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The reticulocytic rat: a model for analysis of methotrexate polyglutamate dynamics in situ
Abstract:
The metabolism of the antifol methotrexate in rat erythrocytes in order to determine the basis for its persistence in this tissue was investigated. By using molecular sieve high-performance liquid chromatography and a methotrexate radioassay, we have detected methotrexate polyglutamates through the heptaglutamate in red cells of rats given a single i.p. injection of 10 mg/kg of methotrexate. Higher concentrations of methotrexate and its polyglutamates were found in blood from animals made reticulocytic with phenylhydrazine than in normal erythrocytes, lending credence to the hypothesis that red cell methotrexate polyglutamates arise from developing erythrocytes in the marrow. This was borne out in discrete populations of red cells and reticulocytes separated by density centrifugation through Percoll. Reticulocytes also were shown to have no significant gamma-glutamylcarboxypeptidase (conjugase) activity. We present the reticulocytic rat as an in vivo model for studying polyglutamate metabolism.
Insights
Methotrexate persists in rat red blood cells due to polyglutamate formation. Developing red blood cells (reticulocytes) accumulate these metabolites, lacking the enzyme to break them down, making the reticulocytic rat a model for studying this metabolism.
Area of Science:
- Pharmacology
- Biochemistry
- Hematology
Background:
- Methotrexate (MTX) is an antifolate drug with known persistence in certain tissues.
- Understanding MTX metabolism in erythrocytes is crucial for explaining its prolonged effects.
Purpose of the Study:
- To investigate the metabolism of methotrexate in rat erythrocytes.
- To determine the basis for methotrexate's persistence in red blood cells.
- To establish an in vivo model for studying polyglutamate metabolism.
Main Methods:
- High-performance liquid chromatography (HPLC) and radioassay were used to detect MTX polyglutamates.
- Density centrifugation through Percoll separated red blood cell populations.
- Phenylhydrazine induced reticulocytosis in rats.
Main Results:
- Methotrexate polyglutamates, up to heptaglutamate, were detected in rat erythrocytes.
- Reticulocytes showed higher concentrations of MTX and its polyglutamates compared to normal erythrocytes.
- Reticulocytes exhibited minimal gamma-glutamylcarboxypeptidase (conjugase) activity.
Conclusions:
- Methotrexate polyglutamate formation occurs in developing erythrocytes (reticulocytes) within the bone marrow.
- The lack of conjugase activity in reticulocytes contributes to MTX polyglutamate persistence.
- The reticulocytic rat serves as a valid in vivo model for studying methotrexate polyglutamate metabolism.