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

J Lipid Res. 2019 May;60(5):1050-1057. doi: 10.1194/jlr.D090233. Epub 2019 Feb 26.


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.

Keywords: cholesterol; familial lecithin:cholesterol acyltransferase deficiency; fish-eye disease; high density lipoprotein; kidney; lecithin:cholesterol acyltransferase; phospholipids.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / blood
  • Filipin / chemistry*
  • Gels / chemistry
  • Humans
  • Lecithin Cholesterol Acyltransferase Deficiency / blood
  • Lecithin Cholesterol Acyltransferase Deficiency / drug therapy*
  • Lecithin Cholesterol Acyltransferase Deficiency / enzymology
  • Lipoprotein-X / blood*
  • Lipoprotein-X / chemical synthesis
  • Lipoprotein-X / chemistry
  • Lipoproteins / blood*
  • Phosphatidylcholine-Sterol O-Acyltransferase / blood*
  • Recombinant Proteins / blood


  • Biomarkers
  • Gels
  • Lipoprotein-X
  • Lipoproteins
  • Recombinant Proteins
  • Filipin
  • Phosphatidylcholine-Sterol O-Acyltransferase