血管紧张肽（素）转化酶动力学测定方法的探讨及其初步临床应用

目的建立血管紧张肽（素）转化酶（ACE）自动化分析方法。方法 ACE能催化底物呋喃酰基-L-苯丙氨酰甘氨酰甘氨酸（FAPGG）,产生呋喃酰基苯丙氨酸（FAP）及双甘氨肽（GG）。在pH值8.2的硼酸缓冲液下,在340 nm处测定产物吸光度值的下降速度可计算出ACE活性。采用本法测定50名正常对照者、30例慢性阻塞性肺部疾病加重期（AECOPD）患者、18例肺癌患者血清ACE的活性。结果本法缓冲液最适pH值为8.2,最适基质浓度为1.0 mmol/L,批内、批间平均变异系数（CV）分别为4.07%、5.50%,米氏常数（Km）为0.09 mmol/L。正常对照组ACE活性为（31.1±18.0）U/L;AECOPD患者ACE活性为（22.4±12.6）U/L,低于正常组（P〈0.000 1）;肺癌患者ACE活性为（28.7±11.4）U/L,稍低于正常对照组,但差异无统计学意义（P〉0.05）。结论该法简便、快捷、精确,可用于自动化分析,适宜临床常规应用。ACE可作为评价肺部疾病的一项指标。     

4种肺部疾病血清血管紧张素Ⅱ转换酶活性的比较

目的探讨血清血管紧张素转换酶(ACE)活性变化对肺部疾病诊断的价值。方法采用紫外光度法检测血清ACE活性。结果肺炎及肺结核患者血清ACE活性分别为(35.1±9.3)U/L和(38.6±11.5)U/L,与对照组(39.4±8.8)U/L相比均无显著性差异(P>0.05)。46例肺癌患者血清ACE活性(26.9±9.2)U/L,显著低于肺炎、肺结核患者及对照组;肺结节病和肺心病患者血清ACE活性分别为(65.8±13.6)U/L和(55.1±7.4)U/L,显著高于对照组、肺癌、肺炎及肺结核组(P<0.001)。结论血清ACE活性测定可作为辅助检测肺癌或肺结节病的重要指标,有助于肺癌、肺结节病、肺心病和肺结核的鉴别诊断。     

血清γ—GT速率测定法的实验条件探讨

本文以γ-谷氨酰对硝基苯胺为底物终浓度为5.0mmol/L。用Tris-甘氨酰甘氨酸(双甘肽)缓冲液(pH8.2),其中双甘肽作γ-谷氨酰基的受体,终浓度为100mmol/L,生成的对硝基苯胺为黄色,在410nm波长处     

甘氨酰脯氨酰二肽氨基肽酶动力学测定方法探讨及初步临床应用

目的建立甘氨酰脯氨酰二肽氨基肽酶(GPDA)自动化分析方法。方法用甘氨酰脯氨酰对硝基苯胺对甲苯磺酸盐为底物,在Tris-HCl-双甘肽缓冲系统下,在405nm处测定产物吸光度的变化,得出GPDA活性。结果最适pH值为8.6,基质浓度为2.0mmol/L。批内、批间平均变异系数分别为3.01%、5.04%。米氏常数(Km)为1.89mmol/L。标本存于4℃冰箱10d内稳定性良好。参考值为(102.8±26.0)U/L。肝癌组该酶范围为(139.9±71.8)U/L,与正常组比较差异有显著性(P<0.05)。胃癌组和慢性胃炎组该酶范围分别为(58.83±18.18)U/L和(61.5±15.8)U/L,与正常组比较差异均有显著性(P<0.001、P<0.005)。结论该法简便、快捷、精确,可用于自动化分析,并可作为评价肝癌、胃癌及慢性胃炎的血清学指标。     

甘氨酰脯氨酰二肽氨基肽酶动力学测定方法探讨及初步临床应用

目的建立甘氨酰脯氨酰二肽氨基肽酶(GPDA)自动化分析方法.方法用甘氨酰脯氨酰对硝基苯胺对甲苯磺酸盐为底物,在Tris-HCl-双甘肽缓冲系统下,在405 nm处测定产物吸光度的变化,得出GPDA活性.结果最适pH值为8.6,基质浓度为2.0 mmol/L.批内、批间平均变异系数分别为3.01%、5.04%.米氏常数(Km)为1.89 mmol/L.标本存于4 ℃冰箱10 d内稳定性良好.参考值为(102.8±26.0) U/L.肝癌组该酶范围为(139.9±71.8) U/L,与正常组比较差异有显著性(P<0.05).胃癌组和慢性胃炎组该酶范围分别为(58.83±18.18) U/L和(61.5±15.8) U/L,与正常组比较差异均有显著性(P<0.001、P<0.005).结论该法简便、快捷、精确,可用于自动化分析,并可作为评价肝癌、胃癌及慢性胃炎的血清学指标.     

鸟氨酸氨基甲酰转移酶速率法测定的初步探讨

目的 建立鸟氨酸氨基甲酰转移酶(OCT)自动化分析的方法.方法 在磷酸盐缓冲液(pH值7.2)条件下,以瓜氨酸为底物,经一系列酶偶联反应,尼克酰胺腺嘌呤二核苷酸(NADH+H+)被氧化为氧化型辅酶Ⅰ(NAD+),340 nm处测定吸光度(A)变化值,A值下降的速率与待测样本中的OCT含量成正比关系,间接求出OCT的活性.结果 本法最适pH值为7.2,最适底物浓度为2.0 mmol/L.批内、批间平均变异系数(CV)分别为3.80%、4.08%.米氏常数(Km)值为0.16 mmol/L.回收率达91.56%.在320 U/L内线性良好.参考范围为0～18 U/L.结论本方法能够快速、简便、准确的测定OCT活性,适用于各类全自动生化分析仪.     

鸟氨酸氨基甲酰转移酶速率法测定的初步探讨

目的建立鸟氨酸氨基甲酰转移酶(OCT)自动化分析的方法。方法在磷酸盐缓冲液(pH值7.2)条件下,以瓜氨酸为底物,经一系列酶偶联反应,尼克酰胺腺嘌呤二核苷酸(NADH+H+)被氧化为氧化型辅酶Ⅰ(NAD+),340 nm处测定吸光度(A)变化值,A值下降的速率与待测样本中的OCT含量成正比关系,间接求出OCT的活性。结果本法最适pH值为7.2,最适底物浓度为2.0 mmol/L。批内、批间平均变异系数(CV)分别为3.80%、4.08%。米氏常数(Km)值为0.16 mmol/L。回收率达91.56%。在320 U/L内线性良好。参考范围为0~18 U/L。结论本方法能够快速、简便、准确的测定OCT活性,适用于各类全自动生化分析仪。     

甘氨酰脯氨酸二肽氨基肽酶的方法学研究

本文研究了甘氨酰脯氨酸二肽氨基肽酶(GPDA)的最佳测定条件。最适pH8.6,Km 值为1.91±0.08mmol/L,选用158mmol/L Tris、64mmol/L 双甘氨肽、10 mmol/L 底物。酶促反应速度在70分钟内呈线性,酶活力在600U/L 内呈线性。高、中,低活力标本批内变异1.7～3.1%,批问变异3.8～6.7%,标本置4℃一周,结果无明显变化。168例健康者GPDA 为150.1±49.8(2SD)U/L,同时研究了测定管、底物空白、对硝基苯胺的光学吸收特性,观察了GPA 试剂在不同pH 和不同时间的变化规律。     

肾实质性高血压患者血管紧张素转换酶基因多态性与血管紧张素转换酶活性的研究

目的研究肾实质性高血压患者血管紧张素转换酶(ACE)基因多态性及与ACE活性的关系。方法运用聚合酶链反应技术检测78例肾实质性高血压患者血管紧张素转换酶插入/缺失基因多态性,采用紫外分光光度法测定ACE活性,以87例正常人为对照。结果①患者组基因型分布与对照组不同(P<0.05),其中缺失型(DD型)频率升高(43.6%vs24.1%,P<0.01);②DD型、缺失/插入型(DI型)、插入型(II型)酶活性在对照组分别为(33.8±12.0)U/L、(18.6±7.2)U/L、(13.4±5.0)U/L,在患者组分别为(31.1±10.2)U/L、(26.3±8.0)U/L、(18.4±7.5)U/L,均表现DD型最高,II型最低(均P<0.01);③患者组ACE活性明显高于对照组(25.9±10.4)U/Lvs(20.7±11.2)U/L(P<0.01);两组ACE活性DD型间差异无统计学意义(P>0.05),而DI型间和II型间患者组明显高于对照组(均P<0.01)。结论①DD基因型在肾实质性高血压患者分布频率升高,是高危因素,与其ACE活性增高有关;②肾实质性高血压患者ACE活性升高,增高程度与基因型有关;因此基因型和ACE活性的检测可作为指导治疗的指标。     

肾病患者尿中二肽基-氨基肽酶Ⅳ的变化

二肽基-氨基肽酶IV(DAP-IV)能使X-脯氨酰-Y-肽分解,产生X-脯氨酸,对氨基端第二位的脯氨酸有高度特异性。作者以7-甘氨酰脯氨酸-4-甲基香豆素酰胺(甘氨酰-脯氨酸-MCA)为底物,用荧光法测定尿中DAP-IV的活性。取试管1支加入pH 8.7的0.15mol/L甘氨酸/氢氧化钠缓冲液40μl,2mmol/L甘氨酰-脯氨酸-MCA水溶液25μl及人尿35μl。另取试管1支以35μl蒸馏水代替人尿作为对照管。将各管置于37℃水浴中保温30分钟,加入1mol/L醋酸盐缓冲液1.0ml     

Methodological aspects of the use of furanacryloyl-phenylalanyl-glycyl-glycine for the photometric detection of angiotensin converting enzyme activity

Photometric evaluation of serum activity of angiotensin-converting enzyme (ACE) making use of synthetic chromogenic substrate N-(3-[2-furyl]acryloyl)-phe-gly-gly (FAPGG) is analyzed. Spectral characteristics of FAPGG are investigated. The Burger-Lambert-Bar dependence is not observed in any of the working concentrations. Extension of the spectral weight band is associated with the appearance of the absorbency/concentration linear function for FAPGG solution. Under such conditions the registered intensity of the analyzer sign (delta OD/min) is in strong correlation with the spectral weight band. As a result, high inter-analyzer variations were observed. This impedes the standardization of the method and prompts the use of attested calibrators. Linear function of analyzer's sign and ACE level is theoretically achieved at FAPGG concentration 1.0 +/- 0.2 mM (Km = 0.35 +/- 0.05 mM, minimum saturation concentration 1.4 +/- 0.2 mM). Synthetic ACE inhibitors are recommended to rule out the interference.     

Uptake of N-[1 (S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro, an inhibitor of angiotensin-converting enzyme by rabbit lungs in situ

Single pass extraction of a new iodinated inhibitor of angiotensin-converting enzyme (ACE) was measured by means of indicator-dilution techniques applied to rabbit lungs, perfused in situ at 20 ml/min with Krebs bicarbonate solution containing 3% bovine serum albumin. A bolus containing the inhibitor, N-[1(S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro (CPAP), and an intravascular marker, [14C]dextran, was injected and extraction calculated at the peak of the resulting venous outflow-time curve. In 13 of 21 lungs used, a synthetic substrate for ACE, [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), was added to the bolus and appearance of its hydrolysis product, [3H]benzoyl-Phe, measured in effluent samples. When low amounts (0.15 nmol) of [125I]CPAP were injected, pulmonary extraction (E) of CPAP was 67 +/- 14% (X +/- S.D; n = 21) and metabolism (M) of BPAP was 56 +/- 9% (n = 13). Addition of unlabeled CPAP (3, 34 or 340 nmol) to the Addition of unlabeled CPAP (3, 34 or 340 nmol) to the injected bolus caused dose-dependent reduction of E(CPAP) and M(BPAP) that was no longer evident 10 min after the largest dose of CPAP. Coadministration of the ACE inhibitor, captopril (3, 6, 8 and 28 nmol), also caused dose-dependent, reversible depression of both E(CPAP) and M(BPAP). Accordingly, extraction of CPAP by perfused rabbit lung is saturable. Inasmuch as CPAP inhibits ACE activity (as reflected by BPAP metabolism) and CPAP uptake is inhibited by captopril (which also inhibits BPAP hydrolysis), it appears that a large portion of this saturable process probably reflects binding to vascular ACE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenobarbital in the genetically obese Zucker rat. I. Pharmacokinetics after acute and chronic administration

We measured both pulmonary and plasma angiotensin converting enzyme (ACE) activity in conscious rabbits before and for 6 days after administration of captopril (2 mg/kg i.v.). Pulmonary ACE activity was measured by means of modified indicator-dilution techniques after bolus injection of [3H]benzoyl-phenyl-alanyl-alanyl-proline ( BPAP , synthetic substrate for ACE). Plasma ACE activity was also determined radiometrically with [3H] BPAP as substrate. In addition, we measured the systemic pressor response to i.v. bolus dose of angiotensin I both before and after captopril. Twenty-four hours after captopril, pulmonary metabolism of BPAP had decreased from control of 73 +/- 5 to 8 +/- 2%; even 6 days after drug treatment there was still evidence of inhibition. In contrast, plasma ACE activity was significantly (P less than .05) reduced within 15 min of treatment (to 20% of control levels) but recovered within 24 hr. The time course of changes in the pressor response to angiotensin I, after captopril, resembled that of plasma ACE activity. Mean systemic arterial blood pressure was 81 +/- 4 torr at control and reached its nadir at 24 hr (64 +/- 2 torr; P less than .05); thereafter a gradual recovery ensued, similar in time course to that of pulmonary ACE. These data suggest that inhibition of plasma ACE and also the pressor response to angiotensin I are unrelated temporally to the hypotensive effects of captopril.     

Angiotensin-converting enzyme determination in plasma during therapy with converting enzyme inhibitor: two methods compared.

For normal and above-normal concentrations of angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity in plasma, results of a manual fluorometric method [with hippuryl-histidyl-leucine (HHL), 5 mmol/L, as substrate] correlated well with those of an automated spectrophotometric method [with 3-(2-furylacryloyl)-L-phenylalanyl-glycyl-glycine (FAPGG), 2 mmol/L, as substrate]. However, for patients receiving converting enzyme inhibitor (CEI) therapy, the spectrophotometric method showed much greater suppression of plasma ACE activity than did the fluorometric method. To determine which of the two methods provided a more reliable indication of ACE inhibition in vivo, we measured plasma ACE, angiotensin I (ANG I), and angiotensin II (ANG II) in patients receiving the CEI perindopril. During perindopril therapy, changes in the ratio of ANG II:ANG I, an index of ACE activity in vivo, showed a close agreement with changes in plasma ACE activity measured with FAPGG as substrate, but not with HHL as substrate. We conclude that measurement of ACE activity in vitro with FAPGG as substrate provides a reliable measure of changes in conversion of ANG I to ANG II in vivo during CEI therapy.     

Angiotensin I-converting enzyme inhibitors block protein kinase C epsilon by activating bradykinin B1 receptors in human endothelial cells

Angiotensin I-converting enzyme (ACE) inhibitors are widely used to treat patients with cardiovascular and kidney diseases, but inhibition of ACE alone does not fully explain the beneficial effects. We reported that ACE inhibitors directly activate bradykinin B1 receptor at the canonical Zn2+ binding site, leading to prolonged nitric oxide (NO) production in endothelial cells. Protein kinase C (PKC) epsilon, a novel PKC isoform, is up-regulated in myocardium after infarction, suggesting a role in the development of cardiac dysfunction. In cytokine-treated human lung microvascular endothelial cells, B1 receptor activation by ACE inhibitors (enalaprilat, quinaprilat) or peptide ligands (des-Arg10-Lys1-bradykinin, des-Arg9-bradykinin) inhibited PKC epsilon with an IC50 = 7 x 10(-9) M. Despite the reported differences in binding affinity to receptor, the two peptide ligands were equally active, even when inhibitor blocked the cleavage of Lys(1), thus the conversion by aminopeptidase. The synthetic undecapeptide (LLPHEAWHFAR) representing the binding site for ACE inhibitors on human B(1) receptors reduced PKC epsilon inhibition by enalaprilat but not by peptide agonist. A combination of inducible and endothelial NO synthase inhibitors, 1400W [N-(3(aminomethyl) benzyl) acetamidine dihydrochloride] and N omega-nitro-L-arginine (2 microM), significantly reduced inhibition by enalaprilat (100 nM), whereas the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (100 microM) inhibited PKC epsilon activity just as the B1 ligands did. In conclusion, NO generated by B1 receptor activation inhibits PKC epsilon.     

Sulphation of colonic and rectal mucin in inflammatory bowel disease: reduced sulphation of rectal mucus in ulcerative colitis.

1. Normal colonic mucin is heavily sulphated and this increases its resistance to degradation by bacterial enzymes. Any defect in mucus sulphation could therefore be important in the pathogenesis of ulcerative colitis. 2. Rectal biopsies taken at colonoscopy from patients with ulcerative colitis (n = 9), patients with Crohn's disease (n = 6) and control subjects (n = 16) were cultured for 24 h in the presence of N-[3H]acetylglucosamine and [35S]sulphate. Mucin was then extracted and purified, and the ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into pure mucin was assessed. 3. The ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into mucin was significantly reduced in rectal biopsies taken from patients with ulcerative colitis (0.463, 0.305-0.703, geometric mean and 95% confidence intervals) compared with control subjects (0.857, 0.959-1.111, P < 0.01). In patients with Crohn's disease the reduction in this ratio (0.559, 0.378-0.829) did not quite reach statistical significance (P = 0.06). There was no difference between the ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into mucin in Crohn's disease and that in ulcerative colitis (P = 0.26). 4. In control subjects the ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into mucin was higher in the rectal biopsies (0.882, 0.618-1.022) than in their paired proximal colonic biopsies (0.602, 0.421-0.861; P < 0.01), but this regional variation was not observed in either ulcerative colitis (rectum: 0.450, 0.262-0.773; right colon: 0.470, 0.321-0.690, P = 0.3) or Crohn's disease (rectum: 0.459, 0.260-0.815; right colon: 0.492, 0.260-0.929, P = 0.8).(ABSTRACT TRUNCATED AT 250 WORDS)     

FK 224, a novel cyclopeptide substance P antagonist with NK1 and NK2 receptor selectivity.

We have discovered a novel cyclopeptide substance P (SP) antagonist, FK 224 (N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N-[3-(2-pentylphenyl )- propionyl]-L-threonyl]tyrosyl-L-leucynyl]-D-phenylalanyl]-L-allo- threonyl]-L-asparaginyl]-L-serine-nu-lactone), which inhibited [3H]SP binding to guinea pig lung membranes in a dose-dependent manner. According to Rosenthal analysis, the inhibitory effect of FK 224 on [3H]SP binding appears to be competitive. In order to clarify the receptor subtype selectivity of FK 224, we have studied the interaction of FK 224 with three tachykinin receptors (NK1, NK2 and NK3) by using receptor binding techniques and in vitro bioassays, and have also compared FK 224 with the novel nonpeptide antagonist, (+/-)-CP-96,345. In binding experiments, FK 224 dose-dependently inhibited [3H]SP binding to rat cerebral cortical membranes (NK1) and [3H]neurokinin (NK) A (NKA) binding to rat duodenum smooth muscle membranes (NK2), but did not affect [3H]eledoisin binding to rat cerebral cortical membranes (NK3). In bioassay experiments, FK 224 inhibited SP-induced contraction of guinea pig ileum (NK1) and NKA-induced contraction of rat vas deferens (NK2) in a dose-dependent manner, but did not affect NKB-induced contraction of rat portal vein (NK3). In contrast, (+/-)-CP-96,345 inhibited SP-induced contraction of guinea pig ileum, but not NKA-induced contraction of rat vas deferens or NKB-induced contraction of rat portal vein. In the presence of FK 224, SP dose-response curves and NKA dose-response curves were shifted to the right in parallel with no depression of the maximal contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of captopril- and enalapril-induced inhibition of pulmonary angiotensin converting enzyme in vivo

The kinetics of angiotensin converting enzyme (ACE) inhibition by captopril (SQ 14225) and enalapril (MK 421) in anesthetized rabbits was investigated. Kinetic parameters, apparent Km, an index of enzyme-substrate affinity and apparent Vmax, a measure of maximal rate of substrate conversion, were determined from indicator dilution measurements of single pass pulmonary metabolism of a synthetic ACE substrate [3H]benzoyl-phenylalanyl-alanyl-proline. Two methods for determination of kinetics in vivo from metabolism data were used. One fit pulmonary venous outflow metabolism data to a nonlinear model of saturable lung metabolic processes. This method required injection of sufficient substrate (benzoyl-phenylalanyl-alanyl-proline) to produce a large range of intravascular substrate concentrations. An alternative method required use of only low intravascular substrate concentrations. Both methods rely primarily on similar Michaelis-Menten assumptions and generated very similar results. Both captopril (10 and 20 nmol/kg) and enalapril (4 and 7 nmol/kg) behaved as noncompetitive ACE inhibitors in vivo. ACE inhibition was characterized by depressed [3H]benzoyl-phenylalanyl-alanyl-proline hydrolysis and apparent Vmax whereas apparent Km was unaffected. Other studies have suggested that these inhibitors act as competitive or mixed competitive and noncompetitive ACE inhibitors in vitro. Significant differences, however, between in vivo and in vitro experimental conditions suggest that the kinetics of enzyme inhibition in vitro may not necessarily reflect the action of the inhibitor in vivo. Additionally, results obtained in vivo may more accurately reflect the therapeutic behavior of these compounds.     

Evidence for a functional tissue renin-angiotensin system in the rat mesenteric vasculature and its involvement in regulating blood pressure

Electrical stimulation of the isolated rat mesenteric vascular bed resulted in a frequency-dependent pressor response, which could be potentiated by increasing concentrations of renin substrate (synthetic tetradecapeptide). This potentiating effect appeared to be mediated by tissue conversion of renin substrate to angiotensin II because the response 1) could be mimicked by angiotensin II, 2) was accompanied by an increase in angiotensin II production and 3) was blocked by the angiotensin converting enzyme (ACE) inhibitor quinaprilat and the angiotensin II receptor antagonist saralasin ([Sar1,Ile5,Ala8]angiotensin II). To assess the role of this tissue renin-angiotensin system in contributing to blood pressure regulation, spontaneously hypertensive rats were administered the prodrug ACE inhibitor quinapril at a dose of 10 mg/kg/day for 7 days. Such administration resulted in a reduction in systolic blood pressure of 48 +/- 3 mm Hg, a greater than 95% inhibition of serum ACE activity, and a significant attenuation of the potentiating effect of renin substrate on electrically evoked contractions of isolated mesenteric beds. Significant reductions in blood pressure and the potentiating effect of renin substrate on the isolated mesenteric vasculature were still observed 24 and 48 hr after the last dose of quinapril. In contrast, serum ACE activity returned to normal levels within 48 hr after the last dose of quinapril. These results suggest that the changes in tissue renin-angiotensin system, and not the circulating system, are closely related to the blood pressure lowering effect of the ACE inhibitor, quinapril.