Assay system for simultaneous measurement of three steroidogenic enzyme activities in rat and human testis--effect of human chorionic gonadotropin |
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| Authors: | S C Sikka R S Swerdloff J Rajfer |
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| Institution: | 1. Division of Urology, Department of Surgery, UCLA School of Medicine, Harbor/UCLA Medical Center, 1000 West Carson Street, Torrance, California 90509 USA;2. Division of Endocrinology, Department of Medicine, UCLA School of Medicine, Harbor/UCLA Medical Center, 1000 West Carson Street, Torrance, California 90509 USA;1. Department of Urology, Imperial Healthcare NHS Trust, Charing Cross Hospital, London, UK;2. Department of Urology, University of Foggia, Foggia, Italy;3. Department of Urology, Foundation IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, University of Milan, Milan, Italy;4. Department of Urology and Andrology, Ospedale di Circolo and Macchi Foundation, Varese, Italy;5. CPUP: Center for Psychology of Porto University, Faculty of Psychology and Educational Sciences, Porto University, Porto, Portugal;6. Department of Urology, Taksim Training & Research Hospital, Istanbul, Turkey;7. Department of Minimally Invasive and Robotic Urologic Surgery and Kidney Transplantation, University of Florence, Florence, Italy;8. Endocrinology Unit, Medical Department, Maggiore-Bellaria Hospital, Bologna, Italy;9. Academic Urology Unit, Institute of Applied Health Sciences, University of Aberdeen, Aberdeen, UK;10. Department of Urology, Aberdeen Royal Infirmary, NHS Grampian, Aberdeen, UK;11. School of Medicine, Department of Urology, Selcuk University, Konya, Turkey;12. Department of Urology, Martha-Maria Hospital Nuremberg, Nuremberg, Germany;13. Centre for Diabetes and Endocrinology, Barnsley Hospital NHS Trust, Barnsley, UK;14. Department of Urology, İstanbul University İstanbul School of Medicine, İstanbul, Turkey;15. Department of Urology, Hospital Universitario Puerta del Hierro Majadahonda, Madrid, Spain;p. Department of Urology, University Hospitals Leuven, Leuven, Belgium;q. Manchester Andrology Centre, Manchester Royal Infirmary, Manchester University Hospitals NHS Foundation Trust, UK;r. Urology Section, Department of Surgery, University of Catania, Catania, Italy;s. Department of Urology, Biruni University School of Medicine, Istanbul, Turkey;t. Department of Medicine and Surgery, Scuola Medica Salernitana, University of Salerno, Fisciano, Italy;u. Division of Experimental Oncology/Unit of Urology, Urological Research Institute, IRCCS Ospedale San Raffaele, Milan, Italy;v. University Vita-Salute San Raffaele, Milan, Italy;1. University of Agder, Jon Lilletuns vei 9, 4879 Grimstad, Norway;2. Multiconsult, Bark Silas vei 7, 4876 Grimstad, Norway;3. Aalborg University, Thomas Manns Vej 23, 9220 Aalborg Ø, Denmark;1. Chair of Separation Science and Technology, Technische Universität Kaiserslautern, Germany;2. Institute of Process Engineering, Johannes Kepler University Linz, Austria;1. Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208, United States;2. Department of Chemistry and International Institute of Nanotechnology, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208, United States |
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| Abstract: | An assay system that measures the enzymatic activities (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) in the delta 4 pathway of testosterone biosynthesis using rat and human testicular homogenate was examined. This system involves the simultaneous separation of the steroid intermediates by a three-step TLC procedure. The observed Rf values were 0.78 for progesterone (P), 0.59 for 17 alpha-hydroxyprogesterone (17 alpha-HP), 0.70 for androstenedione (A), 0.5 for testosterone, 0.64 for dihydrotestosterone, and 0.45 for 3 alpha, 17 beta-androstanediol. The identification of these steroid intermediates was further accomplished by acetylation and rechromatography of the representative samples along with the authentic standards and by recrystallization to constant specific activity until three consecutive crystallizations were within +/- 5% of the mean value. Incubation time up to 30 min and increasing protein concentrations showed a linear relationship with respect to these three enzymatic activities. The optimum temperature for these enzymatic activities varied from 32 to 34 degrees C, with a sharp decline between 37 and 40 degrees C. The Michaelis constants (Km) for the rat testis homogenate samples were 0.17 microM for P, 0.22 microM for 17 alpha-HP, and 2.5 microM for A, while for the human testis the Km values were 1.2, 2.2, and 2.3 microM, respectively, for these substrates. The concentrations of the endogenous steroid substrates present in these homogenate samples did not alter the Km or Vmax values. The effect of human chorionic gonadotropin (hCG) in vitro on these steroidogenic enzyme activities was also studied. In the rat testis, 10 IU of hCG produced a significant rise in all the three enzyme activities whereas in the human testis 10 and 30 IU of hCG showed no significant change in any of these enzymatic activities. However, 100 IU of hCG resulted in a significant increase in 17 alpha-hydroxylase and 17,20-desmolase activities in the human testis. These studies suggest that this assay system for the measurement of these enzymatic activities using a testicular homogenate sample provides consistent and reproducible results. Based on the sensitivities of the measurements and our experience with testicular biopsy technique, we conclude that a routine testicular biopsy in the human should provide sufficient tissue to run these enzymatic assays. |
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