Nonaqueous capillary electrophoresis for analysis of the ethanol consumption biomarker phosphatidylethanol.

Nonaqueous CE (NACE) methodology was developed for the separation and determination of phosphatidylethanol (Peth), a new biomarker of ethanol intake. Peth is an abnormal phospholipid formed in cell membranes only in the presence of ethanol, via the transphosphatidylation reaction of phospholipase D. The NACE separation medium consisted of 80 mM ammonium acetate in 50% ACN, 33% 2-propanol, 12% hexane and 5% methanol. A stacking effect was obtained by reducing the concentration of ammonium acetate in the separation medium for all injected samples. The LOD was estimated to 1 microM (5.6 fmol) of Peth with conventional UV detection, equalling 0.4 micromol/L blood. Peth was successfully determined in extracts of human blood samples. Separation of Peth from other blood lipids in the lipid extract sample was performed in 5 min. The method facilitates smaller sample volumes and performs about ten times faster compared to earlier chromatographical methods.

Ethyl glucuronide discloses recent covert alcohol use not detected by standard testing in forensic psychiatric inpatients.

BACKGROUND: Considerable lives and money could be saved if one could detect early stages of lapsing/relapsing behavior in addicted persons (e.g., in safety-sensitive workplaces) and could disclose harmful drinking in social drinkers. Due to the serious public health problem of alcohol use and abuse worldwide, markers of alcohol use have been sought. Both ethyl glucuronide (EtG) and phosphatidyl ethanol (PEth) appear to have high sensitivity and specificity and a time frame of detection that may elucidate alcohol use not detected by standard testing. Our aim was to assess their potential for detecting recent covert alcohol use under controlled conditions. METHODS: Thirty-five forensic psychiatric inpatients in a closed ward who had committed a substance-related offense ( section sign 64 StGB), were followed for 12 months. The complete time spectrum of possible alcohol consumption was covered by the complementary use of breath and urinary ethanol (hours), urinary EtG (days), %carbohydrate-deficient transferrin (CDT)/PEth (weeks), and gamma-glutamyltranspeptidase (GGT)/mean corpuscular volume (MCV) (weeks-months). RESULTS: Fourteen of the 146 urine samples examined were positive for EtG. In all EtG-positive cases, patients reported alcohol consumption of between 40 and 200 g of ethanol 12-60 hr prior to testing. Urinary and breath ethanol were positive in only one case. In the blood samples, PEth was not positive in any case and %CDT did not exceed the reference value. Isoelectric focusing showed no abnormal Tf subtypes. CONCLUSIONS: The findings emphasize the diagnostic and therapeutic usefulness, specificity, and sensitivity of EtG as a marker of recent alcohol use. Such a test is needed in numerous settings, including alcohol and drug treatment (to detect lapse/relapse), in safety-sensitive work settings where use is dangerous or in other settings where use may be inappropriate (e.g., such as driving, workplace, pregnancy, or monitoring physicians or other professionals who are in recovery and working), or for testing other groups (such as children or those with medical problems) where alcohol use would be unhealthy or unsafe. The health, social and socioeconomic benefits arising from the future use of these markers is hard to overestimate.

Formation of phosphatidylethanol in vitro in red blood cells from healthy volunteers and chronic alcoholics.

Phosphatidylethanol (PEth) is an abnormal phospholipid, formed only in the presence of ethanol via a transphosphatidylation reaction of phospholipase D (PLD). PEth in blood is a promising new marker of alcohol abuse. Blood PEth is found almost exclusively in red cells. This study was performed to investigate a possible PEth formation in human red cells from alcoholics and healthy individuals, at physiologically relevant ethanol concentrations. Blood was drawn from six healthy volunteers (controls) and six chronic inpatient alcoholics. Hematological analyses were performed, and red blood cells were separated and incubated in plasma with ethanol to study PEth formation. Lipids were extracted and PEth analyzed with high pressure liquid chromatography and evaporative light-scattering detection. Incubation of red cells in 50 mM ethanol yielded detectable PEth after 12 hours. Formation of PEth was concentration dependent at 10 to 50 mM ethanol. In vitro formation of PEth was significantly higher (P <.001) in red cells from alcoholics (5.2 +/- 1.1 micromol/l) compared to controls (2.4 +/- 0.6 micromol/l) (mean +/- SD). A significant correlation (P <.01) was observed between initial mean corpuscular volume and accumulated PEth. This study demonstrates that PEth is formed in human red cells at physiologically relevant ethanol concentrations. Alcoholics accumulate about twice as much PEth than controls. The accumulation rate of PEth is slower in red cells compared to rates reported for other tissues.