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Quantitative determination by HPLC of urinary 6β-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs

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Summary

A method for determination of 6β-hydroxycortisol in urine by means of high performance liquid chromatography is described. After extraction of 10–30 ml aliquots of urine with ethylacetate, separation is accomplished on a silica gel column (30 cm, Lichrosorb Si 100) with a special two-phase four-component eluent of methylene chloride, n-hexane, ethanol and water. Complete separation of α- andβ-isomers requires 15 to 20 min. For routine determinations precolumn cleaning by backflush permits injections of samples at minimum time intervals. For quantitative determinations, each injection should contain at least 0.05–0.5 µg of 6β-hydroxycortisol, depending on the detector employed. The mean excretion rate in healthy male adults (26–40 years) was 273 µg/day (SD=74.5; n=12). In patients on long term mono-therapy with rifampicin, 6β-hydroxycortisol excretion had risen fourfold (1166 µg/d; SEM=248; n=7), paralleling the known enzyme-inducing effect of rifampicin. The relatively smaller increase to 498 µg/d observed in patients receiving triple therapy with rifampicin, isoniazid and ethambutol points to possible inhibition by isoniazid. The greatest stimulation of 6β-hydroxycortisol excretion (2352 µg/d) was found in patients receiving antiepileptic therapy (phenytoin and/or carbamazepine and other drugs). The HPLC technique for 6β-hydroxycortisol proved to be a tool routinely applicable to non-invasive evaluation of drug metabolizing enzyme activity in man.

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References

  1. Conney, A. H.: Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev.19, 317–366 (1967)

    Google Scholar 

  2. Roots, I., Saalfrank, K., Hildebrandt, A. G.: Comparison of methods to study enzyme induction in man. In: Cytochromes P-450 and b5. D. Y. Cooper, O. Rosenthal, R. Snyder, and Ch. Witmer (eds.), pp. 485–502. New York: Plenum Press 1975

    Google Scholar 

  3. Hildebrandt, A. G., Roots, I., Speck, M., Saalfrank, K., Kewitz, H.: Evaluation of in vivo parameters of drug metabolizing enzyme activity in man after administration of clemastine, phenobarbital or placebo. Eur. J. Clin. Pharmacol.8, 327–336 (1975)

    Google Scholar 

  4. Frantz, A. G., Katz, F. H., Jailer, J. W.: 6β-hydroxycortisol and other polar corticosteroids: Measurement and significance in human urine. J. Clin. Endocrinol.21, 1290–1303 (1961)

    Google Scholar 

  5. Thrasher, K., Werk, E. E., Choi, Y., Sholiton, L. J., Meyer, W., Olinger, Ch.: The measurement, excretion and source of urinary 6-hydroxycortisol in humans. Steroids14, 455–468 (1969)

    Google Scholar 

  6. Chamberlain, J.: The determination of urinary 6-oxygenated cortisol in evaluating liver function. Clin. Chim. Acta34, 269–271 (1971)

    Google Scholar 

  7. Park, B. K.: A direct radioimmunoassay for 6β-hydroxycortisol in urine. J. Steroid Biochem.9, 963–966 (1978)

    Google Scholar 

  8. Hövermann, W.: Methods of optimization in high pressure liquid chromatography. (Submitted for publication)

  9. Holbe, R., Hövermann, W., Roots, I., Hildebrandt, A. G.: Determination of 6β-hydroxycortisol in urine by high pressure liquid chromatography. Naunyn-Schmiedeberg's Arch. Pharmacol. Suppl. to Vol. 293, R 61 (1976)

  10. Bernstein, S., Littell, R.: C6-Hydroxylated steroids. I. Preparation of 6α- and 6β-hydroxyhydrocortisone and 6α-hydroxyprednisolone. J. Org. Chem.25, 313–314 (1960)

    Google Scholar 

  11. Nigam, S., Roots, I., Hildebrandt, A. G.: Photochemical synthesis of 6β-hydroxycortisol with hydroxyl radicals. (In preparation)

  12. Ohnhaus, E. E., Park, B. K.: Measurement of urinary 6-β-hydroxycortisol excretion as an in vivo parameter in the clinical assessment of the microsomal enzyme-inducing capacity of antipyrine, phenobarbitone and rifampicin. Eur. J. Clin. Pharmacol.15, 139–145 (1979)

    Google Scholar 

  13. Sanghvi, A., Wight, C., Parikh, B., Desai, H.: Urinary 17-hydroxycorticosteroid determination with p-hydrazinobenzenesulfonic acid-phosphoric acid. Am. J. Clin. Pathol.60, 684–690 (1973)

    Google Scholar 

  14. Huber, J. F. K., Hulsman, J. A. M. J.: A study of liquid chromatography in columns. Time of separation. Anal. Chim. Acta38, 305–313 (1967)

    Google Scholar 

  15. Hesse, Chr., Hövermann, W.: Ein Dreikomponenten-Zweiphasensystem für die schnelle Flüssigkeitschromatographie. Chromatographia6, 345–348 (1973)

    Google Scholar 

  16. Ghosh, P. C., Pennington, G. W.: A comparison of gas-liquid chromatography with colorimetric methods for the estimation of extremely polar unconjugated corticosteroids in urine. Steroids13, 247–255 (1969)

    Google Scholar 

  17. Katz, F. H., Lipman, M. M., Frantz, A. G., Jailer, J. W.: The physiologic significance of 6β-hydroxycortisol in human corticoid metabolism. J. Clin. Endocrinol. Metab.22, 71–77 (1962)

    Google Scholar 

  18. Werk, E. E. Jr., MacGee, J., Sholiton, L. J.: Altered cortisol metabolism in advanced cancer and other terminal illnesses: Excretion of 6-hydroxycortisol. Metabolism13, 1425–1438 (1964)

    Google Scholar 

  19. Berthold, K., Staudinger, Hj.: Quantitative Bestimmung von 6β-Hydroxycortisol unter Verwendung der Dünnschichtchromatographie mit Fluoreszenzindikator. Z. Klin. Chemie3, 130–133 (1966)

    Google Scholar 

  20. Yamaji, T., Motohashi, K., Murakawa, S., Ibayashi, H.: Urinary excretion of 6β-hydroxycortisol in states of altered thyroid function. J. Clin. Endocrinol. Metab.29, 801–806 (1969)

    Google Scholar 

  21. Poland, A., Smith, D., Kuntzman, R., Jacobson, M., Conney, A. H.: Effect of intensive occupational exposure to DDT on phenylbutazone and cortisol metabolism in human subjects. Clin. Pharmacol. Ther.11, 724–732 (1970)

    Google Scholar 

  22. Berman, M. L., Green, O. C.: Acute stimulation of cortisol metabolism by pentobarbital in man. Anesthesiology34, 365–369 (1971)

    Google Scholar 

  23. Stevenson, I. H., Browning, M., Crooks, J., O'Malley, K.: Changes in human drug metabolism after long-term exposure to hypnotics. Br. Med. J.1972/IV, 322–324

    Google Scholar 

  24. Smith, S. E., Rawlins, M. D.: Prediction of drug oxidation rates in man: Lack of correlation with serum gamma-glutamyl transpeptidase and urinary excretion of D-glucaric acid and 6β-hydroxycortisol. Eur. J. Clin. Pharmacol.7, 71–75 (1974)

    Google Scholar 

  25. Silber, R. H., Porter, C. C.: The determination of 17, 21-dihydroxy-20-ketosteroids in urine and plasma. J. Biol. Chem.210, 923–932 (1954)

    Google Scholar 

  26. Roots, I., Ley, B., Hildebrandt, A. G.: In vivo parameters of drug metabolism — differences in specificity towards inducing agents. In: Microsomes and Drug Oxidations. V. Ullrich, I. Roots, A.G. Hildebrandt, R.W. Estabrook, A.H. Conney (eds.), pp. 581–588. Oxford: Pergamon Press 1977

    Google Scholar 

  27. Jezequel, A. M., Orlandi, F., Tenconi, T.: Changes of the smooth endoplasmic reticulum induced by rifampicin in human and guinea-pig hepatocytes. Gut12, 984–987 (1971)

    Google Scholar 

  28. Remmer, H., Schoene, B., Fleischmann, R. A.: Induction of the unspecific microsomal hydroxylase in the human liver. Drug Metab. Dispos.1, 224–230 (1973)

    Google Scholar 

  29. Yamada, S., Iwai, K.: Induction of hepatic cortisol-6-hydroxylase by rifampicin Lancet1976/II, 366–367

    Google Scholar 

  30. Raisfeld, I. H., Pfister, L., Finegold, M.: The effect of isoniazid on hepatic endoplasmic reticulum in man and rat. Gastroenterology65, A-42/566 (1973)

  31. Austerhoff, A., Kindler, U., Knop, P., Knieriem, H.-J.: Lebertoxizität einer Rifampicin-Kombinationstherapie. Dtsch. Med. Wochenschr.99, 1182–1188 (1974)

    Google Scholar 

  32. Mitchell, J. R., Thorgeirsson, U. P., Black, M., Timbrell, J. A., Snodgrass, W. R., Potter, W. Z., Jollow, D. J., Keiser, H. R.: Increased incidence of isoniazid hepatitis in rapid acetylators: possible relation to hydrazine metabolites. Clin. Pharmacol. Ther.18, 70–79 (1975)

    Google Scholar 

  33. Passayre, D., Bentata, M., Degott, C., Nouel, O., Miguet, J.-P., Rueff, B., Benhamou, J.-P.: Isoniazid-rifampicin fulminant hepatitis. A possible consequence of the enhancement of isoniazid hepatotoxicity by enzyme induction. Gastroenterology72, 284–289 (1977)

    Google Scholar 

  34. Werk, E. E. Jr., MacGee, J., Sholiton, L. J.: Effect of diphenylhydantoin on cortisol metabolism in man. J. Clin. Invest.43, 1824–1835 (1964)

    Google Scholar 

  35. Roots, I.: Effect of rifampicin on urinary excretion of 6β-hydroxycortisol and glucaric acid in man and animal. Naunyn-Schmiedeberg's Arch. Pharmacol.307, R 72 (1979)

    Google Scholar 

  36. Roots, I., Ley, B., Sctiawan, L., Heinemeyer, G., Hildebrandt, A. G.: Spironolactone. Characterization of its enzyme inducing properties by in vivo parameters. In: Aldosterone Antagonists in Clinical Medicine. G. M. Addison, N. Wirenfeld Asmussen, P. Corvol, P.W.C. Kloppenborg, N. Norman, R. Schröder, J.I.S. Robertson (eds.), pp. 165–176. Amsterdam: Excerpta Medica 1978

    Google Scholar 

  37. Daniilescu-Goldinberg, D., Giroud, C.J.P.: Metabolism of cortisol by the human newborn infant. J. Clin. Endocrinol. Metab.38, 64–70 (1974)

    Google Scholar 

  38. Frantz, A. G., Katz, F. H., Jailer, J. W.: 6-Betahydroxy-cortisol high levels in human urine in pregnancy and toxemia. Proc. Soc. Exp. Biol. Med.105, 41–43 (1960)

    Google Scholar 

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Authors and Affiliations

  1. Department of Clinical Pharmacology, Free University of Berlin, Germany

    I. Roots, R. Holbe, S. Nigam, G. Heinemeyer & A. G. Hildebrandt

  2. Siemens AG, Karlsruhe, Germany

    W. Hövermann

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  1. I. Roots
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  2. R. Holbe
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  3. W. Hövermann
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  4. S. Nigam
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  5. G. Heinemeyer
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  6. A. G. Hildebrandt
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Roots, I., Holbe, R., Hövermann, W. et al. Quantitative determination by HPLC of urinary 6β-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs. Eur J Clin Pharmacol 16, 63–71 (1979). https://doi.org/10.1007/BF00644969

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  • DOI: https://doi.org/10.1007/BF00644969

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