Garcia Erwin, Wolak-Dinsmore Justyna, Wang Zeneng, Li Xinmin S, Bennett Dennis W, Connelly Margery A, Otvos James D, Hazen Stanley L, Jeyarajah Elias J
LipoScience, Laboratory Corporation of America® Holdings, Raleigh, NC, United States.
Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH, United States.
Clin Biochem. 2017 Nov;50(16-17):947-955. doi: 10.1016/j.clinbiochem.2017.06.003. Epub 2017 Jun 15.
Trimethylamine-N-oxide (TMAO) produced by gut microbiota metabolism of dietary choline and carnitine has been shown to be associated with increased risk of cardiovascular disease (CVD) and to provide incremental clinical prognostic utility beyond traditional risk factors for assessing a patient's CVD risk. The aim of this study was to develop an automated nuclear magnetic resonance (NMR) spectroscopy assay for quantification of TMAO concentration in serum and plasma using a high-throughput NMR clinical analyzer.
Key steps in assay development included: (i) shifting the TMAO analyte peak to a less crowded region of the spectrum with a pH buffer/reagent, (ii) attenuating the broad protein background signal in the spectrum and (iii) using a non-negative least squares algorithm for peak deconvolution. Assay performance was evaluated according to Clinical and Laboratory Standards Institute guidelines. A method comparison study was performed to compare TMAO concentrations quantified by NMR and mass spectrometry (MS).
The within-run and within-lab imprecision ranged from 4.3 to 14.5%. Under the acquisition method employed, the NMR assay had a limit of blank, detection and quantitation of 1.6, 3.0 and 3.3μM, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3000μM. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R=0.98).
The NMR based assay provides a simple and accurate measurement of circulating TMAO levels amenable to the high-throughput demands of the clinical chemistry laboratory. Moreover, assay performance enables the levels of TMAO to be quantified in serum or plasma at clinically actionable concentrations for the assessment of cardiovascular disease risks and individualized dietary monitoring.
由肠道微生物群对膳食胆碱和肉碱进行代谢产生的氧化三甲胺（TMAO）已被证明与心血管疾病（CVD）风险增加相关，并且在评估患者CVD风险方面，其能提供超越传统风险因素的额外临床预后效用。本研究的目的是开发一种自动化核磁共振（NMR）光谱分析法，用于使用高通量NMR临床分析仪定量血清和血浆中的TMAO浓度。
分析方法开发的关键步骤包括：（i）使用pH缓冲液/试剂将TMAO分析物峰转移到光谱中较不拥挤的区域，（ii）减弱光谱中宽泛的蛋白质背景信号，以及（iii）使用非负最小二乘法进行峰去卷积。根据临床和实验室标准协会的指南评估分析性能。进行了一项方法比较研究，以比较通过NMR和质谱（MS）定量的TMAO浓度。
批内和批内不精密度范围为4.3%至14.5%。在所采用的采集方法下，NMR分析的空白限、检测限和定量限分别为1.6、3.0和3.3μM。在3.3至3000μM的报告范围内证明了线性关系。使用NMR分析进行的TMAO测量（涉及最少的样品制备）与基于MS的分析获得的值相比良好（R = 0.98）。
基于NMR的分析方法提供了一种简单而准确的循环TMAO水平测量方法，适用于临床化学实验室的高通量需求。此外，分析性能能够在临床可采取行动的浓度下对血清或血浆中的TMAO水平进行定量，以评估心血管疾病风险和进行个性化饮食监测。
