Determination of age-specific reference intervals for ethanolamine plasmalogen species in red blood cells using liquid chromatography tandem mass spectrometry

Plasmalogens are essential membrane components predominantly generated by de novo synthesis beginning in peroxisomes. Hence, patients with peroxisome biogenesis defects exhibit markedly reduced plasmalogens. Moreover, a reduction in plasmalogens has been associated with several degenerative and metabolic disorders, as well as aging. Here, we characterized the distribution and changes over time of the most abundant ethanolamine plasmalogen (PlsEtn) species in packed red blood cells (RBCs) from healthy individuals, and established age-specific reference intervals (RIs). Eighteen PlsEtn species were extracted from RBCs and quantified using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). RIs were established using RBCs from self-reported healthy volunteers and de-identified clinical samples referred for unrelated testing (total n = 441; 218 females, 223 males; range 0 to 88 years). Possible age groups were identified using a model-based clustering algorithm. Initial subgroups were then compared iteratively using the Harris-Boyd method. RIs were determined in the final age groups using parametric or non-parametric statistics (95%, with 90% confidence intervals). Our study demonstrated age-dependent changes in individual PlsEtn species, with most species lower in neonates compared to children and adults. Interestingly, PlsEtn species carrying docosahexaenoic acid were higher at birth and then declined over time. For each analyte, model-based clustering identified, on average, four initial age clusters which were reduced to three by the iterative Harris-Boyd method. Once final age groups were partitioned, we established age-specific RIs for the 18 PlsEtn species and their totals, based on stereospecific numbering-1( sn -1 ) and sn -2 position. We found significant age-dependent changes in RBC PlsEtn requiring separate pediatric and adult RIs. Lacking previously published data, this study is critical to support implementing plasmalogen testing by LC-MS/MS in the clinical laboratory. • Plasmalogens are glycerophospholipids that play essential roles in membrane structure and function. • Low plasmalogen concentrations are a key feature of peroxisome biogenesis disorders, as well as being associated to several neurodegenerative disorders and physiological aging. • Reference intervals (RIs), which are critical for implementing testing in the clinical laboratory, have yet to be established for intact plasmalogen species. • Model-based clustering and the iterative application of the Harris-Boyd method were successfully used to establish RIs for the most abundant plasmalogen species in red blood cells.