Document npMG8MO9JX19n8QKxkexdJEea

226 L. Khvankova. E. Samcova and P. Boiek o /mt uiCAU- V-JO~tv sc sO Electrophoresis 1984,5. 226-230 |4| Gorg. A.. Weser, J.. Weslermeier, R.. Postel. W.. Weidinger, S.. Patutschnick. W. and Cleve, H,, Hum. Genet. 1983, 64. 222-226. 15] Arnaud. P. and Allen. R. C.. in: Allen. R. C. and Arnaud. P. (Eds.), Electrophoresis '81. Walter de Gruyter, Berlin 1981, pp. 495-504. |6| Fagerhol, M. K. and Cox, D. W.,in: Harris. H. and Hirschhorn. K. (Eds.). A dr. Human Genet. 1981,11, 1-62. |7| Constans, J,, Viau, M. and Gouaillard, C., Hum. Genet. 1980, 55. 119-121. 18] Weidinger. S., Cleve, H. and Patutschnick, W,, Z. Rechtsmed. 1982. 88.203-211, Talamo, R. C.. Langley, C. E.. Reed, C. E. and Makino, S.. Science 1973,181. 70-71. Gdrg, A.. Postel, W.. Westermcier. R.. Gianazza. E. and Righetti, P. G..J. Riochem. Biophys. Methods 1980. J, 273-284. Gorg, A.. Postel. W.. Westermeier. R.. Righetti, P. G. and Ek. K., LKB Application Note Nr. 320. 1982. Ritchie. R. F. and Smith, R,, Clin. Chem. 1976.22,497-499. Weidinger, S., Schwarzfischer, F. and Cleve, H.. Proceedings ofthe -10th--International--gnnpwtf. of the Society for. Forensic Haemogenetics. Miinchen 1983, pp. 387-392, CUSTOMER'S COPY COMPLIES WITH COPYRIGHT LAW CHEMICAL ABSTRACTS SERVICE CC CO Ludmil^KrivankoviL^ EvatSamcovij Pett&ocelj rpK 'Institute-of Analytical Chemktryy Jn) Czechoslovak Academy-ef Sciaocies*(tL) Brno 2Department^jf Medical Chemistry dical Faculty of versityTfrague (&cS (o I] M3- Determination of thiodiacetic acid in urine of people exposed to vinyl chloride by analytical capillary isotachophoresis A method is introduced for the determination of thiodiacetic acid in urine by analytical capillary isotachophoresis with column coupling. Thiodiacetic acid is one ofthe final metabolites of carcinogenic vinyl chloride and appears at increased levels in the urine ofpeople exposed to vinyl chloride vapours. The method enables the direct analysis of samples of 10 pi of urine without any pretreatment in about 45 min. The lowest detectable concentration of thiodiacetic acid was about 6 x 10`6 mol/liter and the reproducibility of the analyses in the range of 2 -15 x 1 O'5 mol. per liter was about 3 rel. %. R&S 005194 Introduction The oncogenic properties ofvinyl chloride (VC), the monomer of polyvinyl chloride (PVC), which is one of the most widely used plastics, have been known since 1971 (lj. Thus, exposure to VC vapours must be monitored above all in the production of PVC. where the workers are open to the highest risk. The first step of VC metabolism in the organism is the oxidation by microsomal monooxygenase to chlorethylenoxide, which is then responsible for most of the deleterious effects of VC. One of the final metabolites of VC in the organism is thiodiacetic acid (TDA). The direct correlation between the exposure of the organism to VC and the excretion of TDA in urine has already been proved (2,3 ], Small amounts ofTDA can even be present in the urine of persons not exposed to VC vapours, reflecting the diet of the person. However, for persons working in PVC production, the content ofTDA in urine increases com siderably. Hence, the content ofTDA in urine ofthese workers can be a criterion for health protection in the respective factories. At present TDA in urine is determined by gas chro matography after its preliminary extraction and esterification 12-4]. This multi-stage procedure is laborious, time-consum-' ing and is often subject to considerable errors. Therefore, it was desirable to work out a fast and reliable method for the direct determination ofTDA in the unprocessed urine. Correspondence: Dr, Petr Bocek, Institute of Analytical Chemistry, Czejtoslovak Academy of Sciences, Leninova 82. CS-611 42 Brno, Czecho- W* Abbreviations: VC: Vinyl chloride; PVC: Polyvinyl chloride; TDA: Thio diacetic acid; ITP: Isotachophoresis; LE; Leading electrolyte; TE: Ter minating electrolyte Isotachophoresis (ITP) has already proved to be an expedient analytical method for environmental control and clinical analyses in cases of occupational hazard. Sollenberg and Baldesten [5], Vesterberg and Sollenberg [6], and Zschiesche et al. {7 ] analysed the urine of people exposed to the vapours of styrene, toluene and xylene and determined successfully the metabolites, /. e., mandelic, phenylglyoxylic, hippuric and methylhippuric acids. The concentrations ofthese metabolites in the urine samples were in the order of 10"4 mol/liter and the detection limit of the method was in the order of 10"J mol/liter. 0vrebo et al. (8 ] analysed formic acid in the plasma of people intoxicated with methanol where the concentration level of formic acid was in the order of 10'J mol/liter. These works en couraged us to apply ITP to our problem and we have worked out a method which enables the determination of concentra tions ofTDA in a sample of 1-10 pi of urine during 45 min without any pretreatment of the sample. 2 Materials and methods 2.1 Instrumentation For ITP the CS Isotachophoretic Analyser, Spisska Nova Ves. Czechoslovakia, was used. This instrument is equipped with a column coupling system consisting of two polytetrafiuoroethylene (PTFE) capillaries of 0.8 and 0.3 mm I. D. serving the pre-separation and analytical runs, respectively. Each capillary is equipped with the conductivity detector. The bifurcation block between the two capillaries mentioned above is equipped with a side branch leading to the auxiliary electrode and enables us to select the interesting part ofsample Verlag Chemie GmbH, D-6940 Weinhrim. 1984 0173-0835/84/0408-0226 S2.50/0 Electrophoresis 1984.5.226-230 Isotachophoretic analysis of thiodiacetic acid tn unne 227 r &S 005195 zones (heart-cutting) for further analysis. The migration path between the injection port and the tell-tale detector of the pre separation capillary is about 19 cm. the migration path be tween this detector and the bifurcation point is 5 cm. The pength of the analytical capillary is 17 cm. Analyses were car ried out at room temperature. The stabilized driving currents were 200 pA and 45 pA in the pre-separation and analytical runs, respectively. The time of the analysis in question was dependent on the sample composition and. on average, was about 45 min. 2.2 Electrolytes and chemicals narrow region of the substances to be analysed is then quan titatively trapped into the analytical capillary when passing the bifurcation block. Subsequently, the complete separation and the quantitative detection is achieved in the analytical run. This procedure is analogous to the heart-cutting technique commonly known in chromatography 1121. The range where the suitable electrolyte systems were looked for was restricted to the leading electrolyte ofpH 3-5 with chloride as the leading ion and acetate as the terminating ion. Anions of acids weaker than acetate were expected to migrate in the elution way in the terminator and thus to be eliminated from the system of isotachophoretic zones. The solutions of 10 mmol/liter adjusted with p-alanine to the 3.2 Separations at different pH of the leading electrolyte desired values of pH were used as the leading electrolytes (LEs) in the pre-separation and analytical capillaries. The solutions contained hydroxypropyi-cellulose of 0.2 % w/v. The terminating electrolyte (TE) was 10 mmol/liter acetic acid in all cases. The HC1 and acetic acid used were ofanalyt ical grade (Lachema, Brno, Czechoslovakia); fi-alanine, pure, was from Loba-Chemie, Vienna, Austria; hydroxypropyicellulose was from Ega-Chemie, Albuch, Federal Republic of Germany. Thiodiacetic (thiodiglycolic) acid, p. a., was from Fluka A. G.. Federal Republic of Germany. The other acids used to identify the zones were of analytical grade (Lachema, The sequence of zones of important substances in the range mentioned above follows from Fig. 1 where the relative stepheight (the step-height represents the reciprocal conductivity) vs. pH ofthe LE is plotted. It is obvious from the figure that for pH 3.5-3.7 the zone of TDA migrates together with the phos phate zone and, moreover, at pH 3.9-4.1 the citrate zonejoins them. Hence, the suitable pH range ofthe LE in the pre-separa tion and analytical capillaries seemed to be higher than 4.0 or lower than 3.5. We checked both alternatives. Brno), All chemicals were dissolved in distilled water deioniz ed with mixed-bed ion-exchangers Ostion AD ans KS (Spolchemie, Usti n. L., Czechoslovakia). In Fig. 2, there are records of separations carried out at the higher pH. In accordance with Fig. 1, the zone of TDA migrated before citrate and phosphate together with other more mobile acids (e. g. fumaric acid). For the zones between 2.3 Urine samples ^here was no pretreatment of urine samples. Samples were chlorides and citrates (together with phosphates) the heart-cut was applied and they were further analysed during the ana lytical run in the analytical capillary. For the identification and stored at 0-4 C for 7-10 days and no changes in the con centration of thiodiacetic acid were observed during this period. the quantitative assay ofthe TDA zone, the standard addition technique was used (see Fig, 2c). It is obvious that this proce dure gave good separation in the analytical capillary. Howev er, the sensitivity of analyses was too low due to the low sep aration capacity of the pre-separation capillary. It was tested experimentally that the separation capacity of the isota 3 Results and discussion chophoretic system used was sufficient in this case to sepa rate a maximum of 3 pi of urine. With higher volumes of in 3.1 Interference from bulk anionic cor .>nents jected urine, mixed zones of TDA and citrate were formed in the pre-separation capillary. For samples larger than 3 pi, the dependence of the step-length of TDA on the volume of in TDA is a bivalent acid with pK values.: . 15 and 4.13 [8] in jected urine enriched with TDA was not linear. water and can be easily analysed in the whole pH range prac tically used in anionic ITP. The concentrations of TDA in urine in the order of 1.5-15 x 10"5mol/Iiter also represent no problem for common isotachophoretic instrumentation Therefore, the lower pH range was tested where phosphate migrates in front of TDA and may positively affect the separa- equipped with conductivity, gradient or UV absorption detec tion. However, the analytical problem stems from the sample character. Urine contains on average about 8 g of chlorides, 23 gofurea, 2.1 g ofinorganic phosphates, 1.2 g ofcreatinine, 0.7 gof uric acid, 0.08-0.8 gofhippuricacid [9], 0.05-0.46 g of citric acid [10] etc. in one liter, which corresponds to the concentrations in mol/liter of 0.23, 0.38,0.015, 0.01, 3.47 x 10"3,0.465-4.65 x 10'\ and 0.275-2.4 x 10~J, respectively. Evidently, the concentrations of the bulk anionic components are several orders of magnitude higher than the average con centration of TDA and may strongly interfere with its deter- nation. This was the reason why the working procedure and 30 32 31 36 39 io <-2 U pH Ktrumentation employing the "column coupling technique" Figure 1. The dependences of the relative step-height rj. pH of the LE of [11] was used. The goal of this technique is that good pre-sep some acids present in urine. LE: 10 mmol/liter HC1 + [5-alanine + 0.2 % w/v aration of the interfering bulk components from the analysed hydroxypropyl cellulose. TE: 10 mmol/liter acetic acid. P - phosphates. species is achieved in the pre-separation run, and, further, a C - citrates, T - thiodiacetates, F - fumarates. 228 L. Krivankovi. E. Samcova and P. Bocek Electrophoresis 1984. S, 226-230 R&S 005196 Figure 2. Isotachophoretic analysis of 2 pi of normal urine. LE in both capillaries: 10 mmol/liter HC1 + p-alanine, pH 4.4, 0,2 % w/v hydroxypropyl cellulose. TE: 10 mmol/liter acetic acid, (a) The record of the pre-separation run. Driving current 250 pA, chart speed 0.5 mm/s. Cl chlorides. C - citrates, P - phosphates, A - acetates, (b) The record of the analytical run. Driving current 45 pA, chart speed 1 mm/s. Cl - chlorides, T -thiodiacetates, C - citrates, (c) as in (b), 1 nmole ofthiodiacetic acid add ed. Cl - chlorides, T - thiodiacetates, C - citrates. In the upper part of(b) and (c), the derivative of the detector signal is recorded. tion capacity for the substances migrating behind it [ 13 ]. In the range of pH lower than 3.4, in agreement with the results of Fig. 1, the zone of TDA migrated between phosphate and citrate zones. The part of sample zones between phosphates and citrates was then trapped into the analytical capillary and analysed subsequently. Good separation was reached (see Tig- 3)- The positive effect of the zone of phosphate upon the separation capacity for TDA and substances migrating in its neighbourhood was tested experimentally and it was found that volumes up to 10 pi ofuntreated urine can be successfully analysed. The calibration curve was constructed by employ ing the standard addition technique and the plot of the step length of TDA vs. its amount added to the normal urine was linear in the range 0-3 nmoles. Hence, the LE of pH 3.4 seemed to be suitable for the analyses of TDA in urine. How ever, during practical analyses of the urine of people exposed to VC, another problem appeared. In these samples, some other substances were found, the concentrations and effective mobilities of which were close to those of TDA. It is probable that these substances are chloroderivatives of carboxylic acids. They interfered with the analysis to such an extent that it was necessary to perform the comparison with further analysis of the sample enriched with a known amount of TDA to identify reliably the zone of TDA. Thus, the time of analysis was prolonged to 2 x 45 min. To solve this complicated situa tion we tried to increase the selectivity ofthe separation by us ing the combination of LEs in such a way that LE of pH 3.4 was in the pre-separation capillary and LE ofpH 4.3 was in the analytical capillary. As the heart-cut for the analytical run only the zones of acids between phosphate and citrate were trapped into the analytical capillary. The regions of acids migrating in front of the heart-cut and behind it were eliminated from the separation capillary system by their aigration to the auxiliary electrode (side branch ofthe bifurcaVi block). Using such a procedure, TDA migrated in the Jnalytical capillary in front of citrate together with a small number of well separated zones of other acids and could be easily and unambiguously identified according to its step height in the record. The combination of LE of pH = 3.4 and Figure 3. Isotachophoretic analysis of 5 pi of normal urine containing 1.98 mmol/litcr thiodiacetic acid added. LE: 10 mmol/liter HC1 + p-alanine, pH 3.3 + 0.2 % w/v hydroxypropyl cellulose in both capillaries. TE: 10 mmol/ liter acetic acid, (a) The record of the pre-separation run. Driving current 200 pA, chart speed 0.5 mm/s. Cl - chlorides, P - phophates. T thiodiacetates, C - citrates. A - acetates, (b) The record ofthe analytical run where the heart-cut of the sample zones between phosphates and citrates was analysed. Driving current 40 pA, chart speed 1 mm/s. Cl - chlorides, P - phosphates, F - fumarates, T - thiodiacetates, C - citrates. In the upper part of the figure the derivative of the detector signal is recorded. LE ofpH = 4.3 in the pre-separation and analytical capillaries, respectively, is advantageous and solves the problem of in terfering acids (Fig. 4). 3.3 Calibration curve and reproducibility The calibration curve (step length in the record vs. the amount of TDA added to urine) was linear in the concentration range in question and passed through zero (see Fig. 5; the amount of TDA originally present in urine was subtracted). The depend ence of the step length of thiodiacetic acid on the volume ofthe analysed urine was checked up to the injection of 10 pi and was linear. Hence, the procedure proposed can be satisfactori ly used for the qualitative and quantitative determination of TDA in urine of persons both exposed and not exposed to VC. Considering that the increasing volume of injected urine con siderably prolongs the time needed for the analysis due to the Electrophoresis 1984,5, 226-230 60 *c vci 26 24 22 Uotachophorcttc analyst* of thiodiaceuc acid in urine 229 Figure 4, Analysis of 5 pi of urine of a person exposed to vinyl chloride by employing LE of different pH in the pre-separation and analytical capillaries, (a) The record ofthepre separation run. LE: 10 mmol/liter HC1 + (3-alanine. pH 3.4, * 0.2 i. w/v hydroxvpropy! cellulose. TE: 10 mmol/' liter acetic acid. Driving current 200 pA. chan speed 0.5 mm/s. Cl chlorides. P- phosphates. C- citrates. A - acetates. The section between ar rows indicates the hean-cut for further analysis, b) Analysis ofthe heart-cut in the analytical run. LE: 10 mmol/liter + (3-alanine. pH 4.3 + 0.2 % w/v hydroxypropyl cellulose. TE: 10 mmol/liter acetic acid. Driving current 45 pA, chan speed 2.5 mm/s. Cl - chlorides, T - thiodiacetates. C citrates. A - acetates, (c) Analysis of the heart-cut of the section between chlorides and phosphates m the analytical run. When TDA is to be deter mined. this section ofzones togehter with the zone ofphosphates is cutaway and does not interfere. Conditions as in <b): LE: 10 mmol/liter HCI (3alanine pH 4.3. 0.2 % w/v hydroxypropyl cellulose. TE: 10 mmol/liter acetic acid. Driving current 45 pA, chart speed 2.5 mm/s. Cl - chlorides, P - phosphates. A - acetates. In the upper part of<b) and (c) the derivative of the detector signal is recorded. 60 (- R &s 005197 20b tnioOiocetic ocidlnmoi) Figure 5. The calibration plot of the step-length in the record of the* 10 analytical run vs. the amount of TDA added to normal urine samples. The 375 37 timetminl injected volume of urine with added TDA was 5 pi. Results were corrected to the amount ofTDA present in urine before the standard was added. LE: 10 mmol/liter HCI + (3-alanine + 0.2 % w/v hydroxypropyl cellulose, pH 3,4 and 4.3 in the pre-separation and analytical capillaries, respectively. TE: lOmmol/liter acetic acid. Drivingcurrent:200 pAand45 pA, respectively. Chan speed for the analytical column 2.5 mm/s. Correlation factor 0.99992. exposed to vinyl chloride and concentrations of TDA ranged within 0.025-0.067 mmol/liter. The values found for the sec ond group varied between 0.075 and 0.15 mmol/liter. In both cases, the reproducibility of analyses was approximately the same and amounted to about 3 rel. %. The increase in the con centrations of TDA in urine of the persons exposed to vinyl chloride is evident. 4 Concluding remarks prolonged zone of chlorides, usually 5 pi of urine was analys ed. Under the conditions described above, the step length of TDA in urine of people both exposed and not exposed to VC 1 mm in the record (with chart speed 2.5 mm/s) corresponded can be reliably determined by analytical ITP with the column to 0.12 mm ofthe actual zone length in the analytical capillary coupling technique. The method proposed is rapid and and to the detection limit amounting to 0.061 nmoles ofTDA. samples of 10 pi of urine without any pretreatment can be For the samples of 10 pi it represents the lowest detectable directly analysed. The lowest detectable concentration of concentration of TDA being 6 x 1 O'6 mol/liter. TDA was about 6 x 10'6 mol/liter. Reproducibility was 3 rel, %. The described method may also be the starting determine the reproducibility of the method, the urine analytical procedure for the study of other anionic substances pies of 6 members of our laboratory staff and of 9 persons that appear in the urine of persons exposed to vinyl chloride. from the chemical workroom for PVC production were repeatedly analysed. The first group was considered not to bReeceived March 16, 1984 230 E- H. McConkey and C. Anderson Electrophoresis 1984.5. 230-232 5 References 111 Viola. P. L., Bigotti. A. and Capuio, A- Canccr.Res. 1971. 31. 516-519. |21 Muller. G.. Norpoth. L. and Eckard. R,,Jnt. Arch. Occup. Environ. Health 1976.38.69-75. 131 Muller. G., Norpoth. K.. Kusters. E.. Herweg, K. and Versin. E., Ini. Arch. Occup. Environ. Health 1978.41. J99-205. |4) Muller. G. and Norpoth. K... Naturwissenschaften 1975.62. 541. 151 Sollenberg.J.andBaldesten. A..A Chromatogr. 1977. /32,469-476. I6| Vesterberg, O. and Sollenberg. J,, Excerpta Med. Ini. Congr. Ser. 1977. Ho. 440, pp. 186-189. 17] Zschiesche. W,, Schaller, K. H. ans Gossler. K.. Fresenius Z.Anal. Chem. 1978,290,115. 181 0vrcbo. S-. Jacobsen. D. and Sejersted. O. M.. 3rd Internal. Sym posium on Isotachophoresis. June 1-4, 1982. Goslar. F.R.G.. Sym posium Abstracts, p. 69 191 Sillen. L. G. and Martel!. A. E.. Stability Constants of Metal Ion Complexes. Special Publication Xo. 17. Chemical Society, London 1964. 1101 Harper. H. A.. Review of Physiological Chemistry, (in Czech) Avicenum. Prague 1977, p. 453. 1111 Tschope. W. and Ritz. E.. J. Chromatogr. 1980.221. 59-66. 1121 Evcraerts. F. M.. Verheggen. Th. P. E. M. and Mikkers, F. E. P., J. Chromatogr. 1979.769.21-38. |13l Deans. D. R..J. Chromatogr. 1981.203. 19-28. 114| Bocek. P.. Demi. M., Kaplanova. B. and Janak. J.. J. Chromatogr. 1978.160. 1-9. R&s 005198 Edwin H cCnnlkke^y---4 Catherine^ ndersSo 3 Depjirtmem-ef Molicukr, Celliilax- - and Developmental Biol&gyrUnivfrFstty ef Colorado at-BonfdSr, Boulder, V--' Double-label autoradiography revisited: L-pe rn and "Sfsystem The method of double-label autoradiography described by McConkey (Anal. Biochem, 1979, 96, 39-44) has been updated for the use of the commercial flour, EN3HANCE, and the Kodak films XAR and SB. For gels 0.8 mm thick, the input ratio of 3H dpm/3sS dpm should be 11.1 and the second-stage autoradiogram should be exposed 5.9 x as long as the fluorogram. For gels 0.4 mm thick, the input ratio of ^ ^pm/ 35$ dpm should be 5.3 and the second-stage autoradiogram should be exposed 3.1 x as long as the fluorogram. Introduction Comparison of two-dimensional gel patterns of complex pro tein mixtures, such as those obtained by O'Farrell's method 111, can be both difficult and ambiguous, due to unavoidable minor variations in the first and/or second stage, which cause even replicate samples to give patterns that are not perfectly superimposable. Several years ago, the senior author de scribed a method of double-label autoradiography (2 J which greatly facilitates the detection of one or a few differences in pairs of two-dimensional gel patterns, each of which may contain approximately 1000 co-migrating polypeptides. A sample containing 3H-labeled proteins is mixed with a sample of 14C - or 35S -labeled proteins. Two-dimensional polyacryl amide gel electrophoresis is carried out and the gel is im pregnated with a fluor to make detection of 3H efficient. The dried gel is first autoradiographed on a film that records both 3H and the higher energy isotope, via the production oflight in the flour when beta particles are emitted. Then a second autoradiogram is made, using a film that is relatively insensi tive to the flashes of light. Beta particles from 3H are too weak to reach the film emulsion, but the stronger beta particles produced by MC and 33S produce an image by direct interac tion with silver halide crystals in the film emulsion. When the second stage autoradiogram is placed upon a photographic negative of the fluorogram, any spots that represent poly peptides labeled only with 3H stand out as white spots, in con (respondence: Dr. Edwin H. McConkey, Molecular, Cellular and velopmental Biology, University or Colorado, Campus Box 347 Boulder, CO 80309, USA Abbreviation: PPO: 2. 5, Diphenyloxazoie trast to the gray background of the negative and the detailed pattern of black spots representing MC or 33S-labeled poly peptides. Since the 1979 article was written, there have been several changes in the materials used for double-label autoradio graphy. First, the Kodak company has replaced XR film with XAR film and No Screen film with Direct Exposure film; sec ond. SB film has been recognized as a desirable substitute for No Screen film in autoradiography without fiuors (e. g., (3)); and third, a commercially available fluor. EN3HANCE (New England Nuclear) has largely replaced the use ofPPO dissolv ed in dimethylsulfoxide for detection of 3H in dried polyacryl amide gels. The purpose of this communication is to evaluate each of those new materials and to provide quantitative data that will enable experimenters to use them in double-label autoradiography with maximum efficiency. 2 Materials and methods Calibration gels were made as described previously [2]. In brief, proteins labeled with 3H, HC or 33S were incorporated into 12 % acrylamide gels, which were then fixed, rinsed and impregnated with 20 % w/'w diphenyloxazoie (PPO) in dimethyl sulfoxide or with EN3HANCE. Autoradiography was carried out at -70 C or at room temperature (approx imately 21 C). Optical density of the film at 540 nm was measured in a Zeiss PMQII spectrophotometer, by holding a piece of film over the entrance slit to the sample chamber. All Aj40 values have been corrected for background, determined on a piece of the same film that was not in contact with a gel containing radioactivity. Values of As^o in excess of 1.2 were O Verlag Chcmie GmbH, D-6940 Wcinheim, 1984 0173-0835/84/0408-023012.50/0