Elsevier

Experimental Gerontology

Volume 70, October 2015, Pages 135-143
Experimental Gerontology

Of mice, pigs and humans: An analysis of mitochondrial phospholipids from mammals with very different maximal lifespans

https://doi.org/10.1016/j.exger.2015.08.011Get rights and content

Highlights

  • We compared phospholipid composition and peroxidation index of mitochondria.

  • Mouse mitochondria had high levels of 22:6 and a high peroxidation index.

  • Humans had a lower PUFA content and a low peroxidation index.

  • Values for pigs were intermediate between mice and pigs.

  • This suggests that human longevity may be related to a resistance to oxidative damage.

Abstract

The maximal lifespan (MLS) of mammals is inversely correlated with the peroxidation index, a measure of the proportion and level of unsaturation of polyunsaturated fatty acids (PUFA) in membranes. This relationship is likely related to the fact that PUFA are highly susceptible to damage by peroxidation. Previous comparative work has examined membrane composition at the level of fatty acids, and relatively little is known regarding the distribution of PUFA across phospholipid classes or phospholipid molecules. In addition, data for humans is extremely rare in this area. Here we present the first shotgun lipidomics analysis of mitochondrial membranes and the peroxidation index of skeletal muscle, liver, and brain in three mammals that span the range of mammalian longevity. The species compared were mice (MLS of 4 years), pigs (MLS of 27 years), and humans (MLS of 122 years). Mouse mitochondria contained highly unsaturated PUFA in all phospholipid classes. Human mitochondria had lower PUFA content and a lower degree of unsaturation of PUFA. Pig mitochondria shared characteristics of both mice and humans. We found that membrane susceptibility to peroxidation was primarily determined by a limited number of phospholipid molecules that differed between both tissues and species.

Introduction

Animal longevity has been shown to be inversely correlated with mitochondrial production of reactive oxygen species (ROS) and the degree of fatty acid polyunsaturation in membranes (Hulbert et al., 2007, Barja, 2013). These factors are closely related, as ROS are both a cause and a product of the peroxidation of polyunsaturated fatty acids (PUFA) (Yin et al., 2011). Propagation of peroxidation can adversely affect cell function by forming adducts with DNA and proteins, disrupting the structure of cell membranes, and propagating damage to lipids (Greenberg et al., 2008, Gueraud et al., 2010). The peroxidation of mitochondrial membranes is particularly important as mitochondria are a major source of reactive oxygen species (ROS) within cells (Brown and Borutaite, 2012, Barja, 2013) and mitochondrial failure is an indicator of ageing (López-Otín et al., 2013). Peroxidation of PUFA primarily occurs at bisallylic methylene groups which reside between consecutive carbon–carbon double bonds (i.e.–CH = CH–CH2–CH = CH–) as the reduction potential of these groups is lower than other methylene (Buettner, 1993). Bisallylic methylene groups are present in PUFA, but saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) do not contain these groups and are resistant to peroxidation. The number of bisallylic methylene groups is present in PUFA, and therefore the relative susceptibility of membranes to peroxidation, increases with the degree of unsaturation (Holman, 1954, Cosgrove et al., 1987). Common mammalian PUFA include linoleic acid (18:2) with one bisallylic methylene group, arachidonic acid (20:4) with three groups, and docosahexaenoic acid (22:6) with five groups. These PUFA are differentially distributed across phospholipid classes, being less common in phosphatidylcholine (PC) and more common in phosphatidylethanolamine (PE) and phosphatidylserine (PS) (Marsh, 2010). Some studies have suggested that phospholipid classes may influence membrane peroxidation, but as yet there is no conclusive evidence that this is the case (Schnitzer et al., 2007).

The peroxidation index is a common measure of the relative susceptibility of cellular membranes to peroxidise. The peroxidation index is determined from the relative PUFA composition (per 100 fatty acids) of membranes times experimentally derived rates of peroxidation for each different PUFA type (Pamplona et al., 1998, Hulbert et al., 2007). A strong inverse correlation has been established between peroxidation index and maximal lifespan (MLS) of skeletal muscle and liver mitochondria for a range of mammals (Pamplona et al., 1998, Hulbert, 2008). This relationship is best characterised for mammals, but similar relationships have also been found for birds (Hulbert et al., 2007), molluscs (Munro and Blier, 2012), bees (Haddad et al., 2007), and strains of C. elegans (Reis et al., 2011). Despite the breadth of prior work in this area it is not known: 1) whether muscle mitochondrial membranes correspond to results for whole muscle, 2) if brain mitochondria exhibit a similar relationship between the peroxidation index and MLS as found in skeletal muscle and liver, and 3) how different phospholipid classes and phospholipid molecules contribute to the peroxidation index.

To address these questions, we conducted a lipidomics analysis using three mammal species. The species chosen were the common mouse (Mus musculus, MLS of 4 years), the domestic pig (Sus scrofa, MLS of 27 years), and humans (Homo sapiens, MLS of 122 years). Mice and humans were chosen as examples of the extremes of mammal longevity (AnAge database), and as data for human mitochondria is extremely rare. Pigs were chosen as they have a similar body mass to humans but a much shorter maximal lifespan. We used shotgun lipidomics to determine phospholipid composition of mitochondrial-enriched fractions of skeletal muscle, liver, and brain of each animal, and calculated the fatty acid composition and peroxidation index of mitochondrial membranes from phospholipid values. To the best of our knowledge, this study is the first time that human mitochondria have been examined in such detail.

Section snippets

Materials

Methanol and chloroform (HPLC grade or higher) were supplied by VWR International (QLD, Australia). Analytical grade ammonium acetate was obtained from Crown Scientific (NSW, Australia). Sucrose, Tris, and ethylenediaminetetraacetic acid (EDTA) were supplied by Astral Scientific (NSW, Australia). Pierce BCA Protein Assay Kits were obtained from Thermo Fisher Scientific (VIC, Australia). Butylated hydroxytoluene (BHT) was supplied by Sigma Aldrich (NSW, Australia).

Animals and tissues

Skeletal muscle, liver and

Phospholipid composition of skeletal muscle mitochondria

Greater than 85% of phospholipids found in the mitochondria of skeletal muscle were phosphatidylcholines (PC) and phosphatidylethanolamines (PE), 10% were phosphatidylserines (PS), and less than 5% phosphatidylglycerols (PG) and phosphatidic acids (PA) (Fig. 1A). Mice and pigs possessed a lower percentage of PC and a higher percentage of PE than humans. No significant difference was found in the relative distribution of PS between species. PA was present in mice muscle mitochondria but was

Peroxidation, membrane composition, and maximum life span

Previous work has established an inverse relationship between the maximal lifespan (MLS) and the PUFA content and peroxidation index of membranes in mammals and other animal classes (Haddad et al., 2007, Hulbert et al., 2007, Reis et al., 2011, Munro and Blier, 2012). This relationship has been extensively examined using membrane fatty acid composition, and we are not aware of any previous studies that have investigated this at the level of phospholipid composition. We used shotgun lipidomics

Funding

This investigation was partially supported by the Illawarra Health and Medical Research Institute (IHMRI). TWM is supported by an Australian Research Council Future Fellowship (FT110100249).

Conflicts of interest

None.

The following supplementary data are related to this article.

. Targeted ion scans used to acquired phospholipid data.

Acknowledgements

We would like to thank the generous contribution of those who bequeathed their bodies to the UoW body donation program and made this research possible. We would also like to thank Martin Engel for providing the mice tissue and the Wollondilly Abattoir, Picton, for providing the pig tissue on-site.

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