Exercise-Induced Proteomic Profile Changes in Patients with Advanced Heart Failure.

BACKGROUND/OBJECTIVES: The pathophysiological background of the processes activated by physical activity in patients with heart failure (HF) is not fully understood. Proteomic studies can help to preliminarily identify new protein markers for unknown or poorly defined physiological processes. We aimed to analyse the changes in the plasma proteomic profile of HF patients after a cardiopulmonary exercise test (CPET) to define pathways involved in the response to exercise. METHODS: The study prospectively enrolled 20 male patients with advanced HF (aged 53.3 ± 8.3 years). Blood samples were taken from the patients before and immediately after the CPET to obtain plasma proteomic profiles. Two-sample t-tests (paired or non-paired) were performed with and without false discovery rate (FDR) correction for multiple testing. Enrichment analysis was performed to associate biological processes and pathways with the study results. RESULTS: A total of 968 plasma proteins were identified, of which 722 underwent further statistical analysis. Of these, 236 proteins showed differential expression when comparing all plasma samples collected before and after CPT (p < 0.05), and for 86 of these the difference remained statistically significant after FDR correction. Proteins whose expression changed after exercise are mostly involved in immune response and inflammatory processes, coagulation, cell adhesion, regulation of cellular response to stimulus and regulation of programmed cell death. There were no differences in resting proteomics according to HF etiology (ischemic vs. non-ischemic). CONCLUSIONS: Changes in the proteomic profile revealed a complexity of exercise-induced processes in patients with HF, suggesting that few major physiological pathways are involved. Further studies focusing on specific pathways are needed.

L-arginine and Its Derivatives Correlate with Exercise Capacity in Patients with Advanced Heart Failure.

Methylated arginine metabolites interrupt nitric oxide synthesis, which can result in endothelium dysfunction and inadequate vasodilation. Since little is known about the dynamics of arginine derivatives in patients with heart failure (HF) during physical exercise, we aimed to determine this as well as its impact on the patient outcomes. Fifty-one patients with HF (left ventricle ejection fraction-LVEF ≤ 35%, mean 21.7 ± 5.4%) underwent the cardiopulmonary exercise test (CPET). Plasma concentrations of L-arginine, citrulline, ornithine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) were measured before and directly after CPET. All patients were followed for a mean of 23.5 ± 12.6 months. The combined endpoint was: any death, urgent heart transplantation, or urgent LVAD implantation. L-arginine concentrations increased significantly after CPET (p = 0.02), when ADMA (p = 0.01) and SDMA (p = 0.0005) decreased. The parameters of better exercise capacity were positively correlated with post-CPET concentration of L-arginine and inversely with post-CPET changes in ADMA, SDMA, and baseline and post-CPET SDMA concentrations. Baseline and post-CPET SDMA concentrations increased the risk of endpoint occurrence (HR 1.02, 95% CI 1.009-1.03, p = 0.04 and HR 1.02, 95% CI 1.01-1.03, p = 0.02, respectively). In conclusion, in patients with HF, extensive exercise is accompanied by changes in arginine derivatives that can reflect endothelium function. These observations may contribute to the explanation of the pathophysiology of exercise intolerance in HF.

The diagnostic potential of glutathione S-transferase (GST) polymorphisms in patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers in the world. Despite improvements in screening for early diagnosis, CRC is one of the leading causes of cancer deaths. OBJECTIVES: The aim of the study was to determine a potential association between the frequency of GSTM1 and GSTT1 null genotypes and the risk of CRC in the Polish population. Moreover, we analyzed the clinical parameters with the glutathione S-transferase (GST) gene polymorphisms in patients with CRC. MATERIAL AND METHODS: The study was conducted on 512 Caucasians, including 279 patients (105 women and 174 men) with CRC. DNA from peripheral blood was extracted and the multiplex polymerase chain reaction (PCR) technique was used for glutathione S-transferase theta (GSTT1) and mu (GSTM1) gene deletion genotyping. RESULTS: We found no statistically significant differences in the frequency of the GST gene polymorphisms in patients with CRC and controls. The prevalence of the GSTM1*0 variant in the test subjects was higher than in controls (45.9% vs 42.9%; p > 0.05). The frequency of the GSTT1*0 variant was also higher in patients with CRC compared to the control population (21.1% vs 18.9%; p > 0.05). In addition, the effect of the GSTM1 and GSTT1 polymorphisms on the incidence of CRC was also analyzed. There was a slight, but not statistically significant, increase of the risk of colon cancer for the GSTM1*0 and GSTT1*0 variants. Moreover, we examined the GST genotype due to the cancer TNM classification and the location of the primary tumor. Statistically significant differences in the distribution of the GSTT1*0 and GSTT1*1 genotypes in both the stage and the location of the primary tumor were observed. CONCLUSIONS: It is suggested that the GSTT1 polymorphism may have an impact on the severity of the tumor and its location.

Spectroscopic and Theoretical Studies of Fluorescence Effects in 2-Methylamino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole Induced by Molecular Aggregation.

The article presents the results of fluorescence analyses of 2-methylamino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole (MDFT) in an aqueous environment. MDFT dissolved in aqueous solutions with a pH value in the range from 1 to 4.5 yielded an interesting effect of two clearly separated fluorescence emissions. In turn, a single fluorescence was observed in MDFT dissolved in water solutions with a pH value from 4.5 to 12. As it was suggested in the previous investigations of other 1,3,4-thiadiazole compounds, these effects may be associated with conformational changes in the structure of the analysed molecule accompanied by aggregation effects. Crystallographic data showed that the effect of the two separated fluorescence emissions occurred in a conformation with the -OH group in the resorcyl ring bound on the side of the sulphur atom from the 1,3,4-thiadiazole ring. The hypothesis of aggregation as the mechanism involved in the change in the spectral properties at low pH is supported by the results of (Time-Dependent) Density Functional Theory calculations. The possibility of rapid analysis of conformational changes with the fluorescence spectroscopy technique may be rather important outcome obtained from the spectroscopic studies presented in this article. Additionally, the presented results seem to be highly important as they can be easily observed in solutions and biologically important samples.

Factors related to on-treatment platelet aggregation assessed by multiple electrode aggregometry in percutaneous coronary intervention patients on clopidogrel and aspirin.

INTRODUCTION: There is ongoing controversy concerning the clinical value of platelet function monitoring in patients undergoing percutaneous coronary interventions (PCI). Patients at risk of high on-treatment platelet aggregation (HPR) may benefit most from such monitoring. AIM: To define the factors related to HPR on aspirin and clopidogrel, looking at a wider spectrum of variables than those assessed in some previous studies. MATERIAL AND METHODS: We assessed platelet function in 908 patients on clopidogrel and aspirin after PCI using the multielectrode aggregometry system Multiplate to define which clinical, procedural and laboratory factors are related to on-treatment platelet aggregation in response to aspirin and clopidogrel either as linear values or using established cutoff values for HPR. RESULTS: We found that in PCI patients on clopidogrel and aspirin, age (OR per year 1.06; 95% CI: 1.024-1.097; p = 0.001), gender (OR = 0.319; 95% CI: 0.139-0.731; p = 0.007), active smoking (OR = 2.57; 95% CI: 1.29-5.15; p = 0.008), diabetes (ß = 37.6; 95% CI: 16.5-58.8; p = 0.001) and hypertension (ß = 26.9; 95% CI: 6.73-47.1; p = 0.009) are independently linked to platelet aggregation values treated as linear values and as dichotomous variables at the accepted cutoffs. The same is true for stented segment length (OR per mm 1.033; 95% CI: 1.010-1.057; p = 0.009) and stent inflation pressure (OR per atmosphere 0.862; 95% CI: 0.772-0.963; p = 0.002). CONCLUSIONS: The study shows that, contrary to some earlier data, in the tested cohort women are better clopidogrel responders, but more often aspirin low-responders. Older age, active smoking, diabetes and hypertension all predispose to HPR. A novel finding is that stented segment length is an independent predictor of lower response both to aspirin and clopidogrel, possibly as a marker of more diffuse atherosclerosis.

A Comparison of the Immunochemical Methods, PETINIA and EMIT, With That of HPLC-UV for the Routine Monitoring of Mycophenolic Acid in Heart Transplant Patients.

BACKGROUND: The aim of this study was to evaluate particle enhanced turbidimetric inhibition immunoassay (PETINIA) recently developed for mycophenolic acid (MPA) determination in plasma and to compare it with a reference high-performance liquid chromatography (HPLC) method, using samples from heart transplant recipients. The results are presented in the context of PETINIA being compared with enzyme multiplied immunoassay technique (EMIT). METHODS: PETINIA evaluation was performed using 194 routine trough plasma samples at steady state. EMIT was evaluated using 677 samples from 61 steady-state 12-hour profiles obtained from 35 heart transplant patients. Evaluation was undertaken on a Dimension EXL 200 analyzer (PETINIA) and on a Viva-E analyzer (EMIT). RESULTS: The mean MPA concentration measured by PETINIA was significantly higher than that measured by high-performance liquid chromatography combined with UV detector (2.36 ± 1.30 mcg/mL versus 1.82 ± 1.23 mcg/mL, respectively, P < 0.0001). Bland-Altman analysis revealed a mean bias of 0.54 mcg/mL [95% confidence interval (CI), 0.49-0.59] comprising 33.48% (95% CI, 30.34-36.61). Passing-Bablok regression was: y = 1.100x + 0.38 (95% CI for slope: 1.044-1.154 and for intercept: 0.30-0.47). Regardless of a significant observed correlation (r = 0.9230, P < 0.0001), the statistical analyses showed a significant difference between PETINIA and the reference chromatographic method. The mean MPA concentration measured by EMIT was significantly higher than that measured by HPLC (7.48 ± 8.34 mcg/mL versus 5.57 ± 6.61 mcg/mL, respectively, P < 0.0001) with a mean bias of 1.91 mcg/mL (95% CI, 1.75-2.07) comprising 35.91% (95% CI, 34.37-37.45). The significant difference between EMIT and HPLC was confirmed by Passing-Bablok regression: y = 1.300x + 0.24 (95% CI for slope: 1.279-1.324 and for intercept: 0.18-0.29). The analysis of the determinations, grouped by sampling time, revealed positive bias between EMIT and HPLC ranging from 24.54% to 42.77% and inversely proportional to MPA concentrations with r = 0.9122 (P < 0.001). CONCLUSIONS: The new immunochemical PETINIA method was associated with significantly higher MPA concentrations in routine therapeutic drug monitoring samples from heart transplant patients. The magnitude of the MPA overestimation was similar to that observed by use of the EMIT method.

Platelet Reactivity and Intramyocardial Hemorrhage in Patients With ST-Segment Elevation Myocardial Infarction.

The aim of the study was to analyze the relation between platelet reactivity and intramyocardial hemorrhage (IMH) in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention. Platelet reactivity was measured in 49 patients with means of impedance aggregometry (Multiplate) before reperfusion and repeated in the subacute phase of STEMI. Cardiovascular magnetic resonance was used to detect IMH, which was found in 16 (33%) patients. There were no differences in platelet reactivity between patients with and without IMH before reperfusion. Reassessment in the subacute phase of STEMI demonstrated that patients with IMH had lower thrombin receptor activating peptide (TRAP)-induced platelet aggregation (P = .004) and trends toward lower values of ristocetin and collagen-induced platelet aggregation (P = .09 and P = .07). The TRAP-induced platelet aggregation and initial perfusion grade were the factors independently associated with IMH. Intramyocardial hemorrhage is related to more potent inhibition of platelet aggregation in the subacute phase of STEMI.

Simple HPLC method for cefazolin determination in human serum - validation and stability testing.

The paper presents an HPLC method for cefazolin determination in human serum. The preparation step was based on serum protein precipitation with acetonitrile followed by supernatant evaporation and sample reconstitution in water before injection. The separation of cefazolin and internal standard cefamandole was performed at ambient temperature under isocratic conditions on LiChrosorb RP8-5 column (250mm×4.6mm) using the mixture: CH(3)CN:H(2)O:0.5M KH(2)PO(4) (100:894:6, v/v) as a mobile phase with a flow rate of 1.5mL/min. UV detection was performed at 272nm with LLOQ of 0.2µg/mL. The precision was satisfactory in the whole range tested with RSD of 2.3-12.5% (accuracy: from -2.3% to +3.6%) and of 1.7-7.1% (accuracy: from -3.5% to +1.1%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze-thaw and short- and long-term stability testing. Finally, the procedure described was found resistant to potential human errors.