STUDIES
OF
SMALL
INTESTINE
DURING
DEVELOPMENT
IV.
Digestion
of
Lactose
as
Related
to
Lactosuria
in
the
Rabbit
Velimir V. Sterk, M.D., and Norman Kretchmer, M.D., Ph.D.
Department of Pediatrics, Stan ford University School of Medicine, Palo Alto, California
(Submitted January 9; revision accepted for publication July 15, 1964.)
These investigations have been supported in part by a grants-in-aid from The John A. Hartfori Foundation (SD-134) and a PHS Research Grant (HD 00391) from the National Institute of Child IIealth and Human Development, Public Health Service.
Dr. Sterk held a PHS Foreign Fellowship Award (FF-163R), Office of International Research,
Na-tional Institutes of Health, and then Postdoctoral Fellow, PHS Training Grant (2-Ti HD 49), National Institute of Child Health and Human Development, Public Health Service. He was on leave from 1961-6:3 from the Hadassah Medical School. Present Address: Department of Pediatrics, I-Iadassah Medical School Hebrew University, Jerusalem, Israel.
ADD RESS (N.K.):300 Pasteur Drive, Palo Alto, California.
609
PEDIATRICS, November 1964
ACrOSURIA has been observed in 65% of
premature infants, 28% of full-term
2 3 and also in children with chronic diarrhea who have ingested large
quanti-ties of lactose. The possibility has been
considered that lactosuria is the
conse-quence of an imbalance between intestinal
absorption of lactose and activity of
lac-tase, since, parenterally administered
lac-tose is metabolized slowly or not at all
and subsequently appears in urine. In
sup-port of this hypothesis, children with
“idiopathic lactosuria of infancy” also lack
lactase.
Previous studies of lactase in the
in-testine of the developing mammal have
shown that the activity is elevated
perina-tally and diminishes in adult life.6’7’8 In
order to determine the physiologic
impli-cations of these observations, we have
studied digestion of lactose by slices of
in-testine, isolated intestinal sacs, cell-free
homogenates, and intact animals. Our
in-vestigations emphasize some of the
physi-ologic implications of incomplete
hydroly-sis of lactose, an event dependent upon
the relationship of the activity of lactase
to concentration of lactose in the intestine.
METHODS
For simplicity, the design of each
in-dividual experiment is included in Results.
The following statements have reference
only to chemical methods.
All analyses were carried out on
protein-free filtrates prepared according to
Som-ogyi.#{176}
Glucose was determined colorimetrically
using glucose oxidase, peroxidase, and
0-dianisidine.10 Lactose was determined on
samples which had been pretreated with
an excessive amount of glucose oxidase to
remove free glucose. The glucose oxidase
was inactivated by placing the sample in
a boiling water bath for three minutes.
After cooling, lactose was hydrolyzed with
1-galactosidase obtained from E. coli* and
the amount of glucose liberated was
de-termined.8
Paper chromatography was donehl with
10 p.lsamples of urine using isopropanol
as the solvent. Carbohydrates were
de-tected with benzidine.
EXPERIMENTAL PROCEDURES
AND
RESULTS
Digestion of Lactose in Vivo
New Zealand rabbits from three
differ-ent age groups, 24 hours, 4-5 days, and
35-40 days were fasted about 4 hours and
then fed from 150 to 1,000 mg lactose
per
100 gm body weight by intubation with
polyethylene catheters. The concentration of
* \Ve are indebted to Dr. Boris Rotman of the
25
0
0
.0
E
0
0a)
U) 0
C.)
0’ E
0
I
2
3
TIME
(Hrs.)
FIG. 1. Concentration of glucose in blood following
administration of carbohydrate to rabbits of differ-ent ages: xx signifies a rabbit of 36 days of age given 500 rag of glucose per 100 gin of ani-mal; all other rabbits were given lactose 1 gm per
100 gin afl(l the symbols represent, #{149}-S 24 hours, O-O 4-5 days, and 36 days of
age respectively.
lactose was adjusted so that 5 ml of
solu-tion could be fed per 100 gm body weight.
Glucose was determined in samples of
blood obtained by cardiac puncture before
and after administration of lactose. In some
rabbits simultaneous samples of urine were
obtained for chromatography by applying
suprapubic pressure. In a few animals, total quantitative excretion of lactose was
determined in urine collected for 24 hours
following administration of the
carbohy-drate.
Figure 1 indicates the changes in
con-centration of glucose in blood for each age
group when rabbits were given 1 gm of
lactose per 100 gm of weight. There is a
distinct difference between the shape of
the curve following administration of
glu-cose and those obtained after the feeding
of lactose. Following administration of
lac-tose, the two younger groups show similar
large increases of glucose in blood,
al-though the initial concentration of glucose
was higher in the 4-5-day group. The
in-crease in glucose is much less in the
34-40-day-old animals. In general, the greater the
amount of lactose fed a given age group
the higher the glucose is elevated.
How-ever, the younger animals (24 hr to 5 days)
demonstrated a greater varial)ility in final
concentration of glucose in blood, a
broader maximum (2-3 hr.), and attainment of higher final concentrations.
At the above dosage of lactose lactosuria
was observed in all animals of the
35-36-day-old group but in only 50% of the rabbits
of the 4-5-day-old group (Table I). During
a 24-hour period, the older animals showed
a markedly greater quantitative excretion
of the administered lactose. These data
sug-gest a decreased rate of digestion in the
older animals but permit no conclusion as
to relative rates of absorption of
unhydro-lyzed lactose.
Metabolism of Lactose in Everted
Intestinal Sacs
Everted sacs were prepared from the
upper portion of the jejununi of rabbits
us-TABLE I
LAcTOSUIoA FOLLOWING Oit&i. AI)MINISTIItTION OF LACTOSE TO RABBITS OF 1)IFFEIIENT AGES
A. Qualitative
Age (days) Laeto.se in Urine* lime (hours)
4-S 35-36
()
1/8
0/4
1 3
3/11 6/1!
6/6 6/6
B. Quantitative
Laetose Excreted (mg/24 lir)
Age (days) No.
Control After Lactose
4-5
40
3 <0.1 <0.1
<0.1 l7-4
K K K K x xx x x K E a, 0 a) 0. 0. 0 U) 0 a) U) 0 C.) 0 200.
50- 5 days old
00.
50
ARTICLES 611
ages.
ing a modification of the technique of
Wil-son and Wiseman.’2 Cotton thread (#00)
was inserted through a blunted No.
20-6-inch BD needle. This threaded needle
was gently inserted from one end to the
other of the dissected jejunum, the cotton
was pulled forward, and a ligature placed
on the distal end of the segment of
in-testine and pulled tight to effect closure.
The eversion was completed by rolling the
intestine proximally to distally over the
ligature and the cotton knot which
re-mained on the serosal surface. The serosal
cavity was filled with Krebs Ringer
phos-phate (KRPH) without Ca to which was
added different concentrations of xylose so
as to match initial osmolality of the
solu-tion of the mucosal side of the sac. In each
experiment 50 ml Erlenmeyer flasks
con-taining 10 to 15 ml of 4 X 10\’I lactose
in KRPH were used unless otherwise
in-dicated. The experiments were performed
in a Duhnoff metabolic shaker at 100
oscillation/mm, at 37#{176}C,in an atmosphere
of 95% q to 5% C0.
The hydrolysis of lactose was measured
either by determining the disappearance of
lactose or the appearance of glucose in the
whole system, mucosal medium plus
in-35 days old
0 5 30 45
TIME (Mins.)
FIG. 2. The metabolism of lactose by everted
in-testinal sacs derived from rabbits of two different
400 E x o x a) o_ x 200 a) U) 2 100 C.) 0
B 510 20 30 40
AGE (Days)
Fic. 3. hydrolysis of lactose as a function of age.
testine. The results of both methods were similar, but because of metabolic
utiliza-tion of glucose by intestine, recovery of
glu-cose averaged only 85% (on a molar basis)
of the lactose which had disappeared.
There was considerable hydrolysis of
lac-tose by intestine from animals 5 days old
which continued at a constant rate for at
least 45 minutes when the medium
con-tamed excess lactose (Fig. 2). The activity
of the enzyme in intestine from animals 36
days old was negligible. It was determined
that the concentration of lactose necessary
to maintain saturation of enzyme for one
hour was about 3-4 X 102M regardless of
age. The relationship of age of animal to
hydrolysis of lactose is shown graphically
in Figure 3. The intestine of the newborn
rabbit was able to hydrolyze lactose at an
appreciable rate. The maximal activity was
observed at 3-5 days and decreased to
about one-half by 20 days of age.
There-after it decreased so that by 35 days of
age the values were less than 10% of the
maximum at birth. Hydrolytic activity of
the intestinal sacs was in general
agree-ment with previously published
biochemi-cal data;8 the increase in activity from
birth to 3 days is not as striking.
60 During this period of development there
was no
apparent
change
in the5
0
ii
i
IC-Ic
U)4)
U)
-Ii
70
0
0
I0
Increase
60
125.
0
100 0
75.
0
50
-25
E
a 40
30
20
0
-SB 0 40 50 60
800
400 C
20 30
AGE (Days)
FIG. 6. Relationships between activity of lactase
and growth of intestine in the rabbit. The weight
of the small intestine is indicated by x-x and
the activity of lactase by O-O.
30
pM lactose/mi. MUCOSAL FLUID
FIG. 4. The diffusion of lactose from the mucosal medium to the serosal cavity of everted intestinal
sacs derived from rabbits of different ages. Each symbol signifies rabbits of different ages varying
from 24 hours to 36 days.
serosa was proportional to the
concentra-tion of lactose in the medium bathing the
mucosa but independent of the age of the
animal.
Comparison of Hydrolysis of Lactose by
Homogenates and Intestinal Slices
In order to determine whether a
struc-tural feature of the intestine from animals
of various ages was involved, activity of
lactose of alternate slices of small intestine
were either assayed as intact slices or as
homogenates. The comparisons are
pre-sented in Figure 5. Homogenization of an
intestinal slice from day-old rabbits
re-suIted in an increase of enzymatic activity
which was much less than when slices of
intestine from older animals were
homog-enized. These data may indicate that the
hydrolytic sites for lactose are more readily
accessible in the intestines of the newborn
rabbit.
The entire small intestine of a number of rabbits varying in
age
from5 days
pre-partum to 66 days postpartum was
ho-mogenized and the total activity of lactase
was determined. The hydrolytic activity
increased seemingly exponentially until the
20th day and this increase approximated
the increase of weight of intestine (Fig. 6).
From age 21 days onward there was a
de-crease in total enzymatic activity while the
weight and presumably the absorptive area
for intact lactose continued to increase,
Eli
<24 hrs. 4-21 60-65
days AGE
FIG. 5. The increase in activity of lactase following homogenization of slice of intestine from rabbits
of different ages.
and while the digestive zone proportion-ately decreased.
COMMENT
These experiments were designed to
ie-termine the physiologic basis for lactosuria
and to accumulate additional information
concerning the metabolism of lactose
dur-ing development.
It has been shown previously that the
hydrolysis of lactose, into glucose and
galactose, in the small intestine probably
50
ARTICLES 613
is accomplished in the brush border or in
close proximity to this epithelial
struc-ture.’315
When lactose was administered to
rab-bits, 24 hours to 5 days of age, the
con-centration of glucose in blood was greater
and more sustained than when an
equiv-alent amount of lactose was fed to older
animals. The observation reflects the higher
activity of lactase in younger 816
(see Figs. 2, 3, and 5). It has also been
shown that by 20-30 days of age the growth
of the intestine in the rabbit has greatly
ex-ceeded the increase in activity of lactase.
It is not known whether this dissociation is
the result of an actual decrease in synthesis
of lactase in a given cell type or a change
in the cell population.
The present experiments indicate that
not only is the activity of lactase higher in
the younger intestine, but also cellular
hydrolytic sites may be more available than
in older animals. Although further studies
are necessary to determine the
applicabil-ity of these in vitro studies to actual
physi-ology,
it is clear that the younger animalshould be able to metabolize a larger
con-centration of lactose per area of intestine
in less time than would an older animal.
Lactose can diffuse passively across the
intestinal barrier, as a function of:
concen-tration, availability of surface for diffusion,
and time of contact with this surface.
When lactose permeates the intestinal
barrier as the intact molecule it is not
metabolized in the organism and it will
appear in urine. The ability to hydrolyze
lactose in the intestine is related to age of
the animal. The primary factors in the
pathogenesis of lactosuria and lactosemia
are the concentration of lactose and its
relationship to the activity of intestinal
lactase. Our experiments indicate that
lac-tosuria is a benign condition arising from
an imbalance between diet and hydrolytic
capability of the intestine. The digestion
of lactose in the small intestine may be
de-creased or absent in association with the
age of the individual or with inherentlr,15
or acquiredlB conditions.
SUMMARY
The data indicate that hydrolysis of
lac-tose is maximal perinatally. The increase
in activity of this enzyme is dissociated
from intestinal growth by the time the
rab-bit is 21 days old. There is passive diffusion
of the lactose across an intestinal sac which
is related only to concentration and not
the age of the animal. When lactose is
ad-ministered to rabbits of different ages,
lactosuria is observed mainly in older
ani-mals.
REFERENCES
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2. Haworth,
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Acta, 52:293, 1961.
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Acknowledgment
The authors wish to acknowledge with apprecia-tion the technical assistance of Miss Ruth Hurwitz
