Development of a novel assay system of asymmetric dimethylarginine (ADMA), a risk factor for atherosclerosis.

开发一种新型的不对称二甲基精氨酸 (ADMA) 检测系统，ADMA 是动脉粥样硬化的危险因素。

• 批准号: 15590494
• 负责人: KIMOTO Masumi
• 金额: $ 2.24万
• 依托单位: Okayama Prefectural University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590494/
• 关键词: Nitric oxide; asymmetric dimethylarginine; N^G,N^G-dimethylarginine dimethylaminohydrolase; monoclonal antibody; atherosclerosis; inhibition ELISA; risk factor for cardiovascular disease; 心臓血管動脈硬化症; 組み換え型ヒトDDAH; ハプテン抗原; 組換え型ヒトDDAH

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Elevated ADMA plasma levels have been reported in connection with diseases associated with an impaired endothelial arginine/NO pathway and endothelial dysfunction, such as atherosclerosis, hypercholesterolemia, chronic heart failure, diabetes mellitus, and hypertension. Moreover, ADMA has recently been shown to be a risk factor for cardiovascular diseases. Therefore, there is increasing interest in measuring ADMA levels in human subjects, experimental animals, and cell culture. So far, the quantitative determination of ADMA by high-performance liquid chromatography (HPLC) analysis has been the most widely applied method. We designed specific two assay systems, the first is a method using enzymatic reaction with N^G,N^G-dimethylarginine dimethylaminohydrolase (E.C. 3.5.3.18 : DDAH) that degrades ADMA to citrulline, and the second an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against ADMA. In general, the analytical performance of ELISA is assessed above all by evaluating its specificity, analytical sensitivity, and facility. Therefore, we investigated the development of a novel ELISA assay for ADMA focusing on the second system described above. First we attempted to prepare a monoclonal antibody (mAb) against ADMA using BALB/c mouse immunized with ADMA-MBS-BSA conjugate. The mAb 3C12 obtained reacted specifically ADMA within a variety of amino acids including arginine derivatives and more sensitively to MBS-acylated ADMA than ADMA. We constructed a standard assay system due to the inhibition ELISA using the mAb. The limit of quantitation was 0.4nM, and the mean recovery was 95% after all treatments for the assay system. We determined ADMA levels in human plasma and found good correlation of the values measured by HPLC (r=0.98,p<0.01).

不对称二甲基精氨酸 (ADMA) 是一氧化氮合酶 (NOS) 的内源性竞争性抑制剂。据报道，ADMA血浆水平升高与内皮精氨酸/NO途径受损和内皮功能障碍相关的疾病有关，例如动脉粥样硬化、高胆固醇血症、慢性心力衰竭、糖尿病和高血压。此外，ADMA 最近被证明是心血管疾病的危险因素。因此，人们对测量人类受试者、实验动物和细胞培养物中的 ADMA 水平越来越感兴趣。迄今为止，高效液相色谱（HPLC）分析法定量测定ADMA是应用最广泛的方法。我们设计了特定的两种测定系统，第一种是使用 N^G,N^G-二甲基精氨酸二甲氨基水解酶（E.C. 3.5.3.18：DDAH）进行酶促反应将 ADMA 降解为瓜氨酸的方法，第二种是使用抗 ADMA 单克隆抗体的酶联免疫吸附测定 (ELISA)。一般来说，ELISA 的分析性能首先通过评估其特异性、分析灵敏度和设施来评估。因此，我们研究了针对 ADMA 的新型 ELISA 测定的开发，重点关注上述第二个系统。首先，我们尝试使用用 ADMA-MBS-BSA 缀合物免疫的 BALB/c 小鼠制备抗 ADMA 的单克隆抗体 (mAb)。获得的 mAb 3C12 与多种氨基酸（包括精氨酸衍生物）内的 ADMA 发生特异性反应，并且对 MBS 酰化的 ADMA 比 ADMA 更敏感。由于使用 mAb 进行抑制 ELISA，我们构建了标准检测系统。定量限为 0.4nM，测定系统经过所有处理后平均回收率为 95%。我们测定了人血浆中的 ADMA 水平，发现 HPLC 测量的值具有良好的相关性（r=0.98，p<0.01）。

项目成果

Dimethylarginine dimethylaminohydrolase and endothelial dysfunction in the failing heart.

二甲基精氨酸二甲氨基水解酶和衰竭心脏中的内皮功能障碍。

• 发表时间: 2005
• 期刊: Am.J.Physiol.Heart Circ.Physiol. 289(5)
• 作者: Chen Y.;et al.
• 通讯作者: et al.

Dimethylarginine dimethylaminohydrolase and endotherial dysfunction in the failing heart.

二甲基精氨酸二甲氨基水解酶和衰竭心脏中的内热功能障碍。

• 发表时间: 2005
• 期刊: Am. J. Physiol. Heart Circ. Physiol., 289(5)
• 作者: Chen;Y.;Li;Y.;Zhang;P.;Traverse;J.H.;Hou;M.;Xu;X.;Kimoto;M.;Bache;R.J.
• 通讯作者: R.J.

ADMA regulates angiogenesis : genetic and evidence.

ADMA 调节血管生成：遗传和证据。

• 发表时间: 2005
• 期刊: Vas.Med. 10(1)
• 作者: Achan V.;et al.
• 通讯作者: et al.

ADMA regulates angiogenesis: genetic and metabolic evidence

• DOI: 10.1191/1358863x05vm580oa
• 发表时间: 2005-01-01
• 期刊: VASCULAR MEDICINE
• 影响因子: 3.5
• 作者: Achan, V;Ho, HK;Cooke, JP
• 通讯作者: Cooke, JP

Overexpression of dimethylarginine dimethylaminohydrolase (DDAH) reduces tissue ADMA level and enhances angiogenesis.

二甲基精氨酸二甲氨基水解酶 (DDAH) 的过度表达会降低组织 ADMA 水平并增强血管生成。

• 发表时间: 2005
• 期刊: Circulation, 11(11)
• 作者: Jacobi;J.;Sydow;K.;Degenfeld;G.;Zhang;Y.;Dayoub;H.;Wang;B.Y.;Patterson;A.J.;Kimoto;M.;Blau;H.M.;Cooke;J.P.
• 通讯作者: J.P.