Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers.

The purpose of the research was to identify the role of chronic inflammation in mechanisms of cardiac valve calcification (CVC) in patients undergoing chronic hemodialysis (HD) by determining the relation of inflammatory markers with valve calcification and the correlation of the latter with endothelial damage indices. Methods. The research included 94patients undergoing chronic HD (males, 52, age, (46,4±11,2) years, duration of HD, (28,9±32,4) months). Patients with chronic glomerulonephritis (47,9 %) dominated. All subjects underwent echo– cardiographic examination for detection of CVC. The intensity of the inflammatory process was estimated by the serum content of fibrinogen (FG), amount of circulatory immune complexes (CICs), concentration of C–reactive protein (CRP) and middle molecules (MM). The nitric oxide (NO) production was studied on the plasma content of nitrite–anions (NO2–) by spectrophotometric method, the amount of circulating endothelial cells (CECs) in platelet rich plasma under the method (Hladovec J. et al., 1978), modified by (Susla A.B., Mysula I.R., 2011). Results. The CRP, FG, CICs indices in patients with CVC exceeded in those without the calcification by 44,2 (р=0,009), 18,4 (р<0,001) and 17,2 % (р=0,002) accordingly. The dynamic of the MM with wave length of 254 nm (MM/254) and 280 nm (MM/280) had identical direction. For the first time a group of patients with CVC (n=42) had been identified with correlations between CECs and CRP indices (Rs=0,42, р=0,006), FG (Rs=0,31, р=0,043), CICs (Rs=0,55, р<0,001), MM/254 (Rs=0,36, р=0,018), MM/280 (Rs=0,42, р=0,005) as well as between the values of NO2– and CRP (Rs=–0,55, р<0,001), FG (Rs=–0,41, р=0,007), CICs (Rs=–0,41, р=0,008), MM/254 (Rs=–0,38, р=0,014), MM/280 (Rs=–0,34, р=0,029). Conclusions. The valve calcification in HD patients combines with the activation of chronic inflammation, which manifests itself in accumulation of CRP, FG, CICs and MM, and the inflammation markers tightly correlate with endothelial damage and lack of NO

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Background and Aims The character of endothelial dysfunction (ED), especially of nitric oxide (NO) system, in patients with diabetic kidney disease (DKD) undergoing chronic hemodialysis (HD) was not asserted enough. In this condition not clearly established the relationship of structural and functional activity of endothelium with the presence and severity of cardiac valve calcification (CVC) as independent predictor of cardiovascular morbidity and mortality. The purpose of the current study was to determine the role of ED in mechanisms of mitral (MVC) and aortic (AVC) valve calcification in HD patients with DKD. Method We enrolled 136 patients undergoing HD (male/female, 78/58; age, 53.9±1.0 years; HD duration, 47.6±4.2 month) in this observational cross-sectional study. According to the study design, depending on the presence of type 2 diabetes mellitus with kidney injury patients were divided into two groups: the 1st one – without DKD (n=88); the 2nd one – with DKD (n=48). All subjects underwent echocardiographical examination for detection of CVC; the MVC and AVC degree were scored as follows: 1, no calcification; 2, valve thickening without calcification; 3, valve annulus or cusps calcification. Vasomotional function of endothelium was assessed using a test with reactive hyperemia (brachial artery flow-mediated dilatation (FMD)). Plasma content of nitrites (NO2), circulating endothelial cells (СECs) and serum concentration of C-reactive protein (CRP) were measured as markers of ED. Data are expressed as means±SEM. Used nonparametric statistics methods: Mann-Whitney U-test, χ2-test, Spearman’s rank R correlations. Results In group of HD patients with DKD indices of СECs (22.3±1.3 vs. 14.2±0.7 × 104/L, Z=4.98, p&lt;0.001), CRP (9.94±1.12 vs. 7.07±1.09 mg/L, Z=3.47, p&lt;0.001) were higher, and NO2 (4.16±0.41 vs. 9.01±1.37 umol/L, Z=3.15, p=0.002), FMD (2.27±0.66 vs. 5.13±0.52%, Z=3.26, p=0.001) – lower compared to the group without diabetes. CVC was detected in 66.6% of patients with DKD with predominance of calcification of both valves (35.4%) over isolated MVC (20.8%) and AVC (10.4 %). Combined valve calcification in the HD patients of the 2nd group was observed 2.6 times more often (χ2=8.78, p=0.003) than in the 1st one. For the first time it was established that in DKD the presence of CVC closely associated with indices of FMD (Rs=-0.59, p&lt;0.001), NO2– (Rs=-0.56, p&lt;0.001), СECs (Rs=0.63, p&lt;0.001) and СRP (Rs=0.54, p&lt;0.001). The MVC as well as AVC degree were related with the level of FMD (Rs=-0.47, p&lt;0.001; Rs=-0.43, p=0.003), content of NO2– (Rs=-0.40, p=0.005; Rs=-0.62, p&lt;0.001), СECs (Rs=0.47, p&lt;0.001; Rs=0.62, p&lt;0.001) and СRP (Rs=0.48, p&lt;0.001; Rs=0.41, p=0.004) concentrations respectively. Conclusion (1) HD patients with DKD are combined with damaged endothelium, disturbance of vasoreactivity and lack of NO. (2) Type 2 diabetes mellitus with kidney injury is characterized by the large-scale combined MVC and AVC, which in turn are closely associated with the ED markers. (3) Complex estimation of the character of CVC and endothelium activity in HD patients with DKD permits a better identification of their cardiovascular risk.

Background and Aims The processes of cardiac valve calcification (CVC) in diabetic hemodialysis (HD) patients are not fully understood. In this context, it is reasonable to complex and comprehensively research the activity of chronic inflammation and magnesium (Mg) imbalance as cardiovascular risk factors in end-stage renal disease (ESRD). The main purpose of the current study was to determine the relationship of tumor necrosis factor alpha (TNF-α) and Mg levels with the presence and severity of CVC in diabetic patients with ESRD. Method We enrolled 136 patients undergoing HD (male/female, 78/58;age, 53.9±1.0 years; HD duration, 47.6±4.2 month) in this observational cross-sectional study. The study was performed in accordance with the provisions of the Declaration of Helsinki last revision. Depending on the presence of type 2 diabetes mellitus (T2DM) all subjects were divided into two groups: the 1st one – non-diabetic patients (n=88); the 2nd one – diabetic patients (n=48). Presence of CVC was detected by ultrasound. The mitral (MVC) and aortic (AVC) valve calcification degree were scored as follows: 1, no calcification; 2, valve thickening without calcification; 3, valve annulus or cusps calcification. Serum content of TNF-α as one of the key proinflammatory cytokine was determined by enzyme-linked immunosorbent assay. Serum Mg concentration was estimated by biochemical method. Data are expressed as means±SEM. Used nonparametric statistics methods: Mann-Whitney U-test, χ2-test, Spearman’s rank R correlations. Results In diabetic HD patients TNF-α content was higher (13.86±1.34 vs. 8.73±0.60 ng/L; Z=3.04, p=0.002) whereas Mg concentration (0.87±0.02 vs. 1.00±0.02 mmol/L; Z=4.91, p&lt;0.001) – lower compared to non-diabetic ones, and in 2nd group indices of TNF-α and Mg were related (Rs=-0.68, p&lt;0.001). CVC was detected in 66.6% of T2DM patients with predominance of calcification of both valves (35.4%) over isolated MVC (20.8%) and AVC (10.4%). Combined MVC and AVC in the 2nd group was observed 2.6 times more often (χ2=8.78, p=0.003) than in the 1st one. For the first time it was established that in diabetic patients with ESRD the presence of CVC closely associated with indices of TNF-α (Rs=0.51, p&lt;0.001) and Mg (Rs=-0.57, p&lt;0.001). The MVC as well as AVC degree were related with the content of TNF-α (Rs=0.49, p&lt;0.001; Rs=0.52, p&lt;0.001) and Mg concentration (Rs=-0.47, p&lt;0.001; Rs=-0.50, p&lt;0.001) respectively. Conclusion (1) T2DM in HD patients is characterized with an increase of serum TNF-α activity and simultaneous decreased of Mg content. (2) In diabetic patients with ESRD, both MVC and AVC are closely linked with the TNF-α accumulation and hypomagnesemia. (3) Chronic inflammation and Mg deficiency can be important factors of CVC progression and very high cardiovascular risk in diabetic HD patients.

Introduction and purpose: It is important today to determine the factors that form a very high cardiovascular risk in patients with type 2 diabetes mellitus (DM) with kidney damage on programmed hemodialysis (HD). The aim of the study has been to determine the cardiovascular features of chronic inflammation and endothelial dysfunction (ED) in HD patients with diabetic nephropathy (DN). Material and methods: The study has included 136 patients treated with HD (men, 78; age, 53.9±1.0 year, duration of HD, 47.6±4.2 months). Depending on the presence/absence of type 2 diabetes, they have been divided into two groups: the firstwithout DN (n=88); the secondwith DN (n=48). The intensity of inflammation has been assessed by the serum content of tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), fibrinogen (FG) and albumin. Vasomotor function of the brachial artery (BA) has been determined using a test with reactive hyperemia (endothelium-dependent vasodilation, (EDVD)). The content of nitrite anions (NO2-) and the number of circulating endothelial cells (CECs) in blood plasma have been measured. Results: In patients with DN, TNF-α (p=0.002), CRP (p<0.001) and FG (p=0.008) significantly exceeded those in non-diabetics. The average value of EDVD BA (p=0.001), NO2-(p=0.008) in patients of the second group has been lower, and the number of 145 CECshigher (p<0.001) compared with the first group. Vasoconstriction (EDVD<0%) and undilatation (EDVD=0%) reactions in the case of DN have been registered more often (52.1 vs. 19.1%, p<0.001). In patients with DN for the first time have been established correlations between CECs and TNF-α (Rs=0.73, p<0.001), CRP (Rs=0.53, p<0.001), FG (Rs=0.53, p<0.001), albumin (Rs=-0.43, p=0.002). Conclusions: Chronic inflammation in the constellation with endothelial damage in patients with DN in the case of HD is obviously an important factor in cardiovascular remodeling, a predictor of progression of cardiovascular complications.

Abstract. The generalization of experimental and clinical data currently allows us to confirm the important pathogenetic role of vitamin K deficiency in cardiovascular calcification and atherosclerotic damage in chronic kidney disease (CKD). It was highlighted that, apart from vitamin K, the activity and expression of matrix Gla protein, which strongly inhibits vascular calcification, depended to a considerable extent on vitamin D. The efficacy and safety of the combined intake of vitamin K and D in slowing the progression of ectopic calcification, reducing cardiovascular risk, and improving prognosis in CKD patients need to be confirmed in multicenter randomized controlled trials.