PubMed Health⌕ Search

Biomedical subjects

Dimitrios Tsikas

Publications and source records attributed to Dimitrios Tsikas.

At least 19 recordsLinked to original sources
PubMed2006

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease.

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.

Acetazolamide↗
PubMed2006

Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the L-arginine/nitric oxide area of research.

In the Griess reaction, first reported by Johann Peter Griess in 1879 as a method of analysis of nitrite (NO(2)(-)), nitrite reacts under acidic conditions with sulfanilic acid (HO(3)SC(6)H(4)NH(2)) to form a diazonium cation (HO(3)SC(6)H(4)-N[triple bond]N(+)) which subsequently couples to the aromatic amine 1-naphthylamine (C(10)H(7)NH(2)) to produce a red-violet coloured (lambda(max) approximately 540 nm), water-soluble azo dye (HO(3)SC(6)H(4)-NN-C(10)H(6)NH(2)). The identification of nitrite in saliva has been the first analytical application of this diazotization reaction in 1879. For a century, the Griess reaction has been exclusively used to identify analytically bacterial infection in the urogenital tract, i.e. to identify nitrite produced by bacterial reduction of nitrate (NO(3)(-)), the major nitrogen oxide anion in human urine. Since the discovery of the l-arginine/nitric oxide (l-Arg/NO) pathway in 1987, however, the Griess reaction is the most frequently used analytical approach to quantitate the major metabolites of NO, i.e. nitrite and nitrate, in a variety of biological fluids, notably blood and urine. The Griess reaction is specific for nitrite. Analysis of nitrate by this reaction requires chemical or enzymatic reduction of nitrate to nitrite prior to the diazotization reaction. The simplicity of the Griess reaction and its easy and inexpensive analytical feasibility has attracted the attention of scientists from wide a spectrum of disciplines dedicated to the complex and challenging L-Arg/NO pathway. Today, we know dozens of assays based on the Griess reaction. In principle, every laboratory in this area uses its own Griess assay. The simplest Griess assay is performed in batch commonly as originally reported by Griess. Because of the recognition of numerous interferences in the analysis of nitrite and nitrate in biological fluids and of the desire to analyze these anions simultaneously, the Griess reaction has been repeatedly modified and automated. In recent years, the Griess reaction has been coupled to HPLC, i.e. is used for post-column derivatization of chromatographically separated nitrite and nitrate. Such a HPLC-Griess system is even commercially available. The present article gives an overview of the currently available assays of nitrite and nitrate in biological fluids based on the Griess reaction. Special emphasis is given to human plasma and urine, to quantitative aspects, as well as to particular analytical and pre-analytical factors and problems that may be associated with and affect the quantitative analysis of nitrite and nitrate in these matrices by assays based on the Griess reaction. The significance of the Griess reaction in the L-Arg/NO pathway is appraised.

Animals↗
PubMed2006

Transcoronary delivery of bone marrow cells to the infarcted murine myocardium: feasibility, cellular kinetics, and improvement in cardiac function.

Efficient strategies for labelling and delivery of bone marrow derived stem cells (BMCs) are required to elucidate the cellular kinetics and therapeutic effects after BMC transfer for myocardial infarction (MI). Lineage negative (lin-) BMCs, labelled ex vivo in a simple procedure with the cell tracker dye tetramethyl-rhodamine (TAMRA), were reliably detected by fluorescence microscopy with higher specificity than retroviral enhanced green fluorescence protein (EGFP) marking and detection. Only few cells entered the ischemic myocardium after intravenous (i.v.) application, but this number increased more than 18-fold after transcoronary delivery. Time course and kinetic analysis over 12 h revealed that myocardial colonization seems to be a biphasic process of first order decay with different elimination half-lives. Most cells are eliminated rapidly during the first 2 h (t1/2 40 min), but the remaining cells are retained significantly longer in the ischemic heart (t1/2 5.2 h). In contrast, BMC colonization of the spleen increased rather in a linear fashion. Although transcoronary BMC transfusion did not alter infarct size, it increased capillary density in the infarct border zone and improved LV function 4 weeks after MI. In conclusion, BMCs delivered by transcoronary injection increase capillary density and improve LV function after MI although homing to the ischemic heart is only transient.

Animals↗
PubMed2006

Bone marrow molecular alterations after myocardial infarction: Impact on endothelial progenitor cells.

OBJECTIVE: Standard drugs post-myocardial infarction (MI) such as angiotensin converting enzyme (ACE) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) increase levels of endothelial progenitor cells (EPC). However, potential underlying mechanisms have not yet been investigated. METHODS AND RESULTS: We studied the effects of ACE inhibition or statin treatment on EPC levels and on bone marrow molecular pathways involved in EPC mobilization after MI in rats. Three days post-infarction, acetylated LDL (acLDL)+/Ulex europeus-1 (UEA-1)+/VEGF receptor-2+/eNOS+ EPC levels and formation of endothelial colony forming units (CFU) were reduced to 60+/-12% (p < 0.05) and 68+/-7% (p < 0.05). In bone marrow, extracellular signal-regulated kinase (ERK) phosphorylation and matrix metalloproteinase (MMP)-9 activity were repressed. Endothelial nitric oxide synthase (eNOS) activity was unchanged, whereas reactive oxygen species (ROS) were increased two-fold in bone marrow. ACE or HMG-CoA reductase inhibition resulted in significant increases in EPC levels. ACE inhibition increased bone marrow ERK phosphorylation and MMP-9 activity. Statin therapy enhanced bone marrow VEGF protein levels, Akt phosphorylation, eNOS activity and normalized increased ROS levels. Augmented EPC levels in the early post-infarction phase by ACE inhibition or statin treatment were associated with improved cardiac function and increased capillary density in the peri-infarct area 7 days after MI. Moreover, increased EPC levels in response to ACE inhibition or statin treatment were sustained 10 weeks post-infarction. CONCLUSIONS: Increased ROS and impaired MMP-9 activity in bone marrow likely contribute to reduced EPC mobilization in the early post-infarction phase. ACE inhibition or statin treatment increased EPC levels with distinct drug-specific effects on bone marrow molecular alterations.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2006

Elevated plasma concentrations of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine in citrullinemia.

Citrullinemia is an inborn error of the urea cycle with deficiency of the argininosuccinate synthase. It is characterized by elevated concentrations of l-citrulline and decreased levels of l-arginine in body fluids. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthase that converts l-arginine to l-citrulline and nitric oxide (NO). Asymmetric dimethylarginine is hydrolyzed by the enzyme dimethylarginine dimethylaminohydrolase to l-citrulline and dimethylamine. Elevation of l-citrulline in citrullinemia prompted us to study the l-arginine/NO pathway in this disorder. In 8 children with citrullinemia (3 days to 3 years of age), elevated plasma levels of asymmetric dimethylarginine (P = .028) were found compared with age-matched healthy children. We hypothesize that the l-arginine/NO pathway plays a role in the pathophysiology of citrullinemia.

Arginine↗
PubMed2005

Suppression of endothelial progenitor cells in human coronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine.

OBJECTIVES: We tested the hypothesis that asymmetric dimethylarginine (ADMA) may be an endogenous inhibitor of endothelial progenitor cells (EPCs). BACKGROUND: Endothelial progenitor cells play a pivotal role in regeneration of injured endothelium, thereby limiting the formation of atherosclerotic lesions. Reduced numbers of EPCs may affect progression of coronary artery disease. Regulation of EPC mobilization and function is mediated in part by nitric oxide (NO). Endogenous inhibitors of NO synthases, such as ADMA, contribute to endothelial dysfunction and injury. METHODS: We used flow cytometry and in vitro assays to investigate the relationship between EPC number and function with ADMA plasma levels in patients with stable angina. RESULTS: The plasma concentration of ADMA was related to the severity of coronary artery disease and correlated inversely with the number of circulating CD34+/CD133+ progenitor cells (r = -0.69; p < 0.0001) and endothelial colony forming units (CFUs) (r = -0.75; p < 0.0001). Adjusting for all patient characteristics, we confirmed these findings in multivariate regression analyses. In vitro differentiation of EPCs was repressed by ADMA in a concentration-dependent manner. Compared with untreated cells, ADMA reduced EPC incorporation into endothelial tube-like structures to 27 +/- 11% (p < 0.001). Asymmetric dimethylarginine repressed the formation of CFUs from cultured peripheral blood mononuclear cells to 35 +/- 7% (p < 0.001). Asymmetric dimethylarginine decreased endothelial nitric oxide synthase activity in EPCs to 64 +/- 6% (p < 0.05) when compared with controls. Co-incubation with the hydroxymethyl glutaryl coenzyme A reductase inhibitor rosuvastatin abolished the detrimental effects of ADMA. CONCLUSIONS: Asymmetric dimethylarginine is an endogenous inhibitor of mobilization, differentiation, and function of EPCs. This contributes to the cardiovascular risk in patients with high ADMA levels and may explain low numbers and function of EPCs in patients with coronary artery disease.

Arginine↗
PubMed2005

Specific transport of S-nitrosocysteine in human red blood cells: Implications for formation of S-nitrosothiols and transport of NO bioactivity within the vasculature.

The transport of various S-nitrosothiols, NO and NO donors in human red blood cells (RBC) and the formation of erythrocytic S-nitrosoglutathione were investigated. Of the NO species tested only S-nitrosocysteine was found to form S-nitrosoglutathione in the RBC cytosol. L-Serine, L-cysteine and L-lysine inhibited formation of S-nitrosoglutathione. Incubation of RBC pre-incubated with S-[15N]nitroso-L-cysteine with native plasma or platelet-rich plasma led to formation of S-[15N]nitrosoalbumin and inhibited platelet aggregation, respectively. The specific transporter system of S-nitroso-L-cysteine in the RBC membrane may have implications for formation of S-nitrosoalbumin and S-nitrosohemoglobin and for transport of NO bioactivity within the vasculature.

Adenosine Diphosphate↗
PubMed2005

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake.

3-Nitrotyrosine (NO(2)Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)-tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[(2)H(3)]tyrosine, and GC-tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5+/-7.2) 3-nitrotyrosine levels were determined to be 8.4+/-10.4 nM (range, 1.6-33.2 nM) or 0.46+/-0.49 nmol/mmol creatinine (range, 0.05-1.30 nmol/mmol creatinine). The present GC-tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 microg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.

Administration, Oral↗
PubMed2005

Unusual collision-induced dissociation of fluorated and non-fluorated alpha-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole mass spectrometer under electron-capturing negative-ion chemical ionization conditions.

Unusual collision-induced dissociation (CID) of perfluorated and non-perfluorated alpha-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole (TSQ) mass spectrometer (GC-QqQ-MS) under electron-capturing negative-ion chemical ionization conditions is reported. CID of [M - 1]- of alpha-nitro-2,3,4,5,6-pentafluorotoluene (C6F5CH2-NO2) and alpha-nitro-2,5-difluorotoluene (C6H3F2CH2-NO2) produced an intense ion with m/z 66. By using 15N- or 18O-labelled C6F5CH2-NO2 analogs, we found that this anion has the formula C3NO. By contrast, CID of [M - 1]- of alpha-nitrotoluene (C6H5CH2-NO2) and alpha-nitro-3,5-difluorotoluene (C6H3F2CH2-NO2) produced an anion with m/z 86 with the formula C3H4NO2. The expected CID of the C-N-bond of all alpha-nitrotoluene analogs to form the nitrite anion (NO2-, m/z 46) did not occur. We propose mechanisms for the formation of the anions C3NO and C3H4NO2 in the collision chamber of the TSQ mass spectrometer. The most likely structures for the anion C3NO are :C=C=C=N--O and N triple bond C-C triple bond C--O-. The unique CID behavior of C6F5CH2--NO2 can be utilized to unequivocally identify and accurately quantify nitrite in biological fluids by GC-tandem MS.

Fluorobenzenes↗
PubMed2005

Chronic angiotensin II receptor blockade reduces (intra)renal vascular resistance in patients with type 2 diabetes.

Increased (intra)renal activity of the renin-angiotensin system may cause a persistent increase in renovascular resistance and intraglomerular pressure in patients with diabetes, thus contributing to the development of diabetic renal damage. The effect of chronic angiotensin II subtype 1 receptor blockade on (intra)renal hemodynamics in patients with type 2 diabetes was examined in a double-blind parallel group study. Patients were treated with 40 mg of olmesartan (n = 19) or placebo (n = 16), and renal hemodynamics were assessed before and after 12 wk of treatment using inulin and para-aminohippurate clearance techniques. Olmesartan significantly reduced 24-h ambulatory systolic and diastolic BP (both P < 0.05). In parallel, effective renal plasma flow increased significantly from 602 +/- 76 to 628 +/- 87 ml/min per 1.73 m(2), whereas filtration fraction and renovascular resistance decreased significantly (all P < 0.05). With placebo treatment, effective renal plasma flow decreased and filtration fraction increased significantly (both P < 0.05). GFR was not affected by both treatments. Active plasma renin concentration increased considerably (P < 0.05) with olmesartan therapy but remained unchanged with placebo treatment. Nitric oxide metabolism (plasma nitrate and nitrite) and asymmetric dimethylarginine blood levels were not affected by olmesartan and placebo therapy. In contrast, plasma 8-isoprostane 15(S)-8-iso-prostaglandin F(2a) concentration, a biochemical marker of oxidative stress, decreased significantly (P < 0.05) with olmesartan treatment. Chronic angiotensin II subtype 1 receptor blockade decreases (intra)renal vascular resistance and increases renal perfusion despite significant BP reduction. In addition, it significantly reduces oxidative stress. These effects of angiotensin II receptor antagonists may contribute to their beneficial long-term renal effects in patients with type 2 diabetes.

Aged↗
PubMed2005

Recent methodological advances in the mass spectrometric analysis of free and protein-associated 3-nitrotyrosine in human plasma.

L-Tyrosine and L-tyrosine residues in proteins are attacked by various reactive-nitrogen species (RNS) including peroxynitrite to form 3-nitrotyrosine (NO(2)Tyr) and protein-associated 3-nitrotyrosine (NO(2)TyrProt). Circulating NO(2)Tyr and NO(2)TyrProt have been suggested and are widely used as biomarkers of oxidative stress in humans. In this article the mass spectrometry (MS)-based analytical methods recently reported for the quantification of circulating levels of NO(2)Tyr and NO(2)TyrProt are discussed. These methodologies differ in sensitivity, selectivity, specificity and accessibility to interferences with the latter mainly arising from artifactual formation of NO(2)Tyr and NO(2)TyrProt during sample treatment such as acidification and chemical derivatization. Application of these methodologies to healthy normal humans revealed basal circulating levels for NO(2)Tyr which range between 0.7 and 64 nM, i.e. by two orders of magnitude. Application of gas chromatography-tandem mass spectrometry (GC-tandem MS) methods by two independent research groups by using two different protocols to avoid artifactual nitration of L-tyrosine revealed almost identical mean plasma levels of the order of 1.0 nM in healthy humans. The lower limits of quantitation (LOQ) of these methods were 0.125 and 0.3n M, respectively. This order of magnitude for basal NO(2)Tyr is supported by two liquid chromatography-tandem mass spectrometry (LC-tandem MS) methods with LOQ values of 4.4 and 1.4 nM. On the basis of the data provided by GC-tandem MS and LC-tandem MS the use of a range of 0.5-3 nM for NO(2)Tyr and of 0.6 pmol/mg plasma protein or a molar ratio of 3-nitrotyrosine to tyrosine in plasma proteins of the order of 1:10(6) for NO(2)TyrProt in plasma of healthy humans as reference values appear reasonably justified. Recently reported clinical studies involving 3-nitrotyrosine as a biomarker of oxidative stress are discussed in particular from the analytical point of view.

Humans↗
PubMed2005

Local nitric oxide levels reflect the degree of allergic airway inflammation after segmental allergen challenge in asthmatics.

Nitric oxide (NO) levels are increased in the exhaled air of asthmatics. As NO levels correlate with allergic airway inflammation, NO measurement has been suggested for disease monitoring. In patients with asthma, we previously demonstrated that intrabronchial treatment with a natural porcine surfactant enhanced airway inflammation after segmental allergen provocation. We studied whether local levels of NO reflect the degree of allergic airway inflammation following segmental allergen challenge with or without surfactant pretreatment. Segmental NO, as well as nitrite and nitrate in bronchoalveolar lavage (BAL) fluid, was measured before and after segmental challenge with either saline, saline plus allergen, or surfactant plus allergen in 16 patients with asthma and five healthy subjects. The data were compared with inflammatory BAL cells. Segmental NO levels were increased after instillation of saline (p < 0.05), or surfactant plus allergen in asthmatics (p < 0.05), and values were higher after surfactant plus allergen compared to saline challenge. Nitrate BAL levels were not altered after saline challenge but increased after allergen challenge (p < 0.05) and further raised by surfactant (p < 0.05), whereas nitrite levels were not altered by any treatment. Segmental NO and nitrate levels correlated with the degree of eosinophilic airway inflammation, and nitrate levels also correlated with neutrophil and lymphocyte numbers in BAL. In healthy subjects, NO, nitrite, and nitrate were unaffected. Thus, segmental NO and nitrate levels reflect the degree of allergic airway inflammation in patients with asthma. Measurement of both markers can be useful in studies using segmental allergen provocation, to assess local effects of potential immunomodulators.

Asthma↗
PubMed2005

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids.

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.

Humans↗
PubMed2005

Effects of specific COX-2-inhibition on renin release and renal and systemic prostanoid synthesis in healthy volunteers.

BACKGROUND: The renin-angiotensin system plays a critical role in cardiovascular function, but little is known about the effects of specific cyclooxygenase 2 (COX-2) inhibition on this system in healthy humans under physiologic conditions. METHODS: Twenty-one healthy female volunteers received, in a randomized, double-blind, crossover study, celecoxib 200 mg twice a day, indomethacin 50 mg three times a day, or placebo for 4 days and a single dose, each, on day 5. On day 5 of each treatment, the following parameters were assessed with subjects in an upright position before and after administration of 20 mg furosemide intravenously: plasma renin activity (PRA), plasma aldosterone, serum and urine electrolytes, and creatinine. Index metabolites of prostanoids were analyzed by gas chromatography-tandem mass spectrometry in 24-hour urine on day 4 and in 2-hour urines before and after furosemide administration. RESULTS: Baseline and furosemide-stimulated PRA were reduced to a similar degree by celecoxib and indomethacin. Plasma aldosterone and urinary excretion of potassium showed changes consistent with the alteration of PRA. Urinary excretion rates of prostaglandin E(2), (PGE(2)), 7alpha-hydroxy-5, 11-diketotetranor-prosta-1,16-dioic acid (PGE-M), and 2,3-dinor-thromboxane B(2) (TxB(2)) were not reduced by celecoxib, whereas indomethacin led to a decrease of 40%, 45%, and 80%, respectively. Both active treatments inhibited urinary excretion of 2,3-dinor-6-keto-PGF(1alpha) and 6-keto-PGF(1alpha) by 60% and 40%, respectively. CONCLUSION: Renin-release in healthy humans with normal salt intake is COX-2 dependent. While COX-1 is critical for renal and systemic PGE(2) production, renal prostacyclin synthesis is apparently COX-2 dependent. Finally, the previously demonstrated shift of the thromboxane-prostacyclin balance toward prothrombotic thromboxane by specific COX-2 inhibition is confirmed.

6-Ketoprostaglandin F1 alpha↗
PubMed2004

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). II. Quantitative determination of cis-EODA in human plasma.

Cytochrome P450 dependent epoxidation and non-enzymic lipid peroxidation of oleic acid (cis-9-octadecenoic acid) result in the formation of cis-9,10-epoxyoctadecanoic acid (cis-EODA). This oleic acid oxide has been identified indirectly in blood and urine of humans. Reliable concentrations of circulating cis-EODA have not been reported thus far. In the present article, we report on the first GC-tandem MS method for the accurate quantitative determination in human plasma of authentic cis-EODA as its pentafluorobenzyl (PFB) ester. cis-[9,10-2H2]-EODA (cis-d2-EODA) was synthesized by chemical epoxidation of commercially available cis-[9,10-2H2]-9-octadecenoic acid and used as an internal standard for quantification. Endogenous cis-EODA and externally added cis-[9,10-2H2]-EODA were isolated from acidified plasma samples (1 ml; pH 4.5) by solvent or solid-phase extraction, converted into their PFB esters, isolated by HPLC and quantified by selected reaction monitoring. The parent ions [M-PFB]- at mass-to-charge ratio (m/z) 297 for cis-EODA and m/z 299 for (cis-d2-EODA) were subjected to collisionally-activated dissociation and the corresponding characteristic product ions at m/z 171 and 172 were monitored. In plasma of nine healthy humans (5 females, 4 males), cis-EODA was found to be present at 47.6+/-7.4 nM (mean+/-S.D.). Plasma cis-EODA levels were statistically insignificantly different (P=0.10403, t-test) in females (51.1+/-3.4 nM) and males (43.1+/-2.2 nM). cis-EODA was identified as a considerable contamination in laboratory plastic ware and found to contribute to endogenous cis-EODA by approximately 2 nM. The present GC-tandem MS method should be useful in investigating the physiological role(s) of cis-EODA in humans.

Chromatography, High Pressure Liquid↗
PubMed2004

Effects of carvedilol on oxidative stress in human endothelial cells and healthy volunteers.

OBJECTIVE: Carvedilol is a nonselective beta- and alpha(1)-receptor antagonist with additional antioxidant properties in vitro. In this study, we assessed the antioxidative potential of carvedilol in cell culture and in antihypertensive doses in healthy men. METHODS: In vitro, human cultured endothelial cells were treated with native low-density lipoprotein (LDL), oxidized LDL or tumor necrosis factor (TNF)alpha in the absence and in the presence of carvedilol (40 micro M); 8-iso-prostaglandin (PG)F(2alpha), as parameter of oxidative stress, was determined in the supernatants. In a double-blind, randomized, cross-over study, 17 healthy men received 25 mg carvedilol b.i.d., 100 mg metoprolol b.i.d. or placebo for 6 days. After each treatment, systemic oxidative stress was assessed by measuring urinary excretion of 8-iso-PGF(2alpha) and 2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), and the plasma concentration of 3-nitrotyrosine by means of gas chromatography-tandem mass spectrometry. In addition, thiobarbituric acid-reactive substances (TBARS) in plasma were assessed using spectrophotometry. RESULTS: Native LDL and oxidized LDL induced 8-iso-PGF(2alpha) production in endothelial cells. Carvedilol significantly reduced this effect (e.g., for oxidized LDL: 2.66+/-0.22 pg vs 1.46+/-0.14 pg 8-iso-PGF(2alpha) per micro g protein, P<0.05). In healthy volunteers, carvedilol and metoprolol markedly decreased blood pressure and heart rate, but had no statistically significant effect on any indicator of oxidative stress measured. Remarkably, a trend toward reduction of urinary isoprostanes and 3-nitrotyrosine in plasma by both active treatments was observed, suggesting a non-specific antioxidative effect by beta blockade. CONCLUSIONS: In vitro, the antioxidative potential of carvedilol was confirmed. In healthy men, antihypertensive doses of carvedilol exert no specific inhibition of oxidative stress.

Adrenergic Antagonists↗