Secondary isotope effects in studies using radiolabelled folate tracers

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-aminopurine and several amino acids. Such isotope effects may be more widespread and more important than is generally accepted.