黄杆菌对硫磷水解酶及大肠杆菌过氧化氢酶无水解梭曼活性

黄杆菌对硫磷水解酶及大肠杆菌过氧化氢酶无水解梭曼活性邵煌孙曼霁（军事医学科学院毒物药物研究所，北京１００８５０）黄杆菌（Ｆｌａｖｏｂａｃｔｅｒｉｕｍｓｐ．ｓｔｒａｉｎＡＴＣＣ２７５５１）和缺陷假单胞菌（ＰｓｅｕｄｏｍｏｎａｓｄｉｍｉｎｕｔａＭＧ）的对...     

不同种属动物血浆及红细胞与[~3H]梭曼结合的差异

目的　研究不同种属动物梭曼毒性差异是否与血浆及红细胞与梭曼结合差异有关。方法　 [3 H]梭曼结合饱和方法求出不同种属动物血浆及红细胞的Bmax值。结果　血浆及 1∶3(V/V)红细胞对 [3 H]梭曼的结合具有饱和性 ,血浆与 [3 H]梭曼结合的Bmax值大小依小鼠、大鼠、人及狗顺序递减 ,分别为16 6、36 .1、2 1.9和 10 .7nmol·L- 1。 1∶3(V/V)红细胞悬液与 [3 H]梭曼结合的Bmax值大小依大鼠、小鼠、狗及人顺序递减 ,分别为 73.3、4 8.8、35 .7和 35 .4nmol·L- 1。结论　不同种属动物血浆和红细胞与[3 H]梭曼结合的Bmax值的大小在一定程度上反映了梭曼毒性的种属差异。     

人的基因工程氨酰基脯氨酸二肽酶的多效酶活性(英文)

目的　研究人氨酰基脯氨酸二肽酶除催化水解C端为脯氨酸残基的二肽外 ,是否还有G类有机磷化合物水解酶 (G酶)活性。方法　用基因工程技术克隆及表达人的重组氨酰基脯氨酸二肽酶。氨酰基脯氨酸二肽酶及G酶活性用常规方法测定。结果　COS 7细胞表达的人氨酰基脯氨酸二肽酶催化有机磷化合物梭曼的水解 ,也水解二肽化合物Gly Pro。两种活性比未转染的COS 7细胞高 2倍。比较转染了带有氨酰基脯氨酸二肽酶基因的重组载体的COS 7细胞和对照组细胞中的两种酶活性 ,可以看到有平行的升高趋势及恒定的酶活性比值。结论G酶和氨酰基脯氨酸二肽酶为同一个酶 ,或至少属于同工酶     

人 G类有机磷毒剂水解酶的性质(英文)

观察了部分纯化的人肝 G类毒剂水解酶 ( G酶 )的一些生化性质 .二硫苏糖醇 ( 5mmol·L-1)使G酶活性在 37℃ ,30 min内抑制 35% ;对氯汞苯甲酸 ( 1 - 1 0 0 0 μmol· L-1)不影响 G酶活性 .说明二硫键对酶分子的三维结构至关重要 ,而没有游离巯基参与酶的催化反应 .人肝 G酶专一性地水解带P- F键的有机磷化合物梭曼 ,但不催化带 P- O,P- C或 P- S键的有机磷化合物的反应 .     

人血清对氧磷酶的部分纯化及解毒效应

目的　寻求对氧磷酶 (paraoxonase ,EC 3.1.8.1)作为外源性抗毒剂对抗G类有机磷毒剂的可能性。方法　采用亲和凝胶柱层析进行了人血清对氧磷酶的部分纯化 ,测定了其对于不同底物的米氏常数 (Km)以及钙离子对于酶活性的影响 ,比较了不同种属的对氧磷酶活性 ,采用体外解毒 ,体内染毒的方法检测了对氧磷酶对于不同的底物的作用。结果部分纯化的对氧磷酶对于梭曼和对氧磷均有较好的催化水解作用。以对氧磷为底物时 ,Km 值为 0 .2 2mmol·L- 1,比活性为 375 μmol·min- 1·g- 1蛋白 ;以梭曼为底物时 ,Km 值为 0 .6 0mmol·L- 1,比活性为 4 34μmol·min- 1·g- 1蛋白。不同种属来源的血清对氧磷酶的活性差异较大 ,是造成不同种属动物对有机磷毒剂中毒敏感性差别的原因之一。部分纯化的对氧磷酶在含 1mmol·L- 1CaCl2 的缓冲液中活性良好 ,加入乙二胺四乙酸 (EDTA)后 ,无论以对氧磷或梭曼为底物 ,酶活性均受到明显抑制 ,表明人血清对氧磷酶是钙离子依赖性酶。EDTA的pI50 约为 1.8mmol·L- 1。人血清对氧磷酶能有效地水解对氧磷、梭曼、沙林、塔崩及敌敌畏 ,但对VX及乐果无效 ,表明其对P F、P CN及P O键有选择性 ,对P S键无作用。结论　对氧磷酶可作为G类毒剂的抗毒剂     

体外小鼠、豚鼠、犬及人血液对梭曼的解毒作用

目的　研究不同种属动物及人血液对梭曼的解毒能力与血液中几种梭曼解毒酶活性的关系。方法　测定血液中梭曼残留浓度及解毒酶活性。结果　小鼠、豚鼠、犬、人血浆对梭曼的解毒能力要明显高于其自身红细胞的解毒能力 ,血浆对梭曼的解毒能力依小鼠、豚鼠、犬、人的顺序由高到低依次排列。啮齿类动物血液中羧酸酯酶 (CaE)活性位点数目多且与梭曼结合快 ,因此在梭曼解毒中可发挥重要作用。犬、人血液中乙酰胆碱酯酶 (AChE)在梭曼解毒中占据了重要的解毒地位 ,犬、人血液中CaE基本没有参与梭曼解毒。结论　血液解毒能力的种属差异与种属间解毒酶结合位点数量的多少及梭曼与酶的结合速率密切相关     

酸性染料比色法测定肝尔舒中总生物碱的含量

目的 建立肝尔舒中总生物碱含量的测定方法 . 方法 以辛弗林作为对照品,415 nm作为测定波长,采用酸性染料比色法测定总生物碱. 结果 辛弗林在117.5 ～352.4 μg范围内线性关系良好,平均加样回收率为98.68%(RSD＝1.60%). 结论 该方法简便可靠,可以作为肝尔舒中总生物碱含量测定的方法.     

重组人红细胞生成素对大鼠肝切除后肝再生的影响

目的 探讨重组人红细胞生成素(rhEPO)对大鼠肝切除后肝再生的促进作用.方法 大鼠建立70%肝切除模型后分为两组:实验组术后30 min经尾静脉注射累计4000 IU/kg rhEPO(每天1333 IU/kg rhEPO,连用3 d);对照组给予相同容量的生理盐水.术后1、3、5、7、14 d随机各取5只大鼠处死,称取并计算肝再生率,采用速率法检测血清丙氨酸转氨酶(ALT)和白蛋白(Alb),免疫组化检测肝组织增殖细胞核抗原(PCNA)表达.结果 术后1、3、5、7、14 d实验组肝再生率较实验组明显增高(P＜0.05),实验组血清ALT水平术后5、7、14 d较对照组有降低(P＜0.05),实验组血清Alb术后7、14 d高于对照组(P＜0.05),术后1、3、5、7 d实验组PCNA标记指数均高于对照组(P＜0.05和P＜0.01).结论 rhEPO对大鼠肝切除后肝再生有促进作用.     

反相高效液相色谱法测定体外人肝微粒体中氟西汀及其代谢产物去甲氟西汀

目的:建立同时测定体外肝微粒体中氟西汀及其代谢产物去甲氟西汀的反相高效液相色谱紫外检测法.方法:含微粒体蛋白和NADPH发生系统及氟西汀的孵育液加入冰乙腈酸终止反应后,再加入去甲替林作为内标并以 n-正己烷和乙腈的混合液进行萃取,然后以反相ODS柱分离,在226 nm处以紫外检测器进行检测.结果:孵育体系中没有明显的干扰峰出现,氟西汀和去甲氟西汀洗脱快,分离好,线性范围均为10-800μg/L,最低检测限均为5μg/L,相对回收率为94％-104％,变异系数少于9.1％.结论:本法快速,灵敏,回收率高,且萃取过程简单,可用于体外氟西汀的代谢研究.     

抗人肝微粒体蛋白单克隆抗体细胞系的建立及鉴定

目的 初步建立抗人肝微粒体蛋白单克隆抗体规模化制备技术平台,为制备抗人细胞色素P450(CYP450)亚型蛋白的单克隆抗体奠定基础.方法 按常规方法进行细胞融合,以酶联免疫吸附测定法(ELISA)筛选杂交瘤,以ELISA、免疫组化(IH)、免疫印迹(Western Blot)、免疫沉淀(IP)对单克隆抗体加以鉴定.结果 融合后筛出杂交瘤,其分泌抗体类型为IgG1、IgG2a、IgG2b及IgM.免疫组化(IH)图片上,人肝细胞浆内均可见阳性颗粒.免疫沉淀(IP)和免疫印迹(WesternBlot)结果显示,所制备抗体与人肝微粒体蛋白有特异性结合.结论 筛选制备的单克隆抗体杂交瘤能分泌特异性较强的抗人肝微粒体蛋白单克隆抗体.     

Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes

Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15-35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions.     

Nafamostat is hydrolysed by human liver cytosolic long-chain acyl-CoA hydrolase

Although the authors recently reported that nafamostat, a clinically used serine protease inhibitor, was mainly hydrolysed by carboxylesterase in human liver microsomes, the involvement of human liver cytosol has not been elucidated. The current study examined the in vitro metabolism of nafamostat with human liver cytosols. Kinetic analysis indicated that the V_max and K_m values in the liver cytosols were 9.82?nmol?min^?1?mg^?1 protein and 197?µM for a liver sample HL-1, and 15.1?nmol?min^?1?mg^?1 protein and 157?µM for HL-2, respectively. The V_max/K_m values in both cytosols were at least threefold higher than those in the corresponding microsomes. The liver cytosolic activity for nafamostat hydrolysis was inhibited by phenylmethylsulfonyl fluoride (PMSF) (43% inhibition at 100?µM), whereas diisopropyl fluorophosphate (DFP) and bis(p-nitrophenyl)phosphate (BNPP) failed to inhibit the activity. Furthermore, the hydrolytic activity was also reduced by palmitoyl-CoA (67% inhibition at 100?µM) but not by acetyl-CoA. Effects of PMSF, DFP and BNPP on cytosolic palmitoyl-CoA hydrolytic activity were comparable with those of the cytosolic nafamostat hydrolytic activity. In addition, the palmitoyl-CoA hydrolytic activity was competitively inhibited by nafamostat with the apparent K_i value of 164?µM for the liver cytosol from HL-2. These results suggest that an isoform of long-chain acyl-CoA hydrolase may be responsible for the nafamostat hydrolysis in human liver cytosol.     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

Subchronic physostigmine pretreatment in marmosets: absence of side effects and effectiveness against soman poisoning with negligible postintoxication incapacitation.

Subchronic pretreatment with physostigmine (PHY) (0.0125 mg/kg/h) leading to a blood acetylcholinesterase inhibition of about 30% caused no side effects when applied to marmoset monkeys. This was evident on behavioral parameters and on EEG and cortical visual evoked response. Furthermore, this treatment regime, followed by atropine as postintoxication therapy, protected the marmosets against lethality after a 2 x LD50 dose of soman with negligible postintoxication incapacitation. These findings suggest that a symptom-free pretreatment with subchronic PHY could protect man sufficiently against severe soman intoxication.     

氯沙坦与格列美脲在人肝微粒体中的药物相互作用

目的：研究氯沙坦与格列美脲在人肝微粒体中的药物相互作用。方法：200 μL人肝微粒体孵育体系中加入氯沙坦与格列美脲各1~10 μmol·L^-1,于37℃水浴中孵育30 min,终止反应后的样品经处理,应用UPLC-MS/MS法同时检测氯沙坦和格列美脲代谢产物的生成量,采用Dixon作图并计算格列美脲抑制氯沙坦的K_i值以考察格列美脲和氯沙坦的相互抑制作用。结果：在1~10 μmol·L^-1的浓度范围内,格列美脲对氯沙坦表现出明显的抑制作用,相应的K_i值为（0.407 7±0.086 2）μmol·L^-1;氯沙坦仅在格列美脲浓度为1 μmol·L^-1时表现出抑制作用。结论：在人肝微粒体孵育体系中,格列美脲对氯沙坦的抑制作用强于氯沙坦对格列美脲的抑制作用。氯沙坦和格列美脲在人体内的相互作用有待进一步的人体药代动力学研究。     

山姜素在人肝微粒体的葡萄糖醛酸化反应研究

目的研究体外肝微粒体孵育体系中山姜素的葡萄糖醛酸化代谢情况,鉴定参与山姜素葡萄糖醛酸化代谢的UGT亚型。方法用体外肝微粒体孵育体系,用HPLC-UV检测方法,检测山姜素的葡萄糖醛酸化代谢情况。将代谢产物进行纯化后,用质谱(MS)和核磁共振(NMR)法进一步鉴定其结构。用商业化重组表达的UGT单酶,鉴定代谢产物的结构和归属可能参与山姜素葡萄糖醛酸化代谢反应的葡萄糖醛酸转移酶(UGTs)亚型。结果山姜素葡萄糖醛酸代谢产生一个代谢产物,经结构鉴定为山姜素-氧-单葡萄糖醛酸化产物。人肝微粒体代谢山姜素的动力学行为,符合米方程且动力学参数:Vmax=(101.9±3.0)nmol·min-1·mg-1·pro,Km=(40.6±3.6)μmol·L-1。UGT1A1、UGT1A3、UGT1A9和UGT2B15均参与了山姜素的葡萄糖醛酸化反应。结论山姜素在人肝微粒体孵育体系中会被代谢成为一个单葡萄糖醛酸化产物,且归属了参与的UGT酶。     

Iron concentrations in liver and kidney of cadmium-exposed human subjects

Iron (Fe) concentrations in liver and kidney of cadmium (Cd)-exposed people (11 Itai-Itai patients, 13 persons requiring observation and 4 Cd-exposed persons) were compared with those of nonexposed people. No significant difference in Fe levels in liver was observed between the groups. This fact indicates that anaemia in environmentally Cd-exposed people was not due to iron deficiency in contrast to findings in animal experiments.     

Stereoselective glucuronidation of formoterol by human liver microsomes

AIMS: Formoterol is a beta2-adrenoceptor agonist marketed as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). The drug produces prolonged bronchodilation by inhalation but there is significant interpatient variability in duration of effect. Previous work has shown that in humans formoterol is metabolized by conjugation with glucuronic acid but little is known about the stereoselectivity of this reaction. The aim of the present study was to investigate the glucuronidation of formoterol enantiomers in vitro by human liver microsomes. METHODS: The kinetics of formation of formoterol glucuronides during incubation of racemate and of single formoterol enantiomers with human liver microsomes (n=9) was characterized by chiral h.p.l.c. assay. RESULTS: The kinetics of glucuronidation of the two formoterol enantiomers obeyed the Michaelis-Menten equation. Glucuronidation of formoterol was stereoselective and occurred more than two times faster for (S; S)-formoterol than for (R; R)-formoterol. In incubations with single formoterol enantiomers, the median (n=9) Km values for (R; R)-glucuronide and (S; S)-glucuronide were 827.6 and 840.4 microm, respectively, and the median V max values were 2625 and 4304 pmol min-1 mg-1, respectively. Corresponding values determined in incubations with rac-formoterol were 357.2 and 312.1 microm and 1435 and 2086 pmol min-1 mg-1 for (R; R)- and (S; S)-glucuronide, respectively. Interindividual variation was large with the ratio of V max/Km (S; S/R; R) ranging from 0.57 to 6.90 for incubations with rac-formoterol. CONCLUSIONS: Our study demonstrates that glucuronidation of formoterol by human liver microsomes is stereoselective and subject to high interindividual variability. These findings suggest that clearance of formoterol in humans is subject to variable stereoselectivity which could explain the variation in duration of bronchodilation produced by inhaled formoterol in patients with asthma.     

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes,liver cytosol and plasma preparations

1. The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague–Dawley rats aged 15, 21 and 90 days and from adult humans.

2. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes.

3. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes.

4. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds.

5. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes.

6. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90?day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.