Vitamin D - Deglycosylated Vitamin D Binding Protein Dimer: Positive Synergistic Effects on Recognition, Activation, Phagocytosis and Oxidative Stress on Macrophages.

We have recently shown positive effects in the quality of life in autism and amyloid lateral sclerosis patients using a newly developed 25-OH vitamin D deglycosylated vitamin D binding protein complex (VitD~dgVDBP) by reducing oxidative stress. The question arises whether this reduction of oxidative stress was due to a synergistic effect of the dimer in the recognition and activation of phagocytosis on macrophages combined with a lower oxidative burst compared to the VitD free proteins, namely vitamin D binding protein (VDBP: Gc Protein) and deglycosylated dgVDBP (GcMAF). VDBP sandwich ELISA of equal protein concentration of VDBP, dgVDBP, and VitD~dgVDBP (1 µg/ mL by BCA protein technique) was used to identify immune affinity to polyclonal antibodies raised against human VDBP. The 25(OH) vitamin D levels of VDBP, dgVDBP and VitD~dgVDBP were estimated by a competitive immune assay using a monoclonal antibody. Macrophage phagocytosis and oxidative burst in absence or presence of 400 pg/mL VDBP, 400 pg/mL dgVDBP, and 400 pg/mL VitD~dgVDBP was measured. The recognition of the antibody against VDBP protein was significantly more than 4-fold higher for VitD~dgVDBP (769.2 +/- 35.1%) compared to dgVDBP (186.5 +/- 16.8 %; p < 0.01) and 7-fold higher to VDBP (100 +/- 11.4 %; p < 0.001). 25(OH) vitamin D levels of VDBP (20.7 nmol/mg; p < 0.001) and dgVDBP (28.8 +/- 3.9 nmol/mL; p < 0.001) was significantly lower than of VitD~dgVDBP (324.0 +/- 12.8 nmol/mL). The calculated VitD/ protein ratio showed significantly higher results in favor of VitD~dgVDBP (1.01 +/- 0.12) compared to dgVDBP (0.06 +/- 0.03; p < 0.001) and VDBP (0.05 +/- 0.01; p < 0.001). The estimation of macrophage phagocytosis rate of VitD~dgVDBP (5,864.3 +/- 742.2 cps) was significantly higher compared to dgVDBP (2,789.6 +/- 102.7 cps; p < 0.01) and VDBP (1,134.3 +/- 135.9 cps) whereas the production of macrophage superoxide anion radicals showed significantly higher levels of dgVDBP (255.3 +/- 14.5 cps) in comparison to VDBP (148.6 +/- 24.7 cps, p < 0.01) and VitD~dgVDBP (142.3 +/- 20.0 cps; p < 0.001). Linear regression between VDBP antibody affinity and macrophage phagocytosis of VDBP, dgVDBP and VitD~dgVDBP resulted in a correlation coefficient of r = 0.95 in favor of VitD~dgVDBP. VitD~dgVDBP (Il-42) showed higher macrophage activation and lower oxidative burst than VitD free dgVDBP (GcMaf) and VDBP (Gc) which may result from a synergistic effect by presenting protein bound Vitamin D better to macrophages.