改进HPLC法测定门冬氨酸鸟氨酸原料药中的有关物质

目的:改进测定门冬氨酸鸟氨酸原料药中有关物质的方法。方法:采用高效液相色谱法。色谱柱为Phenomenex Luna NH2(两根串联),流动相为0.02 mol/L磷酸二氢钾缓冲液(p H 5.6)-乙腈(40∶60,V/V),流速为1.0 m L/min,检测波长为205 nm,柱温为35℃,进样量为20μL。结果:马来酸、3-氨基-2-哌啶酮、琥珀酸、苹果酸、富马酸、β-门冬氨酰门冬氨酸检测质量浓度线性范围分别为5.398~26.992μg/m L(r=0.999 1)、5.204~26.021μg/m L(r=0.999 3)、5.149~25.749μg/m L(r=0.999 6)、5.174~25.874μg/m L(r=0.999 3)、5.164~25.823μg/m L(r=0.999 3)、5.194~25.973μg/m L(r=0.999 5);定量限分别为0.22、0.53、51.50、103.50、0.26、8.31 ng,检出限分别为0.13、0.26、10.30、20.70、0.13、2.08 ng;精密度、稳定性、重复性试验的RSD均不超过2%;加样回收率分别为99.30%~102.31%(RSD=1.02%,n=9)、99.88%~100.89%(RSD=0.37%,n=9)、99.36%~101.53%(RSD=0.70%,n=9)、99.13%~102.65%(RSD=1.15%,n=9)、100.18%~101.45%(RSD=0.38%,n=9)、99.39%~100.81%(RSD=0.58%,n=9)。6批样品均未检出马来酸、琥珀酸和β-门冬氨酰门冬氨酸;3-氨基-2-哌啶酮含量为0.006%~0.056%,苹果酸含量为0.014%~0.071%,富马酸含量为0.000 5%~0.003 4%,最大未知杂质含量为0.013%~0.110%,未知杂质总含量为0.013%~0.120%。结论:该方法操作简便,精密度、稳定性、重复性均较好,可用于门冬氨酸鸟氨酸原料药中有关物质的测定。

多巴胺、门冬氨酸钾镁治疗肺心病并多脏器衰竭57例

肺心病急性加重期并多脏器损害（MOF）病情危重,临床表现复杂,预后差,病死率高。如何改善其预后,是目前急救治疗中研究的课题。我院处1993年1月～1996年1月收治了肺心病并MOF57例,对其中28例采用了多巴胺、门冬氨酸钾镁治疗,与常规治疗组比较,预后有明显改善,现将治疗情况分析如下。

一种生物酶制备L-天门冬氨酸钠的方法

本发明涉及一种通过生物酶催化一步制备L-天门冬氨酸钠的方法，将富马酸、氢氧化钠及氨以1：1：1～1：2：2的摩尔浓度比相混合，并调制为pH8．0～pH9．0的底物溶液，然后向此底物溶液中导入含天门冬氨酸酶的发酵液，所述的底物溶液与发酵液的体积比为16：1～16：3，常压条件下于温度37℃-40℃时反应12-20h，经分离得到L-天门冬氨酸钠；本发明生产成本低，且生产过程没有环境污染，有较好的应用前景。

Effects of microcystin-LR on hippocampal N-acetylaspartate and neurobehaviors in rats

The effects of low-doses of microcystin-leucinearginine （ MC-LR ） exposure on neurobehaviors and N-acetylaspartate （NAA） expression in the hippocampus of rats were investigated. After male Sprague-Dawley （SD） rats were treated intra-gastrically with different doses of MC-LR for 90 d, the locomotor activity, spatial learning and memory function were evaluated in the rats after treatment using open field tests and Morris water maze tests. The results show that MC-LR exposure can lead to impairment of the spatial learning capacity and locomotor activity in rats at the dose of 2. 00 p,g/kg. The levels of NAA in the hippocampus were measured by magnetic resonance spectroscopy （MRI）. A significant decrease of NAA/Cr ratio （ P 〈 0. 05） was observed in the hippocampous. This study indicates that intra-gastrical exposure to low-doses of MC-LR has adverse effects on neuronal behavior and NAA levels in the hippocampous.

Effects of Nerve Growth Factor on NMDA Receptor 1 and Neuronal Nitric Oxide Production after Spinal Cord Injury in Rats

Objective:To explore the protective mechanisms of nerve growth factor (NGF) on spinal cord injury (SCI) and provide theoretical basis for its clinical application. Methods: The SCI of Wistar rats was done by Allens weight dropping way by a 10 g×2.5 cm impact on the posterior of spinal cord T 8. NGF (3 g/L, 20 μl) or normal saline was injected through catheter into subarachnoid space 2, 4, 8, 12 and 24 h after SCI. The expression of N-methyl-D-asparate receptor 1 (NMDAR 1) and neuronal constitutive nitric oxide synthase (ncNOS) mRNA in rat spinal cord was detected by in situ hybridization. Results: Abnormal expression of NMDAR 1 and ncNOS mRNA appeared in spinal ventral horn motorneuron in injured rats, as compared with that in control group. The expression of NMDAR 1 and ncNOS mRNA in NGF group was significantly lower than that in saline group (P<0.01). Conclusion: NGF can protect spinal cord against injury in vivo. One of the mechanisms is that NGF can prohibit NMDAR 1 and nitric oxide (NO) production after spinal cord injury.

The Potential Pathway of L-arginine·L-aspartate for Inhibition of Platelet Function

Aim L-Arginine· L-aspartate, a double salt, has been recently reported toinhibit platelet aggregation and thrombosis, but its action mechanism is not clear yet. This studywas conducted to investigate its effect on FITC-PAC-1, an anti-glycoprotein IIb/IIIa monoclonalantibody binding to activated platelets, and on correlative autacoid levels in plasma or inplatelets in order to explore its potential pathway of inhibiting platelet aggregation andthrombosis. Methods Monoclonal antibody binding to activated platelets was assayed by flowcytometry; NO was assessed by colorimetric method. cAMP, TXB_2 or 6-keto-PGF_(1α) levels wereassessed by radioimmunoassay. Results Gavaged 30 mg·kg^(-1) of L-arginine·L-aspartate increasedboth concentration of NO in plasma and 6-keto-PGF_(1) in incubated supernatant of aortic segment ofrats ex vivo (P < 0.05), but it did not influence cAMP content in platelets and the level of TXB_2or 6-keto-PGF_(1) in plasma of rats, whereas ASA significantly lowered TXB_2 or 6-keto-PGF_(1α) inplasma. Both 100 μmol-L^(-1) of L-arginine ·L-aspartate and ASA inhibited FITC-PAC-1 binding toactivated platelets in vitro. Conclusion The increase in NO and PGI_2 release from endo-thelialcells and consequent inhibition of platelet activation may contribute to the inhibition of plateletaggregation and thrombosis by L-arginine· L-aspartate; whereas arachidonic acid or cAMP metabolicpathway is not closely correlative with the studied effect.

用于确定动物的线虫感染抗性的有效重组抗原Aspin在大肠杆菌中的表达

报道了重组蛋白Aspin——由寄生性Trichostronglus colubriformis产生的一种门冬氨酰蛋白酶抑制剂类似物在大肠杆菌中的表达。研究了表达具生物活性、可溶而非形成包涵体的Aspin并使产量最大化的培养条件。高生长速率和表达水平易形成包涵体。在分批发酵中降低生物反应器的温度、溶氧水平和营养物浓度，使表达处于低水平，均能有效增加可溶性Aspin的表达量。分批发酵中，以2g／L的L-阿拉伯糖诱导，培养温度从37℃降至25℃获得了Aspin220mg／L的高表达量。因为最终的酸性pH、低溶氧有利于产生可溶性的Aspin发酵过程，故可不控制pH和溶氧。

治疗肺源性心脏病难治性心衰的疗效观察

1998年1月至2001年10月,我院以酚妥拉明、门冬氨钾镁及参脉注射液联合治疗肺源性心脏病(肺心病)难治性心衰50例.取得较好疗效,现报道如下。1资料与方法1.1病例选择50例患者中,男35例,女15例,年龄40～83岁,平均61.5岁。病程10～40年,所有病例均符合1980年全国第三次肺心病会议修订诊断标准。1.2治疗方法 在常规抗感染、解痉平喘、吸氧。

Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specifi c to rtL180M and rtM204V were selected for YVDD detection. Primer specif ic to rtM204I with an additional 3’-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.RESULTS: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. CONCLUSION: We developed a highly sensitive and specifi c assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.

Metabolic Changes in Rats with Photochemically Induced Cerebral Infarction and the Effects of Batroxobin:A Study by Magnetic Resonance Imaging,~1H-and ^(31)p-Magnetic Resonance Spectroscopy

Metabolic changes in rats with photochemically induced cerebral infarction and the effects of batroxobin were investigated 1, 3, 5 and 7 days after infarction by means of magnetic resonance imaging (MRI), 1H- and 31P-magnetic resonance spectroscopy (MRS). A region of T2 hyperintensity was observed in left temporal neocortex in infarction group and batroxobin group 1, 3, 5 and 7 days after infarction. The volume of the region gradually decreased from 1 day to 7 days after infarction. The ratio of NAA/Cho + Cr in the region of T2 hyperintensity in the infarction group was significantly lower than that in the corresponding region in the sham-operated group 3, 5 and 7 days after infarction respectively (P

Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats

AIM:To investigate the preventive effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on bile duct ligation (BDL)induced liver fibrosis in rats. METHODS:Liver fibrosis in rats was induced by BDL and AcSDKP was infused subcutaneously for 2 wkvia a osmotic minipump (Alzet 2ML4) immediately after BDL operation. After scarifying, serum and liver specimens were collected. Hematoxylin and eosin staining, Sirius red staining, enzyme linked immunosorbent assay, Western blot or real-time polymerase chain reaction were used to determinate liver functions, histological alterations, collagen deposition, mRNA expression of markers for fibroblasts, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). RESULTS:When compared to model rats, chronic exogenous AcSDKP infusion suppressed profibrogenicTGF-β1 signaling, α-smooth muscle actin positivity (α-SMA), fibroblast specific protein-1 (FSP-1) staining and collagen gene expression. Col Ⅰ, Col Ⅲ, matrix metalloproteinase-2, tissue inhibitors of metallopro-teinase-1 and tissue inhibitors of metalloproteinase-2 mRNA expressions were all significantly downregulated by AcSDKP infusion (2.02 ± 1.10vs 14.16 ± 6.50, 2.02 ± 0.45vs 10.00 ± 3.35, 2.91 ± 0.30vs 7.83 ± 1.10, 4.64 ± 1.25 vs 18.52 ± 7.61, 0.46 ± 0.16 vs 0.34 ± 0.12, respectively, P < 0.05). Chronic exogenous AcSDKP infusion attenuated BDL-induced liver injury, inflammation and fibrosis. BDL caused a remarkable increase in alanine transaminase, aspartate transaminase, total bilirubin, and prothrombin time, all of which were reduced by AcSDKP infusion. Mast cells, collagen accumulation, α-SMA, TGF-β1, FSP-1 and BMP-7 increased. The histological appearance of liver specimens was also improved. CONCLUSION:Infusion of exogenous AcSDKP attenu-ated BDL-induced fibrosis in the rat liver. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Aspartate aminotransferase-immunoglobulin complexes in patients with chronic liver disease

AIM: To determine the complex of AST and immunoglobulin and to investigate its clinical significance in patients with liver disease.METHODS: The complex of AST and immunoglobulin was determined by encounter immunoelectrophoresis and its clinical significance was investigated in 128 patients with liver disease.RESULTS: AST was bound to immunoglobulin of antiimmunoglobulin A (IgA) class, but any binding to antiimmunoglobulin G and anti-immunoglobulin M classes was not observed. Although the incidence of ASTimmunoglobulin complex was 41.8% in chronic hepatitis (CH), the incidences in liver cirrhosis and hepatocellular carcinoma were 62.2 and 90.0%, respectively. In alcoholic liver disease with high level of serum IgA, the incidence of the complex was 66.7%, which was higher than that in CH. The ratio of binding to lambda-chain of IgA was higher than that to kappa-chain of IgA. The serum level of IgA and the ratio of AST/alanine aminotransferase (ALT) were significantly higher in patients with AST-IgA complex than in those without complex.CONCLUSION: These results suggest that AST-IgA complex in patients with progressive liver diseases and alcoholic liver injury can lead to elevation of the ratio of AST/ALT.

Disproportional exaggerated aspartate transaminase is a useful prognostic parameter in late leptospirosis

AIM: To evaluate the hepatic dysfunction in leptospirosis is usually mild and resolved eventually. However,sequential follow-up of liver biochemical data remained lacking..METHODS: The biochemistry data and clinical symptoms of 11 sporadic patients were collected and analyzed, focusing on the impacts of leptospirosis upon liver biochemistry tests.RESULTS: The results disclosed that of the 11 cases, 5 or 45% died. The liver biochemistry data in the beginning of the disease course were only mildly elevated.Nevertheless, late exaggerated aspartate transaminase (AST)elevations were noted in three cases who finally died when compared with the typical course. Besides, significant higher AST/alanine transaminase (ALT) ratios (AARs) of the peak levels for transaminase were also noted in the cases who eventually succumbed. The mean±SD of AARs for the survival group and dead group were 5.65±2.27 (n = 5)and 1.86±0.64 (n = 6) respectively (P= 0.006). The ratios of the cases who finally died were all more than 3.0.Conversely, the survival group's ratios were less than 3.0.CONCLUSION: Serial follow-up of transaminase might provide evidence to predict some rare evolutions in leptospirosis. If AST elevated progressively without a concomitant change of ALT, it might indicate an acute disease course with ensuing death. Additionally, AAR is another prognostic parameter for leptospirosis. Once the value was higher than 3.0, a grave prognosis is inevitable.

Alanine and aspartate aminotransferase and glutamine-cycling pathway:Their roles in pathogenesis of metabolic syndrome

Although new research technologies are constantly used to look either for genes or biomarkers in the prediction of metabolic syndrome(MS),the pathogenesis and pathophysiology of this complex disease remains a major challenge.Interestingly,Cheng et al recently investigated possible pathways underlying MS by high-throughput metabolite profiling in two large and well characterized community-based cohorts.The authors explored by liquid chromatography and mass spectrometry the plasma concentrations of 45 distinct metabolites and examined their relation to cardiometabolic risk,and observed that metabolic risk factors such as obesity,insulin resistance(IR),high blood pressure,and dyslipidemia were associated with several metabolites,including branched-chain amino acids,other hydrophobic amino acids,tryptophan breakdown products,and nucleotide metabolites.In addition,the authors found a significant association of IR traits with glutamine,glutamate and the glutamineto-glutamate ratio.These data provide new insight into the pathogenesis of MS-associated phenotypes and introduce a crucial role of glutamine-cycling pathway as prominently involved in the development of metabolic risk.We consider that the hypothesis about the role of abnormal glutamate metabolism in the pathogenesis of the MS is certainly challenging and suggests the critical role of the liver in the global metabolic modulation as glutamate metabolism is linked with aminotransferase reactions.We discuss here the critical role of the "liver metabolism" in the pathogenesis of the MS and IR,and postulate that before fatty liver develops,abnormal levels of liver enzymes,such as alanine and aspartate aminotransferases might reflect high levels of hepatic transamination of amino acids in the liver.

Preparation and enantiosorption of L-aspartic acid pillared hydrotalcites

L-aspartic acid （Asp） pillared hydrotalcites were prepared by direct reaction of the L-Asp anion with layered double hydroxides （LDHs）. The obtained samples were characterized by X-ray diffractometry （XRD）, Fourier transform infrared （FTIR）, and thermogravimetric and differential thermal analysis （TG/DTA）. The results show that the initial interlayer carbonate ions can be completely replaced by the L-Asp anion under the controlled conditions. The pillared hydrotalcites have a crystallized supramolecular structure and thermal stability. The L-Asp pillared LDHs were used in the enantiosorption of enantiopure phenylalanine （Pile）, the results suggest that L-Asp pillared LDHs exhibit an excellent enantiosorption capability for D-Phe, and the adsorption isotherm fits Freundlich equation.

Study of Adsorption Isotherm Curves of Amino Acids on the Kaolinite

This paper studies the adsorption isotherm curves of four amino acids (glycine, aspartic acid, glutamic acid and lysine) and the effects of existences of different concentrations of Cu(II) on them. Both the adsorption isotherm of the amino acids on the kaolinite and that when the copper ion Cu (II) exists belong to the Langmuir type. The slopes of these adsorption isotherm increase with the increasing concentrations of Cu(II) added. It can be explained in terms of formation of the type(I) ternary surface complexes in these systems. The regular pattern of the effects of copper ion Cu(II) on the adsorption isotherm of amino acids on the kaolinite is identical with that of its effects on the exchange and adsorption percentage E (%)-pH on the kaolinite in the same system.

Correlation of Cerebrospinal Fluid Sialic Acid with Various Enzymes in Patients with Meningitis

Meningitis is often associated with cerebral compromise which may be responsible for neurological squeal in nearly half of the survivors. Little is known about the mechanisms of Central Nervous System （CNS） involvement in bacterial meningitis. The present study was undertaken to assess the changes in biochemical parameters including glucose, protein, Sialic Acid （SA） and various enzymes like Aspartate Aminotransferase （AST）, Alanine Aminotransferase （ALT）, Creatine Kinase （CK）, Gamma Glutamyltranspeptidase 7 （GGTP） and Lactate Dehydrogenase （LDH） have been determined in the Cerebrospinal Fluid （CSF） of the meningitis patients （n = 26） and compared with control subjects （n = 26）. Total Protein, CK, SA （P 〈 0.02）, AST （P 〈 0.001） and ALT （P 〈 0.0001） of meningitis patients were significantly higher, whereas, glucose （P 〈 0.05） and Gamma Glutamyltranspeptidase （GGT） （P 〈 0.01 and P 〈 0.05, respectively） were significantly lower than those of the controls. This study suggests that loss of integrity of brain-CSF barriers, sialic acid and enzyme profiles may contribute to the severity and neurological complications of meningitis.

Effect of electroacupuncture on expression of NR2B in prefrontal cortex in morphine-withdrawal rats

Objective To observe the effect of electroacupuncture （EA） on learning and memory abilities and expression of N-methyI-D-aspartate receptor subunit （NR2B） in prefrontal cortex in morphine-withdrawal rats and to investigate the molecular biological mechanisms. Methods Thirty-six male SD rats were randomly divided into four groups, namely control group （group A）, model group （group B）, model with acupuncture group （group C） and model with electroacupunture group （group D）, with 9 in each group. All rats except those in group A were subcutaneously injected with morphine hydrochloride injectio on the back with daily dosage increased day by day. Naloxone was given 3 h after the last injection to establish the models of morphinewithdrawal rats. After the models were established, the rats were treated with acupuncture and electroacupuncture respectively at bilateral ＂Shenshu＂ （肾俞 BL 23） and ＂Zusanli＂ （足三里 ST 36） for 15 min per time, once daily for 6 days. Space learning and memory abilities of the rats were measured by Morris water maze, and protein and gene expression levels of NR2B in prefrontal cortex were measured by Western Blot and RT-PCR. Results In place navigation test, the escape latency in group B, group C and group D was significantly prolonged compared with that of group A （P〈0.01）, the escape latency in group C and group D was significantly shortened compared with that of group B （P〈0.01） and the escape latency in group D was significantly shortened compared with that of group C （P〈0.05）; during spatial probe test, the number of times crossing the platform of group B, group C and group D decreased compared with that of group A （P〈0.01）, and compared with group B, the number of times crossing the platform of group C increased and the number of group D significantly increased （P〈0.01）. Decreased protein expression level of NR2B was found in group B when compared with that of group A （P〈0.01）, increased protein expression levels of NR2B were found in group C and group D when compared with that of group B （P〈0.01）, however, the expression level in group D was higher than that in group C （P〈0.01）. mRNA expression level of NR2B in prefrontal cortex in morphine-withdrawal rats decreased （P〈0.05）, however, compared with that of group B, the expression level increased in group D （P〈0.05）, and there was no statistical significance in increased expression level in group C （P〉0.05）. Conclusion Acupuncture and eletroacupunture can improve space learning and memory abilities of merphine-withdrawal rats, with better efficacy of eletroacupuncture than that of acupuncture, the mechanisms of which may be associated with the regulation of NR2B expression in prefrontal cortex.