Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites.

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Toluene and methylethylketone: effect of combined exposure on their metabolism in rat.

1. Multiple exposures are ubiquitous in industrial environments. In this article, we highlight the risks faced by workers and complete the data available on the metabolic impact of a common mixture: toluene (TOL) and methylethylketone (MEK). 2. Rats were exposed by inhalation under controlled conditions either to each solvent individually, or to mixtures of the two. How the interaction between the two solvents affected their fate in the blood and brain, their main relevant urinary metabolites (o-cresol, benzylmercapturic acid for TOL and 2,3-butanediols for MEK) and their hepatic metabolism were investigated. 3. Although the cytochrome P450 concentration was unchanged, and the activities of CYP1A2 and CYP2E1 isoforms were not additively or synergistically induced by co-exposure, TOL metabolism was inhibited by the presence of MEK (and vice versa). Depending on the relative proportions of each compound in the mixture, this sometimes resulted in a large increase in blood and brain concentrations. Apart from extreme cases (unbalanced mixtures), the amount of o-cresol and benzylmercapturic acid (and to a lesser extent 2,3-butanediols) excreted were proportional to the blood solvent concentrations. 4. In a co-exposure context, ortho-cresol and benzylmercapturic acid can be used as urinary biomarkers in biomonitoring for employees to relatively accurately assess TOL exposure.

Metabolism of inhaled methylethylketone in rats.

Methylethylketone (MEK) is widely used in industry, often in combination with other compounds. Although nontoxic, it can make other chemicals harmful. This study investigates the fate of MEK in rat blood, brain and urine as well as its hepatic metabolism following inhalation over 1 month (at 20, 200 or 1400 ppm). MEK did not significantly accumulate in the organism: blood concentrations were similar after six-hour or 1-month inhalation periods, and brain concentrations only increased slightly after 1 month's exposure. Urinary excretion, based on the major metabolites, 2,3-butanediols (± and meso forms), accounted for less than 2.4% of the amount inhaled. 2-Butanol, 3-hydroxy-2-butanone and MEK itself were only detectable in urine in the highest concentration conditions investigated, when metabolic saturation occurred. Although MEK exposure did not alter the total cytochrome P450 concentration, it induced activation of both CYP1A2 and CYP2E1 enzymes. In addition, the liver glutathione concentration (reduced and oxidized forms) decreased, as did glutathione S-transferase (GST) activity (at exposure levels over 200 ppm). These metabolic data could be useful for pharmacokinetic model development and/or verification and suggest the ability of MEK to influence the metabolism (and potentiate the toxicity) of other substances.

Development of a liquid chromatography-mass spectrometry method for determination of agrimol B in rat plasma: application to preclinical pharmacokinetics.

A sensitive and accurate liquid chromatography coupled with mass spectrometry (LC-MS) method was developed for the determination of agrimol B, a main active ingredient isolated from Agrimonia pilosa Ledeb., in rat plasma. Chromatographic separation was achieved on a Zorbax CN column (150 × 4.6 mm, 5 µm), with isocratic elution consisting of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Agrimol B and dryocrassin ABBA, an internal standard (IS), were analyzed by selected ion monitoring at m/z transitions of 681.3 and 819.4, respectively. This assay exhibited a good linearity with a correlation coefficient >0.99 and showed no endogenous interference with the analyte and IS. The limit of quantification of agrimol B was 8.025 ng/mL with acceptable precision and accuracy. The method was successfully applied in the pharmacokinetic study of agrimol B in rats after intravenous (1 mg/kg) and oral (2, 5 and 10 mg/kg) doses of agrimol B. The absolute bioavailability of agrimol B was 16.4-18.0% in rat. Our study clarifies the pharmacokinetic behavior of agrimol B in animals.

A diclofenac suppository-nabumetone combination therapy for arthritic pain relief and a monitoring method for the diclofenac binding capacity of HSA site II in rheumatoid arthritis.

Diclofenac suppository, a non-steroidal anti-inflammatory drug (NSAID), is used widely in rheumatoid arthritis (RA) patients with severe arthritic pain. As the binding percentage of diclofenac to serum proteins is high, its free (unbound) concentration after rectal administration is low. To increase temporarily the free concentration of diclofenac and to enhance its analgesic effect by inhibiting the protein binding of diclofenac, the analgesic effect of diclofenac was examined before and after the start of an inhibitor administration to RA patients with insufficient control of arthritic pain, and the protein binding capacity of diclofenac was evaluated. Binding experiments were performed by ultrafiltration, and arthritic pain was recorded by the face scale. Free fractions of diazepam and diclofenac were augmented by increasing 6-methoxy-2-naphthylacetic acid (6-MNA; the active metabolite of the NSAID nabumetone) concentrations. The free fraction of diazepam increased after the start of nabumetone administration to RA patients, and arthritic pain relief was observed. These results suggest that 6-MNA has an inhibitory effect on the protein binding of diclofenac and the free fraction of diazepam can be used to evaluate the binding capacity of diclofenac. It is considered that diclofenac suppository-nabumetone combination therapy and the method for protein binding monitoring by diazepam can positively benefit RA patients with insufficient control of arthritic pain.

[Determination of nabumetone and 6-methoxy-2-naphthylacetic acid in plasma using HPLC with UV and MS detection]. / Stanovení nabumetonu a kyseliny 6-methoxy-2-naftyloctové v plazme pomocí HPLC s UV a MS detekcí.

The study aimed to establish and validate an analytical method for the determination of nabumetone and 6-methoxy-2-naphthylacetic acid (6-MNA) in human plasma after a single therapeutic dose of the drug. Two methods based on HPLC with UV and MS detection were compared. Optimal results in sample preparation were achieved using solid phase extraction. The recovery reached approximately 84% and 86-90% for nabumetone and 6-MNA, respectively. A reverse C18 column was used for HPLC separation of the analytes. The limit of UV detection was 50 nM and 0.1 microM for 6-MNA and nabumetone, respectively. The limit of MS detection was 1 microM and 0.5 microM for 6-MNA and nabumetone, respectively. Precision ranged between 4.2-14.4% and 4.6-8.5% using UV and MS detection for nabumetone, respectively. The respective values for 6-MNA were 2.4-12.5% and 2.1-9.4%. Accuracy ranged between 93.4-109.6% in UV detection and 86.2-107.9% using UV and MS detection for nabumetone, respectively. The respective values for 6-MNA were 87.8-107.4% and 86.3-106.4%. The method was subsequently applied to determine the pharmacokinetic parameters of nabumetone and 6-MNA in a group of 24 healthy volunteers.

UHPLC-QqQ-MS/MS method development and validation with statistical analysis: Determination of raspberry ketone metabolites in mice plasma and brain.

Raspberry ketone (RK) (4-(4-hydroxyphenyl)-2-butanone) is the major compound responsible for the characteristic aroma of red raspberries, and has long been used commercially as a flavoring agent and recently as a weight loss supplement. A targeted UHPLC-QqQ-MS/MS method was developed and validated for analysis of RK and 25 associated metabolites in mouse plasma and brain. Dispersion and projection analysis and central composite design were used for method optimization. Random effect analysis of variance was applied for validation inference and variation partition. Within this framework, repeatability, a broader sense of precision, was calculated as fraction of accuracy variance, reflecting instrumental imprecision, compound degradation and carry-over effects. Multivariate correlation analysis and principle component analysis were conducted, revealing underlying association among the manifold of method traits. R programming was engaged in streamlined statistical analysis and data visualization. Two particular phenomena, the analytes' background existence in the enzyme solution used for phase II metabolites deconjugation, and the noted lability of analytes in pure solvent at 4 ℃ vs. elevated stability in biomatrices, were found critical to method development and validation. The approach for the method development and validation provided a foundation for experiments that examine RK metabolism and bioavailability.

Detection and quantification of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) from smoker albumin and its potential as a surrogate biomarker of tobacco-specific nitrosamines exposure and bioactivation.

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2 = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2 = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.

Tobacco-Specific Carcinogens Induce Hypermethylation, DNA Adducts, and DNA Damage in Bladder Cancer.

Smoking is a major risk factor for the development of bladder cancer; however, the functional consequences of the carcinogens in tobacco smoke and bladder cancer-associated metabolic alterations remain poorly defined. We assessed the metabolic profiles in bladder cancer smokers and non-smokers and identified the key alterations in their metabolism. LC/MS and bioinformatic analysis were performed to determine the metabolome associated with bladder cancer smokers and were further validated in cell line models. Smokers with bladder cancer were found to have elevated levels of methylated metabolites, polycyclic aromatic hydrocarbons, DNA adducts, and DNA damage. DNA methyltransferase 1 (DNMT1) expression was significantly higher in smokers than non-smokers with bladder cancer. An integromics approach, using multiple patient cohorts, revealed strong associations between smokers and high-grade bladder cancer. In vitro exposure to the tobacco smoke carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (BaP) led to increase in levels of methylated metabolites, DNA adducts, and extensive DNA damage in bladder cancer cells. Cotreatment of bladder cancer cells with these carcinogens and the methylation inhibitor 5-aza-2'-deoxycytidine rewired the methylated metabolites, DNA adducts, and DNA damage. These findings were confirmed through the isotopic-labeled metabolic flux analysis. Screens using smoke-associated metabolites and DNA adducts could provide robust biomarkers and improve individual risk prediction in bladder cancer smokers. Noninvasive predictive biomarkers that can stratify the risk of developing bladder cancer in smokers could aid in early detection and treatment. Cancer Prev Res; 10(10); 588-97. ©2017 AACR.

Intratissular lymphaticovenous anastomoses demonstrated by perioperative intramuscular injection of 99mTC-colloids.

BACKGROUND: The existence of intratissular lymphaticovenous anastomoses has often been suggested, but it has never been demonstrated. This study aims at demonstrating the presence of such anastomoses. METHODS AND RESULTS: The free flap model was used to investigate the drainage of radiolabeled colloid particles whose size prevents direct passage to the blood vessels. The tracer was injected into the muscle or the skin during the surgical procedure. Blood samples were sequentially drawn from the venous pedicle over the 30 minutes that followed the tracer injection. The blood samples were counted using a gamma well-counter. In all 14 patients, the venous blood radioactivity steadily increased over time. Radiochemical analyses performed on the blood samples demonstrated that the radioactivity is related to the labeled colloids and not to free pertechnetate. Planar imaging performed 24 hours after the surgical procedure showed a significant liver uptake, and no accumulation in the area of normal lymphatic relays. CONCLUSIONS: As, in the free flap model, there is no lymphatic drainage through the classical pathways whatsoever, and since the size of the radiolabeled particles prevents them from directly entering the blood stream, the results strongly suggest the presence of functional intratissular lymphovenous anastomoses.

Quantitative determination of n-butane metabolites in three cases of butane sniffing death.

Butane is an addictive volatile substance like toluene. We report three forensic autopsy cases of sudden death that occurred while sniffing n-butane and isobutane from portable gas cartridges. n-Butane and isobutane were detected in all three cases. In cases 1-3, n-butane concentrations in heart blood were 54.3, 25.5, and 30.7µg/mL, respectively. These concentrations were considered fatal according to the previous reports. In addition, n-butane metabolites (2-butanol and 2-butanone) were detected in cases 1 and 3 but not in case 2. Blood levels of 2-butanol and 2-butanone were 6.5 and 1.8µg/mL, respectively, in case 1, and 6.3 and 5.6µg/mL, respectively, in case 3. According to the police investigation, the decedent in case 1 had misused butane gas for more than 6 months in the period leading up to death. The decedent in case 3 also had a history of chronic misuse of butane gas. There was no history of chronic misuse of butane gas by the decedent in case 2. It was suspected that he attempted suicide via inhalation of butane gas using a plastic bag, leading to a rapid death. The presence or absence of n-butane metabolites might reflect the way of butane inhalation, such as the frequency and duration. Although additional experimental and case studies are necessary to establish the forensic applications of n-butane metabolite detection, it may be a useful method to understand the decedents' pattern of butane sniffing before death.

Penetration of the active metabolite of nabumetone into synovial fluid and adherent tissue of patients undergoing knee joint surgery.

The concentration of 6-methoxy-2-naphthylacetic acid (6-MNA) in plasma, synovial fluid, synovial tissue and fibrous capsule tissue was determined in an open study with 20 patients scheduled for knee joint surgery after oral treatment with nabumetone under steady-state conditions. 6-MNA is the principle metabolite of the prodrug nabumetone arising from an extensive first-pass metabolism in the liver. Patients suffering from rheumatoid arthritis (n = 12) or osteoarthritis stage III or IV (n = 8) received a daily dose of nabumetone 1 g in the evening starting 4 days prior to surgery. On day 1 an additional loading dose of nabumetone 1 g was given in the morning. At the time of surgery (day 5), blood, synovial tissue and fibrous capsule tissue were taken simultaneously. The samples were analysed by high performance liquid chromatography. After 4 days of treatment mean 6-MNA concentration in plasma was 40.76 mg/L, in synovial fluid 34.79 mg/L, in synovial tissue 19.33 mg/g and in fibrous capsule tissue 11.43 mg/g. Under steady-state conditions mean synovial fluid levels of 6-MNA were higher than after administration of a single dose and, in common with levels in synovial tissue, persist in a range sufficient for in vitro cyclo-oxygenase inhibition.

Potentiation of CCl4 hepatotoxicity in rats by a metabolite of 2-butanone: 2,3-butanediol.

The role of ketaone metabolism in 2-butanone-induced potentiaion of carbon tetrachloride (CCl4) hepatotoxicity was studied in rats. The blood concentrations of 2-butanol, 3-hydroxy-2-butanone and 2,3-butanediol detected 4 h after dosing were 3.2 mg/100 ml, 2.4 mg/100 ml and 8.6 mg/100 ml, respectively. Eighteen hours after 2-butanone, the concentration of 2,3-butanediol rose to 25.6 mg/100 ml, while the concentrations of 2-butanol and 3-hydroxy-2-butanone declined to 0.6 mg/100 ml and 1.4 mg/100 ml, respectively. A 16-h pretreatment with either 2-butanone (2.1 ml/kg, p.o.) or 2,3-butanediol (2.12 ml/kg, p.o.) markedly enhanced the hepatotoxic response to CCl4 (0.1 ml/kg, i.p.), as measured by serum glutamic pyruvic transaminase activity and hepatic triglyceride content. In vivo, limited formation of 3-hydroxy-2-butanone occurred after this dose of 2,3-butanediol. These data suggest that the production of 3-hydroxy-2-butanone and 2,3-butanediol via 2-butanone metabolism may participate in the augmented necrogenic effect of CCl4 seen after pretreatment with 2-butanone.

Dose-dependent kinetics of inhaled methylethylketone in man.

Physiologically based stimulation of earlier human inhalation exposure to methylethylketone (MEK) at a concentration of 200 ppm for 4 h suggested that the kinetics were dose-dependent. Two male volunteers were therefore exposed to MEK for 4 h at 3 exposure concentrations: 25, 200 and 400 ppm. Blood MEK concentrations were monitored during and after exposure. The results showed clearly that the kinetics of MEK were dose-dependent at higher exposure concentrations. Simulated exposure to MEK for 8 h suggests that saturation kinetics are reached at an exposure concentration of 50-100 ppm, depending on the physical work load.

Effects of short duration exposures to acetone and methyl ethyl ketone.

Workers are commonly exposed to mixtures or combinations of chemical agents, and these mixtures often consist of solvents. One group of solvents that has been extensively studied for its neurotoxic properties has been the ketones. However, previous research has focused on neuropathies produced by extended exposures and not on the simple pharmacokinetics or the reversible central nervous system (CNS) effects from short-duration exposures. In this research, 137 volunteers were recruited and tested for neurobehavioral performance changes and biochemical indicators during and after a short-duration (4-h) exposure to either acetone at 250 ppm, methyl ethyl ketone (MEK) at 200 ppm, acetone at 125 ppm with MEK at 100 ppm, or a chemical-placebo. Ethanol (95%, 0.84 ml/kg) was used as a positive control. Testing took place in an environmental chamber with four test stations. The computer-controlled test regimen took 10 h, and several measures were collected: (1) biochemical measurements of venous blood and alveolar breath; (2) psychomotor tests of choice reaction time, visual vigilance, dual task (auditory tone discrimination and tracking), and memory scanning; (3) one sensorimotor (postural sway) test; and (4) one psychological (Profile of Mood States [POMS]) test. Blood and breath concentrations during and after exposure did not demonstrate any interaction between the two solvents, nor were statistically significant sex differences present during uptake or elimination. The 250-ppm acetone exposure produced small but statistically significant differences from controls in two measures of the auditory tone discrimination task, and on the anger-hostility scale (males only) of the POMS test. The other chemical exposure conditions, MEK at 200 ppm and combination MEK with acetone, produced no consistent statistically significant results, which suggests there was no potentiation of the acetone effects with the co-exposure to MEK or vice versa under these test conditions. Ethanol at 0.07-0.08% blood alcohol concentration (BAC) caused significant decrements on both the auditory tone and tracking tests in the dual task.

Volatile organic compound testing of a population living near a hazardous waste site.

Accurate measures of individual exposure are critical in reducing misclassification and establishing scientifically valid associations between health outcomes and exposures to environmental contaminants. As part of a community health study, the Agency for Toxic Substances and Disease Registry conducted exposure testing for volatile organic compounds (VOCs) in the blood of people residing near an industrial complex. The purposes of the study were to assess recent exposures to VOCs in this community and to assess the utility of conducting blood VOC testing on populations near hazardous waste sites. One hundred blood specimens from the target area and 106 blood specimens from the control area were collected for analysis. The blood VOC levels in the target-area participants were compared to those in the control area and to a national reference population. Of the 31 separate VOCs for which testing was done, only acetone was statistically significantly (p < 0.05) higher in target-area participants (1,636 parts per billion [ppb]) than in control-area participants (1,353 ppb). 1,1,1-Trichloroethane was found at higher geometric mean levels in the control group (0.169 ppb) than in the target group (0.115 ppb) (p = 0.01). Median blood levels of 2-butanone and 1,4-dichlorobenzene were slightly higher in both target- and control-area groups than in the national reference population, but neither area was statistically significantly higher than the national reference population for any contaminant measured. Overall, there appeared to be no association between residing in the target area and elevated blood VOC levels. Based on the results of this study, blood VOC testing should be limited to populations living near sites where environmental testing has shown recent, elevated VOC exposure, or where unusual circumstances of illness may be attributed to VOC exposure.

Analysis of nabumetone in human plasma by HPLC. Application to single dose pharmacokinetic studies.

A simple and sensitive high performance liquid chromatography method for the determination of nabumetone in human plasma is described. The procedure involves liquid-liquid extraction with ethyl acetate and reversed-phase chromatography with fluorimetric detection (excitation 230 nm, emission 356 nm). The chromatographic conditions and the extraction procedure gave a clean chromatogram for the compound. The limit of quantitation was established as 0.313 ng/ml and the calibration curve was linear up to 20 ng/ml. The within-day and between-day relative standard deviations were less than 10% and the accuracy of the assay was in the range of 99-104%. The suitability of the method is shown for pharmacokinetic studies.

Non-steroidal anti-inflammatory drug, nabumetone, prevents indometacin-induced gastric damage via inhibition of neutrophil functions.

Nabumetone is a non-steroidal anti-inflammatory drug (NSAID). It works as a prodrug and is extensively metabolized to an active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA). It is well known that neutrophil infiltration and activation are critical in the pathogenesis of NSAID-induced gastric injury, and nabumetone shows less incidence of gastrointestinal irritancy. We examined the effects of nabumetone on neutrophil activation and on indometacin-induced gastric damage. In the indometacin-induced gastric mucosal injury, rats were treated with indometacin and then nabumetone or 6MNA was orally administered. Nabumetone prevented gastric damage accompanied by the reduction of neutrophil infiltration into gastric mucosa, but such an effect was not observed with 6MNA. Nabumetone reduced the formyl methionyl leucyl phenylalanine (fMLP)-induced respiratory burst of human neutrophils to 30% of the control level in-vitro, but 6MNA did not. In addition, nabumetone prevented the fMLP-induced migration of neutrophils. Nabumetone did not inhibit O2- generation in the xanthine-xanthine oxidase system. These results suggest that nabumetone prevents gastric damage induced by the active metabolite, 6MNA, via the suppression of neutrophil activation in gastric mucosa.

An unusual source for postmortem findings of methyl ethyl ketone and methanol in two homicide victims.

Contamination from methyl ethyl ketone and methanol utilized in a new technique to visualize fingerprints on human skin was detected postmortem in blood and vitreous humor by toxicological analysis. Two cases which represent the first field trials of the fingerprinting technique in Canada are presented. Details of the toxicological analyses and the nature of the contamination are discussed.

Drinking drivers in Sweden who consume denatured alcohol preparations: an analytical-toxicological study.

In the course of analyzing blood samples from drunk drivers, several low molecular weight volatiles were occasionally identified in addition to ethanol on the gas chromatograms. Among 21, 153 blood specimens analyzed during 1986, 77 contained ethanol as well as other volatile agents at the following mean concentrations: ethanol 2090 mg/L (range 830-3410), methanol 49.6 mg/L (range 20-178), acetone 88.3 mg/L (range 12-307), 2-propanol 32.2 mg/L (range 4-99), 2-butanone 49.2 mg/L (range 5-144), and 2-butanol 23.2 mg/L (range 4-64). A technical alcohol widely available in Sweden, trade name T-red, contains 92% w/w ethanol, 2% w/w acetone, and 5% w/w 2-butanone. A red coloring agent and a substance to impart a bitter taste (bitrex) are added to deter consumption. The drinking drivers who consumed technical alcohol were on average older (43 years compared with 35 years) and had higher mean BAC (2090 mg/L compared with 1767 mg/L). Those who drank denatured alcohol were more often apprehended while driving small motorcycles (mopeds) than were control groups of DWI offenders. The use of technical alcohol for intoxication might reflect, at least in part, the high costs and restricted availability of conventional alcoholic beverages in Sweden.