Turbidimetric measurement of lipase activity--problems and some solutions.

1987 ◽  
Vol 33 (9) ◽  
pp. 1624-1629 ◽  
Author(s):  
N W Tietz ◽  
D F Shuey ◽  
J R Astles
Keyword(s):  
Lipase Activity ◽  
Reaction Rate ◽  
Enzyme Activation ◽  
Lag Phase ◽  
Analytical Sensitivity ◽  
Healthy Individuals ◽  
Turbidimetric Method ◽  
Turbidimetric Measurement ◽  
Ph Stat ◽  
Carboxyl Esterase

Abstract We optimized a commercial turbidimetric method for lipase (EC 3.1.1.3) activity (Boehringer Mannheim Diagnostics) and overcame some of its deficiencies. Increasing the bile salt concentration to 35 mmol/L and the colipase concentration to 6 mg/L and using a continuous recording of the reaction-rate curve greatly improved the reaction kinetics, eliminated false results from increases in absorbance, reduced the lag phase, and increased the analytical sensitivity and accuracy. Differentiation of pancreatitis from nonpancreatitis sera by adding NaCl, 140 mmol/L, to the assay mixture to observe the degree of enzyme activation has important limitations. Sera from patients with pancreatitis and only slight or modest increases in lipase behave like sera from healthy individuals or from patients with nonpancreatic disease. The assay shows no interference by lipoprotein lipase and carboxyl esterase. Results compare well by this optimized method and by an optimized "pH-Stat" titrimetric method.

Evaluation of 3,4-Dinitrophenyl Tetra-N-Acetyl-β-Chitotetraoside as a Substrate for the Measurement of Lysozyme in Normal and Pathological Sera

1979 ◽  
Vol 16 (1-6) ◽  
pp. 51-53 ◽  
Author(s):  
G. A. Turner ◽  
H. Ghneim ◽  
J. Freeman
Keyword(s):  
Reaction Rate ◽  
Colorimetric Method ◽  
Lysozyme Activity ◽  
Group Differences ◽  
Healthy Individuals ◽  
Serum Samples ◽  
Turbidimetric Method ◽  
Egg White Lysozyme ◽  
Inflammatory Bowel ◽  
Assay Precision

Lysozyme was measured using the synthetic substrate 3′,4-dinitrophenyl tetra- N-acetyl-β-chitotetraoside and the LKB Reaction Rate Analyser. This method has been evaluated by comparing levels obtained with serum samples from healthy individuals and patients with either cancer or inflammatory bowel disease with those obtained from the same specimens using a turbidimetric method. In terms of standard egg-white lysozyme, the colorimetric method gave much higher levels for all samples than the turbidimetric method; however, similar group differences were maintained. For individual serum specimens significant correlation between the two methods was found to occur only in the healthy group. Assay precision for the two methods was similar but the turbidimetric method could detect levels of lysozyme activity which were 10 times lower than those detected by the colorimetric method.

Heat Inactivation of Bile Salt-Stimulated Lipase Activity in Human Milk and Colostrum1

1983 ◽  
Vol 46 (6) ◽  
pp. 525-527
Author(s):  
S. H. L. PAN ◽  
C. W. DILL ◽  
E. S. ALFORD ◽  
R. L. RICHTER ◽  
C. GARZA
Keyword(s):  
Human Milk ◽  
Bile Salt ◽  
Lipase Activity ◽  
Bovine Milk ◽  
Heat Inactivation ◽  
Assay Procedure ◽  
Ph Stat

Time-temperature relationships for heat-inactivation of bile salt-stimulated lipase activity in human milk and colostrum were systematically measured using a pH-stat assay procedure with triolein as substrate. The enzyme was not affected in either menstruum at 45°C for 40 min. The enzyme was destroyed almost instantaneously at 60°C, and was slightly more heat-sensitive in colostrum than in milk. The bile salt-stimulated lipase(s) in human milk was more heat sensitive than lipase in bovine milk.

scholarly journals Quantitative Profiling of Platelet-Rich Plasma Indole Markers by Direct-Matrix Derivatization Combined with LC-MS/MS in Patients with Neuroendocrine Tumors

2019 ◽  
Vol 65 (11) ◽  
pp. 1388-1396 ◽  
Author(s):  
Martijn van Faassen ◽  
Grytsje Bouma ◽  
Lotte D de Hosson ◽  
Marloes A M Peters ◽  
Gursah Kats-Ugurlu ◽  
...  
Keyword(s):  
Neuroendocrine Tumors ◽  
Biochemical Characterization ◽  
Solid Phase ◽  
Platelet Rich Plasma ◽  
Chemical Properties ◽  
Analytical Sensitivity ◽  
Healthy Individuals ◽  
Limit Of Quantification ◽  
Hydroxyindoleacetic Acid

Abstract BACKGROUND Currently, several indole markers are measured separately to support diagnosis and follow-up of patients with neuroendocrine tumors (NETs). We have developed a sensitive mass spectrometry method that simultaneously quantifies all relevant tryptophan-related indoles (tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid) in platelet-rich plasma. Direct-matrix derivatization was used to make the chemical properties of the indoles uniform and to improve the analytical sensitivity and specificity of the assay. METHODS In situ derivatization was performed directly in platelet-rich plasma with propionic anhydride at an ambient temperature. The derivatized indoles were extracted by online solid-phase extraction and eluted to the analytical column for separation followed by mass spectrometric detection. The method was validated according to international guidelines. Platelet-rich plasma samples from 68 healthy individuals and 40 NET patients were analyzed for tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. RESULTS The method reproducibly quantified relevant indoles in 8.5 min, including online sample cleanup. Intra- and interassay imprecision, evaluated at 3 different concentrations, ranged from 2.0% to 12% and 1.9% to 13%, respectively. The limit of quantification was sufficient to measure endogenous concentrations of all 4 indoles. Healthy individuals and NET patients had different concentrations of 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid, but tryptophan concentrations were the same. CONCLUSIONS Direct-matrix derivatization in combination with LC-MS/MS is a powerful tool for the simultaneous quantification of all tryptophan-related indoles in platelet-rich plasma. Simultaneous profiling of relevant indoles improves the biochemical characterization and follow-up of NETs.

scholarly journals Adenosine phosphates and the control of glycolysis and gluconeogenesis in yeast

1969 ◽  
Vol 111 (5) ◽  
pp. 609-613 ◽  
Author(s):  
C. Chapman ◽  
W Bartley
Keyword(s):  
Dry Weight ◽  
Enzyme Activation ◽  
Glycolytic Flux ◽  
Lag Phase ◽  
Glycolytic Metabolism ◽  
Alternative Control ◽  
Weight Protein ◽  
Adenosine Phosphates ◽  
Cellular Components ◽  
Rna And Dna

1. Changes in dry weight, protein, RNA and DNA were measured in yeast during adaptation to glycolytic metabolism. 2. Only RNA increased significantly during the lag phase, but during the exponential phase all these cellular components increased in parallel. 3. The concentrations of ATP, ADP, AMP and glucose 6-phosphate were measured in respiring yeast and during the transition to glycolytic metabolism. 4. In respiring cells the concentration of AMP was at its highest and that of ATP was at its lowest; this relationship was reversed in glycolysing cells. 5. ADP concentration was similar in respiring and glycolysing cells, but glucose 6-phosphate concentration was much higher in the glycolysing cells. 6. A possible reason for mitochondrial repression is suggested. 7. It is concluded that adenosine phosphates do not control the direction of glycolytic flux in yeast and an alternative control of glycolysis and gluconeogenesis by enzyme activation and inactivation is suggested.

scholarly journals The pro-urokinase plasminogen-activation system in the presence of serpin-type inhibitors and the urokinase receptor: rescue of activity through reciprocal pro-enzyme activation

2003 ◽  
Vol 371 (2) ◽  
pp. 277-287 ◽  
Author(s):  
Niels BEHRENDT ◽  
Karin LIST ◽  
Peter A. ANDREASEN ◽  
Keld DANØ
Keyword(s):  
Plasminogen Activator ◽  
Enzyme Activation ◽  
Negative Regulator ◽  
Activation Mechanism ◽  
Lag Phase ◽  
Plasmin Activity ◽  
Proteolytic Activation ◽  
Upa Receptor ◽  
Activation System ◽  
Pai 1

The reciprocal pro-enzyme activation system of plasmin, urokinase-type plasminogen activator (uPA) and their respective zymogens is a potent mechanism in the generation of extracellular proteolytic activity. Plasminogen activator inhibitor type 1 (PAI-1) acts as a negative regulator. This system is complicated by a poorly understood intrinsic reactivity of the uPA pro-enzyme (pro-uPA) before proteolytic activation, directed against both plasminogen and PAI-1. We have studied the integrated activation mechanism under the repression of PAI-1 in a purified system. A covalent reaction between pro-uPA and PAI-1 was positively demonstrated but the reaction of PAI-1 with two-chain uPA was found to be at least 1000-fold faster. However, in spite of this very fast inhibition, two-chain uPA still became the dominant plasminogen activator when plasminogen was incubated with pro-uPA and PAI-1. The activity pattern observed under these conditions revealed an initial lag phase, followed by a continuous generation of minute amounts of active two-chain uPA, this uPA having a short lifetime before inhibition but still succeeding to generate new plasmin activity, thus preventing a complete inactivation of the feedback system. This property of the activation system was retained even in the simultaneous presence of PAI-1 and α2-antiplasmin. Addition of soluble uPA receptor to the system did not change the role of pro-uPA and the same pattern was observed when pro-uPA was bound to the uPA receptor on U937 cells. The present mechanism maintains the system at standby level and may be triggered to increased activity without the need for an external initiating event.

scholarly journals Prevalence of Antinuclear Antibodies (ANA) among Healthy Individuals with Different Nationalities in Qatar

2020 ◽  
Author(s):  
Prattyasha Rani Debnath ◽  
Fathima Amanullah ◽  
Gheyath Nasrallah
Keyword(s):  
Antinuclear Antibodies ◽  
Healthy Population ◽  
Specific Marker ◽  
Large Sample Size ◽  
Analytical Sensitivity ◽  
Nuclear Staining ◽  
Healthy Individuals ◽  
Mena Region ◽  
Elisa Kit ◽  
The World

Background: Detection of antinuclear antibodies (ANA) by different immunoassays (usually indirect fluorescence assay IFA) is the most common and sensitive screening test used for the diagnosis of many types of autoimmune disease such as systemic lupus erythematosus (SLE). Moreover, a high titer of ANA can be frequently found in healthy individuals, making it not a very specific marker of autoimmunity. However, the information about the prevalence of these false positive ANA in a healthy individual is not well investigated around the world particularly in Asia and MENA region including Qatar. Objectives: The aim of this study was to (i) estimate the prevalence of ANA among male healthy individuals of local and expatriate communities residing in Qatar and (ii) to evaluate the performance of a new commercial ELISA kit using two quality control parameters; analytical sensitivity (endpoint titration) and positive predictive value (PPV). Methodology: Sera collected from a total of 2965 volunteer donors of age 18 years or older, attending Hamad Medical Corporation between 2013 and 2016, were used to test for the presence of ANA IgG using Bioprobes Srl microplate ELISA kits. All positive ELISA kit results were retested using the IFA IgG for the detection of ANA nuclear staining patterns. Results: ANA prevalence among the healthy population was 0.34%, as only 10 samples out of 2965 were positive by the ANA ELISA. An additional 12 borderline samples were also detected raising the prevalence to 0.74% (22/2965). The Irani nationals had the highest prevalence of ANA with 1.83%. About 70% of the samples had a nuclear-speckled staining pattern. ELISA kit showed excellent performance efficiency, i.e., similar titration score to IFA for positive samples and high PPV (100%). Conclusion: This is the first ANA comprehensive study in the MENA region with such a large sample size. According to literature and our own literature review, the average prevalence of ANA in healthy individuals in a different part of the world is usually about 3-15%; however, in Qatar, it is only 0.34%. We recommend using the new Dia. Pro ELISA kit for ANA screening as this kit showed high efficiency for detecting ANA in comparison to the gold standard IFA assay.

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