RESUMEN
Peripheral blood lymphocytes of healthy volunteers cultured with phytohaemagglutinin in folate-deficient medium exhibit megaloblastic maturation with reduced intracellular folate content. We have employed this in vitro model for megaloblastic maturation to determine accompanying changes in cellular thymidylate cycle activities and deoxynucleotide levels. Folate-deficient cells exhibit a two-fold increase in thymidine kinase and thymidylate synthase activities. These increased activities were reduced to those of folate-replete cells by co-culture of folate-deficient cells with thymidine. Folate deficiency was accompanied by reduced cellular levels of thymidine triphosphate (TTP) and deoxyguanosine triphosphate (dGTP). Exogenous deoxyuridine produced no increase in the reduced levels of TTP of folate-deficient cells but effected a two-fold increase in cellular deoxycytidine triphosphate. Exogenous thymidine increased the reduced TTP levels of folate-deficient cells and corrected the reduced dGTP level; the increase in cellular TTP accompanying exogenous thymidine was more pronounced in folate-deficient cells. These in vitro findings are compatible with a block in de novo thymidylate synthesis and explain in part the reported in vivo changes for the deoxynucleotide pool in megaloblastic marrow cells due to folate or vitamin B12 deficiency.
Asunto(s)
Deficiencia de Ácido Fólico/metabolismo , Activación de Linfocitos/fisiología , Linfocitos/metabolismo , Nucleótidos/metabolismo , Timidina/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Desoxiuridina/farmacología , Relación Dosis-Respuesta a Droga , Ácido Fólico/análogos & derivados , Ácido Fólico/análisis , Humanos , Timidilato Sintasa/análisis , Nucleótidos de Timina/metabolismoRESUMEN
Normal human peripheral blood lymphocytes cultured with phytohemagglutinin in folate-free RPMI 1640 medium supplemented with normal human serum exhibit megaloblastic maturation. These changes are accompanied by a decrease in intracellular folate content, and when compared to folate-replete cells, reveal increased 3H-thymidine incorporation into DNA together with a decreased inhibitory effect of deoxyuridine on 3H-thymidine incorporation. This in vitro system of cellular folate deficiency is a convenient model for analysis of biochemical events accompanying megaloblastic maturation.
Asunto(s)
Eritrocitos Anormales , Deficiencia de Ácido Fólico/sangre , Activación de Linfocitos , Megaloblastos , Fitohemaglutininas/farmacología , Células Cultivadas , Medios de Cultivo , ADN/metabolismo , Hematopoyesis , Humanos , Timidina/metabolismo , Uridina/metabolismoRESUMEN
A rapid radiometric assay for the folate antagonist MTX, based on the inhibition of chicken liver dihydrofolate reductase activity, has been developed. In the system the inhibition by MTX of the reduction of tritiated FH2 is standardized and used to quantitate this antagonist in plasma of subjects receiving high-dose MTX therapy. The assay permits the detection of plasma MTX levels to 5 X 10(-9)M with a coefficient of variation of 15%. The predominating circulating folate, 5 methyltetrahydrofolate, or the rescue agent leucovorin (5 formyltetrahydrofolate) do not significantly interfere with the assay at concentrations attainable with current rescue programs. Analyses of multiple plasma samples with the present assay show close agreement with the competitive protein-binding assay.