e f i c i e n c i e s besides  protein  d e f i c i e n c y have been shown t o a f f e c t the g a s t r o i n t e s t i n a l tract.  When Naiman e t a l . (51) s t u d i e d b i o p s y  samples o f 14  i n f a n t s and c h i l d r e n who were i r o n d e f i c i e n t t h e r e was a high and  incidence of abnormalities function.  shortening  of gastrointestinal structure  The s t u d i e s o f duodenal b i o p s y  and c l u b b i n g  o f the v i l l i ,  i n f i l t r a t i o n o f t h e lamina p r o p r i a .  samples showed  and i n c r e a s e d  cellular  The a b s o r p t i o n o f  x y l o s e was impaired.  When the i r o n d e f i c i e n c y was c o r r e c t e d ,  the xylose absorption  and s t r u c t u r a l a b n o r m a l i t i e s  villi  returned  charidases  t o normal.  Although the i n t e s t i n a l  were not s t u d i e d the f i n d i n g s i n t h i s  of the disac-  specific  experiment demonstrate t h a t n u t r i t i o n a l d e f i c i e n c i e s other than p r o t e i n may be i n v o l v e d i n the damage t o the g a s t r o i n t e s t i n a l structure is  and g a s t r o i n t e s t i n a l f u n c t i o n which  found i n c h i l d r e n s u f f e r i n g from p r o t e i n - c a l o r i e m a l n u t r i -  tion. D.  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 and Carbohydrate  Absorption  i n L a b o r a t o r y Animals. Various  animals have been used t o study the e f f e c t o f  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 on carbohydrate d i g e s t i o n and absorption,  i n c l u d i n g monkeys  (55), and r a t s 1.  (52,53), baboons (54),  pigs  (4,5,6,56,57,58,59,60).  The e f f e c t o f 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 on t h e  i n t e s t i n a l mucosa i n l a b o r a t o r y a n i m a l s .  When the s t r u c t u r e  o f the i n t e s t i n a l mucosa of animals on low p r o t e i n d i e t s was s t u d i e d , changes s i m i l a r t o those found i n c h i l d r e n and a d u l t s  13  s u f f e r i n g from 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 have been noted. Deo  e t a l . (52) working w i t h monkeys, were able t o demonstrate  atrophy of the mucosa a f t e r 8 weeks on a p r o t e i n - f r e e d i e t , when compared to monkeys on a c o n t r o l d i e t c o n t a i n i n g 20% c a s e i n .  shape of the v i l l i  d i d not  change.  a l s o demonstrated by Kumar and  the v i l l i were decreased i n s i z e . included  in  l e s s than  Chase  1%  (53).  on a p r o t e i n - f r e e d i e t f o r p e r i o d s  the e p i t h e l i a l c e l l s  the  Atrophy of the v i l l i  5 weeks (61) were found to have t h i n n e r  in  a  i n the mucosa, however,  the monkey a f t e r 18 weeks on a d i e t c o n t a i n i n g  Rats p l a c e d  a  per square u n i t of mucosa and  decrease i n the number of c e l l s  and  to  In the p r o t e i n d e f i c i e n t monkeys, t h e r e was  decreased number of v i l l i  p r o t e i n was  15  intestinal  up  to  walls  U l t r a s t r u e t u r a l changes  irregular microvilli;  decrease i n the endoplasmic r e t i c u l u m and  RNA  changes i n the m i t o c h o n d r i a l  H i l l et a l .  structures.  a  granules;  and (62)  s t u d i e d the e f f e c t o f p r o t e i n d e p r i v a t i o n on weanling r a t s and  found t h a t the animals which were p r o t e i n d e f i c i e n t f o r  up t o 45 days d i d not i n t e s t i n e s and  had  increase  the  l e n g t h of t h e i r small  s i g n i f i c a n t l y shorter v i l l i  as compared t o the c o n t r o l s .  The  villi  i n the  i n the  ileum  j e j u n a l seg-  ment i n the p r o t e i n d e f i c i e n t animals were t h i n n e r b u t  not  s h o r t e r than i n the c o n t r o l s i n d i c a t i n g t h a t the endogenous protein repair.  i n the lumen i s a v a i l a b l e to the  j e j u n a l mucosa f o r  14  2.  The  e f f e c t of p r o t e i n - c a l o r i e malnutrition  i n t e s t i n a l disaccharidases  i n l a b o r a t o r y animals. The e f f e c t  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 on i n t e s t i n a l has  been s t u d i e d  on  disaccharidases  i n l a b o r a t o r y animals i n order  to c l a r i f y  the problem o f carbohydrate i n t o l e r a n c e i n c h i l d r e n s u f f e r ing from 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 . it  i s p o s s i b l e t o study the  deficiency, on the  i n f e c t i o n , and  In the animal model,  i n d i v i d u a l e f f e c t s of p r o t e i n other n u t r i t i o n a l d e f i c i e n c i e s  i n t e s t i n a l disaccharidases  which cannot be done  on  human s u b j e c t s . Solimano e t a l . (4) i n v e s t i g a t e d the e f f e c t o f p r o t e i n d e f i c i e n c y on the a c t i v i t y o f the i n r a t s and  obtained  jejunal  surprising results.  disaccharidases Sucrase and  maltase  a c t i v i t i e s were s i g n i f i c a n t l y e l e v a t e d a f t e r the animals been fed a p r o t e i n - f r e e or 5% p r o t e i n d i e t f o r 22 and days r e s p e c t i v e l y .  imately  fell  from the p r e v i o u s l y h i g h values  increase  p r o t e i n d e f i c i e n t d i e t s may s i g n i f i c a n t increases t o enzyme i n d u c t i o n by  maltase  t o approx-  the normal l e v e l s found i n the c o n t r o l s .  suggested t h a t the  44  When these r a t s were then g i v e n a p r o t e i n  adequate d i e t f o r 7 days, the l a c t a s e , sucrase and activities  had  The  authors  i n carbohydrate content i n the have been r e s p o n s i b l e  i n sucrase and  f o r the  maltase a c t i v i t i e s  the h i g h p r o p o r t i o n  due  o f carbohydrate  i n the d i e t . The  e x p e r i m e n t a l d i e t s used i n the  i n v e s t i g a t i o n by  Solimano e t a l . (4) were based on a complete i n f a n t food formula t o which c o r n s t a r c h was  added t o g i v e f i n a l  protein  concentrations vitamins  o f 10, 5 and 0.4% i n the t h r e e d i e t s .  and minerals  Extra  were n o t added t o the 5% and 0.4%  p r o t e i n d i e t s so t h a t the low p r o t e i n d i e t s were n o t complete i n a l l the r e q u i r e d n u t r i e n t s .  Also the proportion  c a r b o h y d r a t e i n the t h r e e d i e t s was not the same. the changes i n the i n t e s t i n a l d i s a c c h a r i d a s e  o f the Therefore,  activities  can-  n o t be a t t r i b u t e d o n l y t o the p r o t e i n d e p r i v a t i o n as the authors n o t e d . accharidase Therefore,  A l s o , t h e changes i n the i n t e s t i n a l  dis-  a c t i v i t i e s were o n l y determined i n the jejunum. i t i s n o t known whether the e f f e c t o f 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 has the same e f f e c t on the ileum. Prosper e t a l . (5) i n v e s t i g a t e d the e f f e c t o f 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 on i n t e s t i n a l d i s a c c h a r i d a s e s day  o l d r a t s given  protein.  synthetic diets containing  The i n c r e a s e  i n disaccharidase  27 or 0%  activities  on the p r o t e i n - f r e e d i e t was o n l y s t a t i s t i c a l l y in the t h i r d quarter t o the c o n t r o l s .  o f the s m a l l  i n 22  in rats  significant  i n t e s t i n e when compared  When the animals on t h e p r o t e i n - f r e e d i e t  were f o r c e f e d f o r 20 days so t h a t the c a l o r i c intake would be the same i n both the p r o t e i n - f r e e and t h e c o n t r o l groups, the o n l y s i g n i f i c a n t d i f f e r e n c e s found were an i n c r e a s e i n sucrase a c t i v i t y and  i n the d i s t a l h a l f o f the s m a l l i n t e s t i n e  a decrease i n l a c t a s e a c t i v i t y  the s m a l l  i n the p r o x i m a l h a l f o f  i n t e s t i n e i n the p r o t e i n - f r e e group.  a c t i v i t y o f l a c t a s e , sucrase,  When t o t a l  and maltase was determined,  the l e v e l s o f a c t i v i t y were lower i n the p r o t e i n - f r e e group than i n t h e c o n t r o l group.  When t o t a l enzyme a c t i v i t y was  expressed per 100 grams o f body weight,  t h e r e ware no  c a n t d i f f e r e n c e s between the two groups which had  signifi-  similar  body weights. The e x p r e s s i o n o f t o t a l enzyme a c t i v i t y per 100 grams body weight may  not be v a l i d when comparing h e a l t h y c o n t r o l  animals t o p r o t e i n d e f i c i e n t animals as t h e r e are probably d i f f e r e n c e s i n body composition between the two groups (63). In the study by Prosper e t a l . (5) the d i s a c c h a r i d a s e a c t i v i t i e s were determined  a l o n g the e n t i r e l e n g t h o f the  s m a l l i n t e s t i n e so t h a t a more complete  view o f the e f f e c t  o f the p r o t e i n d e p l e t i o n c o u l d be observed.  However, as i n  the study by Solimano e t a l . (4) the carbohydrate content of the c o n t r o l and p r o t e i n - f r e e d i e t was  not the same.  There-  f o r e , the p o s s i b i l i t y t h a t the higher p r o p o r t i o n o f c a r b o hydrate c o u l d be  i n f l u e n c i n g the d i s a c c h a r i d a s e a c t i v i t i e s  i n the p r o t e i n - f r e e group s t i l l  exists.  In the study by T r o g l i a e t a l . (56) r a t s g i v e n a r e stricted  i n t a k e of r a t chow f o r 5% t o 6% months had e l e v a t e d  l a c t a s e , sucrase and maltase a c t i v i t i e s when compared t o the c o n t r o l groups.  There were 3 c o n t r o l groups i n t h i s  one group of c o n t r o l s which was the r e s t r i c t e d  i n t a k e was  younger than the group w i t h  fed the r a t chow ad 1ibitum u n t i l  the r a t s reached the f i n a l weight o f the r e s t r i c t e d 206 grams; the second c o n t r o l group was r e s t r i c t e d group and was  study:  group,  the same age as the  fed the r a t chow ad_ l i b i t u m ;  the t h i r d c o n t r o l group was  meal f e d .  When t o t a l  d i s a c c h a r i d a s e a c t i v i t i e s were determined,  and  intestinal  only the  calorie  17  r e s t r i c t e d group had s i g n i f i c a n t l y h i g h e r t o t a l activity.  lactase  However, the s p e c i f i c a c t i v i t i e s o f l a c t a s e ,  sucrase and maltase were s i g n i f i c a n t l y h i g h e r i n the c a l o r i e r e s t r i c t e d group than i n the c o n t r o l  groups.  Kumar and Chase (59) produced a s t a t e of u n d e r n u t r i t i o n i n i n f a n t r a t s by a l t e r i n g the l i t t e r s i z e t o 16 animals per mother, 12 hours a f t e r b i r t h . who  A f t e r weaning, the r a t s  had been undernourished r e c e i v e d 8 grams o f an 8% p r o t e i n  d i e t per day as compared t o the c o n t r o l animals who a mean of 16 grams per day o f a 27% p r o t e i n d i e t .  consumed The  disac-  c h a r i d a s e a c t i v i t i e s were s t u d i e d a t v a r i o u s ages s t a r t i n g w i t h 7 days and ending w i t h 115 day t h a t l a c t a s e a c t i v i t y was  old rats.  I t was  s i g n i f i c a n t l y h i g h e r i n the  n o u r i s h e d group a t a l l ages except f o r a temporary a t 21 days when i t was group.  depression  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 i n sucrase  not determined.  r a t was  under-  s i g n i f i c a n t l y lower i n the undernourished  a c t i v i t y between the two groups a t any age. was  found  Maltase  activity  Kumar and Chase (59) concluded t h a t the  not a good e x p e r i m e n t a l model t o use f o r the study of  protein-calorie malnutrition in  man.  As i n the p r e v i o u s s t u d i e s of Solimano e t a l . (4) and Prosper e t a l . (5) the carbohydrate content o f the c o n t r o l and the low p r o t e i n d i e t s i n the study by Kumar and Chase was  not kept c o n s t a n t .  (59)  The p r o t e i n content o f the low p r o -  t e i n d i e t seems t o be too h i g h ( 8 % ) , however, the amount of the d i e t g i v e n each day was undernourished animals was  r e s t r i c t e d so the weight  of the  o n l y 20% o f the weight o f the  c o n t r o l s a f t e r 16 weeks of l i f e .  The  l e v e l s o f the  a r i d a s e a c t i v i t i e s were o n l y determined i n the  disacch-  jejunal sec-  tion . Kumar and  Chase (53)  then s t u d i e d  a low p r o t e i n d i e t (< 1%) o f 10 t o 18 weeks t o see  the e f f e c t o f  feeding  t o young a d u l t monkeys f o r  periods  i f the r e s u l t s resembled those found  i n 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 more c l o s e l y than r e s u l t s o f the s t u d i e s done w i t h r a t s .  the  A f t e r 6 weeks on  low p r o t e i n d i e t , the p r o t e i n d e f i c i e n t monkeys had  the  signifi-  c a n t l y reduced l a c t a s e a c t i v i t y when compared t o the c o n t r o l group which r e c e i v e d a 34% p r o t e i n d i e t . l a c t a s e , sucrase, c a n t l y reduced  and  i n the  maltase a c t i v i t i e s were a l l s i g n i f i low p r o t e i n group.  between the r e s u l t s which Kumar and ing  with rats  A f t e r 10 weeks,  The  differences  Chase obtained  (59) and when working w i t h monkeys (53)  t h a t the e f f e c t o f 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 on i n t e s t i n a l disaccharidases another.  Therefore,  r e s u l t s obtained 3.  The  when work-  one  may  differ  from one  suggest  the  species  to  must e x e r c i s e c a u t i o n when comparing  from d i f f e r e n t s p e c i e s .  e f f e c t o f 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 on absorp  tion of disaccharides  i n laboratory animals.  The e f f e c t 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 on monosaccharide and ide absorption  and  the a c t i v i t y  char idases was  o n l y s t u d i e d by L i f s h i t z e t a l . (6)  used three types of d i e t s : 70% carbohydrate, and  disacchar-  l e v e l s of i n t e s t i n a l  disacThey  a c o n t r o l d i e t w i t h 18% p r o t e i n ,  8% f a t ; a low p r o t e i n d i e t w i t h  p r o t e i n , 70% carbohydrate, and  8%  f a t ; and  4%  a low p r o t e i n ,  low  19  carbohydrate d i e t w i t h 4% p r o t e i n , 45% carbohydrate and fat.  Casein was  were not  used as the  deficient diets.  The the  glucose d e f i c i t s  The  added to  i n the two  protein  r a t s were fed the r e s p e c t i v e  low p r o t e i n d i e t s had  i n t e s t i n a l disaccharidases maltose.  The  s i g n i f i c a n t l y greater w i t h both 45% and  diets  no e f f e c t on the a c t i v i t y and  on  the a b s o r p t i o n  of  rate of glucose absorption  i n the r a t s on the  of  was  low p r o t e i n d i e t s  70% carbohydrate only a f t e r 14 days of  d i e t a r y treatment.  There was  no change i n the a b s o r p t i o n  f r u c t o s e or 3-0-methyl-D-glucose. i n which the p r o p o r t i o n c o n t r o l d i e t was  given  diets  to four weeks.  sucrose and  and  The  i s o c a l o r i c as n o n - n u t r i t i v e c e l l u l o s e was  make up the c a s e i n and  f o r one  source o f p r o t e i n .  8%  T h i s was  the  of carbohydrate i n the  kept the  same, but  first low  of  study  protein  the animals were  the r e s p e c t i v e d i e t s f o r o n l y 4 weeks.  After t h i s  s h o r t p e r i o d o f time, 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 i n the c o n c e n t r a t i o n d e f i c i e n t and  o f mucosal p r o t e i n between the  c o n t r o l animals.  protein  T h i s would i n d i c a t e t h a t  weeks of p r o t e i n d e p r i v a t i o n was  not  4  long enough to demon-  s t r a t e a l l the symptoms 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 . Therefore,  the changes i n d i s a c c h a r i d a s e  changes i n a b s o r p t i o n may  activities  of monosaccharides or  disaccharides  not be apparent a f t e r only 4 weeks o f p r o t e i n 4.  The  e f f e c t of non-protein f a c t o r s on the  of the i n t e s t i n a l d i s a c c h a r i d a s e s  or  in laboratory  deprivation. activity  animals.  The  r e s u l t s o f s t u d i e s d e a l i n g w i t h other f a c t o r s i n c l u d i n g i r o n  20  d e f i c i e n c y or i n f e s t a t i o n by nematodes support the idea the  associated  d e f i c i e n c i e s and i n f e c t i o n s t h a t accompany the  state of p r o t e i n - c a l o r i e malnutrition the d i s a c c h a r i d e  intolerance  may be important i n  found i n c h i l d r e n s u f f e r i n g  from 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 . (64)  that  Hoffbrand and Broitman  found t h a t l a c t a s e , sucrase, and maltase a c t i v i t i e s were  s i g n i f i c a n t l y decreased i n young dogs who were maintained on an the  i r o n d e f i c i e n t d i e t f o r 8 weeks when they were compared t o c o n t r o l group. S r i r a t a n a b a n and Thayer  (57) s t u d i e d  combined i r o n and p r o t e i n d e f i c i e n c y t i n a l disaccharidase i n t o 4 groups:  activities.  the c o n t r o l s  the e f f e c t o f  i n the r a t on the i n t e s -  The animals were d i v i d e d  (27% p r o t e i n ) , low p r o t e i n ( 5 %  p r o t e i n ) , low i r o n , and low iron-low p r o t e i n .  They were  maintained on these d i e t s f o r 75 t o 185 days from the time o f weaning.  As found i n p r e v i o u s s t u d i e s  (4,64) the animals  on the low p r o t e i n d i e t had s i g n i f i c a n t l y h i g h e r l a c t a s e , s u c r a s e , and maltase a c t i v i t i e s than the c o n t r o l s , and the i r o n d e f i c i e n t group had lower l a c t a s e , sucrase, and maltase a c t i v i t i e s when compared t o the c o n t r o l group. t i o n o f i r o n and 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 disaccharidase  The combina-  i n an  increase  a c t i v i t y i n the jejunum and ileum when  compared t o the c o n t r o l s , b u t the l e v e l s o f  disaccharidase  a c t i v i t i e s were lower when compared t o the p r o t e i n d e f i c i e n t group. B o l i n e t a l . (58) s t u d i e d upon d i s a c c h a r i d a s e  activity  the e f f e c t o f worm i n f e s t a t i o n  i n the r a t by i n f e s t i n g p a r t o f  21  a group o f animals on a c o n t r o l d i e t c o n t a i n i n g 30% p r o t e i n w i t h Nippostrongylus caused a d e p r e s s i o n  brasiliensis.  The worm i n f e s t a t i o n  o f j e j u n a l l a c t a s e and maltase a c t i v i t i e s  when compared t o the c o n t r o l group. There a r e many p o s s i b i l i t i e s  f o r f u r t h e r study  on the  e f f e c t s o f i n f e c t i o n s and n u t r i t i o n a l d e f i c i e n c i e s on the i n t e s t i n a l disaccharidase a c t i v i t i e s .  These must be e x p l o r e d  before a d e c i s i o n can be made on the s u i t a b i l i t y o f the r a t as a model f o r s t u d y i n g 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 . E.  O b j e c t i v e s o f the Present From the p r e c e d i n g  Study  review o f the l i t e r a t u r e  i t appears  t h a t w e l l c o n t r o l l e d s t u d i e s a r e needed i n order  to elucid-  a t e f u r t h e r the e f f e c t o f 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 on the d i g e s t i o n and a b s o r p t i o n o f carbohydrates. Therefore, t o study  the primary purpose of t h i s i n v e s t i g a t i o n i s  the e f f e c t o f prolonged  p r o t e i n d e f i c i e n c y and  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 upon the i n v i v o d i s a c c h a r i d e a b s o r p t i o n and i n t e s t i n a l d i s a c c h a r i d a s e a c t i v i t i e s by fe ed in g r a t s w e l l c o n t r o l l e d d i e t s .  The c o n t r o l d i e t w i l l  c o n t a i n 18% l a c t a l b u m i n and w i l l be f e d ad_ l i b i t u m .  The  p r o t e i n d e f i c i e n t d i e t w i l l c o n t a i n 0.5% l a c t a l b u m i n and w i l l a l s o be f e d ad_ l i b i t u m .  The 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  d i e t w i l l c o n t a i n 1% l a c t a l b u m i n and w i l l be g i v e n  in re-  s t r i c t e d amounts (4 grams p e r d a y ) . The  second purpose o f t h i s study  i s t o determine whether  marked i n c r e a s e s o f i n t e s t i n a l sucrase and maltase  activities  p r e v i o u s l y observed i n p r o t e i n d e f i c i e n t r a t s (4) c o u l d have  been i n f l u e n c e d by a h i g h p r o p o r t i o n diet.  T h i s w i l l be  diets  s t u d i e d by  of carbohydrate i n the  feeding r a t s p r o t e i n d e f i c i e n t  (0.5% l a c t a l b u m i n ) c o n t a i n i n g  d i f f e r e n t l e v e l s of  carbohydrate. The  t h i r d purpose of t h i s i n v e s t i g a t i o n  i s t o determine  whether p r o t e i n r e p l e t i o n leads to a r e v e r s a l of p r o t e i n maln u t r i t i o n symptoms to normal.  Rats w i l l be r e f e d a p r o t e i n  adequate d i e t a f t e r being on a p r o t e i n d e f i c i e n t d i e t l a c t a l b u m i n ) f o r 8.5 The a t two  (0.5%  weeks.  i n t e s t i n a l disaccharidase  s i t e s o f the s m a l l  a c t i v i t i e s w i l l be  i n t e s t i n e (jejunum and  i t cannot be assumed t h a t a change a t one  studied  ileum) s i n c e  s i t e w i l l occur a t  a l l p a r t s of the s m a l l i n t e s t i n e . Results  of t h i s i n v e s t i g a t i o n should h e l p  some of the d i g e s t i o n and s u f f e r i n g from p r o t e i n  absorption  deprivation.  in clarifying  problems i n c h i l d r e n  CHAPTER I I MATERIALS A.  AND METHODS  Animals and D i e t s White male r a t s o f the W i s t a r s t r a i n were obtained  the animal u n i t , F a c u l t y o f Medicine, B r i t i s h Columbia.  a t the U n i v e r s i t y o f  T h e i r body weights ranged from 90 grams  to 120 grams w i t h a mean o f 108 - 9 grams. divided  from  The r a t s were  i n t o four groups and fed the f o l l o w i n g d i e t s f o r a  p e r i o d o f 8 t o 9 weeks, w i t h the time p e r i o d depending on l a b o r a t o r y convenience.  One group o f r a t s r e c e i v e d a c o n t r o l  d i e t c o n t a i n i n g 18% l a c t a l b u m i n .  The second group was g i v e n  a 0.5% l a c t a l b u m i n low carbohydrate  d i e t , the t h i r d group  was g i v e n a 0.5% l a c t a l b u m i n h i g h carbohydrate  d i e t , and the  f o u r t h group was g i v e n a 1% l a c t a l b u m i n d i e t r e s t r i c t e d t o 4 grams per day.  P a r t o f the second group o f r a t s , a f t e r  f e d the 0.5% l a c t a l b u m i n low carbohydrate  being  d i e t f o r 8.5 weeks,  was g i v e n the c o n t r o l d i e t c o n t a i n i n g 18% l a c t a l b u m i n f o r 8 weeks.  The composition  o f these d i e t s i s listed i n Table I I .  These d i e t s a r e based on those developed f o r s t u d y i n g kwashiorkor and marasmus  by Edozien  in rats.  (65)  The only  m o d i f i c a t i o n o f the d i e t s f o r use i n the p r e s e n t experiment was the s u b s t i t u t i o n o f c o r n s t a r c h f o r dextrose and s u c r o s e . The  0.5% l a c t a l b u m i n d i e t s w i t h both the h i g h and low  l e v e l s o f carbohydrate  were designed  t o produce  symptoms  c h a r a c t e r i s t i c o f kwashiorkor i n c h i l d r e n which, among o t h e r s , i n c l u d e edema, l o s s i n body weight, f a t t y l i v e r , h a i r d i s -  TABLE I I COMPOSITION OP DIETS  Control 18% l a c t a l b u m i n  0.5% l a c t a l b u m i n Low carbohydrate  0.5% l a c t a l b u m i n High carbohydrate  1% l a c t a l b u m i n (Restricted to 4 grams per day)  Lactalbumin*  18 .0%  0 .5%  0..5%  1 .0%  Cornstrach  66 .0%  66 .0%  83..5%  66 .0%  10 .0%  17 .8%  10..0%  17 .6%  &  Corn o i l Alphacel  3 .4%  9 .7%  &  Salt Mix "®  5 .0%  5 .0%  5..0%  10 .0%  Vitamin Mix*  0 .5%  0 .5%  0..5%  1 .0%  Choline Chloride*  0 .5%  0 .5%  0,.5%  1 .0%  100 .0%  100 .0%  100. .0%  100 .0%  5  •Purchased from N u t r i t i o n a l Biochemical Co., C l e v e l a n d , P u r c h a s e d from General Biochemicals, Chagrin  Falls,  Ohio.  Ohio.  ^Rogers, O.R., and A.E. Harper, "Amino A c i d D i e t s and Maximal Growth i n the Rat," J . Nutr., 87:267-273, 1965.  coloration,  hypoalbuminemia, and  bances (63). the  gastrointestinal  Kwashiorkor i s the  deficiency  of p r o t e i n  d i e t w i t h an adequate intake of c a l o r i e s .  bumin d i e t r e s t r i c t e d to 4 grams per produce the  day  distur-  was  The  of both p r o t e i n  of marasmus i n c h i l d r e n retardation,  designed  and  calories.  The  symptoms  symptoms, f a t t y l i v e r ,  irritability.  However, the  hypoalbuminemia are  severe i n marasmus as they are  i n kwashiorkor  Animals on the  as  (63).  the  animals on  the  other  regimens were kept i n p l a s t i c cages i n groups of  t o f i v e per  talbumin d i e t  cage.  Except for the  1% l a c t a l b u m i n group was a.m.  r a t s on the  ( r e s t r i c t e d to 4 grams per  animals were g i v e n t h e i r d i e t and  and  day),  1%  lac-  a l l other  water ad_ l i b i t u m .  The  fed the 4 grams of d i e t once a  water was  were weighed a t weekly B.  not  1% l a c t a l b u m i n d i e t were housed i n d i v i d -  i n s c r e e n bottom cages, and  a t 9:30  hypo-  gastrointestinal  d i s t u r b a n c e s , f a t t y l i v e r , and  two  to  i n c l u d e muscular wasting, growth  gastrointestinal  albuminemia, and  dietary  lactal-  symptoms c h a r a c t e r i s t i c of marasmus which i s  the d e f i c i e n c y  ually  1%  in  g i v e n ad_ l i b i t u m .  A l l the  day  animals  intervals.  A b s o r p t i o n of D i s a c c h a r i d e s The  a b s o r p t i o n of l a c t o s e ,  sucrose, and  measured i n v i v o u s i n g r a d i o a c t i v e  disaccharides.  were a n e s t h e t i z e d w i t h i n t r a p e r i t o n e a l (Sodium p e n t o b a r b i t o l ) u s i n g 6.0 f o r the  t e s t animals and  f o r the  controls  7.1  mg  as the p r o t e i n  maltose  injections  The  was rats  of Nembutal  mg  per  100  g body weight  per  100  g of body weight  d e p l e t e d animals d i d  not  r e q u i r e as l a r g e a dose o f the a n e s t h e t i c as the c o n t r o l animals.  The Nembutal dose was i n j e c t e d g r a d u a l l y i n t h r e e  t o f i v e p o r t i o n s over a 15 t o 30 minute p e r i o d . The abdomenal w a l l was opened, and approximately a 10 cm j e j u n a l segment d i s t a l t o the ligament o f T r e i t z was t i e d o f f a t b o t h ends. was  Next, 0.5 ml o f the r a d i o a c t i v e  solution  i n j e c t e d i n t o the lumen o f the l i g a t e d j e j u n a l segment.  The e x a c t amount o f r a d i o a c t i v e s o l u t i o n i n j e c t e d i n t o the loop was determined by weighing the i n j e c t i o n .  the s y r i n g e b e f o r e and a f t e r  The i n t e s t i n a l loop was r e t u r n e d t o the  p e r i t o n e a l c a v i t y f o r a 15 minute p e r i o d d u r i n g which the animal was kept warm under a lamp.  A t the end o f t h i s  p e r i o d , the loop was e x c i s e d and r i n s e d w i t h 5 ml o f 0.9% s a l i n e , and the r i n s i n g s were c o l l e c t e d .  The loop was then  weighed, s l i t  open and the mucosa was scraped o f f u s i n g a  glass slide.  The musosal s c r a p i n g s were weighed and then  mixed w i t h 5 ml o f 0.9% s a l i n e u s i n g a g l a s s s t i r r i n g r o d . The  l u m i n a l r i n s i n g s and mucosal s c r a p i n g s were heated i n  b o i l i n g water i n order t o i n a c t i v a t e the enzymes and t o ensure t h a t a l l the r e s i d u a l d i s a c c h a r i d e was d i s s o l v e d i n the s a l i n e s o l u t i o n .  The volume o f the samples was made  up t o 10 ml w i t h 0.9% s a l i n e and mixed t h o r o u g h l y .  The  samples were then c e n t r i f u g e d a t 15,000 RPM f o r 15 minutes. The supernatant f r a c t i o n was removed and immediately f r o z e n . R a d i o a c t i v i t y was determined w i t h i n 1 t o 3 weeks. The r a d i o a c t i v i t y o f the prepared samples was counted i n a P i c k e r Nuclear L i q u i m a t S c i n t i l l a t i o n Counter u s i n g a 0.5 ml a l i q u o t o f the supernatant f r a c t i o n ; 2.0 ml o f N.C.S.  27  Tissue  Solubolizer  (Nuclear Chicago S o l u b o l i z e r , Amersham/  Searle Corp., A r l i n g t o n Heights, I l l i n o i s ) ; and 10.0 ml o f s c i n t i l l a t i o n solvent. up  The s c i n t i l l a t i o n s o l v e n t was made  o f 7.5 gm PPO and 62.5 mg o f POPOP per 1000 ml q . s .  toluene which were a l l o b t a i n e d from the F i s h e r Co.  Scientific  To measure t h e r a d i o a c t i v i t y o f the i n j e c t e d dose,  0.5 ml o f a s o l u t i o n c o n t a i n i n g  approximately 0.01 p C i p e r  ml was used as a s t a n d a r d . The  s c i n t i l l a t i o n samples were counted f o r 2 minutes  w i t h the f o l l o w i n g channel s e t t i n g s on the P i c k e r Nuclear L i q u i m a t 220: A Channel  Upper L i m i t Lower L i m i t  1000 0  B Channel  Upper L i m i t Lower L i m i t  780 80  C Channel  Upper L i m i t Lower L i m i t  1000 200  D Channel  Upper L i m i t Lower L i m i t  1000 320  The  amount o f d i s a c c h a r i d e  subtracting  absorbed was o b t a i n e d by  the r a d i o a c t i v i t y which was r e c o v e r e d i n the  lumen and the mucosa o f the l i g a t e d segment from the t o t a l i n j e c t e d dose.  The p e r cent o f dose absorbed was then  expressed p e r 500 mg wet weight o f the l i g a t e d i n t e s t i n a l segment. C.  Disaccharidase Following  Activities  the d e t e r m i n a t i o n o f d i s a c c h a r i d e  the r e s t of the s m a l l  absorption,  i n t e s t i n e , d i s t a l t o the ligament o f  T r i e t z and up t o the i l e o c e c a l j u n c t i o n was removed, r i n s e d  28  with  i c e - c o l d s a l i n e t o remove the i n t e s t i n a l contents,  p l a c e d on an i c e - c o l d s t a i n l e s s s t e e l t r a y . ine  was  The  then d i v i d e d i n t o four equal p a r t s , and  and  small  intest-  the segment  which had been used f o r the a b s o r p t i o n study was  accounted  for  maltase  i n the  f i r s t quarter.  Lactase,  sucrase, and  a c t i v i t i e s were determined i n the f i r s t and of the i n t e s t i n e . scraped  The  fourth quarters  s e c t i o n s were weighed, the mucosa  o f f w i t h a g l a s s s l i d e , weighed, and  was  immediately  homogenized i n i c e - c o l d s a l i n e s o l u t i o n u s i n g a T e f l o n Homogenized crushed  a t 300 RPM ice.  polystyrene  The  f o r one minute w h i l e b e i n g c o o l e d i n  mucosal samples were then f r o z e n i n s e a l e d  t e s t tubes and  the d i s a c c h a r i d a s e  activities  were determined w i t h i n a week. The  two  s t e p TRIS-glucose oxidase  (66) w i t h s l i g h t m o d i f i c a t i o n s was of l a c t a s e , sucrase and c o n s i s t e d of the use  o f each.  The  used f o r the  maltase a c t i v i t i e s .  of 0.1  ml o f s u b s t r a t e f o r the  method o f D a h l q v i s t  The  determination modifications  ml o f mucosal homogenate and  0.1  i n c u b a t i o n mixture i n s t e a d of 10 jil  absorbance was  measured at 420  mu  against a  reagent b l a n k i n a Coleman H i t a c h i spectrophotometer. Model 101. U n i t s o f d i s a c c h a r i d a s e a c t i v i t i e s were d e f i n e d  as  micromoles of s u b s t r a t e h y d r o l y s e d per minute under the a t i o n c o n d i t i o n s used  (66)  The  s p e c i f i c a c t i v i t y was  i n u n i t s o f a c t i v i t y per gram wet weight o f mucosa and  incub-  expressed per  gram o f mucosal p r o t e i n . ^ T e f l o n Homogenizer, Model K4f. v i l l e Center, New York.  TRI-R Instruments, Rock-  29  D.  Other Assays 1.  Plasma albumin c o n c e n t r a t i o n .  For the plasma albumin  d e t e r m i n a t i o n , b l o o d was drawn from the animal by h e a r t punct u r e and was t r a n s f e r r e d t o t e s t tubes c o n t a i n i n g h e p a r i n . T h i s was done under a n e s t h e s i a .  The b l o o d samples were c e n t r i -  fuged a t 15,000 RPM f o r 15 minutes, and then the plasma was removed and f r o z e n i n s e a l e d t e s t tubes.  The plasma albumin  was determined w i t h i n t h r e e months by the HABA dye method as d e s c r i b e d by Ness e t a l .  (67) and m o d i f i e d by the Technicon  C o r p o r a t i o n f o r use i n the auto a n a l y z e r Methodology, Method F i l e N-15C).  (Auto Analyzer  I t was f u r t h e r m o d i f i e d i n  the p r e s e n t experiment by u s i n g 0.1 ml of plasma and 5 ml o f working HABA dye s o l u t i o n which was made from Stock HABA dye (T21-1079) and Stock Phosphate B u f f e r  (Toi-184) from the  Technicon C o r p o r a t i o n , A r d s l e y , New York.  Bovine Albumin  (Sigma Chemical Co., S a i n t L o u i s , M i s s o u r i ) was used as a standard.  The absorbance was read a t 505 mu i n a Coleman  H i t a c h i spectrophotometer. Model 101. 2.  Mucosal p r o t e i n d e t e r m i n a t i o n . The p r o t e i n o f the  mucosa was determined by the method o f Lowry e t a l . (68) u s i n g a bovine albumin standard (Sigma Chemical Co., S a i n t L o u i s , Missouri).  The absorbance was read a t 500 mu i n a Coleman  H i t a c h i spectrophotometer. Model 101. E.  Materials The r a d i o a c t i v e d i s a c c h a r ides were obtained from the  Amersham/Searle C o r p o r a t i o n , A r l i n g t o n H e i g h t s , I l l i n o i s . The f o l l o w i n g r a d i o - d i s a c c h a r i d e s were used f o r the a b s o r p t i o n  30 studies:  lactose (D-glucose-l-C ), freeze-dried s o l i d , 1 4  s p e c i f i c a c t i v i t y of 10 t o 20 u C i per mmole; sucrose labeled with C^,  uniformly  f r e e z e - d r i e d s o l i d , s p e c i f i c a c t i v i t y of 5  to 15 u C i per mmole; maltose u n i f o r m l y l a b e l e d w i t h  C^,  s p e c i f i c a c t i v i t y 4 t o 10 u C i per mmole. The  0.5  ml of the i n c u b a t i o n mixture  t i o n s t u d i e s contained and d i f f e r e n t of  0.1  amounts o f u n l a b e l e d d i s a c c h a r i d e .  In the  t h e r e was  100 mg  and  l a b e l e d maltose monohydride and the F i s h e r S c i e n t i f i c Co.,  The  reagent grade  sucrose were obtained  F a i r Lawn, New  Jersey.  l a b e l e d b e t a - l a c t o s e , a l s o of reagent grade, was  Glucose  32  Chemical,  Rochester,  New  oxidase, s p e c i f i c a c t i v i t y of  obtained  and peroxidase,  approximately  solution  Triton  ( F o l i n - C i o c a l t e a u ) were obtained Jersey.  sodium p e n t o b a r b i t o l per ml was  from Abbott L a b o r a t o r i e s L t d . , Montreal,  the Model 1130  IBM  2N  from the F i s h e r  c o n t a i n e d 50 mg  student t t e s t was  Saint  base and Phenol reagent  F a i r Lawn, New  and  The g l u -  and  S c i e n t i f i c Co.,  The  and  (horseradish) were  (1 mg glucose per ml)  The TRIS-free  un-  York.  were purchased from the Sigma Chemical Co.,  Louis, Missouri.  from  obtained  from Calbiochem, San Diego, C a l i f o r n i a .  c o s e standard s o l u t i o n X-100  B grade  un-  The  90 u n i t s per ml, A grade, o f f u n g a l o r i g i n , s a l t - f r e e lyophilized  mg  i n the case o f maltose a b s o r p t i o n ,  o f maltose per ml.  from Eastman Organic  case  16 mg o f u n l a b e l e d l a c t o s e  i n the case of sucrose a b s o r p t i o n , t h e r e was  o f sucrose per ml;  absorp-  uCi of r a d i o a c t i v e disaccharide  l a c t o s e a b s o r p t i o n , t h e r e was  per ml;  used f o r the  The Nembutal which purchased  P.Q.  used f o r the s t a t i s t i c a l a n a l y s i s  Computer was  employed.  31  CHAPTER I I I RESULTS A.  Appearance, Body Weight, and Plasma Albumin The changes  Concentration.  i n mean body weight o f the c o n t r o l , p r o t e i n  d e p l e t e d , and p r o t e i n r e p l e t e d r a t s are shown i n F i g u r e The i n i t i a l mean body weight o f a l l the r a t s was grams.  1.  108 ± 9  The c o n t r o l group i n c r e a s e d i n weight t o 359.3 ± 30.6  a f t e r 8.5 weeks on the 18% l a c t a l b u m i n d i e t .  Rats on a l l the  t h r e e p r o t e i n d e f i c i e n t d i e t s l o s t an average o f 45 grams from t h e i r i n i t i a l body weight i n 8.5 weeks, w i t h most o f the l o s s o c c u r i n g d u r i n g the f i r s t no s t a t i s t i c a l l y  t h r e e weeks.  s i g n i f i c a n t d i f f e r e n c e s i n the f i n a l body  weights o f the t h r e e p r o t e i n d e p l e t e d groups. protein depleted r a t s carbohydrate  There were  Refeeding  the  ( r a t s fed the 0.5% l a c t a l b u m i n low  d i e t f o r 8.5 weeks) w i t h the 18% l a c t a l b u m i n  d i e t produced an abrupt g a i n i n body weight, and a f t e r 8 weeks o f r e p l e t i o n , the r a t s had i n c r e a s e d t h e i r mean body w e i g h t t o 341 i 29 grams which was  not s i g n i f i c a n t l y  differ-  e n t from the f i n a l mean body weight f o r the c o n t r o l group (359 i 31 grams).  The  i n i t i a l and f i n a l body weights f o r  each d i e t a r y group are g i v e n i n Table I I I . When the i n i t i a l weights o f each group were compared, t h e r e were no s t a t i s t i c a l l y  s i g n i f i c a n t d i f f e r e n c e s between  the c o n t r o l s and any of the other groups. l a c t a l b u m i n h i g h carbohydrate  However, the  group had a s i g n i f i c a n t l y  0.5% lower  gm  FIGURE 1 CHANGES IN BODY WEIGHTS OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS  •  C o n t r o l s , 18% l a c t a l b u m i n P r o t e i n d e p l e t e d groups: O 0.5% l a c t a l b u m i n , low carbohydrate • 0.5% l a c t a l b u m i n , h i g h carbohydrate A 1% l a c t a l b u m i n ( r e s t r i c t e d t o 4 grams per day)  •  P r o t e i n r e p l e t e d ( r e f e d the 18% l a c t a l b u m i n d i e t a f t e r 8 weeks on the 0.5% l a c t a l b u m i n low carbohydrate d i e t )  TABLE I I I INITIAL AND FINAL BODY WEIGHTS OF THE SURVIVORS IN EACH DIETARY GROUP D i e t a r y Group  Number o f Animals  Weight a t S t a r t of* Weight a t end o f * D i e t a r y Treatment D i e t a r y Treatment (grams) (grams) 107.3 ± 10.0 359.3 ± 30.6  C o n t r o l s , 18% l a c t a l b u m i n  15  0.5%  l a c t a l b u m i n , low carbohydrate  12  113.3 ± 8.8  0.5%  l a c t a l b u m i n high carbohydrate  18  101.9 ± 4 . 2 '  1% l a c t a l b u m i n ( r e s t r i c t e d t o 4 grams per day)  18  109.4 ± 7 . 4 ' '  Repleted  12,  &  a  a  65.0 ± 6.5  62.5 ± 4.8  a  c  d  b  b  64.7 ± 6.0 '  d  62.7 ± 5 . 0 '  d  b  e  b  341.3 ± 28.6  a  * Values are Means i S.D. f o r the number of r a t s shown i n t h e second column. R e f e d the 18% l a c t a l b u m i n d i e t bumin low carbohydrate d i e t . &  f o r 8 weeks a f t e r  from the c o n t r o l group  8.5 weeks on t h e 0.5% l a c t a l -  a  Does not d i f f e r  (P>0.05).  b  Differs  from the c o n t r o l group (P <0.001).  c  Differs  from the 0.5% l a c t a l b u m i n low carbohydrate  group (P <0.001).  Does not d i f f e r from the 0.5% l a c t a l b u m i n low carbohydrate e  Differs  from the 0.5% l a c t a l b u m i n h i g h carbohydrate  group  (P^0.05).  group (P< 0.001).  35  i n i t i a l mean body weight than the 0.5% hydrate  and  the 1% l a c t a l b u m i n  l a c t a l b u m i n low  ( r e s t r i c t e d ) groups  About 40% of the r a t s fed the 0.5% showed a severe  carbo-  (P<0.001).  lactalbumin d i e t s  l o s s of h a i r and became l e s s a c t i v e as  experiment p r o g r e s s e d .  The  animals on the 1%  the  lactalbumin  ( r e s t r i c t e d ) d i e t d i d not show as severe h a i r l o s s e s as on the 0.5%  lactalbumin d i e t s .  The  animals fed the 1%  those lactal-  bumin ( r e s t r i c t e d ) d i e t were h y p e r a c t i v e and a g g r e s s i v e t h e i r behaviour,  whereas the animals on the 0.5%  d i e t s were v e r y p a s s i v e .  Refeeding  in  lactalbumin  the group o f p r o t e i n  d e p l e t e d animals the 18% l a c t a l b u m i n d i e t f o r 8 weeks r e s u l t e d i n a r e v e r s a l of a l l the gross symptoms so t h a t the r e p l e t e d group resembled the c o n t r o l s i n b o t h appearance and F i g u r e s 2 and  3 show the t y p i c a l appearance of the c o n t r o l  and p r o t e i n d e p l e t e d r a t s a t the time of  sacrifice.  Edema, which i s c h a r a c t e r i s t i c o f the f u l l syndrome i n c h i l d r e n , was  kwashiorkor  found i n only a few r a t s on the  l a c t a l b u m i n d i e t s , although,  a number of animals w i t h  obvious edema showed i n c r e a s e d f l u i d accumulation p e r i t o n e a l c a v i t y a t the time o f s a c r i f i c e . at  behaviour.  0.5%  no  i n the  I t was  noted  the time o f s a c r i f i c e t h a t the s m a l l i n t e s t i n e o f the  p r o t e i n d e f i c i e n t r a t s was diameter.  The  very t h i n , f r a g i l e , and s m a l l i n  i n t e s t i n a l mucosal l a y e r was  also very  thin  and p a l e when compared to the c o n t r o l group. The  m o r t a l i t y r a t e o f the r a t s on the three p r o t e i n  d e f i c i e n t d i e t s was experimental  period.  h i g h e s t d u r i n g the l a s t two weeks of  the  The percentage o f s u r v i v o r s f o r each  d i e t a r y group i s shown i n Table  IV.  FIGURE 2 TYPICAL APPEARANCE OF RATS ON THE FOLLOWING THREE DIETS: 1% LACTALBUMIN RESTRICTED TO 4 g PER DAY (LEFT), 18% LACTALBUMIN (CENTER), AND 0.5% LACTALBUMIN HIGH CARBOHYDRATE (RIGHT).  FIGURE 3 A CLOSE-UP OF THE RAT ON THE 0.5% LACTALBUMIN HIGH CARBOHYDRATE DIET SHOWN IN FIGURE 2.  TABLE IV PERCENTAGE OF SURVIVORS ON THE DIFFERENT DIETARY REGIMENS D i e t a r y Group  '  Number of Weeks on the Experimental  Percentage o f S u r v i v o r s  Diet  C o n t r o l s , 18% l a c t a l b u m i n  8.5  100.0%  0.5% l a c t a l b u m i n low carbohydrate  8.5  77.5%  0.5% l a c t a l b u m i n high carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d t o 4 grams per day)  8.0 9.0  86.7% 83.3%  Repleted*  8.0  100.0%  *Refed the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r 8.5 weeks on the 0.5% l a c t a l b u m i n low carbohydrate d i e t .  39  The plasma albumin c o n c e n t r a t i o n s  i n the c o n t r o l s , p r o -  t e i n d e p l e t e d , and p r o t e i n r e p l e t e d r a t s a r e g i v e n i n Table V.  A l l the t h r e e groups o f r a t s on the p r o t e i n d e f i c i e n t  had  s i g n i f i c a n t l y lower plasma albumin c o n c e n t r a t i o n s than the  c o n t r o l group (P< 0.001). t h a t the animals  The low plasma albumin  diets  confirmed  on the 0.5% l a c t a l b u m i n and the 1% l a c t a l b u m i n  ( r e s t r i c t e d ) d i e t s were s u f f e r i n g from p r o t e i n - c a l o r i e malnutrition.  The l e v e l s o f plasma albumin i n the r e p l e t e d and  the c o n t r o l r a t s were almost i d e n t i c a l .  The same was t r u e f o r  the r a t s f e d the 0.5% l a c t a l b u m i n low carbohydrate l a c t a l b u m i n h i g h carbohydrate  diets.  l e v e l s were s i g n i f i c a n t l y higher s t r i c t e d ) group than  and the 0.5%  However, the plasma  i n t h e 1% l a c t a b l u m i n ( r e -  i n the 0.5% l a c t a l b u m i n low carbohydrate  group (P <0.01) . B.  I n t e s t i n a l Disaccharidase The  Activities  j e j u n a l and i l e a l l a c t a s e , s u c r a s e , and maltase  a c t i v i t i e s o f r a t s fed t h e v a r i o u s d i e t s a r e shown i n Tables VI, V I I and V I I I r e s p e c t i v e l y . The  i n t e s t i n a l l a c t a s e a c t i v i t y was  i n a l l the t h r e e p r o t e i n d e f i c i e n t groups low carbohydrate,  significantly  higher  (0.5% l a c t a l b u m i n  0.5% l a c t a l b u m i n high carbohydrate,  and 1%  l a c t a l b u m i n r e s t r i c t e d t o 4 grams p e r day) i n both the jejunum and  ileum when compared w i t h the c o n t r o l s f e d the 18% l a c t a l -  bumin d i e t  (Table V I ) .  When l a c t a s e l e v e l s o f the t h r e e p r o t e i n d e f i c i e n t groups were compared, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s among them i n e i t h e r the j e j u n a l o r i l e a l l a c t a s e a c t i v i t i e s .  When  40  TABLE V PLASMA ALBUMIN CONCENTRATION OF CONTROL, PROTEIN DEPLETED, AND  PROTEIN  REPLETED RATS  D i e t a r y Group  Number o f Rats  Plasma Albumin* mg/l00 ml  C o n t r o l , 18% l a c t a l b u m i n  15  4.5  0.5% l a c t a l b u m i n low carbohydrate  12  2.1 ± 0.6  0.5% l a c t a l b u m i n h i g h carbohydrate  16  2.2 ± 0 . 4 '  b  1% l a c t a l b u m i n ( r e s t r i c t e d t o 4 grams per day)  18  2.7 ± 0 . 4 '  c  Repleted  11  4.4 ± 0.5  &  -  0.5 a  a  a  d  •Values are Means ± S.D. f o r the number o f r a t s shown i n the second column. S'Refed the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r weeks on the 0.5% l a c t a l b u m i n low carbohydrate d i e t . a  D i f f e r s from the c o n t r o l group  8.5  (P <0.001).  •'-'Does n o t d i f f e r from the 0.5% l a c t a l b u m i n low carbohydrate group ( P > 0 . 0 5 ) . D i f f e r s from the 0.5% l a c t a l b u m i n low carbohydrate ( P < 0.01) . c  d  D o e s not d i f f e r from the c o n t r o l group  (P >0.05) .  group  TABLE VI LACTASE ACTIVITIES IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Intestinal Segment  Jejunum  D i e t a r y Group  Number o f Rats  U n i t s per g wet weight  15 12 18 18  0.2 1.3 + 3.9 1.5* + 4.2 1.2 ' + 3.2 0.8 '  Repleted®  12  1.0 + 0.4  C o n t r o l , 18% l a c t a l b u m i n 0.5% l a c t a l b u m i n low carbohydrate 0.5% l a c t a l b u m i n high carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d ) "  15 12 18 18  + 0.1 0.2 0.6 ± 0.4 0.4 + 0 . 3 ' 0.5 + 0 . 2 '  Repleted®  12  0.2 + 0.1  5  9.4 ± 1.1  b  c  b  c  38.2 ± 14.9&45.6 ± 14.7 #c b  32.4 ± 7 . 6 ' b  7.7 ± 3.0a  b  b  b  c  b  c  a  Activity* Units per g mucosal p r o t e i n  +  C o n t r o l , 18% l a c t a l b u m i n 0.5% l a c t a l b u m i n low carbohydrate 0.5% l a c t a l b u m i n high carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d ) 5 1  Ileum  Lactase  2.1 7.6 4.5 5.9  i ± ± ±  2.1 ±  1.0 5.9 3.3 ' 1.9 *b  b  b  l.l  a  *Values a r e Means - S.D. f o r the number o f r a t s shown i n the t h i r d column. R e s t r i c t e d t o 4 grams o f d i e t per day. ®Refed the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r 8.5 weeks on the 0.5% l a c t a l b u m i n low carbohydrate d i e t . a  D o e s n o t d i f f e r from the c o n t r o l group  b  Differs  c  Does n o t d i f f e r from the 0.5% l a c t a l b u m i n low carbohydrate  from the c o n t r o l group  (P>0.05).  (P<0.05). group  (P>0.05).  c  c  c  TABLE V I I SUCRASE ACTIVITIES IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Intestinal Segment  Je junum  Number o f Rats  D i e t a r y Group  U n i t s per g wet weight  U n i t s per g mucosal p r o t e i 54.1 ± 9.0  C o n t r o l , 18% l a c t a l b u m i n  15  7.2 ± 1.3  0.5% l a c t a l b u m i n low  12  15.6 ± 5.4  0.5% l a c t a l b u m i n h i g h carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d ) Repleted®  18 18 12  22.5 ± ' 4 . 2 ' 18.1 ± 3 . 2 ' 6.2 ± 1.2  C o n t r o l , 18% l a c t a l b u m i n 0.5% l a c t a l b u m i n low carbohydrate 0.5% l a c t a l b u m i n high carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d )  15 12 18 18  3.6 2.9 2.7 1.9  Repleted®  12  1.0 ±'o.6*>  carbohydrate &  Ileum  Sucrase A c t i v i t y *  &  151.0 ± 51.8  b  b  c  b  d  b  ± ± ± ±  1.7 1.5 0.7 ' 0.7 ' a  a  d  b  d  241.5 ± 5 1 . l ' 181.6 ± 32.6 ' 46.8 ± 1 1 . l b  b  a  33.7 35.3 28.9 20.9  ± ± ± ±  15.7 24.l 7.1 ' 6.4 '  9.2 ± 3.9  a  a  d  b  d  b  *Values are Means i S.D. f o r the number o f r a t s shown i n the t h i r d column.  Restricted  to 4 grams o f d i e t per day. @Refed the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r 8.5 weeks on the 0 .5% l a c t a l b u m i n low carbohydrate d i e t . D o e s not d i f f e r from the c o n t r o l group ( P > 0 . 0 5 ) . D i f f e r s from the c o n t r o l group (P<0.05). D i f f e r s from the 0.5% l a c t a l b u m i n low carbohydrate group (P< 0.001). D o e s not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P> 0.05) a  b  c  d  b  TABLE V I I I MALTASE ACTIVITIES IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Intestinal Segment  Jejunum  D i e t a r y Group  Number o f Rats  U n i t s per g mucosal p r o t e i n 245 .9 + 23.1 517 .4 + 153.6  15 12  32 .9 + 2. 7 53 .4 + 17 . l  0.5% l a c t a l b u m i n high carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d ) Repleted®  18 18 12  63 .2 + 12 . 2 ' 51 .8 + 13 . 9 ' 18 .2 + 6. Ob  C o n t r o l , 18% l a c t a l b u m i n 0.5% l a c t a l b u m i n low carbohydrate  15 12  0.5% l a c t a l b u m i n high  18  15 .6 + 5.9 25 .6 + 9. 20 .8 + 7. b , d  144 .0 ± 53.0 311 .7 + 152. 6 b,d 223 .6 +  28 .9 + 8. b , d 12 .7 + 4. a 2  320 .3 + 8 7 . 6 '  9  102 .4 + 34. 2  1% l a c t a l b u m i n  carbohydrate  (restricted)  18  &  Repleted® Values  Un i t s per g we t weight  C o n t r o l , 18% l a c t a l b u m i n 0.5% l a c t a l b u m i n low carbohydrate &  Ileum  Maltase A c t i v i t i e s *  12  b  b  d  b  d  g> 3  681 .2 + 1 6 7 . 1 ' 532 .0 ± 132.9 ,<3 139 .2 ± 53.4b b  b  7 4 # 2  b  are Means ± S.D. f o r the number o f r a t s shown i n the t h i r d column.  R e s t r i c t e d t o 4 grams o f d i e t per day. @ Refed the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r 8.5 weeks on t h e 0.5% l a c t a l b u m i n low carbohydrate d i e t . Does not d i f f e r from the c o n t r o l group  b  D i f f e r s from the c o n t r o l group  c  D i f f e r s from the 0.5% l a c t a l b u m i n low carbohydrate  (P>0.05).  d  Does not d i f f e r from the 0.5% l a c t a l b u m i n low carbohydrate  (P<0.05). group  (P<0.01). group  c  b  &  a  b  (P>0.05).  b  d  44  the r a t s on the 0.5%  l a c t a l b u m i n low carbohydrate d i e t were  r e p l e t e d w i t h a p r o t e i n adequate d i e t c o n t a i n i n g 18% bumin, the l a c t a s e a c t i v i t y l e v e l s f e l l h i g h v a l u e s t o approximately The  lactal-  from the p r e v i o u s l y  the l e v e l s found  i n the c o n t r o l s .  l a c t a s e a c t i v i t y i n the jejunum i s markedly h i g h e r  than i n the ileum f o r a l l the d i e t a r y groups and the t e s t d i e t s seem t o have no e f f e c t on the d i s t r i b u t i o n o f l a c t a s e a c t i v i t y between the jejunum and  ileum.  As found w i t h the l a c t a s e a c t i v i t y , the j e j u n a l sucrase activities  i n r a t s on the 0.5%  l a c t a l b u m i n d i e t s w i t h both  h i g h and low l e v e l s o f carbohydrate  and the 1% l a c t a l b u m i n  d i e t were s i g n i f i c a n t l y h i g h e r than the l e v e l s of j e j u n a l s u c r a s e found  i n the c o n t r o l group.  In the ileum, however,  there were no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between the c o n t r o l group and the 0.5% or the 0.5%  l a c t a l b u m i n low  carbohydrate  l a c t a l b u m i n h i g h carbohydrate groups.  In the  l a c t a l b u m i n group, the l e v e l o f i l e a l sucrase a c t i v i t y  1%  was  s i g n i f i c a n t l y lower than i n the c o n t r o l group (Table V T I ) . When the 0.5%  l a c t a l b u m i n low carbohydrate  group  was  compared t o the 0.5%  l a c t a l b u m i n h i g h carbohydrate group, the  sucrase a c t i v i t y was  s i g n i f i c a n t l y h i g h e r i n the animals  on  the h i g h carbohydrate d i e t i n the jejunum, b u t t h e r e were no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between the l e v e l of i l e a l sucrase a c t i v i t y  i n these two groups.  There were a l s o  no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between the  0.5%  l a c t a l b u m i n low carbohydrate and the 1% l a c t a l b u m i n groups. The  j e j u n a l sucrase a c t i v i t i e s o f the c o n t r o l group and  the group which was  r e p l e t e d w i t h 18% l a c t a l b u m i n f o r 8 weeks  45  a f t e r 8.5 weeks o f p r o t e i n d e p r i v a t i o n were s i m i l a r . ever, the i l e a l sucrase a c t i v i t i e s f e l l to  How-  i n the r e p l e t e d group  l e v e l s which were s i g n i f i c a n t l y lower than i n the c o n t r o l  group. The  sucrase a c t i v i t y was always markedly h i g h e r i n the  jejunum than i n the ielum i n the d i e t a r y  groups.  When the i n t e s t i n a l maltase a c t i v i t i e s were compared i n the c o n t r o l and the t h r e e p r o t e i n d e p l e t e d groups (0.5% l a c t albumin and  low carbohydrate,  0.5% l a c t a l b u m i n h i g h  carbohydrate,  1% l a c t a l b u m i n r e s t r i c t e d t o 4 grams p e r d a y ) , i t was  found t h a t b o t h the j e j u n a l and i l e a l l e v e l s o f maltase were s i g n i f i c a n t l y h i g h e r i n the p r o t e i n d e p l e t e d groups than i n the c o n t r o l s , and there was a s i g n i f i c a n t l y h i g h e r l e v e l o f maltase a c t i v i t y  i n the jejunum i n the 0.5% l a c t a l b u m i n h i g h  carbohydrate group than i n the 0.5% l a c t a l b u m i n low c a r b o hydrate group.  There were no s t a t i s t i c a l l y s i g n i f i c a n t  ences between the l a s t two groups i n the i l e a l activities.  differ-  maltase  There were a l s o no s i g n i f i c a n t d i f f e r e n c e s i n  the l e v e l s o f d i s a c c h a r i d a s e s between the groups on the 0.5% l a c t a l b u m i n low carbohydrate  and the 1% l a c t a l b u m i n d i e t s i n  e i t h e r the jejunum or the ileum (Table V I I I ) . The  j e j u n a l and i l e a l l e v e l s o f maltase a c t i v i t y i n the  r e p l e t e d group had f a l l e n t o l e v e l s s i g n i f i c a n t l y lower  than  those of the c o n t r o l group. As found w i t h the l a c t a s e and sucrase a c t i v i t i e s , the maltase  a c t i v i t y was a l s o markedly h i g h e r i n the j e j u n a l  i n the i l e a l  segment i n a l l groups o f r a t s .  than  The mucosal p r o t e i n c o n c e n t r a t i o n s and  i n both the jejunum  ileum of the c o n t r o l , p r o t e i n d e p l e t e d , and p r o t e i n  r e p l e t e d r a t s are shown i n Table IX. c o n c e n t r a t i o n i n the jejunum and l a c t a l b u m i n groups and  The  mucosal p r o t e i n  ileum of the two  0.5%  the 1% l a c t a l b u m i n group were a l l  s i g n i f i c a n t l y lower than the mucosal p r o t e i n c o n c e n t r a t i o n i n the c o n t r o l group.  There was  no s t a t i s t i c a l l y  significant  d i f f e r e n c e between the mucosal p r o t e i n c o n c e n t r a t i o n s o f the c o n t r o l and C.  the p r o t e i n r e p l e t e d groups.  I n t e s t i n a l Absorption o f D i s a c c h a r i d e s i n V i v o The  i n t e s t i n a l a b s o r p t i o n o f d i s a c c h a r i d e s by r a t s fed  the c o n t r o l and Table X.  the p r o t e i n d e f i c i e n t d i e t s i s shown i n  L a c t o s e , sucrose, and maltose a b s o r p t i o n by r a t s  fed the 0.5%  l a c t a l b u m i n low carbohydrate  c a n t l y h i g h e r than a b s o r p t i o n by hydrate d i e t  i n the c o n t r o l group.  the animals  on the 0.5%  d i e t was  signifi-  This greater  l a c t a l b u m i n low  i s c o n s i s t e n t w i t h the h i g h e r l e v e l s o f d i s a c -  c h a r i d a s e s found  i n t h i s group when compared t o the c o n t r o l s .  The comparison o f i n t e s t i n a l l a c t o s e , sucrose, and a b s o r p t i o n i n r a t s f e d the c o n t r o l and the 0.5% h i g h carbohydrate  bumin group.  f o r l a c t o s e a b s o r p t i o n was T h i s i s due  i n the obtained r e s u l t s of the 0.5%  lactal-  not s i g n i f i c a n t l y  l a c t a l b u m i n h i g h carbohydrate  i n the c o n t r o l group.  higher  maltose i n the 0.5%  Lactose a b s o r p t i o n was  the mean v a l u e  maltose  lactalbumin  d i e t demonstrated s i g n i f i c a n t l y  a b s o r p t i o n only f o r sucrose and  i n the 0.5%  carbo-  higher  group even though 8 times higher  than  t o the g r e a t v a r i a b i l i t y l a c t a l b u m i n high  carbo-  TABLE IX MUCOSAL PROTEIN CONCENTRATION IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS D i e t a r y Group  Number o f Rats  mg P r o t e i n per g wet* weight mucosa Jejunum  Ileum  C o n t r o l , 18% l a c t a l b u m i n  15  134.0 ± 8.0  108.4 ± 8.1  0.5% l a c t a l b u m i n low carbohydrate  12  102.7 ± 6.4^  85.5 i 1 4 . 2 '  d  0.5% l a c t a l b u m i n high carbohydrate  18  93.9 ± 1 0 . 3 '  93.5 ± 1 2 . 4 '  d  1% l a c t a l b u m i n ( r e s t r i c t e d t o 4 grams o f d i e t per day)  18  99.1 ± 5 . 9 '  91.1 ± 1 5 . 9 '  d  Repleted  12  133.0 Z 12.6  &  b  b  b  c  d  C  b  b  124.1 ± 25.0  *Values a r e Means - S.D. f o r the number o f r a t s shown i n the second column. R e f e d the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r 8.5 weeks on the 0.5% l a c t a l b u m i n low carbohydrate d i e t . Does n o t d i f f e r from the c o n t r o l group (P;>0.05). D i f f e r s from the c o n t r o l group (P<0.001). D i f f e r s from the 0.5% l a c t a l b u m i n low carbohydrate group (P <0.01). &  a  b  c  d  D o e s not d i f f e r from the 0.5% l a c t a l b u m i n low carbohydrate group  (PJ>0.05).  a  TABLE X LACTOSE, SUCROSE, AND MALTOSE ABSORPTION IN CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Number o f Rats  D i e t a r y Group  Lactose* Absorption  Sucrose* Absorption  Maltose* Absorption  (Percentage a b s o r p t i o n per 500 mg wet weight intestine) 21.9 ± 4.4  C o n t r o l , 18% l a c t a l b u m i n  5  7.7 ± 6.4  0.5% l a c t a l b u m i n low carbohydrate 0.5% l a c t a l b u m i n high carbohydrate 1% l a c t a l b u m i n ( r e s t r i c t e d t o 4 grams d i e t p e r day)  5  46.3 i 18.2  6  55.5 ± 5 1 . 2 '  c  6  72.1 ± 3 8 . 4 '  c  Repleted  4  13.7 ± 6.1  &  *Values  a  b  10.4 ± 5.9  72.5 ± 35.5  b  a  78.8 t  b  57.1 ± 26.8  25.8 '  c  91.6 ± 2 0 . 9 '  c  b  b  20.5 t 4.9  a  b  75.8 ± 4 8 . 6 '  c  75.2 i 2 0 . 7 '  c  b  b  10.0  t  3.3  a  are Means ± S.D. f o r the number o f r a t s shown, i n the second column.  kRefed the 18% l a c t a l b u m i n d i e t f o r 8 weeks a f t e r 8.5 weeks on the 0.5% l a c t a l b u m i n low carbohydrate d i e t . a  D o e s n o t d i f f e r from the c o n t r o l group  (P>0.05) .  b  Differs  c  Does not d i f f e r from the 0.5% l a c t a l b u m i n low carbohydrate  from the c o n t r o l group (P<0.05). group  (P 7-0.05) .  49  hydrate  group as demonstrated by the l a r g e standard  deviation  o f the mean. When the a b s o r p t i o n o f l a c t o s e , sucrose,  and maltose i n  the r a t s f e d the c o n t r o l and the 1% l a c t a l b u m i n d i e t s were compared, the a b s o r p t i o n o f a l l t h r e e d i s a c c h a r i d e s was f i c a n t l y higher The  signi-  i n the 1% l a c t a l b u m i n group than i n the c o n t r o l s .  a b s o r p t i o n values  i n the 1% l a c t a l b u m i n group were 4 t o 10  times h i g h e r than those  i n the c o n t r o l group.  There were no s t a t i s t i c a l l y  significant differences i n  the a b s o r p t i o n o f l a c t o s e , sucrose and maltose between the 0.5% l a c t a l b u m i n low carbohydrate bumin h i g h carbohydrate statistically  group.  group and the 0.5%  lactal-  S i m i l a r l y , 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 between the a b s o r p t i o n  o f the t h r e e d i s a c c h a r i d e s between the 0.5% l a c t a l b u m i n low carbohydrate values  and the 1% l a c t a l b u m i n group.  f o r l a c t o s e , sucrose,  s i s t e n t l y higher  However, the  and maltose a b s o r p t i o n were con-  i n the 1% l a c t a l b u m i n group than i n the 0.5%  l a c t a l b u m i n low carbohydrate  group.  The mean v a l u e f o r l a c t o s e  a b s o r p t i o n f o r the 1% l a c t a l b u m i n group was almost twice o f the 0.5% l a c t a l b u m i n low carbohydrate  group.  The  that  standard  d e v i a t i o n s o f the means i n the 1% l a c t a l b u m i n group were l a r g e and  a statistically  s i g n i f i c a n t d i f f e r e n c e between these  two  groups was n o t demonstrated. The  comparison o f the a b s o r p t i o n o f l a c t o s e , sucrose, and  maltose i n the group of animals f e d the c o n t r o l d i e t (18% lactalbumin)  and the r e p l e t e d group ( f e d the 18% l a c t a l b u m i n  d i e t f o r 8 weeks a f t e r 8.5 weeks o f p r o t e i n d e p l e t i o n )  showed  50  t h a t t h e r e were no s t a t i s t i c a l l y  significant differences in  the a b s o r p t i o n o f the t h r e e d i s a c c h a r i d e s .  51  CHAPTER IV DISCUSSION In s t u d y i n g the e f f e c t of p r o t e i n - c a l o r i e on  malnutrition  i n t e s t i n a l d i s a c c h a r i d a s e s i n r a t s i t i s extremely  t o keep the animals on the p r o t e i n d e f i c i e n t d i e t s extended p e r i o d  the changes a s s o c i a t e d r a t s include  for  of time u n t i l they develop l e s i o n s  with protein-calorie malnutrition.  important an  associated  According t o Edozien  (65),  with p r o t e i n - c a l o r i e malnutrition  in  a marked l o s s of body weight, severe l o s s of h a i r ,  a marked decrease i n plasma albumin c o n c e n t r a t i o n , decrease o f protein  c o n c e n t r a t i o n i n the  t i o n i n the p e r i t o n e a l  i n t e s t i n a l mucosa, f l u i d  c a v i t y , and  accumula-  edema.  In the p r e s e n t study the r a t s on  the  0.5%  lactalbumin  d i e t s developed a l l of the above symptoms which i n d i c a t e s the r a t s had  been on the p r o t e i n d e f i c i e n t d i e t long enough  t o be c o n s i d e r e d p r o t e i n - c a l o r i e d e f i c i e n t .  Also the  differ-  ences i n g r o s s appearance, plasma albumin c o n c e n t r a t i o n b e h a v i o u r between the r a t s on the the  0.5%  1% l a c t a l b u m i n d i e t ( r e s t r i c t e d t o 4 g of d i e t per  assumed t h a t the r a t s on the  p r o t e i n d e f i c i e n t and  0.5%  and  lactalbumin d i e t s  were s i m i l a r t o those r e p o r t e d by E d o z i e n (65). was  that  and day)  Therefore i t  l a c t a l b u m i n d i e t s were  t h a t the r a t s on the  1% l a c t a l b u m i n d i e t  ( r e s t r i c t e d ) were p r o t e i n - c a l o r i e d e f i c i e n t . Solimano e t a l . (4) r e p o r t e d t h a t p r o t e i n d e p r i v a t i o n r a t s produced an  increase  t i n a l d i s a c c h a r i d a s e s and  i n the s p e c i f i c a c t i v i t i e s p a r t i c u l a r l y of sucrase and  of  in  intes-  maltase.  52  They suggested t h a t t h i s  increase i n disaccharidase  activities  was an a d a p t a t i o n t o the h i g h p r o p o r t i o n o f carbohydrates  in  the p r o t e i n d e f i c i e n t d i e t s as the p r o t e i n o f the c o n t r o l d i e t was r e p l a c e d w i t h carbohydrate protein  diets.  The support  obtained  results  the hypothesis  i n the p r e s e n t study  i n the p r o t e i n d e f i c i e n t d i e t can  i n t e s t i n a l disaccharidase  accharidase carbohydrate  a c t i v i t i e s s i n c e the d i s -  l e v e l s i n the r a t s f e d the 0.5% l a c t a l b u m i n d i e t were s t a t i s t i c a l l y s i g n i f i c a n t l y  when compared t o the c o n t r o l s . disaccharidase a c t i v i t i e s hydrate  partially  o f Solimano e t a l . (4) t h a t a high  p r o p o r t i o n o f carbohydrate induce  i n order t o produce the low  high  increased  However, the i n c r e a s e i n the  i n the 0.5% l a c t a l b u m i n high  carbo-  group was n o t e n t i r e l y due t o the h i g h p r o p o r t i o n o f  carbohydrates  i n the d i e t because the r a t s on the 0.5%  l a c t a l b u m i n low carbohydrate  d i e t a l s o showed s t a t i s t i c a l l y  s i g n i f i c a n t i n c r e a s e s i n d i s a c c h a r i d a s e l e v e l s when compared t o the c o n t r o l s although  not t o the same e x t e n t as the 0 . 5 %  l a c t a l b u m i n h i g h carbohydrate  group.  I t i s n o t c l e a r why there i s an i n c r e a s e i n the i n t e s t i n a l disaccharidase a c t i v i t i e s hydrate  i n the 0.5% l a c t a l b u m i n low c a r b o -  group when compared t o the c o n t r o l s .  explanation  One p o s s i b l e  f o r t h i s increase i n disaccharidase l e v e l s could  be a l o s s o f s t r u c t u r a l p r o t e i n s i n the i n t e s t i n a l mucosa. I f there had been a l a r g e r decrease o f s t r u c t u r a l p r o t e i n i n the i n t e s t i n a l mucosa as compared t o the decrease of enzymatic p r o t e i n s , the e x p r e s s i o n  o f enzymatic a c t i v i t y p e r gram o f  p r o t e i n or per gram o f wet weight mucosa would l e a d t o high results.  53  M c N e i l l and Hamilton  (69) r e c e n t l y s t u d i e d t h e e f f e c t o f  f a s t i n g on i n t e s t i n a l d i s a c c h a r i d a s e small ase  activities  i n the r a t  i n t e s t i n e and observed a s t a t i s t i c a l l y s i g n i f i c a n t i n c r e -  in lactase a c t i v i t y .  might have s t i m u l a t e d  They have p o s t u l a t e d  that f a s t i n g  lysosomal b e t a - g a l a c t o s i d a s e .  If a  p r o t e i n d e f i c i e n t d i e t has a s i m i l a r e f f e c t t o f a s t i n g , the increase  i n lactase a c t i v i t y  i n the p r o t e i n d e f i c i e n t r a t s  c o u l d p o s s i b l y be a r e s u l t of the s t i m u l a t i o n o f the lysosomal beta-galactos idase. The  r e s u l t s of the p r e s e n t  study together  w i t h those o f  Solimano e t a l . (4) d i f f e r from the f i n d i n g s by Prosper e t al.  (5) and L i f s h i t z e t a l . (6) i n t h a t they f i n d no marked  alterations  in i n t e s t i n a l disaccharidase  deficient rats.  activities  in protein  I t should be s t r e s s e d , however, t h a t t h e i r  e x p e r i m e n t a l c o n d i t i o n s were very d i f f e r e n t from the p r e s e n t investigation. In the study by Prosper e t a l . (5) the t e s t animals were f e d a p r o t e i n - f r e e d i e t f o r 45 days and the c o n t r o l s were g i v e n a 27% p r o t e i n d i e t . be c o n s i d e r e d  In a d d i t i o n t h e i r f i n d i n g s  should  as i n c o n c l u s i v e on the b a s i s of the f a c t  the d i s a c c h a r i d a s e  that  a c t i v i t i e s were performed on o n l y 3  p r o t e i n d e f i c i e n t r a t s and t h a t there was a l a r g e v a r i a t i o n i n the d i s a c c h a r i d a s e  a c t i v i t i e s w i t h i n both t h e c o n t r o l s and  the t e s t a n i m a l s . L i f s h i t z e t a l . (6) a l s o d i d not observe a s t a t i s t i c a l l y s i g n i f i c a n t increase deficient rats. casein)  in disaccharidase  activity  in protein  They f e d the p r o t e i n d e f i c i e n t d i e t (4%  f o r 1 t o 4 weeks, and a t the end of 4 weeks the  54  mucosal p r o t e i n c o n c e n t r a t i o n was not s t a t i s t i c a l l y from the c o n t r o l s .  different  This would imply t h a t the i n t e s t i n a l  mucosa had n o t y e t undergone changes a s s o c i a t e d w i t h p r o t e i n d e f i c i e n c y because the r a t s were n o t kept long enough on the protein deficient diet. t h a t although  I t should be p o i n t e d out, however,  t h e r e was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e  i n d i s a c c h a r i d a s e l e v e l s between the p r o t e i n d e f i c i e n t r a t s and  the c o n t r o l s i n the study by L i f s h i t z e t a l . (6), the  d i s a c c h a r i d a s e a c t i v i t i e s were a p p r e c i a b l y h i g h e r protein deficient  i n the  rats.  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 between the i n t e s t i n a l disaccharidase a c t i v i t i e s and  i n the 1% l a c t a l b u m i n group  the 0.5% l a c t a l b u m i n low carbohydrate  the r e s t r i c t i o n of d a i l y  group.  Therefore  intake d i d n o t have an e f f e c t on the  a c t i v i t i e s o f l a c t a s e , s u c r a s e , and maltase i n a d d i t i o n t o the e f f e c t caused by p r o t e i n d e p l e t i o n . the  T h i s may be due t o  f a c t t h a t towards the end o f the 9 week  experimental  p e r i o d about o n e - t h i r d o f the animals on the 1% l a c t a l b u m i n d i e t were not e a t i n g the e n t i r e 4 grams o f d i e t which were g i v e n each day.  Therefore,  they  near the end o f the e x p e r i -  mental p e r i o d , these animals were no longer r e s t r i c t e d i n their caloric  intake.  bumin low carbohydrate not monitored.  The amount consumed by the 0.5% l a c t a l group o r the 1% l a c t a l b u m i n group was  However, i t would appear t h a t the f a i l u r e of  some o f the animals on the 1% l a c t a l b u m i n d i e t  (restricted to  4 grams p e r day) t o consume the e n t i r e p o r t i o n o f food d i d not a f f e c t the development o f the c a l o r i c d e f i c i e n c y syndrome as these animals had s i g n i f i c a n t l y d i f f e r e n t plasma albumin  55  c o n c e n t r a t i o n s , gross appearance and from the The effect  l e v e l as  115  0.5%  l a c t a l b u m i n low  s t u d i e s found i n the  r e s t r i c t the i n the  Chase (59)  r e s t r i c t i o n of an days o f age  activity  8%  resulted  i n the  c o n t r o l s who  the  p r e s e n t experiment  l i t t e r size  (16  characteristics carbohydrate  l i t e r a t u r e dealing with intestinal  p r o t e i n content of the  found t h a t the  i n c r e a s i n g the by  the  of c a l o r i c r e s t r i c t i o n on  d i d not  and  animals on  behavior  to as  (1% l a c t a l b u m i n ) .  animals per  day  libitum.  until  the  There  d i f f e r e n c e s i n s u c r a s e a c t i v i t y between the  and  restricted  intake group.  by  lactase  were no the  Kumar  i n t a k e group when compared t o c a s e i n d i e t ad  a  mother) f o l l o w e d  in s i g n i f i c a n t l y higher  r e c e i v e a 27%  low  food a t b i r t h  c a s e i n d i e t t o 8 grams per  restricted  the  disaccharidases  diet  r e s t r i c t i o n of  diet.  control  Maltase a c t i v i t y was  not  determined. Troglia 110  e t a l . (56)  grams who  were then fed a r e s t r i c t e d  (an average of months. jejunal  10.5  At the  time of s a c r i f i c e , the  restricted  results  of the  d i r e c t l y w i t h the e t a l . (56)  as  (1% lactalbumin) was the  results,  f o r 5% to  day)  a c t i v i t y levels  6%  of  maltase were a l l s i g n i f i c a n t l y  group than i n the  c o n t r o l group.  p r e s e n t study cannot be  s t u d i e s of Kumar and  the  and  amount of r a t chow  grams of r a t chow per  l a c t a s e , s u c r a s e , and  h i g h e r i n the The  used r a t s weighing between 90  Chase (59)  p r o t e i n r e s t r i c t i o n i n the much more s e v e r e .  however, i n d i c a t e t h a t the  compared  The  Troglia  p r e s e n t study  similarity  effect  on  t i n a l d i s a c c h a r i d a s e a c t i v i t i e s o f r e s t r i c t i n g the intake of a 1% p r o t e i n d i e t does not  or  the  of intes-  caloric  d i f f e r from the  effect  of  56  r e s t r i c t i n g the c a l o r i c protein  intake of a d i e t c o n t a i n i n g  higher  levels.  In the present  study,  the p r o t e i n d e p l e t e d animals  gained  weight very a b r u p t l y when p l a c e d on the 18% l a c t a l b u m i n and  a t the end o f the 8 week f e e d i n g p e r i o d , they, were n o t  g r o s s l y d i s t i n g u i s h a b l e from the c o n t r o l animals. albumin c o n c e n t r a t i o n was almost i d e n t i c a l and  diet,  the c o n t r o l groups.  activities  The plasma  i n the r e p l e t e d  The l a c t a s e and j e j u n a l sucrase  i n the r e p l e t e d group f e l l  t o the same l e v e l s o f  a c t i v i t y found i n the c o n t r o l group, b u t the j e j u n a l maltase and  i l e a l sucrase and maltase a c t i v i t i e s  were s i g n i f i c a n t l y lower than those group.  fell  t o l e v e l s which  found i n the c o n t r o l  The f a c t t h a t not a l l the i n t e s t i n a l  disaccharidase  a c t i v i t i e s r e t u r n e d t o the same l e v e l as found i n the c o n t r o l group suggests t h a t severe p r o t e i n d e p l e t i o n may cause longed  pro-  o r i r r e v e r s i b l e changes i n i n t e s t i n a l maltase and  sucrase The  activities. a b s o r p t i o n o f l a c t o s e , sucrose,  and maltose was  s i g n i f i c a n t l y enhanced i n the p r o t e i n d e f i c i e n t r a t s when compared t o the c o n t r o l s (Table X) .  T h i s enhancement i n the  a b s o r p t i o n o f the d i s a c c h a r i d e s i s c o n s i s t e n t w i t h the h i g h e r levels of jejunal disaccharidases  i n the p r o t e i n d e f i c i e n t  rats. When the 0.5% l a c t a l b u m i n low carbohydrate l a c t a l b u m i n high carbohydrate cant  groups were compared, s i g n i f i -  i n c r e a s e s i n s u c r a s e and maltase a c t i v i t i e s  carbohydrate  group were found.  and the 0.5%  i n the high  However, there were no d i f f e r -  ences i n the a b s o r p t i o n o f l a c t o s e , sucrose,  or maltose  57  between the 0.5% l a c t a l b u m i n low carbohydrate l a c t a l b u m i n high carbohydrate  groups.  and the 0.5%  Gray and I n g e l f i n g e r  (10,11) had demonstrated t h a t i n human s u b j e c t s the r a t e limiting  step  i n sucrose and maltose a b s o r p t i o n  i s the t r a n s -  p o r t o f the c o n s t i t u e n t monosaccharides a c r o s s the mucosal membrane, and not the l e v e l o f i n t e s t i n a l sucrase and maltase activities.  But i n the case o f l a c t o s e a b s o r p t i o n ,  l a c t a s e a c t i v i t y i s the r a t e - l i m i t i n g  intestinal  s t e p and not the absorp-  t i o n of the c o n s t i t u e n t monosaccharides ( 9 ) .  I t has been  shown t h a t e i t h e r the r e s t r i c t i o n o f d i e t a r y p r o t e i n or s e m i s t a r v a t i o n may i n i t i a l l y r e s u l t  i n an i n c r e a s e d t r a n s p o r t  o f g l u c o s e a c r o s s the mucosal membrane (6,70,71). it  Therefore,  i s p o s s i b l e t h a t the p r o t e i n d e p r i v a t i o n i n c r e a s e d the  a b s o r p t i o n o f sucrose glucose  transport.  and maltose by a f f e c t i n g  the r a t e o f  Lactose a b s o r p t i o n would be i n c r e a s e d i n  response t o the i n c r e a s e i n l a c t a s e a c t i v i t y as t h a t i s the rate-limiting  step i n lactose absorption.  the carbohydrate  The i n c r e a s e i n  content o f the 0.5% l a c t a l b u m i n d i e t  from  66% t o 83.5% r e s u l t e d i n i n c r e a s e s i n sucrase and maltase a c t i v i t i e s b u t not i n sucrose  and maltose a b s o r p t i o n .  would be expected i f the r a t e - l i m i t i n g  This  step o f sucrose and  maltose i s the t r a n s p o r t o f the c o n s t i t u e n t monosaccharides. Most o f the s t u d i e s done w i t h l a b o r a t o r y animals on the effect  o f 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 on d i s a c c h a r i d a s e  a c t i v i t i e s which have been r e p o r t e d  i n the l i t e r a t u r e have  only measured the s p e c i f i c a c t i v i t i e s o f the i n t e s t i n a l d i s a c c h a r i d a s e s without disaccharides.  measuring the a b s o r p t i o n o f the  Only L i f s h i t z e t a l . (6) i n v e s t i g a t e d both  disaccharidase  a c t i v i t i e s and  absorption  of d i s a c c h a r i d e s  and  monosaccharides i n r a t s s u f f e r i n g from p r o t e i n - c a l o r i e malnutr tion.  They found no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s  the a b s o r p t i o n  o f g l u c o s e , f r u c t o s e , 3-0-methyl g l u c o s e ,  s u c r o s e , or maltose between the c o n t r o l s and  the r a t s on  low p r o t e i n d i e t s a f t e r 28 days of d i e t a r y treatment. were a l s o no s i g n i f i c a n t changes i n d i s a c c h a r i d a s e i n the p r o t e i n d e p l e t e d transitory transport  increase  animals a f t e r 28 days.  There was  i n d i c a t i n g t h a t the change was  a  water 14  no change i n the  t r a n s p o r t o f f r u c t o s e or 3-0-methyl glucose d u r i n g  glucose u t i l i z a t i o n  activities  low p r o t e i n d i e t a f t e r  days o f d i e t a r y treatment, but t h e r e was  the  There  i n the r a t e of g l u c o s e , sodium, and  i n the animals on the  p r o b a b l y due  to  that  period  increased  i n the r a t s on the low p r o t e i n d i e t s .  S i m i l a r f i n d i n g s o f an  i n i t i a l compensatory response t o semi-  s t a r v a t i o n i n the r a t which r e s u l t e d i n an t r a n s p o r t had  in  increase  been r e p o r t e d by Kershaw e t a l . (70)  in glucose and  Hind-  marsh e t a l . (71). I t i s d i f f i c u l t t o compare the r e s u l t s obtained i n the p r e s e n t study w i t h the r e s u l t s r e p o r t e d (6) because o f the d i f f e r e n c e s animals were s u b j e c t e d  i n the  t o the low  absorption  i s not  protein-calorie malnutrition  the  (28 days i n  60 days i n the p r e s e n t  However, the r e s u l t s o f b o t h s t u d i e s  disaccharide  The  l e n g t h of time t h a t  protein diets  the study of L i f s h i t z e t a l . (6), and study).  by L i f s h i t z e t a l .  indicate that  impaired i n e x p e r i m e n t a l  i n the r a t .  r e s u l t s o b t a i n e d i n the present i n v e s t i g a t i o n togethe  w i t h those r e p o r t e d  by other authors  (4,5,6,56,59) do  not  59  support the view t h a t d i s a c c h a r i d e deprived  intolerance  i n protein  c h i l d r e n i s the r e s u l t of decreased i n t e s t i n a l  accharidases  dis-  (2,30,32,37) i f we may a p p l y the r e s u l t s f o r r a t s  d i r e c t l y to children.  There a r e s e v e r a l p o s s i b l e  explanations  f o r the l a c k o f agreement between the human and animal response to p r o t e i n - c a l o r i e malnutrition. When the animal models are b e i n g used t o study the e f f e c t of p r o t e i n d e p r i v a t i o n  on i n t e s t i n a l d i s a c c h a r i d a s e  the e x p e r i m e n t a l c o n d i t i o n s  can be c l o s e l y c o n t r o l l e d .  f o r e , the d i e t s can be made adequate i n a l l other b e s i d e s p r o t e i n , whereas developed c o u n t r i e s  activity, There-  nutrients  the d i e t s of the c h i l d r e n i n under-  a r e f r e q u e n t l y d e f i c i e n t i n p r o t e i n as  w e l l as other e s s e n t i a l n u t r i e n t s .  I n a d d i t i o n , the mal-  nourished c h i l d r e n s u f f e r from g a s t r o i n t e s t i n a l i n f e c t i o n s . The  presence or absence o f i n f e c t i o n s can, however, be  controlled  i n the animal experiment.  Sriratanaban  and Thayer  (57) have s t u d i e d the e f f e c t o f  i r o n and combined i r o n - p r o t e i n d e f i c i e n c y on the i n t e s t i n a l disaccharidases  i n the r a t t o h e l p c l a r i f y t h i s p o i n t .  used four groups o f animals i n t h e i r s t u d y :  They  a c o n t r o l (27%  c a s e i n ) , low p r o t e i n  ( 5 % c a s e i n ) , low i r o n (27% c a s e i n ) , and  low  (5% c a s e i n ) .  iron-low  protein  The low i r o n group had  s i g n i f i c a n t l y lower l e v e l s o f l a c t a s e , s u c r a s e , and maltase a c t i v i t y when compared t o the c o n t r o l s .  The p r o t e i n d e f i c i e n t  group had s i g n i f i c a n t l y h i g h e r d i s a c c h a r i d a s e compared t o the c o n t r o l s .  The low iron-low  a c t i v i t i e s when  p r o t e i n group d i d  not demonstrate s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s when compared t o the c o n t r o l s , however, the d i s a c c h a r i d a s e  60  a c t i v i t i e s were somewhat h i g h e r the  jejunum and  duodenum.  ileum and  lower than i n the c o n t r o l s  These r e s u l t s i n d i c a t e t h a t other  d e f i c i e n c i e s may and  than i n the c o n t r o l s i n both  nutritional  a f f e c t i n t e s t i n a l disaccharidase  t h e r e f o r e , may  be c l o s e l y i n v o l v e d w i t h the  decreases i n i n t e s t i n a l disaccharidases  in the  activities,  observed  in children.  This  area needs f u r t h e r work. B o l i n e t a l . (58) has factors besides i d a s e s and nourished  a l s o s t u d i e d the e f f e c t o f  p r o t e i n d e f i c i e n c y on the  found t h a t an  intestinal  disacchar-  i n f e s t a t i o n of a group of adequately  r a t s with Nippostrongylus b r a s i l i e n s i s r e s u l t e d i n  a decrease i n the a c t i v i t i e s of l a c t a s e and t h a t i n f e s t a t i o n may of d i s a c c h a r i d a s e protein-calorie The  other  important f a c t o r i n the e t i o l o g y  depression  and  disaccharide  intolerance in  malnutrition.  study of the  m a l n u t r i t i o n by  be an  maltase showing  i n t e r r e l a t i o n s h i p between i n f e c t i o n and  Scrimshaw e t a l . (72) has  shown t h a t  t i o n g e n e r a l l y a l t e r s the r e s i s t a n c e of the host  malnutri-  to i n f e c t i o n  and  t h a t i n f e c t i o u s d i s e a s e exaggerated e x i s t i n g m a l n u t r i t i o n .  The  mechanisms i n v o l v e d  i n the decreased r e s i s t a n c e to i n f e c -  t i o n caused by m a l n u t r i t i o n i n c l u d e a reduced c a p a c i t y o f host  to form s p e c i f i c a n t i b o d i e s , a decrease i n  a c t i v i t y of microphages and wound h e a l i n g and  inating t i o n and  phagocytic  macrophages, a l t e r a t i o n s i n  c o l l a g e n formation,  i n t e g r i t y , diminished  alterations in tissue  inflammatory response, and  i n a l t e r a t i o n s of i n t e s t i n a l f l o r a  (72).  effects origSince  m a l n u t r i t i o n are so c l o s e l y l i n k e d i t w i l l be  to f u l l y e x p l o r e  the  infecnecessary  the r o l e of i n f e c t i o n i n the development of  61  disaccharide  intolerance  in protein-calorie  malnutrition  b e f o r e the d i f f e r e n c e between the r e s u l t s o f the p r e s e n t study and the symptoms of c h i l d r e n s u f f e r i n g from p r o t e i n calorie malnutrition  a r e understood.  Another p o s s i b l e e x p l a n a t i o n  f o r the d i f f e r e n c e between  the r e s u l t s o b t a i n e d w i t h r a t s and w i t h c h i l d r e n was  suggested  by Kumar and Chase (53). They f e l t t h a t the f a i l u r e o f experiments on 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 decrease i n d i s a c c h a r i d a s e  malnutrition  r a t s t o show a  a c t i v i t i e s may mean t h a t the r a t i s  not an adequate model f o r s t u d y i n g malnutrition.  using  human p r o t e i n - c a l o r i e  When these authors s t u d i e d  protein-calorie  i n monkeys, many o f the same symptoms found i n  c h i l d r e n s u f f e r i n g from 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  were  p r e s e n t i n the monkeys i n c l u d i n g marked v i l l o u s atrophy and decreased i n t e s t i n a l d i s a c c h a r i d a s e of p r o t e i n d e p r i v a t i o n monkeys a l s o s u f f e r e d cause.  a c t i v i t i e s a f t e r 18 weeks  {<Cl% p r o t e i n ) .  The undernourished  from i n t e r m i t t e n t d i a r r h e a  of an unknown  From these r e s u l t s , Kumar and Chase (53) concluded  t h a t the monkey was a b e t t e r model f o r s t u d y i n g malnutrition  protein-calorie  than the r a t .  The r a t cannot be excluded as a s u i t a b l e model f o r p r o t e i n - c a l o r i e malnutrition  u n t i l f u r t h e r work has been done  on the e f f e c t of the non-protein f a c t o r s i n c l u d i n g i n f e c t i o n and  n u t r i t i o n a l d e f i c i e n c i e s on the i n t e s t i n a l  activities.  disaccharidase  62  CHAPTER V SUMMARY AND RECOMMENDATIONS A.  Summary The purpose o f the p r e s e n t  e f f e c t o f prolonged  i n v e s t i g a t i o n was t o study the  experimental  protein-calorie malnutrition  on i n t e s t i n a l d i s a c c h a r i d a s e a c t i v i t i e s and on d i s a c c h a r i d e a b s o r p t i o n , as carbohydrate  i n t o l e r a n c e i s a major problem i n  c h i l d r e n s u f f e r i n g from 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 . Four groups o f r a t a  (90 t o 120 grams) were fed the f o l l o w -  ing d i e t s f o r 8 t o 9 weeks: carbohydrate);  low p r o t e i n low carbohydrate  66% c a r b o h y d r a t e ) ; talbumin,  c o n t r o l (18% l a c t a l b u m i n ,  (0.5% l a c t a l b u m i n ,  low p r o t e i n h i g h carbohydrate  83.5% c a r b o h y d r a t e ) ;  66%  (0.5% l a c -  and low p r o t e i n r e s t r i c t e d ( 1 %  l a c t a l b u m i n , r e s t r i c t e d t o 4 grams p e r d a y ) .  A f t e r 8.5 weeks,  p a r t o f the group on the 0.5% l a c t a l b u m i n low carbohydrate was f e d the c o n t r o l d i e t  diet  (18% l a c t a l b u m i n , 66% carbohydrate)  f o r 8 weeks. At the end o f the feed i n g p e r i o d , the f o l l o w i n g assays were performed: l a c t o s e , sucrose,  1) i n v i v o a b s o r p t i o n o f r a d i o a c t i v e  (^C)  and maltose; 2) a c t i v i t i e s o f i n t e s t i n a l  l a c t a s e , sucrase, and maltase; 3) plasma albumin c o n c e n t r a t i o n s ,and 4) mucosal p r o t e i n c o n c e n t r a t i o n s . a b s o r p t i o n s were expressed  The d i s a c c h a r i d e  as the percentage of the administered  r a d i o a c t i v i t y absorbed p e r u n i t weight o f i n t e s t i n e . accharidase  a c t i v i t i e s were expressed  The d i s -  as the u n i t s of enzyme  a c t i v i t y p e r gram o f wet weight mucosa or p e r gram o f mucosal  p r o t e i n ; the l a t t e r was c o n s i d e r e d  t o be the more s a t i s f a c t o r y  In the t h r e e p r o t e i n d e f i c i e n t groups (0.5% l a c t a l b u m i n low carbohydrate,  0.5% l a c t a l b u m i n h i g h carbohydrate,  and 1%  l a c t a l b u m i n ) , the a c t i v i t y o f b o t h the j e j u n a l and i l e a l accharidases  and the a b s o r p t i o n o f l a c t o s e , sucrose,  dis-  and mal-  t o s e were s i g n i f i c a n t l y higher when compared w i t h the c o n t r o l s The  j e j u n a l sucrase and maltase a c t i v i t i e s were s i g n i f i c a n t l y  higher  i n the 0.5% l a c t a l b u m i n h i g h carbohydrate  t h e 0.5% l a c t a l b u m i n low carbohydrate t i o n o f l a c t o s e , sucrose, 1% l a c t a l b u m i n  group than i n  group, b u t the absorp-  and maltose were a l i k e .  When the  ( r e s t r i c t e d t o 4 grams per day) and the 0.5%  l a c t a l b u m i n low carbohydrate  groups were compared, there were  no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the s p e c i f i c a c t i v i t i e s o f the i n t e s t i n a l d i s a c c h a r i d a s e s or the a b s o r p t i o n o f the d i s a c c h a r i d e s . The  a b s o r p t i o n of l a c t o s e , sucrose, and maltose were  s i m i l a r i n the c o n t r o l s and the p r o t e i n r e p l e t e d group. d i s a c c h a r i d a s e a c t i v i t i e s were a l s o s i m i l a r groups except f o r a s i g n i f i c a n t d e p r e s s i o n and  i l e a l sucrase  and maltase a c t i v i t i e s  i n these  The  two  of j e j u n a l maltase  i n the p r o t e i n  r e p l e t e d group. Therefore,  these r e s u l t s  indicate that p r o t e i n depriva-  t i o n i n r a t s f o r 8.5 weeks causes an i n c r e a s e i n s p e c i f i c a c t i v i t i e s o f the i n t e s t i n a l d i s a c c h a r i d a s e s  i n b o t h the  jejunum and ileum, and t h a t an i n c r e a s e i n the content  carbohydrate  of the p r o t e i n d e f i c i e n t d i e t r e s u l t s i n an i n d u c t i o n  of j e j u n a l sucrase and maltase a c t i v i t i e s .  The h i g h  specific  a c t i v i t y o f the i n t e s t i n a l d i s a c c h a r i d a s e s f o l l o w i n g p r o t e i n -  64  calorie  m a l n u t r i t i o n may b e i n p a r t d u e t o a p r e f e r e n t i a l  of s t r u c t u r a l protein  p r o t e i n r a t h e r than t o an i n c r e a s e  i n the i n t e s t i n a l  The  results  obtained  loss  i n enzymatic  mucosa. on t h e p r o t e i n r e p l e t e d r a t s h a v e  shown t h a t t h e c h a n g e s c a u s e d b y p r o t e i n d e f i c i e n c y a r e o f a reversible The protein  nature.  increase  i n the disaccharidase  deficient  accharide  The i n c r e a s e s  t i o n c o u l d b e due t o t h e h i g h e r  saccharides  i n disaccharide  levels  the  i n the p r o t e i n d e f i c i e n t  rats.  The d e m o n s t r a t i o n  s i g n i f i c a n t d i f f e r e n c e s i n sucrase  0.5% l a c t a l b u m i n increase  i n sucrose  the p r o t e i n d e f i c i e n t  The the  low c a r b o h y d r a t e  view t h a t the h i g h e r  saccharide  absorp-  of disaccharidases or  t a s e a c t i v i t i e s b e t w e e n t h e 0.5% l a c t a l b u m i n  comitant  in dis-  i n t h e t r a n s p o r t o f t h e c o n s t i t u e n t mono-  of s t a t i s t i c a l l y  and  i n the  r a t s i s accompanied by an i n c r e a s e  absorption.  t o an i n c r e a s e  activities  high  and malcarbohydrate  groups, without  or maltose absorption,  absorption  a consupports  o f maltose and s u c r o s e i n  rats i s a result  of increased  mono-  transport.  results obtained  suggestion  i n this  study  are not consistent  that protein-calorie malnutrition i s responsible  for disaccharide  intolerance i n children.  Since c h i l d r e n  suffering  from p r o t e i n - c a l o r i e d e f i c i e n c y a r e u s u a l l y a l s o  deficient  i n other  is  It  n u t r i e n t s and s u f f e r i n g from  not easy t o r e l a t e d i s a c c h a r i d e  levels  of disaccharidases  infections, i t  i n t o l e r a n c e and d e p r e s s e d  to p r o t e i n - c a l o r i e deficiency only.  i s p o s s i b l e , however, t h a t t h e r e  i n the response  with  may b e a s p e c i e s  to protein-calorie deficiency.  difference  65  B.  Recommendations The  f o l l o w i n g recommendations should be c o n s i d e r e d i f  f u r t h e r work i s t o be done on the e f f e c t o f 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 on the  i n t e s t i n a l d i s a c c h a r i d a s e s and  the i n  vivo absorption of disaccharides. Absorption  studies using glucose  should be c a r r i e d  together w i t h the s t u d i e s o f l a c t o s e , sucrose, absorption,  and  out  maltose  i n order t o c o n f i r m whether the i n c r e a s e s i n  a b s o r p t i o n of the t h r e e d i s a c c h a r i d e s i s due d i s a c c h a r i d a s e a c t i v i t i e s or due  to increased  to increased glucose  trans-  port. The  r a t e o f a b s o r p t i o n over a 30 minute p e r i o d  should  be determined r a t h e r than the t o t a l a b s o r p t i o n a f t e r 15 minutes, as the r a t e o f a b s o r p t i o n  i s a more s a t i s f a c t o r y measure f o r  s t u d y i n g the a b s o r p t i o n c a p a c i t y of the s m a l l In the p r e s e n t study, o n l y expressed  intestine.  the d i s a c c h a r i d e a b s o r p t i o n  per u n i t weight of i n t e s t i n e .  Since  a d i f f e r e n c e i n the diameter of the i n t e s t i n e and  was  there i s  the t h i c k -  ness o f the i n t e s t i n a l mucosa between the l a r g e r c o n t r o l animals and  the s m a l l e r p r o t e i n d e p l e t e d animals,  i t would  be more s a t i s f a c t o r y t o express the a b s o r p t i o n r e s u l t s u n i t of s u r f a c e area o f the The DNA  per  intestine.  e x p r e s s i o n o f d i s a c c h a r i d a s e a c t i v i t y per u n i t o f  would c l a r i f y whether there was  a change i n the r a t i o  the s t r u c t u r a l to enzymatic p r o t e i n s i n the of the p r o t e i n d e f i c i e n t I t would be  of  i n t e s t i n a l mucosa  rats.  i n f o r m a t i v e t o study the e f f e c t o f 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 on the i n t e s t i n a l d i s a c c h a r i d a s e s  and  66  d i s a c c h a r i d e a b s o r p t i o n as a f u n c t i o n o f time. a l l o w one t o determine the e a r l i e s t  T h i s would  time a t which the i n t e s -  t i n a l d i s a c c h a r i d a s e a c t i v i t i e s and d i s a c c h a r i d e  absorption  i n c r e a s e , and the temporal r e l a t i o n s h i p s between the i n c r e a s e s . 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