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MINERAL METABOLISM: Edited by Aline Martin and Tamara Isakova

Calciprotein particles: mineral behaving badly?

Smith, Edward R.a,b; Hewitson, Tim D.a,b; Jahnen-Dechent, Willic

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Current Opinion in Nephrology and Hypertension: July 2020 - Volume 29 - Issue 4 - p 378-386
doi: 10.1097/MNH.0000000000000609
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Abstract

INTRODUCTION

The minerals calcium and phosphate are indispensable for cellular metabolism. Both ions typically occur in millimolar concentrations in biological fluids. This produces a transport problem because calcium phosphates, like hydroxyapatite, are only sparingly soluble in water at physiological pH. Although complexation reduces the ion product significantly, levels remain such that only relatively minor perturbations in ionic content or acid–base balance could result in in spontaneous mineralisation of the extracellular matrix. Systems have therefore evolved to regulate body calcium and phosphate balance, to restrict mineralization to bone and teeth and to handle any excess mineral formed as a result of local perturbations in calcium phosphate homeostasis. Although this rationalises the emergence of osseous endocrine functions to regulate organismal balance over hours and days, on a more fundamental level, it necessitated the advent of proteins to bind and chaperone nascent mineral to ensure adequate mineralization in the right place at the right time, over shorter biochemically-relevant timescales. Calciprotein particles (CPP), nano-sized colloidal complexes of mineral and proteins that form spontaneously in protein-containing fluids supersaturated with calcium and phosphate may be one solution to this problem [1,2]. Here we discuss recent work that has shed light on CPP synthesis, metabolism and their role in fostering physiological mineralization while mitigating the risk of ectopic deposition. 

Box 1
Box 1:
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CALCIPROTEIN PARTICLES FORMATION IN VITRO

The term ‘calciprotein particle’ was coined because of structural and functional similarities to lipoprotein particles [3]. Whereas apolipoproteins help organize and solubilize their lipid cargo for transport in the circulation, liver-derived fetuin-A (also known as α2-Heremans-Schmid glycoprotein; ASHG) – assumes the indispensable function of binding to and stabilising nascent mineral domains in CPP. In the absence of fetuin-A, CPP are highly unstable and mineral precipitates within minutes [3,4].

CPP form spontaneously in fetuin-A-containing solutions enriched with supersaturating concentrations of calcium and phosphate. Over several decades of detailed biophysical analysis of mineral nucleating from these solutions has revealed that fetuin-A interacts with mineral species over several orders of scale (Fig. 1). The basic building blocks for CPP are calciprotein monomers (CPM), composed of sub-nanometre-sized clusters of mineral ions tightly bound by monomeric fetuin-A [5,6]. Interaction with fetuin-A does not impact the nucleation of these complexes but controls the subsequent aggregation of clusters into larger entities [7]. CPM can consolidate into spherical aggregates up to 100 nm in diameter. These self-assembling particles lack long-range molecular order and are termed primary CPP (Fig. 1B). By volume each particle consists of ∼25% fetuin-A (several hundred molecules) with the remainder occupied by amorphous calcium phosphate (ACP) [3]. In a final step, the mineral phase can undergo solid–solid phase transition and rearrangement into ordered, more thermodynamically stable crystalline phases like hydroxyapatite forming particles termed secondary CPP. Under supersaturated conditions CPP continue to accrete mineral but only sediment after several days at room temperature.

FIGURE 1
FIGURE 1:
The formation and maturation of calciprotein particles in fetuin-A containing fluids supersaturated with calcium and phosphate. Calcium (Ca) and phosphate (p) ions can densify to form sub-nanometre (∼0.9 nm) sized ion clusters (CaP) with varying, often calcium-deficient, stoichiometries in neutral simulated body fluids [64]. Neighbouring clusters can aggregate, bridged by shared phosphate groups. In physiological fluids mineral-binding proteins like fetuin-A can form tight interactions with these prenucleation ion clusters, chaperoning mineral through nucleation, phase separation and eventual crystallisation. The basic building blocks for CPP are calciprotein monomers (CPM), composed of CaP ion clusters tightly bound and stabilized by monomeric fetuin-A primarily through interactions with acidic residues in its amino-terminal cystatin-like domain 1 (CY1). A computational homology model of human fetuin-A (UniProt#P02765 encompassing G22-Q260) is shown with the surface coloured according to its predicted electrostatic potential (−ve red → +ve blue). Dashed box outlines negatively charged region containing the CY1 domain and is enlarged alongside to depict the array of glutamic and aspartic acid residues (red) that mediate the interaction with mineral complexes and crystals. The 3D model was generated using the SWISS-MODEL workspace [65] based on the recently published 2.3 Å-resolution crystal structure of fetuin-B [66], the closest relative to fetuin-A (Protein Data Bank entry: 6HPV) and visualized using UCSF Chimera [67]. Intrinsically disordered regions such as the C-terminal domain may also be involved in binding regulation (not shown). CPM can consolidate into spherical ∼30–100 nm aggregates (primary CPP; CPP-I), carrying insoluble mineral as amorphous calcium phosphate in diffuse fetuin-A-bound mineral domains. In a final step, the mineral phase can undergo transition and rearrangement into ordered, more thermodynamically stable crystalline phases like hydroxyapatite (secondary CPP; CPP-II), where the mineral is organised in densely-packed needle-shaped lamellae. Morphologically these distinctive crystalloid secondary CPP take the form of prolate ellipsoids, with long axis diameters of ∼100–250 nm. Approximate sizes are shown. Representative cryogenic TEM images of CPP-I and CPP-II are provided in the panels above the schematic (not to scale).

Synthetic formation and endogenous levels in serum

Analyses in mineral-enriched body fluids likewise reveal a stepped process of particle formation and ripening, although the speed of transition from primary to secondary CPP is subject to modulation by a number of factors that are typically present in complex biological fluids [4]. A balance is conceived between factors that promote crystallisation (e.g. phosphate) and those that slow the process – from small molecules operating as crystal disrupters (e.g. pyrophosphates) – to substances that complex ions (e.g. albumin) or adsorb to the mineral surface creating a diffusion barrier that sterically hinders or ‘shields’ crystals from further growth (e.g. fetuin-A) [2]. The transition also appears exquisitely sensitive to changes in temperature and pH. The interplay of all these factors, and other as yet uncharacterised modulators, alters the thermodynamic and kinetic landscape of particle ripening and governs how quickly this transformation occurs. This biophysical transition forms the basis of the T50 test [4], which provides an integrated assessment of the ability of serum to resist de-novo crystal formation. This test holds promise as a clinical tool to refine risk estimates for future adverse events [8–11] and for guiding case management [12,13▪,14].

It is important to stress the distinction between the readout of this ex-vivo functional assay and measurement of endogenous CPP levels, as the two assessments do not necessarily correlate [15]. In vivo, CPP may be formed in environments that differ in composition to serum: thus, not all individuals with high serum CPP levels necessarily have accelerated T50 times. As we discuss below, shortened T50 times ex vivo are also unlikely to manifest in enhanced secondary CPP formation in blood in vivo. Measurement of native CPP in complex biological fluids is challenging because of their low abundance and inherent instability, but recent advances using the fluorescent bisphosphonate-based probe OsteoSense, coupled with nanoparticle flow cytometry [16–18] or gel-filtration-based methods [19] have provided new tools to assess levels in health and disease. To date, only assessment by flow cytometric analysis allows differentiation of CPP from other mineral-containing particles present in the extracellular fluid (ECF). Ascertainment of high molecular weight fractions containing fetuin-A or mineral following centrifugation are not specific for CPP and may yield spurious results [20].

EVIDENCE FOR CALCIPROTEIN PARTICLES IN VIVO

Over the decades, many laboratories have reported formation of CPP-like particles in cultured biological fluids – often coining another name in the process (e.g. bions, nanons, calcifying nanoparticles, mineralo-protein nanoparticles) – but without exception these particles were only observed after prolonged culture or when fluids were ‘seeded’ with additional sources of mineral [20]. Indeed, some investigators mistook observations of apparent ‘self-propagation’ as signs of life, spawning the now debunked concept of nanobacteria [21]. However, far from being exquisite new lifeforms from Mars [22], these where simply the product of mineral and mineral-binding protein constituents of standard culture media [23]. Another concern is that attempts to isolate and characterise these particles might itself result in artefactual synthesis or crystallization [19].

Despite these important caveats, there is evidence to support their existence in vertebrate biology. In humans, arguably the earliest evidence came from the fluid drained from a patient with calcifying peritonitis [24] and in the serum of a dialysis patient with calcific uraemic arteriolopathy [25,26]. In both instances, the particles observed were reminiscent of secondary CPP. To circumvent issues of artefactual generation we developed a more rapid protocol of isolation using fresh serum, but this required scaling up the process and the use of much larger input volumes of serum in order to detect CPP [27▪]. In contrast to earlier studies this revealed that the predominant particle type was amorphous not crystalline and thus primary CPP. In fact, secondary CPP were infrequently observed, out-numbered by primary CPP 10 : 1. On reflection this is perhaps to be expected for several reasons: firstly, considering the many hours in supersaturating conditions needed to permit ripening of primary to secondary CPP ex vivo[4]; and secondly, because precursor forms to secondary CPP are rapidly cleared from the circulation and metabolised [28▪▪]. Thus, the biological plausibility of circulating secondary CPP is low. Fetuin-A-laden mineral complexes that become bound and fixed in tissue may, however, eventually ripen to particles resembling secondary CPP in situ.

When we subsequently developed a flow cytometric-based method to enumerate CPP in biological fluid it became readily apparent that, while modestly elevated in dialysis patients compared to healthy controls [16], CPP remained grossly outnumbered by other particulate species; extracellular vesicles and lipoprotein particles are present in a thousand and a million-fold excess, respectively [29,30]. This has important implications when we come to assess the likely pathophysiological relevance of circulating CPP, but also how we design and interpret models to interrogate function of these particles. It should be pointed out that much of the in vitro work performed to date has used CPP levels typically many orders of magnitude over levels found in vivo[16]: for instance, elevating phosphate from 1 to 3.5 mmol/l generates 1x1011 CPP/ml in standard culture media (10% foetal calf serum in DMEM) over 3 days [31]. Ascribing effects to particular subpopulations of particles (e.g. primary vs. secondary CPP) is also problematic because precursor forms are inherently unstable and therefore difficult to generate and maintain in that form.

ORIGINS OF CALCIPROTEIN PARTICLES IN VIVO

As numerous studies have now looked at CPP in serum one might assume that CPP are made in blood. However, while CPP can form within minutes under permissive conditions in vitro, a combination of kinetic constraints, rapid clearance mechanisms and the ambient activities and turnover of calcium and phosphate ions in plasma, make appreciable spontaneous synthesis in blood unlikely. Consequently one would predict CPM to be the principal species generated intravascularly [32]. Indeed, it is worth recalling that in supersaturated calcium phosphate solutions supplemented with fetuin-A, less than 50% of mineral and less than 5% of fetuin-A is incorporated into CPP with the remainder of the complexed mineral present as CPM [6,33]. To date, the biological import of CPM has largely been neglected but this likely represents a fundamental mechanism by which fetuin-A can stabilise nascent mineral nuclei in vivo, independent of CPP formation. Currently no specific method exists to enumerate this fraction.

Bone

If not formed in blood, what are the other possible origins of circulating CPP? Logically the most likely place for CPP to form is at sites where mineralisation occurs physiologically. Indeed, multiple lines of evidence support the notion that bone could be a major source of CPP. In vivo studies by Price et al. provide compelling evidence that altering the flux of mineral in and out of bone can have marked effects on circulating CPP. Acute blockade of bone mineralization with high-dose etidronate was found to result in a marked elevation in levels within hours [34], whereas administration of inhibitors of bone resorption (e.g. calcitonin, osteoprotegerin and alendronate) resulted in the prompt disappearance of CPP from blood [35]. It should be acknowledged, however, that these quite extreme pharmacological maneuverers also had profound effects on serum biochemistry (e.g. >3–4-fold increase in serum calcium and phosphate concentrations with high-dose etidronate [34]), so it is unclear whether the CPP originated from bone or were made ectopically in blood. Nonetheless, other observational data does support the idea that CPP are formed in bone in situ. Elegant studies using cryogenic tissue processing techniques that are needed to preserve the native mineral architecture reveal the presence of abundant membrane-free ACP-containing particles (i.e. primary CPP) in the extracellular space adjacent to the surface of rapidly forming long bones of chick embryos and caudal fins of zebrafish larvae [36,37]. However, although recent studies in humans also point to a temporal relationship between changes in bone turnover and circulating CPP levels [38▪▪], conclusive evidence that endogenous CPP can traffic to and from bone is currently lacking.

Intestine

Recent reports have also shed light on another potential origin of circulating CPP: following absorption of dietary mineral by the intestine [16,39▪▪]. Despite the large quantities of mineral being absorbed daily, serum calcium and phosphate concentrations fluctuate minimally after meals [40]. Thus, in addition to changes in handling by the bone and kidney, buffering by CPM and CPP may provide a temporary store for excess mineral while the load is safely metabolised and excreted. Congruent with this, in rodents with intact kidney function, we found that chronic dietary phosphate supplementation over several weeks resulted in sustained elevations in primary CPP levels, whilst serum phosphate remained unchanged [16]. In contrast, in animals with superimposed CKD, high-phosphate feeding resulted in a more exaggerated rise in CPP and elevated phosphate levels [16]. Observational studies in humans also support a dietary origin for CPP. Diurnal analyses in patients with diabetes without CKD, revealed a spike in serum CPP levels that coincided with mealtimes [41▪]. Moreover, phosphate lowering with non-calcium-based intestinal phosphate binders also reportedly reduces levels [42▪]. Thus, the weight of evidence suggests that the intestine and bone are the mostly likely sources of CPP circulating in vivo rather than de-novo formation in the vascular space (Fig. 2).

FIGURE 2
FIGURE 2:
Clearance and metabolism of CPP in vivo. The principal sources of circulating CPP are through the intestinal absorption of exogenous mineral loads from the diet or from mineral released by the action of osteoclasts (OC) during bone resorption. At least a proportion of this mineral liberated during bone turnover is likely to be retrieved locally by osteoblasts (OB) and used during bone formation or taken up and metabolized by macrophage (MØ). The remainder may spill over into blood or supply other tissues during periods of low intake. Excessive mineral in the form of CPP or CPM may be sensed by OB and osteocytes (OY) and induce secretion of FGF23. Because systemic clearance is rapid, circulating levels are kept very low unless clearance is chronically overwhelmed or downregulated. Consequently, most mineral in the pathway is found in CPM or CPP-I and not CPP-II, which take much longer to form in appreciable numbers. First-pass metabolism is likely to contribute significantly to the removal of nascently-formed CPP derived from mineral transported from the intestine via the portal circulation. Primary CPP (CPP-I) are rapidly cleared by liver sinusoidal endothelial cells through an uncharacterised receptor whereas secondary CPP (CPP-II) are cleared by phagocytic cells of the reticuloendothelial system in the liver and spleen using the class A scavenger receptor. CPM may be cleared by the kidney although this needs to be experientially verified.

FUNCTIONS OF CALCIPROTEIN PARTICLES IN MINERAL METABOLISM

The primary physiological function of CPM/CPP is to chaperone tiny packets of mineral formed at sites of enhanced mineral ion flux (e.g. bone remodelling compartment, and across epithelia and endothelia in the kidney, small intestine and skin) to sites of recycling or disposal. Beyond this passive transport function, however, recent data also suggests that CPP signal the need to dispose of excess mineral from the body, by stimulating the secretion of fibroblast growth factor 23 (FGF23) - the master phosphatonin - from bone.

Mineral chaperoning and transport

Studies in animals and humans suggest that the highest serum fetuin-A concentrations are attained in utero, with levels approximately twice those in adults [43]. This implies an important role for fetuin-A and CPP formation during foetal development. Interestingly, the human foetus accrues 80% of its mineral content during the final trimester creating a significant demand for mineral [44]. Consequently the foetus maintains higher serum calcium and phosphate concentrations (∼0.3–0.5 mmol/l) than in the mother through enhanced placental transport [45]. Such levels might be considered hazardous in adults and prompt therapeutic interventions in patients with CKD, but appear to occur without obvious sequalae during foetal development. Surprisingly, despite this massive flux in mineral (maximally ∼60-fold greater than loading in adults after a meal per kilogram body weight), little is known about how the very large amounts of calcium and phosphate needed are sequestered, concentrated and transported to bone-forming cells and ultimately deposited in the osteoid scaffold. Indeed, this may exceed the amount of mineral that can be delivered through ions alone. Consistent with a possible role in mineral transport, preliminary reports suggest that circulating CPP levels are elevated in cord blood compared to adult levels, exceeding loads seen in dialysis patients [46▪].

FGF23 secretagogue

Analogous to the manner in which the parathyroid glands secrete PTH when a decrease in the ionised blood calcium is sensed through the calcium-sensing receptor (CaSR), it has long been assumed that cells in bone secrete FGF23, when they sense increased blood phosphate levels through a putative ‘phosphate-sensing receptor’. However, although dietary phosphate loading clearly evokes a phosphaturic homeostatic response [47], uncertainty surrounds both the existence of such a receptor in mammalian bone cells and the nature of the biochemical signal conveyed from gut to bone to induce secretion of FGF23. Intriguingly, studies imply that phosphate sensing in bone is dependent on the presence of both phosphate and calcium above a certain threshold [48,49]. One explanation for this observation is that osteocytes may not sense phosphate itself, but the mineral–protein complexes produced as a result of buffering dietary calcium phosphate.

Now, compelling work in rat osteoblasts has shown that CPP , and CPM in particular, are a more potent secretagogue for FGF23 than ionic phosphate [39▪▪]. Proof-of-principle studies in which mice were given an acute phosphate bolus by oral gavage showed that induction of FGF23 mRNA in bone and subsequent elevations in FGF23 in serum, were preceded by a rapid but transient increase in CPP. Whether these relationships hold true under more physiologically representative conditions remains to be demonstrated. Interestingly, administration of the bisphosphonate alendronate to animals fed a high-phosphate diet, further augmented total FGF23 concentration. Assuming the effects of alendronate in this context are mostly mediated by modulation of CPP formation and ripening, then it implies that mineral–protein precursors to secondary CPP also serve as stronger inducers of FGF23 secretion in vivo. These thought-provoking studies not only confirm a plausible alternative source of CPP, but a further level of complexity in the regulation of total body mineral homeostasis and the linking of humoral and endocrine networks with the diet.

METABOLISM AND CLEARANCE OF CALCIPROTEIN PARTICLES

Studies using fluorescently-labelled fetuin-A as a mineral-binding tracer have demonstrated that clearance of protein–mineral complexes is strongly dependant on their maturation state and crystallinity (Fig. 2). CPM are readily internalized by renal tubular cells, with their remnants rapidly appearing in urine (Jahnen-Dechent W, unpublished data). In contrast, primary CPP are cleared and metabolised within minutes by sinusoidal endothelial cells in the liver [28▪▪]. Crystalline secondary CPP, like other large particulates in blood, are filtered by the mononuclear phagocytic system in the liver and spleen, resulting in a plasma half-life of 10–20 min [28▪▪,50]. Macrophages in atherosclerotic plaques can also accumulate secondary CPP [50] which may theoretically contribute to plaque calcification [31]. Other reports showing that exogenous CPP can extravasate to the bone compartment where osteoblasts reside [39▪▪] further substantiates a role for these particles as a secretagogue for FGF23. Although primary CPP are extremely transient, secondary CPP are retained intracellularly by phagocytic cells for hours [28▪▪]. Thus, as the mineral bound by fetuin-A matures and crystallizes, extraction from the circulation becomes progressively more difficult and the potential for mineral stress increases. The cellular receptors for CPM and primary CPP have yet to be identified, but secondary CPP appear predominantly bound through the class A scavenger receptor [25,50], with apolipoprotein-A1 serving as a possible ligand (Smith ER, unpublished data).

Although clearance kinetics in animals with CKD have yet to be characterized, loss of excretory function in CKD, and the shift towards positive mineral balance is predicted to result in an accumulation of mineral-loaded fetuin-A. Initially this may present as increased CPM, which over time may lead to formation of primary CPP and ultimately the generation of secondary CPP in extreme cases, if clearance mechanisms are overwhelmed or suppressed. Disturbances in clearance may manifest in excessive levels of CPP and pathological sequela [51,52▪,53]. Both primary and secondary CPP appear inflammatory in high doses via activation of the innate immune receptor toll-like receptor 4 and NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome in vitro[28▪▪], but it is uncertain whether such effects manifest at the much lower levels found in vivo.

LIFE WITHOUT CALCIPROTEIN PARTICLES: LESSONS FROM FETUIN-A-DEFICIENT MICE

Genetic models of fetuin-A deficiency provide insight into what happens when the body cannot make or adequately stabilize CPP. Fetuin-A-deficient mice calcify ectopically, but the severity of the phenotype is strongly dependent on the genetic background of the animal. Fetuin-A deficient (Ashg-/-) mice maintained on a DBA/2 background develop one of the most severe phenotypes of ectopic calcification known [54]. Recent studies have revealed that the earliest signs of calcification are in the lumen of the microvasculature suggesting de-novo precipitation of mineral-containing complexes from the fluid phase of blood [55▪▪]. Intraluminal deposition leads to vascular occlusion, ischaemia, necrosis and fibrosis, ultimately triggering further calcification and to grossly expanded deposits that are visible macroscopically.

In stark contrast, fetuin-A-deficient mice on the C57BL/6 background have no ectopic calcification outside the skeleton, with a relatively mild phenotype characterized by epiphysiolysis, distal femur dysplasia and foreshortening of the hindlimbs [56,57]. This phenotype is compatible with a recent report of a child with infantile cortical hyperostosis (Caffey disease), who was found to be completely deficient in fetuin-A because of a homozygous nonsense mutation in the Ashg gene [58▪]. Subsequently, it became clear that genetic differences between the two strains of mice must determine the propensity to calcify when fetuin-A is absent. Concurrent investigations established that the calcification phenotype of Ashg-/- DBA/2 mice was in part because of a compound triple deficiency of pyrophosphate, magnesium and fetuin-A, thus affecting three well described potent regulators of extracellular mineralisation at once [59▪▪]. This has particular relevance to CKD, because levels of pyrophosphate and fetuin-A are often reduced in advanced disease [60,61]. Although magnesium shows a tendency to accumulate in plasma with loss of GFR, a proportion of patients develop inappropriately low levels [62]. Indeed, although estimates suggest that there should still be sufficient fetuin-A present in the ECF of dialysis patients to stabilise CPP – such that intraluminal precipitation should not occur – the interplay of a combined deficiency in other mineralisation inhibitors may engender a tendency towards an enhanced state of calcification and CPP formation. Consistent with the notion that additional stressors are needed to unmask the systemic calcification phenotype in relatively calcification-resistant Ahsg-/- C57BL/6 mice, animals with superimposed CKD (induced by subtotal nephrectomy) fed a high-phosphate diet do indeed develop extensive cardiovascular calcifications [63].

CONCLUSION

Fetuin-A-containing CPP and their precursors allow efficient transport and clearance of bulk calcium phosphate without risk of precipitation. Mineral transport functions during foetal development may be especially important. Circulating CPP may also couple dietary mineral exposure with endocrine control of mineral metabolism by stimulating FGF23 secretion from bone. Conceptual frameworks that consider primary CPP as inert or physiological and secondary CPP as pathological are likely too simplistic, since it is now clear that precursors to the crystalline state are not only themselves bioactive but far more abundant in extracellular fluid. Moreover, while considerable attention has been given to the potentially pro-calcific effects of CPP excess, the inability to make or sufficiently stabilise these particles gives rise to one of the most severe calcification phenotypes known. Hence, the vital homeostatic function performed by this chaperone pathway to mitigate the effects of mineral stress should not be forgotten. Thus, whilst attention is currently skewed towards pathological signalling resulting from chronic CPP excess, these rather enigmatic nanoparticles are best viewed as potential culprit and remedy to phosphate woes; where too little or too much might manifest in disease.

Acknowledgements

None.

Financial support and sponsorship

E.R.S. and T.D.H. are supported by a Royal Melbourne Hospital Home Lottery Project Grant and funding from Sanofi. W.J.D. was supported by grants from Deutsche Forschungsgemeinschaft and the Interdisciplinary Center for Clinical Research of the Medical Faculty of RWTH Aachen University.

Conflicts of interest

W.J.D. is an inventor of the T50-test. Both W.J.D. and E.R.S. hold stock in Calciscon AG, which specializes in performing the T50-test. T.D.H. reports no relevant conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

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      Keywords:

      calciprotein particles; calcium phosphate; colloids; fetuin-A; mineral-binding proteins

      Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.