Novel serum assay measures calcification risk

By Andrew Czyzewski, medwireNews Reporter

Researchers have devised a nephelometer-based assay that measures the risk for calcification in serum samples.

The test also provides a mechanistic basis for calcification regulation, whereby colloidal primary calciprotein particles (CPPs) "ripen" to form secondary CPPs under certain conditions.

"Given the relevance of calcifications for patient morbidity and mortality, it may be a useful tool for the investigation of biomineralization-related issues in both routine clinical use as well as clinical and basic research," Andreas Pasch (University of Bern, Switzerland) and colleagues comment.

Calcium and phosphate concentrations are close to supersaturation in most tissues and body fluids, and are therefore said to be metastable. Under physiologic conditions calcium and phosphate only mineralize in bones and teeth, whereas soft tissue normally does not calcify. This indicates that biomineralization is a tightly regulated, site-specific process, and perturbations, in the blood vessels for example, can lead to cardiovascular disease.

In the current study, the researchers devised a label-free 96-well plate-based assay, which measures the conversion of primary to secondary CPPs by detecting the time-resolved changes of laser light scattering (nephelometry) associated with it.

They performed the assay on serum obtained from knock-out mice deficient in fetuin-A (a major systemic inhibitor of calcification); wild-type mice; 20 human patients on hemodialysis (known to exhibit an increased risk for accelerated vascular and soft tissue calcifications); and 20 healthy volunteers.

The knock-out mice showed a higher propensity for calcification than wild-type mice (as measured by one-half maximal relative nephelometric units [RNU₅₀]), as did the hemodialysis patients relative to controls.

"These observations indicate that the test indeed quantifies the inhibitory potency of calcification," Pasch et al comment.

They also tested the impact of serum-derived proteins and small molecules on the performance of the assay by spiking it with fetuin-A, albumin, lysozyme, calcium, phosphate, or magnesium.

The formation of serum-derived primary CPPs depended on the presence of fetuin-A, the strongest serum-inherent calcification inhibitor, with albumin acting synergistically. By contrast, the timing of the primary to secondary CPP transition largely depended on the presence of small molecules, with phosphate and calcium accelerating and magnesium delaying crystallization.

"When trapped in body compartments, primary CPPs can undergo transition to secondary CPPs, which was shown in a case of progressive calcifying peritonitis and cases of amputated limbs of diabetic patients," Pasch et al comment.

"To the best of our knowledge, we present here the first potentially widely applicable technique with a reasonable throughput capacity for measuring extraosseous calcification propensity," they add.

The research is published in the Journal of the American Society of Nephrology.

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