Application of Isotopic Ratio Mass Spectrometry for the in Vitro Determination of Demethylation Activity in Human Liver Microsomes Using N-methyl-13C-Labeled Substrates

Grand, Florence; Kilinç, İ̇zzet; Sarkis, Albert; Guitton, Jérôme

doi:10.1006/abio.2002.5701

Cited by 7 publications

(3 citation statements)

References 28 publications

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“…Endogenous cholesterol synthesis was determined by IRMS with adult humans who received low doses of D 2 O (Jones et al, 1993; Di Buono et al, 2000). The IRMS method was also successfully applied to in vitro studies in which microsomal desaturase activities were assayed in the brain (Su & Brenna, 1998), and cytochrome P450‐dependent demethylation activity was determined in human liver microsomes (Grand et al, 2002). It has been shown that GC/C/IRMS possesses advantages over scanning MS such as GC/MS in terms of analysis sensitivity.…”

Section: Advances In the Measurements Of Stable Isotope‐labeled Mmentioning

confidence: 99%

Mass spectrometric studies on brain metabolism, using stable isotopes

Ando

Tanaka

2005

Mass Spectrometry Reviews

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In fields related to biomedicine, mass spectrometry has been applied to metabolism research and chemical structural analysis. The introduction of stable isotopes has advanced research related to in vivo metabolism. Stable-isotope labeling combined with mass spectrometry appears to be a superior method for the metabolism studies, because it compensates for the shortcomings of conventional techniques that use radioisotopes. Biomolecules labeled with stable isotopes have provided solid evidence of their metabolic pathways. Labeled large molecules, however, cannot homogeneously mix in vivo with the corresponding endogenous pools. To overcome that problem, small tracers labeled with stable isotopes have been applied to in vivo studies because they can diffuse and attain a homogeneous distribution throughout the inter- and intracellular spaces. In particular, D(2)O-labeling methods have been used for studies of the metabolism in different organs, including the brain, which is isolated from other extraneural organs by the blood-brain barrier (BBB). Cellular components, such as lipids, carbohydrates, proteins, and DNA, can be endogenously and concurrently labeled with deuterium, and their metabolic fluxes examined by mass spectrometry. Application of the D(2)O-labeling method to the measurements of lipid metabolism and membrane turnover in the brain is described, and the potential advantages of this method are discussed in this review. This methodology also appears to have the potential to be applied to dynamic and functional metabolomics.

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Section: Advances In the Measurements Of Stable Isotope‐labeled Mmentioning

confidence: 99%

Mass spectrometric studies on brain metabolism, using stable isotopes

Ando

Tanaka

2005

Mass Spectrometry Reviews

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“…Detection of 13/14 C or 3 H metabolites (H 13/14 CHO and/or 13/14 CO 2 ) of radiolabeled substrates (erythromycin, caffeine, cyclosporine, aminophenazone, diazepam etc. ) is used in some in vitro studies to assess metabolic activity of CYP3A4 [Grand et al 2002, Kenworthy et al 1999. Metabolites may be detected radiochemically, 14 C-formaldehyde may be extracted and detected by radioluminescence in a microplate scintillation counter [Zlokarnik et al 2005].…”

Section: Radiolabeled Substratesmentioning

confidence: 99%

Determination of Cytochrome P450 Metabolic Activity Using Selective Markers

Juřica¹,

Šulcová²

2012

Topics on Drug Metabolism

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The best studied CYP enzyme concerning polymorphisms, enzyme inhibition and dosage individualization according to phenotype is probably CYP2D6, followed by CYP2C9 and CYP2C19. These enzymes are highly polymorphic, contrary to CYP3A4, where the m e t a b o l i c a c t i v i t y m a y v a r y d u e t o d i f f e r e n c e s i n C Y P 3 A 4 g e n e e x p r e s s i o n a n d pharmacokinetic interactions [Ingelman-Sundberg 2004]. Nevertheless, any shift in metabolic capacity of individual CYP enzyme may result either in decreased or increased therapeutic response or intensity of adverse effects. For example, after paroxetine administration to the patients on tamoxifen, a decrease in plasma levels of active metabolite of tamoxifen was detected [Stearns et al. 2003]. This can be crucially important in breast cancer treatment. On the other hand, CYP2D6 ultrarapid metabolizer phenotype may cause failure of pharmacotherapy due to the very low and thus ineffective drug plasma levels [Corruble 2008]. In antipsychotic treatment, an association has been observed between extrapyramidal adverse effects and CYP2D6 genotype [Fleeman et al. 2011]. Significant clinical consequences of CYP2D6 genotype or enzyme inhibition were described also in beta-blockers, antianginal, antiarrythmic drugs, antihistamines and antiemetics. The clinical www.intechopen.com Topics on Drug Metabolism 192 impact of enzyme polymorphism or changes due to inhibition or induction usually depends on the contribution of other CYP forms to the total drug's elimination. By this, the relative therapeutic potency of the parent drug or any of its metabolites may be altered [Zhou 2009]. Methodological approaches such as assessment of metabolic ratio of specific substrate to metabolite(s) in saliva, plasma / serum or urine are most widely used to assess metabolic activity in vivo. Besides determination of the concentrations of probe and metabolite in biological fluids, specific substrates are used in various breath tests. Rarely some other approaches are used, e.g. the pupilometry after opioid administration. It is essentially important to differentiate between in vivo and in vitro metabolic activity assessments, as they show very often discrepant results. One of the greatest advantages of genotype assessment is that it does not need to be repeated because it does not change with time or under the simultaneous influences of drugs and other factors. On the other side the disadvantage of the pharmacogenetic testing is, that genotype does not always correlate with observed metabolic activity recorded using probe drug(s). This discrepancy may be caused by various epigenetic factors as well as by inhibition or induction of enzyme metabolic activity caused by other xenobiotics coadministered. 2.2 Conventional probe substrates for in vivo metabolic activity assessment Metabolic activity of various CYP enzymes is most often assessed using selective substrate of distinct CYP enzyme ("marker of metabolic activity"), i.e. a drug (or substance) which is ideally metabolized by th...

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“…To improve throughput and reduce cost, several HLM CYP inhibition assays, such as radiometric (Rodrigues et al, 1996 ;Zhang and Thomas, 1996 ;Riley and Howbrook, 1997 ;Draper et al, 1998 ;Grand et al, 2002 ;Di Marco et al, 2007 ), luminescence (Garcia et al, 2008 ), and cocktail assays (Bu et al, 2001a,b ;Dierks et al, 2001 ;Zhang et al, 2002 ;Testino and Patonay, 2003 ;Yin et al, 2004 ;Turpeinen et al, 2005 ;He et al, 2007 ;Lin et al, 2007 ;O ' Donnell et al, 2007 ; pharmaceutical industry. Descriptions of detailed method development strategies, reagents used, experimental protocols, and the analytical technologies of these assays are included.…”

Section: Introductionmentioning

confidence: 99%