Constitutional hepatic dysfunction (Gilbert's syndrome)

CLINICAL STUDIES

Constitutional Hepatic Dysfunction (Gilbert's Syndrome) A New Definition Based on Kinetic Studies with Unconjugated Radiobilirubin

PAUL D. BERK, M.D. JOSEPH R. BLOOMER, M.D. ROBERT B. HOWE, M.D.r NATHANIEL I. BERLIN, M.D.

Bethesda, Maryland

From the Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. Requests for reprints should be addressed to Dr. Paul Berk, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. Manuscript received November 20, 1969. ~'Present address: Department of Medicine, University of Minnesota Hospitals, 412 Union Street, S.E., Minneapolis, Minnesota 55455.

296

Plasma clearance studies with unconjugated radiobilirubin were performed in eleven patients who had chronic unconjugated hyperbilirubinemia in the absence of structural liver disease. An abnormal clearance pattern, characterized by increased retention of isotope in the plasma at four hours (24.2 ~+ 8.4 per cent, mean ~ standard deviation; normal = 5.0 ~+ 1.9 per cent) and a reduction in hepatic bilirubin clearance (16 +~ 5 ml/minute; normal -- 47 • 10 ml/minute), was observed in all eleven cases, despite the fact that the mean red cell survival ranged from normal (112 days) to severely reduced (eleven days). Multicompartmental analysis suggests that the reduced hepatic bilirubin clearance is the result of a defect in both hepatic bilirubin uptake and conjugation. Twenty-one studies in a control group with anemia and/or hemolysis were indistinguishable from normal, indicating that neither of these factors was responsible for the abnormalities noted. These studies suggest that current diagnostic criteria for Gilbert's syndrome, which exclude patients with hemolysis, will result in an artificial distinction among patients with the same basic hepatic abnormality. The syndrome of constitutional hepatic dysfunction (Gilbert's syndrome) is characterized by the presence of chronic, low grade unconjugated hyperbilirubinemia in otherwise healthy persons. The diagnosis is usually restricted to patients in whom results of other standard tests of liver function are normal and in whom there is no evidence of overt hemolysis [1]. Because the concentration of bilirubin in the plasma is frequently below the level which causes clinical jaundice, the condition is often discovered fortuitously during the course of routine laboratory examinations, and many cases presumably go unrecognized. Accordingly, its exact incidence in the general population is unknown, although Kornberg [2] observed eight probable cases in a group of 100 medical students and physicians. The hyperbilirubinemia in this condition is presumed to result from an abnormality in the clearance of unconjugated bilirubin from plasma by the liver, and such an abnormality has, in fact, been demonstrated by the injection of either a large dose of unlabeled bilirubin [3] or a tracer dose of bilirubin-'4C [4]. The underlying lesion has been variously attributed to a defect in either hepatic bilirubin uptake [3,5] or glucuronide conjugation within the liver cell [6,7]. Although patients with overt hemolytic disease are usually excluded--by definition--from this diagnostic category, the precise relationship of hemolysis and Gilbert's syndrome remains unclear. Recent reports indicate that a mild hemolytic process which escaped clinical detection could be demonstrated in approximately 50 per cent of the cases of Gilbert's syndrome by means of red blood cell survival studies using radioactive labels [8,9]. This report describes the results of plasma clearance studies with isotopic bilirubin in eleven patients with chronic unconjugated hyperbilirubinemia in whom significant structural liver disease was ruled out by appropriate studies including liver biopsy. A uniform and characteristically abnormal pattern,

The AmericanJournalof Medicine

GILBERT'S SYNDROME: A NEW DEFINITION - - BERK ET AL.

TABLE I

Preliminary Data in Eleven Patients with Chronic Unconjugated Hyperbilirubinemia (Group I)

Case No.

Age (yr) and Sex

Weight (kg)

1

61,M

74.3

2 3 4 5 6a 6b 7

16,M 57,M 40,M 19,M 43,M 44,M 32,M

54.5 81.6 9].0 70.0 71.3 72.2 83.2

13 54 36 15 12 -30

8

18,M

64.1

9

14,M

46.6

10 11a 11b

33,F 46,M 47,M

62.1 66.6 58,7

OnseV~

Palpable Spleen

Mid 30's Negative

Total Red Cell Hemato- Vo]umet crit (cc/kg)

ReticuIocytes (%)

RBC-T89 t (days)

Myeloid: Erythroid Ratio

0.40

25.1

0.4

27.5

1.1:1

Tip Negative Negative Tip Tip Negative Negative

0.46 0.44 0.49 0.42 0.45 0.42 0.42

33.6 36.1 30.5 29.3 28.4 28.2 24.4

0.6 1.0 0.5 1.0 1.7 1.3 1.8

-28.5 32.9 (79)w 18.3 -(58)~

].0:1 1.4:1 2.6:1 "Normal" 2.1:1 -0.9:1

17

Negative

0.39

21.4

2.1

21.0

1.1:1

Birth

Tip

0.37

27.1

5.5

8.5

1.0:1

0.29 0.27 0.42

17.1 18.8 24.9

11.5 11.2 0.8

8.0,(16)w 7.1 19.6,(54)w

1.2:1 0.2:1 --

14 Negativet Childhood 12 cm -Negative-~

RBC Osmotic Fragility Slightly increased $ Normal Normal Normal Slightly increased $

Slightly i ncrea sed .I:. Increased $ Increased

Peripheral Blood Smear Normal Normal Normal Normal Normal Normal Normal Normal Mild polychromatophilia Polychromatophilia Spherocytosis Spherocytosis Spherocytosis

~:'Age at which condition was first detected. tDetermined with ~'Cr-labeled red blood cells (RBC). Abnormality demonstrable only after twenty-four hours incubation in saline solution. w Mean red cell lifespan determined with tritiated diisopropyl fluorophosphate (DFP-3H). c o n s i s t i n g of m a r k e d l y increased r e t e n t i o n of i s o t o p e in the p l a s m a at f o u r hours a n d a reduced h e p a t i c bilirubin clearance, was o b s e r v e d in all eleven p a t i e n t s despite the fact t h a t red cell survival r a n g e d f r o m n o r m a l to severely reduced. C o m p u t e r analysis of the data in t e r m s of a m u l t i c o m p a r t m e n t a l m o d e l of bilirubin m e t a b o l i s m [ 1 0 ] indicates t h a t t h e reduced hepatic bilirubin c l e a r a n c e resulted f r o m defects in both h e p a t i c bilirubin u p t a k e and c o n j u g a t i o n . These studies suggest t h a t the c u r r e n t d i a g n o s t i c criteria for Gilb e r t ' s s y n d r o m e , w h i c h exclude p a t i e n t s with hemolysis, m a y result in an artificial d i s t i n c t i o n a m o n g p a t i e n t s w i t h the s a m e basic defect in hepatic f u n c t i o n .

MATERIALS A N D M E T H O D S Patients. Studies were performed in e~even consecutive patients admitted to the Metabolism Branch of the National Cancer Institute for evaluation of chronic, unconjugated hyperbilirubinemia. These patients are designated group I, and the salient clinical data in each case are presented in Table I. In addition to the data in the table, results of the following studies were normal in all instances: serum glutamic oxa!acetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphatase, serum albumin concentration, protei n electrophoresis and bromsulfalein clearance ( ~ 5 per cent retention of 5 m g / k g dose at forty-five minutes). Two patients were studied twice. In one patient (Case 6) the studies were one year apart, during which time the patient's clinical status was unchanged. The other pa'~ient (Case 11) underwent splenectomy four months after his initial bilirubin-!4C clearance study. A repeat study was performed three months postoperatively. A liver biopsy specimen was obtained from each of the nine adult patients. In seven instances the specimen was obtained percuta-

Volume 49, September 1970

neously, using the Menghini technic. In two patients (Cases 6 and 11) a wedge biopsy specimen was obtained during laparotomy for a coexisting condition. A follow-up biopsy specimen was obtained utilizing the Menghini technic one year after surgery in one of these (Case 6). Bilirubin-'4C clearance data from forty additional studies are presented for comparison with the results obtained in group I. Group II consists of nineteen previously published studies in a group of healthy, young normal volunteer subjects [10]. Group Ill includes twenty-one studies in patients admitted to the Clinical Center of the National Institutes of Health with a variety of hematologic a n d / o r neoplastic diseases. Data obtained in patients known to have significant structural liver disease or who were receiving therapeutic drugs known to be hepatotoxic were excluded from this group. Studies in group III were performed to determine the effects of bilirubin overproduction (from hemolysis or increased ineffective erythropoiesis) a n d / o r anemia on the clearance from the plasma of isotopically labeled bilirubin. Preparation of Isotopic Bilirubin. Unconlugated bilirubin-'4C was prepared biosynthetically in bile-fistula dogs from ~}-aminolevulinic acid-4-'~C as previously described [11]. This material was used in all but two of the fifty-three studies reported. For two studies (study b in Cases 6 and 11) bilirubin-3H was prepared by the same technic from 8-aminolevulinic acid-2,3-3H. The use of this precursor results in a labeled bilirubin in which all tritium atoms are in stable positions on the side chains. Studies have shown that the biologic behavior of bilirubin-3H prepared from this precursor is identical with that of bilirubin-'4C and that there is no loss of tritium from the bilirubin molecule in vivo for more than two weeks after injection [12]. All bilirubin used in these studies was purified by column chromatography and repeated crystallization to a molar extinction coefficient of 58,000 to 61,000. The dose employed ranged from 0.8 to 3.0/~c of bilirubin-'4C or 12 to 35/~c of bilirubin-3H. The actual mass of injected bi]Jrubin never exceeded 500 #g.

297

GILBERT'S SYNDROME: A NEW DEFINITION -- BERK ET AL.

(3)

(1) 31 ~

~//[VASCULAR)

~13

S PLAS MA'X~~12 = ~ POOL ~ /~u

(2)

I [\~\CONJUGATED/ /~

=- HEPAT C

/

X o2

HEPATIC \

~CONJUGATED I \ POOL \

Plasma Bilirubin- 'C and -3H Clearance Studies. Clearance studies with unconjugated radiobilirubin and measurements of fecal and urine radioactivity were performed according to our previously published modification [10] of the method of Barrett et al. [4]. Because of the slower rate of remora of bilirubin from the plasma, we were able to obtain data for a tonger period (forty-eight to ninety-six hours) n group I oatients than iF Subjects with normal bilirubin kinetics given similar doses of isotope (twenty-four to thirty hours). However. the terminal exponential component of the plasma clearance curve n group I was wel defined by thirty hours, so that the additional data obtained in these studies ao not affect the calculated results for comparisons between groups. Total Red Cell Volume and Survival, The ~ Cr method of Sterling and Gray [13] as modified by Read [14] was used to measure the total red cell volume. Plasma volume was calculated from the total red cel volume and peripheral venous hematocrit. After labeling of the red cells, blood samples were drawn three times a week for four weeks to determine the ~ Cr-labeled red cell half ife (T1/2). In four oatients, red blood cell surwva was measured using tritiated diisopropylfluorophosphate (DFP-3H) as a red cel abel E15]. Data Analysis. Data processing was performed on a Univac 1108 d~gital computer, using the SAAM program of Berman and Weiss [16-19]. As previously described [10], the alasma unconjugatea radiobilirubin clearance curves were initially fitted to sums of two three and four exponentials. In a the studies, three exponentials were both necessary and sufficient to describe the data [4,17], From the coefficients and rate constants of the computer-fitted three exponential function and the mean plasma concentration of unconjugated bilirubin (BR), the following physiologic oarameters were calculated [10]: (a), VDBR. the initia distribution volume o1 the rejected labeled bilirubin: (b) M the mass of the intravascular unconlugated bilirubin poot; (c) k,, the fraction of M irreversibly cleared per minute by the liver; (d) CBR,the hepatic bilirubin clearance: (e) the efficiency of bilirubin extraction by the liver and (f) BRP. the bilirubin production rate. The mean red blood cell life span (RBCLS) was calculated from BRP and the tota red cel volume by assuming a 15 per cent contribution to BRP from sources other than the catabolism of the hemoglobin of circulating reo cells [20]. These calculations are independent of any particular compartmental model of bilirubin metabolism [21]. In an attempt to gain further insight into the defects whicn result in the abnormal pattern of bilirubin clearance observed in group I. the olasma clearance data were also used for computer calculation of the parammers of a multicom~)artmental model of bilirubin metabolism (Fig. t). The reasons for the choice of this Darticular model have Deen presented n an earlier oublication [10]. n this system, plasma unconjugated bilirubin (compartment 1) is considered to exchange with an extravascular pool (compartment 3) and a hepat)c pool (compartment 2) of unconjugated bilirubin. Unconjugated bilirubin Is assumed to be eliminated from the system solely from compartment 2. presumab y by conjugation. Alternate minor oathways of bilirubin elimination were not considered. The model can be described by a set of linear first order differential

298

/

/

Fig. 1, Proposed three-compartment model of unconjugated bilirubin metabolism. The fractional transfer rates (~.'s) between the compartments and the sizes of pools 1, 2 and 3 are calculated by computer from the p!asma disappearance curye of u nconjugated radiobilirubin and plasma concentration of unconjugated bilirubin.

equations [10]. From the data, the solutions for the equations and the steady state conditions, values for the following parameters of physiologic interest can be obtained: (a) M,, M 2 and M~--the sizes of the plasma, hepatic and extravascular pools of unconjugated bilirubin; (b) the fractional transfer rates (;~ii) between compartments; (c) ko, C,R and the efficiency of hepatic bilirubin extraction; (d) K2,, the rate of hepatic bilirubin uptake; (e) MCBRL and MCR, estimates of the minimal intrahepatic bilirubin concentration and the liver:plasma concentration gradient; (f) BRP and (g) RBCLS. In addition, the model was used for computer calculation of the isotope content of the hepatic and extrahepatic:extravascular pools, as well as the cumulative fraction of injected radiobilirubin conjugated at various times after injection. Values of M,, ke, CBR, BRP and RBCLS are identical whether calculated directly from the parameters of the bilirubin clearance function or by. compartmental analysis [10]. The values of the model parameters for each of the thirteen studies in group I patients were averaged to get a weighted group mean for each parameter. The weighting procedure has been described previously [22] and has the effect of increasing the contribution of precisely defined data to the calculated mean value while reducing the contribution of data which are less precisely defined9 In all comparisons between groups, Students t test was employed. A difference between values was considered to be significant if the likelihood of its arising by chance was less than 5 per cent (p < 0.05). In several of the studies in group III, data were only available over a six to eight hour period. This led to relatively large uncertain: ties in the computer-calculated values of the derived physiologic parameters. Since the available data in these studies were indistinguishable from normal in terms of slopes, intercepts and the per cent Of injected isotope retained in plasma at four hoUrs, convergence was assisted bY providing the computer with additional information in the form of a variance-cOvariance matrix for the normal population. The equations for this procedure have been published previously [16]. A detailed outline of the mathematical technics employed in the current study as well as a review of the implicit assumptions involved was prepared as an appendix to a previous communication [10] and is available to interested readers. ~ RESULTS Preliminary Data, Eight p a t i e n t s (Cases 1 t h r o u g h 8) m e t the usual d i a g n o s t i c criteria for Gilbert's s y n d r o m e [8]. A l t h o u g h t h e spleen was p a l p a b l e on at least o n e occasion in t h r e e of t h e s e patients, s i g n i f i c a n t s p l e n o m e g a ! y was ruled out in each i n s t a n c e by e i t h e r a p p r o p r i a t e r a d i o i s o t o p e s c a n n i n g pro(:edures or x-ray studies, Or in o n e case, at ':: To obtain this appendix, order NAPS Document 00555 from ASIS, National Auxiliary Publications Service, c/o Information Sciences, Inc., 909 Third Avenue, New York, New York 10022; remitting $1.00 for microfilm or $3.00 for photocopies.

The American Journal of Medicine

GILBERT'S SYNDROME:A NEW DEFINITION-- BERK ET AL.

microscopy revealed no abnormalities of cellular morphology or Iobular architecture, no fibrosis and no evidence of hepatitis in any of the specimens obtained. A uniform, but nonspecific finding was the presence in all cases of moderate amounts of golden brown, granular lipofuscin pigment within the hepatic cord cells. A similar finding has been described previously in several cases of chronic unconjugated hyperbilirubinemia [24,25] and in a variety of miscellaneous Conditions [26-28]. In addition to this material, increased amounts of stainable iron were found in both hepatic cord cells and Kupffer cells in two patients (Cases 10 and 11) in whom overt hemolysis was associated with congenital, spherocytic hemolytic anemia. This is not an uncommon finding in the presence of active hemolysis [29]. The degree of iron deposition was insufficient to produce abnormalities in any of the usual tests of liver function cited and recorded in Table I. In addition, since several of the patients with overt hemolysis but normal bilirubin kinetics (group III) had much more marked hepatic hemosiderosis, it is our opinion that this finding does not explain the abnormalities in bilirubin kinetics observed in these two patients (Cases 10 and 11). A detailed description of the light and electron microscopic studies of this group of liver biopsy specimens will be reported elsewhere, t Kinetic Studies. A representative plasma bilirubin-'"C clearance curve (Case 3) is illustrated in Figure 2. The data for each study (expressed as fraction of dose/ml of plasma) were fitted to a three exponential function of the form q(t) = K(A,,~ -~,t + A,2e--2 t + A,~e-,,t)whereA,, + A,2 + A,3 = 1. K is a proportionality constant equal to the reciprocal of the initial volume of distribution of the injected labeled bilirubin. Values (mean +~ standard deviation) for the A's, ~'s and associated half-times for group I are presented in Table II together with the corresponding normal values. The model-independent parameters derived from these data and from the mean plasma concentration of unconjugated bilirubin are presented in Table III.

laparotomy. Four of these patients had mild shortening of the red cell survival as determined with either DFP-~H or Cr -5' (Table I). In previous reports a similar incidence of mild, compensated hemolysis has been noted in patients thought to have Gilbert's syndrome [8,9]. The remaining three patients in group I had overt hemo!ytic anemia. One patient (Case 9) had an undefined congenital nonspherocytic hemolytic anemia. Extensive evaluation elsewhere had failed to document a red cell enzyme defect. Two patients (Cases 10 and 11) had hereditary spherocytosis. One patient (Case 10) had recurrent hemolysis fourteen years after splenectomy~ presumably due to the presence of an accessory spleen. Another patient (Case 11) was studied on two occasions, before and three months after splenectomy: The effect of splenectomy on his red cell survival is clearly indicated in Table I. Liver Biopsies. The two livers observed at lapar0tomy and all the liver biopsy specimens Were grossly normal. None had the black coloration reported in association with the Dubin-Johnson syndrome [23]. Except for the initiaJ biopsy in one patient (Case 6), ;:' histologic examination under light

".'At the time of his initial biopsy this pa{ient was forty-three yeai's old and had had asymptomatic unconjugated hyperbilirublnemia for about thirty years. In the course of a laparotomy for removal of colonic polyps, a wedge biopsy specimen was obtained from the right lobe of the liver. It showed many focal noncaseating granulomas in addition to the pigment described. A histoplasmin skin test was strongly positive; purified protein derivative (second strength) and blastomycin and coccidioidin skin tests were negative. Cultures of the liver biopsy specimen, bone marrow, blood and urine failed to isolate a pathogen. The patient was observed for one year without specific therapy, during which time he remained well. At the end of this period, a percutaneous liver biopsy specimen obtained from the right lobe was normal except for the nonspecific pigment, with no evidence of granulomatous inflammation. Studies of bilirubin kinetics at the time of the second biopsy (Table III) were virtually identical to those a year earlier. In view of the thirty year history of asymptomatic hyperbilirubinemia, we believe the finding of transient hepatic granulomatosis to be incidental.

fBarth RF, Grimley PM, Berk PD: Histopathology of Gilbert's syndrome. In preparation.

50,0~-

"G I0.0 5.0

_

"5 o

v

Z

.J

Fig. 2. Representative plasma disappearance curve of bilirubin-14C in a patient with Gilbert's disease (Case 3). Solid line represents the computer fit to a three exponential function. Dotted lines are the individual exponential components.

Volume 49, September 1970

(Y)

\

0.1

/~-:0((

minutes

~ T / = 37 minutes \

0.05 0

t

I

I

I

I

I

4

8

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16

20

24

I,

I

28 32 HOURS

I

I

I

I

I

I

36

40

44

48

52

56

299

GILBERT'S SYNDROME:A NEW DEFINITION-- BERK ET AL.

T A B L E II

Parameters of Plasma Unconjugated Radiobilirubin Clearance Curves in Patients with Chronic Unconjugated Hyperbilirubinemia (Group I) and Normal Volunteer Subjects (Group II)

Parameter'~ Air A42 AI3 GI

Group I (n = 13)t 0.47 .+_ 0.15 o.41 .+_ 0.08 0.12 _+_0.09

Group It (n = 19)t 0.66 ~ 0.17 0.31 --- 0.16 0.03 § 0.02

p$ < 0.01 < 0.05 ~ 0.01

T89

0.024 ~ 0.008 rain ' 31.6 • 9.3 min

0.044 +_ 0.013 min ' 17.7 +_ 7.2 rain

< 0.001 < 0.001

T89

0.0040 +__0.0008 min-' 180 ~ 37 rain

0.010 +_ 0.003 rain ' 81 + 39 rain

< 0.001 < 0.001

T89

0.00080 -4- 0.00020 rain ' 926 .+_.273 min

0.0012 • 0.0002 rain ' 578 4- 132 min

< 0.001 < 0.01

OZ2

~3

~'Values presented are means • 1 standard deviation. -in = number of studies. SDetermined with Student's t test.

Because of the wide variation in red cell survival suggested in Table I, the eleven patients in group I were divided into three subgroups as follows: group la (Cases 1 through 4) had normal values for the chromium-labeled red cell T1/2 (s'Cr-RBC-T1/2), group lb (Cases 5 through 8 and Case 11 after splenectomy) had a moderate shortening of the s'CrRBC-T1/2 or the mean red cell life span as measured with DFP3H, and group ic (Cases 9 and 10 and Case 11 before splenectomy) had a marked reduction in red cell survival indicated by a ~'Cr-RBC-T1/2 of less than ten days. Values of the slopes and intercepts of the plasma bilirubin clearance curves, the percentage of isotope retained in the plasma at four hours and all the derived physiologic constants (ke, C~R, efficiency, and ~xi~)for each of the subclasses were compared. In no instance was a statistically significant difference between the subclasses found for any of these values. Accordingly, the thirteen studies of group I were treated as a homogeneous population for comparison with groups II and III. As indicated in Table Ill, ke (the amount of bilirubin irreversibly lost from the system per minute, expressed as a fraction of the plasma pool) equaled 0 . 0 0 4 4 - - - 0 : 0 0 1 4 (mean _+_ standard deviation) in group I. This corresponds to an extraction efficiency of 1.6 • 0.5 per cent and a hepatic bilirubin clearance of 16 ~ 5 cc/minute. All these values are significantly reduced (p < 0.001) compared to groups II and III. The retention of bilirubin in plasma at four hours was correspondingly increased in group I (24.2 • 8.4) compared with groups II or III ( p < 0.001). On the other hand, there were no significant differences in these parameters between group II (normal subjects) and group III (subjects with hematologic and neoplastic diseases). The observed range of plasma radiobilirubin content during the first thirty hours after injection is illustrated for groups I and II in Figure 3. The data for group III all fell within the range observed for the normal volunteer subjects.

300

As indicated by this figure, determination of the isotope retained in plasma four hours after injection is a simple but precise means of distinguishing subjects in group I from subjects with normal bilirubin kinetics. The relationship of the rate of bilirubin production to the plasma concentration of unconjugated bilirubin is shown in Figure 4. In group II and III, for which k~ and the hepatic bilirubin clearance fell within a restricted range of values, the plasma bilirubin concentration increased as a linear function of the rate of bilirubin production. Even at a near maximal BRP of 32 m g / k g / d a y (eight tO nine times the normal mean), the plasma unconjugated bilirubin concentration was only 3.4 mg percent. In contrast, for any given ra{e of bilirubin production, the plasma concentration of unconjugated bilirubin for group I patients was outside the 95 per cent confidence limits defined by groups II and III. Because bilirubin production rates in excess of 9 m g / k g / d a y were often associated with uncompensated hemolytic states, the effect of anemia on hepatic bilirubin clearance must be considered. All the patients in group Ic were anemic (average hematocrit = 0.31; range = 0.27 to 0.37). All but o n e o f the group III patients with corresponding rates of bilirubin production were also anemic (average hematocrit = 0.32; range = 0.21 to 0.45) but nevertheless had normal patterns of bilirubin clearance: Figure 4 and the associated data clearly demonstrate that neither anemia nor bilirubin overproduction, in and of themselves, produce abnormalities of bilirubin kinetics such as those observed in the patients in grouP I. This is als0 indicated by the reproducibility of those parameters reflecting liver function (e.g., CB,, ke) in the duplicate studies in one patient (Case l r , Table III) despite the marked differences in {he i'ates of hemolysis and bilirubin production at the time of the two studies. A similar degree of reproducibility has been observed in patients with normal bilirubin kinetics when studied at different erythropoietic rates. Figure 4 also indicates that the plasma concentration of

The American Journal of Medicine

GILBERT'S SYNDROME: A NEW DEFINITION - - BERK ET AL.

TABLE III

Model Independent Parameters of Hepatic Function and Red Cell Survival "` Plasma Bilirubin Concentration (mg %)t

Case No.

Unconlugated

Total

M, (rag)

ko (min-')

C,, (ml/min)

1 2 3 4

0.80 1.97 1.92 1.37 1.52

0.84 2.24 2.02 1.59 1.67

34.8 63.1 80.0 48.6 56.6

0.0047 0.0035 0.0035 0.0058 0.0044

20.5 11.2 14.7 20.6 16.8

1.7 1.3 1.3 2.1 1,6

5 6a 6b 7 8 11b

1.70 3.23 3.29 2.14 2.26 1.28 2.32

2.00 3.62 3.49 2.49 2.66 1.47 2.62

54.6 135.7 138.4 60.2 89.7 44.9 87.2

Group Ib 0.0056 0.0023 0.0024 0.0058 0.0052 0.0058 0.0045

18.1 9.8 10.0 16.3 18.9 20.3 15.6

9 l0 lla

825 2.98 6.50

8.61 3.58 7.11

194.6 119.1 223.0

Group Ic 0.0021 0.0052 0.0052

5,91

6,43

178.9

0.0042

2.90 (0.80-8.25)$

3.19 (0.84-8.61)$

99.0 (34.8-223.0)$

0.0044• 0.0014

Group I1

047 ~ 0.09

0.47 ~ 0.10

13.2 --- 5.0

Group Ill

0,90 (0,18-3.41)$

1.53 (0.29-4.58)$

32.5 (5.7-90.1)$

Plasma/ 4 Hours Retention (%)

Efficiency (%)

BRP (mg/kg/day)

RBCLS (days)

18.8 28.3 30,8 18.0 24.0

3.2 5.8 5.0 &5 &6

112 81 103 97 98

2.0 0.8 0.0 2.1 1.9 2,1 1.6

21.0 40,9 34.0 18.8 19,8 13.2 24.6

6.3 6.4 6.6 6,1 9,6 6,4 6.9

65 63 61 57 32 55 56

5.0 20.7 17.9

0,8 1.9 1.9

35.8 19.0 16.2

12.8 14,3 25.2

30 17 1]

14,5

1.5

23.7

17.4

19

16 -- 5

1.6 • 0.5

24.2 -- 8.4

8.6 (3.2-25.2)$

60 (]1-112)$

0.015 § 0.004

47 • 10

5.4 •

1.4

5.0 • 1.9

3.8 • 0.6

101 • 13

0.015 • 0.005

54 § 20

5.4 • 1,8

4.4 --- 2.0

11.9 (2.4-32.3)$

45 (8-119)$

Group la

Mean

Mean

Mean

Mean § Standard Deviation (Totals) Group I

':'See text for definition of symbols.

CMean of at least eight determinations.

:t-Observed range. Distribution skewed by variations in BRP.

I.O

0.3

z ---.-J

0.I

(33 t

o~ ~ 0.03 Q::~--

o.oi 0

Fig. 3. Observed ranges of plasma radiobilirubin disappearance curves in patients with chronic unconjugated hyperbilirubinemia (group I, s tippled) and in healthy, young adult volunteer subjects (group II, diagonal shading). The average curve for each group was calculated by computer from the mean values of the intercompartmental rate constants (/X's) of that group. There is no overlap between the two groups for the first sixteen hours after injection.

Volume 49, September 1970

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0.001 _a 13.

0

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301

GILBERT'S SYNDROME: A NEW DEFINITION - - BERK ET AL.

"~ 8.0 -0

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~ 4.0 z

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BILIRUBIN PRODUCTIONRATE(mg/kg/doy) unconjugated bilirubin increases more rapidly with increasing rates of bilirubin production in the patients in group I than in groups II or II1. The resulting higher plasma bilirubin concentration increases the likelihood that the condition will be detected. Thus, more efficient case recognition, rather than an actual biologic association, may explain the high incidence of mild hemolysis recently reported in patients with Gilbert's syndrome [8,9]. Results based on multicompartmental analysis of the data are presented in Figure 5. There were no significant differences in X2,, ~,~ and X02--the fractional transfer rates associated with hepatic bilirubin uptake, reflux to plasma, and elimination of bilirubin from the compartmental system-between groups II and III (Fig. 5). In group I, however, values for ~2, (0.0097 • 0.0018 minute ') and ~0~ (0.0052 • 0.0016 minute -~) were significantly reduced (p ~ 0.001) compared to the normal values (0.0234 • 0.0073 minute-' and 0.0107 • 0.0044 minute-', respectively). Thus the efficiency of both hepatic uptake and conjugation appears to be reduced to 40 to 50 per cent of normal in this group. Attempts to fit the data to the model after fixing either ~2, or ~02 at the mean normal value led to poor convergence, indicating that reductions in both of these parameters were, in fact, necessary to explain the experimental observations. The reduction in X02 is of the same general magnitude as the reduction in hepatic bilirubin UDP glucuronyl transferase activity determined by means of enzyme assay studies in patients with Gilbert's syndrome [4,6,7]. Although kinetic analysis suggests two defects in bilirubin metabolism, it is possible that a single lesion, such as an abnormality in an essential intrahepatic binding protein, could account for the abnormalities in both ~, and ~0~ as well as for the results of the enzyme assay studies.

302

55

Fig. 4. Plasma unconjugated bilirubin concentration as a function of bilirubin production rate. A given rate of bilirubin production results in a higher plasma unconjugated bilirubin concentration in patients with Gilbert's syndrome (group I) than in normal subjects (group II) or patients with anemia and/or increased bilirubin production and normal liver function (group III). The solid line and stippled area represent a regression line +_ 95 per cent confidence limits for the forty studies in groups II and III (subjects with normal liver function).

Although X,2 in group I did not differ appreciably from normal, the ratio ~,2:(Xo2 + X,2) averaged 0.59 ~+ 0.15 in these patients, indicating that 59 per cent of the bilirubin entering the hepatic pool returns to plasma unaltered, whereas only 41 per cent is conjugated and excreted. Corresponding values in normal subjects are approximately the reverse: 63 per cent being conjugated and 37 per cent refluxing to plasma. Finally, the estimated liver : plasma concentration gradient of 2.1 • 0.9 in group I was significantly less than the value of 3.5 • 1.6 observed in the normal volunteer subjects (p <[ 0.01). Group I values for the fractional transfer rates into and out of the extravascular pool, 0.0016 • 0.0006 minute-' (X3,) and 0.0012 I+ 0.007 m i n u t e ' (~,3), are similar to values previously described for the transfer of '3'l-albumin from plasma to the rapidly exchanging extravascular space [30]. The ratio of extravascular to intravascular bilirubin averaged 1.4, a figure also similar to data obtained with radioiodinated albumin. These values are not unexpectedl in view of the tight binding of bilirubin to albumin in vivo [31]. The value for ~3, in group I and the ratio of compartment 3 to compartment 1 are both significantly smaller (p ~ 0.01) than the corresponding values of 0.0047 ~+ 0.0025 and 2.6 ~ 0.8 in normal subjects. We have no explanation for these differences at this time. COMMENTS

Early in this century Gilbert and his colleagues [32] first demonstrated the presence of bilirubin in normal human serum (chol~mie physiologique) and described two syndromes, both of which often had a familial incidence, associated with hyperbilirubinemia. The first group (chol~mie

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GILBERT'S SYNDROME: A NEW DEFINITION -- BERK ET AL

simple familiale) consisted of patients with a modest increase in the serum bilirubin content to about twice normal in the absence of bilirubinuria, minimal alterations in skin color, an absence of hepato- or splenomegaly and a variety of nonspecific complaints ascribed to a "bilious temperament." The second, and less frequently observed, condition was associated with more striking increases in the serum bilirubin concentration, skin and scleral icterus, and frequently with hepatosplenomegaly. This group was labeled "ict~re chronique simple." Although both groups appear to have been heterogeneous, subsequent investigators have concluded that most patients with ict~re chronique simple had various forms of hemolytic anemia [33,34]. The terms, constitutional hepatic dysfunction, Gilbert's syndrome and a number of synonyms emphasizing the nonhemolytic nature of the condition [35,36], have subsequently been applied only to patients of the first type in whom idiopathic unconjugated hyperbilirubinemia is presumably due to abnormal hepatic function. In the presence of obvious hemolysis, hyperbilirubinemia has usually been ascribed entirely to bilirubin overproduction. Although this diagnostic dichotomy recognizes that either hemolysis or abnormal hepatic function may produce an increased serum bilirubin concentration, it makes no provision for the coincident occurrence of both conditions. The over-all transfer of bilirubin from plasma to bile involves a number of discrete processes, some of which have only recently been recognized, and none of which are clearly understood [37]. These include the initial entry of unconjugated bilirubin into the liver cell (whether by active transport or passive diffusion is not known), binding to a number of discrete intracellular proteins [38] which are apparently in equilibrium with binding sites on the microsomal membrane [39,40], conjugation by the microsomal enzyme bilirubin-U DP-glucuronyl transferase [41-44] and active transport of the resultant glucuronide across the canalicular membrane into the biliary radicles [45]. Although each of these processes can, in theory, be defined by a set of physicochemical rate constants, determination of all these parameters is beyond the resolving power of the technics employed in these studies. Our data suggest, however, that the sequence of events including hepatic bilirubin uptake, intracellular transport and conjugation can, for some purposes, be considered as a single first order process. Hence, for subjects in whom the hepatic bilirubin clearance is within a restricted range (e.g., patients in groups II and III) the unconjugated bilirubin concentration in the plasma increases as a linear function of the bilirubin production rate (Fig. 4). Since estimates of bilirubin production obtained from studies of bilirubin kinetics agree closely with those obtained by independent methods [46], we believe that this is a general physiologic relationship which is independent of the method used for measuring bilirubin production. This relationship has a number of important implications. Since it applies over the entire physiologically realizable range of bilirubin turnover (i.e., up to nine times normal), it suggests that the processes involved in the uptake, storage and conjugation of bilirubin have an enormous capacity.

Volume 49, September 1970

.04

)'21

(HepaticUptake) X

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Fig. 5. Fractional transfer rates (X's) for hepatic uptake (~2,), reflux from liver to plasma (~,2) and irreversible removal of unconjugated bilirubin from the system (XoO. Shaded areas represent weighted means • ] standard deviation. Rates of hepatic uptake and "conjugation" for Gilbert's syndrome (group I) are significantly different from those for normal subjects (group II) and patients with anemia and~or bilirubin overproduction (group III).

This conclusion is also indicated by the finding that an injected load of 2 mg/kg of unlabeled unconjugated bilirubin (ten to fifteen times the toal normal circulating pool) is cleared from the plasma in a fashion virtually identical with that observed for labeled tracer doses [3,4]. The mechanisms by which conjugated bilirubin is transferred out of the liver cell presumably have a more restricted capacity [47,48]. A second implication of this relationship is that plasma concentrations of unconjugated bilirubin in excess of 3.0 mg per cent are unlikely, and those in excess of 4.0 mg per cent very unlikely, to result merely from bilirubin overproduction; in such cases a hepatic defect must be sought. Finally, our data indicate the existence of a group of subjects without structural liver disease in whom hepatic bilirubin clearance is reduced to approximately one third of normal. This defect is characterized by an increase in the plasma concentration of unconjugated bilirubin out of proportion to that expected on the basis of the rate of bilirubin production, which may be either normal or increased. When considered in terms of the underlying abnormality in hepatic function, the distinction between patients with minimal or no hemolysis (i.e., chol~mie simple famitiale) and those with marked bilirubin overproduction appears to be of secondary importance. For this reason, and in view of the wide acceptance of the eponym "Gilbert's syndrome," it might be useful to apply this diagnosis (or a more physiologic equivalent) to all patients without structural liver disease who have the characteristic reduction in hepatic bilirubin clearance, regardless of the presence or absence of coincident hemolysis.

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GILBERT'SSYNDROME:A NEWDEFINITION-- BERKETAL.

The prevalence of this condition in the population at large remains uncertain. In the absence of either hemolysis or structural liver disease, we have invariably observed an abnormal pattern of bilirubin kinetics (four hour retention ~ 15 per cent) whenever the plasma concentration of unconjugated bilirubin exceeded 0.7 mg per cent or the total bilirubin 0.8 mg per cent.* Since values between 0.8 and 1.2 mg per cent (total bilirubin) are observed in 10 per cent of an apparently healthy population [49] and are often accepted as being within normal limits [50], the defect may be present in a large number of people. The ability to detect this condition by means of bilirubin kinetic studies in patients with " n o r m a l " plasma bilirubin concentrations may help to resolve the conflicting reports now in the literature concerning the familial incidence and mode of inheritance of Gilbert's syndrome [8,51,52] as well as to fix more precisely the normal range of plasma unconjugated bilirubin concentration. Studies of bilirubin kinetics alone will not distinguish patients with Gilbert's syndrome from those with certain other conditions associated with structural liver disease, since a similar reduction in hepatic bilirubin clearance has been observed in a limited number of patients with Laennec's cirrhosis [3,4] and will presumably be found in other diseases as well. For this reason, needle biopsy of the liver remains an important part of the evaluation of patients who have chronic unconjugated hyperbilirubinemia of a degree not attributable solely to hemolysis [53]. On the other hand, the kinetic abnormalities in Gilbert's syn-

* Although the procedure used in our laboratory for these determinations (Clin Chim Acta 7: 805, 1962) is not in widespread clinical use, the results obtained with it appear to be virtually identical to those obtained with the usual colorimetric methods.

drome, which is associated with a mild defect in hepatic bilirubin glucuronyl transferase, are distinctly different from those observed in each of the two rarer syndromes of chronic nonhemolytic unconjugated hyperbilirubinemia recently reviewed by Arias and his colleagues [54]. These patients appear to have a more severe defect in glucuronide formation involving a number of other substrates in addition to bilirubin. Analysis of radiobilirubin clearance studies from the literature [ 5 5 - 5 7 ] and from our own laboratoryt indicates that the classification of patients with chronic unconjugated hyperbilirubinemia proposed on the basis of clinical and genetic consideration [54] corresponds to observed differences in the rate of radiobilirubin clearance. By providing simultaneous information about the rate of bilirubin production, red cell survival and hepatic function, clearance studies with radioactive unconjugated bilirubin facilitate a more physiologic classification of patients with unconjugated hyperbilirubinemia than has previously been possible. It is expected that further experience with these studies will facilitate more accurate clinical diagnosis in patients of this type and lead to a more detailed understanding of the underlying pathophysiotogy. ACKNOWLEDGMENT

We wish to express our gratitude to the patients and their referring physicians for enthusiastic Cooperation in these studies, to the Nursing Staff of the National Cancer Institute Metabolic Ward and to Mrs. Lona A. Piatigorsky and Mrs. Jeanne G. Waggoner for valuable technical help, The assistance of Mrs. Christine Mathers, Miss Sharon Morgart, Mrs. Gladys Slocum and Mrs. Sandra Carter in the preparation of the manuscript is also appreciated.

t Berk PD, Bloomer JR, Howe RB, Berlin NI: Unpublished data.

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GILBERT'SSYNDROME:A NEW DEFINITION -- BERKET AL.

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37. Arias IM: Hepatic aspects of bilirubin metabolism. Ann Rev Med 17: 257, 1966. 38. Levi A J, Gatmaitan Z, Arias iM: Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in hepatic uptake of bilirubin, sulfobromophthalein and other organic anions. J Clin Invest 48: 2156, 1969. 39. Brown WR, Grodsky GM, Carbone IV: Intracellular distribution of tritiated bilirubin during hepatic uptake and excretion. Amer J Physiol 207: 1237, 1964. 40. Bernstein LH, Benezzer J, Gartner L, Arias IM: Hepatic intracellular distribution of tritium-labeled unconjugated and conjugated bilirubin in normal and Gunn rats. J Clin Invest 45: 1194, 1966. 41. Grodsky GM, Carbone IV: The synthesis of bilirubin glucuronide by tissue homogenates. J Biol Chem 226: 449, 1957. 42. Arias tM, London IM: Bilirubin glucuronide formation in vitro; demonstration of a defect in Gilbert's disease. Science 126: 563, 1957. 43. Schmid R, Hammaker L, Axelrod J: The enzymatic formation of bilirubin glucuronide. Arch Biochem Biophys 70: 285, 1957. 44. Lathe GH, Walker M: Synthesis of bilirubin glucuronide in animal and human liver. Biochemistry 70: 705, 1958. 45. Hanzon V: Liver cell secretion under normal and pathologic conditions studied by microscopy on living rats. Acta Physiol Scand vol 28, supp 101, 1952. 46. Engel R, Berk PD, Rodkey FL, Howe RB, Berlin Nl: Estimation of heme turnover and erythrocyte survival in man from clearance of bilirubin-'4C and from carbon monoxide production (abstract). Clin Res 17: 325, 1969, 47. Arias IM, Johnson L, Wolfson S: Biliary excretion of injected conjugated and unconjugated bilirubin by normal and Gunn rats. Amer J Physiol 200: 1091, 1961. 48. Schalm L, Weber AP: Jaundice with conjugated bilirubin in hyperhaemolysis. Acta Med Scand 176: 549, 1964. 49. Butt HR, Anderson VE, Foulk WT, Baggenstoss AH, Schoenfield L J, Dickson ER: Studies of chronic idiopathic jaundice (Dubin-Johnson syndrome). I1. Evaluation of a large family with the trait. Gastroenterology 51: 619, 1966. 50. With TK: Bile pigments of blood, chap V, Bile Pigments: Chemical, Biological and Clinical Aspects, New York, Academic Press, 1968. 51. Arias IM: Chronic unconjugated hyperbilirubinemia without overt signs of hemolysis in adolescents and adults. J Clin Invest 41: 2233, 1962. 52. Powell LW, Hemingway E, Billing BH, Sherlock S: Idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome). A study of 42 families. New Eng J Med 277: 1108, 1967. 53. Lead Article. Gilbert's syndrome. Brit Med J Ih 256, 1968. 54. Arias IM, Gartner LM, Cohen M, Benezzer J, Levi AJ: Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency: clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Amer J , Med 47: 395, 1969. 55. Schmid R, Hammaker L: Metabolism and disposition of C ~4bilirubin in congenital non-hemolytic jaundice. J Clin Invest 42: 1720, 1963. 56. Billing BH, Gray CH, Kulczycka A, Manfield P, Nicholson TC: The metabolism of ('4C)-bilirubin in congenital non-hemolytic hyperbilirubinemia. Ciin Sci 27: 163, 1964. 57. Crigler JF Jr, Gold N h Effect of sodium phenobarbital on bilirubin metabolism in an infant with congenital non-hemolytic unconjugated hyperbilirubinemia, and kernicterus. J Clin Invest 48: 42, 1969,

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