This method can help answer key questions in the energetics field, such as mitochondrial health, energetic efficiency and energy usage. The main advantage of this technique is that oxygen consumption of isolated mitochondria is measured directly. Results do not need to be corrected for other intracellular metabolic processes.
We first decided to pursue this method in dairy cattle when we wanted to examine how mitochondrial efficiency impacts feed efficiency. Video demonstration of this method is critical as the steps in mitochondrial isolation are difficult to learn because they need to be consistent and precise. As soon as possible after the liver sample is removed from the cow wash the sample in mitochondria isolation media to remove the red blood cells.
Using scissors finely mince the tissue into a chilled beaker that contains enough isolation media to keep the tissue moist. Transfer the minced liver into a 30-milliliter glass vial containing mitochondria isolation media. Add a Teflon pestle that has a clearance of 0.16 millimeters and that has been incubated in ice and homogenize the liver sample at 500 RPM for one minute with four strokes per minute.
After this, transfer the homogenate to a tube containing mitochondria isolation media and place this into an ice-packed beaker. Centrifuge homogenate at 500 times G and four degrees Celsius for 10 minutes. Discard the pellet and transfer the supernatant to a chilled centrifuge tube.
Centrifuge the resulting supernatant at 10, 000 times G and four degrees Celsius for 10 minutes to obtain the mitochondrial pellet. Resuspend and wash the pellet in 10 milliliters of mitochondria isolation media with fatty acid-free BSA. Then centrifuge at 8, 100 times G and four degrees Celsius for 10 minutes.
Discard the supernatant and resuspend the pellet in 200 microliters of isolation media. Place this on ice until ready to use for the oxygen consumption and proton leak kinetic assays. Determine protein concentration of the pellet suspension using a bicinchoninic acid kit per manufacturer's instructions using BSA as the standard.
First, create oxygen consumption media as outlined in the text protocol. Incubate the oxygen consumption media at 30 degrees Celsius. Next, set up the respiration chamber, pump and oxygen electrode for the oxygraph system according to the manufacturer's instructions.
Then place one milliliter of oxygen consumption media into the respiration chamber. Then add 0.35 milligrams of mitochondrial protein to the chamber. Stir vigorously to ensure that the solution becomes saturated with air and maintain a temperature of 30 degrees Celsius.
Record the oxygen consumption for approximately five minutes. When oxygen consumption becomes constant represented by a decreasing straight line, record this as the baseline oxygen consumption. Add 1.25 microliters of a four-millimolar rotenone solution to inhibit complex one, and then add five microliters of one molar succinate solution to reach a final concentration of five millimolar succinate in the respiratory chamber.
This is state IV oxygen consumption. After this, add one microliter of a 100-millimolar ADP solution to reach a final concentration of 100 micromolar in the respiration chamber. Oxygen consumption will decrease for approximately five minutes and then will become a straight line.
Record this oxygen consumption as the state III respiration. Calculate the respiratory control ratio as outlined in the text protocol. Then aspirate all of the solutions out of the respiration chamber.
Rinse the chamber several times with double deionized water. First, prepare a solution of 80 nanograms per milliliter of nigericin in ethanol limiting the amount of ethanol added to less than one microliter. Also, prepare a solution of eight micrograms per milliliter oligomycin in ethanol.
After thoroughly rinsing the chamber with double deionized water, place one milliliter of the oxygen consumption media into the respiration chamber and stir vigorously with a magnetic stir bar. Add 0.35 milligrams of mitochondrial protein to the respiration chamber. Add a methyl triphenylphosphonium-sensitive electrode to the chamber setup making sure that the other end is properly connected to a pH meter.
Add 1.25 microliters of a four-millimolar rotenone solution to inhibit Complex I.Record the respiration for two to five minutes. Record the oxygen consumption value when it becomes constant. Next, add 0.56 microliters of an oligomycin solution to inhibit ADP utilization.
Record the respiration for about two to five minutes. Record the oxygen consumption value when it becomes constant. Then add 0.112 microliters of an 80 nanograms per milliliter nigericin solution to abolish the pH gradient across the mitochondrial inner membrane.
Record respiration for two to five minutes and note the oxygen consumption value when it becomes constant. Add five microliters of 10 millimolar methyl triphenylphosphonium solution to the mitochondrial incubation to begin preparing a standard curve. Repeat this addition four additional times until a total concentration of 2.5.
micromolar has been added. After this, add five microliters of one molar succinate solution to the chamber to initiate respiration. Record the respiration until a stable trace is achieved.
Then add malonate to titrate the system as outlined in the text protocol. In this study, RCR and proton leak kinetics are measured in the milk of Holstein dairy cows 70 days after the dairy cows had been fed with differing levels of copper, zinc, and manganese for 28 days. State IV respiration is seen to be the highest in cows given low levels of manganese, yet is lowest in the control cows, indicating that manganese plays an important role in minimizing proton leak-dependent respiration.
Analysis of the hepatic proton leak-dependent respiration reveals that this respiration is greatest in cows treated with low manganese, and the lowest in control cows. These results confirm that the low manganese group has the greatest state IV respiration while the control group has the lowest. While feed efficiency is higher in Angus steers born from low RFI bulls than high RFI bulls, this was not reflected in the mitochondrial RCR or the proton leak kinetics.
While performing this procedure it's important to keep all vials and beakers on ice while processing the mitochondria. After its development this technique paved the way for researchers in the field of dairy nutrition to explore the role of mitochondria efficiency in feed efficiency and mitochondrial function in liver. Don't forget that working with rotenone can be dangerous, and precautions such as wearing gloves, eye protection and proper PPE should always be taken while performing this procedure.
