Optimization of Single-Step Assay for Circulating Nitrite and Nitrate Ions (NOx) as Risk Factors of Cardiovascular Mortality.

We propose an optimized single-step assay of total nitrite and nitrate concentrations based on serum deproteinization by ultrafiltration. After deproteinization, the residual protein concentration was 0.0800±0.0007 mg/ml, which secured necessary transparency of the specimens in contrast to methanol deproteinization. The optimized assay for serum nitrite and nitrate ions is characterized by high reproducibility and accuracy (variation coefficient <7%) and can be recommended for routine use in clinical practice.

[Combined markers of initial stages of coronary atherosclerosis]. / Kombinirovannye markery nachal'noi stadii ateroskleroza koronarnykh arterii.

Abnormalities in energy metabolism and endothelial dysfunction contribute to signaling processes associated with atherogenesis. The goal of our study was to develop diagnostic tests based on endothelial functional markers and adiponectin to differentiate early stages of coronary lesions during atherogenesis. The cohort included male and female patients from 25 to 86 years of age. All subjects underwent coronary angiography and severity of coronary lesions was quantified by the Gensini score that assigns points depending on location and extent of the lesions. We have estimated associations between the Gensini score and some known primary and secondary diagnostic parameters and have found that the ratio of serum levels of adiponectin to endothelin strongly correlates with severity of coronary lesions and can be used for differentiation of male patients lacking coronary atherosclerosis (despite symptoms of ischemic heart disease) from patients that have severe coronary lesions. Predictive power of adiponectin to endothelin ratio did not depend on drug therapy.

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum.

[Combination of Visual and Metabolic Markers in Assessment of Probability of Presence and Severity of Atherosclerosis of Coronary Arteries].

AIM: to assess possibilities of the use of biochemical markers combined with data of methods of imaging of arterial atherosclerotic lesions for evaluation of risk of presence and severity of coronary atherosclerosis. MATERIALS AND METHODS: We enrolled into this study patients (n=205, 136 men, 69 women, age 33-85 years, 94% on statin therapy) who underwent coronary angiography and carotid artery ultrasound dopplerography. Examination included determination of parameters of lipid profile and carbohydrate metabolism, markers of inflammation and metabolism of visceral adipose tissue. The severity of carotid artery atherosclerosis was estimated using mean common carotid artery intima-media thickness and atherosclerotic plaques presence. Severity of coronary atherosclerosis was evaluated using Gensini score. RESULTS: We found association between a number of biomarkers and severity of coronary artery involvement. Presence of coronary atherosclerosis (Gensini score >0) was associated with male sex, carotid artery stenosis >45%, and adiponectin level <8.0 pg/ml. Overt coronary atherosclerosis (Gensini score more or equal 35) significantly correlated with intima-media thickness more or equal 0.9 mm, C-reactive protein level >3.0 mg/l and adiponectin level <8.0 pg/ml.

[Markers of visceral adipose tissue metabolic abnormalities in patients with coronary atherosclerosis in relation to the presence of type 2 diabetes mellitus]. / Markery narusheniya metabolizma vistseral'noi zhirovoi tkani u patsientov s koronarnym aterosklerozom v zavisimosti ot nalichiya sakharnogo diabeta 2-go tipa.

AIM: To assess the specific features of visceral adipose tissue metabolism in patients with coronary atherosclerosis, complicated or uncomplicated type 2 diabetes mellitus (T2DM). MATERIAL AND METHODS: A cohort of 429 patients (325 men and 104 women; mean age, 61.3±9.4 years) with coronary atherosclerosis who had been admitted to the National Research Centre for Preventive Medicine, Ministry of Health of Russia, to undergo coronarography (CG) and to receive high-tech treatments and met the criteria for being included in and excluded from this investigation, was examined. The Gensini scoring scale was used to estimate the magnitude of coronary atherosclerosis from CG RESULTS: Carotid artery duplex ultrasound scanning estimating the intima-media thickness was performed in 48% of the patients. DM was diagnosed from examination results (fasting plasma glucose ≥7.0 mmol/l and glycated hemoglobin >6.5%) and an endocrinologist's report. 94% of the patients took statins. RESULTS: Overall, 18% of the examinees had DM that was 2.5 times more common in the women than in the men (32.7 and 13.2%, respectively (p=0.000). The diabetic and non-diabetic patients showed no significant differences in age: 62.9±8.3 and 60.9±9.6 years, respectively (p=0.105). There were statistically significant differences in the magnitude of coronary artery atherosclerosis according to the Gensini scale in relation to the presence of T2DM; thus, the median Gensini score was 48 in the diabetic patients and 46 in the persons with no signs of the disease (Mann-Whitney test; p=0.03). Analysis of adipokine levels showed that the median leptin level was significantly higher than that in the male patients with T2DM than in the persons with no signs of the disease. In the patients with T2DM, the median adiponectin level turned out to be significantly lower in both men and women. CONCLUSION: The coronary atherosclerosis severity rated using the Gensini scale is shown to increase in the presence of T2DM. The probability of detecting obvious (>45%) carotid artery lesion is associated with the presence of DM in both men and women. The male patients with T2DM concurrent with coronary atherosclerosis are noted to have an elevated leptin level, but a lower adiponectin concentration was found in both the men and women.

[THE POSSIBILITIES OF APPLICATION OF TECHNOLOGY PROTEIN MICROARRAY (MICROCHIPS) FOR ANALYSIS OF PROTEIN COMPOSITION OF BLOOD SERUM].

The microchip technology represents convenient and relatively economic tool of analyzing specific biomarkers with the purpose to diagnose diseases, to evaluate effectiveness of therapy and to investigate signaling pathways. To analyze protein composition of blood serum certain types of finished microchips which were not applied previously on the territory of Russia. The detection from 2% to 5% out of matrix of chips depending on their variety was managed without preliminary depletion of serum (removal of proteins of major fractions). Hence, partial protein composition of blood serum can be analyzed with microchips even without preliminary removal of proteins of major fractions.

[The influence of afobazole on biochemical and histo-patho-morphological parameters of endothelial function at experimental diabetes mellitis in rats].

Oxidizing stress in rats with experimental diabetes mellitis is accompanied by endothelial dysfunction develops. Its biochemical markers are the increase of concentration of blood MDA, the impairments of cell antioxidant depence and a decrease in concentration of total metabolites of NO and expression of endothelial NO-synthetase (e-NOS). Biochemical changes are considered with histopathomorphologic impairments of aortic endothelium. Treatment with afobazole suppressed free-radical oxidation, increased activity of SOD and concentration of total metabolites of NO and a level of eNOS expression and also restored of a histologic pattern of aortic endothelium due to activation of nucleocytoplasmic regenerative processes.

Effect of antioxidants pQ510 and resveratrol on regulatory function of the endothelium in rats with modeled arterial hypertension.

We studied the effects of antioxidants resveratrol and pQ510 on physiological parameters and the state of endothelial NO-synthase as a marker of the regulatory function of the endothelium in the aorta of rats with modeled arterial hypertension. The antioxidants promoted recovery of stable NO metabolites in rat serum and maintained expression of endothelial NO-synthase at a normal level. These effects were confirmed by correction of blood pressure and endothelium-dependent vascular dilation assessed by endothelial dysfunction coefficient.

[Effects of endotheline and nitric oxide on vascular tonicity in patients with chronic cardiac failure].

AIM: To examine effects of endotheline-1 (ET-1) and nitric oxide (NO) on endothelium-dependent mechanisms of vascular tonicity regulation in patients with chronic cardiac failure (CCF) of FC I-IV (NYHA). MATERIAL AND METHODS: Vascular reactions of 94 patients with CCF of FC I-IV were examined according to D.S. Celermajer method using ultrasound of high resolution. Tissue oxygenation was studied with transcutaneous polarography (TCM-2, Radiometer). Parameters of circulating erythrocytes were studied by impurity spectrum. Endothelial NO-synthase expression was determined with application of monoclonal antibodies to endothelial NO-synthase. NO metabolites were studied with colorimetric method using Griss reagent. ET-1 was measured in plasma by enzyme immunoassay. The control group consisted of 28 healthy men aged 20 to 54 years. RESULTS: Patients with CCF of FC I-IV have reduced endothelium-dependent vascular reactions progressing with aggravation of CCF. The causes of the disorders lie in altered metabolism of ET-1 and NO in CCF. CONCLUSION: CCF patients demonstrate changes in endothelium-dependent mechanisms of vascular tonicity regulation caused by disturbed metabolism of ET-1 and NO developing in abnormal regime of tissue oxygenation and depending on CCF severity.

[Screening as a method for determining the serum level of nitric oxide metabolites].

The paper describes a modified method for determining stable nitric oxide (NO) metabolites, which permits one-stage assay of total nitrates and nitrites after blood serum deproteinization. The assay is based on the simultaneous reduction of nitrates and nitrites by vanadium chloride and by diazotization with subsequent stimulation of tinting whose rate is spectrophotometrically estimated at 540 nm. Assay of 100 microl of the deproteinized serum is made, by using a flat-bottomed 96-well plate. The sensitivity of the method is 1.7 microM. The concentration of total NO metabolites detected by the assay in the sera of volunteers ranged from 37.2 to 87.2 microM. It is suggested that the above modified assay may be used in clinical practice for the measurement of the concentrations of terminal NO metabolites since they are early markers of vascular endothelial NO-forming dysfunction.

The GTP-binding regulatory proteins, Gs and G(i), are altered in erythrocyte membranes of patients with ischemic heart disease resulting from coronary atherosclerosis.

Acute ischemic heart disease is associated with alterations in the cardiac adenylate cyclase system response, although the specificity and mechanism of these events are unknown. We studied the characteristics of inhibitory (G(i)) and stimulatory (Gs) GTP-binding regulatory proteins (G proteins) of adenylate cyclase in erythrocyte membranes of patients (n = 16) with nonacute ischemic heart disease resulting from coronary atherosclerosis. Gs was measured by reconstitution with the resolved catalytic unit of adenylate cyclase and by cholera toxin-catalyzed ADP-ribosylation of a 42-kD protein; G(i) was tested as a 41-kD substrate of pertussis toxin-catalyzed ADP-ribosylation. Gs activity was decreased by 27 +/- 2% in the cholate extract and by 25 +/- 3% in the supernatant of guanosine 5'-(gamma-thio)triphosphate-treated membranes. The amount of cholera toxin substrate was decreased by 33 +/- 3%, and the pertussis toxin substrate was increased by 27 +/- 5% compared with healthy subjects (n = 10). All changes in G-protein characteristics appear to be specific relative to other erythrocyte membrane proteins and hemoglobin. Those patients who have a decreased Gs possess approximately normal Gi, and those with increased G(i) showed no change in Gs. Patients with increased G(i) (normal Gs) exhibited more severe deterioration of their coronary arteries than did patients with decreased Gs (normal G(i)) (P < .05), but these two groups did not differ significantly in serum lipids, hormones, drug therapy, historical data, or baseline assessment (P < 0.05).