Download Activities of gastric, pancreatic, and intestinal brush

Activities of gastric, pancreatic, and intestinal
brush-border membrane enzymes
during postnatal development of dogs
Randal K. Buddington, PhD; Jan Elnif, MS; Christiane Malo, PhD; Jillian B. Donahoo, BS
Objective—To measure activities of digestive
enzymes during postnatal development in dogs.
Sample Population—Gastrointestinal tract tissues
obtained from 110 Beagles ranging from neonatal to
adult dogs.
Procedure—Pepsin and lipase activities were measured in gastric contents, and amylase, lipase,
trypsin, and chymotrypsin activities were measured
in small intestinal contents and pancreatic tissue.
Activities of lactase, sucrase, 4 peptidases, and
enteropeptidase were assayed in samples of mucosa
obtained from 3 regions of the small intestine.
Results—Gastric pH was low at all ages. Pepsin was
not detected until day 21, and activity increased
between day 63 and adulthood. Activities of amylase
and lipase in contents of the small intestine and pancreatic tissue were lower during suckling than after
weaning. Activities of trypsin and chymotrypsin did
not vary among ages for luminal contents, whereas
activities associated with pancreatic tissue decreased between birth and adulthood for trypsin but
increased for chymotrypsin. Lactase and γ-glutamyltranspeptidase activities were highest at birth,
whereas the activities of sucrase and the 4 peptidases increased after birth. Enteropeptidase was detected only in the proximal region of the small intestine
at all ages.
Conclusions and Clinical Relevance—Secretions in
the gastrointestinal tract proximal to the duodenum,
enzymes in milk, and other digestive mechanisms
compensate for low luminal activities of pancreatic
enzymes during the perinatal period. Postnatal
changes in digestive secretions influence nutrient
availability, concentrations of signaling molecules, and
activity of antimicrobial compounds that inhibit
pathogens. Matching sources of nutrients to digestive abilities will improve the health of dogs during
development. (Am J Vet Res 2003;64:627–634)
B
efore proteins, fats, and carbohydrates in feedstuffs can be absorbed, they must be hydrolyzed
by a sequential process that yields the constituent
Received August 14, 2002.
Accepted November 11, 2002.
From the Department of Biological Sciences, College of Arts and
Science (Buddington, Donahoo), and the Department of Basic
Sciences, College of Veterinary Medicine (Buddington),
Mississippi State University, Mississippi State, MS 39762; the
Department of Animal Science and Animal Health, Royal
Veterinary and Agricultural University, DK-1870 Frederiksberg,
Denmark (Elnif); and the Membrane Transport Research Group,
Department of Physiology, Faculty of Medicine, University of
Montreal, Montreal, QC, H3C 3J7, Canada (Malo).
Address correspondence to Dr. Buddington.
AJVR, Vol 64, No. 5, May 2003
amino acids and small peptides, fatty acids and
monoglycerides, and monosaccharides. This is
accomplished by enzymes secreted into the lumen of
the gastrointestinal tract (GIT) and by another set of
enzymes in the brush-border membrane (BBM) of
enterocytes.
Incomplete hydrolysis can limit the availability of
energy and nutrients in feedstuffs. Therefore, postnatal
changes in digestive secretions, including the activities
and relative proportions of the various luminal and
BBM digestive enzymes, can be predicted to influence
the digestion of dietary inputs and availability of nutrients. The best example is the reciprocal shift in the
activities of lactase and sucrase-isomaltase at the time
of weaning and the ability of developing mammals to
tolerate lactose and sucrose, respectively. Although the
pancreas develops before birth, it is functionally immature at the time of birth.1 Corresponding to this, the
activities of pancreatic enzymes are low during early
postnatal development, and most newborn mammals
are characterized as having pancreatic insufficiency.2
Mature functions are not attained until later, generally
at or after the time of weaning. Similarly, activities of
BBM peptidases are lower during suckling, compared
with activities after weaning.3 Consequently, limited
digestive capacities of dogs during suckling may limit
nutrient availability; such limitations have been speculated for cats.4
Patterns of development for the exocrine pancreas
and the BBM are species-specific and set by genetic
determinants with some modulation by dietary inputs
and hormones.1 Consequently, the findings for 1
species or diet regimen may not be applicable for
understanding development of the GIT in another
species or for other conditions.
Dogs have been used extensively for biomedical
research of digestive processes.5 Responses of the
mature digestive tract (including digestive secretions)
to secretogogues or other stimuli6 and to diets with
alternative sources of nutrients7-9 have been evaluated.
However, there is little known about the postnatal
changes in digestive secretions of dogs, despite the
obvious relevance to nutrition and health. The objective of the study reported here was to describe changes
in the activities of enzymes associated with the hydrolysis of proteins, carbohydrates, and fats in Beagles during development. The study involved assaying the
activity of enzymes in contents of the stomach and
small intestine, in pancreatic tissue, and associated
with the BBM of the small intestine. Results of other
studies10-12 in which these dogs were used to examine
characteristics of the small intestine during development have been reported elsewhere.
627
Materials and Methods
Animals—Ninety-five puppies representing 15 litters
and the 15 dams of those litters were used to conduct the
study. Dogs were procured, housed, and fed as described elsewhere.10 Digestive secretions and BBM enzymes were measured in tissues obtained from 6 groups of dogs (unsuckled
neonates within 1 hour after birth [n =14]; 1-day-old
neonates after initial suckling [20]; puppies at 21 [19], 42
[21], and 63 [21] days of age; and adult females [ie, dams of
the puppies; 15]).
Collection of samples—Dogs in each group were euthanatized. Within 5 minutes after each dog was euthanatized, contents of the stomach and mid region of the small intestine were
collected into vials and immediately placed on ice. Mucosa was
scraped from the proximal, mid, and distal regions of the small
intestine (the proximal region represented the proximal portion
of the jejunum, mid region represented the distal portion of the
jejunum, and distal region represented the ileum), rapidly
frozen, and stored at –80oC. The pancreas was removed,
weighed, and stored at –80oC.
Measurement of luminal enzymes—Luminal contents of
the stomach and small intestine were centrifuged (4,000 X g
for 5 minutes). The pH of the supernatants was recorded, and
supernatants were retained on ice for immediate assay of
enzyme activities.
Activity of pepsin (EC 3.4.23.1) in gastric contents was
measured by use of denatured hemoglobin as the substrate.13
Lipase (EC 3.1.1.3) activity in the gastric and small intestinal
contents was measured colorimetrically by use of a diagnostic kita in which 1,2-diglyceride was the substrate for the formation of quinone diimine dye.14 The specific substrates tyrosine-arginine methyl ester and benzoyl tyrosine ethyl ester
were used to quantify the activities of trypsin (EC 3.4.21.4)
and chymotrypsin (EC 3.4.21.1), respectively, in the supernatant from the small intestinal contents. Amylase (EC
3.2.1.1) activity in the intestinal contents was determined by
use of starch as the substrate.15
Measurement of enzymes in pancreatic tissue—Frozen
pancreatic tissue was homogenized in 0.05mM Tris buffer
(pH, 7.4) with 0.02mM CaCl2 (0.025 to 0.05 g of pancreas/mL). Homogenates were centrifuged twice at 4oC
(2,420 X g for 5 minutes, then 16,000 X g for 5 minutes).
After the second centrifugation, the supernatant was divided
into aliquots and stored at –80oC. Supernatants were diluted
(same buffer used for tissue homogenization) so that measured activities of each enzyme would be within the dynamic range of the assay and were comparable to standards with
known activities prepared by use of trypsin,b chymotrypsin,c
amylase,d and lipase.e Activities of trypsin, chymotrypsin, and
lipase were assayed as described previously. Enteropeptidasef
was used to activate trypsinogen and chymotrypsinogen,
which are found in pancreatic tissue as inactive zymogens.
Preliminary studies revealed that maximum trypsin and chymotrypsin activity was obtained by incubating the supernatant with 8.0 units of enteropeptidase at room temperature
(20 to 22oC) for 1 hour and then measuring trypsin and chymotrypsin activities. Amylase activity of the pancreatic tissue
homogenates was measured colorimetrically by use of a diagnostic kitg in which 4,6-ethylene (G7)-α,D-maltoheptaside
was the substrate.16
Enzyme activities in supernatants prepared from the
luminal contents and the homogenates of pancreas tissue
were adjusted on the basis of protein content (specific activity), which was measured by use of the Bradford reagenth
with bovine serum albumin as the standard.
Measurement of BBM enzymes—Specimens of BBM
vesicles (BBMV) were prepared from the samples of frozen
628
mucosa collected from the dogs.11 Activities of the disaccharidases, sucrase (EC 3.2.1.48) and lactase (EC 3.2.1.23),
were assayed in accordance with the method of Kunst et al.17
Leucyl-aminoamidase (LAP; EC 3.4.11.1) was measured by
use of L-leucyl-β-naphthylamide as the substrate.18 Rate of
ρ-nitroanilide release was measured at 410 nm by use of specific substrates to assay for the activities of aminopeptidase A
(EC 3.4.11.7),19 aminopeptidase N (EC 3.4.11.2),20 γ-glutamyltranspeptidase (GGT; [EC 2.3.2.2]),21 dipeptidyldipeptidase IV (EC 3.4.14.5),22 and enteropeptidase (EC
3.4.21.9).23 Activities of the enzymes were adjusted on the
basis of protein content (specific activity) by use of a protein
assay reagenti with bovine serum albumin as the standard.
Analysis of data—Values were reported as mean ± SEM.
Age effects were detected by use of an ANOVAj with specific
differences identified by use of the Duncan test. For all comparisons, values of P ≤ 0.05 were accepted as significant.
Results
Gastric contents—Pepsin activity was not detected in gastric contents obtained from 1-day-old puppies. The pH in gastric contents of 1-day-old puppies
(3.0) did not differ significantly from values recorded
in gastric contents of older dogs, which ranged from
2.5 in adult dogs to 3.9 in 63-day-old puppies. Mean
± SEM pepsin activity did not differ between gastric
contents obtained from puppies at 21 (126 ± 28 U/g of
gastric contents), 42 (601 ± 248 U/g of gastric contents), and 63 (475 ± 202 U/g of gastric contents) days
of age, but values in gastric contents of adult dogs
(2,208 ± 524 U/g of gastric contents) were significantly higher than for gastric contents obtained from the
puppies. Lipase activity was detected in the gastric
contents obtained from dogs of all ages, but values for
suckling dogs (day 1, 10.3 U/g of gastric contents; day
21, 107 U/g of gastric contents; day 42, 48 U/g of gastric contents) were significantly higher than those for
63-day-old puppies (0.29 U/g of gastric contents) or
adult dogs (0.31 U/g of gastric contents).
Contents of the small intestine and pancreatic
enzymes—The pH in the small intestinal contents differed significantly among the groups. It was lowest in
42-day-old puppies (pH, 5.4) and highest in adult dogs
(pH, 6.4), with intermediate values for the other ages.
We were not able to measure pH in 1-day-old puppies
because of the small volume of intestinal contents at
that time.
Protein content of pancreatic tissues decreased significantly from 39 ± 2 mg/g at day 1 to 30 ± 1 mg/g at
day 42. Values increased thereafter and were significantly higher in adult dogs (53 ± 3 mg/g), compared
with values for all other ages.
Trypsin activity of pancreatic tissue was highest
during suckling (days 1 to 42), with the lowest values
measured at day 63 (Fig 1). In contrast, trypsin activity in the luminal contents was significantly lower on
day 1 but did not differ significantly among the other
ages. Chymotrypsin activity of pancreatic tissue
increased between days 1 and 21, with another
increase between day 63 and the adult dogs. Specific
activity for chymotrypsin in the luminal contents was
significantly higher at day 63, compared with activity
at day 1, but had similar values for all other ages.
AJVR, Vol 64, No. 5, May 2003
Figure 1—Mean ± SEM activity of trypsin (A), chymotrypsin (B), amylase (C), and
lipase (D) in the contents of the proximal region of the small intestine (solid circle)
and pancreatic tissue (open circle) of puppies that ranged from 1 to 63 days of age
and adult dogs. Values represent activity adjusted on the basis of wet weight. Trypsin
and chymotrypsin activity of pancreatic tissues was determined after activation with
enteropeptidase. a,b = For intestinal contents, values for each enzyme with different
letters differ significantly (P < 0.05). c,d,e = For pancreatic tissues, values for each
enzyme with different letters differ significantly (P < 0.05).
Amylase activity was not detected in the pancreatic homogenates or luminal contents at day 1 (Fig 1).
Low values were first detected in pancreatic tissue and
luminal contents at day 21. The activity in the pancreatic tissue increased significantly (> 10-fold) between
day 63 and the adult dogs but decreased slightly in the
luminal contents during the same period.
Lipase activity was not detected in pancreatic
homogenates for puppies ≤ 42 days of age, but high
amounts of activity were detected for puppies at day 63
and adult dogs (Fig 1). Low amounts of lipase activity
were detected in intestinal contents of suckling puppies. Activity in the luminal contents peaked in 63day-old puppies, with a decrease in measured activity
in adult dogs.
An additional indicator of age-related changes in
the pancreatic enzymes was determined. Ratios were
calculated for the activities of amylase, lipase, and chymotrypsin relative to trypsin. Amylase-to-trypsin ratios
for pancreatic tissue increased significantly (P < 0.001)
from 0 at day 1 to 0.09 ± 0.01 in the adult dogs. A similar age-related significant (P < 0.001) increase was
AJVR, Vol 64, No. 5, May 2003
Figure 2—Mean ± SEM specific activities of lactase (open symbols) and sucrase (solid symbols) associated with brush-border
membrane vesicles (BBMV) prepared from the proximal (circles), mid (triangles), and distal (squares) regions of the small
intestine of puppies that ranged from birth (day 0) to 63 days of
age and adult dogs.
629
detected for lipase-to-trypsin ratios, but the increase
was after day 42, with the highest ratios at day 63
(0.027 ± 0.002). Chymotrypsin-to-trypsin ratios were
similar from days 1 to 42, then increased significantly
for 63-day-old puppies and adult dogs.
BBM enzymes—Mean values for the enrichment
factors for the BBM enzymes were > 5 for the various
ages and 3 regions of the small intestine. These findings indicated that the BBM had been isolated from the
homogenate. However, mean recovery values were
< 20%, suggesting that only a portion of the BBM was
isolated or, alternatively, that a substantial fraction of
enzyme activity was intracellular and not associated
with the apical membrane.
Lactase activity was detected in homogenates (data
not shown) and BBMV from days 0 to 21 but not thereafter (Fig 2). Sucrase activity was first detected at day
21, but this activity did not increase significantly until
between day 63 and adulthood (Fig 2). When lactase
and sucrase activities were detected, activities
decreased from the proximal to the distal region of the
small intestine, with the exception of lactase at 21
days.
Two patterns of distribution were evident in the
small intestine for the activities of the 4 BBM peptidases (ie, aminopeptidases A and N, LAP, and dipeptidyldipeptidase IV; Fig 3). A proximal-to-distal gradient
was evident in dogs ≥ 21 days old for aminopeptidase
N and LAP but not for aminopeptidase A and dipeptidyl-dipeptidase IV. When all 3 regions were pooled,
the activities of aminopeptidases A and N and dipeptidyl-dipeptidase IV were lower at day 0 with a gradual
increase into adulthood. The activity of LAP was lowest at day 0 but similar at all other ages.
The activity of GGT did not vary among age
groups. It also did not have a consistent pattern of distribution among the 3 regions of the small intestine
(Fig 4). We did not detect Na+-K+-ATPase in BBMV and
homogenates of puppies at day 0 (data not shown),
and there was only low activity detected in BBMV and
homogenates at day 1. The highest activities were
detected at 21 days with lower and similar activities for
Figure 4— Mean ± SEM specific activities of γ-glutamyltranspeptidase (solid symbols) and Na+-K+-ATPase (open symbols) associated with BBMV prepared from the proximal (circles), mid (triangles), and distal (squares) regions of the small
intestine of puppies that ranged from birth (day 0) to 63 days of
age and adult dogs.
puppies at days 42 and 63 and the adult dogs.
Enteropeptidase activity was detected in BBMV
and homogenates at all ages; however, it was detected
only in the proximal region of the small intestine.
Activity was highest at days 0 and 1, compared with
activity for all other ages (data not shown).
Discussion
To our knowledge, the study reported here is the
first in which changes in digestive enzyme activities of
dogs have been measured from birth to adulthood.
Although only a limited number of enzymes were evaluated, the lower activities of some pancreatic and BBM
enzymes revealed the fact that hydrolytic capacities of
suckling puppies differ markedly from those of weaned
and adult dogs. Lower hydrolytic capacities have led to
the contention that neonatal animals have pancreatic
insufficiency despite having a fully formed pancreas at
birth.2 This speculation is corroborated by the decrease
in protein content of the pancreas during early suckling that was reported here, which suggests enzyme
Figure 3—Mean ± SEM specific activities of aminopeptidases A (solid symbols) and
dipeptidase IV (open symbols; A), aminopeptidase N (solid circle), and leucyl-aminopeptidase (open circle; B) associated with BBMV prepared from the proximal (circles), mid
(triangles), and distal (squares) regions of the small intestine obtained from puppies
ranging from birth (day 0) to 63 days of age and adult dogs.
630
AJVR, Vol 64, No. 5, May 2003
synthesis by the pancreas may not be adequate to
match the demands for secretion. The obvious implication is that neonatal and suckling puppies possess
limited abilities for luminal hydrolysis of dietary
inputs. However, the ability of neonatal puppies to
digest milk and grow indicate alternative compensatory digestive processes (eg, other sources of enzymes or
intracellular digestion of macromolecules) similar to
those reported24 for adult dogs with exocrine pancreatic insufficiency.
Age-related increases in the activities of most
digestive enzymes provide growing dogs with
enhanced abilities to hydrolyze feedstuffs. However,
the activities of pancreatic enzymes did not change in
parallel, as evident by the greater proportional increase
after weaning for amylase and lipase compared to
trypsin. In addition, maturation of digestive secretions
was not complete by week 9 based on comparisons
with the adult dogs. Although unknown for Beagles,
the lack of differences between 12 and 20 weeks for the
digestibility and responses of Labrador dogs to different dietsk suggest GIT digestive processes for that breed
mature between weeks 9 and 12.
Lack of pepsin activity in the gastric contents at
day 1 is consistent with the limited secretion of gastric
proteases reported for other suckling mammals,25-27
including humans.28 These findings, in conjunction
with the low activities of trypsin in luminal contents,
suggest that newborn dogs have only limited abilities
to hydrolyze dietary protein. The combination of low
pH of the stomach contents of the dogs reported here,
high protease activities in the gastric mucosa of neonatal dogs29 and pigs,27 high trypsin activity in pancreatic
tissue, and enteropeptidase activity in the proximal
region of the small intestine of the dogs of this study
indicate that low luminal proteolytic activity during
the perinatal period is limited by secretion, not by synthesis or activation. Similarly, peptidase activity associated with the BBM was lower for neonatal puppies,
compared with activity for older puppies or adult dogs.
Because activity was consistently detected in
homogenates at all ages, insertion into the apical membrane, and not a decrease in synthesis, was the probable cause of the low BBM activity. Chymotrypsin activity provided a contrasting pattern, with low activity in
luminal contents and pancreatic tissues throughout all
ages. Despite the age-related increase in chymotrypsin
activity, dogs are more dependent on trypsin for luminal digestion of dietary proteins.30 Interestingly, the
chymotrypsin-to-trypsin ratio in pancreatic tissue
increased nearly 7-fold after weaning, but an age effect
was not detected for the luminal contents. This provides additional evidence that luminal activities are
regulated more by secretion than by synthesis.
The combination of low protease activity in the
luminal contents and BBM during the neonatal period
and protease inhibitors in milk (particularly
colostrum31,32) appears to be adaptive. It has been speculated that limited protease activity allows for the
acquisition of passive immunity and preserves the high
concentrations of signaling molecules contained in
colostrum that may be important for regulating GIT
development and functions. Consequently, neonatal
AJVR, Vol 64, No. 5, May 2003
dogs may be reliant on endocytosis by the small intestine and intracellular digestion of protein. Although
the information is not known for dogs, other species
have postnatal decreases in endocytosis33,34 and a shift
from intracellular digestion to an increasing reliance
on luminal hydrolysis.35
Postnatal changes in protease activities vary
among species36 and enzymes. For example, there are
several forms of gastric proteases that have differing
hydrolytic characteristics.37-40 In the late-fetal and earlysuckling periods of development, the stomach synthesizes and secretes chymosin, which is important for
clotting milk but has weaker hydrolytic properties
compared to the pepsin isoforms secreted by the
mature stomach.41 This may explain the reduced ability of gastric contents from neonatal dogs to hydrolyze
hemoglobin. Conversely, pepsin secreted by the gastric
mucosa of mature dogs is less able to clot milk.42
Apparently, secretion of the pepsin isoforms and other
gastric aspartic proteases that are characteristic of
mature dogs43 increases during suckling, with adult
values achieved sometime after weaning. A similar pattern of postnatal development for the activities and
types of gastric proteases has been reported for cats.44
Low lipase activity in the pancreatic tissue and
luminal contents of the small intestine during early
postnatal development of dogs and ferrets,45 in conjunction with immature bile secretion46 and undeveloped intestinal absorption and recycling of bile acids,47
is not consistent with the high fat content in the milk
of bitches.48 Instead, neonatal and suckling puppies
have a greater dependency on lipase activity in the GIT
proximal to the duodenum.45,49 This includes 3 sets of
enzymes that originate from nonpancreatic tissues,
each of which have unique characteristics and distinct
patterns of development.50
Milk collected from dogs has lipase activities that
are similar to those measured in human milk but that
exceed those in the milk of cats.51 Colostrum has lower
lipase activity, compared with the activity for milk, and
this is consistent with the lower activity measured in
the contents of the stomach and small intestine at day
1, compared with activity measured at day 21. Lipase is
also secreted by the gastric mucosa of suckling dogs29,52
and cats,53 and 10 to 30% of milk fat is hydrolyzed in the
stomach of suckling rats.54 Gastric lipases can be differentiated from those secreted by the pancreas on the
basis of stereospecificity55 and optimal pH.56 The reciprocal decrease in gastric lipase activity and increase in
pancreatic lipase activity at the time of weaning coincided with the loss of milk lipase activity, which indicates that there is a shift in the relative importance of
the various sources of lipase. Although lingual lipase is
secreted by suckling rats,54 the potential contribution to
lipid digestion is unknown for suckling puppies and is
minimal or lacking for adult dogs.29 The shift to a
greater dependency on pancreatic lipase between the
suckling period and adulthood was evident by the >
1,000-fold increase for the lipase-to-trypsin ratio in the
luminal contents of the small intestine.
The source of fat and digestion of fat57 have an
interesting relationship that can have other important
roles in early development. Specifically, hydrolysis of
631
milk fat, but not other lipids, inhibits parasites.58,59
Furthermore, digestion of fat and the release of fatty
acids (eg, oleic acid) stimulates secretion of cholecystokinin and other regulatory peptides60-62 that have
trophic influences on the mucosa.63 It is unknown
whether feeding suckling puppies milk replacers or
solid diets with alternative sources of lipids would
reduce pancreatic secretion, alter development of the
GIT, or influence health of the developing dogs.
The milk of bitches also contains amylase64,65 and
ribonuclease66 activity. This explains the reason that
low but measurable amounts of amylase activity were
detected in the contents of the small intestine at days
1 and 21, despite the virtual lack of amylase activity
in pancreatic tissue at day 1 and the negligible
amount of activity at day 21. The increase in amylase
activity in pancreatic tissue and luminal contents of
the small intestine after day 21 coincided with the
period when the puppies began to eat the weaning
diet, which contained starch and other complex carbohydrates. Another increase in amylase activity in
pancreatic tissue was detected when the dogs were
weaned (day 63) and in the adult dogs30; the maintenance diet for the adult dogs had a higher proportion
of carbohydrate than did the weaning diet. However,
this did not result in higher amylolytic activity in
luminal contents of the small intestine. This further
documents that secretion, not synthesis, limits activity in the luminal contents.
The role of GGT in cysteine absorption and glutathione metabolism is critical for suckling Beagles67
and for function of the GIT.68-70 Activity has been
detected in numerous tissues,71 including the small
intestine of dogs,72 and the higher activities in suckling
puppies, compared with activities for weaned and adult
dogs reported here, is similar to findings73,74 for rodents
and other species.
Although Na+-K+-ATPase activity was detected in
the BBMV, the majority of activity is associated with the
basolateral membrane of enterocytes and colonocytes.75,76 The low amounts of Na+-K+-ATPase activity in
the small intestine of neonatal dogs reported here and
the colon of infant rats reported in another study77 suggest the mechanism of potassium absorption changes
after the neonatal period. The postnatal peak in activity agrees with findings for rodents and with the higher
rates of absorption and potassium requirements for
suckling animals, compared with values for adult animals.75,77
In some animals, patterns of development for the
digestive enzymes reflect a combination of genetic
determinants interacting with dietary inputs.1 The
increase in amylase activity after day 21 is consistent
with starch in the weaning diet but not in milk. This
suggests that dietary inputs play an important role. In
contrast, there was a loss of lactase activity in the BBM
and homogenate between days 21 and 42, even though
the dogs continued to consume milk, which has a relatively constant lactose content.48 In addition, sucrase
activity was initially detected at 21 days, even though
sucrose is not found in milk and the puppies had not
yet started to eat the solid weaning diet. These findings
indicate that genetic determinants, not diet, trigger rec632
iprocal changes in the activities of lactase and sucrase.
Interestingly, specific activity of lactase increased during the first 21 days after birth, whereas rates of aldohexose transport decreased.11 These findings indicate
that the activities of BBM enzymes and the associated
transport functions may not be matched, which is the
situation seen in older animals.78,79
The relative importance of genetic determinants
and dietary inputs in mediating the changes in protease
activities are less obvious, because the protein content
of the weaning diet was not higher than that of milk of
the dams. Instead, the digestibility of proteins in the
plant materials used to formulate diets for dogs is
lower8 and more variable,7 compared with digestibility
of animal tissues, and this may trigger a compensatory
increase in the synthesis and secretion of pancreatic
proteases. It is unlikely that the increase was stimulated by protease inhibitors associated with the plant
ingredients.80
Weaning diets are prepared by use of a combination of animal tissues and plant materials. However,
the age-related changes in digestive secretions may
limit the ability of young dogs to digest some of the
components. A better understanding of the patterns of
development for digestive secretions, associated mechanisms, and signals triggering the changes will assist in
the formulation of diets that are matched to the digestive capacities of specific stages of development in
dogs.
a
Diagnostic kit 805, Sigma Chemical Co, St Louis, Mo.
Trypsin, type III (from bovine pancreas), Sigma Chemical Co, St
Louis, Mo.
c
α-chymotrypsin, type I-S (from bovine pancreas), Sigma Chemical
Co, St Louis, Mo.
d
α-amylase, type VI-B (from porcine pancreas), Sigma Chemical Co,
St Louis, Mo.
e
Lipase, type II (from porcine pancreas), Sigma Chemical Co, St
Louis, Mo.
f
Enteropeptidase (from porcine intestine), Sigma Chemical Co, St
Louis, Mo.
g
Diagnostic kit 577, Sigma Chemical Co, St Louis, Mo.
h
Protein assay dye reagent, Bio-Rad Laboratories, Hercules, Calif.
i
BCA protein assay reagent, Pierce Chemical Co, Rockford, Ill.
j
SAS, version 8, SAS Institute Inc, Cary, NC.
k
Gilham MS, Booles D, Johnson JV, et al. Digestibility in Labrador
Retrievers during growth (abstr), in Proceedings. Nutr Soc
1993;52:294.
b
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