hepatic clearance of unconjugated bilirubin in cholestatic liver diseases

Hepatic Clearance of Unconjugated Bilirubin in Cholestatic Liver Diseases

Joseph R. Bloomer, MD, Paul D. Berk, MD, and Robert B. Howe, MD

The hepatic clearance of unconjugated bilirubin-14C from the plasma of 5 patients with cholestatic liver disease was normal when compared with values obtained in healthy young adults. In 1 patient with benign recurrent cholestasis, clearance was the same during an attack of cholestasis (serum bilirubin concentration 25.7 mg/100 ml) as when the patient was in remission (serum bilirubin concentration 0.65 mg/100 ml). We conclude that the hepatic clearance of unconjugated bilirubin may not be altered in hepatic disorders where cholestasis is the predominant feature and that other explanations should be considered when patients with these disorders manifest an increased plasma concentration of unconjugated bilirubin.

Cholestatic liver disorders have as their prominent abnormality an alteration in bile for- mation, and they are characterized biochemi- cally by serum retention of bile constituents and increased serum alkaline phosphatase. Bio- chemical evidence of hepatic parenchymal dam- age and altered synthetic function is less strik- ing. Although cholestasis should predominantly affect the terminal step of bilirubin excretion, thus causing only conjugated (direct-reacting) hyperbilirubinemia, patients with cholestatic liver disease frequently have elevations of both direct- and indirect-reacting serum bilirubin fractions. The indirect fraction, which is considered to represent unconjugated bilirubin, may be elevated in part due to an artefact of methodology, since Perelli and Watson have shown that the Ducci-Watson modification of the Mal- loy-Evelyn method overreads this fraction in

From the Department of Medicine, Yale University School of Medicine, and the Metabolism Branch, National Cancer Institute, National Institutes of Health.

Address for reprint requests: Dr. Joseph R. Bloomer, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

sera containing mainly conjugated bilirubin (1). However, other factors could produce unconjugated hyperbilirubinemia in patients with cholestatic liver disease. Since these patients may have shortened red cell life spans, increased bilirubin production is one such factor. If the cholestasis is sufficiently long-standing to have caused significant parenchymal liver dam- age, hepatic clearance of unconjugated bilirubin from the plasma may also be impaired. Choles- tasis in itself could affect the hepatic clearance of unconjugated bilirubin by altering microsomal conjugation of bilirubin, the hepatic uptake mechanism, or the intracellular transport pathway.

We have investigated the metabolism of unconjugated bilirubin in a series of patients with different types of cholestatic liver disease by em- ploying a method which quantitates the hepatic clearance of unconjugated bilirubin from plasma (2). Studies were performed in 1 patient with benign recurrent cholestasis both during a cholestatic attack and during remission, per- mitting a direct estimate of the effect of cholestasis on the hepatic clearance of unconjugated bilirubin.

Digestive Diseases, Vol. 19, No. 1 (January 1974) 9

Table 1. Patients Undergoing Bilirubin -x4 C Studies

BLOOMER ET AL

Bilirubin,r Bilirubin,'* unconjugated/

Patient/ Alkaline Cholesterol indirect/direct conjugated Age/Sex Dx SGOT phosphatase (mg/100 ml) (mg/lO0 ml) (mg/100 ml)

AD/15/F Cholestatic hepatitis 100 47 KA 632 1.2/3.6 0.7/3.7 SD/39/F Primary biliar~t cirrhosis 166 226 IU 393 0.9/2.9 0.4/3.3 RB/20/M Benign recurrent 1 13 14 KA - - 0.4/.07 0.5/.15

cholestasis 2 26 327 IU 130 7.4/20.6 5.9/19.8 AB/59/F 2 biliary cirrhosis 120 686 IU 336 1.2/3.7 0.6/3.6 EB/40/M Dubin-Johnson syndrome 17 7 KA 302 1.4/1.0 - -

NORMAL <35 <14 KA <230 <1.0/<0.2 <1.0/<0.15 <70 IU

*Measurement made by an adaptation for an automated system of the method of Jendrassik and Grof. tMeasurement made by the method of Weber and Schalm.

MATERIALS AND METHODS Patients. Studies were performed in 5 patients with cho-

lestatic liver disorders. Basic clinical and laboratory data about each patient are presented in Table 1. In each case the diagnosis was made on the basis of clinical history, laboratory data, histological examination of liver biopsy, and ra- diographic evaluation of the biliary tract. AD had cholestatic hepatitis of undetermined etiology. At the time of the study she had been jaundiced for 9 months. SD had had documented liver disease for 25 years at the time of her study. RB had recurrent bouts of cholestasis characterized by.jaundice and intense pruritus since age I 1. He was studied both during a cholestatic attack and during remission. AB had biliary cirrhosis secondary to an iatrogenic biliary stricture with recurrent bouts of ascending cholangitis. EB, who had the Dubin-Johnson syndrome, cannot be considered to have cholestatic liver disease in the strictest defini- tion of the term, since bile salt excretion is apparently normal in this condition (3). However, he is included because such patients do have defective transport of conjugated bilirubin into bile. Except for AD, who was receiving predni- sone and azathioprine at the time of the study, none of the patients was taking any medication which might affect bilirubin metabolism.

Isotope Studies. The plasma disappearance rate of unconjugated bilirubin-~4C was determined in each patient following the intravenous administration of 1 to 2 #Ci of bilirubin-~4C in 0.5 mg bitirubin, using methods previously described (2), and the following parameters of unconjugated bilirubin metabolism Were calculated: a) hepatic bilirubin clearance (CBR), the volume of plasma irreversibly cleared of unconjugated bilirubin each minute by the liver, and b)

the percent of the administered bilirubin-~4C retained in the plasma in unconjugated form at 4 hours. The data were also analyzed by multicompartmental analysis in terms of a model in which the plasma pool of unconjugated bilirubin was considered to exchange with both a hepatic unconjugated bilirubin pool and an extravascular, extrahepatic pool of unconjugated bilirubin (2). Fractional transfer rates between pools were determined. The relative storage capacity of the liver for conjugated bilirubin (S) was determined by the method of Quarfordt et al (4) from the results of the compartmental analysis. The value for S which we obtained by this technique in 28 healthy volunteers (0.69 • 0.27, mean • SD, mg/(mg/100 ml)/kg body wt) was similar to the value obtained by Raymond and Galambos in healthy volunteers using the classical constant infusion method (5).

RESULTS

Plasma bilirubin concentrations were measured both by the method of Weber and Schalm (6) and by an adaptation for an automated system of the method of Jendrassik and Grof (7) (see Table 1). According to the latter method, each of the 5 patients either had abnormal or borderline elevation of the indirect-reacting serum bilirubin fraction. However, this was true for only patient RB, who was severely jaundiced during his attack of cholestasis, when the method of Weber and Schalm was used. This probably reflects the fact that the method

10 Digestive Diseases, Vol. 19, No. 1 (January 1974)

CHOLESTATIC LIVER DISEASES

Table 2. Parameters of Bilirubin-14 C Clearance

4-hour CBR plasma S

(ml/min/kg retention X21 2t12 X02 (mg/(mg/lO0 Patient body wt) (~ (min -I ) (min -1) (min -1 ) ml)/kg body wt

AD 0.37 5.8 .016 .007 .011 0.33 SD 0.60 3.8 .026 .006 .005 1.13 RB 1 0.37 8.8 .015 .007 .010 0.37

2 0.33 9.3 .036 .009 .003 1.28 AB 0.83 4.3 ,015 .004 .009 0.88 EB 0.66 4.5 .025 .006 .010 0.66 Normal 0.65 5.0 .023 .007 .011 0.69 Mean • 2SD -e0.36 4:3.8 -E.014 ~:.008 4:.008 •

C:BR=The volume of plasma cleared of unconjugated bilirubin by the liver per minute per kilogram body weight.

4-hour plasma retention=the percent of administered bilirubinJ4C retained in the plasma at 4 hours in unconjugated form.

)~21 =the fractional transfer rate of unconjugated bilirubin from plasma pool to hepatic pool (uptake). x12 =the fractional transfer rate of unconjugated bilirubin from hepatic pool to plasma pool (reflux). ,',02 = the irreversible fractional removal rate of unconjugated bilirubin from hepatic pool (conjugation).

S =The relative storage capacity of the liver for unconjugated bilirubin

of Weber and Schalm, which requires separation of unconjugated bilirubin from conjugated bilirubin in the sample before the diazotization reaction is carried out, is a more accurate way of measuring the unconjugated bilirubin fraction in sera containing predominantly con- .jugated bilirubin. The method of Jendrassik and Grof utilizes kinetic analysis for measuring these fractions in a similar manner to the Ducci-Watson method, where it has been demonstrated that the indirect-reacting bilirubin fraction (unconjugated bilirubin) is overread in such sera (1).

Studies with bilirubin-14C in these 5 patients demonstrated that the hepatic clearance of unconjugated bilirubin from plasma (CBR) and the plasma retention of unconjugated bilirubin- 14C at 4 hours were, in every case, within the range found by Berk et al for healthy young adults (see Table 2). Moreover, these values were the same for patient RB during his cholestatic attack as when he was in remission.

Multicompartmental analysis of the plasma bilirubin-14C data further indicated that the fractional transfer rates of unconjugated bilirubin between plasma and liver were also normal for each patient. Notable differences were seen between the 2 studies for patient RB, however. During cholestasis the fractional uptake rate of unconjugated bilirubinJ4C from plasma by the liver (X21) was more than twofold greater than when he was in remission. This was reflected as a greater initial disappearance rate of unconjugated bilirubin-14C from plasma during his cholestasis study (see Figure 1). However, the fractional removal rate of unconjugated bilirubin from his hepatic unconjugated bilirubin pool (X02), which occurs through conjugation, was diminished.

The relative hepatic storage capacity for unconjugated bilirubin (S) was normal in 4 of the patients. In patient RB the value was normal when he was in remission, but it was slightly above the normal range during a cholestatic at-


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BLOOMER ET AL

Fig 1. The early disappearance of unconjugated bil irubin-14C from the plasma of patient RB during a cholestatic attack is compared with that when he was not jaundiced.

tack. SlCr-labeled red cell survivals were measured

in patients AD and AB following the bilirubin- 14C studies and were found to be reduced at 20.0 and 16.0 days, respectively (normal > 26 days). Red cell survival in patient SD was normal at 28 days. Measurements were not made in patients RB and EB, but both had normal he- matocrits and reticulocyte counts.

D I S C U S S I O N

These studies demonstrate that the hepatic clearance of uneonjugated bilirubin from

plasma may remain normal in various forms of cholestatic liver disease. Since only a limited number of patients was studied, it cannot be as- sumed that this will always be the situation, or indeed what the incidence of normal unconjugated bilirubin clearance will be for any par- ticular disease entity. As the liver disease progresses , loss of functioning parenchyma will probably produce a situation in which there is abnormal extraction of unconjugated bilirubin from plasma in all such disorders. Cholestasis by itself, however, does not seem to alter clearance of unconjugated bilirubin by the liver. This is most strikingly seen in the case of RB, in

12 Digestive Diseases, Vol. 19, No. 1 (January 1974)

CHOLESTATIC LIVER DISEASES

whom hepatic clearance of unconjugated bilirubin was the same during an intense degree of chotestasis as when he was not jaundiced. Nota- bly in his type of disorder there is no significant alteration of hepatic morphology other than bile stasis during attacks of jaundice.

Although the overall process of hepatic unconiugated bilirubin clearance remains un- changed, multicompartmental analysis of the plasma bilirubin-~4C data indicates that indi- vidual steps of the hepatic excretory pathway for unconjugated bilirubin may be altered during severe cholestasis. In patient RB both the fractional rate of hepatic uptake of unconjugated bilirubin from plasma and the relative hepatic storage capacity for unconjugated bilirubin were increased during cholestasis, whereas the fractional removal rate of unconjugated bilirubin from the hepatic unconjugated bilirubin pool was diminished.

The physiological alterations produced by severe cholestasis which would cause such changes in the hepatic excretory steps for un- cor~iugated bilirubin have not been defined in patients with cholestatic disorders. Experimen- tal cholestasis, on the other hand, produces sev- eral effects which might alter the metabolism of unconiugated bilirubin. Following bile duct ligation in the rat there is hypertrophy of the hepatic smooth endoplasmic reticulum, accom- panied by decreased activity of the microsomal biotransformation system (8). Prolonged bile duct ligation in the rat causes a reduction in two hepatic cytoplasmic proteins, Y and Z, that bind various organic anions in vivo and in vitro (9). Deconjugation of conjugated bilirubin, with subsequent reentry of the bilirubin molecule into the unconjugated bilirubin pool, has also been demonstrated during experimen- tal obstructive jaundice (10). The net effect of these different occurrences on the hepatic metabolism of unconjugated bilirubin is difficult to predict, however, and their relevance to the cholestatic process in patient RB is uncertain.

The studies presented here thus indicate that an explanation other than abnormal hepatic

function should be considered when a patient with predominantly cholestatic liver disease shows an increased serum concentration of unconjugated (indirect-reacting) bilirubin. The possibility that increased bilirubin production due to red cell hemolysis is contributing to the uneonjugated hyperbilirubinemia should be en- tertained. For example, in our studies both patients AD and AB were documented to have shortened red cell life spans. It is conceivable that there is also increased bilirubin production by the livers of such patients due to enhanced turnover of hepatic heme compounds, since hypertrophy of the hepatic smooth endoplasmic reticulum does occur in the cholestatic ani- mal (8). In many situations the elevation in the serum unconjugated bilirubin concentration (indirect-reacting fraction) will probably reflect the inaccuracy of the method used to measure serum bilirubin, however, and the methodology must be critically appraised before drawing any inferences about unconjugated bilirubin metabolism in cholestatic disorders based on serum measurements.

REFERENCES

1. Perelli WV, Watson C J: Comparison of the Weber-Schalm method with the Ducci-Watson modification of the Malloy-Evelyn method for serum bilirubin determination. Clin Chem 16:239-246, 1970

2. Berk PD, Howe RB, Bloomer JR, Berlin NI: Studies of bilirubin kinetics in normal adults. J Clin Invest 48:2176-2190, 1969

3. Gutstein S, Alpert S, Arias IM: Studies of hepatic excretory function. IV. Biliary excretion of sulfobromophthalein sodium in a patient with the Dubin-Johnson syndrome and a biliary fis- tula. Isr J Med Sci 4:36-40, 1968

4. Quarfordt SH, Hilderman HL, Valle D, Wad- dell E: Compartmental analysis of sulfobromophthalein transport in normal patients and patients with hepatic dysfunction. Gastroenter- ology 60:246-255, 197l

5. Raymond GD, Galambos JT: Hepatic storage and excretion of bilirubin in man. Am J Gas- troentero155:135 144, 197l


BLOOMER ET AL

6. Weber AP, Schalm L: Quantitative separation and determination of bilirubin and conjugated bilirubin in human serum. Clin Chim Acta 7:805-810, 1962

7. Jendrassik L, Grof P: Vereinfacte photomet- rische methodern zur bestimmung des blut bili- rubins. Biochem A 297:81-89, 1938

8. Schaffner F, Bacchin PG, Hutterer F, Scharn- beck HH, Sarkozi L, Denk H, Popper H: Mechanism of cholestasis. 4. Structural and bio- chemical changes in the liver and serum in rats

after bile duct ligation. Gastroenterology 60:888-897, 1971

9. Reyes H, Levi J, Gatmaitan Z, Arias IM: Stud- ies of Y and Z, two hepatic cytoplasmic organic anion-binding proteins: effect of drugs, chem- icals, hormones, and cholestasis. J Clin Invest 50:2242-2252, 1971

10. Acocella G, Tenconi LT, Armas-Merino R, Raia S, Billing BH: Does deeonjugation of bilirubin glucuronide occur in obstructive.jaundice. Lancet 1:68-69, 1968

14 Digestive Diseases, VoL 19, No. 1 (January 1974)

hepatic clearance of unconjugated bilirubin in cholestatic liver diseases

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