Altered clearance of sulphobromophthalein (BSP) in patients with pancreatic disease

Clinw Chimtcu Actu, 138 (1984) 163-173 Elsevier

163

CCA 02813

Altered clearance of sulphobromophthalein patients with pancreatic disease Joan M. Braganza Unrrwsity Department

(BSP) in

*

of Gustroenterology, Munchester Royal Infirmrr~~. Manchester (UK)

(Received

September

12th: revision December

7th. 1983)

Ke_v words: BSP; Pancreattc disease; Mtcrosomul induction

Summary The components of the plasma disappearance curve of sulphobromophthalein K,, K,, K, - were analysed in 26 patients with pancreatic disease. The mean excorrected initial disappearance rate constant, K,, in the patients significantly ceeded the published mean value in controls: all but four patients had a value equal to or higher than the upper limit of the reference range (mean + 2 SD). The mean uncorrected initial disappearance rate constant, K,, in the patients was not significantly different from the mean in controls but the mean of the second exponential, K,, was significantly reduced. At least one abnormality in the test ( Ki, K,, K,) was present in 24 of the 26 patients studied (93%), although clinical evidence of hepatic dysfunction was generally unimpressive. The possible implications of the results are discussed with reference to previous studies.

Introduction Many of the drugs and chemicals that cause proliferation of the hepatic smooth endoplasmic reticulum also enhance the transfer from plasma into liver of the organic anions indocyanine green (ICG) and sulphobromophthalein (BSP) [l-3]. This, together with the recent suggestion that hepatic microsomal induction might be a key factor in the aetiology of pancreatic disease [4,5], prompted a study of BSP clearance in patients with pancreatic abnormalities.

* Address for correspondence: Oxford Road, Manchester, UUUY-8YXl/X4/$03.00

Dr. J.M. Braganza, Ml3 9WL. UK.

Senior Lecturer

0 1984 Elsevier Science Publishers

B.V.

in Gastroenterology.

Royal Infirmary.

Age

Patient

15 54

47

35

49

30

MP* MR*

ER*

PT

JH

GH*

RP, relapsing

22 31

BH LM

_

_.

calculi.

RP and jaundice

M

C, pancreatic

steatorrhoea

diarrhoea

F

M

pain(C)

M

RP pain

+

+ +

+ +(S)

+(S)

_

of other symbols

_ _ _ . -

For explanation

_ +

31

29

34

35

38 31

39 32

38 42 36 33 32 40

g/l (38-48) +

Albumin

40

19

20

8

14 13

15 10

10 11

10

10

11

10

(22)

Bilirubin pmol/l

see legend to Table 1 B.

A’

A2

A3

A2

N A’

-

RP RP

M F

M F

N A2

+ + + + _

+

A+ N A’ N N

-

alcohol/cigarettes cigarettes/tea alcohol/cigarettes cigarettes/coffee printing works/ cigarettes/tea ? Crohn’s/steroids/ azathioprine ? renal transplant/ steroids/azathioprine alcohol/cigarettes/ coffee/pethidine ulcerative colitis/ steroids cigs./coffee/tea/ phenobarbitone alcohol/pethidine

N

Bile duct

?

Liver

RP(C) RP(C) RP(C) steatorrhoea RP

Possible relevant factors

RP

Presentation

F M F M M M

Sex

group

pancreatitis;

Chronic pancreatitis PB * 20 AB 35 MB 54 AC 57 NE 64 HG* 78

(years)

pancreatitis

Chronic

TABLE IA

159

297

485

165

105 110

70 50

71 128 48 29 116 50

U/’ (100)

AP

137

400

111

88

27 20

21 86

17 59 38 59 35 22

U/l (40)

ALT

523

1290

180

71

8 32

19 91

7 135 29 316 450 30

U/’ (65)

yGT

1.4

2.7

2.1

1.1

1.5 3.7

1.7 2.0

0.7 4.9 1.6 1.4 2.0 1.6

(1.8)

TG mmol/l

IB

Age (years)

37 64

EW* DW*

F M

M

Sex

and cancer

chemical works/ tea/cigarettes coffee coffee

diabetes cigarettes/coffee

?

gallstones/tea alcohol ? diabetes ? ‘I

Possible relevant factors

groups

pancreatic metastases pain, wt.loss steatorrhoea Whipple 1981

RP pseudocyst RP pancreatitis RP RP RP RP RP, arthralgia

Presentation

of pancreas

N N N

+ +

A3

N A2 N N N N Caroli

N

Bile duct

++

(S) ++

+

++ _ _ _

_

+

_

Liver

30 38

32

40

42 40 38 41 38 38 37 38

g/l (38-48)

Albumin +

13 8

17

12

19 17 10 16 13 15 8 10

(22)

Bilirubin p mol/l

49 72

141

202

76 94 74 34 62 50 83 96

U/l (100)

AP

71 25

195

68

14 28 21 39 23 58 16 74

U/1 (40)

ALT

200 9

255

365

110 30 6 26 10 23 18 21

U/l (65)

yGT

2.6 1.5

2.9

3.6

1.9 1.8 2.0 4.6 1.5 3.5 1.3 2.1

(1.8)

TG mmol/l

patients, 28-48 g/l for hospitalised patients; RP, relapsing pancreatitis; S. palpable spleen; + , liver palpable; -, liver not palpable; cholangiographic abnormalities: A’, constriction of bile duct in head of pancreas; A2, attenuated biliary radicals; N, normal cholangiogram; AP, alkaline phosphatase; ALT, alanine intrahepatic biliary radicals; A 3, irregular calibre of intrahepatic transferase; yGT, glutamy transpeptidase; TG, triglycerides. Figures in brackets are, for albumin the reference range. and for the other serum constituents the upper limit of the reference ranges.

* Indicates patients on low fat diets for 3 months or more. +. for ambulant

48

JH *

CaIWXr

Acute pancreatitis AH 72 AF 19 EH * 42 AN 59 MB* 51 GJ 26 GK 46 MC 29 DL 54

Patient

Acute pancreatitis

TABLE

166

Materials and methods Patients Thirty patients were investigated, but the results from four tests were omitted from the analysis because of interference in the BSP assay in one study and erratic data in three studies. The diagnosis of chronic pancreatitis in 14 patients was principally based on the presence of pancreatic calculi in four patients, unequivocally abnormal endoscopic pancreatograms [6] in nine patients and grossly reduced bicarbonate output after secretin [7] in one patient. When both pancreatic ductal morphology and function were normal, patients were classified as having acute or recurrent acute pancreatitis [8] (although it is recognised that a few patients with the histological features of early chronic pancreatitis may be wrongly classified by these criteria [7]). Fifteen of the 23 patients with acute or chronic pancreatitis were on their normal diets at the time of study, whilst eight had carefully adhered to the prescribed fat restricted diet for 3 months or more. Three patients had pancreatic cancers that had been verified histologically. One of these patients was fit and asymptomatic 18 months after a Whipple operation and radiation to the tail of the pancreas and received 30 g of medium chain triglycerides each day with reduction in dietary long chain fat: two patients were anorexic for 3 months. Although the liver was palpable in several patients (Table), none had the stigmata of chronic liver disease, only one was icteric (GH), whilst one patient had undergone cholecystjejunostomy to relieve obstructive jaundice 3 years previously (JH). Splenomegaly was present in three patients in whom oesophageal varices were undetectable endoscopically, or by barium meal. Endoscopic cholangiography revealed abnormalities, which were often marginal, in 11 patients (Table). The conditions that have been reported in association with pancreatitis - gall stones, inflammatory bowel disease, diabetes, primary biliary cirrhosis - were present in a few patients. Several patients smoked heavily, a few drank alcohol in excess, others were on immunosuppressants or analgesics, whilst some drank large quantities of theophylline-containing beverages (Table). BSP test This was performed exactly as described by Hacki et al [9] whose study provides the reference ranges for the disappearance rate constants - K,, K, and K,. After an overnight fast, the recumbent patient was given an intravenous injection of BSP (within approximately 30 s) in a dose of 5 mg per kg of body weight. Blood samples were collected from the opposite cubital vein through an indwelling catheter at 3, 5, 7, 10, 15, 20, 25, 30, 35, 40 and 45 min after injection. Time zero was taken as the beginning of the injection. Plasma BSP concentrations were measured spectrophotometrically by a conventional method [lo]. The measurements were adequately reproducible when the plasma BSP concentration exceeded 3 mg/l, but were unreliable at lower concentrations (as also noted by Hacki et al [9]). The BSP disappearance curves were analysed by the peeling-off technique (Fig. 1). The measured BSP concentrations in each patient were plotted on a semilogarithmic scale against time. The uncorrected initial disappearance rate constant, K,, was derived by fitting a straight line through at least three of the four points between 3

and 10 min after the injection. The second exponential, K,, was defined by drawing a straight line through at least 3 of the 4 points between 30 and 45 min after the injection. In either case when a nearly perfect fit of at least three points was unobtainable, the data were omitted (one study). The corrected initial disappearance by plotting the numerical differences between the early rate, K,, was obtained plasma concentrations and the extrapolated straight line used to determine K,. The corresponding points were again fitted by a straight line through at least three of the first four points. The T+ for the uncorrected and corrected disappearance rates and for K, were read off the graph and the respective constants calculated [9] with the formula: 0.693 x 100.

K( %/min)

=

Plasma BSP

mg/

T$ 1

I

0

5

10

15

20

25

30

35

40

45

Mtnutes Fig. 1. A representative BSP plasma disappearance curve after intravenous injection of 5 mg BSP per kg of body weight in patient MR. The lines for K,.and K, were fitted graphically and K, was obtained by the peeling off technique. A, B and C are extrapolated zero time intercepts from which the r: for each component was derived. The disappearance rate constants are expressed in per cent per minute.

168

Student’s t test (2-tailed with test and control groups considered to be different when 2p < O.OS>was used to compare the data with those obtained by Hacki et al [9] in 26 controls with mean age 31 years: a non-parametric test would have been more appropriate but was impossible because the individual values in the controls were not available. (Although Hacki et al’s version of the BSP test is regularly used at Manchester Royal Infirmary to assist in the investigation and management of patients with known or suspected liver disease, studies in healthy volunteers have not been undertaken because of the occasional idiosyncratic fatal reaction to the dye [ll]. However, in a recent local study of haemophiliacs with virtually normal liver histology, the BSP clearance parameters conformed to Hacki et al’s reference range I121). Results The cited reference values are for K,, 12.6 rt 1.6%/min (mean + SD in 26 subjects); K,, 14.3 k l.SW/min (mean + SD in 16 subjects) and K,, 5.3 + 1.9%/min (mean + SD in 16 subjects) f9]. By comparison, the mean K, in patients with pancreatic disease was significantly higher, 26.06 + 12.78W/min (mean + SD in 25 tests, 2p < 0.001); mean K, significantly lower, 3.12 _t 2.43%/min (mean + SD in 25 tests, 2p < 0.005); and mean Ki not significantly different, 15.48 k l.%/min (mean + SD in 26 patients, 0.05 < 2p < 0.10). There were no differences between the mean values in 14 patients with chronic pancreatitis ( Ki, 16.45 of:6.78%/t&r; K,, 2.36 + 2.28%/min; K,, 24.68 rt lO.l9%/min) and the respective values in 9 patients (only 8 patients for K, and K,) with acute pancreatitis (K,, 15.6 & 9.42%/min, NS; K,, 3.78 + 2.27%/min, 0.2 < 2p < 0.3; K,, 32.37 f 16.22%/min, 0.2 < 2p < 0.30). K, was reduced below normal in five patients (Fig. 2): in three of these patients there was evidence of hepatic disease (tender hepatomegaly in a chronic alcoholic, AB; hepatomegaly and jaundice in GH with chronic pancreatitis; multiple hepatic metastases in JH with pancreatic cancer - Table), but there was no reason to suspect hepatic disease in the other two cases (AH and DS in Table). K, was reduced in eight patients: in two (PB and MP) the concentrations of BSP between 30 and 45 min after the injection were very low, 2 mg/l or less, and the results may thus be unreliable, but there was no reason to question the results in the other six patients. The patient with recurrent acute pancreatitis (DL) had hepatosplenomegaly, positive mitochondrial antibody in a titre of 1 : 100, elevated serum IgM at 2.1 g/l, and liver histology compatible with primary biliary cirrhosis. Antimitochondrial antibody was undetectable and serum IgM values were normal in the five patients with chronic pancreatitis and reduced values for K, (derived from reliable BSP measurements). Liver biopsy was performed in three of these patients: in JH and GH the changes of biliary cirrhosis were detected, whilst in PT the changes were ‘compatible with sclerosing cholangitis’. The two patients who did not have a liver biopsy were chronic alcoholics with disturbed serum liver function tests (AB,AC, Table). The corrected initial disappearance rate constant, K,, was above the upper limit of the published reference range (mean + 2 SD, 17.3%/~n) in 19 of the 26 patients studied. Normal values were found in patient DS whose diet was restricted

169

%/min. 60.01 AA A AAA

A

A

t

:A

it.2

P

10.01

4

9 A

A

4

b”

Ai h P

l.Om

$ :

A Chronic pancreatitis A Acute pancreatitis l Cancer of pancreas

O.lW

AAA

Fig. 2. K,, K, and K, in patients with pancreatic ranges (2 SD on either side of the mean) 191.

disease. Hatched

areas represent

the published

reference

in long chain fat and supplemented by medium chain triglycerides, and three patients with pancreatitis who carefully adhered to their restricted fat diet (around 40 g per day) for approximately 6 months (EH with recurrent acute pancreatitis; GH and HG with recurrent chronic pancreatitis). Values of 17.32, 17.32 and 17.30%/min were obtained in patients LM (chronic pancreatitis), AN (acute pancreatitis) and JH (pancreatic cancer): the first two of these patients had been on reduced fat intakes for 4 weeks whilst the third had been profoundly anorexic for 3 months (Table). Discussion

Dyes such as BSP and ICG follow the same general route as does bilirubin into, across and out of the hepatocytes into bile [13,14]. These anions are transported in

170

plasma tightly bound to albumin, traverse the space of Disse and are transferred into hepatocytes by a carrier mechanism that probably involves albumin receptors on the plasma membranes [14]. Once within the cytosol, these anions bind preferentially to glutathione S-transferase B [15,16] formally called Y protein or Ligandin [1.2,17-191 - which also binds drugs, hormones, metabolites and azo-dye carcinogens [17-211. The concentration of Ligandin increases in response to drugs and chemicals that induce proliferation of hepatic endoplasmic reticulum, suggesting that Ligandin is synthesised therein and subsequently released into the cytoplasm [2]. Binding of an anion to Ligandin reduces its reflux into plasma [13,14], which, in the case of bilirubin, amounts to some 30% of that initially taken up into the cells [22]. Thus, although the intracellular concentration of Ligandin has no direct effect on the rate of influx of an anion into hepatocytes, the higher the concentration the less the rate of efflux, with a resultant increase in net uptake of the anion [23]. Binding to Ligandin may also facilitate transport of anions to the hepatic microsomes for conjugation [14]. After conjugation with glutathione, BSP is actively excreted across canalicular membranes: some 60-100% of an intravenously injected dose is recovered in bile in 2 h [24,25]. Extrahepatic elimination of BSP conjugates is probably negligible when the liver is functioning normally, but becomes increasingly important in the presence of obstructive jaundice, or when large doses of BSP are given. Reduced hepatic clearance of BSP has been observed in obese, pregnant or old patients [26], in pyrexial states. and when the serum albumin concentration is very low [27]. With one exception (HG in the Table), none of the patients in the study fell into these categories. The plasma disappearance curve after an intravenous injection of 5 mg of BSP per kg body weight to normal subjects shows two main components [9,25-311, of which the second slower component, K,, largely reflects the maximal canalicular transport capacity for BSP (Tm): it is also influenced, to a small extent, by hepatic storage capacity (S) [9]. Reduced K, values are thus found in diseases which affect the biliary canaliculi, e.g., primary biliary cirrhosis [9]: in non-biliary cirrhosis the fractional biliary clearance of BSP is usually intact 191. It is much more difficult to unravel the various interrelated factors responsible for the initial rapid clearance of BSP from plasma: the status of the hepatic micro-vascular circulation, the integrity of hepatocytes and their complement of membrane carriers and of cytosolic Ligandin all determine the outcome [1,2,13,14]. The initial clearance of BSP is characteristically impaired in patients with hepatic cirrhosis, due to loss of parenchyma and vascular shunts which bypass surviving hepatocytes. Hacki et al [9] suggest that the uncorrected initial disappearance rate constant, K,, provides the most sensitive index of hepatic dysfunction. They also suggest that the corrected initial disappearance rate constant, K,, largely depends on, and reflects hepatic perfusion. This latter suggestion is difficult to accept for two reasons. First it is virtually impossible, using current techniques, to measure effect&e hepatic blood flow (i.e.. only that fraction which flows past functional hepatocytes). Second, studies of bilirubin uptake by isolated perfused livers of rats pretreated with phenobarbitone clearly show that the enhanced net uptake of bilirubin is due to reduced efflux, which in turn is due to enhanced binding by the increased amounts of Ligandin [13]. It seems likely.

171

considering that the transport mechanisms for bihrubin and organic dyes are nearly identical, that a similar phenomenon explains both the enhanced hepatic uptakes of BSP and ICG in rats treated with phenobarbitone and other chemicals [1,2], and the rapid clearance of ICG from plasma in narcotic addicts and patients on anticonv&ants 131. Induction of hepatic Ligandin seems a more plausible explanation than does enhanced blood flow for the increased BSP K, values in patients with pancreatic disease for several other reasons: (1) none of the patients had any clinical features to suggest enhanced hepatic blood flow; (2) the increased concentrations of bilirubin [32], copper [32] and free radical oxidation products (41 in secretin-stimulated bile from patients with pancreatic disease suggest proliferation of the hepatic smooth endoplasmic reticulum [4,5]; (3) hypertriglyceridaemia and increased serum gamma GT levels - both recognised markers of hepatic microsomal enzyme induction [33--351 - are seen in many patients with pancreatic problems (Table); and (4) it is well known that the enzymic composition of cells can be altered by nutritional factors. hormonal influences and by administering drugs or chemicals [36]: a multifactorial aetiology is suspected for both pancreatic cancer (5,373 and pancreatitis [S]. This suspicion, the frequently noted overlap in the demographic, clinical and biochemical features of acute pancreatitis, chronic pancreatitis and pancreatic cancer [5], and now the similarity in the plasma BSP disappearance curves in these diseases, suggests a common denominator in their aetiology and pathogenesis. Whether hepatic microsomal induction strongly supported by the above-cited indirect evidence and currently being directly investigated by studies of hepatic ultrastructure and dynamic tests - is the unifying aetiological factor or merely an epiphenomenon, only time and detailed .studies in animals will tell. Amongst the possible inducers in the patients in this study are cigarettes, alcohol, theophyllinecontaining beverages, steroids, azathioprine, phenobarbitone and occupational exposure to chemicals (Table). In a few patients an inducer was not readily identifiable, but because the four patients with unequivocally normal K, values were on reduced fat intakes for approximately 6 months, and since enhanced microsomal induction occurs in rats given a diet enriched with polyunsaturated fats [38], an increase in dietary fat together with dietary inducers may be relevant. Judging by the BSP K, values, canalicular transport mechanisms were not impaired in the majority of patients studied (Fig. 2): this observation is in keeping with the increased concentrations of products of microsomal metabolism in bile from patients with pancreatic disease [4,32]. Four of six patients in whom the low Kz values were derived from reliable BSP measurements had percutaneous liver biopsies. The histological abnormalities were largely confined to the portal tracts and were indistinguishable from one another: the diagnostic labels of primary biliary cirrhosis (DL), sclerosing cholangitis (PT), or secondary biliary cirrhosis (JH,GH) were applied after consideration of the clinical and biochemical features in each patient. Three of these four patients were on normal diets at the time of study (DL, PT, JH) and all had markedly raised BSP K, values (55.40, 27.60 and 39.6O~/min. respectively). These findings raise the intriguing possibility that the link between primary biliary cirrhosis and chronic pancreatitis [39,40]. and between sclerosing

172

cholangitis and pancreatitis [40,41] might lie at the level of the hepatocyte: the immunological disturbances associated with these hepato-biliary diseases might be secondary events occurring in genetically susceptible individuals [42,43]. Acknowledgments I thank Dr. A. Gowenlock, Head of Pathology Group Services at Manchester Royal Infirmary for allowing the BSP tests to be analysed in his busy department. I thank my colleagues Dr. T.W. Warnes and Dr. A. Smith for discussing their unpublished data, and Dr. H.J. Testa of the Department of Nuclear Medicine for helpful discussions regarding liver blood flow measurements. I thank the Department of Medical Illustration for the Figures and Mrs. Y. Banner and Mrs. V. Tweddle for typing the manuscript. References 1 Reyes H, Levi AJ. Gatmaitan Z, Arias IM. Organic anion-binding protein in rat liver: drug induction and its physiologic consequence. Proc Nat1 Acad Sci 1969; 64: 168-174. 2 Reyes H, Levi AJ, Gatmaitan Z, Arias iM. Studies of Y and 2. two hepatic cytoplasmic organic anion-binding proteins: effects of drugs, chemicals, hormones and cholestasis. J Clin Invest 1971; 50: 2242-2252. 3 Melikian V, Eddy JD, Paton A. The stimulant effect of drugs on indocyanine green clearance by the liver. Gut 1972; 13: 755-758. 4 Braganza JM, Wickens D, Cawood P, Dormandy TL. Lipid peroxidation (free radical oxidation) products in bile from patients with pancreatic disease. Lancet 1983; ii: 375-378. 5 Braganza JM. Hypothesis. Pancreatic disease - casualty of hepatic detoxification. Lancet 1983; ii: 1000-1003. 6 Kasugai T, Kano N, Kizu M. Manometric endoscopic retrograde pancreatocholangiography. Am J Dig Dis 1974; 19: 485-502. 7 Braganza JM, Hunt LP, Warwick F. Relationship between pancreatic exocrine function and ductal morphology in chronic pancreatitis. Gastroenterology 1982; 82: 1341-1347. 8 Sarles H, Sahel J. Chronic pancreatitis. In: Howat HT, Sarles H, eds. The exocrine pancreas. London: WB Saunders Co. Ltd. 1979: 402-439. 9 Hacki W, Bircher J, Preisig R. A new look at the plasma disappearance of sulfobromophthatein (BSP): correlation with the BSP transport maximum and the hepatic plasma flow in man. J Lab Clin Med 1976; 88: 1019-1023. 10 Varley H, Gowenlock AH, Bett M. Practical clinical biochemistry. Voi. I. 5th ed. London: William Heinemann Medical Books Ltd. 1980. II Astin TW. Systemic reaction to bromosulphthalein. Br Med J 1965; 2: 408. 12 Stevens RF, Cuthbert A, Perera P et al. Hepatitis in haemophiliacs - an overstated problem? Br J Haematol 1984; in press. 13 Wolkoff AW. The glutathione S-transferases: their role in the transport of organic anions from blood to bile. In: Javitt NB. ed. Liver and biliary tract physiology, 1, international review of physiology. Vol. 21. Baltimore: University Park Press. 1980: 151-169. 14 Gollan JL, Schmid R. Bilirubin update: formation, transport and metabolism. In: Popper H. Schaffner F, eds. Progress in liver diseases, Vol. VII. New York: Grune & Stratton, 1983: 261-283. 15 Kaplowitz N. Physiological significance of glutathione S-transferases. Am Physiol 1980; 239: G439-G444. 16 Bhargava MM, Arias IM. Ligandin. Trends B&hem Sci 1981; 6: 131-133. 17 Habig WH, Pabst MJ, Fleischner G, Gatmaitan 2, Arias IM, Jakoby WB. The identity of giutathione S-transferase B with Ligandin, a major binding protein of liver. Proc Nat1 Acad Sci USA 1974; 17: 3879-3883.

173 18 Fleischner GM, Robbins JB, Arias IM. Cellular localization of Ligandin in rat, hamster and man, Biochem Biophys Res Commun 1977; 74: 992-1000. 19 Levi AJ. Gatmaitan Z, Arias IM. Two hepatic cytoplasmic protein fractions, Y and 2, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein and other anions, J Clin Invest 1969; 48: 2156-2167. 20 Ketterer B. Ross-Manse11 P, Whitehead JK. The isolation of carcinogen-binding protein from livers of rats given 4-dimethylaminobenzene. Biochem J. 1967; 103: 316-324. 21 Litwack G, Ketterer S, Arias IM. Ligandin: a hepatic protein which binds steroids, bilirubin, carcinogens and a number of exogenous organic anions. Nature 1971; 234: 466-467. 22 Berk PD, Howe RB, Bloomer JR, Berlin NI. Studies of bilirubin kinetics in normal adults, J Clin Invest 1969; 48: 2176-2190. 23 Wolkoff AW. Goresky CA, Selhn J, Gatmaitan 2, Arias IM. Role of Ligandin in transfer of bilirubin from plasma into liver. Am J Physiol 1979; 236: E638-E648. 24 Wheeler HO, Epstein RM, Robinson RR, Snell ES. Hepatic storage and excretion of sulfobromophthalein sodium in the dog. J Clin Invest 1960; 39: 236-247. 25 Quarfordt SH, Hilderman HL, Valle D, Waddell E. Compartmental analysis of sulfobromophthalein transport in normal patients and patients with hepatic dysfunction. Gastroenterology 1971; 60: 246-255. 26 Jablonski P, Owen JA. The clinical chemistry of bromosulphthalein and other cholephilic dyes. Adv Clin Chem 1969; 12: 310-386. 27 Gransz H. Schmid R. Relation between plasma albumin level and hepatic BSP removal. N Engl J Med 1971: 284: 1403-1405. 28 Barber-Riley G, Goetzes AF, Richards IG et al. The transfer of bromosulphthalein from the plasma to the bile in man. Clin Sci 1961; 20: 149-159. 29 Wheeler HO. Meltzer JI, Bradley SE. Biliary transport and hepatic storage of sulfobromophthalein sodium in anaesthetised dog, in normal man and in patients with hepatic disease. J Clin Invest 1960: 39: 1131-1144. 30 Lavers GD, Cole WH, Keeton RW et al. Bromosulphthalein clearance. J Lab Clin Med 1949; 34: 965-972. 31 Inglefinger FJ, Bradley SE, Mendeloff AI et al. Studies with bromosulphthalein; its disappearance from blood after single intravenous injection. Gastroenterology 1948; 11: 646-657. 32 Braganza JM, Klass HJ, Bell M, Sturniolo G. Evidence of altered copper metabolism in chronic pancreatitis. Clin Sci 1981; 60: 303-310. 33 Cucuianu M, Zdrenghea D, Pop M, Opincaru A. Increased serum y-glutamyl transferase in hypertriglyceridaemia: comparison with serum pseudocholinesterase. Clin Chim Acta 1976; 71: 419-427. 34 Martin PJ, Martin JV, Goldberg DM. y-Glutamyl transpeptidase. triglycerides and enzyme induction. Br Med J 1975; i: 17-18. 35 Rosalki SB, Tarlow D, Ram D. Plasma gamma glutamyl transpeptidase elevation in patients receiving enzyme-inducing drugs. Lancet 1971; ii: 3766377. 36 Knox WE. Metabolic adaptation in animals. In: Packer E, ed. Cellular metabolism and infections. New York: Academic Press, 45-60. 37 Lin RS, Kessler II. A multifactorial model for pancreatic cancer in man. J Am Med Assoc 1981; 245: 147-152. 38 Marshall WJ, McLean AEM. A requirement for dietary lipids for induction of cytochrome P450 by phenobarbitone in rats liver microsomal fraction. Biochem J 1971; 122: 569-572. 39 Klass HJ. Braganza JM, Warnes TW. Pancreas in primary biliary cirrhosis. Gut 1980; 21: A 448. 40 Epstein 0. Chapman SWG, Lake-Bakaar G et al. The pancreas in primary biliary cirrhosis and primary sclerosing cholangitis. Gastroenterology 1982; 83: 1177-1182. 41 Shepherd HA. Selby WS. Chapman RWG et al. Ulcerative colitis and persistent liver dysfunction. Quart J Med 1983; 52: 503-514. 42 Wickens DG, Norden AG, Lunec J, Dormandy TL. Fluorescence changes in human gamma globulin induced by free radical activity. Biochim Biophys Acta 1983; 742: 607-616. 43 Anderson R, Donnai D, Dyer P. Braganza JM. HLA-A and B antigens in alcoholic and non-alcoholic chronic pancreatitis; is non-alcoholic chronic pancreatitis an auto-immune disease? Gut 1982; 23: A895.