Curry-Odorants and Their Metabolites Transfer into Human Milk and Urine.

SCOPE: The excretion of dietary odorants into urine and milk is evaluated and the impact of possible influencing factors determined. Furthermore, the metabolic relevance of conjugates for the excretion into milk is investigated. METHODS AND RESULTS: Lactating mothers (n = 20) are given a standardized curry dish and donated one milk and urine sample each before and 1, 2, 3, 4.5, 6, and 8 h after the intervention. The concentrations of nine target odorants in these samples are determined. A significant transition is observed for linalool into milk, as well as for linalool, cuminaldehyde, cinnamaldehyde, and eugenol into urine. Maximum concentrations are reached within 1 h after the intervention in the case of milk and within 2-3 h in the case of urine. In addition, the impact of glucuronidase treatment on odorant concentrations is evaluated in a sample subset of twelve mothers. Linalool, eugenol, and vanillin concentrations increased 3-77-fold in milk samples after treatment with ß-glucuronidase. CONCLUSION: The transfer profiles of odorants into milk and urine differ qualitatively, quantitatively, and in temporal aspects. More substances are transferred into urine and the transfer needs a longer period compared with milk. Phase II metabolites are transferred into urine and milk.

Urinary Methyleugenol-deoxyadenosine Adduct as a Potential Biomarker of Methyleugenol Exposure in Rats.

Methyleugenol (ME), a natural ingredient of several herbs and spices used in the human diet, is hepatocarcinogenic in rodents. Following metabolic activation to the reactive carbocation intermediate, ME can bind covalently to DNA, which is directly associated with its carcinogenicity. In this work, a non-invasive approach to determine ME exposure was established by monitoring the urinary N6-(methylisoeugenol-3'-yl)-2'-deoxyadenosine (ME-dA) adduct. The developed method entails liquid-liquid extraction enrichment of urinary ME-dA, incorporation of deuterated ME-dA as an internal standard, and analysis by liquid chromatography coupled tandem mass spectrometry. Male rats (10-12 weeks, 180-200 g) were treated (p.o.) with ME, and ME-dA was excreted in urine in a dose- and time-dependent manner. The non-invasive approach enabled us to successfully determine exposure to ME-containing herbs and spices. These results suggest that ME-dA can potentially serve as an effective biomarker of ME exposure in rats. It is expected that the developed approach of detecting urinary ME-dA will facilitate the investigation of ME carcinogenesis.

Identification of glutathione and related cysteine conjugates derived from reactive metabolites of methyleugenol in rats.

Methyleugenol (ME), an alkenylbenzene compound, is a constituent of many foods and is used as flavoring agent in foodstuffs and as fragrance in cosmetics. It has been reported that exposure to ME can cause carcinogenicity, cytotoxicity, and genotoxicity. Metabolic activation is suggested to play an important role in ME-induced toxicities. Electrophilic metabolites of ME have been reported to covalently bind to proteins and nucleic acids. The objective of this study was to identify GSH and related cysteine conjugates derived from these reactive metabolites in vivo. Five biliary GSH (M1-M5) and four urinary cysteine conjugates (M6-M9) were detected in rats given ME. M1 and M2 were GSH conjugates derived from the epoxide of ME. M3, M4, and M5 were GSH conjugates possibly generated from the corresponding α,ß-unsaturated aldehyde, carbonium ion, and quinone methide, respectively. The structures of the GSH conjugates were verified by chemical synthesis. Cysteine conjugates M6, M7, M8, and M9 were found to correspond to the respective M1/M2, M3, M4, and M5. The data obtained from the present in vivo work facilitate the understanding of mechanism action of ME toxicities and may provide information suitable for use as biomarkers of exposure to ME.

In vivo and in vitro metabolism of aspirin eugenol ester in dog by liquid chromatography tandem mass spectrometry.

Aspirin eugenol ester (AEE) is a promising drug candidate for treatment of inflammation, pain and fever and prevention of cardiovascular diseases with fewer side effects than its precursor, aspirin. Investigation into its metabolic process in target animal species will help to illustrate its mechanism of action and to establish its residual mark compound to formulate its dosage. Six beagle dogs were orally given a dose of 20 mg kg(-1) of AEE and one dog was used to prepare blank liver microsomes. Their liver microsomes were prepared for in vitro study and their plasma and urine were collected for in vivo metabolic analysis using liquid chromatography tandem mass spectrometry. In this study we identified 10 metabolites, M1, M2, M3, M4, M5 in phase I and M6, M7, M8, M9, M10 in phase II. Based on the metabolites of AEE, the pathways of AEE metabolism in dog were demonstrated.

Analysis of anti-inflammatory dehydrodiisoeugenol and metabolites excreted in rat feces and urine using HPLC-UV.

Dehydrodiisoeugenol (DDIE) is a lignan in the fruit of Myristica fragrans. It can be converted into several metabolites in in vitro and in vivo metabolism. In this study, the excretion of DDIE in urine and feces was investigated after intravenous (i.v.) and intragastric (i.g.) administration to rats. DDIE and its metabolites (M-1 and M-2) were measured using HPLC. The amount of DDIE and its metabolites excreted was higher in feces than in urine, suggesting that DDIE and its metabolites are eliminated primarily in the feces. Significant differences in the excretion levels of DDIE and its metabolites were seen between i.v. and i.g. administration. Greater amounts of DDIE and its metabolites were excreted following i.v. administration, suggesting that DDIE can exert a longer period of anti-inflammatory activity following i.g. administration. The accuracy, precision, recovery and stability of the analytical method in this study were satisfactory for the measurement of DDIE and its metabolites in rat urine and feces. Observations made in this study will contribute to understanding of the absorption, distribution, metabolism and excretion pathway of DDIE and will aid decision-making regarding the best mode of DDIE administration during treatment to maximize its anti-inflammatory effects.

Metabolism of the lignan dehydrodiisoeugenol in rats.

Dehydrodiisoeugenol (DDIE), a major active lignan from the seed and aril of the fruit of Myristica fragrans Houtt., functions as a potential anti-inflammatory agent by inhibiting lipopolysaccharide-stimulated nuclear factor kappa B activation and cyclooxygenase-2 expression in macrophages. However, the metabolism of DDIE remains unknown. This report describes the metabolic fate of DDIE in liver microsomes, urine, and feces of rats treated with DDIE. DDIE metabolites were isolated by sequential column chromatography and high-performance liquid chromatography from liver microsomes incubations, urine, and feces. Nine metabolites ( M-1 to M-9), including 5 new metabolites, were determined spectroscopically using ultra-violet (UV), mass spectrometry (MS), nuclear magnetic resonance (NMR), and circular dichroism (CD). Analysis of the isolated metabolites showed that DDIE undergoes four major pathways of metabolism in the rat: oxidation (including hydroxylation, hydroformylation, and acetylation), demethylation, ring-opening, and dehydrogenation. In contrast to the metabolites from liver microsomes, the major metabolites In vivo were generated from DDIE by multiple metabolic reactions. Given these results, we describe a metabolic pathway for DDIE in the rat that gives insight into the metabolism of DDIE and the mechanism of DDIE bioactivity in humans.

Pharmacokinetics and anesthetic activity of eugenol in male Sprague-Dawley rats.

Eugenol, the principle chemical constituent of clove oil, has recently been evaluated for its anesthetic and analgesic properties in fish and amphibians. The objective of this study was to determine the pharmacokinetic (PK) and anesthetic activity of eugenol in rats. Male Sprague-Dawley rats received single i.v. doses of eugenol (0, 5, 10, 20, 40 and 60 mg/kg) and anesthetic level was evaluated with the withdrawal reflex. For the 20 mg/kg dose level, blood and urinary samples were collected over 1 h for the PK assessment. Plasma and blood concentrations of eugenol, as well as metabolite identification in urine, were determined using a novel dansyl chloride derivatization method with liquid chromatography mass spectrometry (LC/MS/MS). PK parameters were calculated using noncompartmental methods. Eugenol-induced loss of consciousness in a dose-dependent manner, with mean (+/-SEM) recovery in reflex time of 167 +/- 42 sec observed at the highest dose level. Mean systemic clearance (Cl) in plasma and blood were 157 and 204 mL/min/kg, respectively. Glucuronide and sulfate conjugates were identified in urine. Overall, eugenol produced a reversible, dose-dependent anesthesia in male Sprague-Dawley rats.

Disposition and metabolism of isoeugenol in the male Fischer 344 rat.

The primary objective of these studies was to determine the absorption, distribution, metabolism and excretion of isoeugenol following oral and intravenous administration to male Fischer-344 rats. Following a single oral dose of [14C]isoeugenol (156 mg/kg, 50 microCi/kg), greater than 85% of the administered dose was excreted in the urine predominantly as sulfate or glucuronide metabolites by 72 h. Approximately 10% was recovered in the feces, and less than 0.1% was recovered as CO(2) or expired organics. No parent isoeugenol was detected in the blood at any of the time points analyzed. Following iv administration (15.6 mg/kg, 100 microCi/kg), isoeugenol disappeared rapidly from the blood. The t(1/2) was 12 min and the Cl(s) was 1.9 l/min/kg. Excretion characteristics were similar to those of oral administration. The total amount of radioactivity remaining in selected tissues by 72 h was less than 0.25% of the dose following either oral or intravenous administration. Results of these studies show that isoeugenol is rapidly metabolized and is excreted predominantly in the urine as phase II conjugates of the parent compound.

Biological monitoring of environment exposure to safrole and the Taiwanese betel quid chewing.

A rapid and sensitive biological monitoring (BM) method for assessing exposure to the environmental carcinogen safrole has been developed. The method is an isocratic high-performance liquid chromatographic (HPLC) analysis of urinary dihydroxychavicol (DHAB) and eugenol, the urinary metabolites of safrole. Good linearity, precision, and accuracy were demonstrated. A recovery of 98.8 +/- 5.4% (SD, n = 3) was found for DHAB and 84.1 +/- 3.4% (n = 3) for eugenol. The quantitation limits of the method were 8 ng for DHAB and 10 ng for eugenol. The validity of the method was demonstrated by a linear dose-response relationship observed in rats given oral doses of safrole at 30, 75, and 150 mg/kg body weight. The method was also used to monitor the environmental exposure to the Taiwanese betel quid (TBQ) chewing, because TBQ used in Taiwan not only contains areca (betel) nut, slaked lime, and catechu but also Piper betle inflorescence or its leaves. Both of the latter have a high content of safrole. The feasibility of the method to monitor TBQ chewing was demonstrated by an analysis of 153 spot human urine samples. The results showed that the p value of the nonparametric group comparison was < 0.001 for DHAB and 0.832 for eugenol. The TBQ chewers also exhibited a significantly higher rate of urinary DHAB (but not eugenol) than the nonchewers with an odd ratio of 3.47 (95% CI, 1.61-7.51). However, when only the eugenol-positive subjects were taken into analysis, the ratio rose to 24.38 (95% CI, 3.00-197.90).

The metabolism of eugenol in man.

1. The metabolism of eugenol (4-hydroxy-3-methoxy-allylbenzene) was investigated in male and female healthy volunteers. It was rapidly absorbed and metabolized after oral administration and was almost completely excreted in the urine within 24 h. Unmetabolized eugenol excreted in urine amounted to less than 0.1% of the dose. 2. The urine contained conjugates of eugenol and of nine metabolites. The structures of these metabolites, elucidated using g.l.c.-mass spectrometry, and by comparison with synthetic reference compounds, were identified as: eugenol, 4-hydroxy-3-methoxyphenyl-propane, cis- and trans-isoeugenol, 3-(4-hydroxy-3-methoxyphenyl)-propylene-1,2-oxide, 3-(4-hydroxy-3-methoxyphenyl)-propane-1,2-diol, and 3-(4-hydroxy-3-methoxyphenyl)-propionic acid. 3. The structures of the following metabolites were tentatively deduced from mass spectra only, as reference compounds were not available: 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-allylbenzene, 3-(6?-mercapto-4-hydroxy-3-methoxyphenyl)-propane, and 2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-propionic acid. 4. The amounts of the individual metabolites excreted were determined by g.l.c. Some 95% of the dose was recovered in the urine, most of which (greater than 99%) consisted of phenolic conjugates; 50% of the conjugated metabolites were eugenol-glucuronide and sulphate. Other metabolic routes observed were the epoxide-diol pathway, synthesis of a thiophenol and of a substituted propionic acid, allylic oxidation, and migration of the double bond.

Metabolism of alkenebenzene derivatives in the rat. II. Eugenol and isoeugenol methyl ethers.

1. The metabolites of 3,4-dimethoxyallylbenzene (eugenol methyl ether) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.l.c. and g.l.c.-mass spectrometry. 2. The major metabolic reactions of 3,4-dimethoxyallylbenzene were oxidation of the allylic side chain to 2-hydroxy-3-(3,4-dimethoxyphenyl)-propionic acid, 3,4-dimethoxybenzoic acid and 3,4-dimethoxycinnamic acid, the two latter being largely excreted as their glycine conjugates. The formation of the hydroxy acid presumably involved epoxidation of the double bond and subsequent hydration to the diol whereas the formation of 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid involved migration of the double bond and the formation of cinnamoyl intermediates. Other reactions were O-demethylation to 4-hydroxy-3-methoxyallylbenzene (eugenol) and 3-hydroxy-4-methoxyallylbenzene in equal amounts, oxidation to 1-(3,4-dimethoxyphenyl)-2-propen-1-ol, hydroxylation of the benzene ring to a hydroxy-3,4-dimethoxyallylbenzene and oxidation to 3,4-dimethoxyphenylacetic acid. The formation of 1-(3,4-dihydroxyphenyl)propane was found to be carried out by the rat intestinal micro-organisms. A total of at least 63% but as much as 95% dose was accounted for. 3. The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. 4. The biliary metabolites of 3,4-dimethoxyallylbenzene and 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile.