Steina Aradottir

Phosphatidylethanol as a sensitive and specific biomarker: comparison with gamma-glutamyl transpeptidase, mean corpuscular volume and carbohydrate-deficient transferrin.

Phosphatidylethanol (PEth), a direct ethanol metabolite, is detectable in blood for more than 2 weeks after sustained ethanol intake. Our aim was to assess the usefulness of PEth [comparing sensitivity, specificity and the area under the curve (AUC)] as compared with carbohydrate‐deficient transferrin (CDT), gamma‐glutamyl transpeptidase (GGT) and mean corpuscular volume (MCV), calculating the results from sober patients against those from alcohol‐dependent patients during withdrawal. Fifty‐six alcohol‐dependent patients (ICD‐10 F 10.25) in detoxification, age 43 years, GGT 81 U/l, MCV 96.4 fl, %CDT 4.2, 1400 g ethanol intake in the last 7 days (median), were included in the study. Over the time of 1 year, 52 samples from 35 sober forensic psychiatric addicted in‐patients [age 34 years, GGT 16 U/l, MCV 91 fl, CDT 0.5 (median)] in a closed ward were drawn and used for comparison . PEth was measured in heparinized whole blood with a high‐performance liquid chromatography method. GGT, MCV and %CDT were measured using routine methods. A receiver operating characteristic curve analysis was carried out, with ‘current drinking status’ (sober/drinking) as the state variable and PEth, MCV, GGT and CDT as test variables. The resulting AUC was 0.974 (P < 0.0001, confidence interval 0.932–1.016) for PEth. At a cut‐off of 0.36 µmol/l, the sensitivity was 94.5% and specificity 100%. The AUC for CDT, GGT and MCV were 0.931, 0.894 and 0.883, respectively. A significant Spearman’s rank correlation was found between PEth and GGT (r = 0.739), CDT (r = 0.643), MVC (r = 0.639) and grams of ethanol consumed in the last 7 days (r = 0.802). Our data suggest that PEth has potential to be a sensitive and specific biomarker, having been found in previous studies to indicate longer lasting intake of higher amounts of alcohol.

Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes.

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well‐characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal‐like, luminal A, luminal B, HER2‐enriched and normal‐like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2−/Ki67low, ER+/HER2−/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal‐like, triple negative and HER2‐enriched tumors, and high H3K27me3 in luminal A, HER2‐enriched and normal‐like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal‐like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease‐free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer.

Phosphatidylethanol in Human Organs and Blood: A Study on Autopsy Material and Influences by Storage Conditions.

OBJECTIVE Phosphatidylethanol (PEth) is an abnormal phospholipid that is formed and accumulated in mammalian cells that have been exposed to ethanol. PEth has been proposed as a marker of ethanol abuse. This study was conducted to investigate the concentration of PEth in blood and organs obtained during the autopsy of alcoholics. In addition, we performed experiments on rat tissues and human blood to evaluate the effect of various storage conditions on PEth concentrations. METHODS Human tissues and blood from alcoholics and controls were obtained at autopsy and frozen at -20 degrees C until extraction. Blood from healthy donors was incubated with ethanol for 24 hr and thereafter either extracted directly or stored at room temperature, stored at 4 degrees C, frozen at -20 degrees C, or frozen in liquid nitrogen and stored at -80 degrees C before extraction. Rats were given intraperitoneal injections of ethanol and then killed, either while still intoxicated or when sober. Rat organs were homogenized and extracted directly, after a period of storage, and/or after freezing at -20 degrees C. PEth concentration was analyzed using HPLC and verified by mass spectrometry. RESULTS In all rat organs studied, PEth was formed during freezing at -20 degrees C with ethanol present. PEth concentrations of 9 to 205 mumol/liter were observed in the blood obtained at autopsy. The highest value was found in the case with the highest blood alcohol concentration (114 mmol/liter) at the time of death. In the experiments on human blood stored with ethanol present, PEth concentrations were not affected after 72 hr at 4 degrees C or after freezing in liquid nitrogen and storage at -80 degrees C for up to 144 hr but were slightly elevated after 24 hr at room temperature and at -20 degrees C. PEth was found in all organs obtained from the cadavers of alcoholics. Storage of organs at 4 degrees C for 24 hr with ethanol present had no effect on the PEth concentration. The PEth concentration was unaffected when no ethanol was present at the time of freezing. CONCLUSIONS The rat experiments indicated that the very high PEth concentrations found in the organs of the alcoholics were probably largely formed while the organs were frozen at -20 degrees C. Our data suggest that tissue material from bodies that were exposed to ethanol must be stored properly to obtain reliable results from subsequent analysis for PEth. Tissue should not be frozen at -20 degrees C but instead stored refrigerated until extraction, preferably within hours of autopsy, or frozen in liquid nitrogen and stored at -80 degrees C. Blood samples that contain ethanol can be stored refrigerated for up to 72 hr or frozen in liquid nitrogen and stored at -80 degrees C without affecting PEth levels.

Phosphatidylethanol: normalization during detoxification, gender aspects and correlation with other biomarkers and self-reports.

Phosphatidylethanol (PEth) is a direct ethanol metabolite, and has recently attracted attention as biomarker of ethanol intake. The aims of the current study are: (1) to characterize the normalization time of PEth in larger samples than previously conducted; (2) to elucidate potential gender differences; and (3) to report the correlation of PEth with other biomarkers and self‐reported alcohol consumption. Fifty‐seven alcohol‐dependent patients (ICD 10 F 10.25; 9 females, 48 males) entering medical detoxification at three study sites were enrolled. The study sample was comprised of 48 males and 9 females, with mean age 43.5. Mean gamma glutamyl transpeptidase (GGT) was 209.61 U/l, average mean corpuscular volume (MCV) was 97.35 fl, mean carbohydrate deficient transferrin (%CDT) was 8.68, and mean total ethanol intake in the last 7 days was 1653 g. PEth was measured in heparinized whole blood with a high‐pressure liquid chromatography method, while GGT, MCV and %CDT were measured using routine methods. PEth levels at day 1 of detoxification ranged between 0.63 and 26.95 µmol/l (6.22 mean, 4.70 median, SD 4.97). There were no false negatives at day 1. Sensitivities for the other biomarkers were 40.4% for MCV, 73.1% for GGT and 69.2% for %CDT, respectively. No gender differences were found for PEth levels at any time point. Our data suggest that PEth is (1) a suitable intermediate term marker of ethanol intake in both sexes; and (2) sensitivity is extraordinary high in alcohol dependent patients. The results add further evidence to the data that suggest that PEth has potential as a candidate for a sensitive and specific biomarker, which reflects longer‐lasting intake of higher amounts of alcohol and seemingly has the above mentioned certain advantages over traditional biomarkers.

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 and AUDIT scores >or= 8. RESULTS After assessment of the AUDIT, patients were allocated to one of the following groups: patients with AUDIT scores < 8 (n = 52) and with AUDIT scores >or= 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 >or= 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.

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

BackgroundPhosphatidylethanol (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.ResultsMethodological 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.ConclusionMethodological changes make the method more suitable for routine laboratory use, lower the limit of quantification (LOQ) and improve precision.

Emerging Biomarkers : New directions and clinical applications

This article summarizes content proceedings of a symposium held at the 2004 Research Society on Alcoholism Scientific Annual Meeting in Vancouver, Canada. The chairs were Friedrich M. Wurst and Raye Litten. The presentations were (1) Introduction, by Raye Litten; (2) Direct Ethanol Metabolites--On the Threshold From Science to Routine Use, by Friedrich M. Wurst; (3) Sialic Acid Index of Plasma Apolipoprotein J (SIJ) as a Viable Marker for Chronic Alcohol Consumption, by Philippe Marmillot; (4) The Emergence of Ethyl Glucuronide (EtG) Testing as a Tool in Monitoring Healthcare Professionals, by Gregory E. Skipper; (5) Application of Biomarkers for Alcohol Use Disorders in Clinical Practice, by Tim Neumann; (6) Utility of Biomarkers in Assessing the Efficacy of Medications for Treating Alcoholism, by Marty Javors; and (7) Discussion, by Raye Litten.

Phosphatidylethanol formation and degradation in brains of acutely and repeatedly ethanol-treated rats

The formation of the abnormal phospholipid phosphatidylethanol (PEth) was studied in hippocampus, cerebellum and cerebrum of rat brain after intraperitoneal ethanol administration. Prior to analysis by high performance thin layer chromatography PEth was purified. After one injection, PEth levels reached a maximum after 2 h and remained detectable for 14-24 h in all three regions. Repeated injections led to additional accumulation. Maximum in vivo levels of 30-50 nmol/g wet wt. were reached.

World Health Organization/International Society for Biomedical Research on Alcoholism study on state and trait markers of alcohol use and dependence: Back to the future

This article summarizes content proceedings of a symposium held at the 2004 International Society for Biomedical Research on Alcoholism Congress in Mannheim, Germany. The chairs were Boris Tabakoff and Friedrich M. Wurst. The presentations were (1) Genetic associations with alcoholism and affective disorders, by Paula Hoffman; (2) Proteomic analysis of blood constituents in alcoholism, by Boris Tabakoff; (3) Contrasts between the responses of GGT and CDT to high alcohol intake, and a test of their combined use, by John Whitfield; (4) Direct ethanol metabolites such as ethyl glucuronide, fatty acid ethyl esters, phosphatidylethanol and ethyl sulfate: a new line of sensitive and specific biomarkers, by Friedrich Martin Wurst; and (5) Genetic studies of alcoholism subtypes in a Han Taiwanese population, by Ru-Band Lu.

Characterization of Sialic Acid Index of Plasma Apolipoprotein J and Phosphatidylethanol During Alcohol Detoxification-A Pilot Study.

BACKGROUND Apolipoprotein J (ApoJ) is a component of plasma high-density lipoproteins. Previous studies have shown progressive recovery of ApoJ sialic acid content with increased duration of alcohol abstinence. Therefore, the sialic acid index of plasma apolipoprotein J (SIJ) seems to be a promising alcohol biomarker. Phosphatidylethanol (PEth) is a direct ethanol metabolite and has recently attracted attention as a biomarker of prolonged intake of higher amounts of alcohol. The aim of the pilot study was to explore sensitivity, specificity, and normalization of SIJ and PEth in comparison with traditional and emerging biomarkers. METHODS Five male alcohol-dependent patients (International Classification of Diseases 10, F 10.25) were included (median: 40 years old; Alcohol Use Disorders Identification Test value, 30; alcohol consumption in the previous 7 days, 1,680 g). SIJ, PEth, urinary ethyl glucuronide (UEtG), urinary ethyl sulfate (UEtS), and gamma glutamyl-transpeptidase (GGT) were determined at days 1, 3, 7, 10, 14, 21, and 28. RESULTS At study entry, SIJ, PEth, UEtG, and UEtS were positive in all subjects, whereas GGT and mean corpuscular volume were positive in 3 of 5 (60%) of the subjects. Individual SIJ levels increased between day 1 and 28 between 13.7 and 44.3%, respectively. For SIJ and PEth, the ANOVA (p < 0.005) showed a significant trend with the average subjects SIJ and PEth changing 1.22 and 1.02, respectively, per week. CONCLUSIONS Our preliminary data suggest that SIJ and PEth might hold potential as markers of heavy ethanol intake.