T2007 Seattle, Washington

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1 T2007 Seattle, Washington Methanol as an Alcohol Abuse Marker in Forensic Samples Luis Alberto Suarez 1,2, Ernesto Bernal *1 and Adrián Waldo 1 1 Forensic Medical Service of Mexico City, Mexico City, Mexico; 2 National Autonomous University of Mexico, Cuautitlan Izcalli, Mexico ABSTRACT In this study, methanol in post-mortem alcoholic samples with and without ethanol present at the time of death, from social drinkers and teetotallers, was determined with gas chromatography with headspace injection to study the possibility of using methanol as an alcoholism marker in post-mortem samples. Although, a higher methanol concentration was found both in samples from alcoholics and social drinkers than in the teetotallers group, the specificity of methanol as a marker for alcoholism is low, because the difference in the consumption of ethanol between heavy and social drinking cannot be established, consequently it can only be used as a valid alcoholism marker when ethanol is not present. A multiple regression analysis revealed that the factors which impact the methanol concentration the most are a fatty liver and the consumption of ethanol amongst alcoholics, while factors traditionally linked to alcoholism such as cirrhosis do not have an impact on the methanol concentration found. Key words: Alcoholism, Alcoholism Markers, Methanol INTRODUCTION Alcoholism is one of the main health problems in the world [1]; therefore, detecting this condition is important in legal, labor and health areas. Different substances have been proposed as alcoholism markers [2, 3], among which methanol stands out because of the availability of standards and because most of the forensic laboratories have reliable techniques to determine its presence. Several contributors [4, 5] have studied the usefulness of methanol as an alcoholism marker in samples obtained from living people. However, to date no work has been carried out in post-mortem samples. METHODS Chemicals and reagents All chemicals used were obtained from J.T. Baker (Xalostoc, México), and were of an analytical grade. An internal standard calibration of isobutanol with sodium nitrite [6], in a concentration of 75 mg/dl and 180 mm respectively was used with known strength methanol, ethanol and propanol standards. Subjects analized A total of 186 individuals from autopsy cases of the Forensic Medical Service of México City were investigated (Table 1); samples from teetotallers came from children or individuals refered to be teetotallers by relatives; samples for the social drinkers group were from DUI deaths which did not have any biological or testimonial evidence of being alcoholics, while the group clasified as alcoholics with and without ethanol present at the time of death, consisted of individuals with a strong testimonial and biological evidence of being alcoholic.

2 Table 1 Groups studied Studied Group s Teetotallers Social drinkers Alcoholics Alcoholics w/ without ethanol ethanol n Man/woman 58/36 18/4 27/1 38/4 Age range (years) Blood samples were taken from the femoral blood. Blood methanol, ethanol and propanol concentrations were determined by headspace gas cromatography [5]. For the analysis, 0.4 ml of the samples were diluted in 1.6 ml of the internal standard solution, obtaining a final standard concentration of 75 mg/dl. Equipment A HP 6890 gas chromatograph with split/splitless injection and a FID detector, coupled to a HP 7694E headspace injector was used to perform the volatile substance analysis, a 30 meter long, DB-624 capillary column with an internal diameter of 0.32 mm and with a film thickness of 1.8 μm was used with a carrier gas (nitrogen) flow rate of 3 ml/min; air and hydrogen were set to achieve the best FID performance. Temperatures of the chromatograph were set as follows: injector temperature, 200 C, oven temperature 85 C for 2 minutes; followed by a temperature increase of 30 C per minute to reach a final temperature of 120 C, which was sustained two more minutes. Headspace injector operating temperatures were set as follows: sample temperature, 60 C for ten minutes; with the injection loop and the transfer line set at 120 C. Statistics Statistical calculations were made with Statgraphics Centrino and Statgraphics v. 5.1 software. RESULTS AND DISCUSION As expected, a higher methanol concentration was found in samples containing ethanol from alcoholics (Table 2), followed by social drinkers and alcoholics without ethanol. All of these groups had significantly more methanol than the teetotallers group. Table 2 Methanol and ethanol concentrations (mg/dl) in the four groups studied Studied Groups Teetotallers Social drinkers Alcoholics without Alcoholics w/ ethanol ethanol MeOH mean MeOH SD MeOH range EtOH range < < After eliminating some abnormally high values from people suffering metabolic disorders like diabetes in the teetotallers group, a normal distribution was found allowing this group to be considered as a negative control (x= mg/dl, SD=0.248 mg/dl).

3 In contrast to controlled studies [3, 4] in living subjects, more methanol was found in postmortem specimens in all groups studied. In addition, the distribution of results was less homogeneous. A weak correlation (r= ) between methanol and ethanol was found in social drinkers suggesting that methanol found in this group came partially from alcoholic beverages. A similar correlation was not found (r= ) in the alcoholic group (Figure 1). Figure 1. Correlation between EtOH and MeOH in: A.- Alcoholics and, B.- Social Drinkers Methanol concentrations in the social drinkers was not statistically different (Figure 2) from methanol found in the group of alcoholics positive for ethanol, therefore when ethanol is present, the specificity of methanol as a marker for alcoholism is low and consequently it could be used as a valid alcoholism marker only when ethanol is not present. Figure 2. Means and 95.0 percent LSD intervals of the studied groups Methanol concentrations in alcoholics negative for ethanol were particularly heterogeneous ranging for negative to high. These reflect the end of ethanol elimination and different stages of methanol elimination and they can be related to ethanol withdrawal and the time since ethanol was last consumed. Several factors of the forensic cases were used to perform a multifactor analysis of variance for the obtained methanol concentration, to determine which of these factors have a statistically significant effect on methanol concentration (Table 3).

4 Table 3 Analysis of Variance for methanol concentration (mg/dl) - Type III Sums of Squares Source Sum of Squares Df Mean Square F-Ratio P-Value COVARIATES Age (years) 0, , ,08 0,7840 EtOH (mg/dl) 0, , ,04 0,8412 MAIN EFFECTS A:Groups studied 243, , ,12 0,0000 B:Ascitis 7, , ,94 0,3341 C:Cirrhosis 2, , ,27 0,6043 D:Diabetes mellitus 2, ,536 0,32 0,5747 E:Fatty liver 48, ,1235 6,00 0,0153 F:Hemorragic pancreatitis 35, ,72 4,45 0,0363 G:Sex 17, ,5177 2,18 0,1413 RESIDUAL 1347, ,0203 TOTAL (CORRECTED) 2030, All F-ratios are based on the residual mean square error. From the P-values of the studied groups, fatty liver and hemorrhagic pancreatitis are less than 0.05, these factors have a statistically significant effect on the methanol concentration at the 95,0% confidence level, while factors like age and ethanol concentration and others traditionally linked to alcoholism like cirrhosis do not have an effect on methanol concentration. A multiple regression analysis confirms that the factors with more influence on the methanol concentration are the presence of a fatty liver and the ingestion of ethanol in alcoholics, the equation of the fitted model is: MeOH (mg/dl) = 1, ,608251*I1(1) + 2,41139*I1(2) + 0,736054*I1(3) + 1,11911*I2(1) + 0,274388*I3(1) + 0,371979*I4(1) - 1,3365*I5(1) + 1,18144*I6(1) + 0,38731*I7(1) + 0, *years - 0, *EtOH (mg_dl) Where: I1(1) = 1 if Group=alcoholics without EtOH, -1 if Group=Teetotaller, 0 otherwise I1(2) = 1 if Group=Alcoholics with EtOH, -1 if Group=Teetotallers, 0 otherwise I1(3) = 1 if Group=Social Drinkers, -1 if Group=Teetotallers, 0 otherwise I2(1) = 1 if Ascites=NO, -1 if Ascites=SI, 0 otherwise I3(1) = 1 if cirrhosis=no, -1 if cirrhosis=si, 0 otherwise I4(1) = 1 if Diabetes=NO, -1 if Diabetes=SI, 0 otherwise I5(1) = 1 if Fat liver=no, -1 if Fat liver=si, 0 otherwise I6(1) = 1 if Hemorrhagic pancreatitis =NO, -1 if Hemorrhagic pancreatitis =SI, 0 otherwise I7(1) = 1 if Sex=F, -1 if Sex=M, 0 otherwise This equation explains % of the variability in the determined methanol concentration. CONCLUSIONS In contrast to controlled studies in living subjects, more methanol was found in post-mortem specimens in all groups studied. In addition, the distribution of results was less homogeneous.

5 A weak correlation between methanol and ethanol was found in social drinkers suggesting that methanol found in this group came partially from alcoholic beverages. A similar correlation was not found in the alcoholic group. Methanol concentrations in the social drinkers was not statistically different from methanol found in the alcoholic groups therefore when ethanol is present, the specificity of methanol as a marker for alcoholism is low and consequently it could be used as a valid alcoholism marker only when ethanol is not present. The consume of ethanol in alcoholics, a fatty liver and hemorrhagic pancreatitits have a statistically significant effect on the determined methanol concentration, while factors traditionally linked to alcoholism like cirrhosis do not have a significant effect on the determined methanol concentration.. BIBLIOGRAPHY 1.- S. Karch. Handbook of Drug Abuse. CRC Press. Boca Raton, Florida (1998) 2.- F. Musshoff, Th. Daldrup. J. of Chromatogr. B. 713 (1998) F. Musshoff. J. of Chromatogr. B. 781 (2002) B. Brinkmann, H. Köhler, S. Banaschak, A. Berg, B. Eikelmann, A. West, A. Heinecke. Int. J. Legal Medicine. 113 (2000) R. R. Roine, C.J. Eriksson, R. Ylikahri, A. Penttilä, M. Salaspuro. Alcohol. Clin. Exp. Res. 13 (1989) F. Tagliaro, G. Lubli. J. Chromatogr. 580 (1992) ACKNOWNLEDGMENTS We would like to thank Ph.D. Raquel Lopez Arellano from the National Autonomous University of México for her invaluable help in statistical problems and The Forensic Medical Service and The Supreme Court of Justice of México City for its support to the present work..