Rapamycin reverses age-related increases in mitochondrial ROS production at complex I, oxidative stress, accumulation of mtDNA fragments inside nuclear DNA, and lipofuscin level, and increases autophagy, in the liver of middle-aged mice.

Rapamycin consistently increases longevity in mice although the mechanism of action of this drug is unknown. In the present investigation we studied the effect of rapamycin on mitochondrial oxidative stress at the same dose that is known to increase longevity in mice (14mgofrapamycin/kg of diet). Middle aged mice (16months old) showed significant age-related increases in mitochondrial ROS production at complex I, accumulation of mtDNA fragments inside nuclear DNA, mitochondrial protein lipoxidation, and lipofuscin accumulation compared to young animals (4months old) in the liver. After 7weeks of dietary treatment all those increases were totally or partially (lipofuscin) abolished by rapamycin, middle aged rapamycin-treated animals showing similar levels in those parameters to young animals. The decrease in mitochondrial ROS production was due to qualitative instead of quantitative changes in complex I. The decrease in mitochondrial protein lipoxidation was not due to decreases in the amount of highly oxidizable unsaturated fatty acids. Rapamycin also decreased the amount of RAPTOR (of mTOR complex) and increased the amounts of the PGC1-α and ATG13 proteins. The results are consistent with the possibility that rapamycin increases longevity in mice at least in part by lowering mitochondrial ROS production and increasing autophagy, decreasing the derived final forms of damage accumulated with age which are responsible for increased longevity. The decrease in lipofuscin accumulation induced by rapamycin adds to previous information suggesting that the increase in longevity induced by this drug can be due to a decrease in the rate of aging.

Reduced apurinic/apyrimidinic endonuclease 1 activity and increased DNA damage in mitochondria are related to enhanced apoptosis and inflammation in the brain of senescence- accelerated P8 mice (SAMP8).

The senescence- accelerated mouse prone 8 (SAMP8) is a well- characterized animal model of senescence that shows early age- related neurodegeneration with impairment in learning and memory skills when compared with control senescence- resistant mice (SAMR1). In the current study, we investigated whether such impairment could be partly due to changes in mitochondrial DNA (mtDNA) repair capacity and mitochondrial DNA damage in the brain of SAMP8 mice. Besides we studied whether these potential changes were related to modifications in two major processes likely involved in aging and neurodegeneration: apoptosis and inflammation. We observed that the specific activity of one of the main mtDNA repair enzymes, the mitochondrial APE1, showed an age- related reduction in SAMP8 animals, while in SAMR1 mice mitochondrial APE1 increased with age. The reduction in mtAPE1 activity in SAMP8 animals was associated with increased levels of the DNA oxidative damage marker 8oxodG in mtDNA. Our results also indicate that these changes were related to a premature increase in apoptotic events and inflammation in the brain of SAMP8 mice when compared to SAMR1 counterparts. We suggest that the premature neurodegenerative phenotype observed in SAMP8 animals might be due, at least in part, to changes in the processing of mtDNA oxidative damage, which would lead to enhancement of apoptotic and inflammatory processes.

Cysteine dietary supplementation reverses the decrease in mitochondrial ROS production at complex I induced by methionine restriction.

It has been described that dietary cysteine reverses many of the beneficial changes induced by methionine restriction in aging rodents. In this investigation male Wistar rats were subjected to diets low in methionine, supplemented with cysteine, or simultaneously low in methionine and supplemented with cysteine. The results obtained in liver showed that cysteine supplementation reverses the decrease in mitochondrial ROS generation induced by methionine restriction at complex I. Methionine restriction also decreased various markers of oxidative and non-oxidative stress on mitochondrial proteins which were not reversed by cysteine. Instead, cysteine supplementation also lowered protein damage in association with decreases in mTOR activation. The results of the present study add the decrease in mitochondrial ROS production to the various beneficial changes induced by methionine restriction that are reversed by cysteine dietary supplementation.

Carbohydrate restriction does not change mitochondrial free radical generation and oxidative DNA damage.

Many previous investigations have consistently reported that caloric restriction (40%), which increases maximum longevity, decreases mitochondrial reactive species (ROS) generation and oxidative damage to mitochondrial DNA (mtDNA) in laboratory rodents. These decreases take place in rat liver after only seven weeks of caloric restriction. Moreover, it has been found that seven weeks of 40% protein restriction, independently of caloric restriction, also decrease these two parameters, whereas they are not changed after seven weeks of 40% lipid restriction. This is interesting since it is known that protein restriction can extend longevity in rodents, whereas lipid restriction does not have such effect. However, before concluding that the ameliorating effects of caloric restriction on mitochondrial oxidative stress are due to restriction in protein intake, studies on the third energetic component of the diet, carbohydrates, are needed. In the present study, using semipurified diets, the carbohydrate ingestion of male Wistar rats was decreased by 40% below controls without changing the level of intake of the other dietary components. After seven weeks of treatment the liver mitochondria of the carbohydrate restricted animals did not show changes in the rate of mitochondrial ROS production, mitochondrial oxygen consumption or percent free radical leak with any substrate (complex I- or complex II-linked) studied. In agreement with this, the levels of oxidative damage in hepatic mtDNA and nuclear DNA were not modified in carbohydrate restricted animals. Oxidative damage in mtDNA was one order of magnitude higher than that in nuclear DNA in both dietary groups. These results, together with previous ones, discard lipids and carbohydrates, and indicate that the lowered ingestion of dietary proteins is responsible for the decrease in mitochondrial ROS production and oxidative damage in mtDNA that occurs during caloric restriction.

Effects of fasting on oxidative stress in rat liver mitochondria.

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O(2) consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N(epsilon)-malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.

International conference on the healthy effect of virgin olive oil.

1. Ageing represents a great concern in developed countries because the number of people involved and the pathologies related with it, like atherosclerosis, morbus Parkinson, Alzheimer's disease, vascular dementia, cognitive decline, diabetes and cancer. 2. Epidemiological studies suggest that a Mediterranean diet (which is rich in virgin olive oil) decreases the risk of cardiovascular disease. 3. The Mediterranean diet, rich in virgin olive oil, improves the major risk factors for cardiovascular disease, such as the lipoprotein profile, blood pressure, glucose metabolism and antithrombotic profile. Endothelial function, inflammation and oxidative stress are also positively modulated. Some of these effects are attributed to minor components of virgin olive oil. Therefore, the definition of the Mediterranean diet should include virgin olive oil. 4. Different observational studies conducted in humans have shown that the intake of monounsaturated fat may be protective against age-related cognitive decline and Alzheimer's disease. 5. Microconstituents from virgin olive oil are bioavailable in humans and have shown antioxidant properties and capacity to improve endothelial function. Furthermore they are also able to modify the haemostasis, showing antithrombotic properties. 6. In countries where the populations fulfilled a typical Mediterranean diet, such as Spain, Greece and Italy, where virgin olive oil is the principal source of fat, cancer incidence rates are lower than in northern European countries. 7. The protective effect of virgin olive oil can be most important in the first decades of life, which suggests that the dietetic benefit of virgin olive oil intake should be initiated before puberty, and maintained through life. 8. The more recent studies consistently support that the Mediterranean diet, based in virgin olive oil, is compatible with a healthier ageing and increased longevity. However, despite the significant advances of the recent years, the final proof about the specific mechanisms and contributing role of the different components of virgin olive oil to its beneficial effects requires further investigations.

Effect of insulin and growth hormone on rat heart and liver oxidative stress in control and caloric restricted animals.

In order to know if insulin-like signalling is involved in the control of oxidative stress in mammalian tissues in relation to aging, ad libitum-fed and caloric restricted Wistar rats were treated during 2 weeks with GH and insulin. The most consistent effect of the hormonal treatments was an increase in plasma IGF-1 levels. Caloric restriction during 6 weeks decreased ROS generation and oxidative DNA damage in heart mitochondria and this was reversed by insulin treatment. The decrease in oxidative damage to liver nuclear DNA induced by caloric restriction was also reversed by GH and insulin. In the liver, however, insulin and GH decreased mitochondrial ROS generation while they increased oxidative damage to mitochondrial DNA. GH and insulin decreased three different markers of oxidative modification of liver proteins, while they increased lipoxidation-dependent markers. This last result is related to the increase in phospholipid unsaturation induced in the liver by both hormones. The results suggest that the idea that insulin-like signalling controls oxidative stress in mammals cannot be generalized since both prooxidant and protective effects of GH and insulin are observed depending on the particular parameter and tissue selected.

Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart.

Mitochondria are chronically exposed to reactive oxygen intermediates. As a result, various tissues, including skeletal muscle and heart, are characterized by an age-associated increase in reactive oxidant-induced mitochondrial DNA (mtDNA) damage. It has been postulated that these alterations may result in a decline in the content and rate of production of ATP, which may affect tissue function, contribute to the aging process, and lead to several disease states. We show that with age, ATP content and production decreased by approximately 50% in isolated rat mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria. The decline observed in skeletal muscle may be a factor in the process of sarcopenia, which increases in incidence with advancing age. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP content or rate of production in skeletal muscle and had no effect on the heart. 8-Oxo-7,8-dihydro-2'-deoxyguanosine in skeletal muscle mtDNA was unaffected by aging but decreased 30% with caloric restriction, suggesting that the mechanisms that decrease oxidative stress in these tissues with caloric restriction are independent from ATP availability. The generation of reactive oxygen species, as indicated by H2O2 production in isolated mitochondria, did not change significantly with age in skeletal muscle or in the heart. Caloric restriction tended to reduce the levels of H2O2 production in the muscle but not in the heart. These data are the first to show that an age-associated decline in ATP content and rate of ATP production is tissue specific, in that it occurs in skeletal muscle but not heart, and that mitochondrial ATP production was unaltered by caloric restriction in both tissues.

Effect of time of restriction on the decrease in mitochondrial H2O2 production and oxidative DNA damage in the heart of food-restricted rats.

In the present study, the question if medium-term (4 months) caloric restriction (40%) decreases mitochondrial H2O2 production and oxidative DNA damage was investigated. Caloric restriction (CR) is the only experimental manipulation that increases maximum life span. Previous long-term CR studies have showed that CR decreases the mitochondrial rate of free radical production in diverse tissues and species. Those studies agree with the idea that the superior longevity of the restricted animals can be partly due to their lower mitochondrial rate of free radical generation. However, caloric restriction effects strongly depend on implementation time. Previous studies have shown that the decrease induced by CR on oxygen radical generation and oxidative damage to mitochondrial DNA occurs after 1 year but not after 6 weeks of restriction. In the present investigation, mitochondrial H2O2 production did not change in medium-term (4 months) caloric restricted animals, and, in agreement with that, no differences were found in either mitochondrial or nuclear oxidative DNA damage between restricted and ad libitum-fed animals. These results confirm the importance of the time of CR implementation, and show that time longer than 4 months is needed to decrease the mitochondrial rate of free radical generation and the oxidative damage to mtDNA in the rat heart.

Deprenyl protects from MPTP-induced Parkinson-like syndrome and glutathione oxidation in rat striatum.

An intrastriatal injection with 18.8 nmoles of the neurotoxic agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced in rats a progressive parkinsonism characterized by a major loss of striatum dopamine (DA) levels and an increased turnover of this neurotransmitter 96 h after the administration. In addition, the intrastriatal administration of MPTP produced an alteration in various behavioral markers of motor activity. Loss of DA was accompanied by a significant decrease of reduced glutathione (GSH) and an increase in GSH oxidation in the striatum. When deprenyl (10 mg/kg) was i.p. administered 2 h before the intrastriatal injection of MPTP, DA, GSH, glutathione redox status and the indexes of motor activity were not altered. These results show that MPTP increases striatum oxidative stress leading to cellular and in vivo degenerative changes which are prevented by pretreatment with deprenyl.

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart.

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T4) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks. Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA. These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.

Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source.

Oxygen free radicals (ROS) of mitochondrial origin seem to be involved in aging. Whereas in other tissues complexes I or III of the respiratory chain contain the ROS generators, in this study we find that rat liver mitochondria generate oxygen radicals at complexes I, II, and III. Short-term (6 weeks) caloric restriction significantly decreased H2O2 production in rat liver mitochondria. This decrease in ROS production was located at complex I because it occurred with complex I-linked substrates (pyruvate/malate), but did not reach statistical significance with the complex II-linked substrate succinate. The mechanism responsible for the lowered ROS production was not a decrease in oxygen consumption. Instead, the mitochondria of caloric-restricted animals released less ROS per unit electron flow. This was due to a decrease in the degree of reduction of the complex I generator. Furthermore, oxidative damage to mitochondrial and nuclear DNA was also decreased in the liver by short-term caloric restriction. The results agree with the idea that caloric restriction delays aging, at least in part, by decreasing the rate of mitochondrial ROS generation and thus the rate of attack to molecules, like DNA, highly relevant for the accumulation of age-dependent changes.

Influence of hyper- and hypothyroidism on lipid peroxidation, unsaturation of phospholipids, glutathione system and oxidative damage to nuclear and mitochondrial DNA in mice skeletal muscle.

While the biochemical literature on free radical metabolism is extensive, there is little information on the endocrine control of tissue oxidative stress, and in the case of thyroid hormones it is mainly limited to liver tissue and to short-term effects on a few selected biochemical parameters. In this investigation, chronic hypothyroidism and hyperthyroidism were successfully induced in mice, and various oxidative-stress-related parameters were studied in skeletal muscle. In vivo and in vitro lipid peroxidation significantly increased in hyperthyroidism and did not change in the hypothyroid state. The fatty acid composition of the major phospholipid classes was affected by thyroid hormones, leading to a significant decrease in total fatty acid unsaturation both in hypothyroid and hyperthyroid muscle in phosphatidylcholine and phosphatidylethanolamine fractions. In cardiolipin, however, the double bond content significantly increased as a function of thyroid status, leading to a 2.7 fold increase in the peroxidizability index from euthyroid to hyperthyroid muscle. Cardiolipin content was also directly and significantly related to thyroid state across the three groups. Glutathione system was not modified by thyroid state. The oxidative damage marker 8-oxo-7,8-dihydro-2'-deoxyguanosine did not change in mitochondrial DNA, and decreased in genomic DNA both in hypothyroid and hyperthyroid muscle. The results indicate that chronic alterations in thyroid status specially affect oxidative damage to lipids in skeletal muscle, with a probably stronger effect on mitochondrial membranes, whereas the cytosolic redox potential and DNA are better protected possibly due to homeostatic compensatory reactions on the long-term.

Correlation of fatty acid unsaturation of the major liver mitochondrial phospholipid classes in mammals to their maximum life span potential.

Free radical damage is considered a determinant factor in the rate of aging. Unsaturated fatty acids are the tissue macromolecules that are most sensitive to oxidative damage. Therefore, the presence of low proportions of fatty acid unsaturation is expected in the tissues of long-lived animals. Accordingly, the fatty acid compositions of the major liver mitochondrial phospholipid classes from eight mammals, ranging in maximum life span potential (MLSP) from 3.5 to 46 yr, show that the total number of double bonds is inversely correlated with MLSP in both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) (r = 0.757, P < 0.03, and r = 0.862, P < 0.006, respectively), but not in cardiolipin (P = 0.323). This is due not to a low content of unsaturated fatty acids in long-lived animals, but mainly to a redistribution between kinds of fatty acids on PtdCho and PtdEtn, shifting from arachidonic (r = 0.911, P < 0.002, and r = 0.681, P = 0.05, respectively), docosahexaenoic (r = 0.931 and r = 0.965, P < 0.0001, respectively) and palmitic (r = 0.944 and r = 0.974, P < 0.0001, respectively) acids to linoleic acid (r = 0.942, P < 0.0001, for PtdCho; and r = 0.957, P < 0.0001, for PtdEtn). For cardiolipin, only arachidonic acid showed a significantly inverse correlation with MLSP (r = 0.904, P < 0.002). This pattern strongly suggests the presence of a species-specific desaturation pathway and deacylation-reacylation cycle in determining the mitochondrial membrane composition, maintaining a low degree of fatty acid unsaturation in long-lived animals.

Effect of the degree of fatty acid unsaturation of rat heart mitochondria on their rates of H2O2 production and lipid and protein oxidative damage.

Previous comparative studies have shown that long-lived animals have lower fatty acid double bond content in their mitochondrial membranes than short-lived ones. In order to ascertain whether this trait protects mitochondria by decreasing lipid and protein oxidation and oxygen radical generation, the double bond content of rat heart mitochondrial membranes was manipulated by chronic feeding with semi-purified AIN-93G diets rich in highly unsaturated (UNSAT) or saturated (SAT) oils. UNSAT rat heart mitochondria had significantly higher double bond content and lipid peroxidation than SAT mitochondria. They also showed increased levels of the markers of protein oxidative damage malondialdehyde-lysine, protein carbonyls, and N(e)-(carboxymethyl)lysine adducts. Basal rates of mitochondrial oxygen radical generation were not modified by the degree of fatty acid unsaturation, but the rates of H2O2 generation stimulated by antimycin A were higher in UNSAT than in SAT mitochondria. These results demonstrate that increasing the degree of fatty acid unsaturation of heart mitochondria increases oxidative damage to their lipids and proteins, and can also increase their rates of mitochondrial oxygen radical generation in situations in which the degree of reduction of Complex III is higher than normal. These observations strengthen the notion that the relatively low double bond content of the membranes of long-lived animals could have evolved to protect them from oxidative damage.

Effect of aging on mitochondrial and nuclear DNA oxidative damage in the heart and brain throughout the life-span of the rat.

The oxygen radical-induced DNA lesion 8-oxo,7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most commonly measured marker of oxidative DNA damage, which is currently considered a main cause of aging. However, a detailed study of the age-related variations of this marker in both mitochondrial (mtDNA) and nuclear (nDNA) DNA of post-mitotic organs throughout the life span has not been previously performed. In this investigation 8-oxodG steady-state levels were simultaneously measured in mtDNA and nDNA in the heart and brain of Sprague-Dawley rats at up to five different ages covering most of the adult life span, 4, 8, 12, 17 and 24 months of age, using exactly the same digestion of DNA to deoxynucleosides and chromatographic procedures for mtDNA and nDNA. 8-oxodG levels were maintained without changes during young and middle age in all cases, but showed statistically significant increases at the older ages studied in the majority of the kinds of DNA investigated. These age-related increases in oxidative damage occurred in brain nDNA at 17 and 24 months of age, in heart nDNA at 24 months of age, and in brain mtDNA at 24 months of age, whereas no significant age-related changes were detected in heart mtDNA. Besides, 8-oxodG levels were various fold higher in mtDNA than in nDNA, both in brain and heart, at all the ages studied. The results show that oxidative damage to DNA is higher in the mtDNA than in the nDNA of post-mitotic tissues throughout the whole life span of the rat and that and increase in mtDNA and nDNA oxidative stress occurs in most cases in old animals.

The flux of free radical attack through mitochondrial DNA is related to aging rate.

Aging is a progressive and universal process originated endogenously which manifests best in post-mitotic cells. Available data indicate that the relation between oxidative stress and aging is due to the presence of low rates of mitochondrial free radical production and low degrees of fatty acid unsaturation of cellular membranes in the post-mitotic tissues of long-lived animals in relation to those of short-lived ones. Recent research shows that long-lived animals also have lower steady-state levels of oxidative damage in the mitochondrial DNA (mtDNA) of post-mitotic cells than short-lived species. This study shows that the flux of free radical attack to mtDNA is higher in short- than in long-lived animals, and proposes that this is a main determinant of the rate of accumulation of mtDNA mutations, and thus the rate of aging. This implies that aging has been slowed evolutionarily by mechanisms that decrease the generation of endogenous damage rather than try to intercept damaging agents, or to repair the damage already inflicted. The first kind of mechanisms are more efficient and less energetically expensive. Free radicals of mitochondrial origin, oxidative damage to DNA, evolution of aging rate, and possibilities and consequences of their future modification are also discussed.

Effect of thyroid hormones on mitochondrial oxygen free radical production and DNA oxidative damage in the rat heart.

Mitochondria seem to be involved in oxygen radical damage and aging. However, the possible relationships between oxygen consumption and oxygen radical production by functional mitochondria, and oxidative DNA damage, have not been studied previously. In order to analyze these relationships, male Wistar rats of 12 weeks of age were rendered hyper- and hypothyroid by chronic T(3) and 6-n-propyl-2-thiouracil treatments, respectively. Hypothyroidism decreased heart mitochondrial H(2)O(2) production in States 4 (to 51% of controls; P<0.05) and 3 (to 21% of controls; P<0.05). In agreement with this, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) decreased in the heart genomic DNA of hypothyroid animals to 40% of controls (P<0.001). Studies with respiratory inhibitors showed that the decrease in oxygen radical generation observed in hypothyroidism occurred at Complex III (mainly) and at Complex I; that decrease was due to the presence of a lower free radical leak in the respiratory chain (P<0.05). Hyperthyroidism did not significantly change heart mitochondrial H(2)O(2) production since the increase in State 4 oxygen consumption in comparison with control and hypothyroid animals (P<0.05) was compensated by a decrease in the free radical leak in relation to control animals (P<0.05). In agreement with this, heart 8-oxodG was not changed in hyperthyroid animals. The lack of increase in H(2)O(2) production per unit of mitochondrial protein will protect mitochondria themselves against self-inflicted damage during hyperthyroidism.

Low fatty acid unsaturation: a mechanism for lowered lipoperoxidative modification of tissue proteins in mammalian species with long life spans.

Carbonyl compounds generated by the nonenzymatic oxidation of polyunsaturated fatty acids react with nucleophilic groups in proteins, leading to their modification. It has not been tested whether fatty acid unsaturation is related to steady-state levels of lipoxidation-derived protein modification in vivo. A low fatty acid unsaturation, hence a low protein lipoxidation, in tissues of longevous animals would be consistent with the free radical theory of aging, because membrane lipids increase their sensitivity to oxidative damage as a function of their degree of unsaturation. To evaluate the relationship between fatty acid composition, protein lipoxidation, and maximum life span (MLSP), we analyzed liver fatty acids and proteins from seven mammalian species, ranging in MLSP from 3.5 to 46 years. The results show that the peroxidizability index of fatty acids and the sensitivity to in vitro lipid peroxidation are negatively correlated with the MLSP. Based on gas chromatography and mass spectroscopy analyses, liver proteins of all these species contain malondialdehyde-lysine and Nepsilon-carboxymethyllysine adducts, two biomarkers of protein lipoxidation. The steady-state levels of malondialdehyde-lysine and Nepsilon-carboxymethyl lysine are directly related to the peroxidizability index and inversely related to the MLSP. We propose that a low degree of fatty acid unsaturation may have been selected in longevous mammals to protect their tissue lipids and proteins against oxidative damage while maintaining an appropriate environment for membrane function.