Capillary Electrophoresis of Free Amino Acids in Physiological Fluids Without Derivatization Employing Direct or Indirect Absorbance Detection.

Whole blood and/or plasma amino acids are useful for monitoring whole-body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes.Here, we present two protocols for the analysis of free amino acids employing free zone capillary electrophoresis. Main advantage of both methods is an absence of any derivatization procedures that permits the analysis of free amino acid in physiological fluids. The method using direct detection and carrier electrolyte consisting of disodium monophosphate (10 mM at pH 2.90) permits determination of compounds that absorb in UV region (aromatic and sulfur containing amino acids, as well as some peptides such as carnosine, reduced, and oxidized glutathione). The other method use indirect absorbance detection, employing 8 mM p-amino salicylic acid buffered with sodium carbonate at pH 10.2 as running electrolyte. It permits quantification of 30 underivatized physiological amino acids and peptides. In our experience factorial design represents a useful tool for final optimization of the electrophoretic conditions if it is necessary.

Renal transplantation promptly restores excretory function but disturbed L-arginine metabolism persists in patients during the early period after surgery.

The synthesis and whole body metabolism of L-arginine (Arg) are disturbed in renal diseases. Renal transplantation represents the best therapy in the end-stage of these diseases. In the present we compared alterations of plasma Arg and related compounds with renal excretory function in patients with end-stage renal disease, before and after kidney transplantation. Arg, asymmetric dimethylarginine (ADMA), citrulline (Cit), glutamine (Gln), ornithine (Orn), phenylalanine (Phe), tyrosine (Tyr), urea, creatinine, albumin, and nitrate were analyzed in patients before, immediately after (0-time) and 1, 2, 3, 7 and 14 days following living donors kidney transplantation. Healthy subjects were controls. Glomerular filtration rate (GFR) and amino acid molar ratios were calculated. Before transplantation creatinine, urea, Cit, Gln, ADMA, and nitrate were above, while GFR and Arg were below controls, confirming disturbed excretory and metabolic renal functions in patients with renal disease. Renal transplantation promptly normalized creatinine, urea, GFR, Cit, and nitrate. However, regardless of increased molar Phe/Tyr ratios, indicating increased net protein catabolism in peripheral tissues, low Arg and elevated ADMA concentrations persisted throughout the examined period. Alterations of other amino acids also suggest similarly disturbed Arg metabolism in patients after kidney transplantation. In conclusion, renal transplant promptly restored its excretory function, but increased net protein catabolism, disturbed Arg metabolism and endothelial dysfunction in entire body of these patients were not improved throughout the early period after the operation. That has to be considered in their therapy.

Unilateral nephrectomy causes an early abrupt decrease in plasma arginine and simultaneous reduction in glomerular filtration rate in living kidney donors.

OBJECTIVES: Living donor kidney transplantation is regarded as beneficial to allograft recipients and not particularly detrimental to the donors. Recently we have documented a reduced glomerular filtration rate (GFR) in living kidney donors (LKDs). Considering kidneys as essential for l-arginine (Arg) metabolism, in the present study we analyzed plasma Arg and related compounds comparing them with the function of remaining kidney in LKDs after donation. DESIGN AND METHODS: We analyzed GFR, plasma Arg, asymmetric dimethylarginine (ADMA), citrulline (Cit), glutamine (Gln), ornithine (Orn), phenylalanine (Phe), tyrosine (Tyr), thiobarbituric acid reactive substances (TBARS), urea, creatinine, nitrite, nitrate and their sum (NOx) in blood samples taken from LKDs before, immediately after (0-time) and 1, 2, 3, 7 and 14 days following surgery. RESULTS: Gradual and moderate creatinine increase and albumin decrease were associated with decreased GFR. An abrupt decrease in Arg occurred, staying below baseline level throughout the 14 days. Also decreases in Gln, Cit, Orn, increase in Phe and TBARS, and unaltered ADMA, nitrite and NOx concentrations were obtained. Despite increased net protein catabolism (indicated by elevated Phe/Tyr ratios) lack of Arg, suggested by decreased molar Arg/Phe ratios, occurred. Decreased molar Arg/Gln suggests an early but transient decrease in Arg synthesis. CONCLUSION: Unilateral nephrectomy causes an early abrupt decrease in plasma arginine and reduction in glomerular filtration rate in LKDs that was associated with increased net protein breakdown in the peripheral tissues and elevated oxidative damage, which has to be considered in their therapy.

Capillary electrophoresis of free amino acids in physiological fluids without derivatization employing direct or indirect absorbance detection.

Whole blood and/or plasma amino acids are useful for monitoring whole body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes.Here, we present two protocols for the analysis of free amino acids employing free zone capillary electrophoresis. Main advantage of both methods is an absence of any derivatization procedures that permits the analysis of free amino acid in physiological fluids. The method using direct detection and carrier electrolyte consisting of disodium monophosphate (10 mM at pH 2.90) permits determination of compounds that absorb in UV region (aromatic and sulfur containing amino acids, as well as some peptides, such as carnosine, reduced and oxidized glutathione). The other method uses indirect absorbance detection, employing 8 mM p-amino salicylic acid buffered with sodium carbonate at pH 10.2 as running electrolyte. It permits quantification of 30 underivatized physiological amino acids and peptides. In our experience, factorial design represents a useful tool for final optimization of the electrophoretic conditions if it is necessary.

Changes of glomerular filtration after nephrectomy in living donor.

A possible complication after donor nephrectomy is a decrease in glomerular filtration rate. The goal of our investigation is to estimate the function of the remaining donor kidney in the first 6 months after nephrectomy using the equations Cockcroft-Gault, Modification of Diet in Renal Disease 1 (MDRD1) and MDRD2. In addition to basic age and sex data, we collected standard biochemical data from blood: creatinine, blood urea nitrogen, and albumin. Blood samples and diuresis were taken at -1, 0, 1, 2, 3, 7, and 14 days, and after 6 months. Our results show that glomerular filtration rate decreases after nephrectomy and stabilizes after 6 months in values significantly lower compared with predonation values. Both MDRD estimations show that these donors after nephrectomy are patients in the third degree of chronic kidney disease, and we can predict that older donors and those with comorbidities very soon will need a treatment for chronic kidney disease. For glomerular filtration rate estimation, we recommend the MDRD2 equation. All donors must have long-term follow-up and treatment, because there is a possibility of eventual cardiovascular and metabolic diseases.

Increased nitric oxide formation followed by increased arginase activity induces relative lack of arginine at the wound site and alters whole nutritional status in rats almost within the early healing period.

Nitric oxide (NO) production and free amino acid fluxes at the wound side during the first 3 days following cutaneous wound were investigated. Experiments were performed on Albino Oxford rats (n=18) underwent cutaneous implantation of polyvinyl sponges. Intact animals (n=6) were controls. Nitrites, nitrates, free amino acids and urea were measured both in plasma and wound fluids. Inducible nitric oxide synthase (iNOS) gene expressions at wound site were analyzed, too. The highest levels of both iNOS gene expression and its activity (increased wound fluid citrulline and nitrites) were at the first day. Wound fluid nitrates were significantly above plasma levels throughout the whole period, while molar nitrate to nitrite ratio steadily increased. It was associated with gradual increase of both ornithine and urea as well as steadily decreases of arginine and increases of phenylalanine at the wound site. Gradual decrease in glycine to branched-chain molar ratio was observed both in plasma and wound fluids. In conclusion, an early locally induced alterations in Arg metabolism, due to increased NO formation followed by increased arginase activity, produces relative lack of Arg at the wound site and disturbs nutritional status of the whole body almost within early healing period following cutaneous wound in rats. It is likely that NO autoxidation at the wound side is influenced by substrate availability.

Nitrite to nitrate molar ratio is inversely proportional to oxidative cell damages and granulocytic apoptosis at the wound site following cutaneous injury in rats.

Nitric oxide (NO) metabolism in response to the inflammatory cell infiltration and their apoptosis at the wound site, using a model of subcutaneously implanted sponges in Albino Oxford rats, were examined. The injured animals were sacrificed at days 1, 2 and 3 after the injury. Nitrites, nitrates (final products of NO metabolism), malondialdehyde (an indicator of oxidative cell damages), urea (product of arginase activity) and other parameters were measured both in plasma and wound fluid samples. Nitrite to nitrate molar ratio and sum of nitrites and nitrates (NO(x)) were calculated. The total cell numbers were at similar level throughout the examined period, but a gradual decrease of viable granulocytes, mainly due to the increased apoptosis, and the increase of monocyte-macrophage number occurred after the second day. A gradual increase of wound fluid nitrates, NO(x) and malondialdehyde suggested the increases of both NO and free oxygen radicals production. Interestingly, wound fluid nitrites peaked at the first day decreasing to the corresponding plasma levels thereafter. Wound fluid nitrite to nitrate molar ratio gradually decreased and negatively correlated both with the number of apoptotic cells (r= -0.752, p<0.05) and malondialdehyde (r= -0.694, p<0.05) levels. In conclusion, the inversely proportional relation between nitrite to nitrate molar ratio and both malondialdehyde and apoptotic cell number indicated a mutual relationship between NO metabolism, oxidative cell damages and cell apoptosis at the wound site early after the cutaneous wound. Moreover, the obtained findings suggest that measurement of both nitrites and nitrates contribute to better insight into overall wound NO metabolism.

Capillary electrophoresis method optimized with a factorial design for the determination of glutathione and amino acid status using human capillary blood.

Plasma aromatic and sulfur containing amino acids are good indicators of protein anabolism/catabolism, while blood reduced and oxidized glutathione reflect oxidative status in an organism. Using a full factorial design for screening important variables (pH, concentration, temperature) we developed a capillary zone electrophoresis method permitting their measurements in the single run, without any derivatization procedures. The best separations were obtained within less than 30 min employing a 10 mmol/l phosphate buffer, pH 2.8, 18 degrees C, 15 kV voltage. Fairly good precision with a linear relationship between peak area and concentrations (r=0.995-0.999) were obtained. The method was used to analyze human capillary blood.

Correlation of oxidative stress parameters and inflammatory markers in coronary artery disease patients.

OBJECTIVES: In addition to many traditional risk factors for coronary artery disease (CAD) development, enhanced oxidative stress and inflammation are serious conditions that may also be classified as novel risk factors. In the present study, we assessed the relationship between several parameters of oxidative stress status [malonaldehyde (MDA), superoxide anion (O(2)(-)) and plasma and erythrocyte superoxide dismutase (SOD) activities] with high sensitivity C-reactive protein (hsCRP) and fibrinogen as inflammation markers. DESIGN AND METHODS: Oxidative stress status parameters, inflammation markers and lipid status parameters were measured in 385 subjects [188 coronary heart disease (CHD) patients with angiographically diagnosed coronary artery disease (CAD), 141 patients with occlusion >50% in at least one major coronary artery (CAD+) and 47 patients with occlusion less than 50% (CAD-), and 197 CHD-free middle-aged subjects (the control group)]. RESULTS: The plasma MDA concentration and the level of O(2)(-) in plasma were significantly higher in combination with significantly lower SOD activity in the CAD+ group vs. the control group. By using multiple stepwise regression analysis, fibrinogen and hsCRP showed independent correlation with MDA. Binary logistic regression analysis indicated that both MDA and O(2)(-) were significantly associated with CAD development and adjustment for inflammatory markers weakened this association in the case of MDA. CONCLUSIONS: The relationship between oxidative stress parameters and inflammatory species suggest their strong mutual involvement in atherosclerosis development that leads to CAD progression.

Relation between both oxidative and metabolic-osmotic cell damages and initial injury severity in bombing casualties.

BACKGROUND/AIM: We have recently reported the development of oxidative cell damages in bombing casualties within a very early period after the initial injury. The aim of this study, was to investigate malondialdehyde (MDA), as an indicator of lipid peroxidation, and osmolal gap (OG), as a good indicator of metabolic cell damages and to assess their relationship with the initial severity of the injury in bombing casualties. METHODS: The study included the males (n = 52), injured during the bombing with the Injury Severity Score (ISS) ranging from 3 to 66. The whole group of casualties was devided into a group of less severely (ISS < 25, n = 24) and a group of severely (ISS > or = 26, n = 28) injured males. The uninjured volunteers (n = 10) were the controls. Osmolality, MDA, sodium, glucose, urea, creatinine, total bilirubin and total protein levels were measured in the venous blood, sampled daily, within a ten-day period. RESULTS: In both groups of casualties, MDA and OG levels increased, total protein levels decreased, while other parameters were within the control limits. MDA alterations correlated with ISS (r = 0.414, p < 0.01), while a statistically significant correlation between OG and ISS was not obtained. Interestingly, in spite of some differences in MDA and OG trends, at the end of the examined period they were at the similar level in both groups. CONCLUSION: The initial oxidative damages of the cellular membrane with intracellular metabolic disorders contributed to the gradual development of metabolic-osmotic damages of cells, which, consequently caused the OG increase. In the bombing casualties, oxidative cell damages were dependent on the initial injury severity, while metabolic-osmotic cell damages were not.

Very early increase in nitric oxide formation and oxidative cell damage associated with the reduction of tissue oxygenation is a trait of blast casualties.

BACKGROUND: In our previous experimental studies, we found evidence for the increase of nitric oxide (NO) formation immediately after blast injury. In the present study we investigated whether NO overproduction was a trait for the period immediately after blast injury in humans. Concomitant metabolic disturbances were also studied, and compared to the alterations in other traumatized patients. METHODS: Blast casualties (group B, n = 13), surgical patients with the hip replacement or fractures, not exposed to blast effects (group S, n = 7) and healthy volunteers as controls (group C, n = 10), were examined. Both arterial and venous blood samples were taken within 6 hours, and 24 hours after blast injuries or surgical procedures, respectively. Plasma levels of nitrite/nitrate (NOx), superoxyde anion (O2-), sulfhydrils (SH), malondialdehyde (MDA) as well as acid-base status and other biochemical parameters (glucose, urea, creatinine, total proteins, albumin) were measured. RESULTS: Significant, but transient increase in plasma NOx levels occurred only in group B. It was associated with the significant increase of hemoglobin oxygen (sO2) saturation of the venous blood and the concomitant decrease of its arterial--venous difference. In group S the venous sO2 decreased, its arterial--venous difference increased, while NOx levels were within the control limits. In both groups, other parameters of arterial acid-base status were kept within the control limits throughout the examined period. The decrease of SH levels were similar in the examined groups, while the increase of O2- was greater in group B. CONCLUSION: Early NO overproduction was a trait of blast injuries in humans, contributing to the reduction of tissue the oxygenation and intensifying the oxidative cell damage that had to be considered in the therapy of casualties with blast injuries. These alterations were different from those observed in other surgical patients without blast injuries.

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids.

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.