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.

Determination of 25-hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation.

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25-hydroxyvitamin and 1,25-dihydroxyvitamin D(3). The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25-hydroxyvitamin D(3) in human plasma. A method for the measurement of 25-hydroxyvitamin D(3) using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125-4, Purospher RP-18e, 5 microm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra- and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0-103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25-hydroxyvitamin D(3) in a group of blood donors is 62 +/- 26 nmol/L.

Determination of ascorbic acid in human plasma with a view to stability using HPLC with UV detection.

A method for the measurement of ascorbic acid using HPLC with UV detection and investigation into the protein precipitation techniques with regard to stability and recovery are described. The effectiveness of various protein precipitants was tested. Stability of ascorbic acid samples for analysis was investigated over 10 h. Ascorbic acid samples extracted with metaphosphoric acid were stable on a cooled autosampler (4 degrees C) for at least 10 h (with a decline of 1.8% for ascorbic acid solution and 2.8% for plasma). Perchloric acid as protein precipitant for ascorbic acid was unsuitable (with a decline of 36.0% for ascorbic acid solution and 7.3% for plasma). Analytical performance of this method is satisfactory. The intra- and interassay coefficients of variation were 2.1% (n = 10) and 5.8% (n = 12), respectively. The calibration curve was linear with the tested range of 2.0-250.0 micromol/L. The recovery was 96.1% with CV = 4.8% (n = 6) and the LOD was 3 micromol/L. The preliminary reference ranges of ascorbic acid in a group of blood donors are 50.8 +/- 22.4 micromol/L. This assay is a highly sensitive and reproducible HPLC method for the determination of ascorbic acid in human plasma.

Ubiquinol-10/lipids ratios in consecutive patients with different angiographic findings.

OBJECTIVE: Information concerning un-supplemented plasma concentrations of ubiquinol-10 in coronary artery disease patients is still controversial. The aim of this study is to determine the levels of plasma ubiquinol-10 and ratios of ubiquinol-10 to plasma lipids in consecutive patients with different angiographic findings. SUBJECTS AND METHODS: Thirty-six consecutive patients who underwent coronary angiography were split in two groups with different atherosclerotic changes. These patients were un-supplemented with antioxidants and were not treated by lipid-lowering medication. We have measured a plasma level of ubiquinol-10 using high-performance liquid chromatography with coulometric detection. Conventional plasma lipids, markers of oxidative stress and other widely accepted risk factors of atherosclerosis have been determined too. RESULTS: Plasma ubiquinol-10 to low-density lipoprotein cholesterol (LDL-C) ratios in patients with different angiographic findings have been found as 180+/-69 and 132+/-43, respectively (p=0.020). The ubiquinol-10/LDL-C ratio was significantly lower in angiographically positive patients. There were also significant differences in ubiquinol-10 per total cholesterol (109+/-47 and 80+/-26, respectively; p=0.031), per triglycerides (426+/-191 and 237+/-86, respectively; p=0.002) and per the sum of triglycerides and total cholesterol (86+/-35 and 61+/-20, respectively; p=0.013). CONCLUSIONS: There have not been found any significant differences between levels of widely accepted risk factors for genesis and progress of atherosclerotic changes in these two groups of patients. Only the level of triglycerides and the total cholesterol minus high-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio were significantly higher in patients with stenosis. This ratio correlated with the ubiquinol-10/LDL-C ratio, which was significantly lower in patients with stenosis. Our results indicate that the ratio of ubiquinol-10/LDL-C is likely to be a risk factor for atherogenesis.

Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection.

A HPLC method for determination of both reduced (GSH) and oxidized (GSSG) glutathione in plasma, whole blood and rat hepatocytes has been developed and evaluated. Reduced glutathione reacts with orthophthaldehyde (OPA) to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA at pH 12. At measurement of GSSG, GSH was complexed to N-ethylmaleimide. For the separation, reverse phase column Discovery C(18), 150 mm x 4 mm, 5 microm, was used. The mixture of methanol and 25 mM sodium hydrogenphosphate (15:85, v/v), pH 6.0, was used as mobile phase. The analytical performance of this method is satisfactory for both GSH and GSSG. The intra-assay coefficients of variation were 1.8 and 2.1% for whole blood, 2.0 and 1.9% for rat hepatocytes, 4.3 and 5.2% for plasma. The inter-assay coefficients of variation were 5.8 and 6.2% for whole blood, 6.6 and 7.1% for rat hepatocytes, 6.9 and 7.8% for plasma. The recoveries were as follows: 98.2% (CV 3.5%) and 101.5% (CV 4.2%) for whole blood, 99.1% (2.5%) and 102.3 (4.4%) for rat hepatocytes, 94.1% (CV 7.5%) and 103.5 (CV 8.5%) for plasma. The calibration curve was linear in the whole range tested. The limit of detection was 14.0 and 5.6 fmol, respectively. The preliminary reference ranges of reduced and oxidized glutathione in a group of blood donors are (4.69+/-0.93) and (0.28+/-0.12)micromol/gHb for whole blood, (1.82+/-0.55) and (0.154+/-0.044)microM for plasma.

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.

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.

Protective effect of S-adenosylmethionine on cellular and mitochondrial membranes of rat hepatocytes against tert-butylhydroperoxide-induced injury in primary culture.

Accumulating evidence that administration of S-adenosylmethionine (SAMe) protects hepatocytes against oxidative stress-mediated injury led us to evaluate the effect of SAMe on hepatocyte injury induced in culture by oxidant substance tert-butylhydroperoxide (1.5 mM tBHP) with regard to prevent mitochondrial injury. The pretreatment of hepatocyte culture with SAMe in doses of 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 mg/l for 30 min prevented the release of LDH from cells incubated for 30 min with tBHP in a dose dependent manner. The inhibitory effect of SAMe on lipid peroxidation paralleled the effect on cell viability. SAMe also moderated the decrease of the mitochondrial membrane potential induced by tBHP. Our results indicate that the inhibition of lipid peroxidation by SAMe can contribute to the prevention of disruption of both cellular and mitochondrial membranes. While the protective effect of SAMe against tBHP-induced GSH depletion was not confirmed, probably the most potent effect of SAMe on membranes by phospholipid methylation should be verified.

Determination of phenylalanine and tyrosine in plasma and dried blood samples using HPLC with fluorescence detection.

The determination of phenylalanine and tyrosine is presently the most reliable direct approach to the diagnosis of phenylketonuria. An HPLC method for the simultaneous measurement of phenylalanine and tyrosine in samples of dried blood spots and plasma has been developed and evaluated. We have used an inherent fluorescence of both phenylalanine and tyrosine. For the separation, a reverse-phase column LiChroCart 125-4, Purospher RP-18e, 5microm, was used. The mixture of ethanol and deionized water (5:95, v/v) was used as a mobile phase. Analytical performance of this method is satisfactory for both phenylalanine and tyrosine: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma and blood samples were between 92.0 and 102.9%. The limit of detection was 10.0 and 5.0micromol/L, respectively. The preliminary reference ranges of phenylalanine and tyrosine in a group of newborns are 69.3+/-13.1 and 42.7+/-12.9micromol/L, in a group of blood donors are 68.4+/-9.9 and 52.1+/-10.9micromol/L. The presented method is inexpensive and suitable for diagnosis of phenylketonuria.

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection.

BACKGROUND: The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], alpha-keto acids derived from BCAA [BCKA; alpha-ketoisovaleric acid (KIV), alpha-ketoisocaproic acid (KIC), alpha-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. METHODS: Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27-41 years of age and 35 males, 28-43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3-5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 microm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5+/-0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. RESULTS: Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%-110%. CONCLUSIONS: We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Allantoin as a marker of oxidative stress in human erythrocytes.

BACKGROUND: Uric acid is the final product of purine metabolism in humans. It was determined that this compound has important antioxidative properties and it may be oxidized to allantoin by various reactive oxygen species. Therefore, the measurement of allantoin may be useful for the determination of oxidative stress in humans. METHODS: We measured allantoin and uric acid in human plasma and erythrocytes obtained from patients with chronic renal failure before hemodialysis (n=30) and blood donors (n=30). We used a method based on selective isolation of allantoin from deproteinized plasma and erythrocyte lysate samples on AG 1-X8 resin and its derivatization to glyoxylate-2, 4-dinitrophenylhydrazone. Separation of glyoxylate-2, 4-dinitrophenylhydrazone from interfering substances was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. Uric acid was determined by reversed phase HPLC with UV/VIS detection at 292 nm. RESULTS: We found significant differences in allantoin and uric acid concentration between the patients with chronic renal failure and the control group both in plasma (20.5+/-6.5 micromol/L and 323.9+/-62.9 micromol/L vs. 2.1+/-1.1 micromol/L and 270.1+/-62.3 micromol/L, p<0.05) and erythrocytes [82.8+/-39.1 nmol/g hemoglobin (Hb) and 110.7+/-28.8 nmol/g Hb vs. 20.1+/-6.1 nmol/g Hb and 82.1+/-23.7 nmol/g Hb, p<0.05]. CONCLUSIONS: Significant higher levels of allantoin in both plasma and erythrocytes of patients with chronic renal failure indicate that allantoin may be used as a good marker of oxidative stress.