Nitric oxide metabolites are associated with survival in older patients.

OBJECTIVES: To assess the efficacy of various vascular endocrinological substances, such as plasma nitric oxide metabolites (NOx), as surrogate markers of survival in older patients. DESIGN: Prospective cohort, observational. SETTING: Nagoya University Hospital and related hospitals, Japan. PARTICIPANTS: One hundred fifty patients aged 70 and older, recruited consecutively from the outpatient clinics of Nagoya University Hospital and related hospitals. MEASUREMENT: Serum biochemical analyses such as albumin and total cholesterol, various prognostic markers, such as tumor necrosis factor (TNF)-alpha, NOx, activities of daily living (ADLs), and instrumental ADLs (IADLs) were evaluated on enrollment. ADLs, IADLs, and comorbidities, especially depression and impaired cognition, were evaluated on enrollment. The main outcome was survival rate over 2.75 years. RESULTS: Forty-nine patients died during the follow-up period. Mann-Whitney U-test showed that hemoglobin, total protein, serum albumin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high sensitive c-reactive protein, NOx, B-type natriuretic peptide, interleukin-6, and TNF-alpha levels; ADLs; cognitive impairment; and depressive status were significantly different for subjects who survived and those who died. Of the dependent variables in the Cox proportional hazards regression analyses, only ADLs, NOx, and albumin were significantly different. In the Kaplan-Meier analyses of mortality, the prognosis of patients in the third and fourth quartiles of NOx was significantly worse than that of patients in the first or second quartile. The prognosis of patients with impaired ADLs was worse than that of other patients for the overall period. CONCLUSION: Lower levels of NOx may be associated with survival in older patients. It may be an effective marker, like ADLs, which is a well-known marker.

High glucose downregulates the number of caveolae in monocytes through oxidative stress from NADPH oxidase: implications for atherosclerosis.

Atherosclerosis, an inflammatory disease, is closely associated with hyperglycemia, major sign of diabetes mellitus. Caveolae are vesicular invaginations of the plasma membrane that mediate the intracellular transport of lipids such as cholesterol. We evaluated the relationship between the expression of caveolin-1 and the number of caveolae in macrophages under conditions of high glucose concentration. Increased superoxide production, induction of inducible nitric oxide synthase (iNOS), and decreased caveolin-1 were observed in a concentration-dependent manner in THP-1 derived macrophages with high glucose concentrations. Mannitol, used as an osmotic control, showed no effects. Furthermore, co-localization of the NADPH oxidase component, p47(phox), and caveolin was confirmed by confocal microscopy. An atomic force microscopy (AFM) study showed that high glucose concentrations reduced the number and size of the caveolae. The percentage of cells with fragmented DNA was increased in cells grown in hyperglycemic media. Taken together, high glucose concentrations suppress the levels of caveolin-1 expression and reduce the number of caveolae. This might be due to the actions of superoxide via the activation of NADPH oxidase by translocation of its component and uncoupling of induced iNOS in macrophages. Furthermore, the apoptosis of macrophages might occur with high glucose concentrations, leading to the spreading of lipids from macrophages into intracellular spaces in the vessel wall.

Nitric oxide (NO) is a new clinical biomarker of survival in the elderly patients and its efficacy might be nearly equal to albumin.

BACKGROUND: For elderly patients, the consideration of prognostic factors is very important, but there have been few reports about the potential use of vasoactive substances as prognostic markers in the elderly. OBJECTIVE: We assessed endocrinological substances, such as plasma NO(x) (metabolites of NO), as the prognostic marker in elderly. We compared their efficacy with that of such well-known markers as albumin and pro-inflammatory cytokines such as IL-6. METHODS: The patients were recruited consequently from the clinics of Nagoya University Hospital or related home care services facilities. One hundred and twenty seven elderly aged 65 and older were registered. Biochemical analyses such as albumin, total cholesterol, BNP, and NO(x) were measured upon enrollment. The main outcome was the survival rate. RESULTS: Forty-six patients died during the follow-up period. Mann-Whitney's U-test showed that the levels of age, hemoglobin, total protein, serum albumin, serum creatinine, total cholesterol, HDL-cholesterol, LDL-cholesterol, high sensitive CRP, NO(x), IL-6, and TNF-alpha were significantly different between the living and deceased subjects. Among the dependent variables in the logistic regression analyses, only albumin and NO(x) were significantly different. In the Kaplan-Meier analyses of mortality, the prognosis of patients in 3rd and 4th quartile of NO(x) was significantly worse than that in 1st or 2nd quartile. CONCLUSION: NO(x) has potential both as a vascular marker and as a marker for predicting survival in elderly. In the latter role, it may be as effective as albumin.

l-Citrulline and l-arginine supplementation retards the progression of high-cholesterol-diet-induced atherosclerosis in rabbits.

The objective of this study was to evaluate the influence of ingested l-arginine, l-citrulline, and antioxidants (vitamins C and E) on the progression of atherosclerosis in rabbits fed a high-cholesterol diet. The fatty diet caused a marked impairment of endothelium-dependent vasorelaxation in isolated thoracic aorta and blood flow in rabbit ear artery in vivo, the development of atheromatous lesions and increased superoxide anion production in thoracic aorta, and increased oxidation-sensitive gene expression [Elk-1 and phosphorylated cAMP response element-binding protein]. Rabbits were treated orally for 12 weeks with l-arginine, l-citrulline, and/or antioxidants. l-arginine plus l-citrulline, either alone or in combination with antioxidants, caused a marked improvement in endothelium-dependent vasorelaxation and blood flow, dramatic regression in atheromatous lesions, and decrease in superoxide production and oxidation-sensitive gene expression. These therapeutic effects were associated with concomitant increases in aortic endothelial NO synthase expression and plasma NO(2)(-)+NO(3)(-) and cGMP levels. These observations indicate that ingestion of certain NO-boosting substances, including l-arginine, l-citrulline, and antioxidants, can abrogate the state of oxidative stress and reverse the progression of atherosclerosis. This approach may have clinical utility in the treatment of atherosclerosis in humans.

The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase.

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O2- was increased simultaneously. NOS inhibitor, inhibited O2- release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process.

A new HMG-CoA reductase inhibitor, pitavastatin remarkably retards the progression of high cholesterol induced atherosclerosis in rabbits.

BACKGROUND: The remarkable anti-atherosclerotic effects of 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor have not been demonstrated in diet induced severe hyperlipidemia in rabbit model. OBJECTIVE: We have investigated the effect of pitavastatin, a newly developed statin, on atherosclerosis in rabbits. METHODS AND RESULTS: Oophorectomized female NZW rabbits were fed 0.3% cholesterol chow for 12 weeks with or without pitavastatin (0.1mg/kg per day) (Gp.NK and HCD). The level of serum cholesterol was decreased in Gp.NK compared with Gp.HCD (772.8 +/- 70.2 versus 1056.9 +/- 108.3 mg/d), whereas no significant alterations were observed in triglyceride and HDL-cholesterol. NO dependent response stimulated by acetylcholine and calcium ionophore A23187 and tone related basal NO response induced by N(G)-monomethyl-l-arginine acetate were all improved by pitavastatin treatment. Pitavastatin treatment increased the level of cyclic GMP in the aorta of cholesterol fed rabbits. In the aorta, the expression of eNOS mRNA was significantly up regulated and O(2)(-) production was slightly reduced in Gp.NK animals. Atherosclerotic area was significantly decreased in aortic arch and thoracic aorta from Gp.NK compared with those from Gp.HCD ( 15.1 +/- 5.3 versus 41.9 +/- 10.2%, 3.1 +/- 1.1versus 7.9 +/- 1.2% in Gp.NK and Gp.HCD aortic arch and thoracic aorta). Anti-macrophage staining area, the MMP1 or 2 and the nitrotyrosine positive area were decreased in Gp.NK. CONCLUSION: Pitavastatin retards the progression of atherosclerosis formation and it improves NO bioavailability by eNOS up-regulation and decrease of O(2)(-).

Combined effect of testosterone and apocynin on nitric oxide and superoxide production in PMA-differentiated THP-1 cells.

Human inducible nitric oxide synthase (iNOS) is most readily observed in macrophages from patients with inflammatory diseases like atherosclerosis. The aim of the present study was to find out the combined effect of male sex hormone; testosterone and apocynin (NADPH oxidase inhibitor) on cytokine-induced iNOS production. THP-1 cells were differentiated into macrophages by phorbol myristate acetate (PMA). Expression of iNOS was induced by the addition of cytokine mixture? Testosterone was added at different concentrations (10(-6)-10(-12) M) with apocynin (1 mM). Testosterone (10(-8), 10(-10) M) inhibited NOx production in cytokine-added THP-1 cells which was further confirmed by quantikine assay of iNOS protein and RT-PCR analysis. Testosterone treatment decreased 40% of superoxide anion production. Testosterone showed inhibition of NADPH oxidase, especially expression of p67phox and p47phox (cytosol subunits). Addition of testosterone with apocynin further decreased the expression of p67phox and p47phox subunits of NADPH oxidase. The findings of the present study suggest that, testosterone; the male androgen plays an important role in the prevention of atherogenesis. Even though apocynin does not have any role on NO production, addition of apocynin together with testosterone is effective in suppressing iNOS activity.

Gene transfer of endothelial NO synthase, but not eNOS plus inducible NOS, regressed atherosclerosis in rabbits.

The effects of in vivo gene transfer of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) genes on severe atherosclerosis were investigated in rabbits. The recombinant adenoviruses, Ad.eNOS and Ad.iNOS, which respectively express eNOS and iNOS, were constructed. Atherosclerosis was induced by a balloon injury followed by a high cholesterol diet for 12 weeks. The rabbits were divided into six groups: Gp cont (no treatment); Gp null (adenovirus sham-infected); Gp eNOS (Ad.eNOS); Gp iNOS (Ad.iNOS); Gp e+i (Ad.eNOS plus Ad.iNOS); and Gp heNOS (a high dose of Ad.eNOS). Examinations were carried out 7 days after gene transfer. Plasma lipid levels were not significantly changed, but transfection with Ad.eNOS (Gp eNOS and Gp heNOS) decreased the tissue cholesterol concentration and regressed atherosclerotic lesions. Vessels treated with Ad.iNOS (Gp iNOS and Gp e+i) showed iNOS staining in the atheroma, and slight staining at other parts of the vessels; those treated with Ad.eNOS showed eNOS staining in the endothelium and subintima, and slight staining at other parts. Ad.eNOS transfection, but not Ad.iNOS or Ad.eNOS+Ad.iNOS transfection, improved the impaired aortic endothelium-dependent relaxation (EDR) and basal NO-dependent response, increased tissue cyclic GMP (cGMP), and decreased the release of O2- from vessels. eNOS treatment showed a decreasing tendency in regions with peroxynitrite staining, MMP1 staining, and suspected apoptosis. In conclusion, in vivo gene transfer of eNOS, but not iNOS or eNOS plus iNOS, regressed atherosclerosis. The relations among NO, O2-, and peroxynitrite may be critical, and lipid resorption from the lesions may be responsible for the regression.

Plasma adiponectin plays an important role in improving insulin resistance with glimepiride in elderly type 2 diabetic subjects.

OBJECTIVE: We investigated the effect of glimepiride, a third-generation sulfonylurea hypoglycemic agent, on insulin resistance in elderly patients with type 2 diabetes, in connection with plasma adiponectin and 8-epi-prostagrandin F2alpha (8-epi-PGF2alpha), an oxidative stress marker. RESEARCH DESIGN AND METHODS: A total of 17 elderly patients with type 2 diabetes received 12 weeks of treatment with glimepiride. Homeostasis assessment model of insulin resistance (HOMA-IR), homeostasis assessment model of beta-cell function, HbA(1c), C-peptide in 24-h pooled urine (urine CPR), and plasma concentrations of 8-epi-PGF2alpha, tumor necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor type 1, and adiponectin were measured at various times. The metabolic clearance rate of glucose (MCR-g) was also assessed by a hyperinsulinemic-euglycemic clamp. RESULTS: After 8 weeks of glimepiride treatment, significant reductions were observed in HbA(1c) (from 8.4 +/- 1.9 to 6.9 +/- 1.0%), HOMA-IR (from 2.54 +/- 2.25 to 1.69 +/- 0.95%), and plasma TNF-alpha concentrations (from 4.0 +/- 2.0 to 2.6 +/- 2.5 pg/ml). MCR-g was significantly increased from 3.92 +/- 1.09 to 5.73 +/- 1.47 mg. kg(-1). min(-1). Plasma adiponectin increased from 6.61 +/- 3.06 to 10.2 +/- 7.14 micro g/ml. In control subjects, who maintained conventional treatment, no significant changes were observed in any of these markers. CONCLUSIONS: Glimepiride remarkably improved insulin resistance, suggested by a significant reduction in HOMA-IR, an increase in MCR-g, and a reduction in HbA(1c) without changing extrapancreatic beta-cell function and urine CPR. Increased plasma adiponectin and decreased plasma TNF-alpha may underlie the improvement of insulin resistance with glimepiride.

Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS.

The electrospray ionization (ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF) mass spectrometer combined with the nano-HPLC system was utilized to determine the glycosylation site and the glycan structure in glycoprotein TIME-EA4 (EA4) from Bombyx diapause eggs. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730.58 Da. Then, EA4 was digested with trypsin and chymotrypsin to identify the glycosylated peptide. The peptide fragment from G1y21 to Phe25 was found to carry the carbohydrate moiety. LC-MS/MS analysis of this peptide fragment revealed the sequence of the attached oligosaccharide and the glycosylation site at the same time. The present methodology utilizing the combination of the nano-HPLC system and a highly sensitive Q-TOF mass spectrometer is demonstrated to be quite effective for analyses of glycoproteins of relatively low purity and limited availability from natural sources.