Measurement Of Dehydroepiandrosterone Sulfate Research Articles

A liquid chromatography-tandem mass spectrometry method for the analysis of dehydroepiandrosterone sulphate (DHEAs) in serum and plasma with comparison to an immunoassay method in a neonatal population

The measurement of dehydroepiandrosterone-sulphate (DHEAs) is an important second-line test to aid in the diagnosis of premature adrenarche, peripubertal gynaecomastia in males and in identifying the source of elevated androgens in females. Historically, DHEAs has been measured by immunoassay platforms which are prone to poor sensitivity and more importantly poor specificity. The aim was to develop an LC-MSMS method for the measurement of DHEAs in human plasma and serum, develop an in-house paediatric (<6 year old) reference limit and compare the performance against the Abbott Alinity DHEAs immunoassay method.Following pre-treatment with an internal standard, samples were loaded onto EVOLUTE® EXPRESS ABN plate. Analytes were separated with reverse-phase chromatography using ACQUITY® UPLC® HSS T3 2.1 mm × 50 mm, 1.8 μm column. Mass spectrometry detection was performed using a Waters® Xevo TQ-XS in electrospray negative mode. For the paediatric reference range, samples were collected from an inpatient setting (age ≤ 6 years old) with no evidence of adrenal dysfunction or history of/current steroid use. The method comparison was performed using samples from this cohort aged between 0 and 52 weeks.The assay demonstrated linearity up to 15 µmol/L (r2 > 0.99) with a functional sensitivity of 0.1 µmol/L. Accuracy results revealed a mean bias of 0.7% (−14% to 15%) when compared against the NEQAS EQA LC-MSMS consensus mean (n = 48). The paediatric reference limit was calculated as ≤ 2.3 µmol/L (95% C.I. 1.4 to 3.8 µmol/L) for ≤ 6 year olds (n = 38). Comparison of neonatal (<52 weeks) DHEAs with the Abbott Alinity revealed that the immunoassay ran at a 166% positive bias (n = 24) which appeared to lessen with increasing age.Described is a robust LC-MSMS method for the measurement of plasma or serum DHEAs validated against internationally recognised protocols. Comparison of paediatric samples of <52 weeks against an immunoassay platform demonstrated that in the immediate new-born period results generated from the LC-MSMS method offer superior specificity than an immunoassay platform.

Diagnosis of Sertoli-Leydig Cell Tumor of the Ovary Complicated by the Pattern of Hyperandrogenemia and Results of Imaging

Introduction: Virilization in a postmenopausal woman requires evaluation for an androgen-secreting tumor. The differential diagnosis includes adrenal carcinomas and adenomas and Sertoli-Leydig cell tumors, granulosa-theca cell tumors, and hilus-cell tumors of the ovaries. We present a case of virilization in a postmenopausal woman caused by a Sertoli-Leydig cell tumor (SLCT) in which evaluation was complicated by the pattern of androgen elevation, bilateral adrenal nodules, and absence of an adnexal mass. Case: A 64-year-old female was referred for evaluation of hyperandrogenism. Hirsutism, temporal hairline regression, and unusually deep voice were noted on examination. Two total testosterone levels obtained one month apart were 146 ng/dL (2-45), and measurements of dehydroepiandrosterone sulfate (DHEAS) and androstenedione were 299 mcg/dL (12-133) and 1.84 ng/mL (0.130-0.820), respectively. Abdominal CT revealed bilateral adrenal nodules - 2 cm and - 5 Hounsfield units (HU) on the left, and 1.5 cm and 5 HU on the right - but no ovarian masses. Transvaginal ultrasonography also failed to identify a discrete ovarian mass but showed endometrial hyperplasia. Virilization, magnitude of testosterone elevation, and results of imaging were felt to be most strongly indicative of ovarian hyperthecosis, and the patient underwent laparoscopic bilateral salpingo-oophorectomy and hysterectomy. The right ovary was 2.3 cm in largest diameter and approximately 90% replaced by an orange-red mass that showed Sertoli and Leydig cells on microscopy, immunohistochemical staining for the sex cord proteins inhibin and calretinin, and presence of the Leydig cell marker melan A. It was classified as well differentiated. Additional CT imaging and robotic assisted laparoscopy confirmed a stage IA tumor. One month after surgery, hyperandrogenemia had completely resolved (total testosterone < 10 ng/dL, androstenedione 0.379 ng/mL, and DHEAS 99 mcg/dL), and changes of virilization had mostly regressed at an eight months appointment. Discussion: SLCTs are a type of sex-cord stromal ovarian tumor. They constitute < 0.5% of ovarian tumors but account for approximately 75% of testosterone-secreting ovarian masses. This patient’s case was unusual for multiple reasons: 1. Age - most SLCTs are diagnosed in the second or third decade, 2. Imaging - CT and ultrasonography usually show a solid or solid and cystic adnexal mass, and co-existing adrenal nodules are rare, likely due to typical young age of presentation, and 3. Pattern of androgen elevation - DHEAS was more than two-fold elevated, and usually < 10% of DHEA and DHEAS are produced by the ovaries. However, DHEAS fell significantly after oophorectomy. SLCTs are a potential etiology of virilization in postmenopausal women even in the absence of a detectable adnexal mass and when biochemistries and imaging raise the possibility of an adrenal source of androgen.

Measurement of dehydroepiandrosterone sulphate (DHEAS): A comparison of Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) and seven currently available immunoassays

BackgroundDehydroepiandrosterone sulphate (DHEAS) is an important marker of the adrenal gland. Its measurement is required in several adrenal diseases, such as adrenal tumours, adrenal insufficiency and congenital adrenal hyperplasia. Most clinical laboratories measure DHEAS using commercially available immunoassays. The aim of the present study was to investigate the accuracy of currently available DHEAS methods. MethodsSeven commercially available DHEAS assays were compared to Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) by measuring 75 serum samples (concentration range 0.06–20.6μmol/L measured by ID-LC-MS/MS) with each method. Moreover, recovery and linearity experiments were performed. Data from our present study were compared to DHEAS data of the Dutch, German and British External Quality Assessment Schemes (EQAS's). ResultsThree methods agreed well with ID-LC-MS/MS (R between 0.93 and 0.99 and slopes ranging from 0.92 to 1.07) and showed good recoveries. Four methods showed standardization problems (slopes were 0.84, 1.14, 1.20 and 1.28). Linearity was good in all methods. Intra-assay coefficient of variation was 4.1% using ID-LC-MS/MS and below 5.5% in immunometric methods; one assay had an unacceptably high intra-assay coefficient of variation of 18%. Our data are in agreement with data obtained in three EQAS's. ConclusionSome of the commercially available DHEAS methods show standardization problems and/or a high imprecision. These problems may potentially have clinically adverse consequences. We advise the manufacturers to improve their assays and laboratory specialists to scrutinize the DHEAS method they employ.

Development of a method for the measurement of dehydroepiandrosterone sulphate by liquid chromatography-tandem mass spectrometry

Dehydroepiandrosterone sulphate (DHEAS) is a steroid that is increasingly being recognized as a potential drug of abuse in many countries. This is due to its reputation as a hormone that may be able to retard the ageing process. The measurement of DHEAS is useful in the diagnosis of medical conditions such as congenital adrenal hyperplasia and polycystic ovary syndrome. Thus, a liquid chromatography-tandem mass spectrometry method has been developed to determine DHEAS concentrations in human serum. The chromatography was performed using a Waters 2795 Alliance HT LC system coupled to a Mercury Fusion-RP column fitted with a SecurityGuard column. DHEAS and the internal standard, deuterated DHEAS, both had a retention time of 1.5 min. The transition determined by the Micromass Quattro tandem mass spectrometer for DHEAS was m/z 367.3>4 96.7 and for the internal standard m/z 369.3>96.6. The method was linear up to 20 micromol/L; the lower limit of detection and the lower limit of quantitation were both 1 micromol/L. The intra- and interassay imprecision were <11% over a concentration range of 1-18 micromol/L for the in-house quality control and <12% for the intra- and interassay imprecision for the Bio-Rad Lyphocheck QC. The measurement of DHEAS by liquid chromatography-tandem mass spectrometry is robust and has a simple sample preparation procedure with a rapid cycle time of only 4 min.

Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.

The value of dehydroepiandrosterone sulfate measurements in the assessment of adrenal function.

Dehydroepiandrosterone (DHEA) and its sulfated ester (DHEA-S) are corticotropin-dependent adrenal androgen precursors that are uniformly low in treated patients with corticotropin deficiency. There are no data investigating the diagnostic value of DHEA-S measurements in the prospective assessment of adrenal function. This study examined serum DHEA-S levels as possible markers for hypothalamic- pituitary-adrenal (HPA) function in patients with large pituitary adenomas. Patients were characterized to have normal HPA (n = 47) or abnormal HPA (ABN-HPA, n = 35) function based on their respective responses to insulin-induced hypoglycemia. Patients also underwent low-dose Cortrosyn (1 micro g, LDC) and standard-dose Cortrosyn stimulation testing. All patients with ABN-HPA had very low age- and gender-matched serum DHEA-S levels. When the normal response to LDC was set at a cortisol level of at least 18.1 micro g/dl, 10 of 31 patients with ABN-HPA exhibited normal responses. Receiver operating characteristic curves for baseline DHEA-S and for maximal cortisol responses to LDC had areas of 0.984 (confidence interval, 0.962-1.000) and 0.893 (confidence interval, 0.817-0.969), respectively. LDC- or SDC-stimulated serum cortisol levels have significant limitations in defining HPA function. A normal age- and gender-specific serum DHEA-S level makes the diagnosis of corticotropin deficiency extremely unlikely. However, when serum DHEA-S levels are low, further testing is necessary to define HPA function.

Prospective measurements of dehydroepiandrosterone sulfate in a cohort of elderly subjects: relationship to gender, subjective health, smoking habits, and 10-year mortality.

The decrease with age of the adrenal-secreted dehydroepiandrosterone sulfate (DHEAS) in serum has suggested that it may be causally related to longevity. For the PAQUID [People (Personnes) Aged (Agées) About What (Quid, in Latin)] cohort of elderly subjects, we have previously reported higher DHEAS in men than in women, a decrease with age and, among men, a negative correlation between the DHEAS level and mortality at 2 and 4 years. Here, with an 8-year followup in 290 subjects, we show a global decrease of 2.3% per year for men and 3.9% per year for women. However, in approximately 30% of cases, there was an increase of DHEAS. We observed no relationship between the evolution of DHEAS level and functional, psychological, and mental status, possibly because of selection by death. In women, no association was found between mortality and DHEAS level. In men, the relative risk (RR) of death was higher for the lowest levels of DHEAS (RR = 1.9, P = 0.007), with RR = 6.5, P = 0.003 for those under 70 years old, a result indicating heterogeneity of the population. There was an effect of subjective health on mortality that disappeared after adjustment of DHEAS levels, suggesting its relation with these DHEAS levels. Death RR was much higher in smokers with a low DHEAS level than in nonsmokers with high DHEAS (RR = 6.7, P = 0.001). We submit that the involvement of DHEAS is possibly different according to gender, that association between low DHEAS level and mortality only for men under 70 years old possibly reflects heterogeneity of the population, and that DHEAS level is a reliable predictor of death in male smokers.

Adrenal incidentaloma.

Incidentally discovered adrenal masses, or adrenal incidentalomas, have become a common clinical problem owing to wide application of radiologic imaging techniques. This definition encompasses a heterogeneous spectrum of pathologic entities, including primary adrenocortical and medullary tumors, benign or malignant lesions, hormonally active or inactive lesions, metastases, and infections. Once an adrenal mass is detected, the clinician needs to address two crucial questions: is the mass malignant, and is it hormonally active? This article provides an overview of the diagnostic clinical approach and management of the adrenal incidentaloma. Mass size is the most reliable variable to distinguish benign and malignant adrenal masses. Adrenalectomy should be recommended for masses greater than 4.0 cm because of the increased risk of malignancy. Adrenal scintigraphy has proved useful in discriminating between benign and malignant lesions. Finally, fine-needle aspiration biopsy is an important tool in the evaluation of oncological patients and it may be useful in establishing the presence of metastatic disease. The majority of adrenal incidentalomas are non-hypersecretory cortical adenomas but an endocrine evaluation can lead to the identification of a significant number of cases with subclinical Cushing's syndrome (5-15%), pheochromocytoma (1.5-13%) and aldosteronoma (0-7%). The first step of hormonal screening should include an overnight low dose dexamethasone suppression test, the measure of urinary catecholamines or metanephrines, serum potassium and, in hypertensive patients, upright plasma aldosterone/plasma renin activity ratio. Dehydroepiandrosterone sulfate measurement may show evidence of adrenal androgen excess.

The value of dehydroepiandrosterone sulfate measurement in the differentiation between benign and malignant adrenal masses.

Owing to their increasing rate of discovery as incidental findings, the characterization of adrenal masses is an important diagnostic problem which frequently challenges the clinician's skill. The results of dehydroepiandrosterone sulfate (DHEAS) measurement were evaluated in a consecutive series of 107 patients with an adrenal mass (39 men, 68 women aged 15-81 years, median 56 years). DHEAS levels observed in the patients were categorized as reduced, normal or elevated according to sex- and age-adjusted reference ranges obtained by measuring DHEAS in 214 healthy women and 142 healthy men aged 17-93 years. The working hypothesis was that a low DHEAS level is a marker of benignity. In 84 patients, the adrenal mass was discovered serendipitously, while in the remainder the mass was clinically symptomatic. Patients with known extra-adrenal malignancies were excluded. The adrenal masses were categorized as benign or malignant by computerized tomography (CT) criteria. All patients with suspected malignant tumors or with overtly hypersecreting tumors underwent adrenalectomy. The patients with a presumptive benign tumor were followed-up for at least 12 months. In the overall series, the sensitivity and specificity of a low DHEAS level in the identification of a benign lesion were 41% and 100% respectively. Superimposable figures were obtained when considering only adrenal incidentalomas. DHEAS levels in adrenal cancers displayed some overlap with adrenal adenomas, but in only 2/11 adrenal cancers were DHEAS levels normal, while they were elevated in the remainder. The present data suggest that DHEAS measurement may help to differentiate benign from malignant adrenal masses as a complementary test of CT in a clinical research setting. The value of DHEAS measurement in general practice is limited because it may be difficult to differentiate between low and low-normal values, particularly in the elderly.

Hyposecretion of adrenal androgens and the relation of serum adrenal steroids, serotonin and insulin-like growth factor-1 to clinical features in women with fibromyalgia

Neuroendocrine deficiencies have been implicated in fibromyalgia (FM). In the present study, adrenal androgen metabolites and their relationship with health status in FM were investigated. For comparison, serum levels of other implicated neuroendocrine mediators were correlated with health status. Fifty-seven consecutive women with FM completed the Fibromyalgia Impact Questionnaire (FIQ). Fasting blood samples were taken for measurement of dehydroepiandrosterone sulphate (DHEAS), free testosterone (T), cortisol, serotonin and insulin-like growth factor-1. Normal value for DHEAS and T were obtained from 114 controls. DHEAS levels were decreased significantly in pre- and postmenopausal patients ( P<0.0001 and P<0.0005, respectively). T levels were decreased significantly in premenopausal and insignificantly in postmenopausal patients ( P<0.0001 and P=0.06, respectively). The following correlations between neurohormonal levels and FIQ scores were found: DHEAS (after adjustment for age) vs. pain ( P<0.001) and T (after adjustment for age) versus physical functioning ( P=0.002). None of the other neurohormonal levels correlated significantly with any of the FIQ scores. IGF-1 levels were lower in the obese patients as compared to those who were non-obese ( P=0.03). The BMI correlated positively with pain ( P<0.001) and inversely with DHEAS levels ( P=0.006). After further adjustment for BMI, the correlation between age adjusted DHEAS and pain was no longer significant. Hyposecretion of adrenal androgens was documented in FM. This was more pronounced in obese patients. Low serum androgen levels correlated with poor health status in FM. Longitudinal studies are needed to elucidate whether these are cause and/or effect relationships.

Transient Neonatal Hypercholesterolemia

In order to obtain new information about the relationship between transient neonatal hypercholesterolemia and adrenal gland function, we have studied 39 healthy babies found hypercholesterolemic at birth and 39 healthy controls with normal cholesterol levels. The results of this study have shown that levels of dehydroepiandrosterone sulfate in cord blood were not significantly different in the hypercholesterolemic subjects compared with normolipidemic controls (1.4 +/- 0.5 micrograms/dl vs 1.3 +/- 0.5 mu/dl). Moreover correlations between this hormone and levels of total and LDL-cholesterol were not only not statistically significant but even positive (respectively r = +0.207 and +0.195). These data suggest that dehydroepiandrosterone sulfate measurements in cord blood do not give any further information about transient neonatal hypercholesterolemia.

Diagnostic approach to androgen disorders in women: acne, hirsutism, and alopecia

The most common signs of androgen excess in women are acne, alopecia, and hirsutism. Less common manifestations include android obesity, virilization, and acanthosis nigricans. These changes appear to be the result of excessive androgen production or increased target organ sensitivity. To evaluate excessive androgen production, an androgen screening protocol is recommended that includes measurement of dehydroepiandrosterone sulfate, testosterone, androstenedione, prolactin, follicular stimulating hormone, and luteinizing hormone. When androgen excess is confirmed, dexamethasone suppression is recommended to determine the source of the androgen(s). Once excessive androgen production is confirmed, more specific therapies can be administered.

Serum hormones and their receptors in women with normal and inadequate corpus luteum function

The purpose of this study was to attempt to characterize women with a luteal phase defect (LPD) by cytosol estrogen and progesterone (P) levels and peroxidase activity in endometrial biopsy samples. These values were correlated with circulating serum estradiol, P, testosterone, and dehydroepiandrosterone sulfate measurements. Similar data were obtained from women during the proliferative phase of the cycle. Midluteal phase P values were significantly greater in women with normal luteal function than in those with LPD; however, some overlap of values existed. Furthermore, because no significant differences between this group and normal women were seen, we were unable to characterize women with LPD by estrogen or P receptor levels or endometrial peroxidase activity.

The effect of different contraceptive treatments on the serum concentration of dehydroepiandrosterone sulfate

Twenty-six healthy, non-hirsute women had antecubital venous blood obtained before and during treatment with various contraceptive steroids for the measurement of dehydroepiandrosterone sulfate (DHEA-S). Six of the women were randomized to receive Ortho Novum 1/35 (O/N), 7 were assigned to Ovcon 35 (OV), and another five received Nordette (N). The 6 women ingesting O/N as well as the 7 receiving OV had significant decreases in serum concentrations of DHEA-S (p < 0.05). The percentage of decrease in DHEA-S in the O/N group (39 ± 6%) and in the OV group (30 ± 5%) was not statistically different. The group receiving N showed no changes from baseline measurements. Serum samples from another 8 women who had received subcutaneous implants of levonorgestrel (1-Ng) were obtained. No significant changes occurred in serum DHEA-S between baseline and six-month samples. The data that 1-Ng alone and the combination of 1-Ng and ethinylestradiol did not change DHEA-S levels suggest that 1-Ng does not affect adrenal androgen secretion. Because N, O/N and OV contain similar quantities of ethinylestradiol, these results indicate that norethindrone, but not 1-Ng, has a major suppressive effect upon adrenal androgen secretion.

Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.

Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, I) sulfate (Ia) has been characterized in the anterior and the posterior parts of the brain of adult male rats. Its level (1.58 +/- 0.14 and 4.89 +/- 1.06 ng/g, mean +/- SD, in anterior and posterior brain, respectively) largely exceeded that of I in brain (0.42 +/- 0.10 and 0.12 +/- 0.03 ng/g in anterior and posterior brain, respectively) and of Ia in plasma (0.26 +/- 0.13 ng/ml). Brain Ia level did not seem to depend on adrenal secretion; it was unchanged after administration of corticotropin or dexamethasone for 3 days, and no meaningful change occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. In contrast, stress conditions prevailing 2 days after adrenalectomy plus orchiectomy or after the corresponding sham operation resulted in a significantly increased concentration of Ia in the brain. Changes of Ia level in brain occurred irrespective of changes in corresponding plasma samples. It is proposed that Ia formation or accumulation (or both) in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system.

A radioimmunoassay for the estimation of serum dehydroepiandrosterone sulphate in normal and pathological sera

A radioimmunoassay is described for the measurement of dehydroepiandrosterone sulphate in serum. The method requires 0.1 ml of serum that needs no purification other than a single ether extraction. It is both rapid and economical, procedural losses are small and precision and accuracy are acceptable for both normal and pathological sera. Normal values have been established for children and adults which show a relationship to chronological age, values rising steadily as puberty intervenes, peak values occurring between 20 and 27 years, with a decline thereafter. An empirical curve was fitted to the data and approximate normal limits were obtained. The mean value for pregnant women in the age range 20–40 years, was statistically significantly lower than in non-pregnant, premenopausal women. The levels of dehydroepiandrosterone sulphate in cord and newborn blood, were higher than at any other time during childhood until signs of puberty were demonstrable. Elevated levels are reported in six patients with untreated congenital adrenal hyperplasia and in one patient with a tumour of the adrenal cortex. It is suggested that this assay provides a useful and precise index of adrenal androgen secretion.

Radioimmunoassay of Plasma Dehydroepiandrosterone Sulfate

Abstract This report describes a radioimmunoassay method for the direct measurement of dehydroepiandrosterone sulfate (DHEA-S) in diluted plasma. Utilizing a specific DHEA-S antiserum which reacted completely with DHEA, we have measured DHEA-S accurately in small aliquots of diluted plasma. Due to the relatively high concentration of DHEA-S in plasma, the effect of DHEA and other steroids on the assay was negligible. The usable range of the standard curve was between 10 and 500 pg DHEA-S. The range of levels that could be measured per ml of plasma varied from 0.02 to 5.0 pg. The range of values obtained by this method for plasma DHEA-S compared favorably with the ronge of levels observed in adult men and women, using various other techniques.