Analysis of 52 C(19) and C(21) steroids by UPC(2)-MS/MS: Characterising the C11-oxy steroid metabolome in serum.

The C11-oxy androgens have been implicated in the progression of many diseases and endocrine-linked disorders, such as polycystic ovarian syndrome (PCOS), congenital adrenal hyperplasia, specifically 21-hydroxylase deficiency (21OHD), castration resistant prostate cancer (CRPC), as well as premature adrenarche. While the C11-oxy C19 steroids have been firmly established in the steroid arena, the C11-oxy C21 steroids are now also of significance. The current study reports on a high-throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) method for the separation and quantification of 52 steroids in peripheral serum, which include the C11-oxy C19 and C11-oxy C21 steroids. Fifteen deuterium-labelled steroids were included for absolute quantification, which incorporates steroid extraction efficiency, together with one steroid and four non-steroidal compounds serving as quality controls (QC). The 15 min run-time per sample (16 min injection-to-injection time with an 8-step gradient) quantifies 68 analytes in a 2 µL injection volume. A single chromatographic step simultaneously identifies steroids in the mineralocorticoid, glucocorticoid and androgen pathways in adrenal steroidogenesis, together with steroid metabolites produced in the periphery, presenting an analytical method for the application of screening in vivo clinical samples. This study highlights cross-talk between the C11-oxy steroids, and describes the optimisation of multiple reaction monitoring required to measure steroids accurately. The limit of detection for the steroid metabolites ranged from 0.002 to 20 ng/mL and the limit of quantification from 0.02 to 100 ng/mL. The calibration range for the steroids ranged from 0.002 to 1000 ng/mL and for the QC compounds from 0.075 to 750 ng/mL. The method is fully validated in terms of accuracy (%RSD, <13%), precision (including inter-day variability across a three-day period) (%RSD, <16%), recovery (average 102.42%), matrix effect (ranging from -15.25 to 14.25%) and process efficiency (average 101.79%). The dilution protocol for the steroids, internal standards and QC compounds were validated, while the ion ratios of the steroid metabolites (%RSD, <16%) and QC compounds were monitored and the accuracy bias values (%RSD, <9%) were within acceptable limits. The method was subsequently used to quantify steroid levels in a cohort of healthy women. C11-oxy steroid metabolites produced as intermediates in steroidogenic pathways, together with end-products included in the method can potentially characterise the 11β-hydroxyandrostenedione-, C21- and C11-oxy backdoor pathways in vivo. The identification of these C11-oxy C19 and C11-oxy C21 intermediates would allow insight into active pathways, while steroid metabolism could be traced in patients and reference ranges established in both normal and abnormal conditions. Furthermore, conditions currently undefined in terms of the C11-oxy steroids would benefit from the analysis provided by this method, while the C11-oxy steroids could be further explored in PCOS, 21OHD, CRPC and adrenarche.

du Toit T ，van Rooyen D ，Stander MA ，Atkin SL ，Swart AC ... -  《-》

A high-throughput UPC(2)-MS/MS method for the separation and quantification of C(19) and C(21) steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways.

In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method was developed and validated for the determination of 17 C19 and 14 C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately. Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C19 and C21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C19 and C21 steroids, together with their C11-hydroxy and C11-keto metabolites -one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients.

du Toit T ，Stander MA ，Swart AC 《-》

The 11β-hydroxyandrostenedione pathway and C11-oxy C(21) backdoor pathway are active in benign prostatic hyperplasia yielding 11keto-testosterone and 11keto-progesterone.

In clinical approaches to benign prostatic hyperplasia (BPH) and prostate cancer (PCa), steroidogenesis or the disruption thereof is the main thrust in treatments restricting active androgen production. Extensive studies have been undertaken focusing on testosterone and dihydrotestosterone (DHT). However, the adrenal C11-oxy C19 steroid, 11β-hydroxyandrostenedione (11OHA4), also contributes to the active androgen pool in the prostate microenvironment, and while it has been shown to impact castration resistant prostate cancer, the C11-oxy C19 steroids together with the C11-oxy C21 steroids have not been studied in BPH. The study firstly investigated the metabolism of these adrenal steroids in the BPH-1 model. Comprehensive profiles identified 11keto-testosterone as the predominant active androgen in the metabolism of the C11-oxy C19 steroids, and we identified, for the first time, 11β-hydroxy-5α-androstane-3α,17β-diol, a novel steroid in the 11OHA4-pathway. Analysis of the inactivation and reactivation of the metabolites showed that DHT is more readily inactivated than 11keto-dihydrotestosterone (11KDHT). The conversion of 11β-hydroxyprogesterone (11βOHPROG) yielded 11keto-progesterone (11KPROG), while the latter yielded 11keto-dihydroprogesterone (11KDHPROG). BPH tissue analysis identified high levels of 11β-hydroxyandrosterone (4-14 ng/g) and 11keto-androsterone (9-160 ng/g), together with androstenedione (A4; ∼7.5 ng/g). The major C11-oxy C21 steroids detected were 11βOHPROG (∼46 ng/g), 11KPROG (∼130 ng/g) as well as 11KDHPROG (∼282 ng/g). While circulatory 11βOHPROG was detected below the limit of quantification, 11KPROG and 11KDHPROG were detected at 6 and 8.5 nmol/L, respectively. Glucuronide derivatives of both 11KPROG and pregnanetriol were also detected. 11OHA4 was the major free androgen in circulation at 85.9 nmol/L, ±12-fold higher than A4, together with 5α-androstane-3α,17β-diol quantified at 69.3 nmol/L. Circulatory C11-oxy C19 steroids levels were also significantly higher (8-fold) than the C11-oxy C21 steroid levels, while the former were similar to the C19 steroid levels, in contrast to levels in PCa. The study highlights the contribution of adrenal C11-oxy steroids to the androgen pool in BPH underscoring their limited reactivation and elimination, and significant inter-individual variations regarding steroid levels and conjugation. Targeted steroid metabolome analysis is critical to understanding prostate steroidogenesis and disease progression, and analysis of circulatory C11-oxy C19 and C11-oxy C21 steroids, together with intraprostatic levels, add to our current understanding of BPH.

du Toit T ，Swart AC 《-》

Profiling adrenal 11β-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC(2)-MS/MS quantification of 11β-hydroxytestosterone, 11keto-testosterone and 11keto-dihydrotestosterone.

Adrenal C19 steroids serve as precursors to active androgens in the prostate. Androstenedione (A4), 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) are metabolised to potent androgen receptor (AR) agonists, dihydrotestosterone (DHT), 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). The identification of 11OHA4 metabolites, 11KT and 11KDHT, as active androgens has placed a new perspective on adrenal C11-oxy C19 steroids and their contribution to prostate cancer (PCa). We investigated adrenal androgen metabolism in normal epithelial prostate (PNT2) cells and in androgen-dependent prostate cancer (LNCaP) cells. We also analysed steroid profiles in PCa tissue and plasma, determining the presence of the C19 steroids and their derivatives using ultra-performance liquid chromatography (UHPLC)- and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). In PNT2 cells, sixty percent A4 (60%) was primarily metabolised to 5α-androstanedione (5αDIONE) (40%), testosterone (T) (10%), and androsterone (AST) (10%). T (30%) was primarily metabolised to DHT (10%) while low levels of A4, 5αDIONE and 3αADIOL (≈20%) were detected. Conjugated steroids were not detected and downstream products were present at <0.05μM. Only 20% of 11OHA4 and 11OHT were metabolised with the former yielding 11keto-androstenedione (11KA4), 11KDHT and 11β-hydroxy-5α-androstanedione (11OH-5αDIONE) and the latter yielding 11OHA4, 11KT and 11KDHT with downstream products <0.03μM. In LNCaP cells, A4 (90%) was metabolised to AST-glucuronide via the alternative pathway while T was detected as T-glucuronide with negligible conversion to downstream products. 11OHA4 (80%) and 11OHT (60%) were predominantly metabolised to 11KA4 and 11KT and in both assays more than 50% of 11KT was detected in the unconjugated form. In tissue, we detected C11-oxy C19 metabolites at significantly higher levels than the C19 steroids, with unconjugated 11KDHT, 11KT and 11OHA4 levels ranging between 13 and 37.5ng/g. Analyses of total steroid levels in plasma showed significant levels of 11OHA4 (≈230-440nM), 11KT (≈250-390nM) and 11KDHT (≈19nM). DHT levels (<0.14nM) were significantly lower. In summary, 11β-hydroxysteroid dehydrogenase type 2 activity in PNT2 cells was substantially lower than in LNCaP cells, reflected in the conversion of 11OHA4 and 11OHT. Enzyme substrate preferences suggest that the alternate pathway is dominant in normal prostate cells. Glucuronidation activity was not detected in PNT2 cells and while all T derivatives were efficiently conjugated in LNCaP cells, 11KT was not. Substantial 11KT levels were also detected in both PCa tissue and plasma. 11OHA4 therefore presents a significant androgen precursor and its downstream metabolism to 11KT and 11KDHT as well as its presence in PCa tissue and plasma substantiate the importance of this adrenal androgen.

du Toit T ，Bloem LM ，Quanson JL ，Ehlers R ，Serafin AM ，Swart AC ... -  《-》

Inefficient UGT-conjugation of adrenal 11β-hydroxyandrostenedione metabolites highlights C11-oxy C(19) steroids as the predominant androgens in prostate cancer.

Although the adrenal C19 steroids, androstenedione and testosterone, contribute to prostate cancer (PCa) progression the full complement of adrenal androgens, including the C11-oxy C19 steroids, 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) and their androgenic metabolites, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) have, to date, not been considered. This study investigated the contribution of 11OHA4 and 11OHT to the pool of active androgens in the prostate. Steroid profiles were determined in LNCaP, C4-2B and VCaP cell models, in PCa tissue, and in plasma focussing on the inactivation, reactivation and glucuronidation of 11OHA4, 11OHT and their downstream products using ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). The C11-oxy C19 steroids were the predominant steroids with the production of 11KT and 11KDHT in prostate cell models identifying 11β-hydroxysteroid dehydrogenase type 2 activity. Active:inactive steroid ratios indicated efficient inactivation of dihydrotestosterone (DHT) and 11KDHT by 3α-hydroxysteroid dehydrogenases, while the reactivation of DHT by retinol-like dehydrogenases was greater than the reactivation of 11KDHT. In PCa tissue, inactive C11-oxy C19 steroids ranged from 27 to 30 ng/g, whereas inactive C19 steroids were below 1 ng/g. Steroid glucuronidation was impeded: in VCaP cells, the C11-oxy C19 steroids were unconjugated and the C19 steroids fully conjugated; in C4-2B cells, all steroids were unconjugated, except for DHT of which 50% was conjugated; in LNCaP cells only androsterone, 11KT and 11β-hydroxyandrosterone were unconjugated. In PCa patients' plasma 11KDHT was present only in the unconjugated form, with 11KT also predominantly unconjugated (90-95%). Even though plasma and tissue sample numbers were limited, this study serves to demonstrate the abundance of C11-oxy C19 steroids, with notable differences in their metabolism, dictated by steroidogenic enzymes and hampered conjugation, affecting active androgen levels. Larger cohorts are required to analyse profiles in modulated metabolic pathways, in order to shed light on treatment outcomes. The C11-oxy C19 steroids are involved in PCa, with impeded glucuronidation in PCa ascribing a dominant role to these steroids in disease progression.

du Toit T ，Swart AC 《-》