Corticosterone, hydroxylation

The 17- and 21-hydroxylase enzymes are associated with microsomes, whereas the ll- -hydroxylase has a mitochondrial origin. Since the last-named enzyme is not detectable in other steroid-producing tissues, the term 11-oxygenated steroids is considered synonymous with adrenal steroids. Aldosterone synthesis involves an essential 18-hydroxylation step catalyzed by P450d8 with corticosterone as the precursor this reaction also takes place within the mitochondria. 

Within the basic structure of the steroid molecule (Fig. 60.4), a 4,5 double bond and a 3-ketone group are needed for typical steroid activity. A hydroxyl group on Cll is needed for glucocorticoid activity (corticosterone) but is not required for sodium-retaining activity (desoxycorticosterone). The addition of a hydroxyl group on C17, which converts corticosterone to cortisol, also increases glucocorticoid activity. 

Metyrapone (Figure 39-5) is a relatively selective inhibitor of steroid 11-hydroxylation, interfering with cortisol and corticosterone synthesis. 

The synthesis of adrenal steroids is illustrated in Fig. 5.3.1. Cortisol, corticosterone, and aldosterone are formed by sequential hydroxylations and oxidoreductions from pregnenolone and progesterone. 17a-Hydroxypregnenolone (17HP) is a branchpoint constituent because it can be converted to cortisol or adrenal androgens. All of the components of this pathway can be quantified by MS/MS. The steroids around the periphery are urinary metabolites and these are measured by GC-MS following hydrolysis of conjugates and derivatization. 

Patients with 11/1-hydroxylase deficiency present with features of androgen excess, including masculinization of female newborns and precocious puberty in male children. There are two human isozymes that are responsible for cortisol and aldosterone synthesis, respectively. The CYP11B1 enzyme (p45011B) converts DOC to corticosterone (B) and 11-deoxycortisol (S or 11-dihydrocortisol) to cortisol (F). It is also capable of 18-hydroxylating DOC but cannot convert to aldosterone. The latter transformation is carried out by CYP11B2 (also known as aldosterone synthase), which encompasses activity for 18-hydroxylation and subsequent 18-oxidation. When CAH is associated with hypertension, deficient lljS-hydroxylase (CYP11B1) is suspected at this time more than ten mutations have been defined in affected individuals [103]. 

P-450-dependent hydroxylases with NADPH and O2 cofactors. Thus, positions 17, 21 and 11 may be hydroxylated, and the exact order can in fact vary from that shown in Figure 5.114, according to species. It can be seen that production of hydrocortisone from cholesterol actually utilizes cytochrome P-450-dependent enzymes in four of the five steps. The further oxidation of C-18 to an aldehyde via the alcohol allows formation of aldosterone from corticosterone, again involving a P-450 system. 

Once the various steroids have been formed in paticular subcellular compartments, they must be released into the peripheral blood circulation. There is evidence that some steroids are released by passive diffusion, as in the case of corticosterone, but for 18-hydroxylated corticosteroids, Na+/K+-ATPase activity is necessary [6,109]. The situation is more complicated, however, because the presence of proteins in the adrenal cortex, which act as non-classical receptors, may bind C2i steroids to different extents, thus reducing rates of steroid release (see Ref. 6). So far as pregnenolone is concerned, there is no barrier to its efflux from the mitochondria where it is formed from cholesterol [50], During incubation of rat testis [110], pregnenolone was found to travel from the mitochondria, through the ER and cytosol and then out into the medium. The release with time could be resolved into two components, one rapid and the second, much slower. More than 25% of the pregnenolone remained in the tissue after 150 min. incubation. This two-phase release may reflect the presence of two pools of steroid, the initial loss representing passive dif-... 

The pattern of inhibition of 17a-hydroxylase exhibited by metyrapone lends further support to the hypothesis that cytochrome P450 is involved in the enzyme system319 whilst the effect of administration of spironolactone on the cytochrome P450 content and 17a-hydroxylase activity in adrenal tissue has shown that decreases in both of these factors occur only in animals that produce predominantly cortisol rather than corticosterone.320 18-Hydroxylation of deoxycorticosterone has been demonstrated in rat and bovine mitochondrial preparations and in reconstituted systems obtained from these fractions.321 In all cases, 18-hydroxylation was accompanied by 11/8-hydroxylation, and the study indicated that very similar types of cytochrome P450 were involved in both hydroxylation systems. 

The ll-/3-hydroxylase found in the adrenal cortex catalyzes the hydroxylation of 11-deoxycorticosterone to corticosterone. The enzyme requires NAD as a cofactor and contains heme as the prosthetic group. 

The substrate, 11-deoxycorticosterone, was separated from the two reaction products, corticosterone (11-jS-hydroxylation) and 18-hydroxyl-ll-deoxycorticosterone (18-hydroxylation), on reversed-phase HPLC (MicroPak... 

Corynespora melonis229 hydroxylates androst-4-ene-3,17-dione (3) to the 9a, 18-dihydroxy compound 4 while corticosterone (5) is 18-hydroxylated by Corynespora cassiicola3U0. The product 6 can then be chemically converted into 1 l/i,18-epoxy-21-hydroxyprogesterone (7), which can be transformed to aldosterone (8) by the same microorganism. 

In most studies, the selectivities of the MIPs have been estimated by measuring the amount of each ligand required to displace 50% of the binding of radiolabelled imprint species to the MIP (IC50). The first MIA study reported excellent selectivity of the theophylline method for theophylline (1,3-dimethylxanthine) in the presence of the structurally related compound caffeine (1,3,7-trimethylxanthine) [3]. Despite their close resemblance (they differ by only one methyl group), caffeine showed less than 1 % cross-reactivity. A similar level of specificity was recorded for cortisol and corticosterone MIPs, which were able to detect the absence and presence of single hydroxyl groups and double bonds in the steroid structure [13]. 

HSD (possibly substrate specific) acting at a later stage subsequent to hydroxylation at C-17, C-21, and C-11 for the synthesis of cortisol or corticosterone. The possibility of these pathways has been indicated by several workers (for review, see D15), and it seems likely that it is the major mode of synthesis of corticosteroids, at least in guinea pigs, swine, and rats (Yl). 

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