Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors.

Phosphatidylethanol (PEth) is an alcohol biomarker formed from phosphatidylcholine (PC) by the enzyme phospholipase D (PLD) in the presence of ethanol. A drinking study revealed individual differences in maximum PEth levels after drinking to a targeted blood alcohol concentration (BAC) of 0.1%. This seemed to be due to different PLD activities in the tested persons. Furthermore, post-sampling formation of PEth occurred in blood samples, still containing alcohol. Therefore, a standardized in vitro test for measuring individual PEth formation rates was developed. Two PLD inhibitors were tested for their potency to inhibit post-sampling PEth formation. PEth-negative blood samples were collected from a volunteer. Ethanol was added in different concentrations (0.01-0.3% BAC) directly after blood sampling. The specimens were incubated at 37 °C. Aliquots were taken at the start of the incubation, and every hour until 8 h after start of incubation, and one sample was taken on subsequent days over 1 week. PEth 16:0/18:1 and PEth 16:0/18:2 were determined by online SPE-LC-MS/MS. Furthermore, this test system was applied to blood samples of 12 volunteers. For the inhibition tests, fresh blood (spiked with 0.1% ethanol) was spiked with 30, 300, 3000, or 30,000 nM of either halopemide or 5-fluoro-2-indolyl-deschlorohalopemide (FIPI), and incubated at 37 °C. PEth concentrations were determined hourly over 5 h on the first day and once on day 2 and day 3. PEth formation was linear in the first 7 h of incubation and dependent on the alcohol concentration. The formation rates of PEth 16:0/18:1 were 0.002 μmol L-1 h-1 (0.01% BAC), 0.016 μmol L-1 h-1 (0.1% BAC), 0.025 μmol L-1 h-1 (0.2% BAC), and 0.029 μmol L-1 h-1 (0.3% BAC). For PEth 16:0/18:2, the formation rates were 0.002 μmol L-1 h-1 (0.01% BAC), 0.019 μmol L-1 h-1 (0.1% BAC), 0.025 μmol L-1 h-1 (0.2% BAC), and 0.030 μmol L-1 h-1 (0.3% BAC). Maximum concentrations reached 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) at 0.3% BAC after 3 days. Maximum velocity (vmax) was not reached under these conditions. PEth formation in blood of the 12 volunteers ranged between 0.011 and 0.025 μmol L-1 h-1 for PEth 16:0/18:1 and between 0.014 and 0.021 μmol L-1 h-1 for PEth 16:0/18:2. PEth formation in human blood was inhibited by halopemide in a concentration-dependent manner. However, a complete inhibition was not achieved by the applied maximum concentration of 30,000 nM. FIPI showed a better inhibition of PEth formation. A complete inhibition could be achieved by a concentration of 30,000 nM for the first 24 h (for PEth 16:0/18:1) and for 48 h (for PEth 16:0/18:2). Formation of PEth was found to be dependent on the BAC. As a consequence, it is essential to inhibit PLD activity after blood collection to avoid post-sampling formation of PEth in blood samples with a positive BAC. Inhibition of PEth formation was more effective using FIPI, compared to halopemide.