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

这项研究得到了 alltech 和 usda 舱口基金通过加州大学戴维斯分校兽医学院食品动物健康中心的支持。

这里描述的所有方法、协议和研究都得到了加州大学戴维斯分校动物护理和使用机构委员会 (iacuc) 的批准。
1. 从荷斯坦奶牛获得肝脏活检
注: 肝脏活检应由有执照的兽医进行。肝脏活检可以在奶牛所在的乳品现场进行。哺乳奶牛可以继续正常挤奶, 牛奶不需要在手术前或手术后从食物供应中提取。建议至少需要4人在奶牛上进行肝脏活检: 一名兽医进行活检,...

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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. 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该协议中最关键的一点是获得具有代表性的肝脏组织样本, 并在活检后尽快开始分离线粒体。呼吸测量的变化很低 (表 1), 原因是从奶牛到实验室的运输时间很短。为了减少运输时间, 在奶牛场办公室设立了一个小型实验室, 并在收集每个样本时将肝脏样本送到办公室实验室, 以便在活检10分钟内分离线粒体。用 ph 计设置和测试呼吸室和电极 (氧气, tpmp +), 在采集和处理样品的前一天记录?...

线粒体对压力非常敏感, 它们的细胞环境会导致多种代谢疾病。线粒体中的耗氧量和质子泄漏是线粒体健康的指标。本文所描述的基于有质子泄漏和无质子泄漏的耗氧量的 rcr 估计线粒体能量效率的方法。这些结果可以占营养利用1所损失能量的 30%.氧气消耗和质子泄漏的变化可以识别线粒体功能障碍, 从而导致代谢疾病, 并导致能源效率下降。这些方法也可用于检查不同治疗方法对线粒体呼吸的影响。测量线粒体耗氧量和质子泄漏动力学的总体目标是评估线粒体功能和能量效率。
肝线粒体功能障碍与奶牛的几种疾病有关。早期泌乳时, 当面对能量不足时, 细胞代谢在碳水化合物和脂类燃料之间切换的能力受到细胞2中线粒体数量和功能的影响.线粒体适应能量需求增加和β氧化增加的能力存在缺陷, 可导致与胰岛素抵抗相关的细胞内脂质积累, 并可能导致早期哺乳奶牛脂肪肝的形成。线粒体作为酮体生产和使用的场所, 在奶牛3中的酮症中发挥着关键作用。线粒体或线粒体功能障碍的缺乏将影响外围燃料的供应, 并反映在氧气消耗或 rcr 的变化上。
线粒体耗氧量随炎症而变化。7天龄的肉鸡随机分为感染 eimeria 最大值的组和对照组 4。没有经历过球虫病挑战的肉鸡由于质子泄漏而耗氧量较低, rcr 较高表明肝脏线粒体通过增加质子泄漏来应对免疫挑战。虽然质子泄漏和活性氧的产生曾经被认为是线粒体膜功能障碍的表现, 对能量效率不利, 但现在大家都知道, 将蛋白质和钙导入线粒体很重要。, 并为热的产生1。
从呼吸链的电子泄漏使线粒体容易产生活性氧, 并对线粒体膜蛋白、脂质和线粒体 dna 造成氧化损伤。随着线粒体年龄的增加, 损伤尤其会积累到 mtdna 中, 导致线粒体代谢的进一步功能障碍, 并使奶牛对疾病的易感性更强。实际上, 许多牲畜动物被喂养高水平的补充剂, 如铜, 锌和锰, 以提高抗氧化功能。然而, 由于质子泄漏 (状态4呼吸), 摄入高水平的铜、锌和锰会减少牛奶产量, 增加耗氧量.
此前关于线粒体功能在牛能量效率中的作用的研究主要集中在线粒体耗氧量和质子泄漏的变化上。在奶牛中发表的研究很少, 大多数论文将剩余饲料摄入量 (rfi) 的生产效率与肉牛的线粒体功能进行了比较。通过测定哺乳期荷斯坦奶牛和哺乳牛牛 (安格斯、布拉古斯和赫里福德) 肝脏中的3、4和 rcr 状态, 检测线粒体呼吸速率的变异性.研究人员没有发现肉牛线粒体呼吸与生长或挤奶性状有任何相关性, 但确实报告了线粒体呼吸与霍尔斯坦挤奶性状之间的相关性。在两项研究中, 将牛牛的 rfi 与肌肉线粒体 9、10的线粒体呼吸速率 (状态3、状态4和 rcr) 进行了比较。线粒体呼吸率随着 dmi 的变化而变化, 低呼吸率与效率较低的牛肉转向有关。在另一项研究中, 将高或低 rfi 公牛的蒸手的 rfi 与两组后代11之间的线粒体呼吸速率和质子泄漏动力学进行了比较.差异的原因是证实了增益不影响肉牛线粒体呼吸的结论。
本文通过一项实验, 对奶牛饲养3种抗氧化矿物质的肝脏 rcr 进行了研究, 阐述了用方法测量4号和3号州呼吸和 pmf 期间的耗氧量。

<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

耗氧量、质子动力 (pmf) 和质子泄漏是线粒体呼吸的测量, 或线粒体能够将 nadh 和 fadh 转化为 atp 的程度。由于线粒体也是氧气使用和营养氧化的主要场所, 二氧化碳和水, 他们如何有效地使用氧气和产生 atp 直接关系到营养代谢的效率, 动物的营养需求,动物的健康。该方法的目的是检查线粒体呼吸, 可用于检查不同药物、饮食和环境对线粒体代谢的影响。结果包括作为质子依赖性呼吸 (状态 3) 测量的耗氧量和质子泄漏依赖性呼吸 (状态 4)。国家 3/状态4呼吸的比例被定义为呼吸控制比 (rcr), 可以表示线粒体的能量效率。线粒体质子泄漏是通过从 adp 中分离氧化磷酸化来消除线粒体膜电位 (mmp) 的过程, 从而降低了 atp 合成的效率。氧和 trmp + 敏感电极与线粒体底物和电子传输链抑制剂被用来测量状态3和状态4呼吸, 线粒体膜 pmf (或产生 atp 的潜力) 和质子泄漏。这种方法的局限性是肝脏组织必须尽可能新鲜, 所有活检和检测必须在不到10小时内进行。这将一个人一天可以收集和处理的样本数量限制在大约5个。然而, 只需要1克的肝脏组织, 所以在大型动物, 如奶牛, 所需的样本量是小的相对于肝脏大小, 几乎不需要恢复时间。

表 1和图 15分别显示了 rcr 和质子泄漏动力学的阳性结果。本研究7在牛奶中喂养了5个不同水平的铜、锌和锰中的1种, 并在牛奶中测定了荷斯坦奶牛的 rcr 和蛋白质泄漏动力学。状态 4, 最大质子泄漏依赖呼吸, 有倾向于受矿物摄入铜, 锰和锌 (p & lt; 0.1)。状态 3 (最大 atp 刺激呼吸) 和 rcr = 状态 3/状态 4 (呼吸控制率) ?...

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

在这里, 我们分享测量线粒体耗氧量的方法, 营养能量学的一个决定性概念, 和质子泄漏, 这是线粒体产生 atp 效率低下的主要原因。这些结果可以占营养利用损失能量的 30%, 以帮助评估线粒体功能。

荷尔斯坦奶牛肝线粒体耗氧量和质子泄漏动力学评价线粒体呼吸

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

这种方法可以帮助回答能量领域的关键问题，如线粒体健康、能量效率和能量使用。该技术的主要优点是直接测量隔离线粒体的耗氧量。结果不需要纠正其他细胞内代谢过程。当我们想要研究线粒体效率如何影响饲料效率时，我们首先决定在奶牛中采用这种方法。此方法的视频演示至关重要，因为线粒体分离的步骤很难学习，因为它们需要一致和精确。尽快从牛身上取出肝脏样本，在线粒体分离介质中清洗样本，以去除红血球。使用剪刀将组织细细地切碎成冷藏烧杯，该烧杯含有足够的隔离介质，以保持组织湿润。将切碎的肝脏转移到含有线粒体隔离介质的30毫升玻璃瓶中。加入一个间隙为0.16毫米的特氟龙害虫，在冰中孵育，以500 RPM的小时对肝脏样本进行均质化，每分钟四次。在此之后，将同质质转移到含有线粒体隔离介质的管中，并放入冰装烧杯中。在500倍G和4摄氏度的离心均质10分钟。丢弃颗粒，将上一液转移到冷却的离心管中。将产生的上一代在10，000倍G和4摄氏度下离心10分钟，以获得线粒。用无脂肪酸的BSA在10毫升线粒体隔离介质中重新释放和清洗颗粒。然后在8，100倍G和4摄氏度的离心机10分钟。丢弃上一杯，将颗粒重新在 200 微升隔离介质中。将它放在冰上，直到准备用于氧气消耗和质子泄漏动力学测定。使用 BSA 作为标准，根据制造商的说明，使用双辛辛酸试剂盒确定颗粒悬浮液的蛋白质浓度。首先，创建文本协议中概述的耗氧介质。在30摄氏度下孵化耗氧介质。接下来，根据制造商的说明，为氧仪系统设置呼吸室、泵和氧电极。然后将一毫升耗氧介质放入呼吸室。然后向腔室添加0.35毫克线粒体蛋白。大力搅拌，确保溶液与空气饱和，并保持 30 摄氏度的温度。记录氧气消耗约五分钟。当氧气消耗变为由递减的直线表示的常数时，请记录为基准耗氧量。加入1.25微升的四毫摩尔罗酮溶液抑制复杂一，然后加入五微升的一摩尔吸水溶液，以达到呼吸室中5毫摩尔的最终浓度。这是状态IV耗氧量。在此之后，加入100毫摩尔ADP溶液的微升，在呼吸室中达到100微摩尔的最终浓度。氧气消耗将减少大约五分钟，然后将成为一条直线。记录此耗氧量为状态 III 呼吸。计算文本协议中概述的呼吸控制比率。然后从呼吸室中吸出所有溶液。用双脱水冲洗室几次。首先，准备一个溶液，每毫升尼日尔辛80毫微克在乙醇中限制乙醇添加量小于一微升。此外，在乙醇中准备每毫升寡霉素8微克的解决方案。用双脱氧水彻底冲洗腔室后，将一毫升耗氧介质放入呼吸室，用磁搅拌棒大力搅拌。向呼吸室添加0.35毫克线粒体蛋白。将甲基三苯磷敏感电极添加到腔室设置中，确保另一端正确连接到 pH 计。加入1.25微升的四毫摩尔罗泰酮溶液，抑制复杂 I.记录呼吸两到五分钟。记录当氧气消耗值变为常量时。接下来，添加0.56微升的寡霉素溶液，以抑制ADP的利用率。记录呼吸约两到五分钟。记录当氧气消耗值变为常量时。然后添加0.112微升的80毫微克每毫升尼日尔素溶液，以废除线粒体内膜的pH梯度。记录呼吸两到五分钟，并记下氧气消耗值时，它变得恒定。在线粒体孵育中加入5个10毫摩尔甲基三甲磷溶液的微升，开始准备标准曲线。重复此添加四次，直到总浓度为 2.5。已添加微摩尔。在此之后，向腔室添加五微升一摩尔吸华溶液，以开始呼吸。记录呼吸，直到达到稳定的痕迹。然后添加马龙酸酯来滴定系统，如文本协议中所述。在这项研究中，RCR和质子泄漏动力学在荷尔斯泰因奶牛的牛奶中测量70天后，奶牛已经用不同水平的铜，锌和锰喂养了28天。在锰含量低的奶牛中，国IV呼吸被认为是最高的，但控制性奶牛中却最低，这表明锰在尽量减少质子泄漏依赖呼吸方面起着重要作用。对肝质子泄漏依赖性呼吸的分析表明，这种呼吸在用低锰治疗的奶牛中是最大的，在控制奶牛中最低。这些结果证实，低锰组具有最大的状态IV呼吸，而对照组有最低。虽然安格斯的饲料效率高于高RFI公牛从低RFI公牛诞生，但并没有反映在线粒体RCR或质子泄漏动力学中。在执行此过程时，在处理线粒体时，必须将所有小瓶和烧杯放在冰上。该技术开发后，为乳品营养领域的研究人员探索线粒体效率在饲料效率和线粒体功能在肝脏中的作用铺平了道路。不要忘记，使用罗酮可能是危险的，在执行此过程时，应始终采取预防措施，如戴手套、眼睛保护和适当的 PPE。

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

测量来自母猪后代的先前冷冻心脏组织中的线粒体电子转移复合物：评估运动诱导的线粒体生物能量变化的模型

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution (Video) | JoVE 

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals (Video) | JoVE 

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Unveiling Mitochondrial Contact Sites and Architectural Insights (Video) | JoVE

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster (Video) | JoVE

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...