Red Sulfuric Acid Fluorescence of Steroid Hormone

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Yellow-green sulfuric acid fluorescence has been mainly used in currently available fluorometry of C19 or C21 steroid hormones. The procedure for developing the fluorescence is shown in TABLE I (testosterone). The characteristics of yellowgreen fluorescence are: exciting wave length, 470 mμ, emitted wave length, 530 mμ, uncorrected. Although fluorometry using the fluorescence has a high sensitivity, a considerable non-steroidal fluorescence makes it difficult to use the method for routine measurement of hormones, because complicated and intensive purification is usually necessary before the samples are subjected to fluorometry. We have recently discovered that certain C19 and C21 steroid hormones produce red sulfuric acid fluorescence. The procedure for development of the fluorescence is shown in TABLE II. The fluorescence characteristics are: exciting wave length, 595 mμ, emitted wave length, 620 mμ, uncorrected (Fig. 1), This fluorescence gives incredibly low residual fluorescence, probably because the peaks of excitation and emission are located in the long wave length region. The difference in non-steroidal fluorescence between red and yellow-green sulfuric acid fluorescence of testosterone is shown in Fig. 2. Compared with fluorescence intensity of testosterone, reagent blank, thin-layer chromatogram blank and paper-chromatogram blank (before washed with methanol) of the red fluorescence show much lower fluorescence intensity than those of the yellow-green fluorescence. The results clearly indicate that application of red fluorescence of steroid hormones to biological materials is far easier than that of yellow-green fluorescence. Red fluorescence develops gradually, reaches the maximum level in 30 minutes and is Stable through 120 minutes. Steroid hormones which produce red fluorescence of significant intensity are shown in TABLE III. Adrenal corticoids; DOC corticosterone 11-deoxycortisol cortisol Progestins; 20 α OH pregnen-4-en-3-one (20αOHP), 20 βOHP 17 0H-progesterone C19 Steroids; testosterone, epitestosterone Δ4-androstenedione 11-OHΔ4-androstenedione DHEA Estrogens produce no red fluorescence. In the table, several steroid hormones, for instance DOC, corticosterone, 20 α-OHP, 20 β-OHP, give stronger fluorescence than testosterone. This is probably due to the relatively large amount (10μg) used in this study, because 10 μg of testosterone produce considerable yellow-green fluorescence which quenches markedly the intensity of red fluorescence. When we use much smaller amounts, testosterone gives the most intensive red fluorescence among all steroid hormones tested. The minimum detectable amounts of steroid hormones with red fluorescence are: testosterone, epitestosterone 0.1ng 20 α-OHP, 20 β-OHP, DOC, corticosterone 0.5ng 11-deoxycortisol 2ng etc. Molecular structures of the steroid hormones which produce red fluorescence are: C19 or C21 steroids with Δ4-3-keto or Δ5-3βOH structure 11-oxygenation or 18-aldehyde diminishes the fluorescence intensity Red fluorescence is now being successfully applied to measurement of testosterone in rat testicular vein blood, testosterone in mouse testis, testosterone in peripheral plasma of mouse, rat, dog and man, 20 α-OHP in rat ovarian vein blood, 20 α-OHP in peripheral plasma of rat and mouse, 11-deoxycortisol in peripheral plasma of monkey and man. Fluorescence is measured with Shimadzu GSF 16 spectrofluorophotometer.
Japan Society of Clinical Chemistryの論文

Red Sulfuric Acid Fluorescence of Steroid Hormone

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