Abstract
Background
Many hormones display distinct circadian rhythms, driven by central regulators, hormonal bioavailability, and half-life. A set of 11-oxygenated C19 steroids (11-oxyandrogens) and pregnenolone sulfate (PregS) are elevated in congenital adrenal hyperplasia and other disorders, but their circadian patterns have not been characterized.
Participants and methods
Peripheral blood was collected every 2 h over 24 h from healthy volunteer men (10 young, 18–30 years, and 10 older, 60–80 years). We used mass spectrometry to quantify 15 steroids, including androstenedione (A4), testosterone (T), 11β-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11β-hydroxy- and 11-ketoandrostenedione (11OHA4, 11KA4), and 4 ∆5-steroid sulfates. Diurnal models including mesor (rhythm adjusted median), peak, and nadir concentrations, acrophase, and amplitude were computed.
Results
11OHA4 followed a rhythm similar to cortisol: acrophase 8:00 h, nadir 21:00 h and were similar in young and old men. 11KT had similar diurnal patterns, but the peak was lower in older than in young men, as was the case for A4. All four steroid sulfates were higher in young vs older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8:00 and 18:00 h, and nadirs around midnight, while DHEAS and AdiolS displayed minimal diurnal variations. All 4 11-oxyandrogens correlated tightly with cortisol (r from 0.54 for 11OHT to 0.81 for 11OHA4, P < 0.0001 for all), but very weakly with T, supporting their adrenal origin and ACTH governance.
Conclusions
11-Oxyandrogens, PregS, and 17OHPregS display distinct circadian and age variations, which should be accounted for when used as clinical biomarkers.
Introduction
The adrenal gland contributes to the pool of circulating androgens primarily by producing dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). While abundant, these two steroids have no androgenic bioactivity (1) but serve as substrates for potent androgens, such as testosterone (T). In addition to the widely recognized DHEA and DHEAS, the adrenal glands produce a set of 11-oxygenated 19-carbon steroids (11-oxyandrogens), such as 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT), which are further oxidized to 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT), respectively (Fig. 1), a process that occurs in several peripheral tissues (2). Of these, 11KT is an androgen with potency similar to that of T, and it can also be 5α-reduced to 11-ketodihydrotestosterone (11KDHT), which is equipotent with dihydrotestosterone (DHT) (3, 4, 5). In fact, 11KT is the dominant bioactive androgen in pre-pubertal children and 11KT increases in parallel with DHEAS during adrenarche (1). In contrast with the traditional adrenal and gonadal androgens, 11-hydroxyandrogens do not decline with aging (6, 7). Recent studies have shown that 11-oxyandrogens are elevated in patients with congenital adrenal hyperplasia (CAH), polycystic ovary syndrome (PCOS), and premature adrenarche (1, 8, 9).
Steroidogenic pathway focused on 11-oxyandrogen and ∆5-steroid sulfate synthesis. CYP11A1, cholesterol side-chain cleavage enzyme; CYP17A1, 17α-hydroxylase/17,20-lyase; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; CYB5A, cytochrome b5 type A; CYP11B1, 11β-hydroxylase; HSD11B1, 11β-hydroxysteroid dehydrogenase type 1; HSD11B1/2, 11β-hydroxysteroid dehydrogenase type 1 or 2; HSD17B, 17β-hydroxysteroid dehydrogenases; PregS, pregnenolone sulfate; 17OH-Preg, 17α-hydroxypregnenolone; 17OH-PregS, 17OH-Preg sulfate; 17OH-Prog, 17α-hydroxyprogesterone; DHEAS, DHEA sulfate; A4, androstenedione; 11OHA4, 11β-hydroxyandrostenedione; 11OHT, 11β-hydroxytestosterone; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; T, testosterone.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0348
Steroidogenic pathway focused on 11-oxyandrogen and ∆5-steroid sulfate synthesis. CYP11A1, cholesterol side-chain cleavage enzyme; CYP17A1, 17α-hydroxylase/17,20-lyase; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; CYB5A, cytochrome b5 type A; CYP11B1, 11β-hydroxylase; HSD11B1, 11β-hydroxysteroid dehydrogenase type 1; HSD11B1/2, 11β-hydroxysteroid dehydrogenase type 1 or 2; HSD17B, 17β-hydroxysteroid dehydrogenases; PregS, pregnenolone sulfate; 17OH-Preg, 17α-hydroxypregnenolone; 17OH-PregS, 17OH-Preg sulfate; 17OH-Prog, 17α-hydroxyprogesterone; DHEAS, DHEA sulfate; A4, androstenedione; 11OHA4, 11β-hydroxyandrostenedione; 11OHT, 11β-hydroxytestosterone; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; T, testosterone.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0348
Steroidogenic pathway focused on 11-oxyandrogen and ∆5-steroid sulfate synthesis. CYP11A1, cholesterol side-chain cleavage enzyme; CYP17A1, 17α-hydroxylase/17,20-lyase; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; CYB5A, cytochrome b5 type A; CYP11B1, 11β-hydroxylase; HSD11B1, 11β-hydroxysteroid dehydrogenase type 1; HSD11B1/2, 11β-hydroxysteroid dehydrogenase type 1 or 2; HSD17B, 17β-hydroxysteroid dehydrogenases; PregS, pregnenolone sulfate; 17OH-Preg, 17α-hydroxypregnenolone; 17OH-PregS, 17OH-Preg sulfate; 17OH-Prog, 17α-hydroxyprogesterone; DHEAS, DHEA sulfate; A4, androstenedione; 11OHA4, 11β-hydroxyandrostenedione; 11OHT, 11β-hydroxytestosterone; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; T, testosterone.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0348
The hypothalamic–pituitary–adrenal (HPA) axis displays characteristic circadian variations, governed by the suprachiasmatic nucleus located in the hypothalamus (10). All adrenal steroids are produced de novo, as prompted by adrenocorticotropic hormone (ACTH) and other regulators (11). Several adrenal steroids have characteristic diurnal secretory patterns, that reflect not only the ACTH regulation but also their bioavailability and metabolism (12). As we continue to learn about the utility of 11-oxyandrogens and Δ5-steroid sulfates in guiding the management of a diverse set of disorders, we sought to characterize their circadian patterns as an essential prerequisite for enabling clinical interpretation.
Participants and methods
Study participants
Serum samples analyzed for this study were obtained from 20 healthy men, 10 young (median age 24, range 19–29 years), and 10 older (median age 63, range 61–75) years, who participated in a randomized, double-blind placebo-controlled study, in which the primary goal was to evaluate the impact of experimental interleukin 2 (IL-2)-induced inflammatory stress on luteinizing hormone and T secretion in young and older men (13). All participants were non-smokers and had a median BMI of 26.06 (range, 20.1–34.5) kg/m2. Volunteers provided written informed consent for the Mayo Clinic Institutional Review Board-approved protocol. Subjects with glucocorticoid use within the previous 3 months, chronic drug or alcohol abuse, trans-meridian travel (over 3 time zones) within 10 days prior, sleep apnea, and/or major illness were excluded (13). Volunteers were admitted to the Clinical Research Unit at 16:00 h for placement of bilateral forearm IV catheters. Men received a single s.c. injection of either saline or IL-2. Only men who received saline were included in this study, and serum samples obtained every 2 h, beginning with 18:00 h (13 samples per subject), were used to characterize the circadian rhythm of steroids of interest. Participants received a standardized meal 1 h prior to initiation of sampling and subsequently fasted until the end of sampling. Activity was limited to walking on the unit.
Steroid quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
We used LC-MS/MS to quantify 11 Δ4 steroids: cortisol, cortisone, corticosterone, 11-deoxycortisol, 17α-hydroxyprogesterone (17OHP), androstenedione (A4), T, 11OHA4, 11KA4, 11OHT, 11KT; 4 Δ5 -steroid sulfates: PregS, 17OHPregS, DHEAS, and androstenediol-3-sulfate (AdiolS). Steroid extraction and quantification were performed as previously reported (6). Modifications to the chromatography method for Δ5 steroid sulfates are presented in Supplemental Data (14).
Statistical analysis
Loess smoothing curves were fitted using the ggplot2 package in R 3.6.2 software, and models were computed separately for young and old men. These models allowed us to extract the mesor (rhythm adjusted median), peak concentrations, acrophase (time of peak in rhythm), nadir (lowest point of the rhythm), time of nadir, and amplitude (half of the difference between the acrophase and nadir values). The peak and nadir values and corresponding clock times were calculated based on the fitted function, rounded to the nearest minute. Spearman correlations were performed to assess associations between steroids, using and SAS 9.4 (Cary, NC, USA) software. Two-tailed P values < 0.05 were considered statistically significant.
Results
The circadian steroid concentrations in peripheral serum are shown in Fig. 2. The circadian variations of 11OHA4 resembled closely those of cortisol, with an acrophase around 8:00 h and a nadir around 21:00 h, and similar profiles in young and old men (Fig. 2 and Table 1). The circadian profile of 11OHT resembled that of 11OHA4 in young men but displayed blunted excursions in older men. Compared to young men, 11KT had lower morning peaks in older men, but similar nadirs (Fig. 2).
Circadian patterns of Δ4 and steroid sulfates hormones Loess smoothing curves were fitted using data from healthy men (10 young 18–30 years, and 10 older 60–80 years). The central lines represent the fitted medians, and the shaded areas represent the 95% CI. A4, androstenedione; T, testosterone; 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11OHT, 11β-hydroxytestosterone; 11KT, 11-ketotestosterone; 17OHP, 17α-hydroxyprogesterone; DHEAS, DHEA sulfate; PregS, pregnenolone sulfate; 17OHPregS, 17-hydroxypregnenolone sulfate; AdiolS, androstenediol-3-sulfate. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0348.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0348
Circadian patterns of Δ4 and steroid sulfates hormones Loess smoothing curves were fitted using data from healthy men (10 young 18–30 years, and 10 older 60–80 years). The central lines represent the fitted medians, and the shaded areas represent the 95% CI. A4, androstenedione; T, testosterone; 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11OHT, 11β-hydroxytestosterone; 11KT, 11-ketotestosterone; 17OHP, 17α-hydroxyprogesterone; DHEAS, DHEA sulfate; PregS, pregnenolone sulfate; 17OHPregS, 17-hydroxypregnenolone sulfate; AdiolS, androstenediol-3-sulfate. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0348.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0348
Circadian patterns of Δ4 and steroid sulfates hormones Loess smoothing curves were fitted using data from healthy men (10 young 18–30 years, and 10 older 60–80 years). The central lines represent the fitted medians, and the shaded areas represent the 95% CI. A4, androstenedione; T, testosterone; 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11OHT, 11β-hydroxytestosterone; 11KT, 11-ketotestosterone; 17OHP, 17α-hydroxyprogesterone; DHEAS, DHEA sulfate; PregS, pregnenolone sulfate; 17OHPregS, 17-hydroxypregnenolone sulfate; AdiolS, androstenediol-3-sulfate. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0348.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0348
Diurnal steroid concentrations in young and older men. Data represent the extracted medians and interquartile ranges from the fitted loess curves.
| Hormone/age group | Peak (ng/dL) | Acrophase | Nadir (ng/dL) | Time of nadir | Mesor (ng/dL) | Amplitude (ng/dL) |
|---|---|---|---|---|---|---|
| A4 | ||||||
| Young | 78.2 (76.3, 80.1) | 8:19 | 31.3 (29.7, 33.0) | 20:18 | 68.2 (66.2, 70.3) | 23.4 |
| Old | 59.4 (57.8, 61.0) | 8:04 | 27.3 (25.9, 28.6) | 21:34 | 44.2 (42.6, 45.8) | 16.1 |
| T | ||||||
| Young | 388.8 (374.7, 403.0) | 9:36 | 271.6 (250.6, 292.5) | 18:00 | 353.4 (341.0, 365.9) | 58.6 |
| Old | 367.4 (351.3, 383.6) | 10:37 | 275.4 (256.7, 294.1) | 18:55 | 335.4 (312.8, 357.9) | 46.0 |
| 11OHA4 | ||||||
| Young | 259.9 (248.2, 271.5) | 8:10 | 22.1 (12.1, 32.2) | 21:58 | 174.9 (163.3, 186.6) | 118.9 |
| Old | 219.8 (207.6, 231.9) | 8:07 | 52.5 (42.3, 62.7) | 21:00 | 162.8 (150.9, 174.7) | 83.6 |
| 11KA4 | ||||||
| Young | 61.7 (59.1, 64.4) | 8:28 | 12.6 (10.3, 14.9) | 21:47 | 42.6 (40.0, 45.2) | 24.6 |
| Old | 46.1 (42.8, 49.3) | 11:52 | 17.7 (14.7, 20.8) | 22:12 | 34.4 (30.9, 37.9) | 14.2 |
| 11OHT | ||||||
| Young | 17.9 (16.9, 18.8) | 8:06 | 1.1 (0.3, 1.9) | 20:37 | 11.8 (10.8, 12.7) | 8.4 |
| Old | 16.1 (15.7, 16.5) | 12:26 | 6.1 (5.7, 6.5) | 22:08 | 13.8 (13.3, 14.2) | 5.0 |
| 11KT | ||||||
| Young | 41.6 (40.7, 42.6) | 12:36 | 8.6 (7.7, 9.6) | 21:56 | 31.0 (29.9, 32.1) | 16.5 |
| Old | 30.4 (29.7, 31.1) | 12:19 | 12.05 (11.4, 12.7) | 21:02 | 26.2 (25.1, 27.3) | 9.2 |
| Cortisol | ||||||
| Young | 9589.4 (9141.4, 10037.4) | 8:08 | 1243.4 (848.2, 1638.5) | 22:24 | 6945.5 (6495.7, 7395.3) | 4173.0 |
| Old | 10507.1 (10004.6, 11009.6) | 7:36 | 2466.2 (2039.6, 2892.8) | 21:12 | 6509.0 (6016.5, 7001.4) | 4020.4 |
| Cortisone | ||||||
| Young | 1987.2 (1936.8, 2037.5) | 8:10 | 383.5 (339.1, 427.9) | 22:22 | 1457.5 (1405.9, 1509.1) | 801.8 |
| Old | 1614.5 (1561.7, 1667.3) | 8:47 | 580.7 (534.5, 626.8) | 21:38 | 1214.9 (1166.1, 1263.7) | 516.9 |
| Corticosterone | ||||||
| Young | 292.5 (263.0, 321.9) | 6:48 | 27.4 (1.9, 52.9) | 21:18 | 151.1 (125.5, 176.7) | 132.5 |
| Old | 368.9 (344.0, 393.8) | 6:05 | 35.9 (14.2, 57.5) | 20:16 | 137.4 (114.9, 159.9) | 166.5 |
| 11-deoxycortisol | ||||||
| Young | 32.3 (29.9, 34.6) | 11:53 | 7.8 (5.6, 9.9) | 20:51 | 25.3 (22.8, 27.7) | 12.2 |
| Old | 37.1 (35.3, 38.8) | 6:14 | 8.7 (6.7, 10.6) | 18:57 | 22.7 (20.9, 24.4) | 14.2 |
| 17OHP | ||||||
| Young | 129.2 (126.8, 131.7) | 9:39 | 43.8 (40.2, 47.4) | 18:00 | 110.9 (108.4, 113.4) | 42.7 |
| Old | 87.6 (83.2, 92.1) | 6:06 | 41.4 (34.8, 47.9) | 18:00 | 70.6 (66.5, 74.8) | 23.1 |
| PregS | ||||||
| Young | 4565 (4469, 4660) | 18:00 | 2459 (2397, 2521) | 1:05 | 3712 (3648, 3776) | 1052.7 |
| Old | 2396 (2346, 2446) | 8:00 | 1556 (1510, 1602) | 23:43 | 1983 (1933, 2034) | 419.6 |
| 17OHPregS | ||||||
| Young | 515 (498, 532) | 8:21 | 297 (282, 312) | 22:46 | 429 (412, 446) | 109.0 |
| Old | 352 (337, 366) | 6:16 | 197 (176, 218) | 18:00 | 257 (245, 270) | 77.2 |
| DHEAS | ||||||
| Young | 157106 (154348, 159864) | 18:00 | 110156 (108275, 112037) | 02:14 | 124008 (122132, 125884) | 23474.7 |
| Old | 74177 (71987, 76367) | 18:00 | 59171 (57674, 60668) | 3:50 | 68049 (66597, 69501) | 7502.8 |
| AdiolS | ||||||
| Young | 8867 (8782, 8952) | 22:24 | 7724 (7627, 7821) | 9:50 | 8277 (8180, 8374) | 571.6 |
| Old | 5963 (5843, 6083) | 00:15 | 4669 (4481, 4858) | 18:00 | 5283 (5156, 5411) | 646.8 |
11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; 11OHA4, 11β-hydroxyandrostenedione; 11OHT, 11β-hydroxytestosterone; 17OHP, 17α-hydroxyprogesterone; 17OHPregS, 17-hydroxypregnenolone sulfate; A4, androstenedione; AdiolS, Androstenediol-3-sulfate; DHEAS, DHEA sulfate; PregS, pregnenolone sulfate; T, testosterone.
Of the four 11-oxyandrogens, 11OHA4 levels correlated most tightly to cortisol (r = 0.81, P < 0.001), although all had strong positive correlations. In contrast, 11OHT and 11KT correlated weakly with T (r = 0.27 and 0.19, respectively, P < 0.01 for both). Conversely, the two steroids that displayed strong positive correlations with T were A4 and 17OHP (r = 0.70 and 0.57, respectively, P < 0.0001), both known to have partial gonadal origin. 11KA4 and 11KT concentrations correlated most tightly with those of 11OHA4 (r = 0.91, P < 0.001), and 11OHT (r = 0.70, P < 0.001), respectively. In addition, 11KA4 and 11KT showed a strong positive correlation with cortisone (r = 0.75 and 0.64, respectively, P < 0.001 for both).
All four steroid sulfates were higher in younger as compared to older men (Fig. 2). Quantitatively, DHEAS was the dominant steroid in both age groups, followed by AdiolS, PregS, and 17OHPregS (Table 1). Notably, PregS and 17OHPregS displayed distinct declines around midnight, particularly in young men (Fig. 2).
Discussion
Herein, we characterized the circadian rhythms of 11-oxyandrogens, as well as of the understudied steroid sulfates, PregS, and 17OHPregS in healthy men. We show that the circadian pattern of circulating 11OHA4 resembles closely that of cortisol, with similar excursions in young and older men. In fact, of all 15 steroids measured, only cortisone concentrations correlated more tightly to those of cortisol than 11OHA4. 11OHA4 and 11OHT are produced by 11-hydroxylation of A4 and T, respectively, steps executed by the enzyme steroid 11β-hydroxylase (CYP11B1), which also catalyzes the last step in cortisol synthesis (Fig. 1) (11). We have previously shown that 11OHA4 is a major adrenal C19 steroid, with concentrations that far exceed those of its precursor, A4, and that its synthesis responds acutely to ACTH (8, 15, 16).
In contrast with 11OHA4, 11OHT is a minor product of the adrenal gland, with modest levels in the general circulation (7). Nevertheless, the circadian patterns of 11OHT are similar to those of 11OHA4, with similar morning concentrations in young and old men. The downstream metabolites of 11OHA4 and 11OHT, 11KA4 and 11KT, respectively, are produced primarily in peripheral tissues (2). We found that both steroids displayed circadian patterns similar to cortisol and 11OHA4 in young men, but the peak concentrations were lower in older men. These results are consistent with a recent study, in which we found that while 11OHA4 and 11OHT were relatively stable across ages, 11KA4, and 11KT declined modestly in aging men (7).
Although it has been proposed that 11-oxyandrogens might derive from the gonads, the gonadal expression of CYP11B1 is negligible (17). Moreover, while – beginning with puberty – the gonadal output of T is dramatically higher in males than in females, the circulating concentrations of both 11OHT and 11KT are similar in both sexes (7). We herein found that the concentrations of all 11-oxyandrogens correlated tightly with those of cortisol, but only modestly with T, indicating their ACTH dependence and further supporting their adrenal origin. Further, the correlation on 11-ketoandrogens with cortisone supports the common pathway of oxidation at C-11 via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) (18).
Unlike cortisol, DHEAS lacks prominent diurnal excursions, owing to its long half-life (19, 20). DHEAS has been used as a measure of integrated ACTH production, such as in autonomous adrenal cortisol excess (21, 22). Intriguingly, DHEAS is paradoxically low in patients with classic CAH, despite sustained ACTH elevations (23). The upstream steroid sulfates, 17OHPregS, and even more so PregS, accumulate instead in patients with classic CAH, possibly due to an abundance of their Δ5 steroid precursors (8). PregS and 17OHPregS were also found to be associated with surrogate clinical indicators of poor CAH control and sustained ACTH elevations, and both PregS and 17OHPregS correlate more tightly with morning ACTH than DHEAS or AdiolS (24). In this study, we found that while AdiolS and DHEAS had almost flat diurnal patterns, PregS and 17OHPregS concentrations displayed distinct nocturnal declines.
Our study has several limitations. The admission to an inpatient testing unit, frequent sampling, and fasting could have disrupted the natural physiologic rhythm of some steroids. Nevertheless, the pattern of cortisol production during the study period is in line with previous reports (25, 26), suggesting that the circadian rhythm did not suffer major alterations. Due to poor sensitivity without derivatization, we could not simultaneously measure unconjugated ∆5-steroids; however, the circadian patterns of these steroids were previously characterized (27, 28, 29). Additionally, this study was limited to men, and thus, we were not able to derive sex comparisons. The inclusion of men, however, allowed important observations regarding the 11-oxygenated T derivatives with both adrenal and gonadal steroids. Accounting for the diurnal variations of 11-oxyandrogens will be essential for their clinical interpretations, as these steroids are being introduced as biomarkers of several disorders of androgen excess.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.
Funding
This research was conducted with support of grants from NIDDK (1K08DK109116, awarded to A F T, and R01DK069950, awarded to W E R and R J A), and the Michigan Institute for Clinical and Health Research (U064177 awarded to A F T).
Acknowledgements
The authors thank Patrick O’Day and Jianwei Ren for assistance with steroid mass spectrometry assays and all study participants.
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