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Phosphatidylethanol (PEth) is an abnormal phospholipid which is formed in the presence of ethanol, via the action of phospholipase D (PLD). PEth in blood is a potential marker of alcohol abuse. The present study was made to determine the compartmentalization and the elimination rate of PEth in human whole blood. PEth was assayed by an improved HPLC technique, with evaporative light-scattering detection. Blood from six alcoholic males was separated into different blood cell fractions. The PEth concentration in whole blood was 2.5+/-0.9 and 1.9+/-1.1 micromol/l in erythrocytes. Only one subject had detectable PEth in the mononuclear cells. Fifteen patients (13 men, two women) with chronic alcoholism, were followed as inpatients, after admission to an alcohol detoxification clinic. PEth, carbohydrate-deficient transferrin (CDT) and gamma-glutamyltransferase (GGT) were measured on days 1, 3, 5 and 7 after admission. Linear regression analysis of logarithmic PEth values in individuals, with measurable PEth at day 1, gave a good fit (P

Phosphatidylethanol is a "pathological" phospholipid, formed via the action of phospholipase D only in the presence of ethanol. The present study was made to elucidate how different levels and patterns of alcohol intake affect blood levels of phosphatidylethanol in comparison with other markers of abuse. We used a new HPLC-evaporative light-scattering detection technique for phosphatidylethanol quantitation. This method had a total coefficient of variation of

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 muM (5.6 fmol) of Peth with conventional UV detection, equalling 0.4 mumol/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. (Less)

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 substancerelated offense (§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 -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.

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

Phosphatidylethanol (PEth) is an ethanol-phospholipid adduct, formed via non-oxidative metabolism of ethanol. PEth was measured in femoral blood from 85 consecutive forensic autopsies and was detected in 35 of the cases at concentrations ranging from 0.8 to 22.0 micromol/l. Of the PEth positive cases, 12 did not have significant levels of ethanol in the blood. Two cases (both suicides involving hanging) had detectable ethanol, but no PEth present in the blood. We conclude that measurements of PEth provide indications of previous alcohol abuse in cases where this may not otherwise be evident.

As shown in figure 1, ethanol can be metabolized by oxidative and nonoxidative pathways (Lieber, 1995). In the oxidative pathway, ethanol is converted to acetal-dehyde through the action of alcohol dehydrogenases, the microsomal ethanol-oxidizing system, or catalase. Acetaldehyde is then subsequently metabolized to acetate through the action of aldehyde dehydrogenases. In one of the nonoxidative pathways of ethanol metabolism, ethanol undergoes esterification with fatty acids by the action of fatty acid ethyl ester synthase to form fatty acid ethyl esters. The nonoxidative ethanol pathway that is the focus of this review is the pathway leading to the synthesis of phosphatidylethanol. Figure 1 shows how ethanol can be inserted as the head group of a phospholipid to form phosphatidylethanol. The transformation occurs through the activation of phospholipase D, mainly on phosphatidylcholine in the presence of ethanol.

The enzymes needed for galactomannan hydrolysis, i.e., beta-mannanase, alpha-galactosidase and beta-mannosidase, were produced by the filamentous fungus Aspergillus niger. The beta-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the beta-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger beta-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus beta-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.