Distinct Differences in Lipoprotein Particle Number Evaluation between GP-HPLC and NMR: Analysis in Dyslipidemic Patients Administered a Selective PPARα Modulator, Pemafibrate

Abstract

We established a method to evaluate the lipid concentrations, size and particle numbers (PNs) of lipoprotein subclasses by gel permeation chromatography (GP-HPLC). Nuclear magnetic resonance (NMR) is widely used to analyze these parameters of lipoprotein subclasses, but differences of the two methods are unknown. Current study compared the PNs of each lipoprotein subclass measured by GP-HPLC and NMR, and assessed the effect of a selective PPAR modulator, pemafibrate. Methods: Lipoprotein profiles of 212 patients with dyslipidemia who participated in the phase 2 clinical trial of a selective PPAR modulator, pemafibrate, were analyzed by two methods, GP-HPLC and NMR, which were performed with LipoSEARCH (Skylight Biotech) and LipoProfile 3 (LabCorp), respectively. GP-HPLC evaluated the PNs of 18 subclasses, consisting of CM, VLDL1-5, LDL1-6, and HDL1-6. NMR evaluated the PNs of 9 subclasses, consisting of large VLDL & CM, medium VLDL, small VLDL, IDL, large LDL, small LDL, large HDL, medium HDL and small HDL. Results: Three major classes, total CM&VLDL, total LDL and total HDL were obtained by grouping of corresponding subclasses in both methods and PNs of these classes analyzed by GP-HPLC were correlated positively with those by NMR. The correlation coefficients in total CM&VLDL, total LDL and total HDL between GP-HPLC and NMR was 0.658, 0.863 and 0.798 (all p 0.0001), respectively. The PNs of total CM&VLDL, total LDL and total HDL analyzed by GP-HPLC was 249.5 51.7nM, 1,679 359 nM and 13,273 1,564 nM, respectively, while those by NMR was 124.6 41.8 nM, 1,514 386 nM and 31,161 4,839 nM, respectively. A marked difference in the PNs between the two methods was demonstrated especially in total HDL.The number of apolipoprotein (Apo) B molecule per one ApoB-containing lipoprotein particle, total CM&VLDL plus total LDL, was 1.10 0.05 by GP-HPLC, while 1.32 0.18 by NMR. The number of ApoA-I per one HDL particle was 3.40 0.17 by GP-HPLC, but only 1.46 0.15 by NMR, much less than reported previously.From the phase 2 clinical trial, randomizing 212 patients to pemafibrate 0.025-0.2 mg BID, fenofibrate 100 mg QD, or placebo groups, pemafibrate reduced the PNs of CM, large VLDL1-VLDL3 and medium VLDL4, but not small VLDL5 by GP-HPLC. It significantly decreased the PNs of smaller LDL and larger HDL particles, but increased those of larger LDL and smaller HDL particles. In contrast, NMR showed marked variations in the effect of pemafibrate on lipoprotein PNs, and no significant size-dependent changes.Conclusions: GP-HPLC evaluates the lipoprotein PNs more accurately than NMR and can be used for assessing the effects of lipid-lowering drugs on lipoprotein subclasses.

First-in-human autologous implantation of genetically modified adipocytes expressing LCAT for the treatment of familial LCAT deficiency

“… Free cholesterol (A), phospholipid (B), triglyceride (D), cholesteryl ester levels in 20 fractions separated by GP-HPLC are shown. Chylomicron (CM), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) fractions are indicated according to Okazaki and Yamashita [ 26 , 28 ]. CE concentration was calculated according to Okazaki and Yamashita [ 26 ].…”

“…Lipoprotein profiles were analyzed using polyacrylamide disc gel electrophoresis using the LipoPhor system (JOKOH CO., LTD., Tokyo) as described [ 24 ]. An improved HPLC analysis termed GP-HPLC was also performed according to the procedure as described [ 25 , 26 , 27 , 28 ]. Polyacrylamide disc gel electrophoresis and GP-HPLC analysis of the serum samples were performed at BML Inc. (Tokyo, Japan) and Skylight Biotech Inc (Akita, Japan), respectively.…”

The profile of HDL-C subfractions and their association with cardiovascular risk in the Hungarian general and Roma populations

“…They are based on different physicochemical characteristics of the HDL molecules and separate the particles according to size, charge, density, etc. "Same" HDL subfractions isolated by different separation methods may contain HDL particles that differ greatly in their properties 52 . This phenomenon makes it considerably more difficult or even impossible to compare studies using different separation methods.…”

Lipoprotein‐subclass particle numbers in children with abdominal obesity

“…There are several methods to assess the lipoprotein PN profile, but nuclear magnetic resonance spectroscopy (NMR) has been most widely used. Recently, another analytical method using high performance liquid chromatography (HPLC) has been established 16,17 and is reported to be more accurate than NMR 18 …”

“…Recently, another analytical method using high performance liquid chromatography (HPLC) has been established 16,17 and is reported to be more accurate than NMR. 18 The aim of this study was to analyze lipoprotein PNs by HPLC and to examine their association with abdominal adiposity and insulin resistance in Japanese children. In addition, the subclass profile in lipoprotein-PNs was also investigated.…”