A Bacterial Cholesterol Sensor to Assess Cholesterol Accessibility in Red Blood Cells

Date

2016-01-19

Authors

Chakrabarti, Rima Shah

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Abstract

Mammals are able to gain cholesterol from two sources: diet and endogenous synthesis. However, the only means of cholesterol removal is reverse cholesterol transport (RCT), in which cholesterol is transported to the liver and exported into bile. While high density lipoprotein (HDL) is considered to be the major conduit for RCT, studies with HDL-deficient animals reveal no defect in tissue cholesterol balance. We hypothesize that red blood cells (RBCs), which contain 50% of blood cholesterol, also play a role in RCT. To measure accessible cholesterol in RBCs, we developed an assay that utilizes the cholesterol binding properties of the toxin Anthrolysin-O (ALO). We purified and fluorescently labeled domain 4 of ALO (fALOD4). We then incubated fALOD4 with RBCs from 164 subjects and measured fluorescence intensity using flow cytometry. Both intra-assay and intra-individual variability of the assay were less than 10%. In the test population, fALOD4 binding varied 10-fold. fALOD4 binding did not correlate with total RBC cholesterol but did correlate with RBC phosphatidylcholine (PC) (-0.42, p=6e-7) and lyso-phosphatidylcholine (LPC) (0.40, p=6e-6). Increasing the LPC:PC ratio in RBCs with phospholipase A2 (PLA2) increased fALOD4 binding by 3-fold. fALOD4 binding also correlated with plasma HDL (0.30, p=6e-4) and triglycerides (-0.57, p=2e-12). These data suggest that RBC accessible cholesterol varies in a population, is driven by intrinsic RBC phospholipid composition and interacts with known cholesterol transporters in the blood. Future studies will determine if variability in fALOD4 binding is driven by non-lipid RBC membrane components, is genetically determined, or contributes to atherosclerosis.

General Notes

The 54th Annual Medical Student Research Forum at UT Southwestern Medical Center (Monday, January 19, 2016, 2-5 p.m., D1.700)
Each year the Medical Student Research Program awards students for the best oral presentation and the best poster presentation as judged by faculty across campus. This author received an award as one of the best poster presentations at this forum.

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Citation

Chakrabarti, R. S., Radhakrishnan, A., Cohen, J. C., & Hobbs, H. H. (2016, January 19). A bacterial cholesterol sensor to assess cholesterol accessibility in red blood cells. Poster presented at the 54th Annual Medical Student Research Forum, Dallas, TX. Retrieved from https://hdl.handle.net/2152.5/3260

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