Fetuin-A/Albumin-Mineral Complexes Resembling Serum Calcium Granules and Putative Nanobacteria: Demonstration of a Dual Inhibition-Seeding Concept

Abstract

Serum-derived granulations and purported nanobacteria (NB) are pleomorphic apatite structures shown to resemble calcium granules widely distributed in nature. They appear to be assembled through a dual inhibitory-seeding mechanism involving proteinaceous factors, as determined by protease (trypsin and chymotrypsin) and heat inactivation studies. When inoculated into cell culture medium, the purified proteins fetuin-A and albumin fail to induce mineralization, but they will readily combine with exogenously added calcium and phosphate, even in submillimolar amounts, to form complexes that will undergo morphological transitions from nanoparticles to spindles, films, and aggregates. As a mineralization inhibitor, fetuin-A is much more potent than albumin, and it will only seed particles at higher mineral-to-protein concentrations. Both proteins display a bell-shaped, dose-dependent relationship, indicative of the same dual inhibitory-seeding mechanism seen with whole serum. As ascertained by both seeding experiments and gel electrophoresis, fetuin-A is not only more dominant but it appears to compete avidly for nanoparticle binding at the expense of albumin. The nanoparticles formed in the presence of fetuin-A are smaller than their albumin counterparts, and they have a greater tendency to display a multi-layered ring morphology. In comparison, the particles seeded by albumin appear mostly incomplete, with single walls. Chemically, spectroscopically, and morphologically, the protein-mineral particles resemble closely serum granules and NB. These particles are thus seen to undergo an amorphous to crystalline transformation, the kinetics and completeness of which depend on the protein-to-mineral ratios, with low ratios favoring faster conversion to crystals. Our results point to a dual inhibitory-seeding, de-repression model for the assembly of particles in supersaturated solutions like serum. The presence of proteins and other inhibitory factors tend to block apatite nuclei formation or to stabilize the nascent nuclei as amorphous or semi-crystalline spherical nanoparticles, until the same inhibitory influences are overwhelmed or de-repressed, whereby the apatite nuclei grow in size to coalesce into crystalline spindles and films—a mechanism that may explain not only the formation of calcium granules in nature but also normal or ectopic calcification in the body.

Nanoparticle-Based Test Measures Overall Propensity for Calcification in Serum

“…In contrast, when occurring in the presence of lower calcium and phosphate concentrations at room temperature, CPP formation progresses only over weeks to months. 8,9,17 Under the established conditions, the 96-well format along with a measurement time of 10 hours allows the measurement of about 200 samples/d.…”

Hemoglobin aggregates studied under static and dynamic conditions involving the formation of nanobacteria-like structures

“…With the growing number of illnesses supposedly associated with nanobacteria, some researchers believe pharmaceutical agents can help combat nanobacteria buildup (14,15). Biosensors are being developed that can distinguish biocolloids and nanobacteria biogenic particles on the basis of ring formation of polystyrene nanospheres in water drops (16).…”

Clinical implications of calcifying nanoparticles in dental diseases: a critical review

“…Other investigators -Martel and Young, 9 Wu et al, 6 and Raoult et al 10 -did not obtain the same result when they used the same method. Anti-CNP monoclonal antibodies have high sensitivity and low specificity, which explains the failure to achieve cross-reactions with serum protein (albumin and fetuin-A) in other studies.…”

“…Calcifying NP antigens and antibodies were detected significantly more often in CNP-containing diseases compared to controls. 2 Although many studies [5][6][7][8]10 proposed that CNPs might have the ability to form mineral-protein complexes in normal serum in physiological conditions, in reality, Kajander et al reported that this could not be totally true. 37 However, mineral-protein complexes have been shown in a gammairradiated serum study model by Martel and Young.…”

“…One theory, describes CNPs as the smallest-known replicating entities of organic life on earth, while other theories held that CNPs are mineral-protein complexes unrelated to bacteria. [2][3][4][5][6][7][8][9][10][11][12][13] Sommer et al were of the opinion that nanobacteria have unique and unusual characteristics, which preclude placing them into any established evolutionary branch of life. 14 Kajander et al presented a table that compared CNPs, virus, prions, and bacteria using over 20 characteristics or properties, as shown in Table 1.…”