Validation and Application of a Protocol for the Extraction and Quantitative Analysis of Sphingomyelin in Erythrocyte Membranes of Patients with NAFLD

A set of constituents of the erythrocyte membrane lipidome has been proposed to serve as biomarkers for liver disease and acute coronary syndrome. In erythrocytes, sphingomyelin hydrolysis provides ceramide, a signaling lipid necessary for phosphatidylserine exposure and eryptosis. Phosphatidylserine exposure further amplifies hepatic inflammation and fibrosis during non-alcoholic fatty liver disease (NAFLD). In this study, we developed and applied a quantitative TLC for erythrocyte membrane sphingomyelin of NAFLD patients. We also compared 10 extraction methods for the isolation of sphingomyelin from erythrocytes. For quantitative TLC, lipids were separated in Silica gel 60 F254 using a mixture of chloroform/methanol/acetic acid/water (60/50/1/4) (v/v/v/v). The separated lipids were stained in a chamber containing iodine, and the intensity of each of the primary colors (red, green, blue) and the sum of the Red plus Green colors (R+G) was analyzed. The method was linear over a wide range of concentrations, presented acceptable precision (inter-day CV(%) 0.34, 0.006 and 0.44 for 2.5, 5.0 and 10 μg, respectively), good accuracy (recovery range 85.2-97.1%), and excellent limit of detection (0.137 μg/spot) and limit of quantification (0.41 μg/spot). Using this quantitation method, we compared various lipid extraction methods and found that lipid extraction with methanol led to higher yield of erythrocyte sphingomyelin (135.35±1.04% recovery, compared to the Folch method). Application of these methods showed that erythrocytes from NAFLD patients (9 men, 15 women, 57.95±11.11 years old) contained statistically significantly less sphingomyelin (829.82±511.60 vs 1892.08±606.25 μg/ml of packed erythrocytes) compared to healthy controls (4 men, 6 women, 39.3±15.55 years old).