Real-Time Monitoring of Tricarboxylic Acid Metabolites in Exhaled Breath

Abstract

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathway for cellular respiration in aerobic organisms. It provides and collects intermediates for many other interconnecting pathways and acts as a hub connecting metabolism of carbohydrates, fatty acids, and amino acids. Alteration in intracellular levels of its intermediates has been linked with a wide range of illnesses ranging from cancer to cellular necrosis or liver cirrhosis. Therefore, there exists an intrinsic interest in monitoring such metabolites. Our goal in this study was to evaluate whether, at least the most volatile metabolites of the TCA cycle, could be detected in breath in vivo and in real time. We used secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS) to conduct this targeted analysis. We enrolled six healthy individuals who provided full exhalations into the SESI-HRMS system at different times during 3 days. For the first time, we observed exhaled compounds that appertain to the TCA cycle: fumaric, succinic, malic, keto-glutaric, oxaloacetic, and aconitic acids. We found high intraindividual variability and a significant overall difference between morning and afternoon levels for malic acid, oxaloacetic acid, and aconitic acid, supporting previous studies suggesting circadian fluctuations of these metabolites in humans. This study provides first evidence that TCA cycle could conveniently be monitored in breath, opening new opportunities to study in vivo this important metabolic pathway.

Optimizing Secondary Electrospray Ionization High-Resolution Mass Spectrometry (SESI-HRMS) for the Analysis of Volatile Fatty Acids from Gut Microbiome

“…Therefore, the water with 0.1% formic acid ( v / v ) condition was selected for optimizing further parameters. It has been reported that electrosprays of pure solvents can efficiently ionize gas-phase analytes, and several types of electrospray solutions such as water and methanol have been used for various studies [ 30 , 31 , 32 ]. An earlier study by Sinues et al [ 33 ] examined the efficiency of nano-electrospray ionization using three types of spray solvents, including water, water/methanol (1/1), and methanol, to measure amine-containing compounds such as dimethylamine, ethylamine, and trimethylamine.…”

“…Therefore, 0.01% and 0.1% formic acid concentrations in water were chosen as the optimized and least optimized conditions for VFA analysis, respectively, in the later part of this study. Previously, several types of additives, including formic acid, acetic acid, and ammonium formate, have been used to facilitate the interactions between analytes and electrospray charged droplets to enhance the sensitivity of SESI analysis [ 30 , 32 , 34 ]. It has been proposed that gas-phase analytes could be charged by contacting either electrospray droplets or protons that are generated after the droplets have evaporated [ 33 ].…”

“…Notably, the temperature-related signal intensity changes showed that gradually increased signal from 90 °C up to 130 °C, then decreased intensity was observed at 150 °C. The temperature of the sampling line is one of the influential factors which may affect the rate of analytic efficacy by minimizing adsorption of volatile metabolites onto the line and introducing them into a cylindrical SESI-chamber [ 30 ]. Different temperatures of the sample inlet (80‒160 °C) have been adopted in previous researches using SESI-MS [ 5 , 30 , 34 , 36 , 37 ].…”

“…The temperature of the sampling line is one of the influential factors which may affect the rate of analytic efficacy by minimizing adsorption of volatile metabolites onto the line and introducing them into a cylindrical SESI-chamber [ 30 ]. Different temperatures of the sample inlet (80‒160 °C) have been adopted in previous researches using SESI-MS [ 5 , 30 , 34 , 36 , 37 ]. Similarly, a recent study by Weber et al analyzed breath VOC profiles of children with cystic fibrosis by setting a sampling line at 130 °C [ 8 ].…”

Standardization procedures for real-time breath analysis by secondary electrospray ionization high-resolution mass spectrometry

“…The vast majority of the features typically detected by SESI-HRMS in human breath remain to be positively identified. However, over the last years, we have made a substantial effort to systematically identify the molecular structure for some of these metabolites by combining real-time breath MS/MS analysis and UPLC-MS/MS analysis of exhaled breath condensate [47][48][49][50][51][52][53]. Given the clinical importance of aldehydes, as potential surrogates of oxidative stress [54][55][56][57][58][59], we will concentrate in discussing our findings for a series of three classes of fatty aldehydes [48]: 4-hydroxy-2-alkenals (C x H 2x -2 O 2 ), 2-alkenals (C x H 2x − 2 O), and 4-hydroxy-2,6alkadienals (C x H 2x − 4 O 2 ) with chain lengths ranging from C 8 to C 16 .…”