Secondary isotope effects in studies using radiolabelled folate tracers

Abstract

The use of isotopically labelled compounds as metabolic tracers in in vivo and in vitro studies is particularly important in folate biochemistry, where the natural coenzyme forms occur in transient and trace amounts. However, during our studies on the whole body metabolism of radioactive folate tracers, we have now observed that some forms of isotopically labelled folate are biologically distinguishable from the unlabelled carrier molecules by an as yet unidentified secondary isotope effect. Folates excreted in the urine of rats dosed previously with mixtures of ¹⁴C- and ³H-labelled folate derivatives are apparently ³H enriched, that is contain more ³H than ¹⁴C relative to the dosed compound. This apparent ³H enrichment was largely due to the enhanced absorption of ³H-folate from the intestine, confirmed by studies with everted sac preparations of rat jejunum in which it was found that the ³H-labelled folate in the mixture was transported from the mucosal into the serosal compartment at a faster rate than both the ¹⁴C-labelled folate and the unlabelled carrier. Secondary isotope effects were also observed on ion-exchange chromatography, ³H-labelled folates with ³H at the C-9 position eluting fractionally earlier than the corresponding unlabelled or [2-¹⁴C]folate from DEAE-cellulose, a behaviour similar to that reported earlier for isotopically labelled 2-aminopurine¹ and several amino acids². Such isotope effects may be more widespread and more important than is generally accepted.