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Urine as a material for evaluation of exposure to manganese in methcathinone users
Accepted Manuscript Title: Urine as a material for evaluation of exposure to manganese in methcathinone users Author: Magdalena Golasik Grzegorz Wodowski Ewa Gom´ołka Małgorzata Herman Wojciech Piekoszewski PII: DOI: Reference:
S0946-672X(14)00061-3 http://dx.doi.org/doi:10.1016/j.jtemb.2014.04.005 JTEMB 25525
To appear in: Received date: Revised date: Accepted date:
18-2-2014 3-4-2014 25-4-2014
Please cite this article as: Golasik M, Wodowski G, Gom´olka E, Herman M, Piekoszewski W, Urine as a material for evaluation of exposure to manganese in methcathinone users, Journal of Trace Elements in Medicine and Biology (2014), http://dx.doi.org/10.1016/j.jtemb.2014.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Urine as a material for evaluation of exposure to manganese in methcathinone users Magdalena Golasik1, Grzegorz Wodowski2, Ewa Gomółka3, Małgorzata Herman1, Wojciech
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Piekoszewski1,4 Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University,
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Ingardena 3, 30-060 Krakow, Poland
Outpatients Clinic for Drug Prevention and Treatment of Addicts, Św. Katarzyny 3, 31-063,
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Krakow, Poland
Laboratory of Analytical Toxicology and Therapeutic Drug Monitoring, Faculty of Medicine
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Jagiellonian University, Kopernika 15b, 31-501 Krakow, Poland
Laboratory of High Resolution Mass Spectrometry, Regional Laboratory of Physicochemical
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Analysis and Structural Research, Faculty of Chemistry, Jagiellonian University, Ingardena 3,
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30-060 Krakow, Poland
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Corresponding author: Prof. Wojciech Piekoszewski, PhD, DSc Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University
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Ingardena 3, 30-060 Krakow, Poland Phone (+48 12) 663 56 00 e-mail: [email protected]
Short title: Manganese in the urine of methcathinone users
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Abstract Chronic exposure even to low doses of manganese may lead to development of neurological syndrome similar to parkinsonism. The aim of this research is to assess the
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possibility of manganese poisoning based on the level of metal in the urine of long-term methcathinone users from Poland. Graphite furnace atomic absorption spectroscopy
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(GF AAS) was used to determine manganese in urine, while the detection of the psychoactive
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drugs was performed by high-performance liquid chromatography (HPLC).
Results of survey on longitudinal patterns of drug use showed that users of traditional
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illicit drugs now turn to cheaper alternatives, such as methcathinone. Parkinsonian features were observed in almost half of methcathinone users. The subjects had a higher mean level of
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Mn in their urine (8.68±9.27 µg·L-1) than the controls (4.27±1.91 µg·L-1). The presence of numerous psychoactive substances (in unchanged forms and their metabolites) was confirmed
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in all of the samples, with only one exception. The elevated level of manganese in urine (in
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29.2% of patients) can be used as a primary marker of recent methcathinone administration,
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especially in the case of long time intravenous drug users where blood sampling is complicated.
Keywords Methcathinone; Urine; Illicit drugs; Manganese; Parkinson-like syndrome
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Introduction Methcathinone (ephedrone) is an indirect sympathomimetic drug, which has been shown to increase the release of noradrenaline at similar potency to amphetamines [1], and
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has exhibited similar inhibition of noradrenaline reuptake to methamphetamine and ecstasy [2]. It is manufactured by oxidation of ephedrine and pseudoephedrine, usually via
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application of potassium permanganate. Under laboratory conditions, a 1 g of methcathinone
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required the use of 2 g of this oxidizing agent. However, illegal manufacturers or addicts usually add a larger amounts of potassium permanganate [3,4]. One study reported that the
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concentration of methcathinone in a home-made liquid obtained from an addicted person was 2.6 mg∙mL-1 [5].
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The first case of methcathinone intoxication was described 20 years ago [6]. Nowadays, there have been significant increases both in the number of people who use this xenobiotic
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and the cases of poisoning [7]. The occurrence of a Parkinson-like syndrome among
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methcathinone addicts is quite common [8]. This kind of effect of methcathinone was
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observed in many countries worldwide [9-11] and is believed to be related to manganese poisoning brought about by the high concentrations of metal present in the home made methcathinone [12,13]. An extensive review [14] of this phenomenon could suggest that manganese is probably the sole causative agent for the Parkinson’s symptoms in addicts, with negligible contribution to the neurotoxicity being associated with dopamine depletion. Manganese is an essential element, but excessive exposure can be toxic, particularly to the central nervous system [15-17]. The collection of blood from long-term intravenous drug users creates important problems. On the other hand, urine is the basic biological material in diagnostic in clinical toxicology. For that reasons, we decided to assess the applicability of urine for evaluation of methcathinone use in multiple drug addicts on the basis of determination of manganese. 3 Page 3 of 24
Materials and methods Subjects
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Urine, collected from people who have intravenously injected methcathinone (ephedrone), was obtained from the Department of Analytical Toxicology and Therapeutic
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Drug Monitoring, Jagiellonian University, Krakow, Poland. Twenty-four patients addicted to methcathinone were recruited from participants at the Outpatients Clinic for Drug Prevention
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and Treatment of Addicts, Krakow, Poland. Seven patients participated in methadone maintenance treatment. The patients group consisted of 17 men and 7 women with a mean
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age of 38.9±7.9 (age range: 23-59 years). All patients were active smokers (the age of initiation: 16.2±4.7) and drank alcohol (the age of initiation: 14.3±2.2). The results of
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manganese analysis for the patients were compared with those of 25 age-matched healthy volunteers. The control group consisted of 15 men and 10 women with a mean age
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37.7±8.2 (age range: 23-57 years), without a drug abuse history. Among them, 16 subjects
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(64%) were cigarette smokers (the age of initiation: 16.6±1.8), and all volunteers drank
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alcohol (the age of initiation: 14.3±2.2).
All patients completed a drug history questionnaire to document their lifetime and
recent use of alcohol, tobacco, medicines and illicit drugs. Written informed consent was obtained prior to participation. The study was approved
by the Bioethics Committee of the Jagiellonian University in Krakow. Reagents All reagents used in this study were of the highest purity available. All solutions were prepared with the ultrapure deionized water (20 MΩ). Concentrated nitric acid (65%, Suprapur, Merck, Germany) was used for sample digestion. A standard stock solution of Mn (1000 mg·L-1), prepared from Titrisol standard 4 Page 4 of 24
(Merck, Germany) was used fot the preparation of the standard solutions. For GF AAS method, the following modifiers were used: palladium nitrate (c(Pd)=10.0±0.2 g·L-1 (Pd(NO3)2/HNO3 ca. 15%, Merck, Germany) and magnesium nitrate (c=2.6 g·L-1), obtained
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by dissolving proper amount of Mg(NO3)2·6H2O (Merck, Germany) in deionized water. The following reagents were used for HPLC analyses: HPLC-grade acetonitrile and H3PO4
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(Sigma- Aldrich, Germany), HCl and NaOH (POCh, Poland), ethyl acetate (Merck, Germany).
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The accuracy was assessed through the analysis of the certified reference material
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(CRM) of urine (21.2±4.2 μg Mn·L-1, ClinChek Urine Control, Levels II, Recipe, Germany). Analysis of manganese in urine
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A microwave-assisted acid digestion procedure was carried out before quantitative elemental analysis using a Mars X5 microwave system (CEM, USA). One mL of urine or
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urine certified reference material was digested with 6 mL of concentrated HNO3. The process
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of digestion was conducted in three steps, differing in maximum temperature and microwave
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power: 1600C/600W, 1800C/850W, and 2000C/1100W. Each step lasted 8 min (heating to a given temperature for 4 min and holding it for 4 min). After completing the program, the vessels were cooled down (about 30 minutes) and the digested samples solutions were transferred into 10 mL volumetric flasks and diluted with deionized water. A PinAAcle 900Z graphite furnace atomic absorption spectrophotometer with
a transversely-heated graphite tubes with integrated platform (THGATM) and an AS 900 autosampler (Perkin Elmer, USA) was used for the determination of manganese in urine. The major validation parameters of the method were as follow: accuracy (as relative error) 3.46%; precision (CV) - 3.61%; LOD - 0.11 µg·L–1; LOQ - 0.33 µg·L–1; recovery - 94±3%. The method was described in detail in our previous work [18].
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Drug screening in urine For drug screening (amphetamine derivatives, ephedrine, mephedrone, methcathinone, methadone, EDDP), the HPLC method, routinely used in the Laboratory of Analytical
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Toxicology and Therapeutic Drug Monitoring, Faculty of Medicine Jagiellonian University, was applied [19]. Shortly, 0.2 mL of 0.5 M NaOH and 1.2 mL of ethyl acetate was added to
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500 µL of urine and the mixture was vortexed for 3 min. After centrifugation, the organic
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phase was transferred to Eppendorf tube and 50 µL of 0.02 M HCl was added afterwards the solvent was evaporated to dry at 40oC under a stream of nitrogen. The residue was
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reconstituted with a 300 μL of a mixture containing 500 mL of deionized water with 100 µL of concentrated phosphoric acid and acetonitrile (1:1).
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All HPLC analyses were performed in a liquid chromatograph equipped with an Diode Array Detector L-7455, LaChrom System (Merck-Hitachi, Darmstadt, Germany). Separation
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of analytes was conducted in a LiChroCART 125-4 LiChrospher RP-select B (Merck,
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Darmstadt, Germany) column. Separation was performed in gradient conditions (phase A: 500 mL of deionized water with 100 µL of concentrated phosphoric acid; phase B:
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acetonitryle at a flow of 1 mL·min–1. The gradient program (Time [min]/mobile phase A [%]; mobile phase B [%]) was set as 0-8/100;0, 8-10/90;10, 10-15/80;20, 20/70;30, 21/50;50, 25/ 100;0, and 30/100;0, respectively. The results of qualitative analysis of urine sample with standards and the urine of one patient is shown in Figure 1. The screening for opiates (morphine derivatives), cannabinoids, benzodiazepines and
buprenorphine (not used in Poland in maintenance treatment) were performed using enzymelinked immunosorbent assay (ELISA) screening kits from nal von Minden GmbH (Regensburg, Germany) run on a Viva-E analyzer (Siemens Healthcare Diagnostics, Liederbach, Germany). The cut-off were 300, 50, 300 and 10 ng mL-1 for opiates, cannabinoids, benzodiazepines and buprenorphine, respectively. 6 Page 6 of 24
Statistical analyses Statistica 10 (StatSoft Inc., USA) was used for the statistical analyses of data. The normal distribution of the dataset was verified by a Kolmogorov-Smirnov with the Lilliefors
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significance correction test and the Shapiro Wilk test. A basic data analysis was performed to obtain the mean values, range and standard deviation of the mean. Differences between study
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groups were compared with the Mann-Whitney U test. The degree of correlation was
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evaluated from the Pearson correlation coefficients. Significance was set at p<0.05.
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Results
Illicit drugs and medications taken by patients – questionnaire data
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Information obtained from the questionnaire regarding the drugs of abuse is presented in Table 1. The mean age of initiation to drug use was 15.7±2.6 (age range: 13-25). All of the
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patients have used drugs intravenously. Illicit drugs were commonly abused via insufflations
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(79%), smoking (95%) and the oral route (95%). The most popular illicit drugs were opioids
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(91.2% of patients have taken it during their life and 58.3% during the last month), amphetamine (91.2% of patients have taken it during their life and 58.3% during the last month) and THC (91.2% of patients have taken it during their life and 58.3% during the last month).
The majority of the subjects (95%) have administered various types of medications
(average age of initiation: 19.4±5.4). The most popular are benzodiazepines (91.2% of patients have taken it during their life and 58.3% during the last month), opiates (75.0% of patients have taken it during their life and 45.8% during the last month), barbiturates (70.8% of patients have taken it during their life and 8.3% during the last month) and acodine (33.3% of patients have taken it during their life and no one during the last month).
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Methcathinone use - questionnaire data Data concerning methcathinone abuse are presented in Table 2. All of the subjects have administered methcathinone intravenously. In most cases (87.5%), the patients have prepared
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the drug by themselves. Information about methcathinone came from friends (75%) and websites (8.3%). The majority of patients (83.3%) were aware of health problems that may be
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caused by the drug. Among them, 41.7% have reported various symptoms, which probably
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arose from manganese intoxication (but could also be the effect of methcathinone itself), such as impaired physical coordination, balance disorder, slurred speech, difficulties with writing
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and memory, daytime drowsiness, kidney and liver pain, and a swollen face and limbs.
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Licit and illicit drug screening in the urine of the patients
The qualitative results of illicit drugs determinations achieved by HPLC and ELISA
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gave following results: buprenorphine was detected in 9 urine samples (patients 1, 2, 5, 6, 11,
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12, 14, 16, 22), opiates and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine metabolite of methadone) in 7 urine samples (respectively: patients 1, 6, 8, 12, 13, 16, 24, and
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patients 9, 15, 17, 18, 20, 21, 23), methadone in 6 samples (patients 9, 17, 18, 20, 21, 23) and mephedrone in 4 samples (patients 4, 7, 9, 10,). Methcathinone was present in the urine of only 2 subjects (patients 5 and 12). Amphetamine and cannabinoids were only detected in patient n°15, while quetiapine (atypical antipsychotic) – in patient n°18. Only patient n°3 had no illicit substances detected in his urine. It was the person who used methcathinone less than one year and had the lowest concentration of manganese when compared with other addicts. In the urine of patients participated in methadone maintenance treatment, methadone and/or its metabolite EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) were confirmed. Level of manganese in urine 8 Page 8 of 24
The results of determination of manganese in urine for all cases from the group of patients are listed in Table 3. About 29% of addicts had urinary Mn concentration over the level considered normal -
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below 10 μg·L-1 [20]. Recent data showed no gender differences for the urinary excretion of manganese, and also smoking and alcohol consumption have negligible effect on this process
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[21]. The mean level of Mn found in the urine of methcathinone users was 8.68±9.27 µg·L-1, with a very wide range, from 0.92 to 46.57 µg·L-1. This may arise from individual differences
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in the excretion of Mn [21], and the dose and frequency of methcathinone administration. It is also necessary to take under consideration the fact that it is home-made drug, which was
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probably synthesized with the use of the different amount of potassium permanganate. When
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considering the time of last administration of methcathinone, the highest mean concentration of manganese was determined in samples of urine from people taking the drug no longer than one week before sampling (13.82±13.71 µg·L-1). As expected, the lowest level of Mn was
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observed in subjects who had not been used the drug for a year (4.57±3.10 µg·L-1). However, in this group and the group of patients who had taken methcathinone from 1 to 12 months
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ago, there were two patients: n°7 and 14, who had distinctly increased concentration of Mn (10.76 µg·L-1 and 32.16 µg·L-1, respectively). It might be assumed that these individuals had lied about the last administration of the drug and therefore should have been in the first group (people who had taken the drug no longer than one week before sampling). Then, the concentration of Mn in the urine of subjects who had not been used the drug for a year and those who had taken methcathinone from 1 to 12 months ago would be 3.37±1.94 µg·L-1 and 3.69±1.98 µg·L-1, respectively, while the most recent users would have the following level of Mn: 15.35±13.49 µg·L-1. Healthy volunteers have a mean Mn concentration of 4.27±1.91 µg·L-1 (range: 0.65-6.67 µg·L-1). The mean value of Mn concentration in the urine of abusers was statistically significantly higher than in healthy volunteers (p<0.05). The mean
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concentration of Mn in both groups can be considered as a normal for the general population, but the urinary manganese level in some of the methcathinone users was significantly high. Figure 2 shows the dependence of the concentration of manganese in the urine samples of
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patients from the time of last administration of the drug. It was observed that, over time, the concentration of manganese in the urine decreases, until it reaches the physiological level.
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The highest concentration of this element was determined in the urine of a patient who took methcathinone intravenously 30 minutes before the collection of the sample. Correlation
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analysis indicates two strong correlations between the manganese level and the time of the last administration of methcathinone (r=-0.465), as well as the number of times the drug was
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used in the last month (r=0.601). It was found that the number of injections during a life has
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no effect on the concentration of manganese in urine. No correlations were found between the concentration of Mn and the detected drugs or their metabolites in urine.
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Discussion
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Methcathinone, a designer drug known in Russia and Eastern Europe as an ephedrone,
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has become a growing concern across the European Union and United States [22]. It is cheaper than amphetamine or cocaine. In one study, addicts stated that methcathinone caused stronger and longer-lasting toxicological effects than traditional drugs like cocaine, but also made the users psychologically dependent on it [23]. Moreover, it can be prepared in someone's home from over the counter medicines. The procedure of synthesis is rather easy and can be found on numerous websites [8]. Our study examined the longitudinal patterns of methcathinone and other psychoactive substances use, plus the health problems of 24 participants from the Outpatients Clinic for Drug Prevention and Treatment of Addicts, who have taken methcathinone for a long period of time. Similar studies were also conducted in Ukraine [24,25], Russia [16], Estonia [8] and Latvia [12]. According to the best of our knowledge, this is the first such study of 10 Page 10 of 24
methcathinone users in Poland. Close attention was paid to the possibility of manganese intoxication in methcathinone addicts. Urine analyses were conducted to detect the use of methcathinone and other licit and illicit drugs (e.g. methadone, benzodiazepines, opiates),
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plus to determine the level of manganese. Methcathinone was detected only in two samples, despite the fact that some patients
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declared a recent intake of the drug a day before urine sampling. Due to the lack of information about the pharmacokinetics of this drug in humans, it can be assumed that the
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metabolism of methcathinone is similar to cathinone to a very close structurally. Therefore the half-life of both might be comparable, which was found to be 1.5±0.8 h for cathinone
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after chewing of khat leaves [26]. This can explain why methcathinone was detected in the
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urine only in two subjects. Pseudoephedrine, which is a major metabolite of methcathinone, or can be in the drug due to incomplete synthesis [27,28], was not present in the urine of our
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patients.
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The analysis of information obtained from this survey may lead to the conclusion that people with a long history of addiction have given up traditional drugs like cocaine or
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amphetamine for methcathinone. It is probably because they cannot financially afford the traditional drugs and this cheaper analog has a similar effect on them. The main concern of methcathinone abuse is not only the physiological and psychical
effects caused by the drug itself, which are characteristic for amphetamine-type stimulants (e.g., hallucinations, paranoia, restlessness, appetite loss, insomnia) [24], but rather neurological, as the possibility of developing Parkinson’s-like symptoms related to manganese. As mentioned, the reaction of oxidation of pseudoephedrine is often conducted with an excess of potassium permanganate, so the final product is contaminated with manganese. Sikk et al. [8] performed the synthesis of methcathinone according to recipes from their patients and found on the Web, and reported that the obtained mixture contained
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0.6 g Mn·L-1. Managnese is an essential element and its normal concentration ranges between 4 and 12 μg·L-1 in blood [29] and 0.40 to 0.85 μg·L-1 in serum [30]. Only a small amount of manganese is excreted in urine (about 3%), and its level in healthy people ranges from 1 to 10
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μg·L-1 [20], so the use of it as a biomarker for Mn exposure is limited. Usually blood is used for this purpose, but this biological material has important limitations [31]. Collection of
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blood from chronic intravenous drug users is problematic because they usually have
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collapsed veins. Urine collection is much easier and this biological material was chosen in this study.
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Manganese is transported across the blood-brain barrier via many transporter systems, such as the transferrin (Tf) receptor (TfR), the divalent metal transporter 1 (DMT1), the
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magnesium transporter hip14, the divalent metal/bicarbonate ion symporters ZIP8 and ZIP14, the solute carrier-39 (SLC39) family of zinc transporters, the transient receptor potential
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melastatin 7 (TRPM7) channels/transporters, and various calcium channels [15]. Metal
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deposits in the basal ganglia and the globus pallidus of the brain. This area is in the regulation
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of motor control, cognitive and emotional functions, hence manganism may cause neurological symptoms similar to Parkinson’s [14,32]. It is still unknown how the type and length of exposure to Mn, as well as the dose or route of administration lead to development of Parkinson-like syndrome [33]. The first clinical manifestations (speech difficulties and gait abnormality) are observed usually within the first few years after the beginning of methcathinone intake [34].
Many studies reported an elevated concentration of Mn in different biological materials obtained from methcathinone users. Varlibas et al. [35] described three people with movement disorders who have taken intravenously the so-called “Russian Cocktail” (a mixture similar to methcathinone consisting of potassium permanganate, ephedrine and aspirin). Two of them had a high blood concentration of Mn: 2100 μg·L-1 and 3176 μg·L-1. 12 Page 12 of 24
All subjects have received a standard treatment with anti-parkinson drugs and for metal poisoning, but it did not reverse the symptoms of toxicity. Koksal et al. [36] also found that an excess of Mn causes an irreversible neurodegeneration, and that treatment with levodopa
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is not successful. In their study, seven patients who injected a “Russian Cocktail” had an elevated concentration of Mn in serum and urine. The estimated 24 h urinary Mn excretion in
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subjects was as follows: 0.3 μg, 11 μg, 14 μg, 7 μg, 12 μg, 13 μg and 10 μg, while normal value is <0.67 μg. Assuming that an average urine excretion is on the level of 1.5 L per day,
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above results are in the same range as ours. Selikhova et al. [25] have determined Mn in the pubic hair of 9 drug addicts. Three patients with a worsening clinical condition had the
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highest mean level of Mn in hair (2.68±1.75 μg∙g-1), while other subjects had one at 0.74±0.37 μg∙g-1. Healthy volunteers had the concentration reported in other studies as being
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normal at 0.34 ± 0.29 μg∙g-1. Research conducted in Estonia amongst a large amount of
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intravenous methcathinone users has also led to the conclusion that methcathinone users are exposed to manganese poisoning [37]. The addicts had a significantly higher mean
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concentration of Mn in hair (2.9±3.8 mg∙g-1) than the controls (0.82±1.02 mg∙g-1). The Mn
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level in plasma was significantly elevated (11.5±6.2 μg∙g-1) in active rather than in former users (5.6±1.8 μg∙g-1). However, as Smith et al. [31] demonstrated in their study, hair and plasma are not a reliable biomarkers for Mn exposure. Also the standard deviations are higher than mean concentrations in case of hair, what makes this results questionable. To our knowledge, this is the first study that has used urine for evaluation exposure of
methcathinone users to manganese, showing that the mean concentration of Mn in this medium was two-fold higher than in the urine of healthy volunteers, although only in the case of subjects with exposure on several dozen hours before urine collection was the level of Mn over normal value.
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Almost 42% of people from the clinical group reported neurological symptoms (impaired physical coordination, balance disorder, slurred speech, difficulties with writing and memory, daytime drowsiness). As expected, the subjects with a long history of
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methcathinone use (over 1000 injections in life) had the most severe health problems. On the other hand, the last administration of drug did not have any influence on the appearance of
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neurological symptoms. Our survey confirmed that methcathinone is taken as a substitute of more expensive drugs and usually is taken with prescribed medicine (methadone,
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buprenorphine, quetiapine), and illicit drugs, more frequently opiates.
Identifying the cause of an extrapyramidal syndrome in methcathinone abusers could be
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difficult because of the lack of specialist laboratories. In this situation the elevated level of
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manganese can be used as a primary marker of methcathinone use, but individual differences are possible. The most valuable results are obtained from the determination of this element in
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blood, although in the case of long time intravenously drug users, it is very complicated and
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in this situation the concentration of manganese in urine can be helpful. In patients with residential exposure, or those who were previously, but not currently, exposed to manganese,
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the measurement of this element in urine cannot provide a valid history of methcathinone taking.
Acknowledgements
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Environmental Health Perspectives 2010;118:1071-80.
[15] Bowman AB, Kwakye GF, Herrero Hernández E, Aschner M. Role of manganese in
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25:191-203.
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neurodegenerative diseases. Journal of Trace Elements in Medicine and Biology 2011;
[16] Levin OS. “Ephedron” encephalopathy. Zhurnal nevrologii i psikhiatrii imeni S.S.
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Korsakova 2005; 105:12-20.
[17] Guilarte
TR.
Manganese
neurotoxicity:
new
perspectives
from
behavioral,
neuroimaging, and neuropathological studies in humans and non-human primates. Frontiers in Aging Neuroscience 2013;5:23.
[18] Golasik M, Herman M, Piekoszewski W, Gomolka E, Wodowski G, Walas S. Trace Determination of Manganese in Urine by Graphite Furnace Atomic Absorption Spectrometry and Inductively Coupled Plasma - Mass Spectrometry. Analytical Letters 2014, DOI: 10.1080/00032719.2014.888729.
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[19] Gomolka E, Krol A, Gawlikowski T, Amphetamine and its analogues determination in urine from patients hospitalized in the Department of Toxicology Jagiellonian University Collegium Medicum in Krakow. Przeglad lekarski 2009;66:323-5.
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[20] McClatchey KD. Clinical Laboratory Medicine. 2nd ed. Philadelphia: Lippincott Williams & Wilkins, 2002.
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[21] Gil F, Hernández AF, Márquez C, Femia P, Olmedo P, López-Guarnido O, Pla A. Biomonitorization of cadmium, chromium, manganese, nickel and lead in whole blood,
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urine, axillary hair and saliva in an occupationally exposed population. Science of the Total Environment 2011;409:1172–1180.
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[22] Spiller HA, Ryan ML, Weston RG, Jansen J, Clinical experience with and analytical
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confirmation of “bath salts” and “legal highs” (synthetic cathinones) in the United States. Clinical Toxicology 2011;49:499-505.
290.
te
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[23] Miller RL. The Encyclopedia of Addictive Drugs. London: Greenwood Press, 2002. p.
[24] Djamshidian A, Sanotsky Y, Matviyenko Y, O'Sullivan SS, Sharman S, Selikhova M, et Increased
reflection impulsivity in patients with methcathinone-induced
Ac ce p
al.
Parkinsonism. Addiction 2013;108:771-9.
[25] Selikhova M , Fedoryshyn L , Matviyenko Y , Komnatska I , Kyrylchuk M , Krolicki L, et al. Parkinsonism and dystonia caused by the illicit use of methcathinone – a longitudinal study. Movement Disorders 2008;23:2224-31.
[26] Toennes SW, Harder S, Schramm M, Niess C, Kauert GF, Pharmacokinetics of cathinone, cathine and norephedrine after the chewing of khat leaves. British Journal of Clinical Pharmacology 2002;56:125–30. [27] Belhadj-Tahar H, Sadeg N. Methcathinone: A new postindustrial drug. Forensic Science International 2005;153:99–101.
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[28] Marais AA, Laurens JB. Rapid GC–MS confirmation of amphetamines in urine by extractive acylation. Forensic Science International 2009;183:78-86. [29] Šaric M, Lucchini R. Manganese. In: Nordberg GF, Flowler BA, Nordberg M, Friberg
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LT, editors. Handbook on the Toxicology of Metals, 3rd ed. Burlington: Academic Press, 2007. p.645-74.
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[30] Jankovic J. Searching for a relationship between manganese and welding and Parkinson’s disease. Neurology 2005;64:2021-8.
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[31] Smith D, Gwiazda R, Bowler R, Roels H, Park R, Taicher C, et al. Biomarkers of Mn exposure in humans. American Journal of Industrial Medicine 2007;50:801-11.
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[32] Lucchini RG, Martin CJ, Doney BC. From manganism to manganese-induced
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parkinsonism: a conceptual model based on the evolution of exposure. NeuroMolecular Medicine 2009;11:311-21.
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[33] Cersosimo MG, Koller WC. The diagnosis of manganese-induced parkinsonism.
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Neurotoxicology 2006;27:340-6.
[34] Sikk K, Haldre S, Aquillonius SM, Taba P. Manganese-induced parkinsonism due to
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Ephedrone abuse. Parkinson’s Disease 2011:865319.
[35] Varlibas F, Delipoyraz I, Yuksel G, Filiz G, Tireli H, Gecim NO, Neurotoxicity following chronic intravenous use of “Russian cocktail”. Clinical Toxicology 2009;47:157-60.
[36] Koksal A, Baybas S, Sozmen V, Koksal NS, Altunkaynak Y, Dirican A, et al. Chronic manganese toxicity due to substance abuse in Turkish patients. Neurology India 2012;60:224-7. [37] Sikk K, Haldre S, Aquilonius SM, Asser A, Paris M, Roose Ä, et al. Manganeseinduced parkinsonism in methcathinone abusers: bio-markers of exposure and followup. European Journal of Neurology 2013;20:915-20.
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Table 1
Amphetamine - life/last month (n)
24/5
Cocaine - life/last month (n)
21/0
THC - life/last month (n)
23/19
Mephedrone - life/last month (n)
19/3
Hallucinogens - life/last month (n)
22/16
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24/11
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M
an
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Opioids - life/last month (n)
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Details about the past history of illicit drugs abuse in patients.
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Table 2 Selected information about history of methcathinone abuse
50-100
3
100-1000
4
>1000
10
< 1 week
8
1-4 weeks
2
1-12 months
6
> 1 year
8
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Medical problems connected with drug abuse [yes/no]
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Last administration of methcathinone
7
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Methcathinone – number of injections in life
<50
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Number of cases (n)
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10/14
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Table 3 Relationship between the concentration of manganese in the urine of methcathinone users and
6.63±3.76
1-12 months
8.17±11.88
a
te 4.57±3.10
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>1 year
cr
1-4 weeks
9.27 1.64 12.04a 10.88a 3.24 15.20a 45.74a 12.58a 3.97 9.28 1.63 4.66 1.46 3.14 32.16a 5.94 4.06 0.92 10.76a 5.95 5.47 3.50 1.18 4.73
Number of patient 6 10 12 15 19 20 22 23 4 18 2 5 8 13 14 16 1 3 7 9 11 17 21 24
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13.82±13.71
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<1 week
Concentration of Mn [µg∙L-1]
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Mean concentration of Mn±SD [µg∙L-1]
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Last dose of methcathinone
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the time after the last dose of methcathinone administration
Concentration of Mn over the physiological level
21 Page 21 of 24
It is no conflict of interest In behalf of all authors
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an
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Prof. Wojciech Piekoszewski PhD, DSc
22 Page 22 of 24
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Intensity [AU]
1 – phenylpropyloamine (internal standard, tR=12.7 min 2 – ephedrine/pseudoephedrine, tR=14.5 min 3 – methcathinone, tR=14.9 min 4 – amphetamine, tR=15.6 min 5 – mephedrone, tR=18.1 min
Retention time [min]
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Fig 1. HPLC chromatogram of a blank urine sample spiked with standards (400 ng/mL) and an urine sample of the patient (methcathinone and amphetamine).
23 Page 23 of 24
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Fig. 2 Manganese concentration dependence of time of last administration of methcathinone.
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The main plot shows the level of Mn in the urine of all patients (in the range of 50 months). The inside plot shows the concentration of Mn in the urine of selected patients (those who
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had taken methcathinone during four days following the collection of samples). Grey area indicates the range of the normal concentration of manganese in urine. [38]
24 Page 24 of 24
S0946-672X(14)00061-3 http://dx.doi.org/doi:10.1016/j.jtemb.2014.04.005 JTEMB 25525
To appear in: Received date: Revised date: Accepted date:
18-2-2014 3-4-2014 25-4-2014
Please cite this article as: Golasik M, Wodowski G, Gom´olka E, Herman M, Piekoszewski W, Urine as a material for evaluation of exposure to manganese in methcathinone users, Journal of Trace Elements in Medicine and Biology (2014), http://dx.doi.org/10.1016/j.jtemb.2014.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Urine as a material for evaluation of exposure to manganese in methcathinone users Magdalena Golasik1, Grzegorz Wodowski2, Ewa Gomółka3, Małgorzata Herman1, Wojciech
1
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Piekoszewski1,4 Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University,
2
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Ingardena 3, 30-060 Krakow, Poland
Outpatients Clinic for Drug Prevention and Treatment of Addicts, Św. Katarzyny 3, 31-063,
3
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Krakow, Poland
Laboratory of Analytical Toxicology and Therapeutic Drug Monitoring, Faculty of Medicine
4
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Jagiellonian University, Kopernika 15b, 31-501 Krakow, Poland
Laboratory of High Resolution Mass Spectrometry, Regional Laboratory of Physicochemical
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Analysis and Structural Research, Faculty of Chemistry, Jagiellonian University, Ingardena 3,
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30-060 Krakow, Poland
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Corresponding author: Prof. Wojciech Piekoszewski, PhD, DSc Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University
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Ingardena 3, 30-060 Krakow, Poland Phone (+48 12) 663 56 00 e-mail: [email protected]
Short title: Manganese in the urine of methcathinone users
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Abstract Chronic exposure even to low doses of manganese may lead to development of neurological syndrome similar to parkinsonism. The aim of this research is to assess the
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possibility of manganese poisoning based on the level of metal in the urine of long-term methcathinone users from Poland. Graphite furnace atomic absorption spectroscopy
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(GF AAS) was used to determine manganese in urine, while the detection of the psychoactive
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drugs was performed by high-performance liquid chromatography (HPLC).
Results of survey on longitudinal patterns of drug use showed that users of traditional
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illicit drugs now turn to cheaper alternatives, such as methcathinone. Parkinsonian features were observed in almost half of methcathinone users. The subjects had a higher mean level of
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Mn in their urine (8.68±9.27 µg·L-1) than the controls (4.27±1.91 µg·L-1). The presence of numerous psychoactive substances (in unchanged forms and their metabolites) was confirmed
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in all of the samples, with only one exception. The elevated level of manganese in urine (in
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29.2% of patients) can be used as a primary marker of recent methcathinone administration,
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especially in the case of long time intravenous drug users where blood sampling is complicated.
Keywords Methcathinone; Urine; Illicit drugs; Manganese; Parkinson-like syndrome
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Introduction Methcathinone (ephedrone) is an indirect sympathomimetic drug, which has been shown to increase the release of noradrenaline at similar potency to amphetamines [1], and
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has exhibited similar inhibition of noradrenaline reuptake to methamphetamine and ecstasy [2]. It is manufactured by oxidation of ephedrine and pseudoephedrine, usually via
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application of potassium permanganate. Under laboratory conditions, a 1 g of methcathinone
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required the use of 2 g of this oxidizing agent. However, illegal manufacturers or addicts usually add a larger amounts of potassium permanganate [3,4]. One study reported that the
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concentration of methcathinone in a home-made liquid obtained from an addicted person was 2.6 mg∙mL-1 [5].
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The first case of methcathinone intoxication was described 20 years ago [6]. Nowadays, there have been significant increases both in the number of people who use this xenobiotic
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and the cases of poisoning [7]. The occurrence of a Parkinson-like syndrome among
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methcathinone addicts is quite common [8]. This kind of effect of methcathinone was
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observed in many countries worldwide [9-11] and is believed to be related to manganese poisoning brought about by the high concentrations of metal present in the home made methcathinone [12,13]. An extensive review [14] of this phenomenon could suggest that manganese is probably the sole causative agent for the Parkinson’s symptoms in addicts, with negligible contribution to the neurotoxicity being associated with dopamine depletion. Manganese is an essential element, but excessive exposure can be toxic, particularly to the central nervous system [15-17]. The collection of blood from long-term intravenous drug users creates important problems. On the other hand, urine is the basic biological material in diagnostic in clinical toxicology. For that reasons, we decided to assess the applicability of urine for evaluation of methcathinone use in multiple drug addicts on the basis of determination of manganese. 3 Page 3 of 24
Materials and methods Subjects
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Urine, collected from people who have intravenously injected methcathinone (ephedrone), was obtained from the Department of Analytical Toxicology and Therapeutic
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Drug Monitoring, Jagiellonian University, Krakow, Poland. Twenty-four patients addicted to methcathinone were recruited from participants at the Outpatients Clinic for Drug Prevention
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and Treatment of Addicts, Krakow, Poland. Seven patients participated in methadone maintenance treatment. The patients group consisted of 17 men and 7 women with a mean
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age of 38.9±7.9 (age range: 23-59 years). All patients were active smokers (the age of initiation: 16.2±4.7) and drank alcohol (the age of initiation: 14.3±2.2). The results of
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manganese analysis for the patients were compared with those of 25 age-matched healthy volunteers. The control group consisted of 15 men and 10 women with a mean age
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37.7±8.2 (age range: 23-57 years), without a drug abuse history. Among them, 16 subjects
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(64%) were cigarette smokers (the age of initiation: 16.6±1.8), and all volunteers drank
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alcohol (the age of initiation: 14.3±2.2).
All patients completed a drug history questionnaire to document their lifetime and
recent use of alcohol, tobacco, medicines and illicit drugs. Written informed consent was obtained prior to participation. The study was approved
by the Bioethics Committee of the Jagiellonian University in Krakow. Reagents All reagents used in this study were of the highest purity available. All solutions were prepared with the ultrapure deionized water (20 MΩ). Concentrated nitric acid (65%, Suprapur, Merck, Germany) was used for sample digestion. A standard stock solution of Mn (1000 mg·L-1), prepared from Titrisol standard 4 Page 4 of 24
(Merck, Germany) was used fot the preparation of the standard solutions. For GF AAS method, the following modifiers were used: palladium nitrate (c(Pd)=10.0±0.2 g·L-1 (Pd(NO3)2/HNO3 ca. 15%, Merck, Germany) and magnesium nitrate (c=2.6 g·L-1), obtained
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by dissolving proper amount of Mg(NO3)2·6H2O (Merck, Germany) in deionized water. The following reagents were used for HPLC analyses: HPLC-grade acetonitrile and H3PO4
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(Sigma- Aldrich, Germany), HCl and NaOH (POCh, Poland), ethyl acetate (Merck, Germany).
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The accuracy was assessed through the analysis of the certified reference material
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(CRM) of urine (21.2±4.2 μg Mn·L-1, ClinChek Urine Control, Levels II, Recipe, Germany). Analysis of manganese in urine
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A microwave-assisted acid digestion procedure was carried out before quantitative elemental analysis using a Mars X5 microwave system (CEM, USA). One mL of urine or
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urine certified reference material was digested with 6 mL of concentrated HNO3. The process
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of digestion was conducted in three steps, differing in maximum temperature and microwave
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power: 1600C/600W, 1800C/850W, and 2000C/1100W. Each step lasted 8 min (heating to a given temperature for 4 min and holding it for 4 min). After completing the program, the vessels were cooled down (about 30 minutes) and the digested samples solutions were transferred into 10 mL volumetric flasks and diluted with deionized water. A PinAAcle 900Z graphite furnace atomic absorption spectrophotometer with
a transversely-heated graphite tubes with integrated platform (THGATM) and an AS 900 autosampler (Perkin Elmer, USA) was used for the determination of manganese in urine. The major validation parameters of the method were as follow: accuracy (as relative error) 3.46%; precision (CV) - 3.61%; LOD - 0.11 µg·L–1; LOQ - 0.33 µg·L–1; recovery - 94±3%. The method was described in detail in our previous work [18].
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Drug screening in urine For drug screening (amphetamine derivatives, ephedrine, mephedrone, methcathinone, methadone, EDDP), the HPLC method, routinely used in the Laboratory of Analytical
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Toxicology and Therapeutic Drug Monitoring, Faculty of Medicine Jagiellonian University, was applied [19]. Shortly, 0.2 mL of 0.5 M NaOH and 1.2 mL of ethyl acetate was added to
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500 µL of urine and the mixture was vortexed for 3 min. After centrifugation, the organic
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phase was transferred to Eppendorf tube and 50 µL of 0.02 M HCl was added afterwards the solvent was evaporated to dry at 40oC under a stream of nitrogen. The residue was
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reconstituted with a 300 μL of a mixture containing 500 mL of deionized water with 100 µL of concentrated phosphoric acid and acetonitrile (1:1).
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All HPLC analyses were performed in a liquid chromatograph equipped with an Diode Array Detector L-7455, LaChrom System (Merck-Hitachi, Darmstadt, Germany). Separation
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of analytes was conducted in a LiChroCART 125-4 LiChrospher RP-select B (Merck,
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Darmstadt, Germany) column. Separation was performed in gradient conditions (phase A: 500 mL of deionized water with 100 µL of concentrated phosphoric acid; phase B:
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acetonitryle at a flow of 1 mL·min–1. The gradient program (Time [min]/mobile phase A [%]; mobile phase B [%]) was set as 0-8/100;0, 8-10/90;10, 10-15/80;20, 20/70;30, 21/50;50, 25/ 100;0, and 30/100;0, respectively. The results of qualitative analysis of urine sample with standards and the urine of one patient is shown in Figure 1. The screening for opiates (morphine derivatives), cannabinoids, benzodiazepines and
buprenorphine (not used in Poland in maintenance treatment) were performed using enzymelinked immunosorbent assay (ELISA) screening kits from nal von Minden GmbH (Regensburg, Germany) run on a Viva-E analyzer (Siemens Healthcare Diagnostics, Liederbach, Germany). The cut-off were 300, 50, 300 and 10 ng mL-1 for opiates, cannabinoids, benzodiazepines and buprenorphine, respectively. 6 Page 6 of 24
Statistical analyses Statistica 10 (StatSoft Inc., USA) was used for the statistical analyses of data. The normal distribution of the dataset was verified by a Kolmogorov-Smirnov with the Lilliefors
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significance correction test and the Shapiro Wilk test. A basic data analysis was performed to obtain the mean values, range and standard deviation of the mean. Differences between study
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groups were compared with the Mann-Whitney U test. The degree of correlation was
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evaluated from the Pearson correlation coefficients. Significance was set at p<0.05.
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Results
Illicit drugs and medications taken by patients – questionnaire data
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Information obtained from the questionnaire regarding the drugs of abuse is presented in Table 1. The mean age of initiation to drug use was 15.7±2.6 (age range: 13-25). All of the
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patients have used drugs intravenously. Illicit drugs were commonly abused via insufflations
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(79%), smoking (95%) and the oral route (95%). The most popular illicit drugs were opioids
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(91.2% of patients have taken it during their life and 58.3% during the last month), amphetamine (91.2% of patients have taken it during their life and 58.3% during the last month) and THC (91.2% of patients have taken it during their life and 58.3% during the last month).
The majority of the subjects (95%) have administered various types of medications
(average age of initiation: 19.4±5.4). The most popular are benzodiazepines (91.2% of patients have taken it during their life and 58.3% during the last month), opiates (75.0% of patients have taken it during their life and 45.8% during the last month), barbiturates (70.8% of patients have taken it during their life and 8.3% during the last month) and acodine (33.3% of patients have taken it during their life and no one during the last month).
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Methcathinone use - questionnaire data Data concerning methcathinone abuse are presented in Table 2. All of the subjects have administered methcathinone intravenously. In most cases (87.5%), the patients have prepared
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the drug by themselves. Information about methcathinone came from friends (75%) and websites (8.3%). The majority of patients (83.3%) were aware of health problems that may be
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caused by the drug. Among them, 41.7% have reported various symptoms, which probably
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arose from manganese intoxication (but could also be the effect of methcathinone itself), such as impaired physical coordination, balance disorder, slurred speech, difficulties with writing
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and memory, daytime drowsiness, kidney and liver pain, and a swollen face and limbs.
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Licit and illicit drug screening in the urine of the patients
The qualitative results of illicit drugs determinations achieved by HPLC and ELISA
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gave following results: buprenorphine was detected in 9 urine samples (patients 1, 2, 5, 6, 11,
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12, 14, 16, 22), opiates and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine metabolite of methadone) in 7 urine samples (respectively: patients 1, 6, 8, 12, 13, 16, 24, and
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patients 9, 15, 17, 18, 20, 21, 23), methadone in 6 samples (patients 9, 17, 18, 20, 21, 23) and mephedrone in 4 samples (patients 4, 7, 9, 10,). Methcathinone was present in the urine of only 2 subjects (patients 5 and 12). Amphetamine and cannabinoids were only detected in patient n°15, while quetiapine (atypical antipsychotic) – in patient n°18. Only patient n°3 had no illicit substances detected in his urine. It was the person who used methcathinone less than one year and had the lowest concentration of manganese when compared with other addicts. In the urine of patients participated in methadone maintenance treatment, methadone and/or its metabolite EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) were confirmed. Level of manganese in urine 8 Page 8 of 24
The results of determination of manganese in urine for all cases from the group of patients are listed in Table 3. About 29% of addicts had urinary Mn concentration over the level considered normal -
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below 10 μg·L-1 [20]. Recent data showed no gender differences for the urinary excretion of manganese, and also smoking and alcohol consumption have negligible effect on this process
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[21]. The mean level of Mn found in the urine of methcathinone users was 8.68±9.27 µg·L-1, with a very wide range, from 0.92 to 46.57 µg·L-1. This may arise from individual differences
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in the excretion of Mn [21], and the dose and frequency of methcathinone administration. It is also necessary to take under consideration the fact that it is home-made drug, which was
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probably synthesized with the use of the different amount of potassium permanganate. When
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considering the time of last administration of methcathinone, the highest mean concentration of manganese was determined in samples of urine from people taking the drug no longer than one week before sampling (13.82±13.71 µg·L-1). As expected, the lowest level of Mn was
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observed in subjects who had not been used the drug for a year (4.57±3.10 µg·L-1). However, in this group and the group of patients who had taken methcathinone from 1 to 12 months
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ago, there were two patients: n°7 and 14, who had distinctly increased concentration of Mn (10.76 µg·L-1 and 32.16 µg·L-1, respectively). It might be assumed that these individuals had lied about the last administration of the drug and therefore should have been in the first group (people who had taken the drug no longer than one week before sampling). Then, the concentration of Mn in the urine of subjects who had not been used the drug for a year and those who had taken methcathinone from 1 to 12 months ago would be 3.37±1.94 µg·L-1 and 3.69±1.98 µg·L-1, respectively, while the most recent users would have the following level of Mn: 15.35±13.49 µg·L-1. Healthy volunteers have a mean Mn concentration of 4.27±1.91 µg·L-1 (range: 0.65-6.67 µg·L-1). The mean value of Mn concentration in the urine of abusers was statistically significantly higher than in healthy volunteers (p<0.05). The mean
9 Page 9 of 24
concentration of Mn in both groups can be considered as a normal for the general population, but the urinary manganese level in some of the methcathinone users was significantly high. Figure 2 shows the dependence of the concentration of manganese in the urine samples of
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patients from the time of last administration of the drug. It was observed that, over time, the concentration of manganese in the urine decreases, until it reaches the physiological level.
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The highest concentration of this element was determined in the urine of a patient who took methcathinone intravenously 30 minutes before the collection of the sample. Correlation
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analysis indicates two strong correlations between the manganese level and the time of the last administration of methcathinone (r=-0.465), as well as the number of times the drug was
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used in the last month (r=0.601). It was found that the number of injections during a life has
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no effect on the concentration of manganese in urine. No correlations were found between the concentration of Mn and the detected drugs or their metabolites in urine.
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Discussion
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Methcathinone, a designer drug known in Russia and Eastern Europe as an ephedrone,
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has become a growing concern across the European Union and United States [22]. It is cheaper than amphetamine or cocaine. In one study, addicts stated that methcathinone caused stronger and longer-lasting toxicological effects than traditional drugs like cocaine, but also made the users psychologically dependent on it [23]. Moreover, it can be prepared in someone's home from over the counter medicines. The procedure of synthesis is rather easy and can be found on numerous websites [8]. Our study examined the longitudinal patterns of methcathinone and other psychoactive substances use, plus the health problems of 24 participants from the Outpatients Clinic for Drug Prevention and Treatment of Addicts, who have taken methcathinone for a long period of time. Similar studies were also conducted in Ukraine [24,25], Russia [16], Estonia [8] and Latvia [12]. According to the best of our knowledge, this is the first such study of 10 Page 10 of 24
methcathinone users in Poland. Close attention was paid to the possibility of manganese intoxication in methcathinone addicts. Urine analyses were conducted to detect the use of methcathinone and other licit and illicit drugs (e.g. methadone, benzodiazepines, opiates),
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plus to determine the level of manganese. Methcathinone was detected only in two samples, despite the fact that some patients
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declared a recent intake of the drug a day before urine sampling. Due to the lack of information about the pharmacokinetics of this drug in humans, it can be assumed that the
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metabolism of methcathinone is similar to cathinone to a very close structurally. Therefore the half-life of both might be comparable, which was found to be 1.5±0.8 h for cathinone
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after chewing of khat leaves [26]. This can explain why methcathinone was detected in the
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urine only in two subjects. Pseudoephedrine, which is a major metabolite of methcathinone, or can be in the drug due to incomplete synthesis [27,28], was not present in the urine of our
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patients.
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The analysis of information obtained from this survey may lead to the conclusion that people with a long history of addiction have given up traditional drugs like cocaine or
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amphetamine for methcathinone. It is probably because they cannot financially afford the traditional drugs and this cheaper analog has a similar effect on them. The main concern of methcathinone abuse is not only the physiological and psychical
effects caused by the drug itself, which are characteristic for amphetamine-type stimulants (e.g., hallucinations, paranoia, restlessness, appetite loss, insomnia) [24], but rather neurological, as the possibility of developing Parkinson’s-like symptoms related to manganese. As mentioned, the reaction of oxidation of pseudoephedrine is often conducted with an excess of potassium permanganate, so the final product is contaminated with manganese. Sikk et al. [8] performed the synthesis of methcathinone according to recipes from their patients and found on the Web, and reported that the obtained mixture contained
11 Page 11 of 24
0.6 g Mn·L-1. Managnese is an essential element and its normal concentration ranges between 4 and 12 μg·L-1 in blood [29] and 0.40 to 0.85 μg·L-1 in serum [30]. Only a small amount of manganese is excreted in urine (about 3%), and its level in healthy people ranges from 1 to 10
ip t
μg·L-1 [20], so the use of it as a biomarker for Mn exposure is limited. Usually blood is used for this purpose, but this biological material has important limitations [31]. Collection of
cr
blood from chronic intravenous drug users is problematic because they usually have
us
collapsed veins. Urine collection is much easier and this biological material was chosen in this study.
an
Manganese is transported across the blood-brain barrier via many transporter systems, such as the transferrin (Tf) receptor (TfR), the divalent metal transporter 1 (DMT1), the
M
magnesium transporter hip14, the divalent metal/bicarbonate ion symporters ZIP8 and ZIP14, the solute carrier-39 (SLC39) family of zinc transporters, the transient receptor potential
d
melastatin 7 (TRPM7) channels/transporters, and various calcium channels [15]. Metal
te
deposits in the basal ganglia and the globus pallidus of the brain. This area is in the regulation
Ac ce p
of motor control, cognitive and emotional functions, hence manganism may cause neurological symptoms similar to Parkinson’s [14,32]. It is still unknown how the type and length of exposure to Mn, as well as the dose or route of administration lead to development of Parkinson-like syndrome [33]. The first clinical manifestations (speech difficulties and gait abnormality) are observed usually within the first few years after the beginning of methcathinone intake [34].
Many studies reported an elevated concentration of Mn in different biological materials obtained from methcathinone users. Varlibas et al. [35] described three people with movement disorders who have taken intravenously the so-called “Russian Cocktail” (a mixture similar to methcathinone consisting of potassium permanganate, ephedrine and aspirin). Two of them had a high blood concentration of Mn: 2100 μg·L-1 and 3176 μg·L-1. 12 Page 12 of 24
All subjects have received a standard treatment with anti-parkinson drugs and for metal poisoning, but it did not reverse the symptoms of toxicity. Koksal et al. [36] also found that an excess of Mn causes an irreversible neurodegeneration, and that treatment with levodopa
ip t
is not successful. In their study, seven patients who injected a “Russian Cocktail” had an elevated concentration of Mn in serum and urine. The estimated 24 h urinary Mn excretion in
cr
subjects was as follows: 0.3 μg, 11 μg, 14 μg, 7 μg, 12 μg, 13 μg and 10 μg, while normal value is <0.67 μg. Assuming that an average urine excretion is on the level of 1.5 L per day,
us
above results are in the same range as ours. Selikhova et al. [25] have determined Mn in the pubic hair of 9 drug addicts. Three patients with a worsening clinical condition had the
an
highest mean level of Mn in hair (2.68±1.75 μg∙g-1), while other subjects had one at 0.74±0.37 μg∙g-1. Healthy volunteers had the concentration reported in other studies as being
M
normal at 0.34 ± 0.29 μg∙g-1. Research conducted in Estonia amongst a large amount of
d
intravenous methcathinone users has also led to the conclusion that methcathinone users are exposed to manganese poisoning [37]. The addicts had a significantly higher mean
te
concentration of Mn in hair (2.9±3.8 mg∙g-1) than the controls (0.82±1.02 mg∙g-1). The Mn
Ac ce p
level in plasma was significantly elevated (11.5±6.2 μg∙g-1) in active rather than in former users (5.6±1.8 μg∙g-1). However, as Smith et al. [31] demonstrated in their study, hair and plasma are not a reliable biomarkers for Mn exposure. Also the standard deviations are higher than mean concentrations in case of hair, what makes this results questionable. To our knowledge, this is the first study that has used urine for evaluation exposure of
methcathinone users to manganese, showing that the mean concentration of Mn in this medium was two-fold higher than in the urine of healthy volunteers, although only in the case of subjects with exposure on several dozen hours before urine collection was the level of Mn over normal value.
13 Page 13 of 24
Almost 42% of people from the clinical group reported neurological symptoms (impaired physical coordination, balance disorder, slurred speech, difficulties with writing and memory, daytime drowsiness). As expected, the subjects with a long history of
ip t
methcathinone use (over 1000 injections in life) had the most severe health problems. On the other hand, the last administration of drug did not have any influence on the appearance of
cr
neurological symptoms. Our survey confirmed that methcathinone is taken as a substitute of more expensive drugs and usually is taken with prescribed medicine (methadone,
us
buprenorphine, quetiapine), and illicit drugs, more frequently opiates.
Identifying the cause of an extrapyramidal syndrome in methcathinone abusers could be
an
difficult because of the lack of specialist laboratories. In this situation the elevated level of
M
manganese can be used as a primary marker of methcathinone use, but individual differences are possible. The most valuable results are obtained from the determination of this element in
d
blood, although in the case of long time intravenously drug users, it is very complicated and
te
in this situation the concentration of manganese in urine can be helpful. In patients with residential exposure, or those who were previously, but not currently, exposed to manganese,
Ac ce p
the measurement of this element in urine cannot provide a valid history of methcathinone taking.
Acknowledgements
The research was carried out with the equipment purchased thanks to the financial
support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). References [1]
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Cozzi NV, Sievert MK, Shulgin A, Jacob P, Ruoho AE. Inhibition of plasma membrane
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monoamine transporters by β-ketoamphetamines. European Journal of Pharmacology 1999;381:63-9.
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[3]
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Zhingel KY, Dovensky W, Crossman A, Allen A. Ephedrone: 2-methylamino-1-
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[5]
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(ephedrone encephalopathy). In: Anniversary collection: diagnostic and treatment of
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neurological diseases. Medicine 1990, 183–6. Rust KY, Baumgartner MR, Dally AM, Kraemer T. Prevalence of new psychoactive
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substances: A retrospective study in hair. Drug Testing and Analysis 2012;4:402-8. Sikk K, Taba P, Haldre S, Bergquist J, Nyholm D, Zjablov G, et al. Irreversible motor impairment in young addicts–methcathinone, manganism or both? Acta Neurologica Scandinavica 2007;115:385-9.
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Çitçi B, Varlıbaş F, Tutkavul V, Türkoğlu R, Tireli H. Manganese abuse related
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ip t
[12] Stepens A, Logira I, Liguts V, Aldins P, Eksteina I, Platkajis A, et al. A parkinsonian syndrome in methcathinone users and the role of manganese. New England Journal of
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[14] Guilarte TR. Manganese and Parkinson’s disease: a critical review and new findings.
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Environmental Health Perspectives 2010;118:1071-80.
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[22] Spiller HA, Ryan ML, Weston RG, Jansen J, Clinical experience with and analytical
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[23] Miller RL. The Encyclopedia of Addictive Drugs. London: Greenwood Press, 2002. p.
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[25] Selikhova M , Fedoryshyn L , Matviyenko Y , Komnatska I , Kyrylchuk M , Krolicki L, et al. Parkinsonism and dystonia caused by the illicit use of methcathinone – a longitudinal study. Movement Disorders 2008;23:2224-31.
[26] Toennes SW, Harder S, Schramm M, Niess C, Kauert GF, Pharmacokinetics of cathinone, cathine and norephedrine after the chewing of khat leaves. British Journal of Clinical Pharmacology 2002;56:125–30. [27] Belhadj-Tahar H, Sadeg N. Methcathinone: A new postindustrial drug. Forensic Science International 2005;153:99–101.
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[28] Marais AA, Laurens JB. Rapid GC–MS confirmation of amphetamines in urine by extractive acylation. Forensic Science International 2009;183:78-86. [29] Šaric M, Lucchini R. Manganese. In: Nordberg GF, Flowler BA, Nordberg M, Friberg
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LT, editors. Handbook on the Toxicology of Metals, 3rd ed. Burlington: Academic Press, 2007. p.645-74.
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[30] Jankovic J. Searching for a relationship between manganese and welding and Parkinson’s disease. Neurology 2005;64:2021-8.
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[31] Smith D, Gwiazda R, Bowler R, Roels H, Park R, Taicher C, et al. Biomarkers of Mn exposure in humans. American Journal of Industrial Medicine 2007;50:801-11.
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[32] Lucchini RG, Martin CJ, Doney BC. From manganism to manganese-induced
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parkinsonism: a conceptual model based on the evolution of exposure. NeuroMolecular Medicine 2009;11:311-21.
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[33] Cersosimo MG, Koller WC. The diagnosis of manganese-induced parkinsonism.
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Neurotoxicology 2006;27:340-6.
[34] Sikk K, Haldre S, Aquillonius SM, Taba P. Manganese-induced parkinsonism due to
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Ephedrone abuse. Parkinson’s Disease 2011:865319.
[35] Varlibas F, Delipoyraz I, Yuksel G, Filiz G, Tireli H, Gecim NO, Neurotoxicity following chronic intravenous use of “Russian cocktail”. Clinical Toxicology 2009;47:157-60.
[36] Koksal A, Baybas S, Sozmen V, Koksal NS, Altunkaynak Y, Dirican A, et al. Chronic manganese toxicity due to substance abuse in Turkish patients. Neurology India 2012;60:224-7. [37] Sikk K, Haldre S, Aquilonius SM, Asser A, Paris M, Roose Ä, et al. Manganeseinduced parkinsonism in methcathinone abusers: bio-markers of exposure and followup. European Journal of Neurology 2013;20:915-20.
18 Page 18 of 24
Table 1
Amphetamine - life/last month (n)
24/5
Cocaine - life/last month (n)
21/0
THC - life/last month (n)
23/19
Mephedrone - life/last month (n)
19/3
Hallucinogens - life/last month (n)
22/16
cr
24/11
Ac ce p
te
d
M
an
us
Opioids - life/last month (n)
ip t
Details about the past history of illicit drugs abuse in patients.
19 Page 19 of 24
Table 2 Selected information about history of methcathinone abuse
50-100
3
100-1000
4
>1000
10
< 1 week
8
1-4 weeks
2
1-12 months
6
> 1 year
8
an
Medical problems connected with drug abuse [yes/no]
cr
Last administration of methcathinone
7
us
Methcathinone – number of injections in life
<50
ip t
Number of cases (n)
Ac ce p
te
d
M
10/14
20 Page 20 of 24
Table 3 Relationship between the concentration of manganese in the urine of methcathinone users and
6.63±3.76
1-12 months
8.17±11.88
a
te 4.57±3.10
Ac ce p
>1 year
cr
1-4 weeks
9.27 1.64 12.04a 10.88a 3.24 15.20a 45.74a 12.58a 3.97 9.28 1.63 4.66 1.46 3.14 32.16a 5.94 4.06 0.92 10.76a 5.95 5.47 3.50 1.18 4.73
Number of patient 6 10 12 15 19 20 22 23 4 18 2 5 8 13 14 16 1 3 7 9 11 17 21 24
us
13.82±13.71
d
<1 week
Concentration of Mn [µg∙L-1]
an
Mean concentration of Mn±SD [µg∙L-1]
M
Last dose of methcathinone
ip t
the time after the last dose of methcathinone administration
Concentration of Mn over the physiological level
21 Page 21 of 24
It is no conflict of interest In behalf of all authors
Ac ce p
te
d
M
an
us
cr
ip t
Prof. Wojciech Piekoszewski PhD, DSc
22 Page 22 of 24
an
us
cr
ip t
Intensity [AU]
1 – phenylpropyloamine (internal standard, tR=12.7 min 2 – ephedrine/pseudoephedrine, tR=14.5 min 3 – methcathinone, tR=14.9 min 4 – amphetamine, tR=15.6 min 5 – mephedrone, tR=18.1 min
Retention time [min]
Ac ce p
te
d
M
Fig 1. HPLC chromatogram of a blank urine sample spiked with standards (400 ng/mL) and an urine sample of the patient (methcathinone and amphetamine).
23 Page 23 of 24
ip t cr us an M
d
Fig. 2 Manganese concentration dependence of time of last administration of methcathinone.
te
The main plot shows the level of Mn in the urine of all patients (in the range of 50 months). The inside plot shows the concentration of Mn in the urine of selected patients (those who
Ac ce p
had taken methcathinone during four days following the collection of samples). Grey area indicates the range of the normal concentration of manganese in urine. [38]
24 Page 24 of 24