A subscription to JoVE is required to view this content. Sign in or start your free trial.
Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.
Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.
Mitochondria are very sensitive to stress and their cellular environment can contribute to a wide variety of metabolic diseases. Oxygen consumption and proton leak in mitochondria are indicators of mitochondria health. The methods described in this paper estimate mitochondrial energy efficiency using RCR based on oxygen consumption with and without proton leak. These results can account for 30% of the energy lost in nutrient utilization1. Changes in oxygen consumption and proton leak can identify mitochondrial dysfunction which contributes to metabolic disease and results in decreased energy efficiency. These methods can also be used to examine....
All methods, protocol and studies described here were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, Davis.
1. Obtaining a Liver Biopsy from a Holstein Dairy Cow
NOTE: A liver biopsy should be performed by a licensed veterinarian. Liver biopsies can be performed on the dairy site where the cows are located. Lactating dairy cows can continue to be milked normally and milk does not need to be withdrawn from the food supp.......
Positive results showing RCR and proton leak kinetics are shown in Table 1 and Figure 15, respectively. In this study7, RCR and protein leak kinetics were measured in Holstein dairy cows at 70 days in milk after cows had been fed 1 of 5 different levels of Cu, Zn and Mn for 28 days. State 4, maximum proton leak-dependent respiration, had a tendency to be affected by mineral intake of Cu, Mn and Zn (p < 0.1.......
The most critical point in the protocol is obtaining a representative liver tissue sample and beginning the isolation of mitochondria as soon as possible after biopsy. Variation in respiration measurements is low (Table 1) due to a short transport time from cow to laboratory. To reduce transport time, a small laboratory was set up in the office of the dairy, and liver samples were driven to the office laboratory as each was collected so that mitochondria were isolated within 10 min of biopsy. Setup and t.......
The authors have nothing to disclose.
This research was supported by Alltech and USDA Hatch funds through the Center for Food Animal Health at UC Davis School of Veterinary Medicine.
....Name | Company | Catalog Number | Comments |
Liver Biopsy | |||
Equipment | |||
Schackelford-Courtney bovine liver biopsy instrument | Sontec Instruments Englewood CO | 1103-904 | |
Suture | Fisher Scientific | 19-037-516 | |
Suture needles | NA | NA | Included with Suture |
Scalpels | Sigma - Aldrich | S2896 / S2646 | # for handle and blades |
Surgery towels | Fisher Scientific | 50-129-6667 | |
Falcon tubes 50 mL | Fisher Scientific | 14-432-22 | |
Tweezers | Sigma - Aldrich | Z168750 | |
50 mL syringes | Fisher Scientific | 22-314387 | |
Injection needles (22, 2 1/2) | VWR | MJ8881-200342 | |
Cow halter | Tractor Supply Co. | 101966599 | |
Cotton swabbing | Fisher Scientific | 14-959-102 | |
cotton gauze squares (4x4) | Fisher Scientific | 22-246069 | |
Medical scissors | Sigma - Aldrich | Z265969 | |
Chemicals | |||
Coccidiosis Vaccine 0.75 bottle/cow | Provided by Veterinarian | ||
Clostridia Vaccine | Provided by Veterinarian | ||
Liver biopsy antibiotics excenel 2 cc/100 lbs for 3 days | Provided by Veterinarian | ||
Providone Scrub | Aspen Veteterinary Resources | 21260221 | |
Ethanol 70% | Sigma - Aldrich | 793213 | |
Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg/kg bodyweight | Provided by Veterinarian | ||
2% lidocaine HCl (10-15 mL) | Provided by Veterinarian | ||
1 mg/kg IV injection of flunixin meglumine | Provided by Veterinarian | ||
Isolation of Mitochondria (liver) | |||
Equipment | |||
Wheaton vial 30 mL with a Teflon pestle of 0.16 mm clearance | Fisher Scientific | 02-911-527 | |
Homogenizer Motor | Cole Parmer | EW-04369-10 | |
Homogenizer Probe | Cole Parmer | EW-04468-22 | |
Auto Pipette (10 mL) | Cole Parmer | SK-21600-74 | |
Beaker (500 mL) with ice | Fisher Scientific | FB100600 | |
Refrigerated microfuge | Fisher Scientific | 75-002-441EW3 | |
Microfuge tubes (1.5 mL) | Fisher Scientific | AM12400 | |
Chemicals | |||
Bicinchoninic acid (BCA) protein assay kit (microplates for plate reader) | abcam | ab102536 | |
Sucrose | Sigma - Aldrich | S7903-1KG | |
Tris-HCl | Sigma - Aldrich | T1503-1KG | |
EDTA | Sigma - Aldrich | EDS-1KG | |
BSA (fatty acid free) | Sigma - Aldrich | A7030-50G | |
Mannitol | Sigma - Aldrich | M4125-1KG | |
Deionized water | Sigma - Aldrich | 38796 | |
Hepes | Sigma - Aldrich | H3375-500G | |
Use to create mitochondria isolation media: 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 20 mM Tris-HCl, 1 mM EDTA, and 0.1% (w/v) fatty acid free BSA, pH 7.4 at 4 °C, will last 2 days in refrigerator | |||
Mitochondrial Oxygen Comsuption | |||
Equipment | |||
Oxygraph Setup + Clark type oxygen electrode | Hansatech (PP Systems) | OXY1 | |
Thermoregulated Water Pump | ADInstruments | MLE2001 | |
Clark type Oxygen electrode | NA | NA | |
Autopipette (1 mL) | Cole Parmer | SK-21600-70 | Included with Oxy1 |
Small magnetic stir bar | Fisher Scientific | 14-513-95 | |
Micropipette (10 μL) | Cole Parmer | SK-21600-60 | |
pH meter | VWR | ||
Chemicals | |||
KCl | Sigma - Aldrich | P9333-1KG | |
Hepes | Sigma - Aldrich | H3375-500G | |
KH2PO4 | Sigma - Aldrich | P5655-1KG | |
MgCl2 | Sigma - Aldrich | M1028-100ML | |
EGTA | Sigma - Aldrich | E3889-100G | |
Use to make mitochondrial oxygen consumption media: 120 mM KCL, 5 mM KH2PO4, 5 mM MgCl2, 5 mM Hepes and 1 mM EGTA, pH 7.4 at 30 °C with 0.3% defatted BSA | |||
Rotenone (4 mM solution) | Sigma - Aldrich | R8875-5G | |
Succinate (1 M solution) | Sigma - Aldrich | S3674-250G | |
ADP (100 mM solution) | Sigma - Aldrich | A5285-1G | |
Oligomycin (solution of 8 μg/mL in ethanol) | Sigma - Aldrich | 75351 | |
FCCP | Sigma - Aldrich | C2920 | |
Mitochondrial Membrane Potential and Proton Motive Force | |||
Equipment | |||
TPMP electrode | World Precision Instruments. | DRIREF-2 | |
Chemicals-solutions do not need to be fresh but they do need to be kept in a freezer between runs | |||
Malonate (0.1 mM solution) | Sigma - Aldrich | M1296 | |
Oligomycin (8 μg/mL in ethanol), keep in freezer | Sigma - Aldrich | 75351 | |
Nigericin (80 ng/mL in ethanol), keep in freezer | Sigma - Aldrich | N7143 | |
FCCP | Sigma - Aldrich | C3920 | |
TPMP | Sigma - Aldrich | T200 | |
TPMP solution: 10 mM TPMP, 120 mM KCL, 5 mM Hepes and 1 mM EGTA, pH 7.4 at 30 °C with 0.3% defatted BSA |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2025 MyJoVE Corporation. All rights reserved