A.W. Jones Department of Forensic Toxicology, University Hospital, L ink~ping, Sweden. I Abstracl t. Introduction. Materials and Methods

Transcription

1 Lack of Association Between Urinary Creatinine and Ethanol Concentrations and Urine/Blood Ratio of Ethanol in Two Successive Voids from Drinking Drivers A.W. Jones Department of Forensic Toxicology, University Hospital, L ink~ping, Sweden I Abstracl t The relationship between urinary ethanol concentration, urine/blood ratio of ethanol, and urinary crealinine content was investigated by the analysis of two successive voids from 40 individuals apprehended for driving under the influence of alcohol (DUI) in Sweden. The first specimen of urine was collected rain (mean plus or minus standard deviation) before sampling blood, and the second specimen was collected min after blood sampling. The mean blood-alcohol concentration (8AC) was g/l, and the corresponding concentrations in the urine (UAC) were g/l for the first void and g/l for the second void. The mean urine/blood ratios of ethanol were for the first void and for the second void; the difference of was statistically significant (t = 3.08, p < 0.01). The concentrations of creatinine in urine from DUI suspects were 0.72 _ g/l for the first void and g/l for the second void; there was no significant difference (t = 1.45, p > 0.05). The urinary creatinine content in specimens from drunk drivers was significantly less than the first morning voids from 3313 prison inmates ( g/l). No significant correlations existed between UAC and urinary creatinine content (r = -0.14) between urine/blood ratios of ethanol and urinary creatinine (r = -0.1). However, the concentrations of ethanol in blood and urine were highly correlated; they were r = (p < 0.001) for first void and r = (p < 0.001) for the second void. This study demonstrates that the relative dilution of urine specimens, as reflected in creatinine content, is not associated with the concentration of ethanol in the urine samples or with the UAC/BAC ratio. Introduction Measuring urinary creatinine is often included in drugs of abuse screening programs to monitor and control for highly dilute specimens. The concentration of creatinine in urine decrease sharply after drinking water, alcoholic beverages, or taking diuretic drugs (1-3). After increasing the production of urine (e.g., by drinking water), the concentrations of drugs of abuse such as cannabis and its metabolites decrease, making it more likely to obtain results below a certain critical threshold (4,5). Some investigators have suggested that the results of urinary drug testing should be reported per milligram of creatinine instead of per liter of urine whenever unusually dilute specimens are encountered as reflected in the urinary creatinine content (3,6). The analysis of urinary creatinine also provides a check on whether the urine specimens might have been diluted in vitro, that is, after voiding (7). Attempts to adulterate urine specimens in various ways such as by dilution with water or other liquids are not uncommon, especially among drug addicts and prison inmates undergoing rehabilitation (7-). Most analytical systems currently being used for drugs-ofabuse screening in urine make use of various immunoassay technologies, and these procedures can easily be modified to include the determination of urinary ethanol (10-12). There seems to be a growing interest in measuring and reporting the concentration of ethanol in urine along with the concentrations of illicit drugs of abuse (13,14). This raises the question of whether water-induced or alcohol-induced diuresis, as reflected in the concentration of creatinine and the relative dilution of specimens, needs to be considered when the urinary alcohol results are interpreted by medical review officers. Two successive urinary voids were collected from suspected drinking drivers to study the impact of dilution of the urine specimens as reflected in creatinine content on the concentration of ethanol and the urine/blood ratios of ethanol. Urine was voided once before and once after a specimen of venous blood was obtained from each suspected drinking driver. The bloodalcohol (BAC) and urine-alcohol (UAC) concentrations and UAC/BAC ratios were determined and compared with the concentration of creatinine in the urine specimens. Materials and Methods Subjects Specimens of blood and urine were obtained from 40 men apprehended in Sweden for driving under the influence of 184 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

2 alcohol. Because many DUI suspects are not arrested while at the wheel, they have the opportunity to claim consumption of alcohol after driving and before providing body fluids for forensic analysis of alcohol. When this happens, the police must be prepared to obtain two urinary voids in addition to a sample of venous blood. The first specimen of urine should be taken as soon as possible after making the arrest, and the second specimen should be taken approximately 1 h later and close to the time of venous blood sampling. The concentration ratios of alcohol in blood and urine and the magnitude and direction of change in UAC between the two voids provides useful information to establish if recent consumption of ID O Urinary creatinlne (g/l) alcohol has occurred. In this way, the allegation of drinking after the offence can be confirmed or challenged (15). Accordingly, there are long traditions in Sweden for analyzing and interpreting the concentrations of alcohol in blood and urine samples in traffic law enforcement (16). It should be noted that DUI suspects cannot be prosecuted in Sweden on the basis of the UAC alone. The material for this study included 40 cases of DUI selected from those sent to the National Laboratory of Forensic Toxicology (Link6ping, Sweden); both venous blood and two successive urinary voids were available for all cases. Collection of the specimens of blood and urine Blood samples were collected in evacuated tubes (Ivers-Lee Division of Becton Dickinson, West Calclwel], NJ) containing 100 mg sodium fluoride and 25 mg potassium oxalate as preservatives. Two tubes of blood were filled in rapid succession, and aliquots for analysis of ethanol were removed from both of these tubes. The subjects were observed during the collection of urine to ensure that the specimens were not adulterated. The total volume voided was recorded, and approximately 10 ml was transferred into a plastic screwcapped tube that contained a tablet of sodium fluoride (100 rag) as preservative. Urine specimens (N = 3313) were also collected from prison inmates who were mostly healthy individuals not exposed to alcohol and not abusing drugs, and this gives a reference range for urinary creatinine. The first morning void was also collected from one healthy volunteer for 3 consecutive days. >* n Urinary creatlnlne (g/l) Figure 1. Frequency distribution of the concentration of creatinine in 3313 urine specimens from prison inmates (A) that were considered to represent reference values in individuals not exposed to alcohol. Most of the specimens were first morning voids. The mean creatinine was g/l (median 1.70 g/l), and the 5th and 5th percentiles were 0.74 and 3.17 g/i.. The lower plot (B) shows the distribution of urinary creatinine in 3 morning voids from one healthy volunteer mean ( SD) g/l. Determination of urinary ethanol and creatinine content The concentrations of ethanol in blood and urine were determined by headspace gas chromatography (GC) as described in detail elsewhere (17). All determinations of ethanol were made in duplicate (urine) or triplicate (blood), and the mean result was reported to two decimal places as grams ethanol per liter (g/l). The coefficient of variation of the GC method was 0.8% at a mean concentration of 1.0 g/l. Urinary creatinine was determined by the Jaffe method with reagents purchased from Boehringer- Mannheim and run on a Hitachi 717 analyzer. A single determination of creatinine was made on each specimen, and the withinrun coefficient of variation (CV) of the method was 2% at a mean concentration of creatinine of 1.0 g/l. The UAC in the first and second voids were used to estimate the person's BAC by dividing by an assumed UAC/BAC ratio of 1.33:1 (18,1). 185

3 Results Figure 1A shows a histogram of urinary creatinine in 3313 specimens from prison inmates, and these results provide a reference range. The mean concentration of urinary creatinine was 1.78 g/l (median 1.70), and the distribution was somewhat skewed with a coefficient of variation of 41%, a coefficient of skewness of 0.651, and a coefficient of kurtosis of Figure 1B shows a frequency distribution of the concentrations of creatinine in 3 consecutive first morning voids from one healthy male subject. Here the mean was g/l (2 SD), which was not significantly different from the mean value 5.0 = % o I First void I 4.0 ~P ~ Sec~ v~ o o o o ^ _.: Qo if---==--" o g o [ P- 2.0 eo ~o o =-" o o N=tSU 1.0 o o r= (t= -1.3) o o0 u I I I n I u I I Urinary creatinine (g/l) 2.0 I 1.8 First void 1.6.j o Second void '~ 1.4/~ ~ ~ "~ n ( O t o~ o o.sj N= 0.6 ~ r= -0.1 (t= -1.7) ot, i i i i i Urinary creatinine (g/l) Figure 2. Scatter plots showing the lack of correlation between urinary alcohol concentration and urine creatinine (upper plot) and urine/blood ratio of ethanol and urinary creatinine (lower plot) in specimens from 40 drinking drivers. Values are plotted separately for first and second urinary voids that were, on the average, about 65 min apart. for prison inmates (p > 0.05). However, the creatinine content of the urine specimens from DUI suspects (Table I) was significantly less than for the prison inmates and the healthy volunteer (p < 0.001). The change in urinary creatinine between the first and second voids was not statistically significant (t = 1.45, p > 0.05). Table I presents mean values and variability for the concentrations of ethanol in urine, the urine/blood ratios of ethanol, and the concentration of creatinine in both first and second voids from 40 drinking drivers. The mean BAC was g/l, which was in good agreement with the estimated BAC ( g/l) for the first void (UAC/1.33). The mean difference in BAC (observed - estimated) was (2 SE), which was not statistically significant (p > 0.05). The individual differences, however, spanned from g/t, to 1.0 g/l. The mean BAC estimated from the second void was (2 SD), which was less than the actual mean BAC observed (2.20 g/l). The mean difference was g/l (p < 0.05), and the individual differences spanned from to 0.43 g/l. Figure 2 shows scatter plots of urine/blood ratio of alcohol and urinary creatinine (lower plot) and also between UAC and creatinine (upper plot). The lack of association is confirmed by the low and nonsignificant correlation coefficients r = (p > 0.05) for UAC and r = -0.1 (p > 0.05) for UAC/BAC. ' This lack of association between UAC/BAC 3.5 ratio and urinary creatinine is more easily seen from the vertical bar graphs in Figure 3. A small positive correlation was established between urine/blood ratios of ethanol and UAC (r = 0.41, p < 0.05), and the relevant scatter plot is shown in Figure 4. The UAC and BAC values were highly correlated both for the first void (r = 0.4, p < 0.001) and the second void (r = 0.6, p > 0.001) as shown by the scatter plots in Figure 5. The 5% prediction limits for a single new observation are shown, and two outlying values can be identified in the upper plot, which depicts the first urine void. In these individuals, the UAC was less than the BAC at the time of voiding. Discussion Ethanol distributes throughout the total body water after drinking, and any binding to plasma proteins appears to be negligible with only trace amounts entering into lipids and bone. Between 1 and 5% of the dose of ethanol consumed is excreted unchanged in urine, sweat, and exhaled air (20). Most of 186

4 the ethanol is metabolized in the liver to produce acetaldehyde, and this toxic metabolite is oxidized to acetate, which is then oxidized in peripheral tissues to give the end products carbon dioxide and water (21). Because only relatively small amounts of ethanol are excreted unchanged as opposed to being metabolized, large increases in the output of urine, such as might result from Table I. Urinary Ethanol (UAC), Urinary Creatinine, and UAC/BAC Ratios (mean+sd) in Two Consecutive Urinary Voids from 40 Drunk Drivers Estimated* Urine specimen UAC (g/l) UAC/BAC Creatinine (g/l) BAC (~q.) Firstvoid Secondvoid ~ *Estimated as UAC/1.33 and the actual blood-alcohol concentration (mean was 2.20:1:0.76 g/i.. *The time between first and second void was 6 rain (mean "~ 1.2,~ First urinary void Crsatinine (g/l) Figure 3. Lack of association between urine/blood ratios of alcohol and the concentration of creatinine in samples of urine from drinking drivers. Two specimens of urine with creatinine > 1.6 g/l are not included in the plot. drinking water or taking diuretic drugs, will not significantly influence the total amount of alcohol cleared from the body by excretion (20). Ethanol is transported to the kidney via the renal artery at a blood flow of approximately 1.2 L/rain. The high ratio of blood flow to tissue mass in the kidney means that arterial-venous differences for water-soluble drugs like ethanol should be small or negligible (22). Early studies of the urine/blood alcohol relationship were reported by Widmark (23), Miles (24), Eggelton (25), and Haggard and Greenberg (26), and these workers reached the conclusion that alcohol was excreted into urine by a process of passive diffusion and that the kidneys could not concentrate ethanol. If normal urine contains % (w/w) water, the UAC should be closer to the concentration in plasma, which is 2% (w/w) water, rather than the concentration in whole blood, which is 80% (w/w) water. Accordingly, the urine/blood distribution ratio of ethanol should theoretically be approximately 1.23:1 if the specimen was obtained from the ureters that carry urine from each kidney into the bladder. However, the relationship between urinary and blood-alcohol concentration is complirated because of the variable storage time of urine in the bladder before voiding and because a person's BAC is continuously changing. Moreover, if the bladder contained an alcohol-free pool of urine before the person drank alcohol, this urine pool would dilute the concentration of alcohol in freshly produced urine during collection in the bladder (15,27). Under these circumstances, abnormally low urine/blood ratios of ethanol would be expected for the first specimen voided. Because alcohol exerts a diuretic effect, especially during a rising BAC, a person will soon be inclined to urinate so the dilution effect is much less of a problem in subsequent batches of urine (20). Reabsorption of alcohol from the bladder back into the bloodstream is a relatively slow and inefficient process because of the isolated nature of the bladder and its poor blood supply (23,26). Studies have shown that after an evening's drinking, the first morning void might contain relatively high concentrations of alcohol, although the concentration in blood and breath at the time of voiding are below limits of detection by conventional analytical methods (28). Ethanol in the blood is metabolized during the time the person is asleep; therefore, the UAC in the first morning void reflects the average BAC since the bladder was last emptied. Drinking a large volume of water increases the production of urine by inhibition of the antidiuretic hormone vasopressin and this means a more dilute urine in the subsequent void. Creatinine content, osmolality, and specific gravity of the urine decreased markedly after drinking 500 ml water (3). The resulting diuresis would increase the total quantity of ethanol excreted in the urine, although it still remains a small fraction of the total dose ingested (21). Moreover, regardless of diuresis, the concentration of alcohol in the urine will remain more or less unchanged because alcohol and water are completely miscible and are handled in the same way in the kidney tubules by passive diffusion (18,27). The concentrations of creatinine in the urine specimens collected from DUI suspects were low compared with the reference values in the prison inmates as might be expected from the well- 187

5 known diuretic action of ethanol. In the context of urinary drug screening, the rationale for analyzing creatinine is to control for highly dilute specimens and if necessary report the concentration of illicit drug per milligram creatinine instead of per liter of urine (5,6). There was no association between urinary creatinine, UAC, and the UAC/BAC ratio, so making this type of correction for ethanol concentrations in dilute urine specimens is not necessary. Lundquist (2) reported a classic study of the urine/blood alcohol relationship, and he measured creatinine in urine samples from DUI suspects (single voids) and attempted to relate the findings to the person's BAC and diuresis. The highest concentration of creatinine was found in urine from subjects with zero BAC, and the lowest concentration was in those with the highest BAC (2). Lundquist also reported a lack of association between urine flow rate and UAC/BAC ratios based on controlled drinking experiments. When urine flow rate was less than 1.0 ml/min, 1-2 ml/min, or 2-16 ml/min, the mean UAC/BAC ratios were 1.31 (span ), 1.32 (span ), and 1.37 (span ), respectively. Estimating a person's BAC from the concentration measured in urine by assuming a population average UAC/BAC ratio has become a controversial subject in forensic toxicology and especially in traffic law enforcement (18). A randomly timed specimen of urine will not necessarily give an accurate estimate of the concentration of ethanol in the blood at the time of voiding (15,26-28). Instead, the UAC reflects the average BAC since the bladder was last emptied. This problem was recognized in California (18) and in Great Britain (1) when setting statutory concentration limits of. i First void oo 1.0- ~ o o o~ " o Second void r = 0.41 (t = 3.) o T,,,,, Urine ethanol (g/l) Figure 4. Scatter plot of the relationship between urine/blood ratio of alcohol and the urinary alcohol concentration for the first and second voids. The correlation coefficient (r) was r = 0.41 for the whole material and this increased to r= 0.44 when three outliers were eliminated. alcohol in urine for prosecuting drinking drivers. The first void was discarded, and the second urine void, obtained rain after emptying the bladder, was used for quantitative determination of ethanol (18,1). If the second void contained alcohol, there must have been alcohol in the blood during the time interval between samplings, and this BAC is estimated from the relationship UAC/1.33. However, translating UAC into BAC for forensic purposes was not necessary in Great Britain because the threshold UAC for prosecution of drunk drivers was defined by statute. Accordingly, a venous blood-alcohol concentration of 80 rag/100 ml was deemed as being equivalent to a UAC (second void) of 107 mg/dl urine, which implies a urine/blood ratio of 1.33:1, although this conversion factor never became an issue for discussion and debate in DUI litigation (1). The use of urine as a specimen for analysis of ethanol is often criticized because of the risk of ethanol being formed in the specimen after voiding (30). This caution is particularly warranted when urine is obtained from individuals who secrete sugar (e.g., diabetics) or have infections in the bladder or urinary tract (31,32). These two circumstances make it more likely that ethanol could be produced in vitro after voiding or even locally in the bladder by fermentation processes (13,14). The risk of postsampling synthesis of ethanol can be minimized by including NaF in the sampling tubes (at least 1% w/w) and by storing specimens immediately after voiding in a refrigerator at 4~ or, even better, by freezing them (33,34). Various spot tests and color reactions are also available to test for the presence of sugar or bacteria or both in the urine samples before analysis for alcohol. Recent research has shown that the question of ethanol being formed in the urine in vitro after voiding (e.g., by the action of microbes or yeasts) can be resolved by the analysis of urinary metabolites of serotonin. Studies indicate that the ratio of 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5HTOL/5HIAA) in urine increases appreciably after drinking ethanol unless the ethanol was produced in vitro or in the bladder by the action of yeasts or bacteria using glucose as the substrate (35). The analysis of urinary ethanol is useful to demonstrate if a person has been drinking alcohol some time before obtaining the specimen and therefore to monitor abstinence. However, the UAC for a randomly timed sample of urine should not be used to estimate the coexisting BAC or to draw conclusions about the degree of alcohol impairment 5.0 at the time of voiding. Nevertheless, the UAC of a second void rain after emptying the bladder of old urine will bear a close rela- tionship to the coexisting BAC during the collection period, and this is estimated from the ratio UAC/1.33. An increase in the glomerular filtration 188

6 rate after drinking a large volume of water will dilute the urine but not the concentration of ethanol in the subsequent void. If the UAC was lowered after drinking water and the BAC remained unchanged, one would expect to find abnormally low UAC/BAC ratios coinciding with peak diuresis, but this is not the case (23). Filtering more water in the kidney means filtering more ethanol, so the concentration in the urine is virtually unchanged (27). This is supported by the present findings of a lack of association between urinary creatinine, UAC, and UAC/BAC ratio of ethanol. References First urinary void 5.0 I I I I I I J" N = 40..-"', 4.0 UAC = 1.32 BAC r = "",~,/~'...-'" ~ "'"" -""" 2.0,e'"..-"" s,. S" s. "~ 1.0 s ~..~" o~ ''''~..s~ "J~ 0.0 o o o.s 1:0 l:s 2:0 2:s a:o 3.5 Blood-alcohol concentration (g/l) Second urinary void I I I I I l N = 40 UAC = 1.31 BAC r = 0.8.oI"..,".s. "S" o. o'"...o..~'""-~.i...;'"'_~o~"'/f~.. r l " U " I " i I " Blood-alcohol concentration (g/l) Figure 5. Correlations between urine alcohol and blood alcohol concentration for the first (upper plot) and second urinary voids (lower plot). Two outliers shown on the upper plot suggests that these individuals were still absorbing alcohol at the time of sampling. I. S.B. Needleman, M. Porvaznik, and D. Ander. Creatinine analysis in single collection urine specimens. J. Forensic Sci. 37: (12). 2. C. Edwards, M.J. Fyfe, R.H. Liu, and A.S. Walia. Evaluation of common urine specimen adulteration indicators. ]. Anal. Toxicol. 17: (13). 3. P. Lafolie, O. Beck, G. 81ennow, L. Bor~us, S. Borg, C.E.Elwin, L. Karlsson, G. Odelius, and P. Hjemdahl. Importance of creatinine analysis of urine when screening for abused drugs. Ctin. Chem. 37: (11). 4. B.A. Goldberger, B. Loewenthal, W.D. Darwin, and E.J. Cone. Intrasubject variation of creatinine and specific gravity in consecutive urine specimens of heroin users Clin. Chem. 41: (15). 5. R.H. Liu. Important considerations in the interpretation of forensic urine drug test results. Forensic Sci. Ray. 4:51-65 (12). 6. S.L. Mikkelsen and K.O. Ash. Adulterants causing false negatives in illicit drug testing. Clin. Chem. 34" (188). 7. J.E. Mann Interpretation of urinalysis results. In Urine Testing for Drugs of Abuse NIDA Research Monograph 73" (186). 8. A. Warner. Interferences of common household chemicals in immunoassay methods for drugs of abuse. Clin. Chem. 35: (18).. J.T. Cody. Specimen adulteration in drug urinalysis. Forensic Sci. Rev. 2:63-75 (10). 10. S.A. Jortani and A. Poklis. Evaluation of the Syva ETS Plus urine drug and serum ethanol analyzer J. Anal. Toxicol. 17:31-33 (13). 11. S.A. Jortani and A. Poklis. Evaluation of the ADx TM REA assay for determination of ethanol in serum and urine. J. Anal. Toxicol. 17: (13). 12. G.W. Kunsman, J.E. Mann K.R. Cockerham, and B.R. Mann A modification and validation of two urine ethanol procedures for use with the Monarch 2000 chemistry system. J. Anal. Toxicol. 15: (11). 13. L. Kadehjian. Urine alcohol testing: valid and valuable. 5yva Monitor, Fall 11, pp J.B. Flora. Urine as a spc~eje,.n for alcohol testing. Forensic Urine Drug Testing Newsletter, June 12, pp A.W. Jones. Ethanol distribution ratios between urine and capillary blood in controlled experiments and in apprehended drinking drivers. J. Forensic 5ci. 37:21-34 (12). 16. A.W. Jones. Top ten defense challenges among drinking drivers in Sweden. Med. 5cL Law 31: (11). 17. A.W. Jones and J. Schuberth. Computer-aided headspace gas chromatography applied to bloodalcohol analysis: importance of online process control J. ForensicSci. 34: (18). 18. A.A. Biasotti and T.E. Valentine. Blood alcohol concentration determined from urine samples as a practical equivalent or alternative to blood and breath alcohol tests. J. Forensic Sci. 30: (185). 1. A.S. Curry. Advances in Forensic and Clinical Toxicology CRC Press, Boca Rat FL, A.W. Jones. Excretion of alcohol in urine and diuresis in healthy men in relation to their age, the dose administered and the time after drinking. Forensic 5ci. Int. 45: (10). 18

7 21. A.W. Jones. Biochemistry and physiology of alcohol: applications to forensic science and toxicology. In Medicolegal Aspects of Alcohol, 3rd ed. J.C. Garriott, Ed. Lawyers & Judges Publishing Co, Tucson, AZ, 16, pp T.G. Coleman, R.D. Manning, Jr., R.A. Norman, Jr., and A.C. Guyton. Dynamics of water-isotope distribution. Am. J. PhysioL 223: (172). 23. E.M.P. Widmark. Principles and applications of medicolegal alcohol determination (Translation of 132 German monograph). Biomedical Publications, Davis, CA, W.R. Miles. The comparative concentrations of alcohol in human blood and urine at intervals after ingestion. J. Pharmacol. Exp. Ther. 20: (122). 25. M.G. Eggleton. The diuretic action of alcohol in man. J. PhysioL 101: (142). 26. H.W. Haggard, L.A. Greenberg, R.R Carroll, and D.R Miller. The use of the urine in the chemical test for intoxication. J. Am. Med. Assoc. 115: (140). 27. D.J. Blackmore and J.K. Mason. Renal clearance of urea, creatinine, and alcohol. Med. Sci. Law8:51-53 (168). 28. A.W. Jones and A. Helander. Disclosing recent drinking after alcohol has been cleared from the body. J. Anal. Toxicol. 20: (16). 2. F. Lundquist. The urinary excretion of ethanol by man. Acta Pharmacol. Toxicol. 18: (161 ). 30. J.J. Saady, A. Poklis, and H.R Dalton. Production of urinary ethanol after sample collection. J. Forensic 5ci. 38: (13). 31. W. D. Alexander, RD. Wills, and N. Eldren. Urinary ethanol and diabetes mellitus. Diabet. Med. 5: (188). 32. W. Ball and M. Lichtenwalner. Ethanol production in infected urine. N. Engl. J. Meal. 301:614 (17). 33. RS. Lough and R. Fehn. Efficacy of 1% sodium fluoride as a preservative in urine samples containing glucose and candida albicans. J. Forensic 5ci. 38: (13). 34. H.A. Sulkowski, A.H.B. Wu, and Y.S. McCarter. In-vitro production of ethanol in urine by fermentation. ]. Forensic Sci. 40: 0-3 (15). 35. A. Helander, O. Beck, and A.W. Jones. Distinguishing ingested ethanol from nicrobial formation by analysis of urinary 5-hydroxytryptophol and 5-hydroxyindoleacetic acid. J. Forensic Sci. 40: 5-8 (15). Manuscript received May 21, 17; revision received August 12, 17 10