Novel plasma metabolite markers of attention-deficit/hyperactivity disorder identified using high-performance chemical isotope labelling-based liquid chromatography-mass spectrometry

<b>Objectives:</b> Metabolites are the intermediate and final products of biological processes and ultimately reflect the responses of these processes to genetic regulation and environmental perturbations, including those involved in attention deficit/hyperactivity disorder (ADHD). <b>Methods:</b> We identified a quantitative profile of plasma metabolites in 58 ADHD patients (mean age 9.0 years, 77.6% males) and 38 healthy control subjects (mean age 10.2 years, 55.3% males) using the high-performance chemical isotope labelling (CIL)-based liquid chromatography-mass spectrometry (LC-MS). Using a volcano plot and orthogonal projections to latent structure-discriminant analysis (OPLS-DA), we determined nine metabolites with differentially expressed levels in ADHD plasma samples. <b>Results:</b> Compared to the control group, the plasma levels of guanosine, O-phosphoethanolamine, phenyl-leucine, hypoxanthine, 4-aminohippuric acid, 5-hydroxylysine, and L-cystine appeared increased in the ADHD patients, whilegentisic acid and tryptophyl-phenylalanine were down-regulated in the patients with ADHD. We found that the plasma levels of all nine metabolites were able to discriminate the ADHD group from the control group. Levels of O-phosphoethanolamine, 4-aminohippuric acid, 5-hydroxylysine, L-cystine, tryptophyl-phenylalanine, and gentisic acid were significantly correlated with clinical ADHD symptoms. <b>Conclusions:</b> This study is the first to use the CIL-based LC-MS to profile ADHD plasma metabolites, and we identified nine novel metabolite biomarkers of ADHD.

**研究目的**：代谢物（metabolites）是生物过程的中间产物与终末产物，可最终反映此类过程对遗传调控及环境扰动的响应，其中亦包含与注意缺陷多动障碍（attention deficit/hyperactivity disorder, ADHD）相关的扰动。**研究方法**：本研究采用基于高效化学同位素标记（chemical isotope labelling, CIL）的液相色谱-质谱联用技术（liquid chromatography-mass spectrometry, LC-MS），对58名ADHD患者（平均年龄9.0岁，男性占比77.6%）与38名健康对照受试者（平均年龄10.2岁，男性占比55.3%）的血浆代谢物进行定量谱图分析。通过火山图与正交偏最小二乘判别分析（orthogonal projections to latent structure-discriminant analysis, OPLS-DA），本研究在ADHD患者血浆样本中筛选出9个差异表达代谢物。**研究结果**：与健康对照组相比，ADHD患者血浆中鸟苷、O-磷酸乙醇胺、苯基亮氨酸、次黄嘌呤、4-氨基马尿酸、5-羟基赖氨酸及L-胱氨酸的水平显著升高；而龙胆酸与色氨酰苯丙氨酸的水平在ADHD患者体内显著下调。本研究证实，上述9种代谢物的血浆水平均可有效区分ADHD组与健康对照组。进一步分析显示，O-磷酸乙醇胺、4-氨基马尿酸、5-羟基赖氨酸、L-胱氨酸、色氨酰苯丙氨酸及龙胆酸的水平与ADHD临床症状呈显著相关。**研究结论**：本研究首次采用基于CIL的LC-MS技术对ADHD患者血浆代谢物进行谱学分析，并筛选得到9种全新的ADHD代谢物生物标志物。