An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs

Abstract

Introduction Noise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H2) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation. Objectives To present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H2 administration on the perilymph metabolome of noise exposed guinea pigs. Methods The left ear of guinea pigs were exposed to hazardous impulse noise only (Noise, n = 10), noise and H2 (Noise + H2, n = 10), only H2 (H2, n = 4), or untreated (Control, n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF–MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear. Results MVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines. Conclusions A HILIC-UHPLC-Q-TOF–MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H2 treatment.

Comprehensive Peak Characterization (CPC) in Untargeted LC–MS Analysis

“…The algorithm was applied to a dataset from a previously reported study on hydrogen gas attenuation of noise-induced hearing loss in guinea pigs [32]. The full data are available for download via the MetaboLights repository (unique identifier: MTBLS2418).…”

Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics

“…The HILIC method used was adapted from an earlier protocol used for untargeted metabolomics in our lab [ 9 , 10 ]. Adjustments to the method were primarily aimed at minimizing the retention of the compounds that would be collected in the heart-cut in order to minimize the collected volume.…”

“…Unfortunately, validating an untargeted method is a difficult task as it is impossible to ensure that all compounds detectable in the sample are performing to specification. In lieu of untargeted validation methods, a commonly used strategy [ 3 , 9 , 26 , 27 ] is to monitor selected compounds known to be present in the samples. These then serve as proxy measurements of performance for all compounds present.…”

“…The issue of limited sample volume was highlighted to us in a recent study, where we investigated the attenuating effect of hydrogen gas on noise-induced hearing loss (NIHL) in guinea pigs. The sample material used in the study was perilymph, sampled from the inner ear [ 9 , 10 , 11 ]. In the case of perilymph, the maximum volume that can safely be aspirated, without contamination from cerebrospinal fluid (CSF), is less than 1 µL [ 12 ].…”

Caracterização de moléculas com potencial bioativo por espectroscopia de ação e mobilidade iônica acopladas à espectrometria de massas

“…[24][25][26] A possibilidade de analisar misturas complexas, muitas vezes incluindo centenas de analitos, fez com que essas técnicas hifenadas fossem peça fundamental nas estratégias classificadas como ciências ômicas. Temas como metabolômica, 27 proteômica 28 e lipidômica 29 são comuns para cientistas da área de MS e se tornaram destaque ao longo dos últimos anos, fazendo da MS uma das técnicas mais utilizadas para análises clínicas. 30 Recentemente, a técnica de LC-MS foi utilizada com sucesso no desenvolvimento de um método inovador de diagnóstico de pacientes portadores de SARS-COV-2, que causa a doença denominada COVID-19, desenvolvido nacionalmente poucos meses após a confirmação dos primeiros casos no Brasil.…”