5'-Nucleotide phosphodiesterase isoenzymes in human serum: quantitative measurement and some biochemical properties

A method based on native PAGE is used for the quantitative measurement of 5'-nucleotide phosphodiesterase isoenzymes (5'-NPD; EC 3.1.4.1) in human serum. In contrast to other techniques this method works with a commercially available substrate. In sera of healthy donors four isozymes could be separated, designated as 5'-NPD-I, 5'-NPD-II, 5'-NPD-III and 5'-NPD-IV in the reverse order of their electrophoretic mobility. When low amounts of serum were applied to the gel, the separation between all four activities was sufficient enough to allow their quantitation. Higher amounts of serum impaired the separation between the isoenzymes I and II. However, even when using high amounts of serum, the quantitation of these activities was possible when taking advantage of some of their biochemical properties which are described herein.     

Human serum dipeptidyl peptidase IV (DPPIV) and its unique properties

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) has been purified 18,000-fold in a yield of 2.2% from human serum. Serum DPPIV, a serine enzyme with an apparent mass of 250 kDa, consists of two identical subunits with an apparent mass of 100 kDa and is inhibited by DPPIV-specific inhibitor Diprotin A and also by p-chloromercuribenzoate (p-CMB), 2-mercaptoethanol, HgCl₂, CdCl₂, SrCl₂, and ZnCl₂. One of the remarkable properties of DPPIV is that its activity is greatly enhanced by Gly-X (X: especially, Gly, Gln, Glu and Ser) dipeptides. Gly-X dipeptides increase not only an apparent Km of serum DPPIV for glycyl-L-proline 3,5-dibromo-4-hydroxyanilide nearly 10-fold, but also an apparent kcat nearly 4-fold. This mechanism is unclear, but one possibility is that Gly-Pro from substrate might bind amino acids or dipeptides instead of water molecules as DPPIV transpeptidyl activity reported previously. Another remarkable property of DPPIV is the ability to bind adenosine deaminase-I and -II, as is the case with recombinant soluble CD26 (rsCD26). This probably indicates that DPPIV purified from human serum by our method originates from T-lymphocytes. © 1996 Wiley-Liss, Inc.     

Preparation and some properties of highly purified human serum kallikrein.

A 3000--6000-fold purified kallikrein was obtained from human serum in 10--25% yield by chromatography on QAE-Sephadex A-50, Molselect CM-50 and on soybean trypsin inhibitor (SBTI)-AH-Sepharose 4-B. The enzyme had a specific activity of 14--23 U, as measured by BAEE hydrolysis. Some properties of highly purified kallikrein are described.     

一种芳香酯酶检测新方法及其应用

目的建立芳香酯酶检测方法并作出方法学评价,探讨芳香酯酶的临床应用.方法采用有机合成方法合成底物4-乙酰氧基-3-甲氧基-ω-硝基苯乙烯,底物经酶水解生成4-羟基-3-甲氧基-ω-硝基苯乙烯,产物在pH8.6Tris-HCI溶液中显红色.检测520nm处吸光度变化以监测产物生成速度,反映酶活性水平.对90例健康体检者和463例病人血清进行了检测并建立了参考值范围.结果在肝硬化、肝癌、肝炎、胃癌、尿毒症、肾病综合征、肾炎、冠心病(P＜0.01)和高血压(P＜0.05)时芳香酯酶均显著降低.在肝硬化、肝癌、肝炎检测中发现芳香酯酶与白蛋白(ALB)、胆碱酯酶(CHE)和直接胆红素(D-BIL)相关系数分别为0.872、0.840,-0.495,经t检验相关系数均具有高度统计意义(P＜0.01).结论本法简便可靠,可采用自动化和手工两种方法,适合不同条件的单位常规应用.为临床肝硬化、肝癌、肝炎、胃癌、尿毒症、肾病综合征、肾炎、冠心病和高血压的诊断、治疗、预后提供重要信息.     

Preparation and some properties of human serum kallikrein immobilized on ARM-Sepharose.

A highly purified human serum kallikrein immobilized on CH-Sepharose 4-B was obtained. KM values for N-benzoyl-L-arginine ethyl ester and N-tosyl-L-arginine methyl ester hydrolysis of this preparation were 1.10 x 10(-3) M and 3.6 x 10(-4) M, respectively; pH optimum of hydrolysis of these esters were found to be 8.2 and 8.5, respectively. The immobilized kallikrein possessed kininogenase activity and was capable of activating prekallikrein.     

一种芳香酯酶检测新方法及其应用

目的 建立芳香酯酶检测方法并作出方法学评价,探讨芳香酯酶的临床应用。方法 采用有机合成方法合成底物4-乙酰氧基-3-甲氧基-ω-硝基苯乙烯,底物经酶水解生成4-羟基-3-甲氧基-ω-硝基苯乙烯,产物在pH8.6Tris-HCI溶液中显红色。检测520nm处吸光度变化以监测产物生成速度,反映酶活性水平。对90例健康体检者和463例病人血清进行了检测并建立了参考值范围。结果 在肝硬化、肝癌、肝炎、胃癌、尿毒症、肾病综合征、肾炎、冠心病(P<0.01)和高血压(P<0.05)时芳香酯酶均显著降低。在肝硬化、肝癌、肝炎检测中发现芳香酯酶与白蛋白(ALB)、胆碱酯酶(CHE)和直接胆红素(D-BIL)相关系数分别为0.872、0.840,-0.495,经t检验相关系数均具有高度统计意义(P<0.01)。结论 本法简便可靠,可采用自动化和手工两种方法,适合不同条件的单位常规应用。为临床肝硬化、肝癌、肝炎、胃癌、尿毒症、肾病综合征、肾炎、冠心病和高血压的诊断、治疗、预后提供重要信息。     

Human alpha 1-microglobulin: its measurement and clinical significance.

alpha 1-Microglobulin (alpha 1-M), also called protein HC, is a low-molecular-weight (LMW) glycoprotein (about 30 kDa) with unique physicochemical properties. Using purified urinary alpha 1-M a standard and specific antibody against alpha 1-M, an assay system for alpha 1-M was developed, and the clinical significance of this protein was investigated by measuring total levels of alpha 1-M under physiological and pathological conditions. alpha 1-M is distributed in various body fluids: in serum, it consists mainly of free LMW alpha 1-M and monomeric IgA-alpha 1-M complex. The total alpha 1-M level in serum and urine usually reflects LMW alpha 1-M variation sensitively, and its determination is quite useful as an indicator of renal glomerulotubular dysfunction and hepatic dysfunction. Serum levels can vary, depending on IgA-alpha 1-M complex level, in parallel with the IgA concentration. The heterogeneity of alpha 1-M purified from different sources of urine by different procedures and underestimation of IgA-alpha 1-M complex by solid-phase antibody assays can be important causes for the discrepancy of serum levels between assays.     

Macro creatine kinase BB in serum, and some data on its prevalence.

We report the case of a patient with persistently above-normal activity of creatine kinase (CK) in serum, a major fraction of which on electrophoresis moved as a band between the MM and MB isoenzymes and on anion-exchange column chromatography eluted in the MB fraction. Measurements in the presence of specific M or B subunit-inhibitory antibodies indicated that 93% of the activity consisted of B-isomers. From these experiments we conclude that the abnormal CK is of BB nature. Gel filtration and immunoglobulin precipitation showed that the CK-BB was complexed with IgG. Normal CK-BB, when mixed with the patient's serum, was converted to macro CK-BB. In vitro stability of 37 degrees C of the abnormal enzyme was much greater than that of normal BB and MM isoenzymes. Following this finding, we then assessed 310 sera, received for enzyme assay by the clinical laboratory, for electrophoretically abnormally migrating CK isoenzymes. Of these, five (1.6%) contained such enzymes, all being of BB nature. They were of increased molecular mass, and at least three of them were complexed with IgG.     

北京居民血清游离甘油水平及其对甘油三酯水平分类的影响

目的调查血清游离甘油(FG)水平及其与血清甘油三酯(TG)、年龄、性别等的关系，探讨FG对血清TG水平划分的影响。方法用高效液相色谱法测定480例北京居民血清FG，用酶法测定总甘油(1TrG)，分析FG与TG、年龄和性别的关系及FG对TG水平划分的影响。结果480例北京居民血清FG平均值0．082mmol／L，中位数0．075mmol／L；FG／TG平均值8．0％，中位数6．4％；FG和FG／TG女性明显高于男性；TG和TTG高于目前医学决定水平1．69mmol/L(150mg／d1)的个体分别占22％和25％。结论按新的方案对血清TG水平划分，临床TG测定更需具备去除游离甘油的能力。     

The beta-glucuronidase activity in the blood cells and serum of patients with systemic lupus erythematosus]

Beta-glucuronidase (beta-GU) activity and protein concentration were measured in peripheral blood cells (neutrophils and mononuclear cells) and blood serum of patients with systemic lupus erythematosus (SLE). SLE patients were found to tend towards decrease of beta-GU activity in neutrophils and to an appreciable increase thereof (2-fold) in blood serum. The concentration of protein in both neutrophils and mononuclear cells was within normal. Substantial changes in beta-GU activity were seen in SLE patients with trophic disorders (a significant decrease of the enzyme in neutrophils and a rise in blood serum). These patients also manifested a noticeable reduction of protein concentration in mononuclear cells. The measurement of beta-GU activity in blood cells can be used for the choice of adequate therapy in the course of the treatment of SLE patients.     

血清胸苷激酶的检测以及其在前列腺癌早期筛查中的意义

目的前列腺癌是一种老年男性泌尿生殖系统常见的恶性肿瘤,近年其发病率呈上升趋势,已高居泌尿生殖系统肿瘤的前3位,且发病年龄也日趋年轻化。长期以来,人们对前列腺癌的检测手段一直停留在直肠指诊、PSA筛查和穿刺活检上。能否早期诊断、早期治疗前列腺癌在临床上就显得尤为重要。本研究旨在探讨通过检测血清胸苷激酶1（TK）在早期诊断前列腺癌中的可行性和有效性。方法采用免疫印迹-增强化学发光检测法,检测在本院就诊的150例男性患者血清胸苷激酶1水平,其中70例为前列腺癌患者（年龄29～80岁）,40例为前列腺增生患者（年龄50～92岁）,另40例为来本院作健康体检的健康男性（年龄35～50岁）。结果1）前列腺癌组：70例经过病理活检确诊的前列腺癌患者,STK1值（3．66&#177;1．91）pM／L,灵敏度71．5％。2）前列腺增生组：40例同期治疗的病理检查确诊的前列腺增生患者（1．28&#177;0．35）pM／L,特异度85％。3）健康对照组：40例健康人检查结果均无泌尿系统疾病,STK1值（0．79&#177;0．27）pM／L,无一例异常结果。经血清胸苷激酶1检测发现,前列腺癌组与健康男性组间TK1水平差异有统计学意义（P〈0．05）。前列腺癌组与前列腺增生组血清胸苷激酶1水平差异有统计学意义（P〈0．05）。血清胸苷激酶1对前列腺肿瘤TMN分期,T135例,T219例,L10例,T46例的灵敏度分别为：62．85％（22／35）、73．68％（14／19）、80．00％（8／10）、100．00％（6／6）。结论使用血清胸苷激酶检测前列腺癌中有显著的特异性,可作为前列腺癌的早期筛查方法。