CERTAIN STUDIES ON THE DIGESTIVE ENZYME SYSTEMS (SUCRASE, MALTASE AND LACTASE) OF THE SMALL INTESTINE OP THE WISTAR RAT by ROBERT JOHN BOSE B.S.A., The U n i v e r s i t y of B r i t i s h Columbia, 1955 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OP SCIENCE IN AGRICULTURE i i i the d i v i s i o n of Animal Science We accept t h i s t h e s i s as conforming to the required standard Members of the D i v i s i o n THE UNIVERSITY OF BRITISH COLUMBIA October,1957. i i -II. ABSTRACT The primary objective of this study was to establish the nature and extent of the changes with age in sucrase, maltase and lactase activity in the small intestine of the laboratory Wistar rat, raised on a basal diet. The author was, in addition, interested in the possibility of these changes being brought about by the presence or absence of certain specific dietary factors. Analysis have shown marked changes in the activities of these three enzymes with advancing age in Wistar rats weaned at 21 days on to a basal diet. Lactase activity was found to remain at a high level from 18 to 20 days during which time a sharp and uniform drop in activity was indicated. The extent of this drop was found to be great, approximately 80 per cent of the pre-weaned level . In contrast sucrase activity of the small intestine in the pre-weaned rat was negligible and underwent a sharp increase in activity at the weaning age of 21 days. Maltase activity in the pre-weaned Wistar rat was appreciable and was found to increase significantly at weaning. Early weaning at 15 days brought about an earlier decline in lactase activity and a corresponding early rise in sucrase and maltase activities. When early weaning was immedi-ately followed by the feeding of condensed milk no apparent maintenance of lactase activity was noted. Similarily when older rats, 28 days of age, were fed condensed milk no increase - i i i -i n lactase a c t i v i t y was apparent. Neither the condensed milk nor basal d i e t s induced apparent d i f f e r e n c e s on the e f f e c t of ea r l y weaning on sucrase and maltase a c t i v i t y . Apparent maintenance of lactase a c t i v i t y above those l e v e l s demonstrated i n r a t s weaned at 21 days on to a basal d i e t was accomplished by f o s t e r i n g 16 day o l d r a t s on dams which had l i t t e r e d from four to f i v e days p r i o r to t h i s t r a n s f e r and which were assumed to be l a c t a t i n g at a somewhat greater rate than had the o r i g i n a l mothers. I t was noted however that t h i s f o s t e r i n g process had no apparent influence on the extent of the changes i n sucrase and maltase a c t i v i t i e s . The feeding of p u r i f i e d sugar d i e t s containing two d i f f e r e n t l e v e l s of each of the three sugars, sucrose, maltose and l a c t o s e , had l i t t l e e f f e c t on any of the three carbohydrases studied. Growth rates of the r a t s on these p u r i f i e d d i e t s showed marked d i f f e r e n c e s . The author suggests that the maintenance of l a c t a s e a c t i v i t y might be associated with the presence of an inductive component present i n the milk of the f r e s h l y l a c t a t i n g dam, a component not n e c e s s a r i l y present i n the milk of the l a t e r period of l a c t a t i o n , and not n e c e s s a r i l y a component of processed cow's milk. The absence of an adaptive lactase response to the feeding of lactose i n t h i s study and those of other workers suggest that lactose i t s e l f i s u n l i k e l y t h i s inducer. No adap-t i v e response to substrates could be demonstrated f o r e i t h e r sucrase or maltase. I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Hea'el\of my Department o r by h i s r e p r e s e n t a t i v e . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s for// f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . ROBERT JOHN BOSE Department o f THE nTVTRTnw on- awTMar. ^r.rjmr.v, The U n i v e r s i t y o f B r i t i s h C olumbia, Vancouver 8, Canada. Date .TTTT.V jc\ 1957 I. ACKNOWLEDGEMENT The w r i t e r wishes to thank Dean Blythe Eagles, Chairman of the D i v i s i o n of Animal Science, f o r providing the f a c i l i t i e s with which to conduct t h i s experiment. Sincere appreciation i s expressed to Dr. W. D. K i t t s , A s s i s t a n t Professor of Animal Husbandry, f o r sug-gesting t h i s problem and fo r h i s d i r e c t i o n , assistance and c r i t i c i s m during the course of t h i s study. Thanks are expressed to Dr. A. J . Wood and to Dr. J . J . R. Campbell f o r t h e i r advice and c r i t i c i s m s . TABLE OP CONTENTS PAGE I . ACKNOWLEDGEMENT i I I . ABSTRACT i i III. INTRODUCTION 1 IV. HISTORICAL 3 A. Sucrase 3 B. Maltase 5 C. Lactase 7 D. General 8 V. METHODS AND MATERIALS 15 A. Methods 15 1. Housing of the Experimental Animals.. 15 2. Sacrifice and Dissection of the Animals..... 15 3. The Storage of Tissue Samples 16 4. Preparation of the Tissue Homogenates 16 5. Moisture Determination 17 6. Nitrogen Determination 17 7. The Determination of Enzyme Act i v i t y 18 B. Materials 23 1. The Basal Diet 23 2. The Condensed Milk Diet 24 3. The Synthetic Diets .. 25 4. Copper Solution 26 5. Perric Sulfate Solution (According to Bertrand) 26 6. Potassium Permanganate Solution 26 7. Sugar Solutions 26 8. Buffer Solutions 27 VI. RESULTS AND DISCUSSION 28 A. Optima pH for Sucrase, Maltase and Lactase Activity 28 B. The H-ion Concentration of the Small Intestine of the Rat at Various Ages 31 C. The Relationship Between Homogenate Nitrogen Content and Enzyme A c t i v i t y 34 TABLE OF CONTENTS (Continued) PAGE D. The E f f e c t of I n a n i t i o n on Sucrase, Maltase and Lactase A c t i v i t y 37 E. Sucrase, Maltase and Lactase A c t i v i t y From B i r t h to 72 Days of Age. i n the Small Intestine of the Wistar Eat Weaned on to a Basal Diet at 21 Days 39 F. The E f f e c t on Sucrase, Maltase and .. Lactase A c t i v i t y of E a r l y Weaning and the Feeding of Condensed Milk and a Basal Diet at Various Ages i n the Laboratory Wistar Rat 49 G. The E f f e c t on Sucrase, Maltase and Lactase A c t i v i t y of the Small I n t e s t i n e of the Young Wistar Rat of Prolonged Suckling on Foster Dams 57 H. The E f f e c t on Sucrase, Maltase and Lactase A c t i v i t y of Various Levels of the Three Sugars, Sucrose, Maltose and Lactose i n a P u r i f i e d Synthetic Diet .for the Laboratory Wistar Rat. VII. SUMMARY AND CONCLUSIONS 71 VIII. BIBLIOGRAPHY 74 I l l . INTRODUCTION Digestion i n i t s broad sense r e f e r s to the mechanical and chemical degradation of complex d i e t a r y constituents to forms that are s u i t a b l y prepared f o r the absorptive processes of the alimentary t r a c t . The rate at which any animal "breaks down" i t s complex d i e t a r y i s n e c e s s a r i l y a f u n c t i o n of many mechanical and chemical processes. The composition of a mammal's d i e t a r y changes markedly from b i r t h to adult l i f e . The young ingests almost e x c l u s i v e l y i t s mother's milk, the mature or post weaned animal u t i l i z e s e s s e n t i a l l y none of t h i s food f o r i t s d i e t a r y needs. In view of the many hi g h l y s p e c i f i c d i g e s t i v e mechanisms, i t seems reasonable to conclude that accomodation f o r t h i s changing d i e t a r y must n e c e s s a r i l y be made f o r by associated changes i n the d i g e s t i v e complex of the animal. A great number of i n v e s t i g a t o r s have described the numerous enzymes or ferments present i n the various regions of the alimentary t r a c t . Much work has been conducted regarding the d i g e s t i b i l i t y of various d i e t a r y constituents f o r a wide group of species. There i s however, only scanty information on the quantitative measurement of the d i g e s t i v e enzymes. In t h i s i n v e s t i g a t i o n the three carbohydrases, sucrase ( <*-D-glucosidoinvertase), maltase ( oC-D-glucopyranosidase) and lactase (y3 -D-galactosidase) have been assayed q u a n t i t a t i v e l y i n the small i n t e s t i n e of the Wistar r a t at various ages. In addit i o n , experiments have been conducted to demonstrate the presence or absence of enzymatic adaptation to d i e t a r y changes i n these three carbohydrases present i n t h i s d i g e s t i v e organ of the laboratory r a t . The Wistar r a t was employed i n t h i s experiment because of i t s r a p i d growth rate, genetic uniformity, low cost and i t s a v a i l a b i l i t y i n large numbers. The p r a c t i c a l a p p l i c a t i o n s a r i s i n g from information regarding the nature of the changes i n the d i g e s t i v e complex of the laboratory r a t would be the extrapolation of these fundamental p r i n c i p l e s to the feeding of domestic animals such as the p i g , sheep and ox. Inherent changes i n the elaboration of l a c t a s e would necessitate the feeding of lactose at d e f i n i t e ages i n a manner p a r a l l e l i n g the production of t h i s h y d r o l y t i c enzyme i n the di g e s t i v e t r a c t . I f the elaboration of d i g e s t i v e enzymes i s l a r g e l y c o n t r o l l e d by the presence of t h e i r s p e c i f i c substrates i n the ingesta the feeding of starch, f o r example, to young da i r y calves could substitute f o r the more c o s t l y milk d i e t commonly fed these animals. IV. HISTORICAL A b r i e f d e s c r i p t i o n of the three carbohydrases i s f e l t appropriate as an in t r o d u c t i o n to the di s c u s s i o n of the l i t e r a t u r e . A. Sucrase; Sucrase has been known the longest of a l l the carbo-hydrases. In 1828 Dumas and Boullay (14) described the uptake of water during the fermentation of sucrose by yeast. Sucrase has been termed invertase, i n v e r t i n and saccharase. Two types of invertase are described, a fructosidase attacking the fructose end, and a glucosidase attacking: the glucose end of the sucrose molecule (44). Both types are found i n c e r t a i n yeasts. The invertebrates and vertebrates possess the glucoinvertase e x c l u s i v e l y . Sucrase i s reported absent from the s a l i v a and g a s t r i c secretions of mammals (31,7)• I t s presence i n the small i n t e s t i n e of the foetus and s t i l l - b o r n has been reported by Keene et a l (27) and Tachibana (45). Neuberg and Mandl (44) suggest that the elaboration of i n t e s t i n a l sucrase i s inde-pendent on the presence of food i n the i n t e s t i n a l t r a c t . The rough l o c a t i o n of sucrase i n the i n t e s t i n a l structure has been described by Rohmann (42). Figure A represents the path of degradation of sucrose by sucrase. - 4 -CHo0H i d. Sucrase HOH CK -D-glucopyranosido--D-fructofuranoside (sucrose) CH~OH o( - D-glucose p -D-fructose FIGURE A. THE HYDROLYSIS OF SUCROSE BY SUCRASE - 5 -B. Maltase: The enzymatic hydrolysis of maltose to glucose was f i r s t described by Brown and Heron (7) using pancreatic extracts and d r i e d i n t e s t i n a l wall preparations of the p i g . Emil Fi s c h e r (15) > i u 1894, described an extract from d r i e d brewers yeast active not only toward maltose, but also toward methyl-o( -D-glucoside; he termed t h i s enzyme "yeast maltase" (16). Maltase i s widely d i s t r i b u t e d throughout the animal and vegetable kingdoms, u s u a l l y i n as s o c i a t i o n with amylase (44). In mammals maltase has been demonstrated mainly i n preparations of the i n t e s t i n a l t r a c t and blood serum (44). The crypts of Lieberkuhn of the i n t e s t i n a l mucosa are believed to be the s i t e of maltase elaboration i n the i n t e s t i n a l t r a c t . The term maltase r e f e r s to a wide group of enzymes capable of hydrolysing i n ad d i t i o n to t h e i r b i o l o g i c a l substrate maltose, a number of maltose d e r i v a t i v e s and some a l k y l and a r y l o(-D-glucopyranosides (44). Figure B shows the course of degradation of maltose by maltase to two glucose molecules. - 6 --D-glucose FIGURE B. THE HYDROLYSIS OF MALTOSE BY MALTASE - 7 -C. Lactase; Lactase, the enzyme responsible f o r the h y d r o l y t i c cleavage of lactose to glucose and galactose, i s found i n d i f f e r e n t s t r a i n s of b a c t e r i a as well as i n the seeds of a number of plants such as coffee and a l f a l f a (44-). Figure ;C shows the course of the re a c t i o n mentioned above. CH-OH CH~OH Lactase HOH 4- -p-D galactopyranosyl-/^ -D-glucopyranoside (lactose) P -D-galactose FIGURE C. THE HYDROLYSIS OF p -D-glucose LACTOSE BY LACTASE - 8 -D. General L i t t l e literature i s available regarding the quanti-tative measurement of sucrase and maltase a c t i v i t y i n mammals of various ages. Somewhat more information i s available on the presence of lactase i n animals. Early reviews on lactase i n mammalian tissue have been presented by Plimmer (37) and Oppenheimer (35) and later by Cajori (8). In 1899 Weinland (46) described the presence of lactase i n the intestine of the adult rabbit and the fowl when fed lactose. The intestine of adult foul and rabbit fed a lactose-free diet were reported inactive towards the hydrol-ysis of lactose. Portier and Bierry (38) were unable to show consistent lactase adaptability of the duck intestine to the feeding of lactose. Weinland (46), Dastre (13) , Rohmann and Lappe (40) and Fischer and Niebel (17), have described the presence of lactase i n the intestine of the dog, c a l f , ox and horse. Plimmer (37) i n 1906 concluded that i n general the young of most species possess lactase a c t i v i t y i n their intes-t i n a l tissues, while the adult possesses l i t t l e or none of this ferment. The presence of lactase i n the small intestine of young rabbits, oxen, sheep, young and old dogs, young goats and the new-born child was shown i n 1899 by Weinland (46). Adult rabbits, oxen, sheep and fowl possessed l i t t l e or none of this ferment. Caoori (8) observed that water extracts of dog intestinal mucosa are less active than the unextracted tissue and that the succus entericus, the mucous secretions of the intestinal tissue present in the lumen of the gut of the foetus, exhibits only slight activity suggesting that lactase is intimately associated with the mucosal cel ls . Cajori in the same report suggests that the major digestive action, resulting from the elaboration of enzymes in the small intestine would then occur intracellularly or in direct contact with the mucosa. He also observed that maximum enzymatic activity was obtained when fresh, finely ground tissue was used. Jejunal mucosa revealed a 10 to 30 per cent greater lactase activity than duodenal tissues (8). Lactase activity, as determined by Caoori (8) was less than the sucrase and maltase activity of the same tissues in. the dog, as reported by Rohmann and Nagano (41). Cajori (8) concluded that sufficient lactase activity was found to account for almost the complete hydrolysis of lactose i n -jected into the thiry loop of the jejunum. He suggests that the concentration of lactose in the loop in these experiments is somewhat greater than would be the case during ordinary feeding, and that digestion in the small intestine functioning in situ would be somewhat greater. In 1906 Plimmer (37) reported that neither the frog nor the fowl have lactase in their intestinal tracts. He concluded that animals lower than mammals do not possess this ferment. He also stated that of the mammals, the carnivora and omnivora have lactase present throughout their l i f e period, and that the herbivora only when they are young. Plimmer (37) observed no increase in lactase activity in the intestinal - 10 -tis s u e of the ra b b i t and r a t r e s u l t i n g from the feeding of milk and lac t o s e , and also that r a t embryos two days prepart-urium showed no a c t i v i t y while those of 12 hours preparturium showed lacta s e a c t i v i t y . Lactase a c t i v i t y of the pancrease, mucous membranes of the v e n t r i c l e s , small i n t e s t i n e and colon was examined by Heilskov (25) i n 14 cows, 18 r a b b i t s and seven human foetuses. Lactase a c t i v i t y i n cows of a l l ages decreases i n a d i s t a l d i r e c t i o n i n the small i n t e s t i n e , and that the pancreas and the mucous membranes of the colon and v e n t r i c l e do not contain l a c t a s e . I t i s fu r t h e r seen that l a c t a s e a c t i v i t y i n a l l sec-t i o n s of the small i n t e s t i n e decreases with age. A f t e r the age of eight months lactase i s only i n c o n s i s t a n t l y demonstrated i n cows. The r e s u l t s of the examination of 18 r a b b i t s shored e s s e n t i a l l y the same as i n the cow. Lactase was only found i n the mucous membrane of the small i n t e s t i n e i n the r a b b i t . The a c t i v i t y of t h i s enzyme i s found to decrease g r e a t l y a f t e r the age of four weeks. Heilskov (26) permitted eight r a b b i t s to suckle t h e i r mothers to four weeks of age a f t e r which h a l f t h i s number received a la c t o s e - f r e e r a b b i t r a t i o n , the remaining were fed i n a d d i t i o n to t h i s r a t i o n 100 g. milk and ten g. lactose d a i l y , from four to 15 weeks of age. No increase i n lactase a c t i v i t y was found r e s u l t i n g from the lactose feeding. Heilskov (26) suggests that the lactas e a c t i v i t y of the i n -t e s t i n a l contents of the jejunum and duodenum of the r a b b i t and c a l f to be of the same order of magnitude as the a c t i v i t y per g. of mucous membrane of the small i n t e s t i n e of these same animals. animals - 11 -Ammon and Henning (1) stated that the duodenal secretions of 40 human patients showed no a c t i v i t y , or so l i t t l e that i t was without s i g n i f i c a n c e f o r the process of d i g e s t i o n of sucrose, maltose and l a c t o s e . They suggested that the enzymatic hydrolysis of these three disaccharides takes place only a f t e r absorption i n t o the c e l l s of the i n t e s t i n a l mucosa. The recent work of B a i l e y et a l (2) describes i n additio n to l a c t a s e , sucrase and maltase a c t i v i t i e s i n the small i n t e s t i n e of the p i g . Lactase a c t i v i t y i s reported to increase from b i r t h to one week followed by a r a p i d decline from two to four weeks and remained at a low l e v e l to weaning at 50 days. In contrast sucrase and maltase a c t i v i t i e s are reported to be n e g l i g i b l e at b i r t h and to increase r e g u l a r l y to weaning. Data are not presented f o r post weaning sucrase, maltase and lactase a c t i v i t i e s . In an e a r l i e r communication K i t t s et a l (28) report marked increases i n the presence of pancreatic amylase i n the p i g from b i r t h to 40 days. Meyer and Necheles (54) reported age changes i n alimentary enzymes i n the human a f t e r the age of 16 years. S a l i v a r y and pancreatic amylase, pancreatic l i p a s e , t y p s i n and pepsin are a l l reported to decline i n a c t i v i t y with advancing age. F i s c h e r and Sutton (18) reviewed the e f f e c t s of l a c -tose on g a s t r o - i n t e s t i n a l m o t i l i t y . They (18) postulated a hydrogogue e f f e c t of lactose on the contents of the small - 12 -i n t e s t i n e ; i n other words the high molecular weight and non-d i f f u s i b l e nature of lactose i n i t i a t e d the flow of body water into the area of high osmotic pressure set up by the presence of lact o s e i n the gut lumen. They (18) pointed out that an animal may adapt to lactose feeding, that i s diarrhea may become l e s s severe and may cease e n t i r e l y i f the animal con-tinues to ingest large amounts of lactose over a per i o d of time. M i t c h e l l (36) noted that a c i d u r i c i n t e s t i n a l m i c r o f l o r a produced by lactose feeding tended to revert to p u t r i f a c t i v e types as a r e s u l t of continuous lactose feeding. F i s c h e r and Sutton (18) suggest that t h i s might be due to a greater u t i l i z a t i o n of lactose by way of an enhanced production of l a c t a s e . F i s h e r et a l (19) reported the f i n d i n g s of two separate experiments on the e f f e c t of lactose feeding on l a c t a s e production. Groups of r a t s were fed f o r a s i x month period on 31*6 per cent lactose d i e t s supplied from two sources; the f i r s t as f a t - f r e e d r i e d milk, the second as U.S.P. lactose along with a s u i t a b l e corn starch supplement. In the second experiment three groups of r a t s were fed 67 per cent corn starch, 15 per cent U.S.P. l a c t o s e ; and 52 per cent corn starch, and 35 per cent U.S.P. la c t o s e ; and 32 per cent corn starch and 55 per cent U.S.P. la c t o s e . These workers concluded that any s i g n i f i c a n t l y greater lactase a c t i v i t y demonstrated i n response to lactose feeding i s m i c r o b i o l o g i c a l rather than mucosal i n o r i g i n . Both experiments f a i l e d to demonstrate lactase a d a p t a b i l i t y to lactose feeding. F i s c h e r and Sutton (20) reported the e f f e c t of previous lactose feeding upon i n t e s t i n a l absorption of lactose i n the r a t . Lactose was - 13 -found to be absorbed more r a p i d l y from the i n t e s t i n e of r a t s previously fed lactose d i e t s . Rats adapted to lac t o s e feeding within s i x weeks; that i s diarrhea became infrequent. More rec e n t l y Fischer (21) reported on the growth stimulating e f f e c t of lactose on the small i n t e s t i n e of the r a t . Lactose feeding not only increased the small i n t e s t i n e weight per u n i t body weight, but also the lactase a c t i v i t y of the small i n t e s t i n e mucous membrane i n terms of i t s nitrogen content. This i s however not i n agreement with l a t e r experimental work (22) published by the same author i n which she concluded that lactase a c t i v i t y was increased on a u n i t body weight bas i s , but that 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 increase could be demon-strat e d when the h y d r o l y s i s of lactose was expressed i n terms of the nitrogen content of the t i s s u e studied. This example of enzymatic adaptation, as pointed out by F i s c h e r (22) does not however, s a t i s f y the c r i t e r i a of enzymatic adaption defined f o r m i c r o b i o l o g i c a l systems. Bainbridge (9) i n j e c t e d extracts from the mucous membrane of animals fed lactose i n t o animals maintained on a l a c t o s e - f r e e d i e t . Pancreatic l a c t a s e was s a i d to be induced by t h i s technique i n the l a t t e r animals. Carnevale et a l (9) reported the measurement of l i p o l y t i c , amylolytic and p r o t e o l y t i c a c t i v i t y of the duodenal secretions of the suckling, premature and f u l l term i n f a n t to be consistent and independent of stage of development within t h i s age range. Koehler and A l l e n (29) reported that lactose ad-ministered subcutaneously was to a large degree eliminated i n the urine. Rats r a i s e d on sucrose and lactose r e s t r i c t e d - 14 -sugar d i e t s showed good gains on sucrose and poor gains on lactose (29). The n u t r i t i v e value of glucose, l a c t o s e , d e x t r i n and corn starch f o r the newborn p i g was studied using synthetic milk d i e t s by Becker et a l (4). In pigs from seven to 35 days of age equal body weight gains were produced by a l l four carbo-hydrates. Pigs at one and two days of age responded to sucrose feeding with severe diarrhea, r a p i d weight l o s s , u n t h r i f t i n e s s , thinness and death. Neither lactose nor corn starch caused diarrhea. Starch i s reported to r e t a r d s a t i s f a c t o r y growth of two-day o l d pigs ( 1 0 ) . Lloyd et a l suggested that marked changes i n the d i g e s t i v e a b i l i t y of young pigs takes place p r i o r to eight weeks of age ( 3 0 ) . Cunningham and Brisson (11) observed that two-day o l d pigs received no b e n e f i t from supplementing various p u r i f i e d starch-containing d i e t s with amylolytic enzymes. These authors suggested that the f a i l u r e of enzyme supplementation may be due to the p o s s i b l e dena-t u r a t i o n of the ingested enzymes by the secretions of the stomach or the f a i l u r e of the baby p i g to elaborate s u f f i c i e n t maltase to complete the course of degradation. However, these authors suggested from further study (12) that the baby p i g i s capable of u t i l i z i n g some maltose at b i r t h and may be r a i s e d almost as well on maltose as on glucose from one day of age. - 15 -V. METHODS AND MATERIALS A. Methods 1. Housing of the Experimental Animals. In a l l cases laboratory Wistar r a t s were obtained from the Central Depot f o r Experimental Animals, U.B.C. Water and feed were supplied ad l i b i t u m to the animals held i n s i x inch by twelve inch enamel-lined freezer t r a y s . The basal d i e t , U.B.C.-10-55, was supplied as a p e l l e t of a diameter of one inch, and as such was offered loose i n the bottom of the enamel-lined trays along with wood shavings which served as a bedding material. L i q u i d d i e t s were offered ad l i b i t u m i n one h a l f p i n t j a r s , f i t t e d with rubber stoppers and glass d e l i v e r y tubes. S o l i d d i e t s , other than the basal d i e t , were off e r e d i n open 6 oz. glass j a r s placed within the f r e e z e r t r a y s . Depending upon the si z e of animals one to ten Wistar r a t s were placed i n each freezer tray. Water was supplied f r e s h d a i l y and the shavings were renewed twice weekly. 2. S a c r i f i c e and D i s s e c t i o n of the Animals. A uniform procedure of t i s s u e sampling was employed throughout. A l l animals were s a c r i f i c e d without anaesthesia. The animal was f i r s t stunned by d e l i v e r i n g a sharp blow to i t s head. Bleeding followed by c u t t i n g the great vessels of the neck, and allowing the animal to hang with i t s head i n a lowered p o s i t i o n . Bleeding i n t h i s manner lessened to a large - 16 -extent the presence of blood i n the mesenteric t i s s u e . The small i n t e s t i n e was removed by making a s i n g l e i n s c i s s i o n i n the v e n t r a l p e r i t o n e a l w a l l , and separating the t r a c t at the p y l o r i c and l i e o - c e c a l junctions. A l l mesenteric t i s s u e and f a t t y deposits were c a r e f u l l y removed from the small i n t e s t i n e . The i n t e s t i n a l contents were removed by gently f o r c i n g them along the lumen of the t r a c t with the rounded edge of a p a i r of d i s s e c t i o n s c i s s o r s . The small i n t e s t i n e of the youngest animals were too f r a g i l e , and the lumen too small to handle under a stream of running water; f o r t h i s reason the contents were not washed out. Carried along with the exuded lumen contents, was found a variable quantity of mucus and t i s s u e debris. H i s t o l o g i c a l considerations (32) suggest that the c e l l s concerned with i n t e s t i n a l derived h y d r o l y t i c d i g e s t i v e enzymes are buried deep within the lamina p r o p r i a and would u n l i k e l y be s l u f f e d o f f with the desquamated c e l l s of the i n t e s t i n a l mucous membrane. 3 . The Storage of Tissue Samples. The t i s s u e samples thus prepared were placed i n screw-cap 150 mm. culture tubes and frozen immediately. A l l ti s s u e s were held at - 1 5 ° to -20°C u n t i l the measurement of enzyme a c t i v i t y was undertaken. 4. Preparation of the Tissue Homogenates. An aliqu o t of the frozen t i s s u e was placed i n a c o l d three inch mortar with f i n e granular s i l i c a ( S X O 2 ) under the additio n of a suitable volume, u s u a l l y 25 to 35 ml. of b u f f e r - 17 -(Walpole pH 5«67)» and ground to a homogeneous consistancy. This t i s s u e suspension was t r a n s f e r r e d to a 50 ml. f l a s k and the remaining residue i n the mortar ground under the a d d i t i o n of another volume of b u f f e r . The second t i s s u e suspension was added to the f i r s t and the f l a s k t i g h t l y stoppered and shaken on a Kahn s e r o l o g i c a l shaker f o r exactly 15 minutes at a constant rate of 280 cycles per minute. A f t e r shaking, the preparation was allowed to autolyse from 18 to 20 hours at 5°C. The autolysate was f i l t e r e d through a s i n g l e l a y e r of grade 80 s o f t adsorbent cheese c l o t h . A l i q u o t s of the f i l t r a t e were removed f o r the determination of nitrogen by the micro-Kjeldahl technique ( 4 3 ) . The remaining f i l t r a t e was used immediately i n preparing the enzyme-substrate-digestion mixture. Since the t i s s u e homogenate contained p a r t i c l e s which s e t t l e d on standing i t was found necessary to shake t h i s preparation p r i o r to the removal of a l l a l i q u o t s , thus assuring an even d i s t r i b u t i o n of the p a r t i c u l a t e m a t e r i a l . 5 . Moisture Determination. Tissue a l i q u o t s of from one h a l f to one g. were dri e d i n an a i r oven at 100° to 120°C to constant weight i n f l a t aluminum drying dishes, as ou t l i n e d by the A.O.A.C. o f f i c i a l methods of analysis ( 3 3 ) . 6 . Nitrogen Determination. To t a l t i s s u e nitrogen was determined by the Gunning O f f i c i a l method ( 3 3 ) . Approximately one g. a l i q u o t s were removed from frozen minced small i n t e s t i n e at the same time as - 18 -the aliquots for moisture determination and the preparation of the tissue homogenates were obtained. 7. The Determination of Enzyme Activity. The method of Heilskov (25) for the determination of lactase activity was used in this study with certain modi-fications for sucrase and maltase activity. For the estimation of sucrase and maltase activity the digestion mixture contained either sucrose or maltose instead of lactose as the substrate. In this experiment the three carbohydrases were simultaneously measured in three separate reaction mixtures for any given tissue homogenate. Since the three sugars, sucrose, maltose and lactose and their hydrolytic products differ in their reductive capacities, this is the basis upon which measurement of hydrolysis is made. HeoLskov's procedure for reducing sugars differs::, from the classical Fehling's solution method in that he used an acid cupric acetate solution. The greater reducing capacity under these conditions i s demonstrated in Figure A , in which curve (A) is that obtained by Heilskov using added lactic acid in his copper reagent and curve (B) being that of Bertrand's method employing the Fehling's solution ( 5 ) . Preparation of the standard curves: Mixtures of one per cent solution of anhydrous reagent grade lactose,galactose and glucose were mixed in the correct\proportions to give 100 mg. of sugar solution representing the hydrolysis of lactose from zero, 10 , 2 0 , 30 etc. to 100 per cent. Similarily, for the standard curves of sucrose and maltose hydrolysis one - 19 -p e r c e n t m i x t u r e s o f s u c r o s e , f r u c t o s e and g l u c o s e , and g l u c o s e and m a l t o s e were p r e p a r e d , r e s p e c t i v e l y . E a c h m i x t u r e c o n t a i n i n g 100 mg. o f sugar was t r a n s f e r r e d q u a n t i t a t i v e l y to s e p a r a t e 100 mg. v o l u m e t r i c f l a s k s c o n t a i n i n g 50 m l . c o p p e r s o l u t i o n p r e v i o u s l y h e a t e d t o 100°C i n a b o i l i n g water b a t h . B o i l i n g was c o n t i n u e d f o r e x a c t l y 15 m i n u t e s a f t e r which t ime the f l a s k s were r a p i d l y c o o l e d u n d e r r u n n i n g water . T h i s p r e p a r a t i o n was f i l t e r e d t h r o u g h ah a s b e s t o s g l a s s wool f i l t e r p r e p a r e d a c c o r d i n g t o B e r t r a n d ( 5 ) . A p o r t i o n o f the c u p r o u s o x i d e ( C ^ O ) formed as a r e s u l t o f the r e d u c t i o n o f c u p r i c a c e t a t e was c o l l e c t e d on the a s b e s t o s f i l t e r ; the r e m a i n i n g cuprous o x i d e adhered to t h e i n s i d e o f the b o i l i n g f l a s k . The c o n t e n t s o f the b o i l i n g f l a s k were r i n s e d w i t h a volume o f d i s t i l l e d w a t e r and t h i s t o o , was added t o the m a t e r -i a l i n the a s b e s t o s f i l t e r i n g t u b e . The c o n t e n t s o f the f i l t e r were a g a i n r i n s e d , w i t h d i s t i l l e d w a t e r , and t h e n e l u t e d i n t o the o r i g i n a l b o i l i n g f l a s k w i t h s e v e r a l volumes o f h o t f e r r i c s u l p h a t e s o l u t i o n . When the p r e c i p i t a t e d copper had been c o m p l e t e l y e l u t e d a volume o f d i s t i l l e d water was u s e d t o f i n a l l y r i n s e the f i l t e r t u b e . These f l a s k s were h e a t e d i n a w a t e r b a t h t o a s s u r e complete o x i d a t i o n o f the c u p r o u s o x i d e . The r e d u c e d f e r r i c s u l p h a t e r e s u l t i n g was t i t r a t e d a g a i n s t p o t a s s i u m permanagante (KMnO^) p r e v i o u s l y s t a n d a r d i z e d a g a i n s t ammonium o x a l a t e ( ( N H ^ ) 2 C 2 0 ^ ) . The w e i g h t o f c o p p e r o r i g i n a l l y r e d u c e d was c a l c u l a t e d a c c o r d i n g to the f o l l o w i n g e q u a t i o n : Mg. Cu Reduced = ( n o r m a l i t y KMnO^) (mg. K M n 0 4 ) (63 .57) - 20 -The r e s u l t s of these reductions were regressed against the known percentage hydrolysis of each sugar as presented i n Figures 2 , 3 and 4. Substrate Homogenate Reaction Conditions: The r e a c t i o n mixture consisted of f i v e ml. al i q u o t s of the ti s s u e homogenate, added separately to 5 ml. of each of the three 10 per cent sugar solutions (sucrose, maltose and l a c t o s e ) . The tubes were shaken vigorously and then incu-bated f o r s i x hours at 36°C i n a t h e r m o s t a t i c a l l y c o n t r o l l e d water bath. At the end of t h i s incubation period the tubes were removed and heated i n a b o i l i n g water bath f o r f i v e minutes i n order that enzymatic a c t i v i t y be a r r e s t e d . The tubes were then cooled under running water, and 2 ml. aliquotes (100 mg. sugar) were removed from each and t r a n s f e r r e d to clean 15 ml. t e s t tubes and made up to a volume of approximately 10 ml. with d i s t i l l e d water. These a l i q u o t s were t r a n s f e r r e d to 50 ml. of copper s o l u t i o n which had previously been heated i n a b o i l i n g water bath. Each tube was r i n s e d with an a d d i t i o n a l f i v e ml. of d i s t i l l e d water, and the heating of the copper s o l u t i o n was continued f o r exactly 15 minutes as i n the preparation of the standard curves. The reducing capacity of the d i g e s t i o n mixture al i q u o t s was determined as previously outlined, and the percentage hy d r o l y s i s of the p a r t i c u l a r sugars read from the standard curves (Figures 2 , 3 , and 4). Changes i n the presence or a c t i v i t y of any enzyme or s p e c i f i c p r o t e i n i s most probably r e l a t e d to the t o t a l p r o t e i n of the c e l l or t i s s u e , and thus t h i s means of - 21 -300 250 o 200 o § 150 a o 100 50 0 0 10 20 '50 40 50 60 70 80 90 100 PER CENT HYDROLYSIS OF LACTOSE FIGURE 1. THE REDUCTIVE CAPACITY OF LACTOSE AND ITS HYDROLYTIC PRODUCTS USING BOTH ACID CUPRIC ACETATE AND FEHLING'S SOLUTIONS. o 2 o 2 0 10 20 30 40 50 60 70 80 90 100 PER CENT HYDROLYSIS OF LACTOSE FIGURE 2. THE REDUCTIVE CAPACITY OF LACTOSE AND ITS HYDROLYTIC PRODUCTS - 22 -300 P w o 3 o 2 0 FIGURE 3. 10 20 30 40 50 60 70 80 90 100 PER CENT HYDROLYSIS OF SUCROSE ™ D ™ ? S C T I V E C A P A C I T Y O F S U C ROSE AND ITS HYDROLYTIC o 3 o FIGURE 4. 10 20 30 40 50 60 70 80 90 100 PER CENT HYDROLYSIS OF MALTOSE ™Du1?s C T I V E C A P A C I T Y 0 F MALTOSE AND ITS HYDROLYTIC - 23 -comparison has been used i n t h i s presentation. Since the extent of hydrolysis i s known as well as the nitrogen content of the t i s s u e homogenate, the quantity of sugar hydrolysed per u n i t of homogenate nitrogen i s r e a d i l y c a l c u l a t e d according to the following equation: r e a c t i o n mixture sugar x per cent h y d r o l y s i s concentration 100 = rea c t i o n mixture nitrogen concentration 50 mg, X per cent h y d r o l y s i s 100 homogenate nitrogen mg. per ml. mg. sugar hydrolysed mg. nitrogen B. Ma t e r i a l s 1. The Basal D i e t . The composition of the basal d i e t used i n t h i s i n v e s t i g a t i o n i s given below (Table 1). - 24 -TABLE 1 THE COMPOSITION OF BASAL DIET (U.B.C. 10-55) Ground Wheat 750 l b . Wheat Bran 100 l b . Soya Bean Meal (44 per cent) 150 l b . F i s h Meal (70 per cent) 500 l b . Powdered Skim Milk 100 l b . Dried Yeast 25 l b . L i v e r Meal 50 l b . Bone Meal 10 l b . F i s h O i l 5 l b . Molasses 200 l b . Ground Yellow Corn 500 l b . Iodized S a l t 10 l b . 2,000 l b . 2 . The Condensed Milk D i e t . This r a t i o n was prepared f r e s h d a i l y by combining water and canned condensed milk i n the r a t i o s of 1 : 1 . Copper and i r o n were added at l e v e l s of 10 mg. per l i t e r and 100 mg. per l i t e r r e s p e c t i v e l y i n the form of cupric sulphate and f e r r i c c i t r a t e . - 25 -3. The Synthetic D i e t s . These d i e t s were prepared from reagent grade sucrose, maltose, l a c t o s e , and glucose. The s a l t mixture was that prepared by N u t r i t i o n a l Biochemicals Company and designated as s a l t mixture I I . The yeast powder was a product of the same company. The corn o i l was a product of the Canadian Starch Co. L t d . (Mazola). The experimental d i e t contained 64 per cent glucose which was lowered to 24 per cent i n the low sucrose, maltose and lactose d i e t s and omitted i n the d i e t s containing 64 per cent of these sugars. The seven d i e t s were s i m i l a r i n a l l other respe c t s to that of the experimental d i e t whose composition i s given i n Table 2. TABLE 2 THE COMPOSITION OF THE EXPERIMENTAL PURIFIED SUGAR DIET Ingredient Weight glucose 640 g. casein 240 g. s a l t mixture I I 40 g. yeast powder 20 g. corn o i l 120 g. Tota l 1|000 g. These d i e t s were prepared j u s t p r i o r to the ^beginning of the experiment, and were stored at 10°C i n covered containers. - 26 -4. Copper S o l u t i o n . Forty eight g. of cupric acetate (Cu(CH^COO) 2«H 20) were d i s s o l v e d i n about 800 ml. of d i s t i l l e d water. This mixture was heated to b o i l i n g and retained at that temperature f o r one minute. F i f t y ml. of IN l a c t i c a c i d was added and the so l u t i o n b o i l e d f o r an a d d i t i o n a l minute. The s o l u t i o n was zli then cooled to room temperature, f i l t e r e d and made up to one l i t e r . 5. F e r r i c S u l f a t e S o l u t i o n (According to Bertrand). F i f t y g. of f e r r i c s u l f a t e (Fe 2(S0^)^*9H 20) were dissolv e d i n about 600 ml. d i s t i l l e d water under the ad d i t i o n of 200 ml. concentrated reagent grade l s u l f u r i c a c i d . A f t e r the f e r r i c s u l f a t e had dissolv e d completely the s o l u t i o n was f i l t e r e d and made up to a volume of one l i t e r . 6. Potassium Permanganate Solution. F i v e g. potassium permanganate were di s s o l v e d i n about 800 ml. of d i s t i l l e d water at room temperature. An asbestos l i n e d Buckner funnel was prepared using b o i l e d a asbestos f o r f i l t e r i n g the permanganate s o l u t i o n . The permanganate t e t e r was determined against ammonjum oxalate ( ( N H 4 ) 2 C 2 0 4 ) . 7. Sugar Solutions. F i f t y g. each of reagent grade anhydrous sucrose, - 27 -maltose and lactose were dissol v e d separately i n 500 ml. portions of acetate b u f f e r (Walpole pH 5*6). These solutions were preserved with a few drops of toluene and stored at 5°C. 8. Buffer Solutions. An ac e t i c a c i d sodium acetate b u f f e r (0.2M pH 5»6) was prepared according to Walpole (24). Both Sttrenson and Walpole buffers of various pH were prepared at 2M and 15 0.4M strengths r e s p e c t i v e l y f o r the determination of pH optima f o r the three enzyme systems studied. - 28 -VI. RESULTS AND DISCUSSION A, Optima pH f o r Sucrase, Maltase and Lactase A c t i v i t y . Since the three enzymes, sucrase, maltase and lactase were studied using a crude t i s s u e preparation i t was f e l t d esirable to determine the pH optima f o r these systems i n the t i s s u e homogenate of the small i n t e s t i n e of the r a t . In a d d i t i o n to t h i s , there i s inconsistency i n the l i t e r a t u r e regarding the pH optima f o r these three carbohydrases. Sucrase a c t i v i t y i s reported to have pH optima from 4.2 to 7 d i f f e r i n g with o r i g i n (44-). Sucrase from i n t e s t i n a l t i s s u e i s reported to have a higher pH optimum of from pH 6 to pH 8 than the other two enzyme optima. Maltase from p i g i n t e s t i n e i s reported to have a pH optimum of from pH 5.2 to 7.2 while lact a s e has been shown to have maximum a c t i v i t i e s at various pH depending upon i t s source (44). Heilskov (25) reported a pH optimum of 5«5 f o r lactase from the small i n t e s t i n e of the r a b b i t . In t h i s experiment the pH range 4 to 8 was studied employing 10 per cent substrate s o l u t i o n s prepared i n e i t h e r an a c e t i c acid-sodium acetate (Walpole) b u f f e r (0.4M), or a d i b a s i c sodium phosphate (2M/15) (Na2HP0^) and monobasic potassium phosphate (2M/15) (KH 2P0^) bu f f e r . A s i n g l e non-buffered water extract of the small, i n t e s t i n e of r a t s of 16 days of age was prepared f o r lactase determination, and a s i m i l a r extract of the small i n t e s t i n e of 26 day old r a t s f o r - 29 -sucrase and maltase pH optima determination. Table 3 summarizes the r e s u l t s of t h i s experiment. Figure 5 shows g r a p h i c a l l y the pH optima f o r these three enzyme systems i n crude preparations of the small i n t e s t i n e of the r a t . TABLE 3 THE ACTIVITY OF SUCRASE, MALTASE AMD LACTASE IN CRUDE PREPARATIONS OF THE SMALL INTESTINE OF THE RAT AT VARIOUS pH LEVELS Mg. Sugar Hydrolysed Assay per Mg. Nitrogen Number p_H Sucrose Maltose Lactose 1 4 .3 3.02 7.0 4.7 2 5.3 9.5 18.2 13.4 3 5.85 14.2 21.0 16.5 4 5.95 15.2 19.2 15.8 5 6.5 14.7 17.1 9.0 6 6.98 12.4 18.7 7.6 7 7.4 9.5 15.9 6.1 8 7.98 - 12.5 2.4 In these preparations the pH optima f o r sucrase, maltase and lactase were approximately 6.2,6.0, and 5*6 r e s p e c t i v e l y . Since lactase exhibited a pH optimum peak somewhat sharper than e i t h e r sucrase or maltase i t was f e l t d esirable to determine a l l enzyme a c t i v i t i e s at pH 5*6. The e f f e c t of t h i s pH on e i t h e r sucrase or maltase a c t i v i t y would be small i n view of t h e i r father wide pH optima. The r e s u l t s of t h i s experiment are i n agreement with the f i n d i n g of other workers i n which wide pH optima are found f o r sucrase and maltase and a more c r i t i c a l dependence on pH f o r l a c t a s e . Q W CO CO O S3 « o • CO 2 • « P CO t-^ o ce p CO o is En O • hi S • « 5.5 6 .0 6 .5 7 .0 7 .5 pH 8.0 4 . 5 5.0 5.5 6 .0 6 .5 7 .0 7 .5 8.0 pH p 20 CO o K P 10 o c3 . p e> CO s , • « 0 MALTASE o FIGURE 5. THE ACTIVITY OF SUCRASE MALTASE AND LACTASE I N CRUDE PREPARATIONS OF THE SMALL INTESTINE OF THE RAT AT VARIOUS pH LEVELS - 31 -B. The H-ion Concentration of the Small Intestine of the Rat at Various Ages. It was of interest also to estimate the pH of the small intestine at various ages to provide information regarding the possibility of marked shifts in pH with age influencing the activities in situ of sucrase, maltase and lactase. Animals from 10 to 26 days of age were studied. The entire small intestine was removed and the contents recovered in d is t i l led water, f i ltered and read on a Beckman model N pH meter. These preparations were diluted a second time and the pH again measured. Dilution was found to affect the pH only slightly; usually increasing i t 0 .01 to 0 .02 units. Table 4 and Figure 6 summarize these findings. The pH of the intestinal contents was found to be consistently acid ranging from pH 6.08 to pH 6.69* This range is narrow in comparison to published values for a number of species. Fulton (23) suggestedthat the pH of the duodenum rarely goes below five and is most frequently found between 7 and 7»5» This same author suggests that the pH of the small intestine varies widely with the nature and quantity of i ts contents. The pH of the chyme in the small intestine is stabilized to a large degree by the flow of bile and pancreatic secretions. The variation between animals with respect to intestinal pH may be explained on this basis. The limited pH range may be due to a rather consistent " f i l l " of the small intestine in the rat resulting from ad libitum feeding. As - 32 -TABLE 4 . THE pH OP THE SMALL INTESTINE OP THE RAT AT VARIOUS AGES pH of the Assay Number of Age of Body Contents of the number animals animals Weights small i n t e s t i n e 1 1 10 14 g. 6.28 2 1 14 30 6.23 3 2 15 — 6.30 4 1 17 28 6.27 5 1 18 46 6.08 6 1 19 27 6.50 7 1 19 - 6 .22 8 1 20 40 6.29 9 1 20 — 6.49 10 1 21 36 6.24 11 1 22 39 6.14-12 1 22 - 6.53 13 1 23 53 6 .39 14 1 24 - 6.69 15 1 26 59 6 .35 - 33 -P-4 o w ft 00 EH (N CO is M a CO 0 o 0 15 20 25 AGE IN DAYS 30 35 PIGUBE 6. THE pH OP THE SMALL INTESTINE OE THE RAT AT VARIOUS AGES - 34 -pointed out by Prosser (39), the pH optimum f o r a given enzyme i s not n e c e s s a r i l y the same as the p h y s i o l o g i c a l pH at which i t f unctions. By comparing the pH of the small i n t e s t i n e of the r a t with the pH optima f o r sucrase, maltase and lactase a c t i v i t y found i n t h i s study, the statement of Prosser i s confirmed. No s i g n i f i c a n t increase i n pH with advancing age was found i n animals ranging from 10 - 26 days of age. Any marked changes i n sucrase, maltase, and la c t a s e a c t i v i t y i n s i t u would not l i k e l y a r i s e from a s h i f t i n the i n t e s t i n a l pH. C. The Relationship Between Homogenate Nitrogen Content and Enzyme A c t i v i t y . In the c a l c u l a t i o n of enzyme a c t i v i t y the assumption had been made that enzyme a c t i v i t y bore a d i r e c t or l i n e a r rather than an exponential r e l a t i o n s h i p to the nitrogen content of the t i s s u e homogenate. To t e s t t h i s assumption a si n g l e homogenate of the small- i n t e s t i n e of a number of 18 day old ra t s was prepared. A serie s of d i l u t i o n s of t h i s homogenate were made using a pH 5 . 6 acetate (Walpole) b u f f e r , such that the nitrogen content of the solutions ranged from 0.44 mg. of nitrogen per ml. to 2.04 mg. of nitrogen per ml. The measure-ment of enzyme a c t i v i t y was c a r r i e d out according to the method previously described. The r e s u l t s of t h i s experiment (Table 5 , Figure 7 ) suggested a l i n e a r rather than an exponential r e l a t i o n s h i p e x i s t i n g between the rate of h y d r o l y s i s of the three sugars and the nitrogen content of the t i s s u e homogenate. - 35 -Thus the expression of enzyme activity based upon the nitrogen content is valid over the range of 0.44 mg. to 2.04 mg. of nitrogen per ml. TABLE 5. THE RELATIONSHIP BETWEEN HOMOGENATE NITROGEN CONTENT AND ENZYME ACTIVITY Assay number Homogenate nitrogen content (mg. N per ml.) Substrate per cent hydrolysis sucrose maltose lactose 1 2 3 4 5 6 7 2.01 1.64 1.28 1.04 0.85 0.67 0.44 19.9 15 .4 11 .9 10 .4 9 .0 6 . 5 5.0 36 .9 31.7 27 .6 22 .8 19 .2 13 .3 1 1 . 5 25 .2 23 .3 17.5 14.9 12.4 9.8 7.2 37.5 co co 30.0 o g22.5 EH CQ s CO 15.0 7.5 0 0.20 0.50 0.80. 1.10 1.40 1.70 MG. NITROGEN PER ML. HOMOGENATE 2.00 2.30 FIGURE 7. THE RELATIONSHIP BETWEEN HOMOGENATE NITROGEN CONTENT AND ENZYME ACTIVITY - 37 -D. The E f f e c t of I n a n i t i o n on Sucrase, Maltase and Lactase A c t i v i t y , Since no assurance could "be made that " i n t e s t i n a l " f i l l " was at a l l times constant, i t was utherefore necessary to a s c e r t a i n the e f f e c t of i n a n i t i o n on the elaboration of sucrase, maltase and lacta s e i n the small i n t e s t i n e of the r a t . Animals of 18 and 24 days of age were placed i n f r e e z e r trays l i n e d with wood shavings and provided with a constant supply of water. Animals were s a c r i f i c e d f o l l owing 0, 8, 6, 9 , 15* 24, and 48 hours of i n a n i t i o n . The small i n t e s t i n e s were removed and the contents examined. The cleaned t i s s u e s were frozen and l a t e r assayed f o r sucrase, maltase and l a c t a s e a c t i v i t y . The r e s u l t s of these f i n d i n g s are summarized i n Table 6. Included i n t h i s table ar© a l i m i t e d number of values obtained from animals of comparable ages maintained on a non-r e s t r i c t e d basal d i e t (U.B.C.-10-55)• I n a n i t i o n had no depressing a c t i o n on the elaboration of sucrase i n 18 day o l d r a t s as expressed i n terms of u n i t i n t e s t i n a l nitrogen since the gradual increase i n sucrase a c t i v i t y from 18 to 20 days of age i s e s s e n t i a l l y s i m i l a r i n animals fed ad l i b i t u m on a basal d i e t (U.B.C. 10 -55) and those s u f f e r i n g i n a n i t i o n . In 24 day o l d r a t s sucrase a c t i v i t y was found to remain constant, as a r e s u l t of i n a n i t i o n , at somewhat lower l e v e l s than were found i n animals of the same age on " f u l l feed". Changing maltase a c t i v i t y i n 18 day r a t s was found unaffected by periods of i n a n i t i o n up to 24 hours. In older animals of 24 days of age i n a n i t i o n - 38 -TABLE 6. THE EFFECT OF INANITION ON INTESTINAL SUCRASE, MALTASE AND LACTASE IN THE LABORATORY WISTAR RAT Mean small Mg. sugar hydrolysed per Assay Age Mean body intestine Inanition mg. nitro gen number (days) weight (g.) weight (g.) (hours) sucrose maltose lactose 1 18 33.70 0.65 0 1.04 7.64 20.26 *2 18 32.30 1.19 0 0.60 10.40 26.90 *3 18 26.07 1.12 0 0.70 11.30 28.70 4 18 31.26 0.54 3 1.58 4.53 11.30 5 18 31.70 0.56 6 1.86 7.00 13.80 6 18 30.37 0.66 9 1.47 6.59 11.70 7 18 30.70 0.57 15 3.16 8.73 13.90 8 19 26.63 0.57 24 7.09 21.09 18.60 *9 19 44.40 2.27 0 8.10 18.40 18.20 *10 19 36.90 1.92 0 8.40 19.80 17.90 11 20 23.70 0.53 48 14.74 48.74 25.90 *12 20 38.00 2.28 0 13.70 21.90 11.60 *13 20 38.40 1.95 0 13.60 25.00 16.20 14 24 39-00 1.68 0 '6.10 15.82 0.00 15 24 57.35 2.05 3 6.44 17.29 2 .25 16 24 46.00 2.02 6 12.84 21.26 4.22 17 24 47.50 1.89 9 4.73 8.91 0.00 18 24 45.00 1.48 15 8.68 20.38 5.08 19 24 51.85 1.57 24 6.26 11.17 0.00 20 24 39.35 1.20 48 10.12 21.56 2.86 * Wistar rats weaned at 21 days onto a basal diet (U.B.C. 10 -55) - 39 -was not found to a l t e r appreciably the rate of maltase a c t i v i t y . However, a f t e r 48 hours of i n a n i t i o n , maltase a c t i v i t y i n 18 day old r a t s was found to be of the order of 49 mg. sugar hydrolysed per mg. nitrogen as compared to values of approximately 23 mg. found i n animals of the same age fed ad l i b i t u m . Lactase a c t i v i t y i n 24 day o l d r a t s , r e c e i v i n g the basal d i e t , was of a low order and was unaltered by periods of i n a n i t i o n . In younger animals of 18 days of age however, lactase a c t i v i t y was found to be of the order of 20 mg. sugar hydrolysed per mg. nitrogen when suckling. A f t e r three hours of i n a n i t i o n a s l i g h t drop i n lactase a c t i v i t y was experienced!, however i n a n i t i o n of 24 hours had l i t t l e e f f e c t on lactase a c t i v i t y as compared to the control animals. In general, body weight declined as a r e s u l t of i n a n i t i o n and the weight of small i n t e s t i n e per u n i t body weight declined i n 24 day old r a t s and increased i n the 18 day old r a t s . I t was concluded from t h i s study that where ad l i b i t u m feeding was p r a c t i c e d throughout, marked changes i n sucrase, maltase and lact a s e a c t i v i t y could not be a t t r i b u t e d to short periods of i n a n i t i o n . E. Sucrase, Maltase and Lactase A c t i v i t y From B i r t h to 72 Days of Age i n the Small Intestine of the Wistar Rat Weaned on to a Basal Diet at 21 Days. With the exception of the recent work of B a i l e y et a l (2) on the development of the d i g e s t i v e enzyme system - 40 -of the p i g during i t s pre-weaning phase of growth, no sing l e experiment was found i n the l i t e r a t u r e demonstrating the r e l -a t ive a c t i v i t y of sucrase^ maltase and lactase i n the small i n t e s t i n e with advancing age. As previously reviewed, lact a s e a c t i v i t y i n the foetus, young and o l d animals has been estimated f o r a number of d i f f e r e n t species. To v e r i f y and extend the work of B a i l e y et a l (2) using the r a t , i t was proposed to study the enzyme a c t i v i t i e s of animals from b i r t h and at frequent age i n t e r v a l s to 72 days. In t h i s experiment pre-weaned r a t s were obtained d i r e c t l y from the l i t t e r s , s a c r i f i c e d and enzymatic a c t i v i t i e s determined. Since the experimental procedure used required from two to three g. of i n t e s t i n a l tissue i t was necessary to pool the small i n t e s t i n e of as many as 11 r a t s of c e r t a i n age groups i n order to obtain s u f f i c i e n t material f o r a n a l y s i s . Post-weaned animals of known ages were fed ad l i b i t u m the basal d i e t (U.B.C. 10-55) and s a c r i f i c e d at pre-determined age i n t e r v a l s . In a l l instances the small i n t e s t i n e s of the pre-weaned animals were analyzed f o r sucrase, maltase and lactase a c t i v i t y immediately a f t e r s a c r i f i c e . I t was necessary, however, to freeze t i s s u e s obtained from post-weaned animals at -15°C as p r e v i o u s l y o u t l i n e d f o r a short period p r i o r to a n a l y s i s . Live body weights at s a c r i f i c e and small i n t e s t i n e wet weights were recorded f o r each post-weaned animal. I n d i v i d u a l body weights and t o t a l pooled small i n t e s t i n e weights of the several animals of each pre-weaned age group were recorded. - 41 -The r e s u l t s of t h i s experiment (Table 7 , Figures 8, 9 and 10) i n d i c a t e marked changes i n the a c t i v i t i e s of extracts from the small i n t e s t i n e of the laboratory Wistar r a t s towards the i n v i t r o h y d r o l y s i s of sucrose, maltose and l a c t o s e . These changes appear to foll o w d e f i n i t e patterns with advancing age p e c u l i a r to each of the h y d r o l y t i c enzymes. Sucrase a c t i v i t y was found to be n e g l i g i b l e from b i r t h to 18 days of age. An apparent r a p i d increase i n the a c t i v i t y of t h i s enzyme occurred between 18 and 22 days. A peak post-weaning sucrase a c t i v i t y was reached by 30 days followed by a gradual decline to 72 days of age. In post-weaned r a t s sucrase a c t i v i t y was observed to have a c t i v i t i e s from 10 to 24 mg. sugar hydrolysed per mg. i n t e s t i n a l homogenate nitrogen, as compared to 0.4 to 1.5 mg. sucrose hydrolysed per mg. nitrogen f o r r a t s from b i r t h to 18 days of age. Rats 19 days of age had an apparent sucrose a c t i v i t y of from 8 .10 to 8.40 mg. sucrose hydrolysed per mg. nitrogen. In a s i m i l a r manner, maltase a c t i v i t y was found to increase sharply at approximately 18 days of age. There was however, i n contrast to sucrase a c t i v i t y , an appreciable maltase a c t i v i t y from b i r t h to the weaning age of 21 days. Post-weaning small i n t e s t i n e maltase a c t i v i t y was found to be of the order of 25 to 35 nig. maltose hydrolysed i n s i x hours at 36°C per mg. homogenate nitrogen. - 42 -TABLE 7. SUCRASE, MALTASE AND LACTASE ACTIVITY FROM BIRTH TO 72 DAYS OF AGE IN THE SMALL INTESTINE OF THE WISTAR RAT WEANED ON TO A BASAL DIET AT 21 DAYS Mean Number body-Assay Age of weight number (days) animals (g.) Mean Small intestine weight (g.) Mean small intestine nitrogen (mg.per g.) Small intestine Mg. sugar hydrolysed Mg. sugar hydrolysed 1 0 10 6o65 0.21 18.96 2 0 11 6.33 0.20 18.46 3 2 10 6.65 0.26 17.30 4 2 10 6.44 0.24 18.32 5 4 10 7.94 0.26 20.59 6 4 10 8.19 0.26 19.43 7 6 7 11.93 0.37 19.85 8 6 7 12.18 0.38 22.00 9 12 4 18.48 0.79 20.63 10 12 4 16.31 0.68 22.13 11 18 3 32.30 1.19 18.49 12 18 3 26.07 1.12 16.15 13 19 2 44.40 2.27 16.09 14 19 2 36.90 1.92 17.29 15 20 2 38.00 2.28 16.89 16 20 2 38.40 1.95 17.34 17 21 2 52.20 2.32 19.73 18 21 2 46.20 2.34 18.24 19 21 2 47.20 2.23 19.46 20 28 1 61.40 4.00 17.19 21 28 1 62.00 4.20 18.80 22 28 1 67.50 4.99 16.59 23 34 1 87.50 5.66 19.93 24 35 1 95.50 6.48 18.03 25 35 1 92.20 5.64 19.51 26 42 1. 133.00 7.43 19.31 27 42 1 116.80 6.29 19.40 28 42 1 119.00 6.53 18.80 29 49 1 129.60 5.34 19.88 30 49 1 142.50 6.08 20.72 31 50 1 125.50 5.17 20.61 32 50 1 120.00 5.53 20.22 33 50 1 136.20 6.48 20.99 34 50 1 130.50 7.58 16.36 35 57 1 19.20 7.20 21.86 36 57 1 191.00 7.28 21.53 37 57 1 123.30 5.44 21.27 38 66 1 188.00 5.67 22.33 39 66 1 140.5 5.46 20.53 40 66 1 175.0 6.84 20.26 41 72 1 172.0 7.05 20.40 42 72 1 166.5 8.68 18.55 43 72 1 251.0 7.14 21.53 body weight sucrose maltose lactose sucrose maltose lactose 3.16 1.30 13.70 30.50 80 810 1810 3.16 1.10 14.00 28.00 60 830 1670 3.90 1.50 12.00 24.30 100 790 1620 3.73 1.10 12.60 25.80 80 880 1800 3.27 0.50 10.00 21.00 40 680 1430 3.18 0.60 9.60 23.80 40 560 1480 3.19 1.20 7.50 26.90 90 470 1680 3.04 0.90 7.70 29.60 60 520 2000 4.27 0.40 6.70 17.20 40 590 1520 4.16 0.60 8.00 23.50 60 740 2180 3.69 0.70 10.40 26.90 50 710 1840 4.30 0.60 11.30 28.70 40 780 1980 5.11 8.10 18.40 18.20 670 1510 1490 5.20 8.40 19.80 17.80 760 1780 1600 6.00 13.70 21.90 11.60 1380 2220 1170 5.08 13.60 25.00 16.20 1200 2200 1430 4.46 13.40 23.00 11.60 1180 2020 1020 5.07 11.20 21.00 11.20 1040 1940 1030 4.92 10.20 20.00 7.60 960 1880 720 6.50 12.10 22.20 3-10 1340 2510 340 6.77 14.20 25.70 2.70 1810 3270 340 7.40 14.90 26.80 2.20 1830 3280 270 6.48 21.60 27.90 2.80 2780 3600 360 6.79 19.70 24.80 3.20 2400 3030 390 6.12 15.80 25.90 2.60 1880 3090 310 5.59 16.80 26.60 0.90 1810 2870 100 5.38 19.00 26.20 2.10 1990 2740 220 5.50 14.50 23.30 0.20 1490 2410 20 4.00 18.20 27.60 1.80 1460 2220 140 4.28 16.00 26.80 1.30 1420 2370 110 4.12 14.50 24.10 2.90 1230 2050 240 4.60 21.60 34.40 4.40 2010 3200 410 4.75 23.40 31.80 4.40 2340 3180 440 5.82 21.90 31.10 2.90 2090 2950 280 3.78 11.50 24.70 4.20 950 2060 350 3.81 11.90 29.50 5.10 980 2420 420 4.40 14.60 30.30 2.90 1370 2840 270 3.02 18.40 25.90 0.50 1240 2740 30 3.88 12.60 22.20 0.70 1000 1770 50 3.90 9.80 17.40 0.50 770 1380 40 4.10 12.30 24.60 1.70 1030 2060 140 5.20 12.70 24.10 2.50 1230 2330 240 .2.84 11.50 19.40 0.60 700 1190 40 3 0 w CO O u CO 25 w O 20 « w a , Q W CO > H 1-1 o as a H 15 8< 10 8 8 8 FIGURE 8. 40 AGE IN DAYS 50 60 70 80 0 AGE IN DAYS FIGURE 10. - 46 -T i i e i n v i t r o d i g e s t i o n of lactose followed a d i f -ferent pattern. Pre-weaned Wistar r a t s exhibited high lact a s e a c t i v i t y of the order of 20 to 30 mg. lactose hydrolysed per mg. nitrogen. At 20 days of age a p r e c i p i t o u s drop occurred r e s u l t i n g i n post-weaning lacta s e a c t i v i t i e s of a n e g l i g i b l e amount, ranging from 0 . 5 to 5«1 nig. lactose hydrolysed per mg. nitrogen. These enzymatic a c t i v i t y changes when expressed i n terms of body weight were found to follow almost i d e n t i c a l patterns with those previously mentioned, with somewhat poorer agreement between r e p l i c a t e animals of the same age; the greater v a r i a b i l i t y a r i s i n g from v a r i a t i o n s i n nitrogen content of the i n t e s t i n a l t i s s u e s and s i z e of i n t e s t i n e i n r e l a t i o n to body weight. The a c t i v i t i e s expressed by B a i l e y et a l . (2) using e s s e n t i a l l y the same method f o r determining enzyme, a c t i v i t i e s f o r sucrase,, maltase and lactase i n the small i n t e s t i n e of the pre-weaned p i g i n terms of body weight are comparable with the values obtained f o r the r a t . Lactase a c t i v i t y , reported by B a i l e y et a l . (2) per Kg. body weight f o r the new born p i g i s 22.8 g. lactose hydrolysed i n s i x hours, compared to 18.1 g. f o r the new born r a t . Pigs of seven weeks of age are reported to hydrolyse 4.8 g. lactose as compared to a mean value f o r a l l post-weaned r a t s studied at 2 . 5 g. lactose per Kg. body weight. B a i l e y et a l . (2) reported both sucrase and maltase to be i n a c t i v e i n tiss u e homogenates of the new born p i g . In pigs from one to seven weeks of age sucrase a c t i v i t y increased by two f o l d i n the p i g - 47 -and was at a l l times half as active as maltase over the same age period. These results are essentially in agreement with the present findings in the rat over the post-weaning age period. In the post-weaned rat, homogenates hydrolysed maltose at a mean rate of 21.4 g. per Kg. body weight as compared with 15.0 g. per Kg. body weight for the seven week old pig. These same authors report seven week old pigs are capable of hydrolysing 7»35 g« sucrose per Kg. body weight. Post-weaned rats hydrolysed a mean of 15.24 g. sucrose per Kg body weight. Since in both the present study and those conducted by Bailey et a l . a similar method of assaying enzymatic activ-i ty was used, i t seems safe to conclude that the changes in sucrase, maltase andlactase activity in both species are similar. These results are in general agreement with early reports (37, 36, 8, 46, 13,4-0, 26) regarding the presence of lactase in the young and old of a number of species. A rather marked change in the nature of the ingested food was noted after 18 days of age in the suckling rat . In most instances 18 and 20 day old rats were found to be ingesting both the dam's milk and variable amounts of basal diet. The proportion of the ingesta composed of the basal diet increased with advancing age to the 21 day period where only a small proportion of the ingested food appeared to be suckled milk. It is of interest to note that the change in the character of the ingesta appears at a time coincidental to the greatest changes in the activities of the three carbohydrases studied. - 48 -The n u t r i t i v e value of a number of carbohydrates f o r young and o l d animals, reported by several workers (29, 4, 10) provides evidence suggesting that the presence of s p e c i f i c d i g e s t i v e enzymes at various ages imposes c e r t a i n r e -s t r i c t i o n s upon the nature of the d i e t most s u i t a b l e at the various stages of development. The f a i l u r e of amylolytic enzyme supplementation to improve the n u t r i t i v e value of starch-containing d i e t s reported by Cunningham and Brisson (11) may be a t t r i b u t e d to an inadequate supply of maltase i n the two day o l d p i g f o r complete "breakdown" of the starch d i e t . The absence of lactase i n adult r a t s found i n t h i s experiment supports the postulate put f o r t h by F i s c h e r (18) suggesting the i n a b i l i t y of mature r a t s to hydrolyse a lactose d i e t r e s u l t s i n a diarrhea brought about by the hydrogogue e f f e c t of lactose on the i n t e s t i n a l contents. The present study suggests that the nature of the c e l l s of the small i n t e s t i n e change with advancing age i n the r a t , and that these changes r e s u l t i n the changing nature of the secretion products of the small i n t e s t i n e . A t r a n s i t o r y d i g e s t i v e complex of the small i n t e s t i n e i s postulated as a fundamental p h y s i o l o g i c a l phenomena associated with the development and growth of animals. - 4-9 -F. The E f f e c t on Sucrase, Maltase and Lactase A c t i v i t y of E a r l y Weaning and the Feeding of Condensed M i l k and a Basal Diet at Various Ages i n the Laboratory Wistar Rat. In view of the pronounced changes i n sucrase, maltase, and lactase a c t i v i t y at the weaning age of 21 days established i n the previous experiment, i t was of i n t e r e s t to determine the e f f e c t of early weaning on enzyme a c t i v i t y and to investigate the behavior of lactase a c t i v i t y i n response to the prolonged feeding of condensed milk. In t h i s experiment four groups of Wistar r a t s were r a i s e d on e i t h e r a condensed milk d i e t (see materials) or the basal d i e t (U.B.C. 10-55) or both from the age of weaning as given i n the following ta b l e . Group 1 served both as a control group and as a confirmation f o r the previous experiment. TABLE 8. EXPERIMENTAL DESIGN Number of Diet surviving Weaning age Basal d i e t Condensed Group animals (days) (U.B.C. 10-55) Milk d i e t 1 36 21 21 to 40 days . —.. 2 32 15 — 15 to 40 days 3 31 15 28 to 40 rt 15 to 28 " 4 33 15 15 to 28 " 28 to 40 11 - 50 -I t was found necessary to administer the condensed milk to animals i n Groups 2 and 3 with an eye dropper at frequent i n t e r v a l s f o r the f i r s t two days of t h e i r post-weaning l i f e since they f a i l e d to ingest milk provided them i n the h a l f p i n t milk b o t t l e s f i t t e d with rubber stoppers and glass del i v e r y tubes. S i m i l a r d i f f i c u l t i e s were experienced i n feeding 15 day o l d r a t s the s o l i d basal d i e t . Preparing a watery paste of t h i s p e l l e t e d r a t i o n made i t more palatable f o r these immature animals. Because of these inher-ent d i f f i c u l t i e s encountered i n feeding r a t s of 15 days d i e t s other than suckled mother's milk, a c e r t a i n degree of n u t r i t i o n a l inadequacy must n e c e s s a r i l y r e s u l t . Within not more than two days, s u r v i v i n g animals were found to be capable of u t i l i z i n g the condensed milk provided i n the b o t t l e and the basal d i e t as e i t h e r a p e l l e t e d or a ground r a t i o n . A number of animals were s a c r i f i c e d at i n t e r v a l s from each group through to 40 days of age. The i n t e s t i n a l t i s s u e s were frozen and assayed following the conclusion of the experi-mental p e r i o d . The r e s u l t s of t h i s experiment are o u t l i n e d i n Table 9 and Figures 1 1 , 12 and 1 3 . General agreement was found to e x i s t between the r e s u l t s of the previous experiment and the a c t i v i t i e s of sucrase, maltase and lactase estimated f o r the small i n t e s t i n e of r a t s of Group 1 of t h i s experiment. This f a c t lends support to the postulate that f o r any given environmental condition, or s e r i e s of conditions, there e x i s t s , within : - 51 -TABLE 9 . THE EFFECT ON SUCRASE, MALTASE AND LACTASE ACTIVITY OF EARLY WEANING AND THE FEEDING OF CONDENSED MILK AND A BASAL DIET AT VARIOUS AGES IN THE LABORATORY WISTAR RAT. Small Small Mg. sugar Mean body Mean small i n t e s t i n e i n t e s t i n e as hydrolysed Assay Number of weight i n t e s t i n e nitrogen per cent per mg.nitrogen age number animals (g. ) weight(g.) (mg. per g. ) body weight D i e t sucrose maltose l a c t o s e (days) 1 4 5 0 . 5 1 . 2 5 22 .4 4 . 1 0 weaned at 5.00 10.00 1 1 . 8 0 16 2 4 2 9 . 2 1 .35 22 .7 4 . 6 2 21 days — 17.00 1 9 . 6 0 16 5 3 54 .3 1 .35 2 4 . 4 3.94 basal 21 to 6.10 17.50 1 9 . 4 0 18 4 5 57 .5 1 . 4 6 2 4 . 5 3.92 4 0 days 10.20 22.50 2 0 . 4 0 1 8 5 2 4 3 . 0 1.91 25 .2 4 . 4 5 ti 14.50 2 5 . l l 11 .20 20 6 2 4 4 . 2 1.66 2 5 . 4 3.86 11 11.50 2 1 . 4 0 13 .10 20 7 2 4 4 . 7 1.94 2 6 . 4 4 . 3 4 11 1 0 . 8 0 19.90 1 . 8 0 22 8 2 4 8 . 5 1.85 2 6 . 4 3 . 8 2 11 15.30 27 . 8 0 8 . 8 0 22 9 2 5 6 . 5 2 .71 2 4 . 5 4 . 8 0 u 15.70 23.20 2 .70 2 4 10 2 4 5 . 2 1.83 2 6 . 2 4 . 0 5 « 1 4 . 0 0 2 4 . 90 4 . 8 0 24 11 1 6 2 . 6 3 . 2 6 27.1 5.20 it 1 4 . 2 0 22.30 2 . 4 0 26 12 1 59 .7 2 .91 2 9 . 4 4 .88 11 11.00 19.90 2 . 4 0 26 15 1 74 .0 3 .19 2 6 . 5 4 . 3 0 ti 17.70 2 7 . 9 0 6 . 8 0 2 8 1 4 1 7 0 . 2 2 . 7 0 2 8 . 3 3 . 8 4 it 13.20 22.10 2 .90 2 8 15 1 7 6 . 3 3 . 1 8 27 .0 4 .17 11 9-70 19.00 2 .10 32 16 1 6 2 . 2 2 .32 2 9 . 5 3.72 it 13.70 2 4 . 4 0 4 . 0 0 32 17 1 121 .0 4 . 3 4 2 7 . 8 3-58 it 1 4 . 1 0 2 4 . 6 0 2 . 4 0 36 1 8 1 9 5 . 5 3 .55 2 7 . 5 3 . 4 8 n 15.10 29.30 3.50 36 19 1 135 .2 4 . 3 5 27 .6 3 .21 it 15.50 2 6 . 1 0 1.70 4 0 20 1 9 0 . 3 2 . 8 4 2 6 . 8 3 . 1 4 n 17.20 2 5 . 4 0 3 .00 4 0 21 5 4 6 . 3 1 . 4 6 2 4 . 8 3 . 1 6 condensed milk 5.00 2 0 . 8 0 2 6 . 4 0 16 22 6 30 .9 1 . 4 6 21 .0 4 . 7 3 15 to 4 0 days 2 9 . 4 0 3 5 . 4 0 1 4 . 0 0 1 8 25 5 2 4 . 0 1.21 2 4 . 4 5.03 weaned at 2 8 . 5 0 20.10 3.90 20 2 4 5 2 5 . 0 1.25 27 .1 5.00 15 days 23.10 2 8 . 8 0 3 . 4 0 20 25 4 3 2 . 4 1 . 8 0 2 6 . 2 5.56 it 2 3 . 4 0 30.10 4 . 5 0 22 2 6 5 3 0 . 4 1 . 6 2 27 .3 5 .34 ti 1 5 . 4 0 25.20 4 . 5 0 2 4 27 5 2 9 . 4 1.87 2 6 . 4 6 .36 ti 13.70 1 8 . 6 0 1 . 8 0 26 2 8 2 4 2 . 7 2 .49 25 .9 5 . 8 0 u 2 1 . 4 0 31.70 5 . 6 0 2 8 29 2 4 2 . 3 2 .54 25 .8 6 .00 it 1 8 . 1 0 2 6 . 5 0 4 . 7 0 32 50 2 72 .6 3 .71 2 6 . 0 5.10 11 10.00 17.00 3 .00 35 51 1 6 1 . 5 6 . 4 4 27 .6 1 0 . 4 0 tt 12.00 1 7 . 8 0 3.30 39 - 51a -TABLE 9 . (CONTINUED) Assay number Number of animals Mean body weight Mean small i n t e s t i n e weight (g.) Small i n t e s t i n e nitrogen (mg, per g.) Small i n t e s t i n e as per cent body weight Di e t sucrose Mg. sugar hydrolysed per mg. ni t r o g e n maltose l a c t o s e (days) 52 6 2 4 . 7 1.29 20 . 8 0 5.20 weaned at 17 . 60 32.70 17 .00 17 53 3 32 .2 1 . 8 4 22 .8 5.70 15 days 20.50 28.80 4 . 8 0 19 34 3 31 .4 1.83 2 4 . 6 5.83 condensed 16 .00 20.10 3 .20 19 35 4 30 .3 1.70 24 .7 5.61 milk 15 to 17 . 8 0 27.30 2 .30 21 36 3 3 7 . 5 2 .33 27 .4 6 .22 28 days 13.20 17 . 80 3.90 23 37 3 38 .0 2 .56 25 .0 6 .74 basal 28 12.50 16 .10 2 . 4 0 25 38 2 4 7 . 5 3 .21 24 .6 6 .76 to 40 days 19.20 26 .00 4 . 4 0 27 39 2 56 .7 3 .01 2 6 . 4 5.31 ti 13.90 23.30 2 .20 29 40 2 6 0 . 5 3.09 27-9 5.10 11 . 16 .50 25 .80 3 . 4 0 31 4-1 2 7 3 . 4 3 .15 2 6 . 8 4 .29 u 9 . 80 16 .10 1 . 4 0 35 4-2 1 103 .0 4 . 4 2 2 6 . 3 4 . 2 8 it 17.00 25.10 2 . 6 0 39 43 5 3 0 . 5 0 .76 30 . 6 2.50 weaned at 1.10 8 .60 22 .50 16 44 4 30 .2 0 .97 29 .3 3.21 15 days 23 . 40 33 .80 1 4 . 6 0 18 45 3 3 6 . 7 1.77 2 8 . 9 4 . 8 2 basal 15 14 .60 22.90 3 .10 20 46 2 4 8 . 7 1.63 30.90 3 .35 to 28 days 6 . 8 0 14 .10 4 . 0 0 22 47 2 50 .2 1 . 8 0 36 .3 3-58 condensed 9.20 16 .70 2 . 8 0 24 48 2 6 5 . 5 2 .55 31 .2 3.89 milk 28 to 8.90 14.40 1.50 26 49 3 4 5 . 3 2 . 2 6 31 .7 4 .99 40 days 5.90 13.10 2 .00 26 50 3 4 9 . 8 2 . 2 8 30.9 4 .58 11 11.10 18 .20 2 .20 28 51 2 5 5 . 4 1.94 30 .0 3 .50 it 8 . 8 0 15.00 3 .50 30 52 2 58 .8 2 .05 3 2 . 5 3.4-9 ti 6.70 15 .60 2 .20 32 53 2 6 6 . 4 2 . 4 ? 34 .1 3.72 it 9.10 18.30 2 .00 34 54 1 56 .3 2 . 0 4 32 .9 3 .62 n . 5 . 3 0 11.70 1.20 36 55 2 8 7 . 0 2 .85 29 .8 3 . 28 n 5.00 9.30 1.10 38 S25 s EH M CjJ s « W PH P W CO o « p CO o CO WEANING AT 21 LAYS 40 «» BASAL 21 TO 40 DAYS 3 0 20 > 0 O o o j U T T - O — 10 0 0 0 I I I I I I 40 30 20 10 0 15 20 25 30 35 40 45 50 WEANING AT 15 DAYS MILK 15 TO 28 DAYS BASAL 28 TO 40 DAYS - 0 0 o o 1 0 I I I I I p co O « P W co o a o C3 CO 40 WEANING AT 15 DAYS MILK 15 TO 40 DAYS 15 20 25 30 35 40 45 50 40 30 20 10 0 WEANING AT 15 DAYS MILK 15 TO 28 DAYS BASAL 28 TO 40 DAYS ro 15 20 25 30 35 40 45 50 15 20 25 30 35 40 45 50 FIGURE 1 1 . THE EFFECT ON SUCRASE ACTIVITY OF EARLY WEANING AND THE FEEDING OF CONDENSED MILK AND BASAL DIETS AT VARIOUS AGES IN THE RAT EH H c5 « « w (H o « « CO ClJ 40 30 20 10 0 40 30 20 10 0 WEANING AT 21 DAYS BASAL 21 TO 40 DAYS 15 20 2 5 30 35 40 45 50 AGE IN DAYS WEANING AT 15 DAYS MILK 15 TO 28 DAYS BASAL 28 TO 40. DAYS 15 20 25 30 35 40 45 50 AGE IN DAYS 8 E-t s « PM P w CO o a p w w CO 0 0 WEANING AT 15 DAYS MILK 15 TO 40 DAYS 15 20 25 30 35 AGE IN DAYS 40 45 50 WEANING AT 15 DAYS BASAL 15 TO 28 DAYS MILK 28 TO 40 DAYS 1 1 15 20 25 30 35 AGE IN DAYS 40 45 50 FIGURE 1 2 . THE EFFECT ON MALTASE ACTIVITY OF EARLY WEANING AND THE FEEDING OF CONDENSED MILK AND BASAL DIETS AT VARIOUS AGES I N THE RAT AGE IN DAYS AGE IN DAYS FIGURE 13. THE EFFECT ON LACTASE ACTIVITY OF EARLY WEANING AND THE FEEDING OF CONDENSED MILK AND BASAL DIETS AT VARIOUS AGES IN THE RAT - 55 - ' p h y s i o l o g i c a l v a r i a b i l i t y , a f i x e d pattern f o r the a c t i v i t y of each of three carbohydrases, sucrase, maltase and l a c t a s e , with advancing age. In addition the agreement between these two experiments suggested that f o r the length of time the t i s s u e s were frozen, the three carbohydrases r e t a i n t h e i r a c t i v i t i e s , since t i s s u e s of pre-weaned animals were assayed immediately a f t e r s a c r i f i c e i n the previous experiment, and stored at -15°0 f o r varying lengths of time i n the present experiment. In order to t e s t the postulate that the marked changes i n enzymatic a c t i v i t i e s at 21 days of age are associated with the weaning process and not merely c o i n c i -dental to that age period, Groups 2 and 5 were weaned at 15 days of age while Group 1 served as a c o n t r o l , being weaned at 21 days. In t h i s control group and i n the previous experiment lactase a c t i v i t y was found to be maintained to 20 days of age at l e v e l s above 10 mg. lactose hydrolysed per mg. nitrogen. Lactase a c t i v i t y was found to be appreciably lower at t h i s age i n animals i n Groups 2 , 3 and 4 as a r e s u l t of e a r l y weaning (Figure 13)* This decline i n l a c t a s e a c t i v -i t y was e s s e n t i a l l y the same i n both the milk and basal post-weaning d i e t groups, although there i s an apparently higher post-weaning lactase a c t i v i t y i n both Groups 2 and 3 r e -c e i v i n g the condensed milk d i e t immediately a f t e r weaning than i n Group 4 i n which the immediate post-weaning d i e t was the s o l i d U.B.C. 1 0 - 5 5 . The author f e e l s i t hazardous to - 56 -suggest that these differences are a d i r e c t r e s u l t of the absence of lactose i n the basal d i e t and the presence of t h i s substrate i n the condensed milk d i e t . Both sucrase and maltase a c t i v i t i e s increased i n ages 16 to 21 days as a r e s u l t of ea r l y weaning above those a c t i v i t i e s exhibited by Group 1 . In a d d i t i o n early weaning introduced greater v a r i a b i l i t y i n the a c t i v i t i e s of these two carbohydrases. The lower a c t i v -i t i e s found f o r sucrase i n Group 4 are not explainable on the basis of s p e c i f i c substrate since over the age range 28 to 40 days i n Group 2 a s i m i l a r d i e t r e s u l t e d i n appreciably higher sucrase a c t i v i t i e s . The feeding of condensed milk i n no way c o n s i s t e n t l y influenced sucrase a c t i v i t y . The feeding of the basal d i e t immediately a f t e r weaning brought about a drop i n both sucrase and maltase a c t i v i t i e s i n animals beyond 20 days of age. E a r l y weaning brought about an enhancement of maltase a c t i v i t y i n Groups 2 , 3 , and 4 p e c u l i a r to each group and p a r a l l e l l i n g although at higher a c t i v i t i e s the r e s u l t s obtained f o r sucrase a c t i v i t y . The author concludes that the feeding of con-densed milk does not b r i n g about a s i g n i f i c a n t increase i n lactase a c t i v i t y i n a feeding period of 13 days. In add i t i o n , there i s strong evidence to i n d i c a t e that e a r l y weaning brings about an e a r l y s h i f t i n the enzyme patterns estab-l i s h e d i n the previous experiment. - 57 -G. The E f f e c t on Sucrase, Maltase and Lacitase A c t i v i t y of the Small Intestine of the Young Wistar Rat of Prolonged Suckling on Foster Dams, As a r e s u l t of ear l y weaning the patterns of sucrase, maltase and lactase a c t i v i t y , established f o r - -animals weaned at 21 days, were a l t e r e d such that these changes occurred e a r l i e r i n the l i f e of these r a t s . In order to substantiate these findings i t was proposed to delay these changes beyond the ages at which they occur i n r a t s weaned on to a basal d i e t at 21 days by extending the suckling period to 24 days of age. In t h i s experiment four groups of f i v e pre-weaned Wistar r a t s , 15 days of age, were removed from t h e i r dams and placed i n four separate freezer trays l i n e d with wood shavings. Water was provided ad l i b i t u m to these four groups. Eight f r e s h l y l a c t a t i n g dams, a l l of which had l i t t e r e d within four or f i v e days p r i o r to the beginning of t h i s experimental period, served as f o s t e r dams to these suckling 15 day o l d r a t s . Four of these dams were i n i t i a l l y placed with the young l i t t e r s while the remaining four were placed i n s i m i l a r f r e e z e r trays to which had been added the p e l l e t e d basal r a t i o n . A f t e r the f i r s t 12 hours of t h i s experiment and a f t e r every succeeding 12 hour period the dams being suckled were exchanged with the remaining four dams, and allowed to consume the basal d i e t f o r a period of 12 hours. This exchanging procedure was continued throughout the experimental period. - 58 -The suckling r a t s were found to accept r e a d i l y the exchange of dams. The dams however, were e r r a t i c i n t h e i r behavior towards the l i t t e r s that were p r e v i o u s l y associated with the other dams. In some instances c a n n i b a l i s t i c behaviors were observed. Dams that were found to respond i n t h i s manner were removed from the experiment and replaced by other f r e s h l y l a c t a t i n g dams. Within a 56 hour period the dams had been selected such that t h i s d i f f i c u l t y was overcome. In general older dams proved to be the most suit a b l e f o r t h i s experi-ment, since they were found to l a c t a t e more adequately than the younger r a t s as evidenced by the superior rate at which t h e i r l i t t e r s grew. Fewer instances of cannibalism occurred among these older dams. The f o s t e r i n g process continued f o r nine days during which time numbers of suckling r a t s were s a c r i f i c e d at i n t e r v a l s and assayed f o r sucrase, maltase and lacta s e a c t i v i t y . Table 10 summarizes the f i n d i n g s of t h i s experiment. Also included i n t h i s table are data f o r animals of comparable ages weaned on to the basal d i e t at 21 days. Figure 14 shows sucrase, maltase and lactase a c t i v i t i e s of the small i n t e s t i n e of r a t s suckled f o r extended periods of time as well as f o r r a t s weaned on to a basal d i e t at 21 days. The r e s u l t s of t h i s experiment i n d i c a t e that pro-longed suckling a l t e r e d i n s i g n i f i c a n t l y the a c t i v i t i e s of sucrase and maltase established i n r a t s weaned at 21 days on to the basal d i e t . Lactase a c t i v i t y i s maintained at l e v e l s appreciably above those found i n r a t s weaned on to a basal - 59 -TABLE 10. THE EFFECT ON SUCRASE, MALTASE AND LACTASE ACTIVITY OF THE SMALL INTESTINE OF THE WISTAR RAT OF PROLONGED SUCKLING OF FOSTER DAMS Mean small Mean small Small intestine Number Mean intestine intestine weight as per Mg. sugar hydrolysed per mg Assay Age ' of body weight weight nitrogen cent body homoginate nitrogen number (days) Animals (g.) ( K . ) (mg.per g.) weight sucrose maltose lactose 1 17 3 24.00 0.89 26.5 3.70 4.00 13.90 27.70 2 19 3 22.50 0.98 26.8 4 .35 9.30 20.40 27.50 5 21 3 28.30 1.21 26.5 4 .30 8.40 21.50 24.70 4 23 3 24.00 1.14 28.2 4.67 17.00 31.30 14.10 5 24 4 24.20 1.11 23-8 4.60 16.50 29.20 13.20 *6 18 3 32.30 1.19 18.49 3.69 0.70 10.40 26.90 *7 18 3 26.07 1.12 16.15 4.30 0.60 11 .30 28.70 *8 19 2 44.40 2.27 16.09 5.11 8.10 18.40 18.20 *9 19 2 36.90 1.92 17.29 5.20 8.40 19.80 17.80 *10 20 2 38.00 2.28 16.89 6.00 13.70 21.90 11.60 •11 20 2 38.40 1.95 17.34 5.08 13.60 25.00 16.20 •12 21 2 52.20 2.32 19.73 4.46 13.40 23.00 11.60 *13 21 2 46.20 2.34 18.24 5.07 11.20 21.00 11.20 •14 21 2 47.20 2.23 19.46 4 .92 10.20 20.00 7.60 •Suckling on original dams to 21 days, basal diet from 21 days on. 5 I 1 1 I i i i 17 18 19 20 21 22 23 24 AGE IN DAYS FIGURE 14. THE EFFECT ON ENZYME ACTIVITY OF PROLONGED SUCKLING ON FOSTER DAMS - 61 -di e t at 21 days, as a r e s u l t of prolonged suckling of f r e s h l y l a c t a t i n g dams. Rats weaned at 21 days on to the basal d i e t exhibited lactase a c t i v i t i e s of the order of 10 mg. l a c -tose hydrolysed per mg. nitrogen at the weaning age. Rats 21 days of age i n t h i s f o s t e r i n g experiment showed lactase a c t i v i t i e s of the order of 25 mg. lactose hydrolysed per mg. nitrogen (Table 10). The f a i l u r e of lactase a c t i v i t y to be maintained at t h i s high l e v e l throughout t h i s experiment may be due to a s t e a d i l y diminishing rate of l a c t a t i o n i n the f o s t e r dams. 'As pointed out by Brody (6) periods of i n -a n i t i o n of 10 hours brought about cessation of l a c t a t i o n i n the r a t as estimated by the "feed intake" of suckling l i t t e r s ; . The period of i n a n i t i o n imposed on these f o s t e r dams exceeded t h i s 10 hour period. I t can be concluded that f o r at l e a s t part of the suckling period l i t t l e milk was being ingested by the young i n t h i s experiment. One might postulate that a f t e r successive periods of i n a n i t i o n and feeding, a gradual decline i n milk production would ensue such that by the ni n t h exchange of dams none of the dams could be considered to be " f r e e l y " l a c t a t i n g . Any s p e c i f i c lactase inducer funda-mentally responsible f o r the patterns of lactase a c t i v i t y e s tablished may be a component of the colostrum and/or the milk up to the peak of l a c t a t i o n , and as such may predetermine these patterns such that f o s t e r i n g young r a t s on f r e s h l y l a c t a t i n g dams may r e s u l t i n an extended peak of lactas e a c t i v i t y beyond the 21 day weaning age. I t i s of i n t e r e s t to note i n the previous experiment that the decline i n - 62 -lactase a c t i v i t y at the weaning age of 15 days was not as immediate as i n animals weaned at 21 days. One.may postulate that a l a c t a s e inducer may he present i n r e s i d u a l amounts i n the lumen of the gut f o r a period following the 15 day weaning age and not n e c e s s a r i l y i n the i n t e s t i n a l contents a f t e r 21 days of age. H. The E f f e c t on Sucrase, Maltase and Lactase A c t i v i t y of Various Levels of the Three Sugars, Sucrose, Maltose and Lactose i n a P u r i f i e d Synthetic Diet f o r the Laboratory Wistar Rat. The feeding of lactose has been i n c o n s i s t e n t l y shown to increase l a c t a s e a c t i v i t y i n the small i n t e s t i n e of the r a t . The e f f e c t of either sucrose or maltose on the a c t i v i t i e s of t h e i r respective h y d r o l y t i c enzymes has not been described f o r the small i n t e s t i n e of the r a t . In t h i s experiment synthetic p u r i f i e d d i e t s were prepared according to Table 2 . Sucrose, maltose and lactose were f e d at 64- and 40 per cent l e v e l s . Diets containing the 40 per cent l e v e l s of these three sugars had i n additi o n 24 per cent glucose added such that the sugar l e v e l i n a l l d i e t s was brought to 64 per cent. A r a t i o n containing g l u -cose at 64 per cent, as the only sugar, served as a con t r o l d i e t , since presumably no hydrolysis of t h i s sugar would be necessary p r i o r to i n t e s t i n a l absorption. Each of these seven r a t i o n s were fed to seven separate groups of 21 day od old laboratory Wistar r a t s f o r a perio d of 13 days. Numbers - 63 -TABLE 1 1 . THE EFFECT ON SUCRASE, MALTASE AND LACTASE ACTIVITY IN THE SMALL INTESTINE OF THE RAT, OF VARIOUS LEVELS OF SUCROSE, MALTOSE, LACTOSE AND DEXTROSE IN SYNTHETIC DIETS Small Small Assay number Age (days) Number of animals Mean body weight (g.) Mean small i n t e s t i n e weight ( K.) i n t e s t i n e nitrogen (mg.per g.) i n t e s t i n e as per cent body weight Diet Sugar hydrolysed (mg. per mg. nitrogen) sucrose maltose l a c t o s e 1 23 3 4 1 . 5 1.20 30 .2 2 .9 64 per cent 6 . 0 1 2 . 2 1.5 2 25 3 4 6 . 0 1 . 6 7 29 . 6 3.6 dextrose 11 .9 2 1 . 3 4 . 4 3 27 2 52 .0 1.35 2 8 . 5 2 .6 ti 7 .4 1 3 . 2 4 . 6 4 30 1 4 8 . 0 1.5 36.7 3.1 it 7 .2 9 . 8 3 .0 5 34 2 52 .0 1.35 33 .3 2 . 6 it 6 . 2 1 1 . 0 1.3 6 23 3 36.7 1 . 60 31.7 4 . 3 40 per cent 9 . 5 1 6 . 1 3.2 7 25 3 4 2 . 7 1.53 30 .2 3.6 sucrose 8 .8 1 5 . 4 3 .2 8 27 2 43 .0 1.50 29 .0 3 . 5 11 11 .4 1 7 . 5 4 . 3 9 30 1 58 .0 1.20 33 .6 2.1 it 3.3 6 . 8 4 . 4 10 34 2 4 9 . 5 1.35 33 .0 2 .7 ti 8 .3 1 5 . 7 1.8 11 23 3 4 0 . 0 1.33 29 .9 3.3 64 per cent 12 .7 1 9 . 7 4 . 4 12 25 3 4 3 . 3 1.33 31 .0 3.1 sucrose 4 . 6 7 . 4 1.2 13 27 2 4 2 . 5 1.25 33 .0 2 .9 ti 8 .4 1 3 . 7 2 .2 14 30 1 59 .0 1 . 60 33 .4 2 .7 ti 5.0 8 . 3 2 . 4 15 34 2 55.0 1.35 33 .3 2 . 5 it 8.1 1 6 . 3 3.6 16 23 3 4 0 . 0 1.13 31.6 2 .8 40 per cent 9.8 1 5 . 4 4 . 0 17 25 3 4 1 . 6 0.87 31 .8 2.1 maltose 4 . 9 8 . 9 2 . 4 18 27 2 4 7 . 5 1.55 30 .8 3 .3 it 8 .0 1 2 . 2 2 . 8 19 30 2 4 2 . 0 1.55 31 .0 3.7 it 7 .4 15 .1 4 . 6 20 34 1 4 3 . 0 1 . 40 33 .6 3 .3 it 12 . 6 2 5 . 9 6 . 3 21 23 3 4 2 . 6 1.17 32 .2 2 .7 64 perccent 6 . 4 1 1 . 9 2 .9 22 25 3 4 1 . 7 0.98 30 .5 2 . 4 maltose 3.1 6 . 2 1.6 23 27 2 4 0 . 5 1.15 32 .0 2 . 8 11 8 .4 1 6 . 9 4 . 2 24 30 2 39 .0 1.25 30 .6 3.2 u 9-5 1 6 . 7 4 . 2 25 34 1 39 .0 1 . 40 36 . 8 3.6 11 10 .0 2 3 . 3 3.6 26 23 3 4 1 . 0 1.27 30 .0 3.1 40 per cent 1 0 . 4 1 8 . 5 3.6 27 25 3 4 1 . 0 1.87 31 .0 4 . 6 lactose 12 .7 1 9 . 8 2 .9 28 27 2 49 .0 1.35 31 .2 2 .8 it 5.8 9 . 8 1.9 29 30 1 52.0 2 .20 32.6 4 . 2 ti 12 .4 1 7 . 8 4 . 5 30 34 2 4 9 . 5 1 . 80 31.0 3.6 it 10.9 1 7 . 7 1.8 31 23 3 38.0 1 . 07 31.6 2 . 8 64 per cent 8 .7 1 4 . 8 3.7 32 25 3 34 . 6 1 . 63 29 .8 4 . 7 lactose 1 6 . 5 2 4 . 4 4 . 3 33 27 2 4 6 . 5 1 . 80 28 .8 3.9 11 9.1 1 8 . 4 3.6 34 30 1 4 4 . 0 2 . 6 0 29 .8 5.9 it 1 8 . 3 2 7 . 8 2 .2 35 34 2 4 5 . 5 1 . 40 31.2 3.1 n 11 .4 1 5 . 5 3 .4 - 6 4 -O « 35 I-M S3 2 W CJ O K En M S5 CJ3 3 0 64 PER CENT 40 PER CENT 64 PER CENT DEXTROSE 0 SUCROSE 0 SUCROSE £ 23 35 20 o 30 K PH 25 p w CO >H 1-3 o pel P & w CO s o p CO 23 25 27 29 AGE IN DAYS 31 33 40 PER CENT MALTOSE 64 PER CENT MALTOSE 40 PER CENT LACTOSE 64 PER CENT LACTOSE 0 A 25 27 29 AGE IN DAYS FIGURE 15 . THE EFFECT ON SUCRASE ACTIVITY OF VARIOUS SYNTHETIC SUGAR DIETS - 65 -$25 w o « 35 M 64 PES CENT DEXTROSE 0 40 PER CENT SUCROSE • 64 PER CENT SUCROSE f) 23 25 27 29 AGE IN DAYS 31 33 $25 o « EH H 55 35 23 FIGURE 16. 40 PER CENT MALTOSE 64 PER CENT MALTOSE 40 PER CENT LACTOSE 64 PER CENT LACTOSE 25 27 29 AGE IN DAYS 31 33 THE EFFECT ON MALTASE ACTIVITY OF VARIOUS SYNTHETIC SUGAR DIETS - 66 -FIGURE 17. THE EFFECT ON LACTASE ACTIVITY OF VARIOUS SYNTHETIC SUGAR DIETS - 67 -55 bO EH W CiJ 8 /""""\ EH US 50 45 _ 40 21 24 50 45 -40 -21 24 0 64 PER CENT DEXTROSE • 40 PER CENT SUCROSE 64 PER CENT SUCROSE 27 30 AGE IN DAYS 33 0 40 PER CENT MALTOSE • 64 PER CENT MALTOSE 4 40 PER CENT LACTOSE 4 64 PER CENT LACTOSE 27 30 AGE IN DAYS 33 FIGURE 18. BODY WEIGHT GAINS ON LEVELS OF 40 AND 64 PER CENT SUCROS MALTOSE AND LACTOSE, AND 64 PER CENT DEXTROSE - 68 -of animals were s a c r i f i c e d at various age i n t e r v a l s . The t i s s u e s were stored at -15°C u n t i l enzymatic a c t i v i t y was determined. The r e s u l t s of t h i s experiment are presented i n Table 11 and Figures 1 5 , 16, aad 1 7 , and demonstrate sucrase, maltase and lactase a c t i v i t i e s at various ages i n the small i n t e s t i n e of the Wistar rat fed the various synthetic sugar d i e t s . The mean body weights of the s u r v i v i n g animals of each group at the various ages during the course of the experiment are presented i n Figure 18. Lactose feeding at e i t h e r 40 or 64- per cent l e v e l s i n synthetic d i e t s f o r weanling Wistar r a t s 21 to 34 days of age had no apparent lactase inducing e f f e c t as measured by the d i g e s t i o n of lactose i n i n v i t r o studies using t i s s u e homogenates of the small i n t e s t i n e (Figure 1 7 ) . Lactose a c t i v i t y ranged between 0 to 6 mg. lactose hydrolysed per mg. nitrogen, comparable f o r post weaning lactase a c t i v i t i e s f o r Wistar r a t s weaned at 21 days on to the basal (U.B.C. 10-55) r a t i o n . These results, are i n agreement with the f i n d i n g s of F i s c h e r et a l ( 3 3 ) , F i s c h e r ( 3 6 ) , Heilskov (26), and Plimmer ( 1 3 ) , i n which the feeding of lactose i n a number of d i f f e r e n t d i e t s f a i l e d to bring about an enhancement of lactase a c t i v i t y . F i s c h e r and Sutton (34) postulated l a c t a s e a d a p t a b i l i t y to the feeding of l a c t o s e , and i n a l a t e r com-munication Fischer (36) reported l a c t a s e adaptation to the feeding of lactose to r a t s i n terms of enzymatic a c t i v i t y - 69 -per u n i t t i s s u e nitrogen. Neither of the other three sugars apparently" a l t e r e d l a c t a s e a c t i v i t y . Glucose brought about a s l i g h t decline i n maltase a c t i v i t y . Neither sucrose nor lactose at e i t h e r 40 or 64 per cent l e v e l s a f f e c t e d the a c t i v i t y of t h i s enzyme. Maltase a c t i v i t y was increased by the feeding of maltose at both the 40 and 64 per cent l e v e l s . Sucrose at e i t h e r l e v e l f a i l e d to b r i n g about an enhanced a c t i v i t y of i t s h y d r o l y t i c enzyme i n the small i n -t e s t i n e of the r a t above a c t i v i t y l e v e l s found i n animals fed the 64 per cent glucose d i e t . Maltose had no apparent e f f e c t on sucrase a c t i v i t y . Several of the age groups fed 64 per cent lactose exhibited enhanced sucrase a c t i v i t i e s . Rats fed the glucose and sucrose d i e t s gained weight more r e a d i l y than d i d the r a t s f e d the other two sugars. Maltose f a i l e d to give good gains and proved to be somewhat i n f e r i o r at e i t h e r l e v e l i n t h i s regard to l a c t o s e . Rats fed 64 pa? cent maltose gained e s s e n t i a l l y no weight over the 13 day feeding period. Differences i n a c c e p t a b i l i t y of the seven rat i o n s i s proposed to be the major f a c t o r i n de-termining the feeding value of these sugars f o r post weaned r a t s . Rats consuming the ^maltose d i e t were found to waste, by s p i l l i n g , the greater part of the r a t i o n , whereas r a t s fed both the sucrose and glucose d i e t s r e a d i l y consumed - 70 -these sweet r a t i o n s . The lactose d i e t s were consumed more r e a d i l y than were the maltose d i e t s and l e s s r e a d i l y than the sucrose d i e t s . The author concludes that no apparent lacta s e a d a p t a b i l i t y incurred as a r e s u l t of lactose feeding at e i t h e r a 40 or 64 per cent l e v e l . I t would be hazardous to state that e i t h e r sucrase or maltase have been shown to be adaptable i n response to the feeding of t h e i r s p e c i f i c substrates i n :. a p u r i f i e d d i e t i n view of the r e s u l t s of t h i s l i m i t e d ex-periment. - 71 -VII. SUMMARY AND CONCLUSIONS The primary objectives of this study were to investigate the relative activities of intestinal sucrase, maltase and lactase in the laboratory Wistar rat and to study the effect of dietary factors on the activity age pattern of these three carbohydrases. The. results of this study may be summarized as follows: (1) The optima pH for sucrase, maltase and lactase activity of crude preparations of the small intestine of the labor-atory rat were found to be 6.2, 6.0 and 5.6 respectively. (2) The hydrogen ion concentration of the contents of the small intestine weaned at 21 days on to the basal (U.B.C. 10-55) diet was found to range between pH 6.08 and pH 6.65 over the age range 10 to 26 days. No marked change in pH was noted with advancing age. (3) A linear rather than an exponential relationship was found to exist between the enzyme activity of tissue homogen-ates of the small intestine of the rat and the nitrogen content of these preparations. (4) Short periods of inanition had no significant effect on sucrase, maltase and lactase activities in the laboratory Wistar rat. Extended periods of inanition :.of 4-8 hours brought about erratic activities of these three carbohydrases. - 02 -I t was concluded from t h i s study that, where ad l i b i t u m feeding was p r a c t i c e d throughout, marked changes i n sucrase, maltase and lactase a c t i v i t i e s could not be a t t r i b u t e d to short periods of i n a n i t i o n . (5) Sucrasei- maltase and lactase a c t i v i t i e s of the small i n t e s t i n e of the laboratory Wistar r a t weaned on to the basal (U.B.C. 10-55) r a t i o n at 21 days changed dramatically at the weaning age. Lactase i s found to be active i n pre-weaned r a t s and declined sharply at the weaning age to 20 per cent of i t s pre-weaned l e v e l . In contrast pre-weaned r a t s showed l i t t l e i f any sucrase a c t i v i t y , while post-weaned r a t s exhibited high sucrase a c t i v i t y . Maltase of the small i n -t e s t i n e of pre-weaned r a t s was active and increased at weaning to l e v e l s double that of the pre-weaning age group. A t r a n s i t o r y d i g e s t i v e complex i s postulated as a funda-mental p h y s i o l o g i c a l phenomena associated with the develop-ment and growth of animals. (6) E a r l y weaning brought about an e a r l i e r s h i f t i n the enzymatic patterns established f o r the r a t weaned at 21 days. Prolonged feeding of condensed milk f a i l e d to b r i n g about an enhanced lactase a c t i v i t y i n r a t s weaned on to that d i e t at 15 days. Eats weaned on to the basal d i e t at 15 days and fed condensed milk from 28 days on f a i l e d to e x h i b i t increased i n t e s t i n a l lactase a c t i v i t i e s . - 7 3 -( 7 ) Rats 15 days of age, when permitted to suckle freshly lactating dams to 24 days of age, exhibited sucrase and mal-tase activities comparable to those values obtained for rats weaned at 21 days on to the basal diet. Lactase activity however, was significantly higher as a result of prolonged suckling. (8) Lactose fed at both 40 and 64 per cent levels in a synthetic diet for the laboratory Wistar rat failed to bring about increased lactase activity. Maltase activity of the small intestine of the laboratory rat increased as a result of maltose feeding, while sucrase was unaffected by the presence of 40 and 64 per cent dietary levels of i t s specific substrate. 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