Biotin-hydrazide derivatives for the development of steroid enzyme-linked immunoassays.

We are describing the synthesis and use of biotinamidocaproyl-derivatives of 18-oxocortisol-3-carboxymethoxylamine for the development of enzyme labels using the avidin-peroxidase system for the design of enzyme-linked immunoassays (ELISA). An ELISA for 18-oxocortisol and -hydroxycorticosterone was devised which showed improved sensitivity and specificity in comparison to an RIA using a tritiated tracer. The system is easy to prepare and offers the possibility to design immunoassays when no tritiated tracer is available.

Investigation of the salivary 18-hydroxycorticosterone:aldosterone ratio in man using a direct assay.

A method for the direct determination of 18-hydroxycorticosterone (18OHB) in human saliva has been developed and validated. Saliva was collected at 30 min and 1 h intervals between 0600 and 2200 h from healthy men and women for the determination of 18OHB (SHB), aldosterone (SA) and glucocorticoids (SGC = cortisol + cortisone). SHB was highly correlated with SA (r = 0.75; P less than 0.001) but even more highly with SGC (r = 0.89; P greater than 0.001). Multiple regression analysis confirmed that SGC was a more important determinant of SHB than was SA. Though the concentrations of 18OHB and aldosterone were highly correlated there was considerable variation in the 18OHB:aldosterone ratio during the period of saliva collection. This ratio tended to be highest in the morning and lowest in the evening and was weakly correlated with SGC level (r = 0.62; P less than 0.01). The 18OHB:aldosterone ratio in saliva approximates to, and is highly correlated with, that in plasma. We suggest that the fluctuations in SHB:SA ratio correspond to the relative rates of secretion of 18OHB and aldosterone and that this ratio is modulated either by ACTH or by cortisol. Whether this indicates that 18OHB is a by-product of glucocorticoid as well as aldosterone metabolism, or whether this implies a separate physiological role for the steroid remains to be clarified.

The effects of CGS 16949A, an aromatase inhibitor on adrenal mineralocorticoid biosynthesis.

The family of cytochrome P450 enzymes that mediates steroid hydroxylations are distinct but structurally related proteins. Inhibitors of these steroidogenic steps generally exhibit only relative and dose-related specificity. We evaluated an imidazole, cytochrome P450-related inhibitor, CGS 16949A, in postmenopausal patients with metastatic breast cancer. While a relatively specific aromatase inhibitor at daily dosages of 1-2 mg, CGS 16949A significantly blunted cortisol responses to ACTH at a dose of 16 mg daily. To further evaluate other inhibitory effects of this drug, we determined blood levels of aldosterone (ALDO) and 18-hydroxycorticosterone and their respective urinary metabolites, tetrahydroaldosterone and tetrahydro-18-hydroxy-11-dehydrocorticosterone in 16 postmenopausal women receiving CGS 16949A. At a dose of 16 mg/day, CGS 16949A produced significant (P less than 0.001) suppression of both basal and ACTH-stimulated ALDO production. This was accompanied by a significant rise in the blood 18-hydroxycorticosterone/ALDO ratio (11.4 +/- 0.19; normal, less than 2; P less than 0.001), consistent with a corticosterone methyloxidase type II inhibition. A similar significant elevation (7.5 +/- 1.2; normal, less than 5; P less than 0.001) in the urinary tetrahydro-18-hydroxy-11-dehydrocorticosterone/tetrahydroaldosterone ratio was also observed. These results suggest that CGS 16949A is a potent inhibitor of the corticosterone methyloxidase type II enzyme at a dose of 16 mg daily. At doses of 1-2 mg daily, CGS 16949A blocks aromatase without altering basal aldosterone production and, thus, exhibits dose-related specificity.

The determination of 18-hydroxycorticosterone in saliva and plasma: comparison with aldosterone and glucocorticoids.

A radioimmunoassay (RIA) for 18-hydroxycorticosterone (18OHB) is described using antibodies raised against 18OHB-3-CMO conjugated to bovine albumen and an iodine-125 labelled ligand. Extracts of plasma and saliva required thin-layer chromatographic purification prior to RIA. These assays have been validated in terms of precision, accuracy and specificity. The concentration of 18OHB in saliva is approx 20% of that in plasma and the two values are correlated. Thus, in a series of 20 healthy subjects the saliva and plasma concentration were 169 +/- 92 and 904 +/- 621 pmol.l-1 respectively with r = 0.72 (P less than 0.001). When the diurnal patterns of 18OHB, aldosterone and glucocorticoid (cortisol + cortisone) concentrations in saliva were investigated the patterns of 18OHB and aldosterone were usually similar though the 18OHB:aldosterone ratio can vary considerably. These and similar data suggest that the secretion of 18OHB and aldosterone are not completely synchronous. Our results show that saliva is a suitable body fluid in which to estimate 18OHB concentration and this measurement gives a useful reflection of the plasma 18OHB concentration. This technique should facilitate the investigation of the physiological role of this compound.