Background:
17-Hydroxyprogesterone (17-OHP) can be converted to dihydrotestosterone (DHT) via an alternative “backdoor” route that bypasses the conventional intermediates androstenedione and ...testosterone. In this backdoor pathway, 17-OHP is converted to 5α-pregnane-3α,17α-diol-20-one (pdiol), which is an excellent substrate for the 17,20 lyase activity of CYP17A1 to produce androsterone.
Objective and Hypotheses:
The objective of this study was to obtain evidence for the presence of the backdoor pathway in patients with 21-hydroxylase deficiency (21-OHD).
Methods:
We compared urinary steroid hormone profiles determined by gas chromatography-mass spectrometry of 142 untreated 21-OHD patients (age range, 1 d to 25.4 yr; 51 males) with 138 control subjects. The activity of the backdoor pathway was assessed using the ratios of the urinary concentrations of pdiol to those of the metabolites of the classic Δ4 and Δ5 pathways. In contrast to etiocholanolone, which originates almost exclusively from the classic pathways, androsterone may be derived additionally from the backdoor pathway. Therefore, the androsterone to etiocholanolone ratio can be used as an indicator for the presence of the backdoor pathway.
Results:
Untreated 21-OHD subjects showed increased urinary ratios of pdiol to the Δ4 and Δ5 pathway metabolites and a higher androsterone to etiocholanolone ratio.
Conclusions:
The elevated ratios of pdiol to the Δ4 and Δ5 pathway metabolites as well as the higher androsterone to etiocholanolone ratio in patients with 21-OHD indicate postnatal activity of the backdoor pathway with maximum activity during early infancy. Our data provide new insights into the pathophysiology of androgen biosynthesis of 21-OHD.
Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel ...method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R2 > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Biochemical monitoring of treatment in infants with classic congenital adrenal hyperplasia (CAH) is not yet well defined. The aim of this study was to perform a cluster analysis of the urinary ...steroid metabolome for treatment monitoring of infants with classic salt-wasting CAH. We analyzed spot urine samples obtained from 60 young children ≤ 4 years of age (29 females) with classic CAH due to 21-hydroxylase deficiency treated with hydrocortisone and fludrocortisone by targeted gas chromatography-mass spectrometry (GC-MS). Patients were classified into different groups according to their metabolic patterns (metabotypes) using unsupervised k-means clustering algorithms. Three metabotypes could be discovered. Metabotype #1 (N = 15 (25%)) showed high concentrations of androgen and 17-hydroxyprogesterone (17OHP) precursor steroids, metabotype #2 (N = 28 (47%)) revealed balanced metabolic control, and metabotype #3 (N = 17; 28%) demonstrated severe adrenal suppression with low concentrations of androgen and 17OHP precursor steroids. Daily hydrocortisone doses and urinary concentrations of cortisol and cortisone metabolites did not differ between all three metabotypes. Metabotype #2 had highest daily dose of fludrocortisone (p = 0.006). Receiver operating characteristic curve analysis showed that 11-ketopregnanetriol (area under the curve AUC 0.967) and pregnanetriol (AUC 0.936) were most suitable of separating metabotype #1 from #2. For separation between metabotypes #2 vs. #3, the 11-oxygenated androgen metabolite 11-hydroxyandrosterone (AUC 0.983) and the ratio of 11-hydroxyandrosterone to tetrahydrocortisone (AUC 0.970) were most suitable. In conclusion, GC-MS-based urinary steroid metabotyping is a new method to help monitor the treatment of infants with CAH. This method allows classification of under-, over- and adequately treated young children.
•GC-MS-based urinary steroid metabotyping in treated young children with CAH is presented.•Three unique metabotypes could be discovered.•The identification of these different metabolic patterns allows for a truly personalized diagnostic and therapeutic approach.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of ...them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs.
To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group “Sulfated Steroids in Reproduction” was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction.
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•We studied androgen metabolite excretions in treated children with CAH.•Androgen excess was solely due to 11-oxygenated androgens.•11-Oxygenated androgens derive from both, ...androstenedione and 21-deoxycortisol conversion.•11-Oxygenated androgens represent suitable biomarkers of adrenal androgen status and treatment response in CAH.
Adrenal androgen excess is the hallmark of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Recently, 11-oxygenated C19 steroids, a class of highly active adrenal-derived androgens, have been described in patients with CAH.
The aim of our study was to elucidate the significance of 11-oxygenated androgens in children with CAH.
We retrospectively analysed 190 daily urinary excretion rates of glucocorticoid-, 17α-hydroxyprogesterone (17OHP)-, and androgen metabolites determined by gas chromatography-mass spectrometry of 99 children aged 3.0–10.9 years with classic CAH on hydrocortisone and fludrocortisone treatment. Daily urinary steroid metabolite excretions were transformed into z-scores using references of healthy children. Androgen metabolite z-scores were separately calculated for androsterone (AN), the major urinary metabolite of androstenedione (A4), testosterone and 5α-dihydrotestosterone, for urinary metabolites of dehydroepiandrosterone (DHEA), and for 11β-hydroxyandrosterone (11OHAN), the major urinary metabolite of adrenal-derived 11-oxygenated androgens. Multivariate regression analysis was applied to analyse the precursors of 11OHAN synthesis.
11OHAN, cortisol-, and 17OHP metabolite z-scores were elevated in treated children with CAH, whereas AN- and DHEA metabolite z-scores were normalized or suppressed. Multivariate regression analysis revealed that 11OHAN excretion was strongest associated with 21-deoxycortisol (β = 0.379; P =.0006), followed by A4 (β = 0.280; P = .0008)) and 17OHP (β = 0.243; P = .04) metabolite excretion.
Androgen excess in treated children with CAH is solely due to elevated 11-oxygenated androgens that derive in addition to the known conversion from A4 also by direct conversion from 21-deoxycortisol. 11-Oxygenated androgens may represent better biomarkers of adrenal androgen status and treatment response than conventional androgens.
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Treatment of children with classic congenital adrenal hyperplasia (CAH) with glucocorticoids is a difficult balance between hypercortisolism and hyperandrogenism. Biochemical monitoring of treatment ...is not well defined. Achievement of a normal growth rate is the most important therapeutic goal.
We retrospectively evaluated 123 24-hour gas chromatography-mass spectrometry urinary steroid metabolome analyses together with their corresponding 1-year height velocity (HV) z scores in 63 prepubertal children aged 7.2 ± 1.6 years with classic CAH due to 21-hydroxylase deficiency treated with hydrocortisone and fludrocortisone.
Multivariate linear mixed effects model analysis revealed a positive influence of CAH-specific z scores of summed urinary androgen metabolites (B = 0.97 ± 0.20, t = 4.87, P < 0.0001) and a negative influence of the cortisol metabolite tetrahydrocortisol (B = -1.75 ± 0.79, t = -2.20, P = 0.03) on HV z scores. Receiver operating characteristic analysis demonstrated that adrenal androgen excess, defined as HV >1.5 z, was best determined by a z score of all urinary androgen metabolites of >0.512 accuracy, 66.2%; sensitivity, 57.1%; specificity, 74.4%; positive prediction value (PPV), 66.7%; negative prediction value (NPV), 65.9%. Tetrahydrocortisol excretion >1480 µg/m2 BSA/d in conjunction with suppressed urinary androgen metabolites <0.163 z indicated overtreatment, defined as HV < -1.5 z (accuracy, 79.6%; sensitivity, 40.0%; specificity, 94.9%; PPV, 75.0%; NPV, 80.4%).
We established target values for urinary steroid metabolite excretions in children with CAH based on their growth rate. Urinary steroid metabolome analysis represents a highly suitable method for monitoring metabolic control in children with CAH.
To evaluate the impact of anti-tumor necrosis factor-alpha (TNFα: etanercept Etanercept ®) therapy on adrenal activity in juvenile idiopathic arthritis (JIA) .
Eleven JIA patients aged 12 ± 6.2 years ...with a disease duration of 6.3 ± 5.2 years were enrolled. They were treated once weekly with etanercept (0.8 mg/kg) for 3 ± 2.8 years. Urine samples for gas chromatography-mass spectrometry steroid hormone analysis were collected before, and 1 and 3 days after etanercept injection and compared to age- and sex-matched healthy controls.
The levels of 21 of the 31 metabolites were low before etanercept treatment. Those 21 metabolites included 4 C19 steroids (androgens), 5 C C21 steroid hormone intermediates, 10 cortisol metabolites, and 2 corticosterone metabolites. One day after treatment, only 5 of the 21 metabolite levels remained low. They included 2 C19 metabolites, 2 C21 steroid metabolites and 1 cortisol metabolite β -Cortol (β-Cl). Three days after treatment, the only metabolites levels that continued to be low were 2 C19 metabolite, 2 C21 steroid hormone intermediates and 1 cortisol metabolite α-Cortol (a-Cl), while the remaining 15 metabolites had already normalized after 1 day. Dehydroepiandrosterone-sulfate and 17-hydroxypregnenolone metabolite levels were the last ones to recover. Urinary metabolite ratios reflecting cytochrome P450 CYP21A2 (21-hydroxylase) and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzymatic activitieswere lower in JIA patients than in controls, although significant was not reached.
Almost all of the pre-etanercept treatment cortisol urinary metabolite levels were significantly lower than normal, and almost all rose to normal values by 1 day after treatment. The therapeutic effect of anti-TNFα treatment in JIA may be related to its effect on the restoration of adrenal function and cortisol levels.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Treatment of children with classic congenital adrenal hyperplasia (CAH) is a difficult balance between hypercortisolism and hyperandrogenism. Biochemical monitoring of treatment is not well defined.
...Cluster analysis of the urinary steroid metabolome obtained by targeted gas chromatography–mass spectrometry (GC–MS) for treatment monitoring of children with CAH.
We evaluated 24-h urinary steroid metabolome analyses of 109 prepubertal children aged 7.0 ± 1.6 years with classic CAH due to 21-hydroxylase deficiency treated with hydrocortisone and fludrocortisone. 24-h urinary steroid metabolite excretions were transformed into CAH-specific z-scores. Subjects were divided into groups (metabotypes) by k-means clustering algorithm. Urinary steroid metabolome and clinical data of patients of each metabotype were analyzed.
Four unique metabotypes were generated. Metabotype 1 (N = 21 (19%)) revealed adequate metabolic control with low cortisol metabolites (mean: −0.57z) and suppressed androgen and 17α-hydroxyprogesterone (17OHP) metabolites (−0.79z). Metabotype 2 (N = 23 (21%)) showed overtreatment consisting of a constellation of elevated urinary cortisol metabolites (0.62z) and low metabolites of androgens and 17OHP (−0.75z). Metabotype 3 (N = 32 (29%)) demonstrated undertreated patients with low cortisol metabolites (−0.69z) and elevated metabolites of androgens and 17OHP (0.50z). Metabotype 4 (N = 33 (30%)) presented patients with treatment failure reflected by unsuppressed androgen- and 17OHP metabolites (0.71z) despite elevated urinary cortisol metabolites (0.39z).
Metabotyping, which means grouping metabolically similar individuals, helps to monitor treatment of children with CAH using GC–MS urinary steroid metabolome analysis. This method allows classification in adequately-, over-, or undertreated children as well as identification of patients with treatment failure.
•Treatment monitoring of children with CAH is not well defined•Cluster analysis of their urinary steroid metabolome obtained four unique metabotypes•Metabotyping allows classification in adequately-, over-, or undertreated patients•Metabotyping additionally identifies patients with treatment failure
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the ...accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in
, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized
mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•Immunoassays overestimate urinary free cortisol (UFC) due to interfering steroid metabolites.•UFC measurement with established immunoassays correlate with LC–MS/MS.•Measured by ROC AUC, diagnostic ...accuracy of immunoassays is comparable to LC–MS/MS.
24-h urine free cortisol (UFC) is an indicator of integrated cortisol secretion and established screening tool for Cushing’s syndrome (CS). Doubts have been raised regarding specificity of immunoassays, and mass spectrometric techniques have been proposed as an alternative. In the present study we compared diagnostic accuracy of UFC measured with LC–MS/MS vs. immunoassay in patient with CS and patients where CS has been excluded. We examined 24-h urine samples from patients with surgically confirmed CS (n = 77; Cushing’s disease (n = 44), ectopic CS (n = 5), adrenal CS (n = 28)) and patients in whom Cushing’s syndrome was excluded (n = 97) by long-term follow up. UFC was first measured by automated chemiluminescence immunoassays (ADVIA Centaur, Siemens; LIAISON, DiaSorin). Aliquots of all samples were also analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Statistics: Passing-Bablok Regression, Receiver operating characteristic (ROC) analysis with Youden’s index calculation. UFC of CS patients were higher with both immunoassays compared to LC–MS/MS (913 +/- 235 vs. 303 +/- 155 μg/24 h (ADVIA) and 898 +/-216 vs. 399 +/- 196 μg/24 h (LIAISON)). Similarly, UFC were higher with immunoassays than with LC–MS/MS in the control group (223 +/- 10 vs. 23 +/- 2 μg/24 h (ADVIA) and 105 +/- 6 vs. 27 +/- 4 ug/24 h for (LIAISON)). Passing-Bablok regression showed good correlation between LC–MS/MS and ADVIA as well as between LCMS/MS and LIAISON (r = 0.96 and r = 0.99, p < 000.1) but less correlation in controls (r = 0.83 and r = 0.74, respectively, p < 000.1). ROC calculation revealed the highest ROC AUC (0.89) for the LIAISON immunoassay, followed by LC–MS/MS (0.82) and the ADVIA (0.80). In direct comparison, AUCs from LC–MS/MS and immunoassays in the same patient were not statistically different (p < 0,001). Best cut-off concentration to identify patients with CS was 234 μg/24 h (LIAISON), 51 μg/24 h for LC–MS/MS and 330 μg/24 h (ADVIA Centaur).
In summary, UFC values were measured substantially higher by both immunoassays compared to LC–MS/MS. This is most likely due to cross-reactivity from interfering glucocorticoid metabolites. Nevertheless, all three methods correlated well. ROC analysis revealed the highest AUC for one of the immunoassays, although differences between the three methods were not significant. Direct comparison with LC–MS/MS indicates that high diagnostic accuracy can be obtained with suitable immunoassays.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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