Mechanisms of Aging

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Aging is a syndrome of changes that are deleterious, progressive, universal and thus far irreversible. Aging damage occurs to molecules (DNA, proteins, lipids), to cells and to organs. Diseases of old age (diseases which increase in frequency with age, such as arthritis, osteoporosis, heart disease, cancer, Alzheimer's Disease, etc.) are often distinguished from aging per se. But even if the aging process is distinct from the diseases of aging, it is nonetheless true that the damage associated with the aging process increases the probability that diseases of old age will occur.

Some gerontologists prefer to use the word senescence because "aging" implies that the passage of time necessarily results in deterioration (biological entropy) — which is certainly not true during the early, developmental, time of life (before the age of 10 or 12 in humans). I will retain the word "aging" because I believe the association between aging & deterioration is universal as adult years progress and because the distinction between aging & development is very strongly established in conventional language. Also, shorter words make for slightly faster reading.

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One can catalog changes that typically occur with age. For people of developed countries age changes include: A loss of hearing ability, particularly for higher frequencies. There is a decline in the ability to taste salt&bitter (sweet&sour are much less affected). There is a reduction of the thymus gland to 5−10% of its original mass by age 50. Levels of antibodies increase with aging. One third of men and half of women over 65 report some form of arthritis. About half of those aged 65 have lost all teeth. The elderly require twice as much insulin to achieve the glucose uptake of the young. There is reduced sensitivity to growth factors & hormones due to fewer receptors and dysfunctional post-receptor pathways. The temperature needed to separate DNA strands increases with age. Weight declines after age 55 due to loss of lean tissue, water and bone (cell mass at age 70 is 36% of what it is at age 25). Body fat increases to age 60. Muscle strength for men declines 30−40% from age 30 to age 80. Reaction time declines 20% from age 20 to 60. Elderly people tend to sleep more lightly, more frequently and for shorter periods — with a reduction in rapid eye-movement (REM) sleep. Neurogenesis in the hippocampus declines with age. Degree of saturation of fats drops by 26% in the brains of old animals. Presbyopia (reduced ability to focus on close-up objects) occurs in 42% of people aged 52−64, 73% of those 65−74 and 92% of those over age 75. Most people over age 75 have cataracts. About half of those over 85 are disabled (defined as the inability to use public transportation). Over 75% of people over 85 have 3−9 pathological conditions, and the cause of death for these people is frequently unknown.

Aging changes are frequently associated with an increase in likelihood of mortality, but this is not necessarily the case. For example, graying of hair is a symptom of aging, but graying does not increase likelihood of mortality. Aging changes which are not associated with a specific disease, but which are associated with a generalized increase in mortality would qualify as biomarkers of aging — and would distinguish biological age from chronological age. Biomarkers would be better predictors of the increased likelihood of mortality (independent of specific disease) than the passage of time (chronological age). Cross-linking of collagen, insulin resistance and lung expiration capacity have been proposed as candidates but, as yet, no biomarkers of aging have been validated and universally accepted.

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Many scientists have wondered whether a single cause (probably cellular or hormonal) lies behind all aging phenomena — or whether aging is inherently multi-faceted. Differences in lifespan between species raise critical questions, in this regard. Why is a rodent old at 3 years, a horse old at 35 years and a human old at 80 years? Aren't the cells much the same? Why is it that at age 3 about 30% of rodents have had cancer, whereas at age 85, about 30% of humans have had cancer? Some species (such as lobsters, alligators and sharks) show few signs of aging. Cancer cells, stem cells and human germ cells seem "immortal" when compared to other cells.

When discussing aging it is important to distinguish two points on survival curves. Mean lifespan (average lifespan) corresponds to the age at which the horizontal line for 50% survival intersects the survival curve. Maximum lifespan corresponds to the age at which the survival curves touch the age-axis (0% survival) — and this represents the age at which the oldest known member of the species has died. (In animal studies, maximum lifespan is typically taken to be the mean lifespan of the most long-lived 10%.) Curve A as shown is a pure exponential decay curve. Curve B corresponds to the survival of small animals, such as mice or squirrels in a natural environment. Human survival was still close to curve B in ancient Rome when average lifespan was 22 years, but by the mid−1800s the typical North American lived to be 40 — more like curve C. Today, people in the most developed countries have an average lifespan of about 80 — resembling curve D. Reduction of infant mortality has accounted for most of the increased longevity, but since the 1960s mortality rates among those over 80 years has been decreasing by about 1.5% per year. Maximum lifespan for humans, however, has remained about 115−120 all through known history. The longest documented human lifespan has been for Frenchwoman Jean Calment who lived 122.3 years.

Curing specific diseases such as heart disease or cancer can do no more than further "square" the survival curve (toward curve E), with no effect on maximum lifespan. Curing cancer would add about 2 years to human life, whereas eliminating heart disease would add 3 or 4 years. Mean lifespan varies with susceptibility to disease, accident & homicide/suicide, whereas maximum lifespan is determined by "rate of aging". In aging research, maximum lifespan is regarded as a proxy for aging. Chemicals, calorie restriction with adequate nutrition, or other interventions which increase maximum lifespan are said to have slowed the aging process.

If human beings were free of disease & senescence the only causes of death would be accident, suicide & homicide. Under such conditions it is estimated that from a population of one billion, a 12-year-old would have a median lifespan of 1,200 years and a maximum lifespan of 25,000 years.

In 1825 an English actuary named Benjamin Gompertz discovered that likelihood of dying increases exponentially with age after maturity — an empirical observation that has stood the test of time. A 35-year-old is twice as likely to die as a 25-year-old and a 25-year-old is twice as likely to die as a 15-year-old. The exponential increase does not continue past age 80 and death rate may even decline after age 110 [SCIENCE 280:855-860 (1998)]. (Medflies — Mediterranean fruit flies — show a plateau of linear rather than exponential death rate when 20-25% of the population remains). Similarly, the risk of getting Alzheimer's Disease doubles every 5 years past the age of 60 — probably plateauing after age 90 (when over half the population is already demented). Cancer rate increases exponentially with age, but also seems to plateau in the very elderly. One explanation might be that subsets of the population that are considerably more hardy due to genetics or behavior may remain after the more heterogenous majority have died. Another explanation suggests the complete elimination of the forces of natural selection at the oldest ages — which causes subsequent survival to be completely the result of genetic "random drift" [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA) 93:15249-15253 (1996)]. Causes of death in middle-age tend to be due to diseases affecting high-risk individuals (cancer, diabetes, hypertension, etc.), whereas the elderly are more vulnerable to multiple pathologies due to vulnerability of aging organs & tissues [JOURNALS OF GERONTOLOGY 58A(6):B495-B507 (2003)].

Attempts to classify theories of aging have led to the two major classifications programmed aging and wear&tear aging. Programmed aging would be aging due to something inside an organism's control mechanisms that forces elderliness & deterioration — similar to the way genes program other life-stages such as cell differentiation during embryological development or sexual maturation at adolescence. By contrast aging due to wear&tear is not the result of any specific controlling program, but is the effect of the sum effect of many kinds of environmental assaults — ie, damage due to radiation, chemical toxins, metal ions, free-radicals, hydrolysis, glycation, disulfide-bond cross-linking, etc. Such damage can affect genes, proteins, cell membranes, enzyme function, blood vessels, etc.

When Pacific salmon have lived in the ocean for 2 or 3 years, they make an arduous upstream journey against a raging riverswim until they find a place suitable for spawning. After spawning, the adrenal gland releases massive amounts of corticosteroids — leading to rapid deterioration. It would be costly for the species to have salmon that could live another year and repeat the journey — or compete with the offspring for food. Although this process is obviously "programmed", it is inaccurate to describe it as "aging". Programmed death, rather than programmed aging, is a common phenomenon among animals that reproduce only once.

Grazing animals show wear-and-tear to their teeth to the point where they can no longer eat, and they die of starvation. Again, it stretches the point to say the teeth are aging. The teeth of rabbits (like human fingernails) continue to grow as wearing occurs — and in this sense are "programmed" to compensate for "wear&tear". Why don't grazing animals have teeth that continue to grow? Human beings can replace tissue, capillaries and bone in wound-healing, yet cannot regrow a severed limb the way a salamander can. Why isn't human DNA "programmed" to re-grow kidney or liver tissue as it ages? Planarians (flatworms) have a pool of stem cells which can replace any of their fully differentiated cells. Programming that compensates for wear & tear should be distinguished from programming that causes deterioration.

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Russell Wallace, who with Charles Darwin discovered natural selection, speculated that longevity much beyond the age of procreation would be a disadvantage for a species. Parents would threaten their children by competition for resources. This would imply an evolutionary advantage to genetically programmed aging. The programmed self-destruction with corticosteroids by Pacific salmon after spawning — and whose decaying bodies provide nutrient for their offspring — may be severe example indicating the possiblity of programmed senescence. But as biologist Peter Medawar noted, there is circular reasoning in claiming that senescence evolved so that non-senescent individuals could more readily survive. If there were no senescent, poorly-reproducing individuals, there would be no need for replacement.

If aging were the product of evolutionary forces, aging could reasonably be expected to result from programming. But since most animals in the wild die of accident, attack or disease it seems questionable that evolutionary forces determine aging. Robins in the wild, for example, have an estimated 12-year maximum lifespan and a 40% chance of surviving any given year. With a (0.4)12 — or 1 in 60,000 — chance that a robin can avoid accident, attack or disease for 12 years, there would seem to be little opportunity for natural selection to play a role in the evolution of senescence. Against this argument is evidence that early stages of senescence reduce the ability of an animal to survive — thereby causing earlier selection against older animals.

An alternative to the view that senescence is the product of evolution compares genetic programming to the engineering of a fly-by satellite designed to gather data about a planet. The engineering is focused on ensuring that the satellite reaches its destination and performs its data gathering/transmission when passing the planet. Beyond the planet it is a matter of indifference to the engineers how long the satellite continues to function — random decay occurs. Applying the analogy, the satellite passing the planet is like an organism passing its reproductive period. Once the objectives of reproduction & parenting have been achieved the organism decays by random malfunction.

Maximum lifespan of many animal species

The vast range of maximum lifespan differences between species provides convincing evidence that longevity is genetically influenced. An elephant lives about 10−20 times longer than a mouse, yet both animals have roughly the same number of lifetime heartbeats — the elephant at 30 per minute and the mouse at 300 per minute. Both species take about 200 million breaths in a lifetime. And both species have a metabolic potential (total kilocalories used per gram of body weight per lifetime) of about 200 kcal. This figure is much the same for other mammals, but humans are exceptional with a metabolic potential of 800 kcal. Brains use more energy than any other human organ. (Basal metabolic rate for humans is about 80 watts = 70 Calories per hour.) Birds have a metabolic potential of 1,000 to 1,500 kcal.

Gerontologists who compare the longevity of species explain this discrepancy by saying that while body weight correlates well with longevity, there is a better correlation with brain weight for primates. For other species brain size may be more related to motor function than to cognitive capacity.

Flight, like brain weight, also confers a longevity advantage. Finches & robins live about 3 times as long as rodents the same size. Flying squirrels live twice as long as their close relatives the chipmunks. Parrots have a maximum lifespan in excess of 90 years. The Andean condor may be the most long-lived of any bird, but its maximum lifespan has not been confirmed.

Gross attributes of species typically associated with greater longevity are: large size, ability to fly, brainy, a spiny or shelled encasement, and cold-blooded. All but the last attribute reduce vulnerability to predators. Porcupines are the longest-lived rodents. Naked mole rats, by living underground, are also safer from predators and live significantly longer than similarly-sized rats. All adaptations that afford protection from predators and other hazards justify greater developmental resources to build a more durable animal with a longer maximum lifespan.

Opossums evolving on an island free of predators have been shown to have substantially longer lifespans and smaller litters than opossums living on the nearby mainland [JOURNAL OF ZOOLOGY; 229:695-708 (1993)]. Where competition between individuals of a species for mates & resources is more important than survival against predators and other hazards, evolution causes more investment in making a more hardy & durable animal — which includes having fewer offspring on each birthing (but more total offspring over the lifetime) — with each offspring receiving more care and resources. Gene survival can be better promoted (up to a point) by extending lifespan and reproductive period of reproductively successful adults than by creating many more offspring, a signficant number of whom will not survive to become reproductive adults.

Large size also confers protection against predators and confers an improved ability to escape dangerous environments. Metabolic rate decreases proportionally with increases in body size, which allows larger animals to survive longer when food & water are scarce. [For a sphere, surface area S = 4πr2 and volume V = (4/3)πr3, which means that S/V varies inversely with r (radius). Because heat is generated in the volume and dissipates in the surface area, relative dissipation decreases with an increase in radius because of the decrease in S/V.] Large animals are better able to withstand extreme temperatures because of greater body mass. Large animals and birds are more easily able to travel long distances to find food or less harsh environments.

Cold-blooded animals needn't expend energy to maintain body temperature and therefore generate fewer free-radicals. Also, the rate of chemical reactions more than doubles for each 10ºC increase in temperature. Cold-blooded animals may use one-tenth as much energy as warm-blooded animals of the same body weight. The alligator, Galapagose tortoise and lake sturgeon combine large size with cold-bloodedness. Turtles live longer than other reptiles because of the shell which protects against predators. With the combination of hard shell, large size and cold-bloodedness, it is not surprising that the Galagose turtle is probably the most long-lived vertebrate. Hard shell, cold-bloodedness and the ability to reduce metabolic rate allow some bivalves to live nearly four centuries [GERONTOLOGY; Philipp,EER; 56(1):55-65 (2010)].

A short-lived organism would waste metabolic energy by over-investing in anti-oxidant or DNA-repair enzymes when the energy could be spent on rapid growth and reproduction. When a species has fewer predators, evolution invests fewer resources into speedy reproduction and more genetic resources (DNA repair, etc.) into a longer reproductive period (longer life). In the case of birds, the mitochondrial membranes contain more unsaturated fat making them less vulnerable to lipid peroxidation. And the protein complexes of the respiratory chain of mitochondria generate fewer free radicals in birds than in mammals. It is conceivable that an animal with well-engineered cells could live many centuries. Human germ cells have arguably lived for millions of years through an investment in DNA-repair enzymes, antioxidant enzymes and telomerase.

Evolutionary biologists are able to use artificial selection in the laboratory experimentally (rather than passively studying natural selection in the wild) to seek the evolutionary determinates of longevity. Michael Rose at the University of California has shown that Drosophila (fruit-flies) bred for 15 generations by disposing of eggs laid early in life and only using eggs that were laid toward the end of reproductive life achieved maximum lifespans 30% greater than that of controls. The long-lived strains had increased levels of SOD, CAT and xanthine dehydrogenase as well as increased levels of heat shock proteins conferring stress resistance [JOURNALS OF GERONTOLOGY 55A(11):B552-B559 (2000)]. Hsp22 heat shock protein expression was 2−10 times greater in the long-lived strains as compared to controls. Transgenic Drosophila (ie, fruit flies with artificially altered genes) with extra copies of hsp70 genes live nearly 8% longer than controls following heat treatment [NATURE; Tatar,M; 390:30 (1997)].

Dr. Rose has also observed the experimental increase in mortality associated with aging ceases late in life [PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY; Rose;MR; 78(6):869-878 (2005)]. Although mortality rates remain very high in late-life, they plateau. Studies of inbred Drosophila indicate that the plateauing cannot be due to genetic variation. From his evolutionary biology perspective Dr. Rose associates this phenomenon with a late-life end of the force of natural selection. This would imply that senescence is genetically programmed and that studying the genetics of the plateau could be the key to understanding the genetics of longevity.

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In nearly every culture on earth women outlive men — significantly so in the oldest years. But the men who do survive to become elderly are hardier than the women. A US National Institute of Aging study showed that 44% of men over age 80 are "robust and independent" compared with only 28% of women. And the percentage of surviving males increases from 15% at age 100 to 40% at age 105 in the United States.

If aging has been programmed by evolutionary forces, sex could be a very important contributor to the program. The reproductive organs of the human female exhibits a rate of aging that is among the most rapid of body systems. The complete shutdown of female fertility at menopause may be of value in preventing the birth of deformed children or death in childbirth of a mother who has several dependent children. For a species with a lengthy parenting period, it makes sense for fertility to cease long before the debilities of advancing age begin. Gonadotropin hormones from the pituitary gland are controlled by gonadotropin-releasing hormone, a 10-amino-acid peptide originating in neurons located in the arcuate nucleus of the hypothalamus. The two gonadotropin hormones (FSH & LH) are the same for females as for males, although their function is very different. Simplistically, FSH stimulates egg production in females & sperm production in males, whereas LH stimulates estrogen production in females & testosterone production in males.

In fertile females FSH (Follicle-Stimulating Hormone) accelerates the growth of 6−12 primary follicles in the ovary each month — one of which may become a mature ovum. The follicles secrete estrogens, the most powerful of which is estradiol. A sudden increase in LH (Luteinizing Hormone) usually triggers ovulation (follicle rupture with discharge of the ovum) and the conversion of the follicle to the corpus luteum ("yellow body") — which also secretes estrogen, but primarily secretes progesterone. Progesterone stimulate the walls of the uterus to prepare it for implantation of the fertilized ovum. If pregnancy occurs, progesterone inhibits ovulation (by suppressing FSH & LH) and promotes uterine development until the placenta becomes more mature. (Progesterone is so-named because it promotes gestation, ie, the growth of offspring in the womb).



Graph of Female Hormonal Cycles

Aside from their role in the monthly cycle, estrogens are responsible for the development and maintenance of the female sexual organs, cause the deposition of fat in the breast&buttocks (which contributes to the feminine figure) and have a potent effect on bone development.

Menopause is the event in a woman's life when her ovary literally runs out of eggs. The loss of follicles to produce estradiol causes an end to menstrual cycling and production of estrogen & progesterone by the ovary. At age 30, a woman's period is normally 28−30 days, but by age 40 her period is typically closer to 25 days and her rate of egg-loss has accelerated. Further shortening (accompanied by periods when no ovulation occurs) eventually leads to menopause at an average age of 50 (plus or minus 10 years). The menopausal woman often experiences anxiety, irritability and fatigue. Beginning before menopause most women experience "hot flashes", ie, 3 minute surges of blood to the skin of the chest, shoulders & face leading to sudden hotness & sweating. Hot flashes are associated with a pulsatile release of LH from hypothalamic neurons associated with body temperature elevation. Estrogen therapy eliminates hot flashes. The rate of loss of ovarian follicles doubles around age 35, raising the suspicion that a hypothalamic mechanism may be the ultimate cause of menopause [SCIENCE 273:67-70 (1996)].

The most serious complications of menopause are osteoporosis and a decline in cardiovascular health. The Framingham Heart Study demonstrated that between ages 35 to 65 men have 10 times the incidence of heart attack as women — probably because estrogen protects against heart disease. Estrogen elevates HDL cholesterol and reduces LDL cholesterol in the bloodstream.

After menopause, nipples decrease in size and the surrounding alveolar tissue shrinks. Erection of these tissues with external stimulation is more difficult. Vaginal contractions during orgasm is reduced to 4−5 at 0.8-second intervals from 8−12 in young adults.

The testes have been regarded as the source of maleness at least since ancient Rome, where eunuchs & women were not permitted to "testify" (testis is Latin for "witness"). In the male, LH stimulates secretion of testosterone by the interstitial cells of Leydig in the testes. FSH stimulates spermatogenesis in the seminiferous tubules of the testes. Testosterone promotes development of male sexual organs in the foetus. At puberty testosterone stimulates hair growth on the face & pubis, causes enlargement of the larynx to deepen the voice, increases skin thickness, causes a 50% increase in muscle mass, promotes bone growth, increases basal metabolism up to 15% and increases red blood cell concentration.

There is no sudden "andropause" event in males that is comparable to the menopause event of females. Instead, testosterone levels tend to decline gradually with age. This decline occurs most dramatically in those with cardiovascular disease or a predisposition to adult-onset diabetes. Although sperm count declines, fatherhood has been verified for a male as old as 94. Semen production declines in the prostate as a man ages — and the smooth muscle is replaced by overgrowing connective tissue that enlarges the prostate, blocks urine and can lead to cancer. 85% of men over age 50 have symptoms arising from benign prostatic hyperplasia — a noncancerous overgrowth of prostate tissue possibly caused by excessive expression of the anti-apoptosis protein bcl−2 [HUMAN PATHOLOGY 27:668-675 (1996)]. In some tissues testosterone must be converted to dihydrotestosterone (by the enzyme 5−α reductase) in order to act. This occurs most notably in the prostate gland, which produces semen (a mixture of sugars, protein and water). Dihydrotestosterone has also been implicated in baldness. The European drug Permixon (an extract of the saw palmetto berry) inhibits 5−α reductase, and is used to prevent prostate hypertrophy and prostate cancer. The Life Extension Foundation sells saw palmetto berry extracts as a dietary supplement for this purpose.

Testosterone has been used in elderly men for "rejuvenation" — to restore virility & muscle strength. Testosterone increases the risk of cardiovascular disease — by increasing blood pressure, by lowering HDL cholesterol and by elevating LDL cholesterol. These same dangerous side effects are also seen in athletes who attempt to use androgens or other anabolic steroids to improve athletic performance. Eunuchs reportedly live longer, although there have been no controlled clinical trials to prove this observation. Sterilization of a dog or cat (male or female) adds a couple of years to its lifespan. Any reduction in sex hormones would be expected to reduce cell proliferation and hence reduce the probability of cancer.

Male libido peaks in mid-adolescence, and does not correlate exactly with testosterone blood levels. In elderly men it may take from 10 seconds to several minutes to get an erection, in contrast to 3−5 seconds in young men. Contractions of the penile urethra during orgasm is reduced to 1−2 contractions per 0.8-seconds from 3−4 in young adults. Ejaculatory distance is reduced from 12−24 inches to 3−5 inches.

[For more about sex and aging, see Sex Hormone Replacement in Older Adults]

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Aging in the female reproductive system provides the best example of programmed aging in mammals. For many other organs — particularly the heart, brain, lung and kidney — specific disease states associated with aging are of more significance than generalized deterioration. There is wide variation in the health status of specific organs among the elderly.

Skin, lungs, muscles, blood vessels and organ-function in general is adversely affected by protein cross-linking — which is increased in diabetes. Because most of those 65 years of age have at least some symptoms of subclinical diabetes and because most of the symptoms of aging are accelerated in diabetes, diabetes figures strongly when the elderly are described in terms of averages. Generalized reduction in blood flow due to atherosclerosis also has an adverse effect on most organ systems — some more than others. Both protein cross-linking and cardiovascular deterioration are strongly influenced by genetics and environmental influences (diet, smoking, etc.).

With aging there is normally an age-related decrease in insulin sensitivity as well as of resting metabolic rate per unit of fat-free mass. These changes may not occur for those who maintain high levels of aerobic exercise [JOURNAL OF APPLIED PHYSIOLOGY; Clevenger,CM; 93(6):2105-2111 (2002) and AMERICAN JOURNAL OF PHYSIOLOGY; van Pelt,RE; 281(3):E633-E639 (2001)]. A study of very long-lived persons (over age 95) did not show a decline in resting metabolic rate [THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM; Rizzo,MR; 90(1):409-413 (2005)], but another study of those over age 90 did show reduced metabolic rate [JOURNAL OF GERONTOLOGY; Frisard,MI; 62A(7):752-759 (2007)]. Whether survival is due to this trait or whether the trait is a feature of aging cannot be distinguished by cross-sectional studies.

The kidney provides perhaps the most striking example of individual variation in the effects of aging. On average, kidney weight declines about 15% between ages 40 and 80. The kidney's filtering capacity for the average 90-year-old is typically half what it is for the average 20-year-old. But high blood pressure and diabetes are particularly damaging to kidney function. A 20-year longitudinal study showed no change at all among elderly men who had no health problems. If this result can be extrapolated it would mean that within the human maximum lifespan there is no significant kidney deterioration in the absence of disease conditions. (For a discussion of the issue of whether dietary protein can harm kidney function, see my essay Does Excess Protein Cause Kidney Damage?).

Cardiovascular disease is the most frequent cause of death among those over age 85. The left ventricle of the heart increases in size with age (hypertropy) due to an increase in size of the heart muscle cells that must work harder to pump blood through a circulatory system that has narrower channels and reduced elasticity. Lipofuscin content of heart muscle cells increases from about 1% in the young to over 5% in the old. Arteries thicken with age such that about three-quarters of elderly people have increased blood pressure (both systolic & diastolic). But, stated conversely, about a quarter of elderly people do not have elevated blood pressure. According to the Framingham Heart Study, systolic blood pressure is a better predictor of mortality than diastolic blood pressure. Hypertension is defined as a systolic blood pressure greater than 160mm Hg. Hypertension is present in 5% of those aged 60 and nearly one quarter of those aged 75-80. While heart attacks from ischemia account for 43% of deaths for those 65−74 years of age, it accounts for only 8% of deaths for that age group in Japan (where death-rate from stroke is much higher). (For more details concerning cardiovascular disease, risk factors and prevention — see my essays Sudden Cardiovascular Death and Prevention of Cardiovascular Disease.)

Aerobic capacity decline

Aerobic capacity (VO 2 max — liters of oxygen consumed per minute during peak exercise) declines increasingly steeply with age, and declines more steeply in men than in women. Although exercise increases aerobic capacity at any age, exercise does not prevent accelerated decline [CIRCULATION; Fleg,JL; 112(5):674-682 (2005)].

The claim that all people lose about 100,000 neurons per day has not been supported by modern research. 2% of neurons are lost, on average, between ages 20 and 90 (up to 40% of this loss in the frontal cortex). Those over age 86 show an average 10% decline in brain weight from age 20. Between age 30 and 90 brain volume declines an average of 14% in the cerebral cortex, 35% in the hippocampus and 26% in cerebral white matter. But averaging can be misleading, because the elderly include many people with considerable dementia and others with little or none. Nonetheless, a cross-sectional Magnetic Resonance Imaging (MRI) study of healthy volunteers showed age-related declines in the volume of gray matter in association area (rather than sensory areas) of the cerebral cortex, particularly in the prefrontal cortex [CEREBRAL CORTEX; Raz,N; 7(3):268-282 (1997)]. Dementias are more common among the elderly who develop cardiovascular disease. Dramatic reduction in cerebral blood flow and in brain oxygen&glucose utilization is frequently seen after the 8th decade of life. Although most dementias are due to Alzheimer's Disease, at least 20% of dementias are due to stroke(s).

Skeletal muscles are "fast-twitch" or "slow-twitch". Fast-twitch muscles ("white meat") can deliver much power over short periods through energy from anaerobic (oxygen-free) phosphagen (creatine phosphate) and glycogen/lactic-acid metabolism. Slow-twitch muscles ("dark meat") provide endurance with aerobic metabolism — using more mitochondria, more myoglobin and more capillaries per square inch. Sprinters&jumpers have more fast-twitch muscle, whereas marathoners&swimmers have more slow-twitch muscle. Posture is maintained with slow-twitch muscles. Aging results in greater loss of fast-twitch than slow-twitch muscle. Muscle fibers are replaced by fat & connective-tissue. Mitochondria die. Exercise can slow this deterioration because fast-twitch fibers atrophy due to loss of the nerves that innervate them (a loss possibly due to disuse).

Muscles in the iris of the eye atrophy, and pupil size reduces, with age — increasing the need for illumination. The lens thickens and becomes yellowed, reducing green-blue-violet discrimination. (Elderly painters use less violet & dark blue because the colors look the same.)

Collagen & elastin in tendons & ligaments become less resilient and more fragmented as a person grows older, particularly due to glycation (cross-linking of proteins by sugar). Articular cartilage becomes frayed and the synovial fluid between joints becomes "thinner". Decline in circulatory function contributes to this process. Glycation of collagen & elastin is accelerated in diabetics due to high blood sugar.

Hair graying accompanies aging regardless of gender or race. By 50 years of age approximately 50% of people have 50% gray hair [MICRON; Van Neste,D; 35(3):193-200 (2004)].

Aging of skin is commonly divided into "chronological aging" and "photoaging", with up to 80% of skin aging attributed to photoaging in non-smokers. Photoaging is due to ultraviolet (UV) light, which activates inflammatory cytokines & metalloprotein collagenases as well as inducing free radicals [ARCHIVES OF DERMATOLOGY; Fisher,GJ; 138(11):1462-1479 (2002)]. UV radiation generates singlet oxygen which both activates metalloproteinases and causes large scale deletions of mitochondrial DNA [JOURNAL OF BIOLOGICAL CHEMISTRY; Berneburg,M; 274(22):15345-15349 (1999)]. Carotenoids, especially lycopene, are particularly effective quenchers of singlet oxygen [ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS; Di Mascio,P; 274(2):532-538 (1989)].

Collagen & elastin also cross-link in skin, resulting in a loss of elasticity. The protein keratin in fingernails is also a component of the outer layer of skin (epidermis), which provides "water-proofing". The epidermis thins with age, leading to wrinkles. Decreased secretion by sweat glands increases vulnerability to heat stroke. When the melanocytes (cells that produce the skin&hair-coloring substance melanin) associated with hair follicles cease functioning, hair turns white. Partial reduction of melanocyte function results in hair that appears "gray". Yet 90% of Caucasians show increased melanin in the form of brownish spots on the back of their hands ("liver spots").

Although heat waves tend to lead to increased mortality among the elderly, those affected are generally persons with chronic disease conditions and unhealthy lifestyles. There is little alteration of thermoregulation with age among the normal elderly [JOURNAL OF APPLIED PHYSIOLOGY; Kenney;LW; 95(6):2598-2603 (2003)].

Loss of flexibility of the proteins collagen & elastin in the lung results in loss of elastic recoil. It becomes too difficult to fully exhale, which reduces air exchange, reducing the capacity to do work. Oxygen-to-tissue transfer rate is often halved by age 70.

Bone is typically 25% water, 30% soft tissue (cells & blood vessels) and 45% mineral deposits (mostly calcium). Most of the white ash remaining after cremation is calcium, lead, zinc and potassium from bone. Both men & women lose bone mass between the ages of 39 and 70 (osteoporosis), but post-menopausal women (who have reduce estrogen) lose bone mass at twice the rate as men. Decreased growth hormone causes bone loss in both sexes. The physical inactivity & malnutrition (especially for calcium and Vitamins D & C) of so many elderly also worsens bone loss. A reduction of one to three inches in height by age 80 is not unusual, with women shrinking twice as much as men. Young bones have been compared to green tree branches that can bend considerably before breaking — and upon breaking does so with splintering. By contrast, old bone is like a dry stick that snaps upon bending. 20% of hip fractures associated with osteoporosis are fatal in the US.

Joints in the bones of the inner ear calcify, contributing to a loss in the ability to hear higher tones. Loss of sweat glands in the ear causes earwax to become drier & crustier. Wax obstruction reduces the ability to hear low frequencies.

Aging reduces salivary secretion resulting in a drier mouth and decreased protection from bacterial infection of the mouth. Gastric juice volume is reduced 25% by age 60 and there is a 60% decline in pepsin activity. But this does not noticeably affect digestion except in the case of heavy meats. Absorption of Vitamin D (and, hence, calcium absorption), Vitamin B 12 (affected by reduced "intrinsic factor") and folic acid all typically decline with age.

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Atomic nuclei are surrounded by electron orbitals which contain a maximum of two electrons, each having opposite spin. Hydrogen has one outer orbital, but nitrogen, carbon and oxygen have 4 outer orbitals — with a capacity for 8 electrons (an "octet"). Atoms are most stable when they have filled orbitals. Free radicals are highly reactive molecules or atoms that have an unpaired electron in an outer orbital that is not contributing to molecular bonding ("free"). Atoms or small molecules that are free radicals tend to be the most unstable, because larger molecules can have the capacity to form resonance structures.

Electron States of ROS . (dot) indicates free-radical extra electron

Normal molecular oxygen (3O 2 , so-called triplet oxygen) is a very unusual free-radical in that it has two unpaired electrons in outer orbitals (a double radical). Pi−bonds are bonds formed from overlapping p−orbitals. But for 3O 2 , two pi−bonds are formed from two p−orbitals, each containing one electron. The two electrons can have three possible arrangements: two "up"−spin (indicated by two up-arrows in the diagram), two "down"−spin or one spin "up" and one spin "down" — which makes 3O 2 somewhat stable. But by the addition of energy (22.5 kcal/mole), both electrons are move into a single p−orbital, with the electrons having opposite spins — giving singlet oxygen (1O 2 ).

Although singlet oxygen is not a free-radical, the electrons are in an excited state and can thus cause damaging reactions similar to those caused by oxygen free-radicals. On the other hand, if an electron is added to normal triplet oxygen, the new electron completes one orbital, leaving the other orbital with an unpaired electron — resulting in a superoxide anion (.O 2 −), which is a conventional, unitary free-radical. Singlet oxygen is attracted to double-bonds and can react destructively with DNA & proteins. Singlet oxygen is especially reactive with the amino acid histidine — resulting in enzyme denaturation. Singlet oxygen oxidizes the guanine base of DNA to produce 8−OHdG/8−oxoG [JOURNAL OF BIOLOGICAL CHEMISTRY; Ravanat,J; 275(51):40601-50604 (2000)]. Singlet oxygen from ultraviolet light is believed to be the major contributor to "photoaging" of the skin [JOURNAL OF BIOLOGICAL CHEMISTRY; Berneburg,M; 274(22):15345-15349 (1999)].

Lewis structures are structural chemical formulas depicting outer-shell electrons. I use abbreviated Lewis structures showing only relevant outer-shell electrons to explain free radicals — ie, I show a single orbital containing paired or unpaired electrons. Because an orbital containing one (unpaired) electron is not being complemented with an electron of opposite spin, the electron is said to be in an "unstable spin state" (another term for "free radical"). Thus, chemicals that react-with and stabilize free radicals are called spin-trapping substances.

Free radicals can damage nucleic acids, proteins or lipids. For biological systems, oxygen free radicals are the most important, in particular superoxide (.O 2 −), nitric oxide (.NO) and the hydroxyl radical (.OH). About 0.3% of superoxide exists in protonated form (HO 2 .), which is more reactive than superoxide itself. Because the protenated form of superoxide is uncharged, it can penetrate cell membranes more effectively than superoxide. Nitric oxide is a relatively unreactive free-radical which has a half-life of a few seconds, normally reacting quickly with oxygen (O 2 ). But if nitric oxide encounters a superoxide (.O 2 −), it forms peroxynitrite (ONOO−) which can decompose to form a hydroxyl radical (.OH). Peroxynitrite, like the hydroxyl radical, can react directly with proteins and other macromolecules to produce carbonyls (aldehydes & ketones), cross-linking and lipid peroxidation. Only 1−4% of the DNA single-strand breaks caused by peroxynitrite are due to hydroxyl radical (indicating the minor effect decomposition has on total DNA damage by peroxynitrite) [ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS; Roussyn,I; 330(1):216-218 (1996)]. Although hydrogen peroxide (H 2 O 2 ) and hypochlorite (OCl− — the active ingredient in bleach) are not themselves free radicals, these oxygen-containing molecules can facilitate free-radical formation. Moreover, HOCl is estimated to be hundreds of times more toxic than either hydrogen peroxide or superoxide [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Reiter,RJ; 917:376-386 (2000)].

All of these highly reactive oxygen-containing molecules (including singlet oxygen) are described as Reactive Oxygen Species (ROS). ROS attack bases in nucleic acids, amino acid side chains in proteins and double-bonds in unsaturated fatty acids — with the hydroxyl radical being the strongest attacker. ROS attack of macromolecules is often called oxidative stress. Reactive Nitrogen Species (RNS) also cause free radical damage. Peroxynitrite, which does most of its damage to endothelial cells, is nearly as destructive as the hydroxyl radical.

In a neutral water solution about one per 10−7 water molecules will dissociate into two ions, a reaction that can be represented as:

H:O:H => :OH− + H+

However, a water molecule subjected to ionizing radiation might dissociate into two free radicals: a hydroxyl radical & a hydrogen atom. The reaction can be represented as:

H:O:H => .OH + .H

A superoxide ion (.O 2 −) would result from the addition of an electron to a normal oxygen molecule (O 2 ). A more complete Lewis structure of oxygen-containing free-radical molecules (with oxygen & hydroxide ion also illustrated for contrast) showing all outer shell electrons would be:

It would be more accurate to draw resonance structures, but the above representations may be better for explanatory purposes.

The weed-killing herbicide paraquat generates superoxide. Superoxide (.O 2 −) ions are generated in large numbers in the mitochondria. Two superoxide ions are enzymatically converted to hydrogen peroxide (H 2 O 2 ) by the enzyme superoxide dismutase:

.O 2 − + .O 2 − + 2H+ => H 2 O 2 + O 2

The hydroxyl radical (.OH) is typically formed by oxidation of a reduced heavy metal ion (Fe++ or Cu+, usually) by the hydrogen peroxide:

Fe++ + H 2 O 2 => Fe+++ + .OH + :OH−

The last reaction, known as the Fenton Reaction, may be the most dangerous because it can occur in the cell nucleus and lead to DNA damage. The oxidized iron (Fe+++) can then catalyze the Haber-Weiss Reaction between superoxide and hydrogen peroxide to produce more hydroxyl radicals:

.O 2 − + H 2 O 2 => O 2 + .OH + :OH−

At neutral pH the Haber-Weiss reaction occurs only to a negligible extent when no metal ion is available to act as a catalyst. In the human body ascorbic acid is normally beneficial rather than harmful because nearly all iron and copper ions are tightly bound to carrier proteins (transferrin for iron and cearuloplasmin for copper ions), but this is not the case in the Cerebral Spinal Fluid (CSF) or where there is cellular breakdown due to ischemic-reperfusion injury. Bacteria are rich in iron, which is why hydrogen peroxide from macrophages is such an effective bacterial killer.

Metal ions can also react with ascorbate (Vitamin C) to produce singlet oxygen (1O 2 ) from normal triplet oxygen (3O 2 ):

Cu++ + ascorbate + 3O 2 => 1O 2

Unlike iron, copper generates more singlet oxygen than hydroxyl radical upon its reaction with hydrogen peroxide.

Wherever it is produced, the hydroxyl radical is highly reactive and can cause covalent cross-linking or free-radical propagation in a wide variety of biological molecules. A cell's superoxide ions tend to be concentrated in the mitochondria because they are too reactive to travel very far in an unaltered state — and are much less frequently found in the nucleus than in the cytoplasm. Similarly, hydroxyl radicals (which have a billionth-of-a-second half-life) do not drift far from their site of formation. But hydrogen peroxide molecules are more stable and can drift across the nuclear membrane into the nucleus or near cell membranes where hydroxyl radicals can be generated when heavy metal ions are encountered. Hydrogen peroxide can damage proteins directly by the oxidation of −SH groups.

The hydroxyl radical can react with molecules (LH) in membranes to produce lipid molecule radicals (alkyl = .L)

Peroxyl Radical from Alkyl Radical

.OH + LH => .L + H 2 O

These lipid radicals can then react directly with oxygen (autoxidation) in a self-propagating chain reaction forming lipid peroxides (lipid peroxyl radicals, lipid molecules containing paired-oxygen groups −−OO−−):

.L + O 2 => LOO.

LOO. + LH => LOOH + .L

The first reaction is about fifteen hundred times faster with singlet oxygen (1O 2 ) than with normal triplet oxygen (3O 2 ). Singlet oxygen is energetic enough, however, that it can react directly with the double bonds of unsaturated fatty acids, without requiring a free radical intermediate.

The lipid hydroperoxides (LOOH) can promote a Fenton reaction:

Fe++ + LOOH + H+ => Fe+++ + .OL + H 2 O

The lipid alkoxyl radical (alkoxy = alkoxyl = .OL) is more reactive and damaging than the lipid peroxide (peroxyl) radical (peroxy = peroxyl = LOO.). Thus, by a small sequence of steps one free-radical (.L) has become two radicals (.L and .OL) — conditions for an auto-amplifying chain reaction. Nonetheless, if two alkyl, alkoxyl or peroxyl radical molecules collide they will nullify each other, but at the cost of creating a cross-link (covalent bond) between the two lipids.

Free Radical Half-Life at 37ºC Radical Symbol Half-Life Time Hydroxyl .OH one nanosecond Singlet Oxygen 1O 2 one microsecond Superoxide .O 2 − one microsecond Alkoxyl .OL one microsecond Peroxyl LOO. ten milliseconds Nitric Oxide .NO few seconds

The reactivity of free radicals can be quantified by a table of half-life (time taken for half of the remaining radicals to react) values at 37ºC (body temperature). Short half-life corresponds to high reactivity. The one nanosecond half-life of the hydroxyl radical indicates that it is so reactive that it reacts with the first molecule it bumps into.

Outside of the mitochondria, superoxide and hydrogen peroxide can be generated on the endoplasmic reticulum through oxidation processes involving cytochrome P−450 and NADPH−cytochrome c reductase. Abnormal accumulation of normal metabolites such as lactate, pyruvate, acetoacetyl−CoA and glyceraldehyde−3−phosphate can abnormally increase levels of NADH oxidase & reduced flavoenzymes such as xanthine oxidase. In the absence of sufficient electron acceptor substrates these enzymes can directly transfer electrons to O 2 or Fe+++ to form superoxide or Fe++. Ascorbate forms H 2 O 2 on autoxidation (direct combination with oxygen). Both ascorbate & mercaptans (thioalcohols, ie, compounds having "−SH" groups, where sulfur is substituted for the oxygen of alcohol) are capable of reducing Fe+++ & Cu++ to Fe++ & Cu+, thereby promoting Fenton reactions.

Lipid peroxidation of polyunsaturated fatty acids exposed to oxygen leads to rancidity in foods. In living animal cells peroxidized membranes lose their permeability, becoming rigid, reactive and nonfunctional. Lipid peroxidation can produce singlet oxygen, hydroperoxides and lipid epoxides. In addition, many damaging aldehydes are formed during lipid peroxidation, particularly MalonDiAldehyde (MDA, propanedial) & 4−HydroxyNonEnal (4−HNE). MDA is a major metabolite of arachidonic acid (20:4)[fatty acid with 20−carbons & 4 double-bonds]. MDA assays (notably TBARS — ThioBarbituric Acid-Reacting Substances) have been widely used as a measure of cell membrane lipid peroxidation. 4−HNE is also a product of 20:4 fatty acid autoxidation. 4−HNE reacts with cellular components more strongly than MDA. 4−HNE reacts readily with histidine residues, sulfhydryl groups and primary amino groups of proteins [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Uchida,K; 89(10):4544-4548 (1992)]. The fact that 4−HNE is the most toxic known aldehyde produced by lipid peroxidation (much more toxic than MDA) and yet is practically non-reactive with TBA (about 95% of TDA reactivity is due to MDA) points to the deficiency of TBARS as a lipid peroxidation assay [ALCOHOL & ALCOHOLISM 20(2):161-173 (1985)]. F 2 −isoprotanes, produced by oxidation of arachidonic acid, are the best biomarkers of lipid peroxidation [FASEB JOURNAL; Montuschi,P; 18(15):1791-1800 (2004)].



LIPID PEROXIDATION FORMING MDA

Unlike free-radicals, the aldehydes MDA, 4−HNE & other aldehydes are rather long-lived and can drift far from membranes, damaging a wide variety of proteins, lipids & nucleic acids [FREE RADICAL BIOLOGY AND MEDICINE 11:81-128 (1991)]. Such damaged molecules are called Advanced Lipid peroxidation End-products (ALE, which can be as resistant to degradation as AGEs [BRITISH JOURNAL OF PHARMACOLOGY; Negre-Salvayre,A; 153(1):6-20 (2008)]. 4−HNE inactivates glucose−6−phosphate dehydrogenase, an enzyme required for the formation of NADPH and for forming ribose residues for nucleic acid biosynthesis. Aldehyde-bridge formation leads to the protein-protein cross-linking associated with lipofuscin formation. Plasma levels of both MDA and 4−HNE rise significantly with age [FREE RADICAL RESEARCH; Gil,L; 40(5):495-505 (2006)].

Polyunsaturated fatty acids are more vulnerable to free radical oxidation than any other macromolecules in the body — and the sensitivity to free radical damage increases exponentially with the number of double bonds. Studies of the liver lipids of mammals & a bird (pigeon) show an inverse relationship between maximum lifespan and number of double bonds [JOURNAL OF GERONTOLOGY 55A(6):B286-B291 (2000)]. Nonetheless, brain phospholipid unsaturation does not vary much between mammals, probably indicating the importance of unsaturated fatty acids for neural function [COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY Part B 132:515-527 (2002)].

Animal cells contain three important enzymes to deal with the superoxide and hydrogen peroxide: SuperOxide Dismutase (SOD), glutathione peroxidase and CATalase (CAT). A dismutase is an enzyme that catalyzes the reaction of two identical molecules to produce molecules in different oxidative states. In the absense of SOD, two superoxide ions can spontaneously dismutate to produce hydrogen peroxide and singlet oxygen. SOD catalyzes a reaction between two superoxide ions to produce hydrogen peroxide and triplet oxygen.

Catalase catalyzes the formation of water & free oxygen from hydrogen peroxide. CAT is present in membrane-limited organelles known as peroxisomes. Peroxisomes contain enzymes that degrade amino acids & fatty acids — producing hydrogen peroxide as a byproduct.

Glutathione is a tripeptide composed of the amino acids cysteine, glycine and glutamic acid. Glutathione is the major antioxidant in the non-lipid portion of cells (most of the cytoplasm). Glutathione exists in a reduced form (GSH) and an oxidized form (GSSG). Reduced glutathione hydrogen donation can neutralize a hydroxyl radical:

GSH + .OH —> .GS + H 2 O

and then oxidized glutathione radicals can neutralize each other:

.GH + .GH —> GSSG

Glutathione peroxidase neutralizes hydrogen peroxide by taking hydrogens from two GSH molecules — resulting in two H 2 O and one GSSG. The enzyme glutathione reductase then regenerates GSH from GSSG with NADPH as a source of hydrogen.

The elimination of hydrogen peroxide by glutathione can be written as the reaction:

2 GSH + H 2 O 2 => GSSG + 2 H 2 O

Long-lived transgenic fruit flies in which the enzyme which synthesizes GSH was overexpressed showed a maximum lifespan extension of nearly 50% [JOURNAL OF BIOLOGICAL CHEMISTRY; Orr,WC; 280(45):37331-37338 (2005)]. Glutathione levels generally decline with age [JOURNAL OF ANTI-AGING MEDICINE; Lang,CA; 4(2):137-144 (2001)], although no reduction of serum glutathione was seen in elderly women deemed to be in excellent physical and mental health [JOURNAL OF LABORATORY AND CLINICAL MEDICINE; Lang,CA; 140(6):413-417 (2002)]. Free radicals act on lipids to produce peroxides (−O−O− bonds) resulting in mutagenic epoxides and insoluble & non-digestible age pigments such as lipofuscin. Glutathione peroxidase/glutathione destroys fat peroxides in the same way it eliminates hydrogen peroxide:

2 GSH + ROOH => GSSG + ROH + H 2 O

Superoxide dismutase(SOD) is the most abundant anti-oxidant enzyme in animals. The liver, in particular, is very high in SOD. Cellular concentration of SOD relative to metabolic activity is a very good lifespan predictor of animal species. Most mammals experience a lifetime energy expenditure of 200,000 calories per gram, but humans have an amazing 800,000 calories per gram. Humans have the highest levels of SOD — relative to metabolic rate — of all species studied. Oxidative damage to DNA is ten times greater in rats than in humans. Maximum lifespan correlates with lower rate of free-radical production and higher rate of DNA repair [JOURNAL OF COMPARATIVE PHYSIOLOGY B 168(3):149-158 (1998)].

The SOD molecule in the cytoplasm (SOD1) and outside of cells (SOD3) contains copper & zinc atoms (Cu/Zn−SOD), whereas the SOD in mitochondria (SOD2) contains manganese (Mn−SOD).

Superoxide dismutase without glutathione peroxidase or catalase (CAT) to remove hydrogen peroxide is of little value. Insects lack glutathione peroxidase, but experiments have been performed on fruit flies made transgenic by having extra genes for SOD, CAT or both. The flies that were given extra genes for SOD or CAT (but not both) had no more than a 10% increase in mean lifespan, with no increase in maximum lifespan. But flies that had extra genes for both SOD and CAT showed maximum lifespan increase by as much as a third, while showing less protein oxidative damage and better physical performance [SCIENCE 263:1128-1130 (1994)]. But criticisms that the above experiments had been performed on short-lived strains of flies led to later experiments on long-lived strains of flies which showed no lifespan extension for overexpression of Cu/Zn−SOD, Mn−SOD, catalase and thioredoxin [JOURNAL OF BIOLOGICAL CHEMISTRY; Orr,WC; 278(29):26418-26422 (2003)].

Nonetheless, an experiment using SOD/CAT mimetics in nematode worms increased mean lifespan 44% [SCIENCE 289:1567-1569 (2000)]. Selective inbreeding of bread-mold fungus resulted in strains with lifespans more than 6 times longer than wild-type — a change that was shown to be due to increased expression of antioxidant enzymes [FREE RADICAL BIOLOGY & MEDICINE 8:355-361 (1990)]. Females express both more Mn−SOD and more glutathione peroxidase than males, and this has been suggested to be the reason females live longer than males in mammalian species [FEBS LETTERS; Vina,J; 579(12):2541-2545 (2005)]. The maximum lifespan of transgenic mice has been extended about 20% by overexpression of human catalase targeted to mitochondria [SCIENCE; Schriner,SE; 308:1909-1911 (2005)]. Although naked mole rats exhibit high levels of oxidative damage, these levels remain unchanged for over two decades [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Perez,VI; 106(9):3059-3064 (2009)].

Radiation produces the hydroxyl radical, but most of the oxygen free radicals are byproducts of cell metabolism — particularly in the mitochondria, the lysosomes and the peroxisomes. One of the reasons these organelles are surrounded by membranes may be to protect the cell from the free-radicals they generate. DNA may be sequestered in the nucleus, in part, as additional protection against free radicals. Nonetheless, free radicals contribute to DNA damage and mutation.

In addition to enzymes, the animal cell uses many other chemicals to protect against oxygen free-radicals. Vitamin E is the main free-radical trap in the (lipid) membranes. Vitamin C acts as an anti-oxidant in the non-lipid ("watery") portions of cells, between cells and in the bloodstream. Melatonin, a hormone produced by the pineal gland in decreasing quantities with aging, efficiently crosses membranes (including the nucleus) and is effective against hydroxyl radicals.

Uric acid (which is mostly formed from purine degradation) protects Vitamin C from oxidation by divalent ions and can act as an anti-oxidant. Uric acid also protects against free-radical catalysis by binding iron. Humans have higher levels of uric acid than monkeys and other mammals because humans lack the enzyme uricase. But birds typically have twice the plasma uric acid concentration as humans. Birds often live several times as long as comparably sized mammals despite over twice the metabolic rate, 2−6 times the plasma glucose and a 3ºC higher body temperature.

Mammals fed anti-oxidants show up to a 30% increase in average lifespan, but no increase in maximum lifespan. Anti-oxidants are most valuable for animals that are cancer-prone, or subjected to radiation or chemical toxins. There are evidently homeostatic mechanisms in cells that govern the amount of allowable anti-oxidant activity. For example, increased levels of Vitamin E in the diet correlates with reduced levels of glutathione peroxidase activity, and vice versa. Vitamin E was shown to increase catalase in banana fruit-flies — with increasing doses of Vitamin E extending fruit-fly lifespan up to a dose of 5 micrograms/mL, above which increasing doses decreased lifespan [GERONTOLOGY 42:312-321 (1996)].

(For more on anti-oxidants and anti-oxidant enzymes, see my essay General Anti-Oxidant Actions.)

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Mitochondrion organelle

The mitochondria are capsule-shaped cellular organelles that generate energy (ATP molecules) from aerobic (oxygen-utilizing) metabolism utilizing respiratory chain and ATP synthase enzymes. Most animal cells contain between a few hundred and a few thousand mitochondria. The most mitochondria are found in the cells that are most metabolically active: neurons and muscle cells, where mitochondria make up about 40% of cell volume. About 10% of the body weight of a human adult is mitochondria.

A mitochondrion has two membranes. The outer membrane contains small pores (porins, also known as Voltage-Dependent Anion Channels,VDACs) that are freely permeable to ions and other molecules smaller than 10 kiloDaltons in size. The inner membrane is highly impermeable, even to protons (H+ ions). The proton gradient across the inner membrane is used by ATP synthetase enzyme to generate ATP molecules. The region between the outer membrane and the inner membrane is more positively charged (P−phase) because of the higher proton concentration, whereas the inside of the inner membrane is more negatively charged (N−phase, the matrix). It is in the matrix that the Krebs citric acid cycle occurs. There can be tens of thousands of respiratory chain and associated ATP synthase molecules embedded in the inner membrane of a mitochondrion, especially in metabolically active cells that have their inner membranes most highly folded into cristae that increase surface area.

The inner membrane contains a number of active molecule carriers, including a phosphate (P i = H 2 PO 4 -) carrier and the Adenine Nucleotide Transporter (ANT). The ANT imports ADP molecules into the matrix for ATP synthesis in exchange for ATP molecules which are exported for energy use throughout the cell (like portable batteries). The respiratory chain ("electron transport chain") attached to the inner wall of the inner membrane is composed of 4 protein complexes. These protein complexes are identified as Complex I, II, III and IV. Complex II consists of only four peptides, two of which comprise the Krebs citric acid cycle protein succinate dehydrogenase, and two of which anchor the complex to the inner mitochondrial membrane.

Complex I and Complex II independently supply electrons to Complex III, which supplies electrons to Complex IV. Soluble carriers are used to transport electrons to and from Complex III. The soluble carrier transporting electrons from Complex I & II to Complex III is Coenzyme Q (CoQ). The soluble carrier that transports electrons from Complex III to Complex IV is cytochrome−c. For this reason Complex III is also known as cytochrome−c reductase and Complex IV is also known as cytochrome−c oxidase. Complex IV combines its electrons (which are actually hydrogen atoms) with oxygen to form water. The energy released by the oxidations in the respiratory chain are used to pump protons outside the inner mitochondrial membrane.

Protons pumped out of mitochondrial matrix Re-entering protons drive "ATP turbine"

The inner mitochondrial membrane is fairly impermeable to H+ ions ("protons") and thus is able to function much like a hydroelectric dam. Respiratory enzymes (Complex I, III & IV) pump protons out of the inner mitochondrial matrix, building proton pressure outside the "dam" (the membrane). The proton pressure ("proton-motive force") across the inner membrane is composed of two components: a pH difference and an electrical potential (membrane potential), which is the most important component. The pH difference is small, amounting to only about 0.5 pH units. The membrane potential of the mitochondrial membrane is about twice as great as that of a large nerve fiber, amounting to over 200 milliVolts. Complex V (F 0 F 1 −ATP synthase) is the "hydroelectric turbine" that utilizes the energy of the proton flow into the matrix through the "turbine" to synthesize ATP. The ATP synthase (Complex V) "rotary motor" is the smallest known natural nanomachine. It uses proton-motive force to drive the endothermic reaction:

ADP + P i => ATP

The combined result of respiratory (oxidative) steps and the ATP-creation (phosphorylation of ADP) step is called oxidative phosphorylation. Normally respiration (oxygen consumption) and phosphorylation (ATP production) are tightly coupled, ie, the amount of ATP produced corresponds to the amount of oxygen consumed — referred to as state 3 respiration. In the absence of ADP (eg, in a resting state), however, any respiration that occurs will be due to "proton leak" through the inner mitochondrial membrane rather than due to ATP production — referred to as state 4 respiration. (State 1, state 2 and state 5 are experimental conditions of more historical interest than metabolic interest.)

In state 4 respiration protons flowing directly through the inner membrane rather than through the "ATP turbine" (Complex V) produce heat energy rather than ATP energy. Uncoupling proteins are weak acids that dissolve inner membrane lipids thereby increasing the uncoupling of oxidation from phosphorylation. Uncoupling respiration from phosphorylation to produce heat is useful for small rodents, naked newborn babies, and hibernating & cold-acclimated animals, all of which contain "brown fat". Uncoupling is also useful for fever production. UCP1 is the UnCoupling Protein found in "brown fat", fat which has been made brown by high concentrations of mitochondria. UCP2 has broad tissue distribution and seems to function in stress response, but its expression is less than 1% of UCP1. UCP3 is found in muscle and is regulated by thyroid hormone (T 3 ). UCP2 & UCP3 may cause uncoupling for the purpose of reducing mitochondrial superoxide production [FREE RADICAL BIOLOGY & MEDICINE; Echtay,KS; 43(10):1351-1371 (2007)].

The function of UCP1 is to generate heat ("thermogenesis"). Claims have been made that UCP3 generates little heat, but functions to reduce free radical damage by lowering protein gratient during periods of high metabolic activity. Mice with higher UCP3 have shown higher metabolic intensity (17% greater resting oxygen consumption) and 36% longer lifespan [AGING CELL; Speakman,JR; 3(3):87-95 (2004)]. Proton leak has not been shown to be a factor in CRAN (Caloric Restriction with Adequate Nutrition) [AMERICIAN JOURNAL OF PHYSIOLOGY; Ramsey,JJ; 286(1):E31-E40 (2004)]. The fact that dieting-resistant obese subjects have been shown to have smaller amounts of UCP3 [DIABETES; Harper,M; 51(8):2459-2466 (2002)] would seem to indicate that thermogenesis from UCP3 is not negligible.

Compared to the heart & brain, mitochondria in the liver are more tightly coupled and use oxygen more efficiently for ATP production. The heart & brain mitochondria use more oxygen than liver mitochondria, but can produce ATP faster. Brain mitochondria are more geared toward maintaining cell integrity, in contrast to heart mitochondria which are more geared toward preserving cellular energy state [AMERICAN JOURNAL OF PHYSIOLOGY; Cairns,CB; 274(5):R1376-R1383 (1998)].

Increasing insulin levels associated with aging and type−2 diabetes stimulates nitric oxide synthetase resulting in peroxynitrite [THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 34:1340-1354 (2002)]. Lipid peroxidation of the inner mitochondrial membrane by peroxynitrite can increase proton leak independent of uncoupling protein. Peroxynitrite can also degrade function of respiratory enzymes [JOURNAL OF NEUROCHEMISTRY 70:2195-2202 (1998)] and inactivate mitochondrial superoxide dismutase (Mn−SOD) enzyme [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA) 93(21):11853-11858 (1996)].

Mitochondria are the only cellular organelles with their own DNA. (There is no other cellular DNA outside the nucleus apart from the DNA of mitochondria.) Mitochondrial DNA (mtDNA) in humans are circular strands of 16,569 nucleic acids that code for 37 genes — 22 transfer RNAs, 2 ribosomal RNAs and 13 transmembrane proteins. There are nearly 1,500 other gene products in mitochondria, which are coded-for by nuclear DNA (nDNA). In contrast to nDNA, the mtDNA is derived almost entirely from the mother. Each cell contains many mitochondria, but the total mtDNA in a cell represents less than 1% of the amount of DNA found in the nucleus.



Inner Membrane mtDNA-coded Proteins in Complex I, III, IV & V Re-entering protons drive "ATP turbine"

Each mitochondrion contains 2-to-12 identical copies of mitochondrial DNA (2-to-12 circular strands). Each mtDNA strand codes for 13 proteins, all of which are transmembrane subunits of Complex I, III, IV or V. Of the 13 mtDNA proteins, 7 are in Complex I, 1 is in Complex III, 3 are in Complex IV and 2 are in Complex V. A distinctive feature of the 13 proteins coded-for by the mtDNA is that they are hydrophobic (not easily dissolved in water), suggesting that it might be difficult to synthesize & transport them in the watery cytoplasm. For this reason it has seemed improbable that the mtDNA for these proteins could be moved to the nucleus where they would be better protected & repaired. But one of the Complex V (ATPase) mtDNA-coded proteins has been successfully synthesized in the nucleus and utilized in the mitochondria for a mammalian cell [REJUVENATION RESEARCH; Zullo,SJ; 8(1):18-28 (2005)] giving hope to the idea that all 13 mtDNA proteins might eventually be moved to the nucleus. An alternate hypothesis, however, claims that the mtDNA genes are of value in providing rapid local synthesis of proteins required for oxidative phosphorylation. Oxidative stress due to insufficient oxidative phosphorylation capability could signal mitochondrial transcription factors to induce production of mtDNA-coded proteins that are then implanted into the inner membrane where they attract the nDNA-coded proteins required for complete assembly of the complexes [PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY; Allen,JF; 358(1429):19-38 (2003)].

Complex I, which has 7 mtDNA-coded proteins (more than a quarter of all the proteins in the Complex), ages most rapidly. Substantia nigra neurons have increased susceptibility to Complex I defects — which may be responsible for Parkinson's Disease [NEUROBIOLOGY OF AGING; Smigrodzki,R; 25:1273-1281 (2004)]. By contrast, Complex II (which has no mtDNA-coded proteins) and Complex III (which has only one) are relatively unaffected by aging. Cytochrome−c oxidase (between Complex III and Complex IV) activity declines with age, resulting in increased production of superoxide and hydrogen peroxide. Diseases due to mutated mtDNA have the greatest effect on cells producing the most energy — cells of brain and muscle — hence mitochondrial diseases are often encephalomyopathies . A very common syndrome of mitochondrial disease is Mitochondria Encephalomyopathy, Lactic Acidosis & Stroke (MELAS). Homoplasmy describes the original condition of all of a person's mtDNA being the same, but as mtDNA mutations occur and the mutated mtDNA replicates, cells, tissues and even mitochondria can have a mixture of mtDNA types, a condition known as heteroplasmy.

An estimated 1−2% of oxygen used by mitochondria will normally "leak" from the respiratory chain to form superoxide [JOURNAL OF NEUROCHEMISTRY 59:1609-1623 (1992) & JOURNAL OF INTERNAL MEDICINE 238:405-421 (1995)]. The pro-inflammatory cytokine Tumor Necrosis Factor−alpha (TNF−α, associated with the metabolic syndrome) induces increased free radical production from the respiratory chain [AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY; Corda,S; 24(6):762-768 (2001)]. Aging is associated with decreased oxidative phosphorylation coupling efficiency and increased superoxide production. Free radicals can damage the mitochondrial inner membrane, creating a positive feedback-loop for increased free-radical creation. The "viscious cycle" theory that free radical damage to mitochondrial DNA leads to mitochondria that produce more superoxide has been questioned. The most damaged mitochondria are consumed by lysosomes whereas the more defective mitochondria (which produce less ATP as well as less superoxide) remain to reproduce themselves [REJUVENATION RESEARCH; de Grey,A; 8(1):13-17 (2005)]. But the efficiency of lysosomes to consume malfunctioning mitochondria declines with age, resulting in more mitochondria producing higher levels of superoxide. Mitochondria of older organisms are fewer in number, larger in size and less efficient (produce less energy & more superoxide).

Coenzyme Q (CoQ, in humans CoQ 10 ) is also known as ubiquinone, so-called because it is "ubiquitous" (universally-found) in almost all cellular organisms, with the exception of gram-positive bacteria and some fungi. CoQ is an essential component of the mitochondrial respiratory chain. From Complex I or Complex II dehydrogenase CoQ is reduced to CoQH 2 and subsequently oxidized in two steps — first to .CoQ− and then to CoQ. But .CoQ− is unstable and can easily errantly transfer an electron to an O 2 molecule resulting in superoxide ion (.O 2 −).

.O 2 − from Complex III escapes Mitochondria

Complex I has been believed to generate .O 2 − in one of the iron-sulfur clusters, which would go to the mitochondrial matrix where it could be neutralized by Mn−SOD. Experiments on isolated mitochondria identified the site of superoxide generation to be at the flavine mononucleotide moiety of Complex I [JOURNAL OF BIOLOGICAL CHEMISTRY; Kudin,AP; 279(6):4127-4135 (2004)], but claims have been made that experiments on isolated mitochondria are misleading [ACTA BIOCHEMICA POLONICA; Nohl,H; 51(1):223-229 (2004)]. An experiment on isolated synaptosomes indicated that Complex I inhibition increases H 2 O 2 production [THE JOURNAL OF NEUROSCIENCE; Tretter,L; 24(36):7771-7778 (2004)]. Most of the .O 2 − generated from Complex III comes from .CoQ−, with about half going to the matrix to be neutralized and half floating toward the cytoplasm [JOURNAL OF BIOLOGICAL CHEMISTRY; Muller,FL; 279(47):49064-49073 (2004)]. Thus, .O 2 − from Complex I & III can cause lipid peroxidation of the inner mitochondrial membrane and mtDNA damage, whereas .O 2 − from Complex III can damage the whole cell, including nDNA. Membrane potentials below 140 mV (potential resulting from the proton gradients across the inner mitochondrial membrane) are not associated with .O 2 −, but above 140 mV .O 2 − generation increases exponentially with potential. Uncoupling proteins can be a device for reducing proton pressure (membrane potential), thereby reducing superoxide production.



Voltage drops between Complexes

Higher voltage drops between energy states in the Complexes also result in greater capacity for superoxide generation. This may account for the high superoxide production associated with Complex I, which has a high voltage drop in transferring its electrons to Complex III.

Oxidative damage to particular mitochondrial proteins in the flight muscles of houseflies has been identified as a biomarker of aging for those insects. Specifically, adenine nucleotide transferase enzyme in mitochondrial membranes [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Yan,L; 95(22):12896-12901 (1998)] and the citric acid cycle enzyme aconitase [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Yan,L; 94(21):11168-11172 (1997)] are particularly vulnerable to oxidative damage and are used to identify the "physiological age" of houseflies. Aconitase also shows the most significant age-related decline of any citric acid cycle enzyme in mice [MECHANISMS OF AGING AND DEVELOPMENT; Yarian,CS; 127(1):79-84 (2006)]. Aconitase is readily oxidized by superoxide, a process that generates hydroxyl radical [JOURNAL OF BIOLOGICAL CHEMISTRY; Vasquez-Vivar,J; 275(19):14064-14069 (2000)].

CoQ forms an important part of the antioxidant defense against these superoxide radicals [BIOCHEMISTRY AND CELL BIOLOGY 70:390-403 (1992)]. The Mn−SOD (SuperOxide Dismutase) of mitochondria can be induced to higher concentrations by oxidative stress (in contrast to the cytoplasmic Cu/Zn−SOD which is constitutive rather than induced). Heart mitochondria also contains catalase (which is confined to peroxisomes in most other tissues) [BIOSCIENCE REPORTS 17(1):3-8 (1997)].

Associated with aging is a decline in the amount of CoQ in organs. A person 80 years old will typically have about half as much CoQ 10 in the heart, lungs and spleen as a 20-year-old [LIPIDS 24(7):579-584 (1989)]. Declines in functional mitochondria & CoQ 10 with age is most damaging to those organs that have the highest energy demands per gram of tissue, namely: the heart, kidney, brain, liver and skeletal muscle, in that order [JOURNAL OF INTERNAL MEDICINE 238:405-421 (1995)]. Neurons are the largest cells in the body and have the highest metabolic demands, with 70% of ATP produced required to maintain the sodium-potassium pump. Clinically, damage to brain and muscle tissue are the first symptoms of mitochondrial disease. Mitochondria in the brain tissue of Alzheimer's Disease patients is particularly damaged. Therapy has included the B−vitamins that act as coenzymes in the respiratory chain (thiamine, riboflavin, niacinamide) and CoQ 10 [ACTA NEUROLOGICA SCANDINAVIA 92:273-280 (1995)].

mtDNA deletion mutations accumulate in post-mitotic cells with age [BIOCHIMICA ET BIOPHYSICA ACTA 410:183-193 (1999)]. The "mitochondrial theory of aging" postulates that damage to mtDNA and organelles by free radicals leads to loss of mitochondrial function and loss of cellular energy (with loss of cellular function). Mutations in mtDNA occur at 10-20 times the rate seen in nuclear DNA. A significant portion of "photoaging" of the skin may be due to mtDNA deletions from singlet oxygen induced by ultraviolet light [JOURNAL OF BIOLOGICAL CHEMISTRY; Berneburg,M; 274(22):15345-15349 (1999)]. Transgenic mice having high levels of mtDNA point mutations and deletions are models of accelerated aging [CELL METABOLISM; Edgar,D; 10(2):131-138 (2009) and AGING; Edgar,D; 1(12):1028-1032 (2009)]. Unlike nuclear DNA, mtDNA has no protective histone proteins. And DNA repair is less efficient in mitochondria than in the nucleus. These factors account for the more rapid aging seen with Complex I & III as compared to Complex II & IV. Aging mitochondria become enlarged and, if they can be engulfed by lysosomes, are resistant to degredation and contribute to lipofuscin formation [EUROPEAN JOURNAL OF BIOCHEMISTRY; Brunk,UT; 269(8):1996-2002 (2002)].

A comparison of 7 non-primate mammals (mouse, hamster, rat, guinea-pig, rabbit, pig and cow) showed that the rate of mitochondrial superoxide and hydrogen peroxide production in heart & kidney were inversely correlated with maximum life span [FREE RADICAL BIOLOGY & MEDICINE 15:621-627 (1993)]. A similar study of 8 non-primate mammals showed a direct correlation between maximum lifespan and oxidative damage to mtDNA in heart & brain. There was a 4-fold difference in levels of oxidative damage and a 13-fold difference in longevity, supportive of the idea that mtDNA oxidative damage is but one of several causes of aging [THE FASEB JOURNAL; Barja,G; 14(2):312-318 (2000)].

A comparison of the heart mitochondria in rats (4-year lifespan) and pigeons (35-year lifespan) showed that pigeon mitochondria leak fewer free-radicals than rat mitochondria, despite the fact that both animals have similar metabolic rate and cardiac output. Pigeon heart mitochondria (Complexes I & III) showed a 4.6% free radical leak compared to a 16% free radical leak in rat heart mitochondria [MECHANISMS OF AGING AND DEVELOPMENT 98(2):95-111 (1997)]. Hummingbirds use thousands of calories in a day (more than most humans) and have relatively long lifespans (the broad-tailed hummingbird Selasphorus platycerus has a maximum lifespan in excess of 8 years). Birds have less unsaturation (oxidizability) in their mitochondrial membranes and have higher levels of small-molecule antioxidants, such as ascorbate & uric acid. Even for mammals there is a direct relationship between mitochondrial membrane saturation and maximum lifespan [JOURNAL OF LIPID RESEARCH; Pamplona,R; 39(10):1989-1994 (1998)].

Free-radicals from mitochondria result in damage to cellular protein, lipids and DNA throughout the cell. This damage has been implicated as a cause of aging. If the fatty acids entering the mitochondria for energy-yielding oxidation have been peroxidized in the blood, this places an additional burden on antioxidant defenses. The greatest damage occurs in the mitochondria themselves, including damage to the respiratory chain protein complexes (leading to higher levels of superoxide production), damage to the mitochondrial membrane (leading to membrane leakage of calcium ions and other substances) and damage to mitochondrial DNA (leading to further damage to mitochondrial protein complexes). An experiment in yeast that improved the accuracy of mitochondrial protein synthesis demonstrated a 27% longer mean life span [JOURNAL OF GERONTOLOGY 57A(1):B29-B36 (2002)].

Mitochronrial Permaeability

Transition Port (MPTP)

Mitochondria play a key role in apoptosis ("cell suicide"). Release of cytochrome−c from mitochondria into the cytoplasm is the event which initiates apoptotic cell destruction by caspase enzymes. Release of cytochrome−c into the cytoplasm can occur either by a Ca2+−dependent mechanism or a Ca2+−independent mechanism. In the Ca2+−dependent case Ca2+ overload in the mitochondrion triggers opening of the Mitochondrial Permeability Transition Pore (MPTP), which penetrates both the outer and inner membranes making a channel between the mitochondrial matrix and the cytosol outside the mitochondrion. The MPTP is a complex consisting of three proteins, VDAC (porin) of the outer membrane, ANT (Adenine Nucleotide Translocator) of the inner membrane and cyclophilin−D. Cyclophilin−D protein binds to ANT to promote MPTP formation [BIOCHEMICAL JOURNAL; Li,Y; 383(Pt 1):101-109 (2004)], possibly by increasing the sensitivity of the MPTP components to the effects of Ca2+ [CIRCULATION RESEARCH; Weiss,JN; 93(4):292-301 (2003)]. The entry of large solutes and accompanying water into the matrix causes the mitochondrion to swell and burst, releasing cytochrome−c into the cytoplasm.

The Ca2+−independent case requires two separate events for cytochrome−c release: (1) formation of large pores in the outer mitochondrial membrane by Bax/Bak proteins and (2) release of cytochrome−c from the inner mitochondrial membrane [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Ott,M; 99(3):1259-1263 (2002)]. The Ca2+−independent case can lead to apoptosis, whereas the Ca2+−dependent case is invariably associated with necrosis. In apoptosis the MPTP opens only briefly (if it opens at all), whereas in necrosis the MPTP remains open. Apoptosis requires ATP energy, but ATP energy is depleted if the MPTP remains open [NATURE; Halestrap,A; 434:578-579 (2005)]. The threshold amount of Ca2+ which causes MPTP opening in lymphocytes, brain and liver of old mice is significantly lower than that of young mice [BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS; Mather,M; 273(2):603-608 (2000)]. CoEnzyme Q 10 has been shown to reduce apoptosis by direct inhibition of the MPTP [JOURNAL OF BIOLOGICAL CHEMISTRY; Papucci,L; 278(30):28220-28228 (2003)].



Cardiolipin

Cytochrome−c is normally held to the inner mitochondrial membrane by the lipid cardiolipin (diphosphatidylglycerol). Cardiolipin composes 10% of the inner mitochondrial membrane and is present at lower concentrations in the outer mitochondrial membrane (especially near contact sites between the two membranes). This distinctive lipid is found only in mitochondrial membranes. Mitochondrial membrane cardiolipin content declines with age, resulting in a decline in cytochrome−c activity. 40% lower cardiolipin content and 35% lower cytochrome−c activity has been demonstrated in old rats compared to young rats. Restoration of membrane cardiolipin content restored cytochrome−c activity [FEBS LETTERS; Paradies,G; 406(1-2):136-138 (1997)].

Oxidation of cardiolipin releases cytochrome−c from the inner mitochondrial membrane, but cytochrome−c will not be released into the cytoplasm to induce apoptosis without the formation of large pores in the outer mitochondrial membrane by Bax/Bak protein. Bax/Bak membrane permeabilization occurs preferentially at cardiolipin-rich contact sites between the outer and inner mitochondrial membrane [BMC CELL BIOLOGY; Lutter,M; 2:22-30 (2001)]. But Bax/Bak permeabilization of the outer membrane alone may be sufficient to induce apoptosis.

If only one or a few mitochondria release cytochrome−c apoptosis may not occur, but the damaged mitochondria would themselves be degraded. By this means a few aberrant mitochondria which are producing excessive free radicals can be eliminated.

(For more about mechanisms of apoptosis see Cellular Senescence and Apoptosis in Aging)



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Proteins are long chains of amino acids (amino acid polymers, or polypeptides). 20 different amino acids occur in animal proteins. Amino acids are all organic compounds with a protonated amino group [−NH 3 +] and an ionized carboxyl group [−COO−] attached to the same (alpha-position) carbon atom.

Proteins can be damaged both by free-radicals and by glycation. Glycation (also called the Maillard reaction, or non-enzymatic glycosylation) is a reaction by which reducing sugars become attached to proteins without the assistance of an enzyme. (For details on the properties of reducing sugars, see cryopreservation with sugars.) This attachment occurs at the free amine group of lysine or arginine, which is not involved in the peptide bond. The reaction between glucose and a lysine amino acid in a protein molecule can be represented as follows:

In the diagram, glycation is the formation of a double-bond between the glucose aldehyde-group and the lysine amino group with the elimination of a water molecule. The double-bond between the glucose carbon and the lysine nitrogen is an imine (also known as a Schiff base). The imine can quickly re-arrange atoms such that the 2-carbon (2nd carbon) of the glucose loses its two hydrogens — resulting in a carbonyl group (>C=O) and in hydrogen-saturation of the carbon & nitrogen which formerly constituted the imine. This re-arrangement structure is called an Amadori product (a ketoamine). Both glycation and Amadori product formation are completely reversible reactions. But the formation of Advanced Glycation End-products (AGEs) by oxidation of Amadori products is irreversible.

AGEs in tissues increase the rate of free radical production to 50-times the rate of free-radical production by unglycated proteins. Histochemical analysis of the hippocampus of human cadavers shows that chronological age can be estimated by hippocampal AGE levels [HISTOPATHOLOGY; Sato,Y; 38(3):217-220 (2001)]. AGEs attached to LDL-cholesterol accelerates oxidation and subsequent atherosclerosis. The irreversible cross-linked proteins of AGEs in vessel collagen also contributes to atherosclerosis, as well as to kidney failure — conditions worsened in diabetes [DIABETES 46(Suppl 2):S19-S25 (1997)]. Cataracts are composed of urea-insoluble proteins in the lens of the eye. AGEs aggravate protein cross-linking in the plaques & tangles of Alzheimer's Disease, thereby accelerating neuron death [BRAIN RESEARCH REVIEW 23:134-143 (1997)]. AGEs can be formed in the body from glycation & oxidation or can be ingested directly from browned foods (such as fried poultry skin) or tobacco smoke. Approximately one third of absorbed dietary AGEs are excreted in urine and rest is presumably incorporated into body tissues [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Koschinsky,T; 94(12):6474-6479 (1997)]. (For more on the effect of ingested AGEs, see INGESTION OF ADVANCED GLYCATION END-PRODUCTS (AGES).)

The higher glycation rate in diabetics is undoubtedly related to the fact that diabetes greatly resembles accelerated aging. Hemoglobin glycation is often used as a time-integrated (as opposed to instantaneous) measure of blood glucose levels in diabetics. AGEs are universal symptoms of aging — adversely affecting skin, lungs, muscles, blood vessels and organ-function in general. Increased insulin resistance and other symptoms of diabetes are commonly seen features of aging. Diabetes-like atherosclerosis and the resultant generalized reduction in blood flow has an adverse effect on most organ systems.

Although most proteins are short-lived (mouse liver proteins have a half-life of 3 days) some proteins, such as crystallins in the eye lens of mammals, can last a lifetime. Lens crystallines, collagen and basement membrane are the proteins most vulnerable to cross-linking and AGE formation because they are the most long-lived proteins, with a slow rate of turnover.

AGE cross-links can be broken by N−PhenacylThiazolium Bromide (PTB), but 3−phenacyl-4,5-dimethylthiazolum chloride (ALT−711, alagebrium) has proven to be even more effective than PTB in breaking cross-links [CIRCULATION RESEARCH; Candido,R; 92(7):785-792 (2003)]. Alagebrium has proven effective in reducing systolic blood pressure [AMERICAN JOURNAL OF HYPERTENSION; Bakris,GL; 17(12 Pt 2):23S-30S (2004)] and providing therapeutic benefit for patients with diastolic heart failure [JOURNAL OF CARDIAC FAILURE; Little,WC; 11(3):191-195 (2005)]. Carnosine also has anti-glycating effects [CURRENT MEDICINAL CHEMISTRY;Guiotto,A; 12(20):2293-2315 (2005)].

In the extracellular matrix of senescent skin, the major protein cross-link is the arginine-lysine glucose product glucosepane [REJUVENATION RESEARCH; Svantesson,J; 12(3):137-148 (2009)]. In non-diabetic 90-year-olds glucosepane accounts for about 50 times the cross-linking as all other forms of cross-linking, and is more than twice as prevalent in diabetics as in non-diabetics [JOURNAL OF BIOLOGICAL CHEMISTRY; Sell,DR; 280(13):12310-12315 (2005)]. In diabetics, prosclerotic growth factors like TGF−ß (Transforming Growth Factor beta) promote extracellular matrix synthesis.

Collagen accounts for about a third of total body protein in mammals. Collagen cross-linking in skin, muscle and organs throughout the body leads to the sinewy, inelastic tissue characteristic of aging. Cross-linking of proteins makes connective tissue lose elasticity, increases arteriosclerosis, reduces kidney function, slows wound healing, reduces the vital capacity of the lung and contributes to cataracts. Cross-linking also contributes to arteriosclerosis by making LDL-cholesterol unrecognizable to LDL-receptors, thereby increasing LDL in the blood.

Birds have blood glucose levels that are 2−10 times higher, metabolic rates that are more than double and body temperatures 2ºC−4ºC higher — than similarly-sized mammals. Higher temperatures & higher blood glucose would be expected to accelerate glycation & AGE formation in birds — yet their lifespans are considerably longer than those of comparably-sized mammals. Hummingbirds have the highest levels of glycated hemoglobin of any bird, but these levels are lower than those seen in non-diabetic humans, partially because of higher red blood cell turnover and partially because of better membrane control of glucose transport [COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY; Beuchat,CA; 120(Part A): 409-416 (1998)].

Muscle carnosine correlates with mammalian lifespan

Birds have twice the blood concentration of antioxidant uric acid as humans and a much lower rate of free radical production. A study of hens showed less than one hundredth the quantity of Advanced Glycation End-products (AGES) as would be found in humans [JOURNALS OF GERONTOLOGY; Iqbal,M; 54A(4):B171-B176 (1999)]. The dipeptide carnosine (ß−alanyl-L-histidine) both inhibits glycation and has antioxidant metal chelating activity. Skeletal muscle concentrations of carnosine correlate with lifespan in mammals [BRAIN RESEARCH REVIEWS 23:134-143 (1997)]. Vitamin C also has anti-glycation properties as well as antioxidant action [DIABETES; Davie,SJ; 41(2):167-173 (1992)].

Glucose is not the most active sugar for glycation. Galactose is 5 times more reactive than glucose, fructose is 8 times more reactive, deoxyglucose is 25 times more reactive, ribose is 100 times more reactive and deoxyribose is 200 times more reactive. [Sucrose is composed of the two monosaccharides glucose & fructose, whereas lactose (milk-sugar) is composed of the two monosaccharides glucose & galactose.] Mice injected with galactose are models of accelerated aging [MECHANISMS OF AGING AND DEVELOPMENT; Song,X; 108(3):239-251 (1999)]. Some aldehydes produced by lipid peroxidation are more reactive than any of the sugars. Glucose assumes the cyclic conformation more readily than any other monosaccharide, making it the most resistant to both glycation and oxidation of any sugar. It is no evolutionary accident that the least reactive of sugars is the sugar organisms most use for energy.

Lipids as well as proteins are subject to glycation. Lipid glycation of LDL cholesterol increases the LDL oxidation associated with atherosclerosis [ATHEROSCLEROSIS, THROMBOSIS, AND VASCULAR BIOLOGY; Ravandi,A; 20(2):467-477 (2007)]. Vitamin B 6 prevents lipid glycation more effectively than other common anti-glycation agents [JOURNAL OF LIPID RESEARCH; Higuchi,O; 47(5):964-974 (2006)].

(For more information on glycation, see the International Maillard Reaction Society.)

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Not all of the damaging effects of sugar are due to glycation. Glucose & fructose are reduced to sorbitol by the enzyme aldol reductase. Sorbitol is a tissue toxin, contributing to retinopathy, neuropathy, cataracts and kidney disease in diabetes. And not all protein cross-linking is due to glycation. Aldehydes produced by lipid peroxidation, such as MalonDiAldehyde (MDA, propanedial), can cross-link proteins by forming covalent bonds with lysine amino acids.

Cross-linking of protein with lipid peroxidation product MDA

Mitochondria produce nitric acid at a rate comparable to the rate of superoxide production [JOURNAL OF BIOLOGICAL CHEMISTRY; 273(18):11038-11043 (1998)]. Peroxynitrite formed by reaction of nitric oxide with superoxide can irreversibly form covalent bonds with tyrosine amino acids in proteins, thereby blocking phosphorylation. Phosphorylation & dephosphorylation of enzymes by kinases & phosphatases at tyrosine, serine & threonine protein residues play a central role in enzyme activation/deactivation & cell signalling — both of which would be disrupted by nitrotyrosine formation.

Another form of protein damage is racemization, although this kind of protein damage is less serious than glycation. Cells can only make proteins from L−isomer ("left-handed") amino acids. Only L−isomer proteins are functional. Some D−isomer ("right-handed") proteins are not only non-functional, but harmful. Thermal energy causes a small percentage of proteins to spontaneously change from the L−form to the D−form — and this form of molecular deterioration is known as racemization. (Racemization allows for determination of an animal's age from the ratio of D−form to L−form in the dentine of a tooth).

Asparagine and glutamine amino acids on proteins spontaneously deaminate, especially when a glycine is in the adjacent carboxyl position. The rate of deamination of asparagine is 400 times greater than for glutamine. When the enzyme which can repair this protein damage is missing from experimental (knockout) mice, the mice suffer brain damage and die young [JOURNAL OF BIOLOGICAL CHEMISTRY; 276(23):20695-20702 (2001)].

Carbonyl (>C=O, ie, aldehyde or ketone) content of protein is used as a rough measure of protein oxidation. Carbonyl formation is irreversible, so oxidized proteins must be removed by degradation. Carbonyl content of protein in an animal cell increases exponentially with age. At least 30−50% of protein is oxidized in old animals, which correlates well with an estimated 30−50% decrease in enzyme activity in old animals. A study on houseflies showed an association between protein carbonyls and life expectancy, possibly indicative of an effect on rate of aging [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Sohal,RS; 90(15):7255-7259 (1993)].

A study of several species of mammals & a bird (pigeon) indicated a linear relationship between oxidative damage to protein and maximum life span [EXPERIMENTAL GERONTOLOGY 31(3):365-372 (1996)]. Cysteine & methionine are by far more vulnerable to oxidation by reactive oxygen species than any other protein amino acids because of their sulfhydryl groups. Oxidation of cysteine sulfhydryls can result in disulfides that cause protein aggregation and lipofuscin. Naked mole rats (which live at least 9 times longer than mice) have much more oxidative damage to proteins than mice, but maintain that level of damage unchanged for two decades. Although mice have a 12% oxidative decline in cysteine with age, naked mole rats show no age-related change in cysteine for two decades [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Perez,VI; 106(9):3059-3064 (2009)].

Methionine is oxidized to methionine sulfoxide, but methionine sulfoxide reductases enzymatically regenerate methionine [BIOPHYSICA ET BIOCHEMICA ACTA; Lee,BC; 1790 (11): 1471-1477 (2009)]. Additionally, isomerases can reverse the aberrant disulfide bridges — the only known enzymatic repairs of protein oxidation [FREE RADICAL BIOLOGY & MEDICINE; Shringarpure,R; 32(11):1084-1089 (2002)]. Transgenic fruit flies that overexpress methionine sulfide reductase primarily in the nervous system have shown a median lifespan extension of about 70% [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); 99(5): 2748-2753 (2002)]. In one study, methionine sulfoxide reductase knockout-mice showed reduced lifespan and increased carbonyl content on protein [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); 98(23): 12920-12925 (2001)], but another study showed no reduction in lifespan [THE FASEB JOURNAL; Salmon,AB; 23(20):4601-3608 (2009)]. Reducing dietary methionine to a fifth the normal intake has increased the lifespan of rats by 30% [JOURNAL OF NUTRITION; Orentreich,N; 123(2):269-274 (1993)]. Body weight is just over half normal for the rats and there are increased blood as well as decreased tissue levels of glutathione [THE FASEB JOURNAL; Richie,JP; 8(15):1302-1307 (1994)].

Oxidized protein is more vulnerable to cross-linking by lipid peroxidation aldehydes such as MDA & 4−HNE [FEBS LETTERS 405:21-25 (1997)]. Hydroxyl radicals cause protein crosslinking by the formation of dityrosine bridges. Oxidized & cross-linked proteins are resistant to degradation and therefore contribute to the accumulation of damaged proteins in many degenerative diseases.

Cellular proteins are continually being degraded (hydrolyzed) within cells by proteolytic enzymes, both for regulation of cellular processes and for "quality control" of proteins (eliminating malformed or malfunctional ones). Transcription factors and cell-cycle proteins must be quickly eliminated after having served their purpose. The four major classes of cellular proteolytic enzymes are (1) caspases (2) calpains (3) cathepsins and (4) proteasomes.

Caspases are mainly active in apoptosis and are therefore in the category of regulatory proteases. Calpains are Ca2+−dependent, ATP-independent proteases that mainly degrade membrane & cytoskeletal proteins (as well as certain transcription factors). Cathepsins are the major class of proteolytic enzymes found in lysosomes, although there are others. (As well, lysosomes contain enzymes for degrading lipids, carbohydrates and nucleic acids.) Proteasomes are enzymatic, prote