Urinary excretion of homocysteine thiolactone and the risk of acute myocardial infarction in coronary artery disease patients: the WENBIT trial.

OBJECTIVES: No individual homocysteine (Hcy) metabolite has been studied as a risk marker for coronary artery disease (CAD). Our objective was to examine Hcy-thiolactone, a chemically reactive metabolite generated by methionyl-tRNA synthetase and cleared by the kidney, as a risk predictor of incident acute myocardial infarction (AMI) in the Western Norway B-Vitamin Intervention Trial. DESIGN: Single centre, prospective double-blind clinical intervention study, randomized in a 2 × 2 factorial design. SUBJECTS AND METHODS: Patients with suspected CAD (n = 2049, 69.8% men; 61.2-year-old) were randomized to groups receiving daily (i) folic acid (0.8 mg)/vitamin B12 (0.4 mg)/vitamin B6 (40 mg); (ii) folic acid/vitamin B12 ; (iii) vitamin B6 or (iv) placebo. Urinary Hcy-thiolactone was quantified at baseline, 12 and 38 months. RESULTS: Baseline urinary Hcy-thiolactone/creatinine was significantly associated with plasma tHcy, ApoA1, glomerular filtration rate, potassium and pyridoxal 5'-phosphate (positively) and with age, hypertension, smoking, urinary creatinine, plasma bilirubin and kynurenine (negatively). During median 4.7-years, 183 patients (8.9%) suffered an AMI. In Cox regression analysis, Hcy-thiolactone/creatinine was associated with AMI risk (hazard ratio = 1.58, 95% confidence interval = 1.10-2.26, P = 0.012 for trend; adjusted for age, gender, tHcy). This association was confined to patients with pyridoxic acid below median (adjusted HR = 2.72, 95% CI = 1.47-5.03, P = 0.0001; Pinteraction = 0.020). B-vitamin/folate treatments did not affect Hcy-thiolactone/creatinine and its AMI risk association. CONCLUSIONS: Hcy-thiolactone/creatinine ratio is a novel AMI risk predictor in patients with suspected CAD, independent of traditional risk factors and tHcy, but modified by vitamin B6 catabolism. These findings lend a support to the hypothesis that Hcy-thiolactone is mechanistically involved in cardiovascular disease.

Aldosterone modulates blood homocysteine and cholesterol in coronary artery disease patients - a possible impact on atherothrombosis?

Aldosterone plays a key role in maintaining the homeostasis of the whole organism. Under some circumstances, aldosterone can contribute to the progression of cardiovascular diseases, including coronary artery disease. This study demonstrates that aldosterone associates negatively with some lipidogram parameters and positively with the concentration of homocysteine. These associations are characteristic for coronary artery disease and are not present in control subjects. The findings also indicate that in vitro aldosterone stimulates homocysteine production by rat adrenal glands, which may explain the associations observed with coronary artery disease. Moreover, we have found that aldosterone significantly modulates in vitro platelet reactivity to arachidonate and collagen - aldosterone increases the pro-aggregatory action of collagen, but decreases the pro-aggregatory potential of arachidonate. Therefore, the findings of these in vitro and ex vivo experiments indicate the existence of new pathways by which aldosterone modulates lipid- homocysteine- and platelet-dependent atherogenesis.

Nɛ-homocysteinyl-lysine isopeptide is associated with progression of peripheral artery disease in patients treated with folic acid.

OBJECTIVE: Folic acid (FA) administration can reduce plasma total homocysteine (tHcy); however, it fails to decrease cardiovascular events and progression of peripheral artery disease (PAD). Nɛ-homocysteinyl-lysine isopeptide (Nɛ-Hcy-Lys) is formed during catabolism of homocysteinylated proteins. We sought to investigate factors that determine the presence of Nɛ-Hcy-Lys in PAD patients with hyperhomocysteinemia receiving FA. PATIENTS AND METHODS: We studied 131 consecutive PAD patients with tHcy > 15 µmol l(-1) taking FA 0.4 mg d(-1) for 12 months. Serum Nɛ-Hcy-Lys was determined by high-performance liquid chromatography (HPLC). We also measured interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), asymmetric dimethylarginine (ADMA) and 8-iso-prostaglandin F(2α) (8-iso-PGF(2α)). RESULTS: FA administration resulted in a 70.5% decrease in tHcy (p < 0.0001). However, serum Nɛ-Hcy-Lys was detectable in 28 (21.4%) patients on FA who were more frequently current smokers and survivors of ischaemic stroke (p < 0.001). They had higher tHcy by 46.0%, PAI-1 by 51.7%, 8-iso-PGF(2α) by 59.1% and ADMA by 26.4% (all, p < 0.0001). The presence of Nɛ-Hcy-Lys was associated with lower ankle-brachial index (ABI) values (p < 0.001) and higher prevalence of cardiovascular events (p < 0.001) following therapy. CONCLUSION: The presence of Nɛ-Hcy-Lys in one-fifth of hyperhomocysteinemic individuals with PAD despite FA treatment is associated with progression of PAD and with increased ADMA formation, oxidative stress and hypofibrinolysis.

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis.

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.

Novel selenoorganic compounds as modulators of oxidative stress in blood platelets.

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors, or drugs. The effects of five new synthesized selenoorganic compounds (2-(5-chloro-2-pyridyl)-7-azabenzisoselenazol-3(2H)-one; 2-phenyl-7-azabenzisoselenazol-3(2H)-one; 2-(pyridyl)-7-azabenzisoselenazol-3(2H)-one; 7-azabenzisoselenazol-3(2H)-one; bis(2-aminophenyl) diselenide) on oxidative changes in human blood platelets and in plasma were studied in vitro and compared with those of ebselen, a well known antioxidant. Our studies demonstrated that bis(2-aminophenyl) diselenide has distinctly protective effects against oxidative stress in blood platelets and in plasma. It might have greater biological relevance and stronger pharmacological effects than ebselen.

Analysis of urine for cysteine, cysteinylglycine, and homocysteine by high-performance liquid chromatography.

A new analytical method is proposed for simultaneous determination, by liquid chromatography, of the three main urinary thiols-cysteine, cysteinylglycine, and homocysteine. To measure the total amount of these thiols urine is reduced with sodium borohydride, to convert disulfides to thiols which are then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium thiol derivatives formed were achieved by high-performance liquid chromatography with detection at 355 nm. Validation showed the method enabled reliable simultaneous determination of these aminothiols in urine. The calibration graphs for each analyte, obtained by use of normal urine spiked with increasing amounts of cysteine, cysteinylglycine, and homocysteine, were linear (R2 > or = 0.997) over the range covering most practical situations. The recovery of the assay was 98-100% and sensitivity was 0.12-0.25 micromol L(-1). The method was applied to 91 different samples of normal urine to establish reference values for the aminothiols, normalized on creatinine.

Analysis of saliva for glutathione and metabolically related thiols by liquid chromatography with ultraviolet detection.

A method for simultaneous determination of glutathione and its precursors cysteine, cysteinylglycine and homocysteine in saliva is presented. The procedure involves reductive conversion of disulfides to thiols, derivatization to their 2-S-quinolinium derivatives with 2-chloro-1-methylquinolinium tetrafluoroborate and separation and quantitation by reversed-phase ion-pairing high performance liquid chromatography with ultraviolet detection at 355 nm. The calibration performed with saliva samples spiked with thiol disulfides, within the practical concentration ranges, showed linear response of the detector. The method applied to the saliva samples donated by volunteers showed mean concentration (SD, n = 8) of cysteine, cysteinylglycine, glutathione and homocysteine: 26.5 (31.6), 6.05 (5.12), 16.97 (7.68), 3.64 (1.34) nmol/ml respectively.

The protective effects of resveratrol against changes in blood platelet thiols induced by platinum compounds.

Cisplatin (cis-diamminedichloroplatinum II, cisPt) is especially useful in the treatment of epithelial malignancies, however, the use of cisplatin is accompanied by several toxicities including haematological toxicity. Contrary to cisplatin, selenium-cisplatin conjugate ((NH(3))(2)Pt(SeO(3)); Se-Pt) has only a slight toxicity effect on blood platelet function. In the mechanism of platinum compounds action on platelets thiols are involved. The aim of the present studies was to examine in vitro how trans-resveratrol (trans-3,4',5-trihydroxystilbene) acts on the levels of platelet glutathione (GSH) and other thiol-containing compounds and how, as an antioxidant, protecs blood platelets against the oxidative stress caused by platinum compounds (cisPt and Se-Pt). To analyse the level of thiols in human blood platelets treated with platinum compounds and with resveratrol the classical technique HPLC has been used. Blood platelets isolated by differential centrifugation of human blood were incubated (30 min, 37 degrees C) with cisPt or Se-Pt at dose of 10 microg/ml that inhibits platelet function and with resveratrol (25 microg/ml). The obtained results indicate that platinum compounds caused in platelets a decrease of both, reduced glutathione (GSH) and free thiols of cysteine (CSH) and cysteinylglycine (CGSH). The pool of these compounds in unreduced form was increased. Platinum compounds caused the reduction of platelet protein thiols. Resveratrol (after 30 min action) at the concentration of 25 microg/ml partly reduced the platinum compounds induced decrease of platelet thiols, particularly thiols in acid-soluble fraction.

Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection.

The procedure for measurement of different forms of four plasma thiols cysteine, cysteinylglycine, glutathione and homocysteine is proposed. The analytes are derivatized with thiol-specific ultraviolet labeling reagent, 2-chloro-1-methylquinolinium tetrafluoroborate, and separated from each other, reagent excess and plasma matrix constituents by reversed-phase high performance liquid chromatography with detection at 355 nm. Oxidized forms are converted to their thiol counterparts by reductive cleavage with sodium borohydride prior to derivatization step. In order to circumvent the loss of reduced fraction of thiols due to oxidation during sample preparation, the derivatization reagent is added to whole blood immediately after collection and before separation of plasma from erythrocytes. The method measures total thiols, total free thiols, protein-bound thiols and reduced thiols. The method is linear within the physiological and pathological ranges of thiols and is applied for plasma samples donated by apparently healthy volunteers.

Quantitative determination of flavonoids in the flowers and leaves of Prunus spinosa L.

The content of flavonoids in the flowers and leaves of Prunus spinosa L. was determined by spectrophotometric and RP-HPLC method. Determinations included hydrolysis of flavonoid glycosides in extracts from raw materials and then quantitative analysis of the obtained aglycones. Results were calculated for the content of glycosides and statistical analysis of the obtained data was performed.

Facile and sensitive method for the determination of mesna in plasma by high-performance liquid chromatography with ultraviolet detection.

The use of 2-chloro-1-methylquinolinium tetrafluoroborate, an ultraviolet tagging reagent, for the ion-pair, reversed-phase high-performance liquid chromatography of mesna in human plasma is reported. In order to achieve this objective optimization of the two-step procedure, derivatization and separation of mesna S-quinolinium derivative from that of other thiols present in plasma and internal standard, was investigated. The derivatization was optimized in terms of pH, reagent excess and time of the reaction, and the mobile phase in terms of ion-pairing reagent concentration, pH, organic modifier content and temperature. Baseline separation was achieved on an analytical Waters Nova-Pak C18 (150x3.9 mm, 5 microm) column with a mobile phase consisting of pH 2.3 0.05 M trichloroacetic acid-acetonitrile (89:11, v/v) pumped at 1.2 ml/min. The peak height ratios of the mesna derivative to that of the internal standard (thiomalic acid) varied linearly with the concentration of the analyte added to normal plasma with a correlation coefficient of 0.9997. The lower limits of detection and quantitation were 40 pmol/ml (0.8 pmol on-column) and 160 pmol/ml (3.2 pmol on-column), respectively. The intra-run imprecision and inaccuracy were from 1.3 to 2.4 and from 1.3 to 2.0%, respectively.

Determination by liquid chromatography of free and total cysteine in human urine in the form of its S-quinolinium derivative.

A reversed-phase high-performance liquid chromatographic method for the determination of free and total cysteine in urine is described. The method involves reductive conversion of cysteine dimer and cysteine mixed disulphides to their reduced counterpart with the use of tri-n-butylphosphine, ultraviolet-labeling with 2-chloro-1-methylquinolinium tetrafluoroborate, and liquid chromatographic separation with isocratic conditions. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step, and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. The method provides quantitative information on free and total cysteine based on assays with derivatization before and after reduction with tri-n-butylphosphine. The calibration graph, obtained with the use of normal urine spiked with growing amounts of cystine, was linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol detection and quantitation limits, low imprecision and high recovery. The method was validated for urine samples received from several donors. Cystine was chosen as a primary calibrator for these assays.

Liquid chromatographic assessment of total and protein-bound homocysteine in human plasma.

Homocysteine present in human blood plasma is derivatized with thiol selective ultraviolet labelling reagent, 2-chloro-1-methylpyridinium iodide, and separated from other plasma thiol derivatives by high-performance liquid chromatography (HPLC) with detection at 312 nm. The separation is carried out isocratically on LiChrospher RP-18 column using mobile phase consisting of pH 2.5 0.04 M trichloroacetic acid buffer and methanol in the ratio 9:1 (v/v) pumped at 0.5 ml min(-1) at 40 degrees C. The homocysteine S-pyridinium derivative elutes at 6.5 min. To determine total and protein-bound homocysteine it is necessary to cleave disulphide bounds by the use of tri-n-butylphosphine in order to form free sulfhydryl group. The method provides quantitative information on total and protein-bound homocysteine based on assays with derivatization after reduction of whole plasma, and derivatization after reduction of acid precipitated proteins. The calibration graph is linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol sensitivity and is reproducible; intra- and inter-day, relative standard deviation range from 1.79 to 5.09% and from 2.80 to 5.60%, respectively. The method is applied to the determination of total and protein-bound homocysteine in the plasma of healthy individuals.

Urinary excretion measurement of cysteine and homocysteine in the form of their S-pyridinium derivatives by high-performance liquid chromatography with ultraviolet detection.

Several human diseases, in particular metabolic disorders, often lead to the accumulation of characteristic metabolites in plasma, urine and cells. The selected diseases of this type include cystinuria and homocystinuria. In the typical laboratory diagnosis of these two diseases, a positive nitroprusside test is followed by quantitative analysis of urine cysteine and homocysteine in order to differentiate between cystinuria and homocystinuria. A sensitive and reproducible assay for total urine cysteine and homocysteine has been developed. The essential steps in the assay include conversion of disulphides to free thiols with tributylphosphine, conjugation of the thiols with 2-chloro-1-methyl pyridinium iodide, separation of S-pyridinium derivatives of cysteine and homocysteine from other endogenous urine thiol derivatives by reversed-phase high-performance liquid chromatography, and detection and quantitation by spectrophotometry. The method has a sensitivity of 4 pmol and is reproducible, intra- and inter-day coefficients of variation are from 1.37 to 4.14% and from 2.38 to 5.01%, respectively. The mean concentration of total urine cysteine and homocysteine in healthy donors (7 men and 7 women) were for women. 92.0 +/- 45.8 and 16.4 +/- 4.8 respectively, and for men 120.9 +/- 46.6 and 21.5 +/- 7.4 nmol/ml, respectively. Total urine homocysteine represents approximately 17.7% of cysteine in the urine of normal individuals.

Augmentation of oral tissues in rabbit using autogenous fat.

Patients with mild velopharyngeal incompetence (VPI) may have speech disorders, which are not sufficiently severe to warrant extensive surgical intervention, yet may not be amenable to correction by speech therapy alone. Augmentation of the posterior pharyngeal wall to aid in closure of the velopharyngeal sphincter may be beneficial in establishing better speech patterns, especially when combined with speech therapy. A variety of materials and techniques have been used in the past for this purpose. In this setting, autogenous fat may be transplanted without the risks incurred by augmentation with synthetic materials and involves very little donor site morbidity. The literature is somewhat contradictory, however, regarding the stability of the augmentation achieved using autogenous fat and there are no histologic studies describing the fate of fat injected into tissues of the oral cavity. Prior to introduction of this technique into clinical practice, this study was designed to investigate the fate of autogenous fat injected submucosally in the oropharyngeal region. Autogenous fat was injected into the anterior soft palate using the rabbit as a model. Histologic and gross inspections were performed at 2 days, 1, 2, and 4 weeks after injections. At the end of 4 weeks, at least 50% of the injection sites had visible evidence of augmentation, and 90% had histologic evidence of submucosal fat. In some instances most of the fat was resorbed; however, there were no instances of clinical infection or necrosis of the injection site. We conclude that submucosal injection of autogenous fat is a feasible alternative to using synthetic or other biologic materials for augmentation in the oral cavity.

Complication outcomes based on preoperative admission and length of stay for primary palatoplasty and cleft lip/palate revision in children aged 1 to 6 years.

With increasing focus on outcome studies, there is continued need for data about whether same-day admission and reduced hospital stay have adverse effects on surgical treatment, including that for cleft lip and palate. In this study, medical records were inspected for all cleft lip and palate patients, aged 1 to 6 years, who had primary palatoplasty or cleft lip/palate revision in this treatment center between 1978 and 1992 (N = 329). Length of stay for 251 (96.5%) of the 260 subjects admitted the day before surgery was from 4 to 7 days; 9 remained in the hospital longer than 8 days. Length of stay for 67 (97.1%) of 69 patients admitted the day of surgery was from 2 to 3 days; 2 were in the hospital for 7 days, and none for 8 or more days. Thirty-seven instances of surgical complications were reported for the 260 patients admitted the day before surgery (14.2%). Twelve complications (17.4%) were recorded for the 69 patients admitted the day of surgery. There was no significant difference in the number of complications between the two groups of patients (Fisher's exact test, p = 0.5682). There was no significant difference in the types of complications observed between the two groups (Fisher's exact test). Surgery was performed at age 1 year for 61 of the 69 patients admitted on the day of surgery (88.4%). The mean age of this group was significantly younger than that of patients operated on earlier than 1989 and admitted on the day before surgery (Wilcoxon's test, p = .0001, Z = 4.48).(ABSTRACT TRUNCATED AT 250 WORDS)