J. F. Tait

Steroid dynamics under steady-state conditions.

General concepts of steroid dynamics under steady state conditions are presented. In particular steroid dynamic processes in the steady state which relate the secretion of steroids (both precursors and products) to the resultant constant concentration of steroids as measured in the circulating blood are given mathematical expression with the eventual aim of relating these parameters to steroid concentrations in target tissues. Numerous clinical studies are presented to elucidate the theory and methods of 1) metabolic clearance rate; 2) blood production rates; 3) measure of conversion in a single organ (i.e. fraction of secreted precursor converted to product and appearing in efferent circulation of a single organ); and 4) sources and possible functions of precursors. RhoBB a mathematical statement of steroid conversion is calculated for androgens and estrogens from clinical data. Future considerations in steady-state steroid research include development of experimental methods for obtaining knowledge of concentrations and production rates of steroids near target sites and reevaluation of values for secretion rates of interconverting steroids obtained from specific activities of urinary metabolites which have been found to be inaccurate.

THE DISAPPEARANCE OF 7-H3-d-ALDOSTERONE IN THE PLASMA OF NORMAL SUBJECTS*

The study of the disappearance of the radioactivity in plasma after the injection of labeled aldosterone should elucidate the transport and metabolism of this steroid in man. In particular this would allow the calculation of the turnover rate in various clinical conditions. This value, together with a concomitant determination of the secretion rate estimated from the specific activity of a urinary metabolite, would enable the mean blood concentration of aldosterone to be calculated. This is particularly important at the present time, as no practical direct method has yet been reported for the analysis of aldosterone in peripheral blood. After the administration of 16-H13-aldosterone to one normal subject, the labeled hormone had a half-life in plasma of 20 minutes, as calculated from the change in concentration of radioactivity measured specifically as aldosterone between 2 and 3 hours after the injection (1). This indicated that the rate of metabolism of aldosterone was greater than that of cortisol and corticosterone. The data obtained were not sufficient to give a relialle estimate of volumes of distribution. Also, the low specific activity of aldosterone then available made it necessary to administer 2 jug of hormone. This amount represents about 25 per cent of total body pool of hormone. Peterson reported a corresponding half-life of 0.5 to 0.8 hour in normal subjects after injection of tritiated d,l-aldosterone (2). Again in this study large quantities of the hormone were injected. Also it is not known whether the optical isomers of aldosterone are metabolized in an identical manner. The natural hormone is in the d form exclusively.

Metabolic clearance rates and interconversions of estrone and 17β-estradiol in normal males and females

The continuous infusion of (3)H-6,7-estrone and (3)H-6,7-estradiol has been used to study the metabolic clearance rate (MCR), the interconversions, and the red cell uptake of these steroids in normal males and females. The whole blood MCR of estrone is 1,990 +/- 120 liters per day/m(2) (SE) in males and 1,910 +/- 100 liters per day/m(2) in females. The whole blood MCR of estradiol is 1,600 +/- 80 liters per day/m(2) in males and 1,360 +/- 40 liters per day/m(2) in females. The values in females do not vary significantly when studied in the follicular or luteal phase of the cycle. At least 35% of the total estrone metabolism in both sexes is extrasplanchnic and at least 25% of the total estradiol metabolism in males, and 15% in females is extrasplanchnic. The [rho](BB) (2,1) [transfer constant of estradiol to estrone, which is equivalent to the fraction of the precursor (estradiol) converted to the product (estrone) when both the infusion of the precursor and the measurement of the product are in peripheral blood] is 15%; and the [rho](BB) (1,2) [transfer constant of estrone to estradiol, which is equivalent to the fraction of the precursor (estrone) converted to product (estradiol) when both the infusion of the precusor and the measurement of the product are in peripheral blood] is 5% in both males and females. Our findings concerning the radioactivity in whole blood, as measured by our procedure, were the following: 15-20% of estrone in both sexes and 15% of estradiol in males is associated with red cells. Only 2% of the whole blood radioactivity of estradiol in females is associated with red cells. Changes in the distribution of radioactivity between plasma and red cells will influence the MCR as calculated from plasma, but not as calculated from whole blood.

The Metabolism and Secretion of Aldosterone in Elderly Subjects

The secretion rates [34 +/- 6 (SE) mug per day, 9 subjects] and metabolic clearance rates (MCR) [1,288 +/- 120 (SE) L of plasma per day, 9 subjects] of aldosterone in elderly subjects are significantly lower than those of young subjects [77 +/- 7 (SE) mug per day and 1,631 +/- 106 (SE) L per day, respectively]. There is a correlation of the MCR and secretion rate values (p = 0.02), but the calculated plasma concentrations (secretion rate/MCR) are also significantly low in the elderly subjects [2.6 +/- 0.3 (SE) compared with concentrations in the plasma from young subjects of 4.7 +/- 0.6 (SE) mug per 100 ml plasma]. The urinary excretion of radioactivity from oral and intravenously administered labeled aldosterone as aldosterone in the neutral extract, as aldosterone released by acid hydrolysis, and as tetrahydroaldosterone released by incubation with beta-glucuronidase is generally similar for young and elderly subjects except that a larger portion of the oral compared with the intravenous dose is excreted as free aldosterone in the elderly subjects, indicating that the splanchnic extraction is reduced. The calculated splanchnic blood flow (assuming no alteration in extrasplanchnic metabolism) is also slightly lowered. Therefore, as in patients with mild cardiac dysfunction, the lowered MCR of subjects is due to both reduced splanchnic extraction and blood flow. However, unlike the heart failure patients, in the elderly subjects the plasma concentration of aldosterone is also reduced.

The Properties of Adrenal Zona Glomerulosa Cells after Purification by Gravitational Sedimentation

The densities of latex spheres and biological cells can be reliably determined from their sedimentation rate in an albumin gradient under unit gravitational force. The densities of zona glomerulosa and fasciculata cells of rat adrenals were found to be 1.072 ± 0.004 and 1.040 ± 0.001 respectively. Purified zona glomerulosa cells of rat adrenals can be prepared by gravitational sedimentation of dispersed cells from capsule strippings of the gland, which originally contain 3 to10% zona fasciculata contamination. Electron and phase microscopic examination of the sedimented glomerulosa cells and their steroidogenic response to ACTH and cyclic AMP indicate that they are reasonably free of contamination from zona fasciculata cells. Electron microscopic examination of the purified glomerulosa cells indicates that most of them are reasonably normal in structure. Their basal production of corticosterone is decreased after sedimentation. However, their maximal response of corticosterone output to serotonin and potassium and their response to all potassium concentrations is not significantly altered, indicating normal function for the cells producing steroids. Their maximal responses to ACTH, valine angiotensin II and cyclic AMP are decreased, but, at the doses used, steroidogenesis by the zona fasciculata contamination in the unfractionated preparation would be stimulated by these substances. Purified zona glomerulosa cells have about the same maximal response of corticosterone output (about twofold) to potassium, valine and isoleucine angiotensin II, serotonin and ACTH. The maximal response of the purified zona glomerulosa cells to cyclic AMP is similar to that elicited by valine and isoleucine angiotensin II, potassium, serotonin or ACTH. This indicates that if these stimuli act by increasing cyclic AMP output, then the maximal response of corticosterone output (about twofold) is defined by the limited response of the biosynthetic pathways to cyclic AMP.

The metabolic clearance rate of progesterone in males and ovariectomized females.

The metabolic clearance rate (MCR) of a steroid hormone, which is probably most generally defined as the volume of blood cleared completely and irreversibly of steroid in unit time, determines the relationship between plasma production rate and plasma concentration (1). A knowledge of its value may also be important in predicting some of the characteristics of any control system (1-3). It can be shown that after a single injection or continuous infusion of radioactive steroid, the metabolic clearance rate can be calculated as the reciprocal of the integrated plasma concentration of radioactivity measured specifically as the hormone (1). If this integration is performed correctly, the two methods should give the same value for MCR, and a comparison of the values obtained therefore serves as a check on this and other technical requirements for the validity of the calculation (1, 4). In the studies described here, the MCR of progesterone has been measured by application of the single injection and continuous infusion methods in ovariectomized females and in males. Information on volumes of distribution has also been obtained after single injection.

The discovery, isolation and identification of aldosterone: reflections on emerging regulation and function

This paper has a focus on the early history of aldosterone. The Taits take us on a chronological trawl through the history in which they had a first hand role and made a major contribution-their bioassay was in many ways the key. The gifted Swiss chemists made a critical contribution to the scale and isolation of larger amounts. This was international collaboration at its best. Developing technologies were utilised as crucial cutting edge applications in the advancing front, technology transfer before the word was invented. Measurement of aldosterone and angiotensin were crucial advances to the understanding of the regulation of the hormone. In the period 1960-2003, some 30,000 papers mentioned aldosterone as a keyword, even so advances on a larger scale were slow. I have indicated some of my own work with the Howard Florey team using the adrenal autotransplant in the conscious sheep. Recently, the understanding of the role of induced proteins, the flow on from the RALES trial and the development of eplerenone has revitalised the aldosterone field.

The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

The effects of various concentrations of extracellular K+ (3.6 - 13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 μM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p < 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10-6 to 10-4 M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10-4 M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an ‘anomalous’ increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs. Our previous data, obtained after addition of maximally effective amounts of cAMP, indicated that there were also non-cAMP mechanisms involved in the stimulation of steroidogenesis by K+ in z. g. cells. The present data confirm this conclusion, particularly at K+ concentrations above 8.4 mM. They also indicate that at these higher K+ concentrations, by non-cAMP mechanisms increasing intracellular Ca2+ concentrations probably inhibit steroidogenesis. We conclude, however, that in the physiological range of K+ concentrations, the role of cAMP in zona glomerulosa cells is at least comparable in importance to that of non-cAMP mechanisms.

Some theoretical considerations on the significance of the discrepancy in urinary and blood production rate estimates of steroid hormones particularly in those of testosterone in young women

Abstract In some situations, particularly for testosterone production in the female, it is postulated that the amount of hormone synthesized from precursors in a separate anatomical compartment such as the liver is a significant proportion of the total hormone produced in the body. If a urinary metabolite is also formed in the same compartment the calculated urinary and blood production rates may differ. The extent of the discrepancy will depend on the relative amounts of hormone introduced directly into the general circulation and into the outer anatomical compartment, e.g. the liver, and the extraction of the hormone in that compartment. The maximum hepatic extraction of a hormone may be calculated from the value for metabolic clearance rate of the steroid and hepatic blood flow. From this estimate and the values for the urinary and blood production rates, the minimum contribution of the steroid synthesized in the liver to the blood production rate can be calculated. The maximum amount of steroid introduced directly into the general circulation including the hormone secreted can therefore be estimated. For females in the proliferative phase of the cycle, it can be calculated, by this approach, that secretion of testosterone must be a minor component of the total production of the hormone. It can be shown that this must be so even if extrahepatic metabolism of testosterone is significant or testosterone glucuronide is formed extrahepatically. The major assumption in the treatment which could affect the conclusion that secretion is negligible is that the hepatic extraction of testosterone which is synthesized in the liver is equal to that introduced into the organ via the afferent blood supply.

A Comparison of the Metabolism of Radioactive 17-Isoaldosterone and Aldosterone Administered Intravenously and Orally to Normal Human Subjects*

After intravenous and oral administration of radioactive aldosterone to normal subjects, 7.3 +/- 0.4 (SE) and 5.4 +/- 0.5 (SE)%, respectively, of the dose was recovered from a 48-hour collection of urine as aldosterone released by mild acid hydrolysis (from aldosterone 18-glucuronide), and 35 +/- 5 (SE) and 39 +/- 4 (SE)%, respectively, was recovered as tetrahydroaldosterone after incubation with beta-glucuronidase.After intravenous and oral administration of 17-isoaldosterone-4-(14)C to a similar group of subjects, 35 +/- 3 (SE) and 53 +/- 4 (SE)%, respectively, of the dose was recovered as 17-isoaldosterone released by acid and less than 5% as total metabolites after incubation with beta-glucuronidase. No detectable radioactivity (< 0.5%) could be recovered as tetrahydroaldosterone or as a compound with the expected chromatographic properties of tetrahydro-17-isoaldosterone. The total radioactivity in the neutral extracts was also relatively small (< 2%) after administration of either labeled aldosterone or 17-isoaldosterone. The radioactivity as aldosterone in the neutral extract was much lower after oral [0.017 +/- 0.003 (SE)%] than after intravenous [0.21 +/- 0.04 (SE)%] administration of labeled aldosterone. The radioactivity as 17-isoaldosterone in the neutral extract was similar after intravenous [0.20 +/- 0.02 (SE)%] and after oral [0.38 +/- 0.18 (SE)%] administration of 17-isoaldosterone. These results indicated that, due to lack of A-ring reduction of the molecule and the consequent slowing of hepatic clearance, 17-isoaldosterone is converted to an acid-labile conjugate (presumably 17-isoaldosterone 18-glucuronide) as the major metabolite. 17-Isoaldosterone was not secreted or converted to aldosterone to any significant extent in the normal subjects investigated.