Lower levels of F2-isoprostanes in serum and livers of long-lived Ames dwarf mice

F₂-isoprostanes (IsoPs), lipid peroxidation products, are markers that quantitatively measure levels of oxidative stress. IsoP levels increase in tissues and serum of aging animals suggesting an increase in oxidative stress. This supports the Free Radical Theory of Aging, which proposes that elevated levels of reactive oxygen species (ROS) cause macromolecular damage, and is a factor in the age-associated decline in tissue function. Numerous studies have shown that the longevity of long-lived mutant mice correlates with their resistance to oxidative stress. However, although the Ames dwarf (DW) mice show resistance to oxidative stress, it has not been shown that these mice have inherently lower levels of ROS. Our results show that the serum and liver IsoP levels in DW mice are lower at all ages suggesting that the lower levels of endogenous ROS production in DW mice may be a factor in their resistance to oxidative stress and longevity.     

Changes in urinary dinor dihydro F2-isoprostane metabolite concentrations,a marker of oxidative stress,during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this stidy measured 2,3-dinor-5,6-dihydro-15-F_2t-IsoP (F₂-IsoP-M), the major urinary metabolite of 15-F_2t-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F₂-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89–7.8; follow-up: 2.47 ng/Cr mg (1.56–6.86); discharge: 1.42 ng/Cr mg (0.7–4.44); both p<0.01), but not significantly different between admission and follow-up. F₂-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31–1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F₂-IsoP-M concentrations compared to stable asthmatics. F₂-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F₂-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

Urinary 8-epi-PGF2alpha and its endogenous beta-oxidation products (2,3-dinor and 2,3-dinor-5,6-dihydro) as biomarkers of total body oxidative stress

Although measurements of plasma F2-isoprostanes are established markers of oxidative stress, their quantification only reflects acute non-enzymatic lipid peroxidation. In this study, a new approach is described for the rapid isolation and measurement of urinary 8-epi-PGF2alpha and its endogenous beta-oxidation metabolites (2,3-dinor-8-epi-PGF2alpha and 2,3-dinor-5,6-dihydro-PGF2alpha) for use as index of total body oxidative stress. Isoprostanes were partitioned with ethyl acetate and subsequently purified by chromatography on an aminopropyl (NH2) and silica (Si) cartridge. Final analysis of F2-isoprostanes as trimethylsilyl-ester/pentafluorobenzyl ester derivatives was carried out by stable isotope dilution mass spectrometry. Overall recovery of F2-isoprostanes was 80+/-4%. Inter- and intra-assay coefficients of variation were 5% and 7%, respectively. In a group of healthy humans, the mean excretion rates expressed as nmol/mmol creatinine for 2,3-dinor-8-epi-PGF2alpha, 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha, and 8-epi-PGF2alpha were 5.43+/-1.93, 2.16+/-0.71, and 0.36+/-0.16, respectively. Correlations were obtained between 8-epi-PGF2alpha and 2,3-dinor-8-epi-PGF2alpha or 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.998 and r=0.937, respectively). A strong relationship was also seen between 2,3-dinor-8-epi-PGF2 and 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.949). The new technique allows for high sample throughput and avoids the need for HPLC and/or other expensive equipment required for the initial sample preparation. Simultaneous analysis of urinary 8-epi-PGF2alpha and its metabolites should provide unique tool in clinical trials exploring the role of oxidant injury in human disease.     

Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes

The F(2)-isoprostanes are products of free-radical-induced oxidation of arachidonic acid (AA) that are stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)). We describe a method for quantitation of several 15-series PGF isomers (15-PGFs) and AA by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Plasma samples were subjected to alkaline hydrolysis and acidified, and total (free + esterified) 15-PGFs and AA were extracted with organic solvents. The analytes were separated by gradient reverse-phase HPLC and detected by multiple reaction monitoring on a triple-quadrupole mass spectrometer, using deuterated internal standards for quantitation. The assay had a linear range of 1-40 pg of 8-iso-PGF(2alpha) on column and can quantify as little as 40 pg/mL (0.11 nM) in plasma. Outcomes significantly correlated (p < 0.0001) with data obtained by gas chromatography-mass spectrometry GC-MS or enzyme-linked immunosorbent assay. All plasma 15-PGF isomers increased over time with in vitro cigarette smoke exposure and correlated (p < 0.0001) with each other. The same strong inter-15-PGF correlations were observed in plasma from healthy young adult subjects. The coefficients of variation of HPLC-MS-MS measurements (24-32%) were smaller than those obtained by GC-MS (53%). Thus, HPLC-MS-MS potentially offers greater precision and allows quantitation of more compounds with simpler sample preparation than existing methods. Ours is the first validated quantitative assay using HPLC-tandem MS applied to plasma total 15-PGFs.     

Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood

Cord blood is a source of hematopoietic stem cells used in transplantation in which hematopoietic reconstitution is necessary. This transplant modality requires the cryopreservation of hematopoietic stem cells (HSCs). Dimethyl sulfoxide has been used as a cryoprotectant (CPA) in the cryopreservation of HSCs; however, it has been demonstrated that Me₂SO exhibits toxic side effects to the human body. Due to its stability upon freezing, disaccharides such as trehalose have been investigated as a cryoprotectant. This study investigated the hypothesis that a cryopreservation solution containing intracellular and extracellular trehalose improves the recovery of stem cells after cryopreservation. After thawing, the cells were tested for their viability using the 7AAD stain, CD45+/CD34+ cells were assessed using flow cytometry and the MTT viability assay, and the proportion of hematopoietic progenitor cells was measured using the CFU assay. Our results showed the effectiveness of the solution containing intracellular and extracellular trehalose in the cryopreservation of cord blood cells, demonstrating that trehalose may be an optimal cryoprotectant when present both inside and outside of cells.     

Derivatization of F2-isoprostanes with 1-pyrenyldiazomethane and their subsequent determination by fluorescence high-performance liquid chromatography

A novel, sensitive, and specific method is presented for the quantification of endogenous 3-nitrotyrosine in rat plasma based on isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry, using 3-nitro-2,5,6-d(3)-l-tyrosine as an internal standard. The extraction and cleanup method entails three major steps: protein precipitation, solid-phase extraction with an aminopropyl cartridge, followed by derivatization of 3-nitrotyrosine to the corresponding butyl ester. The analysis of the stable butyl ester derivative circumvented matrix interferences, which were encountered on the analysis of the nonderivatized analyte in plasma, and thus significantly improved sensitivity. The mass spectral acquisition of 3-nitrotyrosine butyl ester was done in the positive ion mode using selected reaction monitoring of two specific transitions. The response was linear over the concentration range 1.4-28.5 nM, and the recoveries of spiked 3-nitrotyrosine in rat plasma exceeded 75%. The detection and quantification limits of 3-nitrotyrosine in rat plasma (165 microL equivalent injected) approached 0.43 and 1.4 nM (0.07 and 0.23 pmol, on column), respectively. This study also addresses the potential artifactual formation of 3-nitrotyrosine, which may lead to an overestimation of the background levels of the biomarker. Solid-phase extraction of 3-nitrotyrosine was required prior to esterification to avoid artifactual nitration of tyrosine. In this context, analysis of eight rat plasma samples showed quantifiable levels in only four of the samples of the order of 1.4-1.5 nM.     

Osmotic parameters of red blood cells from umbilical cord blood

The transfusion of red blood cells from umbilical cord blood (cord RBCs) is gathering significant interest for the treatment of fetal and neonatal anemia, due to its high content of fetal hemoglobin as well as numerous other potential benefits to fetuses and neonates. However, in order to establish a stable supply of cord RBCs for clinical use, a cryopreservation method must be developed. This, in turn, requires knowledge of the osmotic parameters of cord RBCs. Thus, the objective of this study was to characterize the osmotic parameters of cord RBCs: osmotically inactive fraction (b), hydraulic conductivity (L_p), permeability to cryoprotectant glycerol (P_glycerol), and corresponding Arrhenius activation energies (E_a). For L_p and P_glycerol determination, RBCs were analyzed using a stopped-flow system to monitor osmotically-induced RBC volume changes via intrinsic RBC hemoglobin fluorescence. L_p and P_glycerol were characterized at 4 °C, 20 °C, and 35 °C using Jacobs and Stewart equations with the E_a calculated from the Arrhenius plot. Results indicate that cord RBCs have a larger osmotically inactive fraction compared to adult RBCs. Hydraulic conductivity and osmotic permeability to glycerol of cord RBCs differed compared to those of adult RBCs with the differences dependent on experimental conditions, such as temperature and osmolality. Compared to adult RBCs, cord RBCs had a higher E_a for L_p and a lower E_a for P_glycerol. This information regarding osmotic parameters will be used in future work to develop a protocol for cryopreserving cord RBCs.     

Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO), but at 37 °C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me₂SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me₂SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

Determination of air pollution-related biomarkers of exposure in urine of travellers between Germany and China using liquid chromatographic and liquid chromatographic-mass spectrometric methods: a pilot study

Objective: The influence of different exposures to PM_2.5 (particulate matter with an aerodynamic diameter below 2.5?μm) on the concentrations of biomarkers of exposure and oxidative stress should be investigated. For this purpose, urine samples from individuals travelling from Germany to China were collected and analysed.

Materials: Robust LC and LC-MS/MS methods were established for the determination of biomarkers including 8-hydroxy-2′-deoxyguanosine, malondialdehyde, F_2α-isoprostanes and hydroxylated polycyclic aromatic hydrocarbons. As a pilot study, nine volunteers travelled from Germany (mean daily concentration of PM_2.5: 21?μg/m³) to China (mean daily concentration of PM_2.5: 108?μg/m³). Urine samples were collected before and after the trip.

Results: In samples collected after return to Germany, the median concentrations of oxidative stress biomarkers were observed to be higher than in samples collected before leaving Germany. Decreasing trends were observed in the sequences of samples collected after return in the following weeks. Correlations were found between exposure and oxidative stress biomarkers.

Conclusion: Travellers are ideal models for PM pollution-induced acute health effects study. Exposure to PM pollution can cause oxidative stress and damage.     

Ubiquinol-10 and ubiquinone-10 levels in umbilical cord blood of healthy foetuses and the venous blood of their mothers

Abstract

Despite their being good markers of oxidative stress for clinical use, little is known about ubiquinol-10 (reduced coenzyme Q₁₀) and ubiquinone-10 (oxidized coenzyme Q₁₀) levels in foetuses and their mothers. This study investigates oxidative stress in 10 healthy maternal venous, umbilical arterial and venous bloods after vaginal delivery by measuring ubiquinol-10 and ubiquinone-10 levels. Serum ubiquinol-10 and ubiquinone-10 levels were measured by HPLC with a highly sensitive electrochemical detector. Maternal venous ubiquinol-10 and ubiquinone-10 levels were significantly higher than umbilical arterial and venous levels (all p < 0.001). However, the ubiquinone-10/total coenzyme Q₁₀ (CoQ₁₀) ratio, which reflects the redox status, was significantly higher in umbilical arterial and umbilical venous blood compared to maternal venous blood (all p < 0.001). The ubiquinone-10/total CoQ₁₀ ratio was higher in umbilical arterial than in umbilical venous blood (p < 0.01). The present study demonstrated that foetuses were under higher oxidative stress than their mothers.     

Isolation and measurement of urinary 8-iso-prostaglandin F2α by high-performance liquid chromatography and gas chromatography–mass spectrometry

8-iso-Prostaglandin F_2α (8-iso-PGF_2α) is a product of free radical-catalyzed peroxidation of arachidonic acid. Measurement of its urinary excretion has been proposed as an index of oxidative status in vivo. A stable isotope dilution method for its quantification by gas chromatography–electron capture chemical ionization mass spectrometry is described. Sample cleanup required the combined use of high-performance liquid chromatography and thin-layer chromatography. The inter-assay R.S.D. in two separate determinations was 1.6 (n=4) and 2.3% (n=4). The accuracy of the assay was evaluated through recovery experiments. The equation of the regression plot correlating the amounts added and recovered was y=0.91x−0.31, r=0.9916 (n=12). The pair of fragment ions ([M−181]⁻) at m/z 569 and m/z 573 was monitored for quantification. The mean 8-iso-PGF_2α excretion rate was 528±127 (S.D.) ng per day in five male volunteers and 730±305 ng per day in six females. Intake of 80 mg of lycopene per day by eleven volunteers for four weeks resulted in a non-significant reduction of 8-iso-PGF_2α excretion.     

Identification of juvenile hormone III in the hemolymph of Blattella germanica adult females by gas chromatography-mass spectrometry

Juvenile hormone III was identified in purified hemolymph extracts of adult females of the cockroach Blatella germanica by gas chromatography-mass spectrometry with chemical ionization and selected ion monitoring. Under these conditions, juvenile hormones I and II were not detected within the sensitivity ranges of this analytical method. For quantification purposes a 5,5-bisdeuterated analog of juvenile hormone III was synthesized and used as an internal standard. In general, juvenile hormone III titers obtained correlated with data on oocyte growth and with hormone titers found from in vitro corpora allata incubations along the first gonotrophic cycle.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

Occurrence of N-acyl-L-homoserine lactones in extracts of some Gram-negative bacteria evaluated by gas chromatography-mass spectrometry

Acylated homoserine lactones (AHLs) are self-generated signal molecules that mediate population density-dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria. These signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. In selected foods AHLs contribute to product spoilage. As different bacterial species produce AHL analogs that differ in length of the N-acyl chain, ranging from 4 to 14 carbons and in the substitution at the C-3 position of the side chain (i.e., oxo or hydroxyl group), the suitability and applicability of a gas chromatography-mass spectrometry direct method for characterizing trace amounts of AHLs was evaluated using N-heptanoyl-homoserine lactone as internal standard. Crude cell-free supernatants of bacterial cultures of Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia enterocolitica, and Serratia liquefaciens were screened for AHL production in selected ion monitoring mode, using the prominent fragment at m/z 143. The observed profiles of distinguishable N-acyl-homoserine lactones occurring in bacterial extracts were compared and discussed. The presence of a labile 3-oxo-hexanoylhomoserine lactone was evidenced but serious difficulties arose in estimating its concentration as thermal degradation occurs during the gas chromatographic separation. Its electron impact mass spectra was, however, given and interpreted.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Enantiomeric determination of ephedrines and norephedrines by chiral derivatization gas chromatography-mass spectrometry approaches

Concerned with variations in abuse potential and control status among various isomers of ephedrines and norephedrines, this study was conducted to develop an effective method for the simultaneous analysis of eight ephedrine-related compounds along with structurally similar cathinones. Among various approaches studied, a 60-m HP-5MS (0.25 mm i.d., 0.25 microm film thickness) was successfully used to characterize the following compounds that were derivatized with (-)-alpha-methoxy-alpha-trifloromethylphenylacetic acid (MTPA): (+)-cathinone, (-)-cathinone, (+)-norephedrine, (-)-norephedrine, (+)-norpseudoephedrine, (+)-ephedrine, (-)-ephedrine, (-)-pseudoephedrine, (+)-pseudoephedrine. (-)-Cathine standard was not available, but should also be resolvable under this analytical procedure. This method was successfully applied to the analysis of selected cold remedies for characterizing the enantiomeric compositions of the compounds present in these samples.     

A phytochemical study of the quinolizidine alkaloids from Genista tenera by gas chromatography-mass spectrometry

Gas chromatography coupled with mass spectrometry has been used to analyse the alkaloids present in the aerial parts of Genista tenera. Anagyrine, cytisine, N-formylcytisine, N-methylcytisine and lupanine were the major compounds, the last two alkaloids being known for their hypoglycaemic activity. Dehydrocytisine, 5,6-dehydrolupanine, rhombifoline, aphylline and thermopsine were the minor alkaloids. The characterisation of the constituents was based on comparison of their Kovats retention indexes and electron impact-mass spectrometric data recorded on-line with those of reference compounds and literature data.     

Determination of isoprostaglandin F2α type III in human urine by gas chromatography–electronic impact mass spectrometry. Comparison with enzyme immunoassay

F₂-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F_2α type III (15-F_2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F_2α type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F_2α type III concentrations determined with gas chromatography–mass spectrometry (GC–MS) did not agree with those determined with EIA. Our results suggest that GC–MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC–MS and EIA should be avoided.