Stability test of six enzymes for internal quality control

The daily quality control for the determination of the catalytic activity concentrations of enzymes is an important aspect in clinical chemistry. Instead of the expensive, commercially available control sera, we have looked for a simple, reliable and cheap method for the quality control of enzyme determinations. Commercially available enzymes were suspended in an albumin solution and ampoules were filled with 1.0 ml of these various solutions. The ampoules were stored at 4 degrees C or -20 degrees C. Once a week, during 10 months, catalytic activities of these enzyme-albumin solutions were determined together with the same activities in freshly reconstituted control sera. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase and gamma-glutamyltransferase were determined at 30 degrees C according to well-described methods. alpha-Amylase was determined with the Phadebas method at 37 degrees C. Except for creatine kinase, the stability and reliability of these enzyme solutions are fully comparable with control sera during the experimental period. The catalytic activity concentration of creatine kinase decreased slowly during the 10 months. The enzyme solutions react in the same manner as commercial test sera on changes in the reaction conditions for the enzyme determinations. The conclusion seems justified that these enzyme solutions can be used for the daily quality control of the enzyme determinations instead of control sera.     

Presence and comparison of angiotensin converting enzyme in commercial cell culture sera

This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes were able to hydrolyze, with different efficiency, angiotensin I, bradykinin and epidermal mitosis inhibiting pentapeptide. The heat inactivation of commercial sera at 56 degrees C for 30 min showed a reduction of ACE activity of about 35-80%. Therefore, the presence of ACE activity in commercial sera can influence the activity of biological peptides tested on cell lines cultured "in vitro."     

Comparative evaluation of three recombinant antigen-based enzyme immunoassays for detection of IgM and IgG antibodies to human parvovirus B19

Background: Diagnosis of acute and past infection with parvovirus B19 is based on detection of IgM and IgG antibodies.Objectives: To evaluate two commercial recombinant antigen-based enzyme immunoassay (EIA) test kits for detection of IgM and IgG antibodies to parvovirus B19 and to compare the commercial EIAs to in-house EIA test procedures.Study design: A panel of 121 sera was used to compare the three IgM EIAs. The panel included 84 sera submitted for parvovirus B19 testing and 37 sera that were IgM positive for other viral pathogens. The same serum panel plus an additional 14 sera submitted for B19 testing was used to compare the three IgG EIAs. The commercial EIAs were performed according to manufacturers' instructions. Using the in-house EIA test procedures as the reference, sensitivity and specificity for each of the commercial EIAs was determined.Results: The commercial B19 IgM EIAs showed agreements of 95.0 and 93.4% to the in-house IgM EIA. Compared to the in-house B19 IgM EIA, the commercial B19 IgM EIAs, were 97.4 and 97.5% sensitive, respectively. Specificities were 93.5 and 91.4%, respectively. Sensitivities for the commercial IgG EIAs, compared to in-house IgG EIA, were 88.0 and 85.2%, respectively, and specificities were 94.1 and 98.0%.Conclusion: We found that the commercial parvovirus B19 IgM and IgG EIAs are comparable to standard in-house EIAs and are suitable for testing for B19 antibodies in human sera.     

Traceability in clinical enzymology

The primary goal of standardisation for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the assay is carried out. In order to pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has established reference systems for the most important clinical enzymes. These systems are based on three requirements: a) reference measurement procedures that are extensively evaluated and carefully described; b) certified reference materials; and c) a network of reference laboratories operating in a highly controlled manner. Using these reference systems and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Clinical laboratories, which use routine procedures with validated calibrators to measure human specimens, can finally obtain values which are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e. they are measuring the same quantity).     

Purine pathway enzymes in a cyst forming strain of Toxoplasma gondii

The activities of purine salvage enzymes in tachyzoites from a cyst-forming strain of Toxoplasma gondii were determined using HPLC. Six enzymes were assayed both in vitro and in vivo: adenosine deaminase, guanine deaminase, purine nucleoside phosphorylase, xanthine oxidase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. In vitro, the tachyzoites were cultured in the human myelomonocytic cell line THP-1, for 24 h to 96 h. Neither guanine deaminase nor hypoxanthine-guanine phosphoribosyltransferase activity was detected in 24 and 96 h cultures. In vivo, in controls and infected animals, the purine nucleoside phosphorylase and adenosine deaminase activities were the most important activities both in sera and cerebral tissue in comparison with the other activities. It was also noted that the infection modified the enzymatic activities of this purine salvage pathway, in particular, the guanine deaminase cerebral activity of infected mice was 20-fold lower than the value of controls. The treatment of mice with 2',3'-dideoxyinosine, a purine analog, at the dose of 100 mg.kg(-1).d for 30 days, induced an important increase of all enzymatic activities in the brains in comparison with control animals. These data suggest that one target of 2',3'-dideoxyinosine is the purine metabolism.     

Modified Hemagglutination-Inhibition Test for Rubella Employing Human Group O Erythrocytes

A hemagglutination-inhibition (HI) test for rubella is described which utilizes human group O, rather than 1-day-old chick, erythrocytes. The test was found to be as sensitive and reproducible for detection of rubella antibody as HI tests employing chick erythrocytes. Advantages to the use of human erythrocytes are (i) they are more available, (ii) it is unnecessary to absorb natural agglutinins from human test sera, and (iii) heparin-MnCl₂-treated sera do not agglutinate human erythrocytes, as is sometimes the case with chick erythrocytes. Factors influencing the reliability of the test are discussed.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.     

Deoxyribonuclease-Inhibitory antibodies in systemic lupus erythematosus

Deoxyribonucleases (DNases) are key enzymes for digesting DNA. Abnormalities in the function of these enzymes may contribute to the development of anti-DNA antibodies in systemic lupus erythematosus (SLE). In this study, we used bovine DNase 1-coated ELISA plates to screen anti-DNase antibodies in SLE patients. About 62% of the sera of SLE patients (63/101) were positive for anti-DNase antibodies compared to only 8% of normal controls (8/98). A positive correlation was also found between the concentrations of anti-DNase and anti-DNA antibodies in sera of SLE patients. Affinity-purified anti-DNase immunoglobulin G (IgG) from pooled sera of SLE patients bound to bovine DNase as well as DNA. A synthetic peptide, corresponding to the catalytic site of DNase, was able to completely inhibit the binding of anti-DNase IgG to DNase. In addition to bovine DNase, the anti-DNase IgG also bound to and inhibited the enzymatic activities of DNase present in streptococcal supernatants and human urine. Immunization of lupus-prone NZB/NZW mice with bovine DNase enhanced the production of anti-DNase and DNA antibodies, and accelerated the occurrence of proteinuria. Taken together, these results suggest that DNase-inhibitory antibodies which recognize a conserved epitope near the catalytic site of DNase may act in the pathogenesis of SLE.     

Lysosomal enzymes in inflammatory synovial effusions.

The concentrations of several polymorphonuclear neutrophilic lysosomal constituents were quantitated by immunochemical and enzymatic assays in 28 inflammatory and 9 noninflammatory synovial fluids. The quantities of lactoferrin, myeloperoxidase, and enzymatically determined lysozyme were covariate with the neutrophil count. Enzymatic activities measured with synthetic substrates developed for the assay of chymotryptic-like cationic protein (cathepsin G) and elastase, along with immunochemically determined lysozyme, were independent of the neutrophil count. Although the latter assays were developed and standardized with human neutrophilic lysosomal constituents, they measure different activities in inflammatory synovial effusions. No elastase was detected if elastin was used as the substrate. Regardless of the source of the enzymes, there was a negative correlation between their concentration and the degree of radiographic destruction of the joint from which the fluid was obtained. Lysosomal enzymes in solution in synovial fluid are not likely to be primarily involved in cartilage destruction.     

Cholinesterase unit establishment and issuing of >Warning Cards< for carriers of suxamethonium sensitive serum butyrylcholinesterase variants

Recognition of butyrylcholinesterase (EC 3.1.1.8) variants in human serum is essential to identify patients who may be susceptible to a prolonged reaction of suxamethonium and mivacurium, short-acting muscle relaxants. Thus they can be given appropriate advice along with their relatives who may be similarly affected. Therefore, Cholinesterase Unit for detection of individuals, carriers of inherited suxamethonium sensitive butyrylcholinesterase variants was established at the Institute for Clinical Chemistry of the Clinical Hospital >Merkur<, Zagreb, Croatia. A study was conducted on sera from patients referred to the Unit. Butyrylcholinesterase variants were determined by measuring the enzyme activity and inhibition by specific inhibitors in the sera of 384 patients and of the members of seven families. Cholinesterase Unit issued >Warning Cards< to the carriers of inherited serum butyrylcholinesterase variants in order to avoid prolonged apnea that suxamethonium might cause.     

Micromethods in single muscle fibers. 1. Determination of catalase and superoxide dismutase

Methods have been developed for the measurements of catalase and superoxide dismutase (SOD) in single, isolated muscle fibers. These fibers are also classified according to fiber type. Catalase is determined using a fluorescent method for the measurement of hydrogen peroxide consumed. SOD measurements are carried out using a modification of established techniques whereby the inhibition of oxidation of epinephrine by SOD is assayed fluorometrically. Both enzymes may be determined in submicrogram samples of dried muscle. This approach avoids the complication of the inclusion of nonmuscle tissue with varying enzymatic activities which is frequently experienced when using homogenates of muscle, particularly diseased muscle. In addition, these techniques can be used to determine the inherent variation in SOD and catalase activities within individual fibers of the same fiber type. The Km and Vmax for catalase, determined using homogenates of human muscle, were found to be 12 mM and 1.45 mumol/min/mg dry wt, respectively. Catalase of muscle was inhibited 50% by 2 microM sodium azide. Mn-SOD contributes less than one-fifth of the total SOD activity. Therefore the activity is largely due to the Cu-Zn form of SOD. These methods are applicable to a wide variety of tissues.     

Microplate-based filter paper assay to measure total cellulase activity

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.     

Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates

Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.     

2020 ESC Guidelines on acute coronary syndrome without ST-segment elevation: Recommendations and critical appraisal from the Dutch ACS and Interventional Cardiology working groups

Recently, the European Society of Cardiology (ESC) has updated its guidelines for the management of patients with acute coronary syndrome (ACS) without ST-segment elevation. The current consensus document of the Dutch ACS working group and the Working Group of Interventional Cardiology of the Netherlands Society of Cardiology aims to put the 2020 ESC Guidelines into the Dutch perspective and to provide practical recommendations for Dutch cardiologists, focusing on antiplatelet therapy, risk assessment and criteria for invasive strategy.     

药物代谢酶特异性探针底物的研发与应用进展

人体内分布数百种结构多样、功能各异的药物代谢酶,其表达和功能易受遗传和环境因素的影响,导致其在不同人种、个体及组织 中的分布都不尽相同。特异性探针底物作为药物代谢酶活性表征的关键工具分子,其在新药发现、药-药相互作用评价、临床药理学及精准 医学检测等领域具有重要的应用价值。围绕人体分布的重要药物代谢酶,综述其特异性探针底物的设计研发及生物医学应用进展,以期为 药物代谢酶的精准检测及相关应用研究提供参考。     

The enzymatic activities of GTP cyclohydrolase, sepiapterin reductase, dihydropteridine reductase and dihydrofolate reductase; and tetrahydrobiopterin content in mammalian ocular tissues and in human senile cataracts

The enzymatic activities of GTP cyclohydrolase, sepiapterin reductase, dihydropterin reductase and dihydrofolate reductase were determined in the ocular tissues of rat, rabbit, calf and human. The enzymatic activities of the pteridine biosynthesis and the content of tetrahydropteridine (BH4) were higher in retina and ciliary body-iris as compared with lens tissue in all mammalian species tested. The activities of the pteridine synthesizing enzymes and BH4 content were decreased in human senile cataracts as compared with age-matched clear human lenses. The loss of BH4 may result in lenticular proteins more susceptible to oxidation and contribute to high molecular weight protein formation in cataracts.     

Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.     

Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass

Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.     

Antioxidant activity levels and oxidative stress as blood markers of allergic response to drugs.

Antioxidant enzymes work together in human blood cells against toxic reactive oxygen species. Although their relationship with several pathophysiologic processes has been stated, not much is known about the connection between antioxidant defence and allergy. This study was designed to determine the enzymatic activities and the oxidative indices in the blood and serum proteins in patients suffering from allergy to drugs. We hypothesize that serum and blood reactions may serve as useful clinical marker for the allergic state. We used enzymatic antioxidant activities, thiobarbituric acid reactive substances, and carbonyl contents of proteins as suitable markers. We determined superoxide dismutases, glutathione peroxidase and catalase activities in each cell type. After antihistaminics plus steroids were given as part of a protocol treatment, enzymatic antioxidant activities, thiobarbituric acid reactive substance levels, and carbonyl contents were used as recovering markers for the disease. We found a relationship between antioxidant enzymatic activities, thiobarbituric acid reactive substance levels, and carbonyl contents for allergic reactions belonging to several type I and type IV allergies, as well as cross-reactive intolerance to nonsteroidal anti-inflammatory drugs and an anaphylactoid reaction to a radiocontrast media. A similar pattern also exists for analogous allergic manifestations and disease-like status.     

A new test for chorismate mutase activity.

Chorismate mutase (EC 5.4.99.5) catalyzes the conversion of chorismic acid to prephenic acid. A continuous test of the enzymatic activity is based on the decrease of the absorption of the substrate chorismate at 274 nm (1). In a sensitive but discontinuous test, prephenic acid, the product of the enzymatic reaction is converted to phenylpyruvic acid. The absorbance of its enolic form is determined in alkaline solution at 320 nm (2,3). Another discontinuous test makes use of the absorption of the phenylpyruvate enol-borate complex (4,5) at 300 nm. The continuous test cannot be used when aromatic compounds are to be tested as modifiers of the enzymatic activity. Similarly, the tests based on the absorption of the enolic form of phenylpyruvic acid cannot be used when compounds which show a high absorbance in the 300 to 320 nm wavelength region are tested. This paper describes a test for chrismate mutase based on the determination of the 2,4-dinitrophenylhydrazone of the α-keto acid, phenylpyruvic acid. In alkaline solution the 2,4-dinitrophenylhydrazone of phenylpyruvic acid shows an absorption maximum at 440 nm, thus allowing one to test compounds like 3-hydroxybenzoic acid and 5-hydroxyisophthalic acid as potential inhibitors of the enzymatic reaction.