Investigating the binding products between human apolipoproteins and oxidized...

A micellar electrokinetic chromatography (MEKC) method has been developed to investigate the binding products between human apolipoproteins (Apos) and oxidized 1-palmitoyl-2-arachidonoyl-sn‑glycero-3-phosphocholine (ox-PAPC) products. The optimal MEKC separation buffer was composed of a solution mixture of 10 mM sodium phosphate, 50 mM bile salts (50 % sodium cholate and 50 % sodium deoxycholate), 30 % (v/v)1-propanol and 70 % (v/v) water, pH 7.4. The optimal MEKC sample buffer was composed of 70 % (v/v) PBS buffer and 30 % (v/v) MeOH. The selected separation voltage was 20 kV, and the capillary temperature was 25℃. The MEKC profiles of ox-PAPC products showed good separations and repeatability. The MEKC profiles of apos and their binding products also showed good repeatability. For the analysis of native PAPC (n-PAPC), the method is linear in the range of 0.00-6.00 mg/mL with a correlation coefficient 0.9984. The concentration limit of detection (LOD) is 0.29 mg/mL. The concentration limit of quantitation (LOQ) is 0.98 mg/mL. The binding reactions between several important human apolipoproteins (Apos A-I, A-II, C-I, C-II, C-III and E) and native PAPC, ox-PAPC products have been investigated. The concentrations of Apos and ox-PAPC products for binding reactions have been examined. The optimal binding buffer selected was 70 % (v/v) PBS buffer and 30 % (v/v) MeOH. The binding reaction was performed at 37 ℃ for 3 hr. The results indicated that ox-PAPC products bound to Apos A-I, A-II, C-I, and E more strongly than n-PAPC. However, both ox-PAPC products and n-PAPC did not bind to Apos C-II and C-III strongly. The results suggested pro-inflammatory properties of Apos A-I, A-II, C-I, and E, and implied one of the molecular mechanisms resulting in dysfunctional HDL particles. This study also demonstrated the feasibility of investigating the binding reactions between human apolipoproteins and ox-PAPC products by MEKC.