1,2-Propylene Glycol: A Biomarker of Exposure Specific to e-Cigarette Consumption

Over the past decade, new emerging tobacco and nicotine-delivery products have changed the tobacco landscape. Especially, electronic cigarettes (ECs) have been suggested to be considered for tobacco harm reduction, reinforcing the need to identify novel biomarkers of exposure (BoE) specific to the EC use as this would complement exposure assessment and product compliance monitoring. Therefore, a sensitive LC-MS/MS method for the quantification of 1,2-propylene glycol (PG) and glycerol (G), the main e-liquid constituents, was established. PG and G were analyzed in plasma and urine samples from a clinical study comparing five nicotine product user groups, users of combustible cigarettes (CC), electronic cigarettes (EC), heated tobacco products (HTP), oral tobacco (OT), and oral/dermal nicotine delivery products (used for nicotine replacement therapy, NRT) with a control group of non-users (NU). Data demonstrate significantly elevated PG levels in urine and plasma in EC users compared to users of CC, HTP, NRT, OT as well as NU. In addition, PG in plasma and urine of vapers significantly correlated with nicotine (plasma) and total nicotine equivalents (urine), biomarkers reflecting product consumption, emphasizing the high specificity of PG as a BoE for EC consumption. We therefore suggest the use of PG as BoE in urine and/or plasma in order to monitor EC use compliance in exposure assessments.

SEEDING to enable sensitive electrochemical detection of biomarkers in undiluted biological samples

The interface between an electrode and a liquid plays a critical role in the overall performance of electrochemical biosensors. Surface morphology and roughness affect key parameters, such as the active area, diffusion profiles, and apparent electron transfer kinetics, whereas porosity may hinder the diffusion of fouling proteins. However, there is no simple and rapid method compatible with photolithographic electrodes to generate both nanostructured and porous surfaces. Herein, we demonstrate the interplay between the preferential etching of chloride and surfactant-assisted anisotropic gold reduction to create homogeneous, nanostructured, and nanoporous substrates on photolithographic gold electrodes within a minute and without using templates. We coined this process, SEEDING, that is, Surfactant-based Electrochemical Etch-Deposit Interplay for Nanostructure/Nanopore Growth. SEEDING on electrodes enhanced the sensitivity and anti-biofouling capabilities, enabling direct analysis of small molecules, proteins, and cancer-derived extracellular vesicles in complex biological fluids such as undiluted plasma and urine samples.

A Pilot Study of miRNA Expression Profile as a Liquid Biopsy for Full-Marathon Participants

Exosomal microRNA (miRNA) in plasma and urine has attracted attention as a novel diagnostic tool for pathological conditions. However, the mechanisms of miRNA dynamics in the exercise physiology field are not well understood in terms of monitoring sports performance. This pilot study aimed to reveal the miRNA dynamics in urine and plasma of full-marathon participants. Plasma and urine samples were collected from 26 marathon participants before, immediately after, 2 h after, and one day after a full marathon. The samples were pooled, and exosomal miRNAs were extracted and analyzed using next-generation sequencing. We determined that the exosomal miRNA expression profile changed under time dependency in full marathon. New uncharacterized exosomal miRNAs such as hsa-miR-582-3p and hsa-miR-199a-3p could be potential biomarkers reflecting physical stress of full marathon in plasma and urine. In addition, some muscle miRNAs in plasma and urine have supported the utility for monitoring physical stress. Furthermore, some inflammation-related exosomal miRNAs were useful only in plasma. These results suggest that these exosomal miRNAs in plasma and/or urine are highly sensitive biomarkers for physical stress in full marathons. Thus, our findings may yield valuable insights into exercise physiology.

1299Association between environmental cadmium exposure and plasma and urinary metabolite profiles in Japanese cohort study

Background The purpose of this study was to identify plasma and urinary metabolites that can be used to better identify the effects of cadmium exposure than N-acetyl-β-D-glucosaminidase (NAG) using capillary electrophoresis - mass spectrometry (CE-MS). Methods Urinary cadmium (U-Cd) was measured as an indicator of cadmium exposure. Fasting plasma and urine samples were collected from 1,412 men and 2,022 women in Tsuruoka Metabolomics Cohort Study. Charged 94 plasma and 123 urinary metabolites were detected and determined. Regression analysis was performed for urinary NAG, plasma, and urinary metabolites as dependent variables and U-Cd in quartiles as an independent variable. Multivariate regression model included age, SBP, smoke, rice intake, BMI, HbA1c, LDLc, alcohol consumption, physical activity, educational history, dietary energy intake, urinary Na/K ratio, and uric acid. Results The mean U-Cd of the population was 2.65 μg/g creatinine (SD: 1.63). NAG was positively associated with U-Cd, but the association was not significant with lower U-Cd quartiles. In the plasma metabolites, 10 metabolites had significantly negative association with U-Cd in all models and Cd quartiles. Among urinary metabolites, 27 metabolites had significantly positive association with U-Cd. Alanine was negatively associated with U-Cd in urinary metabolites. The trend test also showed significant dose-response trends for 9 plasma and all 28 urinary metabolites (p < 0.05). Conclusions We found that the levels of Cd exposure, which did not cause an increase in NAG, caused changes in plasma and urinary metabolites. Key messages This study indicated that metabolomics might be promising and useful as markers of Cd exposure.

Clinical Mass Spectrometry Discovered Human IgG Sialylation as a Potential Biosignature for Kidney Function

Immunoglobulin G (IgG) N-glycosylation was discovered to have an association with inflammation status, which has the potential to be a novel biomarker for kidney diseases. In this study, we applied an ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method to plasma and urine samples from 57 individuals with different levels of kidney function. Natural abundances of total IgG, IgG1, IgG2, and IgG3 subclasses in plasma showed positive correlations to the estimated glomerular filtration rates (eGFRs). Eighteen IgG glycopeptides also showed positive correlations. In contrast, higher IgG amounts were found in urine samples from participants with lower eGFR values. After normalizing IgG glycopeptides from plasma to their respective protein amounts, H4N4F1S1-IgG1 (r = 0.37, p = 0.0047, significant) and H5N4F1S1-IgG1 (r = 0.25, p = 0.063, marginally significant) were the two glycopeptides that still had positive correlations with eGFRs. The results showed that the UHPLC-MS/MS method is capable of investigating IgG profiles, and monitoring IgG and glycosylation patterns is worthy of further clinical application for kidney disease.

Tyrosine Supplementation A Nutraceutical Approach to Counter Heat Stress Induced Cognitive Decline

Supplementation of tyrosine, non-essential amino acid, and precursor of catecholamine was found to ameliorate the heat-induced alterations in latencies of event-related potential P300 and contingent negative variation. Here we present the effect of tyrosine supplementation on heat stress (exposure to ambient temperature 45 oC and relative humidity 30 %) induced alterations in behavior (attention, mood) and levels of plasma monoamines. Ten healthy male participants received a placebo food bar or tyrosine-containing food bar (6.5 g in 50 g) 90 min before heat stress exposure of 90 min. Plasma and urine samples were assayed for catecholamine levels, their precursor, and metabolites using high-performance liquid chromatography. A computer-based automated test battery was used to assess attention and mood by profile of mood states questionnaire. A significantly higher plasma tyrosine (p<0.001) leading to an increased norepinephrine (p<0.05) levels in the tyrosine supplemented group was observed. Selective (p<0.001) and sustained attention (p<0.02) in the tyrosine group were significantly better compared to the placebo group. Reaction time and anger scores decreased (p<0.001) with tyrosine supplementation. It may be concluded that tyrosine supplementation improves heat stress-induced decrement in attention by maintaining the synthesis and turnover of norepinephrine.

Development of an Accurate Mass Retention Time Database for Untargeted Metabolomic Analysis and Its Application to Plasma and Urine Pediatric Samples

Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.

A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

Comparison of the Metabolic Profiles in the Plasma and Urine Samples Between Autistic and Typically Developing Boys: A Preliminary Study

Background: Autism spectrum disorder (ASD) is defined as a pervasive developmental disorder which is caused by genetic and environmental risk factors. Besides the core behavioral symptoms, accumulated results indicate children with ASD also share some metabolic abnormalities.Objectives: To analyze the comprehensive metabolic profiles in both of the first-morning urine and plasma samples collected from the same cohort of autistic boys.Methods: In this study, 30 autistic boys and 30 tightly matched healthy control (HC) boys (age range: 2.4~6.7 years) were recruited. First-morning urine and plasma samples were collected and the liquid chromatography–mass spectrometry (LC-MS) was applied to obtain the untargeted metabolic profiles. The acquired data were processed by multivariate analysis and the screened metabolites were grouped by metabolic pathway.Results: Different discriminating metabolites were found in plasma and urine samples. Notably, taurine and catechol levels were decreased in urine but increased in plasma in the same cohort of ASD children. Enriched pathway analysis revealed that perturbations in taurine and hypotaurine metabolism, phenylalanine metabolism, and arginine and proline metabolism could be found in both of the plasma and urine samples.Conclusion: These preliminary results suggest that a series of common metabolic perturbations exist in children with ASD, and confirmed the importance to have a comprehensive analysis of the metabolites in different biological samples to reveal the full picture of the complex metabolic patterns associated with ASD. Further targeted analyses are needed to validate these results in a larger cohort.