Center for Preventive Cardiology, Knight Cardiovascular Institute, Oregon Health & Science University, 3161 SW Pavilion Loop, Mail Code UHN62, Portland, OR, 97239, USA.
Sci Rep. 2022 Jun 1;12(1):9138. doi: 10.1038/s41598-022-13040-4.
High levels of circulating Lipoprotein (a) [Lp(a)] are an independent risk factor for CVD. One of the major limitations to investigating Lp(a) biology is the need for large volumes of plasma (4-10 mL) for its isolation. We developed an isolation technique requiring only 0.4 mL of plasma yielding an enriched Lp(a) fraction suitable for compositional and functional studies. We collected plasma from patients (n = 9) in EDTA presenting to our Center for Preventive Cardiology for CVD risk management and with circulating Lp(a) > 66 mg/dL. 0.4 mL of plasma was added to 90 µL of potassium bromide (1.33 g/mL) and subjected to our two-step density-gradient ultracentrifugation method. The first step separates VLDL and LDL from the Lp(a) and HDL fractions and the second step further separates VLDL from LDL and Lp(a) from HDL. Lp(a) is then dialyzed for up to 24 h in potassium phosphate buffer. We performed cholesterol gel electrophoresis, immunoblotting and LC-MS/MS proteomics on isolated lipoprotein fractions to confirm fraction enrichment. Functional studies including Lp(a)-dependent induction of macrophage gene expression and cholesterol efflux inhibition were performed on isolated Lp(a) to confirm its preserved bioactivity. Lp(a) yields (264 ± 82.3 µg/mL on average) correlated with Lp(a) plasma concentrations (r = 0.75; p < 0.01) and represented the relative distribution of circulating apo(a) isoforms. Proteomic analyses confirm lipoprotein fraction separation. Functional integrity was confirmed by the findings that isolated Lp(a) inhibited plasminogen-dependent cholesterol efflux in HEK293T cells expressing ABCA1 and increased expressions of Il1b, Nos2 and Ccl2. We developed a small-volume isolation technique for Lp(a) suited for a range of applications used in biomedical research. The use of this technique circumvents volume-dependent limitations and expands our ability to investigate the mysteries of this deleterious lipoprotein.
高水平的循环脂蛋白(a) [Lp(a)]是心血管疾病的独立危险因素。研究 Lp(a) 生物学的主要限制之一是需要大量的血浆（4-10 mL）来分离它。我们开发了一种仅需 0.4 mL 血浆的分离技术，可获得富含 Lp(a) 的部分，适合组成和功能研究。我们从我院预防心脏病学中心接受心血管疾病风险管理的、循环 Lp(a)>66mg/dL 的患者（n=9）收集 EDTA 血浆。将 0.4 mL 血浆加入 90µL 溴化钾（1.33g/mL）中，然后进行我们的两步密度梯度超速离心法。第一步将 VLDL 和 LDL 与 Lp(a) 和 HDL 部分分离，第二步进一步将 VLDL 与 LDL 分离，Lp(a)与 HDL 分离。然后将 Lp(a)在磷酸钾缓冲液中透析长达 24 小时。我们对分离的脂蛋白部分进行胆固醇凝胶电泳、免疫印迹和 LC-MS/MS 蛋白质组学分析，以确认部分富集。对分离的 Lp(a)进行包括 Lp(a)依赖性诱导巨噬细胞基因表达和胆固醇流出抑制在内的功能研究，以确认其保留的生物活性。Lp(a) 的产量（平均 264±82.3µg/mL）与 Lp(a) 血浆浓度相关（r=0.75;p<0.01），并代表循环 apo(a) 同种型的相对分布。蛋白质组学分析证实了脂蛋白部分的分离。通过发现分离的 Lp(a)抑制表达 ABCA1 的 HEK293T 细胞中纤溶酶原依赖性胆固醇流出，并增加 Il1b、Nos2 和 Ccl2 的表达，证实了其功能完整性。我们开发了一种适用于生物医学研究中多种应用的小体积 Lp(a) 分离技术。该技术的使用规避了与体积相关的限制，并扩大了我们研究这种有害脂蛋白奥秘的能力。
