Greilberger J, Herwig R
Clin Lab. 2020 Jan 1;66(1). doi: 10.7754/Clin.Lab.2019.191121.
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 VitDdgVDBP (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 VitDdgVDBP 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 VitDdgVDBP (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 VitDdgVDBP (324.0 +/- 12.8 nmol/mL). The calculated VitD/ protein ratio showed significantly higher results in favor of VitDdgVDBP (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 VitDdgVDBP (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 VitDdgVDBP (142.3 +/- 20.0 cps; p < 0.001). Linear regression between VDBP antibody affinity and macrophage phagocytosis of VDBP, dgVDBP and VitDdgVDBP 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.
我们最近发现，通过降低氧化应激，一种新开发的25-羟基维生素D去糖基化维生素D结合蛋白复合物（VitD~dgVDBP）对自闭症和淀粉样侧索硬化症患者的生活质量有积极影响。问题在于，这种氧化应激的降低是否是由于二聚体在巨噬细胞吞噬作用的识别和激活中具有协同效应，且与游离维生素D蛋白（即维生素D结合蛋白（VDBP：Gc蛋白）和去糖基化的dgVDBP（GcMAF））相比，氧化爆发较低。
使用VDBP夹心ELISA法，对VDBP、dgVDBP和VitDdgVDBP（通过BCA蛋白技术测定为1μg/mL）等蛋白浓度进行检测，以确定其对针对人VDBP产生的多克隆抗体的免疫亲和力。使用单克隆抗体通过竞争性免疫测定法估算VDBP、dgVDBP和VitDdgVDBP的25(OH)维生素D水平。测量在不存在或存在400 pg/mL VDBP、400 pg/mL dgVDBP和400 pg/mL VitD~dgVDBP的情况下巨噬细胞的吞噬作用和氧化爆发。
与dgVDBP（186.5±16.8%；p<0.01）相比，VitDdgVDBP（769.2±35.1%）对VDBP蛋白抗体的识别显著高出4倍以上，与VDBP（100±11.4%；p<0.001）相比高出7倍。VDBP（20.7 nmol/mg；p<0.001）和dgVDBP（28.8±3.9 nmol/mL；p<0.001）的25(OH)维生素D水平显著低于VitDdgVDBP（324.0±12.8 nmol/mL）。计算得出的VitD/蛋白比值显示，与dgVDBP（0.06±0.03；p<0.001）和VDBP（0.05±0.01；p<0.001）相比，VitDdgVDBP（1.01±0.12）的结果显著更高。VitDdgVDBP（5,864.3±742.2 cps）的巨噬细胞吞噬率估计显著高于dgVDBP（2,789.6±102.7 cps；p<0.01）和VDBP（1,134.3±135.9 cps），而巨噬细胞超氧阴离子自由基的产生显示，dgVDBP（255.3±14.5 cps）的水平显著高于VDBP（148.6±24.7 cps，p<0.01）和VitD~dgVDBP（142.3±20.
