Inducing rat brain CYP2D with nicotine increases the rate of codeine tolerance; predicting the rate of tolerance from acute analgesic response

McMillan, Douglas M.; Tyndale, Rachel F.

doi:10.1016/j.bcp.2017.08.015

Cited by 17 publications

(12 citation statements)

References 66 publications

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“…However, this exacerbates the issue of overall lower amounts of microsomal protein available from a single brain, such that pooling of several brains to generate enough material for these assays is necessary [3,4,7]. Other studies [5,6,10] have utilized something called the "total membrane fraction," which is a pooling of both mitochondrial and microsomal membranes, to increase yield. However, these studies [5,6,10] still require long incubation times (2-3 hours) and high protein quantities (1.5-3 mg membrane protein/reaction).…”

Section: Accepted Manuscriptmentioning

confidence: 99%

“…Other studies [5,6,10] have utilized something called the "total membrane fraction," which is a pooling of both mitochondrial and microsomal membranes, to increase yield. However, these studies [5,6,10] still require long incubation times (2-3 hours) and high protein quantities (1.5-3 mg membrane protein/reaction).…”

Section: Accepted Manuscriptmentioning

confidence: 99%

“…1), is a commonly used tool to estimate cytochrome P450 (CYP) 2D6 (CYP2D6) function in humans [1,2]. The DOD assay is also used in animal studies (especially in rats) to compare human CYP2D6 substrate specificities with those in other species [3][4][5][6][7]. In rodents, this activity is more generally assigned to the CYP2D subfamily, given there are multiple isoforms within this subfamily with similar homology and substrate affinities that overlap with the human CYP2D6 isoform [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…In both humans and rats, CYP2D is expressed in both the liver and brain [11][12][13]. Although the liver is considered the main site of drug clearance and metabolism, studies have shown that the expression and activity of CYP2D in the brain can alter pharmacologic responses to centrally-acting drug [5,6,12,[14][15][16][17]. Functional characterization of CYP enzymes in vitro is typically achieved via subcellular fractionation and collection of the small vesicles formed by the endoplasmic reticulum, called microsomes.…”

Section: Introductionmentioning

confidence: 99%

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UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

DuBois

Mehvar

2018

Journal of Chromatography B

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Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d-DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 μL), microsomal protein amount (5 μg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ± SD, n = 4), which showed a maximum velocity of 2.24 ± 0.42 pmol/min/mg and a MM constant of 282 ± 62 μM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.

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Section: Accepted Manuscriptmentioning

confidence: 99%

Section: Accepted Manuscriptmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

DuBois

Mehvar

2018

Journal of Chromatography B

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“…Others (Mann et al, 2008;Toselli et al, 2016;Yue et al, 2008) have relied on methods like RT-PCR and/or Western blotting, instead of enzymatic activity, to estimate changes in the brain CYP2D status. Those studies that have successfully measured functional activity of brain CYP2D utilizing probe substrates, such as dextromethorphan (DXM), tend to either focus on microsomal activity (Asai et al, 2018;Coleman et al, 2000;Jolivalt et al, 1995;Voirol et al, 2000) or something called a "total membrane" fraction (McMillan & Tyndale, 2017;Miksys et al, 2017;Tyndale et al, 1999;Zhou et al, 2013), which is a pooling of all membranes, including microsomes and mitochondria.…”

Section: Introductionmentioning

confidence: 99%

Kinetics of dextromethorphan-O-demethylase activity and distribution of CYP2D in four commonly-used subcellular fractions of rat brain

2019

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1. The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions. 2. Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis-Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot. 3. Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (~ 50-fold) affinity but a higher (~six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes. 4. Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction.

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Cytochrome P450 enzymes and metabolism of drugs and neurotoxins within the mammalian brain

Stocco

Tyndale

2022

Advances in Pharmacology

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Inducing rat brain CYP2D with nicotine increases the rate of codeine tolerance; predicting the rate of tolerance from acute analgesic response

Cited by 17 publications

References 66 publications

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

Kinetics of dextromethorphan-O-demethylase activity and distribution of CYP2D in four commonly-used subcellular fractions of rat brain

Cytochrome P450 enzymes and metabolism of drugs and neurotoxins within the mammalian brain

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