Department of Mental Health and Alcohol Research, National Public Health Institute, POB 33, FIN-00251 Helsinki, Finland and
1 Department of Clinical Neuroscience, Karolinska Institutet, SE-171 76 Stockholm, Sweden
Received 21 October 2002; in revised form 3 February 2003; in revised form 12 February 2003; accepted 12 March 2003
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ABSTRACT |
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INTRODUCTION |
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To identify persons who have been drinking excessively for longer periods, biochemical alcohol markers such as carbohydrate-deficient transferrin (CDT) and -glutamyltransferase (GGT) in serum are often applied (Conigrave et al., 2002
). However, CDT and GGT respond only to repeated heavy alcohol consumption over several weeks or months. Filling the gap between ethanol measurement on the one hand and markers for excessive long-term drinking on the other therefore stands as an important goal in alcohol marker research. In recent years, a number of laboratory methods for acute alcohol intake that show a prolonged detection window compared with ethanol have been introduced. Testing urine for a raised concentration of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) provides a method to reveal alcohol consumption within the preceding
24 h, even for several hours after the ethanol is no longer measurable in body fluids or breath (Helander et al., 1993
, 1994
). To improve the accuracy of this test in routine clinical use, 5-HTOL is expressed as a ratio to the major serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA), because this compensates for variations caused by urine dilution and serotonin turnover (e.g. after dietary intake of serotonin) (Helander et al., 1992
).
Ethyl glucuronide (EtG) is a non-oxidative direct metabolite of ethanol with a longer window of detection than the parent compound (Kamil et al., 1952; Jaakonmaki et al., 1967
; Schmitt et al., 1995
). Besides the two-stage oxidation process via alcohol and aldehyde dehydrogenase that accounts for 9598% of total ethanol elimination, a very small fraction (< 0.1%) of the ingested dose undergoes conjugation with UDP-glucuronic acid to produce EtG, which is eventually excreted in the urine (Dahl et al., 2002
; Goll et al., 2002
). As for the urinary 5-HTOL/5-HIAA ratio, a positive finding of EtG, preferably expressed as the EtG/creatinine ratio to compensate for urine dilution (Dahl et al., 2002
), provides a strong indication that the person was recently drinking alcohol, even when ethanol itself is no longer distinguishable from endogenous levels. While the baseline 5-HTOL/5-HIAA ratio is seemingly not influenced by repeated alcohol consumption (Helander et al., 1996
), EtG has been reported to persist in the urine for up to
7585 h after last intake (Seidl et al., 2001
). This observation, together with reports of consistently elevated levels among alcohol misusers (Schmitt et al., 1997
; Seidl et al., 2001
), suggest that EtG may accumulate upon prolonged drinking.
In contrast to the markers of long-term heavy alcohol intake, an acute alcohol marker should not accumulate on repeated ethanol exposure, as this would complicate the interpretation of single test results. The present study was therefore undertaken to examine the stability and reproducibility of EtG and 5-HTOL levels in urine after single and multiple oral doses of ethanol in healthy human subjects.
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SUBJECTS AND METHODS |
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Study design
The study was a controlled interventional study with a cross-over design. All experiments were started at day 14 of the menstrual cycle (i.e. the seventh day of taking the pill) to compensate for any potential interference by the phase of the menstrual cycle. The menstrual cycle phases and use of oral contraceptives do not influence ethanol metabolism (Mumenthaler et al., 1999), but any likely effects on 5-HTOL and EtG have not been studied. The women were randomly assigned to alcohol or placebo with 4 and 5 subjects in each group and the alcohol (8% ethanol, w/v, in juice) or placebo (an equal volume of juice) was ingested during 8 consecutive days. Fresh ethanol and placebo drinks were provided in sealed containers upon daily visits to the laboratory at 16:00 h for blood ethanol testing. Blood ethanol testing was carried out in order to detect any intake of alcohol besides the prescribed doses; the 0.4 g/kg ethanol dose was metabolized within about 6 h (Sarkola et al., 2001
).
On the first day of the study (day 1), ethanol (0.4 g/kg, corresponding to 22.028.4 g) or placebo was ingested within 15 min at 16:00 h. Spot urine samples (40 ml in vials containing 1.0 g/l sodium azide) were collected at 0 (immediately before intake), 4 and 15 h, and venous blood samples were drawn at 0, 2, 3, 4, 5 and 6 h from intake. During the following 6 days (days 27), the ethanol (0.4 g/kg) or placebo was ingested in the morning (at 06:0007:30 h) and in the evening before going to bed (at 21:0000:00 h; total ethanol dose 0.8 g/kg per day). Urine samples were collected daily in the morning (first void and before ethanol or placebo intake) and blood samples in the afternoon at 16:00 h. On day 8, the ethanol or placebo was ingested in the morning and at 16:00 h. Urine samples were collected in the morning before intake, and urine and blood samples were taken from 16:00 h onwards as detailed above for day 1. Morning urine and afternoon blood sampling was then continued for another 3 days (day 911) with no intake of alcohol or placebo. During the next menstrual cycle, the same procedures were carried out with the ethanol and placebo groups reversed.
The blood samples (10 ml) were collected into Vacutainer tubes containing 22.5 mg of sodium fluoride and 22.5 mg of potassium oxalate as anticoagulants. The blood and urine samples were stored at 70°C until analysed.
The study was conducted in accordance with the guidelines proposed in The Declaration of Helsinki and approved by the Hospital District of Helsinki and Vusimaa, Ethical Committee for Research in Epidemiology and Public Health. Written informed consent was obtained from the participants before their inclusion in the study.
Measurement of ethanol and creatinine
The concentration of ethanol in blood and urine was determined by headspace gas chromatography (Sarkola and Eriksson, 2001). The intra- and inter-assay coefficients of variation (CV) were less than 5%, and the limit of quantification (LOQ) was 0.005 mmol/l. A cut-off of 0.1 mmol/l was chosen to indicate recent alcohol consumption. Urinary creatinine was determined using a VITROS 250 Chemistry System (Ortho-Clinical Diagnostics, Rochester, NY, USA). The intra- and inter-assay CV were less than 5% at a creatinine level of 0.44 mmol/l (n = 7), and the LOQ was 0.08 mmol/l.
Measurement of EtG
The concentration of EtG in urine was determined by a negative ion electrospray liquid chromatographicmass spectrometric (LCMS) method (Stephanson et al., 2002). The ions monitored were m/z 221 for EtG and m/z 226 for EtGd5 (internal standard). The intra- and inter-assay CV of the method were less than 12%, and the LOQ was 0.1 mg/l (Stephanson et al., 2002
). The EtG concentrations of unknown samples were determined from the peak area ratio between EtG and EtGd5 by reference to the calibration curve. The EtG and EtGd5 were purchased from Medichem Diagnostica (Steinenbronn, Germany).
Measurement of 5-HTOL and 5-HIAA
Urinary 5-HIAA was determined by high-performance liquid chromatography (HPLC) and 5-HTOL by gas chromatography mass spectrometry (GCMS) (Helander et al., 1996). For 5-HTOL, which is excreted almost entirely in conjugated form with glucuronic acid (Helander et al., 1995
), enzymatic hydrolysis was made prior to analysis. The intra- and inter-assay CV of the assays were less than 7%, and the LOQ were 1 µmol/l for 5-HIAA and 50 nmol/l for 5-HTOL. To compensate for variations in 5-HIAA and 5-HTOL concentrations caused by urine dilution, the 5-HTOL/5-HIAA ratio was calculated. The reference limit used in clinical practice to discriminate between a normal and an elevated 5-HTOL/ 5-HIAA ratio (i.e. indicative of any recent drinking) has been set at < 15 (nmol/µmol) (Helander et al., 1994
).
Statistics
Results are expressed as mean ± standard deviation (SD). The Wilcoxon test for paired samples was used for statistical evaluation of the data. A two-tailed P value of less than 0.05 was considered statistically significant.
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RESULTS |
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During the ethanol drinking period, the individual 5-HTOL/ 5-HIAA results for the urine samples collected in the morning on days 38, when the 0.4 g/kg ethanol dose had been ingested on the previous evening, were elevated in 6, 8, 7, 7, 7 and 7, respectively, out of the total of 9 subjects (i.e. 78% of the samples were positive for 5-HTOL/5-HIAA on average). This compares with positive ethanol values in 5, 4, 7, 5, 4 and 5 subjects (
56%). In contrast, measurable EtG levels were observed in all 9 urine samples of the 9 subjects (100%). The placebo group consistently showed negative urinary ethanol values and the 5-HTOL/5-HIAA ratios were below the reference value of 15 (nmol/µmol). However, two subjects showed one positive EtG value each (0.60 and 0.12 mg/l, respectively) during the placebo period.
The individual morning urine results were highly variable between subjects during the ethanol drinking period (Fig. 1). Accordingly, the ethanol values ranged from 0 (i.e. below the LOQ of 0.005 mmol/l) to 7.3 mmol/l, 1.471.0 mg/l for EtG, 0.14.5 mg/mmol for the EtG/creatinine ratio, and 2109 nmol/ µmol for the 5-HTOL/5-HIAA ratio. There was also a considerable variation in the results between different days for some individuals (data not shown), which was most likely explained by occasional voiding during night-time (although this was not recorded). The individual results for the six urine samples collected on days 38 yielded CV values of 21137% (mean 69%) for EtG, 24118% (mean 53%) for EtG/ creatinine, and 21126% (mean 65%) for 5-HTOL/5-HIAA. For comparison, the corresponding CV values for 5-HTOL/ 5-HIAA during the placebo period were considerably lower at 1243% (mean 22%).
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The elimination rate of ethanol was significantly increased by 24% on average at the end of the drinking period (day 8: 0.112 ± 0.013 g/kg per hour) compared with the initial experiment (day 1: 0.090 ± 0.013 g/kg per hour; P = 0.006) (Sarkola et al., 2001). The EtG/creatinine and 5-HTOL/5-HIAA ratios obtained during the first and second acute drinking experiments are given in Fig. 2
. At 16:00 h on day 8, the EtG/creatinine and 5-HTOL/5-HIAA values were both elevated, but this resulted from the intake of ethanol on the same morning. However, the urine samples collected at 4 h after intake were not significantly different between the days, whereas at 15 h the EtG/creatinine ratio was significantly (P < 0.05) lower on day 8 (Fig. 2
).
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DISCUSSION |
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In some situations, laboratory tests to indicate any recent consumption of alcohol may be more useful. The introduction of sensitive and specific alcohol markers with a prolonged detection window compared with ethanol, such as the 5-HTOL/ 5-HIAA ratio (Helander et al., 1996) and EtG (Seidl et al., 2001
) in urine, has allowed for new and improved diagnostic and treatment strategies (Helander, 1998
; Spies et al., 1999
). During out-patient treatment, for example, the 5-HTOL/5-HIAA ratio provided a much more sensitive method compared with utilizing breath or blood analysis to detect consumption of even moderate amounts of alcohol within the past
24 h (Helander et al., 1999
). In certain workplaces, tests of acute alcohol consumption may be used upon return to safety-sensitive duties (Hagan and Helander, 1997
), because it is well-known that the after-effects of drinking (i.e. hangover symptoms) can impair behaviour and thereby increase the risk of becoming involved in accidents (Wiese et al., 2000
).
The present study confirmed previous observations that urinary 5-HTOL/5-HIAA and EtG values are increased during acute ethanol intake. The levels for both markers were within the ranges typically observed in urine samples collected during routine screening for recent alcohol consumption (Helander et al., 1999; Stephanson et al., 2002
). The detection time for EtG was found to be somewhat longer than for 5-HTOL/ 5-HIAA. A previous study demonstrated that the detection window for recent drinking by an increased urinary level of 5-HTOL, which is excreted mainly as the glucuronic acid conjugate (Helander et al., 1995
), becomes prolonged at higher turnover of 5-HT (i.e. at higher concentrations of 5-HTOL) (Helander and Some, 2000
). Accordingly, because EtG levels are more than 100-fold higher than 5-HTOL glucuronide levels after an acute ethanol dose (Helander et al., 1993
; Schmitt et al., 1997
), it is possible that the longer detection time observed for EtG compared with 5-HTOL/5-HIAA simply results from the much higher concentrations of EtG.
Given the high sensitivity for EtG in detecting recent intake of very small amounts of ethanol (Stephanson et al., 2002), it cannot be excluded that the two positive EtG results observed during the placebo period resulted from the intake of low-alcohol, or alcohol-free (< 0.5% ethanol), beverages. Endogenous concentrations of ethanol, on the other hand, are not sufficient to produce a detectable EtG (Janda and Alt, 2001
; Stephanson et al., 2002
). However, it should always be considered that self-reports of alcohol consumption may have low accuracy (Helander and Eriksson, 2002
).
In contrast to laboratory tests used to indicate long-term heavy alcohol intake, a marker of acute drinking should preferably cover a well-defined time-window and not accumulate on repeated ethanol exposure, as this would complicate the interpretation of single test results. The present study found no indication of accumulation of either 5-HTOL or EtG in the body upon repeated ethanol exposure at a level of 0.8 g/kg (4457 g) per day for a period of 1 week. For EtG, this is in line with theoretical results from a computer simulation (Droenner et al., 2002
). On the contrary, when the acute ethanol challenge was repeated after the 1-week drinking period, a significantly lower EtG/creatinine ratio was observed at 15 h after intake, suggesting a reduced biological half-life for urinary EtG on repeated ethanol dosage. However, because the ethanol elimination rate was also found to be faster on the second challenge (Sarkola et al., 2001
), the lower EtG/ creatinine ratios were, most likely, related to the lower concentration of the parent compound ethanol.
There were considerable variations in the urinary ethanol, EtG and 5-HTOL/5-HIAA values during the drinking period, both between and within subjects. This was probably due to a combination of biological variation and occasional voiding during night-time. The variable ethanol, 5-HTOL/5-HIAA and EtG results observed in previous studies on mixed populations (Jones and Helander, 1999; Dahl et al., 2002
) were confirmed in the present study, which involved women only. The gender effect for EtG has not yet been evaluated in detail, but no sex difference was apparent in one study (Dahl et al., 2002
). Gender is known not to influence the urinary 5-HTOL/5-HIAA ratio (Helander et al., 1996
). Furthermore, the variability for EtG could not be explained by differences in urine dilution, as it remained high when EtG was expressed as a ratio to creatinine. It should be noted that the study protocol allowed for some hours difference in the time elapsing between drinking the ethanol and collecting the urine sample, which could also have influenced the variability of test values.
In conclusion, the present results confirmed that the urinary excretion of EtG and 5-HTOL become increased following acute ethanol intake, and that levels remain elevated for several hours after the ethanol is no longer measurable in body fluids or breath. Most importantly, no significant accumulation of either compound was demonstrated upon multiple-dose administration of ethanol corresponding to 45 standard drinks per day for 1 week. Accordingly, these results indicate that 5-HTOL/5-HIAA and EtG can be used as sensitive and specific tests to assess recent drinking in light-to-moderate as well as chronic heavy consumers, with a slightly longer detection time for EtG.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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