In Vivo Plasma and Urine Folate Binding after Ingestion of ³H‐Folic Acid and ¹⁴C‐Methyl‐Folate

Abstract

After simultaneous ingestion of equivalent amounts of [3H]folic acid (3H‐PteGlu) and [14C]N5‐methyl‐tetrahydrofolic acid (14C‐CH3‐H4PteGlu) we were able to demonstrate progressive macromolecular binding of radiofolate in plasma, which appeared to be near maximal at 6 h. Bound radiofolate was predominantly of 14C‐CH3H4PteGlu origin, and only at 24 h could 3H incorporation be demonstrated. The binder eluted with albumin from Sephadex DEAE‐A50 columns. In urine a smaller bound radiofolate fraction, with approximately equal amounts of 3H and 14C, appeared after 5.5 h. Plasma chromatography showed radio‐PteGlu (peak 1) to be rapidly converted to CH3‐H4PteGlu (peak 2), with subsequent appearance of two further radiofolate peaks (peaks 3 and 4) the nature of which is as yet unclear. Urine showed similarly placed fractions but their magnitude differed, and urinary peak 3 in particular was much more prominent than its plasma counterpart.

Comparison between the Unsaturated Plasma Folate Binder and <i>in vivo</i> Labelled Plasma Folate Binder

“…In view of the fact that the unsaturated plasma binder preferentially binds PteGlu, while the endogenous (normally saturated) binder has a greater affinity for reduced physiological folates, both in vitro (table I) and in vivo [8,13], this limited study suggests that these two binders are probably not identical. With the relatively small DEAE columns used in this study, we were unable to demonstrate PteGlu protein binding either before or after acid dissociation of the endogenous folate-binder complex.…”

“…The main endogenous binder is chromatographically albumin-associated [8,13,19,20] on DEAE-A50 columns, although minor binders associated with a2-macroglobulins [21] and even transferrin [22] have been suggested. After dissociating folate at acid pH from cord serum binder, Kamen and Caston [9] found a denuded protein with molecular mass of 40,000, a binding preference for PteGlu rather than CH3H4PteGlu, but a binding capacity only 10% of the original serum binder.…”

“…Plasma in heparinized 'Vacutainer' tubes (Beckion-Dickenson) were collected from (i) 4 normal subjects, and (ii) 2 subjects with normal folate status in whom the endogenous binder had been labelled in vivo after ingestion of radiofolate, as pre viously described [8,13], Subjects gave informed consent for this study, which had been accepted by the Medical Faculty's Ethical Research Committee and the Radia tion Control Committee for the Orange Free State (acting on behalf of the Atomic Energy Board). 3 subjects (with normal folate status) took 575 fig u C-CH3H4PteGlu (50 fid ).…”

“…In vivo la belling of the endogenous binder [8] showed N5-methyltetrahydrofolate (5CH3H4PteGlu) to bind in preference to folic acid (PteGlu), whereas in vitro work showed oxydised folates to bind more avidly to the unsaturated binder than did reduced (physiological) folates [4,5]. However, Kamen and Caston [9] making use of acid dissociation of the folate-binder complex [10], reported that the endogenous binder of neonatal serum also binds PteGlu in preference to 5 CH3H4PteGlu.…”

Retained folates in the rat

“…[3H]Folic acid has been used to study folate metabolism in human subjects (e.g. Landon et al, 1975;Retief et al, 1976) and at the end of the experiment (usually several days after administration of [3H]folic acid) a large dose of unlabelled folic acid has been used to 'flush out' any residual folate that may remain in the subject. An appreciable quantity ofthe administered radioactivity was unaccounted for at the end of these experiments.…”