Characterization and quantification of serum lipoprotein subfractions by capillary isotachophoresis: relationships with lipid, apolipoprotein, and lipoprotein levels

Alexandra Schlenck,Bernard Herbeth,Gérard Siest,Sophie Visvikis

doi:10.1016/s0022-2275(20)32436-6

Alexandra Schlenck, Bernard Herbeth + Show 2 more

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https://doi.org/10.1016/s0022-2275(20)32436-6

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Abstract

Human serum lipoproteins are currently defined according to their density as well as according to their electrophoretic mobility. They can be fractionated into discrete subspecies which exhibit variations in their structure and function. Capillary electrophoresis has been suggested to be a potential analytical strategy in understanding metabolic lipoprotein heterogeneity. In a sample of 35 normolipidemic subjects, we analyzed ceramide-labeled serum lipoproteins by capillary isotachophoresis linked to laser-induced fluorescent detection. Capillary isotachophoresis showed advantage to be an automated, rapid (6 min) and reproducible (CV < 7%) separation mode, on-line monitoring lipoprotein subfractions according to net charge. HDL were separated into three subfractions: i) the fast migrating HDL correlated positively with serum apoA-I (P < 0.05) and negatively with triglyceride (P < 0.01) concentrations, ii) the intermediate migrating HDL involved in HDL-cholesterol delivery and inversely related to LDL particles concentration (P < 0.001), and iii) the slow migrating prebeta(1)HDL. Triglyceride level was significantly associated with two fractions: i) the VLDL fraction correlated positively with apoE serum concentration (P < 0.01), and ii) the IDL fraction closely and positively associated with apoC-III-containing lipoprotein level (P < 0.001). Two LDL subfractions were positively related to LDL-cholesterol (0.05 </= P < 0.01) and might characterize, respectively, small dense and large buoyant LDL subfractions: i) the fast migrating LDL, positively linked to apoB concentration and to LpCIII:B (P < 0.01) reflecting altered IDL metabolism, and ii) the slow migrating LDL. Analytical capillary isotachophoresis of fluorescent-stained lipoprotein subfractions might represent an efficient qualitative and quantitative tool which would afford complementary information on lipoprotein metabolism to current clinical lipoprotein analysis.

Highlights

Human serum lipoproteins are currently defined according to their density as well as according to their electrophoretic mobility
As lipoprotein cholesterol content is usually considered the most accurate and informative parameter to quantify lipoprotein groups, Schmitz et al [25] tried to estimate both high density lipoprotein (HDL)-C and low density lipoprotein (LDL)-C by applying serum cholesterol concentration, respectively, to the relative HDL- and LDLfluorescent peaks area. They published high correlation coefficients between usual techniques for HDL-C and LDL-C quantification and capillary isotachophoresis (cITP) calculations, and concluded that ceramide staining was directly proportional to lipoprotein cholesterol content
In our study a high order and statistically significant correlation was observed between LDL-cholesterol calculations according to Friedewald and cITP formulas (r ϭ 0.87, P Ͻ 0.001), and with serum apoB concentration measured by immunoturbidimetric assay (r ϭ 0.80, P Ͻ 0.001)

Summary

Introduction

Human serum lipoproteins are currently defined according to their density as well as according to their electrophoretic mobility. Recent advances in capillary electrophoresis (CE) technology has led several investigators to examine CE as one of the key analytical methodologies for future apolipoprotein and lipoprotein analysis in very small volumes of biological samples [15,16,17,18] It implied generally preparative ultracentrifugation steps and did not allow the detailed distinction of lipoprotein subfractions [19]. Analytical isotachophoresis differs from standard zone electrophoresis by using a discontinuous electrolyte system This system is composed of a leading and a terminating electrolyte, which exhibit, respectively, a higher and a slower electrophoretic mobility than the ionic species contained in the mixed sample. The cITP steady state has a special feature of concentrating effect which may be characterized by a constant concentration throughout each sample zone, and directly proportional to concentration of the leading substance [21]

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