The dried blood spot sampling method in the laboratory medicine.

The minimization is currently expanding in all fields of human life. The smaller size of analytical instruments and their higher sensitivity enables an analysis of small amounts of a biological material with a very low consumption of chemicals, what is economically and also environmentally beneficial. A patient friendly and minimal invasive sample collection is therefore more than required. The dried blood spot (DBS) sampling standardly used in the newborn screening (NS) may be an option. The sample collection is simple, non-invasive, does not require trained medical personnel assistance and also storing and transportation of these samples is much easier in comparison with the whole blood samples. The DBS sampling method is used in the therapeutic drug monitoring or in the diagnosis of infectious diseases and its usage is still spreading. In some cases, it is still only a research object, but it shows a big potential for a future use. It could completely replace the whole venous blood collection in some cases, for example in the metabolic screening of diabetic patients or monitoring of treatment response, and it can overall simplify the sample collection and the transportation process (Tab. 1, Ref. 152). Keywords: dried blood spot, DBS, dried samples, sample collection, laboratory medicine.

Fatty acid profile in erythrocyte membranes and plasma phospholipids affects significantly the extent of inflammatory response to coronary stent implantation.

In coronary heart disease, the treatment of significant stenosis by percutaneous coronary intervention (PCI) with stent implantation elicits local and systemic inflammatory responses. This study was aimed at evaluation of the dynamics of inflammatory response and elucidation of the relationship between the fatty acid profile of red blood cell (RBC) membranes or plasma phospholipids and inflammation after PCI. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), serum amyloid A (SAA), malondialdehyde (MDA) and the fatty acid profiles were determined in patients with advanced coronary artery disease undergoing PCI before, 24 h and 48 h after drug-eluting stent implantation (n=36). Patients after PCI exhibited a significant increase in studied markers (hsCRP, IL-6, SAA, MDA). Many significant associations were found between the increase of IL-6, resp. SAA and the amounts of n-6 polyunsaturated fatty acids (namely linoleic, dihomo-gamma-linolenic, docosatetraenoic and docosapentaenoic acid), resp. saturated fatty acids (pentadecanoic, stearic, nonadecanoic) in erythrocyte membranes. The magnitude of the inflammatory response to PCI is related to erythrocyte membrane fatty acid profile, which seems to be a better potential predictor of elevation of inflammatory markers after PCI than plasma phospholipids.

Amino acid metabolism in human embryos.

The aim of this study was to find some relationship between amino acid metabolism and the embryo morphokinetic parameters studied via time-lapse analysis. Study included 48 human embryo samples and their culture media. Two groups of embryos were identified: embryos reached the 8-cell stage on day 3 (n=34) and embryos failed to develop at any point during the incubation (n=14). Amino acids levels were measured on day 3 of embryo development; using time-lapse analysis, the precise timing of embryo cleavage, synchrony of division, grade of fragmentation etc. were established. No statistically significant differences between dividing and arresting embryos were observed in terms of amino acids production/consumption and turnover. Amino acids which were part of the culture medium did not exhibit any statistically significant correlation with kinetic parameters with the exception of the grade of fragmentation on day 3; there were negative correlation with glutamate, and positive with glutamine, glycine and taurine. In some dividing and in some arresting embryos appeared new amino acids which strongly correlated with each other, with methionine, but not with any other amino acid that is a regular part of the culture medium.

[Changes in the levels of selected metabolites in the culture medium as a possible tool for the embryo selection in assisted reproduction]. / Zmeny hladin vybraných metabolitu v kultivacním médiu jako mozný nástroj pro selekci embryí v asistované reprodukci.

Despite the increasing success of infertility treatment methods of assisted reproduction, it still remains a problem how to select the best embryo that has the potential for further development and implantation. At the present time, embryo selection is based especially on morphological criteria. This approach is subjective; therefore there is a trend to find another more objective and robust method for embryo selection. Embryo metabolism can be used as an indicator of viability. This non-invasive method allows observing changes in the levels of different metabolites in culture medium before and after incubation of the only one embryo. The most mentioned substances are carbohydrates and amino acids as important components of culture medium. Carbohydrates serve predominantly as energy sources, whereas amino acids are precursors of protein and nucleotides, antioxidants, osmolytes, pH regulators etc. Several methods have been proposed for evaluating of embryo metabolic profile of embryo. There are many hypotheses for embryo selection according its metabolic profile.

Determination of seminal plasma malondialdehyde by high-performance liquid chromatography in smokers and non-smokers.

OBJECTIVES: The aim of this study was to establish and validate a high-performance liquid chromatography method for the determination of malondialdehyde in seminal plasma in smokers and non-smokers and to find possible differences between the two groups. BACKGROUND: Malondialdehyde is used as a diagnostic marker of lipid peroxidation and indicator of oxidative stress. Smoking is suspected to be responsible for an increase in its level. Malondialdehyde has been thought to have cytotoxic and damaging effects. METHODS: Semen samples were obtained from male partners of couples requesting a fertility evaluation. Malondialdehyde was derivatized with 2-thiobarbituric acid. The malondialdehyde-2-thiobarbituric acid complex was determined by liquid chromatography with fluorescence detection. The mobile phase consisted of 20% ethanol in 25-mmol/L potassium dihydrogenphosphate (v/v), pH 6.00 ± 0.05. RESULTS: Analytical performance was satisfactory. Malondialdehyde levels were as follows: 1.50 ± 0.55 µmol/L in all patients, 1.40 ± 0.57 µmol/L in smokers, and 1.50 ± 0.53 µmol/L in non-smokers. CONCLUSION: The method presented here is sensitive and accurate for seminal plasma malondialdehyde determination. Our results showed a relationship between sperm motility and the malondialdehyde level in all patients and non-smokers. Malondialdehyde may induce poor sperm functionality and negatively affect the fertilization processes (Tab. 1, Fig. 1, Ref. 23).

Effects of smoking on fatty acid composition of phospholipid sperm membrane and the malondialdehyde levels in human seminal plasma.

The aim of this study was to investigate fatty acids composition of sperm phospholipids, level of lipoperoxidation represented by malondialdehyde and to examine differences between recent smokers and nonsmokers. The levels of malondialdehyde were in the group of all patients 1.51 ± 0.56 µmol l(-1) , in smokers 1.36 ± 0.59 µmol l(-1) and in nonsmokers 1.53 ± 0.55 µmol l(-1) . Total sperm membrane phospholipid fatty acids were profiled into several groups, saturated acids (in smokers 61.86 ± 9.02%, in nonsmokers 61.20 ± 11.66%), polyunsaturated acids n-3 (in smokers 12.62 ± 8.18%, in nonsmokers 14.28 ± 13.65%), polyunsaturated acids n-6 (in smokers 9.13 ± 4.37%, in nonsmokers 10.10 ± 3.79%) and other acids (in smokers 14.36 ± 3.94%, in nonsmokers 13.88 ± 2.31%). Significant correlations were found between the level of malondialdehyde (MDA) and total sperm motility in all patients (r = -0.358, P = 0.013), between both the level of MDA and progressive motility (r = -0.465, P = 0.001) and between the level of MDA and total motility (r = -0.382, P = 0.037) in nonsmokers. There were no statistically significant differences between composition of sperm phospholipid important fatty acids in smokers and nonsmokers. Significant correlations between selected sperm fatty acids and sperm motility and morphology in smokers and nonsmokers were not observed.

A monitoring of allantoin, uric acid, and malondialdehyde levels in plasma and erythrocytes after ten minutes of running activity.

Uric acid is the final product of human purine metabolism. It was pointed out that this compound acts as an antioxidant and is able to react with reactive oxygen species forming allantoin. Therefore, the measurement of allantoin levels may be used for the determination of oxidative stress in humans. The aim of the study was to clarify the antioxidant effect of uric acid during intense exercise. Whole blood samples were obtained from a group of healthy subjects. Allantoin, uric acid, and malondialdehyde levels in plasma and erythrocytes were measured using a HPLC with UV/Vis detection. Statistical significant differences in allantoin and uric acid levels during short-term intense exercise were found. Immediately after intense exercise, the plasma allantoin levels increased on the average of 200 % in comparison to baseline. Plasma uric acid levels increased slowly, at an average of 20 %. On the other hand, there were no significant changes in plasma malondialdehyde. The results suggest that uric acid, important antioxidant, is probably oxidized by reactive oxygen species to allantoin. Therefore allantoin may be suitable candidate for a marker of acute oxidative stress.

[Role of fatty acids in sperm membrane]. / Úloha mastných kyselin v membráne spermií

One of causes of male infertility is reduced sperm motility. Reactive oxygen species (ROS) play significant role for physiological sperm function. Oxidative stress occurs when the production of potentially destructive ROS exceeds the natural antioxidant defences, resulting in cell damage. Sperm phospholipid polyunsaturated fatty acids are particularly susceptible to peroxidative damage by free radicals. Detrimental effects of lipid peroxidation should decrease sperm quality and be responsible of fertility problems. The review deals with sperm membrane composition, importance of fatty acids and prevention possibilities of oxidative cell damage.

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.

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).

[Impact of oxidative stress on male infertility]. / Vliv oxidacního stresu na muzskou plodnost.

Reactive oxygen and nitrogen species (RONS) are necessary for the physiological function of sperm. Its concentration has to be kept on a level without the damage of cells. If this level is overdrawn sperm has pathological effect on many biological structures in the form of oxidative stress. Antioxidants have a key role for keeping of this balance. Oxidative stress is an important factor which causes problems with male fertility. The survey article is complexly concerned with the influence of RONS and antioxidants on male fertility. It outlines some possibilities of treatment and research on this actual issue of assisted reproduction.

Oxidative stress and metabolic syndrome in obese adults with and without controlled diet restriction.

BACKGROUND: Metabolic syndrome (MetS) represents a collection of markers associated with cardiovascular morbidity and mortality. Due to its high prevalence and steady increase of the occurrence, prevention or management of MetS is of paramount importance. The aim of our study was to evaluate MetS occurrence and extent of oxidative stress by comparing obese adults after diet optimization with untreated controls. MATERIAL AND METHODS: Oxidative stress markers (total amount of free radicals, malondialdehyde, allantoin, alpha1-antiproteinase, GSSG/GSH ratio), total antioxidant capacity and lipid standardized alpha-tocopherol were determined in 40 obese people and 48 healthy controls. The obese people were divided into two group A: obese with restricted energy intake with lowered dietary carbohydrates (n=20) and group B: with the same grade of obesity but without following dietary recommendations (n=20). RESULTS: Group A exhibited lower oxidative stress markers than group B; free radicals (5.18+/-1.68 vs 8.43+/-3.66 mmol/l, p<0.01), GSSG/GSH ratio (11.74+/-5.01 vs 15.38+/-5.93%, p<0.05) and higher antioxidants: lipid standardized alpha-tocopherol (3.70+/-0.51 vs 3.35+/-0.60, p<0.05) and ceruloplasmin (0.24+/-0.08 vs 0.21+/-0.03 g/l, p<0.05), in the course of same grade of obesity. Furthermore MetS occurrence was found significantly lower was in group A. CONCLUSION: The energy intake restriction by 2000 kJ, mainly due to carbohydrate limitations, was associated with decreased oxidative stress and simultaneously increased lipid-standardized alpha-tocopherol and ceruloplasmin in obese people. These changes correlated with diminished MetS occurrence by about 50% (Tab. 3, Ref. 32). Full Text (Free, PDF) www.bmj.sk.

Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture.

The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2.5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST 1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS production was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation.

Protective effect of S-adenosylmethionine against galactosamine-induced injury of rat hepatocytes in primary culture.

The protective effect of S-adenosylmethionine (SAMe) on D-galactosamine (GalN)-induced damage to rat hepatocytes was tested in primary cultures. SAMe at concentrations of 50 and 1000 mg/l significantly reduced lactate dehydrogenase release from cells injured by 40 mM GalN after 24 h of incubation. There were no significant changes in urea production after 24 h among tested groups, including control hepatocytes. Exposure of hepatocytes to GalN leads to 3.5-fold decrease in urea synthesis after 48 h in comparison with control cell cultures. Addition of the highest dose of SAMe (1000 mg/l) into the culture media attenuated this decrease by 180 %. None of the tested doses of SAMe (5, 25, 50 and 1000 mg/l) affected considerably the reduced activity of mitochondrial dehydrogenases. The content of reduced and oxidized glutathione in GalN-exposed cells was diminished to 1.5 % and 16 %, respectively, of the control values after 24 h. Using only the highest concentration SAMe increased significantly these contents. SAMe had no effect on dramatically decreased albumin synthesis. These findings indicate beneficial effect of SAMe, especially of the highest concentration, on GalN-induced toxicity to rat hepatocytes in primary culture. This action of SAMe seems to be associated with reduction of plasma membrane damage and increased synthesis of glutathione.

Antioxidant vitamin levels and glutathione peroxidase activity during ischemia/reperfusion in myocardial infarction.

The consequences of increased oxidative stress, measured as the level of malondialdehyde (MDA) during ischemia/reperfusion, were studied in 48 patients in the acute phase of myocardial infarction (AMI) and a control group (21 blood donors). The serum levels of alpha-tocopherol and beta-carotene were followed. Immediately after the treatment onset the level of alpha-tocopherol started to decrease, reaching a plateau after 24 h. The consumption of beta-carotene was delayed by 90 min. Steady decline was detected during the whole time interval studied (48 h). Glutathione peroxidase (GPx) activity, as a representative of antioxidant enzymes, was estimated in whole blood. The influx of oxygenated blood was accompanied by a stimulation of GPx activity, which reached its maximum at the time of completed reperfusion. When comparing the AMI patients with the control group, the levels of MDA were found significantly increased, which indicates that oxidative stress is already increased during ischemia. Lower antioxidant levels found in the patients might either already be the result of vitamin consumption during ischemia or be a manifestation of their susceptibility to AMI. Monitored consumption of alpha-tocopherol and beta-carotene during reperfusion indicated that in the case of patients, whose level of antioxidant vitamins is below the threshold limit, a further substantial decrease of antioxidant vitamins during reperfusion could enhance the oxidative damage of the myocardium.

Selective antioxidant enzymes during ischemia/reperfusion in myocardial infarction.

The study of ischemia/reperfusion injury included 25 patients in the acute phase of myocardial infarction (19 perfused, 6 remained non-reperfused as evaluated according to the time course of creatine kinase and CK-MB isoenzyme activity) and a control group (21 blood donors). Plasma level of malondialdehyde was followed as a marker of oxidative stress. Shortly after reperfusion (within 90 min), a transient increase of malondialdehyde concentration was detected. The return to the baseline level was achieved 6 h after the onset of therapy. The activity of a free radical scavenger enzyme, plasma glutathione peroxidase (GPx), reached its maximum 90 min after the onset of treatment and returned to the initial value after 18 h. The specificity of the GPx response was confirmed by comparing with both non-reperfused patients and the control group, where no significant increase was detected. The erythrocyte Cu,Zn-superoxide dismutase (SOD) did not exhibit significant changes during the interval studied in perfused patients, probably due to the stability of erythrocyte metabolism. In non-reperfused patients, a decrease of SOD was found during prolonged hypoxia. These results help to elucidate the mechanisms of fast activation of plasma antioxidant system during the reperfusion after myocardial infarction.