Plasma levels of 18-hydroxy-11-deoxycorticosterone in essential hypertension.

In order to investigate the role of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) in essential hypertension (EH), the responses of plasma 17-OH-DOC to 7 stimulation tests (furosemide test, adrenal suppression test, angiotensin II infusion test, adrenal stimulation test, metopirone test, saline infusion test and potassium chloride infusion test) and the circadian rhythm were investigated in 18 patients with essential hypertension (low renin group: 8, and normal renin group: 10). From the present study, it micht be thought that plasma 18-OH-DOC does not play an important role in the suppression of PRA in patients with low PRA.     

Serum corticosteroids at the high and low points of the circadian rhythm in rats with regenerating adrenals.

Serum levels of 11-deoxycorticosterone (DOC), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and corticosterone (B) were determined at the high (1800 h) and low (0800 h) points of the circadian rhythm in control rats and in rats with regenerating adrenals. The levels of DOC at 0800 h in quiescent rats with regenerating adrenals were 6.5 times greater than in the control group. The levels of 18-OH-DOC and B, however, were not significantly different between these groups. A circadian rhythm for B, 18-OH-DOC and DOC was evident in control rats with a 12,20 and 3.5 fold increase, respectively, at 1800 h as compared to 0800 h. In animals with regenerating adrenals there was only a minimal change in the levels of B and 18-OH-DOC at 1800 h. There was, however, a 2 fold further increase in the levels of DOC at 1800 h as compared with the elevated levels at 0800 h. These findings show that the decrease in 11β and 18-hydroxylase activity of the regenerating adrenal is most clearly evident at the high point of the circadian rhythm. Furthermore, only by taking into account physiological variations in adrenal activity can an accurate assessment of DOC secretion in the adrenal regeneration model of hypertension be obtained.     

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%.     

Adrenal and liver metabolites of 18-hydroxy-11-deoxycorticosterone in the rat. Identification of reduced compounds (18-OH-TH-DOC)

The presence of reduced metabolites of 18-hydroxy-11-deoxycorticosterone has been investigated in the adrenals of 23 day-old and adult rats and in the liver of adult rats. By thin-layer chromatography a fraction of the adrenal steroid extract migrating like tetrahydro-corticosterone has been isolated. By gas chromatography-mass spectrometry several isomers of 3,18,21-trihydroxy-pregnan-20-one (18-OH-TH-DOC) have been separated in this fraction and identified by comparison with authentic samples which have been chemically and enzymatically synthesized. The major tetrahydrogenated metabolite in the adult and prepuberal rat adrenals is 3β,18,21-trihydroxy-5α-pregnan-20-one (18-OH-TH-DOC II). The 3α,18,21-trihydroxy-5β-pregnan-20-one has been found only in the prepuberal rat adrenal. A third tetrahydrogenated isomer has been tentatively identified as 3α,18,21-trihydroxy-5α-pregnan-20-one. Quantitative measurements by mass fragmentography show that adrenal reductase activity on 18-hydroxy-11-deoxycorticosterone is higher than on corticosterone. The 18-OH-TH-DOC II has been identified in the liver of adult male rat.     

A non-chromatographic radioimmunoassay of 18-hydroxy-11-deoxycorticosterone in human plasma

A method is described for a non-chromatographic assay of 18 hydroxy-11-deoxycorticosterone (18-OH-DOC) using a sensitive and specific antiserum. This direct measurement is assessed in terms of accuracy and precision. The mean 8a.m. plasma 18-OH-DOC levels in the supine position was 10.1 ± 6.5 ng per 100ml in 20 normal subjects and 9.4 ± 4.2 ng per 100ml after two hours of movement. These values are correlated with those obtained in aldosterone. The ACTH-dependency of 18-OH-DOC is demonstrated by diurnal variation and treatment with dexamethasone.     

Decomposition of 11-deoxycorticosterone and corticosterone in soda-lime glass.

When methanolic solutions of tritiated 11-deoxycorticosterone or corticosterone (0.1 to 300 ng) were evaporated to dryness with a stream of nitrogen in soda-lime test tubes only 8-24% of the radioactivity was recovered as the parent steroid. Evaporation in borosilicate test tubes led to a recovery of 90% or more. With ethyl acetate as solvent no decomposition occured in soda-lime test tubes.     

The measurement of 18-hydroxy-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 $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°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 in 18 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.     

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress.

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.     

Development and characterization of antisera to 18-hydroxy-corticosterone 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/HC1 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 × 10¹⁰l/mol and 8.1 × 10⁹l/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.     

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.     

Effect of chronic intracerebroventricular infusion of corticotropin-releasing factor on circadian corticosterone rhythm in the rat

The effects of chronic (14 day) intracerebroventricular infusion of various amounts of ovine corticotropin-releasing factor (oCRF) on the circadian blood corticosterone rhythm in male rats were examined. Control (saline-infused) rats showed distinct blood corticosterone rhythms over 48 h with nadirs at 0900 h and peaks at 2100 h on days 6-7 and 13-14. oCRF at 3 pmol/h did not affect the circadian corticosterone rhythm on these days. When oCRF was infused at a rate of 12 pmol/h, blood corticosterone was increased throughout the 48 h periods. A significant circadian rhythm remained at days 6-7, but continuous infusion for an additional 7 days disrupted the rhythm. Higher doses of oCRF (48 and 240 pmol/h) obliterated the rhythm during both periods; the disruption was characterized by an increase in corticosterone during the lights-on period without a substantial change in the evening maximum. Thus, the blood corticosterone concentration was eventually confined within a narrow range, not exceeding the normal circadian peak, over a wide dose range of centrally administered CRF. Significant effects of oCRF on body and adrenal weight were observed only at the two highest doses used. These findings may provide some insight into the state of the hypothalamic-pituitary-adrenal axis in animals exposed to chronic stress and in patients with depression.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.     

The relation of hepatic in vitro inactivation of corticosteroids to the circadian rhythm of plasma corticosterone.

Plasma corticosterone levels and the in vitro capacity of the liver to reduce the delta(4)3-ketone group of corticosterone were ascertained at 4 hr intervals in male rats maintained on a normal lighting schedule (12L:12D). The rates of delta(4)3-ketone reduction, as well as wet liver weight, were highest during the early portion (08.00 hr) of the light period when liver protein and plasma corticosterone concentrations were low. Shortly (2000 hr) after the beginning of the dark period plasma corticosterone reached peak levels, while hepatic inactivation of corticosterone was markedly depressed. This inverse relationship suggests that the rhythmicity in the capacity of the liver to inactivate corticosterone may contribute to the circadian periodicity of plasma corticosterone.     

Effect of fasting on the circadian rhythm of serum insulin levels.

The effects of 29 hrs of fast on gastric content, serum glucose and IRI levels were studied twice in normal mice. Both experiments showed a circadian rhythm of all parameters studied in control animals. Fasting in both experiments was unable to modify the circadian rhythm of gastric content and serum glucose levels, in spite of substantial modifications in their absolute values. Conversely, while the rhythm of serum IRI levels was not changed in one experiment, the other showed a variation with no level of significance. The latter coincides with the greatest decrease of both gastric content and serum glucose levels. Therefore, circadian IRI levels should be only within some range under the control of food intake. This suggests the possible existence of a threshold for absolute glucose and/or gastric content variation under which serum IRI level rhythm is unable to reach the level of significance.