Monoconjugated Bilirubin Research Articles

A simplified HPLC-based method for measuring unconjugated bilirubin, bilirubin-monoglucuronide, bilirubin-diglucuronide, and delta-bilirubin in plasma with increased conjugated bilirubin

Prolonged icterus is an important and common problem in neonatology and accurate determination of the different bilirubin species, including differentiation between delta-bilirubin and mono- and di-conjugated bilirubin, is useful for diagnostic purposes. However, most bilirubin measurements routinely performed in the clinical laboratory are hampered by the lack of separation of the four bilirubin fractions (unconjugated bilirubin, mono-conjugated bilirubin, di-conjugated bilirubin, and delta-bilirubin). Herein, we propose a high-performance liquid chromatography-based method, independent of commercially available standards or reliable molar absorption coefficients, for the determination of bilirubin fractions in blood samples from icteric patients. The method is a robust and reliable candidate for a semi-automatized setup for measuring the various bilirubin fractions in a specialized laboratory handling samples from clinics with expertise in biliary disease.

A Case of Congenital Dyserythropoietic Anemia Type II, Gilbert's Syndrome and Malleolar Trophic Ulcers

A case of a woman with congenital dyserythropoietic anemia type II (CDA-II), Gilbert's syndrome (GS) and trophic malleolar ulceration is described. The association of CDA-II and GS caused early gallstone formation that led the patient to undergo cholecystectomy at the age of 15. GS is typified by increased production of both unconjugated and monoconjugated bilirubin, which is more lithogenic. The development of ulcers is not typical of CDA-II, even though they are associated with many of the hemolytic anemias, and were thought in our patient to be due to a thrombophilic tendency which manifest with Antithrombin III and Protein C deficiency.

Bile bilirubin pigment analysis in disorders of bilirubin metabolism in early infancy

BACKGROUNDEarly and accurate diagnosis of Crigler–Najjar syndrome, which causes prolonged unconjugated hyperbilirubinaemia in infancy, is important, as orthotopic liver transplantation is the definitive treatment.AIMTo determine whether bilirubin pigment analysis of...

Unconjugated and Conjugated Bilirubin Pigments during Perinatal Development

Objective: To determine the effect of phototherapy on serum conjugated bilirubin fractions, an index of bilirubin conjugation, in hyperbilirubinemic neonates. Method: Serum was sampled from 21 jaundiced (serum diazo total bilirubin ≥274 µmol/l), term, otherwise healthy neonates prior to starting phototherapy, and 24 h after the commencement of treatment. Alkaline methanolysis followed by reverse-phase, high-performance liquid chromatography, specific for determination of unconjugated bilirubin and the monoconjugated and diconjugated fractions of total conjugated bilirubin in serum, was used for the analysis. Prephototherapy values were compared to those at 24 h. Results: Serum total bilirubin and total conjugated bilirubin values decreased during the study period, from 220 (211–239) to 177 (157–213) µmol/l (median (25–75% range)) p = 0.001, for the former, and from 1.4 (0.87–1.57) to 0.93 (0.69–1.84) µmol/l, p = 0.005, for the latter. These parameters decreased by a similar percentage (–16.6 ± 14.8% and –14.0 ± 23.4%, respectively; p > 0.05). Both monoconjugated and diconjugated bilirubin, calculated as a percentage of total conjugated bilirubin, remained constant over the study period (88.2 (72.2–96.4)% before phototherapy and 92.5 (87.3–96.8)% after 24 h, p > 0.05, for monoconjugated bilirubin, and 11.8 (3.6–27.8)% and 7.5 (3.2–12.7)%, respectively, p > 0.05, for diconjugated bilirubin). Conclusions: Serum total conjugated bilirubin values decreased in parallel to serum total bilirubin levels during phototherapy, maintaining a constant relationship between these two parameters. The ratios of monoconjugated and diconjugated bilirubin to total conjugated bilirubin remained constant. These findings imply that phototherapy does not alter bilirubin conjugation in hyperbilirubinemic neonates.

Serum and Bile Bilirubin Pigments in the Differential Diagnosis of Crigler-Najjar Disease

To differentiate between Crigler-Najjar (CN) disease types 1 and 2. The patterns of serum bilirubins, bile pigment composition, and phenobarbital response were studied. Three infants, affected by high serum unconjugated bilirubin concentrations, previously classified as type 1 CN. Serum and bile bilirubin pigment composition, both before and after phenobarbital (PB) treatment, were determined by alkaline methanolysis and high-pressure liquid chromatography. PB was given for at least 3 weeks by oral administration (5 mg/kg bw per day). No diconjugated bilirubin was found either before or after PB treatment in the serum of the three studied infants. In two patients traces of monoconjugated bilirubin were detected before PB therapy, and the ratio of conjugated/total bilirubin (percent) was increased by the PB response. In the third patient, traces of monoconjugated bilirubin appeared only after PB administration. However, the serum unconjugated bilirubin concentration decreased significantly only in the second patient, following the second cycle of PB treatment, leading to the diagnosis of type 2 CN. The analysis of the methyl ester derivatives of bile pigments was also performed on bile samples obtained in two patients by Entero-Test (R) both before and after PB treatment. An absolute increment in monoesterified bilirubin concentration was found after PB administration, although the percent concentration increased in one case and decreased in the other. No diesterified bilirubin was detected in the bile samples. The present results show that in types 1 and 2 CN disease it is possible to detect traces of monoconjugated but not diconjugated bilirubin both in serum and in bile. Whereas PB treatment is effective in slightly increasing the serum monoconjugated bilirubin concentration even in type 1 CN disease, the diagnosis of type 1 or 2 is based on finding a substantial decrease of serum unconjugated bilirubin following PB administration.

Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver.

A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat. Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca(++)-alginate beads. The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures. The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group). Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals. Both bilirubin monoconjugates and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography. Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%). In the test group, the concentration of bilirubin conjugates increase rapidly, attaining median values of 72.26 microM and 92.59 microM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation. In the control group, the levels of either conjugate did not exceed 0.87 microM throughout the experiments. Statistical analysis showed a significant difference between the two groups (p < 0.0023). These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rat's genetic defect. Such a system might be of therapeutic interest in acute liver failure.

Renal clearance of bilirubin conjugates in newborns of different gestational age.

The renal excretion of bilirubin conjugates was analysed in 22 newborns. Bilirubin monoconjugate was the only metabolite detectable in urine samples and its renal excretion correlated with the creatinine excretion rate (r = 0.91). The renal clearance of bilirubin monoconjugates in newborns ranged between 380 and 2160 ml/1.73 m2 per 24 h (median: 790). According to the present findings the renal function should be monitored in newborns and infants with conjugated hyperbilirubinaemia.

The effects of total parenteral nutrition on the hepatic handling of bilirubin in the rat.

Disturbances of bilirubin metabolism such as jaundice or pigment gallstone formation occur during total parenteral nutrition (TPN). We have studied the effects of TPN on bile flow and bile acid secretion and on the hepatobiliary transport of bilirubin in rats. Animals on parenteral nutrition for 5 days received 4.8 g of amino acids and 6.9 g of glucose daily. Controls were orally fed animals. Bile flow and bile acid secretion were not significantly modified by TPN. Serum bile acid and alkaline phosphatase levels were significantly increased in TPN animals when compared with the controls (+98% and +38%, respectively), which points to a relative cholestasis in the TPN rats. The biliary excretion of bilirubin monoconjugates and bilirubin diconjugates was significantly increased (+72% and +78%, respectively). This provides evidence for enhanced production of the pigment. Serum concentration of total bilirubin was enhanced in the TPN rats (+240%). The esterified/total bilirubin ratio in serum increased, whereas the bilirubin diconjugates/bilirubin monoconjugates ratio decreased. These facts, together with the minor reduction of hepatic bilirubin UDP glucuronosyltransferase activity (-12%), suggest that hyperbilirubinemia would be a consequence of both cholestasis and increased bilirubin production. The alterations reported here could contribute to the explanation of hyperbilirubinemia and pigment gallstone formation in patients maintained on parenteral nutrition.

In Vitro Effects of Light on Serum Bilirubin Subfractions Measured by High-Performance Liquid Chromatography: Comparison with Four Routine Methods

The effects of light on serum bilirubin subfractions in vitro were investigated by HPLC and four routine methods for bilirubin analysis. By HPLC, the rate of photodegradation of unconjugated bilirubin (Bu) was nearly twice that of monoconjugated bilirubin (mBc) and threefold that of diconjugated bilirubin (dBc); delta bilirubin (Bd) was most stable against photoirradiation. In the diazo method, the rate of photodegradation of direct bilirubin was almost the same as that of the sum of mBc, dBc, and Bd determined by the HPLC method. However, the rate of photodegradation of indirect bilirubin was significantly lower (P < 0.001) than that obtained by HPLC, because approximately 30% of the bilirubin photoproducts reacted with the diazo reagent as indirect bilirubin. The rate of photodegradation of total bilirubin determined by the direct spectrometric method was lower than that determined by the diazo method, but equal to that of the total peak areas of HPLC. In the Ektachem method, bilirubin photoproducts affected total bilirubin negligibly, and Bc and Bu positively, so that the value of Bd decreased. In the bilirubin oxidase method, bilirubin photoproducts were oxidized enzymatically by both the total and direct bilirubin reagents. We re-emphasize the importance of shielding serum from light to avoid generating bilirubin photoproducts that interfere with the accurate determination of serum bilirubin subfractions. We also recommend HPLC analysis as a standard method for bilirubin measurement.

Thoughts and Progress

Utility of a new method of bilirubin fractionation was evaluated in monitoring the effectiveness of plasma exchange (PE) performed in 10 postoperative cases with liver failure. Fractionation of serum bilirubin by high-performance liquid chromatography demonstrated a higher delta bilirubin (B delta) peak against lower conjugated bilirubin peaks [monoconjugated bilirubin (MCB) and diconjugated bilirubin (DCB)] in the three recovered cases. The calculated ratio of MCB/B delta and the ratio of B delta/(MCB + DCB + B delta) in the recovered cases showed statistically significant different against seven unrecovered cases (p less than 0.01). These results suggest that the recovered cases had a different quality hyperbilirubinemia and a different disease entity before PE as well as a different response to PE, and this novel method for serum bilirubin subfraction is considered a useful marker in selecting a patient responsive to PE.

Pigment Gallstone Disease

Black and brown pigment gallstones are morphologically, compositionally, and clinically distinct. Black stones form primarily in the gallbladder in sterile bile and are associated with advanced age, chronic hemolysis, alcoholism, cirrhosis, pancreatitis, and total parenteral nutrition. Brown stones form not only within the gallbladder but also within the intrahepatic and extrahepatic ducts; they are uniformly infected with enteric bacteria and are usually associated with ascending cholangitis. Brown stones are related to juxtapapillary duodenal diverticula and are the predominant type of de novo common bile duct stones. Cholecystectomy is usually curative in black pigment stone disease, whereas stones often recur after cholecystectomy for brown stone disease. The pathogenesis of black stones is probably related to nonbacterial, nonenzymatic hydrolysis of bilirubin conjugates. At the pH of bile, this results in two monohydrogenated bilirubin anions that precipitate with calcium ions. Bilirubin monoconjugates that are increased in several conditions, such as Gilbert's syndrome and chronic hemolysis, may play a pivotal role in black stone formation as a source of unconjugated monohydrogenated bilirubin and as a possible co-precipitant with calcium. The precipitation of calcium carbonate and phosphate is influenced by local gallbladder factors. Brown pigment stones are formed in bile infected with enteric bacteria that elaborate hydrolytic enzymes: beta-glucuronidase, phospholipase A, and conjugated bile acid hydrolase. The resulting anions of bilirubin and fatty acids form insoluble calcium salts. We used nb/nb mice with a chronic hemolytic anemia as a model of hemolysis-induced black stone disease. The presence of 40% bilirubin monoconjugates in mouse gallstones indicated the importance of this moiety in the pathogenesis of black stones. Other data obtained by marrow transplantation experiments in mice revealed the relative importance of genotype versus the hemolytic anemia on determinants such as biliary bile acid composition and mucin secretory glands in the mouse gallbladder neck. Additional physical chemical studies of the interaction of unconjugated bilirubin in model bile solutions will be helpful in further delineating the pathogenesis of both black and brown pigment gallstones.

Responsiveness to phenobarbital in an adult with Crigler-Najjar disease associated with neurological involvement and skin hyperextensibility

We present the case of a 23-yr-old man who had had since birth marked and sustained unconjugated nonhemolytic hyperbilirubinemia and who had had several attacks of grand mal seizures. Analysis of serum bilirubin by diazoreactive methods showed serum levels of unconjugated bilirubin as high as 445 μmol/L that were not affected by phenobarbital administration. However, analysis of serum bile pigments by high-pressure liquid chromatography demonstrated marked decrease of unconjugated bilirubin after phenobarbital treatment (from 432.4 μmol/L to 291.0 μmol/L) associated with slight increase of bilirubin monoconjugates and diconjugates (from 0.25 μmol/L to 0.42 μmol/L). Furthermore, in the past few years the patient had exhibited striking skin hyperextensibity and diaphragm eventration. This case confirms that alkaline methanolysis—high-pressure liquid chromatography is the most reliable method for assessment of serum fraction bilirubin levels; that clinical parameters such as neurological signs do not unequivocally discriminate between type I and II Crigler-Najjar disease and that response to phenobarbital treatment remains the main diagnostic tool. (HEPATOLOGY 1991;13:213–215).

Responsiveness to phenobarbital in an adult with crigler-najjar disease associated with neurological involvement and skin hyperextensibility

We present the case of a 23-yr-old man who had had since birth marked and sustained unconjugated non-hemolytic hyperbilirubinemia and who had had several attacks of grand mal seizures. Analysis of serum bilirubin by diazoreactive methods showed serum levels of unconjugated bilirubin as high as 445 mumol/L that were not affected by phenobarbital administration. However, analysis of serum bile pigments by high-pressure liquid chromatography demonstrated marked decrease of unconjugated bilirubin after phenobarbital treatment (from 432.4 mumol/L to 291.0 mumol/L) associated with slight increase of bilirubin monoconjugates and disconjugates (from 0.25 mumol/L to 0.42 mumol/L). Furthermore, in the past few years the patient had exhibited striking skin hyperextensibility and diaphragm eventration. This case confirms that alkaline methanolysis-high-pressure liquid chromatography is the most reliable method for assessment of serum fraction bilirubin levels; that clinical parameters such as neurological signs do not unequivocally discriminate between type I and II Crigler-Najjar disease and that response to phenobarbital treatment remains the main diagnostic tool.

Physico-chemical factors predisposing to pigment gallstone formation in liver cirrhosis

Liver cirrhosis is associated with a high prevalence of pigmentary cholelithiasis. The major compound of pigment gallstones is unconjugated bilirubin (UCB) in the form of calcium bilirubinate salts or a black pigment polymer. Most of UCB in bile derives from enzymic or non-enzymic hydrolysis of mono- or diconjugated bilirubin. Changes in the relative ratios between these two bilirubin species have been associated with pigment gallstones. It has also been shown that UCB solubilization in bile depends on its interaction with bile salts. In order to clarify the factors predisposing cirrhotic patients to pigment stone formation, we measured UCB, monoconjugated bilirubin (MCB) and diconjugated bilirubin (DCB) in duodenal bile of 15 patients with cirrhosis, ten patients with chronic active hepatitis (CAH) and ten normal subjects, we also analyzed their relationships with lipids. In cirrhotic patients, the MCB concentration in bile was significantly ( p < 0.05) higher than in normal subjects and was correlated with the severity of the disease. Bile salts and lecithin concentrations were significantly lower in cirrhosis ( p < 0.005 vs. CAH or normals). Cirrhotic patients have a bile salts/UCB molar ratio which is one third that of CAH patients or normal subjects ( p < 0.01). No differences were found between CAH patients and controls in each of the parameters tested. In conclusion, we propose that the very low BS UCB molar ratio and the very high biliary content in MCB represent two independent physico-chemical factors predisposing cirrhotic patients to pigmentary cholelithiasis.

Spontaneous formation of pigmentary precipitates in bile salt-depleted rat bile and its prevention by micelle-forming bile salts

During studies on the effect of bile salt-pool depletion in the bile-fistula rat (adult male Sprague-Dawley), the spontaneous formation of an orange-brown precipitate was noted. The nature of this phenomenon and its relationship to BS and calcium concentration was investigated in depth. Bile from 18 animals was collected in the dark into transparent tubes containing sodium azide, ascorbic acid, and glucaro-1,4-lactone. The tubes were flushed with nitrogen, sealed, and incubated at 37 °C. The pigmentary precipitate formed in all the bile salt-depleted (< 3–5 mM) bile samples (i.e., those collected after 5–7 h of external biliary drainage), but not in bile saltrich biles. It appeared within 30–240 min after collection, both in bile samples collected at room temperature and at 37 °C, initially as a pale flocculation and then slowly sedimenting to form, after centrifugation, a solid, dark-orange pellet. There were no pH changes during incubation, and bile cultures were negative. Under polarizing microscopy, the precipitate appeared amorphous, and there was no evidence of birefringence. High-performance liquid chromatography showed that unconjugated bilirubin was the prevalent pigmentary component, but significant amounts of monoconjugated bilirubin also coprecipitated. Lipid chemistry showed the presence of lecithin (80.1% of total lipids), which was rich in palmitoyl and linoleoyl fatty acids, and of fatty acids (predominantly palmitic and oleic). Infrared spectroscopy and x-ray diffraction showed the presence of calcium bilirubinate and palmitate. In-vivo replenishment of the bile salt pool by intravenous infusion of either taurocholate or taurochenodeoxycholate (1 μmol/min) completely prevented the pigmentary precipitation. In vitro experiments showed inhibition of the precipitate formation by the addition of individual bile salt in concentrations approximating their critical micellar concentration. Precipitate formation was hastened by the addition of calcium chloride (4–12 mM), but only in bile salt-depleted biles. As the composition of the precipitate closely resembles that of human brown-pigment stones and sludge, these findings may provide new insights into an understanding of the pathogenesis of pigment gallstone disease.

Assessment of bilirubin clearance capacity of a newly developed ion‐exchange adsorption column and its possible use as a supportive therapy in hepatorenal syndrome

We assessed the bilirubin reduction capacity of three different types of devices in vitro: a high-permeable membrane column for double-filtration plasmapheresis (DFP) (Evaflux 2A, Kuraray, Japan), and non-coated charcoal column for hemoperfusion (HP) (N-180, Asahi Medical, Japan), and ion-exchange columns for plasma adsorption (PA) (BR-350, Asahi Medical, Japan, and B-001, Kuraray, Japan). A column for DFP reduced the concentration of low-molecular proteins effectively such as plasma bilirubin and bile acids in an albumin-dependent manner. A charcoal column adsorbed low-molecular substances preferentially. But in these two columns, the loss of fibrinogen is a limiting factor for determining the processing plasma volume. Ion-exchange columns for PA adsorbed bile acids, disconjugated bilirubin, and monoconjugated bilirubin more efficiently compared with delta-bilirubin and unconjugated bilirubin. Pretreatment of the column with heparin reduced the loss of fibrinogen to less than 10%. We applied the BR-350 ion-exchange column in vivo for treatment of three patients with hyperbilirubinemia. After treatment, an alcoholic hepatitis patient with the hepatorenal syndrome (HRS) recovered from acute renal failure. However, in a patient with primary biliary cirrhosis and in a patient with fulminant hepatitis, the decrease of serum bilirubin was transient and no obvious beneficial responses were noted. The capacity and ability of the BR-350 column to adsorb plasma bilirubin was shown sufficient to treat deeply jaundiced patients, because 4 liters of the plasma of a patient with 108 mg/dl of initial total bilirubin concentration was able to be processed continuously without an obvious decrease in bilirubin adsorption capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in the relative amount of bilirubin diconjugates in rat bile and serum under infusion of phthaleins and indocyanine green

The effect of organic anions on bilirubin metabolism and excretion was investigated in rats. Biliary excretion of bromosul-fophthalein, bromocresol green and indocyanine green led to a significant decrease in excretion of bilirubin pigments and to an increase of their concentration in serum. This was concomitant with a marked increase in the ratio of bilirubin diconjugates to monoconjugates in bile and serum. These changes were unrelated to either bile flow, biliary lipid output, or hepatic activity of bilirubin UDP-glucuronyl transferase. Following bolus injection of [ 14C]bilirubin, peak excretory rate of radioactivity was markedly delayed in rats infused with bromosulfophthalein, as compared to controls. It is concluded that administration of the organic anions increased the ratio of bilirubin diconjugates to monoconjugates in bile and serum, by slowing down the intrahepatic transit of bilirubin pigments. This, in turn, allowed more efficient enzyme-catalyzed formation of bilirubin diconjugates from the intermediate bilirubin monoconjugates.

Somatostatin inhibits the effect of secretin on bile flow and on hepatic bilirubin transport in the rat.

Increasing amounts of porcine secretion (0.05 to 2.00 clinical units/h/100 g body wt) given to rats during a continuous infusion of bilirubin, increased bile flow and the apparent maximal biliary excretion of bilirubin ('Tm'). This increment was caused by an enhanced biliary output of bilirubin monoconjugates. The effect was dose dependent but maximal at a secretin infusion of 0.80 CU. Somatostatin 0.2 and 0.8 microgram/h/100 g body wt caused a dose related inhibition of the hepatic effects of secretin both on bile flow and on biliary output of bilirubin conjugates. As secretin elicits the release of somatostatin, a feed-back system could be envisaged whereby the somatostatin released stops the effects of secretin.

Lincomycin treatment of guinea pigs causes formation of pigmented phosphate containing gallbladder sludge and stones

We studied the mechanism of gallbladder sludge formation in guinea pigs ( n = 30) treated with lincomycin (80 mg/kg/day) for 7 consecutive days. At sacrifice (day 8) gallbladders of treated animals contained turbid bile, sludge and in one animal a single gallstone. The precipitates were amorphous on X-ray diffraction. Infra-red spectroscopy revealed calcium phosphate as the major component. Compared to saline-treated controls ( n = 15) concentrations of total protein, total phosphate and total bilirubin in gallbladder bile were significantly increased ( P < 0.05). The increase in total phosphate was due to the inorganic component, since phospholipid phosphorus was unchanged. The relative amour's of unconjugated bilirubin and of bilirubin mono- and diconjugates in gall-bladder bile were unaffected by treatment as was β-glucuronidase activity. However, sludge was enriched in unconjugated bilirubin compared to gallbladder bile. This was most probably caused by alkaline hydrolysis of bilirubin monoconjugates. To some extent, disproportionation of bilirubin monoconjugates in bile or sludge, either in vivo or during sample preparation, might also have led to increased unconjugated pigment.

The significance of bile pigment deconjugation by beta-glucuronidase in canine hyperbilirubinemia.

In dogs, the differentiation between haemolytic and cholestatic hepatobiliary diseases cannot be achieved by measuring of the unconjugated:conjugated bilirubin ratio, which is in contrast with generally held clinical concepts. The overlap of the bilirubin ratios between the two groups of icterus-generating diseases might in part be explained by deconjugation of conjugated bilirubin. Enzymatic cleavage by hepatic beta-glucuronidase might result in higher unconjugated bilirubin (UCB) fractions in cholestatic disease. The influence of deconjugation of bilirubins by beta-glucuronidase was investigated in 25 healthy dogs and 35 dogs with spontaneous hyperbilirubinemia due to either hepatobiliary or haemolytic disease. UCB and its mono- and diconjugates were measured by alkaline methanolysis and HPLC in plasma and liver tissue. The activity of beta-glucuronidase was also measured in both liver and plasma. In addition, semiquantitative histochemical quantitation of bilirubins in liver tissue was performed. The concentration and the fraction of UCB in plasma of dogs with hepatobiliary disease were not significantly different from those of dogs with autoimmune haemolytic anaemia. There was a correlation between the fraction of UCB in liver and plasma of jaundiced dogs (r = 0.42, P less than 0.01) and between the histochemically estimated and the biochemically measured total bilirubin concentration in liver tissue. There was no correlation between the beta-glucuronidase activity and either unconjugated or monoconjugated bilirubin in plasma or liver of diseased animals. The fraction and the concentration of UCB in the liver of dogs with hepatic and with haemolytic disease were identical. It is concluded that beta-glucuronidase activity is not the significant factor in explaining the similar levels and fractions of UCB in dogs with hyperbilirubinemia due to either hepatobiliary or haemolytic disease.