Alcohol and Alcoholism Advance Access originally published online on May 25, 2008
Alcohol and Alcoholism 2008 43(4):431-435; doi:10.1093/alcalc/agn035
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Value of Ethyl Glucuronide in Plasma as a Biomarker for Recent Alcohol Consumption in the Emergency Room
1 Department of Anesthesiology and Intensive Care Medicine, Campus Virchow-Klinikum and Campus Charité Mitte, Charité-Universitätsmedizin Berlin, Germany
2 Alcohol Laboratory, Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden
3 Institute for Laboratory Medicine and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Germany
* Corresponding author: Tim Neumann, Charité-Universitätsmedizin Berlin, Campus Charité Mitte, Klinik für Anästhesiologie und Operative Intensivmedizin, Chariteplatz 1, D-10117 Berlin, Germany. Tel: +49-30-450-631-249; Fax: +49-30-450-531-911; E-mail: tim.neumann{at}charite.de
Received 27 September 2007; first review notified 17 December 2007; in revised form 6 February 2008, 17 March 2008; accepted 3 April 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Aim: This emergency department (ED) study compared the value of plasma ethyl glucuronide (EtG) testing with the information about alcohol consumption obtained using the standard alcohol biomarkers gamma-glutamyltransferase (GGT) and carbohydrate-deficient transferring (CDT) and the AUDIT questionnaire. Methods: Minimally injured and clinically non-intoxicated male patients (n = 81) admitted to an ED were screened regarding their alcohol consumption, using the computerized AUDIT questionnaire and a paper-and-pencil assessment including the type, amount and time of alcohol intake. Blood samples were collected for determination of ethanol, EtG (LC-MS) and GGT in plasma and %CDT in serum (Axis-Shield %CDT immunoassay). Results: Out of the 81 patients, 23 (28%) were positive (
8 points) on the AUDIT questionnaire. Only 3 (4%) showed a detectable ethanol concentration (range 0.01–0.07 g/L) but 31 (38%) showed a detectable EtG (0.16–39.5 mg/L). In four patients, EtG was detectable in plasma for >48 h after estimated completed elimination of ethanol. EtG was not correlated with the long-term biomarkers %CDT or GGT, or the AUDIT results, but with the time since estimated completed ethanol elimination. Conclusion: EtG testing in blood was found useful in the ED as a way to detect recent drinking, even in cases of a negative ethanol test, and to confirm abstinence from alcohol. This sensitive and specific short-term biomarker provides valuable additional information about individual drinking habits and might also be helpful to identify an alcohol hangover. |
Introduction |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Patients with alcohol use disorders are frequently seen in emergency rooms (D’Onofrio et al., 2006
; Neumann et al., 2006). Screening for alcohol use disorders after trauma is usually mandatory, because these patients are at increased risk for morbidity and mortality (Neumann and Spies, 2003). Self-report questionnaires such as the Alcohol Use Disorders Identification Test (AUDIT), ethanol testing in breath or body fluids, and biomarkers of chronic heavy alcohol consumption (e.g., carbohydrate-deficient transferrin, CDT, and gamma-glutamyltransferase, GGT) or recent drinking (e.g., ethyl glucuronide, EtG) have been used for this purpose (Neumann and Spies, 2003; Degutis et al., 2004; Neumann et al., 2004; Savola et al., 2004; Runge et al., 2007).
The alcohol screening methods show different pros and cons for the intended use. Questionnaires sometimes provide unreliable information or may not be applicable at all in critically ill patients. The value of breath or blood ethanol testing, on the other hand, is restricted by the rapid elimination rate of ethanol from the body with resulting short detection time (typically <12 h) (Helander et al., 1996). Even intake of 50–80 g ethanol (e.g., a bottle of wine or 3–4 cans of beer) in the evening may not be measurable in the next morning by this method (Bendtsen et al., 1998). Indeed, a substantial number (11–45%) of trauma patients with severe alcohol use disorders are ethanol negative on admission to the hospital (Maio et al., 1997; Soderstrom et al., 1998). Alcohol biomarkers such as CDT and GGT will mainly identify persons engaged in long-term heavy drinking (Spies et al., 1995; Tønnesen et al., 1999; Neumann and Spies, 2003) whereas their sensitivity to detect those who drink less amounts, or only infrequently, is low (Salaspuro, 1999; Neumann et al., 2003).
Use of sensitive short-term alcohol biomarkers with longer detection windows than ethanol testing may add important information about individual drinking patterns (Helander, 2003). Such tests also aid in the diagnosis of hangover, a condition often associated with the occurrence of alcohol-related trauma (Wiese et al., 2000) that is difficult to evaluate in traumatized patients. The common clinical features of an alcohol hangover, including headache, nausea and fatigue (Bendtsen et al., 1998; Wiese et al., 2000), might be related to the trauma.
Measurement of the phase-II ethanol metabolites EtG (Schmitt et al., 1995) and ethyl sulfate (EtS) (Helander and Beck, 2004) are sensitive methods to spot recent drinking. In urine, these tests show positive values for up to 1–3 days after alcohol intake, while the corresponding detection times in the blood are shorter (Hoiseth et al., 2007). However, in clinical settings such as the emergency department (ED), it might not always be practical or even possible to sample urine. In addition, given the long detection times in urine for these sensitive short-term tests after drinking only small amounts of alcohol (Stephanson et al., 2002), blood testing may better display a recent heavy intake.
This ED study compared the value of EtG testing in blood (plasma) with the information about alcohol consumption obtained from the standard alcohol biomarkers CDT and GGT and the AUDIT questionnaire.
|
Materials and Methods |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Subjects
The study was carried out at the ED of a large regional downtown trauma centre, Charité Campus Mitte, University Hospital of Berlin, Germany. All male patients
18 years of age admitted between July 2002 and January 2003 with a primary diagnosis of acute injury were included. Patients were excluded if they were medically unstable or required hospital admission, had severe pain (>3 points on a 10-point numeric rating scale), a second injury, a severe psychiatric condition, did not speak German, were in police custody, members of the hospital staff, if intoxication precluded participation or if alcohol-related data were not fully available or inconsistent. A detailed description of the study design is found elsewhere (Neumann et al., 2006).
Altogether, 81 subcritically injured non-intoxicated male patients with a mean age of 35 years (range 18–76 years) participated in the study. Demographic data and data on injury severity (Injury severity score (ISS), range 0–75 points; >9 is considered a serious injury) (Hoyt et al., 2000) were derived from hospital records or a questionnaire. All patients underwent alcohol screening following written informed consent. Ethical approval was obtained from the Institutional Review Board of the University Hospital Charité, Berlin.
Alcohol questionnaire
After provision of emergency care (including pain management), patients were asked to complete a computerized lifestyle questionnaire using a portable laptop computer. A ‘mouse only’ technique was used that did not require typing. Each question was displayed alone, and a new question was not displayed until 1 s after completion of each item, which prevented question skipping.
The computerized lifestyle questionnaire included the 10 questions of the standardized AUDIT questionnaire. The AUDIT was designed by the World Health Organization to detect a broad range of alcohol problems, ranging from at-risk drinking to dependence (Saunders et al., 1993; Neumann et al., 2004). The standard AUDIT cut-off limit for males at 8 points was used to indicate ‘at-risk drinkers’, whereas those scoring 1–7 points were classified as ‘social drinkers’ and those with 0 points ‘non-drinkers’.
Alcohol consumption data
The weekly average alcohol intake was assessed using a modified quantity–frequency method (Neumann et al., 2006) and a self-administered paper-and-pencil questionnaire. The questions included the type and amount of alcoholic beverage consumed on weekdays and weekends, and the time, type and amount of last intake. Times for trauma and blood sampling were also noted. To adjust for the variable doses of alcohol and different times between drinking and blood sampling, the estimated time since complete ethanol elimination was calculated for each patient, using an ethanol elimination rate of 0.15 g/kg/h and a distribution volume of 0.7 x body weight. To determine if patients met ICD-10 criteria for alcohol dependence or harmful use, trained research fellows working under the supervision of a trained psychiatrist or psychologist conducted a face-to-face diagnostic interview.
Laboratory tests
In the ED, blood was collected and taken for determination of ethanol, EtG and GGT in plasma (heparin) and %CDT in serum. In 80% of the patients, blood sampling was performed within 10 h after trauma and in 50% within 3.5 h. Ethanol was determined by the enzymatic ADH method (Roche Diagnostics, Mannheim, Germany), using a Roche Modular P selective chemistry analyser (Roche Diagnostics). The lower detection limit of this method is 0.1 g/L ethanol, according to information from the manufacturer. Measurement of GGT was performed by the kinetic photometric test at 25°C, using a reference interval of 
28 U/L for males (Szasz, 1974). %CDT analysis was performed using the Axis-Shield %CDT minicolumn immunoassay (Bio-Rad), using an upper reference limit of 3.0% (Helander et al., 2001).
Measurement of EtG in plasma was performed by electrospray liquid chromatography-single mass spectrometry (LC-MS), essentially as described for urine samples (Stephanson et al., 2002), using selected ion monitoring of m/z 221 for EtG and m/z 226 for the penta-deuterated internal standard (EtG-D5). A 200-µL aliquot of plasma was mixed with 1.0 mL methanol containing internal standard and centrifuged at 14.000 g for 10 min. The supernatant was evaporated to dryness under a stream of nitrogen and the final content dissolved in 100 µL of de-ionized water. A 10-µL aliquot was injected into the LC-MS system. A calibration curve for EtG prepared by serial dilution with EtG negative plasma samples was linear (r2 = 0.994, P < 0.0001) in the concentration range 0.1–100 mg/L. The EtG concentration of unknown samples was determined from the peak area ratio between EtG and EtG-D5 by reference to the calibration curve. The limit of quantification was 
0.15 mg/L EtG.
Statistical analysis
Statistical analysis was performed using the SPSS 14 software. Patient characteristics were reported as frequencies, or as the median and quartiles. Frequencies were compared using the chi-square test. The Kruskall–Wallis test, Mann–Whitney U-test and Exact test were used to check for differences between groups. Correlation analysis was done using the Spearman–rho correlation. The Receiver Operating Characteristics (ROC) curve analysis was used to assess the overall accuracy of the test. An area under the ROC curve of 0.8 was chosen as the definition of acceptable screening characteristics, using the following classification: 0.90–1.00 = excellent; 0.80–0.89 = good; 0.70–0.79 = fair; 0.60–0.69 = poor; < 0.60 = fail (Luna-Herrera et al., 2003).
|
Results |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Demographic and alcohol-related characteristics of the 81 non-intoxicated male ED patients with subcritical trauma are shown in Table 1. The majority of patients were young adults and 89% had an ISS score of 1 (total range 1–6) indicating only minor trauma. Based on the results of the AUDIT questionnaire, 9% were classified as non-drinkers (0 points), 63% as social drinkers (1–7 points) and 28% at risk for alcohol-related problems (
8 points). There were no significant differences in age or body mass index (BMI) between the patient groups. The at-risk drinkers consumed twice the amount of alcohol compared with the social drinkers (Table 1). Twelve of the 81 patients (15%) admitted drinking alcohol within 10 h prior to the trauma and 53 (65%) within 24 h.
|
Patients with an ICD-10 diagnosis of alcohol dependence or harmful use were found almost exclusively among the at-risk drinkers. Altogether 11 of 23 (48%) patients who scored
8 points on the AUDIT were also diagnosed with dependence or harmful use, compared with only 1 of 58 (1.7%) for those scoring <8 points. This corresponds to a sensitivity of 83% and a specificity of 92% of the AUDIT questionnaire for either of these alcohol diagnoses. EtG was detected (range 0.16–39.5 mg/L) in 31 of 81 (38%) plasma samples, while only 3 (4%) had a detectable ethanol (Table 1). Positive results for EtG were found among those classified as social drinkers (frequency 39%; range 0.16–11.2 mg/L) and at-risk drinkers (frequency 48%; range 0.33–39.5 mg/L; P = 0.035) but not in non-drinkers. Compared with social drinkers, at-risk drinkers also showed higher median values for %CDT and GGT, but the differences did not reach statistical significance (Table 1).
The time course of EtG in plasma versus the time since calculated completed ethanol elimination is shown in Fig. 1. In seven patients, EtG was detectable in blood collected >24 h after the calculated time for complete ethanol elimination, and in four patients >48 h afterwards, which indicated misreporting. There was a negative correlation between plasma EtG and the time since sobering up (rho = –0.508, P < 0.001), a weak but statistically significant positive correlation with weekly alcohol intake (rho = 0.263, P = 0.018) and serum %CDT (rho = 0.268, P = 0.016), but no significant correlation with GGT (rho = 0.158, P = 0.16) or the results of the AUDIT questionnaire (rho = 0.144, P = 0.20). The overall abilities of EtG, %CDT and GGT to differentiate between a positive and a negative AUDIT result were not markedly different and clinically or statistically not significantly better than chance (Table 2).
|
|
|
Discussion |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This ED study on patients with subcritical trauma compared the extra information about alcohol consumption obtained by the sensitive and specific short-term alcohol biomarker EtG in plasma with that of the conventional long-term biomarkers CDT and GGT and the AUDIT questionnaire. Occasional high alcohol intake is independent to the presence of alcohol use disorders, but excessive drinking is a common cause of trauma and patients with chronic heavy alcohol consumption exhibit a higher incidence of morbidity and mortality following surgery (Spies et al., 1998
). Recognition of these high-risk alcohol patients is therefore important, to allow for intervention and reduction of alcohol-related harm (Spies et al., 1999).
Because the trauma patients that appeared intoxicated at admission were excluded from this study, only few (4%) had a detectable blood ethanol concentration. The finding that four of seven patients that reported very recent alcohol intake had no detectable ethanol further highlights the limited value of ethanol testing in this setting. Nonetheless, a high frequency (38%) of the patients were positive for the ethanol metabolite EtG in plasma, thus confirming that they had consumed alcoholic beverages within the past 12–24 h (Schmitt et al., 1995; Hoiseth et al., 2007). The EtG concentrations were negatively correlated with the self-reported time since last intake, and also with the estimated time since complete ethanol clearance from the body. However, a few positive EtG results were not confirmed by self-report, suggesting that verbal information was not reliable in these cases. EtG testing in blood (plasma or serum) may therefore be a helpful objective tool in the ED, to validate self-reports of drinking and identify high-risk patients engaged in recent heavy drinking, but also to differentiate between alcohol- and trauma-related headache, nausea and fatigue. As expected, because EtG is only detectable in blood for a limited time after each alcohol intake, the correlation of plasma EtG with the long-term measures %CDT, GGT, weekly alcohol intake, and the results of the AUDIT questionnaire was either weak or lacking.
The AUDIT questionnaire was able to detect patients with relevant alcohol use disorders (an ICD-10 diagnosis of alcohol dependence or harmful use). This has previously been demonstrated in different clinical settings including trauma settings (Neumann et al., 2004). It should be kept in mind that paper-and-pencil and computer-based questionnaires are not always applicable in the ED, and obviously not in severely traumatized and heavily intoxicated patients. Based on the results on the AUDIT questionnaire, the patients were classified into 3 alcohol consumption subgroups. EtG was detected in 39% of the plasma samples collected from ‘social drinkers’ and in 48% of samples from ‘at-risk drinkers’. In addition to the higher frequency of positive results among at-risk drinkers, these also showed higher EtG concentrations on average, including the 3 by far highest concentrations (range 31.9–39.5 mg/L). However, 17% of the social drinkers showed elevated biomarkers of chronic consumption (CDT or GGT) and these patients were also positive for plasma EtG in 79% of cases. Likewise, 9 at-risk drinkers did not show positive chronic alcohol biomarkers and, according to the EtG results, had not recently been drinking. The poor ability of conventional alcohol biomarkers to differentiate between patients with and without relevant alcohol use disorders has been described earlier for young trauma patients with a predominant binge-type drinking style (Ryb et al., 1999; Salaspuro, 1999; (Neumann and Spies 2003; Savola et al., 2004). These findings support the need for further clinical evaluation.
This study employed plasma samples for testing of EtG, although EtG is detectable for considerably longer time in urine (Schmitt et al., 1995; Hoiseth et al., 2007) implying higher sensitivity for recent drinking. However, it might not always be feasible to collect urine in the ED. In addition, most patients participating in this study were reluctant to leave urine whereas blood sampling was more tolerated; patients were often afraid to depart from the waiting area to collect a urine sample, because the rest room was out of sight from the treatment area. Many patients agreed to provide a urine sample after being treated but some eventually failed to. Several patients were also afraid that the urine sample would be taken for testing of illicit drugs.
In conclusion, EtG testing in plasma was found useful in the ED as an objective way to detect recent drinking, even in cases of a negative ethanol concentration, and to confirm abstinence from alcohol. Because the EtG results were not correlated with the long-term biomarkers CDT and GGT, or with the results of the AUDIT questionnaire, this sensitive and specific short-term test provides valuable supplementary information about individual drinking habits. However, as a stand-alone test, it should be pointed out that a single positive EtG result cannot distinguish between a recent occasional intake of alcohol and prolonged heavy drinking. Furthermore, if a positive EtG result could lead to legal consequences for the patient, the sample should be taken for confirmatory analysis using LC-MS/MS (Weinmann et al., 2004). Additional studies are warranted to evaluate the effectiveness of this promising test in the context of clinical decision-making, to allocate patients into distinct risk groups for alcohol-related problems, such as at-risk drinkers or excessive harmful drinkers, and in stratifying patients for alcohol intervention (Neumann and Spies, 2003).
|
ACKNOWLEDGEMENTS |
|---|
The study was supported by the German Ministry of Health (BMGS 217-43794-5/5). Financial support was further provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institute.
|
FOOTNOTES |
|---|
a To equal parts.
|
References |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bendtsen P, Jones AW, Helander A. Urinary excretion of methanol and 5-hydroxytryptophol as biochemical markers of recent drinking in the hangover state. Alcohol Alcohol (1998) 33:431–8.
Degutis LC, Rabinovici R, Sabbaj A, et al. The saliva strip test is an accurate method to determine blood alcohol concentration in trauma patients. Acad Emerg Med (2004) 11:885–7.[CrossRef][Web of Science][Medline]
D’Onofrio G, Becker B, Woolard RH. The impact of alcohol, tobacco, and other drug use and abuse in the emergency department. Emerg Med Clin North Am (2006) 24:925–67.[CrossRef][Web of Science][Medline]
Helander A. Biological markers in alcoholism. J Neural Transm Suppl (2003) 66:15–32.[Medline]
Helander A, Beck O, Jones AW. Laboratory testing for recent alcohol consumption: comparison of ethanol, methanol, and 5-hydroxytryptophol. Clin Chem (1996) 42:618–24.
Helander A, Beck O. Mass spectrometric identification of ethyl sulfate as an ethanol metabolite in humans. Clin Chem (2004) 50:936–7.
Helander A, Fors M, Zakrisson B. Study of Axis-Shield new %CDT immunoassay for quantification of carbohydrate-deficient transferrin (CDT) in serum. Alcohol Alcohol (2001) 36:406–12.
Hoiseth G, Bernard JP, Karinen R, et al. A pharmacokinetic study of ethyl glucuronide in blood and urine: Applications to forensic toxicology. Forensic Sci Int (2007) 172:119–24.[CrossRef][Web of Science][Medline]
Hoyt DB, Coimbra R, Potenza BM. Trauma systems, triage, and transport. Moore EE, Feliciano DV, Mattox KL, eds. (2000) 5th ed. New York: McGraw-Hill. 57–83. Trauma.
Luna-Herrera J, Martinez-Cabrera G, Parra-Maldonado R, et al. Use of receiver operating characteristic curves to assess the performance of a microdilution assay for determination of drug susceptibility of clinical isolates of Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis (2003) 22:21–27.[Web of Science][Medline]
Maio RF, Waller PF, Blow FC, et al. Alcohol abuse/dependence in motor vehicle crash victims presenting to the emergency department. Acad Emerg Med (1997) 4:256–62.[Web of Science][Medline]
Neumann T, Spies C. Use of biomarkers for alcohol use disorders in clinical practice. Addict (2003) 98:81–91.[CrossRef]
Neumann T, Neuner B, Gentilello LM, et al. Gender differences in the performance of a computerized version of the Alcohol Use Disorders Identification Test in subscritically injured patients who are admitted to the emergency department. Alcohol Clin Exp Res (2004) 28:1693–701.[CrossRef][Web of Science][Medline]
Neumann T, Neuner B, Weiss-Gerlach E, et al. The effect of computerized tailored brief advice on at-risk drinking in subcritically injured trauma patients. J Trauma (2006) 61:805–14.[Web of Science][Medline]
Runge JW, Hargarten S, Velianoff G, et al. Recommended best practices of emergency medical care for the alcohol-impaired patient: screening and brief intervention for alcohol problems in the emergency department. (2007) Available at: http://www.nhtsa.dot.gov/people/injury/alcohol/EmergCare/recommended.htm. Accessed April 24, 2007.
Ryb GE, Soderstrom CA, Kufera JA, et al. Use of blood alcohol concentration and laboratory tests to detect current alcohol dependence in trauma center patients. J Trauma (1999) 47:874–9.[Web of Science][Medline]
Salaspuro M. Carbohydrate-deficient transferrin as compared to other markers of alcoholism: a systematic review. Alcohol (1999) 19:261–71.[CrossRef][Web of Science][Medline]
Saunders JB, Aasland OG, Babor TF, et al. Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO Collaborative Project on Early Detection of Persons with Harmful Alcohol Consumption-II. Addict (1993) 88:791–804.[CrossRef]
Savola O, Niemela O, Hillbom M. Blood alcohol is the best indicator of hazardous alcohol drinking in young adults and working-age patients with trauma. Alcohol Alcohol (2004) 39:340–5.
Schmitt G, Aderjan R, Keller T, et al. Ethyl glucuronide: an unusual ethanol metabolite in humans. Synthesis, analytical data, and determination in serum and urine. J Anal Toxicol (1995) 19:91–4.[Web of Science][Medline]
Soderstrom CA, Dischinger PC, Kerns TJ, et al. Screening trauma patients for alcoholism according to NIAAA guidelines with alcohol use disorders identification test questions. Alcohol Clin Exp Res (1998) 22:1470–75.[CrossRef][Web of Science][Medline]
Spies CD, Emadi A, Neumann T, et al. Relevance of carbohydrate-deficient transferrin as a predictor of alcoholism in intensive care patients following trauma. J Trauma (1995) 39:742–8.[Web of Science][Medline]
Spies CD, Herpell J, Beck O, et al. The urinary ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid in surgical patients with chronic alcohol misuse. Alcohol (1999) 17:19–27.[CrossRef][Web of Science][Medline]
Spies CD, Kissner M, Neumann T, et al. Elevated carbohydrate-deficient transferrin predicts prolonged intensive care unit stay in traumatized men. Alcohol Alcohol (1998) 33:661–9.
Stephanson N, Dahl H, Helander A, et al. Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography-mass spectrometry. Ther Drug Monit (2002) 24:645–51.[CrossRef][Web of Science][Medline]
Szasz G. gamma-Glutamyltranspeptidase. Bergmeyer HU, ed. (1974) Weinheim: Verlag Chemie. 757. Methoden der enzymatischen Analyse.
Tønnesen H, Carstensen M, Maina P. Is carbohydrate deficient transferrin a useful marker of harmful alcohol intake among surgical patients? Eur J Surg (1999) 165:522–27.[CrossRef][Web of Science][Medline]
Weinmann W, Schaefer P, Thierauf A, et al. Confirmatory analysis of ethylglucuronide in urine by liquid-chromatography/ electrospray ionization/tandem mass spectrometry according to forensic guidelines. J Am Soc Mass Spectrom (2004) 15:188–93.[CrossRef][Web of Science][Medline]
Wiese JG, Shlipak MG, Browner WS. The alcohol hangover. Ann Intern Med (2000) 132:897–902.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||