CROSE TNTAKE DURTNG
UPON THE METABOUC
AND MATURE RATS

THE EFFECT OF A HTGH-SU
GESTATTON AND LACTATTON
PATTERNS OF GROWING

Thesis for the Degree of M. S.
MTCHTGAN STATE UNTVERSTTY

WTLLTAM DAVTDSON EVERS
1976

 

 

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, 2 iv ABSTRACT

THE EFFECT OF A HIGH-SUCROSE INTAKE DURING
GESTATION AND LACTATION UPON THE METABOLIC
PATTERNS OF GROWING AND MATURE RATS
By

William Davidson Evers

The research described in this thesis was conducted to
ascertain whether maternal diet has any long-term effects on
development of offspring. Specifically. does feeding a high-
sucrose diet during pregnancy and lactation alter certain
metabolic processes in the offspring from weaning through
maturity, even though the offspring are not fed a high-
sucrose diet.

Female rats were fed either a 65% cornstarch or a 65$
sucrose diet during pregnancy and lactation. Their offspring
were fed the 65% cornstarch diet from weaning.

At approximately 30, 50 and 110 days of age offspring
were given a glucose load test and their glucose tolerance
was measured. At 30, 60 and 120 days of age. offspring were
killed and the following parameters were analyzed: body
weight, serum triglycerides and cholesterol. relative liver
size, liver glucose-6-phosphate dehydrogenase activity and
liver lipid and cholesterol.

No consistent major differences in body weight. serum
triglycerides. relative liver size. liver enzyme activity.

liver cholesterol or glucose tolerance were noted at any of

William Davidson Evers

the ages. These findings are inconsistent with reports that
body weight and glucose tolerance were elevated and serum
triglycerides were depressed in the offspring from dams fed
a high-sucrose diet.

Serum cholesterol was significantly higher at 120 days
of age in the offspring of sucrose-fed dams, but not at 30
or 60 days of age. Liver lipid was higher at 60 days but
lower at 120 days in the offspring from mothers fed the high-
sucrose diet.

Based upon these results, it was concluded that in the
rat there does not seem to be any consistent long-term meta-
bolic differences in offspring of mothers who had been fed a
high-sucrose diet during gestation and lactation, if the

offspring were fed a cornstarch diet from weaning.

THE EFFECT OF A HIGH-SUCROSE INTAKE DURING
GESTATION AND LACTATION UPON THE METABOLIC
PATTERNS OF GROWING AND MATURE RATS

BY

William Davidson Evers

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements

for the degree of

, MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1976

For Joanne

Ho
Ho

ACKNOWLEDGMENTS

My thanks to my advisor, Dr. Wanda Chenoweth and to
Dr. Maury Bennink for guiding my transit time from neophyte
to mature graduate student; to Dr. Dale Romsos for technical
and procedural assistance as I learned the pathways (labora-
tory and lipogenic): to Dr. Thomas Helmrath for aiding my
human development in the area of statistical procedure; and
to Anne, Wha, Rainie, Will and Ellen for taking time from

their studies to assist me in my laboratory procedures.

iii

TABLE OF CONTENTS

 

 

 

LIST OF TABLES . . . . . . . . . . . v
LIST OF FIGURES. . . . ’. . . . . . . vi
INTRODUCTION . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . 2
Maternal-Fetal Relationships . . . . . . . 2
Sucrose . . . . . . . . . . . . 3
Sucrose and Maternal-Fetal Relationships . . . . 8
MATERIALS AND METHODS . . . . . . . . . 11
Preliminary Study . . . . . . . . . . 11
Main Study. . . . . . . . . . . . 13
RESULTS AND DISCUSSION . . . . . . . . . 16
Preliminary Study . . . . . . . . . . 16
Main Study. . . . . . . . . . . . 18
Litter Size and Weaning Weights . . . . . . 18
Parameters Studied in Dams . . . . . . . 19
Parameters Studied in Offspring . . . . . . 21
SUMMARY AND CONCLUSIONS. . . . . . . . . 35
APPENDIR . . . . . . . . . . . . 37
METHODS OF ANALYSIS . . . . . . . . . 37
BIBLIOGRAPHY . . . . . . . . . . . 50

iv

TABLE
TABLE
TABLE
TABLE
TABLE
TABLE
TABLE
TABLE
TABLE

\OCDVOUI-l-‘UNH

LIST OF TABLES

Diet composition. .

Results of preliminary study.

Litter size and weaning weight

Effect of high-sucrose diets in dams.

Body weight, serum triglycerides and cholesterol.

Liver total lipid and cholesterol

RLS and G6PD activity
Glucose tolerance tests

Glucose removal rate.

12
17
20
20
22
25
27
29

'3A

LIST OF FIGURES
FIGURE 1 Glucose tolerance, 30 and 60 days of age. . 31

FIGURE 2 Glucose tolerance, 120 days of age (2 tests). 32
FIGURE 3 Example of a chromatogram . . . . . 49

vi

INTRODUCTION

This study reviews two areas of human nutrition;
maternal-fetal nutritional relationships, and the effects of
dietary sucrose on metabolic patterns. Then there is an
examination of studies that indicate that dietary sucrose is
a nutritional factor which influences the maternal-fetal
relationship. The question being asked is: Does the feeding
of a high-sucrose diet to a pregnant and lactating rat have
any effect upon the subsequent growth and development of the
I offspring, even though the offspring are fed a normal diet?

The research was conducted to answer this question.

REVIEW OF LITERATURE

Maternal-Fetal Relationships

NMany connections from fetus to mother have been shown
to occur. For example, the pregnant rat during the second
half of gestation shows large increases in food intake and
in insulin secretion, but still loses fat from adipose
tissue because the tissues appear less sensitive to insulin.
This development is considered to be an adjustment to fetal
growth whereby ingested glucose is channelled to the fetus
and the mother draws upon stored lipid for energy(1). The
fetus may act as a parasite during periods of maternal star-
vation, with the continuous consumption of glucose, and
possibly alanine, despite decreased levels in the blood of
the mother. However there are limitations upon this ability
to obtain nutriture from the mother. With prolonged fasting
it would appear that the fetus, like the mother, must
utilize ketones as an alternative source of energy(Z).
Maternal dietary restriction in rats can lead to growth
stunting of offspring(3,#), lending credence to the belief
that the fetus is not always able to extract the nutrients
needed from the maternal blood. Food restriction may demon-
strate another type of interaction. The malnourished fetus

may be less able to withstand other stresses when these

2

3

also occur during pregnancy(5). Intrauterine growth retard—
ation, caused by maternal undernutrition, placental insuffi-
ciency or extrauterine factors; such as disease, maternal
smoking or high altitude living; can lead to small—for-
gestational-age (SGA) infants. The SGA infant in turn may
be more prone to hypoglycemia, have a higher mortality rate
and display a greater incidence of mental-motor retardation
at a later age(6).

The maternal role of nutrient supplier has led many
investigators to explore the effects of maternal diet on
fetal development. Deficiencies of specific nutrients in
maternal diets have been shown to alter fetal development.
For example, zinc deficient diets fed to pregnant rats
result in increased reabsorptions, stillborns and brain and
skull malformations(7-11). A thiamin-deficient diet fed
during pregnancy has been related to reduced number of off-
spring(10). Increased perinatal mortality and uneven hair
development in newborn rats was observed wnen pregnant rats
were fed a diet that was made deficient in nickel(12).
Numerous studies of restricted caloric and/or protein intake
during gestation have shown that growth and possibly brain

development are retarded(3-5,13-16).

Sucrose

Sucrose has been intensively studied over the past few
decades as the consumption of this form of carbohydrate has

risen as a percent of total carbohydrate consumed(17).

Lt

Formerly the general belief was that all metabolizable carbo—
hydrates had the same physiological effects. Now the liter-
ature is replete with studies showing a relationship (causal
or otherwise) between sucrose and the incidence of obesity
(17-20), heart disease and atherosclerosis(17,21-23), serum
triglycerides(24) and serum cholesterol(25), vascular and
neural changes in the eye(17,26) and dental caries(17).
Current research deals with the effects that sucrose
produces on pathways of carbohydrate and lipid metabolism.
Usually the diet containing sucrose is compared with diets
containing starch, high fat, monosaccharides or other disaccha-
rides. Blood and liver lipid concentrations and activities of
lipogenic enzymes in liver and adipose tissue are the parameters
most often examined. High intakes of sucrose have been shown
to cause increases in blood triglycerides in the rat(27-32),
primates(33,3h) and man(35-38); and increases in blood chole-
sterol in the rat(27,31,39) and man(35—38). In other inves-
tigations a rise in blood cholesterol did not occur(25,30,40).
Possible explanations for these findings have been
offered by several groups. Since the most apparent differ-
ence between sucrose and starch, other than chain length, is
the presence of fructose in the former, much of the research
has centered around this monosaccharide. Bar—on and Stein
(#1) compared the effect of glucose and fructose on lipid
metabolism in the rat and found that in the liver more
fructose was converted to triglyceride than was glucose, and

that fructose did not stimulate lipoprotein lipase activity

5

in the adipose tissue. Their explanation was that fructose
was absorbed into the portal circulation and converted in
the liver to cx-glycerophosphate. At the same time the out-
vflow of free fatty acids from the adipose continued, leading
to more triglyceride formation in the liver and its subse-
quent release into the blood. Since lipoprotein lipase
actiVity did not increase, there was a buildup of triglyce-
ride levels in the blood.

Chang et al.(h2) concluded that dietary carbohydrates
affected several enzymes. Depending upon the age and strain
of the rat, they found elevated levels of glucose-6-phosphatase,
glucose-6-phosphate dehydrogenase and aldolase in rats fed a
50% sucrose diet as compared to rats fed 50% glucose or corn-
starch. Other researchers have also reported increased activ—
ity of glucose-6-phosphate dehydrogenase(fl2,u5), glucose—6-
phosphatase(42,h3) and pyruvate kinase(44,u5) in animals fed
a high—sucrose diet.

Antar et al.(36) proposed that the hyperlipidemic effect
of sucrose was dependent upon the percent of total calories as
sucrose or starch, the amounts of saturated and unsaturated fat
and the amount of cholesterol in the diet. They suggested two
possible mechanisms of action: 1) An apparent inability of
fructose to stimulate lipoprotein lipase in adipose tissue,
resulting in an accumulation of blood triglycerides; or 2) the
rapid absorption of simple sugars in comparison to starch
thus causing a flooding effect on the liver and a stimulation

of the release of lipoprotein into the blood.

Macdonald(33) suggested that the type of fat and pro-
tein consumed may also influence the lipid response to a
high-sucrose diet. In studying primates, Macdonald suggested
that presence of fructose in sucrose may be the important
factor. According to Macdonald, fructose does not stimulate
insulin output so lipoprotein lipase activity would not be
stimulated. Therefore serum lipids would increase. Macdonald
(33) and Coltart and Macdonald(34) proposed that the female
sex hormone, estrogen, plays a role after fructose or glucose
has been metabolized to dkglycerophosphate to give the
glycerol moiety of triglycerides.

Naismith and Khan(32), however, reported that a high-
sucrose diet induced a large increase in the activity of
lipoprotein lipase in the adipose tissue, and concluded from
this that in rats the elevated blood triglycerides were not
due to an inability to clear the lipid from the blood.
Naismith et al.(37) also examined the effect of a high-
sucrose diet on humans to determine if the hypertriglyceri-
demia associated with this diet might be caused by some
unrecognized increase in energy intake. Comparing a high
energy-high-sucrose diet to a high energy-starch diet in
humans, they found that only the high-sucrose diet caused
elevated plasma triglycerides, total cholesterol and phospho-
lipids.w They concluded that the elevated blood lipid levels
were induced by the sucrose and not by a change in energy

intake.

In addition, Naismith(29) found that while fructose was
a potent stimulator of hepatic lipogenesis, maltose, which
has no fructose, stimulated lipogenesis as effectively as
did sucrose. Therefore he concluded that fructose could not
be used to explain the hyperlipidemic effect of sucrose.
Instead, he suggested that the primary stimulus was the
flooding of liver cells with large amounts of monosaccharides,
which induced the enzymes of fatty acid synthesis.

Naismith and Rana(27,28) proposed further that the
hyperlipidemic effect of sucrose was due to the rapid rate
of hydrolysis and absorption of sucrose and the increased
output of triglycerides from the liver to the adipose tissue.
It has been recently demonstrated that there may be a
different and more rapid absorption system for sucrose as
compared to more complex carbohydrates(46).

Naismith and Khan(39)showed a rise in blood cholesterol
in rats fed a high-sucrose diet. This was largely confined
to the esterified fraction and there was a concomitant rise
in activity of lecithin:cholesterol acyltransferase which
catalyses the esterification of free cholesterol in the blood.

Using labelled glucose,Bender and Thadani(43) found
that feeding a sucrose diet to rats caused a marked depres-
sion of both glucose oxidation and lipogenesis in the liver.
While they concluded that these metabolic pathways had been
altered, they did not indicate the direction of change of
the pathways. They did suggest that the effect of sucrose

might be a reduction in quantity of glucose-6—phosphate,

which resulted from the stimulation of glucose-é-phosphatase
and by altering the ratio of glucose metabolized through the
pentose pathway, which would occur as a result of the stimu-
lation of the formation of glucose-6-phosphate dehydrogenase.
Moser and Berdanier(47) researched the effects of feeding
a high-sucrose diet during growth. They developed four experi-
mental groups: 1) one fed a 65% starch diet from weaning:
2) one fed a 65% sucrose diet from weaning: 3) one fed a
starch diet from weaning to 50 days of age and then fed a
sucrose diet: and 4) one fed a sucrose diet from weaning to
50 days of age and then fed a starch diet. At 142 days the
animals fed sucrose up to 50 days of age had higher liver
lipid and liver cholesterol than control animals fed starch
from weaning. Differences in activity of several liver
enzymes were also noted, although the differences were not
necessarily attributable to sucrose feeding from weaning to

only 50 days of age.

Sucrose and Maternal-Fetal Relationships

Using the BHE strain of rat, which has been bred to
develop lipemia and diabetes in response to various types of
carbohydrate, Berdanier(48) fed female rats sucrose or starch
diets during pregnancy and lactation, switching some of the
mothers' diets during lactation. Male offspring were then
fed the starch or sucrose diet. This produced six

experimental groups:

Gestation: Starch Sta ch Starch Sucrose Sucrose Sucrose

Lactation: Starch Starch Sucrose Starch Sucrose Sucrose

Growth: Starch Sucrose Starch Starch Sucrose Starch
Grou no ° 1 2 3 4 5 6
p " (control)

At 150 days of age the progeny in groups 3,4 and 6 had
decreased levels of serum triglycerides as compared to con-
trols. Group 2 had elevated Serum triglycerides, similar to
data reported by other researchers(27-32). However, group 5
did not differ from controls in this respect. Group 6 also
showed elevated liver lipid, while groups 2 and 3 had lower
liver lipid, when each was compared to controls. Groups 2
and 5 also showed higher activity of glucose-6-phosphate
dehydrogenase activity when compared to control, but no
difference was noted in any group in the activity of malic
enzyme when compared to controls. From these data, Berdanier
suggested that the post-weaning period is of greater impor-
tance in regards to the lipogenic response of sucrose feeding
than is either gestation or lactation.

Davis et al.(49,50) conducted experiments in mice to
examine the effects of feeding sucrose during gestation.
They increased the sucrose content of the maternal diet by
diluting a regular stock diet with 35% sucrose. At weaning
pups were fed the normal stock diet. Male progeny from dams
fed sucrose during gestation and lactation were heavier in
weight and remained heavier throughout the experiments.

Some of these experiments lasted over 80 weeks. They also

presented results which they interpreted as showing an

10

impaired response to a glucose load by offspring from mice
fed the sucrose diet during gestation and lactation. In
other results they found that the pre-beta lipoprotein
fraction in serum of mice at birth from dams fed the sucrose
diet was over 50% while the control group was less than 5%.
The lipoprotein pattern of the offspring from dams fed the
sucroSe diet is described as similar to that of Type IV
hyperlipoproteinemia. The pre-beta lipoprotein levels were
still elevated at 2 months of age, but had decreased signi-
ficantly by 4 months. They also report higher serum cholest-
erol and body fat in the mice from dams fed the sucrose diet
but do not present any data.

The present study was undertaken to confirm some of the
same parameters used by Berdanier and Davis. However, a
strain of rat was used which does not show any Specific
genetic tendencies toward alterations in carbohydrate as
does the BHE rat. Instead of diluting the diet with sucrose
as was done by Davis and thus reducing the protein, vitamins
and minerals as well, semipurified diets similar to Berdanier's
were used, where the only difference was the type of

carbohydrate.

MATERIALS AND METHODS

Preliminary Study

The following preliminary research was conducted to
decide upon possible parameters to study in future experi-
ments. It is only briefly described here. The more detailed
description given for the main study will elucidate what was
done in this preliminary work.

Eight female Sprague-Dawley rats1

, weighing approxi-

' mately 250 g, were fed a semipurified diet with either corn-
starch (4 rats) or sucrose (4 rats) as the sole source of
carbohydrate (Table 1). These rats were mated and kept on
the same experimental diet. After parturition, litter size
was adjusted to 8 pups per dam.

At weaning, pups were divided into 4 experimental groups

based on diets:

Mothers: Sucrose Sucrose Cornstarch Cornstarch

Weanlings: Sucrose Cornstarch Sucrose Cornstarch

Group No.: 1 2 3 4
(control)

At approximately 9 weeks of age, 2-4 rats from each
group were killed and the fellowing parameters were
examined: body weight, serum cholesterol (51,52), serum

1

 

Spartan Research Animals, Inc., Haslett, MI 48840.

11

12

TABLE 1 Diet composition.

 

Ingredient - Percent of diet
Sodium caseinate1 20.0
Mineral mix2 4.0
Vitamin mix3 1.0
Corn oil“ 5.0
Cellulose 4.0
Choline Cl 0.2
D,L methionine 0.3
Cornstarch or sucrose 65.5

 

1Donated by Quaker Oats Co., Barrington, IL. 2No. 4164
salt mix, Teklad Test Diets, Madison, WI 53713. 3In mg/kg
diet: thiamin HCl, 22: pyridoxine, 22: riboflavin, 22: Ca
pantothenate, 60: p-amino benzoic acid, 110: ascorbic acid,
200: niacin, 100: vitamin B12, 0.3: biotin, 0.6: folic acid,
4.0. In IU/kg diet: vitamin A, 20,000; vitamin E, 100:
vitamin D3, 2,200. ”Mazola.

13

triglycerides (53), liverweight, liver lipid and liver chole-
sterol (51,52). At this same age blood samples from 2—4 of
the remaining rats in each group were drawn from the poster-
ior orbital region of the eye (54). These samples were
analyzed for serum glucose1 and cholesterol.

At 18 weeks of age, blood samples were again taken.
Serum‘glucose, cholesterol and triglycerides were determined.
At 20 weeks of age, the remaining 2-4 rats from each
diet were killed and the same parameters, plus serum glucose,

were studied as with the first group at 9 weeks of age.

Main Study

Thirty female Sprague-Dawley rats2

, weighing approxi-
mately 225 g, were distributed five to a cage. Three groups
were fed the cornstarch diet and three groups the high-sucrose
diet (Table 1). Water was available ad libitum. After 3

days on the experimental diets, a male Sprague—Dawley rat3,
weighing approximately 500-700 g, was placed with each group.
After 8 days, the males were removed, and the females were
separated and weighed every other day. Whenever a rat had a
weight gain of from 5-7 g/day over a 4 day period, it was

placed in a solid bottom plastic cage which contained wood

shavings. Each female remained on the experimental diet that

 

1Glucostat, Worthington Biochemical Corp., Freehold, NJ.

ZSpartan Research Animals, Inc., Haslett, MI 48840.

3These rats were almost one year old. An attempt had
been made to get younger male rats at an age where they were
capable of mating. At that time the supplier was unable to
provide such animals, and it was decided that the older
animals would be used.

14

was originally fed.1 Within 24 hours of delivery, pups were
sexed and litter size was reduced or increased to 8 pups/dam.
If the litter was reduced, the procedure was to maximize the
number of male pups in the litter that remained. Litters
were increased by taking female pups from other dams who had
delivered larger litters within the same 24 hour period.

This latter procedure was used twice. Food and water were
available to the dams at all times and no attempt was made

to prevent the pups from eating from the food jar during the
21 day lactation period.

Pups were weaned at 21 days of age, weighed, placed in
hanging wire cages and fed the cornstarch diet. This produced
two experimental groups: weanlings from dams fed the high-
sucrose diet (the SUC group): and weanlings from dams fed
the cornstarch diet (the CS or control group). Water was
available ad libitum, the room was lighted from 0700 hours
to 2000 hours and room temperature was a constant 22°t1°.
Humidity tended to flucuate with the humidity external to the
building. Food intake was measured whenever food cups were
filled, usually every 3-4 days, and body weights were
recorded weekly.

To develop skill in performing the analytical procedures
and to obtain some initial data, intraperitoneal glucose
tolerance tests, as described by Romsos and Leveille (55),

were conducted on 16 weanlings, 8 from each group (4 males

 

1Since initially only 7 out of the 30 females delivered
pups, the procedure was repeated 3 weeks later with 16 of the
remaining 23 females, 8 of which subsequently had litters.

15

and 4 females), at 30 days of age. Blood glucose concentra-
tion was analyzed by the glucose oxidase procedure.1

At 30-35 days of age, 4 female weanlings from each group
were killed by decapitation. Blood was collected for serum
triglyceride (53) and serum cholesterol (56) determinations.
The liver was rapidly removed and a 1 g sample excised for
the determination of glucose-6-phosphate dehydrogenase
(EC 1.1.1.49, G6PD) activity (57). Protein content was
analyzed using the method of Lowry et al. (58). The rest of
the liver was frozen. After thawing the liver, duplicate 1 g
samples from different lobes were homogenized in 10 ml of
methanol. Total lipids were determined gravimetrically after
two extractions in chloroform:methanol. Liver cholesterol
was assayed after redissolving the lipid material in 10 ml
of chloroform and taking a 3 ml aliquot.

Only male rats were used for the remainder of the exper-
iment. A total of 81 males were weaned, 39 CS weanlings and
42 SUC weanlings. At 53 days of age, glucose tolerance tests
were performed on 8 weanlings from each group, and at 113
days of age on 10 weanlings from each group. Offspring
from several different dams were used each time. At 60 and
120 days of age, one-half of the CS weanlings and one-half
of the SUC weanlings were killed. The same parameters as
those studied at 30-35 days of age were analyzed.

Analyses as used in this study, and percent error, are
described in detail in the Appendix. Student's t test was

used to test the data for significance (59).

 

1Glucostat, Worthington Biochemical Corp., Freehold, NJ.

RESULTS AND DISCUSSION

Preliminary Study

Results from the preliminary study are summarized in
Table 2. Body weight, serum cholesterol and triglycerides,
relative liver size (RLS) and liver total lipid and chole-
sterol are given for the two groups which had the same
experimental design as the groups subsequently used in the
main study. These groups were: the offspring from dams fed
the cornstarch diet during pregnancy and lactation (the CS
or control group): and the offspring from dams fed the high-
sucrose diet during pregnancy and lactation (the SUC group).
All offspring were fed the cornstarch diet after being weaned.

No difference in body weight was observed between the
SUC and control groups either at 66 days of age or 139 days
of age. This finding was not in accordance with results
obtained by Davis et al. (49,50) in mice. Serum cholesterol
was the same at 66 days, but was somewhat elevated at 127 and
139 days of age for the SUC group. Serum triglycerides were
not altered by the eXperimental regimen at either 66 or 127
days of age. At 139 days there was a trend toward lower serum
triglycerides in the SUC animals. This trend in trigly-

cerides is in agreement with results reported by Berdanier (48).

16

17

 

TABLE 2 Results of preliminary study.1
Group
LParameter ii: Eggz
Body weight(g)

66 days 384 365

139 days 539 4 503
Serum cholesterol(mg/lOO ml)

66 days 99 94
127 days 95 120
139 days 105 124

Serum triglycerides(mg/1OO ml)

66 days 61 60
127 days 76 77
139 days 111 87

BLs3(g liver/ 100 g body wt.)

66 days 3.20 3-50

139 days 2.94 2.87
Liver total lipid(mg/g liver)

66 days 55.2 58.2

139 days 49.0 59.0
Liver cholesterol(mg/g liver)

66 days 5.0 5-3

139 days 5.2 6.9

g

 

1No significant differences (p40.05) were found for
any parameter. 2GS = offspring of dams fed cornstarch diet
during pregnancy and lactation; SUC = offspring of dams fed
high—sucrose diet during pregnancy and lactation: all off-
spring fed cornstarch diet after weaning. RLS = relative
liver size.

18

Liver size was not affected by the experimental design
either at 66 or 139 days of age. Total lipid in the liver,
while not different at 66 days, tended to be higher in the SUC
group at 139 days. This apparent elevation at 139 days would
agree with the work of Berdanier(48). For liver cholesterol
no difference was observed at 66 days of age, but the SUC
group results tended to be higher than control at 139 days.

Since some of the results in this preliminary experiment
differed from those found by other investigators, a more
detailed experiment using larger numbers of animals was
undertaken to look at additional parameters related to carbo-
hydrate and lipid metabolism and to see if the present results

could be replicated.

Main Study

Litter Size and Weaning Weights

The feeding of sucrose during pregnancy did not have any
effect on the size of the litter(Table 3). There were no
grossly apparent differences in the physical appearance of
the pups, either at parturition or during lactation. weaning
weights of all pups, and specifically of all male pups, were
not altered by the diet fed the dams during gestation and
lactation(Table 3). The pups in the SUC group consumed some
of the high-sucrose diet during the last week of lactation.
Apparently this brief period of sucrose intake did not have
any immediate effect on body weight and, as will be shown

later, did not affect body weight during the rest of the study.

19

Parameters Studied in Dams

If the parameters being studied were dramatically
different in the mothers, this fact might explain any
differences noted in their offspring. Whether any differences
were due to the experimental design or just random variation
among animals is of some importance. Possibilities might
exist that offspring were exhibiting traits inherited from
the mothers 9; were exhibiting long-term effects that resulted
from the diets fed to the dams during gestation and lactation.
When the dams were killed they had been fed the experimental
diets continuously for an 8-10 week period. All dams had
finished lactation 10-14 days prior to being killed.

Body weights of the dams were similar although the rats
fed the sucrose diet had somewhat lower weights (Table 4).
Serum cholesterol and triglycerides were not significantly
different (Table 4). This result is not in agreement with
other studies (27-32) in which sucrose was fed to mature rats
and elevations of triglycerides were noted. One possible
reason for this difference in results might be the fact that
the rats in this study had recently finished lactating.

The relative size of the liver (RLS) was not different
between the two groups (Table 4). Nor was glucose-6-phosphate
dehydrogenase (G6PD) activity significantly changed by the
experimental design (Table 4). However, for both parameters,
the dams fed the high-sucrose diet had the higher values.

The difference in G6PD activity was rather marked but varia-

tion in activity within the groups was such that this

20

TABLE 3 Litter size and weaning weight.

 

 

Dietary No. of Litter No. of male Weaning
group dams size offspring weights(g)
1. + 2 +
CS . 7 12.3-0.5 39 59-1
SUC1 8 11.4t1.1 42 60f1
1

CS = dams fed cornstarch diet during pregnancy and lac-
tation; SUC = dams fed high-sucrose diet during pregnancy
and lactation. 2Meani’SEM.

 

TABLE 4 Effect of high-sucrose diets in dams.1
CS group SUC group

Parameter n=4 n=
Body weight(g) 313i62 294i?
Serum cholesterol

(mg/ 100 ml) 98i7 11oi8
Serum triglycerides

(mg/100 ml) 180:23 179f36
Relative liver size
(g liver/100 g body wt.) 3.74f0.13 4.39IO.35
G6PD activity3 41.5f12.8 54.3i1o.7
Liver total lipid *4,5
(mg/g fat-free dry matter) 169i10 222:14
Liver cholesterol *
(mg/g fat-free dry matter) 6.0i0.3 7.7:1.0

 

11-2 weeks after termination of lactation: total length
of time on diet was 8-10 weeks. 2MeaniSEM. 3Given as nmoles
substrate converted/ mg protein/min. “Values having an
asterisk are significantly different (p<:0.05) from CS group.
SData for 3 animals.

21

difference could be attributed to a random selection of
animals. Total lipid in the liver was significantly higher
in the mothers fed the high-sucrose diet (Table 4). Other
studies (29,41) have also demonstrated that increases in
liver lipid did occur when a high-sucrose diet was fed.
Liver cholesterol was also significantly higher in the

sucrdSe-fed dams (Table 4).

Parameters Studied in Offspring

Body weight

 

A comparison of body weights revealed no significant
differences between the SUC group and controls either at 60
or 120 days of age (Table 5). The results of Davis et al.
(49,50) in mice, where offspring from sucrose-fed mothers
were found to be heavier then controls throughout the study,
may well be due to other alterations in the diet since the
diet was diluted with sucrose rather then having the sucrose
isocalorically replace different carbohydrates. The species
difference also cannot be disregarded as a possible causative
factor for the difference in weight gain.

Between approximately 80 and 100 days of age, most
animals contracted a respiratory infection of varying degrees

1 in their

of severity. All animals were given an antibiotic
drinking water for 14 days. While some animals experienced

weight loss, by 112 days all were eating amounts equal to or

 

1Cosa-Terramycin (oxytetracycline hydrochloride with
glucosamine HCl and vitamins): Dept. of Veterinary Medicine
Chas. Pfizer and Co., Inc., New York, NY. Approximately 2

tablespoons per liter of water.

22

TABLE 5 Body weight, serum triglycerides and cholesterol.

 

 

Parameter CSlgroup SUC1 group
Body weight(g)
60 days ' 339tu2 334i5
(20)3 (21)
120 days A 450i? 443i?
(19) (21)
Serum triglycerides(mg/1OO ml)
30 days“ 84:10 79I11
(8) (8)
60 days 258i28 219i17
(20) (21)
120 days 135:10 123:12
(19) (21)
Serum cholesterol(mg/lOO ml)
30 days” 107t3 111i4
(8) (8)
60 days 98i4 97i4
(20) (21)
120 days 84i4 9713*5
(19) (21)
1

GS = offspring of dams fed cornstarch during pregnancy
and lactation: SUC = offspring of dams fed high-sucrose diet
during pregnancy and lactation: all offspring fed cornstarch
gist after weaning. 2Meani‘SEM. 3Number of animals in group.

Only female offspring were killed at 30 days of age and only
male offspring were used for the remainder of the study.
5Values with an asterisk are significantly different (p (0.05)
from CS group.

23

greater than before the infection and all were gaining weight.
Although an effect upon weight gain and the other parameters
studied cannot be conclusively ruled out, the close agree-
ment of body weights within groups at 120 days leads to the
supposition that the illness did not have a long—lasting
effect and most likely did not alter the results obtained at

120 days of age.

Serum triglycerides and cholesterol

Contrary to the findings of Berdanier(48), no signifi-
cant differences were noted in serum triglycerides(Table 5).
Since a larger number of animals per group were used in this
study(21 vs 6 for Berdanier's work), these results indicate
that no difference in triglyceride concentration exists. At
all ages when triglycerides were analyzed the SUC rats tended
to have the lower values. This difference might have been
significant if the analyses had been performed on fasted rats
(as was done by Berdanier) instead of nonfasted rats. While
the large variation that is observed may result from not
having fasted the animals, it also should be noted that
Berdanier's results displayed similar or even greater
variations in the findings.

Berdanier offers a possible explanation for the lower
serum triglycerides that she reports. The hypothesis is that
the fetus or suckling rat in the SUC group develops a more
efficient system for clearing the blood triglycerides as a
consequence of an increase in lipoprotein lipase activity

and an increase in the receptor sites for lipoproteins. As

24

long as excess lipids are present in the blood, the increased
enzyme activity and more numerous receptor sites would remain.
Thepuoblem with this hypothesis is that at 60 or 120 days of
age(or 142 days or Berdanier's study), the SUC rats would
not be synthesizing any excess "sucrose-induced" lipids since
they have been fed a cornstarch diet from weaning. Therefore
there should not be any overly large amounts of lipid in the
blood and the two factors would supposedly have returned to
their normal activity or number.

Serum cholesterol was the same in both groups at 30 and
60 days of age (Table 5). At 120 days of age the SUC group
had significantly higher serum cholesterol. Why this increase
occurred is not clear. That the elevation might be due to the
difference in maternal diet appears rather unlikely, for one
would question why this was not observed at 60 days of age.
And yet, given that the only difference between the two
groups was the diets fed to the mothers and given the large n
value for each group, it is difficult to ignore the hypothesis
that the diet fed to the dams during gestation and lactation
might have had some long-term effect on the offspring, at

least with regard to serum cholesterol.

Liver total lipid_and cholesterol

Differences in total lipid in the liver did not show a
consistent pattern (Table 6). At 30 days of age both groups
had similar amounts of lipid in the liver. At 60 days of age
the SUC animals had significantly higher liver lipid, but at
120 days the CS group had significantly higher lipid.

25

TABLE 6 Liver total lipid and cholesterol.

 

Parameter CS1 group SUClgroup
Liver total lipid
(mg/g fat-free dry matter)
30 days2 189+z3 180111
(8) (8)
60 days 213I24 34li23*5
-(20) (21)
120 days 202:8 174i8*
(19) (21)
Liver cholesterol
(mg/g fat-free dry matter)
30 days2 7.3io.4 7.2io.3
(8) (8)
60 days 5.4IO.2 5.7I1.1
(20) (21)
120 days 5.5i0.1 5.6IO.3
(19) (21)

 

1See ffiotnote 1. Table 5.

3MeantSEM. Number of animals in grou
asterisk are significantly different

2See footnote 4, Table 5.

5Values havin. an
($40.05) from C

group.

26

Berdanier reported that at 142 days of age the group compar-
able to the SUC group had significantly higher amounts of
lipid in the liver. Since neither study showed any elevation
of G6PD activity (see following section), nor did Berdanier's
study show any elevation of malic enzyme activity, an increase
in the rate of fatty acid synthesis is not likely. Because
the results of this present study are not consistent from one
age to the next, the reason why liver lipid was different is
not clear.

Liver cholesterol values were very similar at all 3
ages (Table 6). From these results, it would seem that the
maternal diet had no long-lasting effect on the amount of
cholesterol in the liver. That total lipid in the liver
should show changes while cholesterol did not would indicate
that any difference in lipid was in the triglyceride or

phospholipid fraction.

Relative liver size (RLS) and G6PD activity

RLS was lower at 60 and 120 days of age, although the
difference was statistically significant only at 60 days
(Table 7). Berdanier (48) also found a significantly lower
RLS at 142 days of age in the group that is comparable to
the SUC group of this study. Changes in liver size often
result'from changes in total liver lipid. At 60 days in this
study and at 142 days in Berdanier's study, the rats in the
SUC group had a significantly smaller RLS but significantly
mgr; total liver lipid. The greater amount of lipid but

smaller RLS might imply a decrease in water, glycogen or

27

1 2

TABLE 7 RLS and G6PD activity.

Parameter 083 group SUC3 group

 

RLS(g liver/100 g body wt.)

30 days1+ 4.59I0.145 4.58:0.06
(8)6 (8)

60 days 4.34io.06 4.1510.07*7
(20) (21)

120 days 3.54io.06 3.42Io.03
(19) (21)

G6PD activity2

30 days“ 21.3i4.6 20.6iu.o
(7) (7) .

60 days 9.5io.9 1o.4io.7
(20) (21)

120 days 13.9i1.2 14.6t1.o
(19) (21)

 

1RLS = relative liver size. 2Glucose-6—phosphate dehydro-
genase activity expressed as nmoles substrate (NADE) converted
per mg protein per min. See footnote 1, Table 5. See foot-
note , Table 5. JMeaniSEM. 6Number of animals in group. 7Values
having an asterisk are significantly different (p<10.05
from CS group.

28

other nonlipid material. Since Berdanier's rats were fasted,
glycogen would not be present in the liver in any significant
amounts. In this study the fat-free dry matter was not
different between the two groups. The possibility of a
decrease in water content of the liver cannot be ruled out.
A reason for the water content of the liver to be lower is
not known.

G6PD activity was similar in both experimental groups
at all three ages(Table 7). Since the substrate available
was the same for both groups, differences in activity would
not be expected. However, if the hypothesis is that the
maternal diet has some long range effect on lipogenic activi-
ties of the liver, then one would have expected to see some

alteration in the activity of this lipogenic enzyme.

Glucose tolerance

The glucose tolerance test is often administered to
ascertain if diabetes mellitus is present as a metabolic
disease. The concentration of glucose in the blood over time
is an indication of the rates of addition and removal of
blood glucose(60,61). In the present experiment, at the
three ages studied, fasting blood glucose was nearly the same
for both groups(Table 8). Compared to their control, Davis
et al. consistently found significantly higher fasting values
in the mice which were comparable to the SUC group of this
study. At 30 days of age and in the animals tested the
second time at 120 days of age, the CS group had a signifi-
cantly higher peak in bloodgiucose 15-30 minutes after the

29

TABLE 8 Glucose tolerance tests.
No. 3 Blood ngcose (mg/100 ml)

 

Group1 Age2 rats Fast 30 60 120
cs- 30 72155 18616. 8913 7812
(4. 4)3 (4.3) (4.3) (1.4)

SUC 6813 153114”6 8313 7412
(4.4) (4.3) (4.4) (4.4)

as 60 8 9011 231130 246132 176121 12819
SUC 8 8512 280125 284126 182115 12619

cs 120 5 8713 172127 172124 142115 12019

(lst test)7

 

suc 5 8715 185115 208116 192110* 12517
cs 120 5 7 7212 218110 20917 16615 12615
(2nd test) * e * e
500 5 7214 159115 157110 14116 10414
1

See footnote 1, Table 5. 2Days. 3Number of rats varied
at 30 days of age: values in pareflthese represent number of
males and females, respectively. Minutes. 5MeanfsEM. 6Values
having an asterisk are significantly different (p<0. 05)

from cs group at time indicated. 7The first test at 120 days
of age was performed on the rats from females that became
pregnant initially, and the second test was performed on rats
from females that became pregnant 3 weeks later. Two tests
were also performed at 60 days of age, but the data from the
first test was not valid due to problems with the colorimetric
technique that was used.

30

administration of a glucose load(Table 8, Figure 1 and
Figure 2). H0wever at 60 days and in the animals tested

the first time at 120 days of age, the SUC animals had the
higher peak although it was not significantly higher. The
higher peak in glucose concentration in the CS group is in
contrast to the results of Davis et al.(49,50) in which the
mice from mothers fed the sucrose diet always had the higher
mean peak values. .

These investigators also reported that the blood glucose
remained higher for a longer period of time in these mice:
that is, these mice appeared to display a lag time in the
clearance of glucose from the blood. A better understanding
would have resulted if the rate of clearance of glucose from
the blood had been calculated. Amatuzio et al.(60), Scow and
and Cornfeld(61) and Chernick and Scow(62) have developed
methods for finding the rate of removal of glucose from the
blood. The method of Amatuzio et al. requires that a blood
sample be taken very soon after a glucose load is administered.
This was not done here; therefore the formula presented by

Chernick and Scow(62) was utilized. The removal rate(K) is

g1ven as: K: 2'3(1ogtgf%;;2grgifllt where Gt is the

excess glucose concentration at time t: that is, Gt is the

 

blood glucose concentration at time t minus the fasting blood
gmpose concentration. (K x 100) then represents the percentage
decrease in the excess glucose concentration per minute over
time t1 to t2. In this study, t1 was taken as 30 minutes

and t2 as 120 minutes.

31

a. 30 days of age.1

 

 

 

    
  
 

 

 

 

ZOO-
x CS
Blood 0 SUC
150d 0
Glucose
(mg/100 ml) 100_
\
J
50-
i l
FAST 30 60 120
Time (minutes)
b. 60 days of age.
300-
x CS
0 SUC
250-
Blood
200-
Glucose
(mg/100 ml) 150_‘
100
50",
I I
FAST 15 30 60 120

Time (minutes

FIGURE 1 Glucose tolerance, 30 and 60 days of age.

1Male and female rats, see Table 8.

32

a. 120 days of age, lst test.1
250-1

x CS

o SUC

200 - °
0/ \0

150-4 . \
Glucose X‘TT“‘-.~_-R

Blood

 

 

 

 

 

(mg/100 ml) 100
50-1
Fj l I '
FAST 15 30 6.0 120
Time (minutes)
b. 120 days of age, 2nd test.1
250q
x CS
0 SUC
zoo.
Blood
‘0
1 0-
.5 \O
Glucose ‘T“‘--l“‘x
T
50-
I T l I
FAST 15 30 60 120

Time (minutes)

FIGURE 2 Glucose tolerance, 120 days of age (2 tests).

1For description of 2 tests, see footnote 7, Table 8.

33

Table 9 gives the removal rats at the three ages studied.
There is no indication that the removal rate differed between
the two groups at any age. The glucose load administered at
30 days of age was only one-quarter of the amount given at
60 and 120 days of age. This may account for the more rapid
rate of removal.

"The results of Davis et al. might indicate that the
maternal diet somehow altered the fasting glucose values of
the mice. The present study would seem to clearly indicate
that this was not true for the rat. Of course the species
difference is a factor that cannot be ignored when attempting
to compare results.

Since the present study placed greater restrictions on
possible dietary variables other than the carbohydrate source
then did the study conducted by Davis et al., it appears
relatively certain that, at least in the rat, the feeding of
a high-sucrose diet during gestation and lactation does not
alter the reSponse to a glucose load in growing or mature

offspring who have been fed a diet without sucrose.

34

TABLE 9 Glucose removal rate.

 

1 2 No. 3 Removal rate of excess glucose(%/min.)n
Group Age rats 30-120 min.
cs 30 4.010.85
(1.3)3
SUC 3.310-5
(4.3)
as 60 8 1.810.2
SUC 8 1.810.2
cs 120 10 1.010.1
SUC 10 1.21o,2

 

1See footnote 1, Table 5. 2Days. 3See footnote 3,
Table 8. qulOO, see text for derivation of removal
rate, K. 5MeanISEM.

SUMMARY AND CONCLUSIONS

This study was undertaken in an attempt to clarify some
results of investigations previously reported in the litera-
ture (47-50). Various parameters of lipid and carbohydrate
metabolism were evaluated in male offspring of rats fed a
high-sucrose (SUC group) or cornstarch diet (CS or control
group) during pregnancy and lactation. After weaning the
offSpring from both groups of dams were fed a cornstarch
diet. In previous studies the diets were either diluted with
sucrose, thus reducing the protein, vitamins and minerals as
well, or a strain of rat was used that was susceptible to
carbohydrate induced lipemia.

The present study did not show the increase in body
weight or the inability to handle a glucose load by the off-
spring in the SUC group as had been reported in mice by Davis
et al. (49,50). Likewise, a depression in serum triglycerides
and an elevation of liver lipid was not noted in the SUC group,
although these results had been reported by Berdanier (48).
Analyses at 30, 60 and 120 days of age of body weight, serum
triglycerides and cholesterol, relative liver size, liver
G6PD activity, liver lipid and cholesterol, and glucose
tolerance revealed that while some other differences were

apparent, they did not follow the same patterns that had been

35

36

reported previously. Given the large n value of each group,
the results of this study lead one to the conclusion that in
the rat there does not seem to be any consistent long-term
metabolic effects in offspring of mothers that had been fed
a high-sucrose diet during gestation and lactation, if the
offspring were fed a cornstarch diet from weaning. This
conclusion would be true at least for the parameters chosen

for this research.

APPENDIX

APPENDIX
METHODS OF ANALYSIS

Glucose-6-Phosphate Dehydrogenase (G6PD) Activity

1g samples of liver were homogenized in 9 ml of the
following solution: 0.15 M KCl, 10 mM N-acetyl-l-cysteine
and 1 mM Mg012'6 H20. made up in distilled water. This
solution was mixed 2 days prior to use and refrigerated.

The day before the analysis was to be done, the solution was
adjusted to pH 7.6 while still cold. The solution and the
subsequent homogenates were kept on ice throughout the pro-
cedure.

Homogenates were centrifuged for 15 minutes at 31,000 X g
in a Model RCZ—B Sorval refrigerated centrifuge1 (-4° to 0°).
The supernatant was removed using disposable pipettes, with
care being taken to avoid pipetting the top layer of lipid
and the bottom layer of non-cytosolic material. This super-
natant was then centrifuged for 1 hour at 100,000 X g in a
Model L3-40 Beckman centrifuge.2 The supernatant was removed,
with care being taken not to draw off any lipid from the top
or precipitate from the bottom. The supernatant was kept in

tubes on ice throughout the assay.

 

1Ivan Sorval, Inc., Newtown, CT 06470.
2Beckman Instruments, Inc., Palo Alto, CA 94304.

37

38

The reaction mixture consisted of:

(a) 2.7 ml of a solution prepared as follows, for 100 assays:

Tris bufferl, 1 M, pH 7.5, 25° 10 ml
NADP, monosodium salt, 3 mM 10 ml
nglz-6 H20, 200 mM 10 ml
Distilled water 250 ml

(b) 0.1 ml 6-phosphogluconic acid (6PG), trisodium salt, 25 mM.
(0) 0.1 ml glucose-6-phosphate (G6P), monosodium salt. 25 mM.
(d) 0.02 ml sample?

(e) _0.08 ml distilled water.

The initial mixture contained (a), (d) and (e). The
6PG(b) was then added, the solution mixed and then run, 4
samples at a time, on an ACTA CIII Beckman spectrophotometer3
with span set at 0.5 of full range and chart paper speed of
1.2 inches per minute. The temperature was kept constant at
25°. After 4-5 minutes, G6P(c) was added, the solution
rapidly mixed and then once again run. The wavelength was
set at 430 nm. with a slit width of approximately 0.15 mm.

A tungsten lamp was used.

The principle of the assay is based on the reduction of

NADP to NADPH by glucose-6-phosphate dehydrogenase(G6PD) in

the pentose pathway: G6P-—§éEQ——-—a»6-phosphogluconolactone
NADBET‘NADPH
w 6PG -—%Dq-—>D-ribulose -5-phosphate .
H20 NAD NADPH

Centrifuging at 100,000 X g eliminates the probability that
reduction reactions in the mitochondria and microsomes would
be present to interfere with the cytosolic readings. A

change in absorbance due to this conversion of NADP is read

1Sigma Technical Bulletin No. 106 B, Sigma Chemical Co.,
St. Louis, MO 63178.

20.01-0.05 ml samples were used to obtain a good rate
and to check for linearity, and 0.02 ml was chosen as the
best oncentration.

Beckman Instruments, Inc., Scientific Instruments
Division, Fullerton, CA 92634. ‘

 

39

maximally at 430 nm. By adding 6PG first and then waiting
several minutes before adding G6P, a rate can be determined
for the conversion of 6PG to D-ribulose-5-phosphate by the
enzyme, 6-phosphogluconate dehydrogenase (6PGD). The addi-
tion of G6P then will give a rate for both enzymes (G6PD and
6PGD), and the activity of G6PD is found by taking the
difference. This activity is eXpressed in nanomoles of
substrate (NADP) converted per milligram of cytosolic pro-
tein per minute, and is calculated as follows:

1) Determine the rate of change of squares per
minute on the chart paper and the span used. If the
span was 1.0 (full range), then each square would
be equal to 0.01 0D units on chart paper that went
from 0 to 1.00 OD. If the span was 0.5, then each
square would be equal to 0.005 0D units and so on.

2) Record the change in 0D units per minute
(A OD/min) by multiplying the number of squares per
minute by the number of 0D units per square.

. .3) Find the number of micromoles substrate
util1zed per minute. .AODzmin

1 cm ET6.22 cmzflumole

This is based on the fact that absorbance (in 0D
units) is equal to the length of the lightpath
(1 cm in this case) times the concentration of the
substrate times the molar absorptivity (the extinc-
tion coefficient, 5 ) for the substrate. That is,
A = l-c-t. For a 1 molar solution of NADP with a
1 cm lightpath, the molar absorptivity (and conse-
quently the absorbance since the lightpath(l) is 1
and the concentration(c) is 1) is 6.22 x 103. There-
fore a 1 millimolar solution in a 1 cm lightpath
would have a molar absorptivity of 6.22, and the
units of the solution would be millimoles per liter
or micromoles per milliliter. The units cm Ahmole
TH the above equation come from 5: A , when
~ l-c

c=1 millimolar (1ml = lcm3). The equation at the
beginning of 3) is essentially obtained by solving
A = l-c-E for c. Since the concentration is based
upon 1 ml it is then necessary to multiply by 3 ml,
asthis was the volume of the reaction mixture.

4) Divide the value obtained in 3) by the
milligrams of protein in the reaction mixture to
obtain the activity.

X 3 ml

40

Lowry Protein Determination

Reagents
A 2% Na2003 in 0.1 N NaOH, prepared each day
B 0.5% CuSOu-5 H20 in 1% sodium tartrate
C 50 ml A + 1 ml B

Procedure

A stock solution containing 1 mg/ml of protein (bovine
albumin) was used to make standards containing 100, 300 and
600.ug of protein per 1.2 ml of distilled water. To find a
sample dilution that would fall within these standards
(preferably between the 100 and 300 Mg standards), 1:20,
1:50 and 1:100 dilutions (in distilled water) were tried
first. In this study the 1:50 dilution produced the most
satisfactory result. 6 ml of reagent 0 was then added to
1.2 ml of the diluted sample or the standard or blank (dis-
tilled water) and mixed. This was allowed to stand for 10
minutes at room temperature. Then 0.3 ml of reagent E was
added, while the solution was being mixed. This was allowed
to stand at room temperature for 30 minutes. Absorbance was
estimated by reading at 500 nm on a Model 6/20A Coleman
Jr-IIA spectrophotometer.1 For this study, the above method

was found to have a c.v. = 4.7% based on 10 replications.

Glucose Tolerance Tests and Blood Glucose Determinations

Food was withdrawn from the rats between 2200 and 2300

hours the night before the tests. The tests were initiated

 

1Coleman Instruments, Maywood. IL 60153.

41

at approximately 0900 hours. Rats were placed in plastic
restrainers only while blood was drawn. At 30 days of age
the rats were held by hand during blood collections. Between
drawings all animals were kept in their cages. Blood was
drawn by clipping the tail and drawing two 100.Ml samples of
blood into calibrated capillary tubes which had been pre-
‘viouSIy heparinized. After the fasting bloods, subsequent
samples were drawn without clipping the tail further.
Samples were taken for fasting levels, and then at 15, 30,
60 and 120 minutes after injection of the glucose test load.
The glucose load for 53 and 113 day old rats consisted of
800 mg of glucose in 2 ml of normal saline (0.9% NaCl). It
was administered intraperitoneally using a 25 gauge, 5/8 inch
needle and a 2.5 cc syringe. Due to smaller body size, only
0.5 ml of the glucose solution was injected into the 30 day
old rats.

The blood samples were put into test tubes containing
1.9 ml of distilled water. The tubes were set in ice prior
to the blood drawings. Each tube had received one drop of a
NaF solution (10 mg/ml), which was then dried before the
distilled water was added. Keeping the tubes cold and adding
NaF aids in maintaining glucose stability for approximately
24 hours.1

Blood glucose determinations were based on the reactions:

0 glucose oxidase‘

fi-D-glucose+02+-H2 , H202+D-glucono - J-lactone .

 

H202+reduced chromogen-PerOX1dasg:oxidized chromogen+H20.

1From the circular that comes with the Glucostat Reagent
Set, Worthington Biochemical Corp., Freehold. NJ 07728.

 

42

The color produced by the oxidized chromogen is proportional
to the glucose concentration.

It was discovered that the stated concentrations for
the ZnSOu and NaOH deproteinizing steps were not strong
enough to insure complete precipitation of the protein from
whole blood. Therefore, concentrations of 6% ZnSOu and
.46 N NaOH were finally settled upon as being adequate to
completely deproteinize the blood. These were titrated the
day before the tests to be certain that they neutralized each
other. The rest of the deproteinization was carried out
exactly as explained using the semi-micro method.1 It should
be noted that the solutions must be mixed well after the
addition of both the NaOH and the ZnSO4 so that complete
deproteinization will occur. After deproteinization, the
solutions were centrifuged for 20 minutes in a Model U.V.
International centrifuge.2 For the enzymatic reaction, 0.8 ml
of supernatantwas added to 1.2 ml of distilled water. 2.0 ml
of the Glucostat reagent was then added and the solution
mixed. The tube was stoppered and placed in a water bath at
37° for 30 minutes. This allows the reaction to go to
completion and therefore it was necessary to dilute the
supernatant (as described above) before the Glucostat reagent
was added. After removal from the water bath the reaction
was stopped by adding one drop of 4 N HCl and mixing. Each
sample was read at approximately the same time after the
addition of the HCl (usually 1 hour) at 420 nm on the Model

1

 

Glucostat, Worthington Biochemical, Freehold, NJ 07728.
International Equipment Co., Needham. MA.

43

6/20A Coleman Junior IIA spectrophotometer. Based upon 9

replications this author found a coefficient of variation

for this method of 4.7%.

Liver Lipid Extraction

The extraction technique is based on the solubility of
lipidiruachloroform:methanol mixture. Protein, as well as
any carbohydrate, will not dissolve in this mixture and there-
fore the lipid may be quantitatively extracted, after water
is removed by filtering through sodium sulfate.

After thawing, two 1g samples of liver were excised.
Each was completely homogenized in 10 ml of methanol, using
a Model S630 Tri-R-Stir-R homogenizer.1 This solution was
then transferred to a 125-200 ml Erlenmeyer flask. The
homogenizing pestle and tube were washed in 15 ml and then
5 ml of methanol. The washings were added to the flask.
Then 50 ml of chloroform was added and mixed and the flask was
placed in a water bath for 30 minutes at 450-50.. After
cooling,the solution was filtered into 250-300 ml Erlenmeyer
flasks. The 125-200 ml flask was rinsed 3 times with chloro-
form. This was then filtered into the larger flask after
each rinsing. The filter paper was then rinsed twice and
removed and the funnel rinsed once with chloroform. The
filter had been previously weighed, and after filtering the
solution and rinsing the paper, the paper was dried and
placed in a desiccator until a constant weight was obtained

(approximately 48 hours). This fat-free dry matter was used

 

1Tri-R Instruments Inc., Rockville Centre, NY.

44

as a constant for expressing the amount of lipid (and liver
cholesterol) as mg/g fat-free dry matter. The sample and
filter paper were evaporated to dryness in a constant temper-

1 at 50°.

ature drying cabinet
50 ml Erlenmeyer flasks were weighed to the nearest
0.1 mg. Funnels with filter paper were set up and a spatula
of anhydnns sodium sulfate added to remove any moisture in
the extract. Approximately 10 ml of chloroform and some
sodium sulfate were then added to the flasks containing the
lipid material. After mixing well this solution was poured
through the filter paper into the 50 ml flasks. The first
flask was rinsed 3 times, the filter paper rinsed twice and
the funnel once with chloroform. This solution was evaporated
overnight in the drying oven. The flasks were placed in a
desiccator until aconstant weight to the nearest 0.1 mg was
obtained. Total lipid was determined by finding the differ-
ence between the weight of the flask and the weight of the

flask plus the lipid material.

Serum Triglyceride Determination

The method used was exactly the same as described by
Fletcher(53). Glycerides (after removal of phOSpholipids)
were saponified to free glycerol. The glycerol was then
converted to formaldehyde by oxidation. The formaldehyde
was reacted with diacetylacetone and ammonia in a condensa-
tion reaction which yields a fluorescent product (3.5-diacetyl-1,

1

 

Blue M Electric 00., Blue Island, IL.

45

4-dihydrolutidine) absorbing at 405 nm. This procedure was
found to have a coefficient of variation of 5.8% based on 13

replications.

Serum and Liver Cholesterol Determinations

.Gas-liquid chromatography was used for cholesterol
determinations. For serum, the procedure was to make a
standard by adding 0.5 mg cholesterol and 0.2 mg 5-et cholestane
(as the internal standard) to a tube. The solvent was then
evaporated. and the tubes were covered and stored in a
desiccator. When ready to use, 0.1 ml of trimethylsilyl
reagent (TMS) was added. The TMS solution was prepared by
adding, under a hood, 7.7 ml trimethylchlorosilane and 23.1 ml
hexamethyldisilazane to 69.2 ml pyridine (1:3:9).1 Anhydrous
sodium sulfate was placed in the bottom of the jar to absorb
moisture. The purpose of the TMS is to make silyl ether
derivatives of thecflmlesterol, which makes the cholesterol
more volatile and stable for analysis by gas-liquid chroma-
tography. For the serum samples, 0.2 mg 5-a( cholestane(as the
internal standard) was added to a screw cap tube and the
solvent evaporated. Then 0.5 ml of serum was added, followed
by 5 ml of .33 N ethanolic (90% ethanol) NaOH. The tube was
covered, mixed and the solution was saponified at 37° for 3
hours. After cooling, 1 ml of heptane was added and the
solution was shaken vigorously by hand. Then 5 ml of

distilled water was added and again the solution was shaken

 

1Applied Science Catalog 18 (1975), p. 101.

46

vigorously by hand. The top layer of heptane containing the
extracted cholesterol and cholestane was removed and the
heptane evaporated in the drying oven at 50°. After drying,
the samples were stored in a desiccator until a large number
were ready to be injected into the gas chromatograph. At
that time, exactly 0.1 ml of the TMS solution was added.
After addition of TMS the samples or standard were allowed
to sit between 30 and 40 minutes at room temperature before
injection into the gas chromatograph.

The gas chromatograph used was a model No. 900 Perkin
Elmer.1 The column was a 1.8 meter, 2 mm i.d., stainless
steel column packed with 3%-5% SP2401. The chromatographic
conditions were: Column temperature, 230°; injector port
temperature, 285°: detector temperature, 285°: helium
carrier gas flow rate, 30 ml/min. and range. X 100.
Attenuation was usually X 16 and 2‘31 injections were normally
made. The standard was injected initially and after every
8 samples each day.

For the determination of liver cholesterol the procedure
was the same except that the liver lipid was dissolved in
10 ml of choloroform and a 3 ml aliquot was taken. This was
added to a screw cap tube, along with 0.1 mg or 0.2 mg
cholestane, and the solvents evaporated before the saponifi-

cation procedure.

 

1Perkin Elmer, Norwalk, CT.

47

The amount of cholesterol was determined as follows:

1. Take the peak height or the area under the curve
as the proportion for comparison.
2. Find the amount of cholestane in sample injection:

grea(peak) of sample cholestane theoretical
area(peak) of standard cholestane amt. of chole-
tane in sample
injection
3. Determine the factor for cholesterol conversion:

theoretical amt. of cholestane in WHOLE sample
amount of cholestane in sample injection(2T)

4. Repeat 2. for cholesterol to get amount of
cholesterol in sample injection.

5. 3. X 4. = amount of cholesterol in initial serum
sample(0.5 ml in this study) or liver sample
(3 ml aliquot of 10 ml/g liver in this study).

6. Convert serum value to mg% cholesterol and liver
value to mg cholesterol/mg fat-free dry matter.

On 10 replications of serum, this method was found to
have a coefficient of variation of 3.8%. Figure 3 is a
typical example of a chromatogram of a standard and duplicate

sample injections.

48

FIGURE 3 Example of a chromatogram.

A and B, duplicate liver samples: 0, standard.
I, 5-etcholestane: II, cholesterol; III, unknowns.
Chart speed = 1 in./4 min.

49

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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10.

11.

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