Supplementary Material for: Urinary Apolipoprotein C3 Is a Potential Biomarker for Alzheimer’s Disease

<b><i>Introduction:</i></b> Biomarkers of Alzheimer’s disease (AD) that can easily be measured in routine health checkups are desirable. Urine is a source of biomarkers that can be collected easily and noninvasively. We previously reported on the comprehensive profile of the urinary proteome of AD patients and identified proteins estimated to be significantly increased or decreased in AD patients by a label-free quantification method. The present study aimed to validate urinary levels of proteins that significantly differed between AD and control samples from our proteomics study (i.e., apolipoprotein C3 [ApoC3], insulin-like growth factor-binding protein 3 [Igfbp3], and apolipoprotein D [ApoD]). <b><i>Methods:</i></b> Enzyme-linked immunosorbent assays (ELISAs) were performed using urine samples from the same patient and control groups analyzed in the previous proteomics study (18 AD and 18 controls, set 1) and urine samples from an independent group of AD patients and controls (13 AD, 5 mild cognitive impairment [MCI], and 32 controls) from the National Center for Geriatrics and Gerontology Biobank (set 2). <b><i>Results:</i></b> In set 1, the crude urinary levels of ApoD, Igfbp3, and creatinine-adjusted ApoD were significantly higher in the AD group relative to the control group (<i>p</i> = 0.003, <i>p</i> = 0.002, and <i>p</i> = 0.019, respectively), consistent with our previous proteomics results. In set 2, however, the crude urinary levels of Igfbp3 were significantly lower in the AD+MCI group than in the control group (<i>p</i> = 0.028), and the levels of ApoD and ApoC3 did not differ significantly compared to the control group. Combined analysis of all samples revealed creatinine-adjusted ApoC3 levels to be significantly higher in the AD+MCI group (<i>p</i> = 0.015) and the AD-only group (<i>p</i> = 0.011) relative to the control group. <b><i>Conclusion:</i></b> ApoC3 may be a potential biomarker for AD, as validated by ELISA. Further analysis of ApoC3 as a urinary biomarker for AD is warranted.

**引言：** 亟需可在常规健康检查中便捷检测的阿尔茨海默病（Alzheimer's disease, AD）生物标志物。尿液是一种可通过无创方式便捷采集的生物标志物来源。本团队此前曾报道阿尔茨海默病患者尿液蛋白质组的全面图谱，并通过无标记定量（label-free quantification）法鉴定出在AD患者中表达量显著上调或下调的蛋白质。本研究旨在验证本团队此前蛋白质组学研究中鉴定出的、AD组与对照组样本间表达量存在显著差异的三种蛋白质的尿液水平，即载脂蛋白C3（apolipoprotein C3, ApoC3）、胰岛素样生长因子结合蛋白3（insulin-like growth factor-binding protein 3, Igfbp3）以及载脂蛋白D（apolipoprotein D, ApoD）。
**方法：** 本研究采用酶联免疫吸附实验（enzyme-linked immunosorbent assays, ELISAs）对两类尿液样本进行检测：第一类为既往蛋白质组学研究中分析过的同一批患者与对照组样本（18例AD患者、18例健康对照，记为队列1）；第二类为来自日本国立老年医学与老年学研究中心生物样本库的独立队列样本，包含13例AD患者、5例轻度认知障碍（mild cognitive impairment, MCI）患者以及32例健康对照（记为队列2）。
**结果：** 在队列1中，AD组尿液中未经校正的ApoD、Igfbp3水平以及经肌酐校正的ApoD水平均显著高于对照组（分别为*p*=0.003、*p*=0.002和*p*=0.019），与本团队既往蛋白质组学研究结果一致。但在队列2中，AD合并MCI组尿液中未经校正的Igfbp3水平显著低于对照组（*p*=0.028），而ApoD与ApoC3的水平与对照组相比无显著差异。对所有样本的合并分析显示，经肌酐校正的ApoC3水平在AD合并MCI组（*p*=0.015）以及单纯AD组（*p*=0.011）中均显著高于对照组。
**结论：** 经ELISA验证，ApoC3或可成为AD的潜在生物标志物。后续有必要针对ApoC3作为AD尿液生物标志物开展进一步研究。