In Vitro Binding of Folates by Body Fluids

Abstract

It is useful to differentiate between saturated folate binders in serum (carrying endogenous folate) and unsaturated binders (investigated in the present study). These two groups of binders need not necessarily be chemically identical and the unsaturated binder may even be an in vitro artifact, especially when measured with non-physiological folates. Macromolecular binding of radio-active N-5-methyl tetrahydrofolic acid (CH3H4PteGlu) and/or folic acid (PteGlu) by human serum and urine was assessed by means of exhaustive saline dialysis, haemoglobin-coated charcoal adsorption, column chromatography with DEAE-Sephadex A-50, and sucrose gradient analysis. Binding was found to be minimal or absent. Charcoal adsorption showed a mean serum binding capacity of 0 mug/1. for PteGlu and 0.58 mug/1. for CH3H4PteGlu. In pregnancy the mean serum values were 0.23 mug/1. for PteGlu, 0.66 mjg/1. for CH3H4PteGlu, and with folate deficiency 0.30 mug/1. for PteGlu, 0.49 mug/1. for CH3H4PteGlu. Mean urinary folate binding was minimal (less than 0.5 mug/1.), and red cell haemolysate similarly revealed very low binding on exhaustive dialysis. Column chromatography showed that tracer doses of [14C]PteGlu added to serum migrated distally to the protein zone; [14C]CH3H4PteGlu similarly showed no evidence of protein binding. On a sucrose gradient [14C]PteGlu also separated clear of the protein zone.

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

“…Evidence exists that this binder may withhold folate from living cells [5,18], but its true functional significance remains obscure. It is present in normal plasma or serum in minor quantities only, rarely rising above 1 /¿g/1 [5][6][7]12]. It has been suggested that the unsaturaled binder is structurally similar to the saturat ed binder of endogenous folate [9,15,16,24],…”

“…The unsaturated folate-binding capacity of plasma was measured after incuba tion with excess " C-5CH3H4PteGlu and 3H-PteGlu added simultaneously as pre viously described [12], except that free radiofolate was removed by dialysis and not by charcoal adsorption.…”

“…Liquid chromatography with Sephadex DEAE-A50: 20 X 0.8 cm columns were used, 0.5 ml samples applied, and 40-50 X 1 ml fractions eluted stepwise with 7 X5 ml buffer-NaCl solutions at pH 7 [12]. Eluates were measured for radioactivi ty and total protein (UV extinction at 280 nm): eluates 0-10 represent y-globulin and fibrinogen, 10-16 /5-globulin with transferrin, 16-25 a-globulins, 25-45 albu min and prealbumin.…”

“…Radioactivity was measured by liquid scintillation counting on an Intertechnique SL 30 connected to a Multi-9 computer [12].…”