An optimized method for estimating glutaminase activity in biological samples.

Spectrophotometric methodologies have been used to assess glutaminase activity, for which coloured complexes have been developed that measure spectrophotometry across the visible spectrum using different reagents. The present paper describes a precise, simple and reliable procedure for quantifying glutaminase activity, which is a key enzyme in glutamine hydrolysis and also involved in glutamine metabolism regulation. The procedure presented here measures glutaminase activity by incubating glutaminase enzyme at 37 °C for 20 min with a glutamine substrate dissolved in a buffer (pH 8.6). The enzymatic reaction contains suitable activity of glutamate oxidase, which acts to convert glutamate to hydrogen peroxide and 2-oxoglutarate. To terminate the enzymatic activity, a working solution containing pyridine-2,6-dicarboxylic (PDA) acid and ammonium vanadate (AV) was added following incubation. Oxo-peroxo-pyridine-2,6-dicarboxylato-vanadate (OPDV), a stable orange-coloured chelate complex measuring 435 nm spectrophotometrically, was produced by the interaction between the generated hydrogen peroxide and the supplied reagent. Using the response surface methodology (RSM) as an indicator of the assay's accuracy, we employed the Box-Behnken design (BBD) to improve the method's design (the OPDV-Glutaminase assay). Improvement factors were the volume of working reagent solution (PDA/AV), volume of glutamate oxidase solution (GO), and incubation time. In matched samples, this novel method was verified against a Bland-Altman plot assessment of glutaminase activity using the indophenol methodology. A correlation value of 0.99 between the two methods' comparisons showed that the novel protocol was equally applicable to the reference method.

Fluorometric Assessment of Sulfhydryl Oxidase Activity: Optimization by Response Surface Methodology.

Sulfhydryl oxidase was studied using a spectrofluorometric assay. The current protocol operates by using a combination of hemoglobin (HB) and hematin (HT) as a peroxidase mimic to catalyze the H2O2-dependent oxidation of thiamine. The response surface methodology (RSM) is used to optimize the new method. The current method is very accurate, sensitive, and linear up to 200 IU. When compared to the colorimetric method, the method produced a satisfactory correlation. The novel protocol is being used to evaluate asthenospermic patients' and fertile men's seminal sulfhydryl oxidase activity. The current protocol was used to determine reference values for seminal sulfhydryl oxidase activity. Due to the fact the newly developed spectrofluorometric method is more sensitive and precise than other colorimetric methods, and because thiamine is less expensive than other types of probes used in colorimetric and spectrofluorometric methods, it is likely to find widespread use among scientists studying sulfhydryl oxidase activity in biological tissues. The present method's analytical recovery yielded high specific findings.

Effect of Zinc Supplementation on Urate Pathway Enzymes in Spermatozoa and Seminal Plasma of Iraqi Asthenozoospermic Patients: A Randomized Controlled Trial.

BACKGROUND: Uric acid (UA) is crucial for sperm metabolism as it protects seminal plasma against oxidative damage. Zinc also plays a central role in sperm metabolism. The current study was designed to investigate the role of zinc supplementation on qualitative and quantitative properties of seminal fluid, in parallel with the UA level and urate pathway enzymes in the semen of patients with asthenozoospermia. MATERIALS AND METHODS: The study was designed as a randomized controlled trial of 60 asthenozoospermic subfertile men. The current study, which was conducted during one year, involved 60 fertile and 60 asthenozoospermic subfertile men belonging to Hilla City, Iraq. Semen samples were obtained from the participants before and after treatment with zinc supplements. The levels of UA, xanthine oxidase (XO), adenosine deaminase (ADA) and 5'-nucleotidase (5'-NU) activities were determined in spermatozoa and seminal plasma of both groups. RESULTS: UA levels (P=0.034) and 5'-NU activity (P=0.046) were significantly lower but ADA (P=0.05) and XO (P=0.015) activities were significantly higher in infertile men than in healthy men. Treatment with zinc sulfate induced an increase in UA (P=0.001) level and 5'-NU activity (P=0.001), but a decrease in ADA (P=0.016) and XO (P=0.05) activities. CONCLUSION: Zinc supplementation restores UA levels and the activities of enzymes involved in the urate pathway (XO and ADA) in the seminal plasma and spermatozoa of patients with asthenozoospermia, to reference values. Supplementation of Zn compounds enhances the qualitative and quantitative properties of semen (Registration number: NCT03361618).

Effect of Oral Zinc Supplementation on the Thiol Oxido-Reductive Index and Thiol-Related Enzymes in Seminal Plasma and Spermatozoa of Iraqi Asthenospermic Patients.

A thiol group plays an essential role in sperm metabolism and the antioxidative defense state. Zinc is the second most abundant element in the human body, following iron. The present study was conducted to study the effect of zinc supplementation on the characteristics of semen along with thiol and thiol-related enzymes in semen of asthenospermic patients. Semen samples were obtained from 60 fertile and 60 asthenospermic men, from couples who had consulted the infertility clinic of Babil Hospital (Hillah city, Iraq). The subfertile group was treated with zinc; every participant took two 220 mg capsules of zinc sulfate per day for 3 months. Semen samples were obtained (before and after zinc supplementation). The levels of reduced thiol, oxidized thiol, thiol oxido-reductive index, and thiol-related enzymes activities were determined in spermatozoa and seminal plasma of patients and healthy groups. Oxidized thiol levels were significantly higher in the infertile patients compared to that in the fertile group. Conversely, reduced thiol level, sulfhydryl oxidase activity, and glutathione peroxidase activity significantly decreased in the infertile patients compared to that in the fertile group. Oxidized thiol levels, reduced thiol levels, and thiol-related enzymes activities of the infertile patients were restored to normal values after treatment with zinc. However, reduced and oxidized thiol levels in spermatozoa did not change significantly in the group treated with zinc. The quantitative values for RSH/RSSR and thiol-related enzymes may provide useful means to qualitatively express the oxidant/antioxidant balance in clinical and epidemiologic studies. ClinicalTrials.gov Identifier: NCT02985905.

Oral Zinc Supplementation Restores Superoxide Radical Scavengers to Normal Levels in Spermatozoa of Iraqi Asthenospermic Patients.

BACKGROUND: Oxidative stress and decreased antioxidant levels have been projected as potential factors involved in the pathophysiology of diverse male infertility types, including asthenospermia. The present study was conducted to examine the effect of zinc supplementation on the quantitative and qualitative characteristics of semen along with oxido-sensitive index level (superoxide dismutase/xanthine oxidase ratio) in the seminal plasma of asthenospermic patients. METHODS: Semen samples were obtained from 60 fertile (age 31.6 ± 3.3 years) and 60 asthenospermic men (age 32.5 ± 3.23 years) from July 2011 to July 2012, from couples who had consulted the infertility clinic of the Babil hospital of maternity (Hillah, Iraq). The subfertile group was treated with zinc sulfate, every participant took 2 capsules (220 mg each) of zinc sulfate per day for 3 months. Semen samples were obtained (before and after zinc sulfate supplementation). Oxido-sensitive index level, catalase-like activity and various sperm parameters were measured. RESULTS: The value of the oxido-sensitive index of fertile controls (1.28 + 0.31 in seminal plasma and 1.57 + 0.62 in spermatozoa) was significantly higher than that of the infertile patient group (0.56 + 0.48 in seminal plasma and 0.65 + 0.57 spermatozoa) (p = 0.0001). Oxido-sensitive index levels were significantly higher in the infertile group treated with zinc sulfate (1.13 + 0.22 in seminal plasma and 1.15 + 0.16 in spermatozoa) (p = 0.001). Catalase-like activity was increased significantly in spermatozoa and seminal plasma of patients compared to that of healthy controls. Volume of semen, progressive sperm motility and total normal sperm count were increased after zinc supplementation. CONCLUSION: Zinc supplementation restores oxido-sensitive index and catalase-like activity in semen of asthenozoospermic subjects to normal ranges.

Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients.

BACKGROUND: Low concentrations of nitric oxide (NO) are necessary for the biology and physiology of spermatozoa, but high levels of NO are toxic and have negative effects on sperm functions. Although several studies have considered the relationship between infertility and semen NO concentrations, no study on the effects of asthenospermia treatments such as oral zinc supplementation on concentrations of NO, which are important in fertility, has been reported. Studies have shown that oral zinc supplementation develops sperm count, motility and the physical characteristics of sperm in animals and in some groups of infertile men. The present study was conducted to study the effect of zinc supplementation on the quantitative and qualitative characteristics of semen, along with enzymes of the NO pathway in the seminal plasma of asthenospermic patients. METHODS: Semen samples were obtained from 60 fertile and 60 asthenozoospermic infertile men of matched age. The subfertile group was treated with zinc sulfate; each participant took two capsules (220 mg per capsule) per day for 3 months. Semen samples were obtained (before and after zinc sulfate supplementation). After liquefaction of the seminal fluid at room temperature, routine semen analyses were performed. The stable metabolites of NO (nitrite) in seminal plasma were measured by nitrophenol assay. Arginase activity and NO synthase activity were measured spectrophotometrically. RESULTS: Peroxynitrite levels, arginase activity, NO synthase activity and various sperm parameters were compared among fertile controls and infertile patients (before and after treatment with zinc sulfate). Peroxynitrite levels and NO synthase activity were significantly higher in the infertile patients compared to the fertile group. Conversely, arginase activity was significantly higher in the fertile group than the infertile patients. Peroxynitrite levels, arginase activity and NO synthase activity of the infertile patient were restored to normal values after treatment with zinc sulfate. Volume of semen, progressive sperm motility percentage and total normal sperm count were increased after zinc supplementation. CONCLUSIONS: Treatment of asthenospermic patients with zinc supplementation leads to restored peroxynitrite levels, arginase activity and NO synthase activity to normal values and gives a statistically significant improvement of semen parameters compared with controls.

Oral zinc supplementation restores high molecular weight seminal zinc binding protein to normal value in Iraqi infertile men.

BACKGROUND: Zinc in human seminal plasma is divided into three types of ligands which are high (HMW), intermediate (IMW), and low molecular weight ligands (LMW). The present study was aimed to study the effect of Zn supplementation on the quantitative and qualitative characteristics of semen along with Zinc Binding Protein levels in the seminal plasma in asthenozoospermic patients. METHODS: Semen samples were obtained from 37 fertile and 37 asthenozoospermic infertile men with matched age. The subfertile group was treated with zinc sulfate, every participant took two capsules per day for three months (each one 220 mg). Semen samples were obtained (before and after zinc sulfate supplementation). After liquefaction seminal fluid at room temperature, routine semen analyses were performed. For determination of the amount of zinc binding proteins, the gel filtration of seminal plasma on Sephadex G-75 was performed. All the fractions were investigated for protein and for zinc concentration by atomic absorption spectrophotometry. Evaluation of chromatograms was made directly from the zinc concentration in each fraction. RESULTS: A significant high molecular weight zinc binding ligands percentage (HMW-Zn %) was observed in seminal plasma of fertile males compared with subfertile males. However, seminal low molecular weight ligands (LMW-Zn) have opposite behavior. The mean value of semen volume, progressive sperm motility percentage and total normal sperm count were increased after zinc sulfate supplementation. CONCLUSIONS: Zinc supplementation restores HMW-Zn% in seminal plasma of asthenozoospermic subjects to normal value. Zinc supplementation elevates LMW-Zn% in seminal plasma of asthenozoospermic subjects to more than normal value. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01612403.