Isotope Dilution High-performance Liquid Chromatography Research Articles

Association of serum trimethylamine N-oxide levels and bone mineral density in type 2 diabetes mellitus.

The relationship between trimethylamine N-oxide (TMAO) and bone mineral density (BMD) in type 2 diabetes mellitus (T2DM) is unclear. We explore the relationship between TMAO levels and BMD in T2DM. This is a cross-sectional study. 254 T2DM patients were enrolled and divided into three groups by TMAO tertiles, and the clinical data were collected. BMD was determined by dual-energy X-ray absorptiometry (DXA) and serum TMAO levels was determined by stable isotope dilution high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Patients in the highest tertile of TMAO levels (TMAO > 6.72 μmol/L) showed relatively low BMD and a higher number of fracture history, osteoporosis (OP) than those in the lower tertiles. Spearman correlation analysis showed that serum TMAO was negatively correlated with BMD of whole body (WB), lumbar spine (LS) and femoral neck (FN), while TMAO was positive correlated with osteoporotic fracture (p < 0.05). Logistic regression models showed that TMAO was an independent influencing factor of fracture history after adjusting for confounders in TMAO > 6.72 μmol/L group. There is a significant linear correlation between TMAO levels and BMD in T2DM patients. Especially in TMAO > 6.72 μmol/L group, TMAO was negatively correlated with WB, LS, and FN BMD, and was positive correlated with osteoporotic fracture in T2DM patients. The findings suggest that elevated TMAO levels are associated with OP and osteoporotic fracture in T2DM patients.

Short and medium chain acylcarnitines as markers of outcome in diabetic and non-diabetic subjects with acute coronary syndromes

BackgroundCarnitine metabolism produces numerous molecular species of short-, medium-, and long-chain acylcarnitines, which play important roles in energy homeostasis and fatty acid transport in the myocardium. Given that disturbances in the carnitine metabolism are linked to cardiometabolic disease, we studied the relationship of circulating acylcarnitines with outcomes in patients with acute coronary syndromes (ACS) and evaluated differences in circulating levels of these metabolites between diabetic and non-diabetic patients. MethodsHarnessing a prospective multicentre cohort study (SPUM-ACS; NCT01000701), we measured plasma levels of acylcarnitines, carnitine, and carnitine metabolites to assess their relationship with adjudicated major adverse cardiac events (MACE), defined as composite of myocardial infarction, stroke, clinically indicated revascularization, or death of any cause. The SPUM-ACS study enrolled patients presenting with ACS to Swiss University Hospitals between 2009 and 2012. Acetylcarnitine, octanoylcarnitine, proprionylcarnitine, butyrylcarnitine, pentanoylcarnitine, hexanoylcarnitine, carnitine, γ-butyrobetaine, and trimethylamine N-oxide were measured in plasma using stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry. ResultsA total of 1683 patients with ACS were included in the study. All measured metabolites except γ-butyrobetaine and carnitine were higher in diabetic subject (n = 294) than in non-diabetic subjects (n = 1389). On univariate analysis, all metabolites, apart from octenoylcarnitine, were significantly associated with MACE at 1 year. After multivariable adjustment for established risk factors, acetylcarnitine remained an independent predictor of MACE at 1-year (quartile 4 vs. quartile 1, adjusted hazard ratio 2.06; 95% confidence interval 1.12–3.80, P = 0.020). ConclusionCirculating levels of acetylcarnitine independently predict residual cardiovascular risk in patients with ACS.

Novel Isotope Dilution High-Performance Liquid Chromatography Quadrupole–Linear Ion Trap Tandem Mass Spectrometry Method for the Determination of Five Steroid Hormones in Human Serum

Accurate measurement of endogenous steroid hormones in serum is a powerful tool for understanding the status of human adrenal diseases. Isotope dilution–liquid chromatography–tandem mass spectrometry is the most commonly used method for determination of steroids in serum. In this study, we used a laboratory-constructed quadrupole-linear ion trap (Q-LIT) tandem mass spectrometer to develop a new method for the quantification of five steroid hormones in serum. A simple and efficient pretreatment was improved, wherein the serum was extracted once by a mixed solvent of 1.0 mL of ethyl acetate and n-hexane. Using the optimized method, the regression coefficients of the calibration curves were all higher than 0.99, the limits of detection were from 0.31 to 1.25 ng mL−1, and the limits of quantification were between 1.00 and 2.50 ng mL−1. The intra-day (n = 3) relative standard deviations ranged from 4.24% to 13.79%, and the inter-day (n = 3) relative standard deviations from 3.99% to 11.43%. After internal standard correction, the relative recoveries of all steroids were from 86.08% to 106.60%. Twenty-two serum samples were analyzed by Q-LIT, which was compared with commercial triple quadrupole mass spectrometer. The results of the instruments had high consistency. Therefore, Q-LIT accurately quantified steroid hormones in human samples and may be used for clinical diagnosis and monitoring of hormone-dependent diseases.

A potential primary method for peptide purity analysis by gas chromatography-isotope dilution infrared spectrometry.

Here we proposed a method for peptide purity analysis using gas chromatography-isotope dilution infrared spectroscopy. The principle and feasibility of the proposed measurement method were investigated. The derivatization, separation, and infrared detection conditions for amino acids were optimized, and the performance of the method was investigated. Then, the proposed method was used for assessment of [Glu1]-fibrinopeptide B purity, and the results were compared with those obtained by high performance liquid chromatography-isotope dilution mass spectrometry. The average purity of six sub-samples using the proposed method was (0.755 ± 0.017)g/g, which agreed well with that obtained by isotope dilution mass spectrometry (0.754 ± 0.012) g/g. The repeatability of the proposed method was 2.2%, which was similar to that of isotope dilution mass spectrometry (1.7%). The proposed method has a similar principle and had similar accuracy, precision, and linearity to isotope dilution mass spectrometry; however, the developed method had higher limit of detection (LOD) and limit of quantitation (LOQ) values because of the low sensitivity of infrared detection. The results were also Système International d'Unités (SI) traceable. The developed method has the advantage of lower cost compared with isotope dilution mass spectrometry because only one isotope-labeled atom in an analog is required, and several infrared spectra can be extracted, averaged, and used for an amino acid calculation during one run, potentially leading to higher accuracy. This method could be easily expanded to the accurate quantitation of other organic compounds, including proteins. It is expected that the proposed method will be widely used in chemical and biological measurements as a new primary method.

Analysis of gut microbiota in patients with acute myocardial infarction by 16S rRNA sequencing.

An increasing number of studies have shown that gut microbiota are associated with human cardiovascular disease, but the characteristics of intestinal flora in patients with acute myocardial infarction (AMI) are still unclear. In this study, we aimed to investigate the difference of intestinal microflora between patients with AMI and healthy people, and to find the effect of percutaneous coronary intervention (PCI) on intestinal microflora. A total of 60 stool samples and 60 peripheral blood samples were collected from 20 previously diagnosed AMI patients and 20 healthy people serving as controls. Gut microbiota communities were analyzed via 16 ribosomal RNA-sequencing (16S rRNA). Gut microbiota-derived metabolites, trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFA), in the blood were detected using stable isotope dilution high-performance liquid chromatography with on line electrospray ionization tandem mass spectrometry (LC/MS/MS). The results showed that a distinct pattern of gut microbiota was observed in AMI patients compared to healthy controls. AMI patients had lower microbiological richness but no significant change in diversity. Bacteroidetes and Verrucomicobia showed an upward trend, whereas Proteobacteria showed a downward trend in AMI patients. During a longitudinal study to compare the changes in bacteria before and after treatment, we found routine cardiac admission therapy 1 week after PCI surgery had no effect on the microbial community structure in patients. There were significantly higher levels of plasma TMAO in AMI patients' microbiota than that in the control group. Contrarily, there was no obvious change in SCFA. The gut microbiota of patients with AMI differs from that of normal people, and the metabolic products of microflora are more abundant in the plasma of AMI than control cases. Microflora may act on the cardiovascular system through metabolites, and regulation of the microfloral structure may be used in the future treatment of cardiovascular diseases.

Absolute Quantification of Total Hemoglobin in Whole Blood by High-Performance Liquid Chromatography Isotope Dilution Inductively Coupled Plasma-Mass Spectrometry.

Accurate and traceable measurement of hemoglobin (HGB) is of great importance in clinical testing. Although the HiCN method is the internationally accepted conventional reference method for this biomarker and frequently used in clinical routine diagnostics, the HiCN method cannot be traceable to the International System of Units (SI) and thus does not meet highest metrological demands. In this study, an absolute quantitative approach for total HGB in a whole blood sample is proposed based on the determination of natural Fe and S present in the heme-group of HGB by HPLC isotope dilution ICP-MS. IRMM/IFCC-467 is used for method validation, and then clinical blood samples are measured by the established strategy and HiCN method. The measurable ranges of total HGB were 10.0-240.0 g L-1. Limits of detection via Fe and S were 0.01 and 0.07 g L-1, respectively. The intra-assay imprecision CVs via Fe and S were 0.89-1.35 and 0.99-1.56%, and the interassay CVs were 1.19-2.15 and 1.55-2.55%, respectively. Good agreement was achieved in the method validation. In the comparison with HiCN experiments, the ID-ICP-MS assays via Fe and S showed correlations of r2 = 0.991 and 0.970 against HiCN methods. Moreover, the concentration of transferrin (Tf) was also simultaneously measured. This strategy has potential to serve as a reference measurement procedure for total HGB in whole blood, which could be traceable to SI and does not require toxic derivation reagent.

Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices

BackgroundHydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. MethodsAfter synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. ResultsUsing the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0–66.7% reduction with antibiotics). ConclusionsA stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.

Coronary atherosclerotic burden and trimethylamine N-oxide in non-ST-segment myocardial infarction patients: SZ-NSTEMI cohort

Abstract Funding Acknowledgements Type of funding sources: None. Background Trimethylamine N-oxide (TMAO) is reported to accelerate atherosclerosis and adverse cardiac outcomes. Relationship between coronary atherosclerotic burden and TMAO has linked in stable coronary artery disease and ST-segment elevation myocardial infarction, but not in non-ST-segment elevation myocardial infarction (NSTEMI). Objective We examined an association of TMAO with coronary atherosclerosis burden in NSTEMI. Methods In this prospective cohort, two groups including NSTEMI (n = 73) and age-sex matched Healthy (n = 35) individuals were enrolled between 2019 and 2020. The number of diseased coronary vessels and clinical risk scores including SYNTAX and GENSINI were used to stratify coronary atherosclerotic burden. Fasting plasma TMAO was measured by isotope dilution high-performance liquid chromatography. Results The median plasma TMAO levels (0.59 mM; [interquartile range [IQR]: 0.43-0.78) versus (0.42 mM; IQR: 0.33-0.64) were significantly higher in NSTEMI group versus Healthy group, respectively (P = 0.006). Among the patients with NSTEMI group, the plasma TMAO levels were significantly higher in the multivessel disease (MVD) versus single vessel disease (P = 0.002), and intermediate-high risk (score³23) versus low risk (score &amp;lt; 23) of SYNTAX (P = 0.003) and GENSINI (P = 0.005). TMAO levels remained independently associated with MVD (odds ratio [OR]: 5.94, P = 0.005), and intermediate-high risk SYNTAX (OR: 3.61, P = 0.013) and GENSINI scores (OR: 4.60, P = 0.008) following traditional risk factors adjustment. Area under receiver operating characteristic curves (AUC) for TMAO also predicted MVD (AUC: 0.73, 95% confidence interval [Cl]: 0.60-0.86, P = 0.002), and intermediate-high SYNTAX score (AUC: 0.70, 95%Cl: 0.58-0.82, P = 0.003) and GENSINI score (AUC: 0.70, 95%Cl: 0.57-0.83, P = 0.005). Conclusions TMAO levels are independently associated with high coronary atherosclerotic burden in NSTEMI. Abstract Figure. Abstract

Kynurenine to tryptophan ratio as a biomarker of acute stress in fish

The aim of this study was to determine the kynurenine (KYN) to tryptophan (TRP) ratio (KTR) in fish tissue to assess its usefulness as a biomarker of acute stress. Laboratory held rainbow trout (Oncorhynchus mykiss) were subjected to an acute stressor and KYN, TRP and cortisol were measured in liver and brain tissues at 4- and 48-h post-stress. The analytical method used to determine our analytes was based on lyophilization, and liquid-solid extraction followed by isotope dilution high-performance liquid chromatography positive ion electrospray tandem mass spectrometry. The [KYN]/[TRP] ratio (KTR) was greater in fish liver and brain in the 48-h post-stress exposure group (n = 8) relative to controls (n = 8, p < 0.05); a similar increase was not observed in fish in the 4-h post-stress exposure group. Hepatic and brain cortisol levels were also elevated in fish from both stress-induced groups relative to their respective controls implying that cortisol responded more quickly to the stressful stimulus than KYN and TRP. Our results suggest that the KTR is a promising acute stress diagnostic biomarker in fish. Efforts are ongoing to assess whether the KTR can be used as a biomarker for chronic stress in fish exposed to aquatic contaminants and other environmental stressors and if similar assessments can be made on tissues collected via non-lethal approaches.

Development and certification of a reference material for zearalenone in maize germ oil

Zearalenone (ZEN), an estrogenic mycotoxin produced by several species of Fusarium fungi, is a common contaminant of cereal-based food worldwide. Due to frequent occurrences associated with high levels of ZEN, maize oil is a particular source of exposure. Although a European maximum level for ZEN in maize oil exists according to Commission Regulation (EC) No. 1126/2007 along with a newly developed international standard method for analysis, certified reference materials (CRM) are still not available. To overcome this lack, the first CRM for the determination of ZEN in contaminated maize germ oil (ERM®-BC715) was developed in the frame of a European Reference Materials (ERM®) project according to the requirements of ISO Guide 35. The whole process of CRM development including preparation, homogeneity and stability studies, and value assignment is presented. The assignment of the certified mass fraction was based upon an in-house study using high-performance liquid chromatography isotope dilution tandem mass spectrometry. Simultaneously, to support the in-house certification study, an interlaboratory comparison study was conducted with 13 expert laboratories using different analytical methods. The certified mass fraction and expanded uncertainty (k = 2) of ERM®-BC715 (362 ± 22) μg kg−1 ZEN are traceable to the SI. This reference material is intended for analytical quality control and contributes to the improvement of consumer protection and food safety.Graphical abstract

Development of a new matrix certified reference material for metronidazole in egg powder

Due to the potential health risks and hazards associated with the intake of metronidazole, several governments and consumers have become increasingly concerned about the quality and safety of food in the recent years. Since it is not possible to avoid the side effects of metronidazole, immense efforts are being taken to appraise and monitor its concentration in feed in order to ensure the safe consumption of food. However, simple certified reference materials (CRMs) for the quality control and quality assurance of metronidazole are still lacking. Thus, the first matrix CRM for metronidazole was developed from egg powder. This paper presents the complete process of the development of a CRM for metronidazole in egg powder, including sample preparation, homogeneity and stability studies, and the uncertainty assessment. The egg samples were taken from hens that took metronidazole orally. The matrix CRM developed was quantified using isotope dilution high-performance liquid chromatography coupled with tandem mass spectrometry. Concurrently, in order to support the certification study, an interlaboratory contrast study involving eight expert laboratories was implemented. The bulk material was bottled in 4 g portions. While the stability of the CRM was monitored at −20 °C for 13 months, at −4°C, 20 °C, and 40 °C for 9 days. During the monitoring period, the matrix CRM candidate was found to be stable at all temperatures.

Trimethylamine N-oxide is associated with coronary atherosclerotic burden in non-ST-segment myocardial infarction patients: SZ-NSTEMI prospective cohort study.

Trimethylamine N-oxide (TMAO) is reported to accelerate atherosclerosis and the development of adverse cardiac outcomes. Relationship between coronary atherosclerotic burden and TMAO has been examined in stable coronary artery disease and ST-segment elevation myocardial infarction, but not in non-ST-segment elevation myocardial infarction (NSTEMI). We examined the association between TMAO and coronary atherosclerotic burden in NSTEMI. In this prospective cohort study, two groups including NSTEMI (n = 73) and age-sex matched Healthy (n = 35) individuals were enrolled between 2019 and 2020. Coronary atherosclerotic burden was stratified based on the number of diseased coronary vessels and clinical risk scores including SYNTAX and GENSINI. Fasting plasma TMAO was measured by isotope dilution high-performance liquid chromatography. The median plasma TMAO levels were significantly higher in the NSTEMI group than in the Healthy group, respectively (0.59 µM; interquartile range [IQR]: 0.43-0.78 versus 0.42 µM; IQR: 0.33-0.64; P = 0.006). Within the NSTEMI group, higher TMAO levels were observed in the multivessel disease (MVD) versus single vessel disease (P = 0.002), and intermediate-high risk (score ≥ 23) versus low risk (score < 23) of SYNTAX (P = 0.003) and GENSINI (P = 0.005). TMAO level remained an independent predictor of MVD (odds ratio [OR]: 5.94, P = 0.005), intermediate-high risk SYNTAX (OR: 3.61, P = 0.013) and GENSINI scores (OR: 4.60, P = 0.008) following adjustment for traditional risk factors. Receiver operating characteristic curve (AUC) analysis for TMAO predicted MVD (AUC: 0.73, 95% confidence interval [Cl]: 0.60-0.86, P = 0.002), intermediate-high SYNTAX score (AUC: 0.70, 95% Cl: 0.58-0.82, P = 0.003) and GENSINI score (AUC: 0.70, 95% Cl: 0.57-0.83, P = 0.005). In all, TMAO levels are independently associated with high coronary atherosclerotic burden in NSTEMI.

Certification of Amyloid-Beta (Aβ) Certified Reference Materials by Amino Acid-Based Isotope Dilution High-Performance Liquid Chromatography Mass Spectrometry and Sulfur-Based High-Performance Liquid Chromatography Isotope Dilution Inductively Coupled Plasma Mass Spectrometry.

The use of amyloid-beta (Aβ) biomarkers could contribute to an early diagnosis of Alzheimer's disease (AD); however, there are still large variations among results from different assays. This variability can be overcome by standardization of those assays through the use of certified reference materials (CRMs) and the establishment of a traceability chain. In this study, Aβ40 (GBW09874) and Aβ42 (GBW09875) solution CRMs with the certified values and uncertainties of 7.58 ± 0.30 and 7.62 ± 0.30 μg g-1 were developed with high-purity Aβ as raw materials. For the first time, isotope dilution high-performance liquid chromatography mass spectrometry (ID-LC-MS) and high-performance liquid chromatography isotope dilution inductively coupled plasma mass spectrometry (HPLC-ID-ICP-MS) strategies were employed to certify the candidate Aβ solution CRMs. The two candidate CRMs showed good homogeneity, and good stability was also demonstrated for at least 5 days at -20 °C and 14 months at -70 °C. These CRMs are primarily intended to be used for value assignment to secondary calibrators or CRMs with a clinical matrix, which will help in early diagnosis of AD.

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.

Association of bone mineral density with nine urinary personal care and consumer product chemicals and metabolites: A national-representative, population-based study

Personal care and consumer products are extensively used and contain various chemicals that are recognized as endocrine disrupting chemicals. Few epidemiological studies measured the associations of these chemicals and metabolites, especially phenols, with bone health. We measured the associations between nine urinary phenol biomarkers and BMD and the subsequent prevalence of osteopenia/osteoporosis. National representative data from US adults aged over 20 years who participated in the National Health and Nutrition Examination Survey from 2005 to 2010 were used. 2,267 men, 1,145 premenopausal women and 1,033 postmenopausal women were included in the final analyses. Bisphenol A, benzophenone-3, triclosan, butylparaben, ethylparaben, methylparaben, propylparaben, 2,5-dichlorophenol and 2,4-dichlorophenol were measured by high-performance liquid chromatography-isotope dilution tandem mass spectrometry. Paraben concentrations, especially ethyl-, methyl- and propylparabens, were related to greater BMD in the femoral neck and intertrochanter and lumbar spine in men and premenopausal women. 2,4-dichlorophenol concentrations was associated with lower BMD and a higher prevalence of osteopenia + osteoporosis in men. Bisphenol A was associated with a higher prevalence of osteopenia + osteoporosis in the lumbar spine in postmenopausal women. Benzophenone-3 tended to be associated with a higher BMD of femur in men and premenopausal women. In conclusion, certain urinary personal care and consumer product chemicals and metabolites were significantly associated with BMD or osteopenia + osteoporosis. These results further indicate that, in future research, determination of the causal relationships and detection of undelying mechanism could be next important directions.

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.

Urinary Concentrations of Phthalate Metabolite Mixtures in Relation to Serum Biomarkers of Thyroid Function and Autoimmunity among Women from a Fertility Center.

Background:Although previous epidemiological studies have explored associations of phthalate metabolites with thyroid function, no studies to date have assessed associations of mixtures with thyroid function and autoimmunity among potentially susceptible subgroups such as subfertile women.Objective:We aimed to explore associations of mixtures of urinary phthalate metabolites with serum markers of thyroid function and autoimmunity.Methods:This cross-sectional study included 558 women attending a fertility center who provided one spot urine and one blood sample at enrollment (2005–2015). We quantified urinary concentrations of eight phthalate metabolites using mass spectrometry, and biomarkers of thyroid function [thyroid-stimulating hormone (TSH), free and total thyroxine (, ) and triiodothyronine (, ), and autoimmunity [thyroid peroxidase and thyroglobulin antibodies (TPOAb and TgAb, respectively)] in serum using electrochemiluminescence assays. We applied principal component analysis (PCA) and Bayesian kernel machine regression (BKMR) to identify the main patterns of urinary phthalate metabolites. We used linear mixed models to assess the association between PCA-derived factor scores in quintiles and serum thyroid function and autoimmunity, adjusting for age, body mass index (BMI), specific gravity (SG), and, for the PCA, other factor scores.Results:We observed two factors using PCA, one representing the di(2-ethylhexyl) (DEHP) and another non-DEHP metabolites. Compared to women in the lowest quintile of the DEHP factor scores, women in the highest quintile had significantly lower serum concentrations of , , , and [absolute difference: ; 95% confidence interval (CI): , ; ; absolute difference: ; 95% CI: , ; ; absolute difference: ; 95% CI: 0.54, ; ; and absolute difference: ; 95% CI: , ; , respectively]. Using BKMR, we observed that mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was the primary contributor to these negative associations. DEHP and non-DEHP factor scores were not associated with serum TSH, TgAb, or TPOAb.Conclusions:Mixtures of urinary DEHP metabolites were inversely associated with serum biomarkers of thyroid function but not with autoimmunity, which were within normal ranges for healthy adult women. https://doi.org/10.1289/EHP6740

3-Bromopyruvate regulates the status of glycolysis and BCNU sensitivity in human hepatocellular carcinoma cells

Chloroethylnitrosoureas (CENUs) are bifunctional antitumor alkylating agents, which exert their antitumor activity through inducing the formation of dG-dC interstrand crosslinks (ICLs) within DNA double strand. However, the complex process of tumor biology enables tumor cells to escape the killing triggered by CENUs, as for instance with the detoxifying activity of O6-methylguanine DNA methyltransferase (MGMT) to accomplish DNA damage repair. Considering the fact that most tumor cells highly depend on aerobic glycolysis to provide energy for survival even in the presence of oxygen (Warburg effect), inhibition of aerobic glycolysis may be an attractive strategy to overcome the resistance and improve the chemotherapeutic effects of CENUs. Especially, 3-bromopyruvate (3-BrPA), a small molecule alkylating agent, has been emerged as an effective glycolytic inhibitor (energy blocker) in cancer treatment. In view of its tumor specificity and inhibition on cellular multiple targets, it is likely to reduce the chemoresistance when chemotherapeutic drugs are combined with 3-BrPA. In this study, we investigated the effects of 3-BrPA on the chemosensitivity of two human hepatocellular carcinoma (HCC) cell lines to the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the underlying molecular mechanism. The sensitivity of SMMC-7721 and HepG2 cells to BCNU was significantly increased by 2 h pretreatment with micromolar dosage of 3-BrPA. Moreover, 3-BrPA decreased the cellular ATP and GSH levels, and extracellular lactate excreted by tumor cells, and the effects were more effective when 3-BrPA was combined with BCNU. Cellular hexokinase-II (HK-II) activity was also reduced after exposure to the treatment of 3-BrPA plus BCNU. Based on the above results, the effects of 3-BrPA on the formation of dG-dC ICLs induced by BCNU was investigated by stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results indicated that BCNU produced higher levels of dG-dC ICLs in SMMC-7721 and HepG2 cells pretreated with 3-BrPA compared to that without 3-BrPA pretreatment. Notably, in MGMT-deficient HepG2 cells, the levels of dG-dC ICLs were significantly higher than MGMT-proficient SMMC-7721 cells. In general, these findings revealed that 3-BrPA, as an effective glycolytic inhibitor, may be considered as a potential clinical chemosensitizer to optimize the therapeutic index of CENUs.

Glycolytic inhibition by 3-bromopyruvate increases the cytotoxic effects of chloroethylnitrosoureas to human glioma cells and the DNA interstrand cross-links formation

DNA interstrand cross-links (ICLs) are essential for the antitumor activity of chloroethylnitrosoureas (CENUs). Commonly, CENUs resistance is mainly considered to be associated with O6-methylguanine-DNA methyltransferase (MGMT) within tumors. Bypassing the MGMT-mediated resistance, to our knowledge, herein, we first utilized a novel glycolytic inhibitor, 3-bromopyruvate (3-BrPA), to increase the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to human glioma cells based on the hypothesis that blocking energy metabolism renders tumor cells more sensitive to chemotherapy. We found 3-BrPA significantly increased the cell killing by BCNU in human glioma SF763 and SF126 cell lines. Significantly decreased levels of extracellular lactate, cellular ATP and glutathione (GSH) were observed after 3-BrPA treatment, and the effects were more remarkable with 3-BrPA in combination with BCNU. Considering that the role of ATP and GSH in drug efflux, DNA damage repair and drug inactivation, we determined the effect of 3-BrPA on the formation of dG-dC ICLs induced by BCNU using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As expected, the levels of lethal dG-dC ICLs induced by BCNU were obviously enhanced after 3-BrPA pretreatment. Based on these results, 3-BrPA and related glycolytic inhibitors may be promising to enhance the cell killing effect and reverse the clinical chemoresistance of CENUs and related antitumor agents.

Serum folate metabolites, asthma, and lung function in a nationwide US study

In a U.S. nationwide study, a high serum 5-MTHF to UMFA ratio is associated with decreased odds of asthma, increased FEV1 in children and adults, and increased FVC in adults.