Skeletal muscle serves as a major reservoir of nitrate which, after its conversion to nitrite, serves as a source of NO during exercise. Here we present methodology to measure these ions in muscle and other tissues. To begin, prepare the nitrate preserving or stop solution by combining 890 millimolar potassium ferricyanide and 118 millimolar n-methylmaleimide in distilled water, ensuring that no crystals remain in solution, then add a non-ionic surfactant in a 1-to-9 ratio and mix gently to avoid foaming.
Dilute the stop solution in a 1-to-9 ratio with distilled water and add the diluted stop solution to the homogenization tube. Next, thaw the previous isolated and frozen rat muscle tissue on ice. Once the tissue has thawed, remove the remaining fat and connective tissue from the skeletal muscle.
Cut off pieces of the skeletal muscle and blot them on a gauze to dry. Weigh out the appropriate amount of skeletal muscle tissue, then place the pre-weight tissue in the homogenization tubes containing the stop solution. For homogenization in a rotary homogenizer, place the M-type tube containing the pre-weighed skeletal muscle and pre-measured stop solution into the machine.
Homogenize each sample twice, and after each homogenization, place the tube on ice immediately for 5 minutes to cool down. After homogenization, centrifuge the tube briefly. Place the tube back on ice and add the appropriate volume of methanol before vortexing For 15 seconds.
Homogenize the sample again and incubate it on ice for 30 minutes, then centrifuge the sample, aspirate the supernatant, and proceed to measuring the nitrite and nitrate levels. For bead homogenization, place the skeletal muscle tissue in a bead-containing tube and homogenize twice for 45 seconds at the highest speed available on the instrument. After each homogenization, immediately place the tube on ice for 5 minutes to cool down, then centrifuge the tube, place the tube back on ice, and add the appropriate volume of methanol before vortexing For 15 seconds.
Homogenize the sample once again for 45 seconds then incubate it on ice for 30 minutes. Centrifuge the sample, aspirate the supernatant, and proceed to measuring the nitrite and nitrate levels. For pulverizer-based homogenization, prepare tubes containing the diluted stop solution, then weigh and record the weight of the tubes.
After cooling the pulverizer tool on dry ice for 30 minutes, using tweezers chilled in liquid nitrogen, transfer the pre-weighed tissue sample to the pulverizer, then add a small amount of liquid nitrogen to ensure the tissue is at liquid nitrogen temperature. After 95%of the liquid nitrogen has vaporized, place the crushing tool on top of the tissue and press firmly to feel the sample crush, then using a mallet, strike the crushing tool 3 to 5 times. When the whole sample has been pulverized, using a liquid nitrogen-cooled spoon and tweezers, directly transfer the crushed tissue into the pre-weighed tube containing the diluted stop solution.
Next, vortex the tube for 15 seconds, then open the tube to check for any tissue stuck in the lid. If there is, try to dislodge it, then vortex again. Briefly centrifuge the sample for 2 to 3 seconds.
Weigh the tube again and calculate the tissue weight by deducting the original tube weight from this new weight. Place the tube on ice. Add an appropriate volume of methanol to the tube and vortex the tube thoroughly for 15 seconds before incubating it on ice for 30 minutes, then centrifuge the sample, aspirate the supernatant, and proceed to measuring the nitrite and nitrate levels.
Nitrate levels and rat skeletal muscle homogenates prepared using a rotary homogenizer, bead homogenizer, and pulverizer were similar. Interestingly, for nitrite levels, the homogenate sample prepared by a pulverizer showed the highest value, which was statistically different from the other two sample values. A comparison of nitrite and nitrate levels in gluteus homogenates prepared from 20, 50, and 200 milligrams of tissue using a bead homogenizer showed that neither the nitrate nor nitrite levels were significantly different between the muscle sample sizes.
Comparing nitrate levels in different rodent leg skeletal muscle tissues revealed that the gluteus muscle had approximately twofold higher nitrate levels than the other three muscle tissues. However, these differences did not reach statistical significance. Similar to the nitrate levels, the nitrate concentration in the gluteus muscle was also higher than that in the other three muscle tissues and was significantly higher than that in the gastrocnemius sample.
Our method is relatively simple and well-suitable to small samples while preserving nitrate and nitrite during the sample preparation.
