Gilbert's syndrome: diagnosis by typical serum bilirubin pattern

Clinica Chimica Acta, 154 (1986) 41-48 Elsevier

41

CCA 03368

Gilbert’s syndrome: diagnosis by typical serum bilirubin pattern Andreas

Sieg a,*, Adolf Stiehl a, Richard Raedsch a, Dieter Ullrich b, Brigitta Messmer a and Burkhard Kommerell a

a University

of Heidelberg, Department of Medicine, GI - Unit, Heidelberg and b University of Giittingen, Department of Pediatrics, Gijttingen (FRG) (Received June 5th, 1985; revision August 8th, 1985)

Key worak: Gilbert’s syndrome; Bilirubin

Summary

Analysis of serum unconjugated and conjugated bilirubin fractions by routine diazo procedures does not allow a definite diagnosis of Gilbert’s syndrome. By the alkaline methanolysis procedure of Blanckaert followed by thin-layer chromatography we were able to discriminate Gilbert’s syndrome even in the presence of normal serum bilirubin concentrations from healthy subjects, patients with chronic persistant hepatitis and patients with chronic hemolysis. The relative proportion of unconjugated bilirubin in serum was 95 & 2% in patients with Gilbert’s syndrome (n = 28), 84 f 5% in healthy subjects (n = 29) 75 f 6% in patients with chronic persistant hepatitis (n = 7) and 85 f 3% in patients with chronic hemolysis (n = 9). The difference between Gilbert’s syndrome and the control groups with normal or elevated serum bilirubin was highly significant (p < 0.001). In Gilbert’s syndrome, unconjugated bilirubin ranged between 90 and 99%, in healthy subjects between 72 and 90%, in patients with chronic .persistant hepatitis between 68 and 85% and in patients with chronic hemolysis between 81 and 89% of total. An overlap was only seen in one patient with Gilbert’s syndrome and in 2 healthy subjects at the 90% level. We conclude that in most patients with Gilbert’s syndrome provocation tests are no longer necessary.

* To whom correspondence should be addressed at: University of Heidelberg, Medizinische Klinik. Bergheimerstr. 58, D-6900 Heidelberg, FRG. 0009-8981/86/$03.50

0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

42

Introduction

Gilbert’s syndrome is the most common form of familial nonhemolytic unconjugated hyperbilirubinemia with an incidence of 5-7% and a predominance in males [1,2]. The underlying mechanism is defective bilirubin conjugation [3-51 and/or impairment of hepatic uptake [6,7]. The diagnosis of Gilbert’s syndrome so far was based on the demonstration of unconjugated hyperbilirubinemia in patients without evidence of structural liver disease and overt hemolysis [8]. In patients with coincident hemolysis the diagnosis was only possible by evaluation of bilirubin kinetics [6]. Since serum total bilirubin may be normal [9], analysis of bilirubin by routine methods often fails to allow a definite diagnosis of Gilbert’s syndrome. Therefore, provocation tests with nicotinic acid [lo-121 and caloric restriction [13-151 were introduced. The present study was undertaken to evaluate, if diagnosis of Gilbert’s syndrome would be possible by bilirubin analysis with a new thin-layer chromatographic method [16] even in the presence of normal bilirubin concentrations.

Patients and methods

Twenty-eight patients with Gilbert’s syndrome (23 male and 5 female patients aged 15-50 yr; mean, 25) were studied. The diagnosis was based on mild fluctuant unconjugated hyperbilirubinemia, an increase in serum total bilirubin (d&o method) of at least 23.9 pmol/l or 100% after caloric restriction, normal ultrasound scan of the upper abdomen, normal activities of aspartate and alanine amino transferases, alkaline phosphatase, gamma glutamyl transferase, normal total serum bile acids (3.2 f 0.5 pmol/l; normal range, l-9) [17,18] and normal procollagen type-III peptide (7.9 f 0.7 ng/ml; normal, below 15) [19]. Unconjugated hyperbilirubinemia (> 20 pmol/l) was present at least on two occasions over a period of 6 mth. Hemolysis was excluded by normal levels of haptoglobin and lactate dehydrogenase in the serum and the absence of anemia and reticulocytosis. HB,AG was negative in all patients. Blood samples were drawn initially after an overnight fast and after caloric restriction (400 calories/a day) for 48 h. Three groups of subjects with normal or slightly elevated serum bilirubin served as controls. 1. Twenty-nine healthy volunteers (14 females and 15 males aged 16-60 yr; mean, 27) with normal activities of aspartate and alanine amino transferases, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase, normal total and direct serum bilirubin and normal red blood count. There was no history of liver or hematologic disease, excessive alcohol intake or drug consumption. 2. Seven patients (2 females and 5 males, age range 47-63 yr; mean age, 53 yr) with histologically confirmed chronic persistant hepatitis. The activities of aspartate (26 f 12 U/l) and alanine amino transferase (32 f 15 U/l) and of total bilirubin (19.2 f 6.0 pmol/l) in the serum were slightly elevated. 3. Nine patients (3 females and 6 males, age range 45-72 yr, mean age 57 yr) with chronic hemolysis due to

43

mitral or aortic valve prostheses. Right-sided heart failure with hepatic congestion was absent in all patients. Activities of aspartate and alanine amino transferases, alkaline phosphatase and gamma glutamyl transferase in the serum were normal. All patients showed an increased activity of lactate dehydrogenase (379 5 56 U/l, normal < 240), reticulocytosis (14 &-2560) and normal or slightly elevated serum total bilirubin (16.7 f 4.0 pmol/l, normal < 17.1). Total and direct serum bilirubin concentrations were determined by diazo methods [20]. The relative amounts of unconjugated bilirubin and bilirubin conjugates were measured by alkaline methanolysis followed by thin-layer chromatography [16] and expressed as % of total bilirubin. The identity of the reaction products (unconjugated bilirubin, bilirubin monoconjugates and bilirubin diconjugates) was demonstrated by chromatographic analysis of the ethyl anthranilate azo derivatives [16]. The reproducibility of the alkaline methanolysis was reassessed from 10 analyses of a single serum within-day or day-to-day (during 2 wk). For the day-to-day experiments, serum was partitioned into single portions and kept at - 20°C under N,. The concentration of total (10.3 pmol/l) and direct bilirubin (3.4 pmol/l) did not change during storage. In the experiments within-day and day-to-day we found 80 and 82% respectively, of unconjugated bilirubin, 11 and 12%, respectively, of bilirubin monoconjugates and 8 and 78, respectively, of bilirubin diconjugates. The coefficients of variation (CV) for unconjugated bilirubin were 2.6 and 1.5% respectively, for bilirubin monoconjugates 5.7 and 9.78, respectively, and for bilirubin diconjugates 10.5 and 5.4%, respectively (within-day vs. day-to-day). Thus, the precision of this method was even slightly better than that reported for alkaline methanolysis followed by high performance liquid chromatography [21,22]. Interference of carotenoids was excluded by analysis of sera from 4 healthy subjects before and after excessive ingestion of carotenoids (20 mg/day). No significant change of the serum bilirubin fractions was found after intake of carotenoids. Serum total bile acids were measured enzymatically [23]. Procollagen type-III peptide in serum was determined by radioimmunoassay [24]. The rank sum test of Wilcoxon, Mann and Whitney was used for statistical analysis. The results are expressed as mean values + 1 SD, unless otherwise specified. Results

Serum total bilirubin, determined by diazo methods, was 10.9 f 2.6 pmol/l in healthy subjects, 19.2 f 6.0 pmol/l in patients with chronic persistant hepatitis, 16.7 f 4.0 pmol/l in patients with chronic hemolysis, 27.2 + 13.1 pmol/l in patients with Gilbert’s syndrome fasting overnight and 56.0 f 22.0 pmol/l in Gilbert’s syndrome after 48 h caloric restriction (Table I). The bilirubin concentration was normal in 7 out of 28 patients with Gilbert’s syndrome fasting overnight. Unconjugated bilirubin, as calculated from diazo determination of total bilirubin plus fractionation by alkaline methanolysis followed by thin-layer chromatography, was 9.2 f 2.4 pmol/l in normal subjects, 14.5 f 4.8 pmol/l in patients with chronic persistant hepatitis, 14.2 f 3.0 pmol/l in patients with chronic hemolysis, 26.0 k 12.8

44 TABLE

I

Total bilirubin, unconjugated bilirubin and conjugated bilirubina in serum (X f SD) of healthy and patients with chronic persistant hepatitis, chronic hemolysis and Gilbert’s syndrome.

Total bilirubin (pmol/l) Unconjugated bilirubin (umol/) Conjugated bilirubin (pmol/l)

Healthy subjects (n=29)

Chronic persistant hepatitis (n=7)

Chronic hemolysis (n=9)

10.9 + 2.6 *

19.2 f 6.0 “’

16.7+4.0

9.2 f 2.4 *

14.5 f 4.8 **

14.2 f 3.0 *

1.7*0.6

“’

4.7+1.9

*,***

2.5f0.9

**

*,****

subjects

Gilbert’s fasting overnight (n=28)

Gilbert’s after 48 h caloric restriction (n = 28)

27.2k13.1

56.0+22*

26.0 f 12.8

53.6 f 21.9 *

1.2*

0.5

2.2+

0.7 *

a Unconjugated and conjugated bilirubin were calculated from diazo determination of total bilirubin plus fractionation by alkaline methanolysis followed by thin-layer chromatography. * p < 0.001 (vs. Gilbert’s fasting overnight); ** p < 0.01 (vs. Gilbert’s fasting overnight); *** p < 0.001 (vs. controls); **** p < 0.02 (vs. controls); ns, not significant (vs. Gilbert’s fasting overnight).

pmol/l in patients with Gilbert’s syndrome fasting overnight, and 53.6 f 21.9 pmol/l after 48 h caloric restriction (Table I). The mean increase of unconjugated bilirubin in patients with Gilbert’s syndrome after caloric restriction was 116%. The mean conjugated bilirubin concentration, calculated from diazo determination of total bilirubin plus fractionation by alkaline methanolysis followed by thin-layer chromatography, was comparable in Gilbert’s syndrome fasting overnight and healthy subjects (1.2 + 0.5 vs. 1.7 f 0.6 pmol/l, not significant). In patients with chronic persistant hepatitis and chronic hemolysis serum conjugated bilirubin was significantly elevated (4.7 f 1.9 and 2.5 * 0.9 pmol/l, respectively) compared to healthy controls and patients with Gilbert’s syndrome fasting overnight (Table I). After 48 h caloric restriction, serum conjugated bilirubin in Gilbert’s syndrome increased significantly to 2.2 + 0.7 pmol/l (Table I). The relative proportion of unconjugated bilirubin, measured by thin-layer chromatography after alkaline methanolysis was 84 f 5% in healthy subjects, 75 f 6% in patients with chronic persistant hepatitis, 85 k 3% in patients with chronic hemolysis, 95 + 2% in Gilbert’s syndrome fasting overnight, and 96 &-2% in Gilbert’s syndrome after 48 h caloric restriction (Fig. 1). The difference between patients with Gilbert’s syndrome and the control groups (healthy subjects, chronic persistant hepatitis and chronic hemolysis) was highly significant (p < 0.001). The fraction of unconjugated bilirubin in healthy volunteers ranged between 72 and 908, in patients with chronic persistant hepatitis between 68 and 858, in patients with chronic hemolysis between 81 and 89%, and in patients with Gilbert’s syndrome between 90 and 99%. An overlap occurred at the 90% level in 2 healthy subjects and in 1 patient with Gilbert’s syndrome. No correlation was found between the concentration of total serum bilirubin and the fraction of unconjugated bilirubin. In contrast, the relative proportion of unconjugated bilirubin as calculated by the less accurate diazo methods ((TB - DB/TB) X 100) was 72 f 17% in healthy sub-

loo-

5

90

E In

z 3

.. A-d ‘.

..

........... ::

:: .

.:.

So.

3Q

:. :

. . .

.: .

70

..... ..............

4

. . . . . .

.“. . . . . .

-z-.. ...

:

.

. .

. . .

I

-

NORMAL

CtMX4lC

CHRONlC

GILBERT’S

GILBERT’S

SUBJECTS

PERSISTANT

HEMOLYSlS

FASTING

AFTER

OVERNlGHT

CALORlC

HEPATITIS I

I P < 0.001

vs.

I

48h

RESTRlCTlON

GILBERT’S

Fig. 1. Individual and mean values of serum unconjugated bilirubin fractions (UCB, % of total), determined by alkaline methanolysis followed by thin-layer chromatography, in 29 healthy subjects, 7 patients with chronic persistant hepatitis, 9 patients with chronic hemolysis and 28 patients with Gilbert’s syndrome.

jects, 75 f 13% in patients with Gilbert’s syndrome fasting overnight, and 81 * 15% in patients with Gilbert’s syndrome after 48 h caloric restriction. These differences were not significant. Discussion

The fractionation of serum bilirubin into unconjugated and conjugated bilirubin is useful in the evaluation of patients with hyperbilirubinemia. However, fractionation of pigments by diazo procedures is not sufficient accurate to allow a definite diagnosis of Gilbert’s syndrome [25]. Therefore, provocation tests were introduced, and the diagnosis in questionable cases is mainly based upon them [lo-15,261. In the present study, patients with Gilbert’s syndrome were discriminated from healthy subjects and patients with chronic persistant hepatitis and chronic hemolysis by a single bilirubin analysis without the need of provocation tests. The fractionation into unconjugated and conjugated bilirubin was performed by thin-layer chromatography after alkaline methanolysis [16]. An overlap with healthy subjects was only seen in one of 28 patients with Gilbert’s syndrome at the 90% level of the serum unconjugated bilirubin fraction. All other patients with Gilbert’s syndrome had unconjugated bilirubin fractions above 90%, whereas healthy subjects had values between 72 and 908, patients with chronic persistant hepatitis between 68 and 85% and patients with chronic hemolysis between 81 and 89%. The unconjugated bilirubin fraction and the level of total bilirubin did not correlate, and the discrimination between Gilbert’s syndrome and healthy subjects or patients with unconjugated hyperbilirubinemia due to chronic persistant hepatitis and chronic hemolysis was even possible in patients with normal bilirubin concentrations. This

46

discrimination was hitherto impossible because of inaccurate methods for serum bilirubin fractionation [27]. As expected, patients with Gilbert’s syndrome (i.e. defective clearance of unconjugated bilirubin) had higher concentrations of unconjugated bilirubin in serum than healthy controls, but levels of conjugated bilirubin were comparable. After caloric restriction serum unconjugated and conjugated bilirubin increased significantly. The increase of bilirubin conjugates was not detected in studies employing diazo methods [14,25], but is compatible with the finding of conjugated hyperbilirubinemia in fasting rats [28]. It may reflect reduced elimination of bilirubin conjugates by fasting. However, owing to the low concentration of bilirubin conjugates, the significance of this finding is uncertain, although the concentration of pigments was clearly above the limits of detection which are below 0.8 pmol/l. We conclude that the diagnosis of Gilbert’s syndrome should be based on mild unconjugated hyperbilirubinemia with a fraction of serum unconjugated bilirubin above 9055, determined by alkaline methanolysis followed by thin-layer chromatography [16], normal liver enzymes and normal concentrations of serum bile acids [17,18]. Provocation tests seem no longer necessary in the diagnosis of Gilbert’s syndrome. Acknowledgement

This work was supported in part by the Deutsche Forschungsgemeinschaft Si 273).

(Grant

References 1 Foulk WT, Butt HR, Gwen CA, Withcomb FF, Mason HL. Constitutional hepatic dysfunction (Gilbert’s disease): its natural history and related syndromes. Medicine 1959; 38: 25-46. 2 Owens D, Evans J. Population studies on Gilbert’s syndrome. J Med Genet 1975; 12: 152-156. 3 Black M, Billing BH. Hepatic bilirubin UDP-glucuronyltransferase activity in liver disease and Gilbert’s syndrome. N Engl J Med 1969; 280: 1266-1271. 4 Felsher BF, Craig JR, Carpio N. Hepatic bilirubin glucuronidation in Gilbert’s syndrome. J Lab Clin Med 1973; 81: 829-837. 5 Auclair C, Hakim J, Boivin P, Troube H, Boucherot J. Bilirubin and paranitrophenol glucuronyl transferase activities of the liver in patients with Gilbert’s syndrome. Enzyme 1976; 21: 97-107. 6 Berk PD, Bloomer JR, Howe RB, Berlin NI. Constitutional hepatic dysfunction (Gilbert’s syndrome). A new definition based on kinetic studies with unconjugated radiobilirubin. Am J Med 1970: 49: 296-305. 7 Martin JF, Vierling JM, Wolkoff AM, Scharschmidt BF, Vergalla J, Waggoner JG, Berk PD. Abnormal hepatic transport of indocyanine green in Gilbert’s syndrome. Gastroenterol 1976; 70: 385-391. 8 Arias IM. Chronic unconjugated hyperbilirubinemia without overt signs of hemolysis in adolescents and adults. J Clin Invest 1962; 41: 2233-2245. 9 Berk PD. The distribution of serum bihrubin levels in man: implication for the diagnosis of Gilbert’s syndrome. In: Okolicsanyi L, ed. Familial disorders of hepatic bilirubin metabolism. Chichester: J. Wiley and Sons, 1981: 7 ff. 10 Fromke VL, Miller D. Constitutional hepatic dysfunction (CHD; Gilbert’s disease); a review with special reference to a characteristic increase and prolongation of the hyperbilirubinemic response to nicotinic acid. Medicine 1972; 51: 451-464.

47 11 Davidson AR, Rojas-Bueno A, Thompson RPH, Williams R. Reduced caloric intake and nicotinic acid provocation tests in the diagnosis of Gilbert’s syndrome. Br Med J 1975; 2: 480. 12 Rollinghoff W, Paumgartner G, Preisig R. Nicotinic acid test in the diagnosis of Gilbert’s syndrome: correlation with bilirubin clearance. Gut 1981; 22: 663-668. 13 Barret PVD. Hyperbilirubinemia of fasting. J Am Med Assoc 1971; 217: 1349-1353. 14 Owens D, Sherlock S. Diagnosis of Gilbert’s syndrome: role of reduced caloric intake test. Br Med J 1973; 3: 559-563. 15 Felsher BF, Carpio NM. Caloric intake and unconjugated hyperbilirubinemia. Gastroenterology 1975: 69: 42-47. 16 Blanckaert N. Analysis of bilirubin and bilirubin mono- and diconjugates. Determination of their relative amounts in biological samples. Biochem J 1980; 185: 115-128. 17 Briheim G, Fryden A, Tobiasson P. Serum bile acids in Gilbert’s syndrome before and after reduced caloric intake. Scand J Gastroenterol 1982; 17: 877-880. 18 Vierling JM, Berk PD. Hofmann AF, Martin JF, Wolkoff AW, Scharschmidt BF. Normal fasting-state levels of serum cholylconjugated bile acids in Gilbert’s syndrome: an aid to the diagnosis. Hepatology 1982; 2: 340-343. 19 Raedsch R, Stiehl A, Sieg A, Walker S, Kommerell B. Serumprokollagentyp III-Peptidkonzentrationen bei Patienten mit Morbus Gilbert-Meulengracht. Verh Dtsch Ges Inn Med 1984; 90: 1572, 1573 20 Jendrassik L, Grof P. Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins. Biochem Z 1938; 297: 81-89. 21 Blanckaert N, Kabra PM, Farina FA, Stafford BE, Marton LJ, Schmid R. Measurement of bilirubin and its monoconjugates and diconjugates in human serum by alkaline methanolysis and high-performance liquid chromatography. J Lab Clin Med 1980; 96: 198-212. 22 Muraca M. Blanckaert N. Liquid-chromatographic assay and identification of mono- and diester conjugates of bilirubin in normal serum. Clin Chem 1983: 29: 1767-1771. 23 Talalay P. Enzymatic analysis of steroid hormones. Methods Biochem Anal 1960; 8: 119-128. 24 Rohde H, Vargas L, Hahn E. Kalbfleisch H, Bruguera M, Timpl R. Radioimmunoassay for type III-procollagen-peptide and its application to human liver disease. Eur J Clin Invest 1979; 9: 451-459. 25 Thomson HF. Hardt F. Juhl E. Diagnosis of Gilbert’s syndrome. Reliability of the caloric restriction and phenobarbital stimulation tests. Stand J Gastroenterol 1981; 16: 699-703 26 Gentile S, Orzes N, Persico M, Marmo R, Bronzino P, Tiribelli C. Comparison of nicotinic-acid and caloric restriction-induced hyperbilirubinemia in the diagnosis of Gilbert’s syndrome. J Hepatol 1985; 1: 537-543. 27 Killenberg PG. Stevens RD, Wildermann RF, Wildermann NM. The laboratory method as a variable in the interpretation of serum bilirubin fractionation. Gastroenterology 1980: 78: 1011-1015. 28 Rosenthal P, Blanckaert N, Kabra P. Thaler M. Conjugated hyperbilirubinemia of fasting. Gastroenterology 1980; 79: 1050(A).