Formation of phosphatidylethanol from endogenous phosphatidylcholines in animal tissues from pig, calf, and goat

Abstract

In the presence of alcohol, phosphatidylcholine (PC) is transformed to the direct alcohol biomarker phosphatidylethanol (PEth). This reaction is catalyzed by the enzyme phospholipase D (PLD) and dependent on substrate availability. As recent methods have solely focused on the determination of PEth, information about the PC composition was generally missing. To address this issue and monitor PC (16:0/18:1 and 16:0/18:2) and PEth (16:0/18:1 and 16:0/18:2) simultaneously, a reversed phase LC-MS/MS method based on a C8 core-shell column, coupled to a Sciex 5500 QTrap instrument was developed. By application of polarity switching, at first, PC was measured in ESI positive SRM mode, while PEth was determined at a later stage in ESI negative SRM mode. The PEth method was validated for human blood samples to show its robustness and subsequently applied for the investigation of systematic in vitro PEth formation in animal tissue samples (brain, kidney, liver, and blood) from a pig, a calf, and a goat. Homogenized tissue was incubated at 37°C with varying ethanol concentrations from 1 to 7g/kg (determined by HS-GC-FID) for 5h, whereby a sample was taken every 30min. For all tissue samples, an increase in PEth was measurable. PEth concentrations formed in blood remained below the LLOQ, in agreement with literature. Data analysis of Michaelis-Menten kinetics and PC within the tissue provided a detailed insight about PEth formation, as the occurrence of PEth species can be linked to the observed PC composition. The results of this study show that PEth formation rates vary from tissue to tissue and among different species. Furthermore, new recommendations for PEth analysis are presented.

Monitoring of direct alcohol markers in alcohol use disorder patients during withdrawal treatment and successive rehabilitation

“…The finally reported concentrations were determined by taking the mean of both monitored SRM transitions ((SRM1 + SRM2)/2). The reason for using two quantifier transitions can be found in ionization differences which may occur due to the SN1 and SN2 ratio being present within the reference standard and the actual samples …”

“…The reason for using two quantifier transitions can be found in ionization differences which may occur due to the SN1 and SN2 ratio being present within the reference standard and the actual samples. 19…”

Monitoring the use of alcohol—A critical overview of the state‐of‐the‐art biomarkers

“…Phosphatidylethanol is a group of phospholipids that are formed in erythrocyte cell membranes in the presence of ethanol only, which makes them a highly specific direct alcohol biomarker (Hansson et al, 1997). The formation of PEth is catalyzed by the enzyme phospholipase D (PLD) and depends on substrate availability (Luginbuhl et al, 2018). PLD is an enzyme that normally catalyzes the hydrolysis of phospholipids, leading to formation of phosphatidic acid, and choline (Hansson et al, 1997).…”

“…PEth is a promising direct alcohol biomarker because its specificity and sensitivity are higher compared to indirect biomarkers (Table 1). Moreover, the pharmacokinetics provide a longer detection window in biological fluids, compared to other direct alcohol biomarkers such as EtG, EtS, and FAEE (Hill‐Kapturczak et al, 2018; Luginbuhl et al, 2018). A nice example was published by Reisfield et al who studied abstinence in people with and without AUD (Reisfield et al, 2020).…”

Variation in the Relative Isomer Abundance of Synthetic and Biologically Derived Phosphatidylethanols and Its Consequences for Reliable Quantification

“…The internal standard for the PEth determination, PEth 16:0/18:1-d 5 , was synthesized as described elsewhere and diluted in 2-propanol (8). The same internal standard solution, with the same regioisomeric ratio, was used for all the experiments.…”