Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia

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 unconjugated hyperbilirubinemia. We hypothesized that unconjugated bilirubin (UCB) can permeate the intestinal wall and can thus be secreted with the feces. We have previously observed that UCB binds to amorphous calcium phosphate in vitro. Orally ingested amorphous calcium 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 The fecal output of bilirubin was more than doubled in Gunn rats in the first 3 days after the normal diet had been replaced by the high calcium-phosphate diet.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 control diet and 50.9 { 1.4 hours in rats fed the high calcium phosphate diet (P Å .004). After 30 weeks, plasma bilirubin 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, conjugation 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 enzyme needed for this reaction, bilirubin–uridine diphosphate–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 chromatography. 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

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%). Accumulation 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 patients 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 possible 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 bilirubin production rate in Crigler-Najjar patients is equal to that in normal healthy individuals.14 Thus, the amount of bilirubin 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 metabolic 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 monooxygenases in the liver, probably cytochrome P450 IA1. The polar breakdown products are subsequently excreted in bile. Stimulation of this putative pathway is possible by intraperitoneal 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 investigated 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 reduced by oral administration of activated charcoal. Binding of bilirubin to activated charcoal inhibits reabsorption of bilirubin from the gut. However, activated charcoal is not suitable for prolonged use because it will bind a broad range of organic and inorganic compounds, and the daily ingestion of

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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 conditions 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 diffusion 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 administration will lead to a shift of serum bilirubin from the plasma to the gut. This will induce a transient increase in fecal bilirubin 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 present 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, Belgium). 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 aliquots 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 volume 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 residue applied to a Baker silica gel cartridge (J. T. Baker, Inc., Phillipsburg, NJ), which was then eluted with pure chloroform. The yellow 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 microgram 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 chromatography (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, creatinine, g-glutamyl transferase, glucose, lactate dehydrogenase, urea, and phosphate in plasma were determined with routine clinicochemical tests on a Hitachi 747 analyzer (Tokyo, Japan). For bilirubin measurement, blood was protected from light and processed immediately. 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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TABLE 1. Content of the Major Ingredients of the Purified Diets Used in the Calcium Phosphate Study

Diets

Control (mmol/g)

Low Ca (mmol/g)

High Ca (P; CO20 3 ) (mmol/g)

High Ca (P; Cl0) (mmol/g)

Ca P Mg CO3 Cl Na K Cellulose wt/wt

130.8 128.8 21.1 100.0 47.0 93.0 98.5 10.5%

25.0 125.0 21.1 – 51.3 51.3 100.0 11.6%

500.4 317.4 21.1 283.0 51.3 51.3 100.0 5.1%

499.8 317.4 21.1 100.0 316.6 51.3 100.7 5.0%

proximately 150 mg feces was extracted by addition of 10 mL methanol, 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 evaporated 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 residue was dissolved in 400 mL dimethyl sulfoxide, and 20 mL was injected onto a C-18 reversed-phase column (Chrompack, Middelburg, 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-Noctylamine 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 (Spectroflow 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 pigments 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 experimental 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 0 prepared, one containing CO20 3 and the other Cl in addition to phosphate. 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 calcium 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

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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 experiment, 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 3Hlabeled 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)) 3HUCB was injected intravenously via the penile vein. Blood samples were taken at regular intervals by puncture of the tail vein. Twentyfour-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 control 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 transaminase, alanine transaminase, alkaline phosphatase, g-glutamyl transferase, 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 measured 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 performed 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 significantly compared with purified control diet. Plasma calcium, phosphate, and chloride did not differ between groups during the experimental period, but plasma magnesium declined 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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Gunn rats can be influenced by the calcium phosphate content of their diet. This may result from binding of bilirubin to calcium phosphate in the gut, thereby preventing the reabsorption 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 bilirubin 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%. Bilirubin 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 equilibration, the decrease of label in plasma bilirubin followed first-order kinetics, which is in accordance with previous reports.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 excreted 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 decreased 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 compared with turnover in sham-operated rats41 with an intact enterohepatic circulation. In previous experiments, we noticed that the bilirubin level of Gunn rats increases significantly after a major operation, and it takes about 2 weeks before they reach their initial bilirubin levels again (unpublished 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 dehydrogenase were normalized to preoperative values, the bile was

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FIG. 1. Influence of oral calcium phosphate on plasma bilirubin and magnesium 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.

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 bilirubin, and label in blood and feces was collected daily. No difference was found in plasma half-life of 3H-bilirubin between 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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tal period. This indicates that UCB secretion into bile is insufficient 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. Finally, 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

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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 administration 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 administration 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, creatinine 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 tissues. Successive cryocoupes were stained with a conventional hematoxyline-phloxine method. Liver and aorta were negative 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 CriglerNajjar patients have a constant plasma bilirubin level indicates 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 has been shown that reabsorption of UCB can occur.17-19 To overcome the latter problem, we have tried to trap bilirubin in the gut lumen by binding to a matrix. This approach is not new; in the past, different nonabsorbable matrices have been administered to decrease the absorption of bilirubin from the gut. However, inconsistent results were obtained with regard to the effectiveness of both cholestyramine and agar.27-30 Only activated charcoal has a major effect on biliru-

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FIG. 3. Turnover and recovery of 3H-labeled bilirubin in male Gunn rats 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 control diet or a high calcium phosphate diet, male Gunn rats were injected intravenously with 17 kBq 3H-labeled bilirubin. Radioactivity in plasma was determined at the indicated time points after injection, and label was measured 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 calcium phosphate diet. (B) recovery of label in 24-hour feces. Hatched bars: rats in steady state on purified control diet; Filled bars: rats in steady state on purified high calcium phosphate diet.

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FIG. 4. Effect of bile depletion on turnover of 3H-labeled bilirubin in plasma in male Gunn rats. Sixteen hours after the bile was diverted, rats were injected intravenously with 17 kBq 3H-labeled UCB. Radioactivity and bilirubin in plasma were determined at the indicated time points. Circles, bile-diverted rats; squares, rats with an intact enterohepatic circulation. Data represent mean { SD from three animals in each group.

output. This was indeed the case; during the first 3 days after replacement of the purified control diet by a high calcium phosphate diet, fecal UCB was significantly higher than in 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 production. By intravenous injection of 3H-labeled UCB, we demonstrated 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 bilirubin is excreted in the animals’ stool, of which a considerable fraction is converted to oxidative derivatives; only 6% is excreted in urine.14 Therefore, measurement of bilirubin in feces can be used to estimate output of bilirubin despite the fact that it has limited quantitative value. Thus, the beneficial effect of oral calcium phosphate seems to be caused by a shift of the bilirubin pool from the intravascular compartment 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 recovered 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 derivatives by the intestinal flora. Alternatively, the presence of calcium phosphate in the gut might change the intestinal flora and thus diminish breakdown of bilirubin.

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An interesting but unexplained phenomenon was the initial increase in plasma bilirubin level in rats when their diet was changed from the normal undefined lab chow to a purified control diet. Determination of the calcium content of lab chow revealed a somewhat higher content than control purified 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 bilirubin. 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 compared with sham-operated rats with an intact enterohepatic circulation. We were aware of the fact that such an experiment has been performed in the past,14 but a major difference is that, in those early experiments, the rats underwent surgery just before the injection of labeled bilirubin. We observed that, at least in our hands, at that moment, the rats are not in a steady state as far as plasma bilirubin is concerned. Moreover, in these early studies, the rats were placed in restraining 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 expected a decrease in plasma bilirubin in the bile-diverted rats for the following reasons. First, Gunn rats excrete some bilirubin in bile (approximately 4% of the daily production),46

FIG. 5. Long-term effect of high calcium phosphate intake on plasma bilirubin in male Gunn rats. Circles: rats fed purified control diet; Squares, rats fed purified high calcium phosphate diet. Data represent mean { SD from five animals in each group. *P õ .05, **P õ .01.

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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 supposed 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 concentration in the intestine was also lower, e.g., because of cholestasis. However, bile flow, alkaline phosphatase, and g-glutamyl 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 concentrations of calcium, magnesium, and phosphate in Gunn and Wistar rats. Only in Gunn rats with kidneys damaged by bilirubin precipitates were calcifications detected. This probably does not play a role in Crigler-Najjar patients, because, as far as we know, the renal function in Crigler-Najjar patients 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 phenobarbital may be reduced. Acknowledgment: The authors with to thank Adrie Maas and Joost Daalhuisen for expert biotechnical assistance; Diana 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. REFERENCES 1. Crigler JF, Najjar VA. Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics 1952;10:169-179. 2. Chowdhury JR, Wolkoff AW, Arias IM. Hereditary jaundice and disorders of bilirubin metabolism. In: Scriver CR, ed. The metabolic basis of inherited diseases. New York: McGraw-Hill, 1988:1367-1408. 3. Seppen J, Bosma PJ, Goldhoorn BG, Bakker CTM, Roy Chowdhury J, Roy Chowdhury N, Jansen PLM, et al. Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. J Clin Invest 1994;94:2385-2391. 4. Schenker S, Hoyumpa AM, McCandless DW. Bilirubin toxicity to the brain (kernicterus) and other tissues. In: Ostrow JD, ed. Bile pigments and jaundice—molecular, metabolic and medical aspects. New York: Dekker, 1986:395-419. 5. Bratlid D. How bilirubin gets into the brain. Clin Perinatol 1990;17:449465. 6. Amit Y, Fedunec S, Panakkezhum DT, Poznansky MJ, Schiff D. Bilirubinneural cell interaction: characterization of initial cell surface binding leading to toxicity in the neuroblastoma cell line N-115. Biochim Biophys Acta 1990;1055:36-42. 7. McDonagh AF, Lightner DA. Phototherapy and the photobiology of bilirubin. Semin Liver Dis 1988;8:272-283. 8. Ennever JF, Costarino AT, Polin RA, Speck WT. Rapid clearance of a structural isomer of bilirubin during phototherapy. J Clin Invest 1987;79: 1674-1678. 9. Davis DR, Yeary RA, Lee K. The failure of phototherapy to reduce plasma bilirubin levels in the bile duct–ligated rat. J Pediatr 1981;99:956-958. 10. Bloomer JR, Sharp HL. The liver in Crigler-Najjar syndrome, protoporphyria, and other metabolic disorders. HEPATOLOGY 1984;4:18S-21S. 11. Reichen J. Familial unconjugated hyperbilirubinemia syndromes. Semin Liver Dis 1983;3:24-35. 12. Kaufman SS, Wood RP, Shaw BW Jr, Markin RS, Rosenthal P, Gridelli B, Vanderhoof JA. Orthotopic liver transplantation for type I Crigler-Najjar syndrome. HEPATOLOGY 1986;6:1259-1262. 13. Shevell MI, Bernard B, Adelson JW, Doody DP, Laberge JM, Guttman FM. Crigler-Najjar syndrome type I: treatment by home phototherapy followed by orthotopic hepatic transplantation. J Pediatr 1987;110:429-431.

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14. Schmid R, Hammaker L. Metabolism and disposition of 14C-bilirubin in congenital nonhemolytic jaundice. J Clin Invest 1963;42:1720-1734. 15. Kapitulnik J, Ostrow JD. Stimulation of bilirubin catabolism in jaundiced Gunn rats by an inducer of microsomal mixed-function monooxygenases. Proc Natl Acad Sci U S A 1977;75:682-685. 16. Kotal P, Fevery J. Direct permeation of unconjugated bilirubin (UCB) through the intestinal wall and conversion to urobilinogen I (ugen) is a major elimination route in homozygous Gunn rats. HEPATOLOGY 1989;10: 593. 17. Lester R, Schmid R. Intestinal absorption of bile pigments I. The enterohepatic circulation of bilirubin in the rat. J Clin Invest 1963;42:736-746. 18. Brodersen R. Intestinal reabsorption of unconjugated bilirubin: a possible contributing factor in neonatal jaundice. Lancet 1963;1:1242. 19. Gilbertsen AS, Bossenmaier I, Cardinal R. Enterohepatic circulation of unconjugated bilirubin in man. Nature 1962;196:141-142. 20. Lester R, Schmid R. Intestinal absorption of bile pigments II. Bilirubin absorption in man. New Engl J Med 1963;269:178-182. 21. Poland RL, Odell GB. Physiologic jaundice: the enterohepatic circulation of bilirubin. New Engl J Med 1971;284:1-6. 22. Davis DR, Yeary RA, Lee K. Activated charcoal decreases plasma bilirubin levels in the hyperbilirubinemic rat. Pediatr Res 1983;17:208-209. 23. Davis DR, Yeary RA. Activated charcoal as an adjunct to phototherapy for neonatal jaundice. Dev Pharmacol Ther 1987;10:12-20. 24. Ulstrom RA, Eisenklam E. The enterohepatic shunting of bilirubin in the newborn infant. I. Use of oral-activated charcoal to reduce normal serum bilirubin values. J Pediatr 1964;65:27-37. 25. Lester R, Hammaker L, Schmid R. A new therapeutic approach to unconjugated hyperbilirubinemia. Lancet 1962;Dec 15:1257. 26. Odell GB, Gutcher GR, Whitington PF, Yang G. Enteral administration of agar as an effective adjunct to phototherapy of neonatal hyperbilirubinemia. Pediatr Res 1983;17:810-814. 27. Poland RL, Avery GB, Goetcherian E, Odell GB. Treatment of CriglerNajjar syndrome with agar. Pediatr Res 1972;6:377. 28. Caglayan S, Candemir H, Aksit S, Kansoy S, Asik S, Yaprak I. Superiority of oral agar and phototherapy combination in the treatment of neonatal hyperbilirubinemia. Pediatrics 1993;92:86-89. 29. Kemper K, Horwitz RI, McCarthy P. Decreased neonatal serum bilirubin with plain agar: a meta-analysis. Pediatrics 1988;82:631-638. 30. Ostrow JD, Hammaker L, Schmid R. The preparation of crystalline bilirubin-C14. J Clin Invest 1961;40:1442-1452. 31. McDonagh AF, Palma LA, Lauff JJ, Wu TU. Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat. J Clin Invest 1984;74:763-770. 32. Bieri JG, Stoewsand GS, Briggs GM, Phillips RW, Woodard JC, Knapka JJ. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J Nutr 1977;107:1340-1348. 33. Bieri JG. Second report of the ad hoc committee on standards for nutritional studies. J Nutr 1980;110:1726. 34. Vonk RJ, Van Doorn ABD, Strubbe JH. Bile secretion and bile composition in the freely moving, unanaesthetized rat with a permanent biliary drainage: influence of food intake on bile flow. Clin Sci Mol Med 1978;55:253-259. 35. Van der Veere CN, Schoemaker B, Van der Meer R, Groen AK, Jansen PLM, Oude Elferink RPJ. Rapid association of unconjugated bilirubin with amorphous calcium phosphate. J Lipid Res 1995;36:1697-1707. 36. Carey MC. Pathogenesis of gallstones. Am J Surg 1993;165:410-419. 37. Van der Meer R, De Vries HT. Differential binding of glycine- and taurineconjugated bile acids to insoluble calcium phosphate. Biochem J 1985;229: 265-268. 38. Van der Meer R, Govers MJAP. Dietary phosphate does not inhibit the protective effects of calcium on luminal solubility and cytotoxicity of bile acids and fatty acids. Gastroenterology 1991;100:A407. 39. Lapre´ JA, De Vries HT, Termont DSML, Kleibeuker JH, DeVries EGE, Van der Veere R. Mechanism of the protective effect of supplemental dietary calcium on cytolytic activity of fecal water. Cancer Res 1993;53:248-253. 40. Van der Meer R, Welberg JWM, Kuipers F, Kleibeuker JH, Mulder NH, Termont DSML, Vonk RJ, et al. Effects of supplemental dietary calcium on the intestinal association of calcium, phosphate, and bile acids. Gastroenterology 1990;99:1653-1659. 41. Kuipers F, Havinga R, Bosschieter H, Toorop GP, Hindriks FR, Vonk RJ. Enterohepatic circulation in the rat. Gastroenterology 1985;88:403-411. 42. Odell GB, Bolen JL, Poland RL, Seungdamrong S, Cukier JO. Protection from bilirubin nephropathy in jaundiced Gunn rats. Gastroenterology 1974;66:1218-1224. 43. Rungby J, Kassem M, Eriksen EF, Danscher G. The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background. Histochem J 1993;25:446-451. 44. Wiese G, Ballowitz L, Korbmacher C. Pharmacokinetic studies of the enterohepatic circulation in Gunn rats. Klin Paediatr 1985;197:366-370. 45. Anderson KE, Kappas A. Dietary regulation of cytochrome P450. Annu Rev Nutr 1991;11:141-167. 46. Blanckaert N, Fevery J, Heirwegh KPM, Compernolle F. Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats. Biochem J 1977;164:237-249. 47. Haynes RC. Agents affecting calcification: calcium, parathyroid hormone, calcitonin, vitamin D, and other compounds. In: Goodman Gilman A, Rall TW, Nies AS, Taylor P, eds. The pharmacological basis of therapeutics. New York: Pergamon, 1988:1496-1504.

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