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%.     

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.     

Production of 19-hydroxy-11-deoxycorticosterone and 19-oxo-11-deoxycorticosterone from 11-deoxycorticosterone by cytochrome P-450(11)beta

Incubation of 11-deoxycorticosterone with a cytochrome P-450(11)beta-reconstituted system yielded, in addition to corticosterone and 18-hydroxy-11-deoxycorticosterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-11-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-11-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450(11)beta was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-11-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-11-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450(11)beta. Therefore, we next incubated 19-hydroxy-11-deoxycorticosterone with cytochrome P-450(11)beta and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-oxo-11-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and 1H NMR analyses. Its field desorption mass spectrum (FD-MS) showed a M+ peak at m/e 344. The 1H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450(11)beta can catalyze the 19-hydroxylation of 11-deoxycorticosterone, and the 19-hydroxy-11-deoxycorticosterone produced is further oxidized at the C-19 position to 19-oxo-11-deoxycorticosterone.     

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.     

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.     

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.     

19-Hydroxylation of 18-hydroxy-11-deoxycorticosterone by adrenal mitochondria prepared from various animal species

We have recently reported that bovine adrenocortical cytochrome P-45011 beta catalyzes 19-hydroxylation of 18-hydroxy-11-deoxycorticosterone (18(OH)DOC) in addition to 11 beta-hydroxylation of the steroid. In this report, we examine the presence of these two activities in 18(OH)DOC and 11 beta- and 18-hydroxylation activities on deoxycorticosterone (DOC) among the adrenal mitochondria prepared from man, ox, pig, rabbit, guinea-pig and rat. The results indicate that these animals could be classified into three groups with respect of these hydroxylation activities. Mitochondria of the first group comprising ox and pig showed rather high 19- and 11 beta-hydroxylation activities on 18(OH)DOC compared to the hydroxylation activities on DOC. Mitochondria prepared from the second group which comprised rabbit, guinea-pig and man showed low 19-hydroxylation activity on 18(OH)DOC, whereas the 11 beta-hydroxylation of 18(OH)DOC well occurred in these species. The last group comprising rat had very low activity both of 11 beta- and 19-hydroxylations when 18(OH)DOC was used as the substrate, whereas both 11 beta- and 18-hydroxylations of DOC were high in rat adrenal mitochondria. No significant difference of these activities could be found between zona glomerulosa cells and zonae fasciculata-reticularis cells of bovine adrenal cortex, and between adrenal mitochondria from spontaneously hypertensive rat and those from WKY normotensive rat.     

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.     

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.     

Inhibition of aldosterone production in rat adrenal mitochondria by 18-ethynyl-11-deoxycorticosterone: a simple model for kinetic interpretation of mechanism-based inhibitors

A simple mathematical model for studying mechanism-based inhibitors (MBIs) is presented. The mathematical equations are deduced for an experimental protocol consisting of a first incubation of the enzyme in the presence of MBI followed by a washing protocol to eliminate free MBI. Finally enzyme activity (initial velocity) is measured with specific substrate. The representation of the final equation obtained is a straight line, and the MBI-specific association constant of velocity (k) can be calculated from its slope. The mathematical model was then challenged with the effect of 18-ethynyl-11-deoxycorticosterone (18-EtDOC) as an MBI on aldosterone biosynthesis from 11-deoxycorticosterone (DOC) in rat adrenal mitochondria. The last step of the mitochondrial biosynthesis of aldosterone consists of the conversion of DOC into corticosterone (B) or 18-hydroxy-11-deoxycorticosterone (18-OHDOC), and both steroids can then be transformed into aldosterone. The k (mM(-1) x min(-1)) values obtained for 18-EtDOC were: 451 +/- 36 for DOC to aldosterone; 177 +/- 16 for B to aldosterone; 175 +/- 15 for 18-OHDOC to aldosterone; and 2.7 +/- 0.2 for DOC to B. These results show that this MBI practically does not affect the metabolism of DOC to B in our enzyme preparation and that conversions of B and 18-OHDOC into aldosterone are catalyzed by the same enzyme.     

Quantitative evaluation of gamma-turn conformation in proline-containing peptides using 13C N.M.R.

The dependence of the 13C shift difference of proline carbons C beta and C gamma on the dihedral angle psi has been studied using the model peptide acetyl-D-proline N-methylamide. The shift difference delta beta gamma is shown to be correlated with the percent cis isomer about the acetylproline bond, both factors depending strongly on the degree of intermolecular hydrogen bonding. Both the fraction of trans peptide bond and the fractional gamma-turn conformation increase as the sample concentration is decreased in CDCl3. delta beta gamma values have been used to evaluate the fractional gamma-turn probabilities in a number of cyclic and linear peptides including thyrotropin releasing factor and bradykinin. Using this parameter, it is concluded that in bradykinin the gamma-turn probability is low in D2O and not strongly temperature dependent. In contrast, studies of a model peptide for the portion of bradykinin believed to adopt a gamma-turn conformation are consistent with an increased gamma-turn probability inless polar solvents. Data for X-Pro-Y peptides (Y = imino acid) indicate significantly reduced values of delta beta gamma, and this appears to be a useful basis for assigning the Pro C beta resonances corresponding to this sequence.     

Distance geometry analysis of the N.M.R. evidence on the solution conformation of bleomycin

Conformational constraints derived from n.m.r. experiments, X-ray data and the known stereochemistry have been used to investigate by the distance geometry method the range of allowed solution conformations for Cu(II):P-3A (a biosynthetic precursor of bleomycin), Fe(II):bleomycin:carbon monoxide, and Zn(II):bleomycin. The experimental data have been found to be self-consistent and lead to the following observations. 1) Designation of the ligands and the dihedral angles available from vicinal coupling constants are not sufficient to define uniquely the geometry around the metal. 2) When only five bleomycin ligands are invoked (e.g. Cu(II):P-3A or Fe(II):bleomycin:carbon monoxide) there is considerable freedom in the allowed coordination scheme around the metal, but some regions of the molecule have well determined conformation. 3) Introduction of a sixth bleomycin ligand, as in Zn(II):bleomycin, considerably constrains the conformational freedom of the groups coordinated to the zinc. The utility of the distance geometry approach for analysis of data and design of experiments is discussed.     

Carbon-13 N.M.R. study of bleomycin-A2 protonation

The acid-base titration of bleomycin-A2 in D2O solution at 35 +/- 5 degrees has been monitored by 13C n.m.r. spectroscopy at 67.89 MHz. The following pKDa values were obtained: 3.68 +/- 0.05 (secondary amine), 5.29 +/- 0.03 (imidazole), and 8.23 +/- 0.19 (primary amine), where KDa is the dissociation constant in D2O solution. The equilibrium isotope effects (pKDa--pKa in H2O) are: 0.70 +/- 0.06 (secondary amine), 0.28 +/- 0.04 (imidazole), and 0.85 +/- 0.19 (primary amine). Titration of the imidazole group of Bleo-A2 occurs at Npi, i.e. only Ntau is protonated in basic solution. Significant protonation shifts are almost completely limited to carbons of the N-terminal tetrapeptide, suggesting that the C-terminal tripeptide extends into the solvent and interacts to a minimal extent with the rest of the molecule. Long range protonation shifts associated with titration of the imidazole and secondary amine groups indicate that protonation of one or both of these sites is probably accompanied by significant conformational changes. The observed protonation shifts generally fail to correlate with Zn(II) complexation shifts reported by Dabrowiak et al. (1973, Biochemistry 17., 4090) indicating that ligation sites cannot unambiguously be determined from these complexation shifts. The complexation shifts previously attributed to coordination of the imidazole and carbamoyl groups probably result from conformational changes.     

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.