GASTROENTEROLOGY Vol. 60, No.

4
Copyright © 1971 by The Williams & Wilkins Co. Printed in U.S.A.

THE EFFECT OF DIET AND FASTING ON THE SERUM BILIRUBIN

CONCENTRATION IN THE RAT

PETER V. D. BARRETT, M.D.

Division of Gastroenterology, Department of Medicine, Harbor General Hospital,
Torrance, California, and The University of California School of Medicine,
Los Angeles, California

An increase in the total serum bilirubin concentration has been

demonstrated in the congenitally jaundiced (Gunn) rat during partial
or complete caloric deprivation. This suggests that the enzyme uridine
diphosphate-glucuronyl transferase, which is deficient in these mu-
tant rats, is not involved in the hyperbilirubinemia of fasting. Fasting
also increased the total serum bilirubin in normal rats and in rats with
obstructive jaundice. A high fat diet produced a decrease in the total
serum bilirubin; this effect may be the result of an increase in the
caloric intake rather than a specific effect of dietary fat.

The serum bilirubin concentration of
man and rat can be altered by changes in
either the caloric intake or the composition
of the diet. A 48-hr fast has been shown to
double or triple the bilirubin concentra-
tion of both normal subjects and patients
with various types of hepatic dysfunc-
tion.!,2 In congenitally jaundiced (Gunn)
rats, Housset et al. 3 have demonstrated an
inverse relationship between the amount
of fat in the diet and the serum bilirubin
concentration.
In this study, the effect of fasting, semi-
starvation, and a high fat diet on the total
serum bilirubin concentration (TSB) has
been determined in Gunn rats. In addition,
the effect of fasting on the serum bilirubin
Received August 12, 1970. Accepted November
13, 1970.
Address requests for reprints to: Dr. Peter V. D.
Barrett, Harbor General Hospital, 1000 West Carson
Street, Torrance, California 90509.
This investigation was supported by United States
Public Health Service Grant AM·12306, and in part
by Grant GRSG RR-00551 from the National Institu·
tes of Health.
The author is indebted to Claudia M. Silver for
her technical aid, to William Steers, Animal Care
Facilities Manager, for his invaluable help with the
Gunn rats, and to Victoria M. Adkins for her secre·
tarial assistance.
572
concentration of normal rats and rats with
experimental obstructive jaundice has
been examined. The data indicate that
there is an inverse relationship between
the TSB and the caloric intake.
Materials and Methods
The Gunn rats were produced from breeding
stock generously donated by Drs. Rudi Schmid
and Richard L. Swarm. Homozygous female
Gunn rats weighing 150 to 170 g and heter-
ozygous female Gunn rats weighing 240 to 250
g were selected for study. In studies involving
normal rats, female Wistar descendants were
used.
Extrahepatic obstruction was produced in
normal rats by dividing the common bile duct
between two pairs of ligatures at a point one-
third of the distance between the liver and the
duodenum.
The TSB in 0.050 ml of serum was deter-
mined with a micromodification of the method
of Michaelsson,' using a Beckman direct read-
ing DU-2 spectrophotometer. In all studies
with homozygous or heterozygous Gunn rats,
approximately 0.15 ml of blood was withdrawn
from a tail vein into a 1.0-ml syringe containing
a known volume of heparinized saline. Blood
was obtained by cardiac puncture at the end
of fasting experiments in normal rats and in rats
with extrahepatic obstruction. Michaelsson's
diazo method was chosen because of its low
coefficient of variation, which has been re-
April 1971 FASTING AND BILIRUBIN CONCENTRATION
ported to be 0.9% for a serum bilirubin con-
centration of 15 mg per 100 m!. The coefficient
of variation for other standard diazo methods
ranged from 2.4 to 6.8%.' In this laboratory, a
micromodification of Michaelsson's method was
used on six sequential samples of blood ob-
tained from the tail vein of a rat with obstruc-
tive jaundice. Statistical analysis revealed a
mean TSB of 6.88 mg per 100 ml, a standard
deviation of 0.35 mg per 100 ml, and a coeffi-
cient of variation of 5.1 %.' It should be noted
that these results include sampling and dilu-
tion errors as well as the error of the diazo
method itself. To establish the reliability of the
micromodification of Michaelsson's method at
very low concentrations of TSB, seven replicate
determinations of the TSB were performed on
pooled normal rat serum. The tests were done
in a "blind" fashion in that the samples were
coded as coming from different animals. Results
revealed a mean TSB of 0.060 mg per 100 ml,
a standard deviation of 0.012 mg per 100 ml,
and a coefficient of variation of 20%.
For dietary studies, the rats were maintained
in individual metabolic cages. They were al-
lowed to adjust to the cage for 2 weeks prior
to the initiation of the study. The control diet
[General Biochemicals, Chagrin Falls, O. (diet
no . 170610, control) 1 was calculated to contain
4160 cal per kg, of which 17% was lipid. The
high fat diet [National Biochemical Co.,
Cleveland, O. (special basal atherogenic test
diet, modified) 1 was calculated to contain 5689
cal per kg, of which 72% was lipid. The caloric
intake of each rat was determined by weighing
the food daily. The animals were given free
access to water during all studies.
Results
The effect of a 72-hr fast on the TSB of
two homozygous Gunn rats is shown in
figure 1. The average TSB value preceding
the fast was 5.8 mg per 100 m!. During the
fast, the TSB rose progressively, reaching
average values of 12.1 mg per 100 ml at
48 hr and 14.2 mg per 100 ml at 72 hr.
Within 1 day after refeeding, the TSB
fell significantly toward control levels. The
mean hematocrit of these animals increased
from 41 % during the control period to 46%
after fasting for 48 hr.
An attempt was also made to demon-
strate a relationship between fasting and
the TSB in normal and heterozygous Gunn
rats, but the TSB of these animals, all of
573
which weighed over 140 g, was extremely
low and could not be determined reliably.
However, younger rats were noted to have
higher control TSB values, and, therefore,
fasting studies were performed in a group
of normal rats with a mean weight of 60.2
g and a range of 57 to 64 g. The mean
TSB (± standard error) was 0.060 ±
0.007 mg per 100 ml in 6 control rats and
0.149 ± 0.014 mg per 100 ml in 6 animals
after a 48-hr fast. The difference between
the two groups is highly significant (P <
0.001. 5 The mean hematocrits of the con-
trol and fasted rats, determined at the con-
clusion of the experiment, were 33.1 and
40.4%, respectively.
Rats with increased TSB levels caused
by extrahepatic obstruction were studied
in order to examine further the relation-
ship between the TSB and fasting in non-
mutant rats. Twenty-seven normal rats
weighing 140 to 170 g were divided into
three equal groups. Two groups were
fasted for 48 or 24 hr prior to surgery as
well as for 48 hr thereafter. The control
group was allowed an ad libitum diet
before and after surgery. The experiment
was designed in this manner because in
the rat the TSB does not establish a pla-
teau following extrahepatic obstruction;
the TSB rises progressively for approxi-
mately 8 days and then declines to lower
levels. 6 In addition, the caloric intake of
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FIG. 1. Effect of a 72-hr fast on total serum bili-
rubin concentration of 2 homozygous Gunn rats. The
bilirubin concentration increased progressively during
the fast and fell promptly with refeeding.
574 BARRETT
rats with obstructive jaundice is very low
during the 1st week following surgery.
The results of this experiment are shown in
figure 2. Forty-eight hours after surgery the
mean TSB (± standard error) for the group
of rats fasted for 48 hr prior to surgery, 24
hr prior to surgery, or fed an ad libitum
diet were, respectively, 9.56 ± 1.05, 7.27
± 0.50, and 5.90 ± 0.49 mg per 100 ml.
The difference between the group fasted
48 hr preoperatively and the control group
was statistically significant (P < 0.01).
The mean hematocrits of these groups
were, respectively, 45, 45, and 42%.
An attempt was made to follow the TSB
of homozygous Gunn rats during prolonged
fasting, but, because of the death of 2 of
4 rats after 72 hr, caloric restriction was
used instead of a total fast. Mter a 3-week
control period, the daily ration of food of-
fered to a Gunn rat was reduced to 75%
of the average control intake for a period
of 2 weeks and then further reduced to
50% for the next 2 weeks. During the
periods of caloric restriction, the rat con-
sumed all of the food which was offered.
The average TSB was 12.2 mg per 100 ml
for the control period. At the completion
of the period during which the caloric in-
take was 75% of the control period, the
TSB was 17.1 mg per 100 ml, and, during
the last week of caloric restriction to 50%
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2 the TSB. The chronic unconjugated hyper-
o bilirubinemia of these rats has been shown
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48 HOUR FAST 24 HOUR FAST AD LIB DIET
FIG. 2. Effect of preoperative fasting on serum
bilirubin concentration of normal rats with extrahepatic
obstruction. Forty-eight hours after operation, the
mean bilirubin concentrations (± standard error) were
9.6 ± 1.1, 7.3 ± 0.5, and 5.9 ± 0.5 mg per 100 m!.
The difference between the means of the animals
fasted for 48 hr preoperatively and the control ani-
mals is statistically significant (P < 0.01).
Vol. 60, No.4
of the control period, the average TSB
was 18.7 mg per 100 ml. Within 3 days
after the return to an ad libitum diet, the
TSB fell to control levels (fig. 3).
The effect of a high fat diet on the TSB
was examined in 2 Gunn rats with similar
results in both animals; one of these studies
is illustrated in figure 4. The average
daily caloric intakes during the first con-
trol period, the high fat diet, and the sec-
ond control period were, respectively, 23.4,
34.0, and 22.7 cal per 100 g of body weight.
During these same periods the average
TSB values were, respectively, 12.5, 6.5,
a.ld 12.7 mg per 100 ml (the latter value
is the average of four TSB determinations
during the final 2 weeks of the control
period).
Discussion
Previous studies have shown that both
the direct and total serum bilirubin con-
centrations increase in response to fasting
in normal subjects and in patients with
hepatic dysfunction. 1, 2 The mechanism
responsible for this is not well understood,
but diminished renal excretion of bilirubin
and increased production of bilirubin
caused by hemolysis appear to have been
excluded as contributing factors in man. 1
The Gunn rat, a congenitally jaundiced
mutant, seems to be an ideal model for the
study of the effects of diet and fasting on
to result from a deficiency of uridine di-
transferase, the
enzyme necessary for the conjugation and
subsequent excretion of bilirubin. 7 In these
animals, the TSB increased to 212% of the
is similar in magnitude to the mean changes
observed in man after the same period of
fasting: 240% in 5 normal subjects and 194%
in 5 patients with hepatic dysfunction. 1
The demonstration that the hyperbili-
rubinemia of fasting occurs in Gunn rats
suggests that this phenomenon is indepen-
dent of uridine diphosphate-glucuronyl
transferase. This same phenomenon was
observed in normal rats and in rats with
Aprii1971 FASTING AND BILIRUBIN CONCENTRATION 575

FIG. 3. Effect of caloric restriction on the total serum bilirubin concentration of a Gunn rat. After caloric
restriction to 75% of the control level, the bilirubin concentration rose from 12.2 to 17.1 mg per 100 ml; fur-
ther reduction to 50% of the control level produced little additional increase. The daily caloric intake is in-
dicated by the hatched area on the graph .

................ ...................

DAYS
FIG. 4. Effect of a high fat diet on the total serum bilirubin concentration. The bilirubin concentration de-
creased from an average control value of 12.5 to 6.5 mg per 100 ml in association with a high fat diet, and re-
turned to control levels when the normal diet was restored. The average caloric intake during the high fat
diet was almost 50% greater than during the control period.

extrahepatic obstruction of the common
bile duct.
It was found that total caloric depriva-
tion is not essential to produce a significant
elevation of the TSB. In a Gunn rat, after
2 weeks of caloric restriction to 75% of the
control period, the TSB rose from an aver-
age of 12.2 ,to 17.1 mg per 100 ml. Further
restriction of the caloric intake to 50% of
the control period produced little further
increase in the TSB. Felsher et al. 2 re-
cently demonstrated a significant increase
in the serum bilirubin concentration in pa-
tients with Gilbert's syndrome who were
given up to 400 cal per day. The threshold
level of caloric restriction which will pro-
duce elevation in the TSB in man and
animals remains to be determined.
Housset et al. 3 have previously reported
that a high fat diet produced a decrease
in the TSB of the Gunn rat, but the total
caloric intake of the rats was not given and
no explanation for the effect was presented.
One potential mechanism is that unconju-
gated bilirubin which exchanges across
the intestinal mucosa from the plasma
pool may be trapped by lipid in the intes-
tinal lumen, similar to the effect of oral
cholestyramine which has been demon-
strated in Gunn rats. 8 A second possibility
is suggested by the results of the current
study in which the average caloric intake
576 BARRETT
during the high fat diet was almost 50%
higher than the intake during the control
period. Therefore, it is possible that the
decrease in the TSB could be a function of
the increased caloric intake, rather than a
specific effect of fat.
The hematocrits of all the fasted animals
increased, presumably owing to dehydra-
tion, despite the fact that they had free
access to water. The possibility that dehy-
dration contributed to the hyperbilirubi-
nemia of fasting cannot be excluded, but
the experiment in obstructed rats in which
a significant increase in the TSB occurred
with little change in the hematocrit sug-
gests that this is not an important factor.
The fundamental mechanism respon-
sible for the increased TSB which occurs
during caloric deprivation is not yet clear.
However, the fact that the proportional
increase in TSB is similar both in rat and
man and is independent of the bilirubin
concentration and the type of hepatic
pathology suggests that a decreased he-
patic fractional clearance rate of bilirubin
may be responsible. This concept is sup-
ported by recent bilirubin-He plasma
clearance studies in the horse. 9
Vol. 60, No.4
REFERENCES
1. Barrett PVD: Hyperbilirubinemia of fasting: stud-
ies in man and in the congenitally jaundiced (Gunn)
rat (abstr). Gastroenterology 58:926, 1970
2. Felsher BF, Rickard D, Redeker AG: The recip-
rocal relationship between caloric intake and the
degree of hyperbilirubinemia in Gilbert's syn-
drome. New Eng J Med 283: 170-172, 1970
3. H ousset PE, Etienne JP, Petite JP, et al: Interet
d 'un regime alimentaire defini en experimentation
animale. Ann BioI Clin (Paris) 24:771-786, 1966
4. Michaelsson M, Nosslin B, Sjolin S : Plasma bili-
rubin determination in the newborn infant. A
methodological study with special reference to the
influence of hemolysis. Pediatrics 35:925-931, 1965
5. Snedecor GW, Cochran WG: Statistical Methods.
Sixth edition. Ames, Iowa State University Press,
1967
6. Edlund Y: Studies o nthe carbohydrate metabolism
and liver protection therapy in experimental ex-
trahepatic biliary obstruction . Acta Chir Scand
116:1- 187, 1948
7. Schmid R, Axelrod J, Hammaker L, et al: Con-
genital jaundice in rats, due to a defect in glucuro-
nide formation. J Clin Invest 37:1123-1130, 1958
8. Lester R, Hammaker L, Schmid R: A new thera-
peutic approach to unconjugated hyperbilirubi-
nemia. Lancet 2:1257, 1962
9. Gronwall RR, Mia AS: Effect of starvation on
bilirubin metabolism in the horse (abstr). Physi-
ologist 12:241, 1969