Evaluation of sulfate metabolites as markers of topical testosterone administration in Caucasian and Asian populations.

Sulfate metabolites of endogenous anabolic androgenic steroids (EAAS) have been shown to prolong the detection times compared with the conventional urinary markers of the steroid profile for oral and intramuscular administrations of testosterone (T). In this work, the sensitivity of sulfate EAAS markers for the detection of T gel administration has been evaluated in six Caucasian and six Asian male volunteers. Fourteen sulfate metabolites were measured in basal and post-administration samples after multiple doses of T gel (100 mg/day, three consecutive days), and the detection times based on individual thresholds for each volunteer were evaluated. Sulfate concentrations did not show adequate sensitivity, but the results of sulfate ratios were much more promising. Androsterone sulfate/testosterone sulfate (A-S/T-S), epiandrosterone sulfate/epitestosterone sulfate (epiA-S/E-S), epiA-S/T-S, and etiocholanolone sulfate/epitestosterone sulfate (Etio-S/E-S) provided the most consistent detectability for all volunteers and populations, with detection times ranging from 60 to 96 h since the first dose. Additional ratios improved detectability to up to 7 days, but only in particular volunteers. In general, sensitivity was similar to or better than the conventional testosterone/epitestosterone ratio (T/E) of the steroid profile, which further reinforces the conclusion that sulfate EAAS metabolites can be a good complement for the current steroid profile.

Effects of structural characteristics of (un)conjugated steroid metabolites in their collision cross section value.

In this work, the collision cross section (CCS) value of 103 steroids (including unconjugated metabolites and phase II metabolites conjugated with sulfate and glucuronide groups) was determined by liquid chromatography coupled to traveling wave ion mobility spectrometry (LC-TWIMS). A time of flight (QTOF) mass analyzer was used to perform the analytes determination at high-resolution mass spectrometry. An electrospray ionization source (ESI) was used to generate [M+H]+, [M + NH4]+ and/or [M - H]- ions. High reproducibility was observed for the CCS determination in both urine and standard solutions, obtaining RSD lower than 0.3% and 0.5% in all cases respectively. CCS determination in matrix was in accordance with the CCS measured in standards solution showing deviations below 2%. In general, CCS values were directly correlated with the ion mass and allowed differentiating between glucuronides, sulfates and free steroids although differences among steroids of the same group were less significant. However, more specific information was obtained for phase II metabolites observing differences in the CCS value of isomeric pairs concerning the conjugation position or the α/ß configuration, which could be useful in the structural elucidation of new steroid metabolites in the anti-doping field. Finally, the potential of IMS reducing interferences from the sample matrix was also tested for the analysis of a glucuronide metabolite of bolasterone (5ß-androstan-7α,17α-dimethyl-3α,17ß-diol-3-glucuronide) in urine samples.

Potential of desorption electrospray ionization and paper spray ionization with high-resolution mass spectrometry for the screening of sports doping agents in urine.

In this work, desorption electrospray ionization and paper spray ionization both with high-resolution mass spectrometry (DESI-HRMS and PSI-HRMS) were explored for the fast and direct analysis of stimulants and diuretics in urine samples. The analysis was performed at a resolution of 70 000 FWHM (m/z 200) using a quadrupole-Orbitrap mass spectrometer in full scan acquisition mode, detecting stimulants and diuretics in positive and negative ion modes, respectively. The most critical parameters affecting the desorption and ionization efficiencies of compounds were optimized, paying particular attention to the optimization of the spray solvent for PSI-HRMS analysis and to the selection of the DESI sample substrate. For stimulants, the PSI-HRMS method performed better than DESI-HRMS, allowing the direct analysis of raw urine samples with better signal-to-noise ratios than DESI. However, results obtained for diuretics were not as satisfactory as we expected. The PSI-HRMS method was applied to the screening of 52 stimulants for doping control purposes, providing satisfactory detectability for most of them at the Minimum Reporting Level (MRL) in less than 2 minutes for each single analysis. Despite the advantages offered by the PSI-HRMS method, in this study is also included a discussion on the limitations observed because of the presence of interference for some compounds.

Prenatal exposure to mixtures of phthalates and phenols and body mass index and blood pressure in Spanish preadolescents.

BACKGROUND: Pregnant women are simultaneously exposed to several non-persistent endocrine-disrupting chemicals, which may influence the risk of childhood obesity and cardiovascular diseases later in life. Previous prospective studies have mostly examined single-chemical effects, with inconsistent findings. We assessed the association between prenatal exposure to phthalates and phenols, individually and as a mixture, and body mass index (BMI) and blood pressure (BP) in preadolescents. METHODS: We used data from the Spanish INMA birth cohort study (n = 1,015), where the 1st and 3rd- trimester maternal urinary concentrations of eight phthalate metabolites and six phenols were quantified. At 11 years of age, we calculated BMI z-scores and measured systolic and diastolic BP. We estimated individual chemical effects with linear mixed models and joint effects of the chemical mixture with hierarchical Bayesian kernel machine regression (BKMR). Analyses were stratified by sex and by puberty status. RESULTS: In single-exposure models, benzophenone-3 (BP3) was nonmonotonically associated with higher BMI z-score (e.g. Quartile (Q) 3: ß = 0.23 [95% CI = 0.03, 0.44] vs Q1) and higher diastolic BP (Q2: ß = 1.27 [0.00, 2.53] mmHg vs Q1). Methyl paraben (MEPA) was associated with lower systolic BP (Q4: ß = -1.67 [-3.31, -0.04] mmHg vs Q1). No consistent associations were observed for the other compounds. Results from the BKMR confirmed the single-exposure results and showed similar patterns of associations, with BP3 having the highest importance in the mixture models, especially among preadolescents who reached puberty status. No overall mixture effect was found, except for a tendency of higher BMI z-score and lower systolic BP in girls. CONCLUSIONS: Prenatal exposure to UV-filter BP3 may be associated with higher BMI and diastolic BP during preadolescence, but there is little evidence for an overall phthalate and phenol mixture effect.

Elimination profiles of betamethasone after different administration routes: Evaluation of the reporting level and washout periods to ensure safe therapeutic administrations.

Betamethasone (BET) is prohibited in sports competitions when administered by systemic routes, and it is allowed by other routes for therapeutic purposes. In out-of-competition periods, there is no restriction of use. The present work aimed to assess the urinary excretion of BET and its metabolites after allowed and prohibited administrations to verify the suitability of the current reporting level of 30 ng/ml used to distinguish allowed and prohibited administrations and to establish washout periods for oral and intramuscular (IM) administrations when out-of-competition treatments are needed. BET was administered to healthy volunteers by different routes: topical (10 mg/day for 5 days, n = 6 males), intranasal (320 µg/day for 3 days, n = 4 males and 4 females), oral (0.5 mg, n = 8 males) or IM (6 mg, n = 6 males, or 12 mg, n = 4 males and 4 females). Urine and plasma samples collected before and after administration were analysed using liquid chromatography-tandem mass spectrometry. Among all studied metabolites, the parent drug was selected as the best discriminatory marker. After topical administration, BET concentrations were lower than 6.6 ng/ml. However, after intranasal treatment, some samples at concentrations close to or higher than 30 ng/ml were detected, suggesting the need to revise the current reporting level. Urinary concentrations after oral and intranasal administrations were similar, and after IM administration, concentrations were much higher. Taking into account all information, a urinary reporting level of 60 ng/ml is proposed. Washout periods of at least 48 and 96 h are recommended after oral and IM administrations, respectively.

Elimination profiles of prednisone and prednisolone after different administration routes: Evaluation of the reporting level and washout periods to ensure safe therapeutic administrations.

Prednisolone (PRED) and prednisone (PSONE) are prohibited in sports competitions when administered by systemic routes, and they are allowed by other routes for therapeutic purposes. There is no restriction of use in out-of-competition periods. The present study aimed to evaluate the urinary excretion of PRED, PSONE, and their most important metabolites after systemic and nonsystemic treatments in order to verify the suitability of the current reporting level of 30 ng/ml used to distinguish allowed and prohibited administrations and to establish washout periods for oral treatments performed in out-of-competition periods. PRED was studied after dermatological administration (5 mg/day for 5 days, n = 6 males) and oral administration (5 mg, n = 6 males; 10 mg, n = 2 males). PSONE was studied after oral administration (10 mg, n = 2 males; 30 mg, n = 1 male and 1 female). Concentrations in urine were measured using an LC-MS/MS method. Concentrations after dermatological treatment were low for all metabolites. After oral administration, concentrations were very high during the first 24 h after administration ranging from 1.6 to 2261 ng/ml and from 4.6 to 908 ng/ml for PRED and PSONE, respectively. Concentrations of most of the metabolites measured were lower than 30 ng/ml from 24 h after all oral administrations. New reporting levels are proposed for PRED and PSONE considering data of our study and other information published after nonsystemic administrations of the compounds. Washout periods of at least 24 h are recommended to ensure no false positives when oral treatments need to be performed in out-of-competition periods.

Comparison of magnetic bead surface functionalities for the immunopurification of growth hormone-releasing hormones prior to liquid chromatography-high resolution mass spectrometry.

Growth hormone-releasing hormone and its analogues sermorelin, tesamorelin and CJC-1295 are included in the prohibited list of the World Antidoping Agency. These target peptides are found at very low concentrations in urine (at the pg/mL level). For this reason, hyphenated enrichment and purification steps prior to mass spectrometric detection are required. Among different strategies, immunopurification based on magnetic beads is an excellent alternative, as it offers improved selectivity when the immunoreactivity and orientation of the antibody are optimum and non-specific adsorption is minimized. However, choosing the magnetic bead surface functionalities that provide the best recoveries is not so straightforward. In this work, we have evaluated the suitability of magnetic beads with different supports, binding capacities and affinity chemistries prior analysis of human urine samples by liquid chromatography coupled to high resolution mass spectrometry using a Quadrupole-Orbitrap instrument. After optimization of the immunopurification protocol with the magnetic beads that provided better recoveries, the method was fully validated and found to be adequate considering the parameters specificity, intra- and inter-day precision (lower than 15 and 25%, respectively), matrix effect, limit of detection (0.2 ng/mL) and limit of identification (0.5 ng/mL).

Analysis of hydroxylated phenylalkylamine stimulants in urine by GC-APPI-HRMS.

A gas chromatography-atmospheric pressure photoionization-high-resolution mass spectrometry (GC-APPI-HRMS) method was developed for the determination of eight phenylalkylamine stimulants in urine samples. Spiked urine samples were hydrolyzed, processed by solid-phase extraction, and derivatized before analysis. Two derivatization reactions were studied: the formation of trimethylsilyl (TMS) derivatives with N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) and trimethylsilyl/trifluoroacetyl (TMS/TFA) derivatives with MSTFA and N-methyl-bis (trifluoroacetamide) (MBTFA) as derivatization reagents. Gas chromatography of both derivatives was performed with a 100% dimethylsiloxane column and a good separation of all isomeric compounds was achieved. To maximize the signal of the protonated molecule [M+H]+, the APPI most critical parameters were optimized. Three solvents were tested as dopant agents, with acetone yielding the lower in-source collision-induced dissociation (CID) fragmentation. The acquisition was performed in full scan and product ion scan (parallel reaction monitoring, PRM) using a quadrupole-Orbitrap mass analyzer (35,000 FWHM at m/z 200) in positive ion detection mode. At the optimal working conditions, the full scan method was evaluated for the fulfillment of identification requirements in doping analysis. Selectivity, limits of detection, matrix effect, and precision were estimated to validate the method for confirmation purposes and its applicability was tested by the analysis of spiked samples as well as by the analysis of samples obtained after the administration of some of the compounds to healthy volunteers. Results were compared with those obtained by GC-electron ionization-MS, demonstrating that the GC-APPI-HRMS method improved selectivity and sensibility, achieving lower limits of detection and satisfactory reproducibility.

Increased collection efficiency of CD34+ cells after mobilization with preemptive use of plerixafor followed by leukocytapheresis on the same day.

BACKGROUND: Plerixafor should be administered 6 to 11 hours before starting leukocytapheresis. However, we have been using plerixafor followed by leukocytapheresis according to different time schedules since 2007. Our objective was to compare the CD34+ cell collection efficiency (CE1) of the first leukocytapheresis performed after using plerixafor at different time intervals. STUDY DESIGN AND METHODS: Same-day schedule refers to the administration of plerixafor at 10:00 AM and starting the leukocytapheresis on the same day at 4:00 PM (6 hours interval). Next-day schedule refers to the administration of plerixafor at 8:00 PM and starting the leukocytapheresis on the next day (10:00 AM or 4:00 PM; either a 14- or 20-hr interval). Variables that might influence the CE1 of CD34+ cells were analyzed by longitudinal linear regression with a random effects model derived by generalized estimating equations. RESULTS: The median CE1 of CD34+ cells was higher in the group of 30 patients who underwent leukocytapheresis on the same day when compared with the group of 62 patients who underwent leukocytapheresis on the next day (65.8% vs. 56.7%; p < 0.01). In the longitudinal linear regression analysis, only the time from plerixafor administration to leukocytapheresis start was associated with a statistically significant decrease in the CE1 of CD34+ cells (CE1 change -0.034%; p < 0.01). CONCLUSION: Higher CE1 of CD34+ cells was observed when patients underwent leukocytapheresis on the same day after receiving plerixafor in comparison with administering plerixafor and underwent leukocytapheresis on the next day. Larger studies are necessary to confirm present results.

Budesonide use and misuse in sports: Elimination profiles of budesonide and metabolites after intranasal, high-dose inhaled and oral administrations.

Budesonide (BUD) is a glucocorticoid (GC) widely used in therapeutics. In sports, the World Anti-doping Agency (WADA) controls the use of GCs, and WADA-accredited laboratories use a reporting level of 30 ng/mL for 6ß-hydroxy-budesonide (6ßOHBUD) to detect the systemic administration of BUD. In the present work, we examined the urinary excretion profile of 6ßOHBUD, BUD, and 16α-hydroxy-prednisolone (16αOHPRED) after intranasal (INT), inhaled (INH) (at high doses) and oral administrations in male and female volunteers. BUD was administered to healthy volunteers using INT route (256 µg/day for three days, n = 4 males and 4 females), INH route (800 µg/day for three days, n = 4 males and 4 females, and 1600 µg/day for three days, n = 4 males) or oral route (3 mg, n = 8 females). Urine samples were collected before and after administration at different time periods, and were analyzed by liquid chromatography-tandem mass spectrometry. 6ßOHBUD and BUD concentrations were very low after INT treatment (0.0-7.1 and 0.0-8.1 ng/mL, respectively), and higher after INH treatments (0.0-35.4 and 0.0-48.3 ng/mL, respectively). For 16αOHPRED, elevated concentrations were detected after INT and INH treatments (2.6-66.4 and 3.4-426.5 ng/mL, respectively). Concentrations obtained following oral administration were higher than after therapeutic administrations (2.8-80.6, 1.5-36.1, and 10.4-532.2 ng/mL for 6ßOHBUD, BUD, and 16αOHPRED, respectively). After all administrations, concentrations were higher in males than in females. Results demonstrated that 6ßOHBUD is the best discriminatory marker and a reporting level of 40 ng/mL was found to be the best criterion to distinguish allowed from forbidden administrations of BUD.

Additional studies on triamcinolone acetonide use and misuse in sports: Elimination profile after intranasal and high-dose intramuscular administrations.

Triamcinolone acetonide (TA) is a glucocorticoid (GC) widely used in sports medicine. GCs are prohibited in sports competitions by oral, intramuscular (IM), intravenous and rectal administrations, and they are allowed by other routes considered of local action such as intranasal administration (INT). We examined the urinary profiles of TA and its metabolites after INT and high-dose IM administrations. We also measured concentrations of TA and cortisol (CORT) in plasma following IM administration. TA was administered to healthy volunteers using INT route (220 µg/day for 3 days, n = 4 males and 4 females) or IM route (single dose of 40 mg, n = 4 males and 4 females and single dose 80 mg, n = 4 males). Urine and plasma samples were collected before and after administration at different time periods, and were analysed by liquid chromatography-tandem mass spectrometry. TA concentrations in urine were constant during 23 days after IM injection (range 1.4-129.0 ng/mL), and were very low after INT administration (range 0.0-3.5 ng/mL). For 6ß-hydroxy-triamcinolone, the main TA metabolite, higher concentrations were detected (0.0-93.7 ng/mL and 15.7-973.9 ng/mL after INT and IM administrations, respectively). On the other hand, TA was detected in all plasma samples collected during 23 days after IM administration (range 0.2-5.7 ng/mL). CORT levels were largely suppressed after IM injection, and were recovered in a dose-dependent manner. In view of the results obtained, we propose a reporting level of 5 ng/mL for TA to distinguish forbidden from allowed TA administrations in sports. We also suggest that other GCs with faster urinary elimination from the body should be considered for IM therapies in out-of-competition rather than TA, in order to reduce the possibility of reporting false adverse analytical findings.

Elimination profile of triamcinolone hexacetonide and its metabolites in human urine and plasma after a single intra-articular administration.

Triamcinolone hexacetonide (THA) is a synthetic glucocorticoid (GC) used by intra-articular (IA) administration. GCs are prohibited in sports competitions by systemic routes, and they are allowed by other routes considered of local action (IA administration, among others). The aim of the present work was to study the metabolic profile of THA in urine and plasma following IA administration. Eight patients (4 males and 4 females) with knee osteoarthritis received an IA dose of THA (40 mg) in the knee joint. Spot urine and plasma samples were collected before injection and at different time periods up to day 23 and 10 post-administration, respectively. The samples were analysed by liquid chromatography-tandem mass spectrometry. Neither THA nor specific THA metabolites were detected in urine. Triamcinolone acetonide (TA) and 6ß-hydroxy-triamcinolone acetonide were the main urinary metabolites. Maximum concentrations wereobtained between 24 and 48 h after administration. Using the reporting level of 30 ng/mL to distinguish allowed from forbidden administrations of GCs, a large number of false adverse analytical findings would be reported up to day 4. On the other hand, TA was detected in all plasma samples collected up to day 10 after administration. THA was also detected in plasma but at lower concentrations. The detection of plasma THA would be an unequivocal proof to demonstrate IA use of THA. A reversible decrease was observed in plasma concentrations of cortisol in some of the patients, indicating a systemic effect of the drug.

Evaluation of sulfate metabolites as markers of intramuscular testosterone administration in Caucasian and Asian populations.

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed-mode solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A-S/T-S) which prolonged the DT 1.2-2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3-1.5 times in two Asian volunteers. Other ratios between A-S or etiocholanolone sulfate and E-S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T-S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.

Sulfate metabolites improve retrospectivity after oral testosterone administration.

The detection of testosterone (T) misuse is performed using the steroid profile that includes concentrations of T and related metabolites excreted free and glucuronoconjugated, and the ratios between them. In this work, the usefulness of 14 endogenous steroid sulfates to improve the detection capabilities of oral T administration has been evaluated. Quantitation of the sulfate metabolites was performed using solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry. Urine samples were collected up to 144 hours after a single oral dose of T undecanoate (120 mg) to five Caucasian male volunteers. Detection times (DTs) of each marker were estimated using reference limits based on a population study and also monitoring the individual threshold for each volunteer. High inter-individual variability was observed for sulfate metabolites and, therefore, better DTs were obtained using individual thresholds. Using individual threshold limits, epiandrosterone sulfate (epiA-S) improved the DT with respect to testosterone/epitestosterone (T/E) ratio in all volunteers. Androsterone, etiocholanolone, and two androstanediol sulfates also improved DTs for some volunteers. Principal component analysis was used to characterize the sample cohort, obtaining 13 ratios useful for discrimination. These ratios as well as the ratio epiA-S/dehydroepiandrosterone sulfate were further examined. The most promising results were obtained using ratios between sulfates of epiA, androsterone, or androstanediol 1 and E, and also sulfates of epiA or androstanediol 1, and dehydroandrosterone. These selected ratios prolonged the DT of oral T administration up to 144 hours, which corresponded to a significantly higher retrospectivity compared to those obtained using concentrations or the conventional T/E ratio.

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography-electrospray tandem mass spectrometry.

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantitation of endogenous steroid sulfates has been developed to be able to evaluate these metabolites as biomarkers to detect the misuse of endogenous androgenic anabolic steroids in sports. For sample preparation, a mixed-mode solid-phase extraction was optimized to eliminate the glucuronide fraction in the washing step thus obtaining only the sulfate fraction. Chromatographic separation was optimized to achieve adequate resolution between isomers. The electrospray ionization and the product ion mass spectra of the sulfates were studied in order to obtain the most specific and selective transitions. The method was validated for quantitative purposes for 11 steroid sulfates obtaining satisfactory values for linearity, accuracy, and intra- and inter-day precision (relative standard deviation better than 16.2%). Limits of quantitation ranged between 0.5 and 2 ng/mL. Extraction recoveries for sulfate metabolites were between 90 and 94%. Matrix effect ranged from 90 to 110% showing the absence of significant ion suppression/enhancement. Samples were found to be stable after 2 freeze/thaw cycles. The applicability of the method was checked by the analysis of 75 urine samples from healthy volunteers (54 males, 37 Caucasian and 17 Asian, and 21 Caucasian females) to evaluate the concentration levels of endogenous sulfate metabolites in basal conditions.

New approach based on immunochemical techniques for monitoring of selective estrogen receptor modulators (SERMs) in human urine.

Antiestrogenic compounds such as tamoxifen, toremifen and chlomifen are used illegally by athletes to minimize physical impacts such as gynecomastia resulting from the secondary effects of anabolic androgenic steroids, used to increase athletic efficiency unlawfully. The use of these compounds is banned by the World Anti-Doping Agency (WADA) and controls are made through analytical methodologies such as HPLC-MS/MS, which do not fulfil the sample throughput requirements. Moreover, compounds such as tamoxifen are also used to treat hormone receptor-positive breast cancer (ER + ).Therapeutic drug monitoring (TDM) of tamoxifen may also be clinically useful for guiding treatment decisions. An accurate determination of these drugs requires a solid phase extraction of patient serum followed by HPLC-MS/MS. In the context of an unmet need of high-throughput screening (HTS) and quantitative methods for antiestrogenic substances we have approached the development of antibodies and an immunochemical assay for the determination of these antiestrogenic compounds. The strategy applied has taken into consideration that these drugs are metabolized and excreted in urine as the corresponding 4-hydroxylated compounds. A microplate-based ELISA procedure has been developed for the analysis of these metabolites in urine with a LOD of 0.15, 0.16 and 0.63 µg/L for 4OH-tamoxifen, 4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL established by WADA (20 µg/L).

The effect of tea consumption on the steroid profile.

Green tea (GT), along with its flavonol epigallocatechin-3-gallate (EGCG), has shown to inhibit the UGT2B17 isoenzyme, which is highly involved in the glucuronidation of testosterone (T) and its metabolites. Since the steroid profile (SP) is composed of urinary concentrations of T and related metabolites excreted in both the free and the glucuronide fractions, GT consumption could alter the SP, leading to misunderstanding in doping controls. The aim of the present work was to study the effect of GT consumption on the SP. This study was performed with 29 male volunteers, which could be classified in 2 arms depending on their T/E values (0.12 ± 0.02, n = 12; 1.64 ± 0.90, n = 17). The clinical protocol was designed to evaluate the effect of GT administration on the SP biomarkers. Participants were asked to consume GT with a high content of EGCG for 7 days (5 GT beverages along the whole day for days 1-6 and 9 GT beverages on day 7, corresponding to 520 and 936 mg/day of EGCG, respectively). Urine samples were collected before and during GT consumption at different time periods. The SP was measured using gas chromatography-mass spectrometry. The excretion rates of the SP metabolites did not change after GT consumption. Moreover, the individual evaluation of the subject's steroidal biological passport resulted in normal sequences. The results obtained show that GT consumption does not distort the establishment of normal ranges of SP parameters. Therefore, GT consumption does not need to be considered a confounding factor in the SP evaluation.

Exposure to Bisphenol A and Phthalates during Pregnancy and Ultrasound Measures of Fetal Growth in the INMA-Sabadell Cohort.

BACKGROUND: Prenatal exposure to bisphenol A (BPA) and phthalates may affect fetal growth; however, previous findings are inconsistent and based on few studies. OBJECTIVES: We assessed whether prenatal exposure to BPA and phthalates was associated with fetal growth in a Spanish birth cohort of 488 mother-child pairs. METHODS: We measured BPA and eight phthalates [four di(2-ethylhexyl) phthalate metabolites (DEHPm), mono-benzyl phthalate (MBzP), and three low-molecular-weight phthalate metabolites (LMWPm)] in two spot-urine samples collected during the first and third trimester of pregnancy. We estimated growth curves for femur length (FL), head circumference (HC), abdominal circumference (AC), biparietal diameter (BPD), and estimated fetal weight (EFW) during pregnancy (weeks 12-20 and 20-34), and for birth weight, birth length, head circumference at birth, and placental weight. RESULTS: Overall, results did not support associations of exposure to BPA or DEHPm during pregnancy with fetal growth parameters. Prenatal MBzP exposure was positively associated with FL at 20-34 weeks, resulting in an increase of 3.70% of the average FL (95% CI: 0.75, 6.63%) per doubling of MBzP concentration. MBzP was positively associated with birth weight among boys (48 g; 95% CI: 6, 90) but not in girls (-27 g; 95% CI: -79, 25) (interaction p-value = 0.04). The LMWPm mono-n-butyl phthalate (MnBP) was negatively associated with HC at 12-20 pregnancy weeks [-4.88% of HC average (95% CI: -8.36, -1.36%)]. CONCLUSIONS: This study, one of the first to combine repeat exposure biomarker measurements and multiple growth measures during pregnancy, finds little evidence of associations of BPA or phthalate exposures with fetal growth. Phthalate metabolites MBzP and MnBP were associated with some fetal growth parameters, but these findings require replication. CITATION: Casas M, Valvi D, Ballesteros-Gomez A, Gascon M, Fernández MF, Garcia-Esteban R, Iñiguez C, Martínez D, Murcia M, Monfort N, Luque N, Rubio S, Ventura R, Sunyer J, Vrijheid M. 2016. Exposure to bisphenol A and phthalates during pregnancy and ultrasound measures of fetal growth in the INMA-Sabadell cohort. Environ Health Perspect 124:521-528; http://dx.doi.org/10.1289/ehp.1409190.

Prenatal exposure to phthalates and neuropsychological development during childhood.

There is inconsistent evidence regarding the effects of prenatal phthalate exposure on children's neuropsychological development. We evaluate the association between prenatal phthalate exposure and the cognitive, psychomotor and behavioral development of 367 children at repeated ages in a prospective birth cohort study. We measured phthalate metabolites (sum of four DEHP metabolites - Σ4DEHP, MBzP, MEP, MiBP and MnBP) in urine samples collected during the 1st and 3rd trimesters of pregnancy in women participating in the INMA-Sabadell birth cohort study. We assessed cognitive and psychomotor development of their children at 1 and 4 years, and social competence, ADHD symptoms and other behavioral problems at 4 and 7 years. No associations were observed between prenatal phthalate exposure and cognitive and psychomotor scores at the age of 1 year and at the age of 4 years, except for an association between MBzP and lower psychomotor scores (ß=-1.49 [95% confidence interval (CI)=-2.78, -0.21]). Σ4DEHP concentrations were associated with increased social competence scores at 4 years and with reduced ADHD symptoms at age 4 and 7 years. Increasing MEP concentrations were associated with a reduced risk of inattention symptoms at 4 years. No associations were observed for MBzP, MiBP or MnBP in relation to behavioral problems. This study, with multiple phthalate exposure measurements and measures of neuropsychological domains at different ages, suggest that prenatal phthalate exposure does not adversely affect children's cognitive, psychomotor or behavioral development.

Prenatal Phthalate Exposure and Childhood Growth and Blood Pressure: Evidence from the Spanish INMA-Sabadell Birth Cohort Study.

BACKGROUND: Human evidence on the effects of early life phthalate exposure on obesity and cardiovascular disease risks, reported by experimental studies, is limited to a few cross-sectional studies. OBJECTIVES: We evaluated the associations between prenatal phthalate exposure and childhood growth and blood pressure in a Spanish birth cohort study. METHODS: We assessed exposure using the average of two phthalate metabolite spot-urine concentrations collected from the mothers in the first and third pregnancy trimesters (creatinine-adjusted, n = 391). Study outcomes were the difference in age- and sex-specific z-scores for weight between birth and 6 months of age; and repeated age- and sex-specific z-scores for body mass index (BMI) at 1, 4, and 7 years; waist-to-height ratio at 4 and 7 years; and age- and height-specific z-scores for systolic and diastolic blood pressure at 4 and 7 years. RESULTS: The sum of five high-molecular-weight phthalate metabolites (ΣHMWPm) was associated with lower weight z-score difference between birth and 6 months (ß per doubling of exposure = -0.41; 95% CI: -0.75, -0.06) and BMI z-scores at later ages in boys (ß = -0.28; 95% CI: -0.60, 0.03) and with higher weight z-score difference (ß = 0.24; 95% CI: -0.16, 0.65) and BMI z-scores in girls (ß = 0.30; 95% CI: -0.04, 0.64) (p for sex interaction = 0.01 and 0.05, respectively). The sum of three low-molecular-weight phthalates (ΣLMWPm) was not significantly associated with any of the growth outcomes. ΣHMWPm and ΣLMWPm were associated with lower systolic blood pressure z-scores in girls but not in boys. CONCLUSIONS: This study suggests that prenatal phthalate exposure may be associated with postnatal growth and blood pressure in a sex-specific manner. Inconsistencies with previous cross-sectional findings highlight the necessity for evaluating phthalate health effects in prospective studies.