Technical note: Assessing the functional capacity of mitochondria isolated from lactating mammary tissue: Choose your chelating agent wisely

• Published in: Journal of dairy science 2009-05, Vol.92 (5), p.2038-2045
• Main Authors: Hadsell, D.L., George, J., Abraham, P.A., Collier, R.J., Lambert, B.D.
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; animal models; Animal productions; Animals; biochemical pathways; Biochemistry - methods; Biological and medical sciences; bovine serum albumin; cell respiration; chelating agents; Chelating Agents - metabolism; cryopreservation; Cryopreservation - methods; culture media; cytochrome c; Cytochromes c - metabolism; EDTA (chelating agent); Electron Transport Complex IV - metabolism; ethylene glycol tetraacetic acid; Female; Food industries; Fundamental and applied biological sciences. Psychology; lactation; Lactation - metabolism; liver; Liver - enzymology; Liver - metabolism; mammary; mammary glands; Mammary Glands, Animal - enzymology; Mammary Glands, Animal - metabolism; Mice; Milk and cheese industries. Ice creams; mitochondria; Mitochondria - metabolism; Mitochondrial Proton-Translocating ATPases - metabolism; Serum Albumin, Bovine - metabolism; Terrestrial animal productions; Vertebrates
• ISSN: 0022-0302; 1525-3198
• DOI: 10.3168/jds.2008-1360

Previous work has indicated that respiratory activity of mitochondrial preparations prepared from lactating mammary tissue is often much lower than that of mitochondria isolated from other organs such as the liver. Initial studies in our own laboratory also found that mammary mitochondria prepared from lactating mice had much lower ATP synthesis activity than those isolated from liver tissue obtained from the same animals. In this paper, we describe an improved procedure for obtaining coupled mitochondria from the mammary tissue of lactating mice. Using a high-throughput assay for mitochondrial ATP synthesis, we demonstrated that mammary mitochondria, unlike liver mitochondria, are sensitive to the concentration of bovine serum albumin and to the choice of chelating agent used in the preparation and assay buffers. Mammary mitochondria prepared and assayed in buffers containing 1mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′ tetraacetic acid (EGTA) and 0.4% bovine serum albumin have a similar ATP synthesis activity as liver mitochondria. In addition, we show that the chelating agent EDTA ablates the ATP synthesis capacity of mammary mitochondria through a mechanism that does not involve the release of cytochrome c. We also demonstrate that these improved isolation and assay procedures are both scalable and applicable to bovine mammary tissue, and we describe optimal conditions for cryopreservation and recovery of functionally active mitochondria. This work will facilitate future studies aimed at determining the importance of mammary mitochondria to milk production.