Isotope Dilution Tandem Mass Spectrometry Research Articles

Association Between Bisphenol A Exposure and Risk of All-Cause and Cause-Specific Mortality in US Adults

Bisphenol A (BPA) is a major public health concern because of its high-volume industrial production, ubiquitous exposure to humans, and potential toxic effects on multiple organs and systems in humans. However, prospective studies regarding the association of BPA exposure with long-term health outcomes are sparse. To examine the association of BPA exposure with all-cause mortality and cause-specific mortality among adults in the United States. This nationally representative cohort study included 3883 adults aged 20 years or older who participated in the US National Health and Nutrition Examination Survey 2003-2008 and provided urine samples for BPA level measurements. Participants were linked to mortality data from survey date through December 31, 2015. Data analyses were conducted in July 2019. Urinary BPA levels were quantified using online solid-phase extraction coupled to high-performance liquid chromatography-isotope dilution tandem mass spectrometry. Mortality from all causes, cardiovascular disease, and cancer. This cohort study included 3883 adults aged 20 years or older (weighted mean [SE] age, 43.6 [0.3] years; 2032 women [weighted, 51.4%]). During 36 514 person-years of follow-up (median, 9.6 years; maximum, 13.1 years), 344 deaths occurred, including 71 deaths from cardiovascular disease and 75 deaths from cancer. Participants with higher urinary BPA levels were at higher risk for death. After adjustment for age, sex, race/ethnicity, socioeconomic status, dietary and lifestyle factors, body mass index, and urinary creatinine levels, the hazard ratio comparing the highest vs lowest tertile of urinary BPA levels was 1.49 (95% CI, 1.01-2.19) for all-cause mortality, 1.46 (95% CI, 0.67-3.15) for cardiovascular disease mortality, and 0.98 (95% CI, 0.40-2.39) for cancer mortality. In this nationally representative cohort of US adults, higher BPA exposure was significantly associated with an increased risk of all-cause mortality. Further studies are needed to replicate these findings in other populations and determine the underlying mechanisms.

Analysis of per- and polyfluorinated alkyl substances in sub-sampled water matrices with online solid phase extraction/isotope dilution tandem mass spectrometry.

Sorption of PFASs onto surfaces of laboratory materials has been frequently reported. Due to the often complex and poorly understood nature of such sorption, workarounds have often included use of whole samples only, accompanied by sample vessel rinsing to desorb active surfaces. The resulting methods tend to require considerable sample preparation times and preclude typical activities such as aliquoting and dilution of water samples prior to extraction. This manuscript reports an approach for PFAS analysis which uses subsampling of water matrices from vessels including centrifuge tubes and autosampler vials, through the optimized use of solvent to reduce PFAS retention on subsampling vessels. Online solid phase extraction (SPE) using a weak anion exchange resin is then used to concentrate sample aliquots to improve sensitivity and allow for removal of matrix interferences. With the technique of ultra performance liquid chromatography (UPLC) coupled to isotope dilution tandem mass spectrometry, statistically based quantitation limits ranged from sub ng/L to single digit ng/L for carboxylate, sulfonate, and sulfonamide PFASs analytes from C4 to C12. Linear calibration ranges were from 0.25 to 4000ng/L. Matrix effects relevant for drinking water treatment studies, such as cations, organic carbon, and competing PFAS compounds, were evaluated and found to not impact method performance within QC criteria consistent with study data quality objectives.

Associations of Perfluoroalkyl Substances with Incident Natural Menopause: The Study of Women's Health Across the Nation.

Previous epidemiologic studies of per- and polyfluoroalkyl substances (PFASs) and menopausal timing conducted in cross-sectional settings were limited by reverse causation because PFAS serum concentrations increase after menopause. To investigate associations between perfluoroalkyl substances and incident natural menopause. A prospective cohort of midlife women, the Study of Women's Health Across the Nation, 1999-2017. 1120 multiracial/ethnic premenopausal women aged 45-56 years. Serum concentrations of perfluoroalkyls were quantified by high-performance liquid chromatography isotope dilution tandem mass spectrometry. Natural menopause was defined as the bleeding episode prior to at least 12 months of amenorrhea not due to surgery or hormone use. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). Participants contributed 5466 person-years of follow-up, and 578 had incident natural menopause. Compared with the lowest tertile, women at the highest tertile of baseline serum concentrations had adjusted HR for natural menopause of 1.26 (95% CI: 1.02-1.57) for n-perfluorooctane sulfonic acid (n-PFOS) (Ptrend = .03), 1.27 (95% CI: 1.01-1.59) for branched-PFOS (Ptrend = .03), and 1.31 (95% CI: 1.04-1.65) for n-perfluorooctanoic acid (Ptrend = .01). Women were classified into four clusters based on their overall PFAS concentrations as mixtures: low, low-medium, medium-high, and high. Compared with the low cluster, the high cluster had a HR of 1.63 (95% CI: 1.08-2.45), which is equivalent to 2.0 years earlier median time to natural menopause. This study suggests that select PFAS serum concentrations are associated with earlier natural menopause, a risk factor for adverse health outcomes in later life.

Determination of Alternaria Toxins in Sunflower Oil by Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry.

Alternaria toxins have gained attention as a potential health risk and can be classified as emerging mycotoxins. As a result, they are candidates to be regulated by the European Commission. This paper describes a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five Alternaria toxins in sunflower oil, which is a rather different type of sample to those matrices investigated in earlier published papers. An optimal sample preparation condition was achieved when samples were dissolved in n-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation. This study is the first focusing only on this lipophilic matrix and in using all corresponding isotopically labeled internal standards (ISTD) to compensate the matrix effect that strongly influences the LC-MS/MS analysis of toxins. Target compounds were separated on Zorbax Extend C-18 column enabling the analysis at alkaline pH of 8.8 that was necessary to obtain appropriate peak shape of tenuazonic acid and to separate the analytes at baseline. The method was validated according to the EU 2002/657/EC Decision and all the analytical performance characteristics met the requirements. The recovery was between 74% and 122% in fortified sunflower oil samples and the precision varied from 9% to 22%. The method was successfully demonstrated for sunflower seed quality check (QC) samples. Finally, 16 different sunflower oil samples were measured; and tenuazonic acid and tentoxin toxins were detected at levels close to LOQ concentrations.

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems.

Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps—from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus

Background and aimsCardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. MethodsStreptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. ResultsSTZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. ConclusionsIn summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model.

Application of Stable Isotope Dilution and Liquid Chromatography Tandem Mass Spectrometry for Multi-Mycotoxin Analysis in Edible Oils

Abstract Background: Mycotoxin contamination in oils remains an important food safety issue. To monitor the occurrence of mycotoxins in edible oils, it is important to develop analytical methods that can determine multiple mycotoxins in oil products. A stable isotope dilution LC-tandem MS (LC-MS/MS) method for the simultaneous determination of 12 mycotoxins in five edible oil matrixes (canola, corn, olive, peanut, and soybean oil) was developed and validated. Methods: Prior to extraction, the oil samples were fortified with ¹³C uniformly labeled internal standards (¹³C-IS) for 12 target mycotoxins, followed by extraction and LC-MS/MS analysis. Quantitation was achieved using solvent-only calibration standards, relative response factors of ¹³C-IS, and target mycotoxins. Results: The majority of recoveries in oil for ochratoxin A and aflatoxins B1, B2, G1, and G2 fortified at 1, 10, and 100 ng/g as well as deoxynivalenol; fumonisins B1, B2, and B3; T-2 toxin; HT-2 toxin; and zearalenone fortified at 10, 100, and 1000 ng/g ranged from 80 to 120% with RSDs of <20%. The method LOQs ranged from 0.1 ng/g (aflatoxin B1) to 6.4 ng/g (zearalenone). Among 16 U.S. market samples, zearalenone was detected in three corn oil samples at 37, 185, and 317 ng/g, respectively. T-2 toxin was found in two corn oil samples at 7 and 10 ng/g, respectively. Conclusions: The method provides sufficient selectivity, sensitivity, accuracy, and repeatability to screen edible oils for regulated mycotoxins such as aflatoxins at low nanogram per gram concentrations without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

Determination of Cystatin C in human urine by isotope dilution tandem mass spectrometry

This work presents the development of a methodology for the accurate and precise quantification of the renal biomarker Cystatin C in human urine by Isotope Dilution Mass Spectrometry (IDMS). The procedure is based on the addition of a known quantity of the proteotypic peptide ALDFAVG*EYNK labelled with 13C2-glycine to the urine sample followed by protein hydrolysis using trypsin. Then, preconcentration and purification of the isotope diluted peptide was carried out by a selective monoclonal antibody bound to magnetic beads and final measurement was done after injection of the sample in a HPLC-MS/MS triple quadrupole instrument. The isotopic distribution of the isotope diluted proteotypic peptide was measured by low resolution selected reaction monitoring. Using this aquisition mode, the bandpass of the first quadrupole was widened (FWHM =13 u) so the whole isotopic clusters for both the natural abundance and the labelled peptides entered the collision cell. The proposed acquisition mode provided similar accuracy and precision than the regular SRM mode (FWHM =0.7 u) but a higher sensitivity was observed. The purification of the sample by antibody based enrichment of the target peptide was shown to remove interfering compounds more efficiently in comparison with a sample purification based on semipreparative liquid chromatography. Using 5 ng of the labelled peptide it was possible to quantify Cystatin C in human urine in patients with normal and impaired renal function. Recoveries from 100 to 104% were obtained in samples containing from 90 to 700 μg L−1 of Cystatin C with relative standard deviations from 0.5 to 6%. The stability of Cystatin C in urine samples was evaluated under different storage conditions showing that only when the urine samples were stored at room temperature during more than 10 days, a significant degradation of Cystatin C was observed.

Development and validation of an LC-MS/MS method for the simultaneous determination of bedaquiline and rifabutin in human plasma

This article describes the simultaneous determination of bedaquiline fumarate (TMC-207) and rifabutin in human plasma by stable isotope dilution tandem mass spectrometry. The methodology was developed for an investigation of potential drug-drug interactions of the two anti-tuberculosis drugs when given together to healthy human volunteers. Use of the two drugs in combination to treat disease caused by Mycobacterium tuberculosis is contemplated as the bacterium becomes resistant to many currently available drugs.

Urinary bisphenol S concentrations: Potential predictors of and associations with semen quality parameters among men attending a fertility center

BackgroundBisphenol S (BPS) was introduced in the market as a potentially safer alternative to bisphenol A (BPA). However, there are limited studies on health effects of BPS and no epidemiologic studies on its relationship with male reproductive health outcomes, specifically semen quality. ObjectiveTo investigate predictors of urinary BPS concentrations and its association with semen parameters among men attending a fertility center. MethodsThis cross-sectional analysis included 158 men of couples seeking fertility treatment (2011–2017) contributing 338 paired semen and urine samples. At the time of sample collection, men completed a questionnaire on self-reported use of household products and food intake within the previous 24 h. Urinary concentrations of BPA, BPS and bisphenol F were quantified using isotope-dilution tandem mass spectrometry. Semen samples were analyzed following WHO guidelines. Multivariable mixed models were used to investigate predictors of urinary BPS concentrations and to evaluate associations between urinary BPS concentrations and semen parameters, using random intercept to account for correlation in outcomes across multiple observations per man and adjusting for abstinence time, specific gravity, age, body mass index (BMI), year of sample collection and BPA concentrations. Analyses were also stratified by BMI (≥25 vs <25 kg/m2). ResultsMedian (IQR) urinary BPS concentration was 0.30 (0.20, 0.90) μg/L, and 76% of samples had detectable (>0.1 μg/L) concentrations. Self-reported fabric softener and paint/solvent use as well as intake of beef and cheese within 24 h before urine collection were positively associated with BPS concentrations. Men with higher BPS concentrations also had significantly higher BMI. Lower semen parameters were found among men with detectable BPS concentrations, compared to men with non-detectable BPS [2.66 vs. 2.91 mL for volume (p = 0.03), 30.7 vs. 38.3 mil/mL for concentration (p = 0.03), 76.8 vs. 90.0 mil for total count (p = 0.09), 43.7 vs. 47.0% for motility (p = 0.06), and 5.42 vs. 6.77% for morphologically normal sperm (p = 0.24)]. Some associations of BPS with lower semen parameters were only found among men with a BMI ≥ 25 kg/m2. ConclusionsWe identified dietary and lifestyle factors associated with BPS exposure, suggesting potential avenues for reducing exposures. We also observed negative associations between BPS and semen parameters, especially among overweight and obese men.

Melatonin is not stored in platelets

BackgroundMelatonin is one of numerous biologically active compounds reported to be stored in platelets. As melatonin is increasingly linked to several diseases, we wanted to confirm the storage of melatonin in platelets using sensitive liquid chromatography in combination with isotope dilution tandem mass spectrometry (LC-MS/MS). The difference between melatonin levels analyzed in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) served as proxy for platelet levels of melatonin. MethodsMelatonin concentrations were analyzed in PRP and PPP from nineteen healthy volunteers by ELISA and LC-MS/MS. A Wilcoxon signed-rank test was performed to assess if the melatonin levels measured in PRP and PPP were different. Results for melatonin concentrations obtained by LC-MS/MS or ELISA were compared using Passing-Bablok regression. ResultsComparison of the ELISA with the LC-MS/MS method showed poor agreement for melatonin concentrations in PRP and PPP. No indication was found for storage of melatonin in platelets by either LC-MS/MS or ELISA (P = .89 and P = .53 for the LC-MS/MS and ELISA analysis, respectively). ConclusionIn this study we could find no evidence for melatonin storage in platelets.

Complete validation according to current international criteria of a confirmatory quantitative method for the determination of nitrofuran metabolites in seafood by liquid chromatography isotope dilution tandem mass spectrometry

Despite the ban of nitrofurans (NFs) for use in food production in many countries in the 1990s, NF metabolites in food are still regularly detected during import control testing. We have developed a confirmatory routine method for the detection and quantification of NF metabolites in seafood using LC-MS/MS and validated the method according to the strict criteria in European legislation and Codex Alimentarius. Method characteristics were found to fulfill the criteria. We report for the first time a new false positive for 1-amino-2,4-imidazolidinedione (AHD), the metabolite of Nitrofurantoin (NFT). By using optimized washing procedures, the non tissue bound false positives can be minimized. The results from the validation on both lean and fatty fish and crustaceans, results from proficiency tests and routine use over many years, demonstrates that the method is fit for purpose to determine NF metabolites in the seafood category.

Quantification of 16 urinary biomarkers of exposure to flame retardants, plasticizers, and organophosphate insecticides for biomonitoring studies

Chlorinated alkyl and non-chlorinated aryl organophosphate flame retardants (OPFRs) and some brominated flame retardants (FR) were introduced as replacements for polybrominated diphenyl ethers (PBDEs) after PBDEs phase-out in 2004 and 2013. Organophosphorous (OP) insecticides are mainly used in agricultural settings since the Food Quality Protection Act of 1996 phased-out most residential uses of OP insecticides in the United States.Urinary metabolites of FRs and OPs are known exposure biomarkers to FRs and OP insecticides, respectively. For large population-based studies, concurrent quantification of these metabolites using a small urine volume is desirable, but until now was not possible. We developed an analytical approach to quantify in 0.2 mL urine 10 FRs and six OP insecticide metabolites: diphenyl phosphate, bis(1,3-dichloro-2-propyl) phosphate, bis(1-chloro-2-propyl) phosphate, bis(2-chloroethyl) phosphate, dicresyl phosphates, dibutyl phosphate, dibenzyl phosphate, 2,3,4,5-tetrabromobenzoic acid, 2-((isopropyl)phenyl)phenyl phosphate, 4-((tert-butyl)phenyl)phenyl phosphate, dimethyl phosphate, diethyl phosphate, dimethyl thiophosphate, dimethyl dithiophosphate, diethyl thiophosphate, and diethyl dithiophosphate. The method relies on enzymatic deconjugation, automated off-line solid phase extraction, high-performance liquid chromatography, and isotope dilution tandem mass spectrometry. Detection limits ranged from 0.05 to 0.5 ng mL−1, accuracy from 89 to 118%, and imprecision was <10%. .This method is the first to quantify simultaneously trace levels of 16 biomarkers of FRs and OP insecticides in only four drops of urine. We confirmed the method suitability for use in large epidemiological studies to assess background and occupational exposures to these classes of environmental pollutants by analyzing 303 samples collected from the general population and a group of firefighters. FR metabolite and DAPs concentrations in the general population group were lower than in the firefighters group, and within the ranges reported in the U.S. general population and other non-occupationally exposed populations.

Albuminuria, the High-Density Lipoprotein Proteome, and Coronary Artery Calcification in Type 1 Diabetes Mellitus.

Objective- Albuminuria is an important risk factor for cardiovascular disease in diabetes mellitus. We determined whether albuminuria associates with alterations in the proteome of HDL (high-density lipoprotein) of subjects with type 1 diabetes mellitus and whether those alterations associated with coronary artery calcification. Approach and Results- In a cross-sectional study of 191 subjects enrolled in the DCCT (Diabetes Control and Complications Trial)/EDIC study (Epidemiology of Diabetes Interventions and Complications), we used isotope dilution tandem mass spectrometry to quantify 46 proteins in HDL. Stringent statistical analysis demonstrated that 8 proteins associated with albuminuria. Two of those proteins, AMBP (α1-microglobulin/bikunin precursor) and PTGDS (prostaglandin-H2 D-isomerase), strongly and positively associated with the albumin excretion rate ( P<10-6). Furthermore, PON (paraoxonase) 1 and PON3 levels in HDL strongly and negatively associated with the presence of coronary artery calcium, with odds ratios per 1-SD difference of 0.63 (95% CI, 0.43-0.92; P=0.018) for PON1 and 0.59 (95% CI, 0.40-0.87; P=0.0079) for PON3. Only 1 protein, PON1, associated with both albumin excretion rate and coronary artery calcification. Conclusions- Our observations indicate that the HDL proteome is remodeled in type 1 diabetes mellitus subjects with albuminuria. Moreover, low concentrations of the antiatherosclerotic protein PON1 in HDL associated with both albuminuria and coronary artery calcification, raising the possibility that alterations in HDL protein cargo mediate, in part, the known association of albuminuria with cardiovascular risk in type 1 diabetes mellitus. Visual Overview- An online visual overview is available for this article.

Polycyclic aromatic hydrocarbons in foods from the first regional total diet study in Sub-Saharan Africa: contamination profile and occurrence data

As part of the first multi-centre Sub-Saharan Africa Total Diet Study, 660 typical foods from Benin, Cameroon, Mali, and Nigeria were purchased, prepared according to local consumption habits, and pooled into 55 composite samples. These core foods were tested for 15 + 1 EU priority polycyclic aromatic hydrocarbons, which were quantified by isotope dilution and gas chromatography tandem mass spectrometry. The sum of benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthene, and chrysene (PAH4) represented 77% of the 13 genotoxic and carcinogenic PAHs. The highest PAH4 concentration was quantified in sea and fresh water smoked fish (mean: 179.7 μg/kg; max: 560.4 μg/kg) and the PAH4 in all smoked fish composite samples exceeded the EU maximum limit of 12 μg/kg. Further, PAH4 in edible oils (including palm oil and peanut oil) exceeded the EU maximum limit of 10 μg/kg in 50% of the cases (mean 12.0 μg/kg; max: 60.6 μg/kg). These data can be used for assessing the contribution of core foods to dietary exposure and for risk characterization.

Urinary concentrations of bisphenol A, parabens and phthalate metabolite mixtures in relation to reproductive success among women undergoing in vitro fertilization

BackgroundWe have previously investigated whether urinary concentrations of bisphenol A (BPA), parabens, and phthalate metabolites were individually associated with reproductive outcomes among women undergoing in vitro fertilization (IVF) treatment. However, humans are typically exposed to many man-made chemicals simultaneously. Thus, investigating one chemical at a time may not represent the effect of mixtures. ObjectiveTo investigate whether urinary concentrations of BPA, parabens, and phthalate metabolite mixtures are associated with reproductive outcomes among women undergoing IVF. MethodsThis prospective cohort study included 420 women contributing 648 IVF cycles who provided up to two urine samples per cycle prior to oocyte retrieval (N = 1145) between 2006 and 2017 at the Massachusetts General Hospital Fertility Center, and had available urine biomarker data. Urinary concentrations of BPA, parabens, and phthalate metabolites were quantified using isotope-dilution tandem mass spectrometry. Intermediate and clinical end-points of IVF treatments were abstracted from electronic medical records. Principal component analysis (PCA) and Bayesian kernel machine regression (BKMR) were used to identify main patterns of BPA, parabens, and phthalate metabolites concentrations. We used generalized linear mixed models to evaluate the association between PCA-derived factor scores, in quartiles, and IVF outcomes, using random intercepts to account for multiple IVF cycles and adjusting for known confounders. Because of temporal trends in exposure, we conducted a sensitivity analysis restricted to women who underwent IVF cycles in the earlier years of study (2006–2012). ResultsUrinary concentrations of BPA, parabens, and most phthalate metabolites were significantly lower during the second half of the study period (2013–2017) than during the first half (2006–2012). None of the three factors derived from the PCA [di(2-ethylhexyl) phthalate (DEHP), non-DEHP, and paraben] was associated with IVF outcomes in the main analyses. Similarly, BKRM analyses did not identify any associations of individual urinary concentrations of BPA, paraben and phthalate metabolites with IVF outcomes while accounting for correlation between exposures. However, in sensitivity analyses restricted to women who underwent IVF cycles from 2006 to 2012, where concentrations of most phthalates and phenols were higher, there were decreases in implantation, clinical pregnancy, and live birth across quartiles of the DEHP factor. Specifically, women in the highest quartile of the DEHP factor had, on average, lower probabilities of implantation (−22% p, trend = 0.08), clinical pregnancy (−24% p, trend = 0.14), and live birth (−38% p, trend = 0.06) compared to women in the lowest quartile. Among this group of women, BKMR results did not identify any single contributor driving the decreased probabilities of live birth within the DEHP factor. ConclusionsWe confirmed that women undergoing IVF are concurrently exposed to multiple endocrine disrupting chemicals (EDCs). While we found no overall significant associations, we observed diminished pregnancy success with specific clusters of chemicals among women who underwent IVF cycles in earlier years of study, when urinary concentrations of these EDCs were higher.

Identification and quantification of DNA repair protein poly(ADP ribose) polymerase 1 (PARP1) in human tissues and cultured cells by liquid chromatography/isotope-dilution tandem mass spectrometry

Poly(ADP ribose) polymerase 1 (PARP1) is a multifunctional DNA repair protein of the base excision repair pathway and plays a major role in the repair of DNA strand breaks and in replication and transcriptional regulation among other functions. Mounting evidence points to the predictive and prognostic value of PARP1 expression in human cancers. Thus, PARP1 has become an important target in cancer therapy, leading to the development of inhibitors as anticancer drugs. In the past, PARP1 expression levels in tissue samples have generally been estimated by indirect and semi-quantitative immunohistochemical methods. Accurate measurement of PARP1 in normal tissues and malignant tumors of patients will be essential for evaluating PARP1 as a predictive and prognostic biomarker in cancer and other diseases, and for the development and use of its inhibitors in cancer therapy. In this work, we present an approach involving liquid chromatography–isotope-dilution tandem mass spectrometry to positively identify and accurately quantify PARP1 in human tissues and cultured cells. We identified and quantified PARP1 in human normal ovarian tissues and malignant ovarian tumors, and in three pairs of human cell lines, each pair consisting of a normal cell line and its cancerous counterpart. Significantly greater expression of PARP1 was observed in malignant ovarian tissues than in normal ovarian tissues. In the case of one pair of cell lines, the cancerous cell line also exhibited greater expression of PARP1 than in normal cell line. We also show the simultaneous measurement of PARP1 and apurinic/apyrimidinic endonuclease 1 (APE1) in a given protein extract. The approach presented in this work is expected to contribute to the accurate quantitative assessment of PARP1 levels in basic research and clinical studies.

Simultaneous detection of multiple hydroxylated polychlorinated biphenyls from biological samples using ultra-high-performance liquid chromatography with isotope dilution tandem mass spectrometry.

We established a method for the separation and detection of nine hydroxylated polychlorinated biphenyls in whole blood and urine samples using ultra high performance liquid chromatography coupled with electrospray negative ionization tandem mass spectrometry. Clean-up procedures involved a filtration step, and optimization involved a pretreatment step consisting of a simple liquid-liquid extraction using hydrated silica-gel chromatography (5%). Nine hydroxylated polychlorinated biphenyls were separated on an ultra high performance liquid chromatography HSS T3 column using a gradient elution program of 2mmol ammonium formate aqueous solution (A) and methanol (B). Recovery ranged from 84.0 to 105.4% for the nine different hydroxylated polychlorinated biphenyls in urine with three spiked levels of 0.1, 1, and 2ng and from 73.5 to 98.6% for the blood with spiked levels of 0.2, 1, and 2ng. The relative standard deviations were <8.7% (n=6), and the limits of detection in urine and whole blood for the nine hydroxylated polychlorinated biphenyls were in the range of 1.5-4 and 20-100 pg/g, respectively. This analytical method may enable the simultaneous detection of various hydroxylated polychlorinated biphenyls from complex tissue matrices.

Nationwide reconnaissance of five parabens, triclosan, triclocarban and its transformation products in sewage sludge from China

China's rapid growth of both population size and sanitation infrastructure have created a heightened need for responsible management of sewage sludge. We applied liquid chromatography in conjunction with isotope dilution tandem mass spectrometry to measure multiple endocrine disrupting antimicrobials and their transformation products in 100 sewage sludge samples collected across 21 Chinese provinces/districts. Occurrences (detection frequencies) and concentrations (ng/g dry weight) were as follows: triclosan (99%; <4-4870), triclocarban (95%; <3-43,300), 2'-hydroxy-triclocarban (94%; <1-2340), 3'-hydroxy-triclocarban (91%; <1-1250), 3,3',4,4'-tetrachlorocarbanilide (100%; 22-580), dichlorocarbanilide (94%; <2-23,890), monocarbanilide (92%; <2-120), carbanilide (90%; <3-1,340), and five parabens: methyl- (98%; <2-630), ethyl- (96%; <2-170), propyl- (99%; <2-27), butyl- (89%; <2-11) and benzyl-paraben (7%; <2-12). The transformation products of triclocarban were measured for the first time in Chinese wastewater system, and ratios of transformation products to parental triclocarban indicate ongoing triclocarban dechlorination during wastewater treatment. Contaminant profiles and concentrations differed by region, treatment capacity, and wastewater type. Extrapolation of collected data yielded an estimate for the total mass of 13 analytes sequestered in Chinese sewage sludge of 68 t/y with an upper bound of 400 t/y. This China-wide survey established baseline levels of selected antimicrobials in sludges whose current disposal is performed with little regulatory oversight and enforcement.

Secular trends in semen parameters among men attending a fertility center between 2000 and 2017: Identifying potential predictors

BackgroundMultiple meta-analyses have shown sperm count declines in Western countries spanning eight decades. Secular trends in other parameters remain unclear, as are potential predictors of these trends. ObjectiveTo analyze secular trends in semen quality and to evaluate whether factors previously found to be related to semen quality were responsible for these patterns. MethodsThis is a prospective study including 936 men of couples seeking infertility treatment who provided 1618 semen samples at a single center (2000–2017). Self-reported demographic, nutritional and reproductive characteristics were collected using standardized questionnaires. Urinary concentrations of bisphenol A, parabens and phthalates were quantified by isotope-dilution tandem mass spectrometry. Semen samples were analyzed for volume, sperm concentration, count, motility and morphology following WHO guidelines. We estimated the differences in semen parameters over time by fitting generalized linear mixed models with random intercepts to account for repeated samples while adjusting for abstinence time. We also adjusted for demographic, nutritional and environmental factors to investigate these as potential predictors of time trends. ResultsSperm concentration and count declined by 2.62% per year (95% CI −3.84, −1.38) and 3.12% per year (95% CI: −4.42, −1.80), corresponding to an overall decline of 37% and 42%, respectively, between 2000 and 2017. Decreasing trends were also observed for total motility (per year: −0.44 percentage units, 95% CI −0.71, −0.17) and morphologically normal sperm (per year: −0.069 percentage units, 95% CI −0.116, −0.023). These decreases reflected relative percentage declines of 15% and 16% over the 17 year study period, respectively. When reproductive factors were included in the model, the downward trends in sperm concentration and sperm count were attenuated by 29% and 26%, respectively, while the trends in motility and morphology were attenuated by 54% and 53%, respectively. Also, the downward trends in both sperm concentration and sperm morphology over time were attenuated by 19% when including the DEHP and non-DEHP metabolites, respectively. ConclusionsSperm concentration, total count, motility and morphology significantly declined between 2000 and 2017 among subfertile men. These negative trends were attenuated when considering simultaneous changes in reproductive characteristics and urinary phthalates during the course of the study.