Sucrase, Maltase and Lactase Activity in the Small Intestine of the Laboratory Wistar Rat, Raised on a Basal Diet
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 University of British Columbia, 1955
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OP SCIENCE IN AGRICULTURE
iii the division of
Animal Science
We accept this thesis as conforming
to the required standard
Members of the Division
THE UNIVERSITY OF BRITISH COLUMBIA
October,1957. ii -
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 - iii -
in lactase activity was apparent. Neither the condensed milk nor basal diets induced apparent differences on the effect of early weaning on sucrase and maltase activity.
Apparent maintenance of lactase activity above those levels demonstrated in rats weaned at 21 days on to a basal diet was accomplished by fostering 16 day old rats on dams which had littered from four to five days prior to this transfer and which were assumed to be lactating at a somewhat greater rate than had the original mothers. It was noted however that this fostering process had no apparent influence on the extent of the changes in sucrase and maltase activities. The feeding of purified sugar diets containing two different levels of each of the three sugars, sucrose, maltose and lactose, had little effect on any of the three carbohydrases studied. Growth rates of the rats on these purified diets showed marked differences.
The author suggests that the maintenance of lactase activity might be associated with the presence of an inductive component present in the milk of the freshly lactating dam, a component not necessarily present in the milk of the later period of lactation, and not necessarily a component of processed cow's milk. The absence of an adaptive lactase response to the feeding of lactose in this study and those of other workers suggest that lactose itself is unlikely this inducer. No adap• tive response to substrates could be demonstrated for either sucrase or maltase. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Hea'el\of my
Department or by his representative. It is understood that copying or publication of this thesis for// financial gain shall not be allowed without my written permission.
ROBERT JOHN BOSE
Department of THE nTVTRTnw on- awTMar. ^r.rjmr.v,
The University of British Columbia, Vancouver 8, Canada.
Date .TTTT.V jc\ 1957 I. ACKNOWLEDGEMENT
The writer wishes to thank Dean Blythe Eagles,
Chairman of the Division of Animal Science, for providing the facilities with which to conduct this experiment.
Sincere appreciation is expressed to Dr. W. D.
Kitts, Assistant Professor of Animal Husbandry, for sug• gesting this problem and for his direction, assistance and criticism during the course of this study. Thanks are expressed to Dr. A. J. Wood and to Dr. J. J. R.
Campbell for their advice and criticisms. TABLE OP CONTENTS
PAGE
I. ACKNOWLEDGEMENT i
II. ABSTRACT ii
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 Activity 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 Activity 34 TABLE OF CONTENTS (Continued) PAGE D. The Effect of Inanition on Sucrase, Maltase and Lactase Activity 37 E. Sucrase, Maltase and Lactase Activity From Birth to 72 Days of Age. in the Small Intestine of the Wistar Eat Weaned on to a Basal Diet at 21 Days 39 F. 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 49 G. The Effect on Sucrase, Maltase and Lactase Activity of the Small Intestine of the Young Wistar Rat of Prolonged Suckling on Foster Dams 57 H. The Effect on Sucrase, Maltase and Lactase Activity of Various Levels of the Three Sugars, Sucrose, Maltose and Lactose in a Purified Synthetic Diet .for the Laboratory Wistar Rat.
VII. SUMMARY AND CONCLUSIONS 71
VIII. BIBLIOGRAPHY 74 Ill. INTRODUCTION
Digestion in its broad sense refers to the mechanical and chemical degradation of complex dietary constituents to forms that are suitably prepared for the absorptive processes of the alimentary tract. The rate at which any animal "breaks down" its complex dietary is necessarily a function of many mechanical and chemical processes.
The composition of a mammal's dietary changes markedly from birth to adult life. The young ingests almost exclusively its mother's milk, the mature or post weaned animal utilizes essentially none of this food for its dietary needs. In view of the many highly specific digestive mechanisms, it seems reasonable to conclude that accomodation for this changing dietary must necessarily be made for by associated changes in the digestive complex of the animal.
A great number of investigators have described the numerous enzymes or ferments present in the various regions of the alimentary tract. Much work has been conducted regarding the digestibility of various dietary constituents for a wide group of species. There is however, only scanty information on the quantitative measurement of the digestive enzymes. In this investigation the three carbohydrases, sucrase
( <*-D-glucosidoinvertase), maltase ( oC-D-glucopyranosidase) and lactase (y3 -D-galactosidase) have been assayed quantitatively in the small intestine of the Wistar rat at various ages. In addition, experiments have been conducted to demonstrate the presence or absence of enzymatic adaptation to dietary changes in these three carbohydrases present in this digestive organ of the laboratory rat.
The Wistar rat was employed in this experiment because of its rapid growth rate, genetic uniformity, low cost and its availability in large numbers.
The practical applications arising from information regarding the nature of the changes in the digestive complex of the laboratory rat would be the extrapolation of these fundamental principles to the feeding of domestic animals such as the pig, sheep and ox. Inherent changes in the elaboration of lactase would necessitate the feeding of lactose at definite ages in a manner paralleling the production of this hydrolytic enzyme in the digestive tract. If the elaboration of digestive enzymes is largely controlled by the presence of their specific substrates in the ingesta the feeding of starch, for example, to young dairy calves could substitute for the more costly milk diet commonly fed these animals. IV. HISTORICAL
A brief description of the three carbohydrases is felt appropriate as an introduction to the discussion of the literature.
A. Sucrase;
Sucrase has been known the longest of all 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, invertin 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 in certain yeasts.
The invertebrates and vertebrates possess the glucoinvertase exclusively. Sucrase is reported absent from the saliva and gastric secretions of mammals (31,7)• Its presence in the small intestine of the foetus and still-born has been reported by
Keene et al (27) and Tachibana (45). Neuberg and Mandl (44) suggest that the elaboration of intestinal sucrase is inde• pendent on the presence of food in the intestinal tract. The rough location of sucrase in the intestinal 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 first described by Brown and Heron (7) using pancreatic extracts and dried intestinal wall preparations of the pig. Emil
Fischer (15) > iu 1894, described an extract from dried brewers yeast active not only toward maltose, but also toward methyl-o(
-D-glucoside; he termed this enzyme "yeast maltase" (16).
Maltase is widely distributed throughout the animal and vegetable kingdoms, usually in association with amylase (44). In mammals maltase has been demonstrated mainly in preparations of the intestinal tract and blood serum (44). The crypts of Lieberkuhn of the intestinal mucosa are believed to be the site of maltase elaboration in the intestinal tract. The term maltase refers to a wide group of enzymes capable of hydrolysing in addition to their biological substrate maltose, a number of maltose derivatives and some alkyl and aryl 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 for the hydrolytic cleavage of lactose to glucose and galactose, is found in different strains of bacteria as well as in the seeds of a number of plants such as coffee and alfalfa (44-). Figure ;C shows the course of the reaction mentioned above.
CH-OH CH~OH
Lactase
HOH
4- -p-D galactopyranosyl-/^ -D-glucopyranoside (lactose)
P -D-galactose p -D-glucose FIGURE C. THE HYDROLYSIS OF LACTOSE BY LACTASE - 8 -
D. General
Little literature is available regarding the quanti• tative measurement of sucrase and maltase activity in mammals of various ages. Somewhat more information is available on the presence of lactase in animals.
Early reviews on lactase in 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 in 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 in the intestine of the dog, calf, ox and horse. Plimmer (37) in 1906 concluded that in general the young of most species possess lactase activity in their intes• tinal tissues, while the adult possesses little or none of this ferment.
The presence of lactase in the small intestine of young rabbits, oxen, sheep, young and old dogs, young goats and the new-born child was shown in 1899 by Weinland (46). Adult rabbits, oxen, sheep and fowl possessed little 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 cells. 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 in• 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 life period, and that the herbivora only when they are young. Plimmer (37) observed no increase in lactase activity in the intestinal - 10 - tissue of the rabbit and rat resulting from the feeding of milk and lactose, and also that rat embryos two days prepart- urium showed no activity while those of 12 hours preparturium showed lactase activity.
Lactase activity of the pancrease, mucous membranes of the ventricles, small intestine and colon was examined by
Heilskov (25) in 14 cows, 18 rabbits and seven human foetuses.
Lactase activity in cows of all ages decreases in a distal direction in the small intestine, and that the pancreas and the mucous membranes of the colon and ventricle do not contain lactase. It is further seen that lactase activity in all sec• tions of the small intestine decreases with age. After the age of eight months lactase is only inconsistantly demonstrated in cows. The results of the examination of 18 rabbits shored essentially the same as in the cow. Lactase was only found in the mucous membrane of the small intestine in the rabbit. The activity of this enzyme is found to decrease greatly after the age of four weeks. Heilskov (26) permitted eight rabbits to suckle their mothers to four weeks of age after which half this number received a lactose-free rabbit ration, the remaining were fed in addition to this ration 100 g. milk and ten g. lactose daily, from four to 15 weeks of age. No increase in lactase activity was found resulting from the lactose feeding.
Heilskov (26) suggests that the lactase activity of the in• testinal contents of the jejunum and duodenum of the rabbit and calf to be of the same order of magnitude as the activity per g. of mucous membrane of the small intestine of these same animals. - 11 - animals
Ammon and Henning (1) stated that the duodenal secretions of 40 human patients showed no activity, or so little that it was without significance for the process of digestion of sucrose, maltose and lactose. They suggested that the enzymatic hydrolysis of these three disaccharides takes place only after absorption into the cells of the intestinal mucosa.
The recent work of Bailey et al (2) describes in addition to lactase, sucrase and maltase activities in the small intestine of the pig. Lactase activity is reported to increase from birth to one week followed by a rapid decline from two to four weeks and remained at a low level to weaning at 50 days. In contrast sucrase and maltase activities are reported to be negligible at birth and to increase regularly to weaning. Data are not presented for post weaning sucrase, maltase and lactase activities. In an earlier communication
Kitts et al (28) report marked increases in the presence of pancreatic amylase in the pig from birth to 40 days.
Meyer and Necheles (54) reported age changes in alimentary enzymes in the human after the age of 16 years.
Salivary and pancreatic amylase, pancreatic lipase, typsin and pepsin are all reported to decline in activity with advancing age.
Fischer and Sutton (18) reviewed the effects of lac• tose on gastro-intestinal motility. They (18) postulated a hydrogogue effect of lactose on the contents of the small - 12 - intestine; in other words the high molecular weight and non- diffusible nature of lactose initiated the flow of body water into the area of high osmotic pressure set up by the presence of lactose in the gut lumen. They (18) pointed out that an animal may adapt to lactose feeding, that is diarrhea may become less severe and may cease entirely if the animal con• tinues to ingest large amounts of lactose over a period of time. Mitchell (36) noted that aciduric intestinal microflora produced by lactose feeding tended to revert to putrifactive types as a result of continuous lactose feeding. Fischer and
Sutton (18) suggest that this might be due to a greater utilization of lactose by way of an enhanced production of lactase. Fisher et al (19) reported the findings of two separate experiments on the effect of lactose feeding on lactase production. Groups of rats were fed for a six month period on 31*6 per cent lactose diets supplied from two sources; the first as fat-free dried milk, the second as U.S.P. lactose along with a suitable corn starch supplement. In the second experiment three groups of rats were fed 67 per cent corn starch, 15 per cent U.S.P. lactose; and 52 per cent corn starch, and 35 per cent U.S.P. lactose; and 32 per cent corn starch and 55 per cent U.S.P. lactose. These workers concluded that any significantly greater lactase activity demonstrated in response to lactose feeding is microbiological rather than mucosal in origin. Both experiments failed to demonstrate lactase adaptability to lactose feeding. Fischer and Sutton
(20) reported the effect of previous lactose feeding upon intestinal absorption of lactose in the rat. Lactose was - 13 - found to be absorbed more rapidly from the intestine of rats previously fed lactose diets. Rats adapted to lactose feeding within six weeks; that is diarrhea became infrequent. More recently Fischer (21) reported on the growth stimulating effect of lactose on the small intestine of the rat. Lactose feeding not only increased the small intestine weight per unit body weight, but also the lactase activity of the small intestine mucous membrane in terms of its nitrogen content. This is however not in agreement with later experimental work (22) published by the same author in which she concluded that lactase activity was increased on a unit body weight basis, but that no statistically significant increase could be demon• strated when the hydrolysis of lactose was expressed in terms of the nitrogen content of the tissue studied. This example of enzymatic adaptation, as pointed out by Fischer (22) does not however, satisfy the criteria of enzymatic adaption defined for microbiological systems. Bainbridge (9) injected extracts from the mucous membrane of animals fed lactose into animals maintained on a lactose-free diet. Pancreatic lactase was said to be induced by this technique in the latter animals.
Carnevale et al (9) reported the measurement of lipolytic, amylolytic and proteolytic activity of the duodenal secretions of the suckling, premature and full term infant to be consistent and independent of stage of development within this age range.
Koehler and Allen (29) reported that lactose ad• ministered subcutaneously was to a large degree eliminated in the urine. Rats raised on sucrose and lactose restricted - 14 -
sugar diets showed good gains on sucrose and poor gains on
lactose (29).
The nutritive value of glucose, lactose, dextrin and
corn starch for the newborn pig was studied using synthetic milk diets by Becker et al (4). In pigs from seven to 35 days
of age equal body weight gains were produced by all four carbo•
hydrates. Pigs at one and two days of age responded to sucrose
feeding with severe diarrhea, rapid weight loss, unthriftiness,
thinness and death. Neither lactose nor corn starch caused
diarrhea. Starch is reported to retard satisfactory growth of
two-day old pigs (10). Lloyd et al suggested that marked
changes in the digestive ability of young pigs takes place prior to eight weeks of age (30). Cunningham and Brisson (11)
observed that two-day old pigs received no benefit from
supplementing various purified starch-containing diets with
amylolytic enzymes. These authors suggested that the failure
of enzyme supplementation may be due to the possible dena-
turation of the ingested enzymes by the secretions of the
stomach or the failure of the baby pig to elaborate sufficient maltase to complete the course of degradation. However, these
authors suggested from further study (12) that the baby pig
is capable of utilizing some maltose at birth and may be
raised 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 all cases laboratory Wistar rats were obtained from
the Central Depot for Experimental Animals, U.B.C. Water and
feed were supplied ad libitum to the animals held in six inch
by twelve inch enamel-lined freezer trays. The basal diet,
U.B.C.-10-55, was supplied as a pellet of a diameter of one
inch, and as such was offered loose in the bottom of the
enamel-lined trays along with wood shavings which served as a
bedding material. Liquid diets were offered ad libitum in one
half pint jars, fitted with rubber stoppers and glass delivery
tubes. Solid diets, other than the basal diet, were offered
in open 6 oz. glass jars placed within the freezer trays.
Depending upon the size of animals one to ten Wistar rats were
placed in each freezer tray. Water was supplied fresh daily
and the shavings were renewed twice weekly.
2. Sacrifice and Dissection of the Animals.
A uniform procedure of tissue sampling was employed
throughout. All animals were sacrificed without anaesthesia.
The animal was first stunned by delivering a sharp blow to its
head. Bleeding followed by cutting the great vessels of the
neck, and allowing the animal to hang with its head in a
lowered position. Bleeding in this manner lessened to a large - 16 - extent the presence of blood in the mesenteric tissue. The small intestine was removed by making a single inscission in the ventral peritoneal wall, and separating the tract at the pyloric and lieo-cecal junctions. All mesenteric tissue and fatty deposits were carefully removed from the small intestine.
The intestinal contents were removed by gently forcing them along the lumen of the tract with the rounded edge of a pair of dissection scissors. The small intestine of the youngest animals were too fragile, and the lumen too small to handle under a stream of running water; for this reason the contents were not washed out. Carried along with the exuded lumen contents, was found a variable quantity of mucus and tissue debris. Histological considerations (32) suggest that the cells concerned with intestinal derived hydrolytic digestive enzymes are buried deep within the lamina propria and would unlikely be sluffed off with the desquamated cells of the intestinal mucous membrane.
3. The Storage of Tissue Samples.
The tissue samples thus prepared were placed in screw-cap 150 mm. culture tubes and frozen immediately. All tissues were held at -15° to -20°C until the measurement of enzyme activity was undertaken.
4. Preparation of the Tissue Homogenates.
An aliquot of the frozen tissue was placed in a cold three inch mortar with fine granular silica (SXO2) under the addition of a suitable volume, usually 25 to 35 ml. of buffer - 17 -
(Walpole pH 5«67)» and ground to a homogeneous consistancy.
This tissue suspension was transferred to a 50 ml. flask and
the remaining residue in the mortar ground under the addition
of another volume of buffer. The second tissue suspension was added to the first and the flask tightly stoppered and
shaken on a Kahn serological shaker for exactly 15 minutes at
a constant rate of 280 cycles per minute. After shaking, the preparation was allowed to autolyse from 18 to 20 hours at
5°C. The autolysate was filtered through a single layer of
grade 80 soft adsorbent cheese cloth. Aliquots of the
filtrate were removed for the determination of nitrogen by the micro-Kjeldahl technique (43). The remaining filtrate was used immediately in preparing the enzyme-substrate-digestion mixture. Since the tissue homogenate contained particles which settled on standing it was found necessary to shake
this preparation prior to the removal of all aliquots, thus
assuring an even distribution of the particulate material.
5. Moisture Determination.
Tissue aliquots of from one half to one g. were
dried in an air oven at 100° to 120°C to constant weight in
flat aluminum drying dishes, as outlined by the A.O.A.C.
official methods of analysis (33).
6. Nitrogen Determination.
Total tissue nitrogen was determined by the Gunning
Official method (33). Approximately one g. aliquots were
removed from frozen minced small intestine 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 is 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, 20, 30 etc. to 100 per cent. Similarily, for the standard curves of sucrose and maltose hydrolysis one - 19 - per cent mixtures of sucrose, fructose and glucose, and glucose and maltose were prepared, respectively. Each mixture containing 100 mg. of sugar was transferred quantitatively to separate 100 mg. volumetric flasks containing 50 ml. copper solution previously heated to 100°C in a boiling water bath.
Boiling was continued for exactly 15 minutes after which time the flasks were rapidly cooled under running water. This preparation was filtered through ah asbestos glass wool filter prepared according to Bertrand (5). A portion of the cuprous oxide (C^O) formed as a result of the reduction of cupric acetate was collected on the asbestos filter; the remaining cuprous oxide adhered to the inside of the boiling flask. The contents of the boiling flask were rinsed with a volume of distilled water and this too, was added to the mater• ial in the asbestos filtering tube. The contents of the filter were again rinsed, with distilled water, and then eluted into the original boiling flask with several volumes of hot ferric sulphate solution. When the precipitated copper had been completely eluted a volume of distilled water was used to finally rinse the filter tube. These flasks were heated in a water bath to assure complete oxidation of the cuprous oxide.
The reduced ferric sulphate resulting was titrated against potassium permanagante (KMnO^) previously standardized
against ammonium oxalate ((NH^)2C20^). The weight of copper originally reduced was calculated according to the following equation:
Mg. Cu Reduced = (normality KMnO^) (mg. KMn04) (63.57) - 20 -
The results of these reductions were regressed against the known percentage hydrolysis of each sugar as presented in
Figures 2, 3 and 4.
Substrate Homogenate Reaction Conditions: The reaction mixture consisted of five ml. aliquots of the tissue homogenate, added separately to 5 ml. of each of the three 10 per cent sugar solutions (sucrose, maltose and lactose). The tubes were shaken vigorously and then incu• bated for six hours at 36°C in a thermostatically controlled water bath. At the end of this incubation period the tubes were removed and heated in a boiling water bath for five minutes in order that enzymatic activity be arrested. The tubes were then cooled under running water, and 2 ml. aliquotes
(100 mg. sugar) were removed from each and transferred to clean
15 ml. test tubes and made up to a volume of approximately
10 ml. with distilled water. These aliquots were transferred to 50 ml. of copper solution which had previously been heated in a boiling water bath. Each tube was rinsed with an additional five ml. of distilled water, and the heating of the copper solution was continued for exactly 15 minutes as in the preparation of the standard curves. The reducing capacity of the digestion mixture aliquots was determined as previously outlined, and the percentage hydrolysis of the particular sugars read from the standard curves (Figures 2,
3, and 4). Changes in the presence or activity of any enzyme or specific protein is most probably related to the total protein of the cell or tissue, and thus this 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 10 20 30 40 50 60 70 80 90 100 PER CENT HYDROLYSIS OF SUCROSE FIGURE 3. ™D™?SCTIVE CAPACITY OF SUCROSE AND ITS HYDROLYTIC
o
3 o
10 20 30 40 50 60 70 80 90 100 PER CENT HYDROLYSIS OF MALTOSE
FIGURE 4. CTIVE ™Du1?s CAPACITY 0F MALTOSE AND ITS HYDROLYTIC - 23 - comparison has been used in this presentation. Since the extent of hydrolysis is known as well as the nitrogen content of the tissue homogenate, the quantity of sugar hydrolysed per unit of homogenate nitrogen is readily calculated according to the following equation:
reaction mixture sugar x per cent hydrolysis concentration 100 = reaction mixture nitrogen concentration
50 mg, X per cent hydrolysis 100 homogenate nitrogen mg. per ml.
mg. sugar hydrolysed mg. nitrogen
B. Materials
1. The Basal Diet.
The composition of the basal diet used in this investigation is given below (Table 1). - 24 -
TABLE 1 THE COMPOSITION OF BASAL DIET
(U.B.C. 10-55)
Ground Wheat 750 lb.
Wheat Bran 100 lb.
Soya Bean Meal (44 per cent) 150 lb.
Fish Meal (70 per cent) 500 lb.
Powdered Skim Milk 100 lb.
Dried Yeast 25 lb.
Liver Meal 50 lb.
Bone Meal 10 lb.
Fish Oil 5 lb.
Molasses 200 lb.
Ground Yellow Corn 500 lb.
Iodized Salt 10 lb.
2,000 lb.
2. The Condensed Milk Diet.
This ration was prepared fresh daily by combining water and canned condensed milk in the ratios of 1:1.
Copper and iron were added at levels of 10 mg. per liter and
100 mg. per liter respectively in the form of cupric sulphate and ferric citrate. - 25 -
3. The Synthetic Diets.
These diets were prepared from reagent grade sucrose, maltose, lactose, and glucose. The salt mixture was that prepared by Nutritional Biochemicals Company and designated
as salt mixture II. The yeast powder was a product of the
same company. The corn oil was a product of the Canadian
Starch Co. Ltd. (Mazola). The experimental diet contained
64 per cent glucose which was lowered to 24 per cent in the low sucrose, maltose and lactose diets and omitted in the diets containing 64 per cent of these sugars. The seven
diets were similar in all other respe cts to that of the
experimental diet whose composition is given in Table 2.
TABLE 2
THE COMPOSITION OF THE EXPERIMENTAL PURIFIED
SUGAR DIET
Ingredient Weight
glucose 640 g.
casein 240 g.
salt mixture II 40 g.
yeast powder 20 g.
corn oil 120 g.
Total 1|000 g.
These diets were prepared just prior to the ^beginning of the
experiment, and were stored at 10°C in covered containers. - 26 -
4. Copper Solution.
Forty eight g. of cupric acetate (Cu(CH^COO)2«H20) were dissolved in about 800 ml. of distilled water. This mixture was heated to boiling and retained at that temperature for one minute. Fifty ml. of IN lactic acid was added and the solution boiled for an additional minute. The solution was zli then cooled to room temperature, filtered and made up to one liter.
5. Ferric Sulfate Solution (According to Bertrand).
Fifty g. of ferric sulfate (Fe2(S0^)^*9H20) were dissolved in about 600 ml. distilled water under the addition of 200 ml. concentrated reagent grade lsulfuric acid. After the ferric sulfate had dissolved completely the solution was filtered and made up to a volume of one liter.
6. Potassium Permanganate Solution.
Five g. potassium permanganate were dissolved in about 800 ml. of distilled water at room temperature. An asbestos lined Buckner funnel was prepared using boiled a asbestos for filtering the permanganate solution. The permanganate teter was determined against ammonjum oxalate
((NH4)2C204).
7. Sugar Solutions.
Fifty g. each of reagent grade anhydrous sucrose, - 27 -
maltose and lactose were dissolved separately in 500 ml.
portions of acetate buffer (Walpole pH 5*6). These solutions
were preserved with a few drops of toluene and stored at 5°C.
8. Buffer Solutions.
An acetic acid sodium acetate buffer (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 respectively for the determination of pH
optima for the three enzyme systems studied. - 28 -
VI. RESULTS AND DISCUSSION
A, Optima pH for Sucrase, Maltase and Lactase Activity.
Since the three enzymes, sucrase, maltase and lactase were studied using a crude tissue preparation it was felt desirable to determine the pH optima for these systems in the tissue homogenate of the small intestine of the rat.
In addition to this, there is inconsistency in the literature regarding the pH optima for these three carbohydrases. Sucrase activity is reported to have pH optima from 4.2 to 7 differing with origin (44-). Sucrase from intestinal tissue is reported to have a higher pH optimum of from pH 6 to pH 8 than the other two enzyme optima. Maltase from pig intestine is reported to have a pH optimum of from pH 5.2 to 7.2 while lactase has been shown to have maximum activities at various pH depending upon its source (44). Heilskov (25) reported a pH optimum of 5«5 for lactase from the small intestine of the rabbit.
In this experiment the pH range 4 to 8 was studied employing 10 per cent substrate solutions prepared in either an acetic acid-sodium acetate (Walpole) buffer (0.4M), or a dibasic sodium phosphate (2M/15) (Na2HP0^) and monobasic
potassium phosphate (2M/15) (KH2P0^) buffer. A single non- buffered water extract of the small, intestine of rats of 16 days of age was prepared for lactase determination, and a similar extract of the small intestine of 26 day old rats for - 29 -
sucrase and maltase pH optima determination. Table 3
summarizes the results of this experiment. Figure 5 shows
graphically the pH optima for these three enzyme systems in crude preparations of the small intestine of the rat.
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 for sucrase, maltase and lactase were approximately 6.2,6.0, and 5*6 respectively. Since lactase exhibited a pH optimum peak
somewhat sharper than either sucrase or maltase it was felt desirable to determine all enzyme activities at pH 5*6. The effect of this pH on either sucrase or maltase activity would be small in view of their father wide pH optima. The results
of this experiment are in agreement with the finding of other workers in which wide pH optima are found for sucrase and maltase and a more critical dependence on pH for lactase. Q W CO
CO «O S3 o • CO 2 • «
5.5 6.0 6.5 7.0 7.5 8.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 pH pH P CO o p 20 ot-^ MALTASE CO pce
oK P CO o is 10 En o O • c3 . p e> hi S CO s • « , •« 0
FIGURE 5. THE ACTIVITY OF SUCRASE MALTASE AND LACTASE IN 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 distilled water, filtered 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 it 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 its 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 "fill" 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 intestine
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 4-0 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 -
00
EH (N CO
is M 0 o 0 a CO
P-4 o w ft
15 20 25 30 35 AGE IN DAYS
PIGUBE 6. THE pH OP THE SMALL INTESTINE OE THE RAT AT VARIOUS AGES - 34 - pointed out by Prosser (39), the pH optimum for a given enzyme is not necessarily the same as the physiological pH at which it functions. By comparing the pH of the small intestine of the rat with the pH optima for sucrase, maltase and lactase activity found in this study, the statement of Prosser is confirmed. No significant increase in pH with advancing age was found in animals ranging from 10 - 26 days of age. Any marked changes in sucrase, maltase, and lactase activity in situ would not likely arise from a shift in the intestinal pH.
C. The Relationship Between Homogenate Nitrogen Content
and Enzyme Activity.
In the calculation of enzyme activity the assumption had been made that enzyme activity bore a direct or linear rather than an exponential relationship to the nitrogen content of the tissue homogenate. To test this assumption a single homogenate of the small- intestine of a number of 18 day old rats was prepared. A series of dilutions of this homogenate were made using a pH 5.6 acetate (Walpole) buffer, 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 activity was carried out according to the method previously described. The results of this experiment
(Table 5, Figure 7) suggested a linear rather than an exponential relationship existing between the rate of hydrolysis of the three sugars and the nitrogen content of the tissue 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
Homogenate nitrogen Assay content Substrate per cent hydrolysis number (mg. N per ml.) sucrose maltose lactose
1 2.01 19.9 36.9 25.2 2 1.64 15.4 31.7 23.3 3 1.28 11.9 27.6 17.5 4 1.04 10.4 22.8 14.9 5 0.85 9.0 19.2 12.4 6 0.67 6.5 13.3 9.8 7 0.44 5.0 11.5 7.2 37.5
co co 30.0 o
g22.5
15.0 EH CQ s CO 7.5
0 0.20 0.50 0.80. 1.10 1.40 1.70 2.00 2.30 MG. NITROGEN PER ML. HOMOGENATE FIGURE 7. THE RELATIONSHIP BETWEEN HOMOGENATE NITROGEN CONTENT AND ENZYME ACTIVITY - 37 -
D. The Effect of Inanition on Sucrase, Maltase and
Lactase Activity,
Since no assurance could "be made that "intestinal"
fill" was at all times constant, it was utherefore necessary
to ascertain the effect of inanition on the elaboration of
sucrase, maltase and lactase in the small intestine of the
rat. Animals of 18 and 24 days of age were placed in freezer
trays lined with wood shavings and provided with a constant
supply of water. Animals were sacrificed following 0, 8, 6,
9, 15* 24, and 48 hours of inanition. The small intestines were removed and the contents examined. The cleaned tissues were frozen and later assayed for sucrase, maltase and lactase
activity. The results of these findings are summarized in
Table 6. Included in this table ar© a limited number of values
obtained from animals of comparable ages maintained on a non-
restricted basal diet (U.B.C.-10-55)•
Inanition had no depressing action on the elaboration
of sucrase in 18 day old rats as expressed in terms of unit
intestinal nitrogen since the gradual increase in sucrase
activity from 18 to 20 days of age is essentially similar in
animals fed ad libitum on a basal diet (U.B.C. 10-55)
and those suffering inanition. In 24 day old rats sucrase
activity was found to remain constant, as a result of
inanition, at somewhat lower levels than were found in animals
of the same age on "full feed". Changing maltase activity
in 18 day rats was found unaffected by periods of inanition
up to 24 hours. In older animals of 24 days of age inanition - 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 alter appreciably the rate of maltase
activity. However, after 48 hours of inanition, maltase
activity in 18 day old rats was found to be of the order
of 49 mg. sugar hydrolysed per mg. nitrogen as compared to
values of approximately 23 mg. found in animals of the same
age fed ad libitum. Lactase activity in 24 day old rats,
receiving the basal diet, was of a low order and was unaltered by periods of inanition. In younger animals of 18
days of age however, lactase activity was found to be of the
order of 20 mg. sugar hydrolysed per mg. nitrogen when
suckling. After three hours of inanition a slight drop in
lactase activity was experienced!, however inanition of 24 hours had little effect on lactase activity as compared to the
control animals. In general, body weight declined as a result
of inanition and the weight of small intestine per unit body weight declined in 24 day old rats and increased in the 18 day old rats.
It was concluded from this study that where
ad libitum feeding was practiced throughout, marked changes in sucrase, maltase and lactase activity could not be
attributed to short periods of inanition.
E. 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.
With the exception of the recent work of Bailey et al (2) on the development of the digestive enzyme system - 40 - of the pig during its pre-weaning phase of growth, no single experiment was found in the literature demonstrating the rel• ative activity of sucrase^ maltase and lactase in the small intestine with advancing age. As previously reviewed, lactase activity in the foetus, young and old animals has been estimated for a number of different species. To verify and extend the work of Bailey et al (2) using the rat, it was proposed to study the enzyme activities of animals from birth and at frequent age intervals to 72 days. In this experiment pre-weaned rats were obtained directly from the litters, sacrificed and enzymatic activities determined.
Since the experimental procedure used required from two to three g. of intestinal tissue it was necessary to pool the small intestine of as many as 11 rats of certain age groups in order to obtain sufficient material for analysis. Post- weaned animals of known ages were fed ad libitum the basal diet (U.B.C. 10-55) and sacrificed at pre-determined age intervals. In all instances the small intestines of the pre-weaned animals were analyzed for sucrase, maltase and lactase activity immediately after sacrifice. It was necessary, however, to freeze tissues obtained from post- weaned animals at -15°C as previously outlined for a short period prior to analysis. Live body weights at sacrifice and small intestine wet weights were recorded for each post- weaned animal. Individual body weights and total pooled small intestine weights of the several animals of each pre-weaned age group were recorded. - 41 -
The results of this experiment (Table 7, Figures
8, 9 and 10) indicate marked changes in the activities of extracts from the small intestine of the laboratory Wistar rats towards the in vitro hydrolysis of sucrose, maltose and lactose. These changes appear to follow definite patterns with advancing age peculiar to each of the hydrolytic enzymes.
Sucrase activity was found to be negligible from birth to
18 days of age. An apparent rapid increase in the activity of this enzyme occurred between 18 and 22 days. A peak post- weaning sucrase activity was reached by 30 days followed by a gradual decline to 72 days of age. In post-weaned rats sucrase activity was observed to have activities from 10 to 24 mg. sugar hydrolysed per mg. intestinal homogenate nitrogen, as compared to 0.4 to 1.5 mg. sucrose hydrolysed per mg. nitrogen for rats from birth to 18 days of age. Rats 19 days of age had an apparent sucrose activity of from 8.10 to 8.40 mg. sucrose hydrolysed per mg. nitrogen.
In a similar manner, maltase activity was found to increase sharply at approximately 18 days of age. There was however, in contrast to sucrase activity, an appreciable maltase activity from birth to the weaning age of 21 days.
Post-weaning small intestine maltase activity was found to be of the order of 25 to 35 nig. maltose hydrolysed in six 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 Mean Mean small Small Number body- Small intestine intestine Mg. sugar hydrolysed Mg. sugar hydrolysed Assay Age of weight intestine nitrogen number (days) animals (g.) weight (g.) (mg.per g.) body weight sucrose maltose lactose sucrose maltose lactose 1 0 10 6o65 0.21 18.96 3.16 1.30 13.70 30.50 80 810 1810 2 0 11 6.33 0.20 18.46 3.16 1.10 14.00 28.00 60 830 1670 3 2 10 6.65 0.26 17.30 3.90 1.50 12.00 24.30 100 790 1620 4 2 10 6.44 0.24 18.32 3.73 1.10 12.60 25.80 80 880 1800 5 4 10 7.94 0.26 20.59 3.27 0.50 10.00 21.00 40 680 1430 6 4 10 8.19 0.26 19.43 3.18 0.60 9.60 23.80 40 560 1480 7 6 7 11.93 0.37 19.85 3.19 1.20 7.50 26.90 90 470 1680 8 6 7 12.18 0.38 22.00 3.04 0.90 7.70 29.60 60 520 2000 9 12 4 18.48 0.79 20.63 4.27 0.40 6.70 17.20 40 590 1520 10 12 4 16.31 0.68 22.13 4.16 0.60 8.00 23.50 60 740 2180 11 18 3 32.30 1.19 18.49 3.69 0.70 10.40 26.90 50 710 1840 12 18 3 26.07 1.12 16.15 4.30 0.60 11.30 28.70 40 780 1980 13 19 2 44.40 2.27 16.09 5.11 8.10 18.40 18.20 670 1510 1490 14 19 2 36.90 1.92 17.29 5.20 8.40 19.80 17.80 760 1780 1600 15 20 2 38.00 2.28 16.89 6.00 13.70 21.90 11.60 1380 2220 1170 16 20 2 38.40 1.95 17.34 5.08 13.60 25.00 16.20 1200 2200 1430 17 21 2 52.20 2.32 19.73 4.46 13.40 23.00 11.60 1180 2020 1020 18 21 2 46.20 2.34 18.24 5.07 11.20 21.00 11.20 1040 1940 1030 19 21 2 47.20 2.23 19.46 4.92 10.20 20.00 7.60 960 1880 720 20 28 1 61.40 4.00 17.19 6.50 12.10 22.20 3-10 1340 2510 340 21 28 1 62.00 4.20 18.80 6.77 14.20 25.70 2.70 1810 3270 340 22 28 1 67.50 4.99 16.59 7.40 14.90 26.80 2.20 1830 3280 270 23 34 1 87.50 5.66 19.93 6.48 21.60 27.90 2.80 2780 3600 360 24 35 1 95.50 6.48 18.03 6.79 19.70 24.80 3.20 2400 3030 390 25 35 1 92.20 5.64 19.51 6.12 15.80 25.90 2.60 1880 3090 310 26 42 1. 133.00 7.43 19.31 5.59 16.80 26.60 0.90 1810 2870 100 27 42 1 116.80 6.29 19.40 5.38 19.00 26.20 2.10 1990 2740 220 28 42 1 119.00 6.53 18.80 5.50 14.50 23.30 0.20 1490 2410 20 29 49 1 129.60 5.34 19.88 4.00 18.20 27.60 1.80 1460 2220 140 30 49 1 142.50 6.08 20.72 4.28 16.00 26.80 1.30 1420 2370 110 31 50 1 125.50 5.17 20.61 4.12 14.50 24.10 2.90 1230 2050 240 32 50 1 120.00 5.53 20.22 4.60 21.60 34.40 4.40 2010 3200 410 33 50 1 136.20 6.48 20.99 4.75 23.40 31.80 4.40 2340 3180 440 34 50 1 130.50 7.58 16.36 5.82 21.90 31.10 2.90 2090 2950 280 35 57 1 19.20 7.20 21.86 3.78 11.50 24.70 4.20 950 2060 350 36 57 1 191.00 7.28 21.53 3.81 11.90 29.50 5.10 980 2420 420 37 57 1 123.30 5.44 21.27 4.40 14.60 30.30 2.90 1370 2840 270 38 66 1 188.00 5.67 22.33 3.02 18.40 25.90 0.50 1240 2740 30 39 66 1 140.5 5.46 20.53 3.88 12.60 22.20 0.70 1000 1770 50 40 66 1 175.0 6.84 20.26 3.90 9.80 17.40 0.50 770 1380 40 41 72 1 172.0 7.05 20.40 4.10 12.30 24.60 1.70 1030 2060 140 42 72 1 166.5 8.68 18.55 5.20 12.70 24.10 2.50 1230 2330 240 43 72 1 251.0 7.14 21.53 .2.84 11.50 19.40 0.60 700 1190 40 30
25
w O 8 20
« w a, 15 Q W CO >H 8< 1-1 o as a 8 H 10
w CO 8 O u CO
40 50 60 70 80 AGE IN DAYS FIGURE 8.
0
AGE IN DAYS FIGURE 10. - 46 -
Tiie in vitro digestion of lactose followed a dif• ferent pattern. Pre-weaned Wistar rats exhibited high lactase activity of the order of 20 to 30 mg. lactose hydrolysed per mg. nitrogen. At 20 days of age a precipitous drop occurred resulting in post-weaning lactase activities of a negligible amount, ranging from 0.5 to 5«1 nig. lactose hydrolysed per mg. nitrogen. These enzymatic activity changes when expressed in terms of body weight were found to follow almost identical patterns with those previously mentioned, with somewhat poorer agreement between replicate animals of the same age; the greater variability arising from variations in nitrogen content of the intestinal tissues and size of intestine in relation to body weight.
The activities expressed by Bailey et al. (2) using essentially the same method for determining enzyme, activities for sucrase,, maltase and lactase in the small intestine of the pre-weaned pig in terms of body weight are comparable with the values obtained for the rat. Lactase activity, reported by Bailey et al. (2) per Kg. body weight for the new born pig is 22.8 g. lactose hydrolysed in six hours, compared to 18.1 g. for the new born rat. Pigs of seven weeks of age are reported to hydrolyse 4.8 g. lactose as compared to a mean value for all post-weaned rats studied at 2.5 g. lactose per Kg. body weight. Bailey et al. (2) reported both sucrase and maltase to be inactive in tissue homogenates of the new born pig. In pigs from one to seven weeks of age sucrase activity increased by two fold in the pig - 47 - and was at all 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 al. a similar method of assaying enzymatic activ• ity was used, it 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 - 48 - carbohydrases studied.
The nutritive value of a number of carbohydrates for young and old animals, reported by several workers (29, 4, 10) provides evidence suggesting that the presence of specific digestive enzymes at various ages imposes certain re• strictions upon the nature of the diet most suitable at the various stages of development. The failure of amylolytic enzyme supplementation to improve the nutritive value of starch-containing diets reported by Cunningham and Brisson
(11) may be attributed to an inadequate supply of maltase in the two day old pig for complete "breakdown" of the starch diet.
The absence of lactase in adult rats found in this experiment supports the postulate put forth by Fischer (18) suggesting the inability of mature rats to hydrolyse a lactose diet results in a diarrhea brought about by the hydrogogue effect of lactose on the intestinal contents.
The present study suggests that the nature of the cells of the small intestine change with advancing age in the rat, and that these changes result in the changing nature of the secretion products of the small intestine. A transitory digestive complex of the small intestine is postulated as a fundamental physiological phenomena associated with the development and growth of animals. - 4-9 -
F. 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.
In view of the pronounced changes in sucrase, maltase, and lactase activity at the weaning age of 21 days established in the previous experiment, it was of interest to determine the effect of early weaning on enzyme activity and to investigate the behavior of lactase activity in response to the prolonged feeding of condensed milk.
In this experiment four groups of Wistar rats were raised on either a condensed milk diet (see materials) or the basal diet (U.B.C. 10-55) or both from the age of weaning as given in the following table. Group 1 served both as a control group and as a confirmation for the previous experiment.
TABLE 8. EXPERIMENTAL DESIGN
Number of Diet surviving Weaning age Basal diet Condensed Group animals (days) (U.B.C. 10-55) Milk diet
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 -
It was found necessary to administer the condensed milk to animals in Groups 2 and 3 with an eye dropper at frequent intervals for the first two days of their post- weaning life since they failed to ingest milk provided them in the half pint milk bottles fitted with rubber stoppers and glass del ivery tubes. Similar difficulties were experienced in feeding 15 day old rats the solid basal diet. Preparing a watery paste of this pelleted ration made it more palatable for these immature animals. Because of these inher• ent difficulties encountered in feeding rats of 15 days diets other than suckled mother's milk, a certain degree of nutritional inadequacy must necessarily result. Within not more than two days, surviving animals were found to be capable of utilizing the condensed milk provided in the bottle and the basal diet as either a pelleted or a ground ration.
A number of animals were sacrificed at intervals from each group through to 40 days of age. The intestinal tissues were frozen and assayed following the conclusion of the experi• mental period. The results of this experiment are outlined in Table 9 and Figures 11, 12 and 13.
General agreement was found to exist between the results of the previous experiment and the activities of sucrase, maltase and lactase estimated for the small intestine of rats of Group 1 of this experiment. This fact lends support to the postulate that for any given environmental
condition, or series of conditions, there exists, 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 intestine intestine as hydrolysed Assay Number of weight intestine nitrogen per cent per mg.nitrogen age number animals (g. ) weight(g.) (mg. per g. ) body weight Diet sucrose maltose lactose (days)
1 4 50.5 1.25 22.4 4.10 weaned at 5.00 10.00 11.80 16 2 4 29.2 1.35 22.7 4.62 21 days — 17.00 19.60 16 5 3 54.3 1.35 24.4 3.94 basal 21 to 6.10 17.50 19.40 18 4 5 57.5 1.46 24.5 3.92 40 days 10.20 22.50 20.40 18 5 2 43.0 1.91 25.2 4.45 ti 14.50 25.ll 11.20 20 6 2 44.2 1.66 25.4 3.86 11 11.50 21.40 13.10 20 7 2 44.7 1.94 26.4 4.34 11 10.80 19.90 1.80 22 8 2 48.5 1.85 26.4 3.82 11 15.30 27.80 8.80 22 9 2 56.5 2.71 24.5 4.80 u 15.70 23.20 2.70 24 10 2 45.2 1.83 26.2 4.05 « 14.00 24.90 4.80 24 11 1 62.6 3.26 27.1 5.20 it 14.20 22.30 2.40 26 12 1 59.7 2.91 29.4 4.88 11 11.00 19.90 2.40 26 15 1 74.0 3.19 26.5 4.30 ti 17.70 27.90 6.80 28 14 1 70.2 2.70 28.3 3.84 it 13.20 22.10 2.90 28 11 15 1 76.3 3.18 27.0 4.17 9-70 19.00 2.10 32 16 1 62.2 2.32 29.5 3.72 it 13.70 24.40 4.00 32 17 1 121.0 4.34 27.8 3-58 it 14.10 24.60 2.40 36 18 1 95.5 3.55 27.5 3.48 n 15.10 29.30 3.50 36 19 1 135.2 4.35 27.6 3.21 it 15.50 26.10 1.70 40 20 1 90.3 2.84 26.8 3.14 n 17.20 25.40 3.00 40 21 5 46.3 1.46 24.8 3.16 condensed milk 5.00 20.80 26.40 16 22 6 30.9 1.46 21.0 4.73 15 to 40 days 29.40 35.40 14.00 18 25 5 24.0 1.21 24.4 5.03 weaned at 28.50 20.10 3.90 20 24 5 25.0 1.25 27.1 5.00 15 days 23.10 28.80 3.40 20 25 4 32.4 1.80 26.2 5.56 it 23.40 30.10 4.50 22 ti 26 5 30.4 1.62 27.3 5.34 15.40 25.20 4.50 24 27 5 29.4 1.87 26.4 6.36 ti 13.70 18.60 1.80 26 28 2 42.7 2.49 25.9 5.80 u 21.40 31.70 5.60 28 29 2 42.3 2.54 25.8 6.00 it 18.10 26.50 4.70 32 50 2 72.6 3.71 26.0 5.10 11 10.00 17.00 3.00 35 51 1 61.5 6.44 27.6 10.40 tt 12.00 17.80 3.30 39 - 51a -
TABLE 9. (CONTINUED)
Small Small Mg. sugar Mean body Mean small intestine intestine as hydrolysed Assay Number of weight intestine nitrogen per cent per mg. nitrogen number animals weight (g.) (mg, per g.) body weight Diet sucrose maltose lactose (days)
52 6 24.7 1.29 20.80 5.20 weaned at 17.60 32.70 17.00 17 53 3 32.2 1.84 22.8 5.70 15 days 20.50 28.80 4.80 19 34 3 31.4 1.83 24.6 5.83 condensed 16.00 20.10 3.20 19 35 4 30.3 1.70 24.7 5.61 milk 15 to 17.80 27.30 2.30 21 36 3 37.5 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 2.40 25 38 2 47.5 3.21 24.6 6.76 to 40 days 19.20 26.00 4.40 27 39 2 56.7 3.01 26.4 5.31 ti 13.90 23.30 2.20 29 40 2 60.5 3.09 27-9 5.10 11 . 16.50 25.80 3.40 31 4-1 2 73.4 3.15 26.8 4.29 u 9.80 16.10 1.40 35 4-2 1 103.0 4.42 26.3 4.28 it 17.00 25.10 2.60 39 43 5 30.5 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 14.60 18 45 3 36.7 1.77 28.9 4.82 basal 15 14.60 22.90 3.10 20 46 2 48.7 1.63 30.90 3.35 to 28 days 6.80 14.10 4.00 22 47 2 50.2 1.80 36.3 3-58 condensed 9.20 16.70 2.80 24 48 2 65.5 2.55 31.2 3.89 milk 28 to 8.90 14.40 1.50 26 49 3 45.3 2.26 31.7 4.99 40 days 5.90 13.10 2.00 26 50 3 49.8 2.28 30.9 4.58 11 11.10 18.20 2.20 28 51 2 55.4 1.94 30.0 3.50 it 8.80 15.00 3.50 30 52 2 58.8 2.05 32.5 3.4-9 ti 6.70 15.60 2.20 32 53 2 66.4 2.4? 34.1 3.72 it 9.10 18.30 2.00 34 54 1 56.3 2.04 32.9 3.62 n .5.30 11.70 1.20 36 55 2 87.0 2.85 29.8 3.28 n 5.00 9.30 1.10 38 WEANING AT 21 LAYS WEANING AT 15 DAYS 40 «» BASAL 21 TO 40 DAYS 40 MILK 15 TO 40 DAYS
S25 30 s EH M 20 > 0 O CjJ o ojUTT-O — s 10 0 0 « W PH IIIII P 0 I W p CO 15 20 25 30 35 40 45 50 15 20 25 30 35 40 45 50 co ro o « O p WEANING AT 15 DAYS « WEANING AT 15 DAYS MILK 15 TO 28 DAYS P MILK 15 TO 28 DAYS 40 BASAL 28 TO 40 DAYS 40 BASAL 28 TO 40 DAYS CO W co 30 o a 30 o o CO C3 20 CO - 0 20
10 0 o o 0 10
0 1 IIIII 0 15 20 25 30 35 40 45 50 15 20 25 30 35 40 45 50
FIGURE 11. THE EFFECT ON SUCRASE ACTIVITY OF EARLY WEANING AND THE FEEDING OF CONDENSED MILK AND BASAL DIETS AT VARIOUS AGES IN THE RAT WEANING AT 21 DAYS WEANING AT 15 DAYS BASAL 21 TO 40 DAYS MILK 15 TO 40 DAYS 40
30 8 EH E-t H 20 c5 10 s « « 0 PM « 0 15 20 2 5 30 35 40 45 50 P 15 20 25 30 35 40 45 50 w w (H AGE IN DAYS CO AGE IN DAYS o o « a WEANING AT 15 DAYS p WEANING AT 15 DAYS 40 MILK 15 TO 28 DAYS BASAL 15 TO 28 DAYS « BASAL 28 TO 40. DAYS w MILK 28 TO 40 DAYS w CO CO 30
20 ClJ
10
0 0 1 1 15 20 25 30 35 40 45 50 15 20 25 30 35 40 45 50 AGE IN DAYS AGE IN DAYS
FIGURE 12. THE EFFECT ON MALTASE ACTIVITY OF EARLY WEANING AND THE FEEDING OF CONDENSED MILK AND BASAL DIETS AT VARIOUS AGES IN 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 - ' physiological variability, a fixed pattern for the activity of each of three carbohydrases, sucrase, maltase and lactase, with advancing age. In addition the agreement between these two experiments suggested that for the length of time the tissues were frozen, the three carbohydrases retain their activities, since tissues of pre-weaned animals were assayed immediately after sacrifice in the previous experiment, and stored at -15°0 for varying lengths of time in the present experiment.
In order to test the postulate that the marked changes in enzymatic activities at 21 days of age are associated with the weaning process and not merely coinci• dental to that age period, Groups 2 and 5 were weaned at 15 days of age while Group 1 served as a control, being weaned at 21 days. In this control group and in the previous experiment lactase activity was found to be maintained to 20 days of age at levels above 10 mg. lactose hydrolysed per mg. nitrogen. Lactase activity was found to be appreciably lower at this age in animals in Groups 2, 3 and 4 as a result of early weaning (Figure 13)* This decline in lactase activ• ity was essentially the same in both the milk and basal post- weaning diet groups, although there is an apparently higher post-weaning lactase activity in both Groups 2 and 3 re• ceiving the condensed milk diet immediately after weaning than in Group 4 in which the immediate post-weaning diet was the solid U.B.C. 10-55. The author feels it hazardous to - 56 - suggest that these differences are a direct result of the absence of lactose in the basal diet and the presence of this substrate in the condensed milk diet. Both sucrase and maltase activities increased in ages 16 to 21 days as a result of early weaning above those activities exhibited by Group 1.
In addition early weaning introduced greater variability in the activities of these two carbohydrases. The lower activ• ities found for sucrase in Group 4 are not explainable on the basis of specific substrate since over the age range 28 to
40 days in Group 2 a similar diet resulted in appreciably higher sucrase activities. The feeding of condensed milk in no way consistently influenced sucrase activity. The feeding of the basal diet immediately after weaning brought about a drop in both sucrase and maltase activities in animals beyond 20 days of age. Early weaning brought about an enhancement of maltase activity in Groups 2, 3, and 4 peculiar to each group and parallelling although at higher activities the results obtained for sucrase activity.
The author concludes that the feeding of con• densed milk does not bring about a significant increase in lactase activity in a feeding period of 13 days. In addition, there is strong evidence to indicate that early weaning brings about an early shift in the enzyme patterns estab• lished in the previous experiment. - 57 -
G. The Effect on Sucrase, Maltase and Lacitase Activity of
the Small Intestine of the Young Wistar Rat of
Prolonged Suckling on Foster Dams,
As a result of early weaning the patterns of
sucrase, maltase and lactase activity, established for - -
animals weaned at 21 days, were altered such that these changes occurred earlier in the life of these rats. In order
to substantiate these findings it was proposed to delay these changes beyond the ages at which they occur in rats weaned on to a basal diet at 21 days by extending the suckling period to 24 days of age.
In this experiment four groups of five pre-weaned
Wistar rats, 15 days of age, were removed from their dams and placed in four separate freezer trays lined with wood shavings.
Water was provided ad libitum to these four groups. Eight freshly lactating dams, all of which had littered within four or five days prior to the beginning of this experimental period, served as foster dams to these suckling 15 day old rats. Four of these dams were initially placed with the young litters while the remaining four were placed in similar freezer trays to which had been added the pelleted basal ration. After the first 12 hours of this experiment and after every succeeding 12 hour period the dams being suckled were exchanged with the remaining four dams, and allowed to consume the basal diet for a period of 12 hours. This exchanging procedure was continued throughout the experimental period. - 58 - The suckling rats were found to accept readily the exchange of dams. The dams however, were erratic in their behavior towards the litters that were previously associated with the other dams. In some instances cannibalistic behaviors were observed. Dams that were found to respond in this manner were removed from the experiment and replaced by other freshly lactating dams. Within a 56 hour period the dams had been selected such that this difficulty was overcome. In general older dams proved to be the most suitable for this experi• ment, since they were found to lactate more adequately than the younger rats as evidenced by the superior rate at which their litters grew. Fewer instances of cannibalism occurred among these older dams. The fostering process continued for nine days during which time numbers of suckling rats were sacrificed at intervals and assayed for sucrase, maltase and lactase activity. Table 10 summarizes the findings of this experiment. Also included in this table are data for animals of comparable ages weaned on to the basal diet at 21 days.
Figure 14 shows sucrase, maltase and lactase activities of the small intestine of rats suckled for extended periods of time as well as for rats weaned on to a basal diet at 21 days.
The results of this experiment indicate that pro• longed suckling altered insignificantly the activities of sucrase and maltase established in rats weaned at 21 days on to the basal diet. Lactase activity is maintained at levels appreciably above those found in rats 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 - diet at 21 days, as a result of prolonged suckling of freshly lactating dams. Rats weaned at 21 days on to the basal diet exhibited lactase activities of the order of 10 mg. lac• tose hydrolysed per mg. nitrogen at the weaning age. Rats 21 days of age in this fostering experiment showed lactase activities of the order of 25 mg. lactose hydrolysed per mg. nitrogen (Table 10). The failure of lactase activity to be maintained at this high level throughout this experiment may be due to a steadily diminishing rate of lactation in the foster dams. 'As pointed out by Brody (6) periods of in• anition of 10 hours brought about cessation of lactation in the rat as estimated by the "feed intake" of suckling litters;.
The period of inanition imposed on these foster dams exceeded this 10 hour period. It can be concluded that for at least part of the suckling period little milk was being ingested by the young in this experiment. One might postulate that after successive periods of inanition and feeding, a gradual decline in milk production would ensue such that by the ninth exchange of dams none of the dams could be considered to be
"freely" lactating. Any specific lactase inducer funda• mentally responsible for the patterns of lactase activity established may be a component of the colostrum and/or the milk up to the peak of lactation, and as such may predetermine these patterns such that fostering young rats on freshly lactating dams may result in an extended peak of lactase activity beyond the 21 day weaning age. It is of interest to note in the previous experiment that the decline in - 62 - lactase activity at the weaning age of 15 days was not as immediate as in animals weaned at 21 days. One.may postulate that a lactase inducer may he present in residual amounts in the lumen of the gut for a period following the 15 day weaning age and not necessarily in the intestinal contents after 21 days of age.
H. The Effect on Sucrase, Maltase and Lactase Activity of
Various Levels of the Three Sugars, Sucrose, Maltose and
Lactose in a Purified Synthetic Diet for the Laboratory
Wistar Rat.
The feeding of lactose has been inconsistently shown to increase lactase activity in the small intestine of the rat. The effect of either sucrose or maltose on the activities of their respective hydrolytic enzymes has not been described for the small intestine of the rat.
In this experiment synthetic purified diets were prepared according to Table 2. Sucrose, maltose and lactose were fed at 64- and 40 per cent levels. Diets containing the
40 per cent levels of these three sugars had in addition 24 per cent glucose added such that the sugar level in all diets was brought to 64 per cent. A ration containing glu• cose at 64 per cent, as the only sugar, served as a control diet, since presumably no hydrolysis of this sugar would be necessary prior to intestinal absorption. Each of these seven rations were fed to seven separate groups of 21 day od old laboratory Wistar rats for a period of 13 days. Numbers - 63 -
TABLE 11. 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 Mean small intestine intestine Sugar hydrolysed Assay Age Number of Mean body intestine nitrogen as per cent (mg. per mg. nitrogen) number (days) animals weight (g.) weight (K.) (mg.per g.) body weight Diet sucrose maltose lactose
1 23 3 41.5 1.20 30.2 2.9 64 per cent 6.0 12.2 1.5 2 25 3 46.0 1.67 29.6 3.6 dextrose 11.9 21.3 4.4 3 27 2 52.0 1.35 28.5 2.6 ti 7.4 13.2 4.6 4 30 1 48.0 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 11.0 1.3 6 23 3 36.7 1.60 31.7 4.3 40 per cent 9.5 16.1 3.2 7 25 3 42.7 1.53 30.2 3.6 sucrose 8.8 15.4 3.2 8 27 2 43.0 1.50 29.0 3.5 11 11.4 17.5 4.3 9 30 1 58.0 1.20 33.6 2.1 it 3.3 6.8 4.4 10 34 2 49.5 1.35 33.0 2.7 ti 8.3 15.7 1.8 11 23 3 40.0 1.33 29.9 3.3 64 per cent 12.7 19.7 4.4 12 25 3 43.3 1.33 31.0 3.1 sucrose 4.6 7.4 1.2 13 27 2 42.5 1.25 33.0 2.9 ti 8.4 13.7 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 16.3 3.6 16 23 3 40.0 1.13 31.6 2.8 40 per cent 9.8 15.4 4.0 17 25 3 41.6 0.87 31.8 2.1 maltose 4.9 8.9 2.4 18 27 2 47.5 1.55 30.8 3.3 it 8.0 12.2 2.8 19 30 2 42.0 1.55 31.0 3.7 it 7.4 15.1 4.6 20 34 1 43.0 1.40 33.6 3.3 it 12.6 25.9 6.3 21 23 3 42.6 1.17 32.2 2.7 64 perccent 6.4 11.9 2.9 22 25 3 41.7 0.98 30.5 2.4 maltose 3.1 6.2 1.6 23 27 2 40.5 1.15 32.0 2.8 11 8.4 16.9 4.2 24 30 2 39.0 1.25 30.6 3.2 u 9-5 16.7 4.2 25 34 1 39.0 1.40 36.8 3.6 11 10.0 23.3 3.6 26 23 3 41.0 1.27 30.0 3.1 40 per cent 10.4 18.5 3.6 27 25 3 41.0 1.87 31.0 4.6 lactose 12.7 19.8 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 17.8 4.5 30 34 2 49.5 1.80 31.0 3.6 it 10.9 17.7 1.8 31 23 3 38.0 1.07 31.6 2.8 64 per cent 8.7 14.8 3.7 32 25 3 34.6 1.63 29.8 4.7 lactose 16.5 24.4 4.3 33 27 2 46.5 1.80 28.8 3.9 11 9.1 18.4 3.6 34 30 1 44.0 2.60 29.8 5.9 it 18.3 27.8 2.2 35 34 2 45.5 1.40 31.2 3.1 n 11.4 15.5 3.4 - 64 -
O 64 PER CENT DEXTROSE 0 « 35 I- 40 PER CENT SUCROSE 0 M 64 PER CENT SUCROSE £ S3 30 2
23 25 27 29 31 33 AGE IN DAYS
W CJ O 40 PER CENT MALTOSE 0 K En 64 PER CENT MALTOSE M 35 S5 40 PER CENT LACTOSE 64 PER CENT LACTOSE A o 30
K
PH 25 p w CO 20 >H 1-3 o pel P & w CO
so p CCJO3
23 25 27 29 AGE IN DAYS
FIGURE 15. THE EFFECT ON SUCRASE ACTIVITY OF VARIOUS SYNTHETIC SUGAR DIETS - 65 -
$25 w o 64 PES CENT DEXTROSE 0 « 35 40 PER CENT SUCROSE • M 64 PER CENT SUCROSE f)
23 25 27 29 31 33 AGE IN DAYS
$25 o 40 PER CENT MALTOSE « EH 35 64 PER CENT MALTOSE H 55 40 PER CENT LACTOSE 64 PER CENT LACTOSE
23 25 27 29 31 33 AGE IN DAYS
FIGURE 16. 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
50 bO
EH W CiJ 45 _
0 64 PER CENT DEXTROSE • 40 PER CENT SUCROSE 8 64 PER CENT SUCROSE 40
21 24 27 30 33 AGE IN DAYS
0 40 PER CENT MALTOSE • 64 PER CENT MALTOSE 4 40 PER CENT LACTOSE 4 64 PER CENT LACTOSE 50
/""""\
EH US 45 -
40 -
21 24 27 30 33 AGE IN DAYS 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 sacrificed at various age intervals. The tissues were stored at -15°C until enzymatic activity was determined.
The results of this experiment are presented in
Table 11 and Figures 15, 16, aad 17, and demonstrate sucrase, maltase and lactase activities at various ages in the small intestine of the Wistar rat fed the various synthetic sugar diets. The mean body weights of the surviving animals of each group at the various ages during the course of the experiment are presented in Figure 18.
Lactose feeding at either 40 or 64- per cent levels in synthetic diets for weanling Wistar rats 21 to 34 days of age had no apparent lactase inducing effect as measured by the digestion of lactose in in vitro studies using tissue homogenates of the small intestine (Figure 17). Lactose activity ranged between 0 to 6 mg. lactose hydrolysed per mg. nitrogen, comparable for post weaning lactase activities for
Wistar rats weaned at 21 days on to the basal (U.B.C. 10-55) ration. These results, are in agreement with the findings of
Fischer et al (33), Fischer (36), Heilskov (26), and
Plimmer (13), in which the feeding of lactose in a number of different diets failed to bring about an enhancement of lactase activity. Fischer and Sutton (34) postulated lactase adaptability to the feeding of lactose, and in a later com• munication Fischer (36) reported lactase adaptation to the feeding of lactose to rats in terms of enzymatic activity - 69 - per unit tissue nitrogen. Neither of the other three sugars apparently" altered lactase activity.
Glucose brought about a slight decline in maltase activity. Neither sucrose nor lactose at either 40 or 64 per cent levels affected the activity of this enzyme. Maltase activity was increased by the feeding of maltose at both the
40 and 64 per cent levels.
Sucrose at either level failed to bring about an enhanced activity of its hydrolytic enzyme in the small in• testine of the rat above activity levels found in animals fed the 64 per cent glucose diet. Maltose had no apparent effect on sucrase activity. Several of the age groups fed
64 per cent lactose exhibited enhanced sucrase activities.
Rats fed the glucose and sucrose diets gained weight more readily than did the rats fed the other two sugars. Maltose failed to give good gains and proved to be somewhat inferior at either level in this regard to lactose.
Rats fed 64 pa? cent maltose gained essentially no weight over the 13 day feeding period. Differences in acceptability of the seven rations is proposed to be the major factor in de• termining the feeding value of these sugars for post weaned rats. Rats consuming the ^maltose diet were found to waste, by spilling, the greater part of the ration, whereas rats fed both the sucrose and glucose diets readily consumed - 70 - these sweet rations. The lactose diets were consumed more readily than were the maltose diets and less readily than the sucrose diets.
The author concludes that no apparent lactase adaptability incurred as a result of lactose feeding at either a 40 or 64 per cent level. It would be hazardous to state that either sucrase or maltase have been shown to be adaptable
in response to the feeding of their specific substrates in :. a purified diet in view of the results of this limited 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 -
It was concluded from this study that, where ad libitum feeding was practiced throughout, marked changes in sucrase, maltase and lactase activities could not be attributed to short periods of inanition.
(5) Sucrasei- maltase and lactase activities of the small intestine of the laboratory Wistar rat weaned on to the basal (U.B.C. 10-55) ration at 21 days changed dramatically at the weaning age. Lactase is found to be active in pre- weaned rats and declined sharply at the weaning age to 20 per cent of its pre-weaned level. In contrast pre-weaned rats showed little if any sucrase activity, while post-weaned rats exhibited high sucrase activity. Maltase of the small in• testine of pre-weaned rats was active and increased at weaning to levels double that of the pre-weaning age group.
A transitory digestive complex is postulated as a funda• mental physiological phenomena associated with the develop• ment and growth of animals.
(6) Early weaning brought about an earlier shift in the enzymatic patterns established for the rat weaned at 21 days.
Prolonged feeding of condensed milk failed to bring about an enhanced lactase activity in rats weaned on to that diet at
15 days. Eats weaned on to the basal diet at 15 days and fed condensed milk from 28 days on failed to exhibit increased intestinal lactase activities. - 73 - (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 its specific substrate.
In conclusion the author suggests that the character of the cells composing the tissue of the small - intestine undergo a transition with advancing age in respect of their elaboration of sucrase, maltase and lactase, There is, in addition, evidence suggesting that this transition is in some way directed, at least in part, by the changing dietary of the developing animal. Specific enzyme inducers present in the milk of the dam are postulated as a possible mechanism governing these changes. - 74 -
BIBLIOGRAPHY
1. Amnion, R. and U. Henning, Gibst es eine Duodenalsaft- Saccharase und - Maltase, Deutsch Ztschr. Verdauungs U. Stoffwechselkr, 16, 193-197, 1956.
2. Bailey, C. B., W. D. Kitts and A. J. Wood , The Devel• opment of the Digestive Enzyme System of the Pig During Its Pre-weaning Phase of Growth, B. In• testinal Lactase,Sucrase and Maltase, Can. J. Agri. Sci., 26, 51-58, 1956.
3. Bainbridge, F. A., J. Physiol.., £1, 98, (1904), cited by, Knox, W. E., V. H. Auerbach and E. C. C. Lin, Enzymatic and Metabolic Adaptation in Animals, Physiol. Revs., £6, 164-254, 1956.
4. Becker, D. E., D. E. Ullrey and S. W. Terrill, A Comparison of Carbohydrates in A Synthetic Milk Diet for the Baby Pig, Arch. Biochem. Biophys., 48, 178-183, 1954.
5. Bertrandi, G. M., Le Dosage des Sucres Reducteurs, Bull. Soc. Chim., 1285, Paris 1906. 6. Brody, S., "Bioenergetics and Growth", Reinhold, New York, 1945. 7. Brown, H. T. and J. Heron, Ueber die Hydrolytshen Wirkungen des Pankreas und des Duumdarms, Ann., 204, 228-251, 1880.
8. Cajori, F. A., The Lactase Activity of the Intestinal Mucosa of the Dog and Some Characteristics of Intestinal Lactase, J. Biol. Chem., 10°,, 159-168, 1935. 9. Carnevale, A.„ G. Cocozza and P. DeAngelis, Rilievi Sulle Attivita Enzimatiche del Succo Duodenale Nel Lattante, La Pediatria, 62, 3-21, 1954.
10. Cunningham, H. M. and G. J. Brisson, The Utilization of Lard by Baby Pigs, Can. J. Agri. Sci., 371, 1955. 11. Cunningham, H. M. and G. J. Brisson, The Effect of Amylases on the Digestibility of Starch by the Baby Pig, J. Animal Sci., 16, 370-376, 1957. - 75 -
12. Cunningham, H. M. and G.J. Brisson, Unpublished Data, cited in Cunningham, H. M. and G. J. Brisson J. Animal Sci., 16, 570-376, 1957.
13. Dastre, A., Achives de Physiol., 103, 1899; 718, 1887. cited by Plimmer, R. H. A., J. Physiol. 15, 20-31, 1907. 14. Dumas, J. and P. Boullay, Ann. Chim. et Phys., 37, 45, 1828. cited by Sumner, J. B. and K. Myrback, "The Enzymes", Academic Press, New York vol. 1, Part 1, 527, 1951.
15. Fischer, E., Einfluss der Configuration auf die Wirkung der Enzyme, II, Ber., 22, 34-79-3483, 1894. 16. Fischer, E., Einfluss der Configuration auf die Wirkung der Enzyme, III, Ber., 28, 1429-1438, 1895. 17. Fischer, E. and W. Niebel. Zit.Akad. Wiss. Berlin, 73, 1896. cited by Sumner, J. B. and K. Myrback, "The Enzymes", Academic Press, New York, vol. 1, Part 1, 1951.
18. Fischer, J. E. and T. S. Sutton, Effects of Lactose on Gastro-intestinal Motility, A Review, J. Dairy Sci., 22, 139-162, 1949.
19. Fischer, J. E., T. S. Sutton, J. L. Lawrence, H. H. Weiser and G. L. Stahley, The Effect of Lactose Feeding on Lactase Production, J. Dairy Sci., 22, 717-718, 1949.
20. Fischer, J. E. and T. S. Sutton, Effect of Previous Lactose Feeding Upon Intestinal Absorption of Lactose in the Rat, J. Dairy Sci., ^6, 7-10,1953.
21. Fischer, J. E., Small Intestine Growth - Stimulatory Effect of Lactose in Synthetic Diets for the Rat, Fed. Proc, 14, 433, 1955.
22. Fischer, J. E., Effects of Feeding A Diet Containing Lactose Upon B-D-Galactosidase Activity and Organ Development in the Rat Digestive Tract Am. J. Physiol., 188, 49-53, 1957.
23i Fulton, J. P., "A Textbook of Physiology", 17th Ed., W. B. Saunders, Philadelphia, 1955* - 76 -
24. Hawk, P. B., B. L. Oser and W. H. Summerson "Practical Physiological Chemistry", 12th Ed., The Blakiston Company, New York, 1947.
25. Heilskov, N. S. Chr., Studies on Animal Lactase. I. Lactase Activity Determination., Acta Physiol. Scand., 22, 267-276, 1951.
26. Heilskov, N. S. Chr., Studies on Animal Lactase. II. Distribution in Some of the Glands of the Digestive Tract., Acta. Physiol. Scand., 24, 84-89, 1951.'
27. Keene, M. P. L. and E. E. Hewer, Chem. Centr., 1, 3109, 1929. cited by Sumner, J. B. and K. Myrback, "The Enzymes", Academic Press, New York, vol. 1, Part 1, 1951.
28. Kitts, W. P., C. B. Bailey and A. J. Wood, The Development of the Digestive Enzyme System of the Pig During Its Pre-weaning Phase of Growth, A.Pancreatic Amylase and Lipase, Can. J. Agri. Sci., 26, 45-50, 1956.
29. Koehler, A. E., and S. E. Allen, The Nutritive Value of Lactose, J. Nutr., 8, 377-383, 1934. 30. Lloyd, L. E.., E. W. Crampton and V. G. MacKay, The Digestibility of Ration Nutrients by Three-Ve. Seven Week Pigs, J. Animal Sci., 16, 383-388, 1957.
31. Lusk, G., Animal Calorimetry, An Investigation into the Causes of the Specific Dynamic Action of Foodstuffs, J. Biol. Chem., 20, 555, 1915.
32. Maximow, A. A., and W. Bloom, "A Textbook of Histology", 6th ed., W. B. Saunders, Philadelphia, 1952.
33. Methods of Analysis, 7th ed., Association of Official Agricultural Chemists, Washington, 1950.
34. Meyer, J. and H. Necheles, Age Course of Enzymatic Activity in the Human iAlimentary Tract, J. Am. Med. Assoc., llj?, 2050, 1950.
35. Mitchell, H. S., Comparative Physiological Value of Different Amounts of Lactose Based on Growth and Fecal Analysis, Am. J. Physiol., 22, 542-544, 1927. - 77 -
36. Oppenheimer, C, Die Fermente und Ihre Wirkungen, Leipsie, 5th ed., Part 1, p. 628, 1925. cited by Cajori, F. A., J. Biol. Chem., 102, 159-168, 1935.
37. Plimmer, R. H. A., On the Presence of Lactase in the Intestines of Animals and on the Adaptation of the Intestine to Lactose., J. Physiol., 20-31, 1906.
38. Portier and Bierry, Compt. Rend. Soc. Biol., ^2, 423, 1900, £2, 810, 1901. cited by Plimmer, R. H. A., J. Physiol., 20-31, 1906.
39» Prosser, C. L., D. W. Bishop, F. A. Brown, T. L. Jahn, and V. J. Wulff, Comparative Physiology, W. Bi Saunders, Philadelphia, 1950.
40. Rohmann, F. J. Lappe, Ueber die Lactase des Diinndarms Ber. Deut. Chem. Ges., 28, 2506-2507, 1895.
41. Rohmann, F. and J. Nagano, Arch. Ges. Physiol., 95* 535, 1903, cited by Heilskov, N. S. Chr. Acta Physiol. Scand., 24, 84-89, 1951.
42. Rohmann, F., Ueber die Durch Parenterale Rohrzucker- injektionen,, Hervorgelockten,, Fermente des Blutserums von Trachtigen, Biochem. Z., 84, 382-398, 1917.
43. Steyermark, A., "Quantitative Organic Micro-Analysis", Blakiston, New York, 1951.
44. Sumner, J. B. and Myerback, K., "The Enzymes", Academic Press, New York, Vol. 1, Part 1, 1951. 1 45. Tachibana, T., Ber. Ges. Physiol. U. Exptl. Pharmakol., £4, 756, 1930, cited by Sumner, J. B. and K. Myerback, "The Enzymes", Academic Press, New York, Vol. 1, Part 1, 1951.
46. Weinland, E., Z. Biol., £8, 16, 1899. cited by Plimmer, R. H. A., J. Physiol., 20-31, 1906.