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Background: 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).
Methods: 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.
Results: 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.
Conclusions: 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.
DOI: 10.7754/Clin.Lab.2019.191121
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