Method optimisation and applications of phosphatidylethanol, ethyl glucuronide and ethyl sulfate as biomarkers for alcohol consumption

Abstract

Alcohol consumption can lead to major consequences in many clinical and non-clinical settings. Alcohol use disorders, including alcoholic related liver disease and alcohol dependence are two such examples. In many such situations, alcohol abstinence is targeted as a critical endpoint in regards to treatment and/or program completion. Currently, the predominant means in how alcohol consumption information is obtained is through self-report. To supplement self-report data, pathology tests are available that can be utilised to provide further indications for alcohol intake, such as biochemical tests for liver enzymes for alcohol consumption over a period. Yet these tests represent indirect approaches and are non-specific. More recently, there is a growing consensus in the application of direct non-oxidative ethanol metabolites as tests for alcohol intake. Three of these biomarkers are: ethyl glucuronide (EtG), ethyl sulfate (EtS) and phosphatidylethanol (PEth) but these have not been established or optimised for routine clinical use, as standardised cost-effective methodologies are not yet available in this setting

There were three broad aims to this thesis. Firstly, to develop and optimise liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for the analysis of EtG and EtS in serum and PEth in whole blood samples. Secondly, to test and apply these methods in a mixed cohort of individuals and thirdly to demonstrate that PEth concentrations can be influenced by haematocrit levels in blood samples.

The assays that were developed and optimised in this project met the required satisfactory validation parameters in terms of linearity, selectivity, precision, accuracy and recovery. In both EtG/EtS and PEth assays the sample preparation was improved for greater efficiency. Establishing robust LC-MS/MS methods, these biomarkers serve as useful tools for the indication of alcohol consumption and supplement self-report data. These biomarkers were then tested for their presence in a mixed cohort of 231 participants comprised of healthy volunteers, alcohol use disorder patients, including liver disease patients and compared with current ‘gold standard’ self-report measure. There was a significant correlation between these biomarkers and self-reported alcohol consumption. EtG, EtS and PEth were able to provide high discrimination for any alcohol consumption as well as a multitude of different drinking patterns in heavy drinking, very heavy drinking and alcohol misuse and alcohol related harm. These three biomarkers were also found to be superior to the traditional indirect alcohol biomarkers: liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), mean corpuscular volume (MCV) and %carbohydrate deficient transferrin (%CDT). It was also demonstrated that haematocrit levels can influence a PEth result with increases in haematocrit increasing the assayed PEth concentration. Therefore considerations may have to be made when interpreting test results especially in patients who may be severely anaemic which many liver disease patients are.

Overall, EtG, EtS and PEth were found to be very good biomarkers for alcohol consumption.