Determination of enzyme (angiotensin convertase) inhibitors based on enzymatic reaction followed by HPLC

For determination of levels of plasmatic inhibitor of ACE (angiotensin convertase) a simple method was used based on a combination of enzymatic reaction followed by an HPLC determination of its product. The inhibitor (e.g. enalaprilat) was at first separated from the biological material by deproteination (methanol). Then, an aliquot of the sample was added to the reaction mixture containing a commercial ACE enzyme, its specific substrate FAPGG (N-(3-[2-furyl]acryloyl)-Phe-Gly-Gly) and buffer (Tris–HCl, pH 7.5). Degree of inhibition of the conversion of this substrate to FAP (desGlyGlyFAPGG) by the inhibitor present in the sample is related to its amount by a simple dose–response relationship. The amount of the FAP was determined by an HPLC on a RP-18 column with an acetonitril–nonylamine buffer (pH 2.4, adjusted with phosphoric acid) as a mobile phase with detection at 305 nm. Alternatively, the activity of the endogenous ACE present in the plasma was measured. The substrate FAPGG was added to the plasmatic sample containing both the inhibitor and endogenous ACE (as the sample was not deproteinized in this case) and the reaction product was determined as above. Inhibitor concentration has been obtained from a dose–response curve expressing the interaction with inhibitor with an ACE enzyme.     

Use of rapamycin in a transplant patient who developed cyclosporin neurotoxicity

We describe the case of a paediatric kidney transplant patient who developed cyclosporin neurotoxicity on day 7 post-transplant. Consequently, her cyclosporin was stopped and she was commenced on rapamycin. Over the next 3 weeks her creatinine remained elevated and she had several episodes of biopsy proven rejection, despite increasing the initial dose of rapamycin by tenfold. Her whole blood rapamycin levels also remained well below the target range of 10–20 ng/ml. On day 38 post-transplant, the decision was made to add tacrolimus to her immunosuppression. At the same time, phenytoin, which had been commenced during her episode of cyclosporin neurotoxicity, was withdrawn. After this point her rapamycin blood levels rapidly increased to within the therapeutic range and she improved clinically. We propose that phenytoin, as a p450 cytochrome enzyme inducer, increased the metabolism of rapamycin in this patient and hence decreased the initial therapeutic effectiveness of this drug. Received: 8 February 2001 / Revised: 21 May 2001 / Accepted: 21 May 2001     

Angiotensin converting enzyme gene polymorphism in primary vesicoureteral reflux

We studied the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene in 78 patients with primary vesicoureteral reflux (VUR), and examined renal function by dimercaptosuccinate (DMSA) renoscintigraphy and diethylenetriaminepenta-acetic acid (DTPA) renogram in each genotype. Patients were classified into three genotypes according to the ACE gene I/D polymorphisms: 32 in II genotype, 36 in ID, and 10 in DD. The incidence of presumably congenital unilateral small kidneys was high in DD patients (70%). Glomerular filtration rate obtained from DTPA renogram was 120.7±35.7 ml/min (expressed as mean±SD) in II genotype, 111.7±33.3 in ID, and 88.0±18.0 in DD. The total quantitative DMSA tracer uptake of both kidneys was also low in patients with the D allele. This study shows that the D allele of ACE gene is closely related to small congenital kidneys with refluxing ureters in patients with primary VUR, and in accordance with previous reports, this allele is also related to the progression of reflux nephropathy. Received: 27 November 2000 / Revised: 10 April 2001 / Accepted: 10 April 2001     

Persistent arterial hypotension after bilateral nephrectomy in a 4-month-old infant

A patient with congenital nephrotic syndrome underwent bilateral nephrectomy at the age of 4 months. She showed persistent hypotension from the fourth postoperative day until death at the age of nearly 5 months. No cause for the hypotension could be found. It is postulated that, especially in young infants, a deficiency of renin after bilateral nephrectomy may cause persistent hypotension. An explanation for the putative increased risk of this complication in young infants may be their need for a highly active renin-angiotensin system. Until more is known about the incidence of this complication and its predisposing factors, reluctancy towards the performance of bilateral nephrectomy in children under the age of 6 months is warranted. Received: 7 April 2000 / Revised: 11 January 2001 / Accepted: 30 January 2001     

Polymorphism of the angiotensin-converting enzyme gene in patients with cerebral infarction in koreans

The relationship between cerebrovascular disease and an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene is still being debated. The frequency of the DD genotype of the ACE gene was significantly higher in subjects with than those without cerebral infarction in Japan. The aim of the present study was to assess the relationship between ACE gene polymorphism and the development of cerebral infarction in a population from Korea. We examined its possible role as a risk factor in patients with cerebral infarction. The association between ACE gene polymorphism and cerebral infarction was examined in 106 patients with cerebral infarction and 498 controls without cerebral infarction. Frequencies of the genotypes and alleles of the ACE gene were investigated. The ACE genotype was analyzed by the polymerase chain reaction (PCR). The frequency of D allele was 37.7% in patients and 39.1% in controls (X ²=0.128, p=0.720). The frequencies of the genotypes of the ACE gene were II:39.6%, ID:45.3%, and DD:15.1% in patients, and II:37.1%, ID:47.6%, and DD:15.3% in controls (X ²=0.127, p=0.721). There was no significant difference in the frequency of the DD genotype of the ACE gene, and we did not find any association between ACE polymorphism and cerebral infarction. These results indicate that ACE polymorphism is not a risk factor for the development of cerebral infarction in a Korean population.     

Angiotensin-converting enzyme (ACE) gene polymorphisms and familial occurrence of sarcoidosis

OBJECTIVES: The aim of this study was to test for genetic linkage and association between polymorphisms of the angiotensin-converting enzyme (ACE) gene and familial occurrence of sarcoidosis. DESIGN, SETTING AND SUBJECTS: German families with more than one member suffering from sarcoidosis were contacted and a DNA bank was established. Sixty-two families (140 patients, 77 females and 63 males, and 104 unaffected relatives) were genotyped for the ACE gene insertion/deletion (I/D) polymorphism and for two flanking variable sites (ACE A-5466C and ACE 4656(CT)2/3). As controls, 100 DNAs from unrelated resident Caucasians (50 females, 50 males) were analysed. ACE allele and genotype frequencies were determined, and parametric linkage and affected sib pair analyses and transmission disequilibrium tests were performed. RESULTS: There was a striking over-representation of the ACE I/D genotype DD in patients with sarcoidosis and their families as compared with controls of the study and well founded genotype frequencies from the literature. The same was evident for the accompanying genotypes CC and 2,2 of the flanking polymorphisms. Linkage between the segregation of ACE alleles and the disorder within families was clearly excluded for simple models of inheritance. However, there was a suggestive but not significant (P = 0.06) excess of allele sharing amongst affected siblings. There was no transmission disequilibrium for any ACE allele or haplotype. CONCLUSIONS: ACE is involved in the pathogenesis of sarcoidosis, but the ACE polymorphisms are not an inherited main cause of the disease. They are more likely to modify the development of the disorder, and the ACE I/D genotype DD might be a promoter to clinical manifestation.     

结核分支杆菌14KDa蛋白的基因克隆、表达、纯化及其抗原性的研究

目的基因克隆、表达、纯化结核分枝杆菌14KDa蛋白,研究其抗原性,评价其在血清学诊断中的价值。方法以结核分枝杆菌H37Rv基因组DNA为模板,应用PCR技术扩增14KDa蛋白基因片断,将其插入到高效表达载体PET-22b（＋）上,构建重组质粒PET-22b（＋）/14KDa,重组质粒在大肠杆菌中由IPTG诱导表达,表达产物经金属离子鳌合亲和层析方法纯化,免疫印迹和酶联免疫吸附（ELISA）试验分析重组蛋白的抗原性。结果构建了具有正确基因序列的14KDa蛋白重组质粒,在大肠杆菌BL21（DE3）中以可溶性蛋白形式表达,重组蛋白的表达量占菌体蛋白的40.8%,经过一步金属离子鳌合亲和层析后得到纯度为94.3%的目的蛋白。免疫印迹试验结果表明该蛋白能与羊抗结核血清发生特异免疫结合反应。应用ELISA方法对结核血清参考品进行检测,敏感性和特异性分别为75.6%和96.0%。结论获得了能高效表达14KDa蛋白的大肠杆菌工程菌,目的蛋白以可溶性形式表达,该重组蛋白具有良好的免疫原性,可望成为结核血清学的诊断抗原之一。     

温病湿热证大鼠模型肝线粒体K+-Na+-ATP酶活力变化的对比研究

[目的]探讨多因素复合造模方法对模型大鼠能量代谢的影响。[方法]分为正常组、湿热模型组(高脂 高温高湿 大肠杆菌)、模型对照组(普食 高温高湿 大肠杆菌);运用定磷法测肝线粒体Na -K -AT Pase活性。[结果]模型组与模型对照组肝线粒体Na -K -AT Pase活性均较正常组显著降低,但两者比较无显著性差异。[结论]肝线粒体Na -K -AT Pase活性显著降低,是湿热证模型的病理基础之一,与是否高脂饮食无关。     

人工饲养和野生美洲大蠊消化酶活性差异研究

目的比较人工饲养的美洲大蠊和野生美洲大蠊不同虫态主要消化酶的活性。方法解剖人工饲养美洲大蠊和野生美洲大蠊不同虫态的中肠,测定蛋白酶、淀粉酶、脂肪酶和蔗糖酶活性。结果人工饲养的美洲大蠊低龄若虫、高龄若虫、成虫蛋白酶活性分别为56.28、59.75和65.22U,脂肪酶活性分别为47.67、69.91和79.44U,淀粉酶活性分别为51.16、76.40和95.51U,蔗糖酶活性分别为0.60、0.67和0.97U;野生美洲大蠊低龄若虫、高龄若虫、成虫蛋白酶活性分别为31.68、42.49和44.03U,脂肪酶活性分别为56.37、76.38和87.16U,淀粉酶活性分别为47.15、57.90和72.44U,蔗糖酶活性分别为0.91、0.96和1.22U。结论在同一虫态,人工饲养的美洲大蠊蛋白酶和淀粉酶活性均高于野生美洲大蠊,而脂肪酶和蔗糖酶活性低于野生美洲大蠊。在美洲大蠊生长发育阶段,中肠蛋白酶、脂肪酶、淀粉酶和蔗糖酶活性都随其发育而逐渐增强。     

微量元素硒与自由基

硒是基于抗氧化酶的必需组分,它通过消除脂质氢过氧化物,阻断活性氧和自由基的致病作用,而起到防病作用,因此机体硒水平的高低直接影响了机体抗氧化能力,以及对相关疾病的抵抗能力。