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Effect of commonly used organic solvents on aldehyde oxidase-mediated vanillin, phthalazine and methotrexate oxidation in human, rat and mouse liver subcellular fractions.

Xenobiotica; the fate of foreign compounds in biological systems (2014-02-19)
Dayanidhi Behera, Rambabu Pattem, Girish Gudi
ABSTRACT

1. Aldehyde oxidase (AOX) is a cytosolic molybdoflavoprotein enzyme widely distributed across many tissues. In this study, we report the effect of commonly used organic solvents such as dimethyl sulfoxide (DMSO), acetonitrile (ACN), methanol and ethanol on AOX activity in human, rat and mouse liver S9 fractions using vanillin, phthalazine and methotrexate as probe substrates. 2. Methanol was found to be the most potent solvent in inhibiting vanillic acid and 1-phthalazinone formation in comparison to DMSO, ACN and ethanol across the species tested, except 7-hydroxy methotrexate. 3. Treatment with these solvents at approximate IC50 (% v/v) concentrations showed significant reduction in Clint and Vmax of the probe substrates and also resulted in different effects on Km across the species. 4. Marked differences in the activity and affinity towards AOX were observed with different probe substrates with methotrexate showing least activity and affinity as compared to vanillin and phthalazine. 5. Overall, AOX activity seemed to be more resilient to the presence of organic solvents at higher concentrations in human and rodent species. These results suggest that low concentrations of organic solvents are acceptable for in vitro incubations involving AOX-mediated metabolism.

MATERIALS
Product Number
Brand
Product Description

Supelco
Vanillin, Pharmaceutical Secondary Standard; Certified Reference Material
Vanillin, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
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Methanol, ACS spectrophotometric grade, ≥99.9%
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Methanol, Pharmaceutical Secondary Standard; Certified Reference Material
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Ammonium acetate, ≥99.99% trace metals basis
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Methanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8% (GC)
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Ammonium acetate, ACS reagent, ≥97%
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Methanol, ACS reagent, ≥99.8%
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Methanol, BioReagent, ≥99.93%
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Methanol, suitable for HPLC, ≥99.9%
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Methanol, HPLC Plus, ≥99.9%
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Methanol, suitable for HPLC, gradient grade, ≥99.9%
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Methanol, suitable for HPLC, gradient grade, suitable as ACS-grade LC reagent, ≥99.9%
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Methanol, Laboratory Reagent, ≥99.6%
Methotrexate for system suitability, European Pharmacopoeia (EP) Reference Standard
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Methanol, ACS reagent, ≥99.8%
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Methanol, Absolute - Acetone free
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Methanol, puriss., meets analytical specification of Ph Eur, ≥99.7% (GC)
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Magnesium chloride solution, 0.1 M
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Mettler-Toledo Calibration substance ME 51143093, Vanillin, traceable to primary standards (LGC)
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Vanillic acid, certified reference material, TraceCERT®
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Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR
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Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
USP
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Vanillic acid, 97%
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Ammonium acetate solution, for molecular biology, 7.5 M
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Ammonium acetate, BioXtra, ≥98%
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Ammonium acetate, for molecular biology, ≥98%