Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride

OBJECTIVE: To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine. METHODS: The liver injury in the model rat was induced by alpha-naphthylisothiocyanate (ANIT) and carbon tetrachloride (CCl(4) ) respectively, and the effects of Yinchenhao Decoction (, YCHD), a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic, on the two liver injury models were evaluated by analyzing the serum level of alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP), malondialchehyche (MDA), total bilirubin (T-BIL), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) as well as the ratio of liver weight to body weight. The experimental data were analyzed by principal component analytical method of pattern recognition. RESULTS: The ratio of liver weight to body weight was significantly elevated in the ANIT and CCl(4) groups when compared with that in the normal control (P<0.01). The contents of ALT and T-BIL were significantly higher in the ANIT group than in the normal control (P<0.05,P<0.01), and the levels of AST, ALT and ALP were significantly elevated in CCl(4) group relative to those in the normal control P<0.01). In the YCHD group, the increase in AST, ALT and ALP levels was significantly reduced (P<0.05, P<0.01), but with no significant increase in serum T-BIL. In the CCl(4) intoxicated group, the MDA content was significantly increased and SOD, GSH-PX activities decreased significantly compared with those in the normal control group, respectively (P<0.01). The increase in MDA induced by CCl(4) was significantly reduced by YCHD P<0.05). CONCLUSION: YCHD showed significant effects on preventing liver injury progression induced by CCl(4), and the closest or most suitable animal model for damp-heat jaundice syndrome may be the one induced by CCl(4).

Identification and elimination of a matrix-induced systematic error in LA–ICP–MS 206Pb/238U dating of zircon

Black et al. (2004) identified a systematic difference between LA–ICP–MS and TIMS measurements of 206Pb/238U in zircons, which they correlated with the incompatible trace element content of the zircon. We show that the offset between the LA–ICP–MS and TIMS measured 206Pb/238U correlates more strongly with the total radiogenic Pb than with any incompatible trace element. This suggests that the cause of the 206Pb/238U offset is related to differences in the radiation damage (alpha dose) between the reference and unknowns. We test this hypothesis in two ways. First, we show that there is a strong correlation between the difference in the LA–ICP–MS and TIMS measured 206Pb/238U and the difference in the alpha dose received by unknown and reference zircons. The LA–ICP–MS ages for the zircons we have dated can be as much as 5.1% younger than their TIMS age to 2.1% older, depending on whether the unknown or reference received the higher alpha dose. Second, we show that by annealing both reference and unknown zircons at 850 °C for 48 h in air we can eliminate the alpha-dose-induced differences in measured 206Pb/238U. This was achieved by analyzing six reference zircons a minimum of 16 times in two round robin experiments: the first consisting of unannealed zircons and the second of annealed grains. The maximum offset between the LA–ICP–MS and TIMS measured 206Pb/238U for the unannealed zircons was 2.3%, which reduced to 0.5% for the annealed grains, as predicted by within-session precision based on counting statistics. Annealing unknown zircons and references to the same state prior to analysis holds the promise of reducing the 3% external error for the measurement of 206Pb/238U of zircon by LA–ICP–MS, indicated by Klötzli et al. (2009), to better than 1%, but more analyses of annealed zircons by other laboratories are required to evaluate the true potential of the annealing method.

Reactions of simple and peptidic alpha-carboxylate radical anions with dioxygen in the gas phase

alpha-Carboxylate radical anions are potential reactive intermediates in the free radical oxidation of biological molecules (e. g., fatty acids, peptides and proteins). We have synthesised well-defined alpha-carboxylate radical anions in the gas phase by UV laser photolysis of halogenated precursors in an ion-trap mass spectrometer. Reactions of isolated acetate ((center dot)CH(2)CO(2)) and 1-carboxylatobutyl (CH(3)CH(2)CH(2)(center dot)CHCO(2)(-)) radical anions with dioxygen yield carbonate (CO(3)(center dot-)) radical anions and this chemistry is shown to be a hallmark of oxidation in simple and alkyl-substituted cross-conjugated species. Previous solution phase studies have shown that C(alpha)-radicals in peptides, formed from free radical damage, combine with dioxygen to form peroxyl radicals that subsequently decompose into imine and keto acid products. Here, we demonstrate that a novel alternative pathway exists for two alpha-carboxylate C(alpha)-radical anions: the acetylglycinate radical anion (CH(3)C(O)NH(center dot)CHCO(2)(-)) and the model peptide radical anion, YGGFG(center dot-). Reaction of these radical anions with dioxygen results in concerted loss of carbon dioxide and hydroxyl radical. The reaction of the acetylglycinate radical anion with dioxygen reveals a two-stage process involving a slow, followed by a fast kinetic regime. Computational modelling suggests the reversible formation of the C(alpha) peroxyl radical facilitates proton transfer from the amide to the carboxylate group, a process reminiscent of, but distinctive from, classical proton-transfer catalysis. Interestingly, inclusion of this isomerization step in the RRKM/ME modelling of a G3SX level potential energy surface enables recapitulation of the experimentally observed two-stage kinetics.

Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry

Meibum is believed to be the major source of tear film lipids, which are vital in the prevention of excess evaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established. While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometric studies have not detected them. In this study we use electrospray ionisation tandem mass spectrometry to investigate the presence of phospholipids in meibum and provide comparison to the phospholipid profile of tears.Lipids were extracted from human meibum and tear samples using standard biphasic methods and analysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of 35 choline-containing phospholipids were identified in meibum and the profile of these was similar to that observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlight the need for a combination of optimised techniques to enable the identification of the large range of lipid classes in meibum. © 2011 Elsevier Ltd.

Concerted HO2 elimination from alpha-Aminoalkylperoxyl free radicals : Experimental and theoretical evidence from the gas phase NH2 center dot CHCO2- + O-2 reaction

We have investigated the gas-phase reaction of the alpha-aminoacetate (glycyl) radical anion (NH2(sic)CHCO2-) with O-2 using ion trap mass spectrometry, quantum chemistry, and statistical reaction rate theory. This radical is found to undergo a remarkably rapid reaction with O-2 to form the hydroperoxyl radical (HO2(sic)) and an even-electron imine (NHCHCO2-), with experiments and master equation simulations revealing that reaction proceeds at the ion molecule collision rate. This reaction is facilitated by a low-energy concerted HO2(sic) elimination mechanism in the NH2CH(OO(sic))CO2- peroxyl radical. These findings can explain the widely observed free-radical-mediated oxidation of simple amino acids to amides plus alpha-keto acids (their imine hydrolysis products). This work also suggests that imines will be the main intermediates in the atmospheric oxidation of primary and secondary amines, including amine carbon capture solvents such as 2-aminoethanol (commonly known as monoethanolamine, or MEA), in a process that avoids the ozone-promoting conversion of (sic)NO to (sic)NO2 commonly encountered in peroxyl radical chemistry.

Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions

Background Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. Objective We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). Methods The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. Results Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. Conclusion Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.

Discovery of candidate serum proteomic and metabolomic biomarkers in ankylosing spondylitis

Ankylosing Spondylitis (AS) is a common inflammatory rheumatic disease with a predilection for the axial skeleton, affecting 0.2% of the population. Current diagnostic criteria rely on a composite of clinical and radiological changes, with a mean time to diagnosis of 5 to 10 years. In this study we employed nano liquid-chromatography mass spectrometry analysis to detect and quantify proteins and small compounds including endogenous peptides and metabolites in serum from 18 AS patients and nine healthy individuals. We identified a total of 316 proteins in serum, of which 22 showed significant up- or down-regulation (p < 0.05) in AS patients. Receiver operating characteristic analysis of combined levels of serum amyloid P component and inter-α-trypsin inhibitor heavy chain 1 revealed high diagnostic value for Ankylosing Spondylitis (area under the curve = 0.98). We also depleted individual sera of proteins to analyze endogenous peptides and metabolic compounds. We detected more than 7000 molecular features in patients and healthy individuals. Quantitative MS analysis revealed compound profiles that correlate with the clinical assessment of disease activity. One molecular feature identified as a Vitamin D3 metabolite-(23S,25R)-25-hydroxyvitamin D3 26,23-peroxylactone-was down-regulated in AS. The ratio of this vitamin D metabolite versus vitamin D binding protein serum levels was also altered in AS as compared with controls. These changes may contribute to pathological skeletal changes in AS. Our study is the first example of an integration of proteomic and metabolomic techniques to find new biomarker candidates for the diagnosis of Ankylosing Spondylitis

Determination of refractive and volatile elements in sediment using laser ablation inductively coupled plasma mass spectrometry

Wet-milling protocol was employed to produce pressed powder tablets with excellent cohesion and homogeneity suitable for laser ablation (LA) analysis of volatile and refractive elements in sediment. The influence of sample preparation on analytical performance was also investigated, including sample homogeneity, accuracy and limit of detection. Milling in volatile solvent for 40 min ensured sample is well mixed and could reasonably recover both volatile (Hg) and refractive (Zr) elements. With the exception of Cr (−52%) and Nb (+26%) major, minor and trace elements in STSD-1 and MESS-3 could be analysed within ±20% of the certified values. Comparison of the method with total digestion method using HF was tested by analysing 10 different sediment samples. The laser method recovers significantly higher amounts of analytes such as Ag, Cd, Sn and Sn than the total digestion method making it a more robust method for elements across the periodic table. LA-ICP-MS also eliminates the interferences from chemical reagents as well as the health and safety risks associated with digestion processes. Therefore, it can be considered as an enhanced method for the analysis of heterogeneous matrices such as river sediments.

SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

Pyrolysis and combustion of alpha-cellulose: effect of dihydrazinium phosphate (N2H5)2HPO4

A strip of Whatman filter paper (α-cellulose) dipped in an aqueous solution of dihydrazinium phosphate, (N2H5)2HPO4(DHP), and dried, carbonized without flame when ignited. The observed flame retardancy of DHP on α-cellulose has been studied using TG, DTA and mass spectrometry. Dihydrazinium phosphate appears to catalyze the dehydration of α-cellulose, minimizing the depolymerization which produces flammable tars, with the formation of water and char. Flame retardancy of DHP is compared with that of diammonium phosphate and phosphoric acid.

Follicle stimulating hormone secretion and dominant follicle growth during treatment of Bos indicus heifers with intra-vaginal progesterone releasing devices, oestradiol benzoate, equine chorionic gonadotrophin and prostaglandin F-2 alpha

The aim of this study was to investigate the effects on follicle stimulating hormone (FSH) secretion and dominant follicle (OF) growth, of treatment of Bos indicus heifers with different combinations of intra-vaginal progesterone releasing devices (IPRD), oestradiol benzoate (ODB), PGF(2 alpha), and eCG. Two-year-old Brahman (BN; n=30) and Brahman-cross (BNX; n=34) heifers were randomly allocated to three IPRD-treatments: (i) standard-dose IPRD [CM 1.56 g; 1.56 g progesterone (P-4); n = 17]; (ii) half-dose IPRD (CM 0.78 g; 0.78 g p(4); n=15); (iii) half-dose IPRD + 300 IU eCG at IPRD removal (CM 0.78 g+G; n=14); and, (iv) non-IPRD control (2 x PGF(2 alpha); n=18) 500 mu g cloprostenol on Days -16 and -2. IPRD-treated heifers received 250 mu g PGF(2 alpha) at IPRD insertion (Day 10) and IPRD removal (Day -2) and 1 mg ODB on Day -10 and Day -1. Follicular dynamics were monitored daily by trans-rectal ultrasonography from Day -10 to Day 1. Blood samples for determination of P-4 were collected daily and samples for FSH determination were collected at 12 h intervals from Day -9 to Day -2. A significant surge in concentrations of FSH was observed in the 2 x PGF(2 alpha), treatment 12 h prior and 48 h after follicular wave emergence, but not in the IPRD-treated heifers. Estimated mean concentrations of total plasma P-4 during the 8 days of IPRD insertion was greater (P<0.001) in the CM 1.56 g P-4 treated heifers compared to the CM 0.78 g P-4 treated heifers (18.38 ng/ml compared with 11.09 ng/ml, respectively). A treatment by genotype interaction (P=0.036) was observed in the mean plasma P4 concentration in heifers with no CL during IPRD insertion, whereby BN heifers in the CM 1.56 g treatment had greater plasma P-4 than the BNX heifers on Days-9, -7, -6, -5, and -4. However, there was no genotype effect in the CM 0.78 g +/- G or the 2 x PGF(2 alpha) treatment. Treatment had no effect on the DF growth from either day of wave emergence (P=0.378) or day of IPRD removal (P=0.780) to ovulation. This study demonstrates that FSH secretion in B. indicus heifers treated with a combination of IPRD's and ODB to synchronise ovulation was suppressed during the period of IPRD insertion but no significant effect on growth of the DF was observed. (C) 2013 Elsevier B.V. All rights reserved.

Glycoalkaloid Content and Starch Structure in Solanum Species and Interspecific Somatic Potato Hybrids

When genome sections of wild Solanum species are bred into the cultivated potato (S. tuberosum L.) to obtain improved potato cultivars, the new cultivars must be evaluated for their beneficial and undesirable traits. Glycoalkaloids present in Solanum species are known for their toxic as well as for beneficial effects on mammals. On the other hand, glycoalkaloids in potato leaves provide natural protection against pests. Due to breeding, glycoalkaloid profile of the plant is affected. In addition, the starch properties in potato tubers can be affected as a result of breeding, because the crystalline properties are determined by the botanical source of the starch. Starch content and composition affect the texture of cooked and processed potatoes. In order to determine glycoalkaloid contents in Solanum species, simultaneous separation of glycoalkaloids and aglycones using reversed-phase high-performance liquid chromatography (HPLC) was developed. Clean-up of foliage samples was improved using a silica-based strong cation exchanger instead of octadecyl phases in solid-phase extraction. Glycoalkaloids alpha-solanine and alpha-chaconine were detected in potato tubers of cvs. Satu and Sini. The total glycoalkaloid concentration of non-peeled and immature tubers was at an acceptable level (under 20 mg/100 g of FW) in the cv. Satu, whereas concentration in cv. Sini was 23 mg/100 g FW. Solanum species (S. tuberosum, S. brevidens, S. acaule, and S. commersonii) and interspecific somatic hybrids (brd + tbr, acl + tbr, cmm + tbr) were analyzed for their glycoalkaloid contents using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The concentrations in the tubers of the brd + tbr and acl + tbr hybrids remained under 20 mg/100 g FW. Glycoalkaloid concentration in the foliage of the Solanum species was between 110 mg and 890 mg/100 g FW. However, the concentration in the foliage of S. acaule was as low as 26 mg/100 g FW. The total concentrations of brd + tbr, acl + tbr, and cmm + tbr hybrid foliages were 88 mg, 180 mg, and 685 mg/100 g FW, respectively. Glycoalkaloids of both parental plants as well as new combinations of aglycones and saccharides were detected in somatic hybrids. The hybrids contained mainly spirosolanes, and glycoalkaloid structures having no 5,6-double bond in the aglycone. Based on these results, the glycoalkaloid profiles of the hybrids may represent a safer and more beneficial spectrum of glycoalkaloids than that found in currently cultivated varieties. Starch nanostructure of three different cultivars (Satu, Saturna, and Lady Rosetta), a wild species S. acaule, and interspecific somatic hybrids were examined by wide-angle and small-angle X-ray scattering (WAXS, SAXS). For the first time, the measurements were conducted on fresh potato tuber samples. Crystallinity of starch, average crystallite size, and lamellar distance were determined from the X-ray patterns. No differences in the starch nanostructure between the three different cultivars were detected. However, tuber immaturity was detected by X-ray scattering methods when large numbers of immature and mature samples were measured and the results were compared. The present study shows that no significant changes occurred in the nanostructures of starches resulting from hybridizations of potato cultivars.

Seminal plasma proteome of eletroejaculated Bos indicus bulls

The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4±2.3 and 64±3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 D-isomarase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization.

Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

Objectives: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. Design and methods: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated baernoglobin (HbA1c) was measured by HPLC. Results: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. Conclusions: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.