LC-MS/MS analysis of Phosphatidylethanol for abstinence testing and alcohol consumption monitoring in different cohorts, including comparisons to other biomarkers

Abstract

The question of the nature and extent of alcohol exposure of individuals is of substantial importance in forensic assessments and clinical diagnostics. The evaluation is usually based on chemical-toxicological test results. Various alcohol biomarkers have been established for routine testing, but none of them has 100% sensitivity and specificity. Phosphatidylethanol (PEth) has gained increasing recognition as an alcohol consumption marker in recent years. Unlike indirect alcohol markers, which represent biochemical changes in the body (e.g., liver enzyme levels, carbohydrate-deficient transferrin, altered erythrocyte volume), direct alcohol markers are biotransformation products of ethanol, making them highly specific. These include ethyl glucuronide (Etg) and ethyl sulfate (Ets), as well as PEth, an abnormal phospholipid consisting of a glycerol backbone esterified with fatty acid residues at sn-1 and sn-2 positions and with phosphoethanol at sn-3 position. While Etg is measured in urine (detection for about 3 days) and in hair (detection period about 1 to 3 months), PEth is analyzed in blood and provides a detection period of up to several weeks. After successfully establishing a method to quantify six different homologues of PEth by LC-MS/MS, the applicability and diagnostic value of PEth was assessed in two studies with different cohorts. Primary scope was on PEth’s performance in comparison with already established alcohol biomarkers and analysis of the different homologues. In a collective of patients with liver diseases of different etiologies, PEth had a specificity of 100% for alcohol consumption in the last three months, the highest sensitivity (75%) for the detection of consumption in the last week compared to Etg in urine (28%), as well as for consumption in the last three months (53%) compared to Etg in hair (37%). For alcohol uptake in the past four weeks, PEth had a specificity of 98% and a sensitivity of 58%. In a collective of subjects with presumably very high alcohol consumption, PEth and carbohydrate-deficient transferrin (CDT) correlated well (ρ = 0.77 (p < 0.001) for PEth 16:0/18:1), but overall PEth also performed better than the already established markers. PEth analyses were performed from dried blood spots (DBS) generated from venous or capillary blood. The stability of all PEth homologues in blood stored at room temperature proved to be very variable in 62 samples in which PEth concentrations were quantified over a period of 30 days at several time points. Thus, it was concluded that blood samples that are planned to be analyzed for PEth should be sent to the analytical laboratory as soon as possible or DBS should be generated at the site of blood sampling to inhibit decrease of concentration. Overall, results of this work, especially the results of a study with subjects who consumed small amounts of alcohol, led to the conclusion that the currently applied cut-off for PEth should be lowered to improve the sensitivity of this alcohol biomarker. All in all, the compilation of studies on PEth in this work shows why this alcohol biomarker should have a place in routine analysis for abstinence testing and alcohol consumption monitoring, while short comings, that are intensely discussed in this work, need to be considered.