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Biochimica et Biophysica Acta, 444 (1976) 3 3 3 - - 3 3 7 © Elsevier Scient if ic Publ i sh ing C o m p a n y , A m s t e r d a m -- Pr in ted in The N e the r l a nds

BBA 2 8 0 1 2

METABOLISM OF BILIVERDIN

BILIARY EXCRETION OF BILE PIGMENTS AFTER INTRAVENOUS INJECTION OF BILIVERDIN ISOMERS

J A M E S A. B A R R O W M A N , * R A Y M O N D B O N N E T T and P E N E L O P E J. B R A Y

Department of Physiology, London Hospital Medical College, and Department of Chemistry, Queen Mary College, London, E.1 (U.K.)

(Rece ived March 1st, 1976)

Summary

14C-labelled biliverdins IX a, ~, T and 5 have been prepared in vitro from haemoglobin obtained from duck erythrocytes incubated with 5-amino[4-14C] - laevulinic acid. When injected intravenously into rats with biliary fistulae, about 60% of the label was recovered in the bile in 24 h after the a isomer was given, while approximately 10% was recovered with injection of the ~ isomer. The T and 5 isomers gave intermediate values. In each experiment, most of the recovered isotope was found in association with conjugated bile pigment. Thus, the metabolic pathway for bile pigment excretion in the rat handles the IX isomer preferentially but is not specific for it.

Introduct ion

The cleavage of the protoporphyrin ring is a key step in the degradation of haem. In vivo, this appears to occur exclusively at the a methine bridge yielding biliverdin IX a. The reaction is enzymic, being catalysed by a microsomal en- zyme, haem oxygenase [1]. This enzyme activity is demonstrable in rat spleen, liver and kidney, the spleen being the richest source of the activity. Other tis- sues such as chicken macrophages can also produce biliverdin IX a from eryth- rocytes or haemin suspensions [2]. The isomers, biliverdin IX ~, T, and 5 do not appear to be formed in this reaction in vivo. The present investigation was designed to examine the ability of the metabolic pathway for bile pigment ex- cretion to handle all four isomers.

* Present address: Faculty of Medicine Memorial University of Newfoundland, St. John's, New. foundland.

334

Materials and Methods

Preparation of 14C-labelled biliverdin isomers 14C-Labelled haemin was prepared by the method of Custer et al. [3]. 30 ml

of jugular venous blood taken from an anaesthetised White Peking duck, hepa- rinised, treated with streptomycin and penicillin and filtered, was incubated with shaking for 22 h at 37°C, together with ferrous chloride (final concentra- tion 5 • 10 -4 M), 5-amino[4-14C]laevulinic acid (50 pCi; 43 Ci/mol) {Radio- chemical Centre, Amersham, U.K.) and glucose (10 mg/ml). Crystalline haemin (17 rag) was isolated [4] and the isomeric biliverdins were prepared [5]. The crude mixture of 14C-labelled isomeric biliverdin dimethyl esters was separated on silica gel plates (Merck Silica Gel G 0.25 mm developed with 3% acetone in chloroform). The bands were removed, eluted and counted by liquid scintilla- tion spectrometry. The specific activities of the isomers, calculated from ex- tinction coefficients for standard biliverdin isomers [5], were: a, 0.128 Ci/mol; /3, 0.159 Ci/mol; 7, 0.148 Ci/mol; 5, 0.150 Ci/mol. The esters, dissolved in chlo- roform, were hydrolysed with trifluoracetic acid at room temperature. Spectro- scopic examination showed that no breakdown of the linear tetrapyrrole struc- ture had occurred.

Animal experiments Adult male Wistar rats of approximately 280 g body weight, fed on a stan-

dard diet, were anaesthetised with ether and at laparotomy, a flexible poly- thene cannula (PP 10~ Portex Plastics) was placed in the common bile duct. Throughout the following 24 h, the animals remained in restraining cages and were provided with unlimited drinking water. Just before the animals recovered consciousness at the end of the operation, the preparations of isomeric biliver- dins mixed with rat plasma were injected into the tail vein. Bile was collected chilled and in the dark. Whole bile samples were counted by liquid scintillation spectrometry with quench correction by the channels ratio method. To deter- mine with which class of bile pigment the ~4C was associated, bile samples were further analysed by thin layer chromatography using a system of threefold development devised to separate free bilirubin, biliverdin and bilirubin conju- gates into classes. (This system does not separate isomeric compounds.) Silica gel plates were run with standard bilirubin and biliverdin. In the first run, the plates were developed with reagent grade chloroform at 26°C. In this system, standard bilirubin was mobile. Plates were thoroughly dried and developed with the second system, acetone, a solvent in which standard biliverdin was mobile. Finally, after drying, the plates were developed in absolute methanol and a band of strongly pigmented material was mobile in this system. This pigment gave a positive direct d!azo test. It was also eluted and scanned spectrophoto- metrically. This band appeared to correspond to conjugated bilirubin. At each stage in the threefold development procedure, bands corresponding to standard pigments were removed, eluted and counted. The total recovery of radioactivi- ty was calculated as a percentage of that applied to the plate, and the fractions of this recovery in each bile pigment class were also expressed as percentages.

335

Results

Fig. 1 and Table I show the excretion of 14C in the bile following pulse in- jections of labelled pure biliverdin isomers into the tail veins of rats. The amounts of biliverdin injected ranged from approximately 30--70 pg. One ex- periment (with 5 isomer) was terminated at 21/2 h as the animal died suddenly. Otherwise, the animals appeared to be in good health and the bile fistulae flowed freely. Approximately 60% of the injected radioactivity was recovered in bile over 24 h after the a isomer was given. With the ~ isomer, about 10% was recovered over this same period. Intermediate values were obtained for the

T A B L E I

E X C R E T I O N OF 14C IN R A T S W I T H B I L I A R Y F I S T U L A E F O L L O W I N G I N T R A V E N O U S I N J E C T I O N OF 14C-BILIVERDINS IX ~, fl, 7 and 5

I s o m e r nCi in jec ted T ime a f t e r % of dose % of dose Mean r ecove ry in jec t ion r e c ove re d r eco v e red in 24 h (h) in 24 h

Biliverdin 5 .626 2 53.81 IX ~ 3.5 5.77

5 3 .63 24 5.77 68 .98 48 2.11

9 .869 2 40 . 65 4 8.91 6 3 .12

24 6 .82 59 .50

1 4 . 8 2 0 2 39 .00 4 8.51 6 6 .79

24 7 .34 61 .51 63.3

Biliverdin 7 .700 2 5.54 IX fi 4 1 .02

6 0 .66 24 2.91 10 .13

1 4 . 1 4 2 2 7 .34 4 1 .02 6 0 .43

24 2.49 11 .28 10.7

Biliverdin IX 7 1 4 . 1 3 4 2 20 .90

4 4 .72 6 1.86

24 5.47

15 .579 2 32 .75 4 5.12 6 1.76

24 4 .34

Biliverdin 9. 355 2.5 26 .20 I X 5

9 .639 2 21 .04 6 6 .53

2O 2 .14

32 .95

43 .97 38.5

29 .71 (20 h) 29.7 (20 h)

336

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~% 28 2% T,me ofter injection (h)

F i g . 1. C u m u l a t i v e r e c o v e r y o f r a d i o a c t i v i t y in b i le of rats w i t h bi le f is tulae f o l l o w i n g i n t r a v e n o u s i n j ec -

t i o n o f 1 4 C - b i l i v e r d i n s I X a , ~, 7 a n d ~. N u m e r i c a l data are g i v e n in T a b l e I .

T A B L E I I

D I S T R I B U T I O N O F 1 4 C I N B I L I R U B I N , B I L I V E R D I N A N D B I L I R U B I N C O N J U G A T E S ( T H I N L A Y - E R C H R O M A T O G R A P H Y W I T H T H R E E F O L D D E V E L O P M E N T ) I N R A T S

R E C E I V I N G I N T R A V E N O U S B I L I V E R D I N I S O M E R S

B a n d ( i ) c o r r e s p o n d e d to s t a n d a r d b i l i r u b i n a n d b a n d ( i i ) t o s t a n d a r d b i l i v e r d i n , w h i l e b a n d ( i i i ) c o n t a i n e d

b i l i r u b i n g l u c u r o n i d e .

B i l i v e r d i n T o t a l % r e c o v e r y % of r e c o v e r e d r a d i o a c t i v i t y in c l u t e d b a n d s

i s o m e r o f r a d i o a c t i v i t y - - appl i ed to p la te O r i g i n ( i ) ( i i ) ( i i i )

I X c~ 7 6 . 4 1 0 . 0 4 . 3 4 . 0 8 1 . 7 7 8 . 3 1 0 . 1 5 .3 4 . 3 8 0 . 3

I X fi 8 6 . 0 8 .9 3 . 4 4 .1 8 3 . 6 7 9 . 9 6 . 0 4 . 8 4 . 5 8 4 . 7

I X ? 8 4 . 0 1 0 . 3 1 3 . 6 1 0 . 9 6 5 . 2 6 0 . 4 1 1 . 6 3 .2 4 . 8 8 0 . 4

I X 5 7 0 . 3 8 . 5 1 2 . 4 1 7 . 5 6 1 . 6

7 and 5 isomers. Most of the radioactivity recovered was excreted in the bile within 2 h. Table II shows the results of the chromatographic analysis of bile samples according to threefold development system described above. In all cases, the major proportion of 14C was associated with that fraction of the bile containing bilirubin conjugates.

Discussion

These experiments reflect the relative specificity of the haem catabolic path- way towards bile pigments of the ~ series at and after the biliverdin stage. Bill-

3 3 7

rubin of bile is almost exclusively the IX a isomer [6]. This is generally re- garded as a result of the specificity of haem oxygenase, but our results indicate that subsequent steps in bile pigment excretion select the a isomer for prefer- ential handling. Possible sites for this selectivity are (a) the biliverdin reductase step, converting biliverdin to bilirubin; (b) the uptake and intracellular trans- port of unconjugated bilirubin by the hepatocyte ; (c) the conjugation of bili- rubin by the enzyme system of the smooth endoplasmic reticulum of the hepatocyte; or a combinat ion of these.

A preparation of biliverdin reductase from guinea pig liver is reported to have greatest activity towards the IX a isomer with little activity against the

and 5 isomers and no activity against the ~ isomer [7]. Further study of this enzyme preparation suggested that the location of the propionic acid residues in the biliverdin isomers might determine the enzyme specificity [8]. Our re- sults, however, show ~ fairly good excretion of '4C after biliverdin IX 7 was given. This might suggest that there are inter-species differences in the speci- ficity of hepatic biliverdin reductase or that other biliverdin reductases whose specificities differ from those of the guinea pig liver enzyme are also taking part in our system. It should be noted, however, that the chromatographic technique used in this study, while separating unconjugated bilirubin and biliverdin, is not expected to distinguish between conjugated rubins and verdins. Thus, it is possible that some of the isotope excreted in the conjugated pigment fraction was present as a biliverdin conjugate.

There is some indication that the vinyl groups of bile pigments have an in- fluence on the metabolic process. In rather similar experiments with tritiated mesobiliverdin IX ~, Kondo et al. [9] observed massive excretion of radio- activity into the bile in rats (approx. 50% in 2 h), but conversion to mesobili- rubin IX ~ was not established and appears in any case to have been low. The different behaviour observed here with '4C-biliverdin IX ~ (only approx. 6% excretion of radio-isotope in bile in 2 h, but with the activity largely in the con- jugated bilirubin fraction) presumably represents a distinction made between the diethyl and divinyl systems either in transport or in reaction processes.

Acknowledgements

The authors gratefully acknowledge financial support for this investigation from the Medical Research Council.

References

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