Fig. 1. A) Muscle responses (EMG) to electrical stimulation of the vestibular labyrinth; a left, b right abductor indicis muscle, c left, d right neck muscles, e labyrinthine stimulus (intensity of current); A1, A2) cathodal stimulation of the left, A3) of the right labyrinth. A2, A3) The pigeon is blown on by an air jet at its ventral side; v wind velocity. B) Muscles responses to cathodal stimulation of the right labyrinth during a constant air flow to the pigeon's ventral side; a left, b right lateralis caudae muscle, c labyrinthine stimulus, v wind velocity; B1) without, B2) with contact stimuli to the pigeon's feet. C) Muscle responses to anticlockwise rotation of the pigeon in the horizontal plane by means of a rotatory chair; a, ~ left abductor, indicis muscle, b, /~ right neck muscles, c angular velocity ~b of the chair; a, b without, ~, ~ with wind, v wind velocity. D) Muscle responses to clockwise rotation of a black and white striped drum round the pigeon; a left abductor indicis muscle, b right neck muscles, c angular velocity (~ of the drum, d wind velocity v; D1) with wind stimulus, D2) interruption of the wind stimulus

ular nuclei and cerebellum. Contact stimuli to the pigeon's feet counteract the wind stimuli reducing the vestibular and visual system's influence on the wing and tail muscles. We thank Prof. Dr. W. Nachtigall and the Deutsche Forschungsgemeinschaft (Bi 229/1) for support.

Received December 12, 1977

1. Rabin, A.: Exp. Brain Res. 22, 431 (1975) 2. Biederman-Thorson, M., Thorson, J.: J.

Comp. Physiol. 83, 103 (1973) 3. Maeda, M., Magherini, P.C., Precht, W.:

J. Neurophysiol. 40, 225 (1977) 4. Akaike, T., et al. : Exp. Brain Res. 17, 497

(1973) 5. Maeda, M., Maunz, R.A., Wilson,V.J. : ibid.

22, 69 (1975) 6. Gewecke, M., Woike, M. : in prep. 7. Bilo, D. : Biol. Cybernet. 26, 109 (1977)

' Polymer Pigments' in Human Pigment Gallstones U. Wosiewitz and S. Schroebler

Institute of Medical Physics of the University, D-4400 Mtinster

Polymer pyrrolic derivatives are involved in pigment-gallstone formation [1]. In a previous paper [2] we tried to prove that the black 'polymer pigment,' being a chief constituent of human pigment gallbladder stones, is derived from bile pigments that undergo alteration during its accumulation within the gallbladder. This paper will give its special attention to some questions concerning the constitution of the 'poly- mer pigment.' For example, we have to examine if polymerization starts from bile pigments either with a tetra- or with a di- pyrrolic structure. Gallbladder bile samples, obtained by intraoperative punction from patients who underwent cholecystectomy because of (pigment) cholelithiasis were tested for di- pyrrolic compounds by the Stokvis (pent- dyopent) reaction [3], using a spectro- photometer (Beckman Acta C3). The pig- ment stones (airdried) were pulverized and dried for 3 days in a vacuum desiccator with P4Olo, then treated with a 5% aqueous EDTA solution (120~ sealed glass vessel). After 48 h the residue was fil- trated, washed with distilled water and methanol, and extracted with CHC13/ CH3OH (2:1, v/v) in a Soxhlet until the extract became completely colorless. The dried residue was suspended in abso- lute CHsOH (saturated with gaseous HC1) for esterification (4 ~ 12 h). Filtration was followed by another extraction proce- dure (CHC13/CHsOH 2:1, v/v) until the extract became colorless. The entire procedure described above was necessary to separate soluble bile pigments quantitatively from the black 'polymer pigment ' (extraction residue) that finally was subjected to a chromate degradation [4]. Degradation products were analyzed by thin-layer chromatography according to [4]. Biliverdin dimethylester, urobilin di- methylester, esterified polymerized pro- pentdyopent, and esterified fecal pigment were degraded by chromate in the same way. Biliverdin dimethylester was obtained from biliverdin dihydrochloride (Sigma) by treatment with BFs/CH3OH [5]. Uro- bilin dimethylester was prepared from bili- rubin (Baker) by Na/Hg hydrogenation, I2 oxidation and BF3/CHsOH esterifica-

162 Naturwissenschaften 65 (1978) �9 by Springer-Verlag 1978

Table 1. Main products from chromate degradation, separated by t.l.c, on Kieselgel-G plates (Merck). Solvent: CC14/CH3COOC2Hs/cyclohexane (5:3:1). Detection of maleimides by treatment with chlorine, followed by spraying a benzidine solution. Detection of 2,5-pyrrole dialdehydes by spraying a 2,4-dinitrophenylhydrazine solution (see [4])

0"~ \ / ~'0 0 "~ \ / ~0 O~ \ / ~'0 OHC" \ / "CLIO

H H H H

I II III IV

' Polymer pigment' x x x x (pigment gallstones; methylester)

Biliverdin dimethylester x x - •

Urobilin dimethylester - x x x

Polymerized propentdyopent x x - - (methylester)

Fecal pigment ' mesobilifuscin' - x x - (methylester) a

I Methylvinylmaleimide (Rf=0.46) II Hematic acid imide methylester (Rf=0.30) III Methylethylmaleimide (R~=0.44) IV 3-Methyl-4-(methoxycarbonylethyl)-2,5-pyrrole dialdehyde (Rf=0.22)

In addition, 'mesobilifuscin' methylester degradation resulted in a product with a strong C12/benzidine reaction at Rf 0.40. Identification is being worked on

tion [6]. Propentdyopent was prepared from bilirubin by alkaline H202 oxidation [7]. Polymerization was achieved with conc. HC1 [8], and esterification with BF3/ CH3OH. Fecal pigment ( 'mesobilifuscin') was isolated from feces according to [9] and transformed into methylester by treat- ment with absolute CH3OH (saturated with gaseous HC1 at 4 ~ Chromate de- gradation resulted in the main products listed in Table 1. Bile pigments with a tet- rapyrrolic structure will be oxidized to ma- leimides and pyrrole dialdehydes (see oxi- dation of biliverdin and urobilin dimeth- ylester). The 'polymer pigment ' presented the same oxidation pattern. The appear- ance of pyrrole-2,5-dialdehydes during chromate oxidation of 'polymer pigment ' requires monomer units at least with three pyrrole rings linked by two carbon bridges in the 2- and 5:position. However, we really have to assume that polymerization in vivo starts from physiologic tetrapyrrolic bile pigments. Following a suggestion of von Dobeneck [8] about the linkage of dipyrrolic polymers, pyrrole dialdehydes

should not appear during chromate oxi- dation. This fact may be proved by the oxidation pattern of polymerized propent- dyopent and fecal pigment ( 'mesobili- fuscin'), both formed by polymerization of dipyrrolic monomers. From the results of chromate oxidation we cannot exclude that dipyrrolic poly- mers are involved in the formation of the black 'polymer pigment ' but we have to state that the 'polymer pigment ' is not composed entirely of polymer dipyrrolic derivatives. On the other hand, Stokvis reaction ofbiles from patients with pigment stones was either negative or poorly positive. Thus we conclude that the formation of 'polymer pigment ' from human pigment gallstones is based chiefly on the poly- merization of tetrapyrrolic bile pigments, but not ofdipyrrolic fragments from the por- phyrine metabolism or fragments formed by splitting tetrapyrrolic bile pigments during accumulation within the gallbladder. Fur- thermore, the mechanisms concerning the formation o f ' po lymer gallstone pigments,'

though not sufficiently understood, are considered to be different from intestinal fecal pigment formation. Some more ef- forts are required investigating the condi- tions of bile-pigment polymerization in vivo, because this seems to be an important factor in the development of pigment gallstones.

Received November 15, 1977

1. Suzuki, N.: Tohoku J. exp. Med. 85, 396 (1965)

2. Wosiewitz, U., Schroebter, S.: Naturwis- senschaften 64, 340 (1977)

3. Dobeneck, H.v. : Z. Klin. Chem. 4, 137 (1966) 4. R/idiger, W.: Hoppe-Seylers Z. physiol.

Chem. 350, 1291 (1969) 5. Stoll, M.S., Gray, H.C.: Biochem. J. 163,

59 (1977) 6. Stoll, M.S., Gray, H.C.: ibid. 117, 271

(1970) 7. Dobeneck, H.v.: Hoppe-Seylers Z. physiol.

Chem. 269, 268 (1941); 275, 1 (1942) 8. Dobeneck, H.v.: J. Clin. Chem. Clin. Bio-

chem. 14, 145 (1976) 9. Moravec, M. : Z. Klin. Chem. 2, 138 (1964)

Naturwissenschaften 65 (1978) �9 by Springer-Verlag 1978 163