Identification of the Metabolites in Rat Urine after Oral Administration and Elucidation of the Metabolic Process of Naozhenning Granule Using LC-MS.

Naozhenning (NZN) granule, a Chinese herbal formula, is widely used to treat craniocerebral trauma and promote functional recovery. In our previous study, the chemical components, as well as the serum metabolites in the male Sprague-Dawley rats of the NZN granule after oral administration were characterized. In this study, the urine metabolites in the male Sprague-Dawley rats were further investigated by ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry. In order to identify the urine metabolites comprehensively, three sample preparation methods were used, including solid-phase extraction, protein precipitation method and solvent partition. Based on the accurate molecular weight and the fragmentation information from the MS spectra, a total of 76 urine metabolites were identified, which including 17 prototypes and 59 metabolites. The results showed that the detected urine metabolites were different for the different pretreatment methods, as some metabolites could only be detected in the particular pretreatment method. In addition, the metabolic processes of the components from NZN granule to the serum and urine were also elucidated and discussed. The results will provide useful information for further studying the relationship between the chemical components and pharmacological activity of NZN granule.

Biomonitoring data on young adults from the Environmental Specimen Bank suggest a decrease in the exposure to the fragrance chemical 7-hydroxycitronellal in Germany from 2000 to 2018.

7-Hydroxy-3,7-dimethyl-1-octanal, also known as 7-hydroxycitronellal (7-HC, CAS No. 107-75-5) is a synthetic fragrance widely used in cosmetic and hygiene products. Because of its wide spread use and its known sensitizing properties, 7-HC was selected as one of 50 chemicals within the frame of the cooperation project between the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety (BMU) and the German Chemical Industry Association (VCI) to develop a suitable human biomonitoring (HBM) method in order to assess the exposure of the general population in Germany. Within this scope, the recently published analytical method for urinary 7-hydroxycitronellylic acid (7-HCA), the major metabolite of 7-HC, was applied to 329 24h-urine samples of young adults (20 to 29 years) collected between 2000 and 2018 and stored in the Environmental Specimen Bank (ESB). The widespread exposure to 7-HC as already observed in a pilot study with 40 volunteers could be confirmed with quantifiable concentrations of 7-HCA in all 329 study samples (mean: 14.9 ng/mL; median: 8.1 ng/mL). A significant, chronological decrease in 7-HCA levels was found for the monitored years (2000, 2004, 2008, 2012, 2015, 2018). The most pronounced decline occurred between 2000 and 2004 (means: 34.37 versus 23.31, medians: 20.97 versus 12.49 µg/24h; p < 0.01). On average, females exhibited higher levels of urinary 7-HCA compared to males (29.34 versus 17.21 µg/24h, p < 0.05). Based on the urinary 7-HCA excretion, the daily intake (DI) of 7-HC normalized for body weight (bw) was estimated. Over all sampling years, average DI in females was significantly higher compared to males (0.99 versus 0.46 µg/kg bw/d). Assuming dermal exposure as the main route of 7-HC intake, the mean DIs correspond to <0.1% of the derived no effect level (DNEL) of 1,100 µg/kg bw/d defined by the European Chemical Agency (ECHA). The presented results for the exposure to the widely used fragrance 7-HC in Germany can be substantiated by applying the described methodology to the representative cohort of the launched German Environmental Survey in adults (GerES VI).

Simultaneous determination of seven effective components of Tripterygium glycosides in human biological matrices by ultra performance liquid chromatography-triple quadrupole mass spectrometry.

This paper developed a novel, sensitive, and selective ultra-performance liquid chromatography-triple quad mass spectrometry method to simultaneously determine seven effective constituents (triptolide, triptophenolide, celastrol, wilforgine, wilforine, wilfordine and wilfortrine) of Tripterygium glycosides (GTW) in human serum and urine. The chromatographic separation was performed on the C18 column using an ammonium acetate buffer solution-acetonitrile (both containing 0.1% formic acid) in a gradient program with a flow rate of 0.3 mL/min. Monitoring reaction mode was applied to target compounds quantitative analysis in the positive electrospray ionization (ESI) mode. The analysis process took 11 min in total. This method was fully validated with a linear range of 1-200 ng/mL for triptolide, 0.4-80 ng/mL for celastrol, 0.1-20 ng/mL for triptophenolide, wilforgine, wilforine, wilfordine, and wilfortrine. The intra-day and inter-day accuracy and precision of the target compounds all met the 15% criterion in both serum and urine. Extraction recovery, matrix effect, and dilution integrity were also validated. The short-term and long-term stability results indicated that all the constituents were stable in human serum and urine under the investigated storage conditions. 10 patients' specimens were collected and analyzed. Most of the compounds exhibited the tendency of higher concentration in urine than that in serum. The concentration that was detected in the serum and in the urine of alkaloids showed a positive-correlation property. This is the first time that triptophenolide was quantified in human bio-matrices. The method is feasible for multi-components therapeutic monitoring or pharmacokinetics study in clinical pharmaceutical research of Tripterygium glycosides.

A validated UPLC-MS/MS method for biomonitoring the exposure to the fragrance 7-hydroxycitronellal.

7-Hydroxycitronellal is a synthetic fragrance (CAS No. 107-75-5) which is used commonly in cosmetics, washing- and cleaning agents and as flavoring in foods. Due to its broad application in various fields, 7-hydroxycitronellal was selected for the development of a biomonitoring method for the quantitative exposure assessment within the frame of the cooperation project of the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI). For this purpose, an ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) based method was developed for the determination of potential biomarkers of 7-hydroxycitronellal (7-HC) in human urine samples. 7-Hydroxycitronellylic acid (7-HCA) turned out to be the quantitatively most important metabolite of 7-HC in human urine, occurring in 1000 times higher amounts than 7-hydroxycitronellol (7-HCO) or other potential metabolites. Therefore, an analytical method for 7-HCA was developed using stable isotope-labeled 7-HCA as internal standard (IS). The method includes a cleavage step of possible metabolite conjugates with an enzyme mix of ß-glucuronidase and arylsulfatase. Subsequent sample cleanup was performed by liquid-liquid extraction (LLE) with dichloromethane. The method was calibrated by calculating the linear regression between the analyte/IS ratio and the nominal 7-HCA concentrations in water. The method was validated according to approved standard guidelines and proved to be robust, reliable and sensitive for the human biomonitoring of 7-HC. The method was applied to urine samples of 40 adult volunteers from the general population. 7-HCA was quantifiable in urine of all subjects. Thus the developed method proved to be suitable for assessing the background exposure to 7-HC in the general population.

Monocyclic and bicyclic monoterpenes in air of German daycare centers and human biomonitoring in visiting children, the LUPE 3 study.

To investigate the assumed association between indoor air pollution with monoterpenes (MTps) and the internal MTp exposure of occupants, a comparative study was performed in daycare centers in two federal states of Germany. Three well-known monoterpenoid air pollutants, viz. α-pinene (αPN), Δ(3)-carene (CRN), and R-limonene (LMN), were measured in indoor air in 45 daycare centers. Additionally, urine samples of 222 children visiting these facilities were collected in the evening after a full-day stay. Altogether 11 MTp metabolites were analyzed in the urine samples using a novel highly sensitive and selective gas chromatographic-tandem-mass spectrometric procedure. The medians (95th percentiles) of the MTp levels in indoor air were 9.1 µg m(-3) (94 µg m(-3)) for LMN, 2.6 µg m(-3) (13 µg m(-3)) for αPN, and <1.0 µg m(-3) (3.2 µg m(-3)) for CRN. None of the day care centers exceeded the German health precaution or hazard guide value. In spite of the low MTp air exposure, the urine analyses revealed an exposure to the three monoterpenes in almost all children. The median levels of MTp metabolites in urine were 0.11 mg L(-1) for LMN-8,9-OH, 0.10 mg L(-1) for LMN-1,2-OH, 49 µg L(-1) for PA, 2.9 µg L(-1) for POH, 5.2 µg L(-1) for tCAR, and 4.1 µg L(-1) for cCAR (LMN metabolites), 7.2 µg L(-1) for MYR, 19 µg L(-1) for tVER, and 19 µg L(-1) for cVER (αPN metabolites), as well as 8.2 µg L(-1) for CRN-10-COOH (CRN metabolite). Statistically significant and strong correlations among the urinary metabolites of each MTp were found. Moreover, statistical associations between LMN metabolites and the LMN indoor air levels were revealed. However, the weakness of the associations indicates a considerable impact of other MTp sources, e.g. diet and consumer products, on the internal exposure.

Pharmacokinetics of reduced iso-α-acids in volunteers following clear bottled beer consumption.

Reduced iso-α-acids (reduced IAA) consisting of the rho-, tetrahydro- and hexahydro-IAA groups (RIAA, TIAA and HIAA, respectively) are ingredient congeners specific to beer and generally found in clear and also occasionally green bottled beer. Concentrations of reduced IAA were determined in the blood and urine of five volunteers over 6h following the consumption of small volumes of beer containing each of the reduced IAA. The reduced IAA were absorbed and bioavailable with peak concentrations at 0.5h followed by a drop of generally fivefold by 2h. Preliminary pharmacokinetics of these compounds in humans shows relatively small inter-individual differences and an estimated short half-life varying between ∼38 and 46min for the three groups. Comparison of RIAA analyte ratios within the group indicate that some analytes eliminate relatively faster than others and the formation of metabolite products was observed. Preliminary urine analysis showed only unmodified RIAA analytes were detectable throughout 6h and suggests extensive phase I metabolism of TIAA and HIAA analytes. In authentic forensic casework where clear or green bottled beers are consumed, the identification of reduced IAA groups may provide a novel method to target ingredient congeners consistent with beer ingestion and suggest the type of beer consumed.

Pharmacokinetics of iso-α-acids in volunteers following the consumption of beer.

Hop-derived iso-α-acid (IAA) ingredient congeners are specific to beer. Concentrations of IAAs were determined in blood of five volunteers over 6 h following the consumption of small volumes of beer containing relatively high (Pale Ale beer) or low (wheat beer) concentrations of IAAs. IAAs were quickly absorbed with peak trans-IAA concentrations at 0.5 h followed by a drop of generally 10-fold at 2 h and low or not detectable trans-IAA levels at 6 h. However, the qualitative monitoring showed that the cis-IAAs were detected at all time-points. Preliminary pharmacokinetics of these compounds in humans shows relatively small interindividual differences and an estimated short half-life of ∼30 min. Comparison of 0.5 and 2 h blood specimens demonstrated that the trans isomers were eliminated faster than the cis counterparts. Preliminary urine analysis showed only unmodified 'co' analytes detectable throughout the 6 h. In authentic forensic casework where typically large amounts of conventionally hopped beer are consumed, this approach may provide a novel method to target ingredient congeners consistent with beer ingestion.

Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation-tandem mass spectrometry.

A gas chromatographic-positive chemical ionisation-tandem mass spectrometric (GC-PCI-MS/MS) method for the simultaneous determination of 10 oxidative metabolites of the monoterpenoid hydrocarbons α-pinene, (R)-limonene, and Δ(3)-carene ((+)-3-carene) in human urine was developed and tested for the monoterpene biomonitoring of the general population (n=36). The method involves enzymatic cleavage of the glucuronides followed by solid-supported liquid-liquid extraction and derivatisation using a two-step reaction with N,O-bis(trimethylsilyl)-trifluoroacetamide and N-(trimethylsilyl)imidazole. The method proved to be both sensitive and reliable with detection limits ranging from 0.1 to 0.3 µg L(-1). In contrast to the frequent and distinct quantities of (1S,2S,4R)-limonene-1,2-diol, the (1R,2R,4R)-stereoisomer could not be detected. The expected metabolite of (+)-3-carene, 3-caren-10-ol was not detected in any of the samples. All other metabolites were detected in almost all urine samples. The procedure enables for the first time the analysis of trace levels of a broad spectrum of mono- and bicyclic monoterpenoid metabolites (alcohols, diols, and carboxylic acids) in human urine. This analytical procedure is a powerful tool for population studies as well as for the discovery of human metabolism and toxicokinetics of monoterpenes.

No evidence for mevalonate shunting in moderately affected children with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is caused by a genetic deficiency in 7-dehydrocholesterol (7-DHC) reductase (EC 1.3.1.21), the last enzyme of the cholesterol synthetic pathway. In SLOS, plasma cholesterol concentration is reduced and immediate precursor concentration (7-DHC) is elevated. Surprisingly, total sterol synthesis is reduced but HMG-CoA reductase activity, a rate-limiting enzyme in cholesterol synthesis is unaltered as judged by normal urinary excretion of mevalonic acid (MVA) (Pappu et al. J Lipid Res 43:1661-1669, 2002). These findings raise the possibility of increased diversion of MVA into the MVA shunt pathway away from sterol synthesis, by activation of the shunt pathway enzymes. To test this hypothesis, we measured the urinary excretion of 3-methylglutaconic acid (U-3MGC), a by-product of the shunt pathway, in 19 mildly to moderately severely affected SLOS subjects (ten males, nine females) receiving either a cholesterol-free or a high cholesterol diet, and in 20 age- and sex-matched controls. U-3MGC was similar in SLOS and controls, and was unaffected by dietary cholesterol intake. Further, no change in U-3MGC was observed in a subset of SLOS subjects (n = 9) receiving simvastatin. In contrast, U-MVA was reduced by cholesterol supplementation (~54%, p < 0.05) and by simvastatin (~50%, p < 0.04). There was no correlation between U-3MGC and either plasma sterol concentrations, urinary isoprenoids, or the subjects' clinical severity score. However U-3MGC was inversely correlated with age (p < 0.04) and body weight (p < 0.02), and higher in females than in males (~65%, p < 0.025). The data show that DHCR7 deficiency does not result in 3MGC accumulation in SLOS and suggest that the MVA shunt pathway is not activated in patients with the condition.

Environmental tobacco smoke exposure among casino dealers.

OBJECTIVE: This study quantified casino dealers' occupational exposure to environmental tobacco smoke (ETS). METHODS: We measured casino dealers' exposure to ETS components by analyzing full-shift air and preshift and postshift urine samples. RESULTS: Casino dealers were exposed to nicotine, 4-vinyl pyridine, benzene, toluene, naphthalene, formaldehyde, acetaldehyde, solanesol, and respirable suspended particulates. Levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine increased significantly during an 8-hour work shift both with and without adjustment for creatinine clearance. Creatinine-unadjusted cotinine significantly increased during the 8-hour shift, but creatinine-adjusted cotinine did not increase significantly. CONCLUSIONS: Casino dealers at the three casinos were exposed to airborne ETS components and absorbed an ETS-specific component into their bodies, as demonstrated by detectable levels of urinary NNAL. The casinos should ban smoking on their premises and offer employee smoking cessation programs.

Metabolic profiling of GuanXin II prescription based on metabolic fingerprinting and chemical analysis.

A sensitive LC/MS method was established to investigate the in vivo metabolism of GuanXin II prescription, a five-component Chinese herbal medicine formulation. Rat plasma, bile, urine, and feces were collected and analyzed following oral administration of the water decoction. A total of 50 compounds were identified, including 17 prototypes and 33 metabolites underwent methylation, oxidation, hydrolysis, sulfate conjugation, glucuronide conjugation, and glutathion conjugation. In addition, the component herb of the formulation from which the metabolites were derived was also identified. Among the five component herbs, Rhizoma Chuanxiong, Flos Carthami, and Lignum Dalbergiae Odoriferae were actively metabolized, contributing 26 metabolites and 2 prototypes, while Radix Salviae Miltiorrhizae and Radix Paeoniae Rubra underwent less biotransformation, yielding 7 metabolites and 15 prototypes. This is the first study on the metabolic profile of GuanXin II prescription. The results could be valuable to elucidate the material basis of this formulated Chinese medicine.

Single-drop liquid-phase microextraction for the determination of hypericin, pseudohypericin and hyperforin in biological fluids by high performance liquid chromatography.

The analysis of hypericin, pseudohypericin (collectively called in this study hypericins) and hyperforin in biological fluids is reported using single-drop liquid-phase microextraction in conjunction with HPLC-UV-fluorescence detection. A new option for analysis of the active principle constituents in biological samples is proposed, reducing the steps required prior to analysis. There are several parameters which determine the mass transfer such as the extraction solvent, drop and sample volumes, extraction time and temperature, pH and ionic strength, stirring rate and depth of needle tip in the bulk solution. These parameters were chosen to optimize the performance in the current study. The method was validated with respect to precision, accuracy and specificity. The intra-day precision values were below 2.3% for the high concentration level of control samples and 6.2% for the low level. The respective inter-day precision values were calculated to be below 4.4 and 7.1%, respectively, for the two concentration levels. Accuracy of the method, calculated as relative error, ranged from -2.6 to 7.0%. It was demonstrated that as long as the extraction procedure is consistently applied, quantitative analysis is performed accurately and reproducibly in human urine and plasma samples. Limits of quantitation (LOQs) in urine were calculated to be 3, 6 and 12 ng/ml for pseudohypericin, hypericin and hyperforin, respectively. Slightly higher limits were measured in plasma, i.e. 5, 12 and 20 ng/ml, for the respective analytes.

Metabolism and mode of action of cis- and trans-3-pinanones (the active ingredients of hyssop oil).

1. Hyssop oil is an important food additive and herbal medicine and the principal active ingredients are (-)-cis- and (-)-trans-3-pinanones. No information is available on their metabolism or specific mode of action. 2. The metabolites of cis- and trans-3-pinanones were examined from mouse and human liver microsomes and human recombinant P4503A4 with NADPH and on administration to mouse by gas chromatography/chemical ionization mass spectrometry comparison with standards from synthesis. 3. The major metabolite of cis-3-pinanone in each P450 system and in brain of the i.p.-treated mouse in quantitative studies was 2-hydroxy-cis-3-pinanone, and two minor metabolites were hydroxypinanones other than 2-hydroxy-trans-3-pinanone and 4S-hydroxy-cis-3-pinanone. The urine from oral cis-3-pinanone treatment examined on a qualitative basis contained conjugates of metabolites observed in the microsomal systems plus 2,10-dehydro-3-pinanone. 4. Trans-3-pinanone was metabolized more slowly than the cis-isomer in each system to give hydroxy derivatives different than those derived from cis-3-pinanone. 5. Cis- and trans-3-pinanones and hyssop oil act as gamma-aminobutyric acid type A (GABAA) receptor antagonists based on inhibition of 4'-ethynyl-4-n-[2,3-(3)H(2)]propylbicycloorthobenzoate ([(3)H]EBOB) binding in mouse brain membranes (IC(50) of 35-64 microM) and supported by tonic/clonic convulsions in mouse (i.p. LD(50) 175 to >250 mg kg(-1)) alleviated by diazepam. The cis-3-pinanone metabolites 2-hydroxy-cis-3-pinanone and 2,10-dehydro-3-pinanone exhibit reduced toxicity and potency for inhibition of [(3)H]EBOB binding.

Monoterpene and wood dust exposures: work-related symptoms among Finnish sawmill workers.

BACKGROUND: Monoterpenes and wood dust are released into the work environment during sawing of fresh wood. Symptoms related to exposure to monoterpenes and wood dust include irritation of the eyes, mucous membrane, and skin. METHODS: We studied 22 sawhouse workers who process pine and spruce in 1997-99. Exposure to monoterpenes was assessed by determining monoterpenes in air and verbenols in urine by gas chromatography using flame ionization detection. Wood dust was determined gravimetrically. A questionnaire was used to evaluate work-related subjective symptoms. RESULTS: Exposures to monoterpenes (geometric mean, GM) among sawhouse workers were 61-138 mg/m(3) and 2.0-13 mg/m(3) during processing of pine and spruce, respectively. Urinary verbenol correlated well with worker exposure to the alpha-pinene fraction of monoterpenes. The inhalable dust concentration in the breathing zone was 0.5- 2.2 mg/m(3) during pine processing and 0.4-1.9 mg/m(3) during spruce processing. The prevalence of symptoms, in the eyes or respiratory tract, was high during both seasons and in connection with either tree species. CONCLUSIONS: The highest monoterpene concentration (GM), in the breathing zone, measured during processing of pine, was less than one-fourth of the Finnish occupational exposure limit (OEL, 570 mg/m(3)). Verbenol concentrations in postshift urine samples reflected accurately the exposure to monoterpenes. The concentrations of inhalable dust (GM) were less than one-half the Finnish OEL (5 mg/m(3)). No significant differences in dust exposure were observed among tree species processed. Work-related symptoms appeared to correlate with monoterpene exposure during processing of pine and with wood dust exposure during processing of spruce.

Phase I pharmacokinetic trial of perillyl alcohol (NSC 641066) in patients with refractory solid malignancies.

Perillyl alcohol (POH) is a monoterpene with anticarcinogenic and antitumor activity in murine tumor models. Putative mechanisms of action include activation of the transforming growth factor beta pathway and/or inhibition of p21ras signaling, leading to differentiation or apoptosis. In this Phase I trial, 17 patients took POH p.o. three times daily for 14 days of each 28-day cycle. The starting dose of POH was 1600 mg/m2/dose, with escalations to 2100 and 2800 mg/m2/dose in subsequent cohorts. Chronic nausea and fatigue were dose-limiting toxic effects at 2800 mg/m2. Grade 1-2 hypokalemia was common at 2100 and 2800 mg/m2. Although POH could not be detected in plasma, two of its metabolites, dihydroperillic acid (DHPA) and perillic acid (PA), were measured in plasma and urine on days 1 and 15 after the first and last doses of POH, respectively. Both area under the concentration versus time curve and peak plasma concentration (Cmax) values increased with dose and exhibited high intersubject variability. Day 15 DHPA Cmax values ranged from a mean +/- SD of 22.6+/-12 microM at 1600 mg/m2/dose to 42.4+/-15.24 microM at 2800 mg/m2/dose. Corresponding mean +/- SD Cmax values for PA were 433.2+/-245.8 and 774.1+/-439.6 microM. One patient treated at the 2800 mg/m2/dose had markedly prolonged plasma levels of both PA and DHPA and developed grade 3 mucositis. POH treatment did not consistently alter the expression of p21ras, rap1, or rhoA in peripheral blood mononuclear cells obtained from patients treated at the highest dose level. The metabolites PA and DHPA did not change expression or isoprenylation of p21ras in MCF-7 breast or DU145 prostate carcinoma cells at concentrations that exceeded those achieved in patient plasma after POH treatment. We conclude that POH at 1600-2100 mg/m2 p.o. three times daily is well tolerated on a 14-day on/14-day off dosing schedule. Inhibition of p21ras function in humans is not likely to occur after POH administration at safe doses of the present oral formulation.

Hepatoprotective effect of C-phycocyanin: protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicty in rats.

Effect of C-phycocyanin (from Spirulina platensis) pretreatment on carbontetrachloride and R-(+)-pulegone-induced hepatotoxicity in rats was studied. Intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of phycocyanin to rats, one or three hours prior to R-(+)-pulegone (250 mg/kg) or carbontetrachloride (0.6 ml/kg) challenge, significantly reduced the hepatotoxicity caused by these chemicals. For instance, serum glutamate pyruvate transaminase (SGPT) activity was almost equal to control values. The losses of microsomal cytochrome P450, glucose-6-phosphatase and aminopyrine-N-demethylase were significantly reduced, suggesting that phycocyanin provides protection to liver enzymes. It was noticed that the level of menthofuran, the proximate toxin of R-(+)-pulegone was nearly 70% more in the urine samples collected from rats treated with R-(+)-pulegone alone than rats treated with the combination of phycocyanin and R-(+)-pulegone. The possible mechanism involved in the hepatoprotection is discussed.

Terpene exposure and respiratory effects among workers in Swedish joinery shops.

OBJECTIVES: Exposure to monoterpenes (alpha-pinene, beta-pinene and delta 3-carene) in joinery shops was studied in Sweden during the processing of Scot's pine, and the acute respiratory effects among the employees were evaluated. METHODS: A cross-sectional study of 38 workers was carried out in 4 joinery shops. The investigation included personal air sampling of monoterpenes, biological monitoring of metabolites of alpha-pinene in the workers' urine, interviews following a standardized questionnaire, and dynamic spirometry. RESULTS: The personal exposure to monoterpenes in the joinery shops was 10-214 mg/m3. The correlation (correlation coefficient = 0.69) between exposure to alpha-pinene and verbenols (metabolites from alpha-pinene) in urine was relatively good. No acute effects on forced vital capacity or forced expiratory volume during 1 s were detected. The workers had significantly reduced preshift lung function values when compared with the values of a local reference group, even when smokers and ex-smokers were excluded. CONCLUSIONS: Personal exposure to the monoterpenes alpha-pinene, and delta 3-carene in joinery shops may exceed the present Swedish occupational exposure limit of 150 mg/m3 during the winter season when workroom air is commonly recirculated. The determination of metabolites of alpha-pinene (verbenols) in urine can be used as an index of exposure to fumes released during wood-treating processes. The results from the lung function tests indicate chronic rather than acute reactions in the airways. The fact that there were no major changes in lung function over a workshift indicates chronic reaction in the airways.

Identification and characterization of limonene metabolites in patients with advanced cancer by liquid chromatography/mass spectrometry.

Limonene is a farnesyl transferase inhibitor that has shown antitumor properties. The drug had been given orally to cancer patients. Plasma and urine samples collected from the patients were examined by reversed-phase HPLC-atmospheric pressure chemical ionization and electrospray ionization MS. The drug underwent rapid conversion to hydroxylated and carboxylated derivatives. Characterization and structural elucidation of the metabolites were achieved by LC/MS and NMR. Five major metabolites were detected in the plasma extracts, namely limonene-1,2-diol, limonene-8,9-diol, perillic acid, an isomer of perillic acid, and dihydroperillic acid. Urinary metabolites comprised the glucuronides of the two isomers of perillic acid, dihydroperillic acid, limonene-8,9-diol, and a monohydroxylated limonene.

Gas chromatographic-mass spectrometric identification of metabolites from alpha-pinene in human urine after occupational exposure to sawing fumes.

Three metabolites from alpha-pinene (two diols and an alcohol with an aldehyde group) have been identified in human urine after occupational exposure to sawing fumes from pine. Urine was enzymatically hydrolyzed, cleaned up on a C18 micro-column and the metabolites were identified by GC-MS using electron impact (70 eV) and chemical ionization with ammonia or isobutane as the reagent gas. Analysis of underivatized metabolites was performed using a capillary column with a semi-polar phase and trimethylsilylated derivatives were analyzed using a non-polar phase.

Metabolic fate of menthofuran in rats. Novel oxidative pathways.

Metabolic fate of menthofuran (II) in rats was investigated. Menthofuran (II) was administered orally (200 mg/kg of the body weight/day) to rats for 3 days. The following metabolites were isolated from the urine of these animals: p-cresol (VI), 5-methyl-2-cyclohexen-1-one (VII), 3-methylcyclohexanone (VIII), 3-methylcyclohexanol (IX), 4-hydroxy-4-methyl-2-cyclohexen-1-one (V), geranic acid (XI), neronic acid (XII), benzoic acid (XIII), and 2-[2'-keto-4'-methylcyclohexyl]propionic acid (X). Incubation of menthofuran (II) with phenobarbital-induced rat liver microsomes in the presence of NADPH and oxygen resulted in the formation of a metabolite tentatively identified as 2-Z-(2'-keto-4'-methylcyclohexylidene)propanal (III; alpha,beta-unsaturated-gamma-keto-aldehyde). The structure assigned was further supported by trapping this metabolite (III) as a cinnoline derivative. Phenobarbital-induced rat liver microsomes also converted 4-methyl-2-cyclohexenone (IV) to 4-hydroxy-4-methyl-2-cyclohexenone (V) and p-cresol (VI) in the presence of NADPH and oxygen. On the basis of both in vivo and in vitro studies, a possible mechanism for the formation of p-cresol from menthofuran has been proposed.