Development and validation of direct assay for cholesterol content of erythrocytes

Erythrocytes contain a significant amount of membrane cholesterol, which is continuously exchanged with lipoproteins. Recent studies suggest that erythrocyte cholesterol content correlates positively with atherosclerotic cardiovascular disease (ASCVD) severity independent of low-density lipoprotein levels, potentially reflecting residual ASCVD risk. However, conventional methods for measuring erythrocyte cholesterol content require labor-intensive lipid extraction procedures, limiting their clinical applicability. In this study, we developed a novel enzymatic assay that enables direct quantification of erythrocyte total cholesterol content using two denaturants to eliminate hemoglobin interference. This simple method demonstrated high accuracy and precision, as confirmed by intra-assay repeatability, between-day precision, linearity, and spike-recovery tests. Using this assay, we determined the erythrocyte cholesterol content per volume (154.8 ± 2.9 mg/dl in men, 155.9 ± 6.9 mg/dl in women) and per cell (139.0 ± 5.2 fg/cell in men, 140.8 ± 5.3 fg/cell in women) (n = 12, healthy subjects). While erythrocyte cholesterol content per volume correlated with the conventional method, the erythrocyte cholesterol content per cell showed no such correlation. Moreover, neither measure was associated with serum lipid levels, suggesting their potential as independent biomarkers for ASCVD. Additionally, we evaluated erythrocyte cholesterol content across different maturation stages and found that older erythrocytes had significantly lower cholesterol content, consistent with mass spectrometry results. These findings further validated the physiological relevance of the proposed method. In conclusion, we successfully established a simple and clinically applicable enzymatic method for measuring erythrocyte cholesterol content, providing novel insights into erythrocyte cholesterol metabolism and its potential role in ASCVD risk assessment.