128 research outputs found
Commentary on: Differences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods - Response
No full textDifferences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods
No full textBackground: Measurement of 6-thioguanine nucleotide (6-TGN) concentrations in erythrocytes is widely accepted for use in optimization of thiopurine therapy. Various chromatographic methods have been developed for this purpose. In preliminary experiments we observed a considerable difference between 6-TGN concentrations determined with two widely used methods published by Lennard (Lennard L. I Chromatogr 1987; 423:169-78) and by Dervieux and Boulieu (Dervieux T, Boulieu R. Clin Chem 1998;44:551-5). We therefore investigated methodologic differences between the two procedures with respect to hydrolysis of 6-TGNs to 6-thioguanine (6-TG) in more detail. Methods: We analyzed 6-TGNs in erythrocyte preparations (n = 50) from patients on azathioptine therapy by both methods, using the original protocols. In one set of experiments, we replaced the 0.5 mol/L sulfuric acid in the Lennard method with the 1 mol/L perchloric acid used by Dervieux and Boulieu. In a second set of experiments, we investigated the effect of various dithiothreitol (DTT) concentrations on 6-TG recovery with both methods. In a third set of experiments, we determined the effect of hydrolysis time on both protocols. Results: Direct comparison of both methods showed that 6-TGN concentrations were, on average, 2.6-fold higher in the Dervieux-Boulieu method over the concentration range tested, although the correlation (r = 0.99, P <0.001) was good. Replacement of sulfuric acid by perchloric acid reduced this difference to similar to1.4-fold (r = 0.99; P <0.001). Increasing the DTT concentration enhanced 6-TG recovery. The hydrolysis time used in the Lennard method (1 h) was not sufficient to achieve complete hydrolysis. Conclusions: The difference between 6-TGN concentrations measured by the two methods is attributable, at least in part, to differences in the extent of nucleotide hydrolysis. For optimization of thiopurine therapy, method-dependent therapeutic ranges are necessary, which precludes comparison of results from clinical studies derived with these methods. Efforts must therefore be made to standardize the analytical procedures for the determination of 6-TGN. (C) 2003 American Association for Clinical Chemistry
Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid
No full textBackground: Because. mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. Methods: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C-18 column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. Results: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 mug/L, and the method was linear to 1000 mug/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 mug/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 mug/L for free MPA and y 0.98x + 0.03 mg/L for total MPA. Conclusions: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug. (C) 2004 American Association for Clinical Chemistry
Quantification of plasma total MPA and free MPA 80 mass interferences in quantification of using liquid chromatography tandem mass cyclosporine using tandem mass spectrometry spectrometry
No full textQuantification of plasma total MPA and free MPA 80 mass interferences in quantification of using liquid chromatography tandem mass cyclosporine using tandem mass spectrometry spectrometry
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Analytic aspects of monitoring therapy with thiopurine medications
No full textThe thiopurine medications 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and azathioprine are used in treatment of childhood acute lymphoblastic leukemia, amoimmune diseases, and, in the case of azathioprine, in solid organ transplantation. They are converted in vivo to the active 6-thioguanine nucleotides (6-TGN). One person in 300 in white populations has low or undetectable TPMT activity and is at risk for accumulating 6-TGN with the consequence of severe, life-threatening myelosuppression. A rational therapeutic strategy for thiopurine drug use is to first determine TPMT phenotype/genotype and then to adjust the dosage on an individual basis. Determination of erythrocyte 6-TGN levels can further help to optimize therapy. TPMT activity (phenotype) is determined in erythrocytes using radiochemical or HPLC procedures. Recent HPLC procedures show good agreement with the original radiochemical method, while offering simplified sample pretreatment and improved precision. To date, 12 mutant alleles responsible for TPMT deficiency have been published. Restriction fragment length polymorphism PCR and allele-specific PCR have been used for detection of TPMT mutations. Genotyping methods that allow a higher throughput include real-time PCR (LightCycler) and denaturing HPLC. Numerous HPLC methods have been reported for quantification of 6-TGN. The majority involve acid hydrolysis to 6-TG at high temperature. There are substantial differences in the hydrolysis step, extraction procedure, chromatographic conditions and method of detection. Erythrocyte 6-TGN concentrations can vary up to 2.6-fold depending on the HPLC method. The method that has found the greatest application in clinical studies is that of Lennard.(46) This has served as the basis for the establishment of treatment-related therapeutic ranges for thiopurine therapy. These ranges will not necessarily be applicable when other methodology is used. There is an urgent need to harmonize the analytic procedures for 6-TGN
The acyl glucuronide metabolite of mycophenolic acid induces tubulin polymerization in vitro
No full textObjectives: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) forms covalent protein adducts and possesses antiproliferative properties independent of IMPDH inhibition. The underlying mechanism is unknown. Disorganized tubulin polymerization prevents cell cycle progression. We investigated whether AcMPAG interacts with tubulin polymerization. Design and methods: AcMPAG (1.0-100 mu M) was incubated with bovine tubulin in the presence of GTP. Polymerization was followed at 340 nm. The time until onset and the extent of polymerization were determined. MPA (100 mu M), phenolic glucuronide MPAG (100 mu M), and paclitaxel (10 mu M) served as controls. Results: MPAG was without effect. The AcMPAG effect on tubulin polymerization was dose dependent and significantly stronger (about 2.5-fold) than that of MPA (n = 4; p < 0.05), but weaker than paclitaxel. Conclusions: MPA and AcMPAG can induce tubulin polymerization in the presence of GTP with AcMPAG being significantly stronger. This property of AcMPAG may contribute to its IMPDH independent antiproliferative effect. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.DGKL, German
Acyl glucuronide drug metabolites: Toxicological and analytical implications
No full textAlthough glucuronidation is generally considered a detoxification route of drug metabolism, the chemical reactivity of acyl glucuronides has been linked with the toxic properties of drugs that contain carboxylic acid moieties. It is now well documented that such metabolites can reach appreciable concentrations in blood. Furthermore, they are labile, undergo hydrolysis and pH-dependent intramolecular acyl migration to isomeric conjugates of glucuronic acid, and may react irreversibly with plasma proteins, tissue proteins, and with nucleic acids. This stable binding causes chemical alterations that are thought to contribute to drug toxicity either through changes in the functional properties of the modified molecules or through antigen formation with subsequent hypersensitivity and other immune reactions. Whereas in vitro data on the toxicity of acyl glucuronides have steadily accumulated, direct evidence for their toxicity in vivo is scarce. Acyl glucuronides display limited stability, which is dependent on pH, temperature, nature of the aglycon, and so on. Therefore, careful sample collection, handling, and storage procedures are critical to ensure generation of reliable pharmacologic and toxicologic data during clinical studies. Acyl glucuronides can be directly quantified in biologic specimens using chromatographic procedures. Their adducts with plasma or cell proteins can be determined after electrophoretic separation, followed by blotting. ELISA techniques have been used to assess the presence of antibodies against acyl glucuronide-protein adducts. This review summarizes the most recent evidence concerning biologic and toxicologic effects of acyl glucuronide metabolites of various drugs and discusses their relevance for drug monitoring. A critical evaluation of the available methodology is included
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