Monika M. Holbrook

19-Nor-deoxycorticosterone in the neutral fraction of human urine

19-Nor-deoxycorticosterone (19-nor-DOC), in the neutral fraction of human urine, was isolated and quantitated as the acetate derivative using ultraviolet absorption of the peak emerging from a high-pressure liquid chromatographic column. Identification of 19-nor-DOC in a pooled collection of urine after ACTH administration included identical chromatographic mobilities as the parent compound and acetate derivative compared to authentic 19-nor-DOC and mass spectral analysis of the acetate derivative. Values obtained for control and post-ACTH urines were 528 +/- 100 (SE) ng/24 hours and 8851 +/- 824 ng/24 hours, respectively. One patient with primary aldosteronism excreted 1894 ng/24 hours.

Identification of 19-hydroxydeoxycorticosterone in regenerating rat adrenal incubations

19-Hydroxydeoxycorticosterone (19-OH-DOC) was isolated from the incubation medium of enucleated rat adrenal glands during the early sodium retaining phase. Identification included comparison of chromatographic mobility of parent and derivatized compound with standard prepared by the 21-hydroxylation of 19-hydroxyprogesterone and mass spectrometry. The possible role of 19-OH-DOC as a precursor is discussed.

Antihypertensive effects of an aromatase inhibitor in the spontaneously hypertensive rat.

Recent studies from this laboratory have demonstrated that 19-nor-deoxycorticosterone, a potent mineralocorticoid, has been excreted in excess in the urine of young spontaneously hypertensive rats (SHR). Although urinary 19-nor-deoxycorticosterone levels decline before the onset of hypertension, preliminary evidence suggests that 19-nor-deoxycorticosterone is further oxygenated to other steroid products in older SHR. Since 19-hydroxylation is the essential first step in the formation of 19-nor-deoxycorticosterone from deoxycorticosterone and since the mechanism-based aromatase inhibitor 10-propargyl-androst-4-ene,3,17-dione preferentially inhibits 19-hydroxylation, this agent was administered to weanling SHR to determine whether inhibition of 19-nor-deoxycorticosterone formation could modify or prevent hypertension. Accordingly, either 10 mg of 10-propargyl-androst-4-ene,3,17-dione or vehicle (control) was injected daily for several weeks in 4.5 week-old SHR. Injection of 10-propargyl-androst-4-ene,3,17-dione reduced urinary free 19-nor-deoxycorticosterone and retarded the development of hypertension compared with the effect of vehicle injection (p less than 0.05). Mean blood pressure levels in SHR receiving 10-propargyl-androst-4-ene,3,17-dione were lower than those in SHR receiving vehicle for each of the first 8 weeks of treatment (p less than 0.05). These data support the importance of 10-nor-corticosteroids in the pathogenesis of hypertension in SHR.

Antihypertensive effects of an aromatase inhibitor in inbred salt-sensitive rats.

Rats susceptible to the hypertensive effect of dietary salt (SS/Jr) have excess urinary 19-nordeoxycorticosterone compared with salt-resistant control rats (SR/Jr). 19-Nordeoxycorticosterone is a hypertensinogenic mineralocorticoid, but whether it contributes to the salt sensitivity of SS/Jr is unknown. This study sought to evaluate the contribution of 19- nordeoxycorticosterone to the the salt sensitivity of SS/Jr by lowering its production with an aromatase inhibitor, 10-propargyl-androst-4-ener3,17-dione (19-acetylenic-androstenedione, 19-AA). This aromatase inhibitor also preferentially inhibits nonaromatizing adrenal 19- hydroxylation, an essential step in the formation of 19-nordeoxycorticosterone. To test this hypothesis, inhibitor (120 mg) or vehicle pellets were implanted into male and female weanling SS/Jr at 42 days of age. A high salt diet (8% NaCl) was started and two additional pellets were implanted at 52 and 62 days of age. Systolic blood pressure was measured in all animals and urinary corticosteroids in males. Compared with vehicle, the inhibitor lowered blood pressure at 50 days of age (when it could first be measured) until 64 days of age in females and 71 days of age in males. Corticosterone and aldosterone levels were not different between 19-AA- and vehicle-treated SS/Jr. 19-Nordeoxycorticosterone levels, however, were mildly reduced with the inhibitor (0.05 < p<0.10). After 28 days of high salt diet all 23 of the 19-AA-treated SS/Jr were alive, whereas almost one half of the control animals had died. These data demonstrate that 19-AA attenuates the hypertension in SS/Jr, this effect may be through reduction in 19- nordeoxycorticosterone production. This gives support to the contention that 19-nordeoxycorticosterone is involved in the hypertensive effects of dietary salt in SS/Jr.

Relationship of 19-nor-deoxycorticosterone to other mineralocorticoids in low-renin hypertension.

A number of mineralocorticoids have been proposed as etiologic factors in low-renin hypertension. In this study, urinary free 19-nor-deoxycorticosterone (UF 19-nor-DOC) was compared to other mineralocorticoids--aldosterone, deoxycorticosterone (DOC), and 18-OH-DOC, in 11 low-renin hypertensive patients on a controlled diet in a metabolic unit. Results demonstrated that both UF 19-nor-DOC and tetrahydro-DOC (TH-DOC) excretion were elevated (2086 +/- 926, nl = 339-579 ng/day, and 18 +/- 7, nl = 5-15 mcg/day, respectively), and positively correlated (r = 0.95). Neither 18-OH-DOC nor aldosterone secretion rates were elevated, and neither of these hormones correlated with UF 19-nor-DOC, with exception of the supine plasma aldosterone (SPA) (r = 0.86). In conclusion, both UF 19-nor-DOC and TH-DOC were increased and positively correlated in the present series of hypertensives. This association is possibly indicative of a precursor-product relationship between DOC and 19-nor-DOC. 19-Nor-DOC, furthermore, correlated with supine plasma aldosterone (SPA), which could, in part, reflect their shared adrenocorticotropic hormone (ACTH) dependence.

19-Nor-corticosteroids in genetic hypertension. Effects of inhibitors of 11μ,18,19-hydroxylase activity

The long-term objective is to understand the role of the adrenal in altering systemic arterial blood pressure. This paper summarizes research on genetic hypertension in the rat and bears a relationship to several forms of human hypertension in which defects of steroid hydroxylases lead to increased secretion of mineralocorticoids other than aldosterone in genetic and experimental hypertension in rats. We demonstrated that 19-nor-corticosteroids are produced in excess in genetic and experimental hypertension in rats and man. We studied the enzymatic alteration responsible for excessive production of 19-nor-deoxycorticosterone (19-nor-DOC) in the salt-sensitive hypertensive rat S/JR. Biosynthesis of 19-nor-steroids involves hydroxylation of the C-19 methyl group. We characterized the adrenal 11 beta, 18,19-hydroxylase enzyme system in inbred salt-sensitive and resistant rats (R/JR). This system is capable of all three hydroxylations. The Km for 19-hydroxylation was different from S/JR and R/JR but was much greater for 11 beta- and 18-hydroxylation in both. This suggested that the catalytic site for 19-hydroxylation is different from that for 11 beta and 18. The S/JR adrenal enzyme binds the substrate with higher affinity than does the R/JR adrenal enzyme. We were unable to distinguish the cDNAs of the S/JR from the R/JR adrenal enzyme from bovine 11 beta-hydroxylase cDNA by restriction mapping. We were unable to demonstrate restriction length polymorphism. 19-Acetylenic DOC is an inhibitor which preferentially inhibits the 19-hydroxylation of DOC, and does not interfere with the 18- and 11 beta-hydroxylation. This inhibition leads to a reduction in blood pressure in the S/JR Dahl rat. We suggest that an S/JR 19-nor-DOC is involved in the development of salt-sensitivity and hypertension and that inhibition of its formation by acetylenic DOC and other aromatase and non-aromatase inhibitors is associated with reversal of these phenomena.

19-Hydroxylase inhibition of adrenal mitochondrial P450 11β/18/19-hydroxylase by a suicide inhibitor

19-Nor-deoxycorticosterone (19-nor-DOC) is a mineralocorticoid that is increased in some forms of experimental and human hypertension. The pivotal step in 19-nor-DOC biosynthesis is adrenal P450 19-hydroxylase, but this enzyme has not been clearly distinguished from P450 11 beta/18-hydroxylase. This study attempted to specifically inhibit adrenal 19-hydroxylation of deoxycorticosterone (DOC) using a suicide aromatase inhibitor, 19-acetylenic androstenedione (19-AA). Purified bovine P450 11 beta/18/19-hydroxylase was incubated with excess substrate DOC, adrenodoxin, and adrenodoxin reductase in the presence of increasing doses of the inhibitor, 19AA. 11 beta-, 18-, and 19-hydroxylation were measured by quantification of corticosterone, 18-OH-DOC, and 19-OH-DOC respectively. Measurements of these products demonstrated that 11 beta- and 18-hydroxylation was not inhibited whereas 19-hydroxylation was inhibited as manifested by decreased 19-OH-DOC formation (p less than .05). The IC50 of 19-AA was approximately 10(-12) M. The specific inhibition of 19-hydroxylation suggests that the 19-hydroxylase may be an enzyme distinct from the P450 11 beta/18-hydroxylase. This further suggests that 19-nor-DOC biosynthesis may be under independent regulation and may be amendable to specific in vivo inhibition.

Effect of 4-Hydroxyandrostenedione on 19-hydroxylation of deoxycorticosterone in the golden syrian hamster

4-Hydroxyandrostenedione, a known inhibitor of ovarian and peripheral aromatization of androgen (testosterone and androstenedione) to form estrogen, was studied for its inhibitory effect on the 19-hydroxylating enzyme system of the adrenal for the conversion of deoxycorticosterone to 19-hydroxydeoxycorticosterone. In vitro incubation of Golden Syrian hamster adrenal homogenates with tritiated deoxycorticosterone demonstrated (80-85%) reduction in label incorporated into 19-hydroxydeoxycorticosterone in the presence of 4-hydroxyandrostenedione.

Identification of 19-hydroxy-progesterone in human placenta

We have tentatively demonstrated the presence of a 19-hydroxylated C21 steroid, 19-hydroxy-progesterone, in normal human placenta. A 19-hydroxylated steroid such as 19-hydroxy-progesterone, if produced by the placenta, could serve as a precursor for such hypertensinogenic 19-nor-steroids as 19-nor-deoxycorticosterone and 19-nor-progesterone. Freshly delivered, homogenized placental tissue was extracted and subjected to thin layer chromatography. A steroid corresponding to standard 19-hydroxy-progesterone was subsequently purified in HPLC, where authentic 19-hydroxy-progesterone and the sample had the same retention time. The identity of the sample was further confirmed by repeat HPLC after acetylation and mass spectrometry. Our experiment indicates that 19-hydroxy-progesterone is present in term placental tissue, where it appears to be synthesized.

Renal 21-hydroxylation of 19-hydroxy-progesterone to 19-hydroxy-deoxycorticosterone☆

19-Nor-deoxycorticosterone (19-nor-DOC) is a mineralocorticoid present in both rat and human urine, and it is elevated in some forms of experimental and human hypertension. Although the exact steps in the biosynthesis of 19-nor-DOC are uncertain, it is probably produced from a 19-oxygenated derivative of DOC, which undergoes 19-desmolation in the kidney. Since DOC biosynthesis is partly due to renal 21-hydroxylation of progesterone (Prog), we sought to determine whether a parallel pathway could exist for the biosynthesis of 19-hydroxy-DOC, a precursor to 19-nor-DOC. In order to test this hypothesis, authentic 19-hydroxy-progesterone was incubated with homogenized renal tissues from either rat or human sources. Formation of 19-hydroxy-DOC was found to be the major metabolite in both rat and human incubations, as demonstrated by an HPLC retention time identical to authentic 19-hydroxy-DOC. 19-Hydroxy-DOC formation was further verified by GC/MS analysis with highly sensitive selected ion recording. Since it has been demonstrated that the placenta can convert progesterone to 19-hydroxy-progesterone, the renal 21-hydroxylation of 19-hydroxy-progesterone to 19-hydroxy-DOC could be an alternate pathway of 19-nor-DOC production especially during pregnancy.