Influence of Dietary Calcium Phosphate on the Disposition
of Bilirubin in Rats With Unconjugated Hyperbilirubinemia
CHRISTA N. VAN DER VEERE,1 BERRY SCHOEMAKER,1 CONNY BAKKER,1 ROELOF
1
AND RONALD P. J. OUDE ELFERINK
The aim of this study was to test a possible form of
therapy that could be used in the management of uncon-
jugated hyperbilirubinemia. We hypothesized that un-
conjugated bilirubin (UCB) can permeate the intestinal
wall and can thus be secreted with the feces. We have
previously observed that UCB binds to amorphous cal-
cium phosphate in vitro. Orally ingested amorphous cal-
cium phosphate may act as a trapping agent for bilirubin
in the intestine, thereby preventing back-diffusion
across the intestinal wall. In this study, we tested
whether feeding calcium phosphate leads to enhanced
excretion of unconjugated bilirubin in Gunn rats. When
a purified control diet was substituted by a high calcium
phosphate diet, a decrease in bilirubin levels of 30% to
50% in male Gunn rats and of 23% in female rats was
observed. The fecal output of bilirubin was more than
doubled in Gunn rats in the first 3 days after the normal
diet had been replaced by the high calcium-phosphate
diet. The biological half-life of 3H-labeled bilirubin in
blood was 89.8 { 17.2 hours in rats fed the purified con-
trol diet and 50.9 { 1.4 hours in rats fed the high calcium
phosphate diet (P Å .004). After 30 weeks, plasma biliru-
bin levels were still significantly lower in Gunn rats fed
a high calcium phosphate diet. No differences were
found in plasma concentrations of calcium, magnesium,
phosphate, urea, and creatinine in both Gunn rats and
Wistar rats on control or high calcium phosphate diets.
This therapy might be useful in the management of
Crigler-Najjar patients, for example, as an adjunct to
phototherapy. (HEPATOLOGY 1996;24:620-626.)
The Crigler-Najjar syndrome is an inherited disease in
which unconjugated bilirubin, the major breakdown product
of hemoglobin, cannot be excreted by the body in the normal
way and accumulates in blood and tissues.1,2 Normally, conju-
gation of bilirubin with glucuronic acid in the liver leads
to exposure of negative charges, which makes excretion of
bilirubin in bile possible. In Crigler-Najjar patients, the en-
zyme needed for this reaction, bilirubin–uridine diphos-
phate–glucuronosyltransferase, is deficient. Two types of
this disease are distinguished2: type I patients are the most
severely affected because the enzyme activity is absent, and
Abbreviations: UCB, unconjugated bilirubin; HPLC, high-pressure liquid chromatogra-
phy.
From the 1Department of Gastrointestinal and Liver Diseases, Academic Medical Center,
Amsterdam, the Netherlands, 2Department of Nutrition, Netherlands Institute for Dairy
Research, Ede, and 3Department of Hepatology and Gastroenterology, University Hospital,
Groningen, the Netherlands.
Received July 18, 1995; accepted April 22, 1996.
Supported by the Dutch Foundation for Children’s Liver Diseases, ‘‘Het Najjar Fonds.’’
Address reprint requests to: R.P.J. Oude Elferink, Ph.D., Department of Gastrointestinal
Diseases, Academic Medical Center, F0-116, Meibergdreef 9, 1105 AZ Amsterdam, the
Netherlands.
Copyright q 1996 by the American Association for the Study of Liver Diseases.
0270-9139/96/2403-0026$3.00/0
620
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VAN DER MEER,2 PETER L. M. JANSEN,3
type II patients have a less severe form of the disease because
of residual enzyme activity. Genetic analysis of a number of
Crigler-Najjar patients3 has revealed that type I patients
have mutations in both alleles of the UGT1 gene that lead
to complete inactivation or absence of the enzyme, whereas,
in type II patients, the mutations in the gene may be such
that the enzyme has low residual activity (õ5%). Accumula-
tion of unconjugated bilirubin (UCB) is potentially toxic and,
if not treated, can lead to mental retardation and death.4-6
Besides liver transplantation, the only therapy known so far
is phototherapy. Light in the blue range (wavelength range,
400-500 nm) induces formation of photoproducts of bilirubin.
These photoproducts can be excreted in bile.7-9 Type II pa-
tients can be treated with phenobarbital, an agent that is
thought to induce the residual activity of bilirubin–uridine
diphosphate–glucuronosyltransferase. Both therapies have
disadvantages, and phototherapy becomes less effective with
age.10-13 Therefore, the goal of our research was to find possi-
ble new therapies for this disease. Gunn rats have the same
enzyme deficiency as Crigler-Najjar patients and were used
as a model for the disease.
In Gunn rats and in Crigler-Najjar patients, the plasma
bilirubin level is constant. This indicates that an equilibrium
exists between bilirubin production and excretion. The biliru-
bin production rate in Crigler-Najjar patients is equal to that
in normal healthy individuals.14 Thus, the amount of biliru-
bin and bilirubin breakdown products excreted per day will
be the same, despite the fact that the bilirubin pool is much
larger and turnover of bilirubin is much slower in Gunn rats
and Crigler-Najjar patients. Consequently, alternative meta-
bolic pathways for bilirubin must exist in patients with this
syndrome. Two major hypotheses are postulated that may
not be mutually exclusive. First, unconjugated bilirubin is
thought to be oxidized by microsomal mixed-function mono-
oxygenases in the liver, probably cytochrome P450 IA1. The
polar breakdown products are subsequently excreted in bile.
Stimulation of this putative pathway is possible by intraperi-
toneal administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin
in Gunn rats.15 Unfortunately, this drug is carcinogenic and
cannot be used as a therapy for Crigler-Najjar disease. Until
now, no drug has been found that is equally effective but less
toxic. Second, UCB is thought to diffuse from the blood across
the intestinal wall into the intestinal lumen and is excreted
via the feces.14,16 This pathway is not very efficient. From
studies in rats,17 neonates,18 and humans19-21 it is known that
UCB can be reabsorbed from the intestinal lumen. We inves-
tigated the possibility to capture intestinal bilirubin and
thereby make this pathway more efficient so that it can be
used for a possible therapy. It has been shown that plasma
bilirubin levels in Gunn rats22,23 and newborns24 can be re-
duced by oral administration of activated charcoal. Binding
of bilirubin to activated charcoal inhibits reabsorption of bili-
rubin from the gut. However, activated charcoal is not suit-
able for prolonged use because it will bind a broad range of
organic and inorganic compounds, and the daily ingestion of
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HEPATOLOGY Vol. 24, No. 3, 1996
charcoal may alter the availability of drugs and essential
nutrients. In the past, other matrices, like cholestyramine25
and agar,26-28 have been used to lower bilirubin levels in con-
ditions of unconjugated hyperbilirubinemia, but conflicting
conclusions regarding the effectivity of these therapies were
obtained.29
We looked for a compound that is able to bind bilirubin
more specifically and with high affinity, and we described
the association of UCB with amorphous calcium phosphate
in vitro.35 Amorphous calcium phosphate was therefore tested
as a potential therapeutic agent in Gunn rats. If UCB diffu-
sion across the intestinal wall is a relevant mechanism, and
if amorphous calcium phosphate also binds bilirubin in the
intestinal milieu, we expect that calcium phosphate adminis-
tration will lead to a shift of serum bilirubin from the plasma
to the gut. This will induce a transient increase in fecal biliru-
bin output until a new steady state has been reached with a
reduced serum and tissue bilirubin pool. In this situation, we
expect a constant reduction in serum bilirubin level and an
increased turnover of the plasma bilirubin pool. In the pres-
ent study, we report evidence that confirms this hypothesis.
MATERIALS AND METHODS
Materials
Chemicals. The Enzabile kit for analysis of bile acids in serum
was purchased from Sigma Chemical Co. (St. Louis, MO). 3H-Labeled
d-aminolevulinic acid hydrochloride (d-[3,5-3H(N)], 250 mCi, S.A. 1-5
Ci/mmol) was from New England Nuclear Research Products (Du
Pont, CA). Di-N-octylamine was from Aldrich Chemie (Borem, Bel-
gium). Other chemicals were of analytical grade.
Preparation of 3H-Labeled UCB. 3H-Labeled UCB was prepared by
a modification of the procedure described by Ostrow et al.30 as follows
(method kindly provided by J.R. Chowdhury, Albert Einstein College,
NY). The common bile duct of a normal Wistar rat was cannulated
under pentobarbital anesthesia. d-Aminolevulinic acid (250 mCi) was
injected via the penile vein. Bile was collected on ice in 1-hour ali-
quots for 4 to 6 hours. An aliquot of sodium ascorbate was present
in the tubes. NaOH was added to a final concentration of 0.1 mol/L,
and the tubes were kept at room temperature in the dark for 90
minutes. Subsequently, bile was neutralized by adding an equal vol-
ume of 0.1 mol/L HCl and buffered by the addition of HEPES buffer
with a pH of 7.4 to a final concentration of 1 mol/L. Immediately
afterward, the bile was extracted in two volumes of chloroform. The
volume of chloroform was reduced by flushing nitrogen and the resi-
due applied to a Baker silica gel cartridge (J. T. Baker, Inc., Phil-
lipsburg, NJ), which was then eluted with pure chloroform. The yel-
low pigment was collected and the chloroform evaporated. The
bilirubin was purified by thin-layer chromatography (solid phase,
silica gel; fluid phase, chloroform/acetic acid/ethanol 98/1/1). Specific
activity was 130,000 to 153,000 disintegrations per minute per micro-
gram of UCB as determined by using an extinction coefficient of
bilirubin in chloroform of 6.104 mol/L cm01 at 450 nm. The purified
3
H-labeled bilirubin was analyzed by high-pressure liquid chroma-
tography (HPLC) as described. Fractions of 0.5 mL were collected
and counted. Ninety-five percent of the radioactivity coeluted with
the UCB peak. Labeled bilirubin was stored under argon at 0207C
and used within 3 days after preparation.
Analytical Methods
Plasma. Bilirubin, aspartate transaminase activity, alanine
transaminase activity, alkaline phosphatase, calcium, chloride, cre-
atinine, g-glutamyl transferase, glucose, lactate dehydrogenase,
urea, and phosphate in plasma were determined with routine clinico-
chemical tests on a Hitachi 747 analyzer (Tokyo, Japan). For biliru-
bin measurement, blood was protected from light and processed im-
mediately. Magnesium in plasma was determined using flame
spectrophotometry. Bile salts in plasma were measured with the
Enzabile kit using 3a-hydroxysteroid dehydrogenase. 3H was
counted by addition of 100 to 500 mL plasma to 10 mL scintillation
liquid. After mixing, the tubes were counted for 5 minutes in a liquid
scintillation counter.
Feces. Unconjugated 3H-labeled bilirubin was extracted from feces
as follows: feces collected during 24 or 48 hours were homogenized
in a blender. The whole procedure was performed in dim light. Ap-
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VAN DER VEERE ET AL. 621
TABLE 1. Content of the Major Ingredients of the Purified Diets
Used in the Calcium Phosphate Study
High Ca High Ca
Control Low Ca (P; CO20
3 ) (P; Cl0)
Diets (mmol/g) (mmol/g) (mmol/g) (mmol/g)
Ca 130.8 25.0 500.4 499.8
P 128.8 125.0 317.4 317.4
Mg 21.1 21.1 21.1 21.1
CO3 100.0 – 283.0 100.0
Cl 47.0 51.3 51.3 316.6
Na 93.0 51.3 51.3 51.3
K 98.5 100.0 100.0 100.7
Cellulose wt/wt 10.5% 11.6% 5.1% 5.0%
proximately 150 mg feces was extracted by addition of 10 mL metha-
nol, 6 mL chloroform, and 12 mL 0.4 mol/L glycine-HCl buffer (pH
2.4). The tubes were shaken for 10 minutes to extract bilirubin and
subsequently centrifuged for 5 minutes at 2,500g. For HPLC analysis
of bilirubin, 4 mL of the chloroform layer was collected and evapo-
rated under nitrogen at room temperature. The tubes were stored
under argon at 0207C in the dark until analysis was performed,
which was always within 1 week. For counting of label in feces, 50
mL of the chloroform extract and (separately) 500 mL of the water
phase was added to 10 mL of scintillation liquid and counted. For
evaluation of recovery, approximately 100 mg of the homogenized
feces was mixed directly with 10 mL scintillation liquid and counted.
Bilirubin in feces was analyzed by HPLC, by modification31 (kindly
provided by A. F. McDonagh, San Francisco, CA). The bilirubin resi-
due was dissolved in 400 mL dimethyl sulfoxide, and 20 mL was
injected onto a C-18 reversed-phase column (Chrompack, Middel-
burg, the Netherlands). For quantitation, 100-mL bilirubin standard
solutions of 5, 10, 20, and 50 mmol/L were subjected to the same
extraction procedure and applied to the column. The column was
eluted at a rate of 0.7 mL/min with 25% eluens A (24.15 g/L di-N-
octylamine and 6.005 mL/L glacial acetic acid in methanol, pH 8.5)
and 75% eluens B (100 mL bidest, 3.475 mL di-N-octylamine and
eluens A to a final volume of 500 mL, pH 9.2). Two pumps (Spec-
troflow 400; Kratos, Ramsey, NJ) were computer-controlled with
Gynkosoft chromatography data system version 5.21c (Softron and
Gynkotek Gmbh, Germering, Germany). In this system, UCB elutes
at a retention time of 6 minutes. The absorbance of the eluted pig-
ments was monitored at 450 nm with a 1000S Diode Array detector
(Applied Biosystems; Ramsey, NJ), and the area under the peak was
electronically integrated.
Animals. Normal male Wistar rats, weighing 250 to 300 g, were
obtained from Harlan-CPB (Zeist, the Netherlands). Homozygous
Gunn rats (RHA/jj) were obtained from our own breeding colony. In
adult rats, serum bilirubin levels are about 180 to 200 mmol/L in
males and 130 to 150 mmol/L in females. Both male rats, weighing
235 to 365 g, and female rats, weighing 150 to 300 g, were used for
the present work. All animals were fed ad libitum and had free access
to water. Two weeks before the start of the experiments, they were
housed individually, except in long-term feeding experiments where
the animals were caged per group (n Å 5). Offspring from various
couples were randomly distributed between the different experimen-
tal groups.
Diets. All diets were produced by Hope Farms BV, Woerden, the
Netherlands. The standard diet was an undefined lab chow meal
(AM-II). Four chemically defined diets were produced that contained
either 25, 130 (normal; control), or 500 mmol/g calcium (see Table 1
for contents of the diets). Two diets with a high calcium content were
prepared, one containing CO20 0
3 and the other Cl in addition to phos-
phate. This was performed to exclude a possible influence of pH in
the intestinal lumen on bilirubin handling. Because free phosphate
was found to inhibit binding of bilirubin,35 and amorphous calcium
phosphate has a stoichiometry of Ca:P Å 3:2, the same ratio of cal-
cium to phosphate content was used in the diets. The content of K/
and Mg2/ was equal in all diets. The reduction in Ca2/ and Pi in the
low-calcium diet was compensated by cellulose, because we have
observed that feeding cellulose has no effect on serum bilirubin levels
in Gunn rats (unpublished data). Otherwise, all diets were identical,
containing 20% casein, and were prepared according to the American
Institute of Nutrition standards.32,33 All diets were pelleted.
Influence of Different Diets. The effect of calcium phosphate was
hpta WBS: Hepatology
622 VAN DER VEERE ET AL.
evaluated by comparing the purified control diet with one low and
two high calcium phosphate diets. Gunn rats of both sexes were used.
Once a week, body weight was assessed, and blood was sampled by
puncture of the tail vein. Forty-eight-hour intake was evaluated four
times. After 8 weeks, the animals were killed. In a separate experi-
ment, four male Gunn rats received the purified control diet for 1
week, whereas another four rats received the high calcium phosphate
diet. After 1 week, the first group was changed to the high calcium
phosphate diet. From this moment on, daily plasma bilirubin, food
intake, and fecal output were determined for 1 week in both groups.
Additionally, a short experiment was performed using a tracer of 3H-
labeled bilirubin. Male Gunn rats were fed either the high or normal
(control) calcium phosphate diet for 2 weeks (four rats per group).
After 2 weeks, a tracer (approximately 17 kiloBecquerel(kBq)) 3H-
UCB was injected intravenously via the penile vein. Blood samples
were taken at regular intervals by puncture of the tail vein. Twenty-
four-hour intake was measured daily. Feces were collected each 24
hours (first 3 days) or 48 hours (fourth to seventh day). Blood and
feces were analyzed for UCB and label. One week after injection of
the labeled bilirubin, the animals were killed.
To assess the long-term effect of the high calcium phospate diet,
male Gunn and normal Wistar rats received either the purified con-
trol diet or the high calcium phosphate diet for 30 weeks (5 rats per
group). Body weight was assessed and blood samples were taken at
0, 2, 4, 10, 18, and 30 weeks. Forty-eight-hour intake was measured
three times in weeks 3, 11, and 25. At the end of the experiment,
the liver, one kidney, and part of the aorta (including the bifurcatio
to the aa. femorales) were removed for histology.
Bile Salt Depletion. Six male Gunn rats were equipped with a
bile catheter as well as a duodenum catheter. Both catheters were
tunneled under the skin to the head, where they were fixed and
connected by a polyethylene loop. In this way, the enterohepatic
circulation is reestablished.34 After 14 days, when they had recovered
from the surgery and plasma bilirubin levels, aspartate transami-
nase, alanine transaminase, alkaline phosphatase, g-glutamyl trans-
ferase, and lactate dehydrogenase were normalized, and in three
rats, the bile was diverted by connecting the bile cannula to a long
polyethylene tube with insertion of a swivel joint, which allowed the
rats to move freely in their cages.34 The other three rats were used
as a control. Sixteen hours after the bile was diverted, all rats were
intravenously injected with 3H-labeled UCB. Turnover was mea-
sured by repeated analysis of bilirubin, and label in blood and feces
was collected daily.
Statistical Analysis
Results are given as mean { SD. Statistical analysis was per-
formed using the Student’s t test. A level of 0.05, two-tailed, was
used for significance.
RESULTS
When Gunn rats were fed one of the high calcium diets,
bilirubin levels decreased by 30% to 50% in male Gunn rats
(Fig. 1A) and 23% in female rats (Fig. 1B) as compared with
the purified control diet. A significant difference in bilirubin
levels was found between the two high-calcium diets ( P õ
.01). The diet with Cl0 as the additional anion yielded the
best result. A small initial rise in bilirubin levels was present,
especially in female rats, when the animals were changed
from the normal undefined lab chow to the purified control
diet. A low-calcium diet did not influence bilirubin levels sig-
nificantly compared with purified control diet. Plasma cal-
cium, phosphate, and chloride did not differ between groups
during the experimental period, but plasma magnesium de-
clined when rats were fed a high-calcium diet (Fig. 1C; P õ
.01). Forty-eight-hour food intake was comparable between
the three different male groups and two different female
groups, and body weight was constant in the experimental
period.
In addition, a cross-over study was performed, showing
similar differences in plasma bilirubin levels between rats
on high- and low-calcium phosphate diets. Bilirubin levels
reversed within 1 week after changing of the diets (results
not shown).
Thus, plasma bilirubin levels of male as well as female
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HEPATOLOGY September 1996
Gunn rats can be influenced by the calcium phosphate con-
tent of their diet. This may result from binding of bilirubin
to calcium phosphate in the gut, thereby preventing the reab-
sorption of UCB. If this theory is correct, the initial fecal
output of bilirubin should be transiently increased upon
changing to a high calcium phosphate diet. When a new
steady state is reached, output will return to normal values
again, but the turnover of bilirubin will be higher in these
rats because of the fact that the size of the plasma pool has
diminished. In Fig. 2, it is demonstrated that output of biliru-
bin in feces was more than doubled in Gunn rats in the first
3 days after the purified control diet was replaced by the high
calcium phosphate diet. Plasma bilirubin levels decreased
concomitantly from 261 { 8 to 155 { 20 mmol/L (41%) in 1
week.
Quantitative measurement of bilirubin in feces is difficult.
Recovery of bilirubin in feces is only approximately 50%. Bili-
rubin is metabolized in the gut to urobilinogen and probably
dipyrroles and monopyrroles, which are difficult to measure.
Therefore, we decided to perform an additional experiment
in which 3H-labeled UCB was used. Four male Gunn rats
were fed the purified control diet for 2 weeks. In this period,
a steady state is reached with high plasma bilirubin levels
(288 { 26 mmol/L). Another set of four rats was fed the high
calcium phosphate diet for 2 weeks. They showed reduced
plasma bilirubin levels (157 { 14 mmol/L). After 2 weeks, a
tracer amount (approximately 17 kBq) of purified 3H-UCB
was injected intravenously. After an initial phase of equili-
bration, the decrease of label in plasma bilirubin followed
first-order kinetics, which is in accordance with previous re-
ports.14 Half-life of label in blood (during the second phase)
was 89.8 { 17.2 hours in rats fed the purified control diet
and 50.9 { 1.4 hours in rats fed the high calcium phosphate
diet (Fig. 3A; P Å .004). Total recovery of label in feces after
1 week was significantly higher in rats fed the high calcium
phosphate diet, and the majority of the label was excreted in
the first 3 days, whereas in the group fed the purified control
diet the label was excreted later (Fig. 3B). Both groups ex-
creted significant label in the feces passed during the first
day.
In the group fed the high calcium phosphate diet, more
label was recovered in the apolar phase compared with the
group fed the purified control diet (62.0 { 5.3% and 48.9 {
3.1%, respectively; P Å .005) when feces were extracted with
chloroform, methanol, and glycine–hydrochloric acid buffer.
Body weight was similar and constant in both groups.
Bile Depletion. It has been postulated that reabsorption of
UCB in the intestine is dependent on bile salts.36 Bile salts
keep bilirubin solubilized, and this will influence the extent
of reabsorption. On the other hand, some bile salts, especially
glycine-conjugated and unconjugated ones, are precipitated
by calcium phosphate in vitro and in vivo.37-40 Therefore, the
effect of a high dietary intake of calcium phosphate on plasma
bilirubin levels in Gunn rats could partly be caused by de-
creased reabsorption of bilirubin because of a decreased bile
salt concentration. To examine the significance of this effect
for our studies, Gunn rats were equipped with a permanent
bile fistula.34 Turnover of bilirubin in these rats was com-
pared with turnover in sham-operated rats41 with an intact
enterohepatic circulation. In previous experiments, we no-
ticed that the bilirubin level of Gunn rats increases signifi-
cantly after a major operation, and it takes about 2 weeks
before they reach their initial bilirubin levels again (unpub-
lished results). Therefore, Gunn rats (n Å 6) received a bile
catheter connected to a duodenum catheter,41 and thus had
an intact enterohepatic circulation. After 14 days, when they
had recovered from the operation and plasma bilirubin levels,
aspartate transaminase, alanine transaminase, alkaline
phosphatase, g-glutamyl transferase, and lactate dehydroge-
nase were normalized to preoperative values, the bile was
hpta WBS: Hepatology
HEPATOLOGY Vol. 24, No. 3, 1996
diverted in three rats. The other three rats were used as
controls. Sixteen hours after the bile was diverted, all rats
received a tracer amount of 3H-labeled UCB intravenously.
Turnover of label was measured by repeated analysis of bili-
rubin, and label in blood and feces was collected daily. No
difference was found in plasma half-life of 3H-bilirubin be-
tween both groups: 32.3 { 1.3 hours in bile-diverted rats and
33.0 { 1.7 hours in sham-operated rats (Fig. 4). In this figure,
the initial specific activity was normalized to 100 because
different amounts of label were injected. The results indicate
that the effect of oral calcium phosphate on bilirubin levels
in Gunn rats is not caused by a decreased amount or an
altered composition of bile salts in the small intestine. In
addition, we observed no change in serum bilirubin upon bile
diversion in rats on control diet during the entire experimen-
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VAN DER VEERE ET AL. 623
FIG. 1. Influence of oral calcium phosphate on plasma bilirubin and mag-
nesium in male and female Gunn rats. All rats were fed a normal lab chow
for 2 weeks, followed by a purified control diet for 2 weeks, and subsequently
either a purified low calcium phosphate diet (circles) or a purified high calcium
phosphate diet (squares, carbonate as additional anion; triangles, chloride as
additional anion). During the last 2 weeks of the experiment, the animals
received the purified control diet again. (A) Plasma bilirubin in male Gunn
rats. (B) Plasma bilirubin in female Gunn rats. (C) Plasma magnesium in male
Gunn rats. Data represent mean { SD from six animals in each group. *P õ
.05, **P õ .01 as compared with low-calcium diet.
tal period. This indicates that UCB secretion into bile is insuf-
ficient to contribute to overall disposal of UCB. This is in
agreement with previously reported data.14
Long-Term Effects of the High Calcium Phosphate Diet. Fi-
nally, a long-term experiment was performed to determine
the effect on plasma bilirubin and to detect possible adverse
effects of long-term, high calcium phosphate intake. Male
Gunn rats as well as Wistar rats were used because it is
known that Gunn rats have nephropathy, probably secondary
to the high bilirubin levels.42 Therefore, Gunn rats could be
expected to have impaired renal handling of calcium and
phosphate, whereas, in Wistar rats, renal function is normal.
Rather old animals were used (initial body weight, Wistar:
416 { 22 g, Gunn: 274 { 13 g) to increase the sensitivity
for possible tissue calcifications. Body weight in all groups
hpta WBS: Hepatology
624 VAN DER VEERE ET AL. HEPATOLOGY September 1996

bin levels in Gunn rats22 and neonates,24 but, unfortunately,

this agent is not suitable for prolonged use because it is a
nonspecific binder and it causes obstipation.
This study reports a significant effect of oral administra-
tion of calcium phosphate on serum bilirubin levels in the
Gunn rat. The dose of calcium needed to achieve a decrease
of approximately 40% is only four times the normal ingested
amount of calcium. In vitro, the binding capacity of activated
charcoal and amorphous calcium phosphate for bilirubin are
comparable,35 but the effect of amorphous calcium phosphate
is much more specific because it only binds amphipatic
anions. On the basis of our hypothesis, we expected that ad-
ministration of a high calcium phosphate diet would lead to
a shift of UCB from the plasma to the intestinal pool. As a
consequence, there will be a transient increase in fecal UCB

FIG. 2. Plasma bilirubin and 24-hour fecal output of bilirubin in male

Gunn rats on control or high calcium phosphate diets. Data represent mean
{ SD from four animals in each group. *P õ .05, **P õ .01. Open triangles:
plasma bilirubin in rats changed from control to high calcium phosphate diet
on day 0. Open bars: fecal bilirubin output of rats changed from control to
high calcium phosphate diet. Solid triangles, plasma bilirubin in rats 1 week
after changing from normal to high calcium phosphate diet (in steady state).
Filled bars, fecal bilirubin output of rats 1 week after changing from normal
to high calcium phosphate diet. Fecal bilirubin was measured by HPLC as
described in Materials and Methods.

increased, and there was no difference between groups fed

different diets. Similar to the short-term experiments, an
initial increase in bilirubin level was seen in both Gunn rat
groups when their normal lab chow was replaced by a purified
diet. Figure 5 shows that the difference in plasma bilirubin
was maintained for a very long period, although, at 30 weeks,
plasma bilirubin levels in rats on the purified control diet
tended to decrease somewhat ( P Å .03). During the whole
period, plasma calcium, phosphate, magnesium, urea, creati-
nine levels, and 48-hour-intake were similar between
matched groups.
A modified Von Kossa reaction43 using silver lactate was
used for demonstration of possible calcium deposits in tis-
sues. Successive cryocoupes were stained with a conventional
hematoxyline-phloxine method. Liver and aorta were nega-
tive when stained with silver, but some kidney sections were
positive. In Wistar rats, a few small dots were present in rats
fed either diet. In some Gunn rats, large calcium deposits
were present, but only in rats having severe kidney lesions
and fed the high calcium phosphate diet (not shown).

DISCUSSION
The aim of the studies described in this study was to test
a possible form of therapy that could be used for conditions
of unconjugated hyperbilirubinemia. The fact that Crigler-
Najjar patients have a constant plasma bilirubin level indi-
cates that they are capable of secreting UCB or its breakdown
products, albeit at the cost of a severely elevated plasma
bilirubin level. It is our hypothesis that a substantial amount
of UCB permeates the intestinal wall and can thereby be
secreted with the feces. This pathway is inefficient because
it depends on a high plasma concentration, and because it FIG. 3. Turnover and recovery of 3H-labeled bilirubin in male Gunn rats
has been shown that reabsorption of UCB can occur.17-19 To on control or high calcium phosphate diets. Data represent mean { SD from
four animals in each group. **P õ .01. Two weeks after feeding either the
overcome the latter problem, we have tried to trap bilirubin control diet or a high calcium phosphate diet, male Gunn rats were injected
in the gut lumen by binding to a matrix. This approach is intravenously with 17 kBq 3H-labeled bilirubin. Radioactivity in plasma was
not new; in the past, different nonabsorbable matrices have determined at the indicated time points after injection, and label was measured
been administered to decrease the absorption of bilirubin in 24-hour fecal output. (A) Turnover of label in plasma. Circles, rats in steady
state on purified control diet; Squares, rats in steady state on purified high
from the gut. However, inconsistent results were obtained calcium phosphate diet. (B) recovery of label in 24-hour feces. Hatched bars:
with regard to the effectiveness of both cholestyramine and rats in steady state on purified control diet; Filled bars: rats in steady state
agar.27-30 Only activated charcoal has a major effect on biliru- on purified high calcium phosphate diet.

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HEPATOLOGY Vol. 24, No. 3, 1996 VAN DER VEERE ET AL. 625

An interesting but unexplained phenomenon was the ini-

tial increase in plasma bilirubin level in rats when their diet
was changed from the normal undefined lab chow to a puri-
fied control diet. Determination of the calcium content of lab
chow revealed a somewhat higher content than control puri-
fied diet (210 vs. 130 mmol/g). Although this is a moderate
difference, the increase in serum bilirubin upon change from
lab chow to purified control diet might at least be partly
explained by this. Alternatively, undefined compounds (like
those from cabbage-like vegetables) could be present in the
lab chow, which induce cytochrome P450 activities in the
liver45 and thereby increase alternative metabolism of biliru-
bin. In addition, we observed that female rats had a lower
serum bilirubin level than male rats of the same age. We
observed that this is related to body weight rather than to
sex-related factors; young male rats of the same body weight
as older female rats had similar serum bilirubin levels, and
these levels increased with increasing body weight.
The effect of a high dietary intake of calcium phosphate on
plasma bilirubin levels in Gunn rats could partly be caused
by precipitation of bilirubin in the gut caused by a decreased
bile salt concentration. To evaluate the significance of this
effect for our studies, the turnover of labeled bilirubin was
measured in permanently bile-diverted Gunn rats and com-
pared with sham-operated rats with an intact enterohepatic
circulation. We were aware of the fact that such an experi-
ment has been performed in the past,14 but a major difference
FIG. 4. Effect of bile depletion on turnover of 3H-labeled bilirubin in plasma is that, in those early experiments, the rats underwent sur-
in male Gunn rats. Sixteen hours after the bile was diverted, rats were injected gery just before the injection of labeled bilirubin. We observed
intravenously with 17 kBq 3H-labeled UCB. Radioactivity and bilirubin in that, at least in our hands, at that moment, the rats are not
plasma were determined at the indicated time points. Circles, bile-diverted in a steady state as far as plasma bilirubin is concerned.
rats; squares, rats with an intact enterohepatic circulation. Data represent
mean { SD from three animals in each group. Moreover, in these early studies, the rats were placed in re-
straining cages, whereas, in our experiment, the rats could
move freely. Therefore, it is not surprising that our results
differ from those described by Schmid et al.14 in 1963. We
output. This was indeed the case; during the first 3 days after expected a decrease in plasma bilirubin in the bile-diverted
replacement of the purified control diet by a high calcium rats for the following reasons. First, Gunn rats excrete some
phosphate diet, fecal UCB was significantly higher than in bilirubin in bile (approximately 4% of the daily production),46
controls. Thereafter, a new equilibrium was reached with a
reduced plasma (and probably also tissue) bilirubin pool and
a fecal UCB output that returned to control values. It is clear
that, in a steady-state situation, excretion should equal pro-
duction. By intravenous injection of 3H-labeled UCB, we dem-
onstrated that the effect of oral calcium phosphate on plasma
bilirubin was caused by a higher rate of plasma removal of
bilirubin.
The daily production of bilirubin in the Gunn rat has been
estimated to be about 500 nmol per 100 g of body weight.44
In our experiments, the daily recovery of intact UCB in the
feces was approximately 40% of the estimated production.
However, it is known that 85% or more of radiolabeled biliru-
bin is excreted in the animals’ stool, of which a considerable
fraction is converted to oxidative derivatives; only 6% is ex-
creted in urine.14 Therefore, measurement of bilirubin in fe-
ces can be used to estimate output of bilirubin despite the
fact that it has limited quantitative value. Thus, the benefi-
cial effect of oral calcium phosphate seems to be caused by a
shift of the bilirubin pool from the intravascular compart-
ment to the intestinal lumen. Inhibition of reabsorption by
binding to calcium phosphate most probably is the major
mechanism.
When the feces were extracted with chloroform, methanol,
and glycine–hydrochloric acid buffer, more label was recov-
ered in the apolar phase and less in the polar phase from
animals fed the high calcium phosphate diet compared with
animals on the control diet. Possibly UCB bound to calcium
phosphate is unavailable for further metabolism to polar de-
FIG. 5. Long-term effect of high calcium phosphate intake on plasma biliru-
rivatives by the intestinal flora. Alternatively, the presence bin in male Gunn rats. Circles: rats fed purified control diet; Squares, rats fed
of calcium phosphate in the gut might change the intestinal purified high calcium phosphate diet. Data represent mean { SD from five
flora and thus diminish breakdown of bilirubin. animals in each group. *P õ .05, **P õ .01.

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626 VAN DER VEERE ET AL.
which can enter the enterohepatic circulation. When bile is
diverted, this portion of bilirubin is immediately removed
from the body. Second, according to Carey,36 uptake of UCB
in the intestine is dependent on bile salts, and thus we sup-
posed that bilirubin would not be available for reabsorption
when bile salts are absent. But our results show that there
is no difference in the plasma half-life of 3H-bilirubin between
both groups. Because, under these experimental conditions,
the half-life of bilirubin is significantly shorter than in the
experiments with defined, purified diets (33 vs. 90 h), one
could argue that in the sham-operated rats bile salt concen-
tration in the intestine was also lower, e.g., because of chole-
stasis. However, bile flow, alkaline phosphatase, and g-gluta-
myl transferase were normal at the end of the experiment.
Therefore, we think that the shorter biological half-life of
bilirubin is related to the diet of these rats who received the
normal undefined lab chow. As discussed, rats fed this diet
have lower plasma bilirubin levels than rats on a purified
diet. Because bilirubin half-life in rats with an interrupted
enterohepatic circulation is the same as in rats with an intact
cycle, we believe that intestinal bile salts do not play a crucial
role in the secretion of UCB in Gunn rats.
The use of calcium phosphate is well established and has
no major side effects.47 After 30 weeks of administration, no
changes were found in renal parameters and plasma concen-
trations of calcium, magnesium, and phosphate in Gunn and
Wistar rats. Only in Gunn rats with kidneys damaged by
bilirubin precipitates were calcifications detected. This prob-
ably does not play a role in Crigler-Najjar patients, because,
as far as we know, the renal function in Crigler-Najjar pa-
tients is normal. Therefore, this therapy might be useful in
Crigler-Najjar patients. If oral calcium phosphate is effective,
the duration of phototherapy and/or the need for phenobarbi-
tal may be reduced.
Acknowledgment: The authors with to thank Adrie Maas
and Joost Daalhuisen for expert biotechnical assistance; Di-
ana Mulder for taking care of the animals; Rudi de Waard
for HPLC analysis of bilirubin; and Ilse Vogels for advice and
assistance with the preparation and staining of tissues.
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hpta WBS: Hepatology