P. Narasimha Rao

Synthesis of new steroid haptens for radioimmunoassay—part V. 19-O-carboxymethyl ether derivative of testosterone. A highly specific antiserum for immunoassay of testosterone from both male and female plasma without chromatography

Abstract Synthesis of the 19- O -carboxymethyl ether derivative of testosterone and the preparation of its bovine serum albumin (BSA) conjugate is described. By employing this conjugate, antiserum was raised in rabbits which proved to be specific for testosterone. A radioimmunoassay (RIA) procedure is described for measurement of testosterone from both male and female plasma after ether-chloroform (4:1) extraction of the sample without prior chromatography.

Analysis of metabolites of progesterone in bovine liver, kidney, kidney fat, and milk by high performance liquid chromatography

An investigation by high-performance liquid chromatography (HPLC) of the metabolites from the livers of cows and steers treated with [4-14C]-progesterone revealed them to consist principally of 3α-hydroxy-5β-pregnan-20-one and 5β-pregnane-3α,20β-diol. In bovine kidney tissue the principal metabolites were found to be 20β-hydroxypregn-4-en-3-one, 3α and 3β-hydroxy-5α-pregnan-20-one and 5α-pregnane-3β,20β-diol in about equal amount, plus about 15% of the total as unconverted progesterone. In milk the major product was 5α-pregnane-3,20-dione in addition to about 80% of the total as unconverted progesterone. Only traces of compounds other than progesterone were found in kidney fat. These metabolites were further characterized by crystallization to constant specific acitivity, including derivative formation. This study demonstrates for the first time the utility of HPLC for the rapid analysis of a complete mixture of metabolites of progesterone from crude extracts of mammalian tissues.

Antisera for radioimmunoassay of 17α-ethynylestradiol and mestranol

Antisera for radioimmunoassay of 17alpha-ethynyl estradiol and mestr anol were prepared and tested. The antisera were prepared by immunizing rabbits with 6(O-carboxymethyl) oxime-bovine serum conjugates from 6-oxo-17alpha-ethnyl estradiol and 6-oxomestranol. Antisera raised to 17alpha-ethynyl estradiol by employing steroid-protein conjugates coupled at either carbon-6 or -7 proved to be highly specific and showed little cross-reaction with mestranol. Antisera for mestranol also showed little cross-reactivity while the antibody to the 6-linked agent showed only moderate cross-reaction to 6-hydroxymestranol but an avidity for the 6-oxomestranol which exceeded that of the reference compound. This assay has been employed in measuring these substances after ingestion of oral contraceptives.

Metabolites of estradiol-17β in bovine liver: Identification of the 17-β-d-glucopyranoside of estradiol-17α

Abstract An investigation of the unidentified polar metabolites from the livers of steers treated with [4-14-C]-estradiol-17 β or its 3-benzoate revealed them to consist principally of glycosidic derivates of estradiol-17α. The major polar metabolite was the 17-β- d -glucopyranoside of estradiol-17α. The 3-β- d -glucosiduronate of estradiol-17α, and other 17-glycosides of estradiol-17α and estradiol-17β were also characterized. This study demonstrates for the first time the presence of estrogen glycosides in bovine liver tissue.

The conversion of 2-hydroxyestradiol-17β to 2-hydroxy and 2-methoxy metabolites in human urine☆

Abstract Intravenous infusions of 1–200 mg. 2-hydroxyestradiol-17β in plasma were administered to four postmenopausal women. The following urinary metabolites were definitively identified: 2-hydroxyestrone, 2-hydroxyestriol, 2-methoxyestrone, 2-methoxyestradiol-17β, and 2-methoxyestriol. The origin of the urinary estrogen methyl ethers appears to be by way of transmethylation of the 2-hydroxylated compound, or as a result of further metabolism of 2-methoxyestrone.

Synthesis of new steroid haptens for radioimmunoassay—VI. 3-O-carboxymethyl ether derivatives of equine estrogens. Highly specific antisera for measurement of equilin and 17α-dihydroequilin in plasma

Abstract The synthesis of 3-O-carboxymethyl ether derivatives of equilin and 17α-dihydroequilin and the preparation of their bovine serum albumin (BSA) conjugates are described. These BSA-conjugates were employed for the generation of monospecific antisera in rabbits suitable for radioimmunoassay (RIA) of equilin and 17α-dihydroequilin. The preparation of [2,4-3H]-17α-dihydroequilin required as a tracer for the RIA of 17α-dihydroequilin is presented. An RIA procedure is described for the measurement of free and sulpho-conjugated equilin and 17α-dihydroequilin in samples of plasma from women receiving Premarin.

Total synthesis of polymethoxyoestrane compounds. Part I. Synthesis of (±)-2,3,4-trimethoxyoestra-1,3,5(10)-trien-17β-ol and related compounds

By use of general procedures developed for (±)-oestrone, 2,3,4-trimethoxyoestra-1,3,5(10)-trien-17β-ol (7) has been totally synthesized from 3,4-dihydro-5,6,7-trimethoxynaphthalen-1(2H)-one (1). Metal–ammonia reduction of the intermediate 2,3,4-trimethoxyoestra-1,3,5(10),8-tetraen-17β-ol (6) under the usual conditions caused loss of the 3-methoxy-group. Reduction under carefully controlled conditions was necessary to obtain the trimethoxyoestratrienol (7). A series of stereoisomers of (±)-2,3,4-trimethoxyoestranes has been prepared for evaluating biological activity.

Alkylation of brain corticosteroid acetyltransferase by 17-hydroxyprogesterone 17-(9-oxo-10-chlorodecanoate) and related compounds.

Abstract The addition of 17-hydroxyprogesterone 17-(9-oxo-10-chlorodecanoate) (1) at 1.5 μM to a partially purified preparation of corticosteroid acetyl-transferase from the primate brain at pH 7.4, results in a 50% inhibition of enzymatic activity after 30 minutes at 37°. At this concentration the analogous 9-oxo-10-diazodecanoate, 8-carbomethyoxyoctanoate or 8-carboxyoctanoate esters show no effect on the activity of this enzyme. 9-Oxo-10-chlorodecanoate and its methyl ester are respectively 0.44 and 0.07-fold as effective inhibitors as 1. The inhibition by 1 has been shown to be non-competitive and irreversible. There is no reaction of 1 with amino acids, glutathione, or human or bovine serum mercaptalbumin in a pH 7.4 phosphate buffer at 37°, demonstrating the partial specificity of this alkylating agent.

Total synthesis of polymethoxyoestrane compounds. Part II. Synthesis of (±)-2,4-dimethoxyoestra-1,3,5(10)-trien-17β-ol

(±)-2,4-Dimethoxyoestra-1,3,5(10)-trien-17β-ol (7; R1= R3= OMe, R2= H), synthesized from 5,7-dimethoxy-1-tetralone (1), was found to be identical with the demethoxylated product obtained by metal–ammonia reduction of 2,3,4-trimethoxyoestra-1,3,5(10),8-tetraen-17β-ol (6; R1= R2= R3= Me).

Competitive inhibition of primate brain corticosteroid acetyltransferase by triamcinolone and related 16α-hydroxysteroids

Abstract 16α,21-Dihydroxycorticosteroids are acetylated at only 2% of the rate of acetylation of cortisol by primate brain corticosteroid acetyltransferase. However, 50 μM triamcinolone (1) and its 6α-fluoro derivative (2) result in a 60% inhibition of the acetylation of 50 μM cortisol (Ks = 1.23 × 10−6M). This inhibition has been shown to be competitive for 1 with a Ki = 6.40 × 10−6M. Other 16α-hydroxycorticosteroids lacking both the Δ1- and 9α-fluoro- groups are considerably less effective as inhibitors.