New approach for the determination of ortho-phenylphenol exposure by measurement of sulfate and glucuronide conjugates in urine using liquid chromatography-tandem mass spectrometry.

Ortho-phenylphenol (OPP) has been widely used as a fungicide and preservative. Although low-dose studies have demonstrated its low toxicity in animals and humans, high-dose exposure to this contaminant has toxic effects that range from skin irritation to bladder cancer. Thus far, monitoring of OPP exposure in the general population has been performed by measuring OPP after urine hydrolysis with the ß-glucuronidase/arylsulfatase enzyme and sometimes by the use of a mineral acid. We developed a sensitive, accurate, and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to specifically measure two-phase II OPP metabolites excreted in human urine, OPP sulfate (OPP-S), and OPP glucuronide (OPP-G). Comparative analysis of urine samples from 50 volunteers living in the Quebec City area using a direct method and phosphoric acid hydrolysis method previously developed in our laboratory showed no statistically significant difference (p value for paired t test = 0.701) in OPP concentrations. Moreover, a significant difference showed that underestimation (p value for paired t test = 0.025) occurs when ß-glucuronidase/arylsulfatase enzyme deconjugation is used. The LOD achieved by the direct method permits the detection of OPP-S and OPP-G metabolites in urine at the submicrogram per liter level. Graphical abstract ᅟ.

Standardized Procedure for the Simultaneous Determination of the Matrix Effect, Recovery, Process Efficiency, and Internal Standard Association.

The matrix effects (MEs) on the quantification of an analyte can be significant and should not be neglected during development and validation of an analytical method. According to this premise, we developed a standardized procedure based on a set of six tests performed on six different sample matrices to detect and characterize the effects of the matrix for single and multiple analytes methods. The link between the matrix effect, recovery, process efficiency, accuracy, precision, and calibration curve was underscored by calculations performed with peak areas, ratios of standard/internal standard peak area, and concentrations. The terms instrumental ME and global ME were introduced, and the term recovery was subdivided for clarity. The test accounts for the presence of ubiquitous and endogenous analytes through background subtraction. The results showed the necessity for using samples with an original concentration in the same range and that the concentration selected for the addition had a definite impact on the results. The use of six-sample matrices provided a standard deviation on the results, and this information could be inserted in a method performance result to show precision. The tool also allows for testing of different analytes/internal standard combinations, which helps with the selection of the association with minimum MEs. A UPLC-MS/MS method for the quantification of several phthalate metabolites in urine was developed and validated with this test. This methodology responds to a scientific need for homogeneity, clarity, and understanding of the results and facilitates the decision-making process while lowering the required costs and time.

Paraben Concentrations in Maternal Urine and Breast Milk and Its Association with Personal Care Product Use.

Parabens are broad-spectrum antimicrobial preservatives and fragrances used in a wide range of personal care products, pharmaceuticals, and food providing the opportunity for people to be exposed on a daily basis. In 2009-2010, 80 pregnant women from Ottawa Canada participated in the Plastics and Personal-Care Product Use in Pregnancy (P4) Study. A subset of women (n = 31) who provided multiple spot urine samples (n = 542) collected over two 24-h periods had their samples analyzed for methylparaben (MP), n-propylparaben (PP), ethylparaben (EP), butylparaben (BP), isobutylparaben (IBP), and benzylparaben (BzP). These parabens were also measured in breast milk samples collected at approximately 3 months postpartum (n = 56 women). Women kept a diary of products that they used 24 h prior to and during the collection. All parabens measured in maternal urine had moderate to high reproducibility. Women who used lotions in the past 24 h had significantly higher geometric mean paraben concentrations (80-110%) in their urine than women who reported no use in the past 24 h. Women who used shampoo, conditioner, and cosmetics also showed 70-80% higher BP concentrations in their urine. Breast milk samples had >50% detection for MP, PP, and EP.

Stability issues in the determination of 19 urinary (free and conjugated) monohydroxy polycyclic aromatic hydrocarbons.

Data on the stability of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs; metabolites of PAHs) in urine are needed in order to effectively study the effects of PAHs in the body, but the relevant data are not available in the literature. Therefore, in this work, we investigated the stability of OH-PAHs in urine. For each OH-PAH studied, the free form (as opposed to the conjugated form) comprised <10 % of the total OH-PAH in urine samples obtained from a normal population, except for 9-OH-phenanthrene (where the free form represented 22.2 % of the total 9-OH-phenanthrene). 1-Naphthol and 9-OH-phenanthrene were found to be less stable in their free forms in urine than in their conjugated forms when the urine samples were stored at 4 °C or room temperature. Free 3-OH-fluoranthene was also very unstable at 4 °C or room temperature. The conjugated forms of the OH-PAHs were more stable than their corresponding free forms. However, the free and conjugated forms of all the OH-PAHs were stable in urine at -20 °C and -80 °C. A freeze and thaw assay also revealed that freezing and thawing had minimal impact on the stability of the OH-PAHs in urine. For the derivatized extracts, storing the samples under an argon atmosphere at 4 °C was found to maintain sample integrity. In order to measure the stabilities of 19 hydroxylated metabolites of PAHs in urine, we developed a method with sensitivity in the low pg/mL range using nine labeled internal standards. This method combined enzymatic deconjugation with liquid-liquid extraction, derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and gas chromatography/tandem mass spectrometry (GC-MS/MS). Graphical abstract Stability of the conjugated forms of the OH-PAHs versus free forms (e.g. 1-naphthol).

Bisphenol A and phthalate metabolite urinary concentrations: Daily and across pregnancy variability.

Phthalates and bisphenol A (BPA) are high production volume and ubiquitous chemicals that are quickly metabolized in the body. Traditionally, studies have relied on single spot urine analyses to assess exposure; ignoring variability in concentrations throughout a day or over a longer period of time. We compared BPA and phthalate metabolite results from urine samples collected at five different time points. Participants (n=80) were asked to collect all voids in a 24 h period on a weekday and then again on a weekend before 20 weeks of pregnancy. During the second and third trimesters and in the postpartum period, single spot urines were collected. Variability over time in urinary concentrations was assessed using intraclass correlation coefficients (ICCs) and the sensitivity to correctly classify a single sample as high or low versus the geometric mean (GM) of all samples was calculated. We found low reproducibility and sensitivity of BPA and all phthalate metabolites throughout pregnancy and into the postpartum period but much higher reproducibility within a day. Time of day when the urine was collected was a significant predictor of specific gravity adjusted exposure levels. We concluded that, if the interest is in average exposures across pregnancy, maternal/fetal exposure estimation may be more accurate if multiple measurements, collected across the course of the entire pregnancy, rather than a single spot measure, are performed.

Exposure to free and conjugated forms of bisphenol A and triclosan among pregnant women in the MIREC cohort.

BACKGROUND: Bisphenol A (BPA) and triclosan (TCS) are two nonpersistent chemicals that have been frequently measured in spot urine samples from the general population but less so in pregnant women; however, data are limited on the free (bioactive) and conjugated forms of these phenols. OBJECTIVES: The Maternal-Infant Research on Environmental Chemicals (MIREC) Study addressed these data gaps by utilizing stored maternal urine samples from a large multicenter cohort study of Canadian pregnant women. METHODS: Concentrations of free and conjugated forms of BPA and TCS were measured in about 1,890 first-trimester urine samples by ultra performance liquid chromatography-tandem mass spectrometry using isotope dilution. RESULTS: The glucuronides of BPA and TCS were the predominant forms of these chemicals measured (detected in 95% and 99% of samples, respectively), whereas the free forms were detected in 43% and 80% of samples, respectively. The geometric mean urinary concentrations for glucuronides of BPA and TCS were 0.80 µg/L (95% CI: 0.75, 0.85) and 12.30 µg/L (95% CI: 11.08, 13.65), respectively. Significant predictors of BPA included maternal age < 25 vs. ≥ 35 years, current smoking, low vs. high household income, and low vs. high education. For TCS, urinary concentrations were significantly higher in women ≥ 25 years of age, never vs. current smokers, and women with high household income and high education. CONCLUSIONS: The results from this study represent the largest national-level data on urinary concentrations of free and conjugated forms of BPA and TCS in pregnant women and suggest that maternal characteristics predicting elevated urinary concentrations of these phenols largely act in opposite directions.

Maternal and infant exposure to environmental phenols as measured in multiple biological matrices.

BACKGROUND: Results of recent national surveys have shown the high prevalence of exposure to bisphenol A (BPA) and triclosan (TCS) among the general population; however biomonitoring data for pregnant women and infants are limited. METHODS: Women (n=80) were recruited from early prenatal clinics and asked to collect urine samples multiple times during pregnancy and once 2-3 months post-partum. Samples of infant urine and meconium as well as breast milk and infant formula were also collected. Biospecimens were analyzed by GC-MS/MS for BPA, TCS and triclocarban (TCC). RESULTS: Triclosan was detected in over 80% of the maternal urines (geometric mean (GM): 21.61 µg/L), 60% of the infant urines (GM: 2.8 µg/L), 46% of the breast milk and 80% of the meconium samples. Triclocarban was rarely detected in any of the biospecimens. Median total BPA concentrations were 1.21 and 0.24 µg/L in maternal and infant urines, respectively. Free BPA was detected in only 11% of infant urine samples. The meconium of female infants had significantly higher concentrations of total BPA and TCS than those of males, while no differences were observed in infant urine concentrations by sex. CONCLUSIONS: We found widespread exposure among pregnant women and infants to environmental phenols, with large inter-individual variability in exposure to triclosan. These data will contribute to the risk assessment of these chemicals, especially in susceptible sub-populations.

Determination of bisphenol A, triclosan and their metabolites in human urine using isotope-dilution liquid chromatography-tandem mass spectrometry.

Bisphenol A (BPA) and triclosan (TCS) are ubiquitous environmental phenols exhibiting endocrine disrupting activities that may be involved in various health disorders in humans. There is a need to measure separately free forms and conjugated metabolites because only the former are biologically active. We have developed sensitive methods using isotope-dilution liquid chromatography-tandem mass spectrometry for individual measurements of free BPA and TCS as well as their metabolites, BPA glucuronide (BPAG), BPA monosulfate (BPAS), BPA disulfate (BPADS), TCS glucuronide (TCSG) and TCS sulfate (TCSS) in urine. Comparative analyses of urine samples from 46 volunteers living in the Quebec City area using the new methods and a GC-MS/MS method previously used in our laboratory revealed very strong correlations for total BPA (Spearman's rs=0.862, p<0.0001) and total TCS concentrations (rs=0.942, p<0.0001). Glucuronide metabolites were the most abundant BPA and TCS species in urine samples (>94% of total urinary concentrations). Unconjugated TCS concentrations represented a small proportion of total TCS species (median=1.6%) but its concentration was likely underestimated due to losses by adsorption to the surface of polypropylene tubes used for sample storage. To our knowledge, we are the first to report levels of free, sulfated and glucuronidated TCS levels in human urine.

Intravaginal prasterone (DHEA) provides local action without clinically significant changes in serum concentrations of estrogens or androgens.

In order to avoid the risks of non-physiological systemic exposure, serum concentrations of estradiol (E2) and testosterone (as measured by mass spectrometry-based assays) should remain below the 95th centiles measured at 9.3pg/ml and 0.26ng/ml for these respective sex steroids in normal postmenopausal women. To document the possibility of achieving this therapeutic objective, we have measured individual 24h serum E2 and testosterone concentrations in women with vulvovaginal atrophy (VVA) receiving daily intravaginal administration of a clinically effective dose of 6.5mg prasterone (dehydroepiandrosterone, DHEA). Serum E2 and testosterone, as well as DHEA and nine of its other metabolites, were assayed at ten time intervals over 24h on the first and seventh days of daily vaginal administration of 6.5mg prasterone. No significant change from baseline of average 24h serum E2 or testosterone concentrations was observed. Moreover, average 24h serum DHEA remained within the normal postmenopausal range. Estrone sulfate and the androgen metabolites androsterone glucuronide and androstane-3α, 17ß-diol glucuronide did not change, thus confirming the absence of any biologically relevant systemic exposure to estrogens and androgens, respectively. Serum concentrations of metabolites of both estrogens and androgens remain within the normal postmenopausal range following daily intravaginal administration of 6.5mg prasterone. As other studies have shown, local formation of sex steroids in peripheral tissues without significant release of E2 or testosterone in the circulation can be achieved with intravaginal prasterone. Thus, prasterone is a promising physiological and attractive solution to treating VVA symptoms.

Plasma estrone sulfate assay in men: Comparison of radioimmunoassay, mass spectrometry coupled to gas chromatography (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

BACKGROUND: Estrogens are involved in the natural history of the prostate cancer and estrone sulfate, the quantitatively main circulating plasma estrogen in men, has been associated with an aggressive form of this cancer. A convenient and accurate plasma assay of this steroid has become important. METHODS: We simultaneously assayed estrone sulfate in the plasma of one hundred men aged 30-50 years, according to LC-MS/MS, GC-MS after solvolysis of E(1)S, radioimmunoassay after a chromatographic purification step, and a direct RIA commercial kit. RESULTS: Estrone sulfate plasma levels obtained with the first three methods were not significantly different. However, estrone sulfate levels measured by the direct RIA were three-fold higher than those obtained by the first three methods. We showed that the excessively high estrone sulfate levels obtained with the direct RIA kit had two origins: interference by high dehydroepiandrosterone sulfate plasma levels in men, and estrone sulfate inaccurate low concentrations in the standards. CONCLUSION: The LC-MS/MS method can be considered as an optimum option for clinical laboratory. The GC-MS method requires solvolysis to estrone, but allows simultaneous unconjugated steroid measurement. RIA method, with chromatographic purification, is cumbersome, but less expensive. DSL-5400 kit yielded estrone sulfate plasma levels that were too high.

Serum steroid levels during 12-week intravaginal dehydroepiandrosterone administration.

OBJECTIVE: Because a previous 1-week study has shown no or minimal changes in the serum levels of dehydroepiandrosterone (DHEA) and its metabolites after up to daily 1.8% (23.4 mg) intravaginal DHEA, the objective of the present study was to investigate the serum steroid levels during a 12-week daily intravaginal administration of 0%, 0.25%, 0.5%, and 1.0% DHEA (Prasterone) 1.3 mL ovules. METHODS: In a double-blind, placebo-controlled phase III study, 218 postmenopausal women (age range, 42-74 y) were randomized to receive daily one of four DHEA concentrations intravaginally. Serum steroids were measured by a Good Laboratory Practice-validated mass spectrometry technology in samples obtained at time of visit. RESULTS: The serum levels of DHEA and 11 of its metabolites measured at screening, day 1, and weeks 2, 4, 8, and 12 in women showed no or minimal changes during the whole observation period, with all values remaining well within the limits of normal postmenopausal women. No accumulation of the steroid metabolites nor change in DHEA bioavailability was detected. CONCLUSIONS: The present data show that local daily intravaginal DHEA administration at DHEA doses of 3.25-13 mg was able to rapidly and efficiently achieve correction of all the signs and symptoms of vaginal atrophy and improve sexual function and caused no or minimal changes in serum sex steroid levels, which all remain within the normal postmenopausal range, thus avoiding the risks of all estrogen formulations.

Comparable amounts of sex steroids are made outside the gonads in men and women: strong lesson for hormone therapy of prostate and breast cancer.

The objective of this study was comparison of circulating androgens and their metabolites as well as estrogens measured for the first time by a validated mass spectrometry technology in 60-80-year-old men and women of comparable age. Castration in men (n=34) reduces the total androgen pool by only about 60% as indicated by the decrease in the serum levels of the glucuronide metabolites of androgens compared to intact men (n=1302). Such data are in agreement with the 50 to 75% decrease in intraprostatic dihydrotestosterone (DHT) concentration after castration. Most interestingly, the same amounts of androgens and estrogens are found in postmenopausal women (n=369) and castrated men of comparable age. The most significant therapeutic implication of these findings is the absolute need to add a pure (nonsteroidal) antiandrogen to castration in men with prostate cancer in order to block the action of the 25 to 50% DHT left in the prostate after castration. Not adding an antiandrogen to castration in men treated for prostate cancer is equivalent to not prescribing a blocker of estrogens in women suffering from breast cancer because they are postmenopausal and have low circulating estradiol.

Effect of one-week treatment with vaginal estrogen preparations on serum estrogen levels in postmenopausal women.

OBJECTIVE: Approximately 50% of postmenopausal women suffer from vaginal atrophy, and a large proportion of them choose intravaginal estrogen preparations administered for local action to avoid systemic exposure to estrogens and its associated risk of breast and uterine cancer. The primary objective of this study was the evaluation of the systematic bioavailability of estradiol and estrone and the pharmacokinetics of two of the most frequently used intravaginal estrogen preparations, namely Vagifem and Premarin cream. DESIGN: While immunobased assays could not previously provide accurate measurement of serum estrogen concentrations in postmenopausal women, we have used validated mass spectrometry assays to measure the pharmacokinetics of serum estradiol and estrone during the 24 hours following the seventh daily application of 25 microg estradiol (Vagifem) and 1 g (0.625 mg) conjugated estrogens (Premarin) cream in 10 postmenopausal women in each group. RESULTS: Serum estradiol was increased on average by 5.4-fold from 3 to 17 pg/mL during the 24-hour period after daily administration of 25 microg estradiol or 1 g (0.625 mg) conjugated estrogens cream. Serum estrone, conversely, increased 150% with Vagifem and 500% with Premarin cream. CONCLUSIONS: The present data using validated, accurate, and sensitive mass spectrometry assays of estrogens show that the Vagifem pill and Premarin cream, after 1 week of daily treatment, cause an approximately fivefold increase in serum estradiol in postmenopausal women, thus indicating that the effects are unlikely to be limited to the vagina and that systemic actions are expected after application of these intravaginal estrogen preparations.

Effect of intravaginal DHEA on serum DHEA and eleven of its metabolites in postmenopausal women.

The primary objective of this study was measurement of the systemic bioavailability of DHEA and its metabolites following daily intravaginal application of the sex steroid precursor. Forty postmenopausal women were randomized to receive a daily dose of one ovule of the following DHEA concentrations: 0.0%, 0.5%, 1.0% or 1.8%. After only 7 days of treatment, the maturation value of the vaginal epithelial cells was significantly increased while the vaginal pH was significantly decreased at all DHEA doses. These important local effects were observed while the serum concentrations of estradiol and testosterone remained within the values found in normal postmenopausal women at all DHEA doses. Similar observations were made for serum androstenedione, estrone, estrone-sulfate and DHEA-sulfate. Even at the highest 1.8% DHEA dose, serum DHEA was increased at the levels found in normal premenopausal women. The present data show that the intravaginal administration of DHEA permits to rapidly achieve the local beneficial effects against vaginal atrophy without significant changes in serum estrogens, thus avoiding the increased risk of breast cancer associated with the current intravaginal or systemic estrogenic formulations. In addition, the recent observation that DHEA is transformed into both androgens and estrogens in the vagina permits to exert benefits on all the three layers of the vaginal wall.

Changes in serum DHEA and eleven of its metabolites during 12-month percutaneous administration of DHEA.

Healthy postmenopausal women aged 60-65 years (n=150) were randomized to receive twice daily application on the skin of 3g of a 0.3% dehydroepiandrosterone (DHEA) or placebo emulsion for 12 months. Serum DHEA and eleven of its metabolites were measured at screening and on day 1, as well as at 1, 3, 6, 9 and 12 months to study long-term metabolism. While serum DHEA and androst-5-ene-3beta, 17beta-diol (5-diol) increased by 203% and 178%, respectively, on average, during the 12-month period, the sum of concentrations of the metabolites of androgens, namely androsterone glucuronide (ADT-G), androstane-3alpha,17beta-diol-3G and -17G increased by only 71% while usually non statistically significant changes of 30%, 17% and 20% were observed for estrone (E(1)), estradiol (E(2)) and E(1) sulfate (E(1)-S), respectively. Despite the return of serum DHEA to normal premenopausal values with the present DHEA treatment regimen, the 65% decrease in the androgen pool found in this group of postmenopausal women is in fact corrected by only 24%, thus remaining 41% below the values found in normal premenopausal women. In fact, the changes in serum DHEA observed after percutaneous DHEA administration are a 186% overestimate of the true changes in androgen formation while the overestimate of estrogen production is even much higher. On the other hand, the pharmacokinetics of the steroids are stable over the 12-month period with no significant induction or decrease of activity of the enzymatic systems transforming DHEA predominantly into androgens.

Metabolism of DHEA in postmenopausal women following percutaneous administration.

The marked decline in serum dehydroepiandrosterone (DHEA) with age is believed to play a role in health problems associated with aging, these health issues being potentially preventable or reversible by the exogenous administration of DHEA. In the present study, liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) and gas chromatrography/mass spectrometry (GC/MS) were used to measure the serum levels of DHEA and 11 of its metabolites in seventy-five 60-65-year-old Caucasian women who received 3g of 0.1%, 0.3%, 1.0% or 2.0% DHEA cream or placebo applied twice daily on the face, upper chest, arms and legs. The serum levels of DHEA increased 574% over control at the 2.0% DHEA dose while the sum of the androgen metabolites androsterone glucuronide (ADT-G), 3alpha-androstenediol-3G (3alpha-diol-3G) and 3alpha-diol-17G increased by only 231%. On the other hand, serum testosterone and dihydrosterone were increased by 192% and 275%, respectively, above basal levels compared to 139% and 158% for estrone and estradiol. Such data show that the transformation of exogenous DHEA in postmenopausal women is preferentially into androgens rather than into estrogens. On the other hand, the present data indicate that serum DHEA measurements following DHEA supplementation in postmenopausal women are an overestimate of the formation of active androgens and estrogens and suggest a decreased efficiency of transformation of DHEA into androgens and estrogens with aging.

Androgen glucuronides, instead of testosterone, as the new markers of androgenic activity in women.

Despite the long series of cohort studies performed during the last 20 years, the correlation between serum testosterone and any clinical situation believed to be under androgen control in women has remained elusive. This is likely related to the recent finding that the androgens made locally in large amounts in peripheral tissues from the precursor dehydroepiandrosterone (DHEA) act in the same cells where synthesis takes place and are not released in significant amounts in the circulation, thus making unreliable the measurement of serum testosterone as marker of total androgenic activity. The objective is to determine if serum androgen glucuronides can be replaced by testosterone or another steroid as measure of androgenic activity. Since the glucuronide derivatives of androgens are the obligatory route of elimination of all androgens, these metabolites were measured by liquid chromatography tandem mass spectrometry under basal conditions in 377 healthy postmenopausal women aged 55-65 years as well as in 47 premenopausal women aged 30-35 years while testosterone was assayed by gas chromatography mass spectrometry. No correlation was found between the serum concentration of testosterone and that of androsterone glucuronide (ADT-G) or androstenediol glucuronide (3alpha-diol-G), the androgen metabolites which account for the total pool of androgens. The present data show that measurement of the total pool of androgens reflected by the serum levels of ADT-G and 3alpha-diol-G cannot be replaced by serum testosterone or any other steroid, including DHEA or DHEA sulphate. These findings may have implications for women with androgen deficiency involving osteoporosis, obesity, type 2 diabetes, sexual dysfunction, loss of muscular strength and a series of other clinical situations affecting women's health. Measuring ADT-G and 3alpha-diol-G might identify cases of true androgen deficiency and provide an opportunity to offer appropriate androgen therapy.

Are women with Sjögren's syndrome androgen-deficient?

OBJECTIVE: We hypothesize that androgen deficiency is a critical etiologic factor in the pathogenesis of aqueous-deficient and evaporative dry eye in Sjögren's syndrome (SS). We investigated whether women with SS have a deficiency in total androgens. We also examined whether these patients have elevated serum concentrations of estrogens. METHODS: Blood was drawn from women with primary and secondary SS and age matched controls, and analyzed for steroid concentrations by gas and liquid chromatography-mass spectrometry. RESULTS: Our results show that women with SS are androgen-deficient. Concentrations of 5-androstene-3beta,17beta-diol (5-diol), dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), androsterone-glucuronide (ADT-G), and androstane-3a,17beta-diol-G (3alpha-diol-G) were all significantly reduced in SS sera relative to controls. In contrast, SS was not associated with significant alterations in the serum concentrations of testosterone, androstenedione, estrone, or 17beta-estradiol. These overall findings could not be attributed to the use of oral contraceptives or hormone replacement therapy, because the concentrations of 5-diol, DHEA, DHT, ADT-G and 3a-diol-G were also decreased in patients with SS compared to levels in control women who were not taking exogenous estrogens. CONCLUSION: Our results show that women with SS are androgen-deficient.