Methodological modifications on quantification of phosphatidylethanol in blood from humans abusing alcohol, using high-performance liquid chromatography and evaporative light scattering detection

BACKGROUND: Phosphatidylethanol (PEth) is an abnormal phospholipid formed slowly in cell membranes by a transphosphatidylation reaction from phosphatidylcholine in the presence of ethanol and catalyzed by the enzyme phospholipase D. PEth in blood is a promising new marker of ethanol abuse depending on the high specificity and sensitivity of this marker. None of the biological markers used in clinical routine at the present time are sensitive and specific enough for the diagnosis of alcohol abuse. The method for PEth analysis includes lipid extraction of whole blood, a one-hour HPLC separation of lipids and ELSD (evaporative light scattering) detection of PEth. RESULTS: Methodological improvements are presented which comprise a simpler extraction procedure, the use of phosphatidylbutanol as internal standard and a new algorithm for evaluation of unknown samples. It is further demonstrated that equal test results are obtained with blood collected in standard test tubes with EDTA as with the previously used heparinized test tubes. The PEth content in blood samples is stable for three weeks in the refrigerator. CONCLUSION: Methodological changes make the method more suitable for routine laboratory use, lower the limit of quantification (LOQ) and improve precision

Phosphatidylethanol - formation and degradation in blood and organs.

Phosphatidylethanol (PEth) is an abnormal phospholipid formed exclusively by the action of phospholipase D (PLD) in the presence of ethanol. The degradation of PEth is slow and due to its accumulation in some cells the possibility to use PEth as marker of ethanol intake has been proposed. It has also been suggested that PEth mediates some of the damaging effects of ethanol on cells. This study was made to investigate the formation and degradation of PEth, after ethanol exposure, in organs, blood, and cell-lines from animals and humans both in vivo and in vitro. After different in vivo alcohol exposures, the level of PEth accumulated in organs of rats was measured. Blood from humans, rats, a pig and a ferret was also incubated in vitro with ethanol for investigation of the PEth formation. The degradation of PEth was studied in human blood and in two cell-lines in vitro. PEth content in organs and blood from cadavers of alcoholics was measured. The effect of storage conditions on PEth content in blood and organs was studied. PEth was measured in blood from two groups of alcoholic patients and related to the ethanol intake and to three clinically available markers of chronic ethanol intake. PEth was analysed by high performance liquid chromatography (HPLC) and evaporative light scattering detection. The analytical method for PEth was improved by using internal standard, and by modifying the extraction method the limit of quantification could be lowered. Rats exposed to ethanol acutely or chronically formed PEth in most of their organs. The amount of PEth formed in the organs varied, and it was shown that both the differences in type of exposition, and ethanol concentrations, are of importance. Also the rate of PEth degradation varied among the organs. No in vivo PEth formation occurred in the rat blood. PEth was formed in human blood incubated in vitro with ethanol. However, in blood from rat, pig, and ferret, no PEth formation occurred in vitro. PEth formation in human blood in vitro was linear with time and ethanol concentration dependant; individual variation in formation rate was demonstrated. No degradation of PEth could be observed in human blood during 48 hours in vitro. Very high PEth concentrations were found in organs and blood from the autopsied alcoholics, probably due to formation of PEth during freezing at –20°C. Storage of rat organs and human blood at –20°C with ethanol present highly elevates PEth levels. Rat organs and human blood without ethanol present can be frozen at –20°C without affecting PEth levels. In blood from alcoholics PEth had a diagnostic sensitivity of 100% in inpatients and 98% in outpatients. The sensitivity of the other markers varied between 28% and 77%. PEth, CDT, and GGT correlated to ethanol intake with the strongest correlation found for PEth. Thus PEth is highly correlated to ethanol intake and the present results indicate that the diagnostic sensitivity is higher than in previously established alcohol markers

Phosphatidylethanol in rat organs after ethanol exposure.

BACKGROUND: Phosphatidylethanol (PEth) is an abnormal phospholipid formed in mammalian cells that have been exposed to ethanol. It has been suggested that PEth mediates some of the damaging effects of ethanol on cells. This study was performed to investigate the level of PEth in organs of rats after in vivo alcohol exposure. METHODS: Three exposure models were studied: (1) acute, intraperitoneal injection of ethanol (n = 3 x 3); (2) chronic, forced ethanol drinking (n = 6); and (3) chronic, free choice of ethanol (n = 20). PEth was analyzed by high-performance liquid chromatography after lipid extraction of the organs. RESULTS: One acute injection gave detectable PEth levels in most organs analyzed, with maximal levels reached after 2 hr. The highest levels were reached in intestines, stomach, and lung. No PEth was detected in skeletal muscle, pancreas, or testis. The two exposure models for oral intake of ethanol also gave detectable PEth levels in most organs. The highest levels were reached in stomach, lung, and spleen. PEth was detected in muscle only in animals with heavy total alcohol intake. CONCLUSIONS: PEth is formed in most organs of rats exposed to ethanol acutely or chronically. Variations in PEth level and rates of PEth formation and PEth degradation are organ specific

PHosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients

Abstract — Aims: Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol by the enzyme phospholipase D. PEth in blood is a promising new marker for ethanol abuse. None of the biological markers used at the present time is sensitive and specific enough for the diagnosis of alcoholism. Methods: The most frequently used alcohol markers [carbohyd-rate deficient transferrin (CDT), gamma-glutamyltransferase (GGT), and mean corpuscular volume (MCV)] were studied together with PEth in actively drinking alcohol-dependent patients (inpatients and outpatients), with regard to correlation to ethanol intake and diagnostic sensitivity of the markers. The relation between the markers was also studied. Results: PEth, CDT, and GGT correlated to ethanol intake, with the strongest correlation found for PEth. The diagnostic sensitivity for PEth was 99%, and for other markers it varied between 40 and 77%. Only when CDT was combined with GGT was a sensitivity of 94 % reached. PEth correlated to CDT and GGT but not to MCV. CDT did not correlate to GGT or MCV. Conclusions: Blood concentrations of PEth are highly correlated to ethanol intake, and the present results indicate that its diagnostic sensitivity is higher than that for previously established alcohol markers

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnover in neuroblastoma x glioma hybrid cells (NG 108-15)

In neuroblastoma x glioma hybrid cells (NG 108-15) labelled with [32P]-trisodium phosphate, [3H]-inositol and [14C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) while it had no effect on the release of [14C]-arachidonic acid (AA). The effect on PIP2 was time- and dose-dependent with a maximal effect on [3H]-inositol- and [32P]-labelled cells after 10-30 s of stimulation with 10(-6) M bradykinin. However, the hydrolysis of [14C]-AA labelled PIP2 was delayed compared to the effect on [3H]- and [14C]-PIP2 and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [3H]-inositol phosphates (IP) and [32P]- and [14C]-phosphatidic acid (PA) but the time course for PA formation did not follow the time-course for PIP2 hydrolysis. A reduced labelling of [32P]- and [14C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP2. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A2, in NG 108-15 cells

Regulation of phospholipase D activity in neuroblastoma cells

The regulation of phospholipase D was studied in human neuroblastoma cells using phosphatidylethanol as a marker of the enzyme activity. Carbachol induced phospholipase D activity in SH-SY5Y cells. Muscarinic antagonists inhibited the response with potencies suggesting that muscarinic M1 receptors are responsible for the activation. In permeabilized SH-SY5Y cells, both the carbachol- and GTP gamma S-induced Peth formation was inhibited by GDP beta S, indicating that both responses are mediated via a G-protein. The protein kinase C inhibitors, bisindolylmaleimide and staurosporine significantly inhibited the carbachol-induced Peth formation whereas H7 had no effect. Thus, the cholinergic activation of phospholipase D in SH-SY5Y cells is probably mediated via a direct receptor-G-protein coupling but an involvement of protein kinase C cannot be excluded. Calmidazolium, a calmodulin antagonist, induced an increase in phosphatidylethanol formation in both SH-SY5Y and IMR-32 cells. This effect was inhibited by genistein and tyrphostin, indicating a tyrosine kinase dependent pathway for phospholipase D activation in neuroblastoma cells

The usefulness of direct ethanol metabolites in assessing alcohol intake in nonintoxicated male patients in an emergency room setting

Background: A major part of medical pathology in internal medicine is associated with chronic alcoholism. The aim of the current study was to investigate whether screening for Alcohol Use Disorders (AUD) can be improved through determination of direct ethanol metabolites compared to traditional biological state markers, the Alcohol Use Disorders Identification Test (AUDIT) and additional self-reports beyond the detection time period of a positive blood alcohol concentration (BAC). Methods: A total of 74 blood alcohol negative male patients who presented at the emergency room with either thoracic or gastrointestinal complaints were included. Phosphatidylethanol (PEth) was determined in whole blood, and ethyl glucuronide (EtG) in serum and urine samples. Traditional biological state markers [carbohydrate deficient transferrin (%CDT), gamma glutamyl transpeptidase (GGT), mean corpuscular volume (MCV)] were determined. The AUDIT was obtained and furthermore, all patients completed an additional self-report of alcohol consumption. Patients were divided into two (2) groups: AUDIT scores = 8. Results: After assessment of the AUDIT, patients were allocated to one of the following groups: patients with AUDIT scores = 8 (n = 22). Twenty-five percent of the patients with AUDIT scores below the cut-off (n = 13/52) were tested positive for both PEth and UEtG. Of the patients who declared to be sober during the past 12 months, 38.5% were tested positive for PEth and UEtG. PEth discriminated similarly as %CDT for AUDIT scores >= 8 (AUC: 0.672; 95%CI 0.524 to 0.821). Self-reports of alcohol consumption were unreliable. Conclusion: Determination of direct ethanol metabolites such as PEth and UEtG provides additional evidence in screening for AUD in an ER setting. Determination of PEth might be considered complementary with or alternatively to %CDT