Table 2.
Photometric quantification in the filtrate after ultrafiltration of samples after an incubation time of 1 and 12 h during stage 1 and 2 of the CPPs, respectively
| Cutoff | 300 kDa | 3 kDa | |||
|---|---|---|---|---|---|
| Centrifugation | 2000 g, 4°C,Flow direction toward the top | 12,000 g, 4°C,Flow direction toward the bottom | |||
| CPP | − | − | |||
| BF/CPM | + | − | |||
| Mineral ions/clusters | + | + | |||
| Initial concentration | |||||
| BF [mg/mL] | 1 | 0.35 | 2.5 (S3) | 0.5 (S4) | |
| Ca2+ [mM] | 10 | 10 | 10 | 10 | |
| Filtrate analysis after incubation | |||||
| t ≈ 1 h | BF [mg/mL] | 0.78 ± 0.11 | 0.22 ± 0.05 | − | − |
| Ca2+ [mM] | 6.3 ± 0.6 | 6.3 ± 1.0 | 6.5 ± 0.6 | 7.0 ± 0.3 | |
| t ≈ 12 h | BF [mg/mL] | 0.83 ± 0.10 | 0.16 ± 0.02 | − | − |
| Ca2+ [mM] | 2.9 ± 0.3 | 2.8 ± 0.1 | 3.5 ± 0.8 | 3.2 ± 0.4 | |
Samples were ultrafiltrated by centrifugation with 300-kDa (column 2 and 3) and 3-kDa (column 4 and 5) MMCO membranes. Primary and secondary particles are formed after 1 and 12 h of incubation time, respectively. The radius of a primary CPP is at least ∼10 times larger than the radius of a fetuin-A molecule (10). Therefore a 300-kDa membrane retains any CPPs while the ions and BF molecules pass. Only ions and tiny aggregates can pass a 3-kDa membrane while CPPs and BF molecules are retained. Concentrations of the samples: 10 mM CaCl2, 6 mM NaH2PO4, 50 mM Tris pH 7.4, 140 mM NaCl, and 0.35, 0.5, 1, or 2.5 mg/mL BF.