On the chemistry of ‘black’ pigment stones from the gallbladder

1

Clinica Chimica Acta, 89 (1978) l-12 0 Elsevier/North-Holland Biomedical Press

CCA 9187

ON THE CHEMISTRY GALLBLADDER

OF ‘BLACK’ PIGMENT

STONES FROM THE

ULRICH WOSIEWITZ * and STEFAN SCHROEBLER Institute of Medical Physics, University Muenster, Huefferstr.

68, 4400 Muenster (G.F.R.)

(Received March 1 Oth, 1978)

summary Radiolucent (33 cases) and radiopaque (17 cases) black pigment gallstones from patients who underwent cholecystectomy were studied using several spectroscopic and chromatographic methods. Radiolucent pigment stones (mean Ca percentage 2.1%) are composed chiefly of degenerated tetrapyrrolic bile pigments (mean 85.3%) deriving from bilirubin and bilirubinates. Degeneration includes both polymerization and bacterial reduction and leads to products of different grade of polymerization. Final extraction residues (mean 55.5%), called the ‘black pigments’ are considered to be degenerated bile pigments of high molecular weight. The mean percentage of bilirubin (free and inorganic bound bilirubin) was 8.5%, while the percentage of lipids was very low (mean of total lipids -2.7%). Radiopaque black pigment stones (Ca: 12.4%) were composed of ‘black pigments’, too, but contained large amounts of calcium phosphate (carbonate apatite) and/or calcium carbonate. 65% of the radiopaque stones were calcified by calcium phosphate. ‘Black pigments’ were degraded by chromate to maleimides and 2,5-pyrroledialdehyde. These degradation products can be prepared in the same way from normal bile pigments with a tetrapyrrole structure. Polymerized dipyrrelic bile pigments like polymer propentdyopent or ‘mesobilifuscin’ did not give 2,5_pyrroledialdehydes during chromate oxidation. Thus we conclude that the formation of ‘black pigments’ starts from the polymerization of tetrapyrrelic, but not from dipyrrolic units. Accumulation of unconjugated bilirubin and bilirubinates within the gallbladder will precede the development of ‘black pigments’ which play an important role in pigment gallstone formation.

* To whom correepondence should be addreeeed.

2

A. Introduction The frequency of pigment gallstones within cholelithiasis in Japan and North America is reported to be 8-1296. A recent study by Trotman and Soloway even demonstrated a rate of 27% [ 11. The importance of pigment substances is not properly reflected by these values, because bile pigments are more or less important constituents even in cholesterol gallstones. The fact that many cholesterol stones have a pigment nucleus stresses the importance of bile pigments in gallstone formation. The term “pigment stone” is often the result of a subjective gallstone classification. In this study we shall regard concretions as pigment stones if they are composed chiefly (>50%) of bile pigments or bile pigment derivatives which are distributed homogenously within these concretions. Pigment gallstones may be divided into two sub-groups from a phenomenological point of view. First, the reddish brown stones mainly from the common duct, and second the black to dark green, often small, concretions from the gallbladder. These stones are very brittle in the dried state and look like tar products when fractured [ 21. Though these stones, especially from Japanese and North American patients, have been subjected to several analytical investigations, pathogenesis and morphogenesis remained insufficiently understood. This is partly due to the tar-like substances being the chief constituents of most of these pigment stones. The composition and origin of these substances are still under discussion. The present investigations have been undertaken, because there had been no systematic studies of the frequency and composition of pigment stones in Western Europe during the last two decades and because further analytical investigations are required to elucidate the conditions of pigment stone formation. B. Materials Black pigment stones were obtained from patients who underwent cholecystectomy. The concretions were washed with distilled water, dried in a vacuum desiccator and stored in a refrigerator. From preoperative X-ray photographs the material was divided into radiolucent (N = 33) and radiopaque cases (N = 17). Gallbladder bile was obtained from patients with pigment stones by intraoperative puncture. Bile was stored in sterile vessels at -20°C. C. Methods 1. Examination of crude pigment stone powder Pigment stones were pulverized in a vibrating mill. The finely grained, dried powder was studied at first by infrared-spectroscopy (KBr-disk-technique, Perkin Elmer IR 377), followed by X-ray diffraction analysis (Debye-Scherrer technique, Cu K,-radiation) and X-ray emission analysis. X-radiation was excited by the 30 keV electron beam of a scanning electron microscope (Stereoscan MK IIa, Cambridge Ltd., equipped with an Ortec energy dispersive X-ray analyzer). Standard atomic absorption spectroscopy (Perkin Elmer AAS

3

PE 300) was used for the quantitative analysis of Ca, Mg, Cu, Fe and Zn. Dried powdered samples (5-10 mg) were dissolved in a mixture of 65% HN0,/30% Hz02 (3 : 2, v/v). The mixture was carefully heated in a PTFE vessel until dry. The residue was dissolved in 0.1 M HCl, adding 2 ml of 0.1% LaClJlOO ml to mask phosphorus. 2. Determination of lipids and free bilirubin Dried pigment stone powder (20-30 mg) was extracted 5 times each with 10 ml of acidified chloroform/methanol (2 : 1, by vol., 1 ml 1 M HCl/lOO ml) in a 15-ml centrifuge tube which was placed in an ultrasonic glycerine bath. The extracts were recovered by centrifugation and finally transferred to a 50ml volumetric flask. An aliquot volume was evaporated to dryness and hydrolyzed by 5% aqueous NaOH (sealed tube, 120°C). After 7 h of hydrolisis the solution was acidified with 2 M HCl and extracted with diethyl ether. The organic layer was evaporated, the residue dissolved in CH,OH, and 5acholestane was added as internal standard. The whole mixture was then methylated with ethereal diazomethane. Finally the methylated solution was silylated with a mixture of hexamethyldisilazane/trimethylchlorosilane (2 : 1, v/v) and subjected to GLC [ 31. Gas chromatograph: Hewlett Packard GC 5721 A, Integrator 3380, glass column 180 cm, 1.5% Silicon QF-1 on Chromosorb W AW DMCS SO/l00 mesh, temp. program 180-25O”C, 12”C/min. Recovery of lipids was checked subjecting defined amounts of cholesterol, cholesterol palmitate, cholesterol stearate, palmitic and stearic acid, taurocholic and taurodeoxycholic acid to the same hydrolysis, methylation, and silylation procedure, followed by GLC assay. Determination of free bilirubin: Pigment stone powder (10-20 mg) was extracted 5 times each with 10 ml of pure chloroform. After centrifugation the extracts were transplanted to a 50-ml volumetric flask. Bilirubin was quantified by photometric determination of azobilirubin at 545 nm (spectrophotometer Beckman ActaC3) using a 29 mM sulfanilic acid in 0.17 M HCl (Diazo I, 0.2 ml) and a 25 mM NaN02 (Diazo II, 0.05 ml). Formation of azobilirubin was accelerated by an excess of methanol (3-4 ml) [4]. Each standard bilirubin solution was prepared freshly a little while before use. Bilirubin (analytical grade) was dissolved in chloroform. 3. Determination of inorganic bound bilirubin Dried pigment stone powder (20-30 mg) was treated 5 times each with 10 ml of acidified chloroform/methanol and centrifuged. The whole extraction procedure was carried out within 30 min. Each 10 ml supernatant extract was evaporated until dry under reduced pressure immediately after centrifugation. The liberated bilirubin was redissolved in chloroform, transferred to a 50-ml volumetric flask and determinated photometrically (s.a.). Recovery test: Defined amounts of bilirubin were treated in the same way as pigment stone powder (s.a.), finally dissolved in chloroform and subjected to the photometric determination.

4

4. Examination of the acidified chloroform/methanol extract This extract was evaporated until dry under reduced pressure. a. The residue was treated with pure chloroform and the soluble portion was subjected to visual spectroscopy. b. The residue was completely redissolved in chloroform/methanol (2 : 1, by vol.). TLC (solvent I, see below) was applied to separate different constituents. The green-yellow band which did not move was scraped off the plate and degraded with chromate (see below). Degradation products were separated and identified both by TLC and GLC as described below. c. The whole acidified chloroform/methanol extract was evaporated until dry. Methanol and methanolic BF3 was added to give a final concentration of 5% BF3 (by weight). The mixture was refluxed for 30 min. Chloroform and distilled water was added and the mixture was shaken. The chloroform layer was washed with water, saturated sodium acetate, and water, dried by filtration and finally concentrated by evaporation under reduced pressure. Repeated TLC (solvent I, see below) was apIflied for separation and identification of esterified bile pigments being constituents of the acidified chloroform/methanol extract. Standards (for preparation see below) were used as follows: Bilirubin dimethyl ester, biliverdin IIIa-, IXa- and XIIIa-dimethyl ester, biliviolin IIIa-, IXa-dimethyl ester, urobilin dimethyl ester, mesobiliverdin IIIa-, IXa- and XIIIadimethyl ester, mesobiliviolin IIIa-, IXa- and XIIIadimethyl ester and finally isomesobiliviolin IXa dimethyl ester. 5. Examination of final extraction residues (‘black pigments’) The black residue from the CHC1JCH30H/HCl extraction was dried in a vacuum desiccator, then suspended in absolute methanol (saturated with gaseous HCl) to complete esterification and stored at 4°C for about 12 h. After filtration the esterified ‘black pigments’ were exposed to a 1% chromate soluAfter 6-10 h degradation products were tion for oxidative degradation. removed from the chromate solution by extraction with ethyl acetate, dried by filtration and subjected both to TLC and GLC as described below. Solubility of the ‘black pigments’ was tested with several organic and inorganic solvents. In addition the black residues were studied by infrared spectroscopy. 6. Examination of gallbladder bile Gallbladder bile samples from pigment stone patients were tested for dipyrrelic bile pigments by the Stokvis-reaction (pentdyopent-reaction) [ 9,101. 3 ml undiluted bile was adjusted to pH 12 and centrifuged. The supernatant fluid was mixed with 1 ml of a 20% sodium dithionite solution and placed for 2 min in a boiling water bath. The pentdyopent-reaction was studied with a spectrophotometer (s.a.) Chromate degradation: 3 ml undiluted bile were mixed with 5 ml chromate solution (see below) and allowed to stand for 12 h. Bile pigment oxidation products were extracted ‘2 times each with 5 ml of ethyl acetate. The extract was dried by filtration, evaporated to dryness and redissolved in chloroform. The ratio of methylvinylmaleimide : methylethylmaleimide was measured by GLC (see below).

5

7. Chromate degradation, separation and identification of degradation products Chromate degradation was applied chiefly to study the composition of the final extraction residues of black pigment stones. According to Riidiger [5] we used a solution of each 1 g NazCr20, and 1 g KHS04 in 100 ml HzO. Degradation products were extracted with CH3COOC2HS, separated and identified both by thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) and combined GLC-mass spectroscopy (GLC-MS), respectively. TLC: Kieselgel-G plates (Merck), solvent system: CCL4/CH&OOCzH&yclohexane (5 : 3 : 1, v/v/v) [5]. Detection of maleimides by treatment with CIZ/ benzidine [ 51, detection of pyrroledialdehydes by 2,4-dinitrophenylhydrazine [51. GLC: Gas chromatograph Hewlett Packard GC 5721 A, steel column 200 cm, 0.5% neopentyl glycol succinate on Chromosorb G AW DMCS SO/l00 mesh, temp. program 180-25O”C, 12”C/min (for better separation we used a temperature program from 120->25O”C). Degradation products were extracted by CH3COOC2HS. The extract was dried by filtration (Na$O,), evaporated and the residue dissolved in CHC13 for GLC. GLC-MS: Finnigan GC 9500/MS 3100. Column and temp. program s.a.. Polymerized and esterified propentdyopent (PDP-methyl ester) and esterified ‘mesobilifuscin’ (MBF-methyl ester) were subjected to the same degradation procedure. Authentic maleimides and pyrroledialdehydes needed for comparison were obtained from the chromate degradation of mesobiliverdin dimethyl ester and urobilin dimethyl ester. 8. Standards Cholesterol, cholesterol palmitate, cholesterol stearate, palmitic acid and stearic acid were analytical grade and obtained from SERVA (Heidelberg). Taurocholic and taurodeoxycholic acid were from Sigma (Munich). Calcium bilirubinate was prepared as follows: Distilled water was saturated with Ca(OH)* at 4”C, filtered and saturated with Nz. Bilirubin (analytical grade from Serva) was suspended in the Ca(OH), solution. The mixture was stirred at 80°C for 24 h and finally filtrated. The residue was washed with water, methanol and chloroform (3X) to remove free bilirubin. Bile pigments used as standards and prepared in our laboratory were separated and purified by TLC on Kiese1gel-G plates (Merck) with concentration zone (10 X 20 X 0.025). In all cases a repeated development was necessary for sufficient separation. After development, pigments were scraped off the dried plates, dissolved in acetone, centrifuged, and concentrated by evaporation under reduced pressure. Solvent systems for TLC were used as follows: I: benzene/ethanol (10 : 1, by vol.) acid (12 : 2 : 1, by vol.) II: benzene/dioxane/acetic III : methylethylketone/ethylenechloride (2 : 1, by vol.) IV: benzene/benzine/methanol/ethyl acetate (48.5 : 40 : 10.5 : 9, by vol.)

161vi Preparation

of bile pigments

(dimethyl

esters):

Bilirubin

dimethyl

ester:

6

Esterification of bilirubin IXcll with absolute methanol, saturated with gaseous HCl (4°C 12 h). Purification by repeated TLC (solvent I) in the dark and under Nz atmosphere. Biliverdin IIIol-, IXcu- and XIIIadimethyl ester: Oxidation of bilirubin IXry in an acidified solution of dimethyl sulphoxide (DMSO) by 2,3-dichloro-5,6dicyano-p-benzoquinone (DDB) according to Stoll and Gray [ ‘71. Pigments were esterified by methanolic BF3 (5% BF3), refluxing the mixture for 15 min under Nz atmosphere. The biliverdins were extracted with chloroform, washed with water and saturated sodium acetate, dried by filtration (anhydrous sodium sulfate) and concentrated by evaporation under reduced pressure. Purification was achieved by repeated TLC (solvent I and II). Biliviolin IIIa- and IXoldimethyl ester: These pigments were byproducts of the biliverdin preparation, Separation and purification as mentioned above (solvent I). Urobilin dimethyl ester: This bile pigment was prepared from urobilinogen by oxidation with DDB in a methanolic solution 151. Urobilinogen was prepared by hydrogenation of bilirubin IXai in 0.1 M NaOH by liquid (0.5%) sodium amalgam [8]. Urobilin was esterified by methanolic BF3 and purified by repeated TLC (solvent I). Mesobiliverdin III&-, IXol- and XIII@-dimethyl ester: The alkaline urobilinogen solution was acidified with 2 M H$SO+ Urobihnogen was extracted with chloroform. This solution was evaporated until dry and the urob~inogen was redissolved in methanol. 20% FeC13 (w/v) in concentrated HCl was added 171 and the mixture was refluxed for 45 min. Oxidated bile pigments were extracted with chloroform and esterified with methanolic BF3. Separation and purification by repeated TLC (solvent I and III). Mesobiliviolin II&-, IX&- and XII&r-dimethyl ester, isomesobiliviolin IXor dimethyl ester: These pigments were byproducts of the mesobilive~in preparation. The mesobiliviolins were separated and purified by repeated TLC (solvent I and IV). Propentdyopent was prepared by alkaline H,O,-oxidation of bilirubin [9, lo]. Polymerization was carried out in 12 M HCl for 6 h at 80°C [ll]. The black polymerizate was centrifuged, washed several times with distilled water, CH,OH, and finally esterified with absolute CH,OH, catalyzed by BF3 to give PDP-methyl ester. ‘Mesobilifuscin’ (MBF) was isolated from feces [123 and esterified with absolute CH30H (saturated with gaseous HCl at 4°C) to give MBF-methyl ester. D. Results X-ray diffraction studies From 50 investigated pigment stone cases 17 (34%) were radiopaque and 33 (66%) radiolucent. The radiopaque stones contained either CaC03 and/or calcium phosphate. Phase analysis by X-ray diffraction (but also by infrared spectroscopy) demonstrated a relatively high incidence of calcium phosphate in radiopaque pigment stones (in contrast to calcified cholesterol gallstones). 11 (65%) of the 17 radiopaque pigment stones were mainly calcified by carbonate apatite which always crystallizes poorly. CaCO, was detected as calcite,

aragonite identified

or vaterite. Mineral constituents, by means of the ASTM index.

present

in the debyeograms,

were

Infrared spectroscopy studies Within the scope of infrared spectroscopy of black (but not reddish-brown calcium bilirubinate-) pigment stones we distinguished three types of spectra: (a) those with the main absorption bands of calcium bilirubinate (see Fig. la in comparison to synthetic calcium bilirubinate in Fig. lb), but differing from spectra of calcium bilirubinate stones; (b) spectra which deviate to a large extent from the calcium bilirubinate spectrum (see Fig. lc), and (c) spectra from calcified black pigment stones (see Fig. Id). X-ray emission analysis In all cases studied by X-ray emission analysis calcium was the predominant element. Further elements detected by this method: Cu, S, P, Na, K, Mg, Ni, Cl.

I 1700

1300

900 cm”

Fig. 1. IR spectra (‘finger-print region’) of crude pigment stone powder end in vitro prepared calcium bilhbinate. a and c. radiolucent black pigment gallstones; b. in vitro prepared celcium bilhbinate; d, rediopaque black pigment gallstones (calcified by carbonate apetite).

8 TABLE

I

RESULTS Values

OF

QUANTITATIVE

in % related

to initial

METAL dry

Radiolucent

Element

ANALYSIS

BY

ATOMIC

ABSORPTION

SPECTROSCOPY

weight. (N = 33)

Radiopaque

(N = 17)

Mean

Range

Mean

Range

Ca

2.07

0.78-4.41

12.4

4.6-23.0

Mg

0.52

0.22-1.34

2.15

0.36-3.8

CU

0.86

0.19-2.08

0.95

0.2-1.0

Zn



Fe



Results from the examination of the acidified chloroform/methanol extract Pigment stone constituents for the time being dissolved in acidified chloroform/methanol (2 : 1, by vol.) could be redissolved in chloroform only in part after the extract had been evaporated to dryness. The yellow chloroform solution showed one spectral maximum at 450 nm, which was also observed with chloroformic bilirubin solutions. On the other hand, redissolution was achieved with methanolic chloroform (for example CHC1&H30H, 2 : 1, by vol.). Applying TLC to this solution only bilirubin was observed to move on the plate. A dirty-green band remained at the start. Some parts of this band moved with strong tailing when a more polar solvent was used (e.g. chloroform/ methanol/acetic acid, 50 : 10 : 1, by vol.). Scraping off this band and subjecting it to the chromate oxidation procedure we got degradation products like those which we observed when the final extraction residues were treated the same way (see below). The mean ratio of methylvinylmaleimide/methylethylmaleimide, measured by GLC, was 3.4 : 1.

TABLE

II

RECOVERY

OF

LIPIDS

Compound

AND

BILIRUBIN Amount

(mg)

Found

(mg)

Recovery

Cholesterol

7.3

6.9

Cholesterolpalmitate

6.3

3.6

a

2.4

b

93.8

Cholesterolstearate

6.5

3.5

a

90.7

2.7

c

94.4

Palmitic

6.9

6.5

5.3

5.0

5.4

3.3

d

80.7

5.2

3.2

e

81.4

6.1

5.9

Stesxic

acid acid

Taurocholic

acid

Taurodeoxycholic

acid

Bilirubin a Cholesterol. b Palmitic

acid.

’ Stearic

acid.

d Cholic

acid.

’ Deoxycholic

acid.

94.5 93.3

94.3

94.3

96.1

(%,)

9 TABLE III RESULTS FROM THE QUANTITATIVE CONSTITUENTS (N = 33)

DETERMINATION

OF RADIOLUCENT

Constituents

Mean (%) *

Range (46)

Total cholesterol Palmitic acid Stearic acid Cholic acid Deoxycholic acid Total bilirubin Free bilirubin Inorg. bound bilirubin Metals * * Low molecular bile * * * pigment polymers High molecular bile * * * pigment polymers

1.8 0.4 0.3 0.2 0.1 8.5 1.3 1.2 3.5 29.8

0.5-5.3 0.1-0.8 0.1-0.7 0.1-0.5 0.1-0.5 4.7-19.3 1.0-1.7 3.2-12.0 1.2-7.9 11.4-46.1

55.5

37.5-80.1

PIGMENT STONE

* Related to initial dry weight. * * See Table I. * ** Both materials. quantified gravimetrically, may include a few other not identified substances.

This dirty-green material, being the predominant constituent of the acidified chloroform-methanol extract, and not soluble in pure chloroform or other polar solvents, was regarded chiefly as a mixture of low molecular weight bile pigment polymers (see Table III). TLC of esterified bile pigments: we observed yellow (diazo-positive), green and violet bands (with a red fluorescence when spraying a saturated methanolic zinc acetate solution) and identified the bands as bilirubin, bilirubin dimethylester, bilirubin monomethyl ester, biliverdin dimethyl ester (IIIe-, IXa- and XIIIa-series) biliverdin monomethyl ester (IIIa, IXor and XIII&), biliviolin- and isobiliviolin dimethyl ester. No mesoforms were detected except some traces of urobilin dimethyl ester (green fluorescence with zinc acetate). Biliverdin and biliviolin were considered not to be authentic pigment stone constituents but products of bilirubin oxidation or isomerization. When pigment stone powder was extracted briefly and esterification was carried out briefly, also, working under N2 atmosphere, no biliverdins were observed. Final extraction residues After esterification and chromate oxidation we got methylethylmaleimide (I), methylvinylmaleimide (II), hematic acid imide methyl ester (III) and 3-methyl-4-(methoxycarbonylethyl)-2,5-pyrrole dialdehyde (IV). (For compounds I-IV see Fig. 2). The mean ratio of methylvinylmaleimide/methylethylmaleimide, measured by GLC, was about 1 : 3. The final residues were poorly soluble or insoluble in organic solvents (polar or non-polar) but became completely soluble when treated with hot 0.5 M NaOH for several hours. Flocculation and precipitation from the neutralized solution was observed both on adding bi- or trivalent metal cations (Ca’+, Zn’+, Cu2+, La’+) and on acidification (pH 3-4).

10

11

Gallbladder bile The Stokvis reaction of investigated bile samples was either negative or poorly positive: i.e. a weak shoulder (523 nm) of the bilirubin absorption band 450 and 420 nm). (L,, Chromate degradation of bile showed a mean ratio of methylvinylmaleimide/ methylethylmaleimide of 35 : 1. E. Discussion The IR spectra derived from radiopaque and radiolucent black pigment stones (Fig. 1) agree well with those published by Suzuki and coworkers [ 13, 141. Ca is the chief metal element responsible for X-ray absorption in radiopaque pigment stones. The relatively high incidence of calcium phosphate accompanied by an increased Mg percentage in these stones is interesting. The most striking feature of black pigment gallstones is the high percentage of a material hardly soluble in organic solvents called ‘black pigment’. There only exists a little inexact data about the chemistry of these substances. The formation of ‘black pigment’ is a controversial issue. Suzuki [15] considered ‘black pigments’ to be pyrrolic derivatives of different grades of polymerization which, in his opinion, probably originated in bilirubin. Suzuki disproved Miyake’s thesis [16] of an origin from melanin by the comparison of IR and UV spectra of ‘black pigment’ (isolated from human pigment gallstones) and a synthetic ‘black pigment’ (prepared by rigorous degradation of commercial bilirubin and calcium bilirubinate with hydrochloric acid). It is well known that physicochemical methods give a poor characterization of the ‘black pigment’. Published UV and IR data show only broad absorptions without any characteristic maxima. Though very similar they are poorly expressive. The formation of ‘black pigments’ by polymerization of dipyrrolic derivatives (fuscins, protentdyopents) which are considered to be catabolic products of physiological hemoglobin degradation or anabolic byproducts of heme biosynthesis is discussed by With [ 171, though neither of these origins has yet been proven. We have preferred a defined degradation procedure as a chemical means of investigation, because of the difficulties in physicochemical characterization [ 181. The chromate degradation procedure applied to bile pigments for the first time by Riidiger [ 51 allows one to isolate 2,5-pyrroledialdehydes in addition to maleimides (in contrast to other degradation methods, e.g. chromic acid or permanganate degradation). This must be taken into account when deciding whether the ‘black pigment’ is a dipyrrolic polymer. If we accept the proposal of von Dobeneck [ 111 about the C-N linkage of polymer dipyrrolic derivatives, then these substances should not give rise to 2,5-pyrroledialdehydes during chromate oxidation. In fact, 2,5pyrroledialdehydes were not observed when polymer propentdyopent and ‘mesobilifuscin’ (both polymer dipyrrolic compounds) were subjected to chromate oxidation. However, the (esterified) ‘black pigment’ of the pigment gallstones which we examined resulted in 2,5_pyrroledialdehydes during chromate degradation. This is to be expected and can be observed when bile

12

pigments with a tetrapyrrolic structure are subjected to the same degradation procedure (see degradation products of biliverdin- and urobilin dimethyl ester). This stresses the point that tetrapyrrolic bile pigments are involved in the formation of the ‘black pigment’. Dipyrrolic derivatives, both of a catabolic or an anabolic origin, have to be excluded to a large extent because these compounds were found in the corresponding gallbladder bile samples either not at all or in very low concentrations. The latter must not be overestimated because oxidative splitting of bilirubin in biological fluids will occur to some extent even if samples are stored under protection from air and light. With respect to the formation of the ‘black pigment’, we could not establish an analogy to the intestinal bile pigment metabolism by which tetrapyrrolic bile pigments were partly degraded to dipyrroles and finally transformed into polymerized products (‘mesobilifuscins’). The observed relationship between the degradation products methylethylmaleimide (compound I, see Fig. 2) and methylvinylmaleimide (II, see Fig. 2), i.e. cr > err (see Fig. 2c) does not correspond with the ratio of bilirubin to related mesoforms (chiefly urobilin) found in physiological gallbladder bile. The ratio of methylethylto methylvinylmaleimide in chromate degraded gallbladder bile (pigment stone cases) was found to be inverse (cr << cII). The appearance of methylethylmaleimide in the ‘black pigment’, indicating that some hydrogenation process has occurred, may be attributed chiefly to a bacterial reduction of the polymers during their storage within the gallbladder. To sum up it can be said that the investigated pigment stones from the gallbladder are considered to be mixtures of degenerated bile pigments (mostly from bilirubin and bilirubinates). Degeneration includes polymerization and bacterial reduction. Polymers of low molecular weight are soluble in organic polar solvents (e.g. chloroform/methanol or acidified chloroform/methanol). Polymers of high molecular weight are insoluble in organic solvents (see the final extraction residues). The dissolution of the ‘black pigment’ (high molecular pigment polymer) with sodium hydroxide is regarded as the result of the formation of lower molecular fragments which are precipitated in neutralized solution by different bi- or trivalent metal cations. References 1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 17 18

Trotman. B.W. and Soloway, R.D. (1975) Am. J. Dig. Dis. 20, 735 Wosiewitz, U. (1977) Naturwissenschaften 64, 390 Nakayama. F. (1968) J. Lab. Clin. Invest. 72, 602 Malloy. H.T. and Evelyn, K.A. (1937) J. Biol. Chem. 119, 481 Riidiger. W. (1969) Hoppe-Seyler’s Z. Physiol. Chem. 350.1291 Riidiger. W. (1971) Fortschr. Chem. Org. Naturst. 29, 60 Stall, M.S. and Gray, C.H. (1977) Biochem. J. 163.59 Stoll, MS. and Gray. C.H. (1970) Biochem. J. 117, 271 Von Dobeneck, H. (1941) Hoppe-Seyler’s Z. Physiol. Chem. 269. 268 Von Dobeneck, H. (1942) Hoppe-Seyler’s Z. Physiol. Chem. 275.1 Von Dobeneck. H. (1976) J. Clin. Chem. Clin. Biochem. 14.145 Moravec. M. (1964) Z. Klin. Chem. 138 Suzuki, N., Nakamura, Y. and Sate, T. (1975) Tohoku J. Exp. Med. 116. 259 Suzuki. N. and Toyoda, M. (1966) Tohoku J. Exp. Med. 88.353 Suzuki. N. (1966) Tohoku J. Exp. Med. 85,238 Miyake. H., Nagamitsu, S. and Hirayama. F. (1963) Z. Gastroenterol. 1, 367 With, T.K. (1968) Bile Pigments, pp. 45-56, Academic Press. New York Wosiewitz, U. and Schroebler, S. (1977) Naturwissenschaften 64. 340