Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin.

Rima S Chakrabarti,Arun Radhakrishnan,Julia Kozlitina,Jonathan C Cohen,Helen H Hobbs,Austin Gay,Sally A Ingham

doi:10.7554/elife.23355

Rima S Chakrabarti, Arun Radhakrishnan + Show 5 more

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https://doi.org/10.7554/elife.23355

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Abstract

Cholesterol partitions into accessible and sequestered pools in cell membranes. Here, we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals, but varied >10 fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessibility. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans.

Highlights

Cholesterol is an essential component of vertebrate cell membranes (Goldstein et al, 1979)
A recombinant protein consisting of the cholesterol binding domain 4 (D4) of anthrolysin O (ALO) was purified from E. coli and covalently modified with a maleimide-linked fluorescent dye (Alexa Fluor 488) (Gay et al, 2015)
We examined the relationship between fALOD4 binding and plasma lipid and lipoprotein levels after adjusting for age, gender and ethnicity. fALOD4 binding to red blood cells (RBCs) was not related significantly to plasma levels of total cholesterol, low-density lipoprotein-cholesterol (LDL-C) or high-density lipoprotein (HDL)-C levels but was inversely related (p=0.0018) to plasma TG levels (Figure 7)

Summary

Introduction

Cholesterol is an essential component of vertebrate cell membranes (Goldstein et al, 1979). Membrane cholesterol is supplied by the diet or synthesized endogenously from acetyl coenzyme A. All cells in the body except red blood cells (RBCs) synthesize cholesterol. An amount of cholesterol equivalent to the sum of what is made in the cells and acquired from the diet must be removed from the body. The major pathway for cholesterol excretion in humans is via the biliary system. The proteins and processes by which cholesterol is secreted into the bile, either directly, or after conversion to bile acids, have been well characterized (Berge et al, 2000; Russell, 2009). The process by which cholesterol from peripheral tissues is delivered to the liver for excretion remains poorly defined

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