بحوث سكوبس — حوراء سعد حمود الكواز

Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision. © The Author(s) 2024. Published by Oxford University Press.

Neutrophil myeloperoxidase (MPO) is an essential enzyme for the innate immune system. Measuring MPO activity is vital for understanding neutrophil characteristics and functions in various diseases. MPO activity can be measured using several methods, including spectrophotometric and fluorometric protocols. This paper introduces a fluorometric method for specifically quantifying MPO activity based on the H2O2-dependent oxidation of thiamine. We optimized this new method using the robust statistical approach response surface methodology (RSM) and Box Benken Design (BBD). We extensively examined the effects of several experimental parameters using the RSM methodology and determined the best conditions for accurate and sensitive MPO activity measurement. The optimal conditions were determined using the analysis of variance (ANOVA) for second-order polynomial equations. The resulting F-value (4.86) indicated that the model was significant. However, the lack-of-fitness F-value (1.79) suggested it did not differ significantly from the corresponding p-value. The greatest MPO activity (30 ± 2 U L−1) was obtained under optimum conditions, which were 1000 µM of H2O2, 10 min incubation time, and 1000 µM of thiamine. Our results suggest that this advanced fluorometric method has significant accuracy, sensitivity, and linearity up to 60 IU. The new and standard colorimetric methods also showed a good correlation. These results indicate that the new fluorometric method can be dependable and efficient for assessing MPO activity. The new method is characterized by excellent accuracy, sensitivity, and linearity, making it a valuable protocol for researchers and clinicians interested in assessing MPO activity. © 2023 Elsevier Inc.

Asthenospermia is a common cause of male infertility refers to semen samples with spermatozoa that move slowly or immotile. Recent research has implicated oxidative stress-induced ferroptosis in asthenospermia's pathophysiology. Peroxiredoxins are important players in the antioxidant defense to protect cells against oxidative stress. However, some aspects of the antioxidant response necessary for male fertility are not well understood. This case-control study aimed to elucidate the role of peroxiredoxins in regulating oxidative stress in male fertility via their correlation with ferroptosis. It included 90 fertile and 90 asthenospermic subfertile males from Hilla City, Iraq. Total peroxiredoxin activity, peroxiredoxin-6 level, and peroxiredoxin-4 level were measured alongside the ferroptosis biomarkers glutathione peroxidase-4, malondialdehyde, and the reduced/oxidized protein thiol ratio. Infertile males had significantly higher oxidized thiol and malondialdehyde levels than fertile males (p < 0.05). Total peroxiredoxin activities, peroxiredoxin-6 levels, peroxiredoxin-4 levels, glutathione peroxidase-4 levels, and reduced/oxidized protein thiol ratios were significantly lower in infertile males than in fertile males (p < 0.05). Therefore, peroxiredoxin activity and level correlate with reduced/oxidized protein thiol ratio. They are inversely associated with ferroptosis and directly associated with semen quality. These findings suggest that peroxiredoxins are crucial in preventing ferroptosis and have potential implications for treating asthenospermia. © 2023 The Authors

Aspirin, either in combination or alone, is possibly one of the most prescribed medications worldwide. Aspirin hydrolysis is based on enzyme systems found in the liver, intestine, and serum. This work explained and demonstrated the repeatability, accuracy, and precision of a simple spectrophotometric method for assessing aspirin esterase (ASE) activity. In the present assay, ASE activity was determined by incubating enzyme samples with Tris buffer (pH 7.6) containing appropriate quantities of acetylsalicylic acid. At the end of the incubation time, the enzymatic reaction was stopped using zinc sulfate. Finally, the supernatant of the enzymatic reaction was treated with ferric ammonium sulfate (NH4Fe [SO4]2·12H2O). The linkage of the generated salicylic acid to ferric ions yielded a violet-colored ferrisalicylate complex with a wavelength of 540 nm. The Box–Behnken design was utilized to optimize the formation of the violet-colored ferrisalicylate complex. The method’s accuracy was determined using response surface methodology. Bland–Altman plots for ASE activity in matched biological samples were used to validate the new fluorescence protocol. The method proposed in this work does not require hazardous materials, such as mercuric chloride, or high concentrations of strong acids to terminate the ASE reaction. The correlation coefficient between the present and previous procedures was 0.99. This result suggested that the new procedure is very accurate and comparable with previous procedures. Graphical abstract: [Figure not available: see fulltext.] © 2022, Springer-Verlag GmbH Austria, part of Springer Nature.

Trypsin is an enzyme that facilitates digestion. It is found in the small intestine and may also be synthesized by bacteria, plants, and fungi. However, it is mainly synthesized for commercial use from cattle pancreases. Serum trypsin determination seems to be a specific diagnostic for acute pancreatitis. Therefore, the development of improved trypsin activity protocols is essential for clinical pathology and pharmaceutical research. In this study, a simple spectrophotometric procedure for trypsin, a pancreatic protease, was developed. The current protocol utilized a previously known peroxidase-like reaction to estimate the trypsin activity. An additional acidification step is employed to improve the efficiency of the protocol. Trypsin has the ability to preferentially cleave the natural compound cytochrome c into heme-peptide fragments. In our study, the resulting peroxidase-like activity was catalyzed by the oxidation of 3,3ʹ,5,5ʹ-tetramethylbenzidine in the presence of H2O2. Sulfuric acid was added to stop the enzymatic reaction before recording the absorbance at 450 nm. To optimize the formation of the end product, we used the response surface methodology to apply the Box–Behnken design to assess the assay's precision. The reliability of this new method was compared to a Bland–Altman plot analysis of trypsin activity in matched samples using the standard procedure. The protocol allowed for trypsin investigations in the 5–1500 ng/cm3 range, with a detection limit of 0.691 ng/cm3. The protocol demonstrated higher accuracy in the measurement of 500 ng/cm3 trypsin solution, with a relative standard percentage error of 1.4–2.2%. This new protocol was verified against a Bland–Altman plot analysis of trypsin activity in matched samples using the Thiamine–Trypsin test, confirming its potential for application in pharmaceutical development and disease treatment. Our study demonstrated a simple, rapid, low-cost, sensitive, and selective method for assessment of the trypsin enzyme, which can be used to study the clinical importance and pharmaceutical significance of the trypsin enzyme. Graphical abstract: [Figure not available: see fulltext.]. © 2023, Springer-Verlag GmbH Austria, part of Springer Nature.

The enzyme homocysteine thiolactonase (HTase) is involved in the metabolism of homocysteine, an amino acid. As a byproduct of the metabolism of methionine, a secondary amino acid, homocysteine (Hcy), a sulfur-containing amino acid, is produced. Hcy levels in the blood are linked to higher risks of heart disease, stroke, and other disorders. To create Hcy, HTase catalyzes the hydrolysis of homocysteine thiolactone (HTL). This process helps control the body's homocysteine levels and prevents the accumulation of homocysteine in the blood. This essay describes a precise, easy-to-follow procedure for measuring homocysteine thiolactonase (HTase) activity. This protocol (NAM-HTase) determines HTase activity via incubation with γ-thiobutyrolactone, an enzyme substrate, in a pH-appropriate solution at 37 °C for five minutes. It then uses thiol fluorometry to monitor the produced thiol groups. The newly formed thiol groups are reacted with the fluorescent probe N-(9-acridinyl)maleimide (NAM) and quantified using fluorometric intensity at an Ex/Em of 360/432 nm to evaluate HTase activity. The method is linear for 4-mercaptobutyric acid and homocysteine, from 0.005 ± 0.001 to 6 ± 0.04 µM. By comparing the HTase activity in matched samples to that of a standard method, this new protocol was found suitable for clinical applications. Its accuracy compared to other methodologies was shown by a correlation of 0.9995 between its values and those of a standard method. Using the H+ ion releasing method in matched samples, analysis of variance was used to verify the novel method against HTase activity. In conclusion, the proposed NAM-HTase method successfully measured HTase activity with high reliability. © 2023 Elsevier B.V.

Cellulase is a microbial enzyme responsible for degrading the β-1,4 glycoside bond in polysaccharide cellulose, which is abundant in various animal foodstuffs. Cellulase is an important industrial enzyme used for various purposes, including biopolishing textile fibers, softening garments, biostoning denim fabric, and removing excess color from textiles. In the food industry, cellulase is combined with pectinase and hemicellulase. Therefore, the need for a reliable, fast, and inexpensive cellulase activity protocol that could be used with diverse biological and environmental samples is great. This study developed a novel method to quantify cellulase activity using picric acid (PCA), which reacts with generated glucose molecules to produce mahogany red picramic acid. This PCA-cellulase method uses sodium hydroxide instead of sodium carbonate to provide alkalinity in the reaction solution, increasing the stability of picramic acid and the sensitivity and linearity of the reaction. It also overcomes the limitations of previous methods. It is notable for its dependence on few chemicals with low concentrations compared to previous methods that depend on many chemicals with high concentrations. The PCA-cellulase method was optimized using the Box–Behnken design, and its accuracy was determined using a response surface approach. A Bland–Altman cellulase activity graph was used to validate the PCA-cellulase method with a correlation coefficient of 0.9991. Therefore, the novel PCA-cellulase method provides accurate results that are comparable to existing methods. © 2022 Elsevier Inc.

Background: Pre-eclampsia is an idiopathic pregnancy disorder characterized by appearance proteinuria and hypertension, with poorly understood etiology. It has been linked to a variety of system abnormalities, including ion transport disorders in neonatal, maternal, and placental cell lines. A new method was described to evaluate the inhibition percentage of endogenous digitalis in plasma of pre-eclampsia patients compared with normal pregnancies, with the estimation of sensitivity and specificity of the proposed test. Methods: This was a case-control study consisting of 130 cases that were divided into three groups, 55 normal pregnancies (positive control), 30 non-pregnant women (negative control), and 45 preeclampsia (patients). The new method included the estimation of the percentage inhibition of endogenous digitalis by measuring specific enzyme activity of Na-K ATPase for the patient and positive control. The results were analyzed using the statistical package for social sciences (SPSS®) software version 26.0. A p-value of < 0.05 was considered significant. Results: In the pre-eclampsia patient, the specific activity of Na-K ATPase was significantly lower with mean= 0.239 mg/g±0.043 compared to the control group which was 0.397 mg/g±0.021, p< 0.001. While the result of inhibition percentage of endogenous digitalis showed significantly higher in the pre-eclampsia patient (mean= 35.852 mg/g %±2.692%) compared to the control group (mean= 17.964%±1.784), with a p< 0.001. Conclusions: Pre-eclampsia is linked with lower erythrocyte sodium pump activity significantly in pre-eclampsia patients than in normal pregnancies. Also, results show the inhibited percentage of endogenous digitalis elevation in patients with pre-eclampsia compared with normal pregnancy. © 2022. All Rights Reserved.