LC-MS/MS 检测癫痫患者神经递质类氨基酸

目的:建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价.方法:选用AAA-C18柱色谱柱,以乙腈水(含有0.01%七氟丁酸、0.1%甲酸)为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测.疾病组与健康组的统计采用t检验和主成份分析.结果:疾病组和健康组氨基酸测定结果显示:Trp、GABA两组间没有显著性差异(P＞0.05),Arg、Glv、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异(P＜0.05),通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸.结论:试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价.     

野生与豢养长江江豚血清氨基酸含量比较

应用高效液相色谱(HPLC)技术,首次测定了湖北石首长江天鹅州白豚自然保护区野生长江江豚(Neophocaena phocaenoides asiaeorientalis)和中国科学院水生生物研究所白豚馆人工饲养的长江江豚血清中17种氨基酸的含量.结果表明,除了脯氨酸Pro、蛋氨酸Met和组氨酸His外,人工饲养江豚血清中其余14种氨基酸(天门冬氨酸Asp、谷氨酸Glu、丝氨酸Ser、精氨酸Arg、甘氨酸Gly、苏氨酸Thr、丙氨酸Ala、异亮氨酸Ile、亮氨酸Leu、苯丙氨酸Phe、缬氨酸Val、赖氨酸Lys、酪氨酸Tyr、胱氨酸Cys)的含量显著高于野生长江江豚血清中相应氨基酸的含量.野生江豚和人工饲养江豚的血清氨基酸含量均没有显著的性别差异.野生江豚性成熟个体与未成熟个体之间血清氨基酸含量也没有显著性的差异.在所检测的17种氨基酸中,豢养江豚Glu含量最高,其次为Asp和Lys.野生江豚同样是Glu最高,其次是Lys和Asp.豢养和野生江豚都是Met含量最低.野生和豢养江豚必需氨基酸(EAA)和非必需氨基酸(NEAA)之间的比率分别是0.83和0.92,具有极显著的差异(p <0.01).     

HPLC-MS/MS法检测星形胶质细胞兴奋性氨基酸释放

目的:建立一种快速测定星形胶质细胞上清中兴奋性氨基酸释放的高效液相-串联四级杆质谱方法.方法:使用高效液相-串联四极杆质谱的多反应监测(MRM)模式快速测定星形胶质细胞(AST)兴奋性氨基酸(EAAs)的释放量.结果:该方法检测细胞上清液中兴奋性氨基酸释放量能够在4分钟内完成,有很好的精密度(Glu 1.0%,Asp 1.6%),较好的稳定性(组内的变异系数Glu为1.34%,Asp为1.41%;组间变异系数Glu为2.32%,Asp为2.18%)和回收率(Glu 97.1%;Asp 95.8%).结论:该方法定量检测兴奋性氨基酸快速、准确,可用于大批量样品的快速测定.     

异丙酚对全脑缺血/再灌注大鼠海马谷氨酸、抗坏血酸释放的影响

目的：观察异丙酚对全脑缺血／再灌注大鼠海马细胞外谷氨酸（Glu）和抗坏血酸（AA）的影响,探讨异丙酚脑保护作用机制。方法：采用Pulsinelli-Brlerley四血管阻断法制备全脑缺血模型,应用脑微透析技术结合高效液相色谱（HPLc）检测大鼠海马细胞外Glu、AA含量的变化。结果：与缺血／再灌注组各对应时点相比较,异丙酚处理组大鼠海马细胞外Glu、AA含量明显降低,统计结果差异均有显著性（P〈0．05,或〈0．01）。结论：缺血／再灌注早期应用异丙酚不仅减少兴奋性氨基酸释放,还能清除自由基、抑制脂质过氧化反应而产生脑保护作用。     

高效液相荧光法测定大鼠脑内氨基酸类神经递质方法的改良

目的 改良测定大鼠脑组织氨基酸类神经递质的反相高效液相色谱荧光法.方法 改良使用磷酸盐-甲醇-乙腈作为流动相,反相高效液相色谱洗脱,高丝氨酸作为内标,邻苯二甲醛柱前衍生和荧光检测器,检测大鼠大脑皮质、海马、纹状体、中脑、小脑和下丘脑6个脑区中天冬氨酸(Asp)、谷氨酸(Glu)、谷氨酰胺(G1n)、甘氨酸(Gly)、γ-氨基丁酸(GABA)和牛磺酸(Tau)6种氨基酸类神经递质含量.结果 6种氨基酸在20 min内洗脱完全,分离效果良好;在6.25～ 400 μmol/L浓度范围有较好的线性关系,其相关系数不低于0.99;6种氨基酸日内试验精密度范围为1.38％ ～7.59％;日间试验精密度为2.7％～8.68％;6种氨基酸回收率不低于80％.结论 改良后的反相高效液相色谱荧光法灵敏度较高、重复性好,能有效分离检测大鼠脑组织分区中氨基酸类神经递质含量.     

戊四氮致痫大鼠大脑皮质和海马内GFAP和cyclin D1表达的变化及脑和脑脊液天冬氨酸和谷氨酸含量的变化

目的研究癫痫发病时星形胶质细胞的激活及脑和脑脊液兴奋性氨基酸含量的变化.方法将实验SD大鼠随机分为2组:1.生理盐水对照(control)组(n=8);腹腔注射与致痫剂等容量的生理盐水.2.戊四氮(PTZ)组;PTZ 60mg/kg腹腔注射后2h、4h、8h、12h取脑,每个时间点各8只,检测下列指标:(1)用免疫组织化学方法(SABC法)观察大鼠大脑皮质和海马内星形胶质细胞胶质原纤维酸性蛋白(GFAP)含量的变化;(2)用Western blot方法检测大鼠大脑皮质和海马细胞周期素D1(cyclin D1)表达的变化;(3)采用高效液相色谱(HPLC)法测定大鼠大脑皮质和海马组织匀浆及脑脊液中兴奋性氨基酸(天冬氨酸和谷氨酸)含量的变化.结果星形胶质细胞GFAP免疫组织化学染色结果显示癫痫发作4h后在大脑皮质和海马CA3区、CA1区和皮质内GFAP免疫反应明显增强(P<0.05);Western blot检测癫痫发作2h后cyclin D1的表达在皮质和海马均较对照组明显增强(P<0.05);HPLC测定癫痫发作4h后大脑皮质中天冬氨酸(Asp)和谷氨酸(Glu)的含量达到最高峰,分别为8.900±0.540μmol/g protein和17.500±0.526μmol/g protein,与对照组(分别为7.083±0.693μmol/g protein和7.017±0.419μmol/g protein)相比均有显著性差异(P<0.05),在海马内Asp的含量在癫痫发作后2h达到最高峰,为11.425±1.006μmol/g protein,Glu的含量在癫痫发作后4h达到最高峰,为17.698±0.250μmol/g protein,与对照组(分别为7.300±0.824μmol/g protein和10.925±0.323μmol/g protein)比较均有显著性差异(P<0.05),脑脊液中Asp的含量在癫痫发作后2h达到最高峰,为82.65±4.81μmol/L,而Glu的含量在癫痫发作后持续增高,12h后达到72.80±3.66μmol/L.结论戊四氮致痫过程中兴奋性氨基酸含量增多,海马及皮质内星形胶质细胞激活,cyclin D1表达增加,星形胶质细胞增殖,以上变化在癫痫的形成和维持中发挥作用.     

γ-氨基丁酸（GABA）毛叶茶品质成分分析

以毛叶茶为研究对象,通过真空厌氧处理将其制作成γ-氨基丁酸（GABA）毛叶茶,探求毛叶茶经厌氧处理后的品质成分变化。结果表明：（1）厌氧处理后的毛叶茶,其GABA含量显著提高,达到GABA茶标准。游离氨基酸、黄酮和生物碱含量也显著升高,但茶多酚和水浸出物含量降低。同时,真空处理还能促进儿茶素的转化。简单儿茶素含量呈降低趋势,ECG和CG含量显著提高,EGCG、GCG含量及酯型儿茶素总量却先增加后降低,最终总量与对照样无明显差异。（2）毛叶茶中除含有一般的蛋白质氨基酸外,还含有普通茶树品种所特有的特征氨基酸Thea,以及微量的GABA。游离氨基酸中含量较高的有Thea、Glu、Asp,较低的是Met、Cit、α-ABA、Tau、Gly。Cysthi和EOHNH2是GABA毛叶茶中特有氨基酸。在真空厌氧条件下,GABA毛叶茶的游离氨基酸由于蛋白质发生降解而总量增加。其中P-Set、Thr、Ser、Asn、Pro、Gly、Cit、α-ABA、Val、Cysthi、Ile、Leu、Tyr、Phe、GABA、Trp、Lys、His含量上升,Asp、Glu和仪．AAA含量均降低,而Ala和Arg含量却呈现先增后降的趋势,Thea、Cys、Met等游离氨基酸含量在真空处理后无明显变化。     

富硒板党对低氧耐受小鼠兴奋性氨基酸的影响

目的：探讨富硒板党对小鼠低氧耐受形成中脑组织兴奋性氨基酸（EAA）含量的影响。方法：将小鼠用富硒板党处理后,建立小鼠急性重复低氧耐受模型,观察富硒板党对小鼠低氧耐受形成中的脑组织谷氨酸（Glu）、天冬氨酸（Asp）、γ-氨基丁酸（GABA）和甘氨酸（Gly）的影响。结果：富硒板党对正常小鼠脑组织EAA含量无影响,可使低氧耐受形成中脑组织Glu、Asp含量降低,对GABA、Gly无明显影响。结论：富硒板党能明显降低低氧小鼠脑组织中EAA含量,从而发挥对脑损伤的保护作用。     

盐酸戊乙奎醚（长托宁）抑制脑缺血／再灌注时谷氨酸的释放及受体机制研究

目的：观察盐酸戊乙奎醚对全脑缺血／再灌注大鼠易损区海马谷氨酸（Glu）及其受体（NMDAR1）的影响,探讨盐酸戊乙奎醚脑保护作用机制。方法：雄性Wistar大鼠60只,随机分为3组（n=20）：假手术组（A组）;缺血再灌注对照组（B组）;盐酸戊乙奎醚干预组,采用Pulsinelli-Brierley四血管阻断法制备全脑缺血模型,实验分两部分进行,各组随机选择10只大鼠于全脑缺血15min再灌注1h、3h、6h,采用脑微透析技术结合高效液相色谱（HPLC）检测大鼠海马细胞外Glu水平的变化,其余10只大鼠于再灌注3h后灌流固定断头取脑,采用免疫组织化学方法,检测海马CA1区NMDAR1蛋白表达。结果：与缺血／再灌注组各对应时点相比较,盐酸戊乙奎醚干预组大鼠海马细胞外Glu含量明显降低,统计结果差异均有显著性（P〈0．05或P〈0．01）;海马CA1区NMDAR1表达明显受抑制,积分光密度、阳性细胞面积、平均灰度值均存在显著性差异（P〈0．05或P〈0．01）。结论：脑缺血／再灌注早期应用盐酸戊乙奎醚不仅减少兴奋性氨基酸释放,还能抑制NMDAR1的高表达而产生脑保护作用。     

点突变Pro229Ser和Glu243Gly对耐热邻苯二酚2，3—双加氧酶性质的影响

用PCR随机诱变方法,研究氨基酸置换对耐热邻苯二酚2,3-双加养酶性质的影响。比较分析了突变体ro229Ser和Glu243Gly与野生型酶的酶学性质。结果显示点突变Pro229→Ser或Glu243→Gly并未改变酶的最适反应温度（均为60℃）;突变体Pro229Ser（Kcat／Km＝4.89±0.01×10     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Plasma amino acids in 3/sec spike-wave epilepsy

Plasma levels of 14 amino acids were determined in 44 probands with 3/sec spike-wave epilepsy, 27 of their first-degree relatives, and 22 controls. Six ratios of metabolically related amino acids were also calculated. Statistically significant differences were found for 7/20 variables when the experimental and control probands were compared, and for 6/20 variables when the relatives were compared with controls. Tau, Asp, and Tau/Glu were decreased and Glu was increased in both the experimental probands and their first degree relatives. When discriminant analysis was used, age- and sex-matched epileptic probands could be distinguished from the age- and sex-matched control probands with 100% accuracy employing only 3 amino acids (Asp, Glu, Val) and one ratio (Thr/Ser).     

Deracemization of Amino Acids by Partial Sublimation and via Homochiral Self-Organization

Deracemization of a 50/50 mixture of enantiomers of aliphatic amino acids (Ala, Leu, Pro, Val) can be achieved by a simple sublimation of a pre-solubilized solid mixture of the racemates with a huge amount of a less-volatile optically active amino acid (Asn, Asp, Glu, Ser, Thr). The choice of chirality correlates with the handedness of the enantiopure amino acids—Asn, Asp, Glu, Ser, and Thr. The deracemization, enantioenrichment and enantiodepletion observed in these experiments clearly demonstrate the preferential homochiral interactions and a tendency of natural amino acids to homochiral self-organization. These data may contribute toward an ultimate understanding of the pathways by which prebiological homochirality might have emerged.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (Mldl). Structure-functional correlation

Based on three-dimensional model of the bifunctional enzyme Destabilase-Lysozyme (mlDL-Ds2) in complex with trimer of N-acetylglucosoamine (NAG)3 the functional role of the stereochemically based group of amino acids (Glu14, Asp26, Ser 29, Ser31, Lys38, His92), in manifestation of glycosidase and isopeptidase activities has been elucidated. By method of site-directed mutagenesis it has been shown that mlDL glycosidase active site includes catalytic Glu14 and Asp26, and isopeptidase site functions as Ser/Lys dyad presented by catalytic residues Lys38 and Ser29. Thus, among the invertebrate lysozymes mlDL presents first example of the bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

甘肃鼢鼠骨骼19种氨基酸含量的测定

用121型氨基酸分析仪对甘肃鼢鼠(Myospalax cansus)躯干、四肢、头骨骨骼中19种氨基酸含量进行测定,并与高原鼢鼠及其他哺乳动物骨骼中氨基酸含量进行差异显著性分析。结果表明,甘肃鼢鼠各部位骨骼中19种氨基酸含量均为甘氨酸最高,谷氨酸次之,胱氨酸最低。同一氨基酸在甘肃鼢鼠不同骨骼部位含量顺序为躯干>四肢(头骨。甘肃鼢鼠骨骼总氨基酸含量低于塞隆骨,与虎骨相近,高于其他哺乳动物。其中羟脯氨酸含量明显高于虎骨、塞隆骨等。     

Polymers produced by heating an amino acid mixture in sea water enriched with transition elements.

A mixture of eighteen protein amino acids was heated in sea water medium enriched with transition metal ions. Small granules were obtained as precipitates. Both dialyzable polymers and undialyzable polymers were obtained from the supernatant. Dialyzable polymers yielded mainly Glu, Asp, Ser, and Thr on hydrolysis; undialyzable polymers (C, 29.45; H, 3.87; N, 4.87; and ash, 31.5 wt%) yielded Thr, Asp, Glu, Gly, Leu, Ser, Lys, Pro, His, Phe, and a few unidentified ninhydrin positive peaks after acid hydrolysis. Five wt% of the undialyzable polymers con-ist of acid-hydrolyzable protein amino acids.     

2种检测灯盏花素血药浓度方法的比较

目的：建立测定Beagle犬血浆中灯盏花素浓度的反相高效液相色谱法和液相-质谱联用法,并将其进行多参数的两两比较。方法：采用液-液萃取法处理血清,反相高效液相色谱法测定,流动相为甲醇-水（pH3．0）=42：58,EclipseXDB-C18为固定相;将同样的样品用液相-质谱法开展平行检测;将所测得的数据做两两对比,采用SPSS统计软件进行分析。结果：2种方法测定的数据具有良好的相关性（r=0．982,P〉0．05）;最低检测限前者为0．05μg／mL,后者为0．01μg／mL,反相高效液相色谱法的最低检测限和灵敏度不及液相-质谱联用法。结论：反相高效液相色谱法可用于灯盏花素毒代动力学研究,与液相-质谱联用法检测结果无显著差异,且费用较低,不失为-种低成本且行之有效的检测方法。