Raman spectroscopy-based sensitive and specific detection of glycated hemoglobin

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the nonenzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and nonglycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision--with a limit of detection that is a factor of ~15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multicomponent mixtures and potentially in hemolysate and whole blood lysate samples.     

Human hemolysate glycated proteome

Despite continuous advances in hyperglycemia treatments, a precise control through monitoring of glucose and glycated hemoglobin remains in most diabetic patients as the diagnosis/prognosis tool. An alternative perspective could be the discovery and quantitation of new blood glycated proteins formed by nonenzymatic reaction with circulatory glucose. As a result, the human hemolysate is an incomparable source of glycated proteins to further monitor glycemia and interpret changes at the level of this post-translational modification. The human hemolysate is here studied based on the differential labeling of proteins with isotopically labeled-glucose ([(13)C(6)] glucose), named glycation isotopic labeling. Due to the chemoselectivity of glycation, only preferential targets are labeled by this protocol. The approach provides qualitative data through the detection of preferential protein glycation sites as well as quantitative information to evaluate the abundance of this modification. This strategy was applied to human hemolysate samples corresponding to different glycemic states estimated by laboratory-certified concentrations of glycated hemoglobin. The glycation level of each protein can then be employed to interpret the effect of glucose exposition as a consequence of glycemic unbalance. This information should provide new molecular insights into protein glycation mechanisms that might generate a new hypothesis to clinicians to improve the understanding of underlying pathologies associated to prolonged hyperglycemia.     

Determination of the efficiency of gamma detectors from particle-gamma angular correlations

Particle-γ angular correlation technique can be used to determine the efficiency of γ-detectors. This method is demonstrated for 4.44 MeV γ-rays from the reaction ¹²C(α, α′γ).     

Ta掺杂对γ-TiAl基合金块状组织和力学性能的影响

研究了Ti-48Al-2Nb-2Cr-xTa(x=0,1.0,2.0)(原子分数,%)合金α单相区淬火,块状组织(γ_m相)的演变规律,采用EBSD(电子背散射衍射)技术对晶粒取向进行表征。结果表明,在快速冷却条件下,微量Ta元素的添加能够促进γ_m相的析出,促进(体积分数大于50%)或者抑制(体积分数小于50%)γ_m相析出的Ta元素临界含量为2%(原子分数)。Ta元素能够细化γ_m相亚颗粒,同时,不同种类γ_m变体可直接在α₂晶粒内以BOR形核,在生长过程中发生了沿着{111}γ密排面的变体选择,使得第二代γ_m变体偏离BOR。     

Use of home hemoglobin A1c test kits to monitor the effectiveness of diabetes care

BACKGROUND: Periodic measurement of glycated hemoglobin (HbA1c) is highly recommended for people with diabetes to determine whether their blood glucose is adequately controlled. Quality improvement programs initiated by health plans often focus on ensuring that HbA1c is being monitored in members with diabetes. To focus improvement efforts on members with poor blood glucose control, health plans need to know which members have high HbA1c levels. Recent development of home test kits provides another opportunity for health plans to help members measure their HbA1c and to identify members with high levels. METHODS: A sample of members from two health plans who were sent HbA1c self-test kits in January 2000 participated in a telephone interview. To understand why members did or did not use self-test kits sent by their health plans, the survey focused on perceived ease of use, outcomes, and normative beliefs. RESULTS: In the group of 380 members who were interviewed, 170 (45%) used the kit. HbA1c values were > 8 mg/dl in 43%. Among the 170 who used the kit, 160 said that they would use the kit. Their most common reason for using the kit was to find out how well their blood glucose was being controlled (48%). Convenience (12%) was the next most frequent reason for using the kit. Among the 210 members who did not use the kit, 81 members said that they would not or were not sure if they would when interviewed. Their most frequent reason for not using the kit was duplication of tests done by physicians (34%). Others were too busy (12%), wanted to talk with their physician (11%), or had difficulty using the kit (11%). CONCLUSIONS: Because the majority of health plan members did not use the kit and the majority who did use the kit had HbA1c levels < 8 mg/dl, sending home test kits to members did not result in a high yield of members with elevated HbA1c levels. Physicians' support for use of the kits and efforts to make kits easier to use might increase use. Efforts to avoid duplication of physicians' measurements could make this strategy to identify members with poorly controlled levels of blood glucose more cost-effective, although health plans would not know which monitored members might benefit most from programs to improve care of diabetes.     

Selective electrochemical sensing of glycated hemoglobin (HbA1c) on thiophene-3-boronic acid self-assembled monolayer covered gold electrodes

We report a novel concept of sensing glycated hemoglobin, HbA 1c, which is now the most important index for a long-term average blood glucose level, by first selectively immobilizing it on the thiophene-3-boronic acid (T3BA) self-assembled monolayer (SAM)-covered gold electrode by a selective chemical reaction with boronic acid. HbA 1c thus immobilized is then detected by the label-free electrochemical impedance spectroscopic (EIS) measurements with a redox probe, an equimolar mixture of K 3Fe(CN) 6 and K 4Fe(CN) 6, present. The rate of charge transfer between the electrode and the redox probe is shown to be modulated by the amount of HbA 1c in the matrix hemoglobin solution due to the blocking effect caused by the binding of HbA 1c with boronic acid. Both the formation of a well-defined T3BA-SAM on the gold surface and the chemical binding of its boronic acid with HbA 1c in solution were confirmed by quartz crystal microbalance, atomic force microscopy, and EIS experiments.     

Effects of Alloy Elements (C, B, Fe, Si) on the Ti-Al Binary Phase Diagram

1.IntroductionAlloyingisanimportantwaytoimprovetheprop-ertiesoftwofphase(cr2+ry)titaniumaluminideal-loys.Thealloyingelementswhichhavebeenstud-iedcanbesubdividedintothreegroupsX1(V,MnandCr),X2(Nb,Ta,MoandW)andX3(Si,C,B,N,P,Se,Te,NiandFe)['j.Smalladdi-tionsofX3elementsinthealloyshaveparticularlyreceivedmuchattentioninrecentyears['ro,'].ItwasreportedthatadditionofsmallamountsC,B,FeorSirefinesthemicrostructure[3~5?1o]limprovesroom-temperatureductilityl"']andincreasesoxida-tionresistance[…     

A new type face-centered cubic zirconium phase in pure zirconium

So far,only two orientation relationships (OR) between hexagonal close-packed (HCP) (α phase) and face-centered cubic (FCC) structures in zirconium and titanium alloys have been reported.Here a new type FCC phase (named γphase) with OR:< 11(2)0 >αll< 100 >γ and {0001}αll{002}γ was observed for the first time in annealed pure zirconium by means of transmission electron microscopy (TEM) technique.The α→γphase transformation can be accomplished via expansion along[1(1)00]direction and slip of Shockley partial dislocation with 1/3[1(1)00]on (0001) basal planes.     

Nucleation and growth mechanisms of phase with single-unit-cell height in Mg-RE-Zn(Ag) series alloys:a first-principles study

The highly thermal stability precipitates strengthen creep resistance for alloys,which require precipitates to resist coarsen at elevated temperature.In the Mg-RE-Zn (Ag) series alloys,the key strengthening phase-γ'phase,remains its single-unit-cell height throughout aging process.The origin of such extremely thermal stability of γ'phase is still unclear.By using the first-principles calculations,it is found that the formation ofγ'phase introduces compressive strain to its surrounding α-Mg lattice and simultaneously alter charge distribution of α-Mg lattice near the α-Mg/γ'hetero-interface.These two variations over α-Mg lattice lead to lower vacancy formation energies and migration energy barriers for solute atoms near the α-Mg/γ'hetero-interface in comparison with that of bulk condition.Consequently,the variations facilitate rapid diffusion of solute atoms near the γ'phase and promote nucleation rate of other γ'plates around it.On the basis of ledge-thickening model,the origin of nucleation/growth of γ'phase with single-unit-cell height was explained.Thermodynamically and kinetically,the solute atoms are not apt to migrate into the nearest basal plane adjacent to the γ'phase.Moreover,the lower aging temperature(～200 ℃) and almost completely coherent α-Mg/γ'hetero-interface lead to the ledges are extremely hard to nucleate on the hetero-interface.Therefore,γ'plates maintain single-unit-cell height throughout aging process.     

Structure and Stability of TiC/g Interface in Fe-Cr-Ni Base Composite

The morphology, orientation relationship and stability of TiC/γ interface in Fe-Cr-Ni base composite synthesized with a liquid state in-situ process have been studied. The TiC/γ interface in as-cast sample is of coherent feature. Its orientation relationship is (0-20)γ//(2-20)TiC, [001]γ//[001]TiC. During the aging at 1473 K, the TiC/γ interface may dissolve in matrix and lamellar M23C6 compound may precipitate from γ-matrix.     

Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody

Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.     

Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis II (Hunter Syndrome)

We have developed a tandem mass spectrometry based assay of iduronate-2-sulfatase (IdS) activity for the neonatal detection of mucopolysaccharidosis II (MPS-II, Hunter Syndrome). The assay uses a newly designed synthetic substrate (IdS-S) consisting of α-L-iduronate-2-sulfate, which is glycosidically conjugated to a coumarin and a linker containing a tert-butyloxycarbamido group. A short synthesis of the substrate has been developed that has the potential of being scaled to multigram quantities. Sulfate hydrolysis of IdS-S by IdS found within a 3 mm dried blood spot specifically produces a nonsulfated product (IdS-P) which is detected by electrospray tandem mass spectrometry and quantified using a deuterium-labeled internal standard, both carried out in positive ion mode. Analysis of DBS from 75 random human newborns showed IdS activities in the range of 4.8-16.2 (mean 9.1) μmol/(h L of blood), which were clearly distinguished from the activities measured for 14 MPS-II patients at 0.17-0.52 (mean 0.29) μmol/(h L of blood). The assay shows low blank activity, 0.15 ± 0.03 μmol/(h L of blood). The within-assay coefficient of variation (CV) was 3.1% while the interassay CV was 15%.     

GH742中γ′相和γ基体成分随时效时间和温度的变化规律研究

利用透射电镜能谱法(TEM-EDX)研究了GH742合金中γ′和γ基体两相成分随温度和时效时间的变化规律.结果表明:合金在1050℃时效时,γ′相和γ基体的成分在时效初期变化较大,当时效时间超过1440min后,γ′相和γ基体的成份基本稳定.合金在750～1100℃时效时,γ′相和γ基体的成分均随着温度的升高而发生变化,其中γ基体的成分随温度变化较明显.合金中各元素在γ′和γ两相中的偏析率Cγ′/ Cγ变化规律研究表明:Ti,Al,Nb,Ni等γ′形成元素的偏析率均随着时效温度的升高而降低,而Cr,Co,Mo等γ形成元素的偏析率均随着时效温度的升高而增大.     

Microstructure of the Ductile Phase in Intermetallic Compound Ni_(50)Al_(20)Fe_(30)

The microstructure of the single hot extruded and annealed Ni50Al20Fe30Y0.003 intermetallic compound alloys has been examined by means of high resolution electron microscopy (HREM). In these extruded and annealed alloys. the ductile phase is of a mixture of the disordered fcc γ matrix and or dered γ' precipitates. This fact well interprets the reason why the degree of annealing treatment can influence the strength and ductility of these alloys. The HREM observation revealed directly that there was some strain concentration at γ'-γ interfaces, due to the presence of more iron atoms in these two phases. The fixed orientation relationship between the γ phase and γ' precipitates was identified to be {001}γ{00 }γ' and <100 >γ < 100 > γ'     

Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. A reversed-phase liquid chromatography method followed by a neutral loss scan mass spectrometric method was developed for the screening of glycation in proteins. The neutral loss scan was based on a unique sugar moiety neutral loss (-162 Da) that we observed in the fragmentation spectra of glycated peptides on Q-Tof type mass spectrometers. The collision energy was optimized for this neutral loss using a glycated synthetic peptide, and 20 eV was found to be the optimum collision energy. The neutral loss scan experiment was composed of two segments. In the first segment, the glycated peptides were identified based on the signature neutral loss of 162 Da when the collision energy was elevated to 20 eV. In the second segment, the glycated peptides were selected as the parent ions and fragmented at higher collision energy to break the peptide bonds. The fragmentation spectra of the selected glycated peptides revealed both the amino acid sequences and the sites of glycation. This neutral loss scan method was used to study the glycation in human serum albumin (HSA). The glycation sites in HSA were identified based on the retention time shift of glycated peptides, the mass accuracy from the MS scan, the signature neutral loss, and MS/MS information. Using this method, we were able to identify that 31 lysine residues were partially glycated from the glycated HSA sample, which has a total of 59 lysine residues.     

Grain boundary transformation character in Fe-N austenite

The morphology and crystallography of the isothermal transformation of Fe-N austenite at 225 °C were characterized by transmission electron microscopy and scanning electronic microscope. Fe-N austenite was produced by through-nitriding thin pure iron sheets at 640 °C. Lath-shaped and periodically distributed (γ-austenite + γ′-Fe₄N + α-Fe) microstructures formed at the prior austenite grain boundaries, this kind of microstructure was shown to be bainite in nature and kept a Greninge-Troiano orientation relationship between the γ-austenite (γ´-Fe₄N) and α-ferrite. The formation mechanism of the grain boundary microstructure was shown to be the preferential precipitation of the proeutectoid γ′-Fe₄N at the grain boundary, followed by the transformation of the N-depleted austenite into α-ferrite and γ′-Fe₄N, thus forming a periodic γ-γ′-α-γ′-γ-γ′-α-γ′-γ-γ′ arrangement.     

Thrombotic events and markers of oxidation and inflammation in hemodialysis

The goal of this study was to determine whether antioxidant therapy with vitamin E would alter the rate of vascular access complications or other macrovascular complications in hemodialysis (HD) patients. A secondary goal of the study was to explore the relationship between baseline pretreatment markers of oxidative stress (the advanced glycation end product pentosidine and basal levels of vitamin Eα and γ) and the subsequent development of access failure. Thirty‐five stable patients treated by HD were recruited for the study. Patients were provided with vitamin E (800 IU) or placebo capsules to be taken daily. Clinical variables, vascular access function (flow meter access flow measurements), and circulating blood markers were obtained initially and every 3 months throughout the study. Vitamin Eα levels rose in treated patients from 12.7 ± 4.4 to 25.1 ± 15.1 µg/mL at 3 months and 28.6 ± 14.8 µg/mL at 6 months. Vitamin Eγ levels fell in treated patients from 3.9 ± 1.7 to 2.3 ± 1.5 µg/mL at 3 months and 1.7 µg/mL at 6 months. Patients who subsequently developed repeated thrombotic vascular access events were characterized by higher baseline pentosidine content of circulating proteins. Patients who developed a myocardial infarction had higher pentosidine, lower vitamin Eα, and much lower vitamin Eγ than patients who did not develop thrombotic events. These findings lead to the speculation that the anti‐inflammatory effects of vitamin Eγ may play a more important role in thrombotic vascular events than the antioxidant effects of vitamin Eα. Additional studies of these interactions are in progress.     

γ-Fe Nano-particles from Fe(CO)_5 by CW CO_2 Laser-driven

γ-Fe nano-particles with size of 20-40 nm were produced by SF6-sensitized CW CO2 laser-induced gaseous pyrolysis of Fe(Co) 5, The γ-Fe stabte in reaction zone at above 910℃ was formed.The rapid quenching prevents from the γ-Fe transforming to α-Fe as rapidly cooling from high temperature to room temperature, The characteristics of the particles were examined at room temperature by TEM. electron diffraction and XRD. It was proved that about 70% of γ-Fe phase in the particles was present. In addition. the lattice constant of the γ-Fe was 0.364 nm in place of 0.360 nm     

Characterization of nonenzymatic glycation on a monoclonal antibody

We present here an improved analytical method for the analysis of glycation events in proteins. Nonenzymatic glycation of an IgG2 monoclonal antibody was studied using affinity chromatography, mass spectrometry, and chemical derivatization. Analysis of both forced-degraded and bulk-drug substance (BDS) samples showed the presence of glycated protein. A new peptide mapping procedure, incorporating derivatization using sodium borohydride, allowed the development of a sensitive method for detecting and identifying the sites of modification. When combined with tandem mass spectrometry, peptides glycated by glucose showed dramatically improved MS/MS spectra as compared to underivatized controls. Using these methods we were able to map a number of glycation sites in both forced-degraded and BDS samples that were distributed across both light and heavy chain subdomains. The combination of affinity chromatography, high-resolution mass spectrometry, and a simple derivatization procedure should allow the facile analysis of glycation for other antibody and protein samples.