4-Iodobenzonitrile as a fluorogenic derivatization reagent for chromatographic analysis of L-p-boronophenylalanine in whole blood samples using Suzuki coupling reaction.

BACKGROUND: L-p-Boronophehylalanine (BPA) is used in boron neutron capture therapy (BNCT), which is a novel selective cancer radiotherapy technique. It is important to measure BPA levels in human blood for effective radiotherapy; a prompt gamma-ray spectrometer, ICP-AES, and ICP-MS have been used for this purpose. However, these methods require sophisticated and expensive apparatuses as well as experienced analysts. Herein, we propose an HPLC-FL method for the determination of BPA after precolumn derivatization. A new fluorogenic reagent for aryl boronic acid derivatives, namely, 4-iodobenzonitrile, was employed for the fluorogenic derivatization of BPA based on the Suzuki coupling reaction. RESULTS: After the fluorogenic derivatization, a fluorescent cyanobiphenyl derivative is formed with maximum fluorescence at 335 nm after excitation at 290 nm. The developed method showed good linearity (r2=0.997) over the concentration range of 0.5-1000 nmol/L, and the detection limit (S/N = 3) was 0.26 nmol/L. The proposed method is more sensitive than previously reported methods for the determination of BPA, including the ICP-MS. Finally, the proposed method was successively applied to the measurement of BPA in human whole blood samples with a good recovery rate (≥95.7 %) using only 10 µL of blood sample. The proposed method offers a simple and efficient solution for monitoring BPA levels in BNCT-treated patients. SIGNIFICANCE: 4-Iodobenzonitrile was investigated as a new fluorogenic reagent for BPA based on Suzuki coupling. A new HPLC-FL method for BPA in whole blood samples with ultrasensitivity was developed. The developed method is superior in sensitivity to all previously reported methods for BPA. The method requires only a very small sample volume, making it suitable for micro-blood analysis of BPA via fingerstick sampling.

LC-MS/MS analysis of components in smoke from e-cigarettes that use guarana extract as the caffeine source.

Currently, e-cigarette products to inhale caffeine (Caf) are commercially available and widely used. Guarana extract (GE) is used as the caffeine source in some e-cigarette products. In this study, an LC-MS/MS analysis of components in the smoke from e-cigarettes with GE was performed. The concentration ranges of Caf and the minor components theophylline (TP), theobromine (TB), and paraxanthine (PX) in e-liquid and cigarette smoke extract (CSE) of five e-cigarette products were determined. The concentration ranges of e-liquid and CSE were 2.17-8.62 mg/mL and 0.17-1.17 µg/puff for Caf, 0.09-37.58 µg/mL and 0.03-11.88 ng/puff for TB, 50.28-185.26 ng/mL and 0.00-0.05 ng/puff for TP, and 0.44-4.09 µg/mL and 0.03-0.20 ng/puff for PX, respectively. By comparing the peak area ratios of e-liquid and CSE, we clarified that the heat degradation of Caf to its related components in GE products was accelerated. Epicatechin, which is another typical component in GE, was determined for CSE, but not for e-liquid.

Analysis of vaporized caffeine in smoke from e-cigarettes using liquid chromatography-tandem mass spectrometry and clarification of minor components.

PURPOSE: Electronic cigarettes (e-cigarettes) are used widely, and e-cigarettes containing caffeine (Caf) have recently become commercially available. However, no risk evaluation of these Caf-containing products has been performed to date. Such an evaluation requires a sensitive analytical method for quantifying Caf in smoke from e-cigarettes. The aim of this study was to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying vaporized Caf from commercially available e-cigarettes, and to determine minor components related to Caf in cigarette smoke extract (CSE). METHODS: A sampling system for Caf using a suction pump was designed and sampling conditions were optimized. RESULTS: The optimized LC-MS/MS conditions allowed the sensitive determination of Caf in smoke with a limit of detection of 0.03 ng/mL at a signal-to-noise ratio of 3. The method was applied to CSEs from five e-cigarette products and the concentration of Caf ranged from 0.894 ± 0.090 to 3.32 ± 0.14 µg/mL smoke (n = 3). Additionally, minor components related to Caf, such as theobromine, theophylline, and paraxanthine, were detected in CSE and in e-liquid at very low concentrations, indicating that they were impurities in e-liquid and vaporized along with Caf. CONCLUSION: This is the first report to determine the concentration of vaporized Caf using an LC-MS/MS method and to clarify several minor components in smoke from e-cigarettes.

Selective fluorescence labeling of myristicin using Mizoroki-Heck coupling reaction. Application to nutmeg powder, oil, and human plasma samples.

Myristicin [5-allyl-1­methoxy-2,3-(methylenedioxy)benzene] is the major constituent of the seasoning nutmeg oil and powder. Sometimes myristicin is abused via its ingestion at high doses to cause hallucination. In these high doses, myristicin could cause severe adverse health effects, including convulsion, delirium, and palpitation. Hence there is a strong need for a sensitive method for its analysis, such as fluorescence determination. Myristicin has a very weak fluorescence and also lacks derivatizable groups like the carboxylic, hydroxyl, or amino group in its structure, which makes its fluorescence derivatization challenging. In this research, we developed a fluorescence labeling method for myristicin based on the Mizoroki-Heck coupling reaction of its terminal alkene with a fluorescent aryl iodide derivative, 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIB-I). Then, we developed an HPLC fluorescence detection method for the determination of myristicin utilizing this labeling reaction. The developed method showed a good linear response for myristicin (r = 0.995) in the range of 0.01-10 µmol/L with excellent sensitivity down to the detection limit of 2.9 nmol/L (9.6 fmol/injection). Finally, the developed method could be successfully applied to determine myristicin content in nutmeg powder, oil samples, and human plasma with simple extraction methods and good recoveries ranging from 89.3 to 106%.

A sensitive chemiluminescence detection approach for determination of 2,4-dinitrophenylhydrazine derivatized aldehydes using online UV irradiation - luminol CL reaction. Application to the HPLC analysis of aldehydes in oil samples.

Aldehydes are toxic carbonyl compounds that are identified in various matrices surrounding us. For instance, aldehydes could be formed during the cooking and frying of foods which affects the food quality and safety. Derivatization is a must for the determination of aldehydes as they lack intrinsic chromophoric groups. 2,4-Dinitrophenyl hydrazine (DNPH) is the most used derivatizing reagent for aldehydes and the formed hydrazones could be determined by either HPLC-UV or LC-MS. However, UV detection is non-sensitive, and the MS equipment is expensive and not widely available. Thus, herein we report a smart chemiluminescence (CL) detection method for the DNPH aldehydes derivatives. These derivatives are supposed to possess photosensitization ability due to the presence of strong chromophoric structures; nitrobenzene and phenyl hydrazone. Upon their UV irradiation, singlet oxygen is found to be produced which then converts the DNPH-aldehyde derivative into hydroperoxide. Next, the hydroperoxide reacts with luminol in an alkaline medium producing a strong CL. An HPLC system with online UV irradiation and online reaction with luminol followed by CL detection was constructed and used for the determination of aldehydes after their derivatization with DNPH. The developed method showed excellent sensitivity with detection limits down to 1.5-18.5 nM. The achieved sensitivity is superior to that obtained by HPLC-UV and LC-MS detection methods for DNPH-aldehydes derivatives. Additionally, our approach is an chemiluminogenic where the DNPH reagent itself does not produce CL which is an excellent advantage. The method was applied successfully for the determination of aldehydes in canola oil samples using simple liquid-liquid extraction showing good recovery (87.0-106.0%), accuracy (87.2-106.6), and precision (RSD≤10.2%). After analysis of fresh and heated oil samples, it was demonstrated that heating of oil, even for short time, strongly elevated the level of their aldehydes' content. At last, it was found that the results of the analysis of aldehydes in oil samples using the proposed method perfectly matched those obtained by a reference LC-MS method.

A Smart Advanced Chemiluminescence-Sensing Platform for Determination and Imaging of the Tissue Distribution of Natural Antioxidants.

Antioxidants have gained marked attention owing to their ability to prevent the oxidation of biological components and to protect the body from reactive oxygen species, thereby maintaining human health. Thus, antioxidant-rich dietary supplements and natural foods can be effective against oxidative stress and can even act as chemopreventive agents. Therefore, a simple and rapid assay for evaluation of antioxidant capacity and assessment of their distribution profile in natural sources is vital. Herein, we report a rapid, innovative chemiluminescence (CL) platform for evaluation and visualization of antioxidant capacity. We found that intense and long-lasting CL was formed upon the redox reaction of quinones, e.g., menadione, with antioxidants, e.g., l-ascorbic acid, in the presence of luminol. The produced CL intensities were proportional to the antioxidants' concentrations with a detection limit of 0.18 µM for the model antioxidant, l-ascorbic acid. As the formed CL was long-lasting, it could be easily captured and detected with a charge-coupled device (CCD) camera. To evaluate the quantification ability of the CCD camera, we developed a smart and fast microplate-based assay based on photographing the generated CL with a cooled CCD camera. The photographed CL intensities were linearly proportional with the antioxidant concentrations, and then the method was applied for photographing multiple food sample extracts. Ultimately, we utilized our method for the distribution profiling of antioxidant capacity in food cut sections. Samples were dipped in luminol and then in quinone, followed by CCD camera photography, without the need for any pulverization/extraction procedure, giving precise antioxidant distribution information.

Selective, sensitive and comprehensive detection of immune complex antigens by immune complexome analysis with papain-digestion and elution.

Comprehensive identification and profiling of antigens in immune complexes (ICs) in biological fluids, such as serum and cerebrospinal fluid, is useful for developing early diagnostic markers and specific treatments for many diseases. We have developed a method, designated "immune complexome analysis", to comprehensively identify the antigens in ICs. In this method, we first purify ICs from biological fluid by using Protein G- or Protein A-coated beads, then these ICs are subjected to tryptic digestion on the beads and subsequent analysis using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We previously used this method to find specific antigens in circulating ICs (CIC-antigens) in serum for autoimmune diseases, infectious disease and cancers. However, this method detects not only CIC-antigens but also antibodies and proteins bound non-specifically to the beads, which restricts the detection of minor peptides released by the digestion of CIC-antigens whose amounts are generally much less than antibodies and the proteins. To selectively detect CIC-antigens with enhanced sensitivity, in this study we compared three methods (Method A, direct tryptic digestion on the beads; Method B, low-pH elution and tryptic digestion; Method C, papain-digestion, elution, and tryptic digestion) and examined which method selectively elutes CIC-antigens from CICs bound to the beads and selectively detects CIC-antigens using nano-LC-MS/MS. We also compared three types of CIC-capturing beads (Protein G-coated magnetic beads, Protein A-coated magnetic beads and Proceptor™-sepharose beads) to examine if parallel use of these beads aids the comprehensive detection of CIC-antigens in immune complexome analysis. Comparison showed that Method C provided the most selective and sensitive detection of CIC-antigens, without interference by antibodies and proteins non-specifically bound to the beads. In addition, using three types of beads allowed the examination of a wide range of CIC-antigens in immune complexome analysis. Therefore, combining Method C with three types of beads should allow the selective and sensitive identification of IC-antigens present in biological fluids from patients with a variety of diseases. The identification of IC-antigens may lead to the development of diagnostic methods and protocols for specific treatments for these diseases.

Immune complexome analysis reveals the specific and frequent presence of immune complex antigens in lung cancer patients: A pilot study.

Cancer immunotherapies such as antibodies targeting T cell checkpoints, or adaptive tumor-infiltrating lymphocyte (TIL) transfer, have been developed to boost the endogenous immune response against human malignancies. However, activation of T cells by such antibodies can lead to the risk of autoimmune diseases. Also, the selection of tumor-reactive T cells for TIL relies on information regarding mutated antigens in tumors and does not reflect other factors involved in protein antigenicity. It is therefore essential to engineer therapeutic interventions by which T cell reactivity against tumor cells is selectively enhanced (i.e., "focused cancer immunotherapy") based on tumor antigens that are specifically expressed in the tumor of a certain cancer and in many patients with this cancer. Immune complexes (ICs) are the direct and stable products of immunological recognition by humoral immunity. Here, we searched for tumor-specific IC antigens in each of five cancers (lung (n = 28), colon (n = 20), bladder (n = 20), renal cell (n = 15) and malignant lymphoma (n = 9)), by using immune complexome analysis that comprehensively identifies and profiles the constituent antigens in ICs. This analysis indicated that gelsolin and inter-alpha-trypsin inhibitor heavy chains were specifically and frequently detected (at a frequency higher than 80%), and that phosphoproteins (VENTX, VCIP135) were also specifically present in the ICs of lung cancer patients. Immune complexome analysis successfully identified several tumor-specific IC antigens with high detection frequency in lung cancer patients. These specific antigens are required to validate the clinical benefit by further analysis using a large number of patients.

Quinones as novel chemiluminescent probes for the sensitive and selective determination of biothiols in biological fluids.

Altered plasma aminothiol concentrations are thought to be a valuable risk indicator and are interestingly utilized for routine clinical diagnosis and for the monitoring of various metabolic disorders and human diseases, and accordingly there is a need for an accurate and reliable assay capable of simultaneously determining aminothiols including glutathione (GSH), N-acetylcysteine (NAC), homocysteine (Hcys), and cysteine (Cys) in human plasma. Herein, a highly sensitive, selective, and very fast HPLC-chemiluminescence (HPLC-CL) coupled method is reported, exploiting for the first time the strong nucleophilicity and high reactivity of aminothiols toward quinones for a CL assay. The unique redox-cycling capability of quinone and/or Michael addition adducts, thioether-quinone conjugates, was utilized to establish a novel analytical method based on the reaction of adducts with dithiothreitol (DTT) to liberate reactive oxygen species (ROS), which are detected by using a luminol-CL assay. Specimen preparation involved the derivatization of aminothiols with menadione (MQ) for 5 minutes at room temperature. A unique green chemistry synthesis of thioether-quinones in HEPES buffer (pH 8.5) was introduced by using our reaction methodology without needing any hazardous organic solvent or catalyst. The aminothiol-MQ adducts were separated using solid-phase extraction followed by isocratic elution on an ODS column. Linearity was observed in the range of 2.5-500, 5-500, 10-1500, and 20-2000 nM with detection limits (S/N of 3) of 3.8, 4.2, 8, and 16 (fmol per injection) for GSH, NAC, Hcys, and Cys, respectively. The method was successfully applied for the selective determination of aminothiols in human plasma from healthy people and patients with rheumatic arthritis and diabetes mellitus. The obtained results postulated the usefulness of our method for investigating the relationship between aminothiol metabolism and related human disorders.

Characterization of quinone derived protein adducts and their selective identification using redox cycling based chemiluminescence assay.

The cytotoxic mechanism of many quinones has been correlated to covalent modification of cellular proteins. However, the identification of relevant proteins targets is essential but challenging goals. To better understand the quinones cytotoxic mechanism, human serum albumin (HSA) was incubated in vitro with different concentration of menadione (MQ). In this respect, the initial nucleophilic addition of proteins to quinone converts the conjugates to redox-cycling quinoproteins with altered conformation and secondary structure and extended life span than the short lived, free quinones. The conjugation of MQ with nucleophilic sites likewise, free cysteine as well as ɛ-amino group of lysine residue of HSA has been found to be in concentration dependent manner. The conventional methods for modified proteins identification in complex mixtures are complicated and time consuming. Herein, we describe a highly selective, sensitive, simple, and fast strategy for quinoproteins identification. The suggested strategy exploited the unique redox-cycling capability of quinoproteins in presence of a reductant, dithiothreitol (DTT), to generate reactive oxygen species (ROS) that gave sufficient chemiluminescence (CL) when mixed with luminol. The CL approach is highly selective and sensitive to detect the quinoproteins in ten-fold molar excess of native proteins without adduct enrichment. The approach was also coupled with gel filtration chromatography (GFC) and used to identify adducts in complex mixture of proteins in vitro as well as in rat plasma after MQ administration. Albumin was identified as the main protein in human and rat plasma forming adduct with MQ. Overall, the identification of quinoproteins will encourage further studies of toxicological impact of quinones on human health.

Determination of 4-hydroxy-2-nonenal in serum by high-performance liquid chromatography with fluorescence detection after pre-column derivatization using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2, 1,3-benzoxadiazole.

4-Hydroxy-2-nonenal (4HNE) is a major aldehyde generated during lipid peroxidation. The clinical monitoring of 4HNE in biological fluids should be useful for the early diagnosis of several diseases involving lipid peroxidation, such as rheumatoid arthritis, Parkinson's disease and cancer. In this study, an HPLC with fluorescence detection method was developed for the determination of 4HNE in human serum. The proposed method involves the extraction of 4HNE from human serum by sub-zero temperature extraction and fluorescent labeling of 4HNE with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2, 1,3-benzoxadiazole. The lower detection limit (signal-to-noise ratio = 3) of the method was 0.06 µm in serum. The proposed method was successfully applied to the measurement of 4HNE in sera obtained from patients with rheumatoid arthritis.

Determination of 4-hydroxy-2-nonenal in serum by high-performance liquid chromatography with fluorescence detection after pre-column derivatization using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole.

4-Hydroxy-2-nonenal (4HNE) is a major aldehyde generated during lipid peroxidation. The clinical monitoring of 4HNE in biological fluids could be useful for the early diagnosis of several diseases involving lipid peroxidation, such as rheumatoid arthritis, Parkinson's disease and cancer. In this study, an HPLC with fluorescence detection method was developed for the determination of 4HNE in human serum. The proposed method involves the extraction of 4HNE from human serum by subzero temperature extraction and fluorescent labeling of 4HNE with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole. The lower detection limit (signal-to-noise ratio=3) of the method was 0.06 µm in serum. The proposed method was successfully applied to the measurement of 4HNE in sera obtained from patients with rheumatoid arthritis.

Development and validation of the first assay method coupling liquid chromatography with chemiluminescence for the simultaneous determination of menadione and its thioether conjugates in rat plasma.

Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-l-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-l-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5-120 nM for MQ-GS and MQ-NAC and 10-240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.

Monolithic poly(butyl methacrylate-ethylene dimethacrylate-methacrylic acid) column for capillary electrochromatography.

A butyl methacrylate-ethylene dimethacrylate-methacrylic acid (MAA) monolithic column was prepared for capillary electrochromatography. The effect of MAA weight percentage on the EOF mobility and column efficiency was studied. BMA-EDMA-MAA monolith with higher content of MAA provided higher EOF mobility as well as higher efficiency. The effect of mobile-phase composition and buffer pH was also investigated. The monolithic columns exhibited a good repeatability and reproducibility of column preparation with relative standard deviation values below 16% in the studied chromatographic parameters.

Selective chemiluminescence method for monitoring of vitamin K homologues in rheumatoid arthritis patients.

Vitamin K is a fat-soluble vitamin involved in blood coagulation and bone metabolism. The detection and monitoring of vitamin K homologues in rheumatoid arthritis (RA) patients is a challenging problem due to the smaller concentrations of vitamin K and the presence of several interfering medications. Therefore, this study aimed to develop a new highly sensitive and selective chemiluminescence (CL) method designated to quantify vitamin K homologues in plasma of RA patients including phylloquinone (PK, vitamin K(1)), menaquinone-4 (MK-4, vitamin K(2)) and menaquinone-7 (MK-7, vitamin K(2)). The method was based on the unique photochemical properties of vitamin K homologues that were exploited for selective luminol CL reaction. The correlation coefficients of 0.998 or more were obtained in the concentration ranges of 0.1-100 ng mL(-1) vitamin K homologues. The detection limits were 0.03-0.1 ng mL(-1) in human plasma for vitamin K homologues. The developed HPLC-CL system was successfully applied for selective determination of vitamin K homologues in plasma of RA patients. The developed method may provide a useful tool for monitoring vitamin K homologues in different clinical studies such as RA, osteoporosis and hepatocellular carcinoma in which vitamin K is intervented.

Immune complexome analysis of serum and its application in screening for immune complex antigens in rheumatoid arthritis.

BACKGROUND: Analysis of circulating immune complexes (CICs) produced during an immune response may be useful in elucidating some aspects of this process. Identification of antigens incorporated into CICs provides information that may be helpful in developing diagnostic and treatment strategies for autoimmune diseases, infection, cancer, and transplantation therapy, and such information might be more relevant than information on free antigens. Because CICs may contain many antigens, comprehensive identification and profiling of such antigens is more effective than immunoblotting detection. METHODS: We developed a novel proteomic strategy (immune complexome analysis) in which immune complexes (ICs) are separated from serum, digested directly with trypsin, and then subjected to nano-liquid chromatography-tandem mass spectrometry for identifying and profiling antigens in CICs. We applied this strategy to the analysis of CICs in 21 rheumatoid arthritis (RA) patients. Serum samples from 13 healthy donors and 8 osteoarthritis patients were used as controls. RESULTS: CICs containing thrombospondin-1 (TSP-1) and platelet factor 4 (PF4) were found in the serum of 81% and 52% of RA patients, respectively, and in none of the controls. CONCLUSIONS: The ICs in the serum of a majority of the RA patients contained TSP-1 or PF4, and these ICs may have potential as alternative biomarkers. Our technique for immune complexome analysis uses routine clinical samples, simple protocols, and widely available equipment. This method may be generally applicable to the study of the relationship between CICs and certain diseases associated with the immune response in animals and humans.

A toxicoproteomic study on cardioprotective effects of pre-administration of docetaxel in a mouse model of adriamycin-induced cardiotoxicity.

Studies suggest that pre-administration of docetaxel (DOC) in adriamycin (ADR)-DOC combination anticancer therapy results in stronger antitumor effects and fewer ADR-induced cardiotoxic deaths in mouse model, yet no mechanism explaining this effect has been established. The aim of this study was to identify cellular processes in mouse heart tissue affected by different ADR/DOC dosing protocols using a toxicoproteomic approach. We applied fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) - which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry - to the proteomic analysis of heart tissue from control, intermittent-dosing (DOC-ADR), and simultaneous-dosing (ADR&DOC) groups. In DOC-ADR group, ADR was administered 12h after DOC injection; in ADR&DOC group, both drugs were administered simultaneously; in control group, saline was administered at the same timing as ADR injection of other groups. Heart samples were isolated from all mice 1 week after the treatment. The highly reproducible and sensitive method (FD-LC-MS/MS) identified nine proteins that were differentially expressed in heart tissue of control and the two treatment groups; seven of these nine proteins participate in cellular energy production pathways, including glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. Significantly higher expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed in the DOC-ADR group, the group with the fewer cardiotoxic deaths, than in the ADR&DOC group. Therefore, GAPDH may have potential as a drug target for protective intervention and a biomarker for evaluation of the cardioprotective effects in pre-clinical studies.

Automated analysis of the serum antioxidative activities against five different reactive oxygen species by sequential injection system with a chemiluminescence detector.

BACKGROUND: There is a growing evidence that reactive oxygen species (ROS) may cause many pathologic conditions including chronic diseases, neurodegenerative disorders, cancer and aging. There are a number of methods to measure the total antioxidative activity of the serum. However, since the lifetime and oxidative activity of various types of ROS are all different, to measure simply the total antioxidative activity of the serum is not enough. Therefore, to aid the diagnosis and to improve the therapeutic strategy, it is important to develop a simple and reliable method of assaying antioxidative activity of the serum. METHODS: A method that combines sequential injection analysis (SIA) and luminol chemiluminescence (CL) detection was employed for the measurement of antioxidative activities of human serum. We collected sera from healthy subjects (n=42) and patients with diabetes (n=39) and rheumatoid arthritis (n=25) and tested the sensitivity, reproducibility and reliability of our method. RESULTS: Since the operation is automatically controlled by a personal computer, we obtained a satisfactory repeatability without the need of much manpower. The time required for obtaining the antioxidative activity against one ROS for one individual is less than 3min. Although the value of antioxidative activity varies from subject to subject, there were a certain relationship between the disease and the antioxidative values of each type of ROS. The results suggest that the measurement of antioxidative activity against different ROS may provide us with valuable information regarding the disease state. CONCLUSIONS: The evaluation of antioxidative activities against each ROS by the proposed method should be more informative to understand the antioxidative status of biological fluid.

Preparation and characterization of poly(l-phenylalanine) chiral stationary phases with varying peptide length.

Three new chiral stationary phases with different lengths of l-phenylalanine peptide were prepared by solid-phase synthesis with tert-butoxycarbonyl (Boc)-l-phenylalanine on silica. The effect of phenylalanine peptide length on enantioselectivity was studied. The best separation of R/S-warfarin was achieved by the chiral stationary phase with intermediate peptide length. These stationary phases were found to exist mainly in alpha-helical conformation by using FT-IR spectra. The end-capping reagents for the N-terminus of the peptide were also evaluated.

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel.

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.