Suitability of commercial control sera for the quality control of activity determination of alkaline phosphatase.

The suitability of thirteen commercially available control sera for measuring alkaline phosphatase (EC 3.1.3.1; orthophosphoric acid monoester phosphohydrolase, ALP) activity in human serum was tested. Apart from differences in ALP activity observed in some reconstituted commercial sera, the behaviour of control materials towards experimental variables such as the nature and concentration of the substrate, pH and type of buffer (or PO4-acceptor) together with the composition of the isoenzymes present in human serum highlights the problems and difficulties if commercial materials are to be used as control sera. The half-saturation constants in control sera were in all cases smaller than those of ALP isoenzymes from bone and liver. The shape of substrate activity curves and the pH optimum in most of control sera differed from that of human serum. The discrepant kinetic data of control materials and human serum may mask or suggest changes relevant to commercial quality control serum but not to samples of human serum.     

Simple, rapid determination of zinc and acid phosphatase in seminal plasma with an ABA-100 bichromatic analyzer

I describe an automated assay for zinc and acid phosphatase in seminal plasma. These, which are markers of the function of the prostate, were assayed bichromatically with an Abbott ABA-100 analyzer. As many as 25 samples of human seminal plasma can be analyzed sequentially with CVs of 3.1% for zinc and 1.5% for acid phosphatase. The sensitivity, specificity, and speed of this assay system make it practicable for use in investigation of male infertility.     

Investigations on acid phosphatase activity in human plasma and serum

Determination of acid p-nitrophenylphosphatase activity in serum or heparinized plasma gives a wrong picture of the real activity of this enzyme in human plasma. In comparison with the activity in acid-citrate-dextrose (ACD)-treated plasma, activities in serum are too high and in heparinized plasma too low.A possible explanation for the lower activities obtained in heparinized plasma is the globulin-precipitating effect of heparin under the conditions of the assay.The difference in activity of serum and ACD plasma can be attributed to acid p-nitrophenylphosphatase(s) liberated from thrombocytes during the clotting of blood.The acid p-nitrophenylphosphatase from thrombocytes, liberated during the clotting process, is completely inhibited by formaldehyde, considerably inhibited by l-tartrate and to a lesser extent by fluoride. The thrombocytic acid phosphatase also hydrolyses α-naphthyl phosphate.It is concluded, that the most reliable determination of total acid p-nitrophenylphosphatas activity as well as of “prostatic” acid p-nitrophenylphosphatase activity (tartrate-inhibited fraction) is obtained with ACD plasma.     

Some pitfalls in the quality control of alkaline phosphatase activity.

A study of the pH optimum of alkaline phosphatase activity in calibration and control serum preparations reveals a wide variation in pH optima. It is shown that as small an error of +/- 0.05 pH units in preparation of the buffered substrate results in an error in the order of +/- 5% in the response between the calibrating serum and patient specimens. It is recommended that a calibration serum should closely parallel the pH optimum of alkaline phosphatase in human sera and that a quality control serum containing alkaline phosphatase of alternative source to that present in the calibration serum be included with patient samples as a sensitive means of monitoring pH changes in the buffered substrate.     

An automated continuous-monitoring procedure for the determination of acid phosphatase activity in serum.

The method of Hillmann, in which hydrolysis of alpha-naphthyl phosphate by acid phosphatase is coupled to the formation of an alpha-naphthol-Fast Red TR azo-compound, has been adapted for use with the LKB Produkter AB 8600 reaction rate analyzer. Factors which affect the reproducibility of the method are described and its performance is shown to be superior to that of a manual phenyl-phosphate procedure.     

精浆柠檬酸水平与精液质量的关系

目的探讨精浆柠檬酸水平与精液质量间的关系。方法选取2020年3月至2021年3月于该院男性科门诊就诊的124例男性为研究对象,根据精浆柠檬酸水平将研究对象分为降低组(精浆柠檬酸水平降低,64例)和正常对照组(精浆柠檬酸水平正常,60例)。检测并比较两组精液量、精液pH值、精子浓度、精子总数、前向运动精子率(PR)、精子总活力及精浆柠檬酸水平,并分析精浆柠檬酸水平与精液质量相关指标间的相关性。结果与正常对照组相比,降低组精液量、精子总数、PR、精子总活力及精浆柠檬酸水平降低,精液pH值升高,差异有统计学意义(P<0.05)。相关性分析结果显示,精浆柠檬酸水平与精液量、精子总数、PR及精子总活力呈正相关(r=0.510、0.358、0.744、0.678,P<0.05);与精液pH值呈负相关(r=-0.379,P<0.05)。结论精浆柠檬酸水平与男性精液质量具有一定相关性,可作为男性生育能力评估的检测项目。     

不育患者精浆酸性磷酸酶和前列腺特异抗原含量与精子质量的关系

目的探讨不育患者精浆酸性磷酸酶（ACP）和前列腺特异抗原（PSA）含量与精子质量的关系。方法随机选择男性不育症患者122例,采用精子分析仪测定精液参数,同时用酶联免疫吸附试验（ELISA）法检测精浆中PSA水平,用磷酸苯二钠法检测精浆中ACP含量,并分析其与精子密度、活力和黏度的关系。结果精子活力异常组精浆ACP、PSA含量明显低于活力正常组,两组比较差异有统计学意义（P〈0．05）;精液黏度增高组精浆ACP、PSA含量明显低于精液黏度正常组,两组比较差异有统计学意义（P〈0．05）。精子密度正常组ACP高于密度减低组（P〈0．05）,PSA含量两组无明显差异。结论精浆ACP、PSA含量与精子质量有密切关系。     

An optimized continuous-monitoring procedure for semiautomated determination of serum acid phosphatase activity.

A continuous-monitoring method for measuring acid phosphatase activity with alpha-naphthyl phosphate as the substrate was critically evaluated and modified. Using partially purified prostatic acid phosphatase, we show that certain conditions for the assay must be satisfied to ensure linearity. These conditions include maintaining the pH between 5.6 and 5.9 and the addition of detergent to sustain linearity. The results obtained with alpha-naphthyl phosphate have been compared with those obtained by using p-nitrophenyl phosphate as substrate. When used with an automatic rate analyzer, the modified method is as sensitive but more reproducible.     

EB病毒核酸检测质控品制备及其应用

目的制备可模拟真实临床标本的EB病毒(EBV)核酸检测质控品用于上海地区临床实验室室间质评,以评估其EBV DNA的检测能力。方法选择整合有EBV基因组的淋巴细胞(NAMALWA)进行培养,收集细胞后观察细胞原液对国内常用商品化试剂盒的适用性,随后用EBV国际标准品对其定值,经血浆稀释制得含有阴、阳性共5份样本的样本盘,随机编码后发送至参加EBV DNA室间质评的临床实验室,并对回报结果进行分析。结果采用不同公司EBV PCR检测试剂盒检测NAMALWA细胞原液,结果全部阳性。EBV国际标准品对NAMALWA细胞原液定值浓度约为2.13×105IU/m L,部分参评实验室出现假阳性和假阴性问题,高浓度样本(105和104IU/ml)的符合率均为100%,低浓度样本(103和102IU/m L)的符合率分别为92%和40%,阴性样本的符合率为90%。重复样本实验室内检测结果的平均变异系数(CV)为4.1%(1.4%~11.3%),实验室间CV为6%;大多数实验室(75%)的检测结果与预期值呈良好的线性相关。结论成功制备可模拟临床标本的EBV PCR检测质控品,并证实其可有效用于临床实验室室间质评。质评结果显示上海地区参评实验室EBV DNA检测质量整体较好,但个别实验室检测能力尚需提高。     

Xylosyltransferase activity in seminal plasma of infertile men

BACKGROUND: Proteoglycans play an important role during the mammalian conception process and are functionally involved in the preservation of the sperm motility and velocity. Human xylosyltransferase (EC 2.4.2.26, XT) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan chains in proteoglycans. XT activity in body fluids was shown to be an indicator for the actual proteoglycan biosynthesis rate. METHODS: Xylosyltransferase activity was determined in seminal plasma and serum samples of 50 healthy semen donors, 20 infertile men with oligo-, astheno- or teratozoospermia and men after vasectomy. For identification of the XT secreting glands split ejaculates were analyzed. RESULTS: XT activity in seminal plasma samples of infertile men was significantly reduced (mean 1.53 mU/l, 90% range 0.95-2.52 mU/l, p<0.05) in comparison to healthy donors (mean 3.21 mU/l, 90% range 1.82-4.65 mU/l). The analysis of the fractionated ejaculates revealed that the highest XT activity was found in portions secreted by the seminal vesicle glands. CONCLUSIONS: In view of the increasing interest for in vitro fertilization, monitoring the seminal plasma XT activity of men with unexplained infertility is proposed to be an advantageous additional biochemical parameter that could be useful for improving the methods producing pregnancy in some couples.     

不育患者精浆中性α-1,4-糖苷酶活性与精液参数及精子透明质酸酶活性的关系

目的探讨精浆中性α-1,4-糖苷酶(α-1,4-G)活性与精液参数及透明质酸酶(HYD)活性(HYD阳性反应率及HYD活性强度)之间的关系。方法收集52例24~40岁不育患者及12名正常对照者(正常生育组)精液。采用以精子质量分析仪对每份标本进行临床精液常规检查及计算机辅助精液分析;运用酶法测定精浆中性α-1,4-G活性;采用改良固定底物膜法分别检测精子顶体内HYD阳性反应率及HYD活性强度。根据中性α-1,4-G活性检测结果将不育患者分为中性α-1,4-G活性正常组(简称酶活性正常组,39例)和中性α-1,4-G活性异常组(简称酶活性异常组,13例)。结果精浆中性α-1,4-G活性与精子密度、精子活动率、HYD活性(HYD阳性反应率、HYD活性强度)呈正相关(r值分别为0.816、0.890、0.872和0.847,P均0.01);与畸形精子率呈负相关(r=-0.579,P0.01)。不育各组中精子密度、精子活动率和HYD活性(HYD阳性反应率及HYD活性强度)均明显低于正常生育组(P0.01);酶活性异常组的畸形精子率高于酶活性正常组(P0.01)。结论精浆中性α-1,4-G活性对精子密度、精子活动率、畸形精子率和HYD活性(HYD阳性反应率、HYD活性强度)均有明显影响。     

Development of quality control parameters for the standardization of gymnema sylvestre

ObjectiveTo develop a novel qualitative and quantitative technique, which can pave the way for rapid and selective determination of different phytoconstituents of Gymnema sylvestre (G. sylvestre).MethodsPhytochemical test, TLC analysis, solubility study, total phenol and flavonoid content and antimicrobial study were performed in the present investigation. Fingerprint analysis and quantitative analysis of quercetin were also performed through hptlc method.ResultsAlkaloid, saponin, tannin, triterpenoid and flavonoid were found to be present in the G. sylvestre extract. Solubility in water and alcohal, moisture content and gymnemic acid content were found to be 86.36%, 88.24%, 4.20%, and 26.24% w/w. total phenol and flavonoid content were found to be 0.80% and 1.90%. Microbiological assay showed that E. coli and salmonella were found to be absent whereas total bacterial count and yeast & moulds contents were found to be 650 and 60 cfu/g. quantitative analysis through hptlc revealed the presence of 2.95% w/w of quercetin.ConclusionsIn future this study will be helpful for the quantitative analysis of phytoconstituents as well as standardization of the G. sylvestre.     

精浆天门冬氨酸氨基转移酶活性检测及方法学评价

目的建立精浆天门冬氨酸氨基转移酶(AST)活性的检测方法及方法学评价。方法根据检测血清AST活性的方法建立检测精浆AST活性的方法。参考美国临床实验室标准化委员会(NCCLS)的EP5-A和EP6-A文件,观察其精密度、线性检测范围及不同技术人员检测结果之间的差异以评价方法的可接受性。结果低水平样本批内变异系数(CV)为1.93%,批间CV为2.77%,天间CV为3.45%,总CV为4.84%;高水平样本批内CV为0.48%,批间CV为0.53%,天间CV为0.53%,总CV为0.89%。线性检测范围达1 652 U/L。2位技术人员的检测结果差异无统计学意义(P=0.423)。结论通过血清AST检测方法可以建立可接受的精浆AST活性检测方法。     

Placental alkaline phosphatase isoenzyme in quality control materials may be a source of variability in alkaline phosphatase activity

ObjectivesTo determine the cause of discrepancies in the alkaline phosphatase (ALP) activities of quality control (QC) materials in two different analyzers using IFCC method.Design and methodsALP activities of patients' samples and QC materials (QC1 and QC2 from Bio-Rad) measured using TBA-200FR and Synchron LX-20 analyzers were compared and isoenzyme electrophoresis was done. Fractional mixing of bone or liver ALP with placental ALP was performed. ALP activities of QC materials were measured in TBA-200FR with pH-modified reagents.ResultsALP activities of QC materials were significantly lower in TBA-200FR than in LX-20. Placental ALP comprised 57% of QC1 and 95% of QC2. Higher placental ALP proportion in the mixture with bone or liver ALP resulted in lower ALP activities in TBA-200FR. The discrepancy in ALPs of QC materials decreased when measured with pH-modified reagents.ConclusionPlacental ALP in QC materials and differences in reagents' pH caused the discrepancy in ALP activities.     

Interference with the kinetic determination of acid phosphatase

This study advocates cautious use of the kinetic determination method for acid phosphatase with α-naphthylphosphate as substrate and a diazonium salt for colour development. The occurrence of considerable blank rates due to the spontaneous hydrolysis of the substrate was noticed. Of the diazonium salts which were tested as alternatives for Fast Red TR, diazotised p-nitroaniline seemed to be useful. Also the interaction between plasma components and diazonium salts may influence the results of the determination. In this respect a number of possible interfering compounds was investigated and from these a structural element which may lead to side reactions was deduced.     

The effect of pH and temperature on the stability and enzymatic activity of prostatic acid phosphatase. Studies on the optimization of a continuous monitored determination of acid phosphatase, II

The catalytic activity and the stability of prostatic acid phosphatase were studied with respect to pH and temperature: 1. Enzymatic activity in serum decreases with time, and the rate of decrease depends on pH and temperature. Half life times were estimated. 2. To preserve at least 90% of its original activity, serum must be cooled as soon as possible below room temperature and/or the pH must be lowered to 6. 3. Considering the effect of pH on side reactions and kinetic parameters, a pH of 5.2 is recommended for the assay. 4. Between 25 and 37 degrees C, the value for Km app, in the absence of alcohols, is constant within the limits of error. In the presence of alcohols there is a significant increase of Km app at lower temperatures, and higher substrate concentrations are needed to avoid nonsaturation of the enzyme. vmax increases with temperature. Inactivation is observed above 45 degrees C, especially in the presenc of alcohols. 5. The Arrhenius plot shows a strict linear regression between 20 degrees C and 40 degrees C, in the presence or absence of 1,4-butanediol, 1,5-pentanediol or 1,6-hexanediol. 6. Temperature conversion factors for catalytic activity were calculated to be: 1.33 (25 to 30 degrees C), 1.96 (25 to 37 degrees C) and 1.47 (30 to 37 degrees C).