Oldham Dean, Wang Hong, Mullen Juliet, Lietzke Emma, Sprenger Kayla, Reigan Philip, Eckel Robert H, Bruce Kimberley D
Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Front Cardiovasc Med. 2022 Jul 14;9:926631. doi: 10.3389/fcvm.2022.926631. eCollection 2022.
Lipoprotein lipase (LPL) plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides (TGs) in packaged lipoproteins. Since hypertriglyceridemia (HTG) is a major risk factor for cardiovascular disease (CVD), the leading cause of death worldwide, methods that accurately quantify the hydrolytic activity of LPL in clinical and pre-clinical samples are much needed. To date, the methods used to determine LPL activity vary considerably in their approach, in the LPL substrates used, and in the source of LPL activators and inhibitors used to quantify LPL-specific activity, rather than other lipases, e.g., hepatic lipase (HL) or endothelial lipase (EL) activity. Here, we describe methods recently optimized in our laboratory, using a synthetic ApoC-II peptide to activate LPL, and an n-terminal Angiopoietin-Like 4 fragment (nAngptl4) to inhibit LPL, presenting a cost-effective and reproducible method to measure LPL activity in human post-heparin plasma (PHP) and in LPL-enriched heparin released (HR) fractions from LPL secreting cells. We also describe a modified version of the triolein-based assay using human serum as a source of endogenous activators and inhibitors and to determine the relative abundance of circulating factors that regulate LPL activity. Finally, we describe how an ApoC-II peptide and nAngptl4 can be applied to high-throughput measurements of LPL activity using the EnzChek™ fluorescent TG analog substrate with PHP, bovine LPL, and HR LPL enriched fractions. In summary, this manuscript assesses the current methods of measuring LPL activity and makes new recommendations for measuring LPL-mediated hydrolysis in pre-clinical and clinical samples.
脂蛋白脂肪酶（LPL）通过水解包装脂蛋白中的甘油三酯（TGs）在预防血脂异常方面发挥关键作用。由于高甘油三酯血症（HTG）是心血管疾病（CVD）的主要危险因素，而心血管疾病是全球主要死因，因此非常需要能够准确量化临床和临床前样本中LPL水解活性的方法。迄今为止，用于测定LPL活性的方法在其方法、所用的LPL底物以及用于量化LPL特异性活性而非其他脂肪酶（如肝脂肪酶（HL）或内皮脂肪酶（EL）活性）的LPL激活剂和抑制剂来源方面差异很大。在此，我们描述了最近在我们实验室中优化的方法，使用合成的载脂蛋白C-II肽激活LPL，并使用N端血管生成素样4片段（nAngptl4）抑制LPL，提出了一种经济高效且可重复的方法来测量人肝素后血浆（PHP）和来自LPL分泌细胞的富含LPL的肝素释放（HR）组分中的LPL活性。我们还描述了一种基于三油酸甘油酯测定法的改进版本，使用人血清作为内源性激活剂和抑制剂的来源，并确定调节LPL活性的循环因子的相对丰度。最后，我们描述了如何使用EnzChek™荧光TG类似物底物、PHP、牛LPL和富含HR LPL的组分将载脂蛋白C-II肽和nAngptl4应用于LPL活性的高通量测量。总之，本手稿评估了当前测量LPL活性的方法，并对在临床前和临床样本中测量LPL介导的水解提出了新的建议。
