Detection by high-performance liquid chromatography of methotrexate and its metabolites in tumor tissue from osteosarcoma patients treated with high-dose methotrexate/leucovorin rescue.

Methotrexate (MTX) polyglutamates were detected in osteogenic sarcoma tumor samples obtained from patients 24 or 48 h after receiving high-dose MTX/leucovorin rescue therapy. Tumor samples were assayed by high-performance liquid chromatography, and polyglutamyl metabolites, along with MTX, were quantitated using both direct u.v. absorption at 313 nm and an enzyme titration assay. Good agreement between these two methods was found although the more sensitive enzyme assay detected peaks in some samples not detected by u.v. absorbance. A wide variation in MTX:MTX polyglutamate levels (1:1 to 25:1) was found among the six clinical samples studied. Also, no correlation between the extent of polyglutamate formation and plasma levels (determined at the time of tumor sampling) was observed. High intracellular levels of a derivative which appears to be the 7-hydroxy metabolite of MTX were also detected in four of six samples. This material coeluted with authentic standard, showed spectral properties like standard 7-OH-MTX, and did not inhibit dihydrofolate reductase.

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.