Pseudo-equilibrium equations for calcium phosphate precipitation with multi-unit particles.

The chemistry underlying bone mineral formation in vertebrates is the reaction of calcium phosphate precipitation. In a near-neutral solution, an amorphous phase and hydroxyapatite nanoparticles appear successively, and the reaction system containing either of the two kinds of precipitates is in a non-equilibrium state. Here, we propose a pseudo-equilibrium approach to the solution chemistry of the precipitation reactions. We employed two series of reaction systems, collected samples at various stages, and analyzed the solution chemistry data on the basis of a simplified model of reaction. We derived two types of pseudo-equilibrium equations from the two series, respectively. These equations reveal the existence of multiple structural units in a precipitate particle and correlate the ionic product with the surface proportion per structural unit (m). The surface proportion, in turn, is related to the whole particle through a particle-surface equation. Notably, the two types of pseudo-equilibrium constants have the common expression of "Kd = ionic product" if the number of the structural units (u) is large enough. Together, these findings have revealed some aspects of the non-equilibrium thermodynamics of precipitation reactions, indicating the solution chemistry route to the equilibrium state. The concept of the multi-unit particle may shed new light on the study of precipitation reactions of other slightly soluble electrolytes. And the relationship between the ionic product and the surface proportion of a structural unit is not only fundamental in chemistry, but may also apply to non-equilibrium systems in nature and biology, such as marine sedimentation, human vascular calcification, and bone mineral metabolism.

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.

Influences of LaCl3 on the mineral phase transformation during osteoblast mineralization in vitro.

Rat calvarial osteoblasts were treated with lanthanum chloride (LaCl3) to explore its effect on the mineral crystalline phase during the process of osteoblast calcification in vitro. The results confirmed that La was readily deposited in the mineral component of the matrix. Employing high-resolution transmission electron microscopy and Fourier transform infrared microspectroscopy techniques, we demonstrated that features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate, and hydroxyapatite (HAP) were detected in the mineral phases in vitro. Particularly, LaCl3 treatment retarded conversion from DCPD-like phase into HAP during mineralization. In addition, La was introduced in DCPD powder during wet chemical synthesis. When compared with that of La-free DCPD, the dissolution rate of La-incorporated DCPD was lower, thereby leading to a delayed DCPD-to-HAP phase transformation. Thus, it can be concluded that LaCl3 treatment influences the kinetics of inorganic phase transition by decreasing the dissolution rate of DCPD.

[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.

Hyperprogression under treatment with immune-checkpoint inhibitors in patients with gastrointestinal cancer: A natural process of advanced tumor progression?

Immunotherapy has shown great promise in treating various types of malignant tumors. However, some patients with gastrointestinal cancer have been known to experience rapid disease progression after treatment, a situation referred to as hyperprogressive disease (HPD). This minireview focuses on the definitions and potential mechanisms of HPD, natural disease progression in gastrointestinal malignancies, and tumor immunological microenvironment.

Gadolinium inhibits prostate cancer PC3 cell migration and suppresses osteoclast differentiation in vitro.

This study examined whether Gd (gadolinium) could suppress prostate cancer cell migration and prostate cancer cell-induced osteoclast differentiation. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and colony forming assay showed that GdCl3 treatment inhibited both cell viability and colony forming ability in PC3 cells more significantly than that in DU145 cells. Annexin/PI (propidium iodide) staining showed an increase in apoptotic death of PC3 cells in the presence of GdCl3. Wound healing and adhesion assay indicated that GdCl3 suppressed PC3 cell migration. Western-blot analysis demonstrated that GdCl3 treatment inhibited phosphorylation of ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase). Pretreatment with PTx (pertussis toxin), a Gi protein inhibitor, conferred resistance to GdCl3-induced colony formation, ERK and p38 phosphorylation in PC3 cells. Moreover, GdCl3 inhibited PC3 cell-induced osteoclast differentiation. RT-PCR (reverse transcription-PCR) indicated that GdCl3 decreased the expression of RANKL (receptor activator of nuclear factor-κB ligand) in PC3 cells, whereas it increased the expression of OPG (osteoprotegerin) in PC3 and DU145 cells. In conclusion, the present study indicated that GdCl3 inhibited PC3 cell migration mediated by the inactivation of both ERK and p38 MAPK pathways via PTx-sensitive G proteins, and also suppressed PC3 cell-induced osteoclast differentiation via regulating the mRNA expression of OPG and RANKL.

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.

Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomes.

Rhein, an active ingredient extensively found in plants such as Aloe, Cassitora L., rhubarb and so on, has been used for a long time in China. Pharmacological tests revealed that rhein not only had a strong antibacterial action, but also may be useful in cancer chemotherapy as a biochemical modulator. Its therapeutic action and toxicity is still the subject of considerable research. With microsome incubation assays in vitro and HPLC methods, the inhibition of rat liver CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A enzymes by rhein were studied kinetically. The results showed the most inhibition of CYP2E1 by rhein (K(i) = 10 microm, mixed); CYP3A and CYP2C9 were also inhibited by rhein, K(i) = 30 microm (mixed) and K(i) = 38 microm (mixed), respectively; rhein revealed some inhibition of CYP1A2 (K(i) = 62 microm, uncompetitive) and CYP2D6 (K(i) = 74 microm, mixed). Drug-drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in the efficacy of co-administered drugs. Inhibition of the five major CYP enzymes observed for rhein suggested that changes in pharmacokinetics of co-administered drugs were likely to occur. Therefore, caution should be paid to the possible drug interaction of medicinal plants containing rhein and CYP substrates.

Lanthanum enhances in vitro osteoblast differentiation via pertussis toxin-sensitive gi protein and ERK signaling pathway.

Converging lines of evidence suggest that lanthanum tends to deposit in bone. The influence of lanthanum ion (La3+) on osteoblast differentiation and the related mechanism are essential to understanding its effect on bone metabolism. In this study, La3+ treatment enhanced in vitro osteoblast differentiation as evidenced by promoting alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and matrix mineralization. The expressions of osteoblast-specific genes of Cbfa-1, osteopontin (OPN), and bone sialoprotein (BSP) were all increased in the presence of La3+, but no change was observed in that of type I collagen (COL-I). Further studies demonstrated that La3+ treatment enhanced phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 suppressed the effects of La3+ on osteoblast activity. Moreover, pretreatment of the cells with pertussis toxin (PTx), a Gi protein inhibitor, suppressed the La3+-enhanced ERK phosphorylation and osteoblast differentiation. These findings suggest that La3+ exposure enhances in vitro osteoblast differentiation and the effect depends on ERK phosphorylation via PTx-sensitive Gi protein signaling.

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.

Selenoprotein P as the major transporter for mercury in serum from methylmercury-poisoned rats.

Selenium (Se) has been found to promote weight gain, decrease hepatic damage, but redistribute mercury (Hg) in brains and livers in methylmercury (MeHg)-poisoned rats. The aims of the present work were to examine the effects of Se on the levels of Hg in serum and the role of serum selenoproteins in binding with Hg in MeHg-poisoned rats. The concentration of Se, Hg and MeHg were studied using ICP-MS and CVAFS. The Hg- and Se-binding selenoproteins were separated and quantified using affinity chromatography with post-column isotope dilution analysis using both enriched 78Se and 199Hg. It was found that Se treatment reduced Hg levels in serum in MeHg-poisoned rats. Among the three separated selenoproteins, the amounts of SelP-bound Hg and Se increased to 73% and 93.6%, from 64.4% and 89.3% of the total Hg and Se, respectively after Se treatment, suggesting that SelP acts as a major transporter for Hg and pool for Se in serum. Over 90% of the total Hg was MeHg in serum, and the molar ratios of MeHg to Se as 1:4 and 1:9 in the formed MeHg-Se-SelP complex in the control and the Se treatment group, respectively. The elevated Se level binding with SelP facilitated the Hg extraction from tissues and organs, as well as its redistribution in brains and livers through blood circulation in the MeHg-poisoned rats. Together, our findings provide direct evidence that serum SelP is the major Hg transporter in MeHg-poisoned rats.

Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro.

Icariin, a principal flavonoid glycoside in Herba Epimedii, is hypothesized to possess beneficial effects on bone mass. Icariin is metabolized to icariside II and then to icaritin in vivo. In the present study, we investigated the in vitro effects of icariin, icariside II and icaritin on both osteoblasts and osteoclasts. After treatment with these compounds at concentrations 10(-5)-10(-8) mol/l, osteoblasts were examined for proliferation, alkaline phosphatase activity, osteocalcin secretion and matrix mineralization, as well as expression levels of bone-related proteins. The formation of osteoclasts was assessed by counting the number of multinucleated TRAP-positive cells. The activity of isolated rat osteoclasts was evaluated by measuring pit area, actin rings and superoxide generation. Icariside II and icaritin increased the mRNA expression of ALP, OC, COL-1 and OPG, but suppressed that of RANKL. In addition, these compounds reduced the number of multinucleated TRAP-positive cells and the osteoclastic resorption area. Also decreases were observed in superoxide generation and actin ring formation that are required for osteoclast survival and bone resorption activity. These findings suggest that icaritin, which was more potent than icariin and icariside II, enhanced the differentiation and proliferation of osteoblasts, and facilitated matrix calcification; meanwhile it inhibited osteoclastic differentiation in both osteoblast-preosteoclast coculture and osteoclast progenitor cell culture, and reduced the motility and bone resorption activity of isolated osteoclasts.

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.

[Differentiation of HL-60 cells induced by realgar nano-particles].

OBJECTIVE: To investigate the proliferation inhibition and the differentiation effects of realgar (As4S4) nano-particles on human acute myeloid leukemia cell line HL-60. METHOD: Cell viability was determined by MTT and PI-stained cell cycle assays. The realgar induced morphological changes on cells were examined after Wright-Giemsa staining. The cell differentiation was evaluated with NBT and specific cell surface antigen (CD11b and CD14) expression assays. RESULT: HL-60 cells exhibited obvious morphological features of differentiation after the realgar treatment. A 24 h incubation of the cells with 0.25-1.0 micromol x L(-1) realgar caused a great increase in NBT reduction ability. The expressions of CD11b and CD14 were augmented in cells treated with 0.50 micromol x L(-1) realgar for 48 h, and cell cycles were arrested in G1 phase. CONCLUSION: Low dose realgar induces differentiation in human acute myeloid leukemia cell line HL-60.