Determination of Chromium(III)and Chromium(VI) by Electrothermal Atomic Absorption Spectrometry after Separation and Preconcentration with Ion Exchange Resins

A sensitive and selective method for the determination of ppb levels of chromium (III) and chromium (VI) in water, based on preconcentration writh a mixture of finely pulverized chelating exchange resin and anion-exchange resin beads has been investigated by electrothermal atomic absorption spectrometry (ETAAS). A sample solution containing 0.1-2.0μg of both chromium (III) and chromiunl (VI) was placed in a polyethylene beaker; then, 20 mg of anion-exchange resin beads (ARB) and 1ml of finely pulverized chelating exchange resin suspension (CRS) were added at pH 6.　The solution was stirred for 20 min and filtered under suction through a glass filter, and the ARB were placed on the pasted surface of a cel1ophaned tape, fixed by sticking another tape on them, and then divided into several sets of six ARB by cutting with ceramics scissors. A sets of six ARB was inserted into a cup-type cuvette for the determination of chromiunl (VI) by ETAAS. The filtrate was then filtered under suction through a membrane filter. A thin circular resin, which was retained on a membrane filter, was fixed by sticking directly with a cellophaned tape and divided into several 3 mm diameter disks by punching. Each disk was used for the deternination of chromium (III) by ETAAS. The effects of commonly occurring foreign ions were investigated. The proposed method based on preconcentration with the exchange resins will be applied to the determination of chromium (III) and chromium (Vl) in water samples

Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue

3-D structure of extracellular matrix regulates gene expression in cultured hepatic stellate cells to induce process elongation

HSCs showed myofibroblast-like shapes when cultured on polystyrene surface or on type I collagen-coated surface, whereas HSCs cultured on type I collagen gel were induced to elongate cellular processes, suggesting that HSCs recognize 3-D structure of extracellular type I collagen fibrils and change their morphology and function. In this study we examined the differentially regulated gene expression by extracellular matrix (ECM) components by PCR-differential display (PCR-DD) analysis followed by cloning and FASTA homology search, and identified the mRNA species as a transcription factor SP1, breast cancer resistant protein (BCRP), dystonin, and KAP3B. Regulation of dystonin and KAP3B expression was confirmed by RT-PCR analysis. Thus, cell surface-binding to extracellular interstitial collagen may trigger intracellular signaling and alteration in gene expression, and HSCs not only produce various ECM components but also change their morphology and gene expression in response to ECM components adhering to the cells

Intercellular Adhesive Structures Between Stellate Cells – An Analysis in Cultured Human Hepatic Stellate Cells

To investigate whether or not hepatic stellate cells can form intercellular junctions with each other, we cultured human stellate cells (LI90) on different kinds of substrata. Intercellular junctions were detected between these cultured stellate cells by transmission electron microscopy (TEM). The molecular components of the intercellular adhesive structures were identified by immunofluorescence microscopy. Immunofluorescence for cadherin and catenins was detected at the adhesion sites between the cultured stellate cells. Thus, the intercellular junctions were indicated to be adherens junctions at the molecular level. The junctions developed in the cultured stellate cells irrespective of the type of substratum. These data suggest that the junctional formation between the stellate cells occurs in vivo as well as in vitro

Intralobular Distribution of Vitamin A-Storing Lipid Droplets in Hepatic Stellate Cells with Special Reference to Polar Bear and Arctic Fox

We examined the liver of adult polar bears, arctic foxes, and rats by gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, hematoxylin-eosin staining, staining with Masson's trichrome, Ishii and Ishii's silver impregnation, and transmission electron microscopical morphometry. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. These results indicate that the hepatic stellate cells of the polar bears and arctic foxes possess heterogeneity of vitamin A-storing capacity in their liver lobules

Rationale and design of a multicenter randomized study for evaluating vascular function under uric acid control using the xanthine oxidase inhibitor, febuxostat : the PRIZE study

Background: Xanthine oxidase inhibitors are anti-hyperuricemic drugs that decrease serum uric acid levels by inhibiting its synthesis. Xanthine oxidase is also recognized as a pivotal enzyme in the production of oxidative stress. Excess oxidative stress induces endothelial dysfunction and inflammatory reactions in vascular systems, leading to atherosclerosis. Many experimental studies have suggested that xanthine oxidase inhibitors have anti-atherosclerotic effects by decreasing in vitro and in vivo oxidative stress. However, there is only limited evidence on the clinical implications of xanthine oxidase inhibitors on atherosclerotic cardiovascular disease in patients with hyperuricemia. We designed the PRIZE study to evaluate the effects of febuxostat on a surrogate marker of cardiovascular disease risk, ultrasonography-based intima-media thickness of the carotid artery in patients with hyperuricemia. Methods: The study is a multicenter, prospective, randomized, open-label and blinded-endpoint evaluation (PROBE) design. A total of 500 patients with asymptomatic hyperuricemia (uric acid >7.0 mg/dL) and carotid intima-media thickness ≥1.1 mm will be randomized centrally to receive either febuxostat (10–60 mg/day) or non-pharmacological treatment. Randomization is carried out using the dynamic allocation method stratified according to age (<65, ≥65 year), gender, presence or absence of diabetes mellitus, serum uric acid (<8.0, ≥8.0 mg/dL), and carotid intima-media thickness (<1.3, ≥1.3 mm). In addition to administering the study drug, we will also direct lifestyle modification in all participants, including advice on control of body weight, sleep, exercise and healthy diet. Carotid intima-media thickness will be evaluated using ultrasonography performed by skilled technicians at a central laboratory. Follow-up will be continued for 24 months. The primary endpoint is percentage change in mean intima-media thickness of the common carotid artery 24 months after baseline, measured by carotid ultrasound imaging. Conclusions: PRIZE will be the first study to provide important data on the effects of febuxostat on atherosclerosis in patients with asymptomatic hyperuricemia

Febuxostat and carotid atherosclerosis in asymptomatic hyperuricemia

Background An elevated level of serum uric acid (SUA) is associated with an increased risk of cardiovascular disease. Pharmacological intervention with urate-lowering agents, such as the conventional purine analogue xanthine oxidase (XO) inhibitor, allopurinol, has been used widely for a long period of time in clinical practice to reduce SUA levels. Febuxostat, a novel non-purine selective inhibitor of XO, has higher potency for inhibition of XO activity and greater urate-lowering efficacy than conventional allopurinol. However, clinical evidence regarding the effects of febuxostat on atherosclerosis is lacking. The purpose of the study was to test whether treatment with febuxostat delays carotid intima-media thickness (IMT) progression in patients with asymptomatic hyperuricemia. Methods and findings The study was a multicenter, prospective, randomized, open-label, blinded-endpoint clinical trial undertaken at 48 sites throughout Japan between May 2014 and August 2018. Adults with both asymptomatic hyperuricemia (SUA >7.0 mg/dL) and maximum IMT of the common carotid artery (CCA) ≥1.1 mm at screening were allocated equally using a central web system to receive either dose-titrated febuxostat (10–60 mg daily) or as a control-arm, non-pharmacological lifestyle modification for hyperuricemia, such as a healthy diet and exercise therapy. Of the 514 enrolled participants, 31 were excluded from the analysis, with the remaining 483 people (mean age 69.1 years [standard deviation 10.4 years], female 19.7%) included in the primary analysis (febuxostat group, 239; control group, 244), based on a modified intention-to-treat principal. The carotid IMT images were recorded by a single sonographer at each site and read in a treatment-blinded manner by a single analyzer at a central core laboratory. The primary endpoint was the percentage change from baseline to 24 months in mean IMT of the CCA, determined by analysis of covariance using the allocation adjustment factors (age, gender, history of type 2 diabetes, baseline SUA, and baseline maximum IMT of the CCA) as the covariates. Key secondary endpoints included changes in other carotid ultrasonographic parameters and SUA and the incidence of clinical events. The mean values (± standard deviation) of CCA-IMT were 0.825 mm ± 0.173 mm in the febuxostat group and 0.832 mm ± 0.175 mm in the control group (mean between-group difference [febuxostat − control], −0.007 mm [95% confidence interval (CI) −0.039 mm to 0.024 mm; P = 0.65]) at baseline; 0.832 mm ± 0.182 mm in the febuxostat group and 0.848 mm ± 0.176 mm in the control group (mean between-group difference, −0.016 mm [95% CI −0.051 mm to 0.019 mm; P = 0.37]) at 24 months. Compared with the control group, febuxostat had no significant effect on the primary endpoint (mean percentage change 1.2% [95% CI −0.6% to 3.0%] in the febuxostat group (n = 207) versus 1.4% [95% CI −0.5% to 3.3%] in the control group (n = 193); mean between-group difference, −0.2% [95% CI −2.3% to 1.9%; P = 0.83]). Febuxostat also had no effect on the other carotid ultrasonographic parameters. The mean baseline values of SUA were comparable between the two groups (febuxostat, 7.76 mg/dL ± 0.98 mg/dL versus control, 7.73 mg/dL ± 1.04 mg/dL; mean between-group difference, 0.03 mg/dL [95% CI −0.15 mg/dL to 0.21 mg/dL; P = 0.75]). The mean value of SUA at 24 months was significantly lower in the febuxostat group than in the control group (febuxostat, 4.66 mg/dL ± 1.27 mg/dL versus control, 7.28 mg/dL ± 1.27 mg/dL; mean between-group difference, −2.62 mg/dL [95% CI −2.86 mg/dL to −2.38 mg/dL; P < 0.001]). Episodes of gout arthritis occurred only in the control group (4 patients [1.6%]). There were three deaths in the febuxostat group and seven in the control group during follow-up. A limitation of the study was the study design, as it was not a placebo-controlled trial, had a relatively small sample size and a short intervention period, and only enrolled Japanese patients with asymptomatic hyperuricemia. Conclusions In Japanese patients with asymptomatic hyperuricemia, 24 months of febuxostat treatment did not delay carotid atherosclerosis progression, compared with non-pharmacological care. These findings do not support the use of febuxostat for delaying carotid atherosclerosis in this population

Rationale and design of a multicenter randomized controlled study to evaluate the preventive effect of ipragliflozin on carotid atherosclerosis : the PROTECT study

Background: Type 2 diabetes mellitus is associated strongly with an increased risk of micro- and macro-vascular complications, leading to impaired quality of life and shortened life expectancy. In addition to appropriate glycemic control, multi-factorial intervention for a wide range of risk factors, such as hypertension and dyslipidemia, is crucial for management of diabetes. A recent cardiovascular outcome trial in diabetes patients with higher cardiovascular risk demonstrated that a SGLT2 inhibitor markedly reduced mortality, but not macro-vascular events. However, to date there is no clinical evidence regarding the therapeutic effects of SGLT2 inhibitors on arteriosclerosis. The ongoing PROTECT trial was designed to assess whether the SGLT2 inhibitors, ipragliflozin, prevented progression of carotid intima-media thickness in Japanese patients with type 2 diabetes mellitus. Methods: A total of 480 participants with type 2 diabetes mellitus with a HbA1c between 6 and 10 % despite receiving diet/exercise therapy and/or standard anti-diabetic agents for at least 3 months, will be randomized systematically (1:1) into either ipragliflozin or control (continuation of conventional therapy) groups. After randomization, ipragliflozin (50–100 mg once daily) will be added on to the background therapy in participants assigned to the ipragliflozin group. The primary endpoint of the study is the change in mean intima-media thickness of the common carotid artery from baseline to 24 months. Images of carotid intima-media thickness will be analyzed at a central core laboratory in a blinded manner. The key secondary endpoints include the change from baseline in other parameters of carotid intima-media thickness, various metabolic parameters, and renal function. Other cardiovascular functional tests are also planned for several sub-studies. Discussion: The PROTECT study is the first to assess the preventive effect of ipragliflozin on progression of carotid atherosclerosis using carotid intima-media thickness as a surrogate marker. The study has potential to clarify the protective effects of ipragliflozin on atherosclerosis