Examining PEth formation rate with phospholipase-D 1&2 inhibition in ex vivo whole blood
Haidyn G Stark1, Nathalie Hill-Kapturczak1, Brett C Ginsburg1
1Department of Psychiatry and Behavioral Sciences, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
Background:
Phosphatidylethanol (PEth) is formed in human whole blood by phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine in the presence of ethanol. Mammalian PLD exists as two major isoforms (PLD1 and PLD2), yet the primary isoform responsible for PEth formation in whole blood remains unknown. The current study used pharmacological inhibition of PLD1 and PLD2 to identify the isoform that catalyzes PEth formation and to direct subsequent research examining the contribution of these isoforms to inter-individual differences in formation rate.
Methods:
Whole blood from 17 participants was collected via venipuncture. For each participant, blood was divided into separate samples and incubated with an inhibitor for PLD1, PLD2, or nonspecific PLD ranging 0-90% inhibition, and with ethanol ranging 0-0.3% BAC at 37°C. Aliquots were removed from each sample every hour for five hours. PEth 16:0/18:1 concentrations were measured using high performance liquid chromatography with tandem mass spectrometry. PEth formation rates were determined as the slope of linear regressions of PEth concentration over time.
Results:
PEth formation rate varied across participants and decreased with PLD2 inhibition but not with PLD1 inhibition. Inter-participant variability in PEth formation rate was also observed with PLD2 inhibition.
Conclusions:
PLD2 is the primary isoform responsible for PEth formation in human whole blood. These findings provide a mechanistic foundation for future work examining whether differences in PLD2 activity contribute to inter-individual variability in detected PEth concentrations. Future studies should explore whether individual differences in PLD2 activity (via genetic or environmental factors) could contribute to interindividual variability in PEth concentrations.
