Determination of one year stability of lipid plasma profile and comparison of blood collection tubes using UHPSFC/MS and HILIC-UHPLC/MS.

Effects of blood collection tubes, the time period, the sample origin, and the method used on the lipidomic profile are investigated by ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS) and hydrophilic interaction liquid chromatography ultrahigh-performance liquid chromatography - mass spectrometry (HILIC-UHPLC/MS). Heparin plasma samples have been obtained from 99 healthy volunteers at three time points separated by six-month intervals together with one collection for EDTA plasma and serum. Furthermore, lipid concentrations in heparin plasma collected at two different sites are compared. 171 lipid species from eight lipid classes are quantified with UHPSFC/MS, and 122 lipid species from four lipid classes with HILIC-UHPLC/MS. The accuracy of both methods is monitored by the quantitation error using two internal standards (IS) per individual lipid classes. No significant differences in lipid profiles are observed for different time points and types of collection tubes (heparin plasma, EDTA plasma, and serum). Most pronounced lipid concentration differences are observed for the comparison of NIST SRM 1950 human plasma and mean lipid concentrations of the investigated cohort. Furthermore, differences in lipid concentrations are observed between employed methods (UHPSFC/MS vs. HILIC-UHPLC/MS), which can be compensated by the normalization using NIST SRM 1950 human plasma used as the quality control sample.

Very long-term stability of lipid biomarkers in human serum.

The assessment of the stability of biomarkers is very important for epidemiological studies. In this study, the stability of five lipid parameters (total cholesterol, triglycerides, HDL- and LDL-cholesterol and free fatty acids) has been tested in 16 human serum samples after storage at -80 °C up at time points 0, 1, 8 and 13 y. The majority of the lipid biomarkers were stable during storage conditions, except for cholesterol. The correlations between the samples were very good at time points. Therefore, long-term storage of human serum samples allows lipid biomarker determination, provided that the samples are stored at -80 °C.

The effect of fatty acids in red blood cell membranes on the dynamics of inflammatory markers following the coronary stent implantation.

The effect of 20 fatty acids in erythrocyte cell membranes on the extent of inflammatory response and cell oxidative stress was evaluated using multidimensional statistical data analysis in 54 patients suffering from ischemic heart disease undergoing percutaneous coronary intervention with coronary stent implantation using multidimensional statistical data analysis. A systemic inflammatory response was indicated by an increase of C-reactive protein (CRP), serum amyloid A (SAA) and ceruloplasmin 48 h after stent implantation and by an increase of interleukin-6 (IL-6) 24 h after intervention. The increase of malondialdehyde (MDA) after 48 h was used as a marker of cell damage by oxidative stress. Multiple linear regression revealed statistically significant relationships between concentration of some fatty acids and the magnitude of inflammatory response, or oxidative stress, after stent implantation. The most significant relationship with an increase of plasma CRP was found for myristic acid and, to a lesser extent, for oleic acid. Trans octadecenoic acid, and to a lesser extent palmitooleic and nervonic fatty acids were found in inverse correlation with the CRP increase. The increase of IL-6 showed a statistically significant correlation with myristic acid, to a lesser extent with cis-9-eicosenoic acid and to the least extent with docosahexaenoic acid, inversely with pentadecanoic, γ-linolenic and stearic acids. An increase of oxidative stress (MDA) significantly correlated only with γ-linolenic acid. Other studied markers of inflammatory response to coronary stenting were SAA and ceruloplasmin (Cp). Statistical evaluation revealed that SAA and Cp are not suitable markers for assessment relationships between inflammation and erythrocyte membrane fatty acids.

Inflammatory markers in dependence on the plasma concentration of 37 fatty acids after the coronary stent implantation.

Using the regression model building the relationships between the concentration of 37 fatty acids of blood plasma phospholipids of 41 patients with coronary artery disease after coronary stent implantation, the inflammatory response and oxidative stress markers were estimated. The dynamics of the inflammatory response and the oxidative stress was indicated by measuring plasma concentrations of highly sensitive C-reactive protein, interleukin-6, serum amyloid A and malondialdehyde before, 24h after stent implantation. The multiple linear regression analysis was preceded by an exploratory data analysis, principal component analysis, factor analysis and cluster analysis, which proved a hidden internal relation of 37 fatty acids. The concentration of cerotic acid (C26:0) has been positively associated with an increase of malondialdehyde concentration after stent implantation, while the concentrations of tetracosatetraenoic (C24:4 N6) and nonadecanoic (C19:0) acids were associated with decrease of lipoperoxidation. The increase of interleukin-6 during the 24h after implantation was associated with higher levels of pentadecanoic acid (C15:0) and lower levels of α-linolenic acid (C18:3 N3). Regression models found several significant fatty acids at which the strength of the parameter ß for each fatty acid on selected markers of C-reactive protein, malondialdehyde, interleukin-6 and serum amyloid A was estimated. Parameter ß testifies to the power of the positive or negative relationship of the fatty acid concentration on the concentration of selected markers. The influencing effect of the cerotic acid (C26:0) concentration in plasma phospholipids exhibiting parameter ß=140.4 is, for example, 3.5 times higher than this effect of n-3 tetracosapentaenoic acid (C24:5 N3) with ß=40.0. Composition of fatty acids in plasma phospholipids shows spectrum of fatty acids available for intercellular communication in systemic inflammatory response of organism and should affect clinical outcomes.

Long-term stability of oxidative stress biomarkers in human serum.

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at -20 and -80 °C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at -80 °C, but not in samples stored at -20 °C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at -80 °C. ROM and BAP can also be stored at -20 °C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.

Heart failure is the strongest predictor of acute kidney injury in patients undergoing primary percutaneous coronary intervention for ST-elevation myocardial infarction.

BACKGROUND: ST elevation myocardial infarction (STEMI) patients treated by primary percutaneous coronary intervention (PCI) are at higher risk of acute kidney injury (AKI) than patients undergoing PCI in stable clinical conditions. This fact suggests that mechanisms other than contrast nephrotoxicity are involved. AIM: To evaluate the incidence, risk factors, and consequences of AKI in patients undergoing primary PCI for STEMI in current daily practice. METHODS: Analysis of all consecutive patients who underwent primary PCI over a one-year period. AKI was defined as an increase in serum creatinine ≥ 50% or 26.5 µmol/L (AKIN criteria) from the baseline within 48 h. RESULTS: A total of 202 patients were included. AKI occurred in 25 (12.4%) subjects. Baseline characteristics and in-hospital complications of the patients with and without AKI did not differ significantly except for age (69 ± 13 vs. 62 ± 12; p = 0.003), female gender (48.0% vs. 26.6%; p = 0.035), hypertension (88.0% vs. 62.7%; p = 0.013), left ventricular ejection fraction (40% ± 12% vs. 49% ± 14%; p = 0.002), cardiogenic shock (44.0% vs. 5.1%; p < 0.0001), use of intravenous diuretics (76.0% vs. 26.0%; p < 0.0001), ventricular arrhythmias (24.0% vs. 3.4%; p = 0.001), and in-hospital mortality (24.0% vs. 3.4%; p = 0.001). In multivariate analysis heart failure remained the only independent correlate of AKI. CONCLUSIONS: AKI was an frequent and serious complication of STEMI in patients treated by primary PCI. Heart failure was the strongest predictor of AKI. Other risk factors including contrast medium volume, baseline renal function, diabetes, and age failed to predict AKI.

Long-term stability of cancer biomarkers in human serum: biomarkers of oxidative stress and redox status, homocysteine, CRP and the enzymes ALT and GGT.

AIM: Five frequently used biomarkers in cancer research and epidemiological studies were tested for their assay stability upon storage of serum for 12 months at -20 and -70/-80°C. MATERIALS & METHODS: The biomarker assays include reactive oxygen metabolites (ROM), the total thiol levels (TTL), homocysteine (HCy), C-reactive protein (HS-CRP) and two liver enzymes, alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT). RESULTS: The assays for ROM, HCy, HS-CRP and GGT were stable in human serum samples at the two temperatures tested. The two other assays TTL and ALT, however, showed statistically significant differences in their stability between -20 and -80°C. CONCLUSION: Therefore, storage at -80°C is advised to maintain a reliable assay outcome when serum samples have to be stored for longer periods.

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae, Anura).

Using histological techniques and computer-aided three-dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the "ventromedial diverticulum") is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes.

Development of the ethmoidal structures of the endocranium in Discoglossus pictus (Anura: Discoglossidae).

We use histological techniques and computer-aided three-dimensional reconstructions made from serial histological sections to describe the ontogeny of the ethmoidal endocranium of discoglossid frog Discoglossus pictus. We identify a pattern of development for the suprarostral cartilage that differs from previous findings and probably represents the ancestral anuran pattern. The nasal cartilages, including the inferior prenasal cartilage, are de novo adult structures. The only larva-derived structures of the adult nasal capsules are the posterior aspects of the solum nasi and septum nasi. We also identify patterns of development for the ethmoid plate and postnasal wall that occur during early in ontogenesis. These patterns are associated with development events during metamorphic climax. The pattern of timing of chondrification of the anterior nasal cartilages more closely coincides with that of the neobatrachian species than that recorded for the pelobatid frog Spea. In addition, this study supports a sister taxon relationship between Discoglossus and Alytes.

Inverse correlation between plasma Beta-carotene and interleukin-6 in patients with advanced coronary artery disease.

The interrelationships between plasma beta-carotene, alpha-tocopherol, and the level of systemic inflammation and oxidative stress were investigated in patients with advanced coronary artery disease (CAD). Plasma beta-carotene, alpha-tocopherol, malondialdehyde, free radicals, interleukin-6, high sensitive C-reactive protein levels, and other risk factors of CAD were determined in a group of patients with advanced CAD [significant stenosis according to coronarographic examination (n=91) and a control group of examined patients with coronary arteries with no stenosis (n=49)]. Between-group differences in continuous variables were analyzed with the Hotelling T2-test (software NCSS2000), analyses of correlation matrix with the software STATISTICA. Advanced CAD coincided with significantly lower plasma concentrations of high-density lipoprotein (HDL)-cholesterol and beta-carotene as well as with elevated levels of all inflammatory markers, but only with mild increase of oxidative stress. Beta-carotene significantly inversely correlated with interleukin-6. This inverse correlation could suggest potential protective effect of beta-carotene on atherosclerosis due to the inhibition of inflammatory processes.

Evaluation of oxidative stress and inflammation in obese adults with metabolic syndrome.

BACKGROUND: Obesity and metabolic syndrome increase the risk of cardiovascular morbidity and mortality. Oxidative stress seems to be involved in the pathophysiology of diabetes and cardiovascular complications of metabolic syndrome. The aim of our study was to evaluate the level of oxidative stress and inflammation in obese adults with and without metabolic syndrome. METHODS: Oxidative stress and inflammation markers (total amount of free radicals, malondialdehyde, allantoin, alpha1-antiproteinase, oxidized/reduced glutathione ratio, high-sensitive C-reactive protein, fibrinogen), total antioxidant capacity and lipid standardized alpha-tocopherol were determined in obese subjects fulfilling at least three criteria of metabolic syndrome according to the National Cholesterol Education Program-Adult Treatment Panel III guidelines (n=20 patients), in obese subjects without metabolic syndrome (n=20 patients) and in 48 healthy controls. RESULTS: Oxidative stress and inflammation markers were significantly elevated in the obese subjects, especially in those exhibiting metabolic syndrome. According to multidimensional statistical analysis, oxidative stress was independently related to triacylglyceride concentration, abdominal fat, low high-density lipoprotein cholesterol and low lipid standardized alpha-tocopherol in the patients with metabolic syndrome. CONCLUSIONS: High levels of free radicals together with low antioxidant capacity detected in obese adults indicate elevated oxidative stress, which is--together with systemic inflammation--further potentiated in the case of obese patients with metabolic syndrome. This imbalance in oxidative/antioxidative status and subclinical inflammatory state leads to higher risk of atherosclerotic and diabetic complications.

The model of D-galactosamine-induced injury of rat hepatocytes in primary culture.

D-galactosamine (GalN) is a highly selective hepatotoxin that causes liver damage similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. Model of galactosamine hepatotoxicity is frequently used in animal experiments in vitro. The purpose of our study was to establish the model of GalN-induced hepatocyte injury in in vitro conditions using primocultures of rat hepatocytes as an important pre-requisite for further experiments in which we would like to study potential hepatoprotective effect of various substances. Rate of hepatocyte injury was evaluated by morphological changes, changes in cell viability, albumin production, mitochondrial membrane potential, activity of mitochondrial dehydrogenases and glutathione content. Marked dose dependent hepatocyte injury was found after 24-hour incubation with GalN. Based on the results we suggest as an optimal model for short-term toxicity test exposure to GalN for 24 hours in dose of 40 mM.

Monitoring of antioxidant properties of uric acid in humans for a consideration measuring of levels of allantoin in plasma by liquid chromatography.

Uric acid is the end product of purine metabolism in humans. It has been pointed out that uric acid acts as an antioxidant and is capable to react with biologically relevant oxidants to form allantoin. Therefore, measurement of allantoin in humans was proposed as a marker of oxidative stress. We estimated allantoin in human plasma obtained from the patients with chronic renal failure before hemodialysis (n=30), patients with non-insulin dependent diabetes mellitus (n=30) and blood donors (n=30) using a method based on selective isolation of allantoin from deproteinized plasma samples on AG 1-X8 resin and its derivatization to glyoxylate-2,4-dinitrophenylhydrazone. The method is free from urate and glyoxylate interferences. Separation of glyoxylate-2,4-dinitrophenylhydrazone from other hydrazones was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. The analytical performance of this method is satisfactory with intra-assay CV 5.7%, inter-assay CV 8.3% and recovery 94.1%. We have determined other parameters of oxidative stress (malondialdehyde, total antioxidant status, superoxide dismutase and glutathione peroxidase) too. The preliminary reference range of allantoin in a group of blood donors is 4.76+/-2.99 micromol/L. In the patients with chronic renal failure and the patients with non-insulin dependent diabetes mellitus we found allantoin levels in plasma (27.1+/-13.8) micromol/L and (11.08+/-5.90) micromol/L, respectively. It seems that allantoin is a possible indicator of free radical damage in vivo.

[Glutathione and glutathione assays]. / Glutathion a metody stanovení.

Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule.