Ethylated phosphorylcholine – a new marker for ethanol consumption: A proof of concept

Abstract

Aim

During in vitro exposure of HepaRG liver cells to ethanol, untargeted metabolomics revealed the formation of ethylated phosphorylcholine (EtOChoP), a metabolite which was not reported before [1], [2]. To determine its role as a potential new biomarker for alcohol use and its detection window, a single alcohol exposure study was performed on a teetotaler. Whole blood, plasma, and serum were analysed for EtOChoP and compared with levels of established alcohol use biomarkers, such as phosphatidylethanol 16: 0/18: 1 (PEth), ethyl glucuronide (EtG), ethyl sulphate (EtS), and ethanol (EtOH) in the same samples.

Method

For the analysis of EtOChoP in whole blood, serum, and plasma, a 96-well microplate liquid-liquid extraction was developed and validated. Briefly, 100 μL sample and 250 μL of ice cold (−80° C) methanol were added, and subsequently spiked with EtOChoP-D5, internal standard. After vortexing, the 96-well plate was placed on ice, sonicated briefly and left to equilibrate for 20 min. After centrifugation, the supernatant was filtered through a 96-well filtration plate and further evaporated to dryness using nitrogen. The dried extract was reconstituted in 100 μL MeOH: H2O(1: 1) and 5 μL was injected in an Agilent 1290 Infinity LC/6495 C triple quadrupole MS using a Restek Force Biphenyl column set at 60° C. The mobile phase was a mixture of water and 0.1% formic acid as solvent A, and methanol and 0.1% formic acid, as solvent B. The total run time was 8 min. The MS/MS source was operated in positive mode. The monitored m/z transitions were 212.1 > 126.0 (quantifier) and 86.1, 98.1(qualifiers) for EtOChoP, and 217.1 > 126.0 (quantifier) and 86.1(qualifier) for EtOChoP-D5.

Single alcohol exposure study design: after being abstinent for five months, a female volunteer was instructed to abstain from food and beverages (including water) for 12 hours prior to the experiment. The volunteer consumed a dose of 0.5 g ethanol per kilogram body weight in one minute as a 20% (w/v) ethanol solution in water. Whole blood, plasma, and serum were collected at point T0 (before the drinking), T30, T60, T80, T100, T120, T140, T160, T180, T240 min and day 1, 2, 5 and 8 after the drinking experiment.

Results

EtOH, PEth, EtG and EtS, EtOChoP were not detected at T0 in all matrices. EtOH in whole blood showed an increase to 0.76 g/L (T100), followed by a decrease to 0.32 g/L at T240. EtOChoP in whole blood showed an increase in concentration from 0.73 ng/mL (T30) to 1.57 ng/mL (T80) followed by a decrease to 0.55 ng/mL (T240). Even though, serum samples showed a higher concentration, it followed the same pattern as whole blood and plasma samples, an increase from T30 to T80 followed by a decrease until T240. The following days (D1, D2, D5 and D8), EtOH, EtG, EtS and EtOChoP were not detected in any of the matrices. PEth showed an increase in concentration from 6.6 ng/mL (T30) to 18 ng/mL (D1), followed by a decrease in D2, D5 and D8. Nevertheless, the concentrations of PEth did not exceed the 20 ng/mL cutoff value, compatible with abstinence/low alcohol consumption [3].

Conclusion

This proof of concept study shows that EtOChoP is detectable in different matrices after a single alcohol consumption, which demonstrates EtOChoP can be a promising new alcohol biomarker. The presence of EtOChoP up to 240 min and its absence in the following day(s) shows that it could be a short-term alcohol biomarker, together with EtOH, EtG and EtS. Nevertheless, future studies are needed to determine its true potential as an alcohol biomarker, as this study looked only at a controlled single alcohol exposure.