Radioimmunoassay of 18-hydroxy-11-deoxycorticosterone in plasma.

A method is described for the radioimmunoassay of 18-OH-DOC using antibodies generated in rabbits against the carboxymethoxime derivative coupled to bovine serum albumin. The procedure uses 4 ml of plasma with intra and interassay variations of 8 and 9% respectively. Standard 18-OH-DOC added to plasma from an adrenalectomized patient gave a regression equation, Y=0.974X+/-2.210 and a correlation coefficient of 0.999. The only cross reacting steroid, 18-OH-B which may lead to falsely high levels is removed by a single thin layer chromatographic step. Blood levels in normal subjects agree closely with those calculated indirectly by metabolic clearance and secretion rate measurements. ACTH stimulation produced an 18-fold increase in plasma concentration while dexamethasone suppression decreased levels 3-fold. Four hours in the upright position resulted in a decreased plasma concentration while aldosterone increased. No significant response to dietary sodium restriction could be demonstrated.

Serum levels of corticosterone and 18-hydroxy-11-deoxycorticosterone in the female rat at the high and low points of the circadian rhythm.

Serum corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18-hydroxy-DOC) levels were determined in female rats at the high (1800 h) and low (0600 h) points of the circadian rhythm. In order to carry out these studies, a rapid and accurate non-chromatographic radioimmunoassay method was developed for the measurement of 18-hydroxy-DOC in peripheral blood and similar methodology was used for the B assay. In quiescent rats both steroids were dramatically elevated at 1800 h as compared to 0600 h. The serum levels of B and 18-hydroxy-DOC determined at 1800 h fifteen minutes following stress did not differ significantly from the levels determined following a similar stress at 0600 h. There was a good correlation (r = 0.91) between the levels of B and 18-hydroxy-DOC and it appears that both steroids are regulated by ACTH.

Production and characterisation of antisera to 18-hydroxy-11-deoxycorticosterone.

The production of highly sensitive and specific antisera to 18-hydroxy-11-deoxycorticosterone (18,21-dihydroxy-4-pregnene-3,20-dione) is reported. The antisera were generated in rabbits and guinea pigs with a 3-carboxymethoxime derivative of the steroid coupled to rabbit serum albumin. Antibody characteristics were determined by a radioimmunoassay procedure. Only minor differences between the two animal species were observed. Antibody titers ranged from 10 to 8000. Association constants were in the order of 10(8) to 10(10) 1/mole. A minimal amount of 40 pg unlabeled steroid was necessary to displace 50% of the tritiated steroid. Cross reaction with cortisol was 0.0002% to 0.031%, with aldosterone 0.0007% to 1.09%, with corticosterone 0.0025% to 1%, with 18-hydroxy-corticosterone 0.05% to 1% and with progesterone 0.0048% to 1.5%.

Development and characterization of antisera to 18-hydroxycorticosterone and 18-hydroxy-11-deoxycorticosterone and radioimmunoassay for serum 18-hydroxycorticosterone.

A method for the production of the haptens 18-hydroxy-11-deoxycorticosterone-3-(O-carboxymethyl)-oxime (18-OH-DOC-3-CMO) and 18-hydroxycorticosterone-3-(O-carboxymethyl)-oxime (18-OH-B-3-CMO) is described. The formation of the oximes was studied in kinetic experiments. They were prepared at pH 1.6 in methanol/HCl using a short reaction time. Antisera were raised in rabbits using serum albumin conjugates. The highly specific antisera were used at a final dilution of 1:79 000 (18-OH-DOC) and 1:43 000 (18-OH-B); the affinity constants were 1.2 x 10(10) l/mol and 8.1 x 10(9) l/mol, respectively. The radioimmunoassay procedure for 18-OH-B in serum involves purification by paper chromatography. The intra- and interassay precision was 7.3% and 12.3%, respectively. The mean serum 18-OH-B level (+/- S.D.) for normal male and female ambulatory subjects (n = 40) on a normal sodium diet was 0.802 +/- 0.262 nmol/l. After 60 minutes of recumbency, the serum 18-OH-B level was 0.313 +/- 0.061 nmol/l (n = 6) for men.

The measurement of 18-hyroxy-11-deoxycorticosterone in human plasma by radioimmunoassay.

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11 -deoxycorticosterone (18 -OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1-2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a 1/22,000 dilution for 1/2 hour at 37 degrees C and for 2 hours at 4 degrees C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1,2-3H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 +- 8 (S.D)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 +- 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 +- 1.2 ng per 100 ml in18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.