Human Plasma and Amino Acids as Moderators of Uptake and Metabolic Consequences of Antifolates in WIL-2 and Human Leukemia Cells2

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Abstract
Plasma and medium composition significantly affect cellular association of the lipid-soluble antifolate 2,4-diamino-5-(3′,4′-dichlorophenyl)-6-methylpyrimidine (DDMP). This was demonstrated by measuring association of labeled drug with cells and by assaying the antimetabolic effect of DDMP on incorporation of deoxyuridine into DNA. Uptake of both aqueous and lipid-soluble antifolates was substantially reduced in the human lymphoblastoid cell line WIL-2 when Eagle's minimum essential medium (EMEM) was substituted for a basal salts solution containing glucose [DDMP ≈50%, methotrexate (MTX) ≈30%]. Uptake of DDMP, however, was inhibited by the amino acid fraction of EMEM or glutamine alone, whereas MTX uptake was unaffected by amino acids. Further studies with human leukemia cells showed that DDMP was only about 25% as effective an inhibitor of deoxyuridine incorporation in autologous human plasma when compared to its inhibitory effect in RPMI-1640 medium. MTX inhibition of deoxyuridine incorporation in these cells was essentially unaffected by substitution of autologous human plasma for RPMI-1640 medium. Replacing EMEM with pooled human plasma resulted in a 60–70% decrease in DDMP uptake but had only a marginal effect upon MTX uptake. Thus the choice of medium is important in studies of lipidsoluble antifolates such as DDMP that have a high affinity for cellular and medium lipoprotein components.