Method development for routine liquid chromatography–mass spectrometry measurement of the alcohol biomarker phosphatidylethanol (PEth) in blood
Introduction
An abnormal phospholipid originally discovered in organs of rats exposed to ethanol was identified as phosphatidylethanol (PEth) [1], [2]. PEth is formed from ethanol and phosphatidylcholine (PC) in cell membranes by a transphosphatidylation reaction catalyzed by phospholipase D (PLD) [3]. PLD uses water in the hydrolysis reaction that generates phosphatidic acid (PA) and choline. However, because PLD preferentially utilizes ethanol over water, PEth is generated at the expense of PA in the presence of ethanol [4], [5], [6]. In humans, PEth was detected in blood samples [7], [8], [9] and autopsy material [10] collected from chronic heavy drinkers and measurement of PEth in blood was proposed for use as an alcohol biomarker [7], [11], [12], [13]. The blood PEth concentration has been demonstrated to correlate with the amount of alcohol consumed, even though the relationship varies considerably between individuals [14], [15], [16].
Measurement of PEth was originally done by thin-layer chromatography but this technique is less suitable for quantitative analysis and was later replaced by high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) [17], [18]. Recently, methods based on capillary electrophoresis (CE) coupled to UV [19] or mass spectrometric (MS) [20] detection, and HPLC coupled to MS (LC–MS and LC–MS/MS) detection [21], have been introduced. The methodological progress has greatly increased the interest in blood PEth as a routine alcohol biomarker, as reflected in a growing number of clinical and medico-legal evaluations [14], [15], [16], [22], [23], [24], [25], [26], [27], [28].
Being derived from ethanol and PC, an advantage of PEth as alcohol biomarker over liver function tests such as gamma-glutamyltransferase is its high specificity for alcohol. Nonetheless, because the PLD enzyme is also active at low temperature and even on storage at − 20 °C, PEth may be generated post-sampling if ethanol is present [10], [21], [29], [30]. This implies a risk for artifactual formation leading to false indications of prior heavy drinking [11], [23].
PEth is not a single molecule but a group of phospholipids with a common phosphoethanol head onto which two fatty acids of variable carbon chain length and degree of saturation are attached [21]. Accordingly, when HPLC-ELSD and CE-UV methods, measuring a total amount of PEth, are replaced by selective MS-based methods, enabling identification and quantitation of the individual molecular forms [21], [31], it has become necessary to determine which PEth species should be the analytical target. In blood from heavy drinkers, 9 major forms were identified by MS with PEth-16:0/18:1 (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol) and PEth-16:0/18:2 (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol) being predominant [21]. Together PEth-16:0/18:1 and 16:0/18:2 accounted for approximately 60% of the total amount. Later on, the presence of 48 PEth forms was indicated in a blood sample from an autopsy case [32].
The aim of the present work was to further develop LC–MS(/MS) for qualitative and quantitative measurements of PEth in human blood [21] and make it practical for routine laboratory use.
Section snippets
Chemicals
PEth-16:0/18:1 reference material was obtained from Biomol Research Laboratories, PEth-16:0/18:2 (> 99% purity; synthesized on special request), PC-16:0/18:1 (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), PC-16:0/18:2 (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine) and phosphatidylpropanol (PProp-18:1/18:1) were from Avanti Polar Lipids, phospholipase D (PLD type IV from cabbage) from Sigma Aldrich, and deuterated ethanol (ethanol-d6) was from Merck. All other chemicals were of
Diagnostic sensitivity of individual PEth species
To compare the sensitivity as alcohol biomarker of the individual PEth forms, 211 blood specimens with a total PEth level of 0.1–20 μmol/L by ESI-MS were evaluated. For reporting limits at 0.1, 0.3, 0.5, 0.7 and 1.0 μmol/L of total PEth, only PEth-16:0/18:1 was detectable (≥ 0.01 μmol/L) in all specimens (i.e., 100% sensitivity) (Fig. 2). PEth-16:0/18:2 and the combination amounts of PEth-18:0/18:2 and 18:1/18:1 (not separated in the SIM analysis because of identical molecular masses) were both
Discussion
Over the years, several laboratory tests have been evaluated for clinical and medico-legal use as alcohol biomarkers to indicate the approximate amount and duration of drinking [35]. The currently most employed alcohol biomarker is carbohydrate-deficient transferrin (CDT), which is the name for an alcohol-induced temporary change in the glycosylation profile of the iron-transport protein transferrin. Disialotransferrin and asialotransferrin are two normally minor glycoforms that occur in
Acknowledgments
Financial support was provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institutet.
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