SPECIAL CONSIDERATIONS IN THE USE OF BLUE TETRAZOLIUM FOR THE MEASUREMENT OF ALPHA-KETOLS IN MIXTURES WITH OTHER STEROIDS*

Abstract

The influence of different concentrations of alkali on the speed and extent of reduction of blue tetrazolium (BT) by various C21 and C19 steroids was studied over a period of 2 hours at 25[degree]C. When a concentration of 0.022 [image] was used all ketols reacted at the same rate and to the same extent between 30 and 60 minutes. The differences observed were minimal and probably not significant. Non-ketolic steroids with a conjugated 3-keto group caused much less reduction of BT and at a much slower rate. The rate was also influenced by the concentration of alkali but was always much slower than with ketols. When the concentration of base in the reaction mixture was 0.022 [image] the rate was a straight-line function up to 60 minutes. Addition of an 11- and/or 17-keto group enhanced the reaction of the steroids with a conjugated 3-keto group. Addition of a 6-beta-hydroxy or 6-keto group decreased the reactivity of these compounds. An O2 function at carbon 3, 11, 12, 17 or 20 was unreactive unless the delta 4, 3-keto group was also present. Mixtures of ketolic steroids with slowly-reacting non-ketolic steroids gave reduction equivalent to the sums of the separate compounds.