血浆氨基酸代谢谱与糖尿病相关性研究

为了利用代谢组学手段研究血浆氨基酸代谢谱与糖尿病相关性, 采用邻苯二甲醛(OPA)柱前在线衍生反相高效液相色谱法,建立了血浆中21种氨基酸代谢谱的相对定量分析方法.衍生由自动进样器在线自动完成,紫外检测器检测,流动相A为10 mmol/L Na2HPO4-Na2B4O7缓冲液(pH 7.95),B为乙腈-甲醇-水 (45: 45∶ 10,V/V), 线性梯度洗脱,流动相B在30 min内由5%增加至46%,27 min内21种氨基酸全部得到良好分离.用此方法测定了51位临床糖尿病人血浆氨基酸代谢谱.以相对峰面积作为原始数据,按血糖值高低预分组,通过Spss和Matlab软件平台,用线性判别分析、逐步判别分析和得分图进行关联研究,结果表明:血浆中氨基酸代谢谱与血糖值的高低存在相关性,对血糖值低于6.2的志愿者和血糖值高于9.5的志愿者,判别分析的总正判率达到了94.1%.逐步判别分析结果表明,有7种氨基酸(Arg, Cit, Asp, Asn, Thr, Leu, Trp)承载了上述分组的重要信息.其对上述分组的判别分析总正判率为90.2%,可作为反映血糖值高低变化的标志物组,对于糖尿病的早期诊断及深入研究具有潜在的科研及临床价值.     

老年性痴呆患者血浆中氨基酸类神经递质的HPLC荧光检测

采用邻苯二甲醛(OPA)柱前衍生反相高效液相色谱法测定了老年痴呆患者(AD)血浆中4种氨基酸类神经递质(天门冬氨酸、谷氨酸、γ-氨基丁酸、甘氨酸)的含量.用Hanbon反相柱分离,荧光检测器检测;流动相A为四氢呋喃-甲醇-0.05 mol/L磷酸二氢钾(pH 6.0)(体积比2 : 35 : 63);流动相B为90%甲醇;采用二元梯度洗脱.天门冬氨酸、谷氨酸、γ-氨基丁酸、甘氨酸的检出限分别为0.21、0.01、0.05、0.02 pmol/L,在对应的浓度范围内线性关系良好,相关系数为0.997 5 ～0.999 1,峰面积的相对标准偏差分别为1.08%、1.24%、1.12%和1.35%,回收率为98% ～99%.AD患者血浆中天门冬氨酸和谷氨酸的浓度显著高于对照组(P＜0.05),甘氨酸与对照组无显著差异.     

反相高效液相色谱测定依替巴肽注射液中氨基酸的含量

采用异硫氰酸苯酯为柱前衍生化试剂,结合反相高效液相色谱技术,建立了定量分析依替巴肽注射液中氨基酸组分的检测方法。优化色谱条件,选用Agilent XDB-C18色谱柱(5μm,150 mm×4.6 mm),以0.1 mol/L醋酸钠(冰醋酸调至pH 6.0)-乙腈(90∶10)为流动相A,乙腈为流动相B,梯度洗脱方式,检测波长为254 nm,流速为1.0 mL/min,进样体积为5μL,柱温为30℃。结果表明,在优化条件下,各氨基酸能实现有效分离,且在1.23～3.68 mmol/L浓度范围内,各氨基酸的线性关系良好,相关系数(r)为0.999 1～0.999 6。该方法显示了良好的准确性、重复性、稳定性及选择性,能对依替巴肽注射液中的氨基酸实现准确定量分析。     

柱前衍生–高效液相色谱法测定白刺果中氨基酸含量

建立一种快速测定不同产地的白刺果中氨基酸含量的HPLC方法。采用柱前邻苯二甲醛(OPA)和氯甲酸芴甲酯(FMOC)联合在线衍生、二元梯度洗脱(流动相A:40 mmol/L NaH2PO4·H2O,pH 7.8;流动相B:乙腈–甲醇–水的体积比为4.5∶4.5∶1)、反相C18短柱分离(色谱柱:Zorbax Eclipse AAA C18柱,75 mm×4.6 mm,3.5μm)、二极管阵列检测器(检测波长:338 nm;参考波长:390 nm)和荧光检测器(激发波长:340 nm;发射波长:450nm)联合检测,内标法定量。各氨基酸含量在4.5~900μmol/L范围内线性关系良好,相关系数为0.991 2~0.999 8,除了蛋氨酸(部分氧化降解)加标回收率为78.1%外,其它各氨基酸的加标回收率为93.1%~105.1%,相对标准偏差为3.21%~6.23%(n=5)。对产自青海、新疆和内蒙古等3个地区的白刺果中氨基酸含量进行了测定,氨基酸总量分别为11.23,10.47,8.84 g/(100 g),并对各种不同类型氨基酸占氨基酸总量的比例进行了分析。该法适合于白刺果氨基酸含量的测定。     

新型衍生试剂柱前衍生氨基酸的高效液相色谱分析

以邻硝基苯磺酰氯为标记试剂,RP-HPLC为分析模式,建立了一种新的氨基酸衍生化方法.通过液质联用对产物进行定性,研究并确定了最佳衍生化条件:衍生温度25 ℃,缓冲液pH9.0,衍生时间10 min.实验建立了20种氨基酸的HPLC分离方法:选用Kromasil C18柱,流动相A为30 mmol/L的NH4Ac溶液(pH 7.5),B相为乙腈;采用梯度洗脱,检测波长275 nm,室温.20种氨基酸在0.025 ～6.4 mmol/L范围内线性关系良好,相关系数在0.998 0 ～0.999 8之间,检出限为0.01 ～0.113 nmol.     

毛细管电泳-激光诱导荧光用于中药制剂中氨基酸成分的分析

建立了一种用于测定中药制剂中氨基酸成分的毛细管电泳-荧光检测方法. 用含有α-环糊精(α-CD)的硼砂缓冲溶液为背景电解质, 经异硫氰酸荧光素(FITC)衍生的5种氨基酸在50 min内可以得到很好的分离和测定. 考查了各个分离参数对分离的影响, 得到的优化条件为: 含45 mmol/L的α-环糊精的80 mmol/L硼砂缓冲溶液(pH值9.2)作为背景电解质, 分离电压20 kV; 柱温22 ℃. 衍生试剂FITC与单个氨基酸的化学计量比为4∶1时, 能够获得稳定荧光强度的氨基酸衍生物. 在优化条件下, 各氨基酸成分在73.5～2900 nmol/L 的浓度范围内呈良好的线性关系(相关系数r2为0.9906～0.9998). 保留时间和峰面积的相对标准偏差分别为0.8%～3.0%和0.7%～5.7%, 检测限(3倍信噪比)为3.5～35 nmol/L. 该方法准确可靠, 可用于质量控制为目的的中药制剂中氨基酸成分的定量测定.     

高效液相色谱法测定生物体液中γ-氨基丁酸

研究了邻苯二甲醛柱前衍生反相高效液相色谱法测定血浆和脑脊液中γ-氨基丁酸的含量。色谱条件:色谱柱为Nova-pak C18柱,流动相为50 mmol.L-1的乙酸钠(pH 6.8)、甲醇和四氢呋喃(A:82∶17∶1;B:22∶77∶1,V/V),梯度洗脱。方法的线性范围为0.5~100μmol.L-1,检出限为0.04μmol.L-1。日间和日内测定的精密度分别为1.9%~6.8%和1.6%~5.7%;标准加入回收率在92.7%与96.8%之间。采用上述方法测定正常人和癫痫病患者血浆和脑脊液中γ-氨基丁酸含量,结果令人满意。     

高血压患者血浆中游离氨基酸的柱前衍生HPLC法测定

以2,4-二硝基氯苯为衍生化试剂,建立了反相高效液相色谱法测定人血浆中22种游离氨基酸的方法.衍生化产物用Kromasil C18柱分离,梯度洗脱,紫外检测波长为360 nm.在所研究的浓度范围内,22种氨基酸的线性关系良好(r>0.99),日内精密度(RSD)为1.7% ～4.1%,日间精密度(RSD)为2.8% ～6.9%,方法回收率为90% ～106%.用建立的方法对高血压病人和正常人血浆中的游离氨基酸进行测定,并将测定结果进行主成分分析及判别分析,结果表明高血压病人与正常人血浆中的氨基酸存在显著差异,分类判别率为94.4%.     

柱前衍生高效液相色谱测定小儿复方氨基酸注射液中氨基酸

建立了高效液相色谱(HPLC)测定小儿复方氨基酸注射液中各种氨基酸含量的方法.采用Kromasil C18(250×4.6 mm,5 μm)色谱柱,以2,4-二硝基氟苯为衍生试剂,梯度洗脱.流动相A为乙腈∶水(50∶50,V/V),流动相B为0.04 mol/L的磷酸二氢钾溶液(pH=6.8);流速为1.0 mL/min;检测波长为360 nm.所测氨基酸的线性范围为0.008～0.2213 mg/mL,平均回收率在97.85%～102.86%之间.     

对一种分离测定氨基酸方法的改进

 对Waters公司采用 6 氨基喹啉 N 羟基琥珀酰亚胺基 氨基甲酸酯 (AccQ Tag)柱前衍生化测定氨基酸的方法进行了改进。将流动相流速由原来的 1 0mL/min改变为 2 0mL/min ,用AccQ Tag专用柱 (3 9mmi.d .× 15 0mm ,4μm)在 17 5min(原为 35min) (运行周期为 2 2 5min ,原为 45min)内快速分离测定了 18种氨基酸和牛磺酸。用Nova PakC18柱 (3 9mmi.d .× 15 0mm ,4μm) ,Nova PakC18柱 (4 6mmi.d .× 15 0mm ,4μm) ,SymmetryC18柱 (3 9mmi.d .× 15 0mm ,4μm)和WatersXterraRP 18柱等反相C18柱代替AccQ Tag专用柱 ,均可对氨基酸进行快速分离。     

Generation and detection of levuglandins and isolevuglandins in vitro and in vivo

Levuglandins (LGs) and isolevuglandins (isoLGs), formed by rearrangement of endoperoxide intermediates generated through the cyclooxygenase and free radical induced oxidation of polyunsaturated fatty acids (PUFAs), are extraordinarily reactive, forming covalent adducts incorporating protein lysyl ε-amino groups. Because they accumulate, these adducts provide a dosimeter of oxidative injury. This review provides an updated and comprehensive overview of the generation of LG/isoLG in vitro and in vivo and the detection methods for the adducts of LG/isoLG and biological molecules in vivo.     

钙与人体及临床

钙是宏量元素，是人体中含量较高的元素之一，体内99％的钙构成骨骼和牙齿以及维持骨骼结构，1％的钙调节人体重要生理功能。钙的含量过高或过低都与许多疾病有关，只有保持一种平衡状态，才能使机体处于正常环境。     

Coassembly of Nanorods and Nanospheres in Suspensions and in Stratified Films

The entropically driven coassembly of nanorods (cellulose nanocrystals, CNCs) and nanospheres (dye‐labeled spherical latex nanoparticles, NPs) was studied in aqueous suspensions and in solid films. In mixed CNC‐latex suspensions, phase separation into an isotropic latex‐NP‐rich and a chiral nematic CNC‐rich phase took place; the latter contained a significant amount of latex NPs. Drying the mixed suspension resulted in CNC‐latex films with planar disordered layers of latex NPs, which alternated with chiral nematic CNC‐rich regions. In addition, fluorescent latex NPs were embedded in the chiral nematic domains. The stratified morphology of the films, together with a random distribution of latex NPs in the anisotropic phase, led to the films having close‐to‐uniform fluorescence, birefringence, and circular dichroism properties.     

Structure Formation and Molecular Mobility in Water and in Aqueous Solutions

The study of the structure of water and of aqueous solutions has recently received new impetus from the efforts at commercial desalineation of sea water and from developments in molecular biology. The current view that, apart from single molecules, water contains only one type of structural element, namely “flickering” network structures with tetrahedrally hydrogen-bonded water molecules (two-states model) is proving inadequate in the interpretation of new experimental data and in the calculation of thermodynamic functions. After a critical discussion of the basis of this model and of the concept of hydrogen bonds, a second kind of structural element, i.e. a third state, is suggested: small aggregates of molecules containing mainly non-tetrahedral hydrogen bonds as well as some tetrahedral ones, and packed more densely than allowed by the lattice-like structure. These aggregates – dimers to hexamers – can be regarded as the primary products of disruption of the network structures, and displace the latter as structural components in water with increasing temperature or concentration of solutes. This “combined” model allows a consistent interpretation of the properties of water and of the various effects of dissolved substances.     

Advances in amperometric and conductometric detection in capillary and chip-based electrophoresis

Amperometric and conductometric detection are currently the two major electrochemical detection modes in capillary and chip electrophoresis. The ease of miniaturization and integration of electrochemical detection elements offers a high potential for the development of portable analytical devices based on electromigrative separations. The challenges and basic concepts of both detection principles in the context of capillary/chip electrophoresis are shortly introduced and milestones of the methodical developments are summarized from a historical perspective. Recent advances and applications are discussed with more detail. Particular attention is paid to new trends in this area of research such as measurements in short capillaries and the enormous progress and increased popularity of contactless conductivity detection. Correspondence: Frank-Michael Matysik, Institute of Analytical Chemistry, University of Leipzig, Linnéstr. 3, D-04103 Leipzig, Germany     

Femtosecond electron-transfer reactions in mono- and polynucleotides and in DNA

Quenching of redox active, intercalating dyes by guanine bases in DNA can occur on a femtosecond time scale both in DNA and in nucleotide complexes. Notwithstanding the ultrafast rate coefficients, we find that a classical, nonadiabatic Marcus model for electron transfer explains the experimental observations, which allows us to estimate the electronic coupling (330 cm(-1)) and reorganization (8070 cm(-1)) energies involved for thionine-[poly(dG-dC)](2) complexes. Making the simplifying assumption that other charged, pi-stacked DNA intercalators also have approximately these same values, the electron-transfer rate coefficients as a function of the driving force, DeltaG, are derived for similar molecules. The rate of electron transfer is found to be independent of the speed of molecular reorientation. Electron transfer to the thionine singlet excited state from DNA obtained from calf thymus, salmon testes, and the bacterium, micrococcus luteus (lysodeikticus) containing different fractions of G-C pairs, has also been studied. Using a Monte Carlo model for electron transfer in DNA and allowing for reaction of the dye with the nearest 10 bases in the chain, the distance dependence scaling parameter, beta, is found to be 0.8 +/- 0.1 A(-1). The model also predicts the redox potential for guanine dimers, and we find this to be close to the value for isolated guanine bases. Additionally, we find that the pyrimidine bases are barriers to efficient electron transfer within the superexchange limit, and we also infer from this model that the electrons do not cross between strands on the picosecond time scale; that is, the electronic coupling occurs predominantly through the pi-stack and is not increased substantially by the presence of hydrogen bonding within the duplex. We conclude that long-range electron transfer in DNA is not exceptionally fast as would be expected if DNA behaved as a "molecular wire" but nor is it as slow as is seen in proteins, which do not benefit from pi-stacking.     

Carbon chains and rings in the laboratory and in space

Seventy-seven reactive organic molecules of astrophysical interest have been identified in a supersonic molecular beam, 73 in the radio band by Fourier-transform microwave spectroscopy, four in the optical by laser cavity ringdown spectroscopy. Most are linear carbon chains, but six consist of carbon chains attached to the compact, highly polar C3 ring, and two are rhomboidal cyclic configurations of SiC3. The laboratory astrophysics of the radio molecules is complete for the time being, in the sense that essentially all the rotational transitions of current interest to radio astronomy (including hyperfine structure when present) can now be calculated to a small fraction of 1 km s(-1) in equivalent radial velocity; six of the radio molecules have already been detected in space on the basis of the present data. The FTM spectrometer employed in this work is far from fundamental limits of sensitivity, so many more molecules can probably be found by refinements of present techniques. The density of reactive molecules in our supersonic beam is generally high by the standards of laser spectroscopy, and many of the radio molecules probably have detectable optical transitions which we are attempting to find, largely motivated by the long-standing problem of the diffuse interstellar bands. Our most interesting result to date is the detection of a fairly strong molecular band at 443 nm in a benzene discharge, in exact coincidence with the strongest and best known interstellar band. Isotopic shifts measured with partially and totally deuterated benzene suggest that the carrier of the laboratory band is a hydrocarbon molecule with the elemental formula CnH5, with n most likely in the range 3-6.