miRNAs are promising biomarkers but methods for their measurement are not clear. We therefore examined three miRNA detection technologies and considered the analytical characteristics essential for clinical utilization. TaqMan assays, SplintR-qPCR and miREIA were compared for their absolute quantification bias, conformity and robustness. Absolute concentrations of miR-142-5p, miR-23a-3p and miR-93-5p were measured with all three methods using 30 samples. Robustness was evaluated by measurement of miR-21-5p in five uniform experiments. Correlations were miRNA-specific, but we observed a different absolute concentration range in RT-qPCR (fmol/μl) and methods evading the RT process (amol/μl). Consistently, RT-less methods reported better robustness (CV 8-19%) than RT-qPCR (CV 39-50%). The calibration curve in TaqMan Advanced assay was influenced by dilution media. Methods avoiding RT seem to be a promising future alternative for miRNA measurement.
BACKGROUND: Transferrin is synthetized in the liver and is the most important iron-transport carrier in the human body. Severe alcohol consumption leads to alterations in glycosylation of transferrin. Mass spectrometry can provide fast detection and quantification of transferrin isoforms because they have different molecular masses. In this study, we used antibody chips in combination with MALDI-TOF MS for the detection and quantification of transferrin isoforms. METHODS: Protein chips were prepared by functionalization of indium tin oxide glass using ambient ion soft landing of electrosprayed antitransferrin antibody. Two microliters of patient serum was applied on the antibody-modified spots, and after incubation, washing, and matrix deposition, transferrin isoforms were detected by MALDI-TOF MS. Peak intensities of each transferrin form were used to calculate total carbohydrate-deficient transferrin (CDT). The CDT values obtained by the MALDI chip method were compared with the results obtained by a standard capillary electrophoresis (CE). RESULTS: The chip-based MALDI-TOF MS method was used for enrichment and detection of CDT from human serum. A sample cohort from 186 patients was analyzed. Of these samples, 44 were positively identified as belonging to alcoholic patients, whereas 142 were negative by the MALDI chip approach. The correlation of the data obtained by the CE and the chip-based MALDI was r = 0.986, 95% CI. CONCLUSIONS: Functionalized MALDI chips modified by antitransferrin antibody prepared by ambient ion soft landing were successfully used for detection and quantification of CDT from human sera.
- MeSH
- biologické markery krev MeSH
- lidé MeSH
- referenční standardy MeSH
- reprodukovatelnost výsledků MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice přístrojové vybavení MeSH
- studie případů a kontrol MeSH
- transferin analogy a deriváty metabolismus normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. METHODS: MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. RESULTS: The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. CONCLUSIONS: MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.
- MeSH
- chromatografie afinitní MeSH
- fenotyp MeSH
- haptoglobiny analýza imunologie MeSH
- lidé MeSH
- povrchové vlastnosti MeSH
- protilátky imunologie MeSH
- software MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background: Cardiotrophin-1 is newly discovered chemokin with a lot of functions. Aim of our work was to describemost important of them.Methods: systematically scan of available scientifi c resources.Results: Cardiotrophin-1 stimulates the proliferation of cardiomyocytes. Cardiotrophin-1 expression and plasmavalues are elevated in individuals with heart failure and have high diagnostic effi cacy for the heart failure. Plasma valuesare also an independent prognostic factor. Preliminary fi ndings suggest that the determination of plasma cardiotrophin-1may be useful for the follow-up of hypertensive heart disease in routine clinical practice. Cardiotrophin-1 also plays animportant cardioprotective eff ect on myocardial damage, is a potent regulator of signaling in adipocytes in vitro andin vivo and potentiates the elevation the acute-phase proteins. Cardiotrophin-1 may play also an important protectiverole in other organ systems (such as hematopoietic, neuronal, developmental).Conclusion: Cardiotrophin is a newly discovered chemokin with a lot of system eff ects and is stable in systemcirculation hence permitting its development in the routine clinical investigation.
- MeSH
- akutní koronární syndrom diagnóza MeSH
- biologické markery krev metabolismus MeSH
- chemokiny diagnostické užití terapeutické užití MeSH
- cytokiny diagnostické užití terapeutické užití MeSH
- ELISA metody využití MeSH
- kardiomyocyty metabolismus účinky léků MeSH
- kardiovaskulární nemoci diagnóza MeSH
- krevní nemoci diagnóza MeSH
- lidé MeSH
- medicína založená na důkazech MeSH
- metabolický syndrom diagnóza MeSH
- nádory diagnóza MeSH
- nemoci jater diagnóza MeSH
- nemoci nervového systému diagnóza MeSH
- plicní nemoci diagnóza MeSH
- srdeční selhání diagnóza MeSH
- zánět diagnóza MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Moderate alcohol consumption is associated with increased insulin sensitivity and a reduced risk for type 2 diabetes. An important endogenous mediator of insulin sensitivity is adiponectin (AN), an adipokine that displays numerous antiatherogenic, antidiabetogenic and antiinflammatory effects. Recently, acute increase in alcohol consumption has been shown to be associated with increase in plasma adiponectin and, concomitantly, insulin sensitivity. Whether chronic alcohol consumption predicts an increase in plasma AN and whether this is independent of adiposity, markers of liver dysfunction, and plasma adipokines such as tumor necrosis factor (TNF)-alpha is not known. We, therefore, investigated these relationships in 75 men who were diagnosed with liver steatosis using ultrasound/liver biopsy. We examined 75 men, who were diagnosed for having liver steatosis (ultrasound/liver biopsy). Each filled in a questionnaire on alcohol intake. Subjects were divided into two subgroups according to alcohol history and CDT concentrations--drinkers and non-drinkers. All individuals were examined for serum concentrations of AN, glucose, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate transferase (GMT) activity; carbohydrate-deficient transferrin (CDT%) a marker of chronic alcohol consumption, insulin and TNF-alpha. The Quicki insulin sensitivity index was calculated. Forty-eight individuals were found to be moderate drinkers and 27 subjects non-drinkers. Moderate drinkers had significantly higher concentrations of AN (13.8 +/- 3,7 versus 9.1 +/- 5.4 mg/l, means +/- SD, p = 0.012) compared with non-drinkers, independent of adiposity. Plasma AN concentrations in the whole group were positively correlated with TNF-alpha concentrations (r = 0.6; p = 0.0001), CDT (r = 0.26; p = 0.0084), AST/ALT index (r = 0.3, p = 0.009), AST (r = 0.29; p = 0.011) and GMT (r = 0.29; p = 0.011) and negatively with BMI (r = -0.48; p = 0.0002) and glycemia (r = -0.22; p = 0.049). The positive associations of AN with TNF-alpha (0.8; p = 0.001), CDT (0.55; p = 0.017), AST/ALT index (0.55; p = 0.019) and the negative correlation with glycemia (-0.35; p = 0.0158) were independent of BMI. Stratified according to alcohol intake, in moderate drinkers, a positive correlation was found between AN and TNF-alpha concentrations (r = 0.6, p = 0.0001, AST/ALT index (r = 0.34, p = 0.0295) whereas in non-drinkers no such correlations were found. The concentration of AN and BMI displayed a negative correlation in both drinker and nondrinker patients (r = -0.42, p = 0.01 and -0.61; p = 0.012, respectively). We concluded that plasma AN is higher in moderate drinkers compared to non-drinkers, even after correction for BMI. Drinkers suffering from liver steatosis were found to have a positive correlation between AN concentrations, laboratory markers of liver disease and TNF-alpha. Such correlation was absent in non-drinkers suffering from liver steatosis. This suggests that alcohol may modulate the inhibitory effect of TNF-alpha on AN production, and thus, increase its plasma concentrations.
- MeSH
- adiponektin krev MeSH
- alkoholické nemoci jater krev diagnóza MeSH
- biologické markery krev MeSH
- index tělesné hmotnosti MeSH
- inzulinová rezistence MeSH
- lidé MeSH
- pití alkoholu metabolismus MeSH
- ROC křivka MeSH
- senzitivita a specificita MeSH
- TNF-alfa analýza MeSH
- ztučnělá játra krev MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Aim of study was determine if a correlation exists between bone mass density and concentration of osteoprotegerin. We examined the group of 199 patients of mean age of 63 years. Of the group under study, 31 patients had normal bone density (T score > -1 and < 1) and 168 probands had osteopenia or osteoporosis (T < -1). Persons with normal BMD values had median values of OPG 60.8 ng/l, while patients with reduced bone density had median values of 73 ng/l OPG. Cut-off for reduction of bone density was 128 ng/l OPG. We demonstrated that OPG concentrations vary inversely with bone density values (correlation coefficient -0.31). These results suggest that determination of OPG could allow discrimination of individuals with normal bone density and those with reduced bone density.
- MeSH
- dospělí MeSH
- glykoproteiny metabolismus MeSH
- kostní denzita * MeSH
- lidé středního věku MeSH
- lidé MeSH
- metabolické nemoci kostí metabolismus MeSH
- osteoporóza metabolismus MeSH
- osteoprotegerin MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- receptory TNF MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
Osteoprotegerin, RANK (Receptor Activator of Nuclear factor kappa B) and RANKL (Receptor Activator of Nuclear faktor kappa B ligand) became the aim of intensive research. RANK is considered as a hematopoietic surface receptor controlling osteoclastogenesis and calcium metabolism. RANKL may promote osteoresorption by induction of cathepsin K gene expression. The present paper summarizes the most significant data in osteoprotegerin, RANK and RANKL problems obtained.
- MeSH
- glykoproteiny fyziologie MeSH
- lidé MeSH
- ligand RANK MeSH
- membránové glykoproteiny fyziologie MeSH
- osteoklasty fyziologie MeSH
- osteoprotegerin MeSH
- protein RANK MeSH
- receptory cytoplazmatické a nukleární fyziologie MeSH
- receptory TNF MeSH
- transportní proteiny fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
