title: In vivo methotrexate kinetics and metabolism in human hematopoietic cells. Clinical significance of methotrexate concentrations in erythrocytes.
abstract: The author has performed in vivo investigations of the methotrexate (MTX) accumulation, kinetics and polyglutamate metabolism in erythrocytes, neutrophils and myeloid bone marrow cells during clinical MTX therapy of patients with acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma and psoriasis. On the basis of these studies the clinical applicability of monitoring erythrocyte MTX concentrations in children with ALL and adult psoriasis patients have been evaluated. To accomplish this task a set of methods has been developed: 1) An automated enzymatic assay adapted for a centrifugal analyzer was used to measure MTX concentrations between 10 and 60 nmol/l in erythrocytes and serum. 2) For the study of MTX kinetics in myeloid cells, age fractionated erythrocytes and HPLC fractionated methotrexate polyglutamates a sequential radioligand binding assay with a range of 1-8 (and 1-16) nmol/l was employed. 3) Discontinuous Percoll gradients of increasing densities were used to separate myeloid cells and erythrocytes of increasing mean cell age. Declining reticulocyte counts and erythrocyte-aspartate aminotransferase activity were taken as parameters of increasing mean erythrocyte age. 4) In order to study MTX polyglutamate metabolism a high performance liquid chromatography (HPLC) procedure was set up using tetrabutylammonium phosphate in acetonitrile in an automatically generated gradient buffer system. The MTX polyglutamates were separated, and the concentrations determined by the radioligand binding assay. The individual polyglutamates were identified by comparisons with the retention times of MTX polyglutamate standards (MTX-glu1+2+3+4+6+7) which were detected spectrophotometrically at 304 nm. During 24 hour infusions MTX was incorporated predominantly in the proliferating myeloid bone marrow cells before appearing in circulating neutrophils about seven days later. Evidence for MTX incorporation in the erythroid precursors of the bone marrow was provided by demonstrating high MTX content in density fractionated reticulocyte enriched erythrocyte populations. During weekly low dose MTX treatment the erythrocyte MTX concentration reached a constant level (steady state ery-MTX) after 4-6 weeks. MTX concentrations in age fractionated red blood cells and the terminal decline of the ery-MTX and its polyglutamate forms after cessation of MTX administration revealed that maintenance of the steady state ery-MTX depended on three conditions: 1) The amount of MTX added to the circulation via MTX containing reticulocytes. 2) The in vivo efflux of MTX from circulating erythrocytes, and 3) The loss of MTX with age dependent destruction of red blood cells. The in vivo efflux of MTX accounted for a loss of MTX which was 3-4 times greater than the amount that was lost with age dependent erythrocyte destruction.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pediatrics, Aarhus Kommunehospital.
authors.forename: H
authors.initials: H
authors.lastname: Schrøder
mesh_terms: D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000097:blood* / Q000188:drug therapy; D008657:Metabolic Clearance Rate / Q000502:physiology; D008727:Methotrexate / Q000008:administration & dosage / Q000493:pharmacokinetics*; D009369:Neoplasms / Q000097:blood* / Q000188:drug therapy
chemical_list: D008727:Methotrexate