Role of l- and d-Menthol in the Glucuronidation and Detoxification of the Major Lung Carcinogen, NNAL.

Menthol, which creates mint flavor and scent, is often added to tobacco in both menthol and nonmenthol cigarettes. A potent tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is extensively metabolized to its equally carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as (R)- or (S)-NNAL enantiomers. NNAL is detoxified by UDP-glucuronosyltransferase (UGT) enzymes, with glucuronidation occurring on either NNAL's pyridine ring nitrogen (NNAL-N-Gluc) or the chiral alcohol [(R)- or (S)-NNAL-O-Gluc]. To characterize a potential effect by menthol on NNAL glucuronidation, in vitro menthol glucuronidation assays and menthol inhibition of NNAL-Gluc formation assays were performed. Additionally, NNAL and menthol glucuronides (MG) were measured in the urine of smokers (n = 100) from the Southern Community Cohort Study. UGTs 1A9, 1A10, 2A1, 2A2, 2A3, 2B4, 2B7, and 2B17 all exhibited glucuronidating activity against both l- and d-menthol. In human liver microsomes, both l- and d-menthol inhibited the formation of each NNAL-Gluc, with a stereospecific difference observed between the formation of (R)-NNAL-O-Gluc and (S)-NNAL-O-Gluc in the presence of d-menthol but not l-menthol. With the exception of three nonmenthol cigarette smokers, urinary MG was detected in all menthol and nonmenthol smokers, with l-MG comprising >98% of total urinary MG. Levels of urinary NNAL-N-Gluc were significantly (P < 0.05) lower among subjects with high levels of total urinary MG; no significant changes in free NNAL were observed. These data suggest that the presence of menthol could lead to increases in alternative, activating metabolic pathways of NNAL in tobacco target tissues, increasing the opportunity for NNAL to damage DNA and lead to the development of tobacco-related cancers. SIGNIFICANCE STATEMENT: High levels of the major menthol metabolite, menthol-glucuronide, was observed in the urine of smokers of either menthol or nonmenthol cigarettes. The fact that a significant inverse correlation was observed between the levels of urinary menthol-glucuronide and NNAL-N-glucuronide, a major detoxification metabolite of the tobacco carcinogen, NNK, suggests that menthol may inhibit clearance of this important tobacco carcinogen.

Effects of Menthol Flavor Cigarettes or Total Urinary Menthol on Biomarkers of Nicotine and Carcinogenic Exposure and Behavioral Measures.

INTRODUCTION: The effects of either menthol flavor cigarettes or total urinary menthol on nicotine dependence, biomarkers of addictive and carcinogenic exposure, and behavioral measures may inform differences and similarities of these two approaches. METHODS: Stratified recruitment by cigarette (menthol flavor or regular) and race (African American and white) yielded a balanced sample of 136 adult smokers in a 36-hour inpatient protocol. Exposure measures assessed during 24-hour data collection included urinary menthol, total NNAL [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol], 10 polycyclic aromatic hydrocarbon metabolites, baseline plasma cotinine, plasma nicotine pre- and post-smoking, exhaled carbon monoxide pre- and post-smoking, and cigarette puff volumes. The latter three were measured at four specified timepoints throughout the day. RESULTS: There were no significant differences between menthol flavor and regular cigarette smokers in measures of nicotine dependence, biomarkers of addictive and carcinogenic exposures, or behavioral measures. Significant race × cigarette type interaction effects were found for two biomarkers: plasma nicotine and 3-hydroxyphenanthrene. Total urinary menthol was significantly associated with higher levels of nearly all dependent variables including puff volume, exhaled carbon monoxide, plasma nicotine and cotinine, NNAL, and polycyclic aromatic hydrocarbons. The significant effects of total urinary menthol were sustained after adjusting for menthol flavor and regular cigarette type and other covariates (eg, number of cigarettes per day, baseline cotinine, and baseline nicotine). CONCLUSIONS: Urinary menthol is an independent predictive biomarker for nicotine dependence, addictive and carcinogenic exposure, and behaviors. IMPLICATIONS: Comparison of the effects of menthol flavor and total urinary menthol on nicotine dependence, biomarkers of addictive and carcinogenic exposure, and behavioral measures emphasizes the important significant contribution of total urinary menthol concentrations in contrast to no significant associations by dichotomous cigarette type with these biomarkers.

Menthol, a unique urinary volatile compound, is associated with chronic inflammation in interstitial cystitis.

Chronic inflammation is a potential systemic risk factor for many bladder dysfunctions, including interstitial cystitis (IC). However, the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers remains unknown. In this study, we identified odor compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (SPME-GC-TOF-MS) profiling and bioinformatics, we found that levels of urinary volatile metabolites, such as menthol, were significantly reduced in IC patients, compared to healthy controls. In an attempt to understand the mechanistic meaning of our volatile metabolites data and the role of menthol in the immune system, we performed two independent experiments: (a) cytokine profiling, and (b) DNA microarray. Our findings suggest that lipopolysaccharide (LPS)-stimulated inflammatory events, such as the production and secretion of inflammatory cytokines (e.g., TNF-α, IL-6, and IL-1ß) and the activation of NF-κB and associated proteins within a large signaling network (e.g., Akt, TLR1, TNFAIP3, and NF-κB), are suppressed by the presence of menthol. These findings broaden our knowledge on the role of urinary menthol in suppressing inflammatory events and provide potential new strategies for alleviating both the odor and inflammation associated with IC.

Quantitative analysis of menthol in human urine using solid phase microextraction and stable isotope dilution gas chromatography-mass spectrometry.

To accurately measure menthol levels in human urine, we developed a method using gas chromatography/electron ionization mass spectrometry with menthol-d4 stable isotope internal standardization. We used solid phase microextraction (SPME) headspace sampling for collection, preconcentration and automation. Conjugated forms of menthol were released using ß-glucuronidase/sulfatase to allow for measuring total menthol. Additionally, we processed the specimens without using ß-glucuronidase/sulfatase to quantify the levels of unconjugated (free) menthol in urine. This method was developed to verify mentholated cigarette smoking status to study the influence of menthol on smoking behaviour and exposure. This objective was accomplished with this method, which has no carryover or memory from the SPME fiber assembly, a method detection limit of 0.0017µg/mL, a broad linear range of 0.002-0.5µg/mL for free menthol and 0.01-10µg/mL for total menthol, a 7.6% precision and 88.5% accuracy, and an analysis runtime of 17min. We applied this method in analysis of urine specimens collected from cigarette smokers who smoke either mentholated or non-mentholated cigarettes. Among these smokers, the average total urinary menthol levels was three-fold higher (p<0.001) among mentholated cigarette smokers compared with non-mentholated cigarette smokers.

Determination of Menthol in Plasma and Urine by Gas Chromatography/Mass Spectrometry (GC/MS).

Menthol, a monoterpene, is a principal component of peppermint oil and is used extensively in consumer products as a flavoring aid. It is also commonly used medicinally as a topical skin coolant; to treat inflammation of the mucous membranes, digestive problems, and irritable bowel syndrome (IBS); and in preventing spasms during endoscopy and for its spasmolytic effect on the smooth muscle of the gastrointestinal tract. Menthol has a half life of 3-6 h and is rapidly metabolized to menthol glucuronide which is detectable in urine and serum following menthol use. We describe a method for the determination of total menthol in human plasma and urine using liquid/liquid extraction, gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode and menthol-d4 as the internal standard. Controls are prepared with menthol glucuronide and all samples undergo enzymatic hydrolysis for the quantification of total menthol. The method has a linear range of 5-1000 ng/mL, and coefficient of variation <10%.

Urine menthol as a biomarker of mentholated cigarette smoking.

OBJECTIVES: Menthol cigarettes are smoked by 27% of U.S. smokers, and there are concerns that menthol might enhance toxicity of cigarette smoking by increasing systemic absorption of smoke toxins. We measured urine menthol concentrations in relation to biomarkers of exposure to nicotine and tobacco carcinogens. METHODS: Concentrations of menthol glucuronide (using a novel analytical method), nicotine plus metabolites (nicotine equivalents, NE), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and polycyclic aromatic hydrocarbon (PAH) metabolites were measured in the urine of 60 menthol and 67 regular cigarette smokers. RESULTS: Urine menthol was measurable in 82% of menthol and 54% in regular cigarette smokers. Among menthol smokers, urine menthol was highly correlated with NE, NNAL, and PAHs. In a multiple regression model NE but not menthol was significantly associated with NNAL and PAHs. CONCLUSIONS: Urine menthol concentration is a novel biomarker of exposure in menthol cigarette smokers, and is highly correlated with exposure to nicotine and carcinogens. Menthol is not independently associated with carcinogen exposure when nicotine intake is considered.

[Effect of menthol cigarette on rats for metabonomics by liquid chromatography-mass spectrometry].

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the metabonomics study of menthol cigarette effect on rats. Urines from three groups of rats were analyzed, including control rats, rats treated with menthol cigarette and rats treated with normal cigarette, and the data were processed by the method of principal component analysis (PCA). The PCA score plot showed that the metabolic difference between the rats treated with menthol cigarette and the control rats was smaller than that between the rats treated with normal cigarette and the control rats. Based on the PCA score plot, eight important metabolites, for example, kynurenic acid, were found and identified. Their relative concentration changes among the three groups further indicated that menthol cigarette maybe decrease the metabolic effect on rats.

Determination of menthol in plasma and urine of rats and humans by headspace solid phase microextraction and gas chromatography--mass spectrometry.

A method for the determination of menthol and menthol glucuronide (M-G) after enzymatic hydrolysis in plasma and urine of rats and humans was developed using headspace solid phase microextraction and gas chromatography-mass spectrometry in the selected ion monitoring mode (HS-SPME/GC-MS). The assay linearity for plasma ranged from 5 to 1000 ng/ml. The limit of quantification (LOQ) in plasma was 5 ng/ml. The intra- and inter-day precision for menthol and M-G were < or = 18.1% R.S.D. at the LOQ and < or = 4.0% at higher concentrations. Menthol and M-G were determined in rat and human plasma and urine after administration of menthol.

Metabolism of (R)-(+)-pulegone in F344 rats.

(R)-(+)-Pulegone, a monoterpene ketone, is a major component of pennyroyal oil. Ingestion of high doses of pennyroyal oil has caused severe toxicity and occasionally death. Studies have shown that metabolites of pulegone were responsible for the toxicity. Previous metabolism studies have used high, near lethal doses and isolation and analysis techniques that may cause degradation of some metabolites. To clarify these issues and further explore the metabolic pathways, a study of (14)C-labeled pulegone in F344 rats at doses from 0.8 to 80 mg/kg has been conducted. High-pressure liquid chromatography (HPLC) analysis of the collected urine showed the metabolism of pulegone to be extensive and complex. Fourteen metabolites were isolated by HPLC and characterized by NMR, UV, and mass spectroscopy. The results demonstrated that pulegone was metabolized by three major pathways: 1) hydroxylation to give monohydroxylated pulegones, followed by glucuronidation or further metabolism; 2) reduction of the carbon-carbon double bond to give diastereomeric menthone/isomenthone, followed by hydroxylation and glucuronidation; and 3) Michael addition of glutathione to pulegone, followed by further metabolism to give diastereomeric 8-(N-acetylcystein-S-yl)menthone/isomenthone. This 1,4-addition not only took place in vivo but also in vitro under catalysis of glutathione S-transferase or mild base. Several hydroxylated products of the two mercapturic acids were also observed. Contrary to the previous study, all but one of the major metabolites characterized in the present study are phase II metabolites, and most of the metabolites in free forms are structurally different from those previously identified phase I metabolites.

GC profiles of volatile constituents from human urine obtained by closed loop stripping, purge and trap technique and simultaneous stem distillation-extraction.

Different techniques like "closed loop stripping" [CLSA], "purge and trap" [PTI], and continuous steam distillation extraction [SDE] were used to establish GC profiles of major histocompatibility complex-associated volatile constituents of human urine and statistically evaluated for reliability. Of the three methods investigated, PTI appeared to be superior for the detection of very volatile substances, whereas SDE was the most efficient one with respect to yield. A number of short to medium-chain ketones, 4-hydroxy-3-methoxy-styrene, menthol and nicotine were identified in preliminary analyses.

Determination of menthol and menthol glucuronide in human urine by gas chromatography using an enzyme-sensitive internal standard and flame ionization detection.

The rate of peppermint oil absorption and excretion, following peroral administration, was determined by measuring urinary levels of menthol glucuronide. Menthol, a major component of peppermint oil, was liberated from its glucuronide metabolite by treating raw urine with beta-D-glucuronidase (Patella vulgata). Phenyl glucuronide was employed as an enzyme-sensitive internal standard. Menthol and phenol were recovered by ethyl acetate extraction and quantitated by capillary gas chromatography using flame ionization detection. Standard curves were linear between 25 and 250 micrograms/ml with a detection limit (signal-to-noise ratio = 2) of 0.25 microgram/ml. Assay precision was shown to be +/- 1.2% relative standard deviation.