Plasma Of Uremic Patients Research Articles

Are Polyunsaturated Fatty Acid Metabolites, the Protective Effect of 4-hydroxytyrosol on Human Red Blood Cell Membranes and Oxidative Damage (4-hydroxyalkenals) Compatible in Hypertriglyceridemic Patients?

Background:Increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are demonstrated in plasma of uremic patients. A study showed that the comparison of erythrocytes of healthy and diseased patients (obese, hypertensive, and Type 2 diabetics) with age is associated to a disturbed oxidant/antioxidant balance when obesity is associated with hypertension. 4-hydroxytyrosol is shown to significantly protect red blood cells (RBCs) from oxidative damage (4-HNE). In literature, there are partial discussions on the role of lipids and their oxidation products. The products of degradation of membrane proteins are observed as self-consisting products without interrelations with membrane lipids.Objective:The aim of this study is to evaluate the role of polyunsaturated fatty acid (PUFA) metabolites on oxidative damage (4-hydroxy-alkenals) in RBCs of hypertriglyceridemic patients after membrane treatment with 4-hydroxytyrosol.Materials and Methods:The authors optimize the isolation of RBC ghosts and spectrophotometric method to measure free 4-hydroxyalkenals in human RBC membranes and investigated the effect on oxidative damage in human erythrocyte membranes and in vitro 4-hydroxytyrosol treatment to evaluate the membrane lipids reducible by this phenol.Results:Plasma triglyceride levels in patients are clearly higher than in controls. Moreover, total membrane proteins data are similar to previous described. The normalized alkenals levels are significantly enhanced in hyperlipemic patients in comparison to normoglyceridemic controls. After the 4-hydroxytyrosol action, lipid metabolites substantially decrease. The ratio of oxidized lipids (MDA + HNE) and membrane proteins data are similar to previously described ones.Conclusion:According to experimental data, the accumulation of the alkenals in RBC membrane could be produced either by partial PUFA oxidation contained in glycerides and plasma glycerides and by glycerides into plasma membrane recycled RBC.SUMMARY Hypertriglyceridemia induces oxidative stress in human red blood cell (RBC) membranesOxidative stress causes increased plasma membrane total protein concentration and hydroxynonenal and malondialdehyde levelsThe authors optimize the isolation of RBC ghosts and spectrophotometric method to measure free 4-hydroxyalkenals in human RBC membranesAfter the reduction with 4-hydroxytyrosol, oxidized lipid concentration significantly decrease. Abbreviations used: RBC: Red blood cell; MDA: Malondialdehyde; HNE\\HAE: 4-hydroxyalkenals; LPO: Lipid peroxidation; ROS: Reactive oxygen species; ORAC: Oxygen Radical Absorbance Capacity.

Adipokines as Uremic Toxins

The adipose tissue has pleiotropic functions far beyond the mere storage of energy, and it secretes a number of hormones and cytokines, called adipokines, which have biological effects that impact heath and disease. Adipokines are markedly elevated in the plasma of uremic patients, mainly due to decreased renal excretion. They have pluripotent signaling effects on inflammation/oxidative stress (leptin, adiponectin, resistin), protein-energy wasting (leptin, adiponectin), insulin signaling (adiponectin, leptin, visfatin), endothelial dysfunction (visfatin), and vascular damage (adiponectin, leptin, resistin), which are prevalent in uremic patients. Obesity superimposed to uremia may further aggravate hyperadipokinemia, with the exception of adiponectinemia, which is mitigated by adiposity. Among adipokines and until more data become available, only leptin may be considered as a full uremic toxin owing to adverse effects on protein-energy wasting, cardiovascular damage, inflammation, and the immune system, which have been documented both clinically and experimentally. Resistin and visfatin display some features of uremic toxins, but more data are needed to consider these adipokines as true uremic toxins. In contrast, high levels of adiponectin and chemerin seen in uremia appear to be beneficial. Further research is needed to investigate whether selective removal of leptin, resistin, and visfatin and increments of adiponectin and chemerin levels may have clinical relevance in uremic patients.

Association of plasma pentosidine concentrations with renal function in kidney graft recipients

Advanced glycation end-products accumulate in the plasma of uremic patients. We aimed to assess the changes of free (P(free)) and total (P(tot)) plasma pentosidine concentrations in kidney graft recipients, create a model describing their profile and analyze associations with clinical parameters. We measured P(free) and P(tot) in the plasma of 12 non-diabetic patients before and after kidney transplantation by HPLC. P (tot) concentrations were significantly decreasing after transplantation. The changes were well described by the exponential model assuming an asymptotic fall until the steady-state concentration is attained. P(tot) before and after transplantation displayed a strong negative correlation with mean daily diuresis (Rs = -0.64, p < 0.05 before; Rs = -0.94, p < 0.01 after 20 d). The rate of fall of the P(tot) was positively correlated with the mean daily volume of urine passed in the second week after operation (Rs = +0.58, p < 0.05). The rate of fall of the P(tot) after transplantation displays a strong correlation with diuresis, and the P(tot) before transplantation is a potential prognostic factor for diuresis after the operation. Further prospective studies are necessary to demonstrate whether an effort to reduce carbonyl stress and pentosidine concentration before transplantation improves renal outcome.

Measurement of Protein Glycation, Glycated Peptides, and Glycation Free Adducts

Protein glycation adducts, early glycation adducts, such as N(epsilon)-fructosyl-lysine, and advanced glycation end products (AGEs) are uremic toxins. Glycation adducts are found in plasma and tissue proteins (glycation adduct residues), in peptides (glycation adduct peptide residues), and glycated amino acids (glycation free adducts). The latter two analyte groups arise from proteolysis of glycated proteins and glycation of peptides and amino acids. Quantitation of glycation adducts in uremia is difficult because of the presence of many different AGEs at low concentrations in different forms in the presence of many potential interferences. Application of liquid chromatography with tandem mass spectrometric (LC-MS/MS) detection to plasma, urine, and dialysate samples of uremic patients has provided a comprehensive and quantitative analysis of glycation adducts in uremia. Glycation free adducts accumulate markedly in the plasma of uremic patients and are eliminated in the peritoneal dialysate. Multiple glycation adducts, and also protein oxidation and nitration adducts, may be quantified concurrently. Glycation free adducts are the major form of glycation adduct eliminated in dialysate. LC-MS/MS may now be used to quantify concentrations, extents of protein modification, clearances, and excretion rates of glycation adducts in uremia.

Evaluation of Glyoxal and Methylglyoxal Levels in Uremic Patients under Peritoneal Dialysis

Advanced glycation end products (AGEs) accumulate in serum and tissues of patients with chronic renal failure, even in the absence of diabetes, and a different clearance of these species has been observed by hemodialysis and peritoneal dialysis (CAPD). Furthermore, it has been shown that not only AGE but also 1,2-dicarbonyl compounds are formed during heat sterilization of glucose-based peritoneal dialysis fluids. Therefore, we investigated the level of some AGEs (pentosidine and free pentosidine) and dicarbonyl compounds (glyoxal and methylglyoxal) in end-stage renal disease patients subjected to peritoneal dialysis. Samples (20 from healthy subjects, 16 from uremic patients before and after 12 h of peritoneal dialysis) were analyzed, and the plasma and dialysate levels of glyoxal, methylglyoxal, pentosidine, and free pentosidine were determined. In plasma of uremic patients, mean values of pentosidine showed a small decrease after dialysis and were always higher than those of healthy control subjects. An analogous trend was observed for free pentosidine. In the case of peritoneal dialysate, no pentosidine and free pentosidine were found at time zero, whereas both compounds were detected after 12 h of dialysis. Glyoxal and methylglyoxal mean levels showed a decrease in plasma after dialysis even if their values were always higher than those of healthy control subjects. Surprisingly, an analogous trend was observed also in dialysate. These results might indicate that glyoxal and methylglyoxal already present in the dialysis fluid react with the peritoneal matrix proteins, accounting for the gradual loss of peritoneal membrane function that is often observed in patients subjected to CAPD for a long time.

An Unusual Pyridine Nucleotide Accumulating in Erythrocytes: Its Identity and Positive Correlation with Degree of Renal Failure

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An Unusual Pyridine Nucleotide Accumulating in Erythrocytes: Its Identity, and Positive Correlation with Degree of Renal Failure

We have investigated an unusual nucleotide that accumulates, with precursors, in the erythrocytes of patients in uraemia. This nucleotide is related chemically to the NAD breakdown product, N1‐methyl‐2‐pyridone‐5‐carboxamide (Me2Py), found in high concentrations in the plasma of uraemic patients. Both Me2Py and the nucleotide accumulate to high concentrations in the blood during uraemia: our investigations of samples from renal out‐patients have provided information on a plausible link between the two compounds.

Hepatitis intensified oxidative stress, MIP-1β and RANTES plasma levels in uraemic patients

HBV and HCV infections are associated with the increased production of reactive oxygen species (ROS) within the liver that are responsible for the oxidation of intracellular molecules and activation transcription factors. The aim of the present study was to establish whether the presence of hepatitis could be implicated in the elevation of oxidative stress (SOX) and plasma proinflammatory and chemoattractant cytokine levels in uraemic patients. The markers of SOX—autoantibodies to oxidized LDL (OxLDL-Ab); total peroxides; and the major antioxidant enzyme Cu/Zn superoxide dismutase (Cu/Zn SOD); as well as tumor necrosis factor-α (TNF-α); regulated upon activation, normal T cell expressed and secreted (RANTES); and macrophage inflammatory protein-1α (MIP-1α) and β (MIP-1β) levels were measured in the plasma of uraemic patients with hepatitis in comparison to subjects without hepatitis and to healthy volunteers. The values of total peroxide, Cu/Zn SOD, TNF-α, and MIP-1β, were significantly elevated in uraemic patients when compared to the controls, whereas RANTES were decreased. MIP-1α and OxLDL-Ab were similar in the two groups. Cu/Zn SOD, MIP-1β and RANTES concentrations were significantly higher in the hepatitis-positive relative to the hepatitis-negative group. Both MIP-1β and RANTES were directly associated with Cu/Zn SOD levels and the presence of hepatitis. Multiple stepwise regression analysis has shown that the duration of dialysis, followed by the presence of hepatitis, independently and significantly predicted increased Cu/Zn SOD levels, whereas elevated Cu/Zn SOD as an independent variable was significantly associated with both increased both MIP-1β and RANTES in uraemic patients. These results suggest that the presence of viral hepatitis status and liver injury are novel determinants of increased oxidative stress, as well as of increased MIP-1β and RANTES levels in uraemic patients.

Increased level of advanced oxidation protein products in patients with coronary artery disease

Witko-Sarsat et al. previously reported that advanced oxidation protein products (AOPP), which were determined by measurements of optical density (OD) at 340 nm under acidic conditions, were present at high levels in the plasma of uremic patients. We measured AOPP in the plasma of 392 patients who underwent coronary angiography because of suspected coronary artery disease (CAD) and examined the association between plasma AOPP levels and CAD. We also collected plasma from hemodialysis patients and healthy volunteers for AOPP assay. Plasma fractionation by size-exclusion chromatography was performed to determine the weights of the molecules involved in the AOPP activity. The elution patterns obtained after plasma fractionation by size-exclusion chromatography were the similar in both CAD and hemodialysis patients, but differed from those of the healthy volunteers, whose AOPP levels were low. We also found that, the severity score of CAD obtained by the Gensini scoring system had good correlation with AOPP quartiles. Multivariable regression models revealed that the plasma AOPP level was significantly related to CAD status. This is the first report of an association between AOPP and CAD. AOPP was an independent risk factor for CAD. Oxidative stress is speculated to be associated with the pathogenesis of CAD.

Prodrug Metabolites: Implications for Therapeutic Drug Monitoring

Prodrugs are analogs of active drugs that have been developed to improve the bioavailability and/or tolerability of the latter. Fosphenytoin (1) and mycophenolate mofetil (MMF) (2) are two examples of prodrugs where monitoring of the active drug, the anticonvulsant phenytoin and the immunosuppressant mycophenolic acid (MPA), respectively, is used to guide therapy. The successful application of therapeutic drug monitoring to prodrugs requires a reliable analytical methodology, which in general should be specific for the active drug and should provide timely results so that the clinician can, if necessary, institute appropriate dosage adjustments. Although HPLC-based methodology is the most specific procedure for drug measurements, immunoassays are more rapid; they may, however, be subject to interference through cross-reactivity with structurally related compounds (e.g., prodrugs and drug metabolites). This problem often is aggravated in patients with liver dysfunction or renal insufficiency because of extensive accumulation of drug metabolites. The use of prodrugs has the following implications for therapeutic drug monitoring: In this issue of Clinical Chemistry , Annesley et al. (3) report on the isolation and characterization of a novel oxymethylglucuronide metabolite of fosphenytoin, a prodrug of phenytoin. This metabolite has been identified in the plasma of uremic patients …

Multimeric pattern of plasma and platelet von willebrand factor is normal in uremic patients

Several studies have reported increased von Willebrand factor (vWF) levels in plasma of uremic patients, with a normal multimeric pattern. Two recent studies, however, have shown a reduction of the larger multimers of plasma vWF and one has also found low levels of platelet vWF in uremic patients with prolonged bleeding time (BT). We have measured plasma and platelet vWF and analyzed its multimeric pattern in 20 uremic patients, 11 with a prolonged BT and 9 with a normal BT. For Plasma vWF, no difference for vWF:Ag, RiCof, and ristocetin-induced platelet agglutination was found between the two groups with normal and prolonged BT. The multimeric pattern of plasma vWF, as evaluated by densitometric scanning of the electrophoretic gels, was normal in both groups. For platelet vWF, vWF:Ag and RiCof content was similar in the two groups. The multimeric pattern was indistinguishable from that of normal platelets. In conclusion, our study does not confirm the presence of a structural defect of plasma vWF and the reduction of platelet vWF content in uremia.

Isolation of an ultrafilterable Ca(2+)-ATPase inhibitor from the plasma of uraemic patients.

1. Calcium concentration and Ca(2+)-ATPase activity under basal conditions and after maximal stimulation with calmodulin were measured in erythrocytes from 32 patients with end-stage renal failure on haemodialysis and from 27 healthy subjects. 2. In patients with renal failure the Ca2+ concentration in erythrocytes was elevated compared with healthy subjects (4.27 +/- 1.02 versus 2.86 +/- 0.57 mumol/l, P less than 0.05). 3. Basal Ca(2+)-ATPase activity was lower in the patients with renal failure than in healthy subjects (4.62 +/- 1.34 versus 5.43 +/- 1.23 pmol of phosphate min-1 10(-6) erythrocytes). After maximal stimulation, Ca(2+)-ATPase activity reached 6.93 +/- 2.81 pmol of phosphate min-1 10(-6) erythrocytes in the patients with renal failure, whereas in healthy subjects stimulation yielded a Ca(2+)-ATPase activity of 32.54 +/- 8.48 pmol of phosphate min-1 10(-6) erythrocytes. 4. Incubation of erythrocytes from healthy subjects with plasma from uraemic patients caused inhibition of Ca(2+)-ATPase. Likewise, the ultrafiltrate from plasma obtained by haemofiltration treatment inhibited Ca(2+)-ATPase. 5. Gel chromatography of the ultrafiltrate and laser desorption/ionization mass spectroscopy revealed that a fraction containing substances with a molecular mass of about 300 Da inhibited Ca(2+)-ATPase. 6. It is concluded that, in uraemia, a Ca(2+)-ATPase inhibitor accumulates in the plasma, and this could contribute to the toxicity of uraemia by inhibiting cellular Ca2+ transport in erythrocytes and possibly other tissues.

Amino acid metabolism in uremia.

The uremic syndrome is multifactorial, and affects most tissues and organs. Disturbances in protein and amino acid metabolism may play important roles, especially in chronic uremia, either directly or by production of toxic metabolites, with resultant negative nitrogen (N) balance, muscle wasting, reduced protein synthesis, and characteristically abnormal intracellular free amino acid concentrations. There are also grossly abnormal amino acid levels in the plasma of uremic patients, e.g., increases in conjugated amino acids, high levels of several nonessential and low levels of essential amino acids. The ratios of tyrosine/phenylalanine and of valine/glycine are decreased. The low tryptophan levels may contribute to encephalopathy as a result of an imbalance in neurotransmitter synthesis. Citrulline is found in excess; the explanation is unresolved. There are elevated concentrations of the sulfur-containing amino acids: cystine, taurine, cystathionine, and homocysteine. Excess of the latter is implicated in the atherogenesis of renal failure. Disturbed metabolism and interorgan exchange of amino acids in the uremic state explains some of the abnormalities in tissue and plasma concentrations of individual amino acids. Enzymatic defects are involved in the disturbed metabolism of branched chain amino acids (BCAA), with possible antagonism among them, which impairs growth and amino acid utilization. Carbohydrate intolerance, associated with insensitivity of peripheral tissues to insulin and hyperinsulinemia, elicits decreased plasma BCAA. Protein synthesis rates in normal and pathological conditions are more closely related to the intracellular amino acid pool than to plasma amino acid levels. Concentrations of individual amino acids in the plasma pool are poor indicators of their intracellular concentrations. Muscle contains the largest pool of protein and free amino acids in the body. In chronic renal failure patients, the intracellular concentrations of valine, threonine, lysine, and carnosine are low. With low protein diets and in hemodialysis, serine, tyrosine, and taurine often are also low. The low taurine may be related to fatigue and to uremic cardiomyopathies. The commonly used amino acid supplements generally fail to correct the intracellular amino acid deficits. A "New Formula" has been developed to correct these intracellular amino acid abnormalities, and to supplement a low protein diet. It provides more valine than leucine, increased tyrosine and threonine, and less histidine, leucine, isoleucine, lysine, methionine, and phenylalanine than in formulas customarily used for patients with chronic renal failure. It is uncertain whether other ap

Influence of Sodium Balance on Uremic Red Blood Cell Ion Transport

This study was performed to test the effect of dialysis-induced acute modifications of plasma volume and sodium pool on red blood cell (RBC) ion transport in patients with end-stage renal disease. This approach confirmed the presence of an Na-K pump inhibitor in the plasma of uremic patients with extracellular fluid volume expansion. This factor cross-reacts with digoxin antibodies, and its concentration decreases during dialysis; this explains the increased activity of the RBC Na-K pump that is consistently observed during dialysis.

Liquid-chromatographic determination of inosine, xanthine, and hypoxanthine in uremic patients receiving hemodialysis treatment.

We used a liquid-chromatographic method to measure xanthine, hypoxanthine, and inosine in plasma of uremic patients before and after hemodialysis. The concentrations of all three were higher than in normal subjects. After dialysis treatment, the values were significantly lower than before dialysis, but still above normal.

Determination of aspirin and salicylic acid in uremic patients' plasma using reversed-phase high-performance liquid chromatography.

A simple, rapid, and sensitive method for the determination of aspirin and salicylic acid in plasma of uremic patients is reported. After extraction with hexane, aspirin (ASA) and salicylic acid (SA) were quantified by high-performance liquid chromatography (HPLC) with ultraviolet detection at 229 nm. ASA and SA concentrations and peak-height ratios were linearly related up to 20 micrograms/ml. The lower limit of sensitivity was 0.1 microgram/ml for both compounds. The average recoveries of aspirin and salicylic acid were 27 and 54%, respectively. This procedure offers better performance than do previously described methods. The sample clean-up allows quantitation of a low concentration of aspirin in uremic patients' plasma, commonly rich in interfering endogenous substances.

Eco-biotoxicology of chemical pollutants.

The major toxicological problem today is to establish a useful method to cope with a low level and prolonged pollution. The toxicological method under such circumstances should include ecotoxicology and biotoxicology as well as classical toxicology, and is named as eco-biotoxicology. This method includes not only investigations on kinetics and levels of chemicals in the environment, human reaction and adaptation to chemicals or biophysiological and pathophysiological clarification of the effects, but also the establishment of countermeasures to prevent health hazards from chemicals, including biological monitoring and development of exposure parameters. The followings were the results recently obtained in our laboratory in support of the object of eco-biotoxicology. 1. Two low-molecular-weight chromium binding substances were purified from the liver of dogs. Chromium bound to them showed a lower toxicity and higher rates of urinary excretion than inorganic chromium compounds. A low-molecular-weight nickel binding substance was also purified from the liver of rats. Nickel bound to it showed a lower cytotoxity, but a higher malignant transformation induction of cultured cells than inorganic nickel compounds. These results indicate that chemical form of metals and binding protein in the body are important factors in their toxicity. 2. Triphenyl and tributyltins showed to inhibit insulin release from pancreas, collagen-induced aggregation of platelets, O2-production and chemotaxis of leukocytes, histamine release from mast cells, etc. A common mechanism seemed to be an inhibition of the stimulus-response coupling in the cell membrane. 3. Carcinogenic heterocyclic amines were measured in food and biological materials by a newly developed method. Their levels in plasma of uremic patients were higher than normal subjects and decreased by the treatment with hemodialysis. 4. Safety evaluation of bis (tri-n-butyltin) oxide (TBTO) in fishes was carried out according to a toxicological approach. Acceptable daily intake of TBTO in food was estimated to be 1.6 μg/kg/d.

Identification by nuclear magnetic resonance and mass spectrometry of a glucuronic acid conjugate of o-hydroxybenzoic acid in normal urine and uremic plasma.

An endogenous compound (included in the fraction of uremic toxins often called the "uremic middle molecules") was separated from plasma of uremic patients and urine from normal persons. As elucidated by mass spectrometry, enzymatic hydrolysis, and 1H, 13C nuclear magnetic resonance, it appears to be a conjugate of o-hydroxybenzoic acid with glucuronic acid. Its presence in urine of healthy subjects indicates its physiological character.

Improved separation and quantification of the "middle molecule" b4-2 in uremia.

The plasma of uremic patients usually contains high concentrations of the so-called middle molecules (molecular mass, 300 to 1500 Da), which exert various toxic effects. Among these numerous substances, only one, named peak b4-2, has been correlated with uremic neuropathy. We describe our improvement of a two-stage chromatographic method, gel permeation followed by anion-exchange chromatography (J. Chromatogr. 146: 55-65, 1978), for separation and quantification of b4-2 in body fluids. In analyzing more than 300 samples from 43 uremic patients and 12 healthy subjects, we found a linear correlation between peak area at 254 nm and b4-2 concentration in the range 0.8 to 32 mg/L. The coefficient of variation, including data acquired during seven changes of columns, was 9%. Analysis time (80 min) was shorter than required with other methods. Our results confirm previous data for urinary b4-2 excretion by healthy subjects and for b4-2 removal rate in uremic patients undergoing hemodialysis or hemofiltration. Patients treated with continuous ambulatory peritoneal dialysis have a higher b4-2 excretion than do healthy subjects, suggesting a higher production of this solute in uremic patients.

Inhibitor of insulin binding to erythrocytes in plasma of uremic patients.

Previously, we have shown that insulin binding (IB) to erythrocytes was decreased in uremic patients and that hemodialysis corrected the receptor defect. In the present study cross-incubation experiments were performed using uremic and control erythrocytes and plasma. Incubation of control erythrocytes with uremic plasma resulted in a 60% decrease in specific insulin binding, and progressive dilutions of uremic plasma revealed a parallel decrease in degree of inhibition suggesting the presence of an inhibitor of insulin binding in uremic plasma. Plasma obtained from uremic patients exhibiting lower IB to their erythrocytes was more potent in inhibition of IB to control erythrocytes, i.e. predialysis in comparison with postdialysis plasma. The alteration of IB was reversible since incubation of uremic erythrocytes with normal plasma restored IB towards normal values. Subjects having higher IB to their erythrocytes had more efficient plasma in restoring IB to uremic erythrocytes. The data indicate that alteration of insulin binding in uremia is mediated by dialysable plasma inhibitors. The possibility that humoral factors affecting binding of insulin to its receptors can in this way influence the sensitivity of peripheral tissues to insulin is considered.