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1. 发明申请

US20090275012A1 Process For Determining S-Nitrosothiols In Biological Fluids 失效
标题翻译：生物流体中S-亚硝基硫醇的测定方法
公开(公告)号：US20090275012A1
公开(公告)日：2009-11-05
申请号：US12309623
申请日：2007-07-23
申请人： Alfonso Pompella , Aldo Paolicchi , Emillia Bramanti , Renata Barsacchi , Maria Franzini , Cecilia Vecoli
发明人： Alfonso Pompella , Aldo Paolicchi , Emillia Bramanti , Renata Barsacchi , Maria Franzini , Cecilia Vecoli
IPC分类号： C12Q1/00
CPC分类号： C12Q1/37 , C12Q1/48 , G01N33/6815 , G01N33/6842 , G01N33/94 , G01N2333/9108 , G01N2333/96425 , G01N2400/00
摘要： A process for determining S-nitrosothiols, in particular S-nitrosoglutathione, in biological fluids that is easy, selective, cheap with respect to the prior art, which requires the use of equipment commonly available in laboratories, at low cost, which can be used by not qualified operators. The process is based on the hydrolysis of S-nitrosoglutathione (GSNO) by an enzyme, in particular γ-glutamyltranspeptidase (GGT). This enzyme hydrolizes the residual 7-glutamyl of GSNO for giving glutamate (GIu) and S-nitroso-cysteinylglycine (GIyCySNO). In the presence of ions of transition metals GGT speeds up the release of NO since the intermediate that is formed, the GIyCySNO, is is much more sensitive to a metal-dependent decomposition. Advantageously, the amount of nitric oxide present in the sample is measured through a reaction thereof with 4,5 diaminof luorescein (DAF-2), said reaction creating a fluorescent compound in an amount proportional to the S-nitrosothiol amount present in the sample. Alternatively, the amount of released NO can be measured by a chemiluminescence analyser, commercially available. In the presence of biological fluids having complex matrix, the introduction of the enzyme is done after separation of the S-nitrosothiol from the other components of the fluid.
摘要翻译：在生物流体中测定S-亚硝基硫醇，特别是S-亚硝基硫肽素的方法，其相对于现有技术来说容易，选择性，便宜，这需要以低成本使用实验室常用的设备，可以使用由不合格的操作员。该方法基于酶，特别是γ-谷氨酰转肽酶（GGT）水解S-亚硝基谷胱甘肽（GSNO）。该酶水解GSNO的残留的7-谷氨酰谷氨酸（Glu）和S-亚硝基半胱氨酰甘氨酸（GlycySNO）。在过渡金属离子的存在下，由于形成的中间体，GGT加速了NO的释放，因此GlyYSSNO对金属依赖性分解更加敏感。有利的是，样品中存在的一氧化氮的量通过与4,5,4-二氨基荧光素（DAF-2）的反应来测量，所述反应产生与样品中存在的S-亚硝基硫醇量成比例的量的荧光化合物。或者，释放的NO的量可以通过可商购的化学发光分析仪来测量。在具有复杂基质的生物流体的存在下，在将S-亚硝基硫醇与流体的其它组分分离之后，引入酶。

2. 发明申请

US20100227351A1 METHOD OF DETECTING AND SEPARATING SERUM GAMMA-GLUTAMYL TRANSFERASE (GGT) ISOFORMS IN A SAMPLE OF BIOLOGICAL FLUID AND THE ENZYME ISOFORMS THEREBY OBTAINED 失效
标题翻译：检测和分离生物流体样品中的血清葡萄糖 - 谷氨酸转运酶（GGT）的ISOFORMS和获得的酶活性的方法
公开(公告)号：US20100227351A1
公开(公告)日：2010-09-09
申请号：US12665952
申请日：2008-06-24
申请人： Aldo Paolicchi , Alfonso Pompella , Maria Franzini , Renata Barsacchi , Michele Emdin , Emilia Bramanti
发明人： Aldo Paolicchi , Alfonso Pompella , Maria Franzini , Renata Barsacchi , Michele Emdin , Emilia Bramanti
IPC分类号： C12Q1/48
CPC分类号： C12Q1/48
摘要： A method is described for detecting the gamma-glutamyl transferase enzyme isoforms (GGT, EC 2.3.2.2) in a sample of biological fluid, such as for example plasma or serum. The method comprises an HPLC separation step of the sample proteins based on the molecular size and a second step for detecting the GGT isoforms by post-column reaction with a GGT enzyme substrate capable of generating a detectable final product, preferably by spectrophotometric or fluorimetric means. The GGT isoforms can be separated by ultra-centrifugation, thereby obtaining three enzymatic isoforms characterised by molecular weights of approximately 2000, 940, and 140 KDa, respectively.
摘要翻译：描述了用于检测生物流体样品（例如血浆或血清）中的γ-谷氨酰转移酶同种型（GGT，EC 2.3.2.2）的方法。该方法包括基于分子大小的样品蛋白质的HPLC分离步骤，以及用于通过与能够产生可检测的最终产物的GGT酶底物进行柱后反应来检测GGT同种型的第二步骤，优选通过分光光度计或荧光测定方法。可以通过超离心分离GGT同种型，从而分别得到分子量分别为约2000,940和140KDa的三种酶同工型。

3. 发明授权

US08486650B2 Method of detecting serum gamma-glutamyl transferase isoforms in a biological fluid 失效
标题翻译：检测生物体液中血清γ-谷氨酰转移酶同种型的方法
公开(公告)号：US08486650B2
公开(公告)日：2013-07-16
申请号：US12665952
申请日：2008-06-24
申请人： Aldo Paolicchi , Alfonso Pompella , Maria Franzini , Renata Barsacchi , Michele Emdin , Emilia Bramanti
发明人： Aldo Paolicchi , Alfonso Pompella , Maria Franzini , Renata Barsacchi , Michele Emdin , Emilia Bramanti
IPC分类号： C12Q1/48
CPC分类号： C12Q1/48
摘要： A method is described for detecting the gamma-glutamyl transferase enzyme isoforms (GGT, EC 2.3.2.2) in a sample of biological fluid, such as for example plasma or serum. The method comprises an HPLC separation step of the sample proteins based on the molecular size and a second step for detecting the GGT isoforms by post-column reaction with a GGT enzyme substrate capable of generating a detectable final product, preferably by spectrophotometric or fluorimetric means. The GGT isoforms can be separated by ultra-centrifugation, thereby obtaining three enzymatic isoforms characterized by molecular weights of approximately 2000, 940, and 140 KDa, respectively.
摘要翻译：描述了用于检测生物流体样品（例如血浆或血清）中的γ-谷氨酰转移酶同种型（GGT，EC 2.3.2.2）的方法。该方法包括基于分子大小的样品蛋白质的HPLC分离步骤，以及用于通过与能够产生可检测的最终产物的GGT酶底物进行柱后反应来检测GGT同种型的第二步骤，优选通过分光光度计或荧光测定方法。可以通过超离心分离GGT同种型，从而分别得到分子量分别为约2000,940和140KDa的三种酶同工型。

4. 发明授权

US08148099B2 Process for determining S-nitrosothiols in biological fluids 失效
标题翻译：生物体液中S-亚硝基硫醇的测定方法
公开(公告)号：US08148099B2
公开(公告)日：2012-04-03
申请号：US12309623
申请日：2007-07-23
申请人： Alfonso Pompella , Aldo Paolicchi , Emilia Bramanti , Renata Barsacchi , Maria Franzini , Cecilia Vecoli
发明人： Alfonso Pompella , Aldo Paolicchi , Emilia Bramanti , Renata Barsacchi , Maria Franzini , Cecilia Vecoli
IPC分类号： C12Q1/37
CPC分类号： C12Q1/37 , C12Q1/48 , G01N33/6815 , G01N33/6842 , G01N33/94 , G01N2333/9108 , G01N2333/96425 , G01N2400/00
摘要： A process for determining S-nitrosothiols, in particular S-nitrosoglutathione, in biological fluids that is easy, selective, cheap with respect to the prior art, which requires the use of equipment commonly available in laboratories, at low cost, which can be used by not qualified operators. The process is based on the hydrolysis of S-nitrosoglutathione (GSNO) by an enzyme, in particular γ-glutamyltranspeptidase (GGT). This enzyme hydrolizes the residual γ-glutamyl of GSNO for giving glutamate (GIu) and S-nitroso-cysteinylglycine (GIyCySNO). In the presence of ions of transition metals GGT speeds up the release of NO since the intermediate that is formed, the GIyCySNO, is much more sensitive to a metal-dependent decomposition. Advantageously, the amount of nitric oxide present in the sample is measured through a reaction thereof with 4,5 diaminof luorescein (DAF-2), said reaction creating a fluorescent compound in an amount proportional to the S-nitrosothiol amount present in the sample. Alternatively, the amount of released NO can be measured by a chemiluminescence analyser, commercially available. In the presence of biological fluids having complex matrix, the introduction of the enzyme is done after separation of the S-nitrosothiol from the other components of the fluid.
摘要翻译：在生物流体中测定S-亚硝基硫醇，特别是S-亚硝基硫肽素的方法，其相对于现有技术来说容易，选择性，便宜，这需要以低成本使用实验室常用的设备，可以使用由不合格的操作员。该方法基于通过酶，特别是γ-谷氨酰转肽酶（GGT）水解S-亚硝基谷胱甘肽（GSNO）。该酶水解谷氨酸（Glu）和S-亚硝基半胱氨酰甘氨酸（GlycySNO）的GSNO的残留γ-谷氨酰基。在过渡金属离子的存在下，GGT加速了形成的中间体后NO的释放，即GlyYSSiO对金属依赖性分解更加敏感。有利的是，样品中存在的一氧化氮的量通过与4,5,4-二氨基荧光素（DAF-2）的反应来测量，所述反应产生与样品中存在的S-亚硝基硫醇量成比例的量的荧光化合物。或者，释放的NO的量可以通过可商购的化学发光分析仪来测量。在具有复杂基质的生物流体的存在下，在将S-亚硝基硫醇与流体的其它组分分离之后，引入酶。

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