Aging Aging

Xenobiotic-Metabolizing Enzymes 127pH meter the pH of the buffer is adjusted to 7.8 using 3 M HCl. The buffer istransferred to a 1-L volumetric flask and made up to the 1-L mark with distilledwater. The pH of the buffer solution is confirmed at pH 7.8 using a pH meter.3. NADPH (50 mM, Sigma Chemical, Dorset, UK) is dissolved in 1% (w/v) sodiumhydrogen carbonate and kept at 4°C until required. This solution is made freshprior to performing the assays.4. Dicoumarol (20 mM, Sigma Chemical, Dorset, UK) is prepared by dissolvingdicoumarol in 0.1 M Tris-HCl buffer, pH 7.8.5. Spectrofluorometer with excitation wavelength of 510nm and emission wavelengthof 586 nm with excitation and emission slit widths of 10nm and 2.5nm,respectively.6. Positive controls. Samples of rodent liver microsomes that have high activity forethoxyresorufin, methoxyresorufin, or pentoxyresorufin can be obtained fromXentox Limited, Northern Ireland, UK.3. Methods3.1. Preparation of Hepatic Subcellular FractionsMicrosomal fractions are prepared according to the method of Ioannidesand Parke (26).1. Liver sample is weighed and and transferred to a glass beaker with volume capacityat least 5× that of the weight of the liver sample, for example, 10 g of liver ina 50-mL beaker.2. The sample is scissor-minced and transferred to the Potter–Elvehjem homogenizertogether with 3× the liver weight of 1.15% KCl (4°C).3. Homogenize the sample using several up-and-down strokes of the homogenizer.4. The homogenate should be maintained at 4°C during the homogenizationprocess using an ice jacket (metal can filled with ice surrounding the glasshomogenizer).5. The homogenate is transferred to a measuring cylinder and made up to 4× theinitial sample weight with 1.15% (w/v) KCl, for example, 10 g of liver samplemade up to 40 mL of final homogenate volume with 1.15% (w/v) KCl. This is a25% w/v liver homogenate.6. The homogenate is transferred to centrifuge tubes and the tubes balanced forcentrifugation at 9000g for 20 min.7. Following centrifugation at 9000g for 20 min in a refrigerated (4°C) centrifugethe supernatant (S9) is decanted and may be stored at –70°C for up to 6 mo.8. For microsomal preparation the S9 is transferred to ultracentrifuge tubes and balancedfor ultracentrifugation at 105,000g for 60 min at 4°C.9. The supernatant (cytosolic fraction) is discarded and the pellet resuspendend in avolume of 1.15% w/v KCl equal to the volume of S9 initially placed into theultracentrifugation tube.10. The microsomal suspension should be kept on ice and used the same day.