Biochemical Rationale for the Development of a Rapid Spectrophotometric Assay for Diagnosis of Mineral Homeostasis...

Patients with coronary and cerebral atherosclerosis are characterized by increased levels of total serum calcium, ionized calcium, and phosphate, against a background of reduced levels of total serum protein and albumin. Here we aimed to develop a rapid diagnostic assay for mineral homeostasis disorders, based on assessing capacity of the acidic plasma proteins to bind excess calcium and phosphate ions. Plasma from bony fish, amphibians, reptiles, birds, mice, and patients with myocardial infarction was incubated with excess concentrations of calcium and phosphate at 37°C for varying time periods. The following assay readouts were defined: (i) plasma optical density after supersaturation with calcium and phosphate ions, reflecting excessive formation of calciprotein particles (CPPs); and (ii) CPP concentration in plasma. CPPs were formed in all vertebrates. The most pronounced plasma calcification propensity was observed in the human and mouse plasma, suggesting an evolutionary significance of CPP formation as a mechanism for clearance of excess circulating calcium and phosphate ions in mammals. Among the 11 protocols of supersaturation with calcium and phosphate ions, stable increase in plasma optical density at 620 nm wavelength (normalized OD₆₂₀, a measure of plasma calcification propensity) was achieved by adding solutions of CaCl₂ (+2 mmol/L, +50 µL), Na₂HPO₄·12H₂O (+2 mmol/L, +50 µL), and NaCl (+15.4 mmol/L, +20 µL) to plasma (80 µL). Increase in the normalized OD₆₂₀ was consistently detected within 10 min from the reaction onset during incubation in a microplate shaker (37°C), with mild-to-moderate variability across the parallel or sequential measurements and between the different operators. These results support relevance of validating the developed diagnostic assay for assessing mineral homeostasis disorders in the expanded cohorts of patients with myocardial infarction and ischemic stroke.