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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was supported by Alltech and USDA Hatch funds through the Center for Food Animal Health at UC Davis School of Veterinary Medicine.

....

Materials

NameCompanyCatalog NumberComments
Liver Biopsy
Equipment
Schackelford-Courtney bovine liver biopsy instrumentSontec Instruments Englewood CO1103-904
SutureFisher Scientific19-037-516
Suture needlesNANAIncluded with Suture
ScalpelsSigma - AldrichS2896 / S2646# for handle and blades
Surgery towelsFisher Scientific50-129-6667
Falcon tubes 50 mLFisher Scientific14-432-22
TweezersSigma - AldrichZ168750
50 mL syringesFisher Scientific22-314387
Injection needles (22, 2 1/2)VWRMJ8881-200342
Cow halterTractor Supply Co.101966599
Cotton swabbingFisher Scientific14-959-102
cotton gauze squares (4x4)Fisher Scientific22-246069
Medical scissorsSigma - AldrichZ265969
Chemicals
Coccidiosis Vaccine 0.75 bottle/cowProvided by Veterinarian
Clostridia VaccineProvided by Veterinarian
Liver biopsy antibiotics excenel 2 cc/100 lbs for 3 daysProvided by Veterinarian
Providone ScrubAspen Veteterinary Resources21260221
Ethanol 70%Sigma - Aldrich793213
Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg/kg bodyweightProvided by Veterinarian
2% lidocaine HCl (10-15 mL)Provided by Veterinarian
1 mg/kg IV injection of flunixin meglumineProvided by Veterinarian
Isolation of Mitochondria (liver)
Equipment
Wheaton vial 30 mL with a Teflon pestle of 0.16 mm clearanceFisher Scientific02-911-527
Homogenizer MotorCole ParmerEW-04369-10
Homogenizer ProbeCole ParmerEW-04468-22
Auto Pipette (10 mL)Cole ParmerSK-21600-74
Beaker (500 mL) with iceFisher ScientificFB100600
Refrigerated microfugeFisher Scientific75-002-441EW3
Microfuge tubes (1.5 mL)Fisher ScientificAM12400
Chemicals
Bicinchoninic acid (BCA) protein assay kit (microplates for plate reader)abcamab102536
SucroseSigma - AldrichS7903-1KG
Tris-HClSigma - AldrichT1503-1KG
EDTASigma - AldrichEDS-1KG
BSA (fatty acid free)Sigma - AldrichA7030-50G
MannitolSigma - AldrichM4125-1KG
Deionized waterSigma - Aldrich38796
HepesSigma - AldrichH3375-500G
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 electrodeHansatech (PP Systems)OXY1
Thermoregulated Water PumpADInstrumentsMLE2001
Clark type Oxygen electrodeNANA
Autopipette (1 mL)Cole ParmerSK-21600-70Included with Oxy1
Small magnetic stir barFisher Scientific14-513-95
Micropipette (10 μL)Cole ParmerSK-21600-60
pH meterVWR
Chemicals
KClSigma - AldrichP9333-1KG
HepesSigma - AldrichH3375-500G
KH2PO4Sigma - AldrichP5655-1KG
MgCl2Sigma - AldrichM1028-100ML
EGTASigma - AldrichE3889-100G
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 - AldrichR8875-5G
Succinate (1 M solution)Sigma - AldrichS3674-250G
ADP (100 mM solution)Sigma - AldrichA5285-1G
Oligomycin (solution of 8 μg/mL in ethanol)Sigma - Aldrich75351
FCCPSigma - AldrichC2920
Mitochondrial Membrane Potential and Proton Motive Force
Equipment
TPMP electrodeWorld 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 - AldrichM1296
Oligomycin (8 μg/mL in ethanol), keep in freezerSigma - Aldrich75351
Nigericin (80 ng/mL in ethanol), keep in freezerSigma - AldrichN7143
FCCPSigma - AldrichC3920
TPMPSigma - AldrichT200
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

References

  1. Brand, M. D., Divakaruni, A. S. The regulation and physiology of mitochondrial proton leak. Physiology. 26, 192-205 (2011).
  2. Stephenson, E. J., Hawley, J. A. Mitochondrial function in metabolic health: A genetic ....

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