Name: measuring liver mitochondrial oxygen consumption and proton leak kinetics to estimate mitochondrial respiration in holstein dairy cattle
Uploaded: 2018-11-30T13:45:00
Description: Watch this Scientific Journal Video about Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle at JoVE.com

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

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 Lactating dairy cows can continue to be milked normally and milk does not need to be withdrawn from the food supp...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+oxidants+in+biological+systems:+The+superoxide+theory+of+oxygen+toxicity.">Protection against oxidants in biological systems: The superoxide theory of oxygen toxicity.</a> Free Radicals in Biology and Medicine. , 186-187 (1989).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nutrient Requirements of Dairy Cattle. , (2001).</li><li>Ramsey, J. J., Harper, M. E., Weindruch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+of+energy+intake,+energy+expenditure,+and+aging.">Restriction of energy intake, energy expenditure, and aging.</a> Free Radical Biology and Medicine. 29, 946-968 (2000).</li><li>Mehta, M. M., Weinberg, S. E., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+control+of+immunity:+beyond+ATP.">Mitochondrial control of immunity: beyond ATP.</a> Nature. 17, 608-620 (2017).</li><li>Kirby, D. M., Thorburn, D. R., Turnbull, D. M., Taylor, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+assays+of+respiratory+chain+complex+activity.">Biochemical assays of respiratory chain complex activity.</a> Methods in Cell Biology. 80, 93-119 (2007).</li><li>Alex, A. P., Collier, J. L., Hadsell, D. L., Collier, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+yield+differences+between+1x+and+4x+milking+are+associated+with+changes+in+mammary+mitochondrial+number+and+milk+protein+gene+expression,+but+not+mammary+cell+apoptosis+or+SOCS+gene+expression.">Milk yield differences between 1x and 4x milking are associated with changes in mammary mitochondrial number and milk protein gene expression, but not mammary cell apoptosis or SOCS gene expression.</a> Journal of Dairy Science. 98, 4439-4448 (2015).</li><li>Lossa, S., Lionetti, L., Mollica, M. P., Crescenzo, R., Botta, M., Barletta, A., Liverini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high-fat+feeding+on+metabolic+efficiency+and+mitochondrial+oxidative+capacity+in+adult+rats.">Effect of high-fat feeding on metabolic efficiency and mitochondrial oxidative capacity in adult rats.</a> British Journal of Nutrition. 90, 953-960 (2003).</li><li>Boily, G., Seifert, E. L., Bevilacqua, L., He, X. H., Sabourin, G., Estey, C., Moffat, C., Crawford, S., Saliba, S., Jardine, K., Xuan, J., Evans, M., Harper, M. E., McBurney, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SirT1+regulates+energy+metabolism+and+response+to+caloric+restriction+in+mice.">SirT1 regulates energy metabolism and response to caloric restriction in mice.</a> PloS One. 3 (3), e1759 (2008).</li><li>Chen, Y., Hagopian, K., Bibus, D., Villaba, J. M., Lopez-Lluch, G., Navas, P., Kim, K., McDonald, R. B., Ramsey, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+dietary+lipid+composition+on+liver+mitochondria+from+mice+following+1+month+of+calorie+restriction.">The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction.</a> Bioscience Reports. 33, 83-95 (2013).</li><li>Chacko, B. K., Kramer, P. A., Ravi, S., Benavides, G. A., Mitchell, T., Dranka, B. P., Ferrick, D., Singal, A. K., Ballinger, S. W., Bailey, S. M., Hardy, R. W., Zhang, J., Zhi, D., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioenergetic+health+index:+a+new+concept+in+mitochondrial+translational+research.">The bioenergetic health index: a new concept in mitochondrial translational research.</a> Clinical Science. 127, 367-373 (2014).</li></ol>

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...

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 the effect of different treatments on mitochondrial respiration. The overall goal of measuring mitochondrial oxygen consumption and proton leak kinetics is to assess mitochondrial function and energetic efficiency.
Hepatic mitochondrial dysfunction has been associated with several diseases in dairy cattle. The ability of cellular metabolism to switch between carbohydrate and lipid fuels when faced with an energy deficit in early lactation is influenced by the number and function of mitochondria in the cell2. Defects in the ability of mitochondria to adapt to an increased demand for energy and increased &#946;-oxidation can lead to accumulation of intracellular lipid associated with insulin resistance and may lead to the formation of fatty liver in early lactation dairy cows. Mitochondria, as the site of ketone body production and use, can play a key role in ketosis in dairy cows3. A lack of mitochondria or mitochondrial dysfunction will impact fuel availability to the periphery and be reflected in changes in oxygen consumption or RCR.
Mitochondrial oxygen consumption changes in response to inflammation. Seven-day-old broilers were randomly assigned to a group infected with Eimeria maxima and a control group4. Broilers that did not undergo coccidiosis challenge had lower oxygen consumption due to proton leak and higher RCR indicating that liver mitochondria respond to an immune challenge by increasing proton leak. While proton leak and reactive oxygen species production was once considered a sign of mitochondrial membrane dysfunction and detrimental to energetic efficiency, now it is known that it is important for import of proteins and calcium into mitochondria5, and for the generation of heat1.
Electron leak from the respiratory chain makes mitochondria susceptible to reactive oxygen species production and oxidative damage to mitochondrial membrane proteins, lipids and mitochondrial DNA. As mitochondria age, damage can accumulate especially to mtDNA causing further dysfunction in mitochondrial metabolism6 and greater susceptibility of the cow to disease. In practice, many livestock animals are fed high levels of supplements such as Cu, Zn and Mn to boost antioxidant function. However, feeding high levels of Cu, Zn and Mn decreased milk production and increased oxygen consumption due to proton leak (State 4 respiration)7.
Previous research on the role of mitochondrial function in energy efficiency in cattle has focused on changes in mitochondrial oxygen consumption and proton leak. Very few studies have been published in dairy cattle and most papers compare production efficiency in the form of residual feed intake (RFI) to mitochondrial function in beef cattle. Variability in mitochondrial respiration rates were examined by measuring state 3, state 4 and RCR in livers from both lactating Holstein cows and lactating beef cows (Angus, Brangus and Hereford)8. The researchers did not find any correlation in mitochondrial respiration with growth or milking traits for beef cattle but did report a correlation between mitochondrial respiration and milking traits for Holsteins. In two studies, RFI was compared in beef cattle to mitochondrial respiration rates (state 3, state 4 and RCR) in muscle mitochondria9,10. Mitochondrial respiration rates changed in response to DMI and low rates were associated with less efficient beef steers. In another study, RFI of steers from high or low RFI bulls were compared with mitochondrial respiration rates and proton leak kinetics between the two groups of progeny11. Differences were due to gain confirming the conclusion that gain does not impact mitochondrial respiration in beef cattle.
In this paper, an experiment examining liver RCR in response to feeding 3 antioxidant minerals to lactating dairy cattle illustrates the use of methods to measure oxygen consumption during State 4 and State 3 respiration and PMF.

<table>
	<tbody>
		<tr>
			<td>Liver Biopsy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schackelford-Courtney bovine liver biopsy instrument</td>
			<td>Sontec Instruments Englewood CO</td>
			<td>1103-904</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Fisher Scientific</td>
			<td>19-037-516</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture needles</td>
			<td>NA</td>
			<td>NA</td>
			<td>Included with Suture</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Sigma - Aldrich</td>
			<td>S2896 / S2646</td>
			<td># for handle and blades</td>
		</tr>
		<tr>
			<td>Surgery towels</td>
			<td>Fisher Scientific</td>
			<td>50-129-6667</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 mL</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma - Aldrich</td>
			<td>Z168750</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL syringes</td>
			<td>Fisher Scientific</td>
			<td>22-314387</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needles (22, 2 1/2)</td>
			<td>VWR</td>
			<td>MJ8881-200342</td>
			<td></td>
		</tr>
		<tr>
			<td>Cow halter</td>
			<td>Tractor Supply Co.</td>
			<td>101966599</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabbing</td>
			<td>Fisher Scientific</td>
			<td>14-959-102</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton gauze squares (4x4)</td>
			<td>Fisher Scientific</td>
			<td>22-246069</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical scissors</td>
			<td>Sigma - Aldrich</td>
			<td>Z265969</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coccidiosis Vaccine 0.75 bottle/cow</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Clostridia Vaccine</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Liver biopsy antibiotics excenel 2 cc/100 lbs for 3 days</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Providone Scrub</td>
			<td>Aspen Veteterinary Resources</td>
			<td>21260221</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Sigma - Aldrich</td>
			<td>793213</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg/kg bodyweight</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>2% lidocaine HCl (10-15 mL)</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>1 mg/kg IV injection of flunixin meglumine</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Isolation of Mitochondria (liver)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wheaton vial 30 mL with a Teflon pestle of 0.16 mm clearance</td>
			<td>Fisher Scientific</td>
			<td>02-911-527</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Motor</td>
			<td>Cole Parmer</td>
			<td>EW-04369-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Probe</td>
			<td>Cole Parmer</td>
			<td>EW-04468-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Pipette (10 mL)</td>
			<td>Cole Parmer</td>
			<td>SK-21600-74</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (500 mL) with ice</td>
			<td>Fisher Scientific</td>
			<td>FB100600</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microfuge</td>
			<td>Fisher Scientific</td>
			<td>75-002-441EW3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchoninic acid (BCA) protein assay kit (microplates for plate reader)</td>
			<td>abcam</td>
			<td>ab102536</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma - Aldrich</td>
			<td>S7903-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma - Aldrich</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma - Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (fatty acid free)</td>
			<td>Sigma - Aldrich</td>
			<td>A7030-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma - Aldrich</td>
			<td>M4125-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Sigma - Aldrich</td>
			<td>38796</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma - Aldrich</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">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,&#160; pH 7.4 at 4 &#176;C, will last 2 days in refrigerator</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Oxygen Comsuption</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygraph Setup + Clark type oxygen electrode</td>
			<td>Hansatech (PP Systems)</td>
			<td>OXY1</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoregulated Water Pump</td>
			<td>ADInstruments</td>
			<td>MLE2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Clark type Oxygen electrode</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Autopipette (1 mL)</td>
			<td>Cole Parmer</td>
			<td>SK-21600-70</td>
			<td>Included with Oxy1</td>
		</tr>
		<tr>
			<td>Small magnetic stir bar</td>
			<td>Fisher Scientific</td>
			<td>14-513-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette (10 &#956;L)</td>
			<td>Cole Parmer</td>
			<td>SK-21600-60</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma - Aldrich</td>
			<td>P9333-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma - Aldrich</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma - Aldrich</td>
			<td>P5655-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma - Aldrich</td>
			<td>M1028-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma - Aldrich</td>
			<td>E3889-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Use to make mitochondrial oxygen consumption media: 120 mM KCL, 5 mM KH2PO4, 5 mM MgCl2, 5 mM Hepes and 1 mM EGTA,&#160; pH 7.4 at 30 &#176;C with 0.3% defatted BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone (4 mM solution)</td>
			<td>Sigma - Aldrich</td>
			<td>R8875-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate (1 M solution)</td>
			<td>Sigma - Aldrich</td>
			<td>S3674-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP (100 mM solution)</td>
			<td>Sigma - Aldrich</td>
			<td>A5285-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin (solution of 8 &#956;g/mL in ethanol)</td>
			<td>Sigma - Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma - Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Membrane Potential and Proton Motive Force</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TPMP electrode</td>
			<td>World Precision Instruments.</td>
			<td>DRIREF-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals-solutions do not need to be fresh but they do need to be kept in a freezer between runs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malonate (0.1 mM solution)</td>
			<td>Sigma - Aldrich</td>
			<td>M1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin (8 &#956;g/mL in ethanol), keep in freezer</td>
			<td>Sigma - Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Nigericin (80 ng/mL in ethanol), keep in freezer</td>
			<td>Sigma - Aldrich</td>
			<td>N7143</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma - Aldrich</td>
			<td>C3920</td>
			<td></td>
		</tr>
		<tr>
			<td>TPMP</td>
			<td>Sigma - Aldrich</td>
			<td>T200</td>
			<td></td>
		</tr>
		<tr>
			<td>TPMP solution: 10 mM TPMP, 120 mM KCL, 5 mM Hepes and 1 mM EGTA,&#160; pH 7.4 at 30 &#176;C with 0.3% defatted BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring liver mitochondrial oxygen consumption and proton leak kinetics to estimate mitochondrial respiration in holstein dairy cattle

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.

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 &#60; 0.1...

Watch this Scientific Journal Video about Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle at JoVE.com

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

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 ...

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