PROJECT SUMMARY This renewal extends work accomplished during the previous award period and seeks to improve our ability to characterize alcohol drinking through the measurement of blood levels of phosphatidylethanol (PEth). Previously, we showed that: (a) PEth can detect recent alcohol consumption; (b) PEth levels are alcohol-dose dependent; (c) PEth homologs have different rates of synthesis and elimination potentially useful in differentiating recency of drinking; (d) there are no sex differences in PEth synthesis and elimination; and (e) most importantly, there is substantial variability in PEth synthesis and elimination between people, even after the same dose of alcohol is consumed. This variability raises significant concerns over current practices. PEth is now accepted as a valid laboratory test for detecting recent alcohol use in forensic and legal court matters. Distinct cut-offs of PEth levels have been adopted to indicate "light or no consumption", "significant consumption", and "heavy consumption". Unfortunately, because there are significant differences in PEth levels achieved in people given the same dose of alcohol, it could lead to misclassification of drinking. We now propose to identify probable sources of variability in PEth levels and use biological/enzymatic variables to develop equations to improve identification of alcohol consumption in the “real world”. We propose 3 experiments. An in vivo pharmacokinetic study that has 2 phases whereby participants consume 3 different doses of alcohol and blood samples will be collected repeatedly during a 6-hour period to characterize blood alcohol concentration and PEth synthesis. We will then characterize PEth elimination across a 10-day period while remaining alcohol abstinent outside the lab. Second, alcohol-free blood collected from Aim 1 will be examined ex vivo to characterize key biological variables (e.g., enzyme activity, red blood cell count, precursor levels) involved in PEth synthesis and elimination. Equations will be used to evaluate these variables for their ability to explain previously unexplained between-subject differences in the PEth levels formed after the same amount of alcohol is consumed. Lastly, equations will be used to evaluate the value of using these biological/enzyme variables to improve the prediction of 28-days of naturalistic drinking. Our Specific Aims are to: examine the pharmacokinetics of blood alcohol and PEth synthesis/elimination following controlled consumption of alcohol (Aim 1); identify key biological variables (e.g., enzyme activity, RBC count, and/or PEth precursor levels) involved in PEth pharmacokinetics that can account for the previously unexplained between- subject variance observed in vivo (Aim 2); and determine the use of PEth levels, with key biological variables to identify drinking observed naturalistically. Study results will demonstrate that key biological variables can be tested in clinical laboratory studies and found useful ...