RESUMEN
BACKGROUND: The short half-life of the alpha-class glutathione S-transferases (GSTAs) in plasma combined with their even distribution throughout the liver lobule suggests that they may be useful complements to the more traditionally used liver markers. However, the currently available assays for measuring GSTAs in biological fluids have a poor dynamic range and are cumbersome, requiring multiple steps and prolonged incubation times. METHODS: Hybridomas that secrete monoclonal antibodies to human GSTAs were produced and used to develop a rapid one-step immunometric assay for the determination of GSTA in serum. The assay uses a time-resolved immunofluorometric assay (TR-IFMA) format and requires 35 min of incubation. The reference interval was determined using 208 serum samples from healthy blood donors. We also compared our TR-IFMA with a commercially available enzyme immunoassay (EIA) for GSTAS: RESULTS: The assay had a detection limit of 0.07 microg/L with a measuring range up to 625 microg/L. Within-run imprecision (CV) was 1.8-2.6% over the concentrations of GSTA tested (2.5-311 microg/L), with a between-run CV of <5%. In healthy blood donors, the median values and reference intervals were 2.0 microg/L and 0.6-7.2 microg/L for females and 2.6 microg/L and 0.7-9.8 microg/L for males, respectively. GSTA concentrations determined with the TR-IFMA correlated well with those obtained using a commercially available EIA. CONCLUSIONS: This report describes a new assay for monitoring the concentrations of GSTAs in human serum. The method may be useful in further evaluating the potential of monitoring serum GSTAs in the routine clinical setting.
Asunto(s)
Glutatión Transferasa/sangre , Animales , Anticuerpos Monoclonales , Recolección de Muestras de Sangre , Femenino , Fluoroinmunoensayo , Glutatión Transferasa/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Valores de ReferenciaRESUMEN
Subclones of the two well-characterized myeloid cell lines HL-60 and KG1a were selected for doxorubicin resistance by systematic exposure to increased concentrations of the drug in vitro. Both subclones demonstrated a threefold increased resistance to the drug as evident from cell growth in liquid culture and clonogenicity in a semisolid matrix. Both resistant subclones displayed a similar degree of reduced total and nuclear doxorubicin levels. The HL-60 and the KG1a cells differed qualitatively and quantitatively with respect to glutathione (GSH) levels during culture, with markedly elevated concentrations in the resistant HL-60 subclone during 1 week of culture. Total GSH pools in resistant and sensitive KG1a cells were similar, but maximum GSH levels were reached earlier in the resistant KG1a clones than in the parental cells. Northern blot analysis suggests that resistance was accompanied by increased mdr1 expression in the KG1a but not in the HL-60 cells, whereas alterations in the glutathione S-transferase P1-1 and topoisomerase II message was evident in the latter. The results demonstrate the complex, multifactorial mechanisms behind the in vitro induction of even moderate resistance in anthracyclines.