Download Experimental. Subjects. The fetal and new

INTESTINAL
LACTASE, ALKALINE
AND ACID
PHOSPHATASE
IN THE SWINE FETUS AND
NEWBORN
PIG 1
J.
I.
SPRAGUE, 2 D.
E.
ULLREY,
D.
G.
WADDILL, 3 E.
R.
MILLER,
C.
L.
ZUTAUT
AND J. A. HOEFER
Michigan State University, East Lansing ~
E differentiation and growth of the
T Hfetus,
initiated at conception, is destined
to prepare this delicate organism for birth
and a strikingly different postnatal life. This
preparation involves a development of metabolic systems designed to cope with this
change. Among these are the enzymes necessary to promote digestion of food soon to
enter the gastro-intestinal tract.
Much of the research concerned with development of digestive enzymes in mammalian fetuses has been reviewed by Needham
(1931), Driscoll and Hsia (1958) and Fries
(1958). Hartman etal. (1961) surveyed the
literature regarding the digestive enzymes
of the neo-natal pig and the pig through
weaning.
Although considerable information about
intestinal enzymes of the postnatal subject
has accumulated, little similar knowledge of
the fetal pig is available. For that reason this
study was initiated, with the objective of estimating the time of appearance, location and
concentration of intestinal lactase and acid
and alkaline phosphatase as the swine fetus
developed.
considered pregnant and Caesarian sections
were performed at 30, 51, 72 or 93 days
post-breeding or they were allowed to give
birth naturally. In this manner three litters
were examined at 30 days, five at 51 days,
four at 72 days, four at 93 days and three
litters at term. None of the newborn pigs
were allowed to nurse.
Three fetuses or newborn pigs were
selected from each litter for the enzyme study.
In general the first, third and fifth fetuses in
the right uterine horn (counting from the
ovarian end) were used.
The fetuses or pigs were opened by making
a ventral incision exposing the viscera. The
gastrointestinal tract was removed and divided (except for the 30- and 51-day fetuses)
into stomach, cranial duodenum, caudal
duodenum, three equal lengths of jejunumileum, colon and rectum. The segments were
placed separately in glass vials, corked
tightly and immediately frozen at -79 ~ C.
The gastrointestinal tracts of the 51-day
fetuses were analyzed in one section and the
entire bodies of the 30-day fetuses were
analyzed.
T~ssue Preparation for Enzyme Analysis.
M a t e r i a l s and M e t h o d s
Experimental. Subjects. The fetal and newborn pigs were obtained from 19 Yorkshire
first-litter gilts. The gilts were confined to
pens with concrete floors and were offered
water and a natural diet ad libitum (Waddill
et al. 1962). Each gilt was bred to two Yorkshire boars, one boar breeding a gilt on the
first day of estrus followed by a second boar
on the succeeding day. If further estrus was
not observed within 25 days, the gilts were
1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No.
3010.
2 Present address: Department of Animal Science, Colorado
State University, Fort Collins, Colorado.
a Present address: Department of Agriculture, Chico State
College, Chico, California.
Department of Animal Husbandry. Appreciation is expressed
for the assistance of R. W. VanPelt and G. It. Conner of the
Department of Surgery and Medicine in performing the Caesarian sections.
t21
Approximately 1 gin. of thawed wet tissue
was weighed directly into a 2 ml. tared plastic
capsule. The tissue was shredded with a sharp
scissors and the weight adjusted to 1_+0.010
gm. After removal to a cold room (2 ~ C.),
a small quantity of ice water and a hard
plastic bead were placed in the capsule. The
capsule was sealed with a plastic cap and
agitated for 3 min. on a dental amalgamator.
The homogenized tissue was quantitatively
removed from the capsule by washing with
water into a calibrated centrifuge tube. Ice
water was added to dilute the original tissue
homogenate to 10 ml. The tissue was suspended by gentle hand agitation followed by
centrifugation in the cold room for 15 min.
in an Ivan Sorvall Type M centrifuge at
approximately 3000 rpm. The supernatant
was used in the assay.
122
SPRAGUE E T AL.
To allow expression of enzyme concentration on a dry tissue basis, a second weighed
tissue sample was taken to dryness in a
forced draft oven at 105 ~ C. and the percent
dry matter was calculated.
Lactase Assay. The lactase assay was performed according to the method described
by Colowick and Kaplan (1955) except that
a buffer p H of 5.6 was used rather than p H
7.25 since it was experimentally established
that the lower value produced optimum activity. The reaction mixture contained 3.5
ml. of 0.2 M, p H 5.6 acetate buffer, 0.5 ml.
of 0.01 M o-nitrophenyl-fl-D-galactopyranoside and 1.0 ml. of tissue homogenate supernatant. The reaction was allowed to proceed
at 40 ~ C. for 15 min. The o-nitrophenol
released by the enzyme was measured spectrophotometrically.
Alkaline Phosphatase Assay. The phosphatase assays were modifications of the procedure for serum alkaline and acid phosphatase
described in Sigma Technical Bulletin 104
(1958). The reaction mixture for alkaline
phosphatase assay contained 0.5 ml. of
0.1 M, pH 10.5 glycine buffer, 0.5 ml. of
p-nitrophenyl phosphate
substrate,
and
0.1 ml. of tissue homogenate supernatant
(appropriately diluted). This mixture was
incubated at 40 ~ C. for 30 min., following
which 10 ml. of 0.02 N N a O H were added to
stop the reaction. The p-nitrophenol released
by the enzyme was measured spectrophotometrically.
Acid Phosphatase Assay. The reaction
mixture for acid phosphatase assay contained
0.5 ml. of 0.9 M, pH 4.8 citrate buffer, 0.5 ml.
of p-nitrophenyl phosphate substrate and
0.2 ml. of tissue homogenate supernatant.
Following 30 min. of incubation at 40 ~ C.
the reaction was stopped by adding 4 ml. of
0.1 N NaOH. The p-nitrophenol released by
the enzyme was spectrophotometrically determined.
In all assays correction was made for the
optical density contributed by the tissue
homogenate supernatant.
R e s u l t s and D i s c u s s i o n
Enzyme Distribution at Birth. The distribution of intestinal lactase and alkaline and
acid phosphatase at birth is summarized in
table 1.
N o enzyme activity was observed in the
stomach or colon and rectum. The middle
T A B L E 1. E N Z Y M E D I S T R I B U T I O N A T B I R T H
Organ
Stomach
Duodenum, cran.
Duodenum, caud.
Jej.-ileum, cran.
Jej.-ileum, mid.
Jej.-ileum, caud.
Colon a nd rectum
Lactase ~
Alk.
phos. b
0
0.19"
0.38
o. 36
0.36
0.26*
0
0
41"
87
145
174
58*
0
Acid
phos?
0
5.8*
8.0
9.8
9.4
9.4
0
a mM
o-nitrophenol released per 100 rag. dry tissue (15
minutes incubation).
b mlV[ p-nitrophenc~l released per rag. dry tissue (30 minutes incubation).
Significantly (P~.05) less than highest three values.
portions of the small intestine exhibited
greater lactase and alkaline phosphatase activity than did the portions nearer the
stomach or colon ( P ~ . 0 5 ) . The cranial
portion of the duodenum was significantly
( P ~ . 0 5 ) lower in its concentration of acid
phosphatase than were the more caudal portions of the small intestine.
Enzyme Distribution at Fetal Ages. The
differences in enzyme concentration in relation to anatomical location, evident at birth,
prevailed generally at 93 and at 72 days postbreeding and are illustrated in tables 2 and 3.
The duodenal concentration of lactase was
significantly ( ~ . 0 5 ) lower than that of any
section of the jejunum-ileum at 93 days.
At 72 days the duodenal and caudal jejunum-ileum concentration of both alkaline
and acid phosphatase was significantly
( P ~ . 0 5 ) lower than that of the cranial and
middle sections of the jejunum-ileum.
Such differences were not determined in
the 51- and 30-day fetuses.
Enzyme Concentration in Relation to Age.
If one adds the enzyme concentration of all
tissues (showing activity) studied at each age
and divides by the number of observations, an
T A B L E 2. E N Z Y M E D I S T R I B U T I O N I N T H E
F E T U S A T 93 D A Y S P O S T - B R E E D I N G
Organ
Stomach
Duodenum
Jei.-ileum, cran.
Jej.-ileum, mid.
Jei.-ileum, caud.
Colon a nd rectum
Lactase a
Alk.
phos.b
Acid
phos?
0
0.20*
o. 28
0.28
0.24
0
0
7.6
9.0
6.9
8.3
0
0
5.6
6.4
5.6
6.1
0
a mM o-nitrophenol released per 100 rag. dry tissue (15
minutes incubation).
b mM p-nitrophennl released per rag. dry tissue (30 minutes incubation).
Significantly (P~.05) less than highest three values.
LACTASE AND PHOSPHATASE IN SWINE FETUS
TABLE 3. ENZYME DISTRIBUTION IN THE
FETUS AT 72 DAYS POST-BREEDING
Organ
Stomach
Duodenum
Jej.-ileum, cran.
Jej.-ileum, mid.
Jej.-ileum, caud.
Colon and rectum
Lactase =
phosP
Alk.
Acid
phos?
0
0.12
0.16
0.14
0.06
0
0
2.2*
2.7
3.5
2.1"
0
0
5.0"
6.8
6.3
4.1"
0
a mM o-nitrophenol released per 100 rag. dry tissue (15
minutes incubation).
b mM p-nitrophenol released per mg. dry tissue (30 minutes incubation).
Significantly (P~.05) less than highest three values.
estimate of changes occurring, as the fetus
develops, m a y be made. These changes in
enzyme concentration in relation to age are
illustrated in table 4.
T h e concentration of each enzyme studied
was significantly greater a t birth than at a n y
fetal age ( P ~ . 0 5 , lactase and acid phosphatase; P < .01, alkaline p h o s p h a t a s e ) . T h e
concentration of alkaline phosphatase at birth
was 12.6 times t h a t at 93 days post-breeding.
T h e alkaline phosphatase concentration at
93 days post-breeding was significantly
( P < . 0 5 ) greater than at a n y other fetal age.
Several of the changes noted here are
similar to observations made previously in the
chick and the mouse. Moog (1950) found
t h a t the activity of intestinal alkaline phosphatase increased 100 times during the last
2.5 days of embryonic life in the chick. I n the
mouse a similar rise in a c t i v i t y was seen just
before birth (Moog, 1951). Acid phosphatase
concentration was not found to increase as
m a r k e d l y as t h a t of alkaline phosphatase
during the embryonic or fetal period (Moog,
1946).
Lactase has been found a t high concentrations in the pig at birth (Bailey et al., 1956;
Walker, 1959), b u t little information conTABLE 4. ENZYME CONCENTRATION IN
RELATION TO AGE
Days
post-breeding
Lactase ~
Alk.
phosP
30
51
72
93
Birth
0.12
0.21
0.12
0.25
0.31"
4.7
2.8
2.6
8.0*
101.0"*
Acid
phos P
6.1
7.0
5.5
5.9
8.5*
a t o m o-nitrophenol released per 100 rag. dry tissue (15
minutes incubation).
b mM p-nitropbenol released per rag, dry tissue (30 minutes incubation).
Significantly (P~.05) greater than all lower values.
~ Significantly (P~.01) greater than all lower values.
123
cerning the fetus appears to be available.
Fries (1958) in his review of the developm e n t of lactase in h u m a n fetuses reported
t h a t it did not appear until the seventh or
eighth fetal month and often was absent in
p r e m a t u r e infants. I t would a p p e a r t h a t the
last few weeks of fetal life are very important in preparing the developing organism for
gaining sustenance from nutrients soon to be
orally ingested.
Summary
Fifty-seven fetuses or newborn pigs were
obtained from 19 Yorkshire first-litter gilts
for assay of the gastrointestinal concentration of lactase and alkaline and acid phosphatase. T h e fetuses were obtained b y
Caesarian section a t 30, 51, 72 and 93 days
post-breeding. T h e newborn pigs were taken
immediately at birth and were not allowed
to nurse.
N o n e of the enzymes studied were found
in the stomach or colon and rectum. I n general, at 72 and 93 days post-breeding and at
birth the cranial and middle sections of the
jejunum-ileum exhibited greater enzyme
activity, expressed per unit of d r y tissue, than
other portions of the small intestine.
E n z y m e concentration per unit of d r y tissue
was significantly greater at birth than a t a n y
fetal age. Alkaline phosphatase concentration
at 93 days post:breeding was significantly
greater than at a n y earlier fetal period.
Literature Cited
Bailey, C. B., W. D. Kitts and A. J. Wood. 1956.
The development of the digestive enzyme system
of the pi~ during its pre-weaning phase of growth.
B. Intestinal lactase, sucrase and maltase. Can. J.
Agr. Sci. 36:51.
Colowick, S. P. and N. O. Kaplan, ed. 1955. Methods
in Enzymology, Vol. 1. Academic Press Inc., New
York.
Driscoll, S. G. and D. Y: Hsia. 1958. The development of enzyme systems during early infancy.
Pediatrics 22 : 785.
Fries, G. F. 1958. The effect of enzyme-supplementation of milk replacers on the growth of calves.
Ph.D. Thesis. Michigan State University, E. Lansing, Michigan.
Hartman, P. A., V. W. Hays, R. W. Baker, L. H.
Neagle and D. V. Catron. 1961. Digestive enzyme
development in the young pig. J. Animal Sci.
20:114.
Moog, F. 1946. Alkaline and acid phosphomonoesterase activity in chick embryos. J. Cell and
Comp. Physiol. 28:197.
Moog, F. 1950. The functional differentiation of the
124
SPRAGUE
small intestine. I. The accumulation of alkaline
phosphatase in the duodenum of the chick. J. Exp.
Zool. 115:109.
Moog, F. 1951. The functional differentiation of the
small intestine. II. The differentiation of alkaline
phosphomonoesterase in the duodenum of the
mouse. J. E~p. Zool. 118:187.
Needham, J. 1931. Chemical Embryology, Vol. 3.
The University Press, Cambridge, England.
Sigma Technical Bulletin 104. 1958. Determination
E T AL.
of Serum Acid, Alkaline and Prostatic Phosphatase.
Sigma Chemical Co., St. Louis, Mo.
Waddill, D. G., D. E. Ullrey, E. R. Miller, J. I.
Sprague, E. A. Alexander and J. A. Hoefer. 1962.
Blood cell populations and serum protein concentrations in the fetal pig. J. Animal Sci. 21:583.
Walker, D. M. 1959. The development of the digestive system of the young animal. II. Carbohydrase enzyme development in the young pig.
J. Agr. Sci. 52:357.