Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo

Lita A Freeman,Robert D Shamburek,Maureen L Sampson,Edward B Neufeld,Masaki Sato,Sotirios K Karathanasis,Alan T Remaley

doi:10.1194/jlr.d090233

Abstract

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

Highlights

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC)
Synthetic LP-X was prepared in vitro from purified FC and phosphatidylcholine and various dilutions (0.2–200 mg/dl cholesterol) were subjected to electrophoresis on duplicate agarose gels, which were stained with either filipin to detect FC or Sudan Black to detect neutral lipids
We made several potentially important findings related to lipoprotein abnormalities in FLD and have explored the feasibility of using LP-X as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy

Summary

Introduction

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.—Freeman, L. Patients with fish-eye disease (FED) have partial LCAT deficiency, with some residual esterification of FC in apoB-containing lipoproteins (apoB-Lps) but not HDL [1]. These patients have reduced HDL-C and corneal opacities, but do not develop renal disease, presumably because they do not form LP-X [11, 12].

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Conclusion