Unique tryptic peptides specific for bovine and human hemoglobin in the detection and confirmation of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygen-carrying capacity of circulating blood, they are excellent candidates for abuse in human and equine athletes. To deter athletes from blood doping with HBOCs such as Hemopure and Oxyglobin (OXY), a method for detection, confirmation, quantification, and distinguishing of HBOCs from native hemoglobin in test samples is needed. The purpose of this study was to identify unique peptides specific for bovine Hb and human Hb that are useful in the detection and confirmation of HBOCs in test samples. The LC-MS chromatographic peak profiles of tryptic digests from OXY, bovine Hb, human Hb, and equine Hb were compared, and unique tryptic peptides specific for bovine Hb, human Hb, and equine Hb were identified. The peptides specific for bovine Hb and OXY are related to bovine Hb alpha chain residues 69-90 and beta chain residues 40-58. The peptides specific for human Hb are related to human Hb alpha chain residues 63-91 and beta chain residues 42-60 and 68-83. The amino acid sequences of these unique tryptic peptides were confirmed by their characteristic MS/MS spectra. MS/MS spectra, b-ion series and y-ion series, and LC retention time of the tryptic peptides are essential pieces of information for the unequivocal identification, detection, and confirmation of HBOCs. The results of this study provide useful and defensible data on identification, detection, and confirmation of HBOCs of bovine Hb or human Hb origin. In addition, in-ESI-source fragmentation of tryptic peptides was observed in this study. The fragmentation was undesired since it decreased intensities of the trypic peptide ions, but it was helpful to elucidating sequences of the tryptic peptides thanks to the fragment peptide ions produced from the fragmentation.     

Doping control analysis of bovine hemoglobin-based oxygen therapeutics in human plasma by LC-electrospray ionization-MS/MS

Since January 2000, hemoglobin-based oxygen carriers, such as Hemopure, belong to the list of prohibited substances of the International Olympic Committee. Hemopure is based on bovine hemoglobin, which is intra- and intermolecularly cross-linked by glutaraldehyde units causing an average molecular weight of approximately 250,000. Bovine and human hemoglobins differ by 15% in amino acid sequence; hence, tryptic digestion of these proteins generates species-common and -unique peptides. Those specific fragments originate from the alpha- and beta-subunits of hemoglobin, such as bovine Hb peptides alpha(69-90) (2367.2 Da) or beta(40-58) (2089.9 Da). By means of LC-MS/MS, peptides of human and bovine hemoglobin can be separated and identified, enabling the determination of compounds based on Hb of bovine origin and thus the administration of oxygen carriers such as Hemopure. Blank plasma samples were spiked with Hemopure or human or bovine hemoglobin, filtered, enzymatically digested, and analyzed on an Agilent 1100 Series HPLC interfaced to an Applied Biosystems API 2000 triple quadrupole mass spectrometer. In plasma aliquots of 50 microL containing 50 microg of Hemopure (1 mg/mL), peptides of bovine hemoglobin were confirmed, and blank plasma samples as well as 68 specimens of high-performance athletes were tested with the developed procedure.     

Recovery of hemoglobin oxygen saturation and intrinsic fluorescence with a forward-adjoint model

We present two forward-adjoint models for recovering intrinsic fluorescence spectra and hemoglobin oxygen saturation of turbid samples. The first fits measured diffuse reflectance spectra to obtain the absorption and scattering spectra of the medium, and these are then used to correct distortions imposed on the fluorescence spectrum by absorption and scattering. The second fits only the measured fluorescence spectrum to determine simultaneously the amplitudes of absorption and fluorescence basis spectra and scattering parameters. Both methods are validated with Monte Carlo simulations and experimentally in scattering phantoms containing nicotinamide adenine dinucleotide and human erythrocytes. Preliminary measurements from murine tumors in vivo are presented.     

Interaction of bisphenol A with bovine hemoglobin using spectroscopic and molecular modeling methods

The interaction of bisphenol A with bovine hemoglobin (BHb) under physiological conditions was investigated by using fluorescence, ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD), and molecular modeling. The experimental results showed that BPA can bind with BHb to form a complex. The binding constant Ka and the number of binding sites n were calculated to be 1.49 × 10(5) L mol(-1) and 1, respectively. Molecular modeling study revealed that BPA bound into BHb central cavity, and the binding mode of BPA-BHb complex could be hydrogen bonding. The UV-Vis absorption and CD spectra indicated that the secondary structure of BHb was altered, which may affect physiological functions of hemoglobin. This work is helpful for clarifying the molecular toxic mechanism of BPA in vivo.     

Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degrees C for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb alpha chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 microg/mL for HMP in human and equine plasma. The limit of confirmation was 250 microg/mL for OXY in equine plasma, 500 microg/mL for HML in human and equine plasma, and 1000 microg/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 microg/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 microg/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degrees C. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration.     

Microbubble suspension as a carrier of oxygen and acclimated bacteria for phenanthrene biodegradation

The applicability of microbubble suspension made of saponin as a biodegradation-enhancing carrier where oxygen and pollutant-degraders are limited was studied. The saponin-microbubble suspension was used to deliver phenanthrene-degrading bacteria, inorganic nutrients, and oxygen. Bench-scale study was carried out to determine the physical properties of the microbubble suspension and to verify whether the delivered bacteria and oxygen were effectively used to degrade phenanthrene. A concentration of 2g saponin/L H(2)O generated stable microbubble suspension with a long half-drainage time and a high gas hold-up, and the addition of phenanthrene-degraders and inorganic salts to the saponin solution did not affect such properties. The flow of the microbubble suspension through a heterogeneous sand/clay-packed column occurred in two phases, with the liquid front advancing faster and the retarded gas front. The retarded gas front provided oxygen with bacteria, which enables phenanthrene biodegradation. Approximately 30% of the spiked phenanthrene was degraded in 21 days when one pore volume of 2.0 g/L saponin-microbubble suspension was applied whereas no phenanthrene decrease was observed following the application of the same saponin solution without microbubble generation. The decrease mainly occurred at the lower part of the column where the supply of oxygen by the microbubble was concentrated.     

载体对镍基载氧剂化学链燃烧反应特性的作用

采用热重分析仪(TGA)研究了三种镍基载氧剂在化学链燃烧中的循环反应特性,考察了添加不同载体对载氧剂循环反应性能的影响,分析了影响载氧剂转化率的因素.实验结果表明,烧结使得NiO循环反应活性随着循环次数的增加而降低;添加铝酸镍(Nial2O4)和铝酸钙(12CaO·7Al2O3)载体以后,镍基载氧剂比表面积和孔隙率得到提高,载体能有效抑制颗粒之间的烧结,使载氧剂的循环反应活性保持稳定.     

CaSO_4-CuO-Ben载氧体煤化学链燃烧反应特性

以工业CaSO_4、Cu(NO_3)_2及膨润土(Ben)为原料,机械混合法制备CaSO_4-CuO-Ben载氧体;在高温流化床上以水蒸气为气化介质,考察CuO和温度对其煤化学链燃烧反应特性的影响及载氧体的循环反应性能。结果表明:CuO的添加有效增强载氧体的反应活性,提高煤的燃烧效率;850℃、m(CaSO_4)∶m(CuO)为10∶1.5(CaCu1.5)时复合载氧体的反应活性更高,碳转化率达到91.27%,CO_2体积分数达到89.34%;10次还原-氧化实验表明,CaCu1.5载氧体能够保持良好的循环反应活性。     

Structural and functional characterization of glutaraldehyde-polymerized bovine hemoglobin and its isolated fractions

Glutaraldehyde-polymerized bovine hemoglobin (PolyHbBv, trade name Oxyglobin), is a non-site-specific modified hemoglobin-based oxygen-carrying solution, developed for use in veterinary medicine. PolyHbBv was fractionated into four distinct tetrameric and multiple polytetrameric forms ranging in molecular mass (87.2-502.3 kDa) using size exclusion chromatography (SEC) and verified by laser light scattering. We evaluated the structural modification occurring in the fractionated mixture of PolyHbBv and assessed the functionality and redox stability of each fraction in relation to the mixture as a whole. Intramolecular cross-linking evaluation as performed by MALDI-MS and SEC under dissociating conditions revealed no-site-specific tetramer stabilization within the fractions; Intermolecular cross-linking was highly correlated with lysine and histidine modification as determined by amino acid composition analysis. While native unmodified hemoglobin, HbBv, PolyHbBv, and PolyHbBv fractions (F1-F4) revealed significant methionine oxidation, modification, or both, the critical betaMet55 located in the functionally plastic domains (alpha1-beta1 interface) of HbBv was unaltered. Moreover, neither of the two betaCys93 located in the highly plastic alpha1-beta2 interface were modified in PolyHbBv or in F1-F4. Our structural analysis also revealed that the reported loss in sensitivity to chloride in PolyHbBv could not be attributed to direct alteration of chloride ion binding amino acids. Structural modification imparted by glutaraldehyde resulted in nearly identical functional characteristics of PolyHbBv and its fractions with regard to oxygen equilibrium, ligand binding, and autoxidative kinetics.     

A noninvasive method for measuring the oxygen binding-releasing capacity of hemoglobin-loaded polymeric nanoparticles as oxygen carrier

Based on the strong penetration capacity of near infrared lights (NIRs) and different absorption of oxyhemoglobin and deoxyhemoglobin in NIRs region, a novel noninvasive method, with the aid of an airproof-equilibrium apparatus, was developed to determine the oxygen binding-releasing capacity, including oxygen dissociation curve (ODC) and P₅₀, of the hemoglobin-loaded polymeric nanoparticles (HbP) in this study. The measured ODC of the PLA-PEG HbP was very close to that of the native hemoglobin, and the corresponding P₅₀ (26.1 mmHg) was also near to the native precursor protein (27.3 mmHg), indicative of the validity of the method proposed. To further verify the method proposed, the oxygen binding-releasing capacity of the HbPs prepared by PCL, PCL-PEG, PLA were also investigated with human blood as control. These results indicated that the method developed here enabled accurate and noninvasive determination of the oxygen binding-releasing capacity of the biodegradable polymeric oxygen carriers.     

Metal organic chemical vapour deposition of vertically aligned ZnO nanowires using oxygen donor adducts

Vertically aligned zinc oxide (ZnO) nanowires (NWs) have been grown by liquid injection Metal Organic Chemical Vapour Deposition, using oxygen donor adducts of Me2Zn. The growth and characterisation of the nanowires grown using [Me2Zn(L)] where L = monodentate ethers, tetrahydrofuran (C4H8O) (1), tetrahydropyran (C5H10O) (2), furan (C4H4O) (3) and the bidentate ethers, 1,2-dimethoxyethane (C4H12O2,) (4) 1,4-dioxane (C4H8O2) (5) and 1,4-thioxane (C4H8SO) (6) is discussed. Single crystal X-ray structures of (4), (5), (6) have been established and are included here. The ZnO NWs were deposited in the absence of a seed catalyst on Si(111) and F-doped SnO2/glass substrates over the temperature range 350-600 degrees C. X-ray diffraction (XRD) data shows that the nanowires grown from all adduct precursors were deposited in the wurtzitic phase.     

The effect of heparin hydrogel embedding on glutaraldehyde fixed bovine pericardial tissues: Mechanical behavior and anticalcification potential

Heart valve diseases remain common in industrialized countries. Bioprosthetic heart valves, introduced as free of anticoagulation therapy alternatives to mechanical substitutes. Still they suffer from long term failure due to calcification. Different treatment methods introduced to inhibit calcification, have so far been limited in success. Glycosaminoglycans (GAGs) possess properties including high negative charge, anticoagulation and anti-inflammatory activity that make them a potential solution for calcification problem. In this study, heparin hydrogel was prepared and characterized both chemically and mechanically. After that, heparin hydrogel embedded bovine pericardial tissues, fixed with glutaraldehyde, were produced and tested for their mechanical behavior and anticalcifcation potential in vitro using the constant composition model. In the calcification experiments, tissues were divided into three groups: a) Controls without treatment, b) Hydrogel treated tissues and c) Tissues with raw heparin dissolved in the calcification solution. The results showed that embedding of tissue with hydrogel had no stiffening effect on its mechanical behavior. Calcification assessment showed a significant efficacy on inhibition of calcium phosphate deposition of hydrogel treated (second group) in comparison to untreated tissues (control, first group). Calcification inhibition potential was very similar in both the second and raw heparin (third group). Histological data confirmed the obtained results, suggesting that heparin treatment is a promising anticalcification agent.

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铁基载氧体煤化学链制氢的强化反应

为促进铁基载氧体的深度还原提高氢气产量,通过优化反应过程形成强化煤化学链制氢(强化煤CLHG)工艺。采用浸渍法制备铁基载氧体;以梅花井烟煤为原料,在固定床上研究煤与铁基载氧体的质量比对强化煤CLHG的影响;对比了三反应器煤CLHG和强化煤CLHG的制氢过程,对不同阶段的铁基载氧体进行XRD表征;对比二反应器、三反应器以及强化煤CLHG在6次循环实验中的碳转化率和氢气产量。结果表明:当煤与载氧体质量比为1:15时,氢气产量最高达1.74 L/g;强化煤CLHG中的铁基载氧体更多地被还原为FeO或Fe,还原程度加深,同时还原阶段的残炭在蒸汽氧化阶段进一步反应,使得氢气产量比三反应器煤CLHG的高18.4%;在6次循环实验中,强化煤CLHG的碳转化率与三反应器煤CLHG的相差不大,远高于二反应器煤CLHG的;强化煤CLHG的氢气产量始终高于二反应器煤CLHG和三反应器煤CLHG的;强化煤CLHG的单次最高氢气产量为1.76 L/g,循环累计氢气产量为9.54 L。强化煤CLHG缩短制氢时间,制氢能力更优异。     

Magnetic soy protein isolate–bovine serum albumin nanoparticles preparation as a carrier for inulinase immobilisation

Magnetic nanoparticles (NPs) were functionalised with soy protein isolate (SPI) and bovine serum albumin (BSA) for inulinase immobilisation. The results revealed the nanomagnetite size of about 50 nm with a polydispersity index (PDI) of 0.242. The average size of the SPI NPs prepared by using acetone was 80–90 nm (PDI, 0.277), and SPI–BSA NPs was 80–90 nm (PDI, 0.233), and their zeta potential was around −34 mV. The mean diameter of fabricated Fe₃ O₄ @SPI–BSA NPs was <120 nm (PDI, 0.187). Inulinase was covalently immobilised successfully through glutaraldehyde on Fe₃ O₄ @SPI–BSA NPs with 80% enzyme loading. Fourier transform infrared spectra, field emission scanning electron microscopy, and transmission electron microscopy images provided sufficient proof for enzyme immobilisation on the NPs. The immobilised inulinase showed maximal activity at 45°C, which was 5°C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilised enzyme was shifted from 6 to 5.5. Thermal stability of the enzyme was considerably increased to about 43% at 75°C, and K _m value was reduced to 25.4% after immobilisation. The half‐life of the enzyme increased about 5.13‐fold at 75°C as compared with the free form. Immobilised inulinase retained over 80% of its activity after ten cycles.Inspec keywords: magnetic particles, nanoparticles, proteins, molecular biophysics, nanofabrication, enzymes, Fourier transform spectra, infrared spectra, scanning electron microscopy, field emission ion microscopy, transmission electron microscopy, pH, biochemistry, nanobiotechnology, biomagnetism, electrokinetic effects, iron compoundsOther keywords: magnetic nanoparticles, soy protein isolate, bovine serum albumin, inulinase immobilisation, nanomagnetite, polydispersity index, SPI‐BSA NP, zeta potential, inulinase, glutaraldehyde, enzyme loading, Fourier transform infrared spectra, field emission scanning electron microscopy, transmission electron microscopy images, enzyme immobilisation, pH, size 80 nm to 90 nm, temperature 45 degC, temperature 75 degC, Fe₃ O₄      

Wavelet analysis of speckle patterns with a temporal carrier

A novel temporal phase-analysis technique that is based on wavelet analysis and a temporal carrier is presented. To measure displacement on a vibrating object by using electronic speckle pattern interferometry, one captures a series of speckle patterns, using a high-speed CCD camera. To avoid ambiguity in phase estimation, a temporal carrier is generated by a piezoelectric transducer stage in the reference beam of the interferometer. The intensity variation of each pixel on recorded images is then analyzed along the time axis by a robust mathematical tool, i.e., a complex Morlet wavelet transform. After the temporal carrier is removed, the absolute displacement of a vibrating object is obtained without the need for temporal or spatial phase unwrapping. The results obtained by a wavelet transform are compared with those from a temporal Fourier transform.     

Tuning of charge carrier density of ZnO nanoparticle films by oxygen plasma treatment

In this work, the charge carrier density of ZnO nanoparticle films was modified after deposition and annealing by an oxygen plasma treatment. The respective films were utilized as active layers in thin film transistors. For a discussion of the plasma–surface interaction on the molecular level, the electrical behavior of the layers was investigated which in general is highly sensitive to low level variations in defect or doping densities. A treatment with remote oxygen plasma at 400 W for 10 s led to a shift of the turn-on voltage from ?12 to 4 V and a reduction of the off-current by more than two orders of magnitude. A model for the influence of oxygen species adsorbed to ZnO nanoparticle surfaces on electrical characteristics of ZnO nanoparticle thin film transistors is introduced.     

Preparation and characterization of iminodiacetic acid-functionalized magnetic nanoparticles and its selective removal of bovine hemoglobin

In this study, a novel route for the preparation of magnetite (Fe(3)O(4)) nanoparticles (NPs) with immobilized metal affinity ligand iminodiacetic acid (IDA) charged with Cu(2+) was developed. First, magnetite nanoparticles were synthesized by a hydrothermal method. Charged with Cu(2+), the magnetic nanoparticles (MNPs) were applied to separate a model protein mixture of bovine hemoglobin (BHb) and bovine serum albumin (BSA). They could be separated completely and showed low non-specific adsorption. The morphology, structure and composition of the magnetite MNPs were characterized by transmission electron microscopy, power x-ray diffraction, x-ray photoelectron spectrometry and Fourier transform infrared spectroscopy. The resulting magnetite MNPs charged with Cu(2+) show not only a strong magnetic response to externally applied magnetic field, but are also highly specific to protein BHb. It is interesting that MNPs modified with metal ligands showed a property of magnetic colloid photonic crystals. Furthermore, they could efficiently remove the abundant protein bovine hemoglobin from bovine blood. They have potential application in removing abundant protein in proteomic analysis.     

A TDM wattmeter with a 0.5-MHz carrier frequency

The author describes a standard time-division-multiplexing (TDM) wattmeter with a constant average carrier frequency, no inherent phase error, and known systematic magnitude frequency error, as well as efforts to increase frequency bandwidth. The residual error of the wattmeter, after corrections, is below 100 p.p.m. up to 5 kHz. The meter is expected to find application in accurate power measurements under distorted and low power factor conditions, and as a transfer standard for interlaboratory comparisons     

Contribution to carrier densities of the oxygen vacancy in a low-resistivity tin-doped indium oxide film by the hot-cathode plasma sputtering method

In order to clarify the contribution to carrier density by oxygen vacancies in tin-doped indium oxide (ITO) films prepared on glass substrates by the hot-cathode plasma sputtering method, we have investigated the effect of annealing on the electrical properties of an ITO film with a resistivity of 1.0 × 10^− 4 Ω cm. A drastic decrease in carrier density from 2.0 × 10²¹ to 0.88 × 10²¹ cm^− 3 was found with gradual increase in the Hall mobility from 29 to 35 cm² V^− 1 s^− 1 for repeated annealing cycles, when the ITO film was exposed for one hour to 400 °C oxygen gas at atmospheric pressure. The results indicate that the contribution of oxygen vacancies to carrier density was ca. 1.12 × 10²¹ cm^− 3 for the ITO film with an overall carrier density of 2.0 × 10²¹ cm^− 3.     

戊二醛交联淀粉微孔发泡材料(SMCF)的制备及表征

以玉米淀粉为原料,将糊化后的淀粉溶液与戊二醛交联,采用溶剂置换制得高白度、表面带有微孔结构、并具有一定抗水性能的淀粉微孔发泡材料(starch microcellular foam,SMCF).研究了交联反应及溶剂置换各因素对SMCF白度、湿含量及微孔形成的影响,确定最优工艺:交联剂戊二醛的用量为10g戊二醛/100g...