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Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

Ayala DC, Morin D, Buckpitt AR - PLoS ONE (2015)

Bottom Line: Subsequent biotransformation results in urinary elimination of several conjugated metabolites.The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide.The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, United States of America; University of California Davis, Forensic Science, Graduate Program, 1909 Galileo Ct., Suite B, Davis, CA 95618, United States of America.

ABSTRACT
Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

No MeSH data available.


Related in: MedlinePlus

Overview of naphthalene metabolism and excretion.(A) Naphthalene metabolism showing the formation of both naphthol- and glutathione-derived urinary metabolites. (B) Chemical structures of targeted urinary naphthalene metabolites for LC/ESI-MS/MS analysis.
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pone.0121937.g001: Overview of naphthalene metabolism and excretion.(A) Naphthalene metabolism showing the formation of both naphthol- and glutathione-derived urinary metabolites. (B) Chemical structures of targeted urinary naphthalene metabolites for LC/ESI-MS/MS analysis.

Mentions: Several sensitive gas chromatography and gas chromatography-mass spectrometry methods have been developed as a means to effectively assess internal naphthalene exposures in industrial workers through detection of excreted naphthols [6,7]. Furthermore, in the NHANES (National Health and Nutrition Exposure Study) study Li and coworkers found detectable levels of urinary 1- and 2-naphthols in nearly the entire experimental cohort (general population, non-occupationally exposed) by GC-MS [8]. While excreted naphthols indicate naphthalene exposure and metabolism through the epoxide (Fig 1A), they underestimate the total amount of epoxide formed because they do not account for metabolites derived through the glutathione pathway. In vitro studies using human liver microsomes in the absence of glutathione and the GSH transferases demonstrated that the rates of dihydrodiol formation greatly exceed those of 1-naphthol [9]. In animal models, a significant proportion of urinary naphthalene metabolites are derived from the glutathione conjugates (Fig 1A) [10,11]. Yet, downstream metabolites of the glutathione adduct (mercapturic acids) have only been reported in humans using urine spot tests on paper chromatograms from individuals given large (500 mg p.o., approximately 1.4 mg/kg) doses of naphthalene [12]. Interestingly, other work in non-human primates failed to detect mercapturic acids of naphthalene but the reliability of this assay is uncertain because standards were not available [13]. In addition, an important consideration when using urinary naphthols to assess naphthalene exposure is that 1-naphthol excretion is also indicative of exposure to the insecticide, carbaryl [14]. Thus, there is a need to develop and validate quantitative methods for accurately measuring a range of naphthalene metabolites including the mercapturic acids, sulfate and glucuronide derivatives of naphthol, dihydrodiol, and any conjugates arising from naphthoquinones and the diol epoxide.


Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

Ayala DC, Morin D, Buckpitt AR - PLoS ONE (2015)

Overview of naphthalene metabolism and excretion.(A) Naphthalene metabolism showing the formation of both naphthol- and glutathione-derived urinary metabolites. (B) Chemical structures of targeted urinary naphthalene metabolites for LC/ESI-MS/MS analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390350&req=5

pone.0121937.g001: Overview of naphthalene metabolism and excretion.(A) Naphthalene metabolism showing the formation of both naphthol- and glutathione-derived urinary metabolites. (B) Chemical structures of targeted urinary naphthalene metabolites for LC/ESI-MS/MS analysis.
Mentions: Several sensitive gas chromatography and gas chromatography-mass spectrometry methods have been developed as a means to effectively assess internal naphthalene exposures in industrial workers through detection of excreted naphthols [6,7]. Furthermore, in the NHANES (National Health and Nutrition Exposure Study) study Li and coworkers found detectable levels of urinary 1- and 2-naphthols in nearly the entire experimental cohort (general population, non-occupationally exposed) by GC-MS [8]. While excreted naphthols indicate naphthalene exposure and metabolism through the epoxide (Fig 1A), they underestimate the total amount of epoxide formed because they do not account for metabolites derived through the glutathione pathway. In vitro studies using human liver microsomes in the absence of glutathione and the GSH transferases demonstrated that the rates of dihydrodiol formation greatly exceed those of 1-naphthol [9]. In animal models, a significant proportion of urinary naphthalene metabolites are derived from the glutathione conjugates (Fig 1A) [10,11]. Yet, downstream metabolites of the glutathione adduct (mercapturic acids) have only been reported in humans using urine spot tests on paper chromatograms from individuals given large (500 mg p.o., approximately 1.4 mg/kg) doses of naphthalene [12]. Interestingly, other work in non-human primates failed to detect mercapturic acids of naphthalene but the reliability of this assay is uncertain because standards were not available [13]. In addition, an important consideration when using urinary naphthols to assess naphthalene exposure is that 1-naphthol excretion is also indicative of exposure to the insecticide, carbaryl [14]. Thus, there is a need to develop and validate quantitative methods for accurately measuring a range of naphthalene metabolites including the mercapturic acids, sulfate and glucuronide derivatives of naphthol, dihydrodiol, and any conjugates arising from naphthoquinones and the diol epoxide.

Bottom Line: Subsequent biotransformation results in urinary elimination of several conjugated metabolites.The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide.The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, United States of America; University of California Davis, Forensic Science, Graduate Program, 1909 Galileo Ct., Suite B, Davis, CA 95618, United States of America.

ABSTRACT
Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

No MeSH data available.


Related in: MedlinePlus