Protein-bound calcium phosphate in uremic rat serum: a quantitative study.

Protein-bound calcium (prCa) constitutes about 40% of serum total calcium, in which albumin is the most dominant protein. Given the chemical interaction between calcium and phosphate (Pi), the increased serum Pi in chronic kidney disease may cause changes in the composition and structure of the prCa fraction. Here, we report the phosphate binding on the protein-bound calcium in uremic rat serum. Using adenine-fed rats as a uremic model, we separated the calcium and phosphate fractions in rat serum by ultrafiltration, and found that the level of protein-bound phosphate (prPi) in the uremic serum was markedly higher than in control. The elevated prPi level was comparable to the prCa level, consistent with the presence of protein-bound calcium phosphate pr(Ca)j-m(CaPi)m. We then confirmed its presence by ex vivo X-ray absorption near-edge structure spectroscopy, revealing the discrete state of the calcium phosphate clusters associated with protein. Finally, in a quantitative investigation using Ca- and Pi-boosted serum, we discovered the threshold concentration for the Pi binding on prCa, and determined the binding constant. The threshold, while preventing Pi from binding to prCa in normal condition, allows the reaction to take place in hyperphosphatemia conditions. The protein-bound calcium phosphate could act as a link between the metabolism of serum proteins and the homeostasis of phosphate and calcium, and it deserves further investigation whether the molar ratio of (prPi/prCa)⋅100% may serve as a serum index of the vascular calcification status in chronic kidney disease.

Correction to: Protein-bound calcium phosphate in uremic rat serum: a quantitative study.

In the original article, few equations and units were published incorrectly.

Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum.

The total calcium (tCa) in blood serum comprises free Ca2+ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η2-HPO4)(H2O)4 and Ca(η1-HCO3)(H2O)5+, in which HPO42- and HCO3- serve as bidentate and unidentate ligands, respectively. The remained H2O molecules on the coordination sphere of Ca2+ enable these complexes to behave partially like aquated Ca2+ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.

Pseudo-equilibrium equation of calcium phosphate precipitation from aqueous solution.

An X-ray amorphous phase is frequently present at the early stage of calcium phosphate crystallization, and the relevant solution chemistry is essential for understanding the mechanism of reaction. Here, we report a quantitative study of a series of reaction systems at pseudo-equilibrium states. We determined the composition of solutions and the quantities of the precipitate samples, and characterized the long- and short-range order of the precipitate using X-ray diffraction and synchrotron X-ray absorption near-edge structure spectroscopy, respectively. We found that, in a particle with multiple structural units, only a fraction of the units was able to reach pseudo-equilibrium with the solution composition, which represents the average number of surficial clusters per unit. These findings enabled us to propose a general form of the equilibrium constant equation. The equation fits the pseudo-equilibrium data well, and it converts to the "solubility product (Ksp)" and the conventional "reaction quotient" in two limit cases, respectively. Further, using a cube model, we derived a "particle equation" that reveals the connection between the particle structure and the form of equilibrium constant equation. The dependency of the form of pseudo-equilibrium equation on the structure and size of the precipitate reveals a fundamental relation in chemistry, and its applicability remains to be examined in other reaction systems, such as those involving nanocrystals and porous materials.

[Molecular structure and fractal analysis of oligosaccharide].

OBJECTIVE: To propose a calculation method of oligosaccharides' fractal dimension, and to provide a new approach to studying the drug molecular design and activity. METHODS: By using the principle of energy optimization and computer simulation technology, the steady structures of oligosaccharides were found, and an effective way of oligosaccharides fractal dimension's calculation was further established by applying the theory of box dimension to the chemical compounds. RESULTS: By using the proposed method, 22 oligosaccharides' fractal dimensions were calculated, with the mean 1.518 8 ± 0.107 2; in addition, the fractal dimensions of the two activity multivalent oligosaccharides which were confirmed by experiments, An-2 and Gu-4, were about 1.478 8 and 1.516 0 respectively, while C-type lectin-like receptor Dectin-1's fractal dimension was about 1.541 2. The experimental and computational results were expected to help to find a class of glycoside drugs whose target receptor was Dectin-1. CONCLUSION: Fractal dimension, differing from other known macro parameters, is a useful tool to characterize the compound molecules' microscopic structure and function, which may play an important role in the molecular design and biological activity study. In the process of oligosaccharides drug screening, the fractal dimension of receptor and designed oligosaccharides or glycoclusters can be calculated respectively. The oligosaccharides with fractal dimension close to that of target receptor should then take priority compared with others, to get the drug molecules with latent activity.

Lanthanum chloride bidirectionally influences calcification in bovine vascular smooth muscle cells.

Vascular calcification (VC) is frequent prevalence in patients with chronic kidney disease (CKD) and atherosclerosis. Lanthanum carbonate is used as an orally administered phosphate-binding agent to reduce the gastrointestinal absorption of phosphate and ameliorate VC in advanced CKD. In this study, we used bovine vascular smooth muscle cells as a model VC in vitro and studied the effects of lanthanum chloride on calcium deposition. Exposure of cells to LaCl(3) at the concentration of 0.1 µM suppressed the ß-glycerophosphate-induced alkaline phosphatase activity and calcium deposition. Furthermore, LaCl(3) upregulated the ß-glycerophosphate-suppressed expression of calcium-sensing receptor. In contrast to the inhibitory effect of LaCl(3) on calcium deposition, higher level lanthanum (50 µM) was found to promote immediately precipitation of calcium phosphate in cell culture medium. At this concentration, LaCl(3) was found to induce cell apoptosis which involves caspases-9 and -3. These data indicate that the promotory effect of LaCl(3) on calcium deposition is likely mediated by induction of apoptosis. Our in vitro findings do suggest that, in the context of raised lanthanum, greater attention should be paid to potential toxic effects associated to the use of lanthanide-based drugs.

Lanthanum suppresses osteoblastic differentiation via pertussis toxin-sensitive G protein signaling in rat vascular smooth muscle cells.

A major cellular event in vascular calcification is the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. After demonstrating that lanthanum chloride (LaCl(3)) suppresses hydrogen peroxide-enhanced calcification in rat calcifying vascular cells (CVCs), here we report its effect on the osteoblastic differentiation of rat VSMCs, a process leading to the formation of CVCs. Cells were isolated from aortic media of male SD rats, and passages between three and eight were cultured in Dulbeccol's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mM beta-glycerophosphate (beta-GP) in the presence or absence of LaCl(3). Exposure of cells to LaCl(3) suppressed the beta-GP-induced elevations in calcium deposition, alkaline phosphatase (ALP) activity, and Cbfa1/Runx2 expression, as well as the concomitant loss of SM alpha-actin. Furthermore, LaCl(3) activated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and the blockage of either pathway with a specific inhibitor abolished the effects of LaCl(3). In addition, pretreatment of the cells with pertussis toxin (PTx), an inhibitor of G protein-mediated signaling pathway, repealed all the changes induced by LaCl(3). These findings demonstrate that LaCl(3) suppresses the beta-GP-induced osteoblastic differentiation and calcification in rat VSMCs, and its effect is mediated by the activation of both ERK and JNK MAPK pathways via PTx-sensitive G proteins.

Lanthanum chloride suppresses hydrogen peroxide-enhanced calcification in rat calcifying vascular cells.

Lanthanum chloride (LaCl(3)) has been shown to retard the progression of established atherosclerotic lesions in animal models, and used as a calcium channel blocker in various cellular experiments. In this study, we assessed the role of lanthanum chloride (LaCl(3)) in H(2)O(2)-enhanced calcification in rat calcifying vascular cells (CVCs) and examined the involvement of MAPK signaling pathways. H(2)O(2) induced growth inhibition of CVCs, as well as increases in intracellular levels of calcium and reactive oxygen species, ALP activity, apoptosis and calcium deposition. These effects of H(2)O(2) were suppressed by pretreatment of the cells with 1 muM of LaCl(3) for 2 h. In addition, H(2)O(2) activated the phosphorylation of ERK1/2, JNK and p38 MAPK, but only the last two were associated with the ALP activity. Our findings demonstrate that H(2)O(2)-enhanced osteoblastic differentiation and apoptosis are responsible for the increased calcification in rat CVCs, and LaCl(3) can counteract these effects by suppressing the activation of JNK (JNK2, but not JNK1) and p38 MAPK signaling pathway.

Association of oxidative stress with realgar-induced differentiation in human leukemia HL-60 cells.

BACKGROUND: Realgar (arsenic sulfide, As(4)S(4)) has been shown to have clinical efficacy in patients with newly diagnosed and relapsed acute promyelocytic leukemia. Mechanistic studies have demonstrated that realgar is able to induce cell differentiation. METHODS: The oxidative stress in the realgar-induced differentiation was examined with human leukemia HL-60 cells. Cell differentiation was evaluated by the expression of cell surface antigen CD11b and nitroblue tetrazolium assay. The activities of catalase and superoxide dismutase were measured spectrophotometrically. Flow cytometry was used to assess cell cycle distribution and apoptosis, the cellular level of reactive oxygen species (ROS) and glutathione, as well as mitochondrial transmembrane potential (MTP). RESULTS: The realgar-induced differentiation was enhanced by hydrogen peroxide, and preceded with drastic changes in ROS and catalase, as well as small changes in superoxide dismutase and the reduced form of glutathione. MTP values at 24 h were in linear proportion to the CD11b expression at 48 h when no apoptosis was observed. CONCLUSION: Oxidative stress and stress-related MTP decrease are associated with realgar-induced differentiation in HL-60 cells.

Realgar-induced differentiation is associated with MAPK pathways in HL-60 cells.

The clinical efficacy and safety of realgar (arsenic sulfide, As(4)S(4)) in the treatment of acute promyelocytic leukemia in China have given rise to an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiation of HL-60 cells. Treatment with RNPs at 6 microM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductive ability, and elevation of CD11b expression at both mRNA and protein levels. The RNP-induced differentiation was synergized, enhanced and suppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways are closely related to the RNP-induced differentiation in HL-60 cells.

Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by arsenic sulphide: involvement of serine/threonine protein phosphatases.

The clinical efficacy of arsenic sulfide (As(4)S(4)), also known as realgar, in the treatment of leukemia in China is prompting people to explore the underlying mechanism. We examined the realgar-induced differentiation in human promyelocytic leukemia cell line HL-60. Cells exhibited proliferation inhibition when treated with 0.10-1.5 microM of realgar, and underwent monocytic differentiation as indicated by morphological changes, NBT reduction assay, and cytofluorometric analyses of the cell surface antigens, CD11b and CD14. Accompanying the differentiation, the activity of serine/threonine protein phosphatase type 1 (PP1) and type 2A (PP2A) were enhanced, whereas the activity of PP2B remained virtually the same compared to the control. When cells were treated with realgar in the presence of an inhibitor of PP1 and 2A or an inhibitor of PP2B, the differentiation of the cells was partially suppressed as revealed by NBT reduction assay and the expression of CD14. Our data demonstrate that realgar induces monocytic differentiation in HL-60 cells and that some serine/threonine protein phosphatases may be involved in the process.

[Realgar nano-particles induce apoptosis and necrosis in leukemia cell lines K562 and HL-60].

OBJECTIVE: To examine the growth-inhibitory, apoptosis- and necrosis-inducing effects of realgar nano-particles (RNP) in human chronic myelogenous leukemia cell line K562 and acute myeloid leukemia cell line HL-60, and to find out the chemical species with efficacy. METHOD: A "solvent-relay" strategy was used for the preparation of RNP suspension. Cell viability was determined by MTT assay. Cell apoptosis and necrosis were characterized with Annexin V-PI double staining in association with flow cytometry and with morphological examination with Hoechst 33258 staining. Parallel experiments with arsenous acid (H3AsO3), the dominant form of arsenic trioxide in the solution, were conducted for comparison. RESULT: The mean diameter of RNP was 159.0 nm. RNP showed growth-inhibitory effect on both cell lines. The double staining test indicated that RNP induced both apoptosis and necrosis, and this was further confirmed by morphological examination. CONCLUSION: RNP induced both apoptosis and necrosis in leukemia cell lines K562 and HL-60. Thioarsenite species with both As-O and As-S bonds may be the active intermediates in the RNP.

Differentiation-stimulating potency of differentiated HL60 cells after drug treatment.

Differentiation therapy in the treatment of leukemia is often hampered by limitations on using certain pharmaceutical regents or on the required doses due to various reasons, such as drug-resistance and retinoic acid syndrome. To circumvent these problems, a strategy might be developed on the basis of the ability of drug-differentiated cells to stimulate differentiation in leukemia cells. Using the promyelocytic leukemia cell line HL60 as a cell model, we assessed the differentiation-stimulating potency of differentiated granulocytes and monocytes/macrophages after treatments with all-trans retinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. ATRA- and TPA-differentiated cells were able to stimulate differentiation in fresh HL60 cells, accompanied by inhibition on cell growth to various extents. The differentiated cells of the second generation, especially those originated from TPA treatment, were as potent as the drugs themselves in stimulating differentiation in fresh HL60 cells. On the basis of "differentiation induced by differentiated cells", we explored the feasibility of ex vivo therapy.

[Study on the effect of realgar nanoparticles on reducing the respiratory syncytial virus type A (RSV-a) replication in vitro].

This study was to establish a model to explore anti- RSV effect of different administration method of Chinese medicine realgar on respiratory syncytial virus type A (RSV-A) replication in Hep-2 cells. Using high-energy ball milling with distilled water to prepare realgar nanoparticles,the concentration of nanometer realgar was tested by molybdenum blue staining method and the size of realgar nanoparticles was tested on Nano Series. Cell culture with ribavirin as a positive control was applied to observe the effect of anti-respiratory syncytial virus type A replication through prevention, treatment or direct inactivation of three different drug administration methods. Realgar nano-particles was found to be a potential inhibitor of RSV-A in a concentration-dependent manner with the median toxic concentration(TC50) of 0.649 microg/mL in Hep-2 cell culture. The median inhibition concentration (IC50) was 0.20 microg/mL when drug was added before virus infection. The IC50 was 0.13 microg/mL when drug was added after virus infection,and it was 0.16 microg/mL when the drug was mixed with virus and added. The therapeutic index (TI) was 3.18, 4.99 and 4.11, respectively. The results showed realgar nanoparticles could inhibit the replication of the RSV and inactivate the RSV in vitro.

[Study on the effect of realgar nanoparticles anti-adenovirus in vitro].

OBJECTIVE: This study was to establish a model that adenovirus type 3 (HAdV-3) infected on Hep-2 cell in order to explore anti-adenovirus3 (HAdV-3) effect of Chinese medicine realgar in vitro. METHOD: Use high-energy ball milling with distilled water to prepare realgar nanoparticles. The concentration of nanometer realgar was tested by molybdenum blue staining method and realgar nanoparticles' particle size was tested on Nano Series. The technique of cell culture with ribavirin as positiv control was to observe anti-adenovirus effect through prevention, treatment and direct inactivation of three kinds of drug delivery. RESULT: This drug was found to be a potential inhibitor of HAdV-3 in a concentration-dependent manner with the median toxic concentration (TC50) of 0.649 microg/ml in Hep-2 Cell culture. The median inhibition concentration (IC50) was 0.255 microg/ml when drug was added before infection. The IC50 was 0.142 microg/ml when drug was added after virus infection, and it was 0.117 microg/ml as the drug was added after it mixed with virus. The therapeutic index (TI) was 2.55, 4.57 and 5.55 respectively. CONCLUSION: The direct inactivation effect of realgar nanoparticles is the most evident in three drug deliveries manner with the same concentration in vitro.

Gadolinium-promoted precipitation of calcium phosphate is associated with profibrotic activation of RAW 264.7 macrophages.

Gadolinium-based contrast agents are now being linked to nephrogenic systemic fibrosis (NSF). The exact mechanism by which gadolinium species act in the pathogenesis of NSF is not fully understood. In this study, we evaluated the effect of gadolinium chloride (GdCl(3)) on the precipitation of calcium phosphate, and examined the role of the gadolinium-containing precipitates in the profibrotic activation of macrophages. In a free-drift system, the induction time was markedly reduced with increasing concentration of GdCl(3), accompanied by alterations of morphology and composition of the precipitates. In complete cell culture medium, the addition of GdCl(3) resulted in formation of particles around 200-300 nm. In an in vitro cellular model with RAW 264.7 macrophages, GdCl(3) increased the production of TGF-beta1 and IL-6 via the activation of PKC and ERK signaling pathway. Our findings demonstrate that GdCl(3) promotes calcium phosphate precipitation and induces profibrotic activation of macrophages.

Effects of LDL, cholesterol, and their oxidized forms on the precipitation kinetics of calcium phosphates.

BACKGROUND: LDL, cholesterol, and their oxidized forms are known cardiovascular risk factors and are often found in atherosclerotic lesions of various stages. Little is known, however, about whether they are directly involved in the formation of calcium phosphate compounds. METHODS: We used the pH-stat technique to follow the kinetics of calcium phosphate precipitation at pH 7.4, 37 degrees C, and ionic strength 0.150 mol/L, in the presence or absence of LDL, oxidized LDL, cholesterol, cholestane-3beta,5alpha,6beta-triol, and cholesteryl linoleate. The precipitates were characterized by x-ray diffraction, scanning and transmission electronic microscopy coupled with energy-dispersion x-ray analysis, and inductively coupled plasma atomic emission spectroscopy. RESULTS: Under the experimental conditions, LDL (14.8 and 43.1 mg/L protein) had no significant effect on the precipitation kinetics. Oxidized LDL (14.8 and 43.1 mg/L protein) prolonged the nucleation phase and diminished the amount of total precipitate, and both the extent of oxidation and the concentration of the protein affected the kinetics. Cholesterol microcrystals (71.4 and 143 mg/L) made the nucleation phase shorter (300 min vs 390 min for the control), and the precipitated particles had an organic core and a shell composed of calcium phosphates. L-alpha-Phosphatidylcholine vesicles (143 mg/L), cholesterol (71.4 mg/L)/phospholipid (143 mg/L) mixed vesicles, cholesteryl linoleate (143 mg/L), and cholestane-3beta,5alpha,6beta-triol (71.4 mg/L) prolonged the nucleation phase. CONCLUSIONS: LDL is not involved directly in the precipitation of calcium phosphates. Oxidized LDL inhibits both nucleation and crystal growth, possibly by attracting calcium ions in the solution and thus reducing supersaturation. Cholesterol microcrystals serve as seeds for the precipitation of hydroxyapatite, whereas L-alpha-phosphatidylcholine, cholesteryl linoleate, and cholestane-3beta,5alpha,6beta-triol exhibit inhibitive effects on the nucleation of calcium phosphates.