A low-cost, time-efficient, sensitive quantitative thin layer chromatography reveals unaltered exogenous sphingosine utilisation from erythrocytes of MAFLD patients
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BACKGROUND
Erythrophagocytosis constitutes a major pathogenic mechanism of metabolic dysfunction associated fatty liver disease (MAFLD). Our previous research established a quantitative thin-layer chromatography (TLC) technique for sphingomyelin, revealing reduced levels in the red blood cells (erythrocytes) of patients with metabolic dysfunction associated fatty liver disease (MAFLD). This reduction was accompanied by erythrocyte sphingosine accumulation, a driver of pro-inflammatory erythrophagocytosis, though sphingosine 1-phosphate release remained stable. To better understand erythrocyte sphingosine metabolism, we adapted our quantitative TLC method to analyze sphingosine within the erythrocyte-conditioned media (ECM) of MAFLD patients.
Methodology
Separation was performed on 10X10cm Silica gel 60 F254 plates using a mobile phase of chloroform, methanol, acetic acid, and water (60:50:1:4 v/v/v/v). The dynamic range, linearity, and range of linearity were assessed by analysing sphingosine levels from 0.1 to 100μg/spot. We validated the system’s precision and sensitivity by performing triplicate analyses of sphingosine standards (1.25, 2.5, and 5μg). The limits of detection and quantification were derived from the calibration curve’s slope and standard deviation (3.3 XSD/slope for LOD; 10 XSD/slope for LOQ). Accuracy was assessed via recovery tests at 100%, 200%, and 300% of a 2.5μg load. We confirmed specificity by evaluating the retention factors against other lipid species. This protocol was applied to Folch-extracted lipids from the ECM (5 × 10 7 cells/ml) of four MAFLD patients and four healthy controls, spiked with 5μg of sphingosine.
Findings
The calibration model, based on combined Green and Blue color intensities, followed the linear equation y = -11.171x + 353.25(R 2 = 0.94). Interday precision values were 0.21%, 1.65%, and 0.44%, while recovery rates (accuracy) ranged from 94.5% to 98.7%. The measured LOD and LOQ were 0.75μg and 1.21μg, respectively. The sensitivity was calculated at 90ng. Statistical analysis showed no significant variance in sphingosine concentrations in erythrocyte-conditioned media between the MAFLD group and the control group.
Summary
The described thin layer chromatography is accurate, precise, sensitive, with good limits of detection and quantification, and most importantly is low-cost and time-efficient. Using this method, we show that while erythrocytes of MAFLD patients exhibit sphingosine accumulation, the utilisation of exogenous sphingosine from their erythrocytes is not affected. This suggests that the metabolic shift may be driven by increased sphingosine supply from the plasma.
