Na+ ‘pump’ and cell energetics in protein-energy malnutrition

NUTRITION RESEARCH, Vo]. 4, pp. 935-948, 1984 0271-5317/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All r ights reserved.

Na + 'PUMP'AND CELL ENERGETICS IN PROTEIN-ENERGY MALNUTRITION

Suresh S. Kaplay*

N a t i o n a l I n s t i t u t e o f N u t r i t i o n H y d e r a b a d , 50 0007 I n d i a

ABSTRACT

A balance between energy generating and energy utilizing systems is fundamental to the health and disease of a living cell. Attempts have been made to study the derangements in cell e n e r g e t i c s i n p r o t e i n - e n e r g y m a l n u t r i t i o n i n humans and e x p e r i m e n t a l a n i m a l s .

O u a b a i n s e n s i t i v e Na,K A T P a s e - - a b i o c h e m i c a l r e f l e c t i o n o f Na + and K + transport across the cell membrane--is elevated in erythrocyte membrane of children suffering from kwashiorkor and t h e k i d n e y o f p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s . The m e c h a n i s m o f i n c r e a s e i n Na,K ATPase i n e r y t h r o c y t e membrane o f c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r i s ( a ) by i n c r e a s i n g t h e a f f i n i t y f r o m Na § w h i c h a c t i v a t e s p h o s p h o r y l a t i o n s t e p f rom i n s i d e o f t h e membrane and (b) by increasing the total number of pump sites on t h e membrane . The e l e v a t e d Na,K ATPase i n k w a s h i o r k o r p a t i e n t s i s r e v e r s e d t o a n o r m a l l e v e l e i t h e r a f t e r d i e t t h e r a p y o r on s i n g l e a d m i n i s t r a t i o n o f a d i u r e t i c . T h i s i s a s s o c i a t e d w i t h l o s s o f edema f l u i d and a c c u m u l a t i o n o f c e l l Na +. Na + ' p u m p ' i s one o f t h e m a j o r p r o c e s s e s f o r u t i l i z a t i o n o f c e l l e n e r g y g e n e r a t e d by m i t o c h o n d r i a . M i t o c h o n d r i a l r e s p i r a t i o n and i t s k i n e t i c s t u d i e s i n k i d n e y and l i v e r o f p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s show t h a t k i d n e y and l i v e r m i t o c h o n d r i a f r o m p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s a r e more t i g h t l y c o u p l e d and h a v e i n c r e a s e d r a t e s o f s t a t e 3 ( a c t i v e ) r e s p i r a t i o n as compared t o t h o s e o f c o n t r o l r a t s . K idney m i t o c h o n d r i a a l s o h a v e i n c r e a s e d K m ( a p p a r e n t ) f o r ADP as w e l l as P i , p e r h a p s an a d a p t a t i o n t o i n c r e a s e d a v a i l a b i l i t y o f ADP and P i i n t h e c y t o s o l due t o e l e v a t e d Na,K ATPase .

I t i s p r o p o s e d t h a t i n i t i a l l y t h e r e may be an a c c u m u l a t i o n o f i n t r a c e l l u l a r Na i n p r o t e i n - e n e r g y m a l n u t r i t i o n by a m e c h a n i s m w h i c h i s n o t a l t o g e t h e r c l e a r y e t , b u t p e r h a p s due t o s l u g g i s h Na ' p u m p ' c a u s e d by c e l l e n e r g y d e p l e t i o n . I n r e s p o n s e t o t h i s and t o p r e v e n t c o n t i n u e d Na a c c u m u l a t i o n ( w h i c h w i l l be d e t r i m e n t a l t o cell survival) Na 'pump' is stimulated. However, this will i n c r e a s e the demand on c e l l e n e r g y and m i t o c h o n d r i a l r e s p i r a t i o n w i l l h a v e t o be e n h a n c e d t o m e e t t h i s e x c e s s demand on c e l l

*Present address: Department of Cell Physiology, Boston Biomedical Research Institute, 20 Stanford St., Boston, Massachusetts, 02114, U.S.A.

935

936 S.S. KAPLAY

energy . This w i l l l ead to r a p i d d e p l e t i o n of a l r e a d y l i m i t e d c e l l r e s o u r c e s and thus may form the b iochemica l b a s i s f o r the p a t h o g e n e s i s of p r o t e i n - e n e r g y m a l n u t r i t i o n . E l e v a t e d l e v e l s of a l d o s t e r o n e , a n t i d i u r e t i c s hormone and i n s u l i n may p lay an impor tan t r o l e in e l e v a t i o n of Na + 'pump' . These membrane t r a n s p o r t a b n o r m a l i t i e s may not only c o n t r i b u t e to edema of p r o t e i n - e n e r g y m a l n u t r i t i o n but could be s p e c u l a t e d to have i m p l i c a t i o n s on o t h e r membrane f u n c t i o n s l i k e in~eunological and hormonal r e s p o n s i v e n e s s of the c e l l .

KEY WORDS: Sodium pump; Na,K ATPase; Edema; C e l l e n e r g e t i c s ; P r o t e i n - e n e r g y m a l n u t r i t i o n ; O x i d a t i v e p h o s p h o r y l a t i o n .

INTRODUCTION

Biochemica l changes in p r o t e i n - e n e r g y m a l n u t r i t i o n have been the s u b j e c t o f s e v e r a l s t u d i e s over the four decades s i n c e the f i r s t case of kwashiorkor was r e p o r t e d by C e c i l y Wi l l iams (1 ) . These s t u d i e s have been reviewed by many au thors ( 2 - 4 ) . This r ev i ew w i l l not touch upon any of t h e s e , e x c e p t i n g the most r e l e v a n t endoc r ine a s p e c t s . The main aim of the p r e s e n t r ev i ew i s to draw a t t e n t i o n to some of the new approaches and o b s e r v a t i o n s on membrane a b n o r m a l i t i e s - - p a r t i c u l a r l y the ones r e l a t e d to c e l l e n e r g e t i c s - - w h i c h have he lped to improve the unde r s t and ing of the d i s o r d e r to a c e r t a i n e x t e n t .

M u l t i p l i c i t y of r e g u l a t o r y f u n c t i o n s governed by hormonal s i g n a l s and t r a n s p o r t of m e t a b o l i t e s ac ross the b i o l o g i c a l membrane makes i t s s tudy of v i t a l importance f o r the b e t t e r (and perhaps comple te) unde r s t and ing of human d i s o r d e r . T h e r e f o r e , b i o l o g i c a l membranes have been a s u b j e c t o f s tudy in many human d i s o r d e r s of endoc r ine and g e n e t i c o r i g i n . However, u n t i l recently it has not received adequate attention in protein-energy malnutrition. Erythrocyte membrane due to its easy availability and ease with which it can be isolated in pure form, offers itself as an excellent biological membrane for the study of fundamental and applied aspects of membranology.

Reduced d e f o r m a b i l i t y or i n c r e a s e d r e s i s t a n c e to osmot ic l y s i s i s one of the e a r l i e s t e r y t h r o c y t e a b n o r m a l i t i e s to be demons t ra ted in p r o t e i n - e n e r g y m a l n u t r i t i o n ( 5 , 6 ) . This has been conf i rmed in subsequent s t u d i e s by v a r i o u s workers (7-10). However, different mechanisms have been suggested by different workers for this increased resistance of erythrocytes in protein-energy malnutrition to osmotic lysis. According to some, this may be due to increased cholesterol:phospholipid ratio (a direct reflection of area/volume of the membrane) in the erythrocyte membrane of children suffering from protein-energy malnutrition (7). We could neither find a significant elevation in this ratio nor a relationship between osmotic fragility changes and cholesterol:phospholipid ratio in children suffering from kwashiorkor (9). However, we have been able to demonstrate a direct relationship between Ca +2, Mg +2 ATPase activity and osmotic fragility in the erythrocytes of kwashiorkor patients (9). Ca +2 , Mg +2 ATPase is responsible for active Ca +2 efflux from the cell, and its deficiency could lead to accumulation of intracellular calcium. Calcium is known to be one of the factors in the rigidization of the erythrocyte membrane (11). Based on this, we proposed that intracellular accumulation of calcium, due to reduced Ca +2, Ng +2 ATPase, is a major factor in increasing the resistance of erythrocyte membrane to osmotic lysis in kwashiorkor (9). If this indeed is so, then it will have far reaching implications on several of the cellular functions and it may underly many of the cell defects since Ca +2 is known to have an important regulatory role (12).

SODIUM PUMP IN PEM 937

Na+ 'Pump ' in P r o t e i n - E n e r g y N a l n u t r t t i o n

Na,K ATPase (ATP p h o s p h o h y d r o l a s e , sod ium p l u s p o t a s s i u m a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e , EC 3 . 6 . 1 . 3 ) i s a b i o c h e m i c a l r e f l e c t i o n o f Na + and K + ' p u m p ' a c r o s s t h e c e l l membrane ( 1 3 ) . The ' p u m p ' works a g a i n s t t h e u p h i l l c a t i o n g r a d i e n t and u t i l l z e s e n e r g y i n t h e fo rm o f ATP. The m a i n f u n c t i o n o f t h e ' pump ' i s t o m a i n t a i n t h e i o n i c g r a d i e n t a c r o s s t h e c e l l membrane and t h u s h e l p t o m a i n t a i n membrane p o t e n t i a l . The c l o s e l i n k b e t w e e n t h e a c t i v e t r a n s p o r t o f Na + and K + w i t h t h e t r a n s p o r t o f o t h e r important metabolites like sugar and amino acids makes the 'pump' of vital i m p o r t a n c e t o t h e f u n c t i o n o f a l l t h e t i s s u e s . T h i s a l s o makes t h e ' p u m p ' a m a j o r e n e r g y u t i T i z i n g p r o c e s s i n t h e l i v i n g c e l l . I t h a s b e e n e s t i m a t e d t h a t , d e p e n d i n g on t h e t y p e o f c e l l , a l m o s t 30 t o 50~ o f t h e e n e r g y g e n e r a t e d by m i t o e h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n i s u t i l i z e d by Na + ' pump ' ( 1 4 - 1 6 ) . S i n c e t h e g e n e r a t i o n o f c e l l e n e r g y i s c l o s e l y r e l a t e d t o i t s r e q u i r e m e n t i n v a r i o u s c e l l u l a r p r o c e s s e s , Na + ' p u m p ' i s r e c o g n i z e d t o h a v e an i m p o r t a n t r o l e i n t h e c o n t r o l o f m i t o c h o n d r i a l r e s p i r a t i o n ( 1 4 ) . The ' pump ' i s a l s o h i g h l y r e s p o n s i v e t o ho rmones l i k e i n s u l i n , a l d o s t e r o n e , a n t i d i u r e t i c ho rmone , and t h y r o x i n e ( 1 7 ) . I n v i v o o r i n v i t r o p e r t u r b a t i o n s r e s u l t i n g i n c h a n g e s i n i o n i c g r a d i e n t s a c r o s s t h e membrane m o d i f y t h e ' p u m p ' i n an e f f o r t t o r e s t o r e t h e i o n i c g r a d i e n t . Na,K ATPase h a s two s u b u n i t s . The l a r g e r s u b u n i t c a l l e d t h e = s u b u n i t ( a 9 5 , 0 0 0 d a l t o n p o l y p e t i d e ) s p a n s t h e e n t i r e membrane . Na + and ATP s i t e s a r e l o c a l i s e d on t h i s s u b u n i t c l o s e t o e a c h o t h e r on t h e i n n e r s i d e o f t h e membrane . The o u a b a i n and K + s i t e s a r e on t h e e x t e r n a l s u r f a c e o f t h e membrane . The s m a l l e r B s u h u n i t ( a 4 0 , 0 0 0 d a l t o n p o l y p e p t i d e ) i s a g l y c o p r o t e i n ( 1 7 ) .

Na + and K + transport occurs by a two-step mechanism. In the first step t h e enzyme m o l e c u l e i s p h o s p h o r y l a t e d . T h i s s t e p i s a c t i v a t e d by Na + f r o m t h e i n s i d e o f t h e membrane . The p h o s p h o r y l a t e d m o l e c u l e b r i n g s Na + a l o n g w i t h i t t o t h e o u t s i d e o f t h e c e l l . D e p h o s p h o r y l a t i o n o f t h e p r o t e i n on t h e cytoplasmic side is activated by Z + which is carried to the inside of the c e l l by t h e d e p h o s p h o r y l a t e d i n t e r m e d i a t e ( 1 8 ) . O u a b a i n , a p r o t o t y p e o f c a r d i a c g l y c o s i d e i s a s p e c i f i c i n h i b i t o r o f Na,K ATPase. I t b i n d s t o t h e ' pump ' s i t e s s t o i c h i o m e t r i c a l l y on t h e o u t e r s u r f a c e and t h u s p r o v i d e s a d i r e c t e s t i m a t e o f ' pump ' s i t e s on t h e c e l l membrane . Na,K ATPase h a s b e e n s u g g e s t e d t o be a r e c e p t o r f o r c a r d i a c g l y c o s i d e s u s e d i n t h e t h e r a p y o f c a r d i a c d i s o r d e r s and h e n c e i t s b i n d i n g c a p a c i t y t o t h e p l a s m a membrane may be a r e f l e c t i o n o f e f f e c t i v e n e s s o f t h i s d r u g i n t h e s e p a t i e n t s ( 1 9 ) . I n k i d n e y , Na,K ATPase i s l o c a l i s e d on t h e a s c e n d i n g l o o p o f H e n l e and i s c o n s i d e r e d as a s i t e o f a c t i o n o f c e r t a i n d i u r e t i c s l i k e f u r o s e m i d e ( 2 0 ) .

There are two components of Na,K ATPase. The one sensitive to ouabaln is responsible for active cation transport. The precise function of the ouabain insensitive component is not yet clear. In the following discussion Na,Z ATPase refers to only the ouabain sensitive component.

I n t h e e r y t h r o c y t e membrane o f t h e c h i l d r e n who s u f f e r f rom k w a s h i o r k o r (a fo rm o f p r o t e i n - e n e r g y m a l n u t r i t i o n w i t h edema) t h e a c t i v i t y o f Na,K A T P a s e - - e x p r e s s e d e i t h e r as p e r u n i t membrane p r o t e i n o r p e r c e l l , i s e l e v a t e d by a b o u t t w o - f o l d ( 2 1 , 2 2 ) . S p e c i f i c a c t i v i t y o f Na,K ATPase i n t h e e r y t h r o c y t e membrane shows an i n v e r s e r e l a t i o n s h i p w i t h i n t r a c e l l u l a r Na + i n c o n t r o l as w e l l as i n k w a s h i o r k o r c h i l d r e n ( F i g u r e 1 ) . However , t o m a i n t a i n c o m p a r a b l e l e v e l s o f c e l l Na, t h e c e l l i n k w a s h i o r k o r seems t o r e q u i r e a l m o s t t w o f o l d h i g h e r Na,K ATPase a c t i v i t y as compared t o t h e one r e q u i r e d by c o r r e s p o n d i n g c o n t r o l e r y t h r o c y t e s ( F i g u r e 1 ) . No s u c h i n c r e a s e i n Na,K ATPase a c t i v i t y i s o b s e r v e d i n p a t i e n t s w i t h m a r a s m i c c o n d i t i o n ( a fo rm of p r o t e i n - e n e r g y m a l n u t r i t i o n w i t h o u t edema) . T h i s e l e v a t e d Na,K ATPase a c t i v i t y i n t h e e r y t h r o c y t e membrane o f k w a s h i o r k o r p a t i e n t s i s

938 S.S. KAPLAY

r e d u c e d a f t e r d i e t t h e r a p y f o r f o u r t o s i x weeks ( 2 1 ) . The r a t i o o f (Na+K) a c r o s s t h e c e l l membrane a l s o shows s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e two forms of proteln-energy malnutrition ( 2 3 ) . The cell/plasma ratio of (Na+K) i s 0 . 9 0 i n c o n t r o l c h i l d r e n , 0 . 8 3 i n m a r a s m i c and 0 . 7 2 i n k w a s h i o r k o r patients. This reflects an increase in electrolytes and along with that, water in the extra-cellular compartment in kwashiorkor but not in marasmus. It parallels the changes in Na,Z ATPase activity. After dietary therapy in

c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r , j u s t as t h e edema d i s a p p e a r s , t h i s r a t i o r e t u r n s t o n o r m a l ( 0 . 9 6 ) and Na,K ATPase a c t i v i t y i s r e d u c e d c o r r e s p o n d i n g l y ( 2 3 ) . D e s p i t e an i n c r e a s e i n i n t r a - c e l l u l a r Na, m a r a s m i c p a t i e n t s a r e a b l e t o m a i n t a i n t h i s r a t i o w i t h i n a n o r m a l r a n g e ( 2 3 ) . How t h i s i s a c h i e v e d w i t h o u t p e r t u r b a t i o n s i n c a t i o n t r a n s p o r t s y s t e m r e m a i n s u n c l e a r .

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o C O N T R O L r = - 0 , 7 4 , P < O , 0 5

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FIG. 1

I n v e r s e r e l a t i o n s h i p b e t w e e n e r y t h r o c y t e Na and s p e c i f i c a c t i v i t y o f e r y t h r o c y t e membrane Na, K ATPase i n c o n t r o l and c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r . Based on d a t a f rom Kap lay ( 2 1 ) .

When t h e c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r a r e t r e a t e d w i t h a s i n g l e a d m i n i s t r a t i o n o f d i u r e t i c - - f u r o s e m i d e - - t h e r e i s a l o s s o f edema f l u i d , as r e f l e c t e d i n t h e r e d u c t i o n i n t h e body w e i s h t s o f t h e s e c h i l d r e n ( 2 4 ) . The erythrocyte membrane Na,K ATPase activity returns to normal levels and along w i t h t h a t sod ium a c c u m u l a t e s i n t h e c e l l ( 2 4 ) , s i m i l a r t o wha t i s o b s e r v e d


a f t e r d i e t t h e r a p y ( 2 1 ) . T h i s d e m o n s t r a t e s a d i r e c t r e l a t i o n s h i p b e t w e e n edema, t h e e l e v a t e d Na,K ATPase a c t i v i t y and m a i n t e n a n c e o f a p p a r e n t l y n o r m a l l e v e l s o f c e l l Na + i n k w a s h i o r k o r ( 2 1 , 2 4 ) . S t u d i e s o f P a t r i c k on Na + ' p u m p ' i n l e u c o c y t e s o f c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r a l s o show t h a t t h e Na + ' pump ' i s e l e v a t e d i n t h e s e p a t i e n t s and t h a t t h e i n c r e a s e d a c t i v i t y c o u l d be r e v e r s e d by d i u r e t i c t h e r a p y ( 2 5 ) . He a l s o f o u n d t h a t t r e a t m e n t w i t h d i u r e t i c b e f o r e i n i t i a t i o n o f d i e t t h e r a p y i m p r o v e s t h e c h a n c e s o f s u r v i v a l o f t h e p a t i e n t s who h a v e e l e v a t e d Na + ' pump ' i n t h e i r l e u c o c y t e s ( 2 5 ) . T h e s e o b s e r v a t i o n s s u g g e s t t h a t Na + pump i s o f c r u c i a l i m p o r t a n c e i n t h e edema o f p r o t e i n - e n e r g y m a l n u t r i t i o n ( 2 3 , 2 6 ) .

E f f e c t i v e n e s s o f f u r o s e m i d e i n i n h i b i t i n g e r y t h r o c y t e membrane Na,K ATPase seems t o d e p e n d on w h e t h e r o r n o t i t i s i n v o l v e d i n t h e edema. Na,K ATPase does n o t seem t o be i n v o l v e d i n p r e g n a n c y - i n d u c e d edema and h y p e r t e n s i o n s i n c e no a l t e r a t i o n s a r e s e e n i n t h i s enzyme s y s t e m ( 2 7 ) . T r e a t m e n t o f t h e s e p a t i e n t s w i t h f u r o s e m i d e f o r f o u r days n e i t h e r h a s any e f f e c t on e r y t h r o c y t e Na,K ATPase a c t i v i t y n o r on t h e c e l l and p l a s m a electrolytes (27).

I f t h e i n c r e a s e d Na,K ATPase a c t i v i t y i n t h e e r y t h r o c y t e s o f c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r and t h e i n c r e a s e d Na + e f f l u x i n t h e l e u c o c y t e s o f t h e s e p a t i e n t s i s p r e s e n t i n k i d n e y as w e l l , t h e n i n c r e a s e d a c t i v e Na + r e a b s o r p t i o n ( o f w h i c h Na,K ATPase i s a b i o c h e m i c a l r e f l e c t i o n ) w i l l be a m a j o r f a c t o r i n t h e edema o f p r o t e i n - e n e r g y m a l n u t r i t i o n . Such a d i r e c t e v i d e n c e i s d i f f i c u l t t o o b t a i n f rom human s u b j e c t s . Bu t t h e s t r o n g e s t s u p p o r t f o r s u c h a m e c h a n i s m comes f rom r e n a l f u n c t i o n s t u d i e s i n p r o t e i n - e n e r g y m a l n u t r i t i o n . T h e s e s t u d i e s by two i n d e p e n d e n t a u t h o r s h a v e l e d t o t h e s u g g e s t i o n t h a t i n c r e a s e d Na + r e a b s o r p t i o n i n t h e ' l o o p ' o f H e n l e ( a s i t e o f l o c a l i s a t i o n o f Na,K ATPase and s i t e o f a c t i o n o f ' l o o p ' d i u r e t i c s l i k e f u r o s e m i d e ) may be r e s p o n s i b l e f o r edema i n p r o t e i n - e n e r g y m a l n u t r i t i o n ( 2 8 , 2 9 ) . I n a d d i t i o n , s t u d i e s i n e x p e r i m e n t a l a n i m a l s h a v e shown an i n c r e a s e i n k i d n e y Na,K ATPase i n p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s ( 3 0 ) .

Mechan i sm o f I n c r e a s e d Na,K ATPase i n P r o t e i n - E n e r g # M a l n u t r i t i o n

An i n c r e a s e i n enzyme a c t i v i t y c o u l d be e i t h e r due t o m o d i f i c a t i o n i n i t s a f f i n i t y f o r t h e s u b s t r a t e o r an a b s o l u t e i n c r e a s e i n t h e enzyme c o n t e n t s . C o m b i n a t i o n o f t h e s e f a c t o r s h a s b e e n f o u n d t o be r e s p o n s i b l e f o r t h e i n c r e a s e d Na,K ATPase i n k w a s h i o r k o r . The K m ( a p p a r e n t ) f o r Na, w h i c h a c t i v a t e s p h o s p h o r y l a t l o n o f Na,K ATPase f rom i n s i d e t h e membrane i s s i g n i f i c a n t l y l o w e r e d i n t h e e r y t h r o c y t e s o f c h i l d r e n s u f f e r i n g f r o m kwashiorkor. The K m (apparent) for the other two substrates E + and ATP, however, is unchanged (31). This indicates an increased affinity for Na +. The i n c r e a s e i n a f f i n i t y n e e d n o t n e c e s s a r i l y mean a m o d i f i c a t i o n i n t h e enzyme m o l e c u l e b u t c o u l d be due t o a l t e r e d m i c r o e n v i r o n m e n t i n t h e membrane i n t h e v i c i n i t y o f t h e a c t i v e s i t e . I n a d d i t i o n , t h e r e a l s o seems t o h e an i n c r e a s e i n t h e number o f ' p u m p ' s i t e s on t h e e r y t h r o c y t e membrane i n t h e c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r . The e r y t h r o c y t e s o f t h e s e p a t i e n t s h a v e 891 o u a b a i n s i t e s p e r c e l l as compared t o 316 s i t e s on t h e e r y t h r o c y t e s o f m a r a s m i c and 385 s i t e s i n c o n t r o l e r y t h r o c y t e s ( 3 2 ) . The d i s s o c i a t i o n c o n s t a n t Kd c a l c u l a t e d f r o m t h e S c a t c h a r d p l o t a n a l y s i s o f t h e o u a b a i n b i n d i n g i s n o t d i f f e r e n t i n t h e s e g r o u p s . The i n c r e a s e i n ' p u m p ' s i t e s c o u l d e i t h e r r e f l e c t an a b s o l u t e i n c r e a s e i n enzyme u n i t s o r an e x p o s u r e o f e x i s t i n g s i t e s m a k i n g t h e m a v a i l a b l e t o o u a b a i n . The l a t t e r p o s s i b i l i t y i s s u p p o r t e d by e x p e r i m e n t s i n k i d n e y m l c r o s o m a l p r e p a r a t i o n s f r o m p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s ( 3 0 ) . T r e a t m e n t o f m i c r o s o m e s w i t h d e o x y c h o l a t e , a d e t e r g e n t , c a u s e s e x p o s u r e o f ' p u m p ' s i t e s w h i c h a r e b u r i e d i n t h e membrane ( 3 3 ) . When k i d n e y m i c r o s o m a l p r e p a r a t i o n s f r o m w e l l - f e d

940 S.S. KAPLAY

control rats are treated with deoxycholate, Na,K ~TPase activity i s increased by 65%. On t h e c o n t r a r y , s i m i l a r t r e a t m e n t o f k i d n e y m i c r o s m a l p r e p a r a t i o n s f rom p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s r e s u l t s i n o n l y a b o u t 20~ i n c r e a s e i n Na,K ATPase ( 3 0 ) . T h i s i n d i c a t e s t h a t t h e e x i s t i n g Na + ' pump ' s i t e s on t h e p l a s m a membrane i n p r o t e i n - e n e r g y m a l n u t r i t i o n a r e e x p o s e d due t o membrane m o d i f i c a t i o n . I t i s p o s s i b l e t h a t a s i m i l a r m e c h a n i s m c o u l d be o p e r a t i v e i n t h e i n c r e a s e o f e r y t h r o c y t e membrane ' p u m p ' s i t e s i n k w a s h i o r k o r a l s o . However , f r om t h e m a g n i t u d e o f i n c r e a s e i n ' pump ' s i t e s i n t h e e r y t h r o c y t e s o f c h i l d r e n s u f f e r i n g f rom k w a s h i o r k o r , i t w i l l a p p e a r t h a t e x p o s u r e o f e x i s t i n g s i t e s c o u l d o c c u r i n c o m b i n a t i o n w i t h a n e t i n c r e a s e i n t h e number o f s i t e s .

Vmax/K m i s a c l o s e r e f l e c t i o n o f t h e p h y s i o l o g i c a l c a p a c i t y o f an enzyme s y s t e m ( 3 4 ) . T h i s c a p a c i t y f o r Na,K ATPase e x p r e s s e d p e r c e l l i n t h e e r y t h r o c y t e membrane o f k w a s h i o r k o r c h i l d r e n i s a b o u t 260% o f t h a t i n t h e control ( T a b l e I).

I t i s g e n e r a l l y b e l i e v e d t h a t t h e l i v i n g c e l l m a i n t a i n s i t s e n e r g y b a l a n c e by g e n e r a t i n g j u s t enough e n e r g y t o mee t i t s e n e r g y demands . I t i s o f i n t e r e s t t o f i n d o u t as t o how t h e c e l l i n k w a s h i o r k o r i s m e e t i n g t h e s e i n c r e a s e d demands f o r c e l l e n e r g y , r e q u i r e d i n t h e p r o c e s s o f e n h a n c e d Na + ' p u m p ' . The m a j o r s u p p l y o f e n e r g y f o r Na + ' p u m p ' comes f rom m i t o c h o n d r i a l o x i d a t i v e p h o s p h o r y l a t i o n ( 1 4 - 1 6 ) . However , i t i s n o t p o s s i b l e t o c a r r y o u t many o f t h e s e d e t a i l e d s t u d i e s i n humans and h e n c e an e x p e r i m e n t a l mode l o f t h e d i s o r d e r i s f o u n d u s e f u l .

C e l l E n e r g e t i c s - S t u d i e s i n E x p e r i m e n t a l P r o t e i n - E n e r g y M a l n u t r i t i o n

S t u d i e s i n e x p e r i m e n t a l a n i m a l s p r o v i d e c o n s i d e r a b l e e v i d e n c e w h i c h f a v o u r s a g e n e r a l i z e d i n c r e a s e i n t h e Na + d e p e n d e n t a c t i v e t r a n s p o r t and i n t u r n i n c r e a s e d demand f o r c e l l e n e r g y i n v a r i o u s t i s s u e s i n p r o t e i n - e n e r g y malnutrition. Apart from being directly responsible for cation transport, Na § ' p u m p ' c o n t r i b u t e s t o t h e a c t i v e t r a n s p o r t o f o t h e r m e t a b o l i t e s l i k e sugar and amino acides (17) and also to thermogenesis (35).

Intestinal sugar transport ( 3 6 ) , amino acid transport (37) and kidney Na,K ATPase have been found to be higher in protein-energy malnourished rats as compared to the control. (In the experimental model of protein-energy m a l n u t r i t i o n employed i n o u r s t u d i e s , t h e p r o t e i n and e n e r g y i n t a k e s o f r a t s were as f o l l o w s : p r o t e i n , 1 g , 0 . 6 g and 0 . 0 5 g; e n e r g y , 2 4 . 5 k c a l , 1 4 . 5 k c a l , and 24 .7 k c a l p e r 100 g body w e i g h t f o r c o n t r o l , e n e r g y r e s t r i c t e d and p r o t e i n r e s t r i c t e d a n i m a l s r e s p e c t i v e l y . E x p e r i m e n t s were c a r r i e d o u t b e t w e e n 7 t o 9 weeks on t h e s e d i e t s c h e d u l e s . At t h i s t i m e , r a t s i n p r o t e i n d e f i c i e n t g r o u p s s i m u l a t e d many o f t h e s i g n s o f human k w a s h i o r k o r ( 3 0 ) . Hove recently, cellular thermogenesis is also shown to be enhanced in protein d e f i c i e n t r a t s and p i g s ( 3 8 ) .

I n a c c o r d a n c e w i t h t h e s e o b s e r v a t i o n s , m i t o c h o n d r i a l r e s p i r a t i o n i n t h e k i d n e y and l i v e r o f p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s i s a l s o f o u n d t o be e l e v a t e d ( 3 9 - 4 1 ) . The e x t e n t o f i n c r e a s e i n m i t o c h o n d r i a l s t a t e 3 ( a c t i v e ) r e s p i r a t i o n i s more o r l e s s o f t h e same m a g n i t u d e as t h e i n c r e a s e i n Ns,K ATPase, a t l e a s t i n t h e c a s e o f k i d n e y ( 4 1 ) . The l i v e r and k i d n e y m i t o c h o n d r i a f rom p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s a r e much more t i g h t l y c o u p l e d as compared t o t h o s e i s o l a t e d f rom c o n t r o l , w e l l - f e d r a t s ( 4 0 , 4 1 ) . F u r t h e r c h a r a c t e r i z a t i o n o f k i d n e y m i t o c h o n d r i a f rom t h e s e r a t s shows t h a t there is significant increase in the K m (apparent) for ADP and Pi in protein-energy malnourished rats (41). This indicates an adaptation to the increased availability of ADP and Pi in the cytosol due to increased h y d r o l y s i s ( u t i l i z a t i o n ) o f ATP. A l l t h e s e o b s e r v a t i o n s d e m o n s t r a t e


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i n c r e a s e d e f f i c i e n c y o f e n e r g y g e n e r a t i o n by m i t o c h o n d r i a l r e s p i r a t i o n t o m e e t t h e i n c r e a s e d demands f o r c e l l e n e r g y f o r a c t i v e t r a n s p o r t .

P o s s i b l e S t i m u l u s f o r E n h a n c e d Na+ 'Pump ' i n P r o t e i n - E n e r g y M a l n u t r i t i o n

Due t o t h e c o m p l e x i t y o f t h e d i s o r d e r , i t i s d i f f i c u l t t o i d e n t i f y one s i n g l e f a c t o r w h i c h c o u l d m o s t p o t e n t l y c a u s e e n h a n c e m e n t o f Na ' pump ' i n p r o t e i n - e n e r g y m a l n u t r i t i o n . The s u g g e s t i o n by P a t r i c k t h a t d e f f i c i e n c y o f v a n a d i u m , an i n h i b i t o r o f Na,K ATPase , may be r e s p o n s i b l e f o r t h i s phenomenon ( 4 2 ) , a l t h o u g h n o t a l t o g e t h e r i m p r o b a b l e , seems t o be a f a c t o r o f r a t h e r weak i m p o r t a n c e i n l i g h t o f t h e s u p e r i m p o s i n g h o r m o n a l p r o f i l e w h i c h emerges i n p r o t e i n - e n e r g y m a l n u t r i t i o n . The e l e v a t e d l e v e l s o f a l d o s t e r o n e ( 4 3 ) , a n t i - d i u r e t i c hormone ( 4 4 ) , and i n s u l i n (45) i n k w a s h i o r k o r a r e a l l known t o s t i m u l a t e t h e Na + pump ( 1 7 ) . R e c e n t s t u d i e s d e m o n s t r a t e a r e l a t i o n s h i p b e t w e e n d i e t a r y c a r b o h y d r a t e and r e n a l Ha r e a b s o r p t i o n l e a d l n g t o edema and h y p e r t e n s i o n . S u c r o s e i s b e l i e v e d t o i n c r e a s e i n s u l i n s e c r e t i o n w h i c h i n t u r n s t i m u l a t e s Na r e a b s o r p t i o n i n t h e k i d n e y , l e a d i n g t o f l u i d a c c u m u l a t i o n (46 , 4 7 ) . M e t c o f f and a s s o c i a t e s h a v e i m p l i c a t e d d i e t a r y c a r b o h y d r a t e and i n s u l i n i n t h e p a t h o g e n e s i s o f p r o t e i n - e n e r g y m a l n u t r i t i o n ( 4 8 ) . Coward and W h i t e h e a d c o u l d p r o d u c e edema i n b a b y b a b o o n s o n l y when s u c r o s e was i n c l u d e d i n t h e d i e t ( 4 9 ) . W h i t e h e a d and Lunn h a v e a l s o shown an a s s o c i a t i o n b e t w e e n p r o g r e s s i v e i n c r e a s e i n se rum i n s u l i n and t h e i n c i d e n c e o f edema i n Gsmbian c h i l d r e n ( 4 5 ) . I n o u r own e x p e r i e n c e , when two g r o u p s o f r a t s a r e m a i n t a i n e d on 1% p r o t e i n d i e t , one w i t h 80% s t a r c h and a s e c o n d w i t h 40% s t a r c h p l u s 40% s u c r o s e , o n l y t h e r a t s r e c e i v i n g s u c r o s e show edema and f a t t y i n f i l t r a t i o n o f t h e l i v e r . M a g n i t u d e o f r e d u c t i o n i n se rum a l b u m i n i s , h o w e v e r , o f t h e same o r d e r i n b o t h t h e g r o u p s a t t h e end o f 7 to 8 weeks (50). All these studies strongly point to the link between n a t u r e o f t h e c a r b o h y d r a t e , i n s u l i n s e c r e t i o n , i n c r e s a s e d a c t i v e Na + r e a b s o r p t i o n i n t h e k i d n e y ( o f w h i c h Na,K ATPase i s a b i o c h e m i c a l r e f l e c t i o n ) and edema o f p r o t e i n - e n e r g y m a l n u t r i t i o n .

I n f a c t , i n t r a c e l l u l a r a c c u m u l a t i o n o f t h e Na + a c t s as a v e r y p o t e n t s t i m u l u s f o r Ha,K ATPase. E r y t h r o c y t e s f rom m a r a s m i c c h i l d r e n h a v e s i g n i f i c a n t a c c u m u l a t i o n o f c e l l Na + ( 2 1 ) . Then , i s t h e r e a c h a n c e t h a t t h o s e m a r a s m i c c h i l d r e n who e n h a n c e t h e i r Na + ' pump ' d e v e l o p edema and t u r n i n t o k w a s h i o r k o r ? T h i s , h o w e v e r , i s s p e c u l a t i v e s i n c e n o t e v e r y k w a s h i o r k o r c h i l d goes t h r o u g h a m a r a s m i c c o n d i t i o n . The s i t u a t i o n i s c e r t a i n l y more complex t h a n one would be i n c l i n e d t o v i s u a l i z e .

T h e s e o b s e r v a t i o n s on t r a n s p o r t and m i t o c h o n d r i a l r e s p i r a t o r y c h a n g e s l e n d t h e m s e l v e s t o a c e r t a i n p o s s i b l e s e q u e n c e o f m e t a b o l i c e v e n t s w h i c h may fo rm t h e b i o c h e m i c a l b a s i s f o r t h e p a t h o g e n e s i s o f p r o t e i n - e n e r g y m a l n u t r i t i o n .

P o s s i b l e S e q u e n c e o f M e t a b o l i c E v e n t s i n P r o t e i n - E n e r g y M a l n u t r i t i o n : A H y p o t h e s i s

The i n c r e a s e d c e l l Ha i n m a r a s m i c s u b j e c t s (21) r a i s e s t h e p o s s i b l i t y t h a t a c c u m u l a t i o n o f c e l l Na c o u l d be one o f t h e p r i m a r y e v e n t s i n t h e e v o l u t i o n o f edema o f k w a s h i o r k o r . Though m e c h a n i s m of t h i s c e l l Ha a c c u m u l a t i o n i s n o t y e t c l e a r , an i n i t i a l d e p l e t i o n o f c e l l e n e r g y c a u s i n g s l u g g i s h Ha + ' pump ' c o u l d be one o f t h e p o s s i b i l i t i e s . S i n c e t h i s w i l l be d e t r i m e n t a l t o c e l l s u r v i v a l , Ha + ' pump ' w i l l be s t i m u l a t e d (by one o f t h e m e c h a n i s m s d i s c u s s e d a b o v e ) t o p r e v e n t c o n t i n u e d a c c u m u l a t i o n o f c e l l Na +. I t i s b e l i e v e d t h a t a l i v i n g c e l l a d a p t s t o a s t r e s s s i t u a t i o n by r e s p o n d i n g t o e n s u r e i t s i m m e d i a t e s u r v i v a l . The l o n g - t e r m c o n s e q u e n c e s o f s u c h an a d a p t i v e m e c h a n i s m may o r may n o t be c o n d u c i v e t o i t s p e r m a n e n t h e a l t h y s u r v i v a l . E n h a n c e m e n t o f Na + ' p u m p ' i n p r o t e i n - e n e r g y m a l n u t r i t i o n a p p e a r s


to be one such adaptation. This mechanism, while relieving the cell of Na +, will cause edema and also impose additional demands on cell energy. This will require generation of more &TP by mltochondrla. Since it will not be p o s s i b l e f o r a c e l l i n p r o t e i n - e n e r g y m a l n u t r i t i o n to a c h i e v e t h i s i n c r e a s e i n t h e r a t e o f r e s p i r a t i o n by s y n t h e s i z i n g more m i t o c h o n d r i a l a s s e m b l i e s , t h i s w i l l h a v e t o he a c h i e v e d by f i n e r r e g u l a t o r y m e c h a n i s m s . E x t e n s i v e s t u d i e s h a v e b e e n c a r r i e d o u t by W i l s o n and h i s a s s o c i a t e s on t h e c o n t r o l o f m i t o c h o n d r i a l r e s p i r a t i o n and t h e s u b j e c t ha s b e e n r e c e n t l y r e v i e w e d ( 5 1 ) . C y t o s o l i c p h o s p h o r y l a t i o n p o t e n t i a l [ATP] / [ADP][P i ] h a s been shown to bear an inverse relationship to mitochondrial r e s p i r a t i o n ( 5 2 ) . I n a c e l l w i t h e n h a n c e d Na,K ATPase , t h e r e w i l l be i n c r e a s e d g e n e r a t i o n o f ADP and P i and more d e p l e t i o n o f ATP. T h i s w i l l r e s u l t i n l o w e r i n g o f p h o s p h o r y l a t i o n p o t e n t i a l and may p r o v i d e a t r i g g e r f o r m i t o c h o n d r i a l r e s p i r a t i o n . T h i s i n d e e d seems t o be t h e c a s e i n k i d n e y m i t o c h o n d r i a f rom p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s . I n c r e a s e d m i t o c h o n d r i a l r e s p i r a t i o n i n k i d n e y i s a s s o c i a t e d w i t h i n c r e a s e i n t h e K m ( a p p a r e n t ) f o r ADP as w e l l as P i ( 4 1 ) , m o s t l i k e l y an i n d i c a t o r o f i n c r e a s e d a v a i l a b i l i t y o f ADP as w e l l as P i i n t h e c y t o s o l .

E n e r g y c h a r g e ( 1 / 2 [ADP] + [ATP]/[AMP] + [ADP] + [ATP]) i s a n o t h e r r e g u l a t o r o f ATP s y n t h e s i s by m i t o c h o n d r i a . N o r m a l l y , e n e r g y c h a r g e i s m a i n t a i n e d a r o u n d 1 i n a c e l l . However , i n t h e e v e n t o f s e v e r e e n e r g y d e p l e t i o n , e n e r g y c h a r g e f a l l s . U s i n g human s p l e e n l y m p h o b l a s t c e l l c u l t u r e s , i t h a s b e e n r e c e n t l y shown t h a t a f a l l i n e n e r g y c h a r g e b e l o w 0 . 6 s t i m u l a t e s c y t o p l a s m i c AMP d e a m i n a s e and 5 ' n u c l e o t i d a s e . T h i s r e s u l t s i n r a p i d d e g r a d a t i o n o f AMP and e n e r g y c h a r g e s t a r t s r e c o v e r i n g ( 5 3 ) . T h i s t h e n e s t a b l i s h e s a new e n e r g y b a l a n c e f o r t h e c e l l u n d e r c o n d i t i o n s o f r a p i d e n e r g y u t i l i z a t i o n .

From t h e work i n human h e p a t o m a s and r e g e n e r a t i n g r a t l i v e r , y e t a n o t h e r m e c h a n i s m f o r i n c r e a s e d ATP p r o d u c t i o n i s o b s e r v e d ( 5 4 ) . T h e s e a u t h o r s s u g g e s t t h a t i n c r e a s e d m i t o c h o n d r i a l r e s p i r a t i o n i s i n d i c a t i v e o f d e l i c a t e e n e r g y b a l a n c e i n a c e l l . I n t h e i r s t u d i e s , i n c r e a s e d m i t o c h o n d r i a l r e s p i r a t i o n i s a s s o c i a t e d with i n c r e a s e d c y t o c h r o m e ( C + C l ) / c y t o c h r o m e ( a+a 3) r a t i o ( 5 4 ) . I n a s t r i k i n g l y s i m i l a r s i t u a t i o n , i t i s s i g n i f i c a n t t h a t i n c r e a s e d l i v e r m i t o c h o n d r i a l r e s p i r a t i o n i n p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s i s a s s o c i a t e d w i t h a 50~ i n c r e a s e i n t h i s r a t i o o f c y t o c h r o m e s ( 5 5 ) . However , t h i s does n o t a p p e a r t o be t h e c a s e i n t h e k i d n e y m i t o c h o n d r i a f r o m t h e s e r a t s s i n c e no s u c h a l t e r a t i o n i n t h e c y t o c h r o m e p r o f i l e h a s b e e n o b s e r v e d ( S e m i u d d i n and K a p l a y , u n p u b l i s h e d ) . K idney m i t o c h o n d r i a seems t o h a v e r e s p o n d e d t o a l t e r e d ADP and Pi i n t h e c y t o s o l by i n c r e a s i n g t h e K m v a l u e s f o r t h e s e m e t a b o l i t e s ( 4 0 ) . I f some o f t h e s e f i n e r e g u l a t o r y m e c h a n i s m s were t o be o p e r a t i v e i n p r o t e i n - e n e r g y m a l n u t r i t i o n , t h e n i t i s n o t d i f f i c u l t t o v i s u a l i z e how t h e s e c e l l s i n c r e a s e e n e r g y g e n e r a t i o n d e s p i t e t h e i r d e p l e t e d r e s o u r c e s .

W h a t e v e r i s t h e m e c h a n i s m u s e d t o g e n e r a t e more e n e r g y t o mee t t h e i n c r e a s e d demands f rom e n h a n c e d Na + ' p u m p ' , t h i s e x t r a d r a i n on e n e r g y w i l l d e p l e t e t h e c e l l much f a s t e r o f i t s l i m i t e d r e s o u r c e s and may t h u s fo rm t h e b a s i s f o r p a t h o g e n e s i s of p r o t e i n - e n e r g y m a l n u t r i t i o n .

P o s s i b l e C o n s e q u e n c e s o f Membrane A b n o r m a l i t i e s : A S p e c u l a t i o n

The membrane a b n o r m a l i t i e s f o u n d t o o c c u r i n p r o t e i n - e n e r g y m a l n u t r i t i o n a r e l i k e l y t o be o f f a r - r e a c h i n g c o n s e q u e n c e t o some o f t h e c r i t i c a l c e l l u l a r f u n c t i o n s . Two o f them d e s e r v e s p e c i a l m e n t i o n .

T h e r e i s v o l u m i n o u s i n f o r m a t i o n on t h e complex e n d o c r i n e p r o f i l e t h a t emerges i n p r o t e i n - e n e r g y m a l n u t r i t i o n . I t i s known t h a t d i e t a r y m o d i f i -

944 S.S. KAPLAY

c a t i o n o f a b i o l o g i c a l membrane a l t e r s i t s r e s p o n s i v e n e s s t o ho rmones ( 5 6 ) . I n t h a t c a s e , i t w i l l n o t o n l y be o f a c a d e m i c i n t e r e s t b u t a l s o o f p r a c t i c a l r e l e v a n c e t o l e a r n how a c e l l f r om a p r o t e i n - e n e r g y m a l n o u r i s h e d c h i l d r e s p o n d s t o d i f f e r e n t h o r m o n e s .

A n o t h e r a r e a o f i m m e d i a t e r e l e v a n c e t o t h e s e a b n o r m a l i t i e s i s t h e i m m u n o l o g i c a l s t u d i e s i n t h e s e p a t i e n t s . I t h a s b e e n amply d e m o n s t r a t e d t h a t i m m u n o l o g i c a l f u n c t i o n i s i m p a i r e d i n p r o t e i n - e n e r g y m a l n u t r i t i o n . One o f t h e e a r l i e s t e v e n t s i n l y m p h o c y t e t r a n s f o r m a t i o n on s t i m u l a t i o n by p h y t o - h e m a g g l u t i n i n (PHA) and C o n c o v a l i n A i s t h e e n h a n c e m e n t o f Na + 'pump* by i n c r e a s i n g t h e o u a b a i n s i t e s ( 5 7 ) . I f Na + ' pump ' s i t e s i n t h e p r o t e i n - e n e r g y m a l n o u r i s h e d c e l l s a r e e i t h e r i n c r e a s e d i n a b s o l u t e number o r due t o e x p o s u r e o f e x i s t i n g s i t e s due t o membrane m o d i f i c a t i o n , as i s s e e n i n t h e e r y t h r o c y t e membrane ( 3 2 ) , l e u c o c y t e s ( 2 6 ) , and k i d n e y ( 3 0 ) , t h e n t h e i r r e s p o n s e t o PHA o r Con A w i l l n o t be l i k e i n t h e n o r m a l c e l l and may fo rm t h e b a s i s o f i m m u n o l o g i c a l d e f e c t s i n t h e s e p a t i e n t s . Such a p o s s i b i l i t y i s s t r e n g t h e n e d by t h e e x p e r i m e n t s i n k i d n e y m i c r o s o m a l p r e p a r a t i o n s f r o m p r o t e i n - e n e r g y m a l n o u r i s h e d r a t s , whe re i t i s n o t p o s s i b l e t o b r i n g o u t a f u r t h e r i n c r e a s e i n t h e number o f ' p u m p ' s i t e s on d e o x y c h o l a t e t r e a t m e n t l i k e i n t h e c a s e o f membranes f rom c o n t r o l r a t s ( 3 0 ) .

Studies to understand the biochemical basis of cell injury in one human disorder could also find usefulness in understanding the response of an organism to other stress conditions, at least to a limited extent. The complex nature of protein-energy malnutrition continues to evade its complete understanding. Efforts to understand this enigmatic disorder, however, will have to continue since in this could be an answer to the improved under- s t a n d i n g o f a l i v i n g c e l l .

ACKNOWLEDGEMENTS

My t h a n k s a r e due t o Dr s . S. G. S r i k a n t i a and B. S. N a r a s i n g a Rao f o r t h e i r i n t e r e s t . T h i s work would n o t h a v e b e e n p o s s i b l e w i t h o u t t h e a v a i l a b i l i t y o f c l i n i c a l m a t e r i a l p r o v i d e d by D r s . V i n o d i n i Reddy and P. B h a s k a r a m . C r i t i c a l reading of the manuscript by Drs. J. B. Hughes, Saro~ Joshi and Mira Kaplay is highly appreciated. Mr. S. Ranganathan and Mrs. Meenakshi Subramaniam p r o v i d e d e x c e l l e n t t e c h n i c a l a s s i s t a n c e d u r i n g t h i s work .

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Accepted for publication July 2, 1984.

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Na+ ‘pump’ and cell energetics in protein-energy malnutrition