Levels of circulating endothelial progenitor cells are related to uremic toxins and vascular injury in hemodialysis patients.

BACKGROUND: Patients suffering from chronic kidney diseases (CKD) exhibit cardiovascular diseases and profound endothelial dysfunction. CKD patients have reduced numbers of endothelial progenitor cells, but little is known about the factors influencing these numbers. OBJECTIVES: Among these factors, we hypothesized that uremic toxins and vascular injury affect endothelial progenitor cells. PATIENTS/METHODS: Thirty-eight hemodialysis patients were investigated and compared with 21 healthy controls. CD34+CD133+ immature progenitors, CD34+KDR+ endothelial progenitors cells (EPC) and myeloid EPC (mEPC) were counted in peripheral blood. Levels of uremic toxins beta(2)-microglobulin, indole-3 acetic acid, indoxylsulfate, p-cresylsulfate and homocysteine were measured. Vascular injury was assessed in hemodialysis (HD) patients by measuring aortic pulse wave velocity and plasma levels of endothelial microparticles. In vitro experiments were performed to study the effect of uremic toxins on apoptosis of progenitor cells. RESULTS AND CONCLUSIONS: CD34+CD133+ immature progenitor cell number was negatively correlated with the levels of uremic toxins beta(2)-microglobulin and indole-3 acetic acid. In vitro, indole-3 acetic acid induced apoptosis of CD133+ cells. These data indicate uremic toxins have a deleterious role on progenitor cells, early in the differentiation process. Moreover, mEPC number was positively correlated with markers of vascular injury-pulse wave velocity and endothelial microparticle levels. This suggests that vascular lesions could stimulate progenitor cell mobilization, even in a context of reduced EPC induced by CKD. In conclusion, uremic toxins and vascular injury appear to affect endothelial progenitor cell biology in CKD.

Antioxidant status of neonates exposed in utero to tobacco smoke.

To investigate the influence of maternal smoke exposure on neonatal and maternal antioxidant status, 39 mothers who were active smokers, 14 mothers exposed to environmental tobacco smoke (ETS), 17 controls, and their newborns were included in a prospective, controlled study. Plasma total antioxidant capacity, measured as total radical-trapping antioxidant parameter (TRAP) and ferric reducing antioxidant power (FRAP), and concentrations of specific antioxidants were measured in cord and in maternal blood. A similar, significant increase in ceruloplasmin concentration was observed in neonates born to actively smoking mothers and in those born to ETS exposed mothers. Uric acid and TRAP concentrations were significantly increased in ETS-exposed newborns and their mothers, compared to newborns and mothers from the active smoking and no-exposure groups with a trend towards increased uric acid, TRAP and FRAP concentrations being observed in the active smokers group. Neonatal and maternal antioxidant concentrations correlated significantly, except for ceruloplasmin. Cord blood vitamin A, E and C concentrations were unaffected by smoke exposure. These results show that maternal active smoking as well as ETS exposure significantly affect neonatal and maternal antioxidant status.

Sterol and fatty acid composition of Candida lusitaniae clinical isolates.

The sterol and fatty acid compositions of four amphotericin B-resistant and of two amphotericin B-susceptible Candida lusitaniae clinical isolates were determined. A flow cytofluorometric susceptibility test (FCST) with a membrane potential-sensitive cationic dye was used as a complement to the conventional method for selecting the isolates. Compared to susceptible isolates, resistant ones showed a greatly reduced ergosterol content and changes in sterol composition, consistent with a defect in Delta8-->7 isomerase. Within each group, no correlation between the sterol or fatty acid pattern or composition and both the degree of in vitro susceptibility and FCST MIC was found.

Automated strong cation exchange extraction of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol from oil samples for routine quantification by HPLC-APCI/MS/MS.

Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol are currently considered the best chemical markers of toxic oils related to the Spanish toxic oil syndrome. Recent research in this area has undertaken the exhaustive and quantitative characterization of these compounds in oils collected during the epidemic outbreak. Current methods developed in this laboratory are based on solid phase extraction (SPE) using SCX cartridges followed by HPLC-APCI/MS/MS quantification. To circumvent the long and tedious extraction procedure, the SPE protocol was adapted for automatic extraction and the problems derived from the use of the immiscible solvents required for the SCX extraction were solved. Linearity of the analytical method was found in the same range as for the manual method. Extraction recoveries were 87 and 75% for 2-hydroxy-3-(N-phenylamino)propyl linoleate and 2-(linoleyloxy)-3-(N-phenylamino)propyl linoleate, respectively, and the corresponding coefficients of variation were approximately 1%, greatly improving reproducibility over manual procedures.

Determination of aniline derivatives in oils related to the toxic oil syndrome by atmospheric pressure ionization-tandem mass spectrometry.

In 1981, an unknown disease appeared in Spain, the Spanish Toxic Oil Syndrome. Nowadays and despite all efforts, the etiological agent is still unknown. Early studies showed a link between this illness and the consumption of denatured rapeseed oil fraudulently processed and marketed as edible oil. Two families of aniline derivatives present in these oils (fatty acid anilides and acylated phenyl amino propanediol derivatives or PAPs) were found to be good chemical markers of toxic oils. In this work, a new method has been developed to analyze these aniline derivatives in oil samples by HPLC-MS and HPLC-MS/MS with an API source. For their quantification, three different internal standards were used, one for anilides and two for PAPs. Quantification limits were 8 ppm for anilides and 0.2 ppm for PAPs. Anilides and PAPs were found in marker-positive samples at levels up to 50,000 and 330 ppm, respectively. The relative abundance of the different fatty acid anilides and PAPs correlates with the fatty acid composition of the oils. More than 2,600 different samples were analyzed by this method in the most exhaustive screening of suspected toxic oils carried out to date.

Storage time and deodorization temperature influence the formation of aniline-derived compounds in denatured rapeseed oils.

In 1981 an epidemic, named Toxic Oil Syndrome, occurred in Spain as a result of ingestion of rapeseed oil denatured with 2% aniline, which had been imported for industrial use but was fraudulently diverted and processed for human consumption. Two groups of chemical compounds have been identified in the ingested toxic oil: fatty acid anilides and amino-propanediol derivatives. The objective of this work was to assess the effect of several refining process variables on the formation of 3-(N-phenylamino)-1,2-propanediol (PAP) esters. The amount of PAP esters in aniline-denatured oil increased dramatically when oil was heated from 250 degrees C to 300 degrees C. However, the ones formed when 300 degrees C was reached were lost during processing at that temperature. The level maintained during the operation time at 300 degrees C was higher in denatured samples stored for 3 weeks before refining than in denatured samples stored only for 1 week. Anilides were also analyzed. We found that anilides decreased very little with distillation time. In this paper we discuss the influence of storage time prior to refining and of elevated refining temperature, such as temperatures that might occur in close proximity to a deodorizer coil.

Absorption and effects of 3-(N-phenylamino)-1,2-propanediol esters in relation to toxic oil syndrome.

Toxic Oil Syndrome (TOS) was an epidemic disease related to the consumption of rapeseed oil denatured with aniline that made its sudden appearance in Spain in 1981. The fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP), which is a chemical class of by-products resulting from the reaction of aniline with oil components, have shown a strong association with TOS-related oils. These compounds also show some structural similarities to platelet-activating factor (PAF). In search of a toxic agent that could explain the widespread systemic effects observed in TOS patients, we investigated the intestinal absorption and biotransformation of the different PAP esters found in TOS-related oil samples and the possible pathophysiological effect of these mediators and their metabolic products if acting as PAF analogs. Results indicate that PAP esters are absorbed in the gastrointestinal tract and are distributed and stored in different organs, particularly in the liver and brown adipose tissue. PAP in these organs showed different patterns of fatty acids, indicating the ability of the gastrointestinal tract to modify the fatty acid composition of the parent PAP. Thus, the fatty acid profile of the PAP esters found in intestine appears to be related to the type of oil used as vehicle. Some of these PAP esters, when a long acyl chain was present in the sn-1 position of the molecule, showed an inhibitory effect on the PAF synthesis. This is an important observation in line with the systemic nature of the disease.

Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods.

We present a new analytical method for thiol quantification in plasma, based on the use of capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze 6-iodoacetamidofluorescein derivatives. Quantitative results of homocysteine, glutathione, cysteinylglycine, and cystationine are presented. A comparison of the quantitation of homocysteine in plasma, using high performance liquid chromatography/fluorescence detection and fluorescence polarization immunoassay is proposed. The results indicate that these techniques for plasma total homocysteine (tHcy) determination can be used interchangeably. The major advantage of CE-LIF is that it can quantitate the thiols in one run while keeping the price of consumables reasonable.

Quantitative electrospray LC-MS and LC-MS/MS in biomedicine.

The use of electrospray LC-MS and LC-MS/MS for the quantitative determination of two low molecular weight (< 500 Da) organic compounds in human plasma (Lovastatin) and cell supernatants (Arachidonic acid) and medium molecular weight (> 2000 Da) endogenous peptides (Endothelins) in supernatants of human umbilical vein endothelial cell cultures is reported. These methods make use either of deuterium labelled or structurally similar molecules as internal standards for quantitation and one or more pre-purification steps previous the LC-MS analysis. Linear calibration curves and detection limits around 50 pg ml(-1) were obtained in all three cases.

Quantitative analysis of mevinolinic acid in human plasma by high-performance liquid chromatography coupled with negative-ion electrospray tandem mass spectrometry.

Mevinolinic acid (MVA), the major active metabolite of Lovastatin in human blood, is analysed by microbore high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Quantification is carried out by using methylmevinolinic acid (MMVA) as internal standard. Positive- and negative-ion mass spectra of these compounds are shown. Because of the higher sensitivity obtainable, the negative-ion mode is selected for the analysis. Solid-phase extraction cartridges are used off-line to prepurify and concentrate the sample and a microbore (1 x 100 mm) reversed-phase column is used for chromatography. Tandem mass spectrometry is carried out in the precursor-ion mode using a selected-reaction monitoring procedure. The [M-H]- precursor ions for MVA and MMVA are selected in the first quadrupole analyser and the second analyser is focused on the common product ion at m/z 319. Detection and quantification limits are ca. 50 pg/mL and ca. 200 pg/mL respectively. An example of the application of the method to the routine analysis of samples from a bioequivalence study is also shown.

Rapid determination of desmosine and isodesmosine in tissue hydrolysates by isocratic high performance liquid chromatography and precolumn derivatization.

A rapid and sensitive isocratic high performance liquid chromatographic method has been developed for the single and specific determination of low concentrations of desmosine (Des) and isodesmosine (Ide), the major specific crosslink aminoacids in elastin.Samples of isolated elastin or whole tissue were hydrolysed in 6N HCl, and the hydrolysates were prefractionated on cellulose CF1. Des, Ide,γ-glutamyl-glutamic acid as internal standard were dansylated and derivatives were extracted from reaction mixture by ethylacetate. Their separation on a Lichrosphere 100-NH2 column, using methanol-water as mobile phase containing acetic acid and 0.25 M sodium acetate, final pH 6.5, was followed by fluorescence detection (340-510 nm). The overall reproducibility was 5.9% for Des and 5.0% for Ide. The limits of detection were 2.2 pmol and 2.5 pmol, respectively. The method was successfully applied for the determination of Des and Ide in normal pig aortas.

[Inhibitors of angiotensin-converting enzyme and processus of intima proliferation in atherosclerosis]. / Les inhibiteurs de l'enzyme de conversion de l'angiotensine et les processus de prolifération intimale dans l'athérosclérose.

The renin angiotensin system is a negative feed-back system of blood pressure control. A number of concordant experimental and clinical results indicate that the angiotensin family has a trophic effect on the vessel wall. These properties of the angiotensins favorise the proliferation of the cells which make up the vessel wall and also amplify the vascular dysfunction in the absence of the inhibitory regulations. Angiotensin converting enzyme inhibitors could be a valuable therapeutic method of counteracting these deleterious effects on the composition and function of the vessel wall.

Heart and liver membrane phospholipid homeostasis during acute administration of various antitumoral drugs to the rat.

The purpose of this study was to investigate in the rat heart and liver the effects of an acute administration of three anthracyclines, doxorubicin, epirubicin and pirarubicin, and an anthracenedione, mitoxantrone, on the membrane peroxidative status, which was estimated by the composition of polyunsaturated fatty acids (PUFA), and on the activities of the enzymes involved in membrane repair processes and lipid hydroperoxide detoxification. Rats were injected for four consecutive days with the drugs or saline (control) and killed 24 hr after the last injection. All the drugs induced an increase in plasma thiobarbituric reactive substances and alpha-tocopherol concentrations, both expressed per milligram of plasma lipids. Plasma vitamin A was decreased by about a factor of two by all the drugs. The fatty acid profile in the heart lipids showed that the polyunsaturated species (20:4 n-6, 22:6 n-3) remained at the same or even higher levels after anthracycline treatment. This can be explained by the fact that the activities of the enzymes involved in either the recycling of membrane phospholipids, such as phospholipases A1 and A2 (EC 3.1.1.4 and EC 3.1.1.32), lysophospholipases (EC 3.1.1.5) and acylCoA:lysophosphatidylcholine acyltransferases (EC 2.3.1.23), or hydroperoxide detoxification, such as selenium-dependent glutathione peroxidase (GSH-PX, EC 1.11.1.9) and glutathione S-transferases (GSH-T, EC 2.1.5.18), were maintained at the same level of activity after the antitumoral treatment. In liver, membrane phospholipid levels of PUFA were maintained as well as the activities of phospholipid-metabolizing enzymes. GSH-PX activity was not affected whereas that of GSH-T was slightly lowered by the drugs. These results suggest that during acute antitumoral-induced lipid peroxidation of membranes, the multi-enzymatic complex of the immediate processes of repair and detoxification is fully operational, allowing the membrane to rapidly recover its functional status. The results are discussed in the context of the equivocal relationships between antitumoral-induced lipid peroxidation and cardiac disturbances.

[The homocysteinemia vascular risk factor. Methodologies and application to a clinical case]. / L'homocystéinémie facteur de risque vasculaire. Méthode de mise en évidence, application à un cas clinique.

Early onset vascular disease unexplained until today by usual risk factors (hyperlipidemia, hypertension, tobacco, stress), can now find an explanation in sulfur amino acid metabolism defect. By transsulfuration, alimentary methionine leads to homocysteine, which is itself turn into cysteine, or remethylated into methionine. Several abnormalities of these different pathways lead to plasma accumulation of homocysteine, which will be responsible of arterial or venous occlusive lesions, concerning peripheral or deep vessels. Homocysteine stays in plasma upon several forms: 75% being linked by disulfide bounds to proteins, 22% as disulfide, homocystine (homocysteine-homocysteine) or mixed-disulfide (homocysteine-cysteine), and less than 3% as free reduced homocysteine. Plasma reduction allows total homocysteine evaluation with amino acid autoanalyzer. The basal plasma homocysteine level is less than 14 microMl. However, levels near this basal value can be found in patients with latent abnormality, which needs to be revealed by a methionine loading test. This study concerns two methodologies and their application to the exploration of a patient with unidentified neurologic disorders. The first one describes a new galenic oral form of methionine. Other authors use the methionine load of 100 mg/kg dissolving it in a fruit juice glass. In order to obtain a complete dissolution of this weakly soluble substance and to ensure its total absorbtion by the patient, we prepare a granular form aimed to give in water a perfect flavoured suspension. The second methodology concerns methionine loading test and amino acid analysis. After 10 hours fasting, a 100 mg/kg peroral methionine load is realized performing 5 EDTA blood samples before and 4, 8, 12 and 24 hours after loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Inverse modifications of heart and liver alpha-tocopherol status by various dietary n-6/n-3 polyunsaturated fatty acid ratios.

The effect of dietary n-6/n-3 fatty acid ratio on alpha-tocopherol homeostasis was investigated in rats. Animals were fed diets containing fat (17% w/w) in which the n-6/n-3 ratio varied from 50 to 0.8. This was achieved by combining corn oil, fish oil, and lard. The polyunsaturated to saturated ratio and total alpha-tocopherol remained constant in all diets. Results showed that enrichment of n-3 polyunsaturated fatty acids in the diet, even at a low amount (3.9% w/w), resulted in a dramatic reduction of blood alpha-tocopherol concentration, which, in fact, is the result of a decrease in plasma lipids, since the alpha-tocopherol to total lipids ratio was not significantly altered. The most striking effect observed was a considerable alpha-tocopherol enrichment (x 4) of the heart as its membranes became enriched with n-3 polyunsaturated fatty acids. This process appeared even with a low amount of fish oil (3.9% w/w) added to the diet. Accordingly, a strong positive correlation was found between heart alpha-tocopherol and docosahexaenoic acid (r = 0.86) or docosahexaenoic acid plus eicosapentaenoic acid levels (r = 0.84). Conversely, the liver alpha-tocopherol level dropped dramatically when n-3 polyunsaturated fatty acids were gradually added to the diet. It is concluded that fish oil intake dramatically alters the alpha-tocopherol homeostasis in rats.

Effect of increasing amounts of dietary fish oil on brain and liver fatty composition.

Increasing dietary fish oil in rat had the following effect on brain lipids: Arachidonic acid regularly decreased; eicosapentanenoic acid, normally nearly undetectable, was present; 22:5(n - 3), dramatically increased but remained below 1% of total fatty acids; cervonic acid was increased by 30% at high fish oil concentration. Saturated and monounsaturated fatty acids were not affected regardless of chain-length. In contrast, in the liver, nearly all fatty acids (saturated, monounsaturated and polyunsaturated) were affected by high dietary content of fish oil, but liver function was normal: serum vitamin A and E, glutathione peroxidase, alkaline phosphatase, transaminases were not affected. Serum total cholesterol, unesterified cholesterol and phosphatidylcholine were slightly affected. In contrast, triacylglycerols were dramatically reduced in proportion to the fish oil content of the diet.

Vitamin A, vitamin E, retinol binding protein (RBP), and prealbumin in digestive cancers.

The existence of a relation between vitamin A and vitamin E and human cancers is supported by epidemiologic investigations. The aim of this study is to link the level of these vitamins to those of plasmatic protein carriers like retinol binding protein (RBP) and prealbumin (TTR), in three groups of subjects: healthy patients (n = 78), polyp (n = 34) and digestive cancer patients (n = 70). A paired t-test did not reveal any significant variation in any parameter between the polyp group and controls, but did evidence a significant decrease in serum levels of retinol (p less than 2.10(-4], RBP (p less than 2.10(-4), TTR (p less than 10(-5), and alpha-tocopherol (p less than 2.10(-3), in cancer cases as against control subjects. Comparison of RBP renal clearance and retinol tissue clearance in cancer and healthy patients indicates that the decrease in circulating retinol levels cannot be attributed to an increase in peripheral consumption. The simultaneous reduction of RBP and TTR serum levels is to be considered as a sign of protein denutrition. Thus our results suggest that the decrease serum levels of vitamins A and E observed in digestive cancers are a consequence of this nutritional deficiency.

Effect of dietary salmon oil feeding on rat heart lipid status.

For 2 mo rats were fed a salmon oil diet (12.5%, wt/wt) supplemented with 4.5% (wt/wt) corn oil, a corn oil diet (17%, wt/wt) or a low fat diet (4.4%, wt/wt). Cardiac lipids were analyzed and fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined. Ventricular biopsies were taken for ultramicroscopic examination. Serum cholesterol, triglyceride, phospholipid and vitamin E concentrations were significantly lower in rats fed salmon oil than in those fed the other two diets, whereas serum transaminases and vitamin A were not significantly affected. Cardiac protein, phospholipid, triglyceride and cholesterol concentrations were unaffected by diet. Cardiac phospholipid composition remained unchanged and no significant changes in lyso-PC or lyso-PE levels were observed. However, the salmon oil diet produced a markedly lower n-6/n-3 ratio in both PE and PC than in the other two diets. This was the result of replacement of n-6 polyunsaturated fatty acids (PUFA), primarily 20:4n-6 with n-3 PUFA, primarily 22:6n-3. The unsaturation index of PC and PE was higher with the salmon oil diet than with the other two diets. Ventricular biopsies of rats fed salmon oil showed mild lipid accumulation associated with some lipofuscin-like material. It is suggested that, in rat heart, fish oil led to a moderate accumulation of lipids, the composition of which may include long-chain monounsaturated fatty acids and a degradative form of peroxidized lipids.

Prealbumin-retinol-binding-protein-retinol complex in hemodialysis patients.

In hemodialysis (HD) patients, serum prealbumin (TBPA) is correlated to nutritional status and outcome despite usually elevated serum levels. The purpose of this work was to study the role of TBPA-retinol-binding-protein (RBP)-retinol complex changes in the elevation of serum TBPA in HD patients. Serum TBPA, RBP, and retinol were measured in 30 otherwise healthy HD patients (15 men, 15 women) and in 30 healthy volunteers (15 men, 15 women). The dependence of TBPA on RBP was studied by covariance and regression methods. TBPA (p less than 0.05), RBP (p less than 0.01), and retinol (p less than 0.05) were elevated in HD patients. Elevated TBPA was associated with a decrease of TBPA free from RBP (p less than 0.01). The decrease of free TBPA may explain the reduction of TBPA breakdown and its elevation in HD patients.