0270-9 1~9/84/040~i-04ii4$02.

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HEPATOLOW Vol. 4, NO.3, pp. 454-460, 1984
Copyright '(:) 1984 hy the American Association for the Study of Liver Diseases Printed in U.S.A.

The Effects of Nutrition on Unconjugated

Plasma Bilirubin Concentrations in
Squirrel Monkeys
OSCARW. PORTMAN,MANFREDALEXANDER,CHARLESE. CORNELIUS,
R O Y CHOWDHURY,
JAYANTA NAMITAR O Y CHOWDHURY AND IRWIN M. ARIAS

Oregon Regional Primate Research Center, Beaverton, Oregon 97006; California Primate
Research Center, Davis, California 95616; and Liver Research Center, Albert Einstein College of
Medicine, Bronx, New York 10461
Bolivian squirrel monkeys (Suimiri sciureus) have fasting unconjugated hyperbilirubinemia
(males: 2.0 2 0.14; females: 3.0 -I. 0.26 mg per dl) which resembles that of humans with Gilbert's
syndrome. Closely related Brazilian squirrel monkeys have fasting levels (males: 0.29 2 0.045;
females: 0.36 2 0.073 mg per dl) similar to normal people. The purpose of this study was to identify
the underlying mechanisms and the nutritional factors involved. Both Bolivian and Brazilian
squirrel monkeys had higher plasma bilirubin concentrations after an 18 hr fast than 4 hr after
feeding. The development of fasting hyperbilirubinemia was progressive for at least 24 hr. Both
populations that received a semipurified diet containing 5% fat had lower fasting and postprandial
plasma bilirubin concentrations than did animals receiving 0.3%fat but much lower than those
receiving 20%fat. The emulsified complete meal, or glucose, sucrose, casein, or lactalbumin alone
when given by intragastric tube lowered the plasma bilirubin levels of Bolivian monkeys to less
than one-half of fasting values within 1 to 4 hr. Water or butter did not have a significant effect.
Glucose or fructose, when given intravenously, lowered the plasma bilirubin levels to less than
half of fasting values; the fat emulsion, Intralipid, did not have a statistically significant effect.
Subcutaneous epinephrine increased plasma glucose concentrations and reduced plasma bilirubin
concentrations. When glucose or glucose plus butter were given by stomach tube to Bolivian
squirrel monkeys for 48 hr, a very low plasma bilirubin concentration resulted whereas butter
given alone resulted in high values.
Fasting Bolivian squirrel monkeys had impaired plasma clearance of [3H]bilirubin; intravenous
glucose normalized the pattern. Glucose administration markedly increased excretion of bilirubin
into bile. Glucose or bilirubin given intravenously or sucrose administered by gastric tube lowered
plasma bile acid concentrations. The complete ration or fat given alone by gastric tube raised
plasma bile acid concentrations.
Thus carbohydrate, but not fat, lowered fasting hyperbilirubinemia and increased bilirubin
excretion in Bolivian squirrel monkeys. Exogenous glucose may increase the availability of UDP-
glucuronate for hepatic conjugation of bilirubin in the fasting subject.

Bolivian squirrel monkeys (Saimiri sciureus), unlike al., unpublished observations-some of this information
Brazilian squirrel monkeys, have a syndrome which re- has appeared in abstract form (Gastroenterology 1982;
sembles Gilbert's syndrome in man [(l); Chowdhury et 82:1225 and Hepatology 1982; 2:723)]. Similarities in-
clude nonhemolytic unconjugated hyperbilirubinemia,
fasting hyperbilirubinemic response, decreased plasma
Received October 11, 1983; accepted December 5, 1983. clearance of bilirubin, reduced hepatic uridinediphos-
This is Publication 1299 of the Oregon Regional Primate Research phoglucuronate glucuronyl transferase activity and re-
Center.
This work was supported in part by the National Institutes of Health duced bilirubin diglucuronide to monoglucuronide ratio
Grants HI,-09744, AM-2019, AM-32297, RR-00163, RR-05694 and RR- in bile (2). Serum aminotransferases and alkaline phos-
00169. phatase activities and liver histology are similar in Bo-
Dr. d. Roy Chowdhury is a recipient of a Research Career Award of livian and Brazilian squirrel monkeys (1).
the National lnstitutes of Health. How nutritional factors influence serum bilirubin lev-
Address reprint requests to: Oscar W. Portman, M.D., Oregon Re-
gional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, els is not precisely known (3-5). In this study, we dem-
Oregon 97006. onstrated that intravenous or intragastric administration
454
Vol. 4,No. 3, 1984 NUTRITION AND PLASMA BILIRUBIN IN SQUIRREL MONKEYS
of carbohydrate, but not lipid, reduced fasting plasma
unconjugated bilirubin concentration within 1 hr and
increased bilirubin removal from blood and secretion into
bile.
MATERIALS AND METHODS
EXPERIMENTAL ANIMALSAND DIETS
Twenty-eight Brazilian squirrel monkeys (Sairniri sci-
ureus) and 36 Bolivian squirrel monkeys were used. With
the exception of monkeys born at the Oregon Primate
Center of Tarpon Zoo parents, the Brazilian squirrel
monkeys were secured from a Leticia, Colombia, collect-
ing station via the Tarpon Zoo, Tarpon Springs, Florida,
or from Dr. T. B. Clarkson’s laboratory-raised colony a t
the Bowman-Gray School of Medicine. Bolivian mon-
keys were obt,ained from Santa Cruz, Bolivia, through
Primate Imports, Port Washington, NY, or were later
born at the Oregon Primate Center. The place of origin
of the two monkey populations agreed well with expected
hair color patterns, particularly in the supraorbital area.
For the purposes of this study, the geographic origin of
each monkey or its predecessors was the primary crite-
rion for subspecies classification.
Monkeys were fed either ground Purina Monkey
Chow-25’ supplemented with vitamins, water and ba-
nanas, or a semipurified ration containing 19.5% butter
and 0.68% corn oil (w/w) that supplied 37.8% of calories
as fat (6). Other diets also contained 0.3% corn oil (low
fat) or 0.6% corn oil plus 4.5% butter (5% fat). These
diets had ratios of protein, vitamins and minerals to
calories identical to the 20% fat diet. All foods were
molded into cubes, and 280 kcal per day were fed in two
meals; there was substantial wastage of food. Twelve
Bolivian and 10 Brazilian squirrel monkeys were sequen-
tially given each of these diets for 1 week; the sequence
was 20, 0.3, 5, 20 and 5% fat. Six Bolivian squirrel
monkeys consumed 20% fat throughout and served as
controls.
P R E PARATI 0N OF [“HIBILIRLJBI
N As reported previously (l),Bolivian squirrel monkeys
Unconjugated [“Hlbilirubin (200 Ci per mole) was
prepared by hydrolysis of pigments excreted in rat bile
after intravenous administration of 10 moles (600 pCi)
of [:3H-2,3]-6-aminolevulinic
acid (7) and was purified by
high-pressure liquid chromatography as described pre-
viously (8).
EXPERIMENTAL PROCEDURES WITH MONKEYS
Fasting plasma bilirubin levels were determined
around 9 a.m. after food had been removed 18 hr previ-
ously and in animals from which food was withheld for
18 hr followed by feeding for 4 hr.
The biliary excretion of bilirubin and bile acids was
studied in 6 monkeys. Squirrel monkeys which were
fasted for 18 hr were treated with atropine (0.025 mg)
intramuscularly and anesthetized with 1%halothane and
1%nitrous oxide. Surgery was completed in less than 20
min. Each animal was prepared with a femoral vein and
a femoral artery catheter in different legs. The cystic
duct was tied prior to insertion of a cannula into the
common bile duct. Sedation during the subsequent pro-
455
cedure was obtained with acepromazine maleate (Ayerst
Lab, NY).
No sedation was used in animal procedures that did
not involve biliary cannulation, and the monkeys were
only restrained during injection, intubation or with-
drawal of blood. Test carbohydrates were obtained from
Sigma Chemical Co. (St. Louis, Mo.), and Intralipid
(Vitrum), an intravenous fat emulsion with 10% soybean
oil and 1.2% egg yolk phospholipid, was obtained from
Cutter Laboratories (Berkeley, Calif.).
Disappearance of an intravenous dose of [3H]bilirubin
was measured in Bolivian and Brazilian squirrel monkeys
which received 5 ml of saline intravenously and in Boliv-
ian monkeys given 5 ml of glucose 15 min prior to [3H]
bilirubin injection and 2 hr subsequently. The O-time
value for plasma bilirubin disappearance curves was de-
termined from the antilog of the Y intercept obtained
from a mathematical best fit of the logarithms of plasma
radioactivities at 1, 2 and 3 min.
Direct (conjugated) and indirect (total) plasma biliru-
bin concentrations were measured by the method of
Jendrassik and Grof (9). In some samples, conjugated
and unconjugated bilirubins were separated by the sol-
vent partition method of Weber and Schalm (10). Over
95% of the total plasma bilirubin concentrations was
unconjugated bilirubin as confirmed by high-pressure
liquid chromatography (8). Biliary bile acids were meas-
ured by the 3a-dehydrogenase method of Mashige et al.
(11)and plasma bile acids by a radioimmunoassay (12).
Plasma glucose concentrations were determined utilizing
glucose oxidase with a Beckman Glucose Analyzer and
Beckman Instructions 015-083513F.
RESULTS
PLASMA CONCENTRATIONS OF UNCONJUCATED
BILIRUBININ BOLIVIANAND BRAZILIAN SQUIRREL
MONKEYS
invariably have elevated plasma unconjugated bilirubin
levels following fasting, whereas Brazilian squirrel mon-
keys have minimal increases. Only 3 of 28 fasting Bra-
zilian monkeys had values greater than 0.5 mg of biliru-
bin per dl whereas 34 of 36 Bolivian monkeys had fasting
concentrations greater than 0.5 mg per dl; in 29 the level
exceeded 1.0 mg per dl. Mean fasting total plasma bili-
rubin concentrations in 18 male and 11 female Bolivian
monkeys and in 15 male and 9 female Brazilian monkeys
which were maintained on the semipurified diet with
butter (20%) for 18 months were 2.00 & 0.14,2.98 k 0.26,
0.29 k 0.045, and 0.36 f 0.073 mg per dl, respectively
(near f S.E.).
SHORT-TERM
EFFECTSOF FEEDINGA N D FASTING
Regardless of which population of squirrel monkeys
was studied or whether the diet was semipurified or a
commercial preparation, refeeding quickly lowered fast-
ing plasma bilirubin concentrations (Figure 1). The de-
crease was greatest in Bolivian monkeys with the highest
fasting values; thus, the percentage of decline was less
456 PORTMAN ET AL.
variable than the absolute decline. Fed and fasting Bra-
zilian monkeys had low plasma bilirubin levels which
also decreased within 1hr after feeding.
In three Bolivian monkeys, the plasma bilirubin levels
rose much more slowly during fasting than they declined
during refeeding following an 18-hr fast (Figure 2).
EFFECTSOF DIETARYFAT AND CARBOHYDRATE O N
PLASMA BILIRUBINCONCENTRATIONS
Bolivian squirrel monkeys which received semipurified
food had higher fasting plasma concentrations of uncon-
jugated bilirubin than did Bolivian monkeys which re-
ceived Purina Monkey Chow (Figure 1: males 1.97 & 0.15
vs. 1.08 rf: 0.23 mg per dl, p < 0.01; females 3.49 rf: 0.28
vs. 1.42 rf: 0.16 mg per dl, p < 0.001).
Fed and fasting plasma bilirubin concentrations of
Semipurified Chow
20% Fat
A--ABrazilian
-Bolivian
0 +i 6 +h
Hours After Fast
FIG. 1. The effect of fasting and refeeding on the plasma total
bilirubin concentrations in Brazilian (A-- -A)and Bolivian (W)
squirrel monkeys that consumed either a semipurified diet that con- 4 hr. The plasma glucose levels after fructose, mannose
tained 20% fat (left) or Purina Monkey Chow-25 (right). Zero time or galactose were near 60 mg per dl initially and slowly
values were for monkeys which had been fasted overnight (18 hr); +4-
hr values were for the same monkeys which were subsequently refed.
“1 t plasma bilirubin concentrations after 1and 2 hr, and an
Hours
FIG. 2. The effect of duration of fasting on the total bilirubin
concentration in plasma of three Bolivian squirrel monkeys. Zero time
was the end of a 4-hr feeding period.
HEPATOLOGY
Bolivian and Brazilian squirrel monkeys were also influ-
enced by the amount of fat in the semipurified ration
(Table 1).Fasting plasma bilirubin concentrations were
lowest at the end of the 5% fat periods and highest after
the 20% fat periods ( p < 0.001). Plasma bilirubin levels
after refeeding were lower in monkeys which received
the 0.3 or 5% fat diet than in those which consumed 20%
fat ( p < 0.01 for Bolivians; C0.001 to 0.05 for Brazilians).
SHORT-TERM EFFECTSOF INTRAGASTRIC AND
INTRAVENOUS FEEDINGO N PLASMA BILIRUBIN
CONCENTRATIONS
To determine which dietary ingredients were respon-
sible for the rapid decrease in plasma bilirubin concen-
trations during refeeding, the effect of several nutrients
given intravenously or intragastrically were tested in
Bolivian squirrel monkeys. These monkeys regularly
consumed the high-fat semipurified food and were tested
after an 18-hr fast (Table 2). An aqueous suspension of’
the complete semipurified ration when administered or-
ally or intragastrically produced a rapid decrease in
plasma bilirubin concentrations, as did the intragastric
feeding of 25 kcal of sucrose, glucose or lactalbumin
hydrolysate. Fasting plasma bilirubin levels were in-
creased after administration of butter in two monkeys
and were unchanged by water. Five milliliters of 50%
intravenous glucose lowered (p < 0.001 at 15 min, 1 and
2 hr) and 5 ml of the soybean oil emulsion, Intralipid,
resulted in an insignificant (p = 0.14) increase in the
plasma concentration. Administered glucose produced
blood glucose levels over 600 mg per dl with levels above
200 mg per dl after 4 hr; a substantial reduction in plasma
bilirubin was obtained with as little as 1 ml (0.5 g) of
50% glucose (results not shown). Intravenous fructose
was as effective as glucose. Mannose or galactose infu-
sions resulted in greater reductions than saline only after
rose to 200 mg per dl after 4 hr.
Intravenous administration of isotonic saline also re-
sulted in a small but statistically significant reduction in
increase in the plasma glucose concentrations (from 62
to 80 mg per dl). Subcutaneously administered epineph-
rine (0.5 ml of 1:10,000 solution) also increased fasting
plasma glucose concentrations at 15 min and 1 hr in
Bolivian and Brazilian squirrel monkeys (Table 3). Iso-
tonic saline administered subcutaneously was almost as
effective as epinephrine. There was no difference be-
tween fasting glucose levels in Bolivian and Brazilian
squirrel monkeys.
EFFECTSO F REPEATEDGASTRICINTUBATIONS OF
CARBOHYDRATE AND/OR FAT
Three male and three female Bolivian squirrel mon-
keys were fed for 48 hr by gastric intubations to test
long-term effects of single nutrients or mixtures. After
an 18-hr overnight fast, the monkeys received 60 kcal of
glucose or butter, or a mixture of the two, in a balanced
salt solution. This procedure was repeated after 3 hr.
The same feeding regimen was followed on the second
Vol. 4, No. 3, 1984 NUTRITION AND PLASMA BILIRUBIN IN SQUIRREL MONKEYS 457

TABLE
1. THE EFFECT OF THE BACKGROUNDDIET ON THE CONCENTRATION OF PLASMA TOTAL BILIRUBINCONCENTRATION AFTER
AN OVERNIGHT (18-HR) FASTAND 4 HR AFTER REFEEDING
Level of fat
20% 0.3% 5% 20% 5%
Fasted
Bolivian"
Male 2.12 i 0.62 1.94 f 0.42 0.54 f 0.12 1.98 f 0.33 1.35 f 0.19 (6)
Female 3.99 f 0.11 2.94 f 0.79 0.88 t 0.25 2.68 ? 0.36 1.37 f 0.32 (6)
ra lT
Male and Female 2.92 l 0 . 5 1 2.33 f 0.45
bJ1 '0'14
2.34 f 0.26
b
1.36 j?
l
0.16 (12)

Brazilian d
Male 0.35 rt 0.10 0.12 f 0.03 0.07 k 0.04 0.33 2 0.06 0.19 k 0.02 (4)
Female 0.44 f 0.22 0.38 f 0.24 0.10 f 0.06 0.34 k 0.06 0.26 f 0.13 (6)

Male and Female 0.37 1.0.08 7b f 0.15

0.27 la-
0.08 f 0.03 rd-&&
0.34 f 0.04 0.05 (10)

Fed
Bolivian"
Male 0.56 f 0.10 0.13 f 0.05 0.32 f 0.21 0.38 f 0.07 0.18 f 0.05
Female 1.32 & 0.35 0.31 f 0.20 0.14 f 0.06 0.40 f 0.12 0.13 _t 0.05
ICIC1
Male and Female 0.89 f 0.21 0.22 f 0.10 0. 3 k 0.11 0.39 f 0.07 0.17 f 0.04
-C-
Brazilian
Male 0.11 -c 0.01 0.05 k 0.02 0.07 f 0.03 0.12 f 0.02 0.04 f 0.02
Female 0.14 rt 0.02 0.04 f 0.01 0.04 f 0.02 0.12 f 0.02 0.03 f 0.02

Male and Female 0.12 40.01 d vf 0.01

0.04 Id-
0.05 f 0.02 0.12 f 0.01
I a 7 0 4 f 0.01
L a -
Values are mean f S.E. of total plasma bilirubin concentrations. a, p < 0.001; b, p < 0.005; c, p < 0.01; d, p < 0.05.
Six additional male Bolivian monkeys which were fed only 20% fat diet throughout the trial were tested during each of the five test periods
indicated above. The mean plasma bilirubin concentrations of this group during the fasting were 1.79 f 0.18, 1.52 f 0.25, 1.83 f 0.30, 2.17 f
0.28, and 2.06 f 0.26 mg f dl, respectively.

2. EFFECTS OF DIFFERENTINTRAGASTRIC OR 1.v. INFUSIONS (ADMINISTEREDAS A BOLUSWITHIN 2 MIN) ON PLASMA TOTAL

TABLE
BILIRUB~NCONCENTRATIONS I N MALE BOLIVIANSQUIRREL MONKEYSTHAT HAD BEEN PREVIOUSLY FASTED 18 HR
Fasting Plasma bilirubin concentration as
bilirubin per cent of 0 level
Treatment __
(mg/dl)
at 0' 15' 1 hr 2 hr 4 hr

HzO gastric (4) 1.62 ? 0.41 108.4 k 5.7 160.5 f 24.6

Diet oral (6) 1.44 f 0.24 58.8 f 12.7 52.3 f 13.4
Diet gastric (4) 1.32 f 0.25 46.5 f 5.9 72.8 f 9.2
Sucrose gastric (8) 2.45 f 0.38 32.6 f 6.2 39.0 f 6.7
Glucose gastric (2) 2.16 f 0.52 60.5 f 16.9 31.5 f 0.8
Lactalbumin gastric (4) 2.75 f 0.57 63.4 f 13.0 76.9 f 21.8
Butter gastric (2) 1.58 f 0.42 114.0 f 17.8 151.8 f 41.4
Saline i.v. (9) 2.06 k 0.28 89.9 f 5.7 81.5 f 2.2 92.5 rt 0.9 105.8 f 5.1
Intralipid i.v. (4) . 1.02 f 0.16" 161.5 k 32.9 191.3 f 49.2 146.9 f 30.7 106.8 rt 6.2
Glucose i.v. (11) 2.23 k 0.35 65.2 C 3.6 41.0 f 3.9 55.6 f 7.4 82.1 i 9.1
Fructose i.v. (6) 2.56 f 0.30 48.3 -C 9.6 33.6 f 2.2 47.3 i 2.7 67.7 f 6.1
Mannose i.v. (8) 2.50 f 0.49 76.4 -C 7.0 78.8 f 9.0 80.2 k 9.0 85.0 f 4.4
Galactose i.v. (6) 2.40 f 0.41 71.6 -C 3.3 73.8 f 6.6 69.8 f 8.1 69.0 f 4.3
Unconjugated only-lower layer Weber-Schalm (plasma too lipemic after Intralipid to use the method of Jendrassik-Grof).

day. As shown in Figure 3, A and B, plasma bilirubin EFFECTSOF INTRAVENOUS GLUCOSEON THE
concentrations in animals receiving only glucose or glu- REMOVAL FROM THE BLOODOF A TRACER DOSE
cose and butter were considerably less than fasting bili- OF BILIRUBIN
rubin levels. The plasma bilirubin concentrations of
monkeys receiving an emulsion of butter in a balanced After an 18-hr fast, Brazilian squirrel monkeys cleared
salt solution were not changed from fasting concentra- an intravenous tracer dose of ['Hlbilirubin more rapidly
tions. than did Bolivian squirrel monkeys (Figure 4). Intrave-
458 PORTMAN ET AL. HEPATOLOGY

3. EFFECTS
TABLE (0.5 ML 1:10,000) OR SALINE (0.5 ML) INJECTED SURCUTANEOUSLY
OF EPINEPHRINE AT 0' O N FASTINGPLASMA
BILIRUBIN A N I ) GLUCOSECONCENTRATIONS
Bilirubin (mg/dl) Glucose (mg/dl)
1 -~ ~ _ _ _ ~ ~

0' 15' 60' 0' 15' 60 '

Bolivian epinephrine 8 1.95 f 0.36 1.48 +- 0.29" 1.40 f 0.27 *

69.0 5.7 107.0 k 10.4" 110.4 f 7.6"
Bolivian saline 4 1.72 ? 0.39 1.54 f 0.37 1.44 Ifr 0.30" 63.0 k 6.9 91.0 t 10.4" 78.2 -t 9.0"
Brazilian epinephrine 8 0.271 f 0.081 0.158 f 0.042" 0.312 f 0.082 *
63.1 3.6 86.4 ? 6.4" 87.9 f 11.2"
Brazilian saline 4 0.113 f 0.016 0.087 k 0.028 0.129 f 0.037 *
64.8 4.0 82.0 f 6.7" 69.2 k 2.2
Comparisons are between 15' and O', and 60' and 0'.
'p 5 0.05 by paired analysis of differences for individual animals.

I I I=lntragastric
meal

/' '\
/' \

I"leCll0"
%line or Dexlrore

- 1
- 1 -I
I 2 3 4 5 6
HOUR5

FIG. 4. The rates of disappearance of tracer doses of ['Hlbilirubin

(0.5 ml) from the plasma of Bolivian and Brazilian squirrel monkeys
which were injected intravenously with saline and of Bolivian monkeys
that were injected with 50% glucose (5 ml). Values are mean & S.E.

__________ ---+--

n 1 , I I I I
3 6 9 12 15 18 22 &' 3b
B Hours
F I G . 3. T h e effects of repeated intragastric meals of glucose, glucose
and butter, or butter in balanced salt solution on the bilirubin concen-
tration in plasma of Bolivian male (A) or female (B) squirrel monkeys.
Monkeys were fasted for 18 hr, then bled at time 0, and given the first
intragastric meal. Each line represents a single monkey, but the changes
are consistent with those for single treatments of larger series of
monkeys (Table 2). The male monkey receiving glucose had an atypi-
cally low bilirubin concentration at time 0.
nous administration of 5 ml of 50% glucose 15 min prior
to administration of ["Hlbilirubin and 2 hr thereafter
accelerated the rate of disappearance of plasma radioac-
tivity frorn fasting Bolivian squirrel monkeys as com-
pared to monkeys receiving saline. A more rapid decrease
in radioactivity occurred at 2 hr (the time of the second
glucose injection) and further emphasized the effective-
ness of glucose in accelerating bilirubin clearance from
the circulation.
EFFECTSOF INTRAVENOUS GLUCOSEON THE
BILIARY EXCRETION OF BILIRUBIN
Since the concentration of plasma bilirubin was rap-
idly reduced in fasting Bolivian monkeys by intragastric
0 I 2 3 4
Hours
F I G . 5. The effect of 5 ml of intravenous saline (S) or glucose (D)
(two boluses of each) on biliary bilirubin excretion in two Bolivian
squirrel monkeys. Common duct cannulas were inserted 20 min prior
to time 0. Two other monkeys gave similar results.
or intravenous glucose as reflected by increased clearance
of [3H]bilirubin from the circulation, it appeared proba-
ble that intravenous glucose might also increase bilirubin
secretion into the bile. In the four monkeys used to test
this hypothesis, the rate of biliary bilirubin excretion fell
from an initial peak immediately after cannulation. They
were tested after that period. Intravenous saline had no
effect on the pattern of bilirubin excretion into bile, but
glucose caused a brief (20 min) reduction in bile flow and
bilirubin excretion followed by increased bilirubin excre-
tion (Figure 5). Monkeys receiving two injections of
glucose had a double peak of bilirubin excretion into bile.
The interval between the two peaks of bilirubin excretion
VOI. 4, NO. 3 , 1984 NUTRITION AND PLASMA BILIRUBIN IN SQUIRREL MONKEYS 459

related to that interval between the two glucose injec- DISCUSSION

tions. Variations in caloric intake are associated with day-
In Figure 6, the patterns of bilirubin and bile acid to-day fluctuations in the plasma concentration of un-
excretion into bile for a Bolivian squirrel monkey which conjugated bilirubin (13). Fasting produces mild eleva-
received continuous saline or glucose infusions (2 ml/hr) tions in plasma unconjugated bilirubin levels in normal
with and without sodium taurocholate (2%) is presented. individuals and in patients with hemolytic disease (14,
Infusion of 50% glucose without sodium taurocholate 15).An exaggerated increase in plasma bilirubin concen-
caused a higher rate of bilirubin excretion than did saline trations is observed after fasting in certain animals and
or 5% glucose infusions and decreased bile acid excretion. humans with recognized familial syndromes with uncon-
Addition of sodium taurocholate was associated with jugated hyperbilirubinemia. These syndromes include:
high bilirubin and bile acid excretion regardless of mutant Gunn rats (16); Crigler-Najjar Type I1 (17, 18)
whether saline, 5% glucose or 50% glucose were used. and Gilbert’s syndromes in man (19); and mutant South-
When the 50% glucose plus sodium taurocholate infusion down sheep (20, 21). Reduced caloric intake has been
was discontinued and a single intravenous injection of 2 used diagnostically in Gilbert’s syndrome when plasma
ml of 50% glucose was administered, the biliary excretion unconjugated bilirubin levels are in the normal range (3,
of bile acids dropped precipitously, but the excretion rate 22, 23). Horses, unlike other species studied to date,
of bilirubin remained elevated. A second monkey (results markedly increase plasma unconjugated bilirubin levels
not shown) was infused with saline followed by saline after fasting (24).
plus taurocholate. This monkey responded to taurocho- There is a consistent appearance in fasting Bolivian
late with an immediate rise in the biliary excretion of squirrel monkeys of unconjugated bilirubin concentra-
bilirubin and a delayed rise in bile acid excretion. tions, ranging from 1 to 5 mg per dl, which is unusual
for other nonhuman primates and does not occur in the
BILE ACID (:ONCENTRATIONS IN PERIPHERAL
closely related population of Brazilian squirrel monkeys.
BLOOD As a model of Gilbert’s syndrome, Bolivian monkeys
Intragastric administration of a complete diet or butter appear superior to other animal models due to their close
increased bile acid concentrations of fasted Bolivian similarity to man. Heterozygous Gunn rats also have
squirrel monkeys a t 1 and 4 hr (Table 4). Intragastric reduced hepatic glucuronyl transferase activity for bili-
sucrose decreased plasma bile acid concentrations at 1 rubin but have normal plasma bilirubin levels (25).
hr. Intravenous glucose caused no change in bile acid Southdown sheep with unconjugated hyperbilirubinemia
concentrations after 5 min but a decrease to 12% of the do not have lowered hepatic glucuronyl transferase activ-
initial value a t 1 hr. Similarly, a load (12 mg per kg) of ity for bilirubin (26).
bilirubin intravenously did not immediately alter the Whitmer and Gollan (4) and Okolicsanyi et al. (27)
plasma bile acid concentration, but after 1 hr, the mean have recently reviewed the factors and possible mecha-
concentration was only 34% of preinjection values. nisms involved in the plasma bilirubin response to fast-
ing in humans with Gilbert’s syndrome. Gollan et al. (23)
observed that 2,400 kcal as intravenous glucose produced
hyperbilirubinemia a t least as severe as that induced by
-0 dietary restriction but that 20% Intralipid administered
m intravenously or ingestion of 2500 kcal with 9 or 29%
cu from fat returned plasma bilirubin concentrations to
u)
0
0 minimum baseline levels. In contrast, Felsher (28), how-
-a ever, found that diets with adequate calories but low in
cu 0
-C
.- carbohydrate, fat (3.7% of calories) or protein were
Q
0 In equally effective in reducing the fasting (prebreakfast)
level of plasma unconjugated bilirubin below that which
700t--l-I-l-I- 1-1 I I occurred with a low calorie diet in Gilbert patients.
Whitmer and Gollan (4) found that dietary fat must be
reduced to an extremely low level (perhaps to 0.6% of
the total caloric intake) to produced hyperbilirubinemia
in Gilbert’s subjects. Lipid-free diets caused unusually
high plasma bilirubin concentrations in Gunn rats which
are reduced by small lipid supplements (29). In contrast
to those observations in humans and rats, fasting plasma
bilirubin concentrations in Bolivian squirrel monkeys
fed 5% fat (11%of calories) were lower than those fed
very low- (0.3%) or high-fat (20%) diets. The large and
rapid decreases in the plasma bilirubin concentrations of
Bolivian squirrel monkeys which receive carbohydrate
Hours with or without fat, but not fat alone also differs from
Gollan’s findings in Gilbert patients (4, 23).
FIG. 6. The effect of continuous intravenous infusion of different Glucose, administered intravenously, was as active as
solutions (2 ml per hr) on biliary bilirubin and bile acid excretion in a
Bolivian squirrel monkey. Common duct cannulas were inserted 20 fructose and more active than galactose or mannose in
min prior to time 0. Similar results were observed in another monkey. reducing the unconjugated hyperbilirubinemia of fasting
460 PORTMAN E T AL. HEPATOLOGY

TABLE
4. RELATIONSHIPS
BETWEENPLASMA BILE ACIDS A N D BILIRUBININ BOLIVIANSQUIRREL MONKEYS“

Plasma bile acids Plasma bilirubin

_____
Treatment Zero time = 100 Zero time = 100
pM ___ mg/d - ___.____
1’ 5’ 20’ 60’ 4hr 1‘ 5’ 20’ 60’ 4 hr

Complete diet intragastric (a = 2) 11.3 - - - 129 269 1.11 - - - 53 70

Sucrose iiitragastric (2) 26.0 - - - 55 108 1.60 - - - 28 44
Butter intragastric (2) 9.4 - - - 175 334 1.58 - - - 114 152
Glucose i.v. ( 3 ) 10.6 - 107 - 12 - 2.43 - 66 - 56 -
Bilirubin i.v. (12/mg/kg) (2) 10.0 111 - 85 34 - 0.93 2,940 - 2,150 1,840 -
‘The mean plasma concentrations of bile salts and total bilirubin are given for each group of monkeys prior to the intragastric administration
of a complete diet or sucrose or butter or the i.v. administration of glucose or bilirubin. The concentrations of bile salts and bilirubin at various
intervals after treatment are given relative to 100 for pretreatment concentrations.

Bolivian squirrel monkeys. The findings that excitement
and probable endogenous epinephrine release, as well as
exogenous epinephrine, produce a mild hyperglycemia
and reduce plasma bilirubin concentrations in fasting
monkeys further support the hypothesis that glucose,
and not lipid, is crucially involved in the control of
plasma bilirubin levels in Bolivian squirrel monkeys.
There was no difference between plasma glucose con-
centrations in Brazilian (normal) and Bolivian (Gilbert-
like) populations of squirrel monkeys during fasting or
after injection of glucose. Bolivian squirrel monkey liver
has lower levels of UDP-glucuronyl transferase activity
for bilirubin than does Brazilian squirrel monkey liver
(1); however, exogenous carbohydrate caused a decline
in fasting plasma bilirubin concentrations of both pop-
ulations. Although the underlying mechanism is uncer-
tain, glucose could affect hepatic bilirubin metabolism
by increasing the formation of UDP-glucuronate. During
the hour after intravenous glucose administration, bili-
rubin clearance from plasma increased (Figure 4) and
excretion into bile increased within 20 min (Figures 5
and 6). This must be associated with increased bilirubin
conjugation, but most likely is too rapid to result from
increases in hepatic concentrations of glucuronate acti-
vating or transferase enzymes. Perhaps, fasting humans
whose plasma bilirubin concentrations reportedly do not
respond to glucose (4)have higher rates of UDP-glucu-
ronate formation than do Bolivian squirrel monkeys. The
remarkable difference between the two populations of
squirrel monkeys in fasting hyperbilirubinemia, and the
distinct effects of dietary carbohydrate on plasma bili-
rubin levels and biliary excretion provide important areas
for further investigation on bilirubin metabolism.
Acknowledgments: We thank the Surgical Group at the
Oregon Center for some preparations.
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