Gordana Žunić

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.

Simple and rapid method for the measurement of nitrite and nitrate in human plasma and cerebrospinal fluid by capillary electrophoresis

Nitrite and nitrate levels in physiological fluids are commonly used as an index of nitric oxide production. We developed simple and rapid method for the determination of these anions by capillary zone electrophoresis employing borate buffer (pH 10, 100 mmol/l) as running electrolyte. The anions were analyzed in plasma and cerebrospinal fluid (CSF) without deproteinization of the samples. Electrophoresis was carried out in a capillary (36.5 cm x 75 microm) at a potential of 15 kV, with on-column UV detection at 214 nm. Mean retention times for nitrite and nitrates were 4.631 and 5.152 min, respectively. The method was linear (r=0.999) within a 1-500 micromol/l concentration range. Physiological levels of nitrate in plasma (40.2 micromol/l) and CSF (15.3 micromol/l) could be determined with good precision (coefficients of variation <6%) and accuracy (recoveries of added nitrate to plasma and CSF were 97.4 and 104.5%, respectively). Measurements of the physiological levels of nitrite in plasma (6.1 micromol/l) and CSF (0.9 micromol/l) were less precise and accurate.

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.

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.

The effect of physostigmine on acid-base status in arterial and venous blood of anaesthetized rabbits following hypovolemic shock

1. The effects of physostigmine (70 micrograms kg-1, intravenously) on acid-base status in arterial and venous blood were studied in anaesthetized rabbits subjected to hemorrhagic hypovolemia. 2. Hemorrhagic shock was produced using intermittent bleeding of 50% of the estimated blood volume, during 30 min. Experimental group was treated with physostigmine (70 micrograms kg-1 body mass, intravenously) and the control group with the same volume (0.1 ml) of saline, immediately after bleeding. Blood samples were taken before and after bleeding (0, 15 and 60 min). 3. It was found that physostigmine increased the mean arterial blood pressure, did not change the heart rate, and improved survival of the animals. 4. These effects of physostigmine were associated with significant decrease in venous pH, produced mainly by increased PCO2. This can partly be explained in terms of additional vasoconstriction due to physostigmine action. 5. In arterial blood decreased pH, decreased standard bicarbonate, negative values of excess base and decreased PCO2 were observed both in physostigmine-treated and the control group of animals, indicating partly respiratory compensated metabolic acidosis. These findings indicate that the hypertensive effect of physostigmine in shock was not accompanied by more severe disturbance in arterial acid-base status than was observed in hypovolemic shock alone.

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.

Rapid ion-exchange method for the determination of 3-methylhistidine in rat urine and skeletal muscle

A method for the determination of 3-methylhistidine using an automatic amino acid analyser has been developed. A single column system with lithium buffer (pH 3.950, 0.500 mol/l lithium and 0.067 mol/l citrate) was used for elution. The standard amino acid mixture of basic amino acids and some dipeptides usually present in physiological fluids was analysed for the development of the method. 3-Methylhistidine eluted in 46.7 +/- 0.049 min and the peak area coefficient of variation for the same sample was 1.07%. As 3-methylhistidine is completely resolved from the other basic amino acids and some dipeptides (anserine and carnosine), this method is suitable for the analysis of urine and muscle extracts as well as skeletal muscle protein hydrolysates where this amino acid is present in much lower concentrations than other amino acids.

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.

URINARY EXCRETION OF 3-METHYLHISTIDINE IN INJURED RATS

Publisher Summary This chapter describes the urinary excretion of 3-methylhistidine in injured rats. The chapter presents a study, the aim of which was to see the effect of two different kind of local injuries on the excretion of 3-methylhistidine (3-MeH), which is thought to be a useful index of muscle protein catabolism. Under ether anesthesia, nonlethal hind-limb ischemia or full-thickness scalding were produced in rats. At different times after injury, urine was collected during 24 h period. In both kinds of injuries, maximal excretion of 3-MeH was found at the second day after their infliction. The excreted amount of 3-MeH decreased thereafter, but the values were not normal at the 14th day. Scalding and limb ischemia resulted in similar urinary excretion of 5-MeH, although its content in normal skin was small as compared to muscles. The posttraumatic muscle protein catabolism is part of general rather than local response to injury.