Relationship between iron, calcium and phosphate during experimental cutaneous calcinosis

Metabolism, 2008

Hemochromatosis is a known cause of osteoporosis in which the pathophysiology of bone loss is largely unknown and the role of iron remains questionable. We have investigated the effects of iron on the growth of hydroxyapatite crystals in vitro on carboxymethylated poly(2hydroxyethyl methacrylate) pellets. This noncellular and enzyme-independent model mimics the calcification of woven bone (composed of calcospherites made of hydroxyapatite crystals). Polymer pellets were incubated with body fluid containing iron at increasing concentrations (20, 40, 60 μmol/L). Hydroxyapatite growth was studied by chemical analysis, scanning electron microscopy, and Raman microscopy. When incubated in body fluid containing iron, significant differences were observed with control pellets. Iron was detected at a concentration of 5.41-to 7.16-fold that of controls. In pellets incubated with iron, there was a ∼3to 4-fold decrease of Ca and P and a ∼1.3to 1.4-fold increase in the Ca/P ratio. There was no significant difference among the iron groups of pellets, but a trend to a decrease of Ca with the increase of iron concentration was noted. Calcospherite diameters were significantly lower on pellets incubated with iron. Raman microspectroscopy showed a decrease in crystallinity (measured by the full width of the half height of the 960 Δcm −1 band) with a significant increase in carbonate substitution (measured by the intensity ratio of 1071 to 960 Δcm −1 band). Energy dispersive x-ray analysis identified iron in the calcospherites. In vitro, iron is capable to inhibit bone crystal growth with significant changes in crystallinity and carbonate substitution.

Journal of nephrology

Understanding the mechanism of calcium deposition in soft tissues is of great importance in a variety of pathological conditions such as chronic kidney disease. The present study examined the role of phytate and osteopontin during the development of soft tissue calcification in an animal model. Male Wistar rats (16 rats per treatment) were fed with a diet (AIN-76A) in which phytate is undetectable (non-phytate-treated group), or with a phytin-enriched AIN-76A diet (phytate-treated group). After 21 days on the respective diets, all rats were subjected to calcinosis induction by subcutaneous injection with KMnO4 at 2 sites on either side of the interscapular region. At 2, 5, 8 and 10 days after the calcinosis induction, 4 rats of each group were sacrificed, and the injured tissues were removed for histological analysis and for calcium determination. Calcification was notably and significantly reduced in phytate-treated rats compared with non-phytate-treated rats. Calcified deposits ap...

Arthritis & Rheumatism, 1984

Synthetic hydroxyapatite (HA) crystals in 1 % serum stimulated ' H thymidine uptake into quiescent canine synovial fibroblasts and human foreskin fibroblast cultures, as did 10% serum. The onset of stimulation and peak uptake of thymidine after crystal addition were delayed by 2-3 hours as compared with the effects produced by 10% serum. Stimulation of 'H thymidine uptake was proportional to the serum concentration used. HA crystals (50 pglml) stimulated nuclide uptake at each serum concentration used. 3H thymidine uptake was also proportional to the dose of HA or calcium pyrophosphate dihydrate crystals, although larger doses of the latter crystal were required to produce equivalent effects. Not all particulates were effective mitogenic agents. Latex beads and diamond crystals had no effect. Monosodium urate crystals modestly stimulated and calcium urate crystals markedly stimulated nuclide uptake. The more complex crystals found in a naturally occurring condition (calcinosis) were as mitogenic as the pure synthetic HA. The synovial cell hyperplasia sometimes associated with crystals might be explained in part by their mitogenic activity.

Cell Structure and Function, 2009

Ectopic calcification occurs in the skeletal muscle of mdx mice, a dystrophin-deficient animal model of Duchenne muscular dystrophy. The purpose of this study was to clarify the mechanism of the calcification. The calcified deposits were identified as hydroxyapatite, a crystallized form of calcium phosphate, and the serum inorganic phosphate (Pi) level in the mdx mice was approximately 1.4 times higher than that in the normal B10 mice, suggesting that Pi plays a critical role in the ectopic calcification. When C2C12 mouse myoblasts were cultured under high-Pi conditions, myogenic differentiation was retarded while the expression of osteogenic markers such as osteocalcin and Runx2 were upregulated. This was followed by the generation of calcium deposition. Moreover, ectopic calcification reduced to an undetectable level in most of the mdx mice fed a Pireduced diet. We therefore conclude that the Pi-induced osteogenesis of muscle cells is responsible for ectopic calcification in the skeletal muscle of mdx mice.

IOP Conference Series: Materials Science and Engineering, 2017

One of important aspects in bone healing process is physiological level of calcium (Ca), and phosphorus (P) that can be altered by implantation of biodegradable porous iron. Therefore, this study aims to investigate the concentration of Ca, P and Ca/P ratio in the peripheral blood during the implantation period up to 4 months. Forty adult male Sprague Dawley rats were used and divided into 3 groups receiving different pore size of iron implants (pore size 450, 580, 800μm) and one group of sham. The implants (5x2x0.5mm) were inserted into flat bone defects at latero-medial of femoral bone. Blood sample was taken from ventral tail artery before and after 4 month of implantation. Calcium and P concentrations in the blood were determined by BA-88A Semi-Auto Chemistry Analyzer. Results showed that concentration of Ca and P are slightly higher after implantation than before implantation, except for the 450µm group. The Ca/P ratio before and after implantation was increased in the sham group, and decreased in the 450 and 800µm groups. Concentration of Ca, P and Ca/P ratio insignificantly change between before and 4 months after surgery in some groups.

Journal of Structural Biology, 2008

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin-Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where b-glycerophosphate (bGP) was added as an exogenous organic source of phosphate, bGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.

British Journal of Pharmacology and Chemotherapy, 1968

Compounds which produce tissue calcification at the site of injection in otherwise untreated rats are known as calcergens or direct calcifiers. These include lead acetate, potassium permanganate, indium trichloride, the chlorides of rare earth metals and zinc chloride. On the other hand, numerous other metallic compounds-for example, aluminium trichloride, chromium chloride, ferric chloride, scandium trichloride-are inactive in this respect (Selye, 1962; Padmanabhan, Tuchweber & Selye, 1963; Gabbiani, Jacqmin & Richard, 1966). Calcergy must be distinguished from calciphylaxis, in which tissue calcification can be produced by certain challengers (for example, aluminium trichloride, ferric chloride, chromium chloride, etc.) only after suitable sensitization with systemic factors such as vitamin D compounds or parathyroid hormone (Selye, 1962). While all calcergens are calciphylactic challengers, the reverse is not true. Among the calcergens, lead acetate given intravenously prepares the rat for the

Journal of Nephrology, 2014

Background Medial vascular calcification is a specific complication in chronic kidney disease (CKD) patients although its pathogenesis is poorly understood. The administration of iron (Fe), generally used for the treatment of anemia in CKD patients, induces oxidative stress. Fe loading possibly affects the progress of vascular calcification in uremia. We investigated the effect of Fe on vascular calcification and its mechanism in uremic rats. Method Thirty-two rats were divided into four groups: untreated rats (controls), rats fed a standard diet with Fe administration (Fe group), rats fed an adenine-enriched diet (uremic group), and rats fed an adenine-enriched diet with Fe administration (uremic ? Fe group). Iron dextran was administered once a week for 5 weeks intraperitoneally. Morphological alterations and vascular calcification-associated factors in the aortic wall were evaluated. Results No aortic calcification was observed in the control group although uremic rats developed severe vascular calcification. Fe loading suppressed vascular calcification in the uremic groups. Expressions of runt-related transcription factor 2 (Runx2), single-strand (ss)DNA and phosphate transporter (Pit)-1 were increased in the uremic rats compared to the control rats. In the uremic group, Fe administration did not show any effect on ssDNA expression, but reduced Runx2 and Pit-1 expressions. Conclusion Fe suppressed the development of vascular calcification through the prevention of Pit-1 and vascular smooth muscle cell osteoblastic transdifferentiation.

Journal of The American Ceramic Society, 2003

The nucleation sites of calcium phosphate crystals during collagen mineralization were studied by Fourier transform infrared spectrometry and transmission electron microscopy. It was found for the first time that there is another nucleation site, i.e., carbonyl (>C=O) on collagen, besides the previous reported nucleation site of carboxyl (–COOH). By comparing the IR spectra of collagen not only with collagen/calcium phosphate but also with collagen/Ca2+, it was observed that the peak intensities of amides I, II, and III of collagen decreased significantly after mineralization. The decrease of the amide I peak intensity was mainly due to blockage of the C=O stretch. Furthermore, the peak for amide I gradually shifted to a lower wavenumber during collagen mineralization. This shift indicated that chemical interaction between carboxyl groups and Ca2+ ions formed in the mineralization.

Life Sciences, 2004

Myo-inositol hexaphosphate (InsP 6 ) is an abundant component of plant seeds. It is also found in significant levels in blood and mammalian tissues, but they are totally dependent on their dietary intake. In the present paper, we describe studies on the effect of InsP 6 on a model of dystrophic calcification, which was chemically induced by subcutaneous injection of a 0.1% KMnO 4 solution. Male Wistar rats were randomly divided into four groups for treatment over 31 days. A: animals consuming a purified diet in which InsP 6 was absent but to which 1% of InsP 6 (as sodium salt) was added. In this group, the InsP 6 plasma levels (0.393 F 0.013 AM) were similar to those observed in rats consuming a standard diet. B: animals consuming only the purified diet in which InsP 6 was absent. In this case the InsP 6 plasma levels decreased (0.026 F 0.006 AM); C: animals consuming the same purified diet as group B but received daily subcutaneous injections of 50 Ag kg -1 etidronate during the last 14 days. In this case the InsP 6 plasma levels were also very low (0.025 F 0.007 AM); D: animals consuming the same diet as group B but a 6% of carob germ (InsP 6 rich product) was added. The InsP 6 plasma levels (0.363 F 0.035 AM) were also similar to those observed in rats consuming a standard diet. After 21 days plaque formation was induced. Calcification plaques were allowed to proceed for 10 days, after which the plaque material present was excised, dried and weighed. It was found that the presence of myo-inositol hexaphosphate (phytate) in plasma at normal concentrations (0.3-0.4 AM) clearly inhibited the development of dystrophic calcifications in soft tissues. These results demonstrates that myo-inositol hexaphosphate acts as an inhibitor of calcium salt crystallization.

AJP: Cell Physiology, 2010

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca2+ and 2 mM Pi. CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × Pi concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × Pi mM2 but not with 2 × 1 or 1 × 4 Ca × Pi mM2. Incubation with 4 mM Pi without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron m...

Biochemical and Biophysical Research Communications, 2003

Pathological calcifications are associated with many medical conditions including diabetes, breast cancer, and crystals-associated osteoarthritis. The deposition of calcium-containing crystals on cells induces detrimental cellular effects and speeds up the progression of associated diseases. We carried out the present study to test the hypotheses that calcium-containing crystals may stimulate the influx of other molecules existing in the extracellular fluid disturbing normal molecular signaling and that anti-calcification agent will inhibit such endocytotic process. We found that basic calcium phosphate (BCP) crystals greatly stimulated the endocytotic activity of cells by rendering the cells more permeable and that the anti-calcification agent phosphocitrate and several others inhibited the crystals-mediated endocytosis. This is the first study reporting that the endocytotic activity of cells is affected by BCP crystals and that such endocytotic activity can be inhibited by anti-calcification agents. Since calcium-containing crystals are associated with many human diseases and in many circumstances are associated with apoptotic bodies, extracellular and matrix vesicles where DNA fragments, small peptides, and minerals are released into extracellular space, the findings reported here are important for our understanding of the complex biological effects and the potential pathological role of calcium-containing crystals in crystals-associated diseases, and for the development of disease modifying agents as well.

Data in Brief, 2016

This data article contains data related to the research article entitled, "Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice" [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) . Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation.

Journal of Bone and Mineral Research, 2009

The shape, the typical orientation, and the average size of mineral crystals in different types of mineralized tissues were investigated by means of small-angle x-ray scattering (SAXS). To rule out eventual artifacts due to sample preparation, four different standard preparation techniques were used and a comparison showed that the SAXS results were identical for all four methods. In mineralized turkey leg tendon, a frequently used model system for bone, the crystals were found to be typically plate-like with a thickness of the order of 2 nm. This stands in contrast to the case of bone (calvaria, femur, and iliac crest) from mouse, rat, and dog, where mainly needle-like crystals were found. The thickness of these crystals ranged from 3 to 4 nm but was remarkably constant for different bones of a given animal. The preferred orientation of the needle-like crystals was along the main axis of the femur and within the surface of the calvaria (for mouse, rat, and dog). The mineral plates in turkey leg tendon were located inside the hole zone and oriented along the fibril axis. Finally, no periodic arrangement of the crystals inside the hole zone of the collagen could be found.

Biochemical Pharmacology, 1997

Asbestos exposure causes pulmonary fibrosis by mechanisms that remain uncertain. There is increasing evidence that iron from asbestos is responsible for many of its effects. In this paper, we investigated the effect of iron mobilized from crocidolite asbestos on collagen content in rat lung fibroblast cultures under serum-free conditions. Crocidolite (2,4, 6 pg/cm* well) increased collagen content in a dose-dependent manner (+42 2 8, +92 ? 10, and +129 5 13 / '0 vs controls).