Determination of Thiopurine Methyltransferase Activity in Human Erythrocytes by High-Performance Liquid Chromatography: Comparison With the Radiochemical Method

Menor, César; Fueyo, Jesús; Escribano, Óscar; Cuite, Cara L.; Fernández‐Moreno, María Dolores; Domingo, Alberto; Guijarro, Luis G.

doi:10.1097/00007691-200110000-00007

Cited by 30 publications

(18 citation statements)

References 11 publications

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“…Moreover, the radiochemical assay in Singapore Chinese produced results that were approximately 3 times higher than those obtained from this study and Mayo Clinic. The TPMT activity values measured by HPLC were reported to be in close agreement with those measured by the radiochemical assay [19,22]. It has been shown that differences in assay conditions can cause significant differences in TPMT activity measured in different laboratories [5].…”

Section: Discussionsupporting

confidence: 58%

Phenotyping and genotyping study of thiopurine S‐methyltransferase in healthy Chinese children: A comparison of Han and Yao ethnic groups

Zhang¹,

Guan²,

et al. 2004

Brit J Clinical Pharma

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AimsEthnicity is an important variable influencing drug response. Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. Previous population studies have identified ethnic variations in both phenotype and genotype of TPMT , but limited information is available within Chinese population that comprises at least 56 ethnic groups . The current study was conducted to compare both phenotype and genotype of TPMT in healthy Han and Yao Chinese children. MethodsTPMT activity was measured in healthy Chinese children by a H PLC assay ( n = 213, 87 Han Chinese and 126 Yao Chinese). Allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) were used to determine the frequency of TPMT mutant alleles ( TPMT * 2 , TPMT * 3 A, TPMT * 3B and TPMT * 3C ) in these children. ResultsThere was no significant difference in the mean TPMT activity between Han and Yao Chinese children. A unimodal distribution of TPMT activity in Chinese children was found and the mean TPMT activity was 13.32 ± 3.49 U ml -1 RBC. TPMT activity was not found to differ with gender, but tended to increase with age in Yao Chinese children. TPMT * 2, TPMT * 3B and TPMT * 3A were not detected, and only one TPMT * 3C heterozygote (Han child) was identified in 213 Chinese children. Erythrocyte TPMT activity of this TPMT * 3C heterozygote was 12.36 U ml -1 RBC. The frequency of the known mutant TPMT alleles was 0.2% [1/426] in Chinese children. ConclusionThe frequency distribution of RBC TPMT activity was unimodal. The frequency of the known mutant TPMT alleles in Chinese Children is low and TPMT * 3C appears to be the most prevalent among the tested mutant TPMT alleles in this population.J.-P. Zhang et al. 16458 :2 Br J Clin Pharmacol

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Section: Discussionsupporting

confidence: 58%

Phenotyping and genotyping study of thiopurine S‐methyltransferase in healthy Chinese children: A comparison of Han and Yao ethnic groups

Zhang¹,

Guan²,

et al. 2004

Brit J Clinical Pharma

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“…The lysate was centrifuged at 13,000 g for 10 min and the supernatant was used immediately for enzyme assays or stored at -85 • C; in this condition, the enzyme remains stable for several weeks. RBC TPMT activity was measured by a radiochemical method as previously described [15]. This procedure is based on the conversion of 6-MP to 6-methylmercaptopurine, using S-adenosyl-l-[methyl-3H]methionine as methyl donor.…”

Section: Analysis Of Tpmt Activitymentioning

confidence: 99%

Thiopurine Methyltransferase Activity in Spain: A Study of 14,545 Patients

et al. 2007

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We sought to assess the activity of thiopurine methyltransferase (TPMT) in 14,545 Spanish patients with different diseases amenable to treatment with azathioprine/6-mercaptopurine (6-MP), and to evaluate the proportion of patients with low TPMT activity and therefore a higher risk of myelotoxicity with these drugs. TPMT activity in red blood cells (RBCs) was measured by a radiochemical method. The association between several clinical variables and TPMT activity was assessed by multiple linear regression. We included 14,545 patients: autoimmune hepatitis (n=359 patients), inflammatory bowel disease (n=7,046), multiple sclerosis (n = 814), myasthenia gravis (n=344), pemphigus (n=133), and other diseases (n=5,849). Mean TPMT activity was 20.1 +/- 6 U/mL, but differed depending on the disease (P<.001). TPMT distribution was low (<5) in 0.5%; intermediate (5.0-13.7) in 11.9%; or high (>or=13.8) in 87.6%. Only when TPMT activity was considered separately in each disease did it reveal a normal distribution. In the multivariate analysis, gender, hematocrit, and treatment with 5-aminosalicylates/steroids/azathioprine/6-MP statistically influenced TPMT activity, although, probably, in a clinically irrelevant manner. This study shows, in a large sample of 14,545 patients, that 0.5% had low TPMT activity, indicating a higher risk of myelotoxicity with azathioprine/6-MP, a figure similar or slightly higher than that reported in other areas. Nevertheless, the trimodal distribution of TPMT activity varied depending on disease, and the proportion of patients with low activity values ranged from 0-0.8%. The drugs prescribed for the treatment of autoimmune diseases, including azathioprine/6-MP, modified TPMT activity, but the magnitude of this effect was very small and the differences found are probably irrelevant from the clinical point of view.

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“…When Aza enters the cell, it is transformed into 6-MP by an unknown cytosolic isoform of GST using GSH as a cosubstrate, which depletes the cytosolic GSH pool. After this, the following metabolic pathways are proposed for 6-MP: 1) methylation by thiopurine-methyltransferase to produce 6-methyl-mercaptopurine (Menor et al, 2001); 2) oxidation by XO to yield thiouric acid (Lewis and Olibier, 1977); 3) "the salvage pathway", whereby 6-MP would be incorporated into the intracellular purine nucleotide pool by the participation of hypoxantine phosphoribosyl transferase (Aarbakke et al, 1997); or 4) interaction with MPTP. The Aza/6-MP detoxificating role is attributable to thiopurine-methyl-transferase and XO-catalyzed reactions, whereas the antimitotic effect observed for both drugs corresponds to their incorporation into the 6-thioguanine nucleotide pool.…”

Section: Azathioprine Acts On Hepatocyte Mitochondria Involving Mapk 673mentioning

confidence: 99%

Azathioprine Acts upon Rat Hepatocyte Mitochondria and Stress-Activated Protein Kinases Leading to Necrosis: Protective Role of N-Acetyl-L-cysteine

Menor¹,

Fernández‐Moreno²,

Fueyo³

et al. 2004

J Pharmacol Exp Ther

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Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release.The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

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Determination of Thiopurine Methyltransferase Activity in Human Erythrocytes by High-Performance Liquid Chromatography: Comparison With the Radiochemical Method

Cited by 30 publications

References 11 publications

Phenotyping and genotyping study of thiopurine S‐methyltransferase in healthy Chinese children: A comparison of Han and Yao ethnic groups

Phenotyping and genotyping study of thiopurine S‐methyltransferase in healthy Chinese children: A comparison of Han and Yao ethnic groups

Thiopurine Methyltransferase Activity in Spain: A Study of 14,545 Patients

Azathioprine Acts upon Rat Hepatocyte Mitochondria and Stress-Activated Protein Kinases Leading to Necrosis: Protective Role of N-Acetyl-L-cysteine

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