Nanoparticle-based test measures overall propensity for calcification in serum

Abstract

Vascular and soft tissue calcification contributes to cardiovascular morbidity and mortality in both the general population and CKD. Because calcium and phosphate serum concentrations are near supersaturation, the balance of inhibitors and promoters critically influences the development of calcification. An assay that measures the overall propensity for calcification to occur in serum may have clinical use. Here, we describe a nanoparticle-based assay that detects, in the presence of artificially elevated calcium and phosphate concentrations, the spontaneous transformation of spherical colloidal primary calciprotein particles (CPPs) to elongate crystalline secondary CPPs. We used characteristics of this transition to describe the intrinsic capacity of serum to inhibit the precipitation of calcium and phosphate. Using this assay, we found that both the sera of mice deficient in fetuin-A, a serum protein that inhibits calcification, and the sera of patients on hemodialysis have reduced intrinsic properties to inhibit calcification. In summary, we developed a nanoparticle-based test that measures the overall propensity for calcification in serum. The clinical use of the test requires evaluation in a prospective study.

Figure 1.

Test principle. (A) Three-dimensional dynamic light scattering (3D-DLS) measurement of calcium (10 mM) and phosphate (6 mM) precipitating in the presence of fetuin-A (1 g/L); primary calciprotein particles (CPPs; R_h of 75 nm) undergo spontaneous transition to secondary CPPs (R_h of 120 nm). (B) Immediate precipitation of calcium and phosphate in the absence of serum and delay of particle formation (primary and secondary CPPs) in the presence of serum; this delay reflects the intrinsic calcification-inhibitory forces of serum. (C) Precipitation of calcium and phosphate in the presence of serum monitored by 3D-DLS. (D) Schematic illustration of nephelometry, a method capable of detecting (nano) particles by measuring light scattering. (E) Nephelometric measurement of the primary to secondary CPP transformation step over time in the presence of serum. (F) Exemplary readout of serum measurements performed in 96-well plate format with the Nephelostar nephelometer (BMG Labtech). R_h, hydrodynamic radius; RNU, relative nephelometric units.

Figure 2.

Particle characterization. (A) Pellets after sharp centrifugation (16,000×g for 120 minutes at 20°C) of the solutions shown in Figure 1A. (B) Scattering electron microscopy (SEM) and transmission electron microscopy (TEM) of primary CPPs. (C) SEM and TEM of secondary CPPs. (D) Coomassie blue stain of protein contents of primary and secondary CPPs. (E) Albumin and fetuin-A Western blots of primary and secondary CPPs. (F) Decrease of phosphate concentrations from supernatant solution on formation of primary and secondary CPPs. Scale bars, 1 μm for SEM in B and C; 500 nm for TEM in B and C.

Figure 3.

Serum measurements. (A) Nephelometry assay using sera from adult 10- to 16-month-old noncalcifying wild-type DBA/2 mice (green), noncalcifying heterozygous fetuin-A^+/− knockout mice having half-normal serum fetuin-A (red), and heavily calcifying fetuin-A–deficient homozygous fetuin-A^−/− knockout mice (black). (B) Nephelometry assay with sera from 20 hemodialysis patients (black) and 20 healthy volunteers (green).

Figure 4.

Impact of selected serum components on test readout. (A) Nephelometry assay in the absence of serum. Spiking with fetuin-A delayed the transition from primary to secondary CPPs. and decreased RNU. NaCl indicates a reference precipitation curve without spiking. (B) Nephelometry assay in the presence of serum. Note that fetuin-A, albumin, and magnesium inhibited and lysozyme, phosphate, and calcium promoted precipitation. The red, nonspiked reference curve is the pooled serum from healthy volunteers. (C) Alternative presentation of T₅₀ and RNU₅₀ values taken from Figure 4B. Minerals mainly influence T₅₀, whereas proteins influence both T₅₀ and RNU₅₀. Quantities spiked in A and B: fetuin-A: 0.0625, 0.125, 0.25, 0.375, and 0.5 g/L (normal serum concentration is about 0.5 g/L); albumin: 3.125, 6.25, 12.5, 25, and 50 g/L (normal serum concentration is 35–52 g/L); lysozyme: 3.125, 6.25, 12.5, 25, and 50 g/L (normally not present in serum); Ca²⁺: 0.25, 0.75, 1.0, 1.5, and 2.0 mmol/L (normal serum concentration is 2.1–2.55 mmol/L); PO₄: 0.25, 0.75, 1.0, 1.5, and 2.0 mmol/L (normal serum concentration is 0.84–1.45 mmol/L); Mg²⁺: 0.25, 0.5, and 0.75 mmol/L (normal serum concentration is 0.75–1.0 mmol/L).

Figure 5.

Schematic illustration of test principle. The addition of 10 mM calcium and 6 mM phosphate to serum triggers the formation of primary CPPs (<100 nm), reflecting a serum-intrinsic defense mechanism against calcification. Primary CPPs contain fetuin-A and albumin as well as amorphous calcium phosphate. Primary CPPs undergo spontaneous transformation into secondary CPPs, which contain crystalline calcium phosphate and an extended spectrum of serum proteins. The transformation time (T₅₀) is specific for individual sera and depends on—among other factors—the concentration of the serum constituents indicated in the figure. The transformation from primary to secondary CPPs is detected by time-resolved nephelometry and provides an estimate of the integrated calcification-inhibitory capacity present in this particular serum sample.