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. To maintain cholesterol balance, 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). In contrast, the process by which cholesterol from peripheral tissues is delivered to the liver for excretion remains poorly defined.

A mechanism for the centripetal trafficking of cholesterol from extrahepatic tissues to the liver, which has been termed reverse cholesterol transport, was first conceptualized by Glomset (1968). That work, and subsequent studies, have implicated high density lipoproteins (HDL) as an essential component of this pathway (Bailey, 1964) (Figure 1). In the first step of the Glomset model, HDL acquires free cholesterol from cell membranes in peripheral tissues. Free cholesterol is transferred from cells to HDL by ATP binding cassette, subfamily A, member 1 (ABCA1) (Lawn et al., 1999). The free cholesterol in the HDL particle is then esterified with long-chain fatty acids by lecithin-cholesterol acyltransferase (LCAT) (Glomset, 1968) and sequestered in a hydrophobic core below the phospholipid surface of HDL (Stoffel et al., 1974). This step is critical to the delivery of cholesterol to the liver; sequestration in the HDL core decreases the propensity of cholesterol to exchange with cell membranes, thus limiting the retrograde transfer of cholesterol from HDL back to the tissues. The cholesteryl ester is then delivered to the liver either directly via a cell surface receptor, scavenger receptor class B member 1 (SCARB1) (Acton et al., 1996), or indirectly after transferring the cholesterol to Apolipoprotein (Apo)B-containing lipoproteins, which then undergo endocytosis by the low density lipoprotein receptor (LDLR) (Figure 1). Several components of the HDL-mediated reverse cholesterol transport pathway have been supported by studies in cultured cells and in vivo (Lewis and Rader, 2005).

Figure 1

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Despite data implicating HDL as a central component of the reverse cholesterol transport pathway, some observations in murine models of HDL deficiency are not compatible with an essential role for HDL in the transport of cholesterol from peripheral tissues to the liver and bile (Xie et al., 2009; Jolley et al., 1998) (Figure 1). Mice lacking ABCA1, or ApoA1, the major structural protein of HDL, have very low HDL levels and yet have no alterations in cholesterol turnover in peripheral tissues (Xie et al., 2009; Jolley et al., 1998).

Glomset proposed that RBCs may participate in the transport of cholesterol from tissues to the liver (Glomset, 1968). Approximately 50% of circulating cholesterol is carried in RBC membranes and tracer studies in humans indicate that the magnitude of the cholesterol flux through RBCs is comparable to the total efflux of free cholesterol from tissues (Turner et al., 2012). Cholesterol exchanges freely between RBCs, plasma, circulating lipoproteins, and cells (Hagerman and Gould, 1951) (Figure 1). More recently, Smith and colleagues (Hung et al., 2012) showed that reducing the number of RBCs in ApoA1 knockout mice, which have little to no circulating HDL, decreases the transfer of radiolabeled cholesterol from macrophages to feces.

For RBCs to participate in reverse cholesterol transport, the cholesterol must be located in the outer leaflet of the RBC membrane, and thus accessible for transfer to proteins and cells. The accessibility of cholesterol in a membrane is related to its chemical activity (McConnell and Radhakrishnan, 2003), alternatively referred to as its fugacity (tendency to flee). The chemical activity of cholesterol generally increases with increasing cholesterol concentration. However, at lower concentrations, complex formation with phospholipids can suppress the chemical activity of cholesterol below what would be expected in the absence of lipid interactions. At higher concentrations, when phospholipids become limiting, the chemical activity of cholesterol rises. Sharp rises in chemical activity have been detected in model membranes by measurements of cholesterol accessibility, as assayed by cyclodextrin extraction (Radhakrishnan and McConnell, 2000; Litz et al., 2016), oxidation by cholesterol oxidase (Ahn and Sampson, 2004; Lange et al., 2013), and binding of cholesterol-dependent cytolysins such as perfringolysin O (PFO) or anthrolysin O (ALO) (Heuck et al., 2000; Gay et al., 2015). A sharp rise in the chemical activity of RBC membrane cholesterol has been observed using the cholesterol oxidase assay to assess cholesterol accessibility (Lange et al., 1980). In all of these cases, the increase in cholesterol accessibility with increasing cholesterol concentration was sigmoidal, rather than linear, with little change until a threshold concentration was reached. Once the cholesterol content of the membrane exceeded this threshold, accessibility of membrane cholesterol increased sharply.

The partitioning of cholesterol between accessible and sequestered pools may allow RBCs to serve as vehicles for cholesterol transport from peripheral tissues to the liver. As a first step towards elucidating the relative contributions of the accessible and sequestered pools of RBC cholesterol to cholesterol transport, we have developed an assay using ALO, a cholesterol-binding toxin produced by Bacillus anthracis (Bourdeau et al., 2009), to measure the relative amount of accessible cholesterol in RBC membranes.

Using this assay, we show that RBC cholesterol accessibility is stable within individuals, but varies over a more than ten-fold range between individuals. To address the basis for the inter-individual variation in accessible RBC cholesterol, we examined the effect of demographic, physiologic, and biochemical differences among individuals on this newly described trait. Whereas age and gender had modest effects on accessible RBC cholesterol, significant ethnic differences were apparent in RBC cholesterol accessibility. The variation in cholesterol accessibility was not related to the cholesterol content of the membrane or to pronase-accessible proteins in the RBC membrane, but rather to the content of other membrane lipids and to levels of circulating triglycerides (TG). Finally, we find that a group of patients on hemodialysis, who have more coronary heart disease than anticipated based on known risk factors (Levey et al., 1998), have higher RBC cholesterol accessibility.

Here, we describe a new assay that measures the relative amount of accessible cholesterol in the membranes of RBCs. The assay is highly reproducible and captures a new quantitative trait in humans. The major finding of the study is that the amount of accessible cholesterol in RBC membranes is stable within an individual but varies >10 fold among individuals. The high degree of inter-individual variation was unrelated to total cholesterol content of the RBC membrane and modestly affected by pronase-sensitive changes in protein content of the RBC membrane. Differences in RBC phospholipid composition of the RBC membrane accounted for 17.6% of the inter-individual variability (Table 5). Cholesterol accessibility in RBCs varied significantly between ethnic groups and correlated indirectly with plasma levels of TG (Figure 7), but not with the morphology or hemoglobin content of the RBCs (Figure 6). Thus, we have shown that the level of accessible cholesterol in RBCs in the population varies significantly among individuals in a stable fashion and we have identified several factors, both intrinsic and extrinsic to the RBC membrane, that contribute to the variation in RBC cholesterol accessibility. The significance of this variability on the trafficking of cholesterol among plasma components and tissues or if it relates to human disease awaits further study.

Among individuals, relative fALOD4 binding to RBCs did not correlate with the total cholesterol content of the RBC membranes (Figure 4C). Thus, the cholesterol in the RBC membrane that is accessible to fALOD4 represents a distinct pool (or state) of cholesterol, which is not reflected by direct assays of RBC cholesterol mass. The striking reproducibility of relative fALOD4 binding to RBCs within a given individual (Figure 3) indicates that the factors that govern RBC cholesterol accessibility must be relatively stable.

The lack of a relationship between cholesterol content and cholesterol accessibility in RBCs is similar to what has been observed among membranes from different organelles in mammalian cells, For example, plasma membranes (PMs) have a cholesterol content of ~40 mole%, whereas the cholesterol content of the membranes from the endoplasmic reticulum (ER) is only ~5 mole% (Sokolov and Radhakrishnan, 2010). Despite this wide difference in cholesterol content, the chemical activity of cholesterol in these two membranes may be similar (Radhakrishnan and McConnell, 2000). This equivalence could be achieved if PM phospholipids interact more strongly with cholesterol than ER phospholipids, a plausible possibility based on extensive fractionation analysis of PM and ER lipids (Colbeau et al., 1971; Comte et al., 1976; Schroeder et al., 1976) and biophysical studies of the varying affinity of cholesterol for different phospholipids (McConnell and Radhakrishnan, 2003; Wattenberg and Silbert, 1983; Finean, 1953). Fluctuations in membrane composition lead to changes in the chemical activity of cholesterol and could result in cholesterol transport between membranes, either internally or externally. In the case of the RBC, which has no internal organelle membranes, changes in cholesterol activity in the plasma membrane must equilibrate with cholesterol in lipoproteins, other plasma proteins, or with other cells, such as circulating leukocytes or endothelial cells (Lange and Steck, 2016).

We found that treatment of RBCs with either PLA2 or SMase increased cholesterol accessibility (Figure 9), a finding that is consistent with prior studies (Subbaiah et al., 2003; Slotte et al., 1989). Non-cholesterol lipids contributed significantly to inter-individual differences in cholesterol accessibility (Table 4), but identifying exactly which lipid species contributed to the observed variation in cholesterol accessibility was not straightforward. The complex population of phospholipids in the RBC membrane and the high degree of correlation between different phospholipid classes confounded the analysis. In the multivariate model adjusted for age, sex, lipid and lipoprotein levels, only LPE, SM and PA were associated with fALOD4 binding. Taken together, differences in these membrane lipids accounted for 17.6% of the variability in fALOD4 binding. It is also possible that differences in phospholipid composition alter the distribution of cholesterol between the two leaflets of the RBC membrane. Further studies in which the RBC membrane content of individual phospholipids can be selectively altered will be required to elucidate the effect of membrane phospholipids on cholesterol accessibility.

Why should RBC cholesterol accessibility be maintained at levels that closely bracket the threshold point of cholesterol accessibility? At cholesterol concentrations below the inflection point, the capacity of the membrane to incorporate cholesterol into complexes with phospholipids is high and the propensity for cholesterol to leave the membrane is low, therefore cholesterol will tend to accumulate in the RBC. At cholesterol concentrations above the inflection point, the capacity of the membrane to accommodate additional cholesterol in complexes with phospholipids is low, and cholesterol is poised to leave the RBC membrane when suitable acceptors become available. Thus, the cholesterol content of the RBC membranes is maintained close to equilibrium with respect to cholesterol exchange.

It remains possible that differences in the complement of proteins in RBC membranes contribute to the observed inter-individual variability in fALOD4 binding. However, depletion of membrane proteins by pronase digestion had only a modest effect on fALOD4 binding (Figure 8A). We cannot exclude the possibility that in our experiments pronase treatment failed to digest a protein that contributes to the inter-individual differences in cholesterol accessibility. No association was found between ABO blood groups and fALOD4 binding (Figure 8B, upper panel), but fALOD4 binding was reproducibly lower in those who were negative for the Rh antigen, a transmembrane protein of unknown function (Huang et al., 2000) (Figure 8B, lower panel). The effect of the Rh antigen on fALOD4 binding may be direct or indirect. Rh antigen may compete with or directly inhibit fALOD4 binding. Alternatively, the antigen may alter the cholesterol accessibility in the RBC membrane. More extensive studies of the relationship between Rh antigenicity and cholesterol accessibility will be needed to elucidate the basis for this relationship.

Factors that are extrinsic to the RBC were also found to be associated with differences in RBC cholesterol accessibility. Lipoprotein lipids accounted for 5.4% of the variation in fALOD4 binding in a multivariate model that included gender and race (Table 2). Plasma levels of TG and RBC cholesterol accessibility were inversely correlated. This correlation may be a direct result of movement of accessible cholesterol from RBCs to very low density lipoproteins (VLDL) (Chung et al., 1986). It is also possible that circulating lipoproteins influence RBC cholesterol accessibility by transferring phospholipids to (or from) the RBC membrane (Reed, 1968).

It is possible that ABCA1 transfers cholesterol directly from cells to RBCs, and thus contributes to the flux of cholesterol from extrahepatic tissues to the liver (Figure 1). RBCs do not have access to peripheral tissues unless there is loss of vascular integrity, but they can interact indirectly with peripheral tissues through cells, such as macrophages, and lipoproteins that enter the tissues and return to the circulation. RBCs have been shown to be excellent acceptors of free cholesterol from macrophages (Ho et al., 1980). Brown and Goldstein found that RBCs were 6-fold more potent than whole serum as cholesterol acceptors from macrophages (Ho et al., 1980). Lipoprotein-depleted plasma is as efficient an acceptor as whole serum or plasma from cholesterol-loaded macrophages (Ho et al., 1980). Although here we focused on the relationship between RBC accessible cholesterol and lipoprotein levels, it is also possible that blood components other than lipoproteins contribute to differences in cholesterol accessibility of the RBC membrane (Ho et al., 1980). RBCs may also exchange cholesterol with endothelial cells, thus indirectly transporting cholesterol from the tissues. It is unlikely that RBCs deliver cholesterol directly to hepatocytes since they are too large to enter the space of Disse.

Significant and systematic differences in RBC cholesterol activity were found among individuals of European, African, and Hispanic descent. Median fALOD4 binding was highest in African-Americans (1.18 nRFU), intermediate in Hispanics (0.86 nRFU) and lowest in European-Americans (0.77 nRFU) (Figure 5). These differences were seen in two independent samples collected at different times (Figure 5 and Table 4). The increased RBC accessibility in Blacks was not fully explained by the measured differences in RBC lipid composition or by the plasma lipoprotein levels. In linear regression analysis, adjusted for these factors, the proportion of variability in fALOD4 binding explained by ethnicity was reduced from 32% to 22.4%, but remained highly statistically significant (Table 5). Dietary differences between the ethnic groups may also contribute to differences in RBC cholesterol accessibility. It would be informative to measure fALOD4 binding to RBCs in a larger multiethnic sample and use genetic association to identify potential factors accounting for the observed differences between both individuals and ethnic groups.

Interethnic differences in cholesterol accessibility may be the consequence(s) of historic differences in selective pressure among the three ethnic groups. RBCs with higher fALOD4 binding would be anticipated to be more susceptible to microorganisms that produce cholesterol-binding toxins. ALO, produced by Bacillus Anthracis, is a member of the cholesterol-dependent cytolysin family, which is a family of pore-forming toxins produced by a myriad of different gram positive bacteria, including species of Streptococcus, Listeria and Clostridium (Palmer, 2001). Perhaps individuals with lower fALOD4 binding are less susceptible to the hemolytic effects of these toxins, thus providing them with a survival advantage.

Genetic and pharmacological studies have provided compelling evidence that increased circulating levels of HDL-cholesterol, widely viewed as the vehicle for reverse cholesterol transport, do not confer protection against coronary heart disease (Siddiqi et al., 2015). RBCs may be a conduit for transporting cholesterol from peripheral tissues to the liver. Mice made anemic had reduced fecal excretion of cholesterol from macrophages (Hung et al., 2012), and anemia has been shown to be associated with increased coronary artery disease (Hazard Ratio of 1.41 over 6 years) (Sarnak et al., 2002). To determine if RBC cholesterol accessibility provides a clinically relevant biomarker, we measured cholesterol accessibility in patients with chronic renal failure on dialysis, who have a dramatically increased risk of atherosclerosis that is not explained by known risk factors (Levey et al., 1998). We found a systematic increase in cholesterol accessibility in RBCs in patients on dialysis when compared to healthy age- and race-matched controls. The cause of these differences and whether they are related to the increase in atherosclerosis seen in this population is not known at this time and will require further study.

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Author details

1. Rima S Chakrabarti

   Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   RSC, Conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article

   Contributed equally with

   Sally A Ingham

   Competing interests

   No competing interests declared.

2. Sally A Ingham

   Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   SAI, Conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article

   Contributed equally with

   Rima S Chakrabarti

   Competing interests

   No competing interests declared.

3. Julia Kozlitina

   Departments of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   JK, Analysis and interpretation of data, drafting or revising the article

   Competing interests

   No competing interests declared.

4. Austin Gay

   Departments of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   AG, Conception and design, design and acquisition of data, analysis and interpretation of data, drafting or revising the article

   Competing interests

   No competing interests declared.

5. Jonathan C Cohen

   Departments of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   JCC, Conception and design, analysis and interpretation of data, drafting or revising the article

   For correspondence

   jonathan.cohen@utsouthwestern.edu

   Competing interests

   No competing interests declared.

6. Arun Radhakrishnan

   Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   AR, Conception and design, analysis and interpretation of data, drafting or revising the article

   For correspondence

   arun.radhakrishnan@utsouthwestern.edu

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336

7. Helen H Hobbs

   1. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
   2. Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   HHH, Conception and design, analysis and interpretation of data, drafting or revising the article

   For correspondence

   helen.hobbs@utsouthwestern.edu

   Competing interests

   HHH: Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-8700-9897

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