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An investigation of folate compounds in the kidney, red blood cells, and urine of rats was made by tracing the radioactivity 24 hr after the injection of 20μCi [ 3 H]folic acid. Column chromatography of tissue folates with standard marker compounds on Sephadex G-15 and G-25, and DEAE-cellulose columns shows the following results: (1) 75%-80% of kidney folates consists of reduced derivatives of pteroylpentaglutamate; (2) ca. 95% of total folates in red blood cells are 5-methyl derivatives of reduced pteroylpolyglutamates, mainly as pentaglutamate (ca. 60%) and hexaglutamate (ca. 35%); (3) more than 90% of urine folates are identified as derivatives of pteroylmonoglutamate.

[3H]Methotrexate was metabolized mainly to 4-amino-4-deoxy-10-methylpteroic acid in resting cells of Lactobacillus casei and a small amount of methotrexate remained inside the cells after 24 hours of incubation. Preincubation of L. casei with methotrexate inhibited the incorporation of [3H]folic acid completely and l-5-formyl-[3H]tetrahydrofolic acid partially into pteroyloctaglutamate and increased the amount of pteroyltetraglutamate and lower polyglutamates. In rats, methotrexate was metabolized partially to lower methotrexate polyglutamates; however, its uptake was much lower than folic acid and no pentaglutamate of methotrexate was found. Pretreatment of rats with 100 µg of methotrexate daily for 5 days decreased the uptake of [3H]folic acid into liver considerably (∼20 to ∼8%) and the radioactivity in liver was mainly reduced derivatives of pteroylpentaglutamate. With treatment with 300 µg of methotrexate per day for 6 days, however, not only the uptake of [3H]folic acid decreased to ∼4%, but also pteroylpentaglutamate comprised only ∼50% of total radioactivity in liver and the rest was lower polyglutamate and unmetabolized monoglutamate. Column chromatography with Sephadex G-25 and DEAE-cellulose provides a good method for the identification of methotrexate-related compounds.

The separation of folate compounds has been achieved by gel chromatography and the effect of buffer concentration on the elution pattern of folate derivatives was investigated by Sephadex G-15 and G-25 column chromatography. Highly conjugated polyglutamates appear in the effluent considerably sooner when eluted with water than would be expected from their molecular weights. Monoglutamates, except 10-formyltetrahydropteroylglutamic acid, are retarded on G-15 and G-25 in phosphate buffer (pH 7.0) solvent system; the situation becomes more apparent as the buffer concentration is increased. Chromatography on Sephadex G-25 with 0.1 m phosphate buffer, pH 7.0, containing 0.2 m 2-mercaptoethanol gives a satisfactory separation of pteroylpolyglutamates containing 2 to 7 glutamic acid residues.