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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., University of 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 in the D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia A p r i l , 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department The University of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT The purpose of the present investigation was to study the e f f e c t of prolonged p r o t e i n - c a l o r i e malnutrition on i n t e s t i n a l disaccharidase a c t i v i t i e s and on disaccharide absorption, as carbohydrate intolerance is a major problem in children s u f f e r -ing from p r o t e i n - c a l o r i e malnutrition. Four groups of rats (90 to 120 grams) were fed the f o l -lowing d i e t s for 8 to 9 weeks: control (18% lactalbumin, 66% carbohydrate); low protein low carbohydrate (0.5% l a c -talbumin, 66% carbohydrate); low protein high carbohydrate (0.5% lactalbumin, 83.5% carbohydrate); and low protein r e s t r i c t e d (1% lactalbumin, r e s t r i c t e d to 4 grams per day). After 8.5 weeks, part of the group on the 0.5% lactalbumin low carbohydrate d i e t was fed the control d i e t (18% l a c t a l -bumin, 66% carbohydrate) for 8 weeks. At the end of the feeding period, the following assays were performed: 1) iri vivo absorption of radioactive ("*"4C) lactose, sucrose, and maltose; 2) a c t i v i t i e s of i n t e s t i n a l lactase, sucrase, and maltase; 3) plasma albumin concentra-tions; and 4) mucosal protein concentrations. In the three protein d e f i c i e n t groups (0.5% lactalbumin low carbohydrate, 0.5% lactalbumin high carbohydrate, and 1% lactalbumin), the a c t i v i t y of both the jejunal and i l e a l disaccharidases and the absorption of lactose, sucrose and maltose were s i g n i f i c a n t l y higher when compared with the control s . The jejunal 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 in the 0.5% lactalbumin high carbo-i i hydrate group than in the 0.5% lactalbumin low carbohydrate group, but the absorption of lactose, sucrose and maltose were a l i k e . When the 1% lactalbumin ( r e s t r i c t e d to 4 grams per day) and the 0.5% lactalbumin 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 differences in the s p e c i f i c a c t i v i t i e s of the i n t e s t i n a l disaccharidases or the absorption of the disaccharides. The absorption of lactose, sucrose, or maltose were si m i l a r in the controls and the protein repleted group. The disac-charidase a c t i v i t i e s were also s i m i l a r in these two groups except for a s i g n i f i c a n t depression of jejunal maltase and i l e a l sucrase and maltase a c t i v i t i e s in the protein repleted group. Therefore, these results indicate that protein depriva-t i o n in rats for 8.5 weeks causes an increase in s p e c i f i c a c t i v i t i e s of the i n t e s t i n a l disaccharidases in both the jejunum and ileum, and that these changes caused by protein depletion may be reversed by feeding a d i e t high i n pr o t e i n . Also, an increase in the carbohydrate content of the protein d e f i c i e n t d i e t results in an induction of jejunal sucrase and maltase a c t i v i t i e s . The high s p e c i f i c a c t i v i t y of the i n t e s t i n a l disaccharidases following p r o t e i n - c a l o r i e malnutrition may be in part due to a p r e f e r e n t i a l loss of s t r u c t u r a l proteins rather than to an increase in enzymatic protein in the i n t e s t i n a l mucosa. The increase in the disaccharidase a c t i v i t i e s in the protein d e f i c i e n t rats i s accompanied by an increase in di s a c -charide absorption which could be due to the higher levels of disaccharidases or to an increase in the transport of the constituent 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 differences in sucrase and maltase a c t i v i t i e s between the 0.5% lactalbumin high carbohydrate and the 0.5% lactalbumin low carbohydrate groups without a con-comitant increase in sucrose and maltose absorption, supports the view that the higher absorption of sucrose and maltose in the protein d e f i c i e n t rats is a r e s u l t of increased monosac-charide transport. The r e s u l t s of t h i s study are not consistent with the suggestion that p r o t e i n - c a l o r i e malnutrition is responsible for disaccharide intolerance in c h i l d r e n . ACKNOWLEDGEMENT The author would l i k e to sincerely thank her advisor, Dr. J . Leichter, for his d i r e c t i o n and help throughout the study; Dr. M. Lee and Dr. J.C. Brown for t h e i r advice in the preparation of the manuscript; and Miss U s e Borgen for her kind assistance in the laboratory. This study was supported by Grant No. A6249 from the National Research Council of Canada. V TABLE OF CONTENTS CHAPTER PAGE I LITERATURE REVIEW 1 A. Introduction 1 B. Digestion and Absorption of Carbo-hydrates 2 1. Review of digestion and absorp-t i o n of carbohydrates 2 v 2. Location of the i n t e s t i n a l disaccharidases 3 3. Changes in i n t e s t i n a l d i s a c -charidase a c t i v i t i e s with age.... 5 4 . E f f e c t of dietary carbohydrate on a c t i v i t y l e v e l s of i n t e s t i n a l disaccharidases 5 C. Protein-Calorie Malnutrition and Carbohydrate Absorption in Man........ 6 1. The e f f e c t of protein-calorie malnutrition on the i n t e s t i n a l mucosa in man 6 2. The e f f e c t of pr o t e i n - c a l o r i e malnutrition on the i n t e s t i n a l disaccharidases in man 7 3. The e f f e c t of pr o t e i n - c a l o r i e malnutrition on disaccharide absorption i n man 7 4 . The r e l a t i o n s h i p between lactose intolerance in pr o t e i n - c a l o r i e malnutrition and the incidence of primary lactase deficiency 9 5. The e f f e c t of infections 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 of the i n t e s t i n a l disaccharidases 10 v i D. Protein-Calorie Malnutrition and Carbohydrate Absorption in Laboratory Animals 12 1. The e f f e c t of prot e i n - c a l o r i e malnutrition on the i n t e s t i n a l mucosa i n laboratory animals 12 2. The e f f e c t of prot e i n - c a l o r i e malnutrition on i n t e s t i n a l d i sac-char idases i n laboratory animals. 14 3. The e f f e c t of pr o t e i n - c a l o r i e malnutrition on absorption of disaccharides in laboratory animals..... 18 4. The e f f e c t of non-protein factors on the a c t i v i t y of the i n t e s t i n a l disaccharidases i n laboratory animals.... 19 E. Objectives of the Present Study 21 II MATERIALS AND METHODS 23 A. Animals and Diets 23 B. Absorption of Disaccharides 25 C. Disaccharidase A c t i v i t i e s 27 D. Other Assays 29 1. Plasma albumin concentration 29 2. Mucosal protein determination 29 E. Materials 29 III RESULTS 31 A. Appearance, Body weight, and Plasma Albumin concentration 31 B. I n t e s t i n a l Disaccharidase A c t i v i t i e s . . . . 39 C. I n t e s t i n a l Absorption of Disaccharides in Vivo 46 v i i IV DISCUSSION 51 V SUMMARY AND RECOMMENDATIONS 62 A. Summary 62 B. Recommendations 65 VI BIBLIOGRAPHY 67 v i i i LIST OP TABLES TABLE PAGE I Digestion and Absorption of Carbohydrates.... 4 II Composition of Diets 24 III I n i t i a l and F i n a l Body Weights of the Survivors in Each Dietary Group 3 4 IV Percentage of Survivors on the Different Dietary Regimens... 38 V Plasma Albumin Concentration of Control, Protein Depleted, and Protein Repleted Rats.. 40 VI Lactase A c t i v i t i e s i n Jejunum and Ileum of Control, Protein Depleted and Protein Repleted Rats 41 VII Sucrase A c t i v i t i e s in Jejunum and Ileum of Control, Protein Depleted, and Protein Repleted Rats 42 VIII Maltase A c t i v i t i e s in Jejunum and Ileum of Control, Protein Depleted, and Protein Repleted Rats 43 IX Mucosal Protein Concentration i n Jejunum and Ileum of Control, Protein Depleted, and Protein Repleted Rats 47 X Lactose, Sucrose, and Maltose Absorption in Control, Protein Depleted, and Protein Repleted Rats 48 ix LIST OP FIGURES FIGURE PAGE 1. Changes in Body Weight of Control, Protein Depleted, and Protein Repleted Rats 33 2. Typical Appearance of Rats on the Following Three d i e t s : 1% Lactalbumin, Restricted to 4 g per Day (Left); 18% Lactalbumin (Center); and 0.5% L a c t a l -bumin High Carbohydrate (Right) 36 3. A Close-up of the Rat on the 0.5% L a c t a l -bumin High Carbohydrate Diet Shown in Figure 2 37 1 CHAPTER I LITERATURE REVIEW A. Introduction Protein-calorie malnutrition is s t i l l one of the major problems in developing countries. In children, i t has been c l e a r l y shown to be associated with depressed a c t i v i t i e s of i n t e s t i n a l disaccharidases (1) which i s probably a consequence of a l t e r a t i o n s of i n t e s t i n a l structure (2). Bowie et a l . (3) noted that 60% of children with pro t e i n - c a l o r i e malnutrition suffered from malabsorption of carbohydrates. However, i t i s not clear whether the disaccharide intolerance and diminished disaccharidase a c t i v i t i e s are primarily due to protein depriv-ation, or to 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 d e f i c i e n c i e s of nutrients other than protein. In order to c l a r i f y t h i s problem of carbohydrate i n t o l -erance in p r o t e i n - c a l o r i e deficiency, a number of i n v e s t i g a -t o r s have studied the e f f e c t of protein d e f i c i e n t diets on i n t e s t i n a l disaccharidase a c t i v i t i e s in young rats ( 4,5). The r e s u l t s obtained in those studies were su r p r i s i n g because protein deprivation i n rats produced an increase in the s p e c i f i c a c t i v i t i e s of disaccharidases and p a r t i c u l a r l y of maltase and sucrase. I t has been suggested, therefore, that t h i s increase i n disaccharidase a c t i v i t i e s in rats was an adaptation to the high proportion of carbohydrate in the protein d e f i c i e n t diets since most of the investigators 2 replaced the protein portion of the control d i e t with carbo-hydrate. I t should also be pointed out that in the experiments designed to show the e f f e c t of protein d e f i c i e n t d iets on i n t e s t i n a l disaccharidase a c t i v i t e s , the experimental animals have not been kept on the protein d e f i c i e n t diets long enough to develop edema which i s so c h a r a c t e r i s t i c of the f u l l kwashiorkor syndrome in children ( 4 , 5 , 6 ) . Almost a l l of the studies on the e f f e c t of protein mal-n u t r i t i o n on carbohydrate absorption reported in the l i t e r a -ture determined changes in i n t e s t i n a l disaccharidase a c t i v i t i e s without measuring the absorption of disaccharides. Depressed l e v e l s of disaccharidases can be implicated as being func-t i o n a l l y s i g n i f i c a n t only i f variations in disaccharidase leve l s are r e f l e c t e d in changes i n disaccharide absorption as well ( 7 ) . Also, i t is important to study the absorption of the disaccharides by an in_ vivo technique as procedures which separate the intestine from the animal create a condition in which the blood vessels are no longer present to carry away the absorbed material. With the preceding ideas in mind, i t seemed necessary to further study the e f f e c t of long-term p r o t e i n - c a l o r i e deprivation on i n t e s t i n a l disaccharidase a c t i v i t i e s and on in vivo absorption of disaccharides. B. Digestion and Absorption of Carbohydrates 1. Review of digestion and absorption of carbohydrates. Carbohydrates are ingested in complex forms which must be broken down to t h e i r simpler components before they can be 3 absorbed. The process of digestion and absorption of dietary carbohydrates i s shown in Table I. The digestion of carbo-hydrates occurs in the lumen of the small intestine with the action of amylase on the polysaccharides, amylose and amylo-pectin, and at the surface of the mucosal c e l l s of the small intestine with the action of the disaccharidases (lactase, sucrase, and maltase) on the disaccharides (lactose, sucrose, and maltose). The f i n a l products of disaccharide hydrolysis are the monosaccharides: glucose, fructose, and galactose. The monosaccharides are then transported across the mucosal membrane by either active transport, as i n the case of glucose and galactose, 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 of fructose (8). When the complete process of disaccharide hydrolysis and monosaccharide absorption were studied in man, the action of lactase was found to be the r a t e - l i m i t i n g step in the absorption of lactose (9), whereas the mucosal trans-fer of glucose was the r a t e - l i m i t i n g step in sucrose and maltose absorption (10,11). 2. Location of the i n t e s t i n a l disaccharidases. The a c t i v i t i e s of i n t e s t i n a l disaccharidases, lactase, sucrase, and maltase, are not uniformly d i s t r i b u t e d along the length of the small intestine with the highest a c t i v i t y being found in the jejunal section and the lowest a c t i v i t y being found in the i l e a l section i n rats (12), pigs (13), and man (14). The lactase, sucrase and maltase a c t i v i t i e s are located in the brush border of the mucosa (15) with most of the a c t i v i t y being found along the sides of the v i l l i and not in the crypts (16). The a c t i v i t i e s of sucrase and maltase TABLE I* DIGESTION AND ABSORPTION OF CARBOHYDRATES CARBOHYDRATE SITES OF DIGESTION Intraluminal Muscosal ABSORPTION (MODE OF TRANSPORT) Starch amylose amylopectin Sucrose Lactose amylase maltose maltotriose maltose amylase maltase glucose ^ maltotriose alpha dextr inase alpha dextr ins -> glucose sucrase -> lactase glucose fructose glucose 'galactose active active active f a c i l i t a t e d d i f f u -sion active active *Taken from: Ward A. Olsen, "Carbohydrate Digestion and Absorption," Post Graduate Medicine, 51:149-152, 1972. 5 have been associated with small knobs on the outer edge of the m i c r o v i l l i (17,18). 3. Changes i n i n t e s t i n a l disaccharidase a c t i v i t i e s with  age. At the time of b i r t h in the r a t , the l e v e l s of lactase a c t i v i t y are high and decrease during the immediate post weaning period to the low levels c h a r a c t e r i s t i c of the adult animal. Sucrase and maltase a c t i v i t i e s are low at b i r t h , and by 4 to 5 weeks of age, increase to le v e l s found in adult rats (19,20). In man, sucrase and maltase a c t i v i t i e s (except for maltase I) at b i r t h are the same as in adult l i f e (14). In the case of lactase, the l e v e l of a c t i v i t y found at b i r t h may ei t h e r remain the same throughout the l i f e of the in d i v i d u a l or decrease within the f i r s t decade of l i f e to a lower l e v e l of a c t i v i t y which would then be permanent. The populations in which a decrease in lactase a c t i v i t y a f t e r e a r l y childhood has been shown to be very prevalent are l i s t e d by Herber (14). 4. E f f e c t of dietary carbohydrate on a c t i v i t y l e v e l s of i n t e s t i n a l disaccharidases. The a c t i v i t y of the i n t e s t i n a l disaccharidases can be influenced by the carbohydrate content of the d i e t . I t has been demonstrated that a f t e r a 3 day fast, a 70% sucrose or maltose d i e t causes increased jejunal sucrase and maltase a c t i v i t i e s in the r a t when compared to animals fed a carbohydrate-free d i e t a f t e r the fast (7,21). In the case of lactase, some researchers have found an increase i n i n t e s t i n a l lactase a c t i v i t y i n rats in response to high l e v e l s of lactose in the d i e t a f t e r weaning, (20, 6 22,23) while others were not able to confirm these findings (12,19,24). Studies done on the e f f e c t of dietary carbohydrate on the a c t i v i t i e s of i n t e s t i n a l disaccharidases i n man (25,26) have shown that an increase in dietary sucrose and fructose r e s u l t s , a f t e r 3 to 5 days, in s i g n i f i c a n t increases in sucrase and maltase a c t i v i t i e s . When subjects were given d i e t s containing 0 to 80% sucrose or glucose, jejunal sucrase and maltase a c t i v i t i e s increased in the subjects receiving the sucrose or glucose in the d i e t as compared to the carbo-hydrate-free group (27). The increases in sucrase, and maltase a c t i v i t i e s were proportional to the l e v e l s of sucrose and glucose i n the d i e t . C. Protein-Calorie Malnutrition and Carbohydrate Absorption  in Man 1. The e f f e c t of p r o t e i n - c a l o r i e malnutrition on the  i n t e s t i n a l mucosa in man. In p r o t e i n - c a l o r i e malnutrition, the changes that take place in the mucosal tissue of the g a s t r o i n t e s t i n a l t r a c t include thinning of the i n t e s t i n a l w all and mucosa; broadening and atrophy of the v i l l i ; increased c e l l u l a r i t y of the lamina propria; and decreased height of the e p i t h e l i a l c e l l s . There is also the presence of lymphocytic 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 on the surface of the v i l l i (28). These changes have been noted in 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 malnutrition by Stanfield et a l . (2), Burman (29), Barbezat et a l . (30), 7 Brunser et a l . (31), and Berkel et a l . (32) and i n mal-nourished adults by Tandon et a l . (33), and Mayoral et a l . (34,35) . The renewal of the i n t e s t i n a l mucosa as expressed by the mitotic index (which is the percentage of the e p i t h e l i a l c e l l s undergoing mitosis in the crypt of Lieberkuhn (36) has been shown to be lower for marasmic patients than in kwash-iorkor patients and both are lower than i n healthy i n d i v i d -uals (31,36). 2. The e f f e c t of pro t e i n - c a l o r i e malnutrition on the  i n t e s t i n a l disaccharidases i n man. The r e s u l t s of studies on the a c t i v i t y of i n t e s t i n a l disaccharidases in children s u f f e r -ing from protein-calorie malnutrition in various areas of the world including Uganda (2), South A f r i c a (30), and Turkey (32) have demonstrated s i g n i f i c a n t l y lower levels of the three disaccharidases, lactase, sucrase, and maltase, when compared to the lev e l s of a c t i v i t y for these enzymes i n healthy children of s i m i l a r age. James (37) studied Jamaican children with kwashiorkor and marasmus and found that 8 of the 19 c h i l d r e n had decreased lactase, sucrase, and maltase a c t i v i t i e s in the jejunal biopsy specimens. The childr e n diagnosed as marasmic had higher lev e l s of lactase, sucrase, and maltase a c t i v i t i e s than the children diagnosed as marasmic-kwashiorkor or kwashiorkor. 3. The e f f e c t of pro t e i n - c a l o r i e malnutrition on d i s - acchar ide absorption in man. Bowie et a l . (38) proposed that the diarrhea, which i s often a serious complication of 8 p r o t e i n - c a l o r i e malnutrition, i s caused by intolerance to disaccharides. To t e s t t h i s hypothesis, they removed carbo-hydrates from the d i e t of 13 malnourished African children suffering from diarrhea. After 3 days on the carbohydrate-free d i e t , there was a s i g n i f i c a n t decrease in sto o l weight and a decrease i n f e c a l l a c t i c acid in 9 of the 13 c h i l d r e n . Lactose, glucose/galactose, or glucose tolerance tests performed on 3 of the children showed an intolerance to lactose but not to the glucose/galactose mixture or glucose alone. Further studies by Bowie et a l . (3,39) of malnourished childre n with diarrhea showed that the use of a carbohydrate-f r e e formula corrected the diarrhea in 60 to 65% of the c h i l d r e n . The children who improved on the carbohydrate-free d i e t demonstrated an intolerance to a lactose load but not to an equivalent load of either sucrose, maltose or glucose plus galactose. Lactase a c t i v i t y in the jejunal biopsy samples was below normal in 8 out of 20 malnourished children, suc-rase a c t i v i t y was below normal in 3, and maltase a c t i v i t y was below normal in one. Prinsloo e t a l . (40) studied the e f f e c t s of various dietary monosaccharides and disaccharides in the co n t r o l of diarrhea i n children with pr o t e i n - c a l o r i e malnutrition. They divided 120 malnourished Bantu children into 6 groups and gave each group a d i f f e r e n t source of dietary carbohydrate in t h e i r formula. Five of the groups were given the same basic formula except that the carbohydrate source was either 9 lactose, glucose, sucrose, dextrin-maltose, or carbohydrate-f r e e . The s i x t h group of children was fed a full-cream powdered milk formula. The children on the formula with lactose or the full-cream powdered milk suffered the most severe diarrhea and had the highest l a c t i c acid excretion i n the s t o o l s . The severity of the diarrhea was about the same i n the groups of children with glucose, sucrose, or dextrin-maltose in their diets and the carbohydrate-free group. The a c t i v i t i e s of i n t e s t i n a l lactase, sucrase, and maltase for the malnourished children in these studies by Prinsloo et a l . (40) were lower than those for normal children with lactase a c t i v i t y being the most affected. James (41,42,43) studied malnourished Jamaican infants using an i n t e s t i n a l perfusion technique, and demonstrated a generalized defect in glucose, sucrose, and lactose absorp-t i o n . 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 rate of hydrolysis of the disaccharides and the l e v e l of disaccharidase a c t i v i t y in the jejunal mucosa. Treatment for 2 to 4 months with a high protein d i e t resulted in an increase in the absorption of glucose, sucrose, and lactose. 4. The re l a t i o n s h i p between lactose intolerance in  prot e i n - c a l o r i e malnutrition and the incidence of primary  lactase d e f i c i e n c y . Lactose intolerance i n adults due to primary lactase deficiency e x i s t s with varying frequency in d i f f i c u l t ethnic groups as reviewed by Herber (14), and many of the population groups with high prevalence of primary lactase deficiency are populations i n which pr o t e i n - c a l o r i e 10 malnutrition i s also prevalent. Wharton et a l . (44) and Cook e t a l . (45) studied the incidence of disaccharide intolerance in the Ugandan Bantu population and suggested that the i n t o l -erance to lactose which did not improve with the treatment f o r p r o t e i n - c a l o r i e malnutrition may be due to the underlying genetic deficiency of lactase a c t i v i t y . The presence of hereditary lactase deficiency which was v e r i f i e d by family h i s t o r y was reported from India by Chandra et a l . (46). Of the 100 infants studied, 50% had low pH (<pH 6.0) s t o o l s . When lactose, sucrose, and maltose t o l e r -ance tests were performed on those 50 children, 39 were intolerant to lactose, 25 to sucrose, and 16 to maltose. After treatment with a high protein d i e t for 3 months, 4 of the 50 c h i l d r e n were s t i l l intolerant to lactose and were considered to have primary lactase deficiency. Therefore, i t has been well documented that disaccharide intolerance and depression of disaccharidase a c t i v i t i e s i s a problem for man^f children who are suffering from p r o t e i n -c a l o r i e malnutrition. However, i t is not c l e a r whether p r o t e i n - c a l o r i e malnutrition is the cause of these problems. 5. The e f f e c t of infections and n u t r i t i o n a l d e f i c i e n c - ies on the a c t i v i t y of the i n t e s t i n a l disaccharidases. Protein deficiency usually does not occur without 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 without the complica-tion of an accompanying i n f e c t i o n . In children with p r o t e i n -c a l o r i e malnutrition, admission to the hospital may be the r e s u l t of an acute episode of diarrhea of unknown cause. 11 James (43) concluded from his survey on the e f f e c t s of protei n - c a l o r i e malnutrition on i n t e s t i n a l absorption that the relevant importance of acute infections or of malnutri-t i o n i t s e l f on the changes which occur in the i n t e s t i n a l function and structure have not been determined. In the study by Barbezat et a l . (30), there was some c o r r e l a t i o n between the presence of Giardia Iambiia i n f e s t a t i o n and the depression of lactase and sucrase a c t i v i t i e s . In the studies by Prinsloo et a l . (40) and Bowie et a l . (3,39), however, there was no c o r r e l a t i o n between the presence of pathogens and the sev e r i t y of the diarrhea. Mata et a l . (47) reported that the studies performed at the c l i n i c a l unit at INCAP (Institute of N u t r i t i o n of Central America and Panama) on 13 childre n with kwashiorkor or marasmus showed that there was more bac t e r i a in the stomach and jejunum of malnourished children with diarrhea than i n malnourished children who were not su f f e r i n g from diarrhea. A decrease in i n t e s t i n a l disaccharidase a c t i v i t i e s may occur due to damage of the i n t e s t i n a l mucosa caused by i n f e s t a t i o n . Hookworms are an example of t h i s (48,49). Tripathy et a l . (50) noted abnormal r e s u l t s of D-xylose absorption tests and mucosal damage i n children with heavy Ascaris i n f e c t i o n s . Therefore, the re s u l t s of these studies which have just been discussed show that the role of in f e c t i o n in the etiology of disaccharide intolerance i n prote i n - c a l o r i e malnutrition i s not completely understood and needs to be studied 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 deficiency have been shown to a f f e c t the g a s t r o i n t e s t i n a l t r a c t . When Naiman et a l . (51) studied biopsy samples of 14 infants and children who were iron d e f i c i e n t there was a high incidence of abnormalities of g a s t r o i n t e s t i n a l structure and function. The studies of duodenal biopsy samples showed shortening and clubbing of the v i l l i , and increased c e l l u l a r i n f i l t r a t i o n of the lamina propria. The absorption of xylose was impaired. When the iron deficiency was corrected, the xylose absorption and s t r u c t u r a l abnormalities of the v i l l i returned to normal. Although the i n t e s t i n a l d i s a c -charidases were not studied the findings i n t h i s s p e c i f i c experiment demonstrate that 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 protein may be involved in the damage to the gastro-i n t e s t i n a l structure and g a s t r o i n t e s t i n a l function which i s found in children suffering from pro t e i n - c a l o r i e malnutri-t i o n . D. Protein-Calorie Malnutrition and Carbohydrate Absorption  in Laboratory Animals. Various animals have been used to study the e f f e c t of pro t e i n - c a l o r i e malnutrition on carbohydrate digestion and absorption, including monkeys (52,53), baboons (54), pigs (55), and rats (4,5,6,56,57,58,59,60). 1. The e f f e c t of protein-calorie malnutrition on the  i n t e s t i n a l mucosa in laboratory animals. When the structure of the i n t e s t i n a l mucosa of animals on low protein diets was studied, changes s i m i l a r to those found in children and adults 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 malnutrition have been noted. Deo et a l . (52) working with monkeys, were able to demonstrate atrophy of the mucosa after 8 weeks on a protein-free diet, when compared to monkeys on a control d i e t containing 15 to 20% casein. In the protein d e f i c i e n t monkeys, there was a decreased number of v i l l i per square unit of mucosa and a decrease in the number of c e l l s in the mucosa, however, the shape of the v i l l i did not change. Atrophy of the v i l l i in the monkey afte r 18 weeks on a d i e t containing l e s s than 1% p r o t e i n was also demonstrated by Kumar and Chase (53). Rats placed on a protein-free d i e t for periods up to 5 weeks (61) were found to have thinner i n t e s t i n a l walls and the v i l l i were decreased in s i z e . U l t r a s t r u e t u r a l changes in the e p i t h e l i a l c e l l s included i r r e g u l a r m i c r o v i l l i ; a decrease in the endoplasmic reticulum and RNA granules; and changes i n the mitochondrial structures. H i l l et a l . (62) studied the e f f e c t of protein deprivation on weanling rats and found that the animals which were protein d e f i c i e n t for up to 45 days did not increase the length of t h e i r small intestines and had s i g n i f i c a n t l y shorter v i l l i in the ileum as compared to the controls. The v i l l i in the jejunal seg-ment in the protein d e f i c i e n t animals were thinner but not shorter than in the controls indicating that the endogenous protein in the lumen i s a v a i l a b l e to the jejunal mucosa for r e p a i 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 on  i n t e s t i n a l disaccharidases in laboratory animals. The e f f e c t of protein c a l o r i e malnutrition on i n t e s t i n a l disaccharidases has been studied in laboratory animals in order to c l a r i f y the problem of carbohydrate intolerance i n children s u f f e r -ing from pr o t e i n - c a l o r i e malnutrition. In the animal model, i t i s possible to study the i n d i v i d u a l e f f e c t s of protein deficiency, i n f e c t i o n , and other 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 disaccharidases which cannot be done on human subjects. Solimano et a l . (4) investigated the e f f e c t of protein deficiency on the a c t i v i t y of the jejunal disaccharidases in rats and obtained surprising r e s u l t s . 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 elevated a f t e r the animals had been fed a protein-free or 5% protein d i e t for 22 and 44 days re s p e c t i v e l y . When these rats were then given a protein adequate d i e t for 7 days, the lactase, sucrase and maltase a c t i v i t i e s f e l l from the previously high values to approx-imately the normal l e v e l s found in the controls. The authors suggested that the increase in carbohydrate content in the protein d e f i c i e n t d i e t s may have been responsible for the s i g n i f i c a n t increases i n sucrase and maltase a c t i v i t i e s due t o enzyme induction by the high proportion of carbohydrate in the d i e t . The experimental d i e t s used i n the investigation by Solimano et a l . (4) were based on a complete infant food formula to which cornstarch was added to give f i n a l protein concentrations of 10, 5 and 0.4% in the three d i e t s . Extra vitamins and minerals were not added to the 5% and 0.4% protein diets so that the low protein diets were not complete in a l l the required nutrients. Also the proportion of the carbohydrate in the three diets was not the same. Therefore, the changes in the i n t e s t i n a l disaccharidase a c t i v i t i e s can-not be at t r i b u t e d only to the protein deprivation as the authors noted. Also, the changes in the i n t e s t i n a l d i s -accharidase a c t i v i t i e s were only determined in the jejunum. Therefore, i t is not known whether the e f f e c t of prot e i n -c a l o r i e malnutrition has the same e f f e c t on the ileum. Prosper et a l . (5) investigated the e f f e c t of prot e i n -c a l o r i e malnutrition on i n t e s t i n a l disaccharidases in 22 day old r a t s given synthetic diets containing 27 or 0% prote i n . The increase i n disaccharidase a c t i v i t i e s in rats on the protein-free d i e t was only s t a t i s t i c a l l y s i g n i f i c a n t in the t h i r d quarter of the small intestine when compared to the contro l s . When the animals on the protein-free d i e t were force fed for 20 days so that the c a l o r i c intake would be the same in both the protein-free and the control groups, the only s i g n i f i c a n t differences found were an increase in sucrase a c t i v i t y i n the d i s t a l h a l f of the small intestine and a decrease in lactase a c t i v i t y in the proximal h a l f of the small intestine in the protein-free group. When t o t a l a c t i v i t y of lactase, sucrase, and maltase was determined, the l e v e l s of a c t i v i t y were lower i n the protein-free group than in the control group. When t o t a l enzyme a c t i v i t y was expressed per 100 grams of body weight, there ware no s i g n i f i -cant differences between the two groups which had si m i l a r body weights. The expression of 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 healthy control animals to protein d e f i c i e n t animals as there are probably differences i n body composition between the two groups (63). In the study by Prosper et a l . (5) the disaccharidase a c t i v i t i e s were determined along the entire length of the small intestine so that a more complete view of the e f f e c t of the protein depletion could be observed. However, as in the study by Solimano et a l . (4) the carbohydrate content of the control and protein-free d i e t was not the same. There-fore, the p o s s i b i l i t y that the higher proportion of carbo-hydrate could be influencing the disaccharidase a c t i v i t i e s in the protein-free group s t i l l e x i s t s . In the study by T r o g l i a et a l . (56) rats given a r e -s t r i c t e d intake of rat chow for 5% to 6% months had elevated lactase, sucrase and maltase a c t i v i t i e s when compared to the c o n t r o l groups. There were 3 control groups in t h i s study: one group of controls which was younger than the group with the r e s t r i c t e d intake was fed the r a t chow ad 1ibitum u n t i l the rats reached the f i n a l weight of the r e s t r i c t e d group, 206 grams; the second control group was the same age as the r e s t r i c t e d group and was fed the r a t chow ad_ libitum; and the t h i r d control group was meal fed. When t o t a l i n t e s t i n a l disaccharidase a c t i v i t i e s were determined, only the c a l o r i e 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 higher t o t a l lactase a c t i v i t y . However, the s p e c i f i c a c t i v i t i e s of lactase, sucrase and maltase were s i g n i f i c a n t l y higher in the c a l o r i e r e s t r i c t e d group than in the control groups. Kumar and Chase (59) produced a state of undernutrition in infant rats by a l t e r i n g the l i t t e r size to 16 animals per mother, 12 hours a f t e r b i r t h . After weaning, the rats who had been undernourished received 8 grams of an 8% protein d i e t per day as compared to the control animals who consumed a mean of 16 grams per day of a 27% protein d i e t . The d i s a c -charidase a c t i v i t i e s were studied at various ages s t a r t i n g with 7 days and ending with 115 day old r a t s . I t was found that lactase a c t i v i t y was s i g n i f i c a n t l y higher in the under-nourished group at a l l ages except for a temporary depression at 21 days when i t was s i g n i f i c a n t l y lower in the undernourished group. There were no s i g n i f i c a n t differences in sucrase a c t i v i t y between the two groups at any age. Maltase a c t i v i t y was not determined. Kumar and Chase (59) concluded that the r a t was not a good experimental model to use for the study of prot e i n - c a l o r i e malnutrition in man. As in the previous studies of Solimano et a l . (4) and Prosper et a l . (5) the carbohydrate content of the control and the low protein diets i n the study by Kumar and Chase (59) was not kept constant. The protein content of the low pro-t e i n d i e t seems to be too high (8%), however, the amount of the d i e t given each day was r e s t r i c t e d so the weight of the undernourished animals was only 20% of the weight of the controls a f t e r 16 weeks of l i f e . The l e v e l s of the disacch-aridase a c t i v i t i e s were only determined in the jejunal sec-t i o n . Kumar and Chase (53) then studied the e f f e c t of feeding a low protein d i e t (< 1%) to young adult monkeys for periods of 10 to 18 weeks to see i f the results resembled those found in human pro t e i n - c a l o r i e malnutrition more c l o s e l y than the r e s u l t s of the studies done with r a t s . After 6 weeks on the low protein d i e t , the protein d e f i c i e n t monkeys had s i g n i f i -c antly reduced lactase a c t i v i t y when compared to the control group which received a 34% protein d i e t . After 10 weeks, lactase, sucrase, and maltase a c t i v i t i e s were a l l s i g n i f i -cantly reduced in the low protein group. The differences between the r e s u l t s which Kumar and Chase obtained when work-ing with rats (59) and when working with monkeys (53) suggest that the e f f e c t of p r o t e i n - c a l o r i e malnutrition on the i n t e s t i n a l disaccharidases may d i f f e r from one species to another. Therefore, one must exercise caution when comparing r e s u l t s obtained from d i f f e r e n t species. 3. The e f f e c t of protein-calorie malnutrition on absorp  ti o n of disaccharides in laboratory animals. The e f f e c t of p r o t e i n - c a l o r i e malnutrition on monosaccharide and disacchar-ide absorption and the a c t i v i t y l e v e l s of i n t e s t i n a l d i s a c -char idases was only studied by L i f s h i t z et a l . (6) They used three types of d i e t s : a control d i e t with 18% protein, 70% carbohydrate, and 8% f a t ; a low protein d i e t with 4% protein, 70% carbohydrate, and 8% f a t ; and a low protein, low 19 carbohydrate d i e t with 4% protein, 45% carbohydrate and 8% f a t . Casein was used as the source of protein. The diets were not i s o c a l o r i c as non-nutritive c e l l u l o s e was added to make up the casein and glucose d e f i c i t s in the two protein d e f i c i e n t d i e t s . The rats were fed the respective diets for one to four weeks. The low protein diets had no e f f e c t on the a c t i v i t y of the i n t e s t i n a l disaccharidases and on the absorption of sucrose and maltose. The rate of glucose absorption was s i g n i f i c a n t l y greater in the rats on the low protein diets with both 45% and 70% carbohydrate only a f t e r 14 days of dietary treatment. There was no change in the absorption of fructose or 3-0-methyl-D-glucose. This was the f i r s t study i n which the proportion of carbohydrate in the low protein and control d i e t was kept the same, but the animals were given the respective diets for only 4 weeks. After t h i s short period of time, there were no s i g n i f i c a n t differences in the concentration of mucosal protein between the protein d e f i c i e n t and control animals. This would indicate that 4 weeks of protein deprivation was not long enough to demon-strate a l l the symptoms of protein-calorie malnutrition. Therefore, the changes in disaccharidase a c t i v i t i e s or changes in absorption of monosaccharides or disaccharides may not be apparent a f t e r only 4 weeks of protein deprivation. 4. The e f f e c t of non-protein factors on the a c t i v i t y  of the i n t e s t i n a l disaccharidases in laboratory animals. The r e s u l t s of studies dealing with other factors including iron 20 deficiency or i n f e s t a t i o n by nematodes support the idea that the associated d e f i c i e n c i e s and infections that accompany the state of pr o t e i n - c a l o r i e malnutrition may be important i n the disaccharide intolerance found in children s u f f e r i n g from prot e i n - c a l o r i e malnutrition. Hoffbrand and Broitman (64) found that lactase, 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 in young dogs who were maintained on an iron d e f i c i e n t d i e t for 8 weeks when they were compared to the control group. Sriratanaban and Thayer (57) studied the e f f e c t of combined iron and protein deficiency in the r a t on the in t e s -t i n a l disaccharidase a c t i v i t i e s . The animals were divided i n t o 4 groups: the controls (27% protein), low protein (5% protein), low iron, and low iron-low protein. They were maintained on these diets for 75 to 185 days from the time of weaning. As found in previous studies (4,64) the animals on the low protein d i e t had s i g n i f i c a n t l y higher lactase, sucrase, and maltase a c t i v i t i e s than the controls, and the iron d e f i c i e n t group had lower lactase, sucrase, and maltase a c t i v i t i e s when compared to the control group. The combina-t i o n of iron and protein deficiency resulted in an increase in disaccharidase a c t i v i t y in the jejunum and ileum when compared to the controls, but the level s of disaccharidase a c t i v i t i e s were lower when compared to the protein d e f i c i e n t group. Bol i n e t a l . (58) studied the e f f e c t of worm i n f e s t a t i o n upon disaccharidase a c t i v i t y in the r a t by in f e s t i n g part of 21 a group of animals on a control d i e t containing 30% protein with Nippostrongylus b r a s i l i e n s i s . The worm in f e s t a t i o n caused a depression of jejunal lactase and maltase a c t i v i t i e s when compared to the control group. There are many p o s s i b i l i t i e s for further study on the ef f e c t s of infections 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 explored before a decision can be made on the s u i t a b i l i t y of the r a t as a model for studying p r o t e i n - c a l o r i e malnutrition. E . Objectives of the Present Study From the preceding review of the l i t e r a t u r e i t appears that well controlled studies are needed in order to e l u c i d -ate further the e f f e c t of pr o t e i n - c a l o r i e malnutrition on the digestion and absorption of carbohydrates. Therefore, the primary purpose of thi s investigation i s to study the e f f e c t of prolonged protein deficiency and pr o t e i n - c a l o r i e malnutrition upon the in vivo disaccharide absorption and i n t e s t i n a l disaccharidase a c t i v i t i e s by feeding rats w e l l controlled d i e t s . The control d i e t w i l l contain 18% lactalbumin and w i l l be fed ad_ libitum. The protein d e f i c i e n t d i e t w i l l contain 0.5% lactalbumin and w i l l a l s o be fed ad_ libitum. The prote i n - c a l o r i e malnutrition d i e t w i l l contain 1% lactalbumin and w i l l be given in r e -s t r i c t e d amounts (4 grams per day). The second purpose of t h i s study i s to determine whether marked increases of i n t e s t i n a l sucrase and maltase a c t i v i t i e s previously observed in protein d e f i c i e n t rats (4) could have been influenced by a high proportion of carbohydrate i n the d i e t . This w i l l be studied by feeding rats protein d e f i c i e n t diets (0.5% lactalbumin) containing 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 this investigation i s to determine whether protein repletion leads to a reversal of protein mal-n u t r i t i o n symptoms to normal. Rats w i l l be refed a protein adequate d i e t a f t e r being on a protein d e f i c i e n t d i e t (0.5% lactalbumin) for 8.5 weeks. The i n t e s t i n a l disaccharidase a c t i v i t i e s w i l l be studied at two s i t e s of the small intestine (jejunum and ileum) since i t cannot be assumed that a change at one s i t e w i l l occur at a l l parts of the small i n t e s t i n e . Results of this investigation should help in c l a r i f y i n g some of the digestion and absorption problems in children s u f f e r i n g from protein deprivation. CHAPTER II MATERIALS AND METHODS A. Animals and Diets White male rats of the Wistar s t r a i n were obtained from the animal unit, Faculty of Medicine, at the University of B r i t i s h Columbia. Their body weights ranged from 90 grams to 120 grams with a mean of 108 - 9 grams. The rats were divided into four groups and fed the following diets for a period of 8 to 9 weeks, with the time period depending on laboratory convenience. One group of rats received a control d i e t containing 18% lactalbumin. The second group was given a 0.5% lactalbumin low carbohydrate d i e t , the t h i r d group was given a 0.5% lactalbumin high carbohydrate d i e t , and the fourth group was given a 1% lactalbumin d i e t r e s t r i c t e d to 4 grams per day. Part of the second group of ra t s , a f t e r being fed the 0.5% lactalbumin low carbohydrate d i e t for 8.5 weeks, was given the control d i e t containing 18% lactalbumin for 8 weeks. The composition of these diets i s listed in Table I I . These diets are based on those developed by Edozien (65) for studying kwashiorkor and marasmus in r a t s . The only modification of the diets for use in the present experiment was the su b s t i t u t i o n of cornstarch for dextrose and sucrose. The 0.5% lactalbumin diets with both the high and low level s of carbohydrate were designed to produce symptoms c h a r a c t e r i s t i c of kwashiorkor in childre n which, among others, include edema, loss in body weight, f a t t y l i v e r , hair d i s -TABLE II COMPOSITION OP DIETS 1% lactalbumin Control 0.5% lactalbumin 0.5% lactalbumin (Restricted to 18% lactalbumin Low carbohydrate High carbohydrate 4 grams per day) Lactalbumin* 18 .0% 0 .5% 0. .5% 1 .0% Cornstrach & 66 .0% 66 .0% 83. .5% 66 .0% Corn o i l 10 .0% 17 .8% 10. .0% 17 .6% A l p h a c e l & 9 .7% 3 .4% Salt Mix 5"® 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% •Purchased from N u t r i t i o n a l Biochemical Co., Cleveland, Ohio. Purchased from General Biochemicals, Chagrin F a l l s , Ohio. ^Rogers, O.R., and A.E. Harper, "Amino Acid Diets and Maximal Growth in the Rat," J . Nutr., 87:267-273, 1965. coloration, hypoalbuminemia, and g a s t r o i n t e s t i n a l d i s t u r -bances (63). Kwashiorkor i s the deficiency of protein i n the d i e t with an adequate intake of c a l o r i e s . The 1% l a c t a l -bumin d i e t r e s t r i c t e d to 4 grams per day was designed to produce the symptoms c h a r a c t e r i s t i c of marasmus which is the deficiency of both protein and c a l o r i e s . The symptoms of marasmus in children include muscular wasting, growth retardation, g a s t r o i n t e s t i n a l symptoms, f a t t y l i v e r , hypo-albuminemia, and i r r i t a b i l i t y . However, the g a s t r o i n t e s t i n a l disturbances, f a t t y l i v e r , and hypoalbuminemia are not as severe in marasmus as they are in kwashiorkor (63). Animals on the 1% lactalbumin d i e t were housed i n d i v i d -u a l l y in screen bottom cages, and the animals on the other dietary regimens were kept i n p l a s t i c cages in groups of two to f i v e per cage. Except for the rats on the 1% l a c -talbumin d i e t ( r e s t r i c t e d to 4 grams per day), a l l other animals were given t h e i r d i e t and water ad_ lib i t u m . The 1% lactalbumin group was fed the 4 grams of d i e t once a day at 9:30 a.m. and water was given ad_ li b i t u m . A l l the animals were weighed at weekly i n t e r v a l s . B. Absorption of Disaccharides The absorption of lactose, sucrose, and maltose was measured in vivo using radioactive disaccharides. The rats were anesthetized with intraperitoneal injections of Nembutal (Sodium pentobarbitol) using 6.0 mg per 100 g body weight for the t e s t animals and 7.1 mg per 100 g of body weight for the controls as the protein depleted animals did not require as large a dose of the anesthetic as the control animals. The Nembutal dose was injected gradually in three to f i v e portions over a 15 to 30 minute period. The abdomenal wall was opened, and approximately a 10 cm jejunal segment d i s t a l to the ligament of T r e i t z was t i e d o f f at both ends. Next, 0.5 ml of the radioactive solution was injected into the lumen of the lig a t e d jejunal segment. The exact amount of radioactive solution injected into the loop was determined by weighing the syringe before and a f t e r the i n j e c t i o n . The i n t e s t i n a l loop was returned to the peritoneal cavity for a 15 minute period during which the animal was kept warm under a lamp. At the end of t h i s period, the loop was excised and rinsed with 5 ml of 0.9% sal i n e , and the rinsi 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 using a glass s l i d e . The musosal scrapings were weighed and then mixed with 5 ml of 0.9% saline using a glass s t i r r i n g rod. The luminal r i n s i n g s and mucosal scrapings were heated i n b o i l i n g water in order to inactivate the enzymes and to ensure that a l l the residual disaccharide was dissolved in the saline s o l u t i o n . The volume of the samples was made up to 10 ml with 0.9% saline and mixed thoroughly. The samples were then centrifuged at 15,000 RPM for 15 minutes. The supernatant f r a c t i o n was removed and immediately frozen. Radioactivity was determined within 1 to 3 weeks. The r a d i o a c t i v i t y of the prepared samples was counted in a Picker Nuclear Liquimat S c i n t i l l a t i o n Counter using a 0.5 ml aliquot of the supernatant f r a c t i o n ; 2.0 ml of N.C.S. 27 Tissue Solubolizer (Nuclear Chicago Solubolizer, Amersham/ Searle Corp., Arlington Heights, I l l i n o i s ) ; and 10.0 ml of s c i n t i l l a t i o n solvent. The s c i n t i l l a t i o n solvent was made up of 7.5 gm PPO and 62.5 mg of POPOP per 1000 ml q.s. toluene which were a l l obtained from the Fisher S c i e n t i f i c Co. To measure the r a d i o a c t i v i t y of the injected dose, 0.5 ml of a solution containing approximately 0.01 pCi per ml was used as a standard. The s c i n t i l l a t i o n samples were counted for 2 minutes with the following channel settings on the Picker Nuclear Liquimat 220: A Channel Upper Limit 1000 Lower Limit 0 B Channel Upper Limit 780 Lower Limit 80 C Channel Upper Limit 1000 Lower Limit 200 D Channel Upper Limit 1000 Lower Limit 320 The amount of disaccharide absorbed was obtained by subtracting the r a d i o a c t i v i t y which was recovered i n the lumen and the mucosa of the l i g a t e d segment from the t o t a l injected dose. The per cent of dose absorbed was then expressed per 500 mg wet weight of the li g a t e d i n t e s t i n a l segment. C. Disaccharidase A c t i v i t i e s Following the determination of disaccharide absorption, the r e s t of the small intestine, d i s t a l to the ligament of T r i e t z and up to the i l e o c e c a l junction was removed, rinsed 28 with ice-cold saline to remove the i n t e s t i n a l contents, and placed on an ice-cold s t a i n l e s s s t e e l tray. The small i n t e s t -ine was then divided into four equal parts, and the segment which had been used for the absorption study was accounted for in the f i r s t quarter. Lactase, sucrase, and maltase a c t i v i t i e s were determined in the f i r s t and fourth quarters of the i n t e s t i n e . The sections were weighed, the mucosa was scraped o f f with a glass s l i d e , weighed, and immediately homogenized in ice-cold saline solution using a Teflon Homo-g e n i z e d at 300 RPM for one minute while being cooled i n crushed i c e . The mucosal samples were then frozen in sealed polystyrene t e s t tubes and the disaccharidase a c t i v i t i e s were determined within a week. The two step TRIS-glucose oxidase method of Dahlqvist (66) with s l i g h t modifications was used for the determination of lactase, sucrase and maltase a c t i v i t i e s . The modifications consisted of the use of 0.1 ml of mucosal homogenate and 0.1 ml of substrate for the incubation mixture instead of 10 jil of each. The absorbance was measured at 420 mu against a reagent blank in a Coleman Hitachi spectrophotometer. Model 101. Units of disaccharidase a c t i v i t i e s were defined as micromoles of substrate hydrolysed per minute under the incub-ation conditions used (66) The s p e c i f i c a c t i v i t y was expressed i n units of a c t i v i t y per gram wet weight of mucosa and per gram of mucosal protein. ^Teflon Homogenizer, Model K4f. TRI-R Instruments, Rock-v i l l e Center, New York. 29 D. Other Assays 1. Plasma albumin concentration. For the plasma albumin determination, blood was drawn from the animal by heart punc-ture and was transferred to t e s t tubes containing heparin. This was done under anesthesia. The blood samples were c e n t r i -fuged at 15,000 RPM for 15 minutes, and then the plasma was removed and frozen i n sealed t e s t tubes. The plasma albumin was determined within three months by the HABA dye method as described by Ness e t a l . (67) and modified by the Technicon Corporation for use in the auto analyzer (Auto Analyzer Methodology, Method F i l e N-15C). I t was further modified i n the present experiment by using 0.1 ml of plasma and 5 ml of working HABA dye solution which was made from Stock HABA dye (T21-1079) and Stock Phosphate Buffer (Toi-184) from the Technicon Corporation, Ardsley, New York. Bovine Albumin (Sigma Chemical Co., Saint Louis, Missouri) was used as a standard. The absorbance was read at 505 mu in a Coleman Hitachi spectrophotometer. Model 101. 2. Mucosal protein determination. The protein of the mucosa was determined by the method of Lowry et a l . (68) using a bovine albumin standard (Sigma Chemical Co., Saint Louis, Missouri). The absorbance was read at 500 mu in a Coleman Hita c h i spectrophotometer. Model 101. E. Materials The radioactive disacchar ides were obtained from the Amersham/Searle Corporation, Arlington Heights, I l l i n o i s . The following radio-disaccharides were used for the absorption 30 studies: lactose (D-glucose-l-C 1 4), freeze-dried s o l i d , s p e c i f i c a c t i v i t y of 10 to 20 uCi per mmole; sucrose uniformly labeled with C^, freeze-dried 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 uCi per mmole; maltose uniformly labeled with C^, s p e c i f i c a c t i v i t y 4 to 10 uCi per mmole. The 0.5 ml of the incubation mixture used for the absorp-t i o n studies contained 0.1 uCi of radioactive disaccharide and d i f f e r e n t amounts of unlabeled disaccharide. In the case of lactose absorption, there was 16 mg of unlabeled lactose per ml; in the case of sucrose absorption, there was 32 mg of sucrose per ml; and in the case of maltose absorption, there was 100 mg of maltose per ml. The reagent grade un-labeled maltose monohydride and sucrose were obtained from the Fisher S c i e n t i f i c Co., F a i r Lawn, New Jersey. The un-labeled beta-lactose, also of reagent grade, was obtained from Eastman Organic Chemical, Rochester, New York. Glucose oxidase, s p e c i f i c a c t i v i t y of approximately 90 units per ml, A grade, of fungal o r i g i n , s a l t - f r e e and l y o p h i l i z e d and peroxidase, B grade (horseradish) were obtained from Calbiochem, San Diego, C a l i f o r n i a . The g l u -cose standard sol u t i o n (1 mg glucose per ml) and T r i t o n X-100 were purchased from the Sigma Chemical Co., Saint Louis, Missouri. The TRIS-free base and Phenol reagent 2N solu t i o n (Folin-Ciocalteau) were obtained from the Fisher S c i e n t i f i c Co., F a i r Lawn, New Jersey. The Nembutal which contained 50 mg sodium pentobarbitol per ml was purchased from Abbott Laboratories Ltd., Montreal, P.Q. The student t t e s t was used for the s t a t i s t i c a l analysis and the Model 1130 IBM Computer was employed. 31 CHAPTER III RESULTS A. Appearance, Body Weight, and Plasma Albumin Concentration. The changes in mean body weight of the control, protein depleted, and protein repleted rats are shown i n Figure 1. The i n i t i a l mean body weight of a l l the rats was 108 ± 9 grams. The control group increased i n weight to 359.3 ± 30.6 gm a f t e r 8.5 weeks on the 18% lactalbumin d i e t . Rats on a l l the three protein d e f i c i e n t diets l o s t an average of 45 grams from t h e i r i n i t i a l body weight in 8.5 weeks, with most of the loss occuring during the f i r s t three weeks. 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 differences i n the f i n a l body weights of the three protein depleted groups. Refeeding the protein depleted rats (rats fed the 0.5% lactalbumin low carbohydrate d i e t for 8.5 weeks) with the 18% lactalbumin d i e t produced an abrupt gain in body weight, and a f t e r 8 weeks of re p l e t i o n , the rats had increased t h e i r mean body weight to 341 i 29 grams which was not s i g n i f i c a n t l y d i f f e r -ent from the f i n a l mean body weight for the control group (359 i 31 grams). The i n i t i a l and f i n a l body weights for each dietary group are given in Table I I I . When the i n i t i a l weights of each group 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 differences between the controls and any of the other groups. However, the 0.5% lactalbumin high carbohydrate group had a s i g n i f i c a n t l y lower FIGURE 1 CHANGES IN BODY WEIGHTS OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS • Controls, 18% lactalbumin Protein depleted groups: O 0.5% lactalbumin, low carbohydrate • 0.5% lactalbumin, high carbohydrate A 1% lactalbumin ( r e s t r i c t e d to 4 grams per day) • Protein repleted (refed the 18% lactalbumin d i e t a f t e r 8 weeks on the 0.5% lactalbumin low carbohydrate diet) TABLE III INITIAL AND FINAL BODY WEIGHTS OF THE SURVIVORS IN EACH DIETARY GROUP Dietary Group Number of Animals Weight at Start of* Weight at end of* Dietary Treatment Dietary Treatment (grams) (grams) Controls, 18% lactalbumin 15 107.3 ± 10.0 359.3 ± 30.6 0.5% lactalbumin, low 12 113.3 ± 8.8 a 62.5 ± 4.8 b carbohydrate 0.5% lactalbumin high 18 101.9 ± 4.2 a' c 64.7 ± 6.0 b' d carbohydrate 1% lactalbumin (restricted 18 109.4 ± 7.4 a' d' e 62.7 ± 5.0 b' d to 4 grams per day) Repleted & 12, 65.0 ± 6.5b 341.3 ± 28.6 a * Values are Means i S.D. for the number of rats shown in the second column. &Refed the 18% lactalbumin d i e t for 8 weeks afte r 8.5 weeks on the 0.5% l a c t a l -bumin low carbohydrate d i e t . aDoes not d i f f e r from the control group (P>0.05). b D i f f e r s from the control group (P <0.001). c D i f f e r s from the 0.5% lactalbumin low carbohydrate group (P <0.001). Does not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P^0.05). e D i f f e r s from the 0.5% lactalbumin high carbohydrate group (P< 0.001). 35 i n i t i a l mean body weight than the 0.5% lactalbumin low carbo-hydrate and the 1% lactalbumin (restricted) groups (P<0.001). About 40% of the rats fed the 0.5% lactalbumin d i e t s showed a severe loss of hair and became less active as the experiment progressed. The animals on the 1% lactalbumin (restricted) d i e t did not show as severe hair losses as those on the 0.5% lactalbumin d i e t s . The animals fed the 1% l a c t a l -bumin (restricted) d i e t were hyperactive and aggressive in t h e i r behaviour, whereas the animals on the 0.5% lactalbumin d i e t s were very passive. Refeeding the group of protein depleted animals the 18% lactalbumin d i e t for 8 weeks resulted in a reversal of a l l the gross symptoms so that the repleted group resembled the controls i n both appearance and behaviour. Figures 2 and 3 show the t y p i c a l appearance of the control and protein depleted rats at the time of s a c r i f i c e . Edema, which i s c h a r a c t e r i s t i c of the f u l l kwashiorkor syndrome in children, was found i n only a few rats on the 0.5% lactalbumin d i e t s , although, a number of animals with no obvious edema showed increased f l u i d accumulation in the peritoneal cavity at the time of s a c r i f i c e . I t was noted a t the time of s a c r i f i c e that the small intestine of the protein d e f i c i e n t rats was very t h i n , f r a g i l e , and small in diameter. The i n t e s t i n a l mucosal layer was also very thin and pale when compared to the control group. The mortality rate of the rats on the three protein d e f i c i e n t diets was highest during the l a s t two weeks of the experimental period. The percentage of survivors for each dietary group i s shown in 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 CARBO-HYDRATE DIET SHOWN IN FIGURE 2. TABLE IV PERCENTAGE OF SURVIVORS ON THE DIFFERENT DIETARY REGIMENS Dietary Group ' Number of Weeks on the Percentage of Survivors Experimental Diet Controls, 18% lactalbumin 8.5 100.0% 0.5% lactalbumin low carbohydrate 8.5 77.5% 0.5% lactalbumin high carbohydrate 8.0 86.7% 1% lactalbumin (restricted to 4 grams 9.0 83.3% per day) Repleted* 8.0 100.0% *Refed the 18% lactalbumin d i e t for 8 weeks after 8.5 weeks on the 0.5% lactalbumin low carbohydrate d i e t . 39 The plasma albumin concentrations in the controls, pro-t e i n depleted, and protein repleted r a t s are given i n Table V. A l l the three groups of rats on the protein d e f i c i e n t diets had s i g n i f i c a n t l y lower plasma albumin concentrations than the control group (P< 0.001). The low plasma albumin confirmed that the animals on the 0.5% lactalbumin and the 1% lactalbumin (restricted) diets were suf f e r i n g from p r o t e i n - c a l o r i e mal-n u t r i t i o n . The level s of plasma albumin in the repleted and the control rats were almost i d e n t i c a l . The same was true for the rats fed the 0.5% lactalbumin low carbohydrate and the 0.5% lactalbumin high carbohydrate d i e t s . However, the plasma l e v e l s were s i g n i f i c a n t l y higher in the 1% lactablumin (re-stri c t e d ) group than in the 0.5% lactalbumin low carbohydrate group (P <0.01) . B. I n t e s t i n a l Disaccharidase A c t i v i t i e s The jejunal and i l e a l lactase, sucrase, and maltase a c t i v i t i e s of rats fed the various diets are shown in Tables VI, VII and VIII r e s p e c t i v e l y . The i n t e s t i n a l lactase a c t i v i t y was s i g n i f i c a n t l y higher in a l l the three protein d e f i c i e n t groups (0.5% lactalbumin low carbohydrate, 0.5% lactalbumin high carbohydrate, and 1% lactalbumin r e s t r i c t e d to 4 grams per day) i n both the jejunum and ileum when compared with the controls fed the 18% l a c t a l -bumin d i e t (Table VI). When lactase lev e l s of the three protein d e f i c i e n t groups were compared, there were no s i g n i f i c a n t differences among them in ei t h e r the jejunal or i l e a l lactase 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 Dietary Group Number of Plasma Albumin* Rats mg/l00 ml Control, 18% lactalbumin 15 4 .5 - 0.5 0.5% lactalbumin 12 2.1 ± 0.6a low carbohydrate 0.5% lactalbumin 16 2.2 ± 0.4 a' b high carbohydrate 1% lactalbumin ( r e s t r i c t e d 18 2.7 ± 0.4 a' c to 4 grams per day) Repleted & 11 4.4 ± 0.5 d •Values are Means ± S.D. for the number of rats shown in the second column. S'Refed the 18% lactalbumin d i e t for 8 weeks a f t e r 8.5 weeks on the 0.5% lactalbumin low carbohydrate d i e t . a D i f f e r s from the control group (P <0.001). •'-'Does not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P>0.05). c D i f f e r s from the 0.5% lactalbumin low carbohydrate group (P< 0.01) . dDoes not d i f f e r from the control group (P >0.05) . TABLE VI LACTASE ACTIVITIES IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS I n t e s t i n a l Segment Jejunum Dietary Group Ileum Number of Rats Lactase A c t i v i t y * Units per g Units per g wet weight mucosal protein Control, 18% lactalbumin 15 1.3 + 0.2 9.4 ± 1.1 0.5% lactalbumin low carbohydrate 12 3.9 + 1.5* 38.2 ± 14.9&-0.5% lactalbumin high carbohydrate 18 4.2 + 1.2 b' c 45.6 ± 14.7b#c 1% lactalbumin ( r e s t r i c t e d ) 5 1 18 3.2 + 0.8 b' c 32.4 ± 7.6 b' c Repleted® 12 1.0 + 0.4b 7.7 ± 3.0a Control, 18% lactalbumin 15 0.2 + 0.1 2.1 i 1.0 0.5% lactalbumin low carbohydrate 12 0.6 ± 0.4b 7.6 ± 5.9 b 0.5% lactalbumin high carbohydrate 18 0.4 + 0.3 b' c 4.5 ± 3.3 b' c 1% lactalbumin (restricted) 5" 18 0.5 + 0.2 b' c 5.9 ± 1.9b*-c Repleted® 12 0.2 + 0.1 a 2.1 ± l . l a *Values are Means - S.D. for the number of rats shown i n the t h i r d column. R e s t r i c t e d to 4 grams of d i e t per day. ®Refed the 18% lactalbumin d i e t for 8 weeks a f t e r 8.5 weeks on the 0.5% lactalbumin low carbohydrate d i e t . aDoes not d i f f e r from the control group (P>0.05). b D i f f e r s from the control group (P<0.05). cDoes not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P>0.05). TABLE VII SUCRASE ACTIVITIES IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS In t e s t i n a l Dietary Group Number of Sucrase A c t i v i t y * Segment Rats Units per g Units per g wet weight mucosal protei Je junum Control, 18% lactalbumin 15 7.2 ± 1.3 54.1 ± 9.0 0.5% lactalbumin low carbohydrate 12 15.6 ± 5.4 b 151.0 ± 51.8 b 0.5% lactalbumin high carbohydrate 18 22.5 ±'4.2 b' c 241.5 ± 5 1 . l b ' 1% lactalbumin ( r e s t r i c t e d ) & 18 18.1 ± 3.2 b' d 181.6 ± 32.6 b' Repleted® 12 6.2 ± 1.2b 46.8 ± 1 1 . l a Ileum Control, 18% lactalbumin 15 3.6 ± 1.7 33.7 ± 15.7 0.5% lactalbumin low carbohydrate 12 2.9 ± 1.5 a 35.3 ± 2 4 . l a 0.5% lactalbumin high carbohydrate 18 2.7 ± 0.7 a' d 28.9 ± 7.1 a' d 1% lactalbumin ( r e s t r i c t e d ) & 18 1.9 ± 0.7 b' d 20.9 ± 6.4 b' d Repleted® 12 1.0 ±'o.6*> 9.2 ± 3.9 b *Values are Means i S.D. for the number of rats shown in the t h i r d column. R e s t r i c t e d to 4 grams of die t per day. @Refed the 18% lactalbumin d i e t for 8 weeks aft e r 8.5 weeks on the 0 .5% lactalbumin low carbohydrate d i e t . aDoes not d i f f e r from the control group (P>0.05). b D i f f e r s from the control group (P<0.05). c D i f f e r s from the 0.5% lactalbumin low carbohydrate group (P< 0.001). dDoes not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P> 0.05) TABLE VIII I n t e s t i n a l Segment Jejunum Ileum MALTASE ACTIVITIES IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Dietary Group Number of Maltase A c t i v i t i e s * Rats Un i t s per g Units per g we t weight mucosal protein Control, 18% lactalbumin 15 32 .9 + 2. 7 245 .9 + 23.1 0.5% lactalbumin low carbohydrate 12 53 .4 + 17 . l b 517 .4 + 153.6 b 0.5% lactalbumin high carbohydrate 18 63 .2 + 12 .2 b' d 681 .2 + 167.1 b' c 1% lactalbumin ( r e s t r i c t e d ) & 18 51 .8 + 13 .9 b' d 532 .0 ± 132.9b,<3 Repleted® 12 18 .2 + 6. Ob 139 .2 ± 53.4b Control, 18% lactalbumin 15 15 .6 + 5. 9 144 .0 ± 53.0 0.5% lactalbumin low carbohydrate 12 25 .6 + 9. g> 311 .7 + 152. 6 b 0.5% lactalbumin high carbohydrate 18 20 .8 + 7. 3b,d 223 .6 + 7 4 # 2 b , d 1% lactalbumin ( r e s t r i c t e d ) & 18 28 .9 + 8. 2b,d 320 .3 + 87.6 b' d Repleted® 12 12 .7 + 4. 9 a 102 .4 + 34. 2 b Values are Means ± S.D. for the number of rats shown in the t h i r d column. & Restricted to 4 grams of d i e t per day. @ Refed the 18% lactalbumin d i e t for 8 weeks a f t e r 8.5 weeks on the 0.5% lactalbumin low carbohydrate d i e t . a Does not d i f f e r from the control group (P>0.05). b D i f f e r s from the control group (P<0.05). c D i f f e r s from the 0.5% lactalbumin low carbohydrate group (P<0.01). d Does not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P>0.05). 4 4 the rat s on the 0.5% lactalbumin low carbohydrate d i e t were repleted with a protein adequate d i e t containing 18% l a c t a l -bumin, the lactase a c t i v i t y l e v e l s f e l l from the previously high values to approximately the lev e l s found in the controls. The lactase a c t i v i t y i n the jejunum i s markedly higher than i n the ileum for a l l the dietary groups and the t e s t d i e t s seem to have no e f f e c t on the d i s t r i b u t i o n of lactase a c t i v i t y between the jejunum and ileum. As found with the lactase a c t i v i t y , the jejunal sucrase a c t i v i t i e s i n rats on the 0.5% lactalbumin diets with both high and low l e v e l s of carbohydrate and the 1% lactalbumin di e t were s i g n i f i c a n t l y higher than the l e v e l s of jejunal sucrase found in the control 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 differences between the control group and the 0.5% lactalbumin low carbohydrate or the 0.5% lactalbumin high carbohydrate groups. In the 1% lactalbumin group, the l e v e l of i l e a l sucrase a c t i v i t y was s i g n i f i c a n t l y lower than i n the control group (Table VTI). When the 0.5% lactalbumin low carbohydrate group was compared to the 0.5% lactalbumin high 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 higher in the animals on the high carbohydrate d i e t in the jejunum, but 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 differences between the l e v e l of i l e a l sucrase a c t i v i t y in these two groups. There were also 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 differences between the 0.5% lactalbumin low carbohydrate and the 1% lactalbumin groups. The jejunal sucrase a c t i v i t i e s of the control group and the group which was repleted with 18% lactalbumin for 8 weeks 45 aft e r 8.5 weeks of protein deprivation were s i m i l a r . How-ever, the i l e a l sucrase a c t i v i t i e s f e l l in the repleted group to 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 control group. The sucrase a c t i v i t y was always markedly higher in the jejunum than in the ielum in the dietary 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 control and the three protein depleted groups (0.5% l a c t -albumin low carbohydrate, 0.5% lactalbumin high carbohydrate, and 1% lactalbumin r e s t r i c t e d to 4 grams per day), i t was found that both the jejunal and i l e a l l e v e l s of maltase were s i g n i f i c a n t l y higher i n the protein depleted groups than i n the controls, and there was a s i g n i f i c a n t l y higher l e v e l of maltase a c t i v i t y in the jejunum i n the 0.5% lactalbumin high carbohydrate group than i n the 0.5% lactalbumin low carbo-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 d i f f e r -ences between the l a s t two groups in the i l e a l maltase a c t i v i t i e s . There were also no s i g n i f i c a n t differences in the l e v e l s of disaccharidases between the groups on the 0.5% lactalbumin low carbohydrate and the 1% lactalbumin d i e t s in eit h e r the jejunum or the ileum (Table V I I I ) . The jejunal and i l e a l l e v e l s of maltase a c t i v i t y in the repleted group had f a l l e n to l e v e l s s i g n i f i c a n t l y lower than those of the control group. As found with the lactase and sucrase a c t i v i t i e s , the maltase a c t i v i t y was also markedly higher in the jejunal than in the i l e a l segment in a l l groups of r a t s . The mucosal protein concentrations in both the jejunum and ileum of the control, protein depleted, and protein repleted rats are shown in Table IX. The mucosal protein concentration i n the jejunum and ileum of the two 0.5% lactalbumin groups and the 1% lactalbumin group were a l l s i g n i f i c a n t l y lower than the mucosal protein concentration in the control group. There 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 difference between the mucosal protein concentrations of the control and the protein repleted groups. C. I n t e s t i n a l Absorption of Disaccharides in Vivo The i n t e s t i n a l absorption of disaccharides by rats fed the control and the protein d e f i c i e n t d i e t s i s shown in Table X. Lactose, sucrose, and maltose absorption by rats fed the 0.5% lactalbumin low carbohydrate d i e t was s i g n i f i -cantly higher than i n the control group. This greater absorption by the animals on the 0.5% lactalbumin low carbo-hydrate d i e t i s consistent with the higher l e v e l s of disac-charidases found in t h i s group when compared to the controls. The comparison of i n t e s t i n a l lactose, sucrose, and maltose absorption in rats fed the control and the 0.5% lactalbumin high carbohydrate d i e t demonstrated s i g n i f i c a n t l y higher absorption only for sucrose and maltose in the 0.5% l a c t a l -bumin group. Lactose absorption was not s i g n i f i c a n t l y higher in the 0.5% lactalbumin high carbohydrate group even though the mean value for lactose absorption was 8 times higher than in the control group. This i s due to the great v a r i a b i l i t y in the obtained r e s u l t s of the 0.5% lactalbumin high carbo-TABLE IX MUCOSAL PROTEIN CONCENTRATION IN JEJUNUM AND ILEUM OF CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Dietary Group Number of Rats Control, 18% lactalbumin 15 0.5% lactalbumin low carbohydrate 12 0.5% lactalbumin high carbohydrate 18 1% lactalbumin (re s t r i c t e d to 18 4 grams of die t per day) Repleted & 12 mg Protein per g wet* weight mucosa Jejunum 134.0 ± 8.0 102.7 ± 6.4^ 93.9 ± 10.3 b' c 99.1 ± 5.9 b' d 133.0 Z 12.6C Ileum 108.4 ± 8.1 85.5 i 14.2 b' d 93.5 ± 12.4 b' d 91.1 ± 15.9 b' d 124.1 ± 25.0 a *Values are Means - S.D. for the number of rats shown i n the second column. &Refed the 18% lactalbumin d i e t for 8 weeks after 8.5 weeks on the 0.5% lactalbumin low carbohydrate d i e t . aDoes not d i f f e r from the control group (P;>0.05). b D i f f e r s from the control group (P<0.001). c D i f f e r s from the 0.5% lactalbumin low carbohydrate group (P <0.01). dDoes not d i f f e r from the 0.5% lactalbumin low carbohydrate group (PJ>0.05). TABLE X LACTOSE, SUCROSE, AND MALTOSE ABSORPTION IN CONTROL, PROTEIN DEPLETED, AND PROTEIN REPLETED RATS Dietary Group Number of Rats Control, 18% lactalbumin 5 0.5% lactalbumin low 5 carbohydrate 0.5% lactalbumin high 6 carbohydrate 1% lactalbumin (restricted 6 to 4 grams die t per day) Repleted & 4 Lactose* Absorption Sucrose* Absorption Maltose* Absorption (Percentage absorption per 500 mg wet weight intestine) 7.7 ± 6.4 46.3 i 18.2 b 55.5 ± 51.2 a' c 72.1 ± 38.4 b' c 13.7 ± 6.1a 21.9 ± 4.4 72.5 ± 35.5 b 78.8 t 25.8 b' c 91.6 ± 20.9 b' c 20.5 t 4.9 a 10.4 ± 5.9 57.1 ± 26.8 b 75.8 ± 48.6 b' c 75.2 i 20.7 b' c 10.0 t 3.3a *Values are Means ± S.D. for the number of rats shown, in the second column. kRefed the 18% lactalbumin d i e t for 8 weeks a f t e r 8.5 weeks on the 0.5% lactalbumin low carbohydrate d i e t . aDoes not d i f f e r from the control group (P>0.05) . b D i f f e r s from the control group (P<0.05). cDoes not d i f f e r from the 0.5% lactalbumin low carbohydrate group (P 7-0.05) . 49 hydrate group as demonstrated by the large standard deviation of the mean. When the absorption of lactose, sucrose, and maltose in the rats fed the control and the 1% lactalbumin diets were compared, the absorption of a l l three disaccharides was s i g n i -f i c a n t l y higher in the 1% lactalbumin group than i n the controls. The absorption values i n the 1% lactalbumin group were 4 to 10 times higher than those i n the control 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 differences i n the absorption of lactose, sucrose and maltose between the 0.5% lactalbumin low carbohydrate group and the 0.5% l a c t a l -bumin high carbohydrate group. Sim i l a r l y , 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 differences between the absorption of the three disaccharides between the 0.5% lactalbumin low carbohydrate and the 1% lactalbumin group. However, the values for lactose, sucrose, and maltose absorption were con-s i s t e n t l y higher i n the 1% lactalbumin group than in the 0.5% lactalbumin low carbohydrate group. The mean value for lactose absorption for the 1% lactalbumin group was almost twice that of the 0.5% lactalbumin low carbohydrate group. The standard deviations of the means in the 1% lactalbumin group were large and 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 difference between these two groups was not demonstrated. The comparison of the absorption of lactose, sucrose, and maltose i n the group of animals fed the control d i e t (18% lactalbumin) and the repleted group (fed the 18% lactalbumin d i e t for 8 weeks af t e r 8.5 weeks of protein depletion) showed 50 that 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 differences in the absorption of the three disaccharides. 51 CHAPTER IV DISCUSSION In studying the e f f e c t of p r o t e i n - c a l o r i e malnutrition on i n t e s t i n a l disaccharidases in rats i t is extremely important to keep the animals on the protein d e f i c i e n t d i e t s for an extended period of time u n t i l they develop lesions associated with p r o t e i n - c a l o r i e malnutrition. According to Edozien (65), the changes associated with p r o t e i n - c a l o r i e malnutrition in rats include a marked loss of body weight, severe loss of h a i r , a marked decrease in plasma albumin concentration, decrease of p r o t e i n concentration i n the i n t e s t i n a l mucosa, f l u i d accumula-t i o n in the peritoneal cavity, and edema. In the present study the rats on the 0.5% lactalbumin d i e t s developed a l l of the above symptoms which indicates that the rats had been on the protein d e f i c i e n t d i e t long enough to be considered 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 d i f f e r -ences i n gross appearance, plasma albumin concentration and behaviour between the rats on the 0.5% lactalbumin diets and the 1% lactalbumin d i e t ( r e s t r i c t e d to 4 g of d i e t per day) were s i m i l a r to those reported by Edozien (65). Therefore i t was assumed that the rats on the 0.5% lactalbumin diets were pr o t e i n d e f i c i e n t and that the rats on the 1% lactalbumin d i e t (restricted) were prote i n - c a l o r i e d e f i c i e n t . Solimano et a l . (4) reported that protein deprivation i n r a t s produced an increase in the s p e c i f i c a c t i v i t i e s of i n t e s -t i n a l disaccharidases and p a r t i c u l a r l y of sucrase and maltase. 52 They suggested that t h i s increase in disaccharidase a c t i v i t i e s was an adaptation to the high proportion of carbohydrates in the protein d e f i c i e n t diets as the protein of the con t r o l d i e t was replaced with carbohydrate in order to produce the low protein d i e t s . The obtained results in the present study p a r t i a l l y support the hypothesis of Solimano et a l . (4) that a high proportion of carbohydrate in the protein d e f i c i e n t d i e t can induce i n t e s t i n a l disaccharidase a c t i v i t i e s since the d i s -accharidase l e v e l s in the rats fed the 0.5% lactalbumin high carbohydrate 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 increased when compared to the controls. However, the increase in the disaccharidase a c t i v i t i e s in the 0.5% lactalbumin high carbo-hydrate group was not e n t i r e l y due to the high proportion of carbohydrates in the d i e t because the rats on the 0.5% lactalbumin low carbohydrate d i e t also 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 increases in disaccharidase lev e l s when compared to the controls although not to the same extent as the 0.5% lactalbumin high carbohydrate group. I t i s not cle a r why there i s an increase i n the i n t e s t i n a l disaccharidase a c t i v i t i e s in the 0.5% lactalbumin low carbo-hydrate group when compared to the controls. One possible explanation for t h i s increase in disaccharidase levels could be a loss of s t r u c t u r a l proteins i n the i n t e s t i n a l mucosa. I f there had been a larger decrease of s t r u c t u r a l protein in the i n t e s t i n a l mucosa as compared to the decrease of enzymatic proteins, the expression of enzymatic a c t i v i t y per gram of protein or per gram of wet weight mucosa would lead to high r e s u l t s . 53 McNeill and Hamilton (69) recently studied the e f f e c t of fa s t i n g on i n t e s t i n a l disaccharidase a c t i v i t i e s in the r a t small intestine 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 -ase in lactase a c t i v i t y . They have postulated that f a s t i n g might have stimulated lysosomal beta-galactosidase. I f a protein 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 to fasting, the increase i n lactase a c t i v i t y in the protein d e f i c i e n t rats could possibly be a r e s u l t of the stimulation of the lysosomal beta-galactos idase. The r e s u l t s of the present study together with those of Solimano et a l . (4) d i f f e r from the findings by Prosper e t a l . (5) and L i f s h i t z e t a l . (6) in that they f i n d no marked alt e r a t i o n s in i n t e s t i n a l disaccharidase a c t i v i t i e s in protein d e f i c i e n t r a t s . I t should be stressed, however, that t h e i r experimental conditions were very d i f f e r e n t from the present i n v e s t i g a t i o n . In the study by Prosper et a l . (5) the t e s t animals were fed a protein-free d i e t for 45 days and the controls were given a 27% protein d i e t . In addition their findings should be considered as inconclusive on the basis of the f a c t that the disaccharidase a c t i v i t i e s were performed on only 3 protein d e f i c i e n t rats and that there was a large v a r i a t i o n in the disaccharidase a c t i v i t i e s within both the controls and the t e s t animals. L i f s h i t z et a l . (6) also did 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 in disaccharidase a c t i v i t y in protein d e f i c i e n t r a t s . They fed the protein d e f i c i e n t d i e t (4% casein) for 1 to 4 weeks, and at the end of 4 weeks the 54 mucosal protein concentration was not s t a t i s t i c a l l y d i f f e r e n t from the contro l s . This would imply that the i n t e s t i n a l mucosa had not yet undergone changes associated with protein deficiency because the rats were not kept long enough on the protein d e f i c i e n t d i e t . I t should be pointed out, however, that although there 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 difference in disaccharidase levels between the protein d e f i c i e n t rats and the controls in the study by L i f s h i t z et a l . (6), the disaccharidase a c t i v i t i e s were appreciably higher in the pr o t e i n d e f i c i e n t r a t s . There were no s i g n i f i c a n t differences between the i n t e s -t i n a l disaccharidase a c t i v i t i e s i n the 1% lactalbumin group and the 0.5% lactalbumin low carbohydrate group. Therefore the r e s t r i c t i o n of d a i l y intake d i d not have an e f f e c t on the a c t i v i t i e s of lactase, sucrase, and maltase in addition to the e f f e c t caused by protein depletion. This may be due to the fact that towards the end of the 9 week experimental period about one-third of the animals on the 1% lactalbumin d i e t were not eating the en t i r e 4 grams of d i e t which they were given each day. Therefore, near the end of the experi-mental period, these animals were no longer r e s t r i c t e d in th e i r c a l o r i c intake. The amount consumed by the 0.5% l a c t a l -bumin low carbohydrate group or the 1% lactalbumin group was not monitored. However, i t would appear that the f a i l u r e of some of the animals on the 1% lactalbumin d i e t ( r e s t r i c t e d to 4 grams per day) to consume the en t i r e portion of food did not a f f e c t the development of the c a l o r i c deficiency 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 concentrations, gross appearance and behavior c h a r a c t e r i s t i c s from the animals on the 0.5% lactalbumin low carbohydrate d i e t . The studies found in the l i t e r a t u r e dealing with the e f f e c t of c a l o r i c r e s t r i c t i o n on the i n t e s t i n a l disaccharidases did not r e s t r i c t the protein content of the d i e t to as low a l e v e l as i n the present experiment (1% lactalbumin). Kumar and Chase (59) found that the r e s t r i c t i o n of food at b i r t h by increasing the l i t t e r s i z e (16 animals per mother) followed by r e s t r i c t i o n of an 8% casein d i e t to 8 grams per day u n t i l 115 days of age resulted in s i g n i f i c a n t l y higher lactase a c t i v i t y in the r e s t r i c t e d intake group when compared to the controls who receive a 27% casein d i e t ad l i b i t u m . There were no differences in sucrase a c t i v i t y between the control and the r e s t r i c t e d intake group. Maltase a c t i v i t y was not determined. Tr o g l i a et a l . (56) used rats weighing between 90 and 110 grams who were then fed a r e s t r i c t e d amount of rat chow (an average of 10.5 grams of r a t chow per day) for 5% to 6% months. At the time of s a c r i f i c e , the a c t i v i t y levels of jejunal lactase, sucrase, and maltase were a l l s i g n i f i c a n t l y higher in the r e s t r i c t e d group than in the control group. The r e s u l t s of the present study cannot be compared d i r e c t l y with the studies of Kumar and Chase (59) or Troglia et a l . (56) as the protein r e s t r i c t i o n in the present study (1% lactalbumin) was much more severe. The s i m i l a r i t y of the r e s u l t s , however, indicate that the e f f e c t on the i n t e s -t i n a l disaccharidase a c t i v i t i e s of r e s t r i c t i n g the c a l o r i c intake of a 1% protein d i e t does not d i f f e r from the e f f e c t of 56 r e s t r i c t i n g the c a l o r i c intake of a d i e t containing higher protein l e v e l s . In the present study, the protein depleted animals gained weight very abruptly when placed on the 18% lactalbumin d i e t , and at the end of the 8 week feeding period, they, were not grossly distinguishable from the control animals. The plasma albumin concentration was almost i d e n t i c a l in the repleted and the control groups. The lactase and jejunal sucrase a c t i v i t i e s in the repleted group f e l l to the same level s of a c t i v i t y found in the control group, but the jejunal maltase and i l e a l sucrase and maltase a c t i v i t i e s f e l l to levels which were s i g n i f i c a n t l y lower than those found in the control group. The fac t that 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 returned to the same l e v e l as found in the control group suggests that severe protein depletion may cause pro-longed or i r r e v e r s i b l e changes in i n t e s t i n a l maltase and sucrase a c t i v i t i e s . The absorption of lactose, sucrose, and maltose was s i g n i f i c a n t l y enhanced in the protein d e f i c i e n t rats when compared to the controls (Table X) . This enhancement in the absorption of the disaccharides is consistent with the higher leve l s of jejunal disaccharidases in the protein d e f i c i e n t r a t s . When the 0.5% lactalbumin low carbohydrate and the 0.5% lactalbumin high carbohydrate groups were compared, s i g n i f i -cant increases in sucrase and maltase a c t i v i t i e s i n the high carbohydrate group were found. However, there were no d i f f e r -ences in the absorption of lactose, sucrose, or maltose 57 between the 0.5% lactalbumin low carbohydrate and the 0.5% lactalbumin high carbohydrate groups. Gray and Ingelfinger (10,11) had demonstrated that i n human subjects the r a t e -l i m i t i n g step in sucrose and maltose absorption is the trans-port of the constituent monosaccharides across the mucosal membrane, and not the l e v e l of i n t e s t i n a l sucrase and maltase a c t i v i t i e s . But i n the case of lactose absorption, i n t e s t i n a l lactase a c t i v i t y i s the r a t e - l i m i t i n g step and not the absorp-tion of the constituent monosaccharides (9). I t has been shown that eit h e r the r e s t r i c t i o n of dietary protein or semistarvation may i n i t i a l l y r e s u l t in an increased transport of glucose across the mucosal membrane (6,70,71). Therefore, i t i s possible that the protein deprivation increased the absorption of sucrose and maltose by a f f e c t i n g the rate of glucose transport. Lactose absorption would be increased in response to the increase i n lactase a c t i v i t y as that i s the r a t e - l i m i t i n g step in lactose absorption. The increase in the carbohydrate content of the 0.5% lactalbumin d i e t from 66% to 83.5% resulted in increases in sucrase and maltase a c t i v i t i e s but not in sucrose and maltose absorption. This would be expected i f the r a t e - l i m i t i n g step of sucrose and maltose is the transport of the constituent monosaccharides. Most of the studies done with laboratory animals on the e f f e c t of pro t e i n - c a l o r i e malnutrition on disaccharidase a c t i v i t i e s which have been reported in 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 of the i n t e s t i n a l disaccharidases without measuring the absorption of the disaccharides. Only L i f s h i t z et a l . (6) investigated both disaccharidase a c t i v i t i e s and absorption of disaccharides and monosaccharides in rats s u f f e r i n g from pr o t e i n - c a l o r i e malnutr t i o n . 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 differences in the absorption of glucose, fructose, 3-0-methyl glucose, sucrose, or maltose between the controls and the rats on the low protein diets a f t e r 28 days of dietary treatment. There were also no s i g n i f i c a n t changes in disaccharidase a c t i v i t i e s in the protein depleted animals a f t e r 28 days. There was a transitory increase in the rate of glucose, sodium, and water transport in the animals on the low protein d i e t a f t e r 14 days of dietary treatment, but there was no change in the transport of fructose or 3-0-methyl glucose during that period indicating that the change was probably due to increased glucose u t i l i z a t i o n in the rats on the low protein d i e t s . Similar findings of an i n i t i a l compensatory response to semi-st a r v a t i o n in the rat which resulted in an increase in glucose transport had been reported by Kershaw et a l . (70) and Hind-marsh et a l . (71). I t is d i f f i c u l t to compare the r e s u l t s obtained i n the present study with the r e s u l t s reported by L i f s h i t z et a l . (6) because of the differences in the length of time that the animals were subjected to the low protein diets (28 days i n the study of L i f s h i t z et a l . (6), and 60 days in the present study). However, the r e s u l t s of both studies indicate that disaccharide absorption i s not impaired in experimental pr o t e i n - c a l o r i e malnutrition in the r a t . The r e s u l t s obtained in the present investigation togethe with those reported by other authors (4,5,6,56,59) do not 59 support the view that disaccharide intolerance i n protein deprived children is the r e s u l t of decreased i n t e s t i n a l d i s -accharidases (2,30,32,37) i f we may apply the r e s u l t s for rats d i r e c t l y to ch i l d r e n . There are several possible explanations for the lack of agreement between the human and animal response to pr o t e i n - c a l o r i e malnutrition. When the animal models are being used to study the e f f e c t of protein deprivation on i n t e s t i n a l disaccharidase a c t i v i t y , the experimental conditions can be c l o s e l y c o n t r o l l e d . There-fore, the diets can be made adequate in a l l other nutrients besides protein, whereas the diets of the children in under-developed countries are frequently d e f i c i e n t in protein as well as other e s s e n t i a l nutrients. In addition, the mal-nourished children 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 of infections can, however, be cont r o l l e d in the animal experiment. Sriratanaban and Thayer (57) have studied the e f f e c t of iron and combined iron-protein deficiency on the i n t e s t i n a l disaccharidases in the r a t to help c l a r i f y this point. They used four groups of animals in t h e i r study: a control (27% casein), low protein ( 5 % casein), low iron (27% casein), and low iron-low protein (5% casein). The low iron group had s i g n i f i c a n t l y lower levels of lactase, sucrase, and maltase a c t i v i t y when compared to the controls. The protein d e f i c i e n t group had s i g n i f i c a n t l y higher disaccharidase a c t i v i t i e s when compared to the contro l s . The low iron-low protein group did 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 differences when compared to the controls, however, the disaccharidase 60 a c t i v i t i e s were somewhat higher than i n the controls in both the jejunum and ileum and lower than in the controls in the duodenum. These results indicate that other n u t r i t i o n a l d e f i c i e n c i e s may a f f e c t i n t e s t i n a l disaccharidase a c t i v i t i e s , and therefore, may be c l o s e l y involved with the observed decreases in i n t e s t i n a l disaccharidases in c h i l d r e n . This area needs further work. Bol i n et a l . (58) has also studied the e f f e c t of other factors besides protein deficiency on the i n t e s t i n a l disacchar-idases and found that an i n f e s t a t i o n of a group of adequately nourished rats with Nippostrongylus b r a s i l i e n s i s resulted in a decrease in the a c t i v i t i e s of lactase and maltase showing that i n f e s t a t i o n may be an important factor in the etiology of disaccharidase depression and disaccharide intolerance in p r o t e i n - c a l o r i e malnutrition. The study of the 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 malnutrition by Scrimshaw et a l . (72) has shown that malnutri-t i o n generally a l t e r s the resistance of the host to i n f e c t i o n and that infectious disease exaggerated e x i s t i n g malnutrition. The mechanisms involved in the decreased resistance to i n f e c -t i o n caused by malnutrition include a reduced capacity of the host to form s p e c i f i c antibodies, a decrease in phagocytic a c t i v i t y of microphages and macrophages, alterations in wound healing and collagen formation, a l t e r a t i o n s in tissue i n t e g r i t y , diminished inflammatory response, and e f f e c t s o r i g -inating in 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). Since i n f e c -t i o n and malnutrition are so c l o s e l y linked i t w i l l be necessary to f u l l y explore the r o l e of i n f e c t i o n in the development of 61 disaccharide intolerance i n pr o t e i n - c a l o r i e malnutrition before the difference between the results of the present study and the symptoms of children suffering from p r o t e i n -c a l o r i e malnutrition are understood. Another possible explanation for the difference between the r e s u l t s obtained with rats and with children was suggested by Kumar and Chase (53). They f e l t that the f a i l u r e of exper-iments on pro t e i n - c a l o r i e malnutrition using rats to show a decrease in disaccharidase a c t i v i t i e s may mean that the r a t is not an adequate model for studying human pr o t e i n - c a l o r i e malnutrition. When these authors studied p r o t e i n - c a l o r i e malnutrition in monkeys, many of the same symptoms found in children suffering from protein-calorie malnutrition were present in the monkeys including marked v i l l o u s atrophy and decreased i n t e s t i n a l disaccharidase a c t i v i t i e s a f t e r 18 weeks of protein deprivation {<Cl% p r o t e i n ) . The undernourished monkeys also suffered from intermittent diarrhea of an unknown cause. From these r e s u l t s , Kumar and Chase (53) concluded that the monkey was a better model for studying protein-calorie malnutrition than the r a t . The rat cannot be excluded as a suitable model for p r o t e i n - c a l o r i e malnutrition u n t i l further work has been done on the e f f e c t of the non-protein factors including infection 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 . 62 CHAPTER V SUMMARY AND RECOMMENDATIONS A. Summary The purpose of the present investigation was to study the e f f e c t of prolonged experimental p r o t e i n - c a l o r i e malnutrition on i n t e s t i n a l disaccharidase a c t i v i t i e s and on disaccharide absorption, as carbohydrate intolerance is a major problem in children 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. Four groups of rata (90 to 120 grams) were fed the follow-ing diets for 8 to 9 weeks: control (18% lactalbumin, 66% carbohydrate); low protein low carbohydrate (0.5% lactalbumin, 66% carbohydrate); low protein high carbohydrate (0.5% l a c -talbumin, 83.5% carbohydrate); and low protein r e s t r i c t e d (1% lactalbumin, r e s t r i c t e d to 4 grams per day). After 8.5 weeks, part of the group on the 0.5% lactalbumin low carbohydrate d i e t was fed the control d i e t (18% lactalbumin, 66% carbohydrate) for 8 weeks. At the end of the feeding period, the following assays were performed: 1) in vivo absorption of radioactive (^C) lactose, sucrose, and maltose; 2) a c t i v i t i e s of i n t e s t i n a l lactase, sucrase, and maltase; 3) plasma albumin concentrations ,-and 4) mucosal protein concentrations. The disaccharide absorptions were expressed as the percentage of the administered r a d i o a c t i v i t y absorbed per unit weight of i n t e s t i n e . The d i s -accharidase a c t i v i t i e s were expressed as the units of enzyme a c t i v i t y per gram of wet weight mucosa or per gram of mucosal protein; the l a t t e r was considered to be the more s a t i s f a c t o r y In the three protein d e f i c i e n t groups (0.5% lactalbumin low carbohydrate, 0.5% lactalbumin high carbohydrate, and 1% lactalbumin), the a c t i v i t y of both the jejunal and i l e a l d i s -accharidases and the absorption of lactose, sucrose, and mal-tose were s i g n i f i c a n t l y higher when compared with the controls The jejunal 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 in the 0.5% lactalbumin high carbohydrate group than i n the 0.5% lactalbumin low carbohydrate group, but the absorp-t i o n of lactose, sucrose, and maltose were a l i k e . When the 1% lactalbumin ( r e s t r i c t e d to 4 grams per day) and the 0.5% lactalbumin 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 differences in the s p e c i f i c a c t i v i t i e s of the i n t e s t i n a l disaccharidases or the absorption of the disaccharides. The absorption of lactose, sucrose, and maltose were si m i l a r i n the controls and the protein repleted group. The disaccharidase a c t i v i t i e s were also s i m i l a r in these two groups except for a s i g n i f i c a n t depression of jejunal maltase and i l e a l sucrase and maltase a c t i v i t i e s i n the protein repleted group. Therefore, these results indicate that protein depriva-t i o n in rats for 8.5 weeks causes an increase in s p e c i f i c a c t i v i t i e s of the i n t e s t i n a l disaccharidases in both the jejunum and ileum, and that an increase i n the carbohydrate content of the protein d e f i c i e n t d i e t r e s u l t s in an induction of jejunal sucrase and maltase a c t i v i t i e s . The high s p e c i f i c a c t i v i t y of the i n t e s t i n a l disaccharidases following protein-6 4 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 r a t h e r than t o an in c r e a s e 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 r e s u l t s o b tained on the p r o t e i n r e p l e t e d r a t s have shown t h a t the changes caused by p r o t e i n d e f i c i e n c y are of a r e v e r s i b l e n a t u r e . 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 . The i n c r e a s e s i n d i s a c c h a r i d e absorp-t i o n c o u l d be due t o the higher l e v e l s o f d i s a c c h a r i d a s e s or 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 mono-sa c c h a r i d 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 . 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 mal-tase 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 and 0.5% l a c t a l b u m i n low carbohydrate groups, without a con-comitant i n c r e a s e i n sucrose or maltose a b s o r p t i o n , supports the view t h a t the hi g h e r a b s o r p t i o n o f maltose and sucrose 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 mono-s a c c h a r i d e t r a n s p o r t . The r e s u l t s o b t a i n e d i n t h i s study are not c o n s i s t e n t w i t h the s u g g e s t i o n t h a t 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 r e s p o n s i b l e f o r d i s a c c h a r i d e i n t o l e r a n c e i n c h i l d r e n . Since 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 d e f i c i e n c y are u s u a l l y a l s o d e f i c i e n t i n other n u t r i e n t s and s u f f e r i n g from i n f e c t i o n s , i t i s not easy t o r e l a t e d i s a c c h a r i d e i n t o l e r a n c e and depressed l e v e l s o f d i s a c c h a r i d a s e s t o p r o t e i n - c a l o r i e d e f i c i e n c y o n l y . I t i s p o s s i b l e , however, t h a t t h e r e may be a s p e c i e s d i f f e r e n c e i n the response t o p r o t e i n - c a l o r i e d e f i c i e n c y . 65 B. Recommendations The following recommendations should be considered i f further work i s to be done on the e f f e c t of p r o t e i n - c a l o r i e malnutrition on the i n t e s t i n a l disaccharidases and the in  vivo absorption of disaccharides. Absorption studies using glucose should be carried out together with the studies of lactose, sucrose, and maltose absorption, in order to confirm whether the increases i n absorption of the three disaccharides is due to increased disaccharidase a c t i v i t i e s or due to increased glucose trans-port. The rate of absorption over a 30 minute period should be determined rather than the t o t a l absorption a f t e r 15 minutes, as the rate of absorption is a more s a t i s f a c t o r y measure for studying the absorption capacity of the small i n t e s t i n e . In the present study, the disaccharide absorption was only expressed per unit weight of i n t e s t i n e . Since there i s a difference i n the diameter of the intestine and the t h i c k -ness of the i n t e s t i n a l mucosa between the larger control animals and the smaller protein depleted animals, i t would be more s a t i s f a c t o r y to express the absorption r e s u l t s per u n i t of surface area of the i n t e s t i n e . The expression of disaccharidase a c t i v i t y per u n i t of DNA would c l a r i f y whether there was a change in the r a t i o of the s t r u c t u r a l to enzymatic proteins in the i n t e s t i n a l mucosa of the protein d e f i c i e n t r a t s . I t would be informative to study the e f f e c t of protein-c a l o r i e malnutrition on the i n t e s t i n a l disaccharidases and 66 disaccharide absorption as a function of time. This would allow one to determine the e a r l i e s t time at which the i n t e s -t i n a l disaccharidase a c t i v i t i e s and disaccharide absorption increase, and the temporal relationships between the increases. I t would also demonstrate whether there were further changes in the i n t e s t i n a l disaccharidase a c t i v i t i e s or disaccharide absorption as the animals become moribund. 67 CHAPTER VI BIBLIOGRAPHY 1. Cook, G.C, and F.D. Lee. The jejunum afte r kwashiorkor. Lancet 2:1263-1267, 1966. 2. Stanfield, J.P., M.S.R. Hutt and R. T u n n i c l i f f e . Intes-t i n a l biopsy i n kwashiorkor. Lancet 2:519-523, 1965. 3. Bowie, M.D., G.O. Barbezat and J.D.L. Hansen. Carbohydrate absorption in malnourished c h i l d r e n . Am. J . C l i n .  Nutr. 20:89-97, 1967. 4. Solimano, G., E. A. Burgess and B. Levin. Protein-calorie malnutrition: e f f e c t of d e f i c i e n t d iets on enzyme le v e l s of jejunal mucosa of r a t s . B r i t . J . Nutr. 21:55-68, 1967. 5. Prosper, J., R.L. Murray and F. Kern, J r . Protein starva-t i o n and the small i n t e s t i n e . I I . Disaccharidase a c t i v i t i e s . Gastroenterology 55:223-228, 1968. 6. L i f s h i t z , F., R. L. Hawkins, S. 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