The present studies show that the previously reported ability of fetuin to inhibit the precipitation of hydroxyapatite from supersaturated solutions of calcium and phosphate in vitro is accompanied by the formation of the fetuin-mineral complex, a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein that was initially discovered in the serum of rats treated with etidronate and that appears to play a critical role in inhibiting calcification in vivo. Rat serum potently inhibited the precipitation of calcium phosphate mineral when the concentration of calcium and phosphate were increased by 10 mM each, and the modified serum was incubated at 37°C for 9 days; in the absence of serum, precipitation occurred in seconds. Large amounts of the fetuin-mineral complex were generated in the first 3 h of this incubation and remained throughout the 9-day incubation. Purified bovine fetuin inhibited the precipitation of mineral for over 14 days in a solution containing 5 mM calcium and phosphate at pH 7.4 at 22°C, whereas precipitation occurred in minutes without fetuin. There was a biphasic drop in ionic calcium in the fetuin solution, however, from 5 to 3 mM in the first hour and from 3 to 0.9 mM between 20 and 24 h; these changes in ionic calcium are due to the formation of complexes of calcium, phosphate, and fetuin. The complex found at 24 h to 14 days is identical to the fetuinmineral complex found in the serum of etidronatetreated rats, whereas the complex found between 1 and 20 h is less stable.The present studies were carried out to better understand the mechanisms responsible for the generation of the serum fetuin-mineral 1 complex in the rat. The fetuin-mineral complex is a complex of a calcium phosphate mineral phase and the proteins fetuin and matrix Gla protein (MGP) that appears in the blood of rats treated with doses of etidronate that acutely inhibit bone mineralization (1). The appearance of this complex in serum following etidronate injection causes a 4-fold increase in total serum calcium without any increase in ionic calcium levels. The fetuin-mineral complex is quite stable in serum at 37°C, with no evidence for the growth, aggregation, and precipitation of the mineral component of the complex, which suggests that the previously reported calcification inhibitory 2 activities of fetuin (2, 3) and MGP (4 -7) may be related to their ability to form stable complexes with nascent mineral nuclei.The dominant protein component of the fetuin-mineral complex, fetuin, is a 59-kDa glycoprotein that consists of two Nterminal cystatin domains and a smaller C-terminal domain. Fetuin is synthesized in the liver and is found at high concentrations in mammalian serum (8, 9) and bone (10 -15). The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (9). Fetuin is one of the most abundant noncollagenous proteins found in bone (10 -15), with a concentration of about...

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The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril therefore provides the aqueous compartment in which mineral grows. Although knowledge of the size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding the mechanism of bone mineralization, there have been as yet no studies on the size exclusion properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we developed a gel filtration-like procedure that uses columns containing collagen from tendon and bone. The elution volumes of test molecules show the volume within the packed column that is accessible to the test molecules, and therefore reveal the size exclusion characteristics of the collagen within the column. These experiments show that molecules smaller than a 6-kDa protein diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa protein are excluded from this water. These studies provide an insight into the mechanism of bone mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all water within the fibril and so can directly impact mineralization. Although molecules larger than a 40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril.

One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism "mineralization by inhibitor exclusion," the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.

BackgroundEpidemic outbreaks of multi-drug resistant (MDR) Acinetobacter baumannii (AB) in intensive care units (ICUs) are increasing. The incidence of MDR AB bacteremia, which develops as a result of colonization, is increasing through widespread dissemination of the pathogen, and further colonization. We sought to determine risk factors for MDR AB bacteremia in patients colonized with MDR AB in the ICU.MethodsWe conducted a retrospective, observational study of 200 patients colonized with MDR AB in the ICU at Severance Hospital, South Korea during the outbreak period between January 2008 and December 2009.ResultsOf the 200 patients colonized with MDR AB, 108 developed MDR AB bacteremia, and 92 did not. APACHE II scores were higher in bacteremic than non-bacteremic patients at the time of ICU admission and colonization (24.0 vs. 21.6; P = 0.035, 22.9 vs. 16.8; P < 0.001, respectively). There was no difference between the two groups in the duration of time from ICU admission to colonization (7.1 vs. 7.2 days; P = 0.923), but the duration of time at risk was shorter in bacteremic patients (12.1 vs. 6.0 days; P = 0.016). A recent invasive procedure was a significant risk factor for development of bacteremia (odds ratio = 3.85; 95% CI 1.45-10.24; P = 0.007). Multivariate analysis indicated infection and respiratory failure at the time of ICU admission, maintenance of mechanical ventilation, maintenance of endotracheal tube instead of switching to a tracheostomy, recent central venous catheter insertion, bacteremia caused by other microorganism after colonization by MDR AB, and prior antimicrobial therapy, were significant risk factors for MDR AB bacteremia.ConclusionsPatients in the ICU, colonized with MDR AB, should be considered for minimizing invasive procedures and early removal of the invasive devices to prevent development of MDR AB bacteremia.