Journal of Chromatography B (v.1019, #C)
Editorial Board (i).
Analytical tools and protocols in oxidative stress by Dimitrios Tsikas; Georgios Theodoridis (1-3).
Chromatographic and mass spectrometric techniques in studies on oxidative stress in autism by Joanna Kałużna-Czaplińska; Jagoda Jóźwik-Pruska (4-14).
Healthy body is characterized by the presence of a dynamic and balanced equilibrium between the production of reactive oxygen species (ROS) and the antioxidant capacity. In oxidative stress this balance is switched to reactions of oxidation leading to increased production of ROS, exceeding the capacity of physiological antioxidant systems. Oxidative stress is known to be linked to many disturbances, disorders and diseases. One of these is the autism spectrum disorder (ASD). ASD is a neurodevelopmental disorder manifested by abnormalities in social communication and interaction, as well as by occurrence of repetitive, restricted patterns of behavior or activities. It is believed that adequate knowledge about the oxidative stress biomarkers and the possibility of their reliable measuring could be useful in broadening knowledge on various diseases including ASD. A high number of compounds have been proposed as biomarkers of oxidative stress. Some of these are connected with the severity of ASD. The present review gives a summary of the chromatographic techniques used for the determination of biomarkers for oxidative stress in autism, and of other compounds important in this context. The first part of the review focuses on the correlation between oxidative stress and autism. The second part describes applications of chromatographic and mass spectrometric methods to the analysis of different metabolites connected with oxidative stress in biological fluids of autistic children. Advantages as well as disadvantages of the application of these methods for the analysis of different types of oxidative stress biomarkers are discussed.
Keywords: Autism; Biomarkers; Chromatography; Mass spectrometry; Oxidative stress;
Role of dimethyl fumarate in oxidative stress of multiple sclerosis: A review by Suneetha A.; Raja Rajeswari K. (15-20).
Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS affecting both white and grey matter. Inflammation and oxidative stress are also thought to promote tissue damage in multiple sclerosis. Recent data point at an important role of anti-oxidative pathways for tissue protection in chronic MS, particularly involving the transcription factor nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2). Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for MS treatment. Oxidative stress and anti-oxidative pathways are important players in MS pathophysiology and constitute a promising target for future MS therapy with dimethyl fumarate. The clinical utility of DMF in multiple sclerosis is being explored through phase III trials with BG-12, which is an oral therapeutic agent. Currently a wide research is going on to find out the exact mechanism of DMF, till date it is not clear. Based on strong signals of nephrotoxicity in non-humans and the theoretical risk of renal cell cancer from intracellular accumulation of fumarate, post-marketing study of a large population of patients will be necessary to fully assess the long-term safety of dimethyl fumarate. The current treatment goals are to shorten the duration and severity of relapses, prolong the time between relapses, and delay progression of disability. In this regard, dimethyl fumarate offers a promising alternative to orally administered fingolimod (GILENYA) or teriflunomide (AUBAGIO), which are currently marketed in the United States under FDA-mandated Risk Evaluation and Mitigation Strategy (REMS) programs because of serious safety concerns. More clinical experience with all three agents will be necessary to differentiate the tolerability of long-term therapy for patients diagnosed with multiple sclerosis. This write-up provides the detailed information of dimethyl fumarate in treating the neuro disease, multiple sclerosis and its mechanism involved via oxidative stress pathway. The rapid screening methods are also need to be developed to estimate DMF in biological samples to perform and proceed for further investigations.
Keywords: Multiple sclerosis; Oxidative stress; Dimethyl fumarate;
Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room by Daniela Giustarini; Dimitrios Tsikas; Graziano Colombo; Aldo Milzani; Isabella Dalle-Donne; Paolo Fanti; Ranieri Rossi (21-28).
Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.
Keywords: Glutathione; Glutathione disulfide; Oxidative stress; Protocols;
The functional interactome of GSTP: A regulatory biomolecular network at the interface with the Nrf2 adaption response to oxidative stress by Desirée Bartolini; Francesco Galli (29-44).
Glutathione S-transferase P (GSTP), and possibly other members of the subfamily of cytosolic GSTs, are increasingly proposed to have roles far beyond the classical GSH-dependent enzymatic detoxification of electrophilic metabolites and xenobiotics. Emerging evidence suggests that these are essential components of the redox sensing and signaling platform of cells. GSTP monomers physically interact with cellular proteins, such as other cytosolic GSTs, signaling kinases and the membrane peroxidase peroxiredoxin 6. Other interactions reported in literature include that with regulatory proteins such as Fanconi anemia complementation group C protein, transglutaminase 2 and several members of the keratin family of genes. Transcription factors downstream of inflammatory and oxidative stress pathways, namely STAT3 and Nrf2, were recently identified to be further components of this interactome. Together these pieces of evidence suggest the existence of a regulatory biomolecular network in which GSTP represents a node of functional convergence and coordination of signaling and transcription proteins, namely the “GSTP interactome”, associated with key cellular processes such as cell cycle regulation and the stress response. These aspects and the methodological approach to explore the cellular interactome(s) are discussed in this review paper.
Keywords: Glutathione S-transferase P; Interactome; Regulatory network; Nrf2; Interaction proteomics; Stress adaption response;
Validation of a liquid chromatography tandem mass spectrometry method to measure oxidized and reduced forms of glutathione in whole blood and verification in a mouse model as an indicator of oxidative stress by Sang-Guk Lee; Jisook Yim; Yein Lim; Jeong-Ho Kim (45-50).
As a possible marker of oxidative stress, many studies have measured whole blood reduced glutathione (GSH) and oxidized glutathione (GSSG). However, large differences in GSH and GSSG levels reported in different studies, calls for a reliable standardized method. In this study, we validate not only analytical performance of new measurement method for GSH and GSSG, but also the clinical utility of these markers in a mouse model with chronic oxidative stress. Twenty mice were randomized into four treatment groups according to iron burden: 0 mg, 5 mg, 10 mg, or 15 mg of iron were injected into the peritoneum per day over 4 weeks. To prevent artifactual GSH auto-oxidation, we pretreated the sample with N-ethylmaleimide (NEM) immediately after sample collection. After protein precipitation using sulfosalicylic acid, GSSG and GSH-NEM were measured using liquid chromatography-tandem mass spectrometry (LCMS/MS). The mean GSH/GSSG ratios of the mouse model were 163.1, 31.0, 27.9, and 12.8 for control, 5 mg, 10 mg, and 15 mg injection groups, respectively, showing a decrease in the GSH/GSSG ratios according to the amount of oxidative stress induced. Inter-assay coefficients of variation were 4.1% for GSH-NEM and 7.3% for GSSG. Recoveries were 98.0105.9% for GSH-NEM and 98.0107.3% for GSSG. No ion suppression was observed at the retention time for GSH-NEM and GSSG. This study suggests an accurate method that can be used for glutathione measurement using LCMS/MS, and showed that GSH/GSSG ratio could provide an assessment of the degree of oxidative stress.
Keywords: Glutathione oxidative stress; Mouse model; Tandem mass spectrometry;
Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides by Daniela Giustarini; Federico Galvagni; Maurizio Orlandini; Paolo Fanti; Ranieri Rossi (51-58).
Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at 80 °C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.
Keywords: Thiols; Disulfides; Blood; Red blood cells; Platelets; Plasma;
Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry by Muneki Isokawa; Tatsuo Shimosawa; Takashi Funatsu; Makoto Tsunoda (59-65).
Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction–oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2–4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization–mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH]−) upon tandem MS spectrometric analysis.
Keywords: Amide column; Oxidative stress; SBD-F; Thiol metabolism;
Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors by Patrícia Mazureki Campos; Fabíola Silva Garcia Praça; Maria Vitória Lopes Badra Bentley (66-71).
Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 μm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 μg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 μg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 μg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 μg/mL, respectively, for HPLC/ELS for the added LA concentration (10 μg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.
Keywords: Lipoic acid; HPLC-ultraviolet; HPLC-electrochemical; HPLC-evaporative light scattering; Validation; Skin samples;
Evidence by chromatography and mass spectrometry that inorganic nitrite induces S-glutathionylation of hemoglobin in human red blood cells by Anke Böhmer; Andreas Pich; Mario Schmidt; Arash Haghikia; Dimitrios Tsikas (72-82).
Previously we found by HPLC with fluorescence detection that inorganic nitrite induces oxidation of glutathione (GSH) to its disulfide (GSSG) in intact and more abundantly in lyzed red blood cells (RBCs) from healthy humans. In the present work, we performed MS-based protein analysis and observed that nitrite (range, 020 mM) induces formation of S-glutathionyl hemoglobin (HbSSG) at cysteine (Cys) β93 and β112 of oxyhemoglobin (HbO2) in lyzed human RBCs (range, 68 mM HbO2). Hemoglobin species were isolated from incubation mixtures of nitrite in lyzed RBCs by ultrafiltration or affinity chromatography and analyzed by HPLC and LCMS/MS. The mechanism likely involves inhibition of catalase activity by nitrite (IC50, 9 μM), which allows H2O2 to accumulate and oxidize Cys moieties of oxyhemoglobin and erythrocytic GSH to form HbSSG in addition to GSSG. In freshly prepared hemolysate samples, nitrite induced release of superoxide and molecular oxygen. In the presence of paracetamol and nitrite in hemolysate samples, 3-nitro-paracetamol was detected. Nitrite also induced S-nitroso hemoglobin (HbSNO) formation in low yield (i.e., 0.1%). Synthetic cysteine (Cys), glutathione (GSH), N-acetylcysteine (NAC) and N-acetylcysteine ethyl ester (NACET) inhibited nitrite-induced modifications of oxyhemoglobin including methemoglobin, HbSSG (CysSH >> NACET >> GSH NAC; thiol concentration, 50 μM) and HbSNO. Nitrite-induced oxidative modifications may alter physiological hemoglobin functions and may require alternative treatments for conditions associated with oxidized hemoglobin like in nitrite-induced methemoglobinemia. Accumulation of soluble Cys in RBCs via oral administration of NACET could be a new promising strategy to prevent nitrite-induced methemoglobinemia by nitrite and other oxidants.
Keywords: Catalase; Erythrocytes; S-Glutathionylation; Hemoglobin; Mass spectrometry; Methemoglobin; Nitrite; Thiols;
Identification of the anti-oxidant components in a two-step solvent extract of bovine bile lipid: Application of reverse phase HPLC, mass spectrometry and fluorimetric assays by Namrata Singh; Debasish Bhattacharyya (83-94).
An ether extract of nine different bacterial metabolites in combination with two solvent extract (ether followed by ethanol) of bile lipids from ox gall bladder is used as an immune stimulator drug. Over the years bile acids are discussed regarding their anti-oxidant and lipid peroxidation properties. Since some of the bile acids are known to be potent antioxidants, presence of similar activity in the solvent extract of ox bile lipid was investigated using TLC and reverse phase HPLC systems. Fractions from HPLC were analyzed with mass spectrometry using electrospray ionization. The presence of twelve different bile acids along with other substances in small proportions including fatty acids, sulfate conjugates and bile pigments were confirmed. The twelve separated peaks had similar retention times as those of tauroursodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid. Subsequently, all fractions were tested for their anti-oxidative property on HepG2 cells exposed to H2O2 that served as an oxidative injury model. Four fluorescent dyes H2DCF DA, MitoSOX red, Amplex red and DAF-2 DA were used for estimation of reactive radicals in the HepG2 cells. Among the separated bile acids, tauroursodeoxycholic acid, glycoursodeoxycholic acid and ursodeoxycholic acid prevented the HepG2 cells from H2O2-induced oxidative stress.
Keywords: Ox bile; Separation and identification of bile acids; Reverse-phase HPLC; Mass spectrometry; Oxidative stress; HepG2 cells; Anti-oxidant assay by fluorescence probes;
Development, validation and biomedical applications of stable-isotope dilution GC–MS and GC–MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2α and nitric oxide (•NO) by Dimitrios Tsikas; Sabine Rothmann; Jessica Y. Schneider; Maria-Theresia Suchy; Arne Trettin; Darko Modun; Nadine Stuke; Norbert Maassen; Jürgen C. Frölich (95-111).
Malondialdehyde (MDA, CH2 ―(CHO)2) is one of the best investigated and most frequently measured biomarkers of lipid peroxidation in biological fluids, a constituent of the so called thiobarbituric acid reactive substances (TBARS). The reaction of thiobarbituric acid with MDA and other carbonyl compounds is the basis for the batch TBARS assay, one of the most commonly and widely used assays of oxidative stress. Yet, the TBARS assay lacks specificity even if combined with HPLC separation prior to visible absorbance or fluorescence detection. In this article, we report highly specific and sensitive stable-isotope dilution GC–MS and GC–MS/MS methods for the quantitative determination of MDA in human plasma (0.1 mL). These methods utilize the acidity (pK a, 4.46) of the two methylene H protons of MDA in aqueous solution, which are as acidic as acetic acid. Endogenous MDA in native plasma and the externally added internal standard [1,3-2H2]-MDA (d2-MDA, CH2 ―(CDO)2) are derivatized in aqueous acetone (400 μL) with pentafluorobenzyl (PFB) bromide (10 μL). The reaction products were identified as C(PFB)2 ―(CHO)2 (molecular weight, 432) and C(PFB)2 ―(CDO)2) (molecular weight, 434), respectively. After solvent extraction with toluene (1 mL) quantification is performed by selected-ion monitoring (SIM) in GC–MS and by selected-reaction monitoring (SRM) in GC–MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode. In the SIM mode, the anions [M − PFB]– at m/z 251 for MDA and m/z 253 for d2-MDA are detected. In the SRM mode, the mass transitions m/z 251 to m/z 175 for MDA and m/z 253 to m/z 177 for d2-MDA are monitored. The method was thoroughly validated in human plasma. Potential interfering substances including anticoagulants and commercially available monovettes commonly used for blood sampling were tested. The lowest MDA concentrations were measured in serum followed by heparinized and EDTA plasma. The GC–MS and GC–MS/MS methods were found to be specific, precise, accurate and sensitive. Thus, the LOD of the GC–MS/MS method was determined to be 2 amol (2 × 10−18 mol) MDA. The GC–MS/MS method is exceedingly useful in clinical settings. We report several biomedical applications and discuss the utility of circulating MDA as a biomarker of lipid peroxidation, especially in long-term clinical studies, and its relation to the F 2-isoprostane 15(S)-8-iso-prostaglandin F 2α and nitric oxide (•NO).
Keywords: Biomarker; CH-acidity; Derivatization; Lipid peroxidation; Oxidative stress; 15(S)-8-iso-prostaglandin F2α; Sample storage;
Oxidative stress during bacterial growth characterized through microdialysis sampling coupled with HPLC/fluorescence detection of malondialdehyde by Keng-Chang Hsu; Pi-Fu Hsu; Ya-Ching Chen; Hsin-Chieh Lin; Chih-Chang Hung; Po-Chih Chen; Yeou-Lih Huang (112-116).
Organisms that grow aerobically are routinely exposed to oxidative stress in the form of reactive oxygen species. Monitoring the dynamic variations of oxidative stress allows us to understand its role in basic cellular function and determine mechanisms of antioxidation. In this study, microdialysis (MD) sampling was employed for continuous monitoring of the formation of malondialdehyde (MDA) in a bacterium-inoculated culture broth. To test the practicality of this approach, oxidative stress was induced by cadmium and then a 60-min interval was selected to collect sufficient amounts of dialysate for high-performance liquid chromatography with fluorescence (HPLC-FL) detection. After optimization of this simple-to-operate, simultaneous, and continuous method for dynamic monitoring of MDA during periods of bacterial growth, a retrodialysis technique and a no-net-flux method were used to assess the probe recovery and analytical performance of the proposed system. The mean probe recovery of MDA was 78.6 ± 0.9%, with intra- and interday precisions of 2.7–6.1 and 3.5–7.6%, respectively. To evaluate the practicality of this method, the dynamic variations in the concentrations of MDA in standardized bacterial species (Staphylococcus aureus, ATCC® 29213™) were monitored continuously for 24 h. The analytical results confirmed that this MD sampling technique combined with HPLC-FL detection can be used to accurately and continuously monitor the levels of MDA in microbially inoculated culture broths.
Keywords: Microdialysis; HPLC-FL; Culture broth; Dynamic monitoring; Malondialdehyde;
Airborne aldehydes in cabin-air of commercial aircraft: Measurement by HPLC with UV absorbance detection of 2,4-dinitrophenylhydrazones by Wolfgang Rosenberger; Bibiana Beckmann; Renate Wrbitzky (117-127).
This paper presents the strategy and results of in-flight measurements of airborne aldehydes during normal operation and reported smell events on commercial aircraft. The aldehyde-measurement is a part of a large-scale study on cabin-air quality. The aims of this study were to describe cabin-air quality in general and to detect chemical abnormalities during the so-called smell-events. Adsorption and derivatization of airborne aldehydes on 2,4-dinitrophenylhydrazine coated silica gel (DNPH-cartridge) was applied using tailor-made sampling kits. Samples were collected with battery supplied personal air sampling pumps during different flight phases. Furthermore, the influence of ozone was investigated by simultaneous sampling with and without ozone absorption unit (ozone converter) assembled to the DNPH-cartridges and found to be negligible. The method was validated for 14 aldehydes and found to be precise (RSD, 5.510.6%) and accurate (recovery, 98103 %), with LOD levels being 0.30.6 μg/m3. According to occupational exposure limits (OEL) or indoor air guidelines no unusual or noticeable aldehyde pollution was observed. In total, 353 aldehyde samples were taken from two types of aircraft. Formaldehyde (overall average 5.7 μg/m3, overall median 4.9 μg/m3, range 0.444 μg/m3), acetaldehyde (overall average 6.5 μg/m3, overall median 4.6, range 0.390 μg/m3) and mostly very low concentrations of other aldehydes were measured on 108 flights. Simultaneous adsorption and derivatization of airborne aldehydes on DNPH-cartridges to the Schiff bases and their HPLC analysis with UV absorbance detection is a useful method to measure aldehydes in cabin-air of commercial aircraft.
Keywords: Aircraft; Aldehydes; Cabin-air quality; HPLCUV; Oxidative stress; Smell-events;
Two-dimensional gel electrophoretic detection of protein carbonyls derivatized with biotin-hydrazide by Jinzi Wu; Xiaoting Luo; Siqun Jing; Liang-Jun Yan (128-131).
Protein carbonyls are protein oxidation products that are often used to measure the magnitude of protein oxidative damage induced by reactive oxygen or reactive nitrogen species. Protein carbonyls have been found to be elevated during aging and in age-related diseases such as stroke, diabetes, and neurodegenerative diseases. In the present article, we provide detailed protocols for detection of mitochondrial protein carbonyls labeled with biotin-hydrazide followed by 2-dimensional isoelectric focusing (IEF)/SDS-PAGE and Western blotting probed with horse-radish peroxidase-conjugated streptavidin. The presented procedures can also be modified for detection of carbonylation of non-mitochondrial proteins.
Keywords: Biotin-hydrazide; Protein carbonyls; Carbonylation; IEF/SDS-PAGE; Mitochondria; Streptavidin; Protocol;
Development, validation and application of a fast analytical methodology for the simultaneous determination of DNA- and RNA-derived urinary nucleosides by liquid chromatography coupled to tandem mass spectrometry by Encarnación Rodríguez-Gonzalo; Leticia Herrero-Herrero; Diego García-Gómez (132-139).
In the present work we report the development and validation of a fast liquid chromatography–mass spectrometry method for the simultaneous determination of endogenous nucleosides derived from DNA and RNA in urine. The target compounds were 2′-deoxyguanosine and 8-hydroxy-2′-deoxyguanosine, derived from DNA, and the analogue 8-hydroxyguanosine, derived from RNA, together with adenosine, 1-methyladenosine, 7-methylguanosine and inosine. The method is based on the use of a chromatographic column packed with superficially porous particles for high-efficiency separation; further detection by MS/MS was accomplished with a triple quadrupole-mass spectrometer for analyte identification and accurate quantification. As a preliminary purification step, we developed a new procedure based on solid-phase extraction (SPE) with a mixed-sorbent prepared from three polymeric materials that facilitated the isolation of modified nucleosides, such as 2-deoxynucleosides, that are not retained by phenylboronic acid-based SPE. The proposed approach (SPE prior to LC–MS/MS) was validated in human urine in terms of linearity, the limit of detection, the limit of quantification, accuracy, recovery, repeatability, reproducibility and matrix-effects. For the SPE step, intra-day and inter-cartridge reproducibility were evaluated in natural and spiked urine samples, being ±16.9% or below, with recoveries in the 74–125% range. No significant matrix effects were found in further MS/MS detection. The application of the present method to urine from healthy smoker and non-smoker volunteers is also reported in order to test its usefulness as a tool for clinical and toxicological trials.
Keywords: Urinary nucleosides; Smokers; Non-smokers; Mixed-sorbent; Fast liquid chromatography; Superficially porous particles; Tandem mass spectrometry;
Determination of the oxidative stress biomarker urinary 8-hydroxy-2⿲-deoxyguanosine by automated on-line in-tube solid-phase microextraction coupled with liquid chromatographytandem mass spectrometry by Hiroyuki Kataoka; Keisuke Mizuno; Eri Oda; Akihiro Saito (140-146).
8-Hydroxy-2⿲-deoxyguanosine (8-OHdG) is a marker of oxidative DNA damage in human urine. 8-OHdG can be simultaneously analyzed with creatinine to normalize urine volume by automated on-line in-tube solid-phase microextraction (SPME) coupled with stable isotope-dilution liquid chromatographytandem mass spectrometry (LCMS/MS).Display OmittedA simple and sensitive method for the determination of 8-hydroxy-2⿲-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage in human urine, was developed using automated on-line in-tube solid-phase microextraction (SPME) coupled with stable isotope-dilution liquid chromatographytandem mass spectrometry (LCMS/MS). Creatinine was also analyzed simultaneously to normalize urine volume by the in-tube SPME LCMS/MS method, and 8-OHdG and creatinine were separated within 3 min using a Zorbax Eclipse XDB-C8 column. Electrospray MS/MS for these compounds was performed on an API 4000 triple quadruple mass spectrometer in the positive ion mode by multiple reaction monitoring. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Carboxen 1006 PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The calibration curve for 8-OHdG using its stable isotope-labeled internal standard was linear in the range of 0.0510 ng/mL, and the detection limit was 8.3 pg/mL. The intra-day and inter-day precision (relative standard deviations) were below 3.1% and 9.6% (n = 5), respectively. This method was applied successfully to the analysis of urine samples without any other pretreatment and interference peaks, with good recovery rates above 91% in spiked urine samples. The limits of quantification of 8-OHdG and creatinine in 0.1 mL urine samples were about 0.32 and 0.69 ng/mL (S/N = 10), respectively. This method was utilized to assess the effects of smoking, green tea drinking and alcohol drinking on the urinary excretion of 8-OHdG.
Keywords: 8-Hydroxy-2⿲-deoxyguanosine; Oxidative stress biomarker; In-tube solid-phase microextraction; Liquid chromatographytandem mass spectrometry;
Identification of dityrosine cross-linked sites in oxidized human serum albumin by Andrea Annibal; Graziano Colombo; Aldo Milzani; Isabella Dalle-Donne; Maria Fedorova; Ralf Hoffmann (147-155).
Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3⿲-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAVYHHFISDGVR, TEVSSNHVLIYLDK, and LVAYYTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.
Keywords: Collision induced dissociation (CID); Cross-linked protein; Dityrosine; Mass spectrometry; Protein oxidation;
Stable isotope-assisted LCMS/MS monitoring of glyceryl trinitrate bioactivation in a cell culture model of nitrate tolerance by Elizabeth R. Axton; Elizabeth A. Hardardt; Jan F. Stevens (156-163).
The nitric oxide (NO) metabolites nitrite (NO2 ) and nitrate (NO3 ) can be quantified as an endpoint of endothelial function. We developed a LCMS/MS method of measuring nitrite and nitrate isotopologues, which has a lower limit of quantification (LLOQ) of 1 nM. This method allows for isotopic labeling to differentiate newly formed nitrite and nitrate from nanomolar to micromolar background levels of nitrite and nitrate in biological matrices. This method utilizes 2,3-diaminonaphthalene (DAN) derivatization, which reacts with nitrite under acidic conditions to produce 2,3-naphthotriazole (NAT). NAT was chromatographically separated on a Shimadzu LC System with an Agilent Extend-C18 5 μm 2.1 ÿ 150 mm column and detected using a multiple reaction monitoring (MRM) method on an ABSciex 3200 QTRAP mass spectrometer operated in positive mode. Mass spectrometry allows for the quantification of 14N-NAT (m/z 170.1) and 15N-NAT (m/z 171.1). Both nitrite and nitrate demonstrated a linear detector response (1 nM 10 μM, 1 nM 100 nM, respectively), and were unaffected by common interferences (Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phenol red, and NADPH). This method requires minimal sample preparation, making it ideal for most biological applications. We applied this method to develop a cell culture model to study the development of nitrate tolerance in human endothelial cells (EA.hy926).
Keywords: Nitrite; Nitric oxide; Nitrate tolerance; LCMS/MS; Glyceryl trinitrate; Cell culture;
Evaluation of the antioxidant activity and capacity of some natural N 6-substituted adenine derivatives (cytokinins) by fluorimetric and spectrophotometric assays by Andrea Brizzolari; Cristina Marinello; Marina Carini; Enzo Santaniello; Pier Antonio Biondi (164-168).
Four natural N 6-substituted adenine derivatives (cytokinins) were evaluated for the first time in vitro for they antioxidant capacity by using fluorimetric and spectrophotometric assays, i.e., the oxygen radical absorbance capacity (ORAC), trolox equivalence antioxidant capacity (TEAC) and the 2-deoxyribose degradation (2-DRA) assays. The results from the TEAC assay show that only N 6-(4-hydroxybenzyl)adenine (p-topolin) shows an electron transfer capacity due to the presence of a phenolic moiety in the N 6-position. The results from the ORAC test show that the antioxidant activity of N 6-furfuryladenine (kinetin, K) is the highest up to a concentration of 1 μM, whereas at concentrations higher than 1 μM p-topolin is the most efficient antioxidant. Analysis of the kinetic data suggests that, compared to the other cytokinins, more sites of the molecular structure of p-topolin are available for the quenching of peroxyl radicals. The hydroxyl radical scavenger ability, as measured by the 2-DRA assay, showed that all tested cytokinins react in this test and that N 6-(Δ2-isopentenyl)adenine is slightly more potent, probably because of the allylic methylene group present in the N 6-isopentenyl moiety. Our data suggest that a part of the biological activity of the evaluated cytokinins is likely to be related to an intrinsic antioxidant capacity.
Keywords: Antioxidant activity; Cytokinins; 2-Deoxyribose degradation assay; Oxygen radical absorbance capacity (ORAC); Trolox equivalent antioxidant capacity (TEAC);
Comparison of antioxidant and antiproliferative activity between Kunlun Chrysanthemum flowers polysaccharides (KCCP) and fraction PII separated by column chromatography by Siqun Jing; Wenjie Chai; Gai Guo; Xiaoming Zhang; Jun Dai; Liang-Jun Yan (169-177).
The aim of the present study was to compare the antioxidant and antiproliferative effects on cancer cells between Kunlun Chrysanthemum flowers polysaccharides (KCCP) and its fraction PII that were separated by Biologic low pressure (LP) chromatography system followed by DEAE cellulose column chromatography. Results of in vitro experiments showed that the reducing power and the scavenging capacity of KCCP towards hydroxyl radicals (•OH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radicals increased in a concentration dependent manner and were stronger than that of fraction PII. Results of the antiproliferative effect of KCCP and fraction PII on cervical cancer HeLa cells, esophagus cancer Eca109 cells, and mouse ascites hepatomas H22 cells indicated that both KCCP and its fraction PII possessed inhibitory activity on all the tested cancer cells at a dose- and time-dependent manner, with KCCP showing higher inhibitory activity than that of fraction PII. The present study demonstrates that KCCP and its fraction PII have antioxidant properties that may help fight cancers.
Keywords: KCCP; Fraction PII; Analytical; Antioxidant; Antiproliferative effect;
A step-by-step protocol for assaying protein carbonylation in biological samples by Graziano Colombo; Marco Clerici; Maria Elisa Garavaglia; Daniela Giustarini; Ranieri Rossi; Aldo Milzani; Isabella Dalle-Donne (178-190).
Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience.
Keywords: Protein carbonylation; 2,4-Dinitrophenylhydrazine; Biotin-hydrazide; Aldehyde-reactive probe; Fluorescein-5-thiosemicarbazide;
Protocols for the measurement of the F2-isoprostane, 15(S)-8-iso-prostaglandin F2α, in biological samples by GC–MS or GC–MS/MS coupled with immunoaffinity column chromatography by Dimitrios Tsikas; Maria-Theresia Suchy (191-201).
Arachidonic acid, the origin of the eicosanoids family, occurs in biological samples as free acid and as ester in lipids. Free arachidonic acid is oxidized to numerous metabolites by means of enzymes including cyclooxygenase (COX). Arachidonic acid esterified to lipids is attacked by reactive oxygen species (ROS) to generate numerous oxidized arachidonic acid derivatives. Generally, it is assumed that ROS-derived arachidonic acid derivatives are distinct from those generated by enzymes such as COX. Therefore, ROS-generated eicosanoids are considered specific biomarkers of oxidative stress. However, there are serious doubts concerning a strict distinction between the enzyme-derived eicosanoids and the ROS-derived iso-eicosanoids. Prominent examples are prostaglandin F2α (PGF2α) and 15(S)-8-iso-prostaglandin F2α (15(S)-8-iso-PGF2α) which have been originally considered to exclusively derive from COX and ROS, respectively. There is convincing evidence that both COX and ROS can oxidize arachidonic acid to PGF2α and 15(S)-8-iso-PGF2α. Thus, many results previously reported for 15(S)-8-iso-PGF2α as exclusive ROS-dependent reaction product, and consequently as a specific biomarker of oxidative stress, require a careful re-examination which should also consider the analytical methods used to measure 15(S)-8-iso-PGF2α. This prominent but certainly not the only example underlines more than ever the importance of the analytical chemistry in basic and clinical research areas of oxidative stress. In the present work, we report analytical protocols for the reliable quantitative determination of 15(S)-8-iso-PGF2α in human biological samples including plasma and urine by mass spectrometry coupled to gas chromatography (GC–MS, GC–MS/MS) after specific isolation of endogenous 15(S)-8-iso-PGF2α and the externally added internal standard [3,3′,4,4′-2H4]-15(S)-8-iso-PGF2α by immunoaffinity column chromatography (IAC). 15(S)-8-iso-PGF2α esterified to plasma lipids is hydrolysed by KOH. 15(S)-8-iso-PGF2α and [3,3′,4,4′-2H4]-15(S)-8-iso-PGF2α are analyzed as pentafluorobenzyl ester trimethylsilyl ether derivatives in the electron-capture negative-ion chemical ionization mode.
Keywords: Biological samples; Immunoaffinity column chromatography; Mass spectrometry; Oxidative stress; Protocols; Stable isotopes;
Cholesterol as a natural probe for free radical-mediated lipid peroxidation in biological membranes and lipoproteins by Albert W. Girotti; Witold Korytowski (202-209).
We describe a relatively convenient and reliable procedure for assessing the magnitude of free radical-mediated (chain) lipid peroxidation in biological systems. The approach is based on use of radiolabeled cholesterol ([14C]Ch) as a probe and determination of well-resolved oxidation intermediates/products ([14C]ChOX species), using high performance thin layer chromatography with phorphorimaging detection (HPTLC-PI). In a lipid hydroperoxide-primed liposomal test system treated with ascorbate and a lipophilic iron chelate, the following well-resolved [14C]ChOX are detected and quantified: 7α/7β-OOH, 7α/7β-OH, and 5,6-epoxide, their levels increasing with incubation time at 37 °C. [14C]Ch also serves as an excellent probe for lipid peroxidation in lipoproteins and plasma membranes of mammalian cells. Because this approach utilizes Ch as a natural in situ probe, it eliminates potential artifacts associated with artificial probes such as spin traps and fluorophores.
Keywords: Cholesterol; Cholesterol oxides; Free radicals; Lipid peroxidation; Thin layer chromatography; Phosphorimaging;