A procedure is described for the chemical quantitation of three different pools of one-carbonsubstituted, reduced folates in rat liver with the simultaneous determination of their corresponding poly-y-glutamyl chain lengths. Pool 1 is made up of 5,10-methylenetetrahydrofolates and unsubstituted tetrahydro-and dihydrofolates. Pool 2 is made up solely of 5-methyltetrahydrofolates and pool 3 includes the folates with one-carbon substituents at the formyl level of oxidation. Seventy-two hours after an intraperitoneal injection of ['Hlfolic acid into rats, the label was incorporated predominantly into pentaglutamates in pools 1 and 2. The hexaglutamates represented about one-third or less and the pentaglutamates approximately two-thirds of these pools. In contrast, in pool 3 the proportion of hexaglutamates was approximately equal to that of pentaglutamates. The proportions of radioactivity present in pools 1, 2, and 3 were 22.8 + 0.4, 27.3 + 3.0, and 49.9 f 3.1%, respectively.
In a previous communication
(1) we have presented and validated the principle of a new analytical procedure designed to quantitate three different pools of one-carbonsubstituted, reduced folates.' Briefly, the method is based on the fact that the naturally occurring folates can be grouped in three pools according to their response to acid treatment and subsequent air oxidation: Pool 1 is made up of 5,10-methylenetetrahydrofolates (5, lo-CH2-H4-PteGlu,)' and unsubstituted tetrahydro-and dihydrofolates (H,-PteGlu,, H,-PteGlu,).
Pool 2 is made up solely of 5-methyltetrahydrofolates (5-CH,-H,-PteGlu,)
and pool 3 includes ' The term folates will be used to indicate folic acidderived coenzymes differing from each other in the nature of the one-carbon substituent (if present) and/or in the chain length of the poly-y-glutamyl chain. The abbreviation PteGlu. will be used to indicate a polyy-glutamyl derivative of pteroylglutamic acid with 'W' number of y-glutamyl residues.