Lipid peroxidation is a common feature of liver diseases, especially non-alcoholic fatty liver disease (NAFLD). There are limited validated tools to study intra-hepatic lipid peroxidation, especially for small specimen. We developed a semi-quantitative, fully automated immunohistochemistry assay for the detection of 4-hydroxynoneal (4-HNE) protein adducts, a marker of lipid peroxidation, for adaptation to clinical diagnostics and research. We used Hep G2 cells treated with 4-HNE to validate specificity, sensitivity, and dynamic range of the antibody. Staining and semi-quantitative automated readout were confirmed in human needle-biopsy liver samples from subjects with NAFLD and normal liver histology. The ability to detect changes in lipid peroxidation was tested in paired liver biopsies from NAFLD subjects, obtained before and after 4 weeks of treatment with the antioxidant vitamin E (ClinicalTrials.gov NCT01792115, n=21). The cellular calibrator was linear and NAFLD patients had significantly higher levels of 4-HNE adducts compared to controls (p=0.02). Vitamin E treatment significantly decreased 4-HNE (p=0.0002). Our findings demonstrate that 4-HNE quantification by immunohistochemistry and automated image analysis is feasible and able to detect changes in hepatic lipid peroxidation in clinical trials. This method can be applied to archival and fresh samples and should be considered for use in assessing NAFLD histology.

脂质过氧化是肝脏疾病，尤其是非酒精性脂肪肝疾病（NAFLD）的常见特征。有有限的经过验证的工具来研究肝内脂质过氧化，特别是对于小样本。我们开发了一种半定量，全自动免疫组织化学测定法，用于检测脂质过氧化的标记物4-羟基壬醛（4-HNE）蛋白加合物，以适应临床诊断和研究。我们使用经4-HNE处理的Hep G2细胞来验证抗体的特异性，敏感性和动态范围。在来自NAFLD和正常肝组织学受试者的人穿刺活检肝样本中证实了染色和半定量自动读数。在来自NAFLD受试者的成对肝活检中测试了检测脂质过氧化变化的能力，n = 21）。细胞校准物是线性的，并且NAFLD患者的4-HNE加合物水平明显高于对照组（p = 0.02）。维生素E治疗显着降低了4-HNE（p = 0.0002）。我们的发现表明，通过免疫组织化学和自动图像分析对4-HNE进行定量分析是可行的，并且能够在临床试验中检测肝脂质过氧化的变化。此方法可用于存档和新鲜样品，应考虑用于评估NAFLD组织学。