Detecció dels metabòlits del plastificant di(2-etilhexi)l ftalat com a marcadors de l'ús de transfusions en l'esport

El di(2-etilhexil) ftalat (DEHP) és un plastificant que s’afegeix als productes de clorur de polivinil (PVC) per a dotar-los de més flexibilitat. El material mèdic fet de PVC, i en particular els dispositius i bosses que s’utilitzen en les transfusions de sang, conté el DEHP com additiu. Així, el receptor d’una transfusió està altament exposat a aquest compost. L’objectiu de la tesi va ser estudiar els metabòlits del DEHP en orina com a possibles marcadors de la pràctica d’una transfusió de sang en l’esport. Es va desenvolupar i validar un mètode d’anàlisi per cromatografia líquida acoblada a espectrometria de masses en tàndem per a la quantificació dels principals metabòlits del DEHP en orina humana: mono-(2-etilhexil) ftalat (MEHP), mono-(2-etil-5-hidroxihexil) ftalat (MEHHP), mono-(2-etil-5-oxohexil) ftalat (MEOHP), mono-(2-carboximetilhexil) ftalat (2cx-MMHP) i mono-(2-etil-5-carboxipentil) ftalat (5cx-MEPP). El mètode es va aplicar a mostres procedents de voluntaris sans (grup control), de pacients hospitalitzats que havien rebut una transfusió de sang i de pacients hospitalitzats sotmesos a tractaments mèdics amb materials de PVC i no a transfusions. Es van obtenir diferències significatives en les concentracions dels tres metabòlits estudiats (MEHP, MEHHP, MEOHP) entre les mostres dels pacients transfosos respecte els altres dos grups de població. El mètode també es va aplicar a mostres d’orina de vint-i-cinc voluntaris sans que s’havien sotmès a un procediment d’autotransfusió. Els resultats van indicar concentracions elevades dels cinc metabòlits del DEHP en orina fins a les 48 hores després d’haver rebut la sang. Finalment, es van determinar les concentracions dels cinc metabòlits de DEHP en una població d’esportistes i es van calcular límits de referència que permetessin sospitar d’una transfusió. Així doncs, els resultats indiquen que la mesura dels metabòlits de DEHP en orina pot ser usada com una eina pel cribatge de l’ús de transfusions en l’esport.The plasticizer di(2-ethylhexyl)phthalate (DEHP) is used in polyvinyl chloride products (PVC) to increase its flexibility. Medical devices made of PVC, especially blood bags used in blood transfusions, contain DEHP as additive. Therefore, subjects submitted to blood transfusion are widely exposed to this compound. The aim of the project was to evaluate DEHP metabolites in urine as possible markers of the use of a blood transfusion in sports. An analytical method was developed and validated to quantify the main DEHP metabolites mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP), mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP) and mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), in human urine by liquid chromatography tandem mass spectrometry. The methodology was applied to samples belonging to healthy volunteers (control group), hospitalized patients subjected to blood transfusions and hospitalized patients subjected to medical treatments involving plastic material different to blood transfusions. Significant differences were obtained in the concentrations of the three metabolites studied (MEHP, MEHHP, MEOHP) between transfused patients samples’ and the other two population groups. The method was also applied to urine samples from twenty-five healthy volunteers who were subjected to an autologous blood transfusion. The results indicated high concentrations of the five DEHP metabolites in urine up to 48 hours after the blood transfusion. Finally, the concentration of the five DEHP metabolites were evaluated in a sportsmen population and reference limits to allow suspicion of blood transfusion were calculated. Thus, the results indicate that the DEHP metabolites could be used as markers of blood transfusions in sports

Screening method for stimulants in urine by UHPLC-MS/MS: identification of isomeric compounds.

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between-day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti-Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented.The financial support received from Consell Català de l’Esport and DIUE (2014 SGR 692) (Generalitat de Catalunya; Spain) is acknowledged. Technical assistance of Noemi Haro is gratefully acknowledge

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 6β-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

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 6β-hydroxy-budesonide (6βOHBUD) to detect the systemic administration of BUD. In the present work, we examined the urinary excretion profile of 6βOHBUD, 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. 6βOHBUD 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 6βOHBUD, BUD, and 16αOHPRED, respectively). After all administrations, concentrations were higher in males than in females. Results demonstrated that 6βOHBUD 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

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: β = 0.23 [95% CI = 0.03, 0.44] vs Q1) and higher diastolic BP (Q2: β = 1.27 [0.00, 2.53] mmHg vs Q1). Methyl paraben (MEPA) was associated with lower systolic BP (Q4: β = -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.We thank all study participants for their generous collaboration. INMA-Sabadell: This study was funded by grants from Instituto de Salud Carlos III (Red INMA G03/176; CB06/02/0041; PI041436; PI081151 incl. FEDER funds), CIBERESP, Generalitat de Catalunya-CIRIT 1999SGR 00241, Generalitat de Catalunya-AGAUR 2009 SGR 501, Fundació La marató de TV3 (090430), EU Commission (261357, H2020 No 874583, the ATHLETE project, and No 825712, the OBERON project). Maribel Casas holds a Miguel Servet fellowship (MS16/00128) funded by Instituto de Salud Carlos III and co-funded by European Social Fund “Investing in your future”. We acknowledge support from the Spanish Ministry of Science and Innovation and the State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. INMA-Gipuzkoa: This study was funded by grants from Instituto de Salud Carlos III (FIS-PI13/02187 and FIS-PI18/01142 incl. FEDER funds), CIBERESP, Department of Health of the Basque Government (2015111065), and the Provincial Government of Gipuzkoa (DFG15/221) and annual agreements with the municipalities of the study area (Zumarraga, Urretxu, Legazpi, Azkoitia y Azpeitia y Beasain). INMA-Valencia: This study was funded by Grants from UE (FP7-ENV-2011 cod 282957 and HEALTH.2010.2.4.5-1), Spain: ISCIII (G03/176; FIS-FEDER: PI11/01007, PI11/02591, PI11/02038, PI12/00610, PI13/1944, PI13/2032, PI14/00891, PI14/01687, PI16/1288, and PI17/00663; Miguel Servet-FEDER MS11/00178, MS15/00025, and MSII16/00051), Generalitat Valenciana (AICO/2021/182, and FISABIO: UGP 15-230, UGP-15-244, and UGP-15-249), and Alicia Koplowitz Foundation 2017

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.This study was funded by grants from RecerCaixa (2010ACUP 00349), Instituto de Salud Carlos III (Red INMA G03/176, CB06/02/0041, postdoctoral grant CD12/00563), Spanish Ministry of Health (FIS-PI041436, FIS-PI081151), Generalitat de Catalunya-CIRIT (Comissió Interdepartamental de Recerca i Innovació Tecnològica) (1999SGR00241), and Fundación Roger Torné

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.This study was funded by grants from RecerCaixa (2010ACUP 00349), Instituto de Salud Carlos III (Red INMA G03/176, CB06/02/0041, postdoctoral grant CD12/00563), Spanish Ministry of Health (FIS-PI041436, FIS-PI081151), Generalitat de Catalunya-CIRIT (Comissió Interdepartamental de Recerca i Innovació Tecnològica) (1999SGR00241), and Fundación Roger Torné