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Archives Internationales de Physiologie et de Biochimie, 1985, 93, 255-256 255
R q u le 20 juin 1985.
Communication breve
P. GENARD, M. PALEM-VLJERS, M. R. LOMBA-PIGNON, L. CHRISTIAENS('), and J. DE GRAEVE [DPpartement de Clinique et de Pathologie MPdicales (Professeur H. Van Cauwenberghe), Universitk de Lisge, H6pital de Bavi&re, B-4020 Likge]
Discovery of a natural spirolactone derivative in man and animal (1 figure).
In previous papers (GENARD et al., 1977; GENARD & PALEM-VLIERS, 1981) we reported that during the estimation of urinary aldosterone and 18-hydroxy- deoxycorticosterone (1 8-OH-DOC) in man by gas liquid chromatography (GLC) an unknown compound more polar than aldosterone was detected. Analytical and spec- troscopic data indicate a structure similar to that of canrenone I . However the spot of canrenone in thin layer chromatography (TLC) was very far from that of the in- vestigated substance. We started then with the hypothesis that the natural compound was not canrenone itself but a derivated structure substituted at C,-C, possibly with two hydroxyl groups (6,7-dihydroxy-6,7-dihydrocanrenone, DHC), this natural com- pound being probably thermally transformed into canrenone on the GLC column at 250 "C.
4 6 1 -
FIG. 1.
(') Service de Chimie Organique HPtProcyclique (Profeseur M. Renson), Institut de Chimie B6 Sart
v) Laboratoire de Chimie MPdicale (Professeur J. Gielen), Institut de Pathologie B23, Sart Tilman, Tilman, B-4000 Liege.
8-4000 Liege.
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256 GENARD, PALEM-VLIERS, LOMBA-PIGNON, CHRISTIAENS AND DE GRAEVE
In the present paper we report the results of our preliminary investigations of that unknown compound. Having in mind the starting hypothesis, we have been able to synthetize some isomeric 6,7-dihydroxy-6,7-dihydrocanrenones. Starting from canrenone 1 reaction on one hand with osmium tetroxide leads to the two isomeric DHC & and and on the other hand with periodic acid gives the Pa derivative a.
The structure of these steroids has been established by spectroscopic measurements and will be published elsewhere.
The unknown compound was investigated in 212 patients ranging in age from 28 to 65 years without any treatment. One spot was isolated in TLC on silicagel of an extract of 24-h urine collection prepared as described elsewhere (GENARD et a/., 1977). This spot has nearly the same R, as 6p, 7a-, 601, 701- or 60, 7P-DHC in four eluents of increasing polarity. In the same conditions, the R, of monohydroxylated 6,7-dihydrocanrenone (601, 6P or 7a) are quite different. The spot isolated by TLC was analysed through heptafluorobutyrylation. Under these conditions of improved sensitivity three peaks were detected in GLC with retention times totally different of that of 6,7-dihydrocanrenone monohydroxylated at C, or C,. Pure synthetic dihydroxylated materials submitted to the same analysis also furnish three peaks at the same retention times as the natural products.
Due to the low concentration of the natural product, a pool of ten litres of human urine from subjects without any treatment was extracted, and then purified by high performance liquid chromatography (HPLC). The fraction corresponding to the DHC was collected and analysed by GLC and mass spectrometry. The GLC retention time of the product after heptafluorobutyrylation as well as its mass spectrum are the same as for synthetic materials.
Thus, these experiences confirm the presence in man of natural dihydroxylated spirolactone. We have been able to detect the same natural product in rat and sheep and to show that as well in man as in rat, sodium loading conditions result in a decrease of urinary DHC. On the contrary sodium depletion increases the DHC concentra- tion. Further experiments will allow to determine the correct isomeric structure at C,C, and to acute the biological activity.
Summary
The presence of 6,7-dihydroxy-6,7-dihydrocanrenone (DHC) in man and in animal has been shown. Sodium loading results in a decrease of urinary DHC. On the con- trary, sodium depletion increases its concentration.
Acknowledgments. - We thank Roussel UCLAF, Searle for generous gift of spirolactones, and Pro- fessor M. RENSON for fruitful discussion. This research was supported in part by grant of the FRSM (Belgium).
References
GENARD, P. & PALEM-VLIERS, M. (1981) Ann. Endocrinol. 42, 77 (Abstr.). GBNARD, P., PALEM-VLIERS, M. & EECHAUTE, W. (1977) Res. Steroids, 8, 309-311.
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