B-260 Prevalence and Relative Distributions of Major Phosphatidylethanol (PEth) Homologs in a Large-Scale Clinical Study
Authors: Dui W , Smith M P , Bartock S H 
Published: October 2024

Abstract

Background
Ethanol abuse remains a significant public health challenge in the United States. Detection of ethanol exposure is crucial for preventive, diagnostic, and therapeutic endeavors. Phosphatidylethanol (PEth) is a specific and direct ethanol biomarker. Compared to other metabolites, such as ethyl glucuronide and ethyl sulfate in urine (average 2-3 days), PEth in blood has a longer window of detection (average 1-2 weeks). Among the known 48 PEth homologs, PEth 16:0/18:1 (POPEth) and PEth 16:0/18:2 (PLPEth) are the most abundant species accounting for 37% to 46% and 26% to 28%, respectively. PLPEth is synthesized at a greater rate than POPEth, but POPEth has a longer half-life than PLPEth. The purpose of this study was to understand the prevalence and levels of POPEth and PLPEth in a large number of clinical specimens.

Methods
Briefly, lavender-top whole blood specimens were diluted with isotopically labeled internal standard (IS) in an organic buffer to precipitate proteins on an automated Hamilton platform. The mixture was centrifuged, and a supernatant portion isolated for injection onto a Sciex QTRAP 5500 liquid chromatography tandem mass spectrometry (LC-MS/MS) system using electrospray ionization (ESI) in negative mode. The assay was fully validated under CLIA regulations for lab developed tests: the analytical measurement range (AMR) was 10-400 ng/mL; the administrative reporting threshold for PEth positivity was 20 ng/mL based on clinical literature and a consensus among other United States laboratory methods.

Results
Precision of 4 QC levels was evaluated with between-run CV% (n=25) ranging from 1.6-6.3%, and accuracy across the AMR was achieved with 87-110%. No significant matrix effects were observed; variation in different whole blood specimens was corrected with IS normalization. No significant interferences were observed from 137 commonly seen prescription and nonprescription drugs and 6 other PEth homologs. Of the clinical specimens, most (20,833, 71.3%) were negative (&lt;20 ng/mL) for both analytes. Meanwhile, 8,222 specimens (28.3%) were positive with either POPEth, or PLPEth or both ≥ 20ng/mL. There were 150 specimens excluded from the statistics due to interference challenges. Interestingly, 757 specimens (2.6%) were PLPEth negative and POPEth positive (mean value 40.1 ng/mL). Conversely, 62 specimens (0.2%) were POPEth negative and PLPEth positive (mean value 24.6 ng/mL). Among the specimens with both POPEth and PLPEth levels within the AMR, the average POPEth/PLPEth ratio was 1.4 supporting the overall prevalence of higher POPEth concentrations than PLPEth. However, the POPEth/PLPEth ratio varied widely, from 0.3 to 8.8, reflecting patient differences in PEth metabolism patterns.

Conclusions
To help verify probable ethanol abstinence, we have developed, validated, and deployed a high-throughput clinical assay for the major PEth homologs, POPEth and PLPEth. In a retrospective analysis of 29,205 clinical specimens: 71.3% of results were consistent with abstinence; 28.3% of the results were positive suggesting moderate alcohol consumption within the last 1-2 weeks. With POPEth most prevalent, yet variation observed, there were 62 specimens with PLPEth positive and negative for POPEth. This clinical PEth study including both POPEth and PLPEth provides greater sensitivity to identify ethanol exposure than only testing a single homolog.