Characterization of Granulations of Calcium and Apatite in Serum as Pleomorphic Mineralo-Protein Complexes and as Precursors of Putative Nanobacteria

Abstract

Calcium and apatite granulations are demonstrated here to form in both human and fetal bovine serum in response to the simple addition of either calcium or phosphate, or a combination of both. These granulations are shown to represent precipitating complexes of protein and hydroxyapatite (HAP) that display marked pleomorphism, appearing as round, laminated particles, spindles, and films. These same complexes can be found in normal untreated serum, albeit at much lower amounts, and appear to result from the progressive binding of serum proteins with apatite until reaching saturation, upon which the mineralo-protein complexes precipitate. Chemically and morphologically, these complexes are virtually identical to the so-called nanobacteria (NB) implicated in numerous diseases and considered unusual for their small size, pleomorphism, and the presence of HAP. Like NB, serum granulations can seed particles upon transfer to serum-free medium, and their main protein constituents include albumin, complement components 3 and 4A, fetuin-A, and apolipoproteins A1 and B100, as well as other calcium and apatite binding proteins found in the serum. However, these serum mineralo-protein complexes are formed from the direct chemical binding of inorganic and organic phases, bypassing the need for any biological processes, including the long cultivation in cell culture conditions deemed necessary for the demonstration of NB. Thus, these serum granulations may result from physiologically inherent processes that become amplified with calcium phosphate loading or when subjected to culturing in medium. They may be viewed as simple mineralo-protein complexes formed from the deployment of calcification-inhibitory pathways used by the body to cope with excess calcium phosphate so as to prevent unwarranted calcification. Rather than representing novel pathophysiological mechanisms or exotic lifeforms, these results indicate that the entities described earlier as NB most likely originate from calcium and apatite binding factors in the serum, presumably calcification inhibitors, that upon saturation, form seeds for HAP deposition and growth. These calcium granulations are similar to those found in organisms throughout nature and may represent the products of more general calcium regulation pathways involved in the control of calcium storage, retrieval, tissue deposition, and disposal.

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.…”

“…4,5 We and others have identified the serum protein fetuin-A as a major systemic inhibitor of calcification. [6][7][8][9] Together with additional blood components, fetuin-A prevents the supersaturated calcium and phosphate from precipitating by forming soluble colloidal protein-mineral nanoparticles. Calcification takes place when this humoral line of defense, inherent in small molecules (particularly pyrophosphate and magnesium) and proteins (particularly fetuin-A and albumin), is overwhelmed.…”

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

“…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.…”

“…39,35,36 Many findings and data oppose the hypothesis that CNPs are mineral-protein complexes. Although the formation of complexes of minerals and protein serum and other biological liquid under homeostasis was proposed by Martel and Young, 9 Wu et al, 6 Young et al, 7,8 and Raoult et al, 10 Kutikhin et al 2 believed that the presence of CNPs in an organism is clearly a pathological process. In addition to their pathogenicity, these proteins may have a specific immunological reaction in forming specific antibodies.…”

“…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.…”