Ameliorative Effects of Testosterone Administration on Renal Redox Homeostasis in Naturally Aged Rats

Assay of tyrosine-derived oxidation marker

Analysis of protein-bound DT was performed using the spectrofluorimetric method according to Sadowska-Bartosz et al. ²⁵ with some modifications in the pre-analytical step. The pre-treatment of supernatant samples was done to avoid the possible fluorescence interference derived from tissue constituents that were processed in the following way. Proteins of the supernatant samples were precipitated with 30 mM perchloric acid for 20 min on ice. After centrifugation for 15 min at 5000×g, the pellets were re-dissolved and diluted (1:20) in phosphate-buffered saline (PBS) buffer (pH 7.4), and then used for fluorescence spectroscopy. Fluorescence intensity was expressed in fluorescence units (FU), and the results were expressed as FU/mg protein. Supernatant fractions were diluted (1:20) with PBS (pH 7.4), and fluorescence intensity was recorded at the emission maximum (415 nm) upon excitation at 330 nm. The coefficients of intra- and inter-assay variations for DT assay were 4.2% (n=8) and 5.6% (n=8), respectively.

Assay of tryptophan-derived oxidation markers

KYN and N-FKYN contents were analyzed using the spectrofluorimetric method according to Sadowska-Bartosz et al. ²⁵ with some modifications in the pre-analytical steps. The pre-treatment of supernatant samples was to avoid the possible fluorescence interference derived from tissue constituents. It was performed as follows. Supernatant proteins of the homogenates were precipitated with 30 mM perchloric acid for 20 min on ice. After centrifugation for 15 min at 5000×g, the final protein pellets were re-dissolved and diluted (1:20) in PBS buffer (pH 7.4), and they were used for fluorescence spectroscopy. Fluorescence intensity was recorded at the emission maximum (480, 434 nm) upon excitation at 365, 325 nm, respectively. Fluorescence intensity was expressed in FU, and the results were given as FU/mg protein.

Protein carbonyl groups

The PCO assay was performed as previously described by Reznick and Packer ²⁶ with some slight modifications to apply small volumes of supernatant samples. PCO groups react with 2,4-dinitrophenylhydrazine (DNPH) reagent (100 μL of supernatant:400 μL of DNPH) to form chromophoric dinitrophenylhydrazones. DNPH was dissolved in HCl, and after the DNPH reaction proteins were precipitated with an equal amount of 20% (wt/vol) trichloroacetic acid (TCA) and washed three times with 400 μL of an ethanol/ethyl acetate mixture (1:1). The washing procedure was performed by mechanical disruption of pellets, and the re-pelleting process was accomplished by centrifugation at 3000×g for 5 min. Finally, the protein precipitates were dissolved in a 200 μL of 6 M guanidine-HCl solution. The final absorbance values were recorded at 360 nm using the molar extinction coefficient of DNPH, ε=22,000 M⁻¹cm⁻¹ The coefficients of intra- and inter-assay variations for the modified PCO assay were 3.6% (n=8) and 6.1% (n=8), respectively. The PCO-BSA–positive control and un-treated BSA were both prepared according to the method of Lenarczyk et al. ²⁷ and tested according to the PCO assay protocol.

Protein hydroperoxides

P-OOHs were analyzed with the ferric-xylenol orange (FOX) method, which was established by us. Our P-OOH assay is based on the oxidation of Fe²⁺ by P-OOHs in the presence of the dye xylenol orange (XO) that gives a colored complex with the Fe³⁺ ions. The Fe⁺⁺–XO complex can be analyzed in the visible absorbance range (560 nm). Proteins were precipitated from 25 μL of supernatant by adding 125 μL of 30 mM ice-cold perchloric acid. Precipitated samples were kept on ice for 5 min and centrifuged at 5000×g. The protein pellet was then washed twice with chloroform containing 4 mM butylated hydroxytoluene solution. The precipitated proteins were then dissolved in 150 μL of PBS. Fifty-microliter aliquots of dissolved proteins were transferred into microcentrifuge reaction vials. The FOX reagent was prepared as previously described, ²⁸ 950 μL was added to the tubes, and the samples were mixed by vortexing. After incubation with FOX2 (ferrous oxidation with xylenol orange, version 2) reagent at room temperature for 30 min, the samples were centrifuged at 3000×g at 20°C for 10 min. The resulting supernatant was carefully transferred into microplate wells. Absorbance values were recorded at 560 nm against a reagent blank. The molar concentration of P-OOHs in the final medium was calculated from the standard curve prepared by hydrogen peroxide (H₂O₂) in methanol. The coefficients of intra- and inter-assay variations for P-OOH assay were 3.0% (n=8) and 4.9 % (n=8), respectively. The P-OOH-BSA–positive control and untreated BSA were both prepared in vitro according to the method of Peskin et al. ²⁹ and analyzed according to the assay protocol.

Advanced oxidation protein products

Spectrophotometric analysis of AOPP concentrations was performed with modifications of the Hanasand method. ³⁰ Samples were prepared as in the following way. A 1-μL amount of supernatant and 200 μL citric acid (20 mmol /L) were mixed in a microplate. After 1 min, 10 μL of 1.16 M potassium iodide was added to the microplate wells, and the absorbance of the reaction mixture was recorded at 340 nm against a reagent blank. All readings were performed within 2 min after potassium iodide addition to avoid uncontrollable color development, which leads to possible deviation from the chloramine standard curve. The absorbance of chloramine-T standards was run in duplicate to increase precision of the AOPP assay at 340 nm. The related absorbance values were linear within the range of 0–100 μmol/liter. AOPP concentration was expressed as micromoles per liter of chloramine-T equivalents. The coefficients of intra- and inter-assay variations were 1.3% (n=8) and 2.0% (n=8), respectively. The AOPP-BSA–positive control and untreated BSA were both prepared in vitro and tested according to the AOPP assay protocol. ³¹

Protein thiol groups

The concentration of the thiol (–SH) group containing redox status biomarkers such as T-SH, NP-SH, and P-SH was analyzed by using the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) method as described by Sedlak and Lindsay. ³² We made some slight modifications from previously described –SH methods to apply small volumes of supernatant samples. A 20-μL amount of the supernatant was mixed in a 1.5-mL test tube with 400 μL of 0.2 M Tris buffer (pH 8.2) and 20 μL of 0.01 M DTNB for the determination of T–SH groups. Samples for NP-SH determination were assayed as follows. A 20-μL amount of supernatant was mixed in 400 μL of 50% TCA. The tubes were mixed intermittently for 10 min and centrifuged at 3000×g for 15 min. Supernatant fractions were assayed as for T-SH. The absorbance values of the resulting samples were recorded at a 412-nm wavelength against a reagent blank. The value of molar extinction coefficient of –SH groups at wavelength 412 nm is ε=13,100 M⁻¹ cm⁻¹. The P-SH groups were calculated by subtracting the NP-SH from T-SH. The coefficients of intra- and inter-assay variations were 1.5% (n=8) and 3.4% (n=9), respectively.

Malondialdehyde

MDA is considered as one of the major secondary reactive products of lipid peroxidation. MDA, along with other by-products, reacts with thiobarbituric acid (TBA) to generate a colored product, which absorbs maximally at 535 nm, representing the color produced by all the TBA-reactive substances. The rate of lipid peroxidation was determined by the procedure of Buege and Aust with slight modifications comprising only volumetric changes proportionally. ³³ The pre-treatment of supernatant samples to avoid the possible interference derived from tissue constituents was performed according to Lykkesfeldt. ³⁴ Ten microliters of supernatant was placed in a microcentrifuge tube; 500 μL of 42 mmol/L H₂SO₄ was added and mixed gently, after which 125 μL of 100 g/L phosphotungstic acid was added and mixed by vortexing. After 5 min at room temperature, this mixture was centrifuged (3 min at 6000×g). MDA is associated with the lipoprotein and consequently was contained in the pellet. The resulting pellet was re-suspended in 750 μL of 0.75 % TBA and 500 μL of 30% TCA boiled for 15 min at 95°C and cooled. The mixture was centrifuged at 3000×g for 5 min, and the absorbance of supernatant was read at 532 nm against a reagent blank. Intra-assay variation of the MDA assay is controlled by duplicate samples so that more precise test results can be obtained. The concentration of MDA in supernatant was calculated using the molar extinction coefficient (ε=31,500 M⁻¹cm⁻¹) and it is expressed as μmol /mg protein. The coefficients of intra- and inter-assay variations for MDA assay were 3.4% (n=10) and 4.2% (n=10), respectively.

Lipid hydroperoxides

L-OOHs levels were determined spectrophotometrically by the method that uses the oxidation of ferrous ions with xylenol orange FOX2. ²⁸ L-OOHs oxidized ferrous to ferric ions selectively in dilute acid, and the resultant ferric ions were determined by using ferric-sensitive dye, which represents the concentration of L-OOHs. XO binds to ferric ions with high selectivity to produce a colored (blue–purple) complex. Fifty-microliters aliquot of homogenates were transferred into microcentrifuge reaction vials. FOX2 reagent (950 μL) was then added, and the samples were mixed by vortexing. After incubation with FOX2 reagent at room temperature for 30 min, the samples were centrifuged at 3000×g at 20°C for 10 min, the final supernatant was carefully transferred into microplate wells and absorbance was recorded at 560 nm against reagent blank The coefficients of intra- and inter-assay variations were 2.1% (n=8), and 2.0% (n=11), respectively.

Conjugated dienes

CD levels were determined with the CDs method of Francesco and Sebastiano. ³⁵ We made some volumetric modifications of the previously established method that gave increased sensitivity and high reproducibility to the CD assay. At first, tissue lipids were isolated from kidney tissue samples according to the method of Folch et al. ³⁶ The supernatants were mixed with chloroform/methanol (2v/1v) reagent to a final volume 20 times the volume of the sample at first. Then the whole mixtures were placed in an orbital shaker at room temperature for 15 min. Resulting samples were centrifuged to recover the liquid phase. The solvents were washed with 4 mL of 0.9% NaCl. After vortexing for a few seconds, the mixtures were centrifuged at 2000×g to separate two phases. Upper phases were removed, and the lower chloroform phases containing lipids were evaporated under a nitrogen stream. After the evaporation stage, the samples were dissolved in cyclohexane. All of the samples were subjected to spectral scanning between 230 and 250 nm to determine wavelength peaks between 230–260 nm. Both CDc,t and CDt,t present distinct absorbance at 242 nm and 233 nm, respectively. CDs detected in tissue lipid extracts were improved by second derivative spectrophotometry by using the following formula: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}^{ \rm n} { \rm D}_{{\rm x}\lambda} = { \rm d}^{ \rm n} { \rm A} / { \rm d} \lambda^{ \rm n} = { \rm f} ( \lambda ) \quad { \rm or} \quad ^{ \rm n} { \rm D}_{ \rm x , v} = { \rm d}^{ \rm n} { \rm A} / { \rm dv}^{ \rm n} = { \rm f} ( \rm v)\end{align*} \end{document}

expressing: n, derivative order; ⁿD_xλ or ⁿD_x,v, value of an order derivative of an analyte (x); λ, analytical wavelength; v, wavelength number; and A, absorbance. The second derivative spectra showed fine spectral structures with at least three negative peaks. The wavelengths of their minima are different for the geometric isomers CDc,t and CDt,t, allowing determination of each individual isomer in a mixture without any separation.

Protein-bound advanced glycation end products

Determination of protein-bound AGEs (i.e., some fluorescent products from the family of AGEs) was realized on the basis of the spectrofluorimetric method according to Munch et al. ³⁷ The pre-treatment of supernatant samples was done to avoid the possible fluorescence interference derived from tissue constituents as follows. Supernatant proteins of the homogenates were precipitated with 100 grams/L TCA for 20 min on ice. After centrifugation for 15 min at 5000×g and three washing steps with 100 grams/L TCA, the pellets were re-dissolved and diluted (1:20) in PBS buffer (pH 7.4) and then used for fluorescence spectroscopy. Fluorescence intensity was expressed in FU, and the results were given as FU/mg protein. Supernatant was diluted (1:20) with PBS (pH 7.4), and fluorescence intensity was recorded at the emission maximum (440 nm) upon excitation at 350 nm. The coefficients of intra- and inter-assay variations for protein- bound AGE assay were 3.8% (n=8) and 5.3% (n=8), respectively. The AGE-BSA–positive control and untreated BSA were both prepared according to the method of Wrobel et al. ³⁸ and tested according to the assay protocol.

Ferric reducing anti-oxidant power assay

The anti-oxidant status of supernatant was evaluated using the ferric reducing anti-oxidant power (FRAP) assay. ³⁹ The modified FRAP assay uses reductant anti-oxidants in a redox-linked colorimetric method. In this assay, at low pH, a ferric–2,4,6-tripyridyl-s-triazine (Fe^III–TPTZ) complex was reduced to the ferrous form, which was blue in color and monitored by measuring the change in absorption at 593 nm. The change in absorbance is directly proportional to the reducing power of the electron-donating anti-oxidants present in the sample. Three hundred mmol/L of acetate buffer (pH 3.6), 10 mmol/L TPTZ in 40 mmol/L HCl, and 20 mmol/L FeCl₃ ċ 6H₂O in the ratio of 10:1:1 give the working FRAP reagent. Fe(II) standards were used. Fe(II) (1000 μmol/L) is equivalent to 1000 μmol/L of FRAP. A 750-μL amount of working FRAP reagent was mixed with 25 μL of supernatant or standard in a test tube. The absorbance at 593 nm was recorded against the reagent blank. The absorbance change was converted into a FRAP value as mM by relating the change of absorbance at 593 nm of the test sample to that of a standard solution of known FRAP value (3–0.375 mM). The coefficients of intra- and inter-assay variations for modified FRAP assay were 1.6% (n=8) and 2.8 % (n=8), respectively.

Cu-Zn SOD activity

Cu-Zn SOD activity (Cu-Zn SOD) (EC 1.15.1.1) activity was measured by using the method of Sun and Oberley ⁴⁰ with some volumetric modifications. This method involves the inhibition of nitroblue tetrazolium (NBT) reduction, with xanthine oxidase used as a superoxide anion generator. Enzyme activity was determined by measuring the inhibition rate of substrate hydrolysis in the assay mixture containing 0.3 mmol/L xanthine, 0.6 mmol/L Na₂EDTA, 150 μmol/L NBT, 400 mmol/L Na₂CO₃, and 1 gram/L BSA. The pH value of the assay mixture was adjusted to pH 10.2. A 972-μL amount of assay mixture and 13 μL of xanthine oxidase (167 U/L) were added into 25 μL supernatant sample. At the end of the 20-min incubation period, 250 μL of 0.8 mmol/L CuCl₂ was added to the microplate wells to terminate the reaction. The final absorbance was recorded at 560 nm against a reagent blank. The percent inhibition rate was calculated according to the following equation: A_blank − A_sample/A_blank.100. One unit of Cu-Zn SOD is defined as the amount of enzyme needed to exhibit a 50% dismutation of superoxide radical anion. The coefficients of intra- and inter-assay variations for modified Cu-Zn SOD assay were 3.8% (n=8) and 4.2% (n=8), respectively.

Catalase activity

CAT activity (E.C.1.11.1.6) was determined by the method described by Aebi. ⁴¹ The decomposition rate of the substrate H₂O₂ was monitored at 240 nm. A molar absorptivity of 43.6 L mol⁻¹ cm⁻¹ was used to calculate the activity. One unit is equal to 1 μmol of H₂O₂ decomposed/min. CAT activity was expressed as U/mg protein.

Xanthine oxidase activity

Xanthine oxidase (E.C.1.17.3.2) activity was measured by using the method of Prajda and Weber. ⁴² This assay was based on oxidation of xanthine to uric acid by xanthine oxidase. The amount of uric acid was stabilized by using 100% TCA, and the absorbance was reading at 293 nm. The supernatant was pre-incubated for 40 min at 37°C and then added to the reaction mixture, which contained in final concentrations xanthine (0.17 mM), phosphate buffer (33 mM, pH 7.5), and a suitable amount of enzyme (supernatant). The reaction was carried out at 37°C and was stopped at 0 and 20 min by addition of 0.1 mL of 100% (wt/vol) TCA. The mixture was centrifuged at 10,000×g for 15 min. In the clear supernatants, the uric acid produced from the substrate, xanthine, was measured by the increase in absorbancy at 293 nm.