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

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.DOI: http://dx.doi.org/10.7554/eLife.23355.001

Transverse distribution of plasma membrane bilayer cholesterol: Picking sides

“…There is good evidence for the assumption that, at least in the human erythrocyte plasma membrane, all of the phosphatidylcholine and sphingomyelin is localized to the outer leaflet, while the other major phospholipid classes are in the inner leaflet . This would mean that the outer leaflet might contain 55% or more of the phospholipids while the inner leaflet species would comprise the other 45% or less . (The literature values for the plasma membranes of nucleated cells were too diverse to be useful for this calculation.)…”

“…PFO and its derivatives do not bind uniformly to the cholesterol present in membranes and therefore do not report its quantity directly. Rather, these toxins only associate with the available (accessible or chemically active) fraction of the cholesterol, because the bulk of the sterol is complexed stoichiometrically with the bilayer phospholipids and is thus competitively inhibited from binding the probe . The membrane cholesterol concentration dependence of probe binding therefore has a threshold that reflects the relative affinities of the phospholipids and the probes for the sterol .…”

“…A separate issue arises from the authors' study of red cells . The mole ratio of cholesterol to total sphingomyelin in these membranes is ~2.5:1, with only about one‐third of the fatty acid substituents in the sphingomyelin being C24:0 . It follows that each C24:0 chain of the sphingomyelin in the outer‐leaflet would have to constrain multiple cholesterol molecules in the inner leaflet, a striking implication.…”

“…Rather, these toxins only associate with the available (accessible or chemically active) fraction of the cholesterol, because the bulk of the sterol is complexed stoichiometrically with the bilayer phospholipids and is thus competitively inhibited from binding the probe. 22,[57][58][59] The membrane cholesterol concentration dependence of probe binding therefore has a threshold that reflects the relative affinities of the phospholipids and the probes for the sterol. [60][61][62] Calibration with synthetic vesicles of the appropriate phospholipid composition is therefore critical.…”

“…49 The mole ratio of cholesterol to total sphingomyelin in these membranes is 2.5:1, with only about one-third of the fatty acid substituents in the sphingomyelin being C24:0. 59,106 It follows that each C24:0 chain of the sphingomyelin in the outer-leaflet would have to constrain multiple cholesterol molecules in the inner leaflet, a striking implication. A possible test of the C24 sphingomyelin hypothesis would be to examine the transmembrane distribution of cholesterol in the red cells of ungulates (where sphingomyelin replaces phosphatidylcholine and comprises half of the total phospholipids) with rat or mouse red cells, which have minimal sphingomyelin, given that these various species contain about the same abundance of cholesterol per phospholipid.…”