HPLC-electrospray Ionization Tandem Mass Spectrometry Research Articles

Assessment of Rhamnus alaternus Leaves Extract: Phytochemical Characterization and Antimelanoma Activity.

Rhamnus alaternus (Rhamnaceae) has been used as a laxative, purgative, diuretic, antihypertensive, and depurative. However, few scientific research studies on its antimelanoma activity have been reported. This study aimed to investigate the in vitro antimelanoma effect of an enriched total oligomer flavonoid (TOF) extract, from R. alaternus, and to identify its phytochemical compounds. The chemical composition of TOF extract was assessed by HPLC-electrospray ionization tandem mass spectrometry (HPLC/ESI-MS2) analysis. Antimelanoma activity was determined on cultured tumor cell B16F10 by the crystal violet assay, the alkaline comet assay, acridine orange/ethidium bromide (AO/EB), annexin V-fluorescein isothiocyanate/ propidium iodide (V-FITC/PI) staining, the cell cycle distribution, and the wound healing assay. Regarding chemical composition, a mixture of quercetin diglucoside, quercetin-3-O-neohesperidoside, kaempferol-3-O-(2G-α-L-rhamnosyl)-rutinoside, rhamnetin hexoside, kaempferol-3-O-rutinoside, rhamnocitrin hexoside, pilosin hexoside, apigenin glucoside, and kaempferol-3-O-glucoside was identified as major phytochemical compounds of the extracts. TOF extract inhibits melanoma B16F10 cell proliferation in dose-dependent manner. The induction of apoptosis was confirmed by comet assay, AO/EB, and annexin V-FITC/PI test. TOF extract could also induce S phase cell cycle, inhibit, and delay the cell migration of B16F10 cells. The findings showed that TOF extract from R. alaternus could be a potentially good candidate for future use in alternative antimelanoma treatments.

Short communication: Space allocation in intensive Mediterranean buffalo production influences the profile of functional biomolecules in milk and dairy products

The aim of the present study was to determine if space allocation influenced the concentration of biomolecules in buffalo milk and dairy products. Intensively housed buffaloes (n = 96) were randomly assigned to 2 groups according to days in milk, parity, and milk yield: group S10 had a space allocation of 10 m2 per buffalo and group S15 had a space allocation of 15 m2 per buffalo. Individual milk yield was recorded daily. Twice a month, a bulk milk sample was collected for each group, as well as whey, ricotta, and mozzarella cheese, to assess cheese yield and to conduct HPLC-electrospray ionization-tandem mass spectrometry, milk antioxidant activity, and cell viability analyses. We tested milk extracts from the 2 groups in vitro to evaluate their efficacy in counteracting endothelial oxidative damage induced by high glucose. We evaluated reproductive function in 28 buffaloes from each group using the Ovsynch-timed artificial insemination program. We observed no differences in milk quantity or quality in terms of fat, protein, or lactose, and reproductive function did not differ between the 2 groups. Compared with group S10, group S15 had higher concentrations of carnitine (56.7 ± 1.1 vs. 39.8 ± 0.7 mg/L in milk and 40.9 ± 0.8 vs. 31.7 ± 0.7 mg/L in whey), acetyl-l-carnitine (51.9 ± 0.3 vs. 39.7 ± 0.7 mg/L in milk and 41.1 ± 1.7 vs. 28.7 ± 2.6 mg/L in whey), propionyl-l-carnitine (34.8 ± 1.0 vs. 21.0 ± 0.9 mg/L in milk and 26.9 ± 0.8 vs. 17.6 ± 1.2 mg/L in whey), glycine betaine (23.1 ± 1.9 vs. 13.5 ± 1.6 mg/L in milk and 10.7 ± 0.4 vs. 7.9 ± 0.5 mg/L in whey), and δ-valerobetaine (24.2 ± 0.5 vs. 16.7 ± 0.5 mg/L in milk and 22.0 ± 0.9 vs. 15.5 ± 0.7 mg/L in whey). Group S15 also had higher total antioxidant activity than group S10 (56.7 ± 1.9 vs. 46.4 ± 1.13 mM Trolox equivalents). Co-incubation of high-glucose-treated endothelial cells with milk extracts from group S15 improved cell viability compared with cells treated with high glucose only; it also reduced intracellular lipid peroxidation (144.3 ± 0.4 vs. 177.5 ± 1.9%), reactive oxygen species (141.3 ± 0.9 vs. 189.3 ± 4.7 optical density units), and cytokine release (tumor necrosis factor-α, IL-1β, IL-6). Greater space allocation was associated with higher levels of biomolecules in buffalo milk. This could have been the result of improved welfare in buffaloes that were allocated more space.

A Validated HPLC-MS/MS Method for Simultaneous Determination of Militarine and Its Three Metabolites in Rat Plasma: Application to a Pharmacokinetic Study.

A rapid, reliable, and sensitive HPLC-electrospray ionization-tandem mass spectrometry (HPLC-MS/MS) method was established and validated for simultaneous determination of militarine and its three metabolites (gastrodin, α-isobutylmalic acid, and gymnoside I) in rat plasma. Plasma was acidified with formic acid, and protein was precipitated with methanol. MS/MS with ESI and multiple reaction monitoring at m/z 725.3→457.3, 457.1→127, 304.3→107.2, 189→129, and 417.1→267.1 was used for determination of militarine, gastrodin, α-isobutylmalic acid, gymnoside I, and puerarin (internal standard), respectively. Chromatographic separation was conducted using an ACE UltraCore SuperC18 (2.1 × 100 mm, 2.5 μm) column with gradient mobile phase (0.1% formic acid in water and acetonitrile). The lower limits of quantitation for militarine, gastrodin, α-isobutylmalic acid, and gymnoside I were 1.02, 2.96, 1.64, and 0.3 ng/mL, respectively. The relative standard deviations of intra- and interday measurements were less than 15%, and the method accuracy ranged from 87.4% to 112.5%. The extraction recovery was 83.52%-105.34%, and no matrix effect was observed. The three metabolites (gastrodin, α-isobutylmalic acid, and gymnoside I) were synchronously detected at 0.83 h, suggesting that militarine was rapidly transformed to gastrodin, α-isobutylmalic acid, and gymnoside I. Moreover, the area under the curve (AUC) and Cmax of militarine were significantly lower than those of gastrodin and α-isobutylmalic acid, showing that militarine was largely metabolized to gastrodin and α-isobutylmalic acid in vivo. The studies on pharmacokinetics of militarine and its three metabolites were of great use for facilitating the clinical application of militarine and were also highly meaningful for the potential development of militarine.

Canis familiaris tissues are characterized by different profiles of cytokinins typical of the tRNA degradation pathway.

Cytokinins (CKs) are a group of phytohormones essential to plant growth and development. The presence of these N6-modified adenine derivatives has also been documented in other groups of organisms, including bacteria, fungi, and insects. Thus far, however, only a single CK, N6-(Δ2-isopentenyl) adenine-9-riboside (iPR), has been identified in mammals. In plants, the nucleotide form of isopentenyladenine [iPR (either mono-, di-, or tri-) phosphate (iPRP)] is the first form of CK synthesized, and it is further modified to produce other CK types. To determine if a similar biosynthesis pathway exists in mammals, we tested for the presence of 27 CKs in a wide selection of canine organs using HPLC electrospray ionization-tandem mass spectrometry. Seven forms of CK were detected in the majority of the analyzed samples, including iPR, iPRP, cis-zeatin-9-riboside, cis-zeatin-9-riboside-5' (either mono-, di-, or triphosphate), 2-methylthio-N6-isopentenyladenine, 2-methylthio-N6-isopentenyladenosine, and 2-methylthio-zeatin. Total CK concentrations ranged from 1.96 pmol/g fresh weight (adrenal glands) to 1.40 × 103 pmol/g fresh weight (thyroid). The results of this study provide evidence that mammalian cells, like plant cells, can synthesize and process a diverse set of CKs including cis- and methylthiol-type CKs.-Seegobin, M., Kisiala, A., Noble, A., Kaplan, D., Brunetti, C., Emery, R. J. N. Canis familiaris tissues are characterized by different profiles of cytokinins typical of tRNA degradation pathway.

Trichloroethylene and Parkinson's Disease: Risk Assessment.

This study was conducted to investigate the mechanism of action and extent of selective dopaminergic neurodegeneration caused by exposure to trichloroethylene (TCE) leading to the endogenous formation of the neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in rodents. Beginning at 3months of age, male C57BL/6 mice received oral TCE dissolved in vehicle for 8months. Dopaminergic neuronal loss was assessed by nigral tyrosine hydroxylase (TH) immunoreactivity. Selective dopaminergic neurodegeneration was determined based on histological analysis of non-dopaminergic neurons in the brain. Behavioral assays were evaluated using open field activity and rotarod tests. Mitochondrial complex I activity, oxidative stress markers, and microglial activation were also examined in the substantia nigra. The level of TaClo was detected using HPLC-electrospray ionization tandem mass spectrometry. Dopaminergic neurotoxicity of TaClo was determined in midbrain organotypic cultures from rat pups. Following 8months of TCE treatment, there was a progressive and selective loss of 50% of the dopaminergic neurons in mouse substantia nigra (SN) and about 50% loss of dopamine and 72% loss of 3,4-dihydroxyphenylacetic acid in the striatum, respectively. In addition, motor deficits, mitochondrial impairment, oxidative stress, and inflammation were measured. TaClo content was quantified in the brain after TCE treatment. In organotypic cultures, TaClo rather than TCE induced dopaminergic neuronal loss, similar to MPP+. TCE exposure may stimulate the endogenous formation of TaClo, which is responsible for dopaminergic neurodegeneration. However, even prolonged administration of TCE was insufficient for producing a greater than 50% loss of nigral dopamine neurons, indicating that additional co-morbid factors would be needed for mimicking the profound loss of dopamine neurons seen in Parkinson's disease.

Stimulation of diuresis and natriuresis by renomedullary infusion of a dual inhibitor of fatty acid amide hydrolase and monoacylglycerol lipase.

The renal medulla, considered critical for the regulation of salt and water balance and long-term blood pressure control, is enriched in anandamide and two of its major metabolizing enzymes, cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH). Infusion of anandamide (15, 30, and 60 nmol·min-1·kg-1) into the renal medulla of C57BL/6J mice stimulated diuresis and salt excretion in a COX-2- but not COX-1-dependent manner. To determine whether endogenous endocannabinoids in the renal medulla can elicit similar effects, the effects of intramedullary isopropyl dodecyl fluorophosphate (IDFP), which inhibits the two major endocannabinoid hydrolases, were studied. IDFP treatment increased the urine formation rate and sodium excretion in a COX-2- but not COX-1-dependent manner. Neither anandamide nor IDFP affected the glomerular filtration rate. Neither systemic (0.625 mg·kg-1·30 min-1 iv) nor intramedullary (15 nmol·min-1·kg-1·30 min-1) IDFP pretreatment before intramedullary anandamide (15-30 nmol·min-1·kg-1) strictly blocked effects of anandamide, suggesting that hydrolysis of anandamide was not necessary for its diuretic effect. Intramedullary IDFP had no effect on renal blood flow but stimulated renal medullary blood flow. The effects of IDFP on urine flow rate and medullary blood flow were FAAH-dependent as demonstrated using FAAH knockout mice. Analysis of mouse urinary PGE2 concentrations by HPLC-electrospray ionization tandem mass spectrometry showed that IDFP treatment decreased urinary PGE2 These data are consistent with a role of FAAH and endogenous anandamide acting through a COX-2-dependent metabolite to regulate diuresis and salt excretion in the mouse kidney.

High performance liquid chromatography (HPLC) fingerprints and primary structure identification of corn peptides by HPLC-diode array detection and HPLC-electrospray ionization tandem mass spectrometry

Corn peptides (CPs) are reported to have many biological functions, such as facilitating alcohol metabolism, antioxidation, antitumor, antihypertension, and hepatoprotection. To develop a method for quality control, the high-performance liquid chromatography (HPLC) system was applied. Twenty-eight common peaks were found in all the CPs of corn samples from Enshi, China, based on which, a fingerprinting chromatogram was established for use in quality control in future research. Subsequently, the major chemical constituents of these common peaks were identified respectively using the HPLC-diode-array detection electrospray ionization tandem mass spectrometry (DAD-ESI-MS/MS) system, and 48 peptide fractions were determined ultimately. This was the first time for the majority of these peptides to be reported, and many of them contained amino acids of glutamine (Q), L and A, which might play an important role in the exhibition of the bioactivities of CPs. Many peptides had a similar primary structure to the peptides which had been proven to be bioactive such as facilitating alcohol metabolism, scavenging free radicals, and inhibiting lipid peroxidation. This systematical analysis of the primary structure of CPs facilitated subsequent studies on the relationship between the structures and functions, and could accelerate holistic research on CPs.

Development of metallomics research on environmental toxicology

Metallomics is newly coined terms and defined as a comprehensive analysis of the entirety of metal and metalloid species within a cell or tissue type. Then, metallome is defined as the entire category of metalloproteins and any other metal-containing biomolecules. Metallomics and research on metallome require analytical techniques that can provide information on the identification and quantification of metal/metalloid-containing biomolecules. This concept has been called speciation, and the acquisition of data according to the concept is performed using hyphenated techniques involving both separation and detection methods. In this review, the author intends to present several applications of complementary use of HPLC-inductively coupled plasma mass spectrometry (ICP-MS) and HPLC-electrospray ionization tandem mass spectrometry for identification of unknown selenium-containing metabolites, and also to present a newly developed technique, capillary LC-ICP-MS to be used for the analysis of metal-binding proteins.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Quantification of acylglycines in human urine by HPLC electrospray ionization-tandem mass spectrometry and the establishment of pediatric reference interval in local Chinese

Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of metabolisms (IEMs). However, without metabolic decompensation, these parameters are often not helpful. On the other hand, in cases of IEM, acylglycines are consistently raised even when patients appear to be in remission. This study aims to set-up a simple liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method for the determination of urine acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM. In addition, local reference intervals for various acylglycines are established by using this method. Acylglycines were isolated by solid-phase extraction, derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS. Acylglycines were quantified with deuterated internal standards. Mean recoveries of acylglycines ranged from 90.2 to 109.3%. Within- and between-run imprecisions for all acylglycines have CVs less than 10%. Linear regression coefficients were greater than 0.99. Reference intervals were established according to CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age. The distributions of AG in the “normal” urine were skewed towards the right. After log transformation, all the results were normally distributed. Partitioning into age group reference intervals was not indicated, according to the Harris and Boyd approach. In this context, a single reference interval for each acylglycine could be used. This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urine organic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.

The Identification of Raptor as a Substrate for p44/42 MAPK

The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.

Intestinal transit and systemic metabolism of apple polyphenols

Apples are the most widely consumed fruits in Germany and various other countries. Positive health effects of apple-derived polyphenols in vivo depend on their absorption, metabolism, distribution, and elimination from the body after consumption. Data on the metabolism of these polyphenols in humans are scarce. In order to study the intestinal transit and metabolism of apple polyphenols in humans, a variety of experiments were carried out. Polyphenols were incubated with saliva (for 5 min), simulated gastric or duodenal juice (4 or 10 h, respectively), or rat hepatocytes (4 h) under aerobic conditions, and with ileostomy fluid under aerobic conditions for 10 h. The polyphenol profile in human serum (8 h later) and renal elimination in urine (24 h later) were also investigated after consumption of 1 L apple juice. Polyphenols and their metabolites were identified and quantified by high-performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-electrospray ionization-tandem mass spectrometry (ESI-MS/MS), and gas chromatography (GC)-MS. In the presence of native saliva or ileostomy fluid, β-glycosides of phloretin and quercetin were hydrolyzed, to varying degrees depending on the sugar moiety, and to much lesser degrees in the presence of antibiotics. In the gastric milieu, almost complete degradation of procyanidin B(2) to (-)-epicatechin was observed. In the presence of artificial duodenal juice flavan-3-ol epimerization occurred. Quercetin was completely converted to phloroglucinol, 3,4-dihydroxybenzoic acid, and 2,4,6-trihydroxybenzoic acid. Formation of isomeric products of hydroxycinnamic acid esters and their corresponding methyl esters was also observed, and similar results were obtained after incubation with rat hepatocytes. Products of phase II metabolism, two phloretin O-glucuronides and eight (methyl) quercetin O-glucuronides, were identified in the hepatocyte samples. Following enzymatic hydrolysis, 5-caffeoylquinic acid, 4-p-coumaroylquinic acid, caffeic acid, (-)-epicatechin, phloretin, and quercetin were recovered in both serum and urine (5.3% and 3.5% of the amounts consumed, respectively). In addition, 19.5% of the polyphenols consumed were identified in the urine in the form of hydroxylated phenolic and hippuric acids. The findings relating to the absorption, metabolism, and systemic availability of polyphenols in vivo should contribute to our understanding of their biological effects, and the characterization of newly formed metabolites should facilitate further studies.

Proteome of the Large Pseudomonas Myovirus 201φ2-1: DELINEATION OF PROTEOLYTICALLY PROCESSED VIRION PROTEINS

Pseudomonas chlororaphis phage 201 phi 2-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 201 phi 2-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions.

Liquid chromatography/mass spectrometry-based structural analysis of new platycoside metabolites transformed by human intestinal bacteria

Platycosides, the main active constituents of Platycodi Radix, have been thoroughly studied for the characterization of their potent biological activities. However, metabolism of platycosides has not yet been characterized. A HPLC electrospray ionization-tandem mass spectrometry (LC/ESI-MS n ) approach was applied to new complex platycoside metabolites transformed by human intestinal bacteria to identify their structures and determine metabolic pathway. The molecular weights of metabolites were identified by LC/ESI-MS analysis in both positive and negative modes. Structures for the platycoside metabolites were proposed by the molecular weights and the expected enzymatic activity of intestinal microbes on platycoside. In the second step, successive LC–MS n analysis was used to demonstrate the proposed structures. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M+Na] + ions exclusively showed signals, consistent with the cleavage of glycoside bonds, rearrangement and some cross-ring cleavage, thus allowing the rapid identification of platycoside metabolites. The metabolites identified in the time-dependent metabolism experiments enable us to propose several microbial pathways for platycosides. Even though the metabolites of some platycosides may have unknown structures and low levels, the analytical tools presented in this study made it possible to obtain a rapid and complete characterization of new metabolites and their metabolism pathway in human intestinal bacteria.

Evaluation of a Deuterium-Labeled Internal Standard for the Measurement of Sirolimus by High-Throughput HPLC Electrospray Ionization Tandem Mass Spectrometry

Matrix effects in HPLC-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS)1 can cause differences in the ionization of an internal standard (IS) compared with the analyte of interest. Unless sample cleanup or chromatographic conditions eliminate or minimize ion suppression or enhancement, variability in interpatient matrices may cause erroneous results. A stable isotope-labeled IS can be used to minimize analytical interpatient variation. We used protein precipitation and HPLC-ESI-MS/MS to quantify sirolimus (SIR) with both desmethoxyrapamycin (DMR) and deuterium-labeled sirolimus (SIR-d(3)) as the IS to analyze a range of whole-blood and extraction-matrix samples, and to estimate recovery, matrix effects, process efficiency, and interpatient variation. We also analyzed a series of blood samples from 72 patients taking SIR, including external proficiency-testing samples, with these ISs. The range of interpatient assay imprecision (CV) values for the SIR assay was consistently lower with SIR-d(3) (2.7%-5.7%) than with DMR (7.6%-9.7%). The results obtained with the 2 different ISs for the patient samples showed a linear relationship, but the results were higher with DMR as the IS than with SIR-d(3). The use of SIR-d(3) as the IS in the high-throughput HPLC-ESI-MS/MS assay of SIR yielded improved results compared with the use of DMR. SIR-d(3) appears to be less affected by differences in the ionization of SIR and its IS caused by the variability of interpatient matrices. The IS-related difference in SIR estimation needs further investigation.

Quantitative analysis of the experimental cytotoxic drug cyclopentenyl cytosine and its metabolite in plasma with HPLC tandem mass spectrometry

The cytotoxic drug cyclopentenyl cytosine (CPEC) is currently being investigated in early clinical trials. Monitoring of plasma levels is required for pharmacokinetic analysis and management of toxicity. This paper describes the analysis of CPEC and cyclopentenyl uracil (CPEU) in plasma by HPLC-electrospray ionization tandem mass spectrometry. The calibration curves for CPEC and the metabolite CPEU were linear up to 10 microm with correlation coefficients of 0.997 (SD = 0.002, n = 10) and 0.997 (SD = 0.004, n = 10), respectively. The detection limit for CPEC was 0.03 and 0.12 microM for CPEU. The intra- and interassay coefficients of variation for CPEC and CPEU were less then 10%. The usefulness of the method was demonstrated by analyzing CPEC and CPEU in plasma of a patient treated with CPEC. HPLC with electrospray ionization tandem mass spectrometry allowed rapid and sensitive determination of CPEC and CPEU levels in plasma.

Clozapine causes oxidation of proteins involved in energy metabolism: a possible mechanism for antipsychotic-induced metabolic alterations

Although atypical antipsychotics are widely known to induce alterations in lipid and glucose metabolism, the mechanisms by which these alterations occur remain unknown. Several recent studies have shown that atypical antipsychotics induce oxidative stress and oxidative cell injury by increasing levels of lipid and protein oxidation. In this study, a novel proteomic approach was used to identify specific proteins oxidized after clozapine treatment. Differentiated neuroblastoma SKNSH cells were treated with 0, 5 or 20 mum clozapine for 24 h and protein extracts were labelled with 6-iodoacetamide fluorescein (6-IAF). The lack of incorporation of 6-IAF to cysteine residues is an indicator of protein oxidation. Labelled proteins were exposed to 2D electrophoresis, and differential protein labelling was assessed. Increased oxidation after clozapine treatment was observed in 10 protein spots (p<0.05), although only four of them remained significant after correcting for analysis with two drug concentrations. Five proteins, corresponding to nine of the spots, were identified by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) as mitochondrial ribosomal protein S22, mitochondrial malate dehydrogenase, calumenin, pyruvate kinase and 3-oxoacid CoA transferase. The latter four proteins play important roles in energy metabolism. These results suggest that oxidative stress may be a mechanism by which antipsychotics increase the risk for metabolic syndrome and diabetes.

Comparative Proteomic Analysis of Radiation-Induced Changes in Mouse Lung: Fibrosis-Sensitive and -Resistant Strains

To determine whether comparative proteomics could detect differential protein expression after lung irradiation in two mouse strains with different radiation responses, lung proteins were subjected to two-dimensional orthogonal liquid-phase separations, with chromatofocusing in the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second. Five weeks after 12 Gy whole-lung irradiation, 15 and 31 proteins had significantly altered expression levels in C3H/HeJ (less likely to develop lung fibrosis) and C57BL/6J mice (more likely to develop lung fibrosis), respectively. These proteins were analyzed by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and identified by matching sequences in a peptide database. The proteins are associated with redox, energy consumption, glycolysis, or chromatin/ RNA structure formation. Five of the six redox-related proteins, including superoxide dismutase 1 (SOD1), cytochrome c oxidase, glutamate dehydrogenase, biliverdin reductase, peroxiredoxin and carbonyl reductase, were down-regulated in the irradiated C57BL/6J mice, whereas SOD1, sulfurtransferase and carbonyl reductase increased in the irradiated C3H/ HeJ mice. Thus decreased antioxidant proteins in the irradiated C57BL/6J mice may be correlated with increased early lung toxicity. Changes in SOD1 and 8-hydroxydeoxy-guanosine (8-OHdG, an oxidative stress marker) were further confirmed by immunohistochemistry and/or Western blot analysis. These data suggest that a proteomics approach has the potential to detect protein changes relevant to early lung toxicity after irradiation.

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration

A single tryptophan (W 334(I)) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W 334(I) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS–PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W 334(I) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W 48(IV) and W 19(VIIc), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003–1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.

Identification of selenohomolanthionine in selenium-enriched Japanese pungent radish

Sulfur (S) and selenium (Se) are same-group elements that share, in part, a common metabolic pathway. Because a radish variant having pungent flavor (Raphanus sativus L. cv. ‘Yukibijin’) has a unique S metabolic pathway, we expected that it would also produce unique Se compounds through that metabolic pathway. Se metabolites in the radish were assigned by speciation techniques, i.e., HPLC-inductively coupled plasma mass spectrometry (ICP-MS) and HPLC-electrospray ionization tandem mass spectrometry (ESI-MS-MS). 68.5% of total Se in the selenized Japanese pungent radish was water-extractable, and the water extract contained selenate (around 45% of total Se), Se-methylselenocysteine (around 11%), an unknown Se compound tentatively named KD-1 (around 5%), and others (around 1.2%). KD-1 was assigned by ESI-MS-MS as 4,4′-selenobis[2-aminobutanoic acid] or Se-(3-amino-3-carboxypropyl)-homocysteine, commonly known as selenohomolanthionine (SeHLan). This Se compound was first identified in nature. The metabolic pathway of SeHLan was suggested to involve the phosphorylation of homoserine to produce O-phosphohomoserine, and the subsequent reaction of O-phosphohomoserine with selenohomocysteine (SeHCys).