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

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 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 β-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 mitochondria^9,10. 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.

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 supply before or after the procedure. It is recommended that at least 4 people are needed to perform the liver biopsy on a dairy cow: a veterinarian to perform the biopsy, an animal handler to stand at the cow's hip to protect the biopsy area and veterinarian, a lab technician on the outside of the pen to transfer tools, materials and biopsy sample to and from the veterinarian and maintain the clean area, which can be in the back of a vehicle (Figure 1), and a technician to retrieve the liver sample and begin mitochondrial isolation.

1. One month prior to liver biopsies, give cows a clostridia vaccination. Create surgical packs by autoclaving surgical towels, biopsy instrument, scalpel holders and surgical equipment.
2. One day before the liver biopsy, inject the cow with Ceftiofur Hydrochloride 0.044 mL/kg bodyweight subcutaneously in the neck. Monitor cow temperature, intake and fecal scores to use as a baseline for normal function.
3. Create mitochondria isolation media (MIM) containining 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 1 mM EDTA, and 0.1% (w/v) fatty acid free BSA, pH 7.4 at 4 °C. Approximately 30 mL per sample will be needed.
4. Restrain cow physically utilizing a headlock with a halter as needed (Figure 2). Using the halter, tie her head to the left side of the stanchion. If necessary, a chemical restraint (Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg / kg bodyweight) can be used.
5. The area of the biopsy is found at the right 10 - 11th intercostal space (Figure 3). Draw a straight line from the right tuber coxae to the point of the right shoulder. The biopsy site is where this line intersects with the 10-11th intercostal space. Sterilize the area of the cow to be biopsied by shaving a 10 cm square area (Figure 4). Wash area with 10% providone scrub (Figure 5) using a circular motion. Spray area with 70% ethanol solution (Figure 6). Repeat providone and ethanol washes.
   NOTE: The liver is in a slightly different position in Holstein dairy cattle compared to beef cattle.
6. Inject 2% lidocaine HCl (10-15 mL) locally to the area to provide anesthesia of the skin and underlying muscle and connective tissue (Figure 7). Repeat providone and 70% ethanol washes.
   NOTE: The nerve endings are in the skin and muscle, but not internal organs, so only a local anesthestic is needed. At most, the cow should only feel some pressure and no pain during the biopsy procedure.
7. Make a 1-2 cm stab-incision through the skin of the 10-11th intercostal space (Figure 8). Pass a Schackelford-Courtney bovine liver biopsy instrument through the skin and direct the biopsy instrument in a slight cranial direction while continuing through the diaphragm and into the liver (Figure 9, Figure 10). Obtain a 1 g sample of the liver and remove the instrument (Figure 11). Close the skin with suture placement (Figure 12).
8. Place liver sample in a conical tube with enough MIM to cover sample, on ice for immediate mitochondria isolation
9. Check incision for any redness, swelling, heat, or pain within 24 hours of biopsy and inject the cow with Ceftiofur Hydrochloride 0.044 mL/kg bodyweight subcutaneously in the neck once a day for the next 3 days (Figure 13). Monitor the cow's temperature, intake and fecal scores daily for 1 week after liver biopsy. If a fever develops, continue antibiotics at the discretion of the veterinarian.
   NOTE: If a cow is exhibiting signs of pain, such as kicking at the incision site, recumbancy, redness, heat, or reaction to touch within 1 h after the liver biopsy, a 1 mg/kg bodyweight IV injection of flunixin meglumine can be used to alleviate pain and inflammation. A second injection can be administered if necessary.
10. Remove sutures 7 days after biopsy.

2. Isolating Mitochondria from Dairy Cow Liver

1. As soon as possible after the liver sample is removed from the cow, wash the liver sample in MIM (step 1.3) to remove red blood cells and finely mince the sample with scissors. The liver should be minced in a chilled beaker containing enough isolation media to keep the tissue moist.
2. Place the minced liver into a 30 mL glass vial with a teflon pestle of 0.16 mm clearance incubated in ice and containing MIM (1:4 w/v).
3. Homogenize liver sample in teflon pestle at 500 rpm for a min with 4 strokes/min.
   NOTE: The liver homogenate is kept in an ice-packed beaker in MIM during the entire process, and all of the following centrifugation steps are completed at 4 °C
4. Centrifuge homogenate at 500 x g for 10 min, discard pellet, transfer supernatant to a chilled centrifuge tube and then centrifuge the resulting supernatant at 10,000 x g for 10 min to obtain the mitochondrial pellet.
5. Resuspend and wash the pellet in 10 mL of MIM with fatty acid free BSA and centrifuge at 8100 x g for 10 min. Discard supernatant.
6. Resuspend and wash the pellet in 10 mL of MIM without fatty acid free BSA and centrifuge at 8100 x g for 10 min. Discard supernatant.
7. Suspend the pellet in 200 µL of isolation media and place on ice until used for oxygen consumption and proton leak kinetic assays.
8. Determine protein concentration of the pellet suspension (1/100 dilution) using Bicinchoninic acid (BCA) kit per manufacturer's protocol with BSA as the standard. All protein is considered to be mitochondrial protein.

3. Measuring Mitochondrial Oxygen Consumption (State 3 and State 4)

1. Create oxygen consumption media (OCM) from 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. Approximately 3 mL per sample will be needed. Also prepare a solution of 8 μg/mL oligomycin in ethanol.
2. Incubate OCM at 30 °C. Set up respiration chamber, pump and oxygen electrode according to manufacturer directions (oxygraph system). The oxygraph software should already be installed on the computer.
3. Place 1 mL of OCM into the respiration chamber and stir vigorously. This will help insure that the solution becomes saturated with air.
4. Add 0.35 mg protein of mitochondrial protein to the respiration chamber and maintain temperature at 30 °C.
5. Record oxygen consumption for approximately 5 min. The oxygraph system records oxygen concentration so as respiration increases, oxygen concentration decreases. When oxygen consumption becomes constant (a decreasing straight line), record oxygen consumption (slope of line = concentration of oxygen/time). This is baseline oxygen consumption.
6. Add 1.25 µL of 4 mM rotenone solution to inhibit Complex I and then add 5 µL of 1 M succinate solution to reach a final concentration in the respiratory chamber of 5 mM succinate. This is state 4 respiration.
7. Add 1 µL of 100 mM ADP solution to reach a final concentration in the respiration chamber of 100 μM. Oxygen concentration will decrease (increased respiration) and then after approximately 5 min becomes a straight line. Record oxygen consumption (slope of line = concentration of oxygen/time). This is State 3 respiration.
8. Optional: At the end of the run, add FCCP (0.2 μM total volume) to induce maximal respiration. Record respiration for about 5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption. This is maximal oxygen consumption.
9. Calculate Respiratory Control Ratio (RCR) using the equation State 3 oxygen consumption / State 4 oxygen consumption.
10. Aspirate all of the solutions out of the respiration chamber. Rinse the chamber several times with double deionized water.

4. Measuring Mitochondrial Membrane Potential (MMP) and Proton Motive Force (PMF)

1. Prepare solution of 80 ng/mL nigericin in ethanol.
   NOTE: These chemicals are dissolved in ethanol, and every effort should be made to limit the amount of ethanol that is added to less than 1 μL, since ethanol can uncouple the electron transport system and cause mitchondrial dysfunction.
2. After thoroughly rinsing the chamber with double deionized water, place 1 mL of the OCM into the respiration chamber and stir vigorously with a magnetic stir bar. This will help insure that the solution becomes saturated with air. Add methyl-triphenyl-phosphonium (TPMP+) sensitive electrode to chamber setup. TPMP+ electrode should be connected to a pH meter and values are read from the pH meter.
3. Add 0.35 mg of mitochondrial protein to the respiration chamber.
4. Add 1.25 µL of 4 mM rotenone solution to inhibit Complex I. Record respiration for 2-5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption.
5. Add 0.56 μL of 8 μg/mL oligomycin solution for a final concentration of 2.8 µg oligomycin /0.35 mg of mitochondrial protein to inhibit ADP utilization. Record respiration for 2-5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption.
6. Add 0.112 μL 80 ng/mL nigericin solution to abolish the pH gradient across the mitochondrial inner membrane. Record respiration for 2-5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption.
   NOTE: Rotenone and oligomycin are used to block electron transport chain at Complex I and ATP synthase, respectively. Nigericin is added to convert transmembrane H+ gradient to a K+ gradient that can be measured with an electrode.
7. Prepare a standard curve for TPMP+ by adding 5 µL of 10 mM TPMP+ solution to the mitochondrial incubation. Repeat this step four more times until a total concentration of 2.5 μM TPMP+ has been added.
8. Initiate respiration by adding 5 μL of 1M succinate to the chamber.
9. Record respiration until you have achieved a stable trace, and then titrate the system by adding malonate. Additions of malonate should be 0.5 µL, 1 µL, 1.5 µL, 3.0 µL, 6.0 µL, 9.0 µL, then 12.5 µL of 0.1 mM Malonate solution to achieve successive additions of malonate concentrations in the incubation chamber of 0.1, 0.2, 0.3, 0.6, 1.2, 1.8, and 2.5 mM.
10. Collect data from the two electrodes (oxygen and TPMP⁺). Data acquisition software from the oxygraph system can be used to collect simultaneous measurements of mitochondrial oxygen consumption and mitochondrial membrane potential and observe changes in oxygen consumption in real time. Figure 14 shows how the oxygraph system records oxygen consumption as the experiment progresses.
11. Calculate MMP in mV based on the Nernst equation:
    MMP = 61.5 log ([TPMP+] added – external [TPMP+]) x TPMP+ binding correction/ (0.001 x mg of protein/mL x [TPMP+])
    A TPMP+ binding correction of 0.4 µL/mg of mitochondrial protein^-1 is used.
    Example calculation based on concentrations in protocol:
    MMP = 61.5 x log (5 µM – 2 µM) x 0.4/ (0.001 x 0.35 mg mitochondrial protein/mL x 2 µM)
    MMP = 198.9 mV
12. Estimate PMF by plotting a graph of MMP vs. oxygen consumption (Figure 15). PMF is reported as oxygen consumption at a membrane potential of 165 mV.
    NOTE: Titrating the electron transport chain with malonate (0.1 to 2.5 mM) shows the kinetic response of proton leak to MMP. Then, plotting MMP against oxygen consumption determines proton leak kinetics. PMF is determined by calculating oxygen consumption at a common membrane potential (165 mV).
13. At the end of the last run of the sample, add FCCP (0.2 μM total volume) to induce maximal respiration and release TPMP+ for baseline correction.
14. Aspirate all of the solutions out of the respiration chamber. Rinse the chamber several times with double deionized water. At the end of the day, the chamber should also be rinsed a few times with ethanol.

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...

Materials

List of materials used in this article

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