Method For Determination Of Activity Research Articles

Clinical, Biochemical and Genetic Analysis of Biotinidase Deficiency in Iranian Population.

Biotinidase deficiency (BTD) is an autosomal recessive disorder of biotin metabolism. Biotin is a coenzyme that enhances the action of the four enzymes that play an important role in carbohydrates, amino acid, and fatty acid metabolism. Defects in these pathways cause severe metabolic disorder in the body. In general, biotinidase deficiency can be classified into two levels: partial and profound. The incidence of BTD is 1:40,000 to 1:60,000 births in the world, even though no convincing statistical data on the prevalence of this disorder exist in Iran. In this study, we aimed to set up a test for determining biotinidase activity among the Iranian population and report BTD mutations. The quantitative method for the determination of biotinidase activity was set up in the National Biochemistry Reference Laboratory (NBRL) of Pasteur Institute of Iran in Tehran. To detect mutations in BTD, polymerase chain reaction (PCR) was performed followed by DNA sequencing. The biotinidase activity range values were 3.81 - 8.25 nmol/min/mL. We identified 8 BTD patients out of 47 cases with neurologic signs. We detected two mutations, c.98-104del7ins3 and p.Arg79Cys, in 5 patients with profound BTD, and one p.Asp444His mutation in 3 patients with partial BTD. Infants suffering from BTD seem healthy during their first months of life. At present, the screening program for metabolic disorders such as BTD is in progress. The patients that are BTD deficient benefit from the availability of the tests, and consequently receive the Biotin supplements before being clinically affected.

Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase.

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate-active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper-dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.

Pozzolanic Activity Diagnostics of Fly Ash for Portland Cement

The work deals with the analysis and evaluation of different methods of pozzolanic activity determination in order to rationally use ash in cement. The most optimum diagnostic method has been suggested. The possibility of increasing fluidal fly ash activity has been established using the synthetic nanosilica.

Determination of 238Pu, 239+240Pu and 241Am in air filters

The aim of this work is to present the method for sequential plutonium and americium activity determination in air filters using chromatographic radionuclide separation and alpha spectrometry measurement. The developed method may be employed for the purposes of workplace monitoring and as an indicator of the need of introducing the individual monitoring as well as a useful complementation of individual monitoring. Basic parameters describing the developed method such as values of chemical recoveries and minimum detectable activities for plutonium and americium isotopes have been determined. Applied counting efficiency was obtained using Monte Carlo calculation method.

Comparative assessment of antioxidant activity of chicory products

There are various methods for determining the antioxidant activity. They are differing by the source of oxidation, oxidized compounds and methods for the measurement of oxidized compounds. Methods for determination of antioxidant activity provide a wide range of results, so the evaluation of the antioxidant activity should be carried out in several ways, and the results should be interpreted with caution. In assessing antioxidant capacity is necessary to consider not only the nature and content of the reducing agent in the test facility, but also the possibility of their mutual influence. The objects of research in this research work were Instant chicory (Leroux), Fried chicory (Leroux), Instant chicory (LLC Favorit), Instant chicory (LLC SlavKofe), Instant chicory with hawthorn (Iceberg Ltd and K), Instant chicory (LLC Flagistom), Instant chicory (Ltd. Around the World), Instant chicory (LLC Beta +), Chicory flour (Leroux), Chicory flour. Antioxidant activity of the products of chicory determined by spectrophotometric (using adrenaline and 2,2 – diphenyl- 1-picrylhydrazyl), and amperometric methods. These results indicate that chicory products possess antioxidant activity but not give fully correlated with each other results. The highest antioxidant activity have fried products from chicory – Instant chicory (LLC SlavKofe) and Instant chicory with hawthorn (Iceberg Ltd and K). No fried products from chicory (Chicory flour (Leroux)) have lower antioxidant activity then fried products from chicory. Because during frying new compounds are formed – chikoreol, which comprises acetic and valeric acid, acrolein and furfural, furfuryl alcohol, diacetyl. Best convergence results provide spectrophotometric methods using epinephrine and 2,2 – diphenyl-1-picrylhydrazyl.

Label-free nanopore single-molecule measurement of trypsin activity.

Trypsin is the most important digestive enzyme produced in the pancreas, and is a useful biomarker for pancreatitis. In this work, a rapid and sensitive method for the quantitative determination of trypsin activity is developed by using a biological alpha-hemolysin protein nanopore. Due to its much larger molecular diameter than the narrow pore constriction, trypsin itself cannot transport through the alpha-hemolysin channel. Hence, an indirect trypsin detection method is developed by monitoring its proteolytic cleavage of a lysine-containing peptide substrate. Based on the current modulations produced by the translocation of the substrate degradation products in the nanopore, the activity levels of trypsin could be determined. The method is rapid and highly sensitive, with picomolar concentrations of trypsin detected in minutes. In addition, the effects of cation and temperature on the sensor sensitivity, trypsin inhibition, and serum sample analysis are also investigated.

A new near-infrared method for simultaneous determination of caffeic acid phenethyl ester and antioxidant activity of propolis samples

The problem of standardization of propolis is a complex issue that has been approached by different researchers based on the elucidation of chemical composition and its correlation with biological activity. Due to variability of the matrix and the particular characteristics of the various types of propolis, this work is complex and time consuming, requires the availability of several analytical techniques, and generally leaves out the interactions between the different compounds that constitute the matrix. A complete analysis of the matrix together with the selective determination of some compounds with relevant biological activity could establish a quality parameter of the samples that helped us to develop a control system that facilitates their standardization. Caffeic acid phenethyl ester (CAPE) is a derivative of caffeic acid that has been extensively studied, because it has various types of biological activity beneficial to human health. In addition, it is present in different proportions in all the samples studied. In this work, a new method based on near-infrared spectroscopy (NIR) to estimate equivalents of CAPE in propolis samples is proposed. Simultaneously, a new method for estimating antioxidant activity expressed as EC50 is also presented. A multivariate partial least squares (PLS) method was used for calibration. These models were previously calibrated by measuring the content of CAPE by HPLC-EC, and antioxidant activity by UV–vis spectroscopy with the high-performance microplate reader method. PLS models were studied by cross-validation and external validation. The NIR methods developed proved to be a non-destructive, simple, fast, and inexpensive to use. This study model can be used as a quality parameter of in situ raw propolis samples, without any preprocessing, and contributes to solve the problem of standardization of propolis.

Method development and validation for determination of antihemolytic activity of eculizumab (Soliris)

Bioanalytical methods are applied at the development and test of drugs as well as at the step of pharmaceutic products issue. Drugs and excipients quality estimation is made by means of precise and reproducible methods. Accuracy and reproducibility of a method is established during validation which is obligatory for medicine registration. The principal task of method validation is experimental evidence of its suitability for the objectives to be achieved. Validation of bioanalytical methods is one of the elements of the whole medicine production validation [1, 2]. The aim of research : to validate a method for determination of specific anti-hemolytic activity of eculizumab developed in LCC “IBC Generium”. Materials and methods: eculizumab, antibody-sensitized chicken erythrocytes, complement-containing human serum. Results. We demonstrated the specificity of the method and its correspondence to criteria of accuracy (103.0±1.4)%, robustness (CV – 11.5%), repeatability (CV – (4.9±0.9)%), reproducibility (CV – (3.5±0.4)%), and linearity (k -1.0275; R2 – 0.9975) during validation. The system validity (equipment, materials, analytical operations and analyzed samples) was confirmed for true results obtaining during validation. Results discussion. Experimental evidence of suitability of the method for eculizumab specific activity assessment was obtained in course of validation. The simplicity of the method allows obtaining accurate results in other laboratories. The developed method can be used not only for specific activity of Soliris determination but also for other pharmaceutical substances and drugs based on antibodies specific to human complement C5.

Comparative evaluation of antibacterial activity of caffeic acid phenethyl ester and PLGA nanoparticle formulation by different methods

The aim of the present study was to evaluate the antimicrobial activity of nanoparticle and free formulations of the CAPE compound using different methods and comparing the results in the literature for the first time. In parallel with this purpose, encapsulation of CAPE with the PLGA nanoparticle system (CAPE-PLGA-NPs) and characterization of nanoparticles were carried out. Afterwards, antimicrobial activity of free CAPE and CAPE-PLGA-NPs was determined using agar well diffusion, disk diffusion, broth microdilution and reduction percentage methods. P. aeroginosa, E. coli, S. aureus and methicillin-resistant S. aureus (MRSA) were chosen as model bacteria since they have different cell wall structures. CAPE-PLGA-NPs within the range of 214.0 ± 8.80 nm particle size and with an encapsulation efficiency of 91.59 ± 4.97% were prepared using the oil-in-water (o–w) single-emulsion solvent evaporation method. The microbiological results indicated that free CAPE did not have any antimicrobial activity in any of the applied methods whereas CAPE-PLGA-NPs had significant antimicrobial activity in both broth dilution and reduction percentage methods. CAPE-PLGA-NPs showed moderate antimicrobial activity against S. aureus and MRSA strains particularly in hourly measurements at 30.63 and 61.25 μg ml−1 concentrations (both p < 0.05), whereas they failed to show antimicrobial activity against Gram-negative bacteria (P. aeroginosa and E. coli, p > 0.05). In the reduction percentage method, in which the highest results of antimicrobial activity were obtained, it was observed that the antimicrobial effect on S. aureus was more long-standing (3 days) and higher in reduction percentage (over 90%). The appearance of antibacterial activity of CAPE-PLGA-NPs may be related to higher penetration into cells due to low solubility of free CAPE in the aqueous medium. Additionally, the biocompatible and biodegradable PLGA nanoparticles could be an alternative to solvents such as ethanol, methanol or DMSO. Consequently, obtained results show that the method of selection is extremely important and will influence the results. Thus, broth microdilution and reduction percentage methods can be recommended as reliable and useful screening methods for determination of antimicrobial activity of PLGA nanoparticle formulations used particularly in drug delivery systems compared to both agar well and disk diffusion methods.

Calorimetric method for determination of 51Cr neutrino source activity

Experimental study of nonstandard neutrino properties using high-intensity artificial neutrino sources requires the activity of the sources to be determined with high accuracy. In the BEST project, a calorimetric system for measurement of the activity of high-intensity (a few MCi) neutrino sources based on 51Cr with an accuracy of 0.5–1% is created. In the paper, the main factors affecting the accuracy of determining the neutrino source activity are discussed. The calorimetric system design and the calibration results using a thermal simulator of the source are presented.

Identification of bioactive phytochemical from two Punica species using GC-MS and estimation of antioxidant activity of seed extracts.

Punica species are medicinally important plants belonging to the family Lythraceae. The pomegranate is widely reported to exhibit antiviral, antioxidant, anticancer, anti-proliferative activities. In the present study the ethanolic extract of the peel seeds of two species of Punica (Punica granatum and Punica protopunica) were subjected to GC–MS analysis. Twenty-one and 14 compounds were identified in P. granatum and P. protopunica peel seeds, respectively. The main chemical constituents in P. granatum-peel seeds were propanoic acid, benzenedicarboxylic acid, methoxypropionic acid and methyl amine. The corresponding constituents of P. protopunica peel seeds were benzenedicarboxylic acid, benzoic acid and propanoic acid. Moreover, the antioxidant effects of the aqueous ethanolic extracts were estimated in vitro. The two tested extracts contained significantly different phenolic and total flavonoid contents in P. granatum than in P. protopunica. Different in vitro methods of antioxidant activity determination produced varying results. In malondialdehyde (MDA), hydrogen peroxide (H2O2) scavenging and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays, the two peel seed extracts exhibited very high antioxidant activities, with higher activity observed for the P. granatum extract.

A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)- d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection.

Estimation of specific activity of 177Lu by 'saturation assay' principle using DOTA as ligand.

Lutetium-177 is a widely used therapeutic radionuclide in targeted therapy and it is important to know its specific activity at the time of radiopharmaceutical preparation, especially for radiolabeling peptides. However, there are no direct methods for the experimental determination of the specific activity which can be readily applied in a hospital radiopharmacy. A new technique based on the 'saturation assay' principle using DOTA as the binding agent for the estimation of specific activity of (177)Lu is reported. The studies demonstrate the proof of principle of this new assay technique. The method is general and can be modified and applied for the estimation of specific activity of other metallic radionuclides by using DOTA or other suitable chelating agents.

Trophic patterns of functioning and microbial profile of the evolutionally established associated kefir grains culture

The associated culture of kefir grains was analyzed by molecular methods for determination of the functional activity of microbial isolates and molecular genetic techniques for their identification. A combination of 16S rRNA analysis and denaturing gradient gel electrophoresis was used to determine the microbial profile of kefir grains and to reveal lactic acid bacteria of two physiological groups, differing in their ability to use lactose for lactic acid fermentation. The role of inducible β-galactosidase of lactic acid bacteria for the functional stability of the microbial community was shown in the study of the functional activity and microbial profile of the kefir grains after long-time cultivation (over 4 years) on lactose-free milk. The results obtained improve our understanding of the possible trophic interactions in such microbial communities and may be used to develop the algorithm for experimental production of a stably associated culture of kefir grains.

A Sensitive and Robust Method for Direct Determination of Lipolytic Activity in Natural Milk Environment

Microbial lipases may be produced during milk storage and processing. This can lead to organoleptic changes in the corresponding dairy products. Thus, monitoring of lipase activity in milk is desirable. Turbidity of milk prevents a direct photometric measurement of lipase activity using chromophore- or fluorophore-based assays. Laborious pretreatments or alternative analytical methods normally have to be used. With the method for the determination of lipolytic activity (MeDeLi) proposed here, it is possible to measure lipase activity directly in the natural milk utilizing tailored fluorometric substrates. Only a defatting step is carried out initially for the MeDeLi. Then, the conversion of added lipase substrate is carried out in the unmodified milk without addition of any solutions or any enzyme extraction procedure which may influence the enzyme activity. Thereafter, the milk sample is treated with two solutions to remove the turbidity of milk by dissolution. A valid and sensitive fluorometric measurement is then possible. The applicability of the MeDeLi was demonstrated in comparison with tests published previously: The limit of detection for lipolytic activity measured by MeDeLi was the lowest, with 41 pkat/mL. Raw milk, milk products, and spoiled milk samples were also investigated with the MeDeLi.

Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models.

The cytosolic 5'-nucleotidase (cN-II) has been shown to be involved in the response of cancer cells to cytotoxic agents, and the quantification of its activity in biological samples is of great interest. In this context, we developed and validated an analytical method for determination of cN-II activity in cultured cancer cells. This non-radioactive method, using a Hypercarb column as stationary phase, was validated with a lower limit of quantification of 0.1 μM inosine. We used it to characterize cell line models with modified cN-II expression obtained with stable transfections. We show that the short hairpin RNA (shRNA)-mediated inhibition of cN-II expression in various malignant blood cells is associated with decreased protein expression and enzymatic activity (1.7-6.2-fold) as well as an increased sensitivity to cytotoxic agents (up to 14-fold). On the other hand, expression of green fluorescent protein (GFP)-fused wild type or hyperactive mutant (R367Q) cN-II increased the activity and also decreased the sensitivity to nucleoside analogues. Our results confirm the biological relevance of modulating cN-II in cancer cells, and we present a straightforward validated method for the determination of cN-II activity in cellular samples.

Improved green coffee oil antioxidant activity for cosmetical purpose by spray drying microencapsulation

The oil extracted by cold pressing unroasted coffee beans, known as green coffee oil, has been widely used for cosmetic purposes. The objective of this work was to prepare and characterize microcapsules containing green coffee oil and to verify its antioxidant activity under the effect of light, heat and oxygen. The encapsulating material was arabic gum and the microcapsules were obtained by spray drying an oil-in-water emulsion containing green coffee oil. The characterization of the microcapsules was performed by laser diffraction, scanning electron microscopy, differential scanning calorimetry and the antioxidant activity. The antioxidant activity was determined by a modified active oxygen method with light irradiation, heating and oxygen flux. The microparticles were effectively produced by the proposed spray drying method, which resulted in green coffee oil loads of 10 and 30%. The morphological evaluation of microcapsules showed spherical shape with smooth and non-porous surfaces, demonstrating the adequacy of arabic gum as encapsulating material. Calorimetric analysis of individual components and microcapsules with 10 and 30% green coffee oil showed diminished degradation temperatures and enthalpy, suggesting a possible interaction between arabic gum and green coffee oil. The antioxidant activities for pure green coffee oil and its microcapsules with loads of 10 and 30% showed high activity when compared to the reference antioxidant alfa-tocopherol. Microcapsules containing 10 and 30% of oil showed 7-fold and 3-fold increase in antioxidant activity when compared to pure green coffee oil. The new method for antioxidant activity determination proposed here, which applies heat, light and oxygen simultaneously, suggests a high improvement in encapsulated green coffee oil when compared to this active alone. The results showed herein indicate a promising industrial application of this microencapsulated green coffee oil.

Development of method for the mineral water catalase activity determination

Introduction. The availability in Ukraine of diversified unique natural mineral water (MW) resources does necessitate a detailed study of that water properties in order of its widespread use in the balneological and extra-resort practice, considering the MW as an important factor of population health preserving and improving [1]. The mineral water rational use is largely determined by its physical and chemical composition, sanitary and microbiological status, the presence of autochthonous microorganisms and those microorganisms’ ability to influence the water organoleptic characteristics and biological properties. In MW practical use the key importance is tribute to controlling the water quality parameters for the prediction of water safety degree and the detection of biological exposure. The MW biological effect depends on the mineralization degree, chemical composition, presence of biologically active components and compounds, as well as on their microbial cenosis’ metabolic products. Analyzing the mineral water microorganisms’ list we reveal their active biochemical effect [1]. The numerous microorganisms contributing to the mineral water’s autochthonous microflora do often include significant concentrations in saprophytic microorganisms producing the catalase as an enzyme of oxidoreductases class, a heme-containing chromoproteid making part of the cell’s antioxidant system and acting as an antiperoxidant protector agent [2]. Peroxide mechanisms may occur in the brain and in other tissues during aging, when exposure to poisons and toxins, when cerebral apoplexy, at nervous system injuries, and others processes. The biological role of catalase refers to the complex enzymatic protection of cell membranes against hydrogen peroxide-caused degradation. The catalase does metabolize the hydrogen peroxide, preventing its accumulation in the cell through producing water and oxygen. The most relevant catalase application relates to the treatment of diseases requiring an effective antioxidant protector: cardiovascular, rheumatic diseases, allergies, central and autonomic nervous systems’ diseases, gastrointestinal diseases, oncology, plastic surgery, etc. [2]. Given the important physiological role of catalase, it is necessary to determine the mineral water’s enzymatic activity. At present the methodology for MW enzymatic activity determination is still absent, so such methods’ elaboration problem is top priority one. Analysis of recent researches and publications. Methods for catalase activity quantitative determination are based on the estimation of enzyme-cleaved hydrogen peroxide quantity when enzymatic activity is assessed by the catalase index. The reference sources describe several methods of catalase activity assessment, but these methods relate to the biological objects (plasma, serum, etc.). To develop methods for determining the MW catalase activity we proceed to review the available approaches. Known is the enzymatic activity determining method by Bach and Oparin based onto titration [3], through assessing the quantity of hydrogen peroxide decomposed during incubation with the enzyme. The amount of hydrogen peroxide in the reaction mixture is assessed by titration with a Potassium permanganate solution in an acidic environment. However, this method has such drawbacks as a high labour intensity, results’ poor reproducibility and low sensitivity.

Estimation of operators' fatigue using optical methods for determination of pupil activity.

The purpose of this study was to develop and initially validate an objective, yet quick, method for assessment of human fatigue. This aim can be achieved by the use of an optical, non-contact method of analysis of pupil activity. The study involved 2 phenomena typical for the behavior of the human pupil: pupillary reflex and pupillary movements. The 1st phenomenon is related to the pupillary light reflex (PLR), which presents the results of physiological adaptation mechanisms of the human eye. The pupillary unrest index (PUI) is an additional parameter referring to the tendency for instability of the pupil. Indicators of these 2 mechanisms were assessed under the same experimental laboratory conditions. Assessment was conducted on a group of volunteers (N = 10) during 4 controlled series of measurements performed at night. Pupillary reflex parameters associated with PLR and pupillary unrest index (PUI) were recorded using F²D Fit-For-Duty, a commercially available system made by AMTech Pupilknowlogy GmbH. Baseline pupil diameter, oscillations, reflex latency, maximum reaction time, pupil constriction time, pupil dilation time, and constriction amplitude were recorded. As a result of the study, we were able to demonstrate correlation and confirm the usefulness of PLR and PST methods in the assessment of sleep deprivation. Parameters of PLR that may indicate human fatigue were identified. The effect of light impulse sequences on the dynamics of pupillary reflex and the relationship between PUI (pupillary unrest index) measurement duration and sleepiness assessment validity, were assessed. The results of the pilot studies were sufficient to develop minimum requirements for a PLR sensor that would be capable of estimating the level of fatigue with accuracy of a PUI method, but at a 5 times faster rate.

ANALYSIS OF OXIDIZING POWER IN MICROORGANISMS OF SLUDGE BIOFILM BY MEASURING THEIR DEHYDROGENASE ACTIVITY

The general oxidizing capacity of active sludge microorganisms occurring in a bioreactor in the form of attached flora (biofilm) has been determined. We used the method of dehydrogenase activity (DHA) determination based on the application of methylene blue as an indicator. A non-linear character of kinetic curves of a substrate (glucose) biological oxidation was observed; putatively, this is a result of the peculiarities of the accumulation of respiratory mediators in the biomass. The reserves of those mediators of redox reactions was assessed, and the data on the biofilm DHA activity (~ 150 nmole/min/mg of dry biomass) are represented. This value testifies to a high oxidation potential of the active sludge biofilm in bioreactors.