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Background: Thiopurine methyltransferase (TPMT) catalyses the S-methylation of 6-thiopurine drugs, which are commonly used in the treatment of autoimmune diseases, leukaemia and organ transplantation. TPMT activity is polymorphic as a result of gene mutations. Ethnic variations in phenotype and genotype have been identified in previous population studies, but no information was available within Latin-American populations., Aim: To establish the genetic polymorphism of TPMT in an Argentine population., Methods: TPMT enzymatic activity of 147 healthy Argentine subjects was measured using a high-performance liquid chromatography method. The genotyping assay for nine defective alleles (TPMT*2 - *8) was based on restriction fragment length polymorphism polymerase chain reaction and allele-specific polymerase chain reaction methods., Results: All subjects had detectable TPMT activity. Twelve individuals with low to intermediate activity were heterozygous for one of the mutant alleles: nine were TPMT*1/*3A, two TPMT*1/*2 and one TPMT*1/*4. All examined subjects with normal activity had wild-type genotype (TPMT*1/*1)., Conclusion: Variant TPMT alleles were present in 8.2% of the examined subjects, which is in accordance with other studies. The frequency of TPMT*3A, TPMT*2 and TPMT*4 was 3.1%, 0.7% and 0.3%, respectively. TPMT*3A was the most prevalent allele, which is in accordance with results from Caucasian populations. This study provides the first analysis of TPMT activity and allele frequency distribution in Argentina, South America.

Background: Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity., Methods: We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate., Results: The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found., Conclusion: The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.

Background: Methotrexate (MTX) followed by 6-mercaptopurine (6MP) is one of the best known combinations for the treatment of childhood acute lymphoblastic leukaemia. Tiazofurin (TF) and 6-thioguanine (TG) are also used as chemotherapy agents in the treatment of malignancies. We have examined the induction of apoptosis by combinations of these drugs to gain more insights into their efficacy in the treatment of malignancies., Methods: The induction of apoptosis was examined in Molt-4, a human malignant acute lymphoblastic T-cell line. The cells were exposed to increasing drug concentrations at various exposure times. Annexin V/FITC and propidium iodide (PI) were used as markers for apoptosis and cell death. Annexin V/FITC positive and PI positive cells were detected by flow-cytometric analysis., Results: Sequential 24-h exposure with MTX (0.005-0.02 micro mol) followed by 6MP (1-10 micro mol) and 24-h exposure with TF (5-20 micro mol) followed by TG (0.5-2 micro mol) showed a more than additive induction of apoptosis compared with single-drug exposure. Simultaneous administration of the drugs does not show an additive effect on apoptosis., Conclusions: The results of this study indicate that sequential administration of MTX before 6MP and of TF before TG may be essential for therapeutic success in the treatment of leukaemia.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). A deficiency of DPD is increasingly being recognized as the cause of an important pharmacogenetic syndrome. The importance of DPD deficiency in the aetiology of unexpected severe 5FU toxicity has been demonstrated by the fact that, in 39-59% of cases, decreased DPD activity could be detected in peripheral blood mononuclear (PBM) cells. It was observed that 55% of the patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of the patients with a normal DPD activity (P = 0.01). Furthermore, toxicity developed significantly earlier in patients with low DPD activity than in patients with normal DPD activity (10.0 +/- 7.6 versus 19.1 +/- 15.3 days, P < 0.05). In patients suffering from severe 5FU-associated toxicity, 11 mutations have been identified in DPYD, including one splice-site mutation (IVS14 + 1G-->A), one nonsense mutation (E386X), four missense mutations (M166V, V335L, I560S, D949V) and five polymorphisms (C29R, R21Q, S534N, I543V, V732I). Considering the common use of 5FU in the treatment of cancer patients, the severe 5FU-related toxicities in patients with a low DPD activity and the high prevalence of the IVS14 + 1G-->A mutation, analysis of the DPD activity in PBM cells or screening for the IVS14 + 1G-->A mutation should be routinely carried out prior to the start of treatment with 5FU.

In a patient with microcephaly, feeding problems and restlessness, moderately increased serum and urine citrulline concentrations were observed. Protein and allopurinol loading did not result in additional indications for a urea cycle defect. The diagnosis of citrullinaemia was made at both the enzyme and DNA level, resulting from a novel mutation in the argininosuccinate synthetase gene. The fact that the patient has not suffered from severe deterioration, and that there were only minor abnormalities in metabolite concentrations, suggests that the argininosuccinate synthetase capacity was less affected in vivo than in vitro. In vitro nuclear magnetic resonance investigation suggested an active acetylation mechanism for citrulline. This case illustrates the importance of performing extensive biochemical and molecular investigations in order to reach a definitive diagnosis, particularly in instances of moderate citrullinaemia.

The effects of inhibition of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide de novo synthesis, on cell growth, cell viability, endogenous nucleotide concentrations and concentrations of extracellular nucleosides and bases were studied in Molt F4 human malignant lymphoblasts. Mycophenolic acid (MPA) was used as a specific inhibitor of the enzyme activity. IMPDH activity was maximally inhibited with 0.5 microM MPA. After a 2 h exposure of the cells to 0.5 microM MPA, guanine nucleotides were depleted to approximately 50% of control values, whereas 5-phosphoribosyl-1-pyrophosphate levels increased to approximately 200%. Under these conditions, cytotoxicity became obvious after 24 h. Depletion of guanine nucleotides and cytotoxicity were prevented by addition of guanosine to MPA treatment. Daily supplements of guanosine were required to prevent MPA cytotoxicity during the entire incubation period of 72 h. We conclude that depletion of guanine nucleotides, induced by treatment with MPA, induces a severe and rapid cytotoxicity in Molt F4 cells.