Purpose: Biochemical/metabolic: To investigate whether Ecstasy use is associated with an increase in indicators of oxidative stress, or with changes in erythrocyte acetylcholinesterase (enzyme that breaks down the neurotransmitter acetylcholine). Specific Hypothesis Tested -- that antioxidant enzyme activity and erythrocyte acetylcholinesterase levels would be lower in Ecstasy users than in non-drug user controls, and that lipoperoxidation (sign of oxidative stress) would be higher in Ecstasy users than in non-drug user controls.

Design: Retrospective (non-experimental) between-subjects study, with drug use (Ecstasy use versus no drug use) serving as a between-group variable, and with all participants undergoing blood draws used for assessing erythrocyte acetylcholinesterase levels, plasma and erythrocyte lipoperoxidation, and levels of antioxidant enzyme activities.

Subjects: 120 Ecstasy users and 120 non-user controls apparently residing in or near Hangzhou, China. No information was provided concerning means of recruiting subjects. Matching -- No specific information is provided on group matching. Nevertheless, examining group demographics, and given authors’ statement of using "random control" study design suggests that healthy volunteers may have been selected to match Ecstasy users on a specific demographic variable, such as age or gender. (Findings relating to lipoperoxidation, superoxide dismutase (SOD) and catalase activity have already appeared in a previous publication (Zhou et al. 2003) in the journal Free Radical Research.)

Criteria for Inclusion -- Ecstasy users -- Having used Ecstasy (possibly for at least one month), with Ecstasy use detected via urinary analysis and through self-report. Non-users -- Never having used Ecstasy or other psychoactive drugs, including sedatives or hypnotics. All groups -- Absence of major medical illness (especially those known to alter blood antioxidant levels), no use of vitamins or supplements (including no beta-carotene supplements), not employed in jobs that exposed them to radiation, pesticides or "intoxicating materials," absence of tobacco use or alcohol abuse (undefined). There are no statement requiring participants to abstain from psychoactives for a given period of time, nor is any information provided on length of abstinence from Ecstasy, if such a period existed.

Drug Use Parameters -- No information is provided on lifetime Ecstasy consumption; a rough estimate of range of lifetime consumption is between 23.2 tablets (assuming high-dose tablets and use twice per month) to 116 tablets (assuming low-dose tablets and use twice per month). Average dose per use, in mg, was 80.75 ± 19.96 mg (range = 40 to 120 mg). (Text refers to this as "daily use," but probably refers to average dose per use, and not dose used "per day," as Ecstasy users in the US and Europe rarely, if ever, take Ecstasy on a daily basis.) Estimating from figures for average dose per use and MDMA content per tablet, estimated average number of tablets per use is 2-10 tablets, depending on tablet contents. Duration of use, in months, was 5.8 ± 2.9 months, (range = 1 to 12 months), and no information is provided concerning frequency of use. No information is provided about time since last Ecstasy use. Tablet MDMA content ranged from 8.75-47.53 mg (authors do not state whether this information was gathered through chemical analyses of tablets or by requesting estimates from users). Other drugs – Minimal information is provided about use of other drugs, but the authors state that both Ecstasy users and healthy volunteers did not abuse alcohol (presumably defined differently from use) and did not use tobacco. Given knowledge of drug use behavior outside China, it is difficult to believe that Ecstasy users did not use other substances in addition to Ecstasy.

Group Demographics and Matched Variables -- No information is provided concerning sample matching, but it appears that samples were matched on gender, age and tobacco use. Gender, as M/F Ratio – Ecstasy users, 67/53, non-user controls, 60/60. Age – Average, in years, Ecstasy users = 23.5 ± 3.4 (range = 18-35), non-user controls = 23.6 ± 3 (range = 20-35). No information is provided about educational attainment in either sample. Other variables – Average body mass index, Ecstasy users = 23.18 ± 1.26, non-user controls = 23.25 ± 1.33. Plasma hemoglobin and albumen -- Ecstasy users, hemoglobin = 134.94 ± 6.96, albumen = 42.62 ± 2.3, non-users hemoglobin = 135.66 ± 7.19, albumen = 42.85 ± 2.4 Systolic blood pressure, diastolic blood pressure -- Ecstasy users SBP = 110.82 ± 11.19, DBP = 75.57 ± 6.73, non-users, SBP = 110.10 ± 10.52, DBP = 75.29 ± 6.15. Ecstasy users may have consumed less food, including antioxidant-rich food, owing to lack of appetite from Ecstasy use.

Measures: All assessments were made in blood samples taken In fasting subjects, apparently only drawn once (time of day unspecified). Erythrocyte acetylcholinesterase (AchE) was assessed via electrophotospectrometry, apparently using color change in acetylcholine chloride-alkalescent hydroxylamine-ferric chloride, expressed as mcg/g * Hb. Erythrocyte lipoperoxide (LPO) was assessed via spectrometry (spectrophotometry) with thiobarbituric acid. Erythrocyte SOD activity was assessed via spectrometry involving inhibiting pyrogallol auto-oxidation, and expressed as mcg/g * Hb. Erythrocyte catalase activity was detected via spectrophotometry using hydrogen peroxide, and acetic acid-potassium dichromate was used to determine catalase activity expressed as kg/g *Hb. Erythrocyte glutathione peroxidase (GPX) activity was assessed using Hafeman’s spectrophotometry, and expressed in mcg/mg * Hb.

Analyses: Independent-sample t tests, chi-square analyses and reliability analyses were used to compare AchE and antioxidant enzyme activity in Ecstasy with levels in non-drug using controls, with p. set at 0.05, and power at 0.80. Separate regression and correlational analyses were performed on data from Ecstasy users in order to detect any possible relationships between erythrocyte AchE and enzyme activities.

Results-Significant Differences Found: Ecstasy users had lower levels of erythrocyte AchE than non-drug user controls. Glutathione peroxidase activity was also lower in Ecstasy users when compared to controls. As previously reported, SOD and catalase activity in Ecstasy users was lower than in non-drug user controls. As previously reported, lipoperoxidase was higher in Ecstasy users than in controls.

AchE Levels and Enzyme and lipoperoxidation levels – There was an inverse (negative) association between erythrocyte AchE levels and lipoperoxidation, with higher AchE levels associated with less lipoperoxidation. There was a positive correlation between AchE levels and enzyme activity for SOD, catalase and GPX, with higher AchE levels associated with higher antioxidant enzyme levels.

Results-No Significant Differences: No non-significant findings were reported. The authors do not report any relationship between erythrocyte AchE levels and drug use parameters, though a previous publication detailed relationships between drug use parameters and antioxidant levels. The absence of information concerning possible relationships between drug use parameters and antioxidant enzyme activity or AchE levels may either reflect a failure to perform the analyses or a failure to find a significant relationship.

Overall Effects: Antioxidant enzyme activity was lower in Ecstasy users than in non-drug using controls, including SOD, catalase and glutathione peroxidase activity. Erythrocyte acetylcholinesterase (AchE) was lower in Ecstasy users than in non-drug users, and AchE levels were correlated with enzyme activity levels. As previously reported, lipoperoxidation was higher in Ecstasy users than in non-drug user controls. AchE levels were negatively associated with lipoperoxidation, with greater AchE levels found when lipoperoxidation was lower. The authors’ hypothesis was confirmed; levels of AchE and antioxidant enzymes were lower in Ecstasy users than in non-drug using controls, and lipoperoxidation was higher in Ecstasy users. While most study findings imply higher levels of oxidative stress in Ecstasy users than in non-users, there are alternative interpretations for reduction in erythrocyte AchE levels.

Comments: This is the second of two papers published by the same research team reporting findings of oxidative stress in Ecstasy users, and the first report of lower erythrocyte AchE in Ecstasy users. The authors describe lower AchE levels as resulting from "another neurotoxicity," without providing further information on the nature of this toxicity. A search of the PubMed database on August 14 2003 uncovered some studies supporting peripheral AchE as a marker for oxidative stress (Alchgari et al. 2003) while other studies failed to find a link between AchE levels and oxidative stress (el-Demerdash et al. 2003). The authors do not discuss alternative explanations for changes in AchE, including changes directly related to MDMA’s central activity, changes in behavior (such as nutrition) or direct effects of MDMA on acetylcholine metabolism. The same criticisms of the previous publication apply to this report as well; the authors fail to provide essential information about drug use parameters in their samples, including time elapsed since last Ecstasy use and lifetime Ecstasy consumption, and they fail to describe subject recruitment. Hence the findings, while provocative, are incomplete and difficult to interpret. The authors acknowledge that lack of appetite, an acute effect of MDMA, may have direct effects on AchE. Other study limitations include retrospective design and incomplete information on drug use parameters and the possibility that samples were not properly matched for use of other drugs.