甲胺及其代谢产物甲醛在动物体内的药代动力学

目的进一步了解血浆氨基脲敏感型胺氧化酶(SSAO)活性与内源性甲醛的生理学与病理学意义。方法采用高效液相色谱法测定小鼠、大鼠和兔血浆中SSAO活性和静脉给予甲胺后甲胺及其代谢产物甲醛浓度。结果小鼠、大鼠和兔血浆中SSAO活性分别为(4.1±1.0),(2.0±0.3)和(325.8±3.9)μmol·h-1·L-1。3种动物单次iv甲胺后,甲胺的分布和代谢迅速。小鼠单次iv4.2mg·kg-1甲胺后甲醛浓度在代谢初期缓慢上升,在20~40min达峰后缓慢消除。而大鼠单次iv4.2mg·kg-1甲胺后血浆甲醛浓度无明显改变。兔单次iv2.3,9.6和36.8mg·kg-1甲胺后,甲胺AUC与剂量不成比例,Cl随剂量增加明显减少,呈非线性动力学特征。甲醛在兔体内消除较快,甲醛AUC与甲胺剂量的比值和全身清除率Cl无明显差异,但ke随给予甲胺剂量的增大而减少,t1/2显著延长。结论甲胺在3种动物间代谢情况相似,其代谢产物甲醛则明显不同。甲胺给予剂量的不同可能导致甲胺代谢动力学的改变,进而可能影响甲醛的代谢。     

卡托普利对蛋氨酸所致血管内皮功能损伤的保护作用与对氧磷酶1的关系

目的探讨卡托普利对高蛋氨酸饮食所致大鼠血管内皮功能损伤的保护作用及其机制。方法将蛋氨酸通过灌胃的方法,1次.d-1,连续4周,诱导大鼠血管功能损伤,治疗组同时给予卡托普利、依那普利、N-乙酰半胱氨酸灌胃。4周后处死动物,检测血清一氧化氮(nitric oxide,NO)、丙二醛(malondialdehyde,MDA)含量、对氧磷酶(paraoxonase 1,PON1)、超氧化物歧化酶(superoxide dismutase enzyme,SOD)、血管紧张素转换酶(an-giotensin-converting enzyme,ACE)活性。取胸主动脉检测由乙酰胆碱(acetylcysteine,Ach)诱导的血管内皮依赖性舒张反应。结果高蛋氨酸损伤组大鼠血管内皮依赖性舒张反应显著减弱,血清中MDA浓度升高,PON1活性、血浆NO浓度与SOD活性降低;卡托普利、N-乙酰半胱氨酸和依那普利能显著改善血管内皮依赖性舒张反应、降低MDA浓度、提高血清中的PON1活性、SOD活性和NO浓度。结论卡托普利能够改善高蛋氨酸引起的血管内皮功能的损伤,该作用可能与保护PON1活性及其抗氧化作用、促进内皮细胞释放NO有关。     

2-溴乙胺改善动脉粥样硬化兔血管内皮舒张功能

目的利用兔动脉粥样硬化实验模型及离体血管功能检测系统探讨SSAO抑制剂2-溴乙胺是否能改善动脉粥样硬化兔内皮依赖性血管舒张功能及减轻其动脉粥样斑块形成,观察SSAO底物甲胺对血管内皮功能的影响。方法雄性新西兰白兔24只等分为4组：①正常饲料组（CC组）;②1%胆固醇饲料组（HC组）;③1%胆固醇及0.2%甲胺饲料组（MA组）;④1%胆固醇及0.2%甲胺饲料并于9、10、11周时连续3次耳缘静注射2-溴乙胺（10mg·kg^-1）,动物喂养共12周。实验结束时抽取耳中央动脉血,作生化分析,剥离主动脉（自起始部到髂动脉分叉处）起始段4mm制成动脉环用于内皮依赖性血管舒张功能研究,其余部分作斑块面积测定。结果同CC组相比,HC组、MA组和2-BEA的血清总胆固醇（T-CHO）和低密度脂蛋白胆固醇（LDL-C）均明显增高（P〈0.01）,但3组间上述指标相比无明显差异;HC组和MA组动物主动脉对乙酰胆碱的内皮依赖性血管舒张功能明显损害,而2-BEA组动物主动脉对乙酰胆碱的内皮依赖舒张功能损害较轻;2-BEA组动物的动脉粥样斑块面积显著少于HC组和MA组。结论2-溴乙胺能够改善实验性动脉粥样硬化兔的内皮依赖性血管舒张功能,减少动脉粥样斑块的形成。     

Cariporide对糖基化终末产物所致血管内皮损伤的保护作用

目的观察选择性Na /H 交换蛋白1(NHE-1)抑制剂Cariporide对外源性糖基化终末产物(AGEs)所致大鼠血管内皮功能损伤的保护作用。方法将体外制备的糖基化牛血清白蛋白(AGEs-BSA)通过尾静脉注射的方法,1次.d-1,连续4周,诱导大鼠血管功能损伤,治疗组同时给予Cariporide(0.1、1 mg.kg-1.d-1)灌胃。4周后处死动物,取胸主动脉用于血管内皮依赖性舒张功能的检测,主动脉弓做NF-κB-p65免疫组化检测,并测定血清NO及MDA含量。结果大鼠注射AGEs-BSA后,主动脉对乙酰胆碱诱导的内皮依赖性舒张反应明显降低,大鼠血清MDA水平增加,NO水平降低;Cariporide呈剂量依赖性改善AGEs-BSA所致大鼠胸主动脉内皮依赖性舒张反应降低,抑制AGEs-BSA引起的血清MDA浓度升高和血清NO的减少;给予AGEs-BSA后大鼠血管内皮NF-κB活性明显增加,Cariporide能明显抑制AGEs-BSA诱导的NF-κB活化。结论Cariporide对外源性AGEs诱导的大鼠血管内皮功能损伤具有保护作用,其保护作用可能与降低氧化应激、抑制血管内皮细胞的NF-κB活化有关。     

2型糖尿病大鼠主动脉内皮细胞氧化损伤及缬沙坦的保护作用

目的探讨2型糖尿病大鼠氧化应激与主动脉内皮细胞损伤的关系,观察缬沙坦对两者的影响。方法SD大鼠,用长期高能量饮食加小剂量注射链脲佐菌素(STZ)的方法复制模型。注射STZ12wk末,将大鼠分为3组:正常组、糖尿病组、缬沙坦治疗组(24mg·kg-1·d-1,灌胃给药8wk)。在注射STZ12和20wk末,检测大鼠的内皮依赖性血管舒张反应及主动脉内皮形态,血清超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,丙二醛(MDA)和一氧化氮(NO)含量,以及主动脉一氧化氮合酶(NOS)基因表达情况。结果①12wk末,糖尿病大鼠主动脉对低浓度乙酰胆碱(ACh)舒张反应减弱,局部内皮隆起,血清SOD、GSH-Px活性增强,MDA和NO含量增加,主动脉iNOS mRNA表达明显上调,eNOS mRNA表达无明显改变。②20wk末,糖尿病大鼠主动脉对各浓度ACh的反应性均减弱,主动脉内皮变性、坏死,血清SOD、GSH-Px活性减弱,MDA含量进一步增加,NO含量下降,主动脉iNOS mRNA表达仍升高,eNOS mRNA表达降低,缬沙坦治疗后能减轻主动脉病变,改善血清SOD、GSH-Px、MDA、NO及主动脉NOS mRNA表达的异常。结论糖尿病大鼠的氧化应激和NO系统的紊乱参与了主动脉病变过程,增强机体抗氧化能力及调节NO生成可能是缬沙坦发挥主动脉保护作用的机制之一。     

槲皮素对高同型半胱氨酸血症家兔血管内皮细胞的保护作用

目的　评估槲皮素对高同型半胱氨酸血症家兔血管内皮细胞的影响。方法　制造家兔高同型半胱氨酸损伤血管内皮细胞模型 ,同时灌胃给予槲皮素 5 0、10 0和 2 0 0mg·kg-1,30d后采血进行循环内皮细胞计数 ,测定血清MDA、SOD、NO和血浆ET ,取胸主动脉作血管反应性测定 ,并进行组织学检查。结果　与损伤模型组相比 ,槲皮素给药组循环内皮细胞降低 ,MDA、ET水平降低 ,而NO和SOD水平升高 ,对ACh的内皮依赖性舒张反应趋近正常 ,对NTG的内皮非依赖性舒张反应无影响 ,组织学检查也证实槲皮素有效地对抗高同型半胱氨酸造成的血管内皮细胞损伤。结论　槲皮素对高同型半胱氨酸损伤家兔血管内皮细胞有保护作用。     

补体旁路激活导致内皮细胞活化和损伤

目的研究补体替代途径的激活对血管内皮细胞活化和损伤的作用。方法采用眼镜蛇毒因子(CVF)与人血清孵育,特异激活补体替代途径。将孵育物作用于人微血管内皮细胞,采用ELISA检测内皮细胞P-selectin、E-selectin、ICAM-1、MCP-1和IL-8的表达,采用酶活性测定法检测乳酸脱氢酶(LDH)活性,化学发光法检测内皮细胞caspase-8活化信号,MTT法检测内皮细胞增殖活性,并检测内皮细胞释放NO的变化。结果补体旁路激活导致内皮细胞瞬时表达P-selectin,并进而使内皮细胞上调表达E-selectin、ICAM-1、MCP-1和IL-8。内皮细胞经补体激活物刺激后,LDH释放增加、凋亡信号caspase-8活化上调以及NO释放下调,同时,细胞增殖也受到抑制。结论补体旁路激活能诱导内皮细胞活化和损伤,介导血管内皮的生理结构与功能发生变化,从而可能导致相应的炎症和组织损伤。     

龙血竭胶囊抗慢性血瘀证作用的研究

目的 研究龙血竭及其总黄酮类成分对慢性血瘀证兔模型的血液流变学指标及心主动脉内皮细胞等的影响。方法 建立慢性血瘀证兔模型, 造模同时各给药组每天给药1次, 干预4周后对各组血液流变学各项指标以及血清一氧化氮(NO)和内皮素(ET)水平进行检测, 并通过HE染色病理切片对兔心主动脉内皮细胞形态学变化进行观察比较。结果 与模型组相比, 龙血竭及其总黄酮类成分均能降低慢性血瘀证兔模型全血黏度、血浆黏度、红细胞压积、红细胞聚集指数和卡森黏度, 能升高红细胞变形指数, 对血小板聚集有抑制作用;同时能降低慢性血瘀证兔模型血清ET水平, 升高NO水平;此外, 各给药组兔心主动脉HE染色病理切片内皮细胞较模型组结构完整而光滑, 排列平行有序, 损失程度有所改善。结论 龙血竭及其总黄酮类成分均能改善慢性血瘀证兔模型的血液流变学特性, 调节NO/ET平衡, 并对其心主动脉内皮细胞有保护作用。     

氨基胍改善高果糖对大鼠主动脉内皮依赖性舒张功能损伤的保护作用及机制

目的探讨氨基胍对高果糖致大鼠离体胸主动脉环内皮依赖性舒张反应损伤的保护作用及机制。方法用离体血管环灌流装置,观察不同浓度氨基胍对高果糖引起血管内皮依赖性舒张反应损伤的保护作用;并分析血管壁中NO含量以及eNOS、Akt和ERK1/2蛋白磷酸化的表达。结果高果糖抑制乙酰胆碱诱导的内皮依赖性舒张,降低血管壁中NO的含量和eNOS活性,并促进ERK1/2磷酸化水平上调,但对Akt磷酸化无显著影响;用氨基胍与高果糖共同孵育后,明显改善高果糖对内皮依赖性舒张反应的损伤并恢复血管壁NO含量和eNOS活性,同时降低ERK1/2磷酸化水平。结论氨基胍能改善高果糖对离体胸主动脉环内皮依赖舒张反应的损伤作用。其保护机制与抑制ERK1/2磷酸化和恢复eNOS活性有关,而与AKT磷酸化活性无关。     

卡托普利对大鼠血管内皮功能损伤的保护作用

目的探讨卡托普利对烟碱所致大鼠血管内皮功能损伤的影响及可能机制。方法将30只大鼠分为3组，即正常对照组、烟碱损伤组（2mg／kg，腹腔注射）、卡托普利保护组（烟碱2mg／kg，腹腔注射＋卡托普利3mg／kg，静脉注射），4周后检测各组肠系膜动脉环内皮依赖性舒张（EDR）反应及主动脉一氧化氮（NO）含量，一氧化氮合成酶（NOS）和超氧化物歧化酶（SOD）活性变化。结果烟碱损伤组肠系膜动脉环EDR明显降低，并伴随主动脉NO含量及NOS，SOD活性的下降；卡托普利保护组血管EDR得到了明显改善，并且抑制了烟碱诱导的主动脉NO含量及NOS，SOD活性的下降。结论卡托普利对烟碱所致血管内皮功能损伤有明显保护作用，该作用与其清除氧自由基、促进内皮细胞合成、释放NO有关。     

Methylamine metabolism to formaldehyde by vascular semicarbazide-sensitive amine oxidase.

The capacity of the vascular enzyme, semicarbazide-sensitive amine oxidase (SSAO), to metabolize methylamine to the potentially toxic product, formaldehyde, was tested using rat aortic homogenates and purified porcine aortic SSAO. Formaldehyde production in incubations of enzyme source with methylamine (1 mM) was detected by high performance liquid chromatography and product was confirmed by desorption chemical ionization mass spectrometry (DCI-MS). Inhibitor studies using the specific SSAO inhibitor semicarbazide and the monoamine oxidase inhibitor pargyline indicate that SSAO is responsible for metabolism of methylamine to formaldehyde. These results suggest the possibility that elevated methylamine found in several pathologic states (such as uremia and diabetes mellitus), or generated from exogenous sources, could result in overproduction of formaldehyde in tissues with high SSAO activity, especially blood vessels.     

2-Bromoethylamine as a potent selective suicide inhibitor for semicarbazide-sensitive amine oxidase

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of methylamine and aminoacetone to produce toxic aldehydes, i.e. formaldehyde and methylglyoxal, as well as hydrogen peroxide and ammonia. An increase of SSAO activity was detected by different laboratories in patients suffering from vascular disorders, i.e. diabetes and myocardial infarction. The enzyme has been suggested to play a role in vascular endothelial damage and atherogenesis. To date, there are no selective SSAO inhibitors. In the present study, 2-bromoethylamine (2-BrEA) was found to be a highly effective and selective inhibitor of SSAO obtained from different sources. The inhibition was irreversible and time dependent. It was competitive when the enzyme was not preincubated with the inhibitor, but became noncompetitive after incubation of the enzyme with 2-BrEA. The aldehyde trapping agent o-phenylenediamine was capable of preventing 2-BrEA-induced inhibition of SSAO activity. An aldehyde product was detected to be an initial product of 2-BrEA after it was incubated with SSAO. The inhibition, therefore, is mechanism-based. The SSAO inhibitory effects of eight structural analogues of 2-BrEA were assessed. It was concluded that a bromine atom at the beta position is quite important for exerting high potency of SSAO inhibition. The inhibition of SSAO activity by 2-BrEA was also demonstrated in vivo. It increased the urinary excretion of methylamine, an endogenous substrate for SSAO, in mice. 2-BrEA can be employed as a very useful tool in the investigation of SSAO.     

Alterations of intracellular calcium concentration and nitric oxide generation in pulmonary artery endothelium after subarachnoid hemorrhage of the rabbit

The present study was designed to investigate whether endothelial intracellular calcium concentration ([Ca(2+)](i)), endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) generation altered in association with impaired endothelium-dependent relaxation (EDR) in pulmonary artery (PA) specimens from experimental subarachnoid hemorrhage (SAH) rabbits. Injecting non-heparinized autologous arterial blood into cisterna magna induced the SAH. Simultaneous measurements of endothelial [Ca(2+)](i) and isometric tension of PA specimens were performed using fura 2. The subjects included normal control rabbits (group N), SAH rabbits with normal EDR (group A) and with impaired EDR (group B). When treated with 10(-7) M acetylcholine (ACh), endothelial [Ca(2+)](i) was significantly lower in group B (74.1+/-8.5 nM) than that in groups A (153.0+/-28.0 nM, p<0.05) and N (184.8+/-27.8 nM, p<0.01). Basal and ACh-stimulated cyclic GMP productions as a marker of NO generation were also significantly (p<0.005) decreased in group B as compared to those in the other two groups. Meanwhile, there were no differences in eNOS activity per se among the three groups. These results suggest that the attenuated endothelial [Ca(2+)](i) elevation leads to the impaired NO generation in PA endothelium, which in turn impairs the EDR and possibly increases the vascular resistance of PA following SAH.     

Determination of human serum semicarbazide-sensitive amine oxidase activity: a possible clinical marker of atherosclerosis.

Semicarbazide-sensitive amine oxidase (SSAO) is present in the plasma membrane of several human tissues, e.g. vascular smooth muscle cell adipocytes, and is also found in human serum. Some previous studies on cultured endothelial cells indicate that cytotoxic metabolites (e.g. hydrogen peroxide, formaldehyde, acrolein) formed by serum SSAO may cause endothelial injury and subsequently induce atherosclerosis. To investigate the role of this enzyme in the pathogenesis of macrovascular complications in diabetes, a simple and sensitive radiometric procedure was adapted for human serum measurements. Serum SSAO activity of 35 patients with non-insulin dependent diabetes mellitus (NIDDM) and that of 30 controls was determined using [14C]-benzylamine as substrate. The severity of atherosclerosis was assessed by carotid sonography. Diabetic patients with atherosclerosis exhibited a higher SSAO activity compared to diabetic patients without complications (212.91 +/- 90.54 pmol/mg protein/h versus 133.17 +/- 65.40 pmol/mg protein/h, P <0.04). In diabetic patients without complications, serum SSAO activity was elevated compared to control subjects (133.17 +/- 65.40 pmol/mg protein/h versus 91.79 +/- 31.70 pmol/mg protein/h, P <0.01). These results suggest that determination of human serum SSAO activity might be a useful marker in the prognostic evaluation of diabetic angiopathy and atherosclerosis.     

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.     

Therapeutic use of citrulline in cardiovascular disease

L-citrulline is the natural precursor of L-arginine, substrate for nitric oxide synthase (NOS) in the production of NO. Supplemental administration L-arginine has been shown to be effective in improving NO production and cardiovascular function in cardiovascular diseases associated with endothelial dysfunction, such as hypertension, heart failure, atherosclerosis, diabetic vascular disease and ischemia-reperfusion injury, but the beneficial actions do not endure with chronic therapy. Substantial intestinal and hepatic metabolism of L-arginine to ornithine and urea by arginase makes oral delivery very ineffective. Additionally, all of these disease states as well as supplemental L-arginine enhance arginase expression and activity, thus reducing the effectiveness of L-arginine therapy. In contrast, L-citrulline is not metabolized in the intestine or liver and does not induce tissue arginase, but rather inhibits its activity. L-citrulline entering the kidney, vascular endothelium and other tissues can be readily converted to L-arginine, thus raising plasma and tissue levels of L-arginine and enhancing NO production. Supplemental L-citrulline has promise as a therapeutic adjunct in disease states associated with L-arginine deficiencies.     

Methylamine but not mafenide mimics insulin-like activity of the semicarbazide-sensitive amine oxidase-substrate benzylamine on glucose tolerance and on human adipocyte metabolism

It has been reported that benzylamine reduces blood glucose in rabbits, stimulates hexose uptake, and inhibits lipolysis in mouse, rabbit, and human adipocytes. In the presence of vanadate, benzylamine is also able to improve glucose disposal in normoglycaemic and diabetic rats. Such insulin-mimicking properties are the consequence of hydrogen peroxide production during benzylamine oxidation by semicarbazide-sensitive amine oxidase (SSAO). The aim of the study was to determine whether other SSAO-substrates could share such potential antidiabetic properties. Thus, mafenide, a synthetic antimicrobial sulfonamide structurally related to benzylamine, and which has been recently reported to interact with SSAO, was tested in the above mentioned models, in parallel with methylamine, a proposed endogenous SSAO-substrate. All tested amines stimulated glucose uptake and inhibited lipolysis in rat and mouse fat cells. Methylamine and benzylamine, but not mafenide, reduced the hyperglycaemic response during a glucose tolerance test in rabbits while the three amines tested were devoid of insulin-releasing activity under both in vivo and in vitro conditions. In human adipocytes, mafenide did not stimulate glucose transport since it was not a high-affinity substrate for SSAO and generated less hydrogen peroxide than benzylamine or methylamine. Therefore, mafenide could not be considered as an antidiabetic drug despite being oxidized and exhibiting insulin-mimicking effects in rat and mouse adipocytes. By contrast, the endogenous substrate methylamine improved glucose utilization in all in vitro and in vivo models, leading to consider novel SSAO substrates as drugs with potential anti-hyperglycaemic properties.