Abstract
The mitochondrial free radical theory of aging is currently one of the more widely accepted theories to explain the aging process. It posits that aging results from free radical damage to mitochondrial DNA that is caused by reactive oxygen species (ROS) generated within the mitochondria during complex I electron transport. Vulnerability to ROS peroxidation, and thus aging, varies with the quantity of polyunsaturated fatty acids incorporated into cellular membranes. The current data in support of this theory suggest that antioxidant intake has little impact on increasing maximal longevity and also that intake of polyunsaturated fatty acids may be associated with faster aging. These implications are relevant to clinical practice.
Introduction
An estimated 300 theories have been put forth over the years to explain why aging occurs; these can be sorted into 3 main groups: genetic mutation theories, wear-and-tear theories, and cell waste accumulation theories.1 This article focuses on the most widely accepted current theory, the mitochondrial free radical theory of aging (MRFTA), which does not fully fit into any one of these categories.
A number of basic aspects of aging must be explained by any theory that we consider: “The natural aging process has four characteristics: it is progressive, endogenous, irreversible, and deleterious for the individual. The progressive character of aging suggests that causes of aging are present during an organism’s whole lifespan, both at young and old ages. That aging is an endogenous process suggests exogenous factors are not causes of the intrinsic aging process, although they may interact with endogenous causes and either enhance or diminish their effects.”2
Lifespans vary greatly among animal species from only a few days to hundreds of years.* The challenge for any theory of longevity is that it must explain why longevity varies so widely: 200-fold from shrews to whales or 30-fold from mice to men. Longevity is closely regulated within a species yet widely variable between them. For an individual, life expectancy depends on environment more than on genes, but longevity (ie, the maximum achievable age) depends on genotype.
The beginnings of the free radical stress theories of aging can be dated all the way back to Paul Bert, who in 1878 described the toxic effects of oxygen at high concentration, in particular on warm-blooded animals. He was able to demonstrate that oxygen’s toxicity to animals varied with ambient temperature in cold-blooded animals.3
In 1908 Max Rubner observed that longevity of mammals increases with body size and that the rate of metabolism of mammals decreases with increases in body size. He combined these 2 measurements for 5 mammal species (ie, guinea pigs, cats, dogs, cattle, horses) and described their “lifetime energy potential.” This calculated potential turned out to be fairly constant across these diverse species.4
In 1928 Raymond Pearl reported, in an experiment reminiscent of Bert’s, that one could vary the lifespan of fruit flies by simply changing the temperature of their cages and thus shifting their metabolic rates or, to use the term he originated, their “rate of living.”5
The Mitochondrial Free Radical Theory of Aging
What we now call the mitochondrial free radical theory of aging (MFRTA) goes back to Denham Harman’s 1956 proposal that “aging and the degenerative diseases associated with it are attributed basically to the deleterious side attacks of free radicals on cell constituents and on the connected tissues.”6,** He expanded the theory in the 1970s to suggest that the mitochondria are the source of these free radicals.7 As most chronic diseases, including heart disease, cancer, and diabetes, are associated with aging, understanding and learning to influence this aging process has significant implications.
The early versions of this theory led to the hypothesis that consuming more antioxidants, either as supplements or as foods, could, because of antioxidants’ ability to quench free radicals, slow the aging process and prevent chronic diseases.8 Many people accept this hypothesis as fact.
Ongoing research over the last half century has forced the MRFTA to adapt, and it is necessary that we review the evolution of this theory; some of the early assumptions that underlie our practices have been disproven.
Many of the following ideas are summarized from articles written by Gustavo Barja; greater details are available in his 2013 comprehensive review.9
After decades of research, only 2 factors clearly correlate with longevity in vertebrates:
1. The rate of production of reactive oxygen species (ROS) in the mitochondria. (mtROSp)
2. The degree of fatty acid unsaturation of tissue cellular membranes.
The longer a species lives, the lower both these values are. That is the opposite of widely accepted beliefs about longevity.
Antioxidants
The MRFTA focused on the effects of antioxidants for decades. Antioxidant levels are relatively easy to measure, and it fit our paradigm that antioxidant levels would correlate positively with longevity. Indeed, Lopez-Torres reported in 1993 that tissue antioxidants were correlated across a wide range of vertebrate species with longevity. The association though is negative.10
Longer-lived animals have lower levels of antioxidants within their tissues than shorter-lived animals.11 For example, hamsters make 20 times as much glutathione peroxidase in their livers as humans do.12 Pérez-Campo et al looked at 27 correlations between antioxidant production and longevity; of these 21 showed a negative association, and 6 did not show significant association. There was not a single example of a positive association between higher endogenous antioxidants and longer life expectancy.11
More recently, Pamplona and Constantini (2011) reported that in comparing endogenous antioxidants with life expectancy in 78 different species, the associations were negative in 72, no different in 6,† and positive in just 1 species.13 Animals that live longer do not do so by producing more antioxidants.
A hypothesis based on early versions of the free radical theory of aging predicted that adding dietary antioxidants would reduce oxidative damage and influence lifespan positively. Multiple studies were performed during the 1970s and 1980s in the hope of confirming this prediction. Greater longevity was seen only in about half of them.9
The addition of antioxidants did not increase what was hoped for though: maximum longevity. Antioxidants, when they had an effect, increased mean lifespan only in relatively short-lived animals (lifespan < 3 years). This has been interpreted to say that when living conditions were not ideal, the antioxidants protected the animal from early or premature death. Graphs of survival curves look more rectangular with antioxidants, but survival curve ends at the same age; maximum age was unchanged. This is what has happened to humans in the last century. Our life expectancy has increased but our rate of aging remains constant.
Taking antioxidants can protect short-lived species (<3 years) from diminished survival when they live in less-than-optimal environments, but antioxidants do not seem to increase longevity. They protect against the chemical and oxidative insults brought on by life, but they do not slow the clock of aging.14
It should be noted that in 1 invertebrate, the nematode Caenorhabditis elegans, antioxidants increased longevity in some (but not all) studies. If this proves true, these worms are the exception to a general rule.15,16
In his 2013 review, Barja wrote, “In any case, a final goal of gerontology is to increase human longevity, and it is now reasonably clear that antioxidants do not increase longevity in mammals.”9
Mitochondrial ROS Production
This information gave rise to an alternate hypothesis, that the rate of production of mtROSp in an animal is the critical factor in aging.17 Producing antioxidants is a metabolically energy-expensive process. Long-lived animals economize by limiting ROS production.
The association of low mtROSp and greater longevity has since been demonstrated in a wide range of animals, in particular in comparisons between short-lived rodents with birds that live 7 to 9 times longer but are of similar weight and metabolism.xviii
This association was seen both in species that follow Pearl’s 1928 ‘rate of living law’ (ie, the lower the whole body weight-specific metabolic rate, the longer the longevity) and also in those that do not. Three groups of vertebrates have greater longevity than predicted by Pearl’s law: birds, bats, and primates. The relationship between the mtROSp and longevity in all these species does follow the MFRTA.19
In the 1970s, it was thought that this ROS production occurred in complex III of the mitochondrial electron transport chain, but it was later determined that this actually occurs in complex I.20,21
Within the mitochondria the site in which ROS are generated lies in close proximity to the mitochondria’s DNA (mtDNA). While other sites within the cell may produce ROS, it is the quantities of ROS produced in close proximity to the mtDNA that are most correlated with lifespan.
Researchers have correlated measurements of 8-Oxo-2'-deoxyguanosine (8-oxodG), a chemical that reflects levels of DNA damage and that can be measured easily in blood tests, with longevity and with levels of mitochondrial DNA damage, but notably not with the amount of DNA damage found in the cell nucleus.22
Why is this so counterintuitive? How would something as localized as mitochondrial ROS production be so important an influence on longevity while systemic antioxidants have so little effect?
The answer is likely that our view of a cell’s interior is oversimplified; we see cells as balloons of undifferentiated cytoplasm, when in fact a cell’s contents are highly compartmentalized. Measurements of the global oxidative stress of homogenized cells does not tell us what is actually going on when it comes to aging, even if it does help us predict near-term survival. Aging increases the probability of death, but it is not death. Old people have aged over time, but they are still alive; their odds of dying increase with advancing age.
So the answer to our quandary is probably that the concentration of ROS varies in particular cell compartments, particularly in the mitochondria and in this location, the proximity to complex I reactions that generate ROS, outweighs general antioxidant levels in the cell.
We need to view these ROS production processes on the micro level, not the cellular level, and certainly not on the organism level. It is damage to the mtDNA that determines age, and antioxidants appear to have little protective effect against locally generated ROS. Antioxidants may have global protective effect, but we age locally.
Fatty Acid Saturation
The second aspect of the longevity discussion that should catch us by surprise is the association between cellular membrane fatty acid saturation and longevity. The degree of fatty acid unsaturation in cellular membranes correlates with longevity. This is firmly established; it has been studied many times and the study results are consistent. The longer a species lives, the smaller the number of fatty acid double bonds in its cell membranes. The fewer unsaturated fatty acids, the more resistant these membranes are to lipid peroxidation, a highly destructive process that produces both mutagenic and toxic metabolites within the cell.
In 1996, Pamplona et al compared liver mitochondrial membranes from rats, pigeons, and humans. “Liver mitochondrial membranes of especially long-lived species show both a low level of free radical production and a low degree of fatty acid unsaturation as important constitutive protective traits to slow down aging.”23 Since then at least 23 additional studies examining a variety of species have reported similar findings.24,25
Couture and Hulbert reported in 1995 that membrane fatty acid composition varies in a systematic manner with body size in mammals. By comparing tissue extracts from mammals that varied in body mass by 9,000-fold (mice up to cattle), it was found that “there were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (22:6 omega 3) in heart and skeletal muscle.” [The greater the mass, the lower the DHA levels.]
“In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI [unsaturation index] than larger mammals.… The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 22:6 omega 3.”26,9
That there is an “exponential relationship between docosahexaenoic acid of cardiac phospholipids and the heart rate” was first reported in 1978.27 This relationship is not restricted to heart tissue but exists in most important mammalian tissues. The bigger an animal is, the lower the DHA content in its membranes: DHA content of cell membranes decreases by 12% to 24% for each doubling of body mass in mammals.28
By 2007 these concepts of membrane unsaturation had been incorporated into the free radical theory of aging. Hulbert et al wrote that year, “The fatty acid composition of cell membranes varies systematically between species, and this underlies the variation in their metabolic rate. When combined with the fact that 1) the products of lipid peroxidation are powerful reactive molecular species, and 2) that fatty acids differ dramatically in their susceptibility to peroxidation, membrane fatty acid composition provides a mechanistic explanation of the variation in maximum lifespan among animal species.”29
ROS injure many types of molecules within cells. They attack protein and modify DNA, but perhaps of greater consequence, they damage intracellular organelle membranes. How vulnerable the fats in a cell are to oxidative damage is determined by 2 things. First is that oxygen and other free radicals are more soluble in lipid membrane bilayers than in aqueous solution. The second is that not all fatty acid chains are equally susceptible to damage. It is this latter consideration that is key to understanding these relationships.29
Docosahexaenoic acid (DHA) is a highly polyunsaturated omega-3 polyunsaturated fatty acid (PUFA). With 6 double bonds, DHA is very vulnerable to oxidative attack. More than half a century ago, Holman reported that DHA is 8 times more vulnerable to peroxidation than linoleic acid (LA), which has only 2 double bonds. DHA is 320 times more susceptible to peroxidation than the monounsaturated oleic acid (OA).30
The current view is that long-lived species have evolved a strategy to reduce the amount of PUFAs in their cell membranes to make them more resistant to ROS injury.24
The MRFTA has, in recent years, incorporated data on both calorie-restricted diets and methionine depletion and found this new information congruent with the general theory and relevant as these dietary strategies may provide a key to manipulating the key determinants of longevity as outlined by the theory.
It is established that cutting calorie intake extends lifespan in many animals.31 While there are many competing theories to explain this effect, most are related to mitochondrial oxidative stress.32
In mice and rats, caloric restriction significantly decreases the rate of mtROS generation in many organs, consistent with the MRFTA.33 Similar changes are seen with methionine depletion without caloric restriction. Severe methionine restriction may also change membrane unsaturation, but this only appears to occur in long-lived animals.34 Levels of 8-oxodG, a measure of DNA damage, were significantly lower in tissues of calorie-restricted rats in which mtROSp was also shown to have diminished.35
Long-term calorie-restricted diets will not work for most patients, but moderate methionine restriction may provide many of the same benefits, according to a rodent study.
Caloric restriction appears to be more complex than we thought; the source of the calories matters. Every other day feeding increases longevity independent of insulin-like growth factor (IGF) effects; it appears to do so by lowering mtROSp.36 Restricting only carbohydrates or only fats does not seem to increase longevity.37 Protein restriction when examined alone appears to be responsible for about half of the life-extending effects of caloric restriction. Attention is now focused on depleting specific amino acid depletion while using isocaloric diets, especially on methionine depletion.
Current data suggest that methionine depletion alone may account for half the increase in longevity seen in calorie-restricted diets. It is interesting to note that glutathione levels decrease in the livers of methionine-restricted animals; this antioxidant is not involved in the antiaging effect of methionine depletion. It is also worth noting that restricting other amino acids aside from methionine does not have similar effects on mtROSp or longevity.38
This information certainly has clinical relevance. Long-term calorie-restricted diets will not work for most patients, but moderate methionine restriction may provide many of the same benefits, according to a rodent study.39
Beta Blockers, Fish Oil, and Other Points
It has been reported that the β-blocker drug atenolol extends maximal lifespan in laboratory mice. This effect has recently been attributed to atenolol reducing PUFA in cell membranes, “leading to lower lipid peroxidation and decreased lipoxidation-derived damage to cellular proteins.”40,41 The older explanation though, that the benefit derives from reducing sympathetic driven stress responses, still seems reasonable.
It might also be noted that decreased levels of unsaturated fatty acids are present in patients with both psychiatric disorders and cardiovascular disease and that it has been established that increasing levels of PUFA, particularly omega-3 fatty acids, are associated with benefits.42
Fish oils
The current version of the MFRTA that equates higher PUFA levels with shorter lifespan obviously raises the question as to whether supplementation with fish oil or other highly unsaturated fats may have detrimental effects, accelerating aging. In 2014 Kelley et al reviewed 22 published human studies to determine whether omega-3 PUFA supplementation affected lipid peroxidation levels: “nine found no change, eight a decrease, and five an increase in markers of LPO [lipid peroxidation].”
These studies were limited by methodology, and most importantly they did not appear to focus in on mitochondrial membrane peroxidation, the area of concern. Still the majority showed either no increase or a reduction in lipid peroxidation.43 The brains of all species have similar levels of unsaturated fats despite variations in lifespan. The brain’s distinctive requirements for high PUFA may explain in part fish oil’s benefit for age-related cognitive decline.44
The Okinawan ‘Blue Zone’ question
In recent years a theory has been popularized in the media that people living in such diverse locales as Okinawa and Sardinia live longer because of their diets—in particular because of higher fish consumption. Such areas were given the name ‘Blue Zones’ by Dan Buettner in a cover story written for National Geographic magazine in November 2005.45
A detailed analysis of long-lived individuals in Sardinia did not find that longevity in the region resulted from dietary factors, but instead gave credit to various social factors: “that the high rate of inbreeding determined by frequent marriages between consanguineous individuals and low immigration rates have progressively decreased the variability of the genetic pool and facilitated the emergence of genetic characteristics that protect individuals from diseases that are major causes of mortality particularly in older individuals.”46
In other words, the secret to long life in these ‘Blue Zones’ may not be specific foods, good living, or any of the traits Buettner and others would have us aspire to, but instead to social patterns that result in what many would call inbreeding.
This assumption of greater longevity with fish consumption lacks support. An 8.5-year follow-up of an Italian study of aging reported that while higher monounsaturated (MUFA) intake increased survival, increasing unsaturated fatty acids was associated with increased mortality.47
Feeding mice fish oil instead of safflower oil shortens their lifespan, an effect blamed on “strong oxidative stress that caused hyperoxidation of membrane phospholipids and a diminished antioxidant defense system.”48
The focus on certain aspects of the Okinawan diet in popular media and the interpretation of this information by some health practitioners now appears to be an oversimplification.49 There are many aspects to this diet—in particular the high percentage of total calories derived from carbohydrates—that are often ignored. Current explanation for Okinawan longevity is the caloric restriction experienced by many in the population earlier in life and the high consumption of foods that mimic the biological effects of caloric restriction. The phenotypic appearance of many in this long-lived population (short stature, low body weight, and lean BMI) as well as the reduction in age-related chronic diseases and longer lifespan are consistent with long-term effects seen from caloric restriction early in life.50
These considerations aside, it appears that higher fish consumption reduces mortality in some51 but not all populations.52
Clinical Implications
While the MFRTA is well accepted among researchers, few of these basic concepts have reached us in clinical practice. Our appreciation for both antioxidants and polyunsaturated fatty acids are in part based on earlier versions of the MRFTA that have since been disproven. There are several assumptions we often make in practice that need to be considered.
We should be mindful that any benefits of antioxidants on increasing average lifespan were seen only in short-lived species and not in animals that survive longer than 3 years. This should be kept in mind when translating research findings from animal studies to clinical practice.
The hypothesis that supplemental antioxidants will increase maximal lifespan has been solidly disproven, yet it remains firmly entrenched in general belief. The value of antioxidants may only be to compensate against damage caused by suboptimal living conditions. Antioxidants may extend life for those who might otherwise die prematurely but may not act as the panacea to slow the process of aging.
We should question the universal high regard for polyunsaturated fatty acids, in particular DHA. If the current MFRTA proves true, high doses of PUFAs may, because they increase vulnerability to oxidation of cellular membranes, leave us more susceptible to aging.
Another concept worth highlighting is that caloric restriction and possibly methionine restriction (and atenolol) are the only reported strategies that may significantly change aging. All the many products and behaviors we encourage patients to take may have relatively less effect in comparison.
These may prove to be controversial ideas, but they certainly warrant consideration and discussion.
* Arctica islandica, a species of mud clam, one of which was an estimated 507 years old, is currently considered the longest-living species of animal.
** "Harman proposed that physiological iron and other metals would cause ROS to form in the cell via Haber-Weiss chemistry as a byproduct of normal redox reactions. The ROS would damage nearby structures like mitochondrial DNA. Harman predicted that administering compounds that are easily oxidized, such as cysteine, would slow down the aging process, a suggestion recently reviewed by Dröge. Harman's proposal constitutes the basis of the free radical theory of aging, which is supported by a large body of scientific evidence. The discovery of the superoxide dismutase enzyme in living organisms led to serious consideration of the role of free radicals in biology. If an enzyme that decomposed oxygen radicals inside the body was highly conserved during evolution, oxygen free radicals were likely important in biological systems.” From Life and Death: Metabolic Rate, Membrane Composition, and Lifespan of Animals
