Roman Kanďár

The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.

Determination of retinol and α-tocopherol in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Retinol and α‐tocopherol have an important role in the spermatozoa defences against oxidative stress. A method is described here for the simultaneous determination of retinol and α‐tocopherol in human seminal plasma with a suitable sample preparation procedure to prevent retinol and α‐tocopherol degradation. After adequate sample preparation, the samples were determined by reversed‐phase column chromatography with UV detection. The analytical performance of this method was satisfactory. The intra‐assay and inter‐assay coefficients of variation were below 10%. The recoveries were as follows: 90.7% (CV 8.1%) for retinol and 98.2% (CV 4.8%) for α‐tocopherol. No significant differences in both retinol and α‐tocopherol concentration between the smokers and nonsmokers (15 ± 7 nm and 1.86 ± 0.29 μm versus 15 ± 6 nm and 1.93 ± 0.45 μm) were found. A selective high‐performance liquid chromatography method for the determination of retinol and α‐tocopherol in human seminal plasma was developed.

The ratio of oxidized and reduced forms of selected antioxidants as a possible marker of oxidative stress in humans.

Oxidative stress is an imbalance between reactive oxygen species exposure and the ability of organisms to detoxify the reactive intermediates and to repair the oxidative damage of biologically important molecules. Many clinical studies of oxidative stress unfortunately provide conflicting and contradictory results. The ability of antioxidant systems to adequately respond to oxidative stress can be used in laboratory diagnostics. In the present review, methods using the ratio of reduced and oxidized forms of uric acid, ascorbic acid, glutathione and coenzyme Q10 as suitable indicators of oxidative stress are discussed. From the mentioned publications it is evident that suitable sample preparation prior to analysis is crucial.

An Assay of Selected Serum Amino Acids in Patients with Type 2 Diabetes Mellitus

BACKGROUND Amino acids are the building blocks of proteins. In case of insulin resistance, which is typical for type 2 diabetes mellitus (T2DM), proteolysis is increased and protein synthesis is decreased; therefore, we can observe changes in the levels of amino acids in diabetics vs. non-diabetics. OBJECTIVES The aim of this study was to find differences in the levels of selected amino acids between patients with diabetes (type 2) and a control group. MATERIAL AND METHODS Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of potassium cyanide to form fluorescent 1-cyanobenz(f)isoindole product. Amino acids derivatives were measured using a high-performance liquid chromatography with fluorescence detection. The serum levels of glucose were determined using an automatic biochemistry analyzer, glycated hemoglobin HbA1c was measured by cation exchange chromatography. RESULTS A total of 19 serum amino acids in T2DM patients and non-diabetics were measured. There were 9 amino acids, which were significantly different in these groups (p<0.05). Significantly decreased levels of arginine, asparagine, glycine, serine, threonine and significantly increased levels of alanine, isoleucine, leucine, valine in diabetics were found. CONCLUSIONS Significant difference in metabolism of amino acids between diabetics and non-diabetics were observed. The altered levels of amino acids in diabetic patients could be a suitable predictor of diabetes.

Assay of total glutathione and glutathione disulphide in seminal plasma of male partners of couples presenting for a fertility evaluation

A method is described here for the determination of total glutathione (TGSH) and glutathione disulphide (GSSG) in the seminal plasma of the male partners of couples requesting a fertility evaluation. A suitable sample preparation procedure prior to high‐performance liquid chromatography analysis is discussed. After adequate sample preparation, the samples were derivatised with ortho‐phthaldialdehyde to form a stable, highly fluorescent tricyclic derivative. Reversed‐phase column chromatography was used for the separation, and the effluent was monitored with a fluorescence detector at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The analytical performance of this method was satisfactory. The intra‐assay and inter‐assay coefficients of variation were below 10%. The recoveries were as follows: 94.1% (CV 2.3%) for TGSH and 93.2% (CV 4.0%) for GSSG. No significant differences were found in either TGSH or GSSG concentration between the smokers and nonsmokers (2.07 ± 1.28 μm versus 1.56 ± 1.20 μm, P = 0.431 and 95 ± 56 nm versus 112 ± 138 nm, P = 0.825).

An HPLC method for the determination of selected amino acids in human embryo culture medium

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP18e or Ascentis Express C18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.

Determination of selected fatty acids in dried sweat spot using gas chromatography with flame ionization detection

A method is described for the determination of fatty acids in dried sweat spot and plasma samples using gas chromatography with flame ionization detection. Plasma and dried sweat spot samples were obtained from a group of blood donors. The sweat was collected from each volunteer during exercise. Sweat was spotted onto collection paper containing butylated hydroxytoluene. Fatty acids were derivatized with acetyl chloride in methanol to form methyl esters of fatty acids. The fatty acids in dried sweat spot samples treated with butylated hydroxytoluene and stored at -20°C were stable for 3 months. Our results indicate that sweat contains, among fatty acids with short chain, also fatty acids with long chain and unsaturated fatty acids. Linear relationships between percentage content of selected fatty acids in dried sweat spot and plasma were observed.

Determination of Total Glutathione in Dried Blood Spot Samples Using a High-Performance Liquid Chromatography

A method is described for the determination of total glutathione (TGSH) in dried blood spot (DBS) samples using high-performance liquid chromatography with fluorescence detection. Whole blood and DBS samples were obtained from a group of blood donors. After GSH reduction with dithiothreitol and protein precipitation with ethanol, the samples were derivatized with naphthalene-2,3-dicarboxaldehyde to form a very stable, highly fluorescent derivative. For the separation, a reversed phase HPLC method was used. The mixture of ethanol and deionized water (8 : 92, v/v) was used as a mobile phase. The analytical performance of this method was satisfactory: the intra- and interassay coefficients of variation were below 10%. Quantitative recoveries from spiked DBS samples were between 98.3 and 103.6%. The presented method is inexpensive and suitable for clinical testing purposes.

AN ASSAY OF TOTAL GLUTATHIONE AND GLUTATHIONE DISULFIDE IN HUMAN WHOLE BLOOD AND PLASMA USING A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

A method is described here for the determination of total glutathione and glutathione disulfide in human whole blood and plasma with a suitable sample preparation procedure to prevent glutathione oxidation and glutathione disulfide hydrolysis. Whole blood samples were obtained from a group of blood donors. After adequate sample preparation, the samples were derivatized with ortho-phthaldialdehyde to form a stable, highly fluorescent tricyclic derivative. Reverse-phase column chromatography was used for the separation and fluorimetric detection to monitor the effluent. The analytical performance of this method was satisfactory for the determination of total glutathione and glutathione disulfide. The intra-assay and inter-assay coefficients of variation were below 10%. The recoveries were as follows: 96.1% (CV 1.4%) and 104.3% (CV 2.4%) for whole blood, 92.3% (CV 2.4%) and 107.4% (CV 2.6%) for plasma. We found no significant differences in both total glutathione and glutathione disulfide concentration between the women and men. We developed a selective high-performance liquid chromatography method for the determination of total glutathione and glutathione disulfide in human whole blood and plasma. Our sophisticated sample preparation procedure prevents significant glutathione oxidation and glutathione disulfide hydrolysis.

Association of fatty acid profile in plasma lipid fractions with HbA1c in type 2 diabetic patients

The aim of this pilot study has been the comparison of fatty acid profiles of diabetic and healthy subjects in order to evaluate the relationship between fatty acid profiles in plasma lipid fractions and glycated haemoglobin (HbA1c) in type 2 diabetes (T2D) patients. The fatty acid composition of fasting plasma lipid subfractions has been analyzed in patients (n = 26) diagnosed with T2D and in corresponding control group (n = 26) of healthy voluntary blood donors. Five subfractions containing phospholipids (PLs), diglycerides (DGs), free fatty acids (FFAs), triglycerides (TGs), and cholesterol esters (CEs) were isolated from plasma samples and separated by thin-layer chromatography. Fatty acid composition of these subfractions was analyzed by GC/FID. Significant changes in fatty acid profiles were found in all lipid fractions from T2D patients in comparison with the control group. HbA1c correlated negatively with delta 9 desaturation (9D) index. Significantly positive correlation of palmitic acid levels and negative correlation of oleic acid levels with HbA1c concentration were found in PL and TG fractions with higher significance in TGs. This pilot study has shown possible associations of HbA1c, common parameter measured in routine laboratories, with lipid metabolism. The strongest correlation was found in plasma TGs, especially in case of palmitic and oleic acids. This is the first report showing that metabolic control assessed by HbA1c is negatively associated with delta 9D index.