Isotope Dilution Mass Research Articles

Polychlorinated biphenyl (PCB) concentrations and breakpoint trends across the waters of the Great Lakes by isotope-dilution high-resolution mass spectroscopy.

Water samples were collected during each of the 2012-2019 Cooperative Science and Monitoring Initiative (CSMI) cruises aboard the U.S. EPA R/V Lake Guardian as part of the Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) lower food web contaminant assessment. The CSMI rotates around each of the Great Lakes in a 5-yearcycle providing top-to-bottom biological, chemical, and physical environmental assessments, including dissolved-phase surface water studies at two sample locations. Average polychlorinated biphenyl (∑PCB) concentrations across the Great Lakes was 268pg/L with a station range of 72pg/L (Keweenaw Point-Lake Superior) to 834pg/L (Middle Bass Island-Lake Erie). The highest average Great Lakes concentration (pg/L-sample year) were measured in Lake Erie (645pg/L-2014) and decreased in the order of Lake Huron (378pg/L-2012)>Lake Erie (364pg/L-2019)>Lake Ontario (300pg/L-2013)>Lake Michigan (125pg/L-2015)>Lake Huron (123pg/L-2017)>Lake Superior (74pg/L-2016). Lake Erie registered a 44% reduction over the 2014-2019 period, attributed to sediment remediation in the late-1990's on the Detroit River, whereas Lake Huron exhibited a 67% decrease over the 2012-2017 sample period. Our results indicate that dissolved-phase PCB water concentrations in Lake Ontario have significantly increased, rebounding from a low-point in the late-1990's likely due to the bioenergetic diversion of dissolved- and particulate-phase PCBs into the benthic food web by invasive zebra and quagga mussel colonization. Trend analyses uncovered breakpoints in the early 1990's documenting significantly slowing rates of PCB declines for both Lakes Superior and Michigan.

A sterol panel for rare lipid disorders: sitosterolemia, cerebrotendinous xanthomatosis, and Smith-Lemli-Opitz syndrome

Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoring of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements is limited in many parts of the world. A simple and rapid method to extract freesterols from human serum and quantitate theirconcentration using isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization was developed. The method was designed to be compatible with routine workflows (e.g., 96-well format) in a clinical lab and extensively validated. Serum from at least 125 controls were analyzed and used to estimate the upper reference limits for sitosterol, campesterol, stigmasterol, desmosterol, 7-dehydrocholesterol (7DHC), lathosterol, and cholestanol. Serum from patients with the rare lipid disorders sitosterolemia (n=7), Smith-Lemli-Opitz syndrome (SLOS; n=1), and cerebrotendinous xanthomatosis (CTX; n=1) were analyzed. All seven sitosterolemia patients had greatly elevated levels of free plant sterols (sitosterol, campesterol, and stigmasterol) compared to the controls. The SLOS serum contained massively increased concentrations of 7DHC. CTX serum contained greatly increased concentrations of cholestanol, as well as 7DHC and lathosterol. Spiking experiments indicated that the method is likely also useful for the diagnosis of desmosterolosis and lathosterolosis. The reported method is a relatively simple and fast LC-MS/MS method capable of quantitating diagnostically important sterols and differentiated patients with three rare lipid disorders from controls.

Discrimination of overheated pasteurized milk using mass spectrometry-based proteomics

Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides—INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK—were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

A Cross-Sectional Exploratory Study of Cardiovascular Risk Biomarkers in Non-Obese Women with and without Polycystic Ovary Syndrome: Association with Vitamin D.

Vitamin D is proposed to have a protective effect against cardiovascular disease, though the mechanism is unclear. Vitamin D deficiency is common in polycystic ovary syndrome (PCOS), where it is strongly related to obesity, insulin resistance (IR) and risk of cardiovascular disease. To determine if the inherent pathophysiology of PCOS or vitamin D levels are linked to dysregulation of cardiovascular risk proteins (CVRPs), a study in non-obese women with PCOS and without IR was undertaken. Our hypothesis was that the levels of vitamin D3 and its active metabolite would be associated with CVRPs comparably in women with and without PCOS. In women with PCOS (n = 29) and controls (n = 29), 54 CVRPs were determined by Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement and correlated to 25-hydroxyvitamin D3 (25(OH)D3) and the active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) measured by gold standard isotope-dilution liquid chromatography tandem mass spectrometry. Women with PCOS had comparable IR and systemic inflammation (normal C-reactive protein) to control women, though had higher free androgen index and anti-Mullerian hormone levels. 25(OH)D3 and 1,25(OH)2D3 levels did not differ between groups. Nine CVRPs were higher in PCOS (p < 0.05) (Galectin-9, Brother of CDO, C-motif chemokine 3, Interleukin-18 receptor-1, Thrombopoietin, Interleukin-1 receptor antagonist protein, Programmed cell death 1 ligand-2, Low-affinity immunoglobulin gamma Fc-region receptor II-b and human growth hormone), whilst 45 CVRPs did not differ. 25(OH)D3 correlated with five CVRPs in PCOS and one in controls (p < 0.05). Despite the women with PCOS not exhibiting overt systemic inflammation, 9 of 54 CVRPs were elevated, all relating to inflammation, and 5 of these correlated with 25(OH)D3, suggesting an ongoing underlying inflammatory process in PCOS even in the absence of obesity/IR.

PFAS fate using lysimeters during degraded soil reclamation using biosolids.

Carbon- and nutrient-rich biosolids are used in agriculture and land reclamation. However, per- and polyfluoroalkyl substances (PFAS) typically present in biosolids raise concerns of PFAS leaching to groundwater and plant uptake. Here, we investigated PFAS persistence and leaching from biosolids applied to a site constructed artificially to mimic degraded soils. Treatments included biosolids and biosolids blended with mulch applied at different rates to attain either one and five times the agronomic N rate for vegetable crops and a control treatment with synthetic urea and triple superphosphate fertilizer. Leachates were collected for a 2-year period from 15-cm depth zero-tension drainage lysimeters. Soils were analyzed post biosolids application. PFAS were quantified using isotope-dilution, solid-phase extraction and liquid chromatography tandem mass spectrometry. Leachate profiles exemplified an initial high total PFAS concentration, followed by a sharp decline and subsequent small fluctuations attributed to pre-existing soil conditions and rainfall patterns. Quantifiable PFAS in leachate were proportional to biosolids application rates. Short-chain perfluoroalkyl acids (CF2<6) were dominant in leachate, while the percentage of longer chains homologues was higher in soils. A 43% biosolids blend with mulch resulted in 21% lower PFAS leachate concentrations even with the blend application rate being 1.5 times higher than biosolids due to the blend's lower N-content. The blending effect was more pronounced for long-chain perfluoroalkyl sulfonic acids that have a greater retention by soils and the air-water interface. Biosolids blending as a pragmatic strategy for reducing PFAS leachate concentrations may aid in the sustainable beneficial reuse of biosolids.

Development of an insulin-like growth factor-1 certified reference material by SI-traceable isotope-dilution mass spectrometry

In this study, an insulin-like growth factor-1 (IGF-1) certified reference material (CRM) was developed by the National Institute of Metrology (NIM), and two different principles for evaluating the IGF-1 CRM were established. After optimisation of the acid hydrolysis conditions (110 °C, 36 h), quantitative determination of peptide purity, and chromatographic separation and mass spectrometric detection, amino acid analysis-based high-performance liquid chromatography combined with isotope-dilution tandem mass spectrometry (AAA-HPLC-IDMS/MS) and peptide analysis-based HPLC-IDMS/MS (Peptide-HPLC-IDMS/MS) were used for certified value assignment; the results obtained were 136.28 and 135.01 μg/g, respectively, which were in good agreement. These results were subjected to the normal distribution test, outlier test, and method consistency test. The homogeneity and stability of the reference materials were also examined, and the uncertainty introduced in the experimental process was calculated. The final certified value was (136 ± 15) μg g−1 (k = 2). The CRM was found to be stable for at least six months when stored at −70 °C and for 7 d when stored at higher temperatures (−20 °C, 4 °C, 25 °C, or 40 °C). The CRM is expected to be used as a primary calibrator for quality control in biopharmaceutical production and clinical diagnostics.

Initial Validation of a Soil-Based Mass-Balance Approach for Empirical Monitoring of Enhanced Rock Weathering Rates.

Enhanced rock weathering (ERW) is a promising scalable and cost-effective carbon dioxide removal (CDR) strategy with significant environmental and agronomic co-benefits. A major barrier to large-scale implementation of ERW is a robust monitoring, reporting, and verification (MRV) framework. To successfully quantify the amount of carbon dioxide removed by ERW, MRV must be accurate, precise, and cost-effective. Here, we outline a mass-balance-based method in which analysis of the chemical composition of soil samples is used to track in situ silicate rock weathering. We show that signal-to-noise issues of in situ soil analysis can be mitigated by using isotope-dilution mass spectrometry to reduce analytical error. We implement a proof-of-concept experiment demonstrating the method in controlled mesocosms. In our experiment, a basalt rock feedstock is added to soil columns containing the cereal crop Sorghum bicolor at a rate equivalent to 50 t ha-1. Using our approach, we calculate rock weathering corresponding to an average initial CDR value of 1.44 ± 0.27 tCO2eq ha-1 from our experiments after 235 days, within error of an independent estimate calculated using conventional elemental budgeting of reaction products. Our method provides a robust time-integrated estimate of initial CDR, to feed into models that track and validate large-scale carbon removal through ERW.

Microbe-derived uremic solutes enhance thrombosis potential in the host.

Alterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

Simultaneous determination of ergothioneine, selenoneine, and their methylated metabolites in human blood using ID-LC-MS/MS.

Ergothioneine and selenoneine are structurally related dietary antioxidants and cytoprotectants that may help prevent several chronic diseases associated with inflammation and aging. Both compounds share pharmacokinetic characteristics such as cellular uptake through the ergothioneine transporter, accumulation in red blood cells, and biotransformation to methylated metabolites. A rapid, sensitive, specific, precise, and cost-effective analytical method is required to further investigate the potential health benefits of these compounds, individually or combined, in large epidemiological studies. We developed and validated an isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous specific quantification of these analytes in human blood following a simple sample preparation consisting of dilution in aqueous dithiothreitol followed by centrifugal filtration. Chromatographic separation of the analytes is achieved using a reversed-phase chromatography within an 8-min run. Analyte detection is performed using triple quadrupole mass spectrometry in multiple reaction monitoring mode. Each analyte is quantified against its corresponding isotopically labeled internal standard either commercially available or synthesized in-house (77Se-labeled selenoneine compounds). The validated method demonstrates excellent linearity and very good precision (all CV < 10%). Matrix effects are minimal, suggesting that this method could easily be adapted to other matrices. Freeze/thaw cycles have little effect on methylated metabolites but significantly reduced concentrations of the parent compounds. The method was successfully applied to a small set of volunteer blood samples containing low levels of the analytes. The developed ID-LC-MS/MS method opens new avenues for exploring the roles of these bioactive compounds and their metabolites in human health and disease.

Urinary Glyphosate, 2,4-D and DEET Biomarkers in Relation to Neurobehavioral Performance in Ecuadorian Adolescents in the ESPINA Cohort.

Herbicides are the most used class of pesticides worldwide, and insect repellents are widely used globally. Yet, there is a dearth of studies characterizing the associations between these chemical groups and human neurobehavior. Experimental studies suggest that glyphosate and 2,4-dichlorophenoxyacetic acid (2,4-D) herbicides can affect neurobehavior and the cholinergic and glutamatergic pathways in the brain. We aim to assess whether herbicides and insect repellents are associated with neurobehavioral performance in adolescents. We assessed 519 participants (11-17 years of age) living in agricultural communities in Ecuador. We quantified urinary concentrations of glyphosate, 2,4-D, and two N,N-diethyl-meta-toluamide (DEET) insect repellent metabolites [3-(diethylcarbamoyl)benzoic acid (DCBA) and 3-(ethylcarbamoyl)benzoic acid (ECBA)] using isotope-dilution mass spectrometry. We assessed neurobehavioral performance using 9 subtests across 5 domains (attention/inhibitory control, memory/learning, language, visuospatial processing, and social perception). We characterized the associations using generalized estimating equations and multiple imputation for metabolites below detection limits. Models were adjusted for demographic and anthropometric characteristics, urinary creatinine, and sexual maturation. Mediation by salivary cortisol, dehydroepiandrosterone, , and testosterone was assessed using structural equation modeling. The mean of each neurobehavioral domain score was between 7.0 and 8.7 [standard deviation (SD) range: 2.0-2.3]. Glyphosate was detected in 98.3% of participants, 2,4-D in 66.2%, DCBA in 63.3%, and ECBA in 33.4%. 2,4-D was negatively associated with all neurobehavioral domains, but statistically significant associations were observed with attention/inhibition [score difference per 50% higher metabolite concentration 95% confidence interval (CI): , ], language [ (95% CI: , )], and memory/learning [ (95% CI: , 0.01)]. Glyphosate had a statistically significant negative association only with social perception [ (95% CI: , )]. DEET metabolites were not associated with neurobehavioral performance. Mediation by gender and adrenal hormones was not observed. This study describes worse neurobehavioral performance associated with herbicide exposures in adolescents, particularly with 2,4-D. Replication of these findings among other pediatric and adult populations is needed. https://doi.org/10.1289/EHP11383.

A-214 Modified CDC Beta-Quantification Reference Method for HDL-C and LDL-C Determinations Using Isotope-Dilution Mass Spectrometry—Pilot Report

Abstract Background It is known that there is average positive 1.6% bias of total cholesterol by modified Abell-Levy-Brodie-Kendall (AK) colorimetric method, which have been the traceable reference measurement procedure (RMP) used by most of clinical guidelines, comparing to those by isotope-dilution mass spectrometry (ID-MS), the most accurate RMP. The CDC beta-quantification (BQ) reference method is currently the only RMP registered JCTLM database for high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) using the AK colorimetric assay after ultracentrifugation. We compared the modified BQ method using ID-MS assay after ultracentrifugation with the original CDC BQ reference method of HDL-C or LDL-C. Methods For this study, 18 commutable serum pools according to the CLSI 37A and 6 external quality assessment survey materials operated by Cholesterol Reference Method Laboratory Network (CRMLN) were used. We used the same ultracentrifugation procedure as in the original CDC BQ reference method, and the bottom fraction cholesterol (BFC) and HDL-C from supernatant after apo B-containing lipoprotein precipitation using heparin/manganese were measured for each sample using both AK and ID-MS methods, then LDL-C was calculated as the difference between BFC and HDL-C. The comparability analysis between methods at multiple medical decision levels (MDL), and Passing-Bablok regression analysis for each analyte were performed. Results Each Passing-Bablok regression equation of modified CDC BQ reference method vs. original CDC BQ reference method for three fractions, BFC, HDL-C, and LDL-C, were as follows; BFC was y (IDMS) = 0.996x (AK) - 0.342 (R = 0.999), for HDL-C was y (IDMS) = 1.007x (AK)-1.037 (R = 0.995), and for LDL-C was y (IDMS) = 0.990x (AK) + 0.860 (R = 0.999). Both cholesterol results by two methods showed excellent correlation and agreement of the three fractions showing regression R larger than 0.995 and slope or intercept containing 1 and 0 in their 95% confidence interval. The mean differences of IDMS against AK method were −0.56% for BFC, −0.51 mg/dL for HDL-C, and −0.35% for LDL-C, respectively. When the regression prediction at MDLs, 40/50/60 mg/dL for HDL-C and 100/130/160 mg/dL for LDL-C were estimated, all the differences were confirmed to be within allowable level claimed by CRMLN member laboratory accreditation criteria, ±1mg/dL for HDL-C and ±2.0% for LDL-C, showing equivalent results against CDC BQ reference method, but average negative bias of −0.72%, −1.59%, and −0.28% for BFC, HDL-C, and LDL-C, respectively are still present. These results of the modified BQ method using ID-MS assay may be promising, because the tedious and less accurate AK-assay can be switched to the ID-MS assay, not sacrificing trueness LDL-C or changing clinical guidelines, but quite large negative bias of HDL-C for some samples should require further investigation. Conclusion We got comparable HDL-C and LDL-C results using modified BQ method using ID-MS assay with those by original CDC BQ reference method using AK colorimetric assay. The quite large negative bias of HDL-C for some samples require large-scale additional further multi-center study among CRMLN network laboratories to evaluate the results observed in our study.

Dose-response formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine in liver and urine correlates with micronucleated reticulocyte frequencies in mice administered safrole oxide

Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the most abundant SAFO-induced DNA adduct, N7-(3-benzo[1,3] dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SAFO-G), in mouse urine using a well-developed isotope-dilution high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (ID-HPLC-ESI-MS/MS) method. This study further elucidated the genotoxic mode of action of SAFO in mice treated with SAFO 30, 60, 90, or 120 mg/kg for 28 days. The ID-HPLC-ESI-MS/MS method detected N7γ-SAFO-G with excellent sensitivity and specificity in mouse liver and urine of SAFO-treated mice. Our data provide the first direct evidence of SAFO-DNA adduct formation in rodent tissues. N7γ-SAFO-G levels in liver were significantly increased by SAFO 120 mg/kg compared with SAFO 30 mg/kg, suggesting rapid spontaneous or enzymatic depurination of N7γ-SAFO-G in tissue DNA. Urinary N7γ-SAFO-G exhibited a sublinear dose response. Moreover, the micronucleated peripheral reticulocyte frequencies increased dose-dependently and significantly correlated with N7γ-SAFO-G levels in liver (r = 0.8647; p < 0.0001) and urine (r = 0.846; p < 0.0001). Our study suggests that safrole-mediated genotoxicity may be caused partly by its metabolic activation to SAFO and that urinary N7γ-SAFO-G may serve as a chemically-specific cancer risk biomarker for safrole exposure.

Urinary Profile of Alkylated DNA Adducts and DNA Oxidative Damage in Sulfur Mustard-Exposed Rats Revealed by Mass Spectrometry Quantification.

Alkylation reagents, represented by sulfur mustard (SM), can damage DNA molecules directly as well as lead to oxidative stress, causing DNA lesions indirectly. Correspondingly, two types of biomarkers including alkylated DNA adducts and oxidative DNA adducts are commonly involved in the research of DNA damage evaluation caused by these agents. However, the correlations and differences of the occurrence, duration, severity, and traceability between alkylation and oxidation lesions on the DNA molecular level reflected by these two types of biomarkers have not been systematically studied. A simultaneous determination method for four alkylated DNA adducts, i.e., N7-(2-hydroxyethylthioethyl)2'-guanine (N7-HETEG), O6-(2-hydroxyethylthioethyl)-2'-guanine (O6-HETEG), N3-(2-hydroxyethylthioethyl)-2'-adenine (N3-HETEA), and bis(2-ethyl-N7-guanine)thioether (Bis-G), and the oxidative adduct 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in urine samples by isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) was built with a lower limit of detection of 0.02 ng/mL (except Bis-G, 0.05 ng/mL) and a recovery of 79-111%. The profile of these adducts was simultaneously monitored in urine samples after SD rats' dermal exposure to SM in three dose levels (1, 3, and 10 mg/kg). The time-effect and dose-effect experiments revealed that when exposed to SM, DNA alkylation lesions would happen earlier than those of oxidation. For the two types of biomarkers, alkylated DNA adducts showed an obvious dose-effect relationship and could be used as internal exposure dose and effect biomarkers, while 8-OH-dG did not show a correlation with exposure dose, demonstrating that it was more suitable as a biomarker for DNA oxidative lesions but not an indicator for the extent of cytotoxicity and internal exposure.

Changes in Kidney Function Among Malaysian Adolescents and Its Determinants

The health and wellbeing of adolescents are often neglected, including the knowledge of chronic kidney disease (CKD), especially in its early stages. A total of 607 adolescents were recruited from the Malaysian Health and Adolescents Longitudinal Research Team (MyHeART) study, a prospective cohort study conducted from March 2012 to May 2016 that explored the noncommunicable diseases (NCDs) risk factors among 13 to 17 years old students in 3 states of Peninsular Malaysia. Students who participated in all 3 data collection periods in 2012, 2014, and 2016 with kidney function assessment across all 3-time points were included in the current study. The students' estimated glomerular filtration rate (eGFR) was calculated from isotope-dilution mass spectrometry-traceable Schwartz's equation and categorized based on Kidney Disease: Improving Global Outcomes (KDIGO) classification. Changes in kidney function were examined, and the longitudinal relationship between eGFR and multiple NCD risk factors was analyzed using the generalized estimating equation (GEE). The prevalence of decreased eGFR (60-89 ml/min per 1.73 m2) among the students increased from 6.1% (2012) to 30.0% (2014) and 40.2% (2016). Based on the GEE, the student's eGFR decreased over time, with a steeper decline during early to midadolescence. Males and rural students had lower eGFR compared to their counterparts. Students who are morbidly obese had lower eGFR than those with normal body mass index (BMI). Protein consumption also has a potential moderating effect on eGFR in adolescents. Kidney function changes can be detected as early as adolescence and are likely attributable to multiple NCD risk factors. Therefore, more comprehensive prevention efforts from various stakeholders are needed to identify health issues like CKD.

A targeted isotope dilution mass spectrometry assay for osteopontin quantification in plasma of metastatic breast cancer patients.

Osteopontin (OPN) is a secreted glycophosphoprotein that derives its name from its high abundance in bone and secretion by osteoblasts. It is also secreted by a number of immune cells and, therefore, is present in human plasma at nanogram per millilitre levels where it affects cell adhesion and motility. OPN is involved in several normal physiological processes; however, OPN dyregulation leads to overexpression by tumor cells leading to immune evasion and increased metastasis. Plasma OPN is primarily measured by enzyme-linked immunosorbent assay (ELISA). However, due to the complexity of the various OPN isoforms, conflicting results have been obtained on the use of OPN as a biomarker even in the same disease condition. These discrepant results may result from the difficulty in comparing ELISA results obtained with different antibodies that target unique OPN epitopes. Mass spectrometry can be used to quantify proteins in plasma and, by targeting OPN regions that do not bear post-translational modifications, may provide more consistent quantification. However, the low (ng/mL) levels in plasma present a significant analytical challenge. In order to develop a sensitive assay for plasma OPN, we explored a single-step precipitation method using a recently developed spin-tube format. Quantification was performed using isotope-dilution mass spectrometry. The concentration detection limit of this assay was 39 ± 15 ng/mL. The assay was applied to the analysis of plasma OPN in metastatic breast cancer patients, where levels from 17 to 53 ng/mL were detected. The sensitivity of the method is higher than previously published methods and sufficient for OPN detection in large, high grade tumors but still requires improvement in sensitivity to be widely applicable.

LC-MS/MS analysis of elastin crosslinker desmosines and microscopic evaluation in clinical samples of patients with hypertrophy of ligamentum flavum

Ligamentum flavum (LF) pathologies often lead to severe myelopathy or radiculopathy characterized by reduced elasticity, obvious thickening, or worsened ossification. Elastin endows critical mechanical properties to tissues and organs such as vertebrae and ligaments. Desmosine (DES) and isodesmosine (IDES) are crosslinkers of elastin monomers called tropoelastin. These crosslinkers are potential biomarkers of chronic obstructive pulmonary disease. As a biological diagnostic tool that supplements existing symptomatic, magnetic resonance imaging scanning or radiological imaging diagnostic measures for LF hypertrophy and associated pathologies, an isotope-dilution liquid chromatography-tandem mass spectrometry method with selected reaction monitoring mode for the quantitation of DESs in human plasma, urine, cerebrospinal fluid (CSF), and yellow ligamentum was investigated. Isotopically labeled IDES-13C3,15N1 was used as an internal standard (ISTD) for DES quantitation for the first time. The samples plus ISTD were hydrolyzed with 6 N hydrochloric acid. Analytes and ISTD were extracted using a solid phase extraction cellulose cartridge column. The assays were repeatable, reproducible, and accurate with % CV ≤ 7.7, ISTD area % RSD of 7.6, and % AC ≤ (101.2 ± 3.90) of the calibrations. The ligamentum samples gave the highest average DES/IDES content (2.38 μg/mg) on a dry-weight basis. A high percentage of the CSF samples showed almost no DESs. Urine and plasma samples of patients showed no significant difference from the control (p-value = 0.0519 and 0.5707, respectively). Microscopy of the yellow ligamentum samples revealed dark or blue-colored zones of elastin fibers that retained the hematoxylin dye and highly red-colored zones of collagen after counterstaining with van Gieson solution. Thus, we successfully developed a method for DES/IDES quantitation in clinical samples.

An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification of γ-H2AX in human peripheral blood lymphocytes.

Exposure to environmental ionizing radiation (IR) is ubiquitous, and large-dose exposure to IR is known to cause DNA damage and genotoxicity which is associated with an increased risk of cancer. Whether such detrimental effects are caused by exposure to low-dose IR is still debated. Therefore, rapid and early estimation of absorbed doses of IR in individuals, especially at low levels, using radiation response markers is a pivotal step for early triage during radiological incidents to provide adequate and timely clinical interventions. However, there is currently a crucial shortage of methods capable of determining the extent of low-dose IR exposure to human beings. The phosphorylation of histone H2AX on serine 139 (designated γ-H2AX), a classic biological dosimeter, can be used to evaluate the DNA damage response. We have developed an estimation assay for low-level exposure to IR based on the mass spectrometry quantification of γ-H2AX in blood. Human peripheral blood lymphocytes sensitive to low-dose IR, maintaining low temperature (4°C) and adding enzyme inhibitor are proven to be key steps, possibly insuring that a stable and marked γ-H2AX signal in blood cells exposed to low-dose IR could be detected. For the first time, DNA damage at low dose exposures to IR as low as 0.01 Gy were observed using the sensitive variation of γ-H2AX with high throughput mass spectrometry quantification in human peripheral blood, which is more accurate than the previously reported methods by virtue of isotope-dilution mass spectrometry, and can observe the time effect of DNA damage. These in vitro cellular dynamic monitoring experiments show that DNA damage occurred rapidly and then was repaired slowly over the passage of post-irradiation time even after exposure to very low IR doses. This assay was also used to assess different radiation exposures at the in vitro cellular level. These results demonstrate the potential utility of this assay in radiation biodosimetry and environmental risk assessment.

Inflammatory Markers in Non-Obese Women with Polycystic Ovary Syndrome Are Not Elevated and Show No Correlation with Vitamin D Metabolites.

Introduction. Chronic low-grade inflammation is a characteristic of women with polycystic ovary syndrome (PCOS), although this may be obesity-driven rather than an intrinsic facet of PCOS; furthermore, vitamin D deficiency, another common feature of PCOS, is reported to have an association with increased inflammation. Therefore, circulating inflammatory protein levels and circulating levels of vitamin D may be linked in PCOS, though it is unclear which vitamin D metabolites may be important. Methods. We measured plasma levels of 24 inflammatory proteins and 12 matrix metalloproteinases (proteins modulated by the inflammatory process) by slow off-rate modified aptamer (SOMA)-scan plasma protein measurement in weight and aged-matched non-obese non-insulin resistant PCOS (n = 24) and control (n = 24) women. Inflammatory proteins and matrix metalloproteinases were correlated to 25-hydroxy vitamin D3 (25(OH)D3), its epimer 25-hydroxy-3epi-vitamin D (3epi25(OH)D) and the active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) as measured by gold standard isotope-dilution liquid chromatography tandem mass spectrometry. Results. PCOS women had both an elevated free androgen index and circulating anti-mullerian hormone, though insulin resistance was comparable to controls. C-reactive protein, as a standard circulatory marker of inflammation, was comparable between cohorts. Levels of circulating inflammatory proteins and matrix metalloproteinases were not different between the PCOS and control women, with no correlation of 25(OH)D3, 1,25(OH)2D3 or 3epi25(OH)D with any of the inflammatory proteins. Conclusion. In a non-obese PCOS population matched for age and insulin resistance, circulating inflammatory proteins and matrix metalloproteinases were not elevated and did not correlate with 25(OH)D3, its epimer 3epi25(OH)D or 1,25(OH)2D3 in either control or PCOS women, indicating that the inflammatory response is absent and the vitamin D-metabolite independent in non-obese women with PCOS.

Determination of Vitamin B12 in Milk and Dairy Products by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry

An isotope-dilution liquid chromatography tandem mass spectrometry method was established for the determination of vitamin B12 in milk and dairy products. The samples were spiked with stable isotope-labeled vitamin B12 and digested by pepsin and amylase. The various forms of cobalamin were transformed to cyanocobalamin by potassium cyanide after they were released from the enzymatically digested samples. Cyanocobalamin was extracted and purified by an immunoaffinity SPE cartridge and then measured in multiple reaction monitoring mode (MRM). The linear correlation coefficient (R2) of this method was greater than 0.999 in the range of 2–100 ng/mL. The detection limit and the quantification limit were 0.5 μg/kg and 1.0 μg/kg, respectively. The spiked recoveries ranged from 92.0% to 99.4% at the three spiked levels with the relative standard deviation (RSD) between 1.89% and 4.51%. The measured results of NIST SRM1849a and NIST SRM1869a by the current method are all within the reference value range. The Z value was 0.8 during participating in the FAPAS proficiency test using the developed method in 2021. The method is simple, rapid, accurate, and sensitive, and it is suitable for the determination of vitamin B12 in different types of milk and dairy products such as whey powder, whole milk powder, pure milk, fermented milk, infant formula, and prescription food for special medical purposes.

Towards a novel application of wastewater-based epidemiology in population-wide assessment of exposure to volatile organic compounds

In this study, we investigated the feasibility of detecting 35 urinary biomarkers of volatile organic compounds (VOCs) exposure in community wastewater. 24-h composited municipal wastewater samples were collected from two communities (n = 8) in the southeastern US. Using isotope-dilution liquid chromatography-tandem mass spectrometry, results showed 16 metabolites were detected in wastewater samples, including indicators of exposure to acrolein, acrylonitrile, 1,3-butadiene, crotonaldehyde, n,n-dimethylformamide (DMF), ethylbenzene, nicotine, propylene oxide, styrene, tetrachloroethylene, toluene, and xylene. Additional metabolites qualitatively identified exposure to acrylamide and trichloroethylene. Community 1 (closer proximity to manufacturing facilities) had a greater number of detects (n = 36) and higher VOC loadings, 22,000 mg day−1 per 1000 people, as compared to Community 2 (n = 28), 7100 mg day−1 per 1000 people. Normalizing to nicotine consumption biomarkers to account for differences in smoking behaviors, Community 1 continued to have higher levels of propylene oxide, crotonaldehyde, DMF, and acrylonitrile exposures, VOCs generally sourced from manufacturing activities and vehicle emissions. This is the first study to utilize wastewater to detect urinary biomarkers of VOCs exposure. These preliminary results suggest the WBE approach as a potentially powerful tool to assess community health exposures to indoor and outdoor air pollutants.