To determine the metabolic pool changes during acute ACTH infusion, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, corticosterone and cortisol were measured by RIA in subcellular fractions from rat adrenal glands. The in vivo effect of ACTH on steroid biosynthesis was deduced from correlations between subcellular fraction measurements and serum concentrations values, permitting a dynamic interpretation of results. Twenty six Sprague-Dawley male rats, received 0.1 I.U./g of body weight of ACTH I.P. while eighteen (control group) received isotonic saline solution. Rats in both groups were decapitated immediately and at 5, 10 and 20 min after injection. Results showed the highest serum concentrations of corticosterone at time zero. Pregnenolone and corticosterone concentrations were highest in the mitochondrial fraction while progesterone was in the microsomal fraction. Exogenous ACTH depleted-pregnenolone and progesterone pools increasing the corticosterone pool as well as its serum concentration, with minor modifications in other steroids. Its maximal effect occurred at 10 min. In contrast with previous reports, it was found that 17-hydroxylated compounds such as cortisol, revealing 17-hydroxylase enzyme activity. It is concluded that ACTH not only stimulates pregnenolone synthesis but also increases the enzymatic activity of other systems which use this compound as a substrate to produce corticosterone.

Most of the attempts to produce steroid antibodies have utilized one of the functional groups of the steroid molecule to form the corresponding haptene. We have prepared 19-hemisuccinate derivatives of androstenedione, 5α-dihydrotestosterone and testosterone, bound to BSA via the carbodiimide reaction to obtain antibodies with high specificity. With the hemisuccinate linked at the C-19 position the disturbance of the functional groups is reduced, introducing minimal changes and yielding the highest possible resemblance between the antigenic complex and the original steroid molecule. Furthermore, with the hemisuccinate chain in the axial position, the carboxyl group is located far enough away to avoid intramolecular interactions between the protein and the free functional groups. In this form the specificity improved significantly. After 12 weeks of injecting the haptenes into rabbits, we obtained the following antisera: for androstenedione at a dilution of 1:12000, with crossreactivity to androstanedione and epiandrosterone with a displacement effect around 50% of the 3H-androstenedione to dehydroepiandrosterone (DHEA) and testosterone was less than 25%; for dihydrotestosterone the antiserum titres were 1:6000 with crossreaction to testosterone, 5α-androstane 3–17-dione and androstenediol but less than 50% bound and minimal to DHEA and androstenedione; the testosterone antiserum in dilution 1:2000 revealed similar binding for dihydrotestosterone and very low for DHEA and androstenedione. We concluded: 1.—The ring D as well as the A of the steroid molecule were determinants in conferring specificity to the antibodies 2.—The coupling of the protein in the C-19 position near to the ring A together with 3-ketone group and the double bond in C-4 impeded the selective antibody production, decreasing specificity. In view of this 3.—The coupling length chain will be modified to achieve better specificity.

CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) are steroidogenic enzymes which mediate the final step (11β-hydroxylation) in cortisol synthesis and the final three steps (11β-hydroxylation, 18-hydroxylation, and 18-oxidation) in aldosterone synthesis, respectively. The enzymes share 93% identity in amino acid sequence and are encoded by two structurally similar genes which are located in tandem on chromosome 8q22, approximately 40 kb apart. Expression of the aldosterone synthase gene (CYP11B2) is limited to the zona glomerulosa of the adrenal cortex, thereby limiting the synthesis of aldosterone to that zone, where it is principally regulated by plasma levels of angiotensin II and potassium. The 11β-hydroxylase gene (CYP11B1) is expressed in the zona fasciculata, the zone which also expresses a 17-hydroxylase activity, where it mediates cortisol synthesis under the control of ACTH. Genetic recombination involving a mispairing of the two CYP11B genes can lead to duplications and deletions of the genes, creation of hybrid genes of several forms, or transfer of coding and regulatory sequences from one gene to the other. Since the two genes have related but different activities, are normally expressed in different zones, and respond to different physiological signals, such recombination has the potential to generate a variety of inherited disorders of steroid production. In this paper we review the range of mutations which can occur and the resulting disorders of steroid biosynthesis, and suggest some novel mutations which might be sought in variants of these endocrinological syndromes.