Concentration and physicochemical characterisation of unsaturated cobalamin binding proteins in amniotic fluid

Indirect evidence for the presence of intrinsic factor in amniotic fluid has been provided recently, using either a radioisotope binding assay or a radioimmunoassay. We have determined the unsaturated cobalamin binding capacity and the physicochemical properties of the 3 cobalamin binding proteins in 59 amniotic fluids using radioisotope binding assay, gel filtration and isoelectrofocussing. A good correlation with gestational age was found for the total unsaturated Cbl binding capacity (r = 0.735) and for the concentration of unsaturated haptocorrin (r = 0.746), but not for the concentration of unsaturated intrinsic factor (r = 0.003). When their binding capacities were expressed as a percentage of the total unsaturated Cbl binding capacity, the percentage of intrinsic factor, transcobalamin II and transcobalamin III (the less acidic fraction of haptocorrin) decreased and the percentage of haptocorrin increased in function of gestational age. The physicochemical properties of intrinsic factor in amniotic fluid were close to those in gastric juice: the molecular mass was estimated to 49,200 +/- 4,900 Da (n = 24) in Sephacryl S 300 gel filtration, the cobalamin-protein complex was resolved in 2-6 isoproteins isoelectric at a pH range of 4.6-5.8 and with a mean isoelectric point of 5.18 +/- 0.16 (n = 5) in isoelectrofocusing and it crossreacted with anti-intrinsic factor autoantibodies (from a Biermer anaemia serum). Amniotic fluid collected at 13 wk of gestational age was found to contain intrinsic factor and haptocorrin with less acidic isoproteins than those usually observed in gastric juice and serum. This could indicate that sialic acid is less involved in the composition of the carbohydrate core of cobalamin binding glycoproteins in this period of the gestational age than later on and that cobalamin binding proteins have mainly a foetal origin.