Mavel Sylvie, Lefèvre Antoine, Bakhos David, Dufour-Rainfray Diane, Blasco Hélène, Emond Patrick
UMR 1253, iBrain. Université de Tours, Inserm, Tours, France.
Service ORL et Chirurgie Cervico-Faciale, CHRU de Tours, Blvd Tonnellé, 37044, Tours, France.
Hear Res. 2018 Sep;367:129-136. doi: 10.1016/j.heares.2018.05.016. Epub 2018 May 22.
Although there is some data from animal studies, the metabolome of inner ear fluid in humans remains unknown. Characterization of the metabolome of the perilymph would allow for better understanding of its role in auditory function and for identification of biomarkers that might allow prediction of response to therapeutics. There is a major technical challenge due to the small sample of perilymph fluid available for analysis (sub-microliter). The objectives of this study were to develop and validate a methodology for analysis of perilymph metabolome using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Due to the low availability of perilymph fluid; a methodological study was first performed using low volumes (0.8 μL) of cerebrospinal fluid (CSF) and optimized the LC-HRMS parameters using targeted and non-targeted metabolomics approaches. We obtained excellent parameters of reproducibility for about 100 metabolites. This methodology was then used to analyze perilymph fluid using two complementary chromatographic supports: reverse phase (RP-C18) and hydrophilic interaction liquid chromatography (HILIC). Both methods were highly robust and showed their complementarity, thus reinforcing the interest to combine these chromatographic supports. A fingerprinting was obtained from 98 robust metabolites (analytical variability <30%), where amino acids (e.g., asparagine, valine, glutamine, alanine, etc.), carboxylic acids and derivatives (e.g., lactate, carnitine, trigonelline, creatinine, etc.) were observed as first-order signals. This work lays the foundations of a robust analytical workflow for the exploration of the perilymph metabolome dedicated to the research of biomarkers for the diagnosis/prognosis of auditory pathologies.
虽然有一些来自动物研究的数据，但人类内耳液的代谢组仍然未知。对内淋巴代谢组的表征将有助于更好地理解其在听觉功能中的作用，并有助于识别可能预测治疗反应的生物标志物。由于可用于分析的内淋巴液样本量很小（亚微升），存在重大技术挑战。本研究的目的是开发和验证一种使用液相色谱-高分辨率质谱（LC-HRMS）分析内淋巴代谢组的方法。由于内淋巴液的可获得性较低，首先使用少量（0.8μL）脑脊液（CSF）进行了方法学研究，并使用靶向和非靶向代谢组学方法优化了LC-HRMS参数。我们获得了约100种代谢物的出色重现性参数。然后使用两种互补的色谱载体：反相（RP-C18）和亲水相互作用液相色谱（HILIC）来分析内淋巴液。两种方法都非常稳健，并显示出它们的互补性，因此增强了结合这些色谱载体的兴趣。从98种稳健的代谢物（分析变异性<30%）中获得了指纹图谱，其中氨基酸（如天冬酰胺、缬氨酸、谷氨酰胺、丙氨酸等）、羧酸及其衍生物（如乳酸、肉碱、胡芦巴碱、肌酐等）被观察为一级信号。这项工作为探索内淋巴代谢组奠定了稳健分析流程的基础，该代谢组致力于研究听觉疾病诊断/预后的生物标志物。
