Journal of Chromatography B (v.851, #1-2)
Editorial Board (iii).
Analysis of the l-arginine/NO pathway by Dimitrios Tsikas (1-2).
Microarray-based gene expression profiling to elucidate cellular responses to nitric oxide—A review from an analytical and biomedical point of view by Thomas Thum; Johann Bauersachs (3-11).
Nitric oxide (NO) produced by NO synthases (NOS) regulates a wide range of cellular functions. Analysis by gene arrays provides valuable information for identifying important elements of the cellular responses to NO. Such screening tools might be useful to elucidate NO-responsive regulators, which play a central role in mediating NO effects. Although the final importance of a particular gene is determined by the encoded protein and further protein modifications, measurements of RNA levels have proven to be partly valuable in identifying the molecular changes that occur in cells. Microarray technology permits large-scale and genome-wide analysis of gene expression from multiple samples. We review the current knowledge of the use of microarray gene expression screening in elucidating the effects of NO on various cells and tissues. We also point out the limitations of general microarray-based gene expression analyses and especially when investigating the effects of NO.
Keywords: Reviews; Microarray; Gene expression; Endothelium; Nitric oxide; Transcriptome; Sensitivity;
Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide by Andrei L. Kleschyov; Philip Wenzel; Thomas Munzel (12-20).
Nitric oxide (NO) is a free radical species with multiple physiological functions. Because of low concentrations and short half-life of NO, its direct measurement in living tissues remains a difficult task. Electron paramagnetic resonance (EPR) spin trapping is probably one of the best suitable platforms for development of new methods for quantification of biological NO. The most reliable EPR-based approaches developed so far are based on the reaction of NO with various iron complexes, both intrinsic and exogenously applied. This review is focused on the current state and perspectives of EPR spin trapping for experimental and clinical NO biology.
Keywords: Reviews; Nitrosyl–iron complexes; Electron paramagnetic resonance;
HPLC analysis of ADMA and other methylated l-arginine analogs in biological fluids by Tom Teerlink (21-29).
Post-translational methylation of arginine residues in proteins leads to generation of N G-monomethylarginine (MMA) and both symmetric and asymmetric dimethylarginine (SDMA and ADMA), that are released into the cytosol upon proteolysis. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Plasma concentrations of ADMA and SDMA are very low, typically between 0.3 and 0.8 μM, making their quantification by HPLC an analytical challenge. Sample preparation usually involves a cleanup step by solid-phase extraction on cation-exchange columns followed by derivatization of amino acids into fluorescent adducts. Because ADMA and SDMA concentrations in healthy subjects show a very narrow distribution, with a between-subject variability of 13% for ADMA and 19% for SDMA, very low imprecision is an essential assay feature. Procedures for sample cleanup, derivatization, and chromatographic separation of arginine and its methylated analogs are the main topics of this review. In addition, important aspects of method validation, pre-analytical factors, and reference values are discussed.
Keywords: Reviews; Asymmetric dimethylarginine; Monomethylarginine; Symmetric dimethylarginine; Nitric oxide synthase; Cardiovascular disease; Pre-column derivatization; Solid-phase extraction;
Chromatographic-mass spectrometric methods for the quantification of l-arginine and its methylated metabolites in biological fluids by Jens Martens-Lobenhoffer; Stefanie M. Bode-Böger (30-41).
l-Arginine (Arg) and its methylated metabolites play a major role in the synthesis of the cell signaling molecule nitric oxide (NO). Arg serves as a substrate for the enzyme NO synthase (NOS), which produces NO, whereas monomethylarginine (l-NMMA) and asymmetric dimethylarginine (ADMA) act as competitive inhibitors of NOS. Symmetric dimethylarginine (SDMA) has virtually no inhibitory effect on NOS activity, but shares the pathway for cell entry and transport with Arg and ADMA. Accurate and reliable quantification of these substances in various biological fluids is essential for scientific research in this field. In this review, chromatographic-mass spectrometric methods for Arg and its methylated metabolites ADMA and SDMA are discussed. Mass spectrometric detection provides an intrinsic higher selectivity than detection by means of UV absorbance or fluorescence. Taking advantage of the high selectivity, approaches involving mass spectrometric detection require less laborious sample preparation and produce reliable results. A consensus emerges that the concentration values in plasma of young healthy volunteers are about 65 μM for Arg, 0.4 μM for ADMA and 0.5 μM for SDMA.
Keywords: Reviews; Arginine; Dimethylarginine; Mass spectrometry; HPLC; GC;
An overview of plasma concentrations of asymmetric dimethylarginine (ADMA) in health and disease and in clinical studies: Methodological considerations by John D. Horowitz; Tamila Heresztyn (42-50).
Recent studies among patients including those with known coronary disease demonstrate that small elevations in asymmetric dimethylarginine (ADMA) concentrations in plasma are predictive of adverse outcomes. The precision of current methodologies for quantitation of ADMA such as HPLC, MS and ELISA is discussed with respect to many reports which appear to over-estimate ADMA levels and quote broad concentration ranges. While plasma ADMA concentrations tend to increase with age, the mean for a healthy population is between 0.4 and 0.6 μM. ADMA levels may fluctuate in normal subjects, and this needs to be considered in light of the relatively small differences in ADMA concentration between healthy normal subjects and patients.
Keywords: Reviews; ADMA; HPLC; ELISA; Mass spectrometry; Clinical studies;
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 by Dimitrios Tsikas (51-70).
In the Griess reaction, first reported by Johann Peter Griess in 1879 as a method of analysis of nitrite (NO2 −), nitrite reacts under acidic conditions with sulfanilic acid (HO3SC6H4NH2) to form a diazonium cation (HO3SC6H4–N N +) which subsequently couples to the aromatic amine 1-naphthylamine (C10H7NH2) to produce a red–violet coloured (λ max ≈ 540 nm), water-soluble azo dye (HO3SC6H4–N＝ N–C10H6NH2). 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 (NO3 −), 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.
Keywords: Reviews; Diazotization; Fluorescence; HPLC; Interferences; Quantitation; Reduction; Spectrophotometry;
Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography by Wenjuan S. Jobgen; Scott C. Jobgen; Hui Li; Cynthia J. Meininger; Guoyao Wu (71-82).
Various analytical techniques have been developed to determine nitrite and nitrate, oxidation metabolites of nitric oxide (NO), in biological samples. HPLC is a widely used method to quantify these two anions in plasma, serum, urine, saliva, cerebrospinal fluid, tissue extracts, and fetal fluids, as well as meats and cell culture medium. The detection principles include UV and VIS absorbance, electrochemistry, chemiluminescence, and fluorescence. UV or VIS absorbance and electrochemistry allow simultaneous detection of nitrite and nitrate but are vulnerable to the severe interference from chloride present in biological samples. Chemiluminescence and fluorescence detection improve the assay sensitivity and are unaffected by chloride but cannot be applied to a simultaneous analysis of nitrite and nitrate. The choice of a detection method largely depends on sample type and facility availability. The recently developed fluorometric HPLC method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) and the enzymatic conversion of nitrate into nitrite, offers the advantages of easy sample preparation, simple derivatization, stable fluorescent derivatives, rapid analysis, high sensitivity and specificity, lack of interferences, and easy automation for determining nitrite and nitrate in all biological samples including cell culture medium. To ensure accurate analysis, care should be taken in sample collection, processing, and derivatization as well as preparation of reagent solutions and mobile phases, to prevent environmental contamination. HPLC methods provide a useful research tool for studying NO biochemistry, physiology and pharmacology.
Keywords: Reviews; Nitric oxide; Plasma; Serum; Urine; Clinical and animal studies;
Measurement of the NO metabolites, nitrite and nitrate, in human biological fluids by GC–MS by Steve M. Helmke; Mark W. Duncan (83-92).
In this article we critically review the development and application of gas chromatography–mass spectrometry (GC–MS) techniques to the measurement of the nitric oxide (NO) metabolites, nitrite and nitrate, in human biological fluids. Our focus is on the issue of the fitness of any analytical strategy to its intended purpose and the validity of the analytical results generated. The accuracy, precision, recovery, selectivity and sensitivity of the various methods are evaluated and the potential pitfalls, both specific to the methods, and general to the area, are considered. Several examples of the applications of these techniques to clinical investigations of NO physiology are also critically evaluated.
Keywords: Reviews; Nitration; Alkylation; Stable isotopes; Clinical investigations; Mass spectrometry;
Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence by Peter H. MacArthur; Sruti Shiva; Mark T. Gladwin (93-105).
Considerable disparities in the reported levels of basal human nitrite and S-nitrosothiols (RSNO) in blood have brought methods of quantifying these nitric oxide (NO) metabolites to the forefront of NO biology. Ozone-based chemiluminescence is commonly used and is a robust method for measuring these species when combined with proper reductive chemistry. The goal of this article is to review existing methodologies for the measurement of nitrite and RSNO by reductive chemiluminescence. Specifically, we discuss in detail the measurement of nitrite and RSNO in biological matrices using tri-iodide and copper(I)/cysteine-based reduction methods coupled to chemiluminescence. The underlying reaction mechanisms, as well as the potential pitfalls of each method are discussed.
Keywords: Reviews; Nitric oxide; S-Nitrosohemoglobin; Tri-iodide; Copper(I)/cysteine;
Recent methodological advances in the analysis of nitrite in the human circulation: Nitrite as a biochemical parameter of the l-arginine/NO pathway by Marijke Grau; Ulrike B. Hendgen-Cotta; Paris Brouzos; Christine Drexhage; Tienush Rassaf; Thomas Lauer; André Dejam; Malte Kelm; Petra Kleinbongard (106-123).
Nitric oxide (NO) plays a pivotal role in the modulation of multiple physiological processes. It acts as a messenger molecule within the cardiovascular system. NO is a highly unstable free radical in circulating blood and is oxidized rapidly to nitrite and nitrate. Recent studies suggest that nitrite has the potential to function as a surrogate of NO production under physiological and pathophysiological conditions and could therefore be of high relevance as a biochemical parameter in experimental and clinical studies. Under hypoxic conditions nitrite is reduced to bioactive NO by deoxyhemoglobin. This mechanism may represent a dynamic cycle of NO generation to adapt the demand and supply for the vascular system. Because of these potential biological functions the concentration of nitrite in blood is thought to be of particular importance. The determination of nitrite in biological matrices represents a considerable analytical challenge. Methodological problems often arise from pre-analytical sample preparation, sample contamination due to the ubiquity of nitrite, and from lack of selectivity and sensitivity. These analytical difficulties may be a plausible explanation for reported highly diverging concentrations of nitrite in the human circulation. The aim of this article is to review the methods of quantitative analysis of nitrite in the human circulation, notably in plasma and blood, and to discuss pre-analytical and analytical factors potentially affecting accurate quantification of nitrite in these human fluids.
Keywords: Reviews; Nitrite analysis; Human circulation; Clinical studies; Biochemical parameter; Validation;
Detection of S-nitrosothiols in biological fluids: A comparison among the most widely applied methodologies by Daniela Giustarini; Aldo Milzani; Isabella Dalle-Donne; Ranieri Rossi (124-139).
Many different methodologies have been applied for the detection of S-nitrosothiols (RSNOs) in human biological fluids. One unsatisfactory outcome of the last 14 years of research focused on this issue is that a general consensus on reference values for physiological RSNO concentration in human blood is still missing. Consequently, both RSNO physiological function and their role in disease have not yet been clarified. Here, a summary of the values measured for RSNOs in erythrocytes, plasma, and other biological fluids is provided, together with a critical review of the most widely used analytical methods. Furthermore, some possible methodological drawbacks, responsible for the highlighted discrepancies, are evidenced.
Keywords: Reviews; Nitric oxide; S-Nitrosoalbumin; S-Nitrosohemoglobin; S-Transnitrosation reactions;
S-Nitrosothiol measurements in biological systems by Andrew Gow; Allan Doctor; Joan Mannick; Benjamin Gaston (140-151).
S-Nitrosothiol (SNO) cysteine modifications are regulated signaling reactions that dramatically affect, and are affected by, protein conformation. The lability of the S―NO bond can make SNO-modified proteins cumbersome to measure accurately. Here, we review methodologies for detecting SNO modifications in biology. There are three caveats. (1) Many assays for biological SNOs are used near the limit of detection: standard curves must be in the biologically relevant concentration range. (2) The assays that are most reliable are those that modify SNO protein or peptide chemistry the least. (3) Each result should be quantitatively validated using more than one assay. Improved assays are needed and are in development.
Keywords: Reviews; S-Nitrosoglutathione; S-Nitrosylation; S-Nitrosohemoglobin; Signaling; Cysteine;
Proteomic methods for analysis of S-nitrosation by Nicholas J. Kettenhofen; Katarzyna A. Broniowska; Agnes Keszler; Yanhong Zhang; Neil Hogg (152-159).
This review discusses proteomic methods to detect and identify S-nitrosated proteins. Protein S-nitrosation, the post-translational modification of thiol residues to form S-nitrosothiols, has been suggested to be a mechanism of cellular redox signaling by which nitric oxide can alter cellular function through modification of protein thiol residues. It has become apparent that methods that will detect and identify low levels of S-nitrosated protein in complex protein mixtures are required in order to fully appreciate the range, extent and selectivity of this modification in both physiological and pathological conditions. While many advances have been made in the detection of either total cellular S-nitrosation or individual S-nitrosothiols, proteomic methods for the detection of S-nitrosation are in relative infancy. This review will discuss the major methods that have been used for the proteomic analysis of protein S-nitrosation and discuss the pros and cons of this methodology.
Keywords: Reviews; S-Nitrosothiols; Proteomics; Nitrosation; Nitric oxide; Thiols;
Chromatographic and mass spectrometric methods for quantitative determination of 3-nitrotyrosine in biological samples and their application to human samples by Henrik Ryberg; Kenneth Caidahl (160-171).
The permanent modification of soluble and protein-associated tyrosine by nitration results in the formation of 3-nitrotyrosine, which can be used as a marker of “nitro-oxidative” damage to proteins. Based on the analysis of patient materials, over 40 different diseases and/or conditions have been linked to increased nitration of tyrosine. They include many cardiovascular diseases, conditions associated with immunological reactions and neurological diseases. In this article we review the existing chromatographic and mass spectrometric methods for quantitative measurements of 3-nitrotyrosine in different human biological samples including plasma, either from the free amino acid pool or from hydrolyzed proteins from different matrices.
Keywords: Reviews; HPLC; Mass spectrometry; Plasma; Urine; Cerebrospinal fluid;
Methods using stable isotopes to measure nitric oxide (NO) synthesis in the l-arginine/NO pathway in health and disease by H.M.H. van Eijk; Y.C. Luiking; N.E.P. Deutz (172-185).
Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid l-arginine by the action of nitric oxide synthases (NOS) in what is called the l-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, l-arginine and its simultaneously generated product, l-citrulline. Considering only a fraction of the endogenous l-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of l-arginine and/or l-citrulline alone. Instead, to estimate NO-related changes in the l-arginine and/or l-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of l-arginine to NO and l-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.
Keywords: Reviews; Nitric oxide; Stable isotope; l-Arginine; Mass spectrometry;
Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis by Dmitri Y. Boudko (186-210).
This review briefly summarizes recent progress in fundamental understanding and analytical profiling of the l-arginine/nitric oxide (NO) pathway. It focuses on key analytical references of NO actions and the experimental acquisition of these references in vivo, with capillary electrophoresis (CE) and high-performance capillary electrophoresis (HPCE) comprising one of the most flexible and technologically promising analytical platform for comprehensive high-resolution profiling of NO-related metabolites. Another aim of this review is to express demands and bridge efforts of experimental biologists, medical professionals and chemical analysis-oriented scientists who strive to understand evolution and physiological roles of NO and to develop analytical methods for use in biology and medicine.
Keywords: Reviews; Nitrite; Peroxynitrite; 3-Nitrotyrosine; N-Nitrosotryptophan; ADMA;
High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma by Edzard Schwedhelm; Renke Maas; Jing Tan-Andresen; Friedrich Schulze; Ulrich Riederer; Rainer H. Böger (211-219).
The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. l-Arginine is converted to NO and l-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-μl aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74 ± 19 (SD), 0.46 ± 0.09, and 0.37 ± 0.07 μM in healthy humans (n = 85), respectively, and 44 ± 14, 0.72 ± 0.23, and 0.19 ± 0.06 μM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166 ± 50 and 85 ± 22, respectively.
Keywords: Arginine; Asymmetric dimethylarginine (ADMA); Nitric oxide; LC-tandem MS; Stable isotopes; Symmetric dimethylarginine (SDMA);
A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue by Renke Maas; Jing Tan-Andreesen; Edzard Schwedhelm; Friedrich Schulze; Rainer H. Böger (220-228).
The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the hydrolysis of asymmetric dimethylarginine (ADMA) to l-citrulline and dimethylamine. DDAH is currently investigated as a promising target for therapeutic interventions, as ADMA has been found to be elevated in cardiovascular disease. In many tissues continuous endogenous formation of ADMA and l-citrulline poses considerable limitations to the presently used assays for DDAH activity, which are commonly based on the measurement of ADMA or l-citrulline. We therefore developed a stable-isotope-based assay suitable for 96-well plates to determine DDAH activity. Using deuterium-labeled ADMA ([2H6]-ADMA) as substrate and double stable-isotope labeled ADMA ([13C5-2H6]-ADMA) as internal standard we were able to simultaneously determine formation and metabolism of ADMA in renal and liver tissue of mice by LC–tandem MS. Endogenous formation of ADMA could largely be abolished by addition of protease inhibitors, while metabolism of [2H6]-ADMA was not significantly altered. The intra-assay coefficient of variation for the determination of endogenous ADMA and [2H6]-ADMA was 2.4% and 4.8% in renal and liver tissue, respectively. The inter-assay coefficient of variation for DDAH activity based on degradation of [2H6]-ADMA determined in separate samples from the same organs was determined to be 8.9% and 10% for mouse kidney and liver, respectively. The present DDAH activity assay allows for the first time to simultaneously determine DDAH activity and endogenous formation of ADMA, SDMA, and l-arginine in tissue.
Keywords: l-Arginine; ADMA; SDMA; Assay; Stable isotopes; Mass spectrometry;
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 by Dimitrios Tsikas; Thomas Thum; Thomas Becker; Vu Vi Pham; Kristina Chobanyan; Anja Mitschke; Bibiana Beckmann; Frank-Mathias Gutzki; Johann Bauersachs; Dirk O. Stichtenoth (229-239).
Dimethylamine [DMA, (CH3)2NH)] 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 (CD3)2NH 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 (CD3)2NH in the electron ionization mode. GC–MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264 ± 173 μM equivalent to 10.1 ± 1.64 μmol/mmol creatinine. GC–tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3 ± 15.3 μM corresponding to 1.35 ± 1.2 μmol/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 μmol/mmol creatinine) and ADMA (3.85 ± 1.65 μmol/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 μmol/mmol creatinine) and ADMA (5.6 ± 1.5 μmol/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.
Keywords: l-Arginine; Clinical study; DDAH; Nitric oxide; Quality control;
Accurate quantification of dimethylamine (DMA) in human plasma and serum by GC–MS and GC–tandem MS as pentafluorobenzamide derivative in the positive-ion chemical ionization mode by Kristine Chobanyan; Anja Mitschke; Frank-Mathias Gutzki; Dirk O. Stichtenoth; Dimitrios Tsikas (240-249).
Dimethylamine (DMA) circulates in human blood and is excreted in the urine. Major precursor for endogenous DMA is asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is hydrolyzed to DMA and l-citrulline by dimethylarginine dimethylaminohydrolase (DDAH). In previous work, we reported a GC–MS method for the quantification of DMA in human urine. This method involves simultaneous derivatization of endogenous DMA and the internal standard (CD3)2NH by pentafluorobenzoyl chloride (PFBoylCl) and extraction of the pentafluorobenzamide derivatives by toluene. In the present work, we optimized this derivatization/extraction procedure for the quantitative determination of DMA in human plasma. Optimized experimental parameters included vortex time and concentration of PFBoylCl, carbonate and internal standard. The GC–MS method was thoroughly validated and applied to measure DMA concentrations in human plasma and serum samples. GC–MS quantification was performed by selected-ion monitoring of the protonated molecules at m/z 240 for DMA and m/z 246 for (CD3)2NH in the positive-ion chemical ionization mode. Circulating DMA concentration in healthy young women (n = 18) was determined to be 1.43 ± 0.23 μM in serum, 1.73 ± 0.17 μM in lithium heparin plasma, and 9.84 ± 1.43 μM in EDTA plasma. DMA was identified as an abundant contaminant in EDTA vacutainer tubes (9.3 ± 1.9 nmol/monovette, n = 6). Serum and lithium heparin vacutainer tubes contained considerably smaller amounts of DMA (0.42 ± 0.01 and 0.95 ± 0.01 nmol/monovette, respectively, each n = 6). Serum is recommended as the most appropriate matrix for measuring DMA in human blood. The present GC–MS method should be useful for the determination of systemic and whole body DDAH activity by measuring circulating and excretory DMA in experimental and clinical studies.
Keywords: ADMA; Contamination; DDAH; Derivatization; Nitric oxide; Validation;
Arginine, citrulline and nitrate concentrations in the cerebrospinal fluid from patients with acute hydrocephalus by Iván Pérez-Neri; Elvira Castro; Sergio Montes; Marie-Catherine Boll; Juan Barges-Coll; José Luis Soto-Hernández; Camilo Ríos (250-256).
Citrulline and nitric oxide (NO) are synthesized by NO synthase (NOS) in a 1:1-stoichiometry. In this study, we determined by HPLC arginine and citrulline concentrations by fluorescence detection and nitrate levels by UV absorbance detection in the cerebrospinal fluid (CSF) from patients with acute hydrocephalus that underwent ventricular drainage. We found increased citrulline concentration (50.6 ± 17.2 versus 20.9 ± 2.0 μM) and decreased arginine/citrulline molar ratio (0.42 ± 0.11 versus 1.12 ± 0.16) in hydrocephalus patients, while arginine and nitrate concentrations and citrulline/nitrate molar ratio remained with little change. Citrulline has been determined as a marker of NOS activity in some studies, but it remains to be determined the extent at which this statement holds true, since other biochemical pathways also regulate the concentration of this amino acid. Our results suggest that citrulline is primarily synthesized from NOS in acute hydrocephalus. The evaluation of sample deproteinization by addition of methanol for the analysis of amino acids in CSF is also reported.
Keywords: Sample pretreatment; Inflammation; Cerebrospinal fluid drainage; Argininosuccinate synthetase;
Comparison of nitrite/nitrate concentration in human plasma and serum samples measured by the enzymatic batch Griess assay, ion-pairing HPLC and ion-trap GC–MS: The importance of a correct removal of proteins in the Griess assay by Federica Romitelli; Stefano Angelo Santini; Eleonora Chierici; Dario Pitocco; Barbara Tavazzi; Angela Maria Amorini; Giuseppe Lazzarino; Enrico Di Stasio (257-267).
Mass spectrometry-based approaches are the reference techniques for the determination of nitrite and nitrate in plasma and serum. However, due to their simplicity and rapidity, assays based on the Griess reaction or HPLC are generally used in clinical studies, but they generate diverging values for nitrite/nitrate concentration. In this study, particular attention is paid to the optimization of the deproteinization procedure for plasma and serum samples prior to nitrite/nitrate analysis by an enzymatic batch Griess assay, HPLC and GC–MS. A method is reported to verify completeness of deproteinization and to correct for nonspecific contribution to the absorbance of the diazo dye at 540 nm. With the application of such optimized procedures, we were able to significantly improve the correlation between Griess and HPLC method or the GC–MS technique for nitrite + nitrate concentrations in human serum and plasma. Despite remaining potentially interfering pre-analytical and analytical factors, the procedures reported in the present study may be helpful in a critical evaluation of limits and possibilities of the enzymatic batch Griess assay as a large-scale method for nitrite/nitrate determination in human serum in clinical studies.
Keywords: Nitric oxide; Nitrite; Nitrate; Griess assay; Mass spectrometry; High-performance liquid chromatography;
A comparative study of proteolysis methods for the measurement of 3-nitrotyrosine residues: Enzymatic digestion versus hydrochloric acid-mediated hydrolysis by Thierry Delatour; François Fenaille; Véronique Parisod; Janique Richoz; Jacques Vuichoud; Pascal Mottier; Timo Buetler (268-276).
A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC–MS(/MS) or LC–MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6 M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.
Keywords: 3-Nitrotyrosine; Enzymatic digestion; Acidic hydrolysis; Mass spectrometry; LC–MS/MS; BSA;
Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting by Ann-Sofi Söderling; Lena Hultman; Dick Delbro; Peter Højrup; Kenneth Caidahl (277-286).
We noted differences in the antibody response to 3-nitrotyrosine (NO2Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inflammatory stimulated rat abdominal, liver and lung tissue homogenates caused no immunoreactivity, in contrast to a polyclonal nitrotyrosine antibody applied in fixed and non-fixed tissues. Western blot studies using both mono- and polyclonal antibodies showed a temperature- and heme group-dependent reduction of NO2Tyr in nitrated rat and bovine serum albumin incubated with dithiothreitol. Mass spectrometric analyses of a nitrated peptide angiotensin II revealed under similar conditions a positive temperature effect between 56 and 70 °C on reduction of NO2Tyr to 3-aminotyrosine which is not detected by anti-NO2Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO2Tyr during sample preparation might conceal the actual nitration of proteins.
Keywords: Angiotensin II; Mass spectrometry; 3-Nitrotyrosine; Temperature; Western blot;
9- and 10-Nitro-oleic acid do not interfere with the GC–MS quantitative determination of nitrite and nitrate in biological fluids when measured as their pentafluorobenzyl derivatives by Anja Mitschke; Frank-Mathias Gutzki; Dimitrios Tsikas (287-291).
The nitrated lipids 9-nitro-oleic acid (9-NO2-OA) and 10-nitro-oleic acid (10-NO2-OA) have been reported to be present in blood of healthy humans. Free and esterified forms of 9-NO2-OA and 10-NO2-OA have been detected in human plasma at about 600 and 300 nM, respectively. These concentrations are of the same order of magnitude of circulating nitrite. In theory, 9-NO2-OA and 10-NO2-OA may interfere with the analysis of circulating nitrite and nitrate. In the present study, we investigated a possible interference of 9-NO2-OA and 10-NO2-OA with the GC–MS method of analysis of nitrite and nitrate involving derivatization by pentafluorobenzyl (PFB) bromide in aqueous acetone at 50 °C for 5 min (nitrite) or for 60 min (nitrite and nitrate). Our results show that 9-NO2-OA and 10-NO2-OA do not interfere with the GC–MS analysis of nitrite and nitrate as PFB derivatives in plasma and phosphate buffered saline when added to these matrices at supraphysiological concentrations of 1–10 μM. Thus, nitrated lipids such as 9-NO2-OA and 10-NO2-OA can be excluded as potential interfering substances in the GC–MS quantitative determination of nitrite and nitrate as their PFB derivatives.
Keywords: Circulation; Derivatization; GC–MS; Interferences; Nitrated lipids; Pentafluorobenzyl bromide;