| The free radical theory of aging
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| The most widely accepted chemical theory of aging is the free radical theory of aging (FRTA). This theory treats aging as the result of cumulative oxidative damage to biomolecules: DNA, RNA, protein, lipids, and glycoconjugates. From the viewpoint of the FRTA, longer-lived organisms have lower rates of production of reactive oxygen species (ROS; Chapter 35), better antioxidant defenses, and more efficient repair or turnover processes. While generally considered a chemical theory, the FRTA does not ignore the importance of genetics and biology in limiting the production of ROS, and in antioxidant and repair mechanisms. It also interfaces with other theories of aging, such as the rate of living theory because the rate of generation of ROS is considered a function of the overall rate and/or extent of oxygen consumption, and the crosslinkage theory because some products of ROS damage crosslink protein. Finally, as a chemical hypothesis, the FRTA does not exclude cumulative chemical damage, independent of ROS, such as racemization, deamidation and alkylation reactions, but focuses on ROS as the primary source of damage and the fundamental cause of aging.
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| The FRTA is supported by the inverse correlation between basal metabolic rate (rate of oxygen consumption per unit weight) and maximum lifespan of mammals, and by evidence of increased oxidative damage to proteins with age. Protein carbonyl groups, such as glutamic and aminoadipic acid semialdehyde, formed by oxidative deamination of arginine and lysine, respectively, are formed in proteins exposed to ROS. The steady-state level of protein carbonyls in intracellular proteins increases logarithmically with age and at a rate inversely proportional to the lifespan of species. Protein carbonyls are also much higher in fibroblasts from patients with Werner's or Hutchinson-Gilford syndromes, compared to age-matched subjects. Similar concentrations of protein carbonyls are also present in tissues of old rats and elderly humans, arguing that similar changes occur at old age in a range of organisms, regardless of the difference in their lifespans.
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Figure 42.5 Accumulation of amino acid oxidation products in human skin collagen with age. Methionine is oxidized to methionine sulfoxide (MetSO) by HOCl or H2O2; ortho-tyrosine is a product of hydroxyl radical addition to phenylalanine. Despite a 100-fold difference in their rate of accumulation in collagen, levels of MetSO and o-tyrosine correlate strongly with one another, indicating that multiple ROS contribute to oxidative damage to proteins (adapted from Wells-Knecht MC, et al.: Age-dependent accumulation of ortho-tyrosine and methionine sulfoxide in human skin collagen is not increased in diabetes: evidence against a generalized increase in oxidative stress in diabetes. J Clin Invest 1997;100:839-846.) |
| Figure 42.5 illustrates the accumulation of two relatively stable amino acid oxidation products in human skin collagen: methionine sulfoxide and ortho-tyrosine. These compounds are formed by different mechanisms involving different ROS
(Chapter 35) and are present at significantly different concentrations in skin collagen, but increase in concert with age. Other amino acid modifications that accumulate in skin collagen with age include advanced glycoxidation and lipoxidation end-products (AGE/ALEs), such as carboxymethyllysine and pentosidine (Fig. 42.6), and d-aspartate. The rate of accumulation of these modifications depends on the rate of turnover of the collagens (Fig. 42.7) and is accelerated in diabetes and hyperlipidemia. The increase in AGE/ALEs in collagen is thought to impair the normal turnover and contribute to the thickening of basement membranes with age. Increased age-adjusted levels of AGE/ALEs in collagen are implicated in the pathogenesis of complications of diabetes and atherosclerosis.
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| AGE/ALES: BIOMARKERS OF OXIDATIVE STRESS AND AGING |
| Advanced glycoxidation and lipoxidation end-products (AGE/ALEs) are carbohydrate- and lipid-derived chemical (nonenzymatic) modifications and crosslinks in protein. They are formed by reaction of proteins with products of oxidation of carbohydrates and lipids (Fig. 42.6). Some compounds, such as Nε-(carboxymethyl)lysine (CML), may be formed from either carbohydrates or lipids; others, such as pentosidine, are formed only from carbohydrates, and others, such as the malondialdehyde adduct to lysine, are formed exclusively from lipids. Carbohydrate sources of AGEs include glucose, ascorbate, and glycolytic intermediates; polyunsaturated fatty acids in phospholipids are considered the primary source of ALEs. Lysine, histidine and cysteine residues are the major sites of AGE/ALE formation in protein. Over 20 different AGE/ALEs have been detected in tissue proteins, and many of these are known to increase with age. AGE/ALEs are useful biomarkers of the aging of proteins and their exposure to oxidative stress. |
| d-aspartate is a non-oxidative modification of protein that is formed by spontaneous, age-dependent racemization of l-aspartate, the natural form of the amino acid in protein. The more rapid turnover of skin, compared to articular, collagen yields a lower rate of accumulation of d-aspartate in skin collagen and also explains the difference in rates of accumulation
of AGE/ALEs in skin versus articular collagen. AGE/ALEs are even higher in lens crystallins, which have the slowest rate of turnover among proteins in the body. Deamidation of asparagine and glutamine is another non-oxidative chemical modification that increases with age in proteins; it has been described primarily in intracellular proteins.
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| Figure 42.6 Structure of major advanced glycoxidation and lipoxidation end-products (AGE/ALEs). (A) the AGE/ALE, Nε-(carboxymethyl)lysine (CML), which is formed during both carbohydrate and lipid peroxidation reactions; (B) the AGE pentosidine, a fluorescent crosslink in proteins; (C) the ALE, malondialdehyde-lysine (MDA-Lys), a reactive ALE that may proceed to form aminoenimine (RNHCH=CHCH=NR) crosslinks in proteins. |
| Figure 42.7 Accumulation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs) and d-aspartate in collagens with age. Nε-(carboxymethyl)lysine (CML) is formed by oxidative mechanisms from glycated proteins or reaction of glucose, ascorbate or lipid peroxidation products with protein. The fluorescent crosslink pentosidine is formed by oxidative reaction of glucose or ascorbate with proteins. d-Aspartate is formed non-oxidatively by racemization of l-aspartate residues in protein. Differences in rate of accumulation of both oxidative and non-oxidative biomarkers are attributable to differences in rates of turnover of collagens. (adapted from Verzijl N, et al.: Effect of collagen turnover on the accumulation of advanced glycation endproducts. J Biol Chem 2000;275:39027-39031.) |
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| AGING OF THE CIRCULATORY SYSTEM |
| The extracellular matrix of the aorta and major arteries becomes thicker and more highly crosslinked with age, contributing to both the decrease in elasticity and the capacity of the endothelium to dilate blood vessels in response to physical and chemical stimuli. These changes occur naturally with age, independent of pathology, but may account for the increase in cardiovascular risk in the elderly. AGEs and ALEs are implicated in the crosslinking of the vascular extracellular matrix, explaining the increase in arterial crosslinking in diabetes and dyslipidemia. A new class of drugs, known as AGE-breakers, is being evaluated clinically for reversal of increased vascular stiffness in aging and disease. |
| The fluorescent age-pigment lipofuscin is considered the accumulated debris of reactions between lipid peroxides and proteins. Lipofuscin accumulates in granular form, possibly residual bodies derived from lysosomes, in the cytoplasm of post-mitotic cells at a rate that is inversely related to species lifespan. It may account for 10-15% of the volume of cardiac muscle and neuronal cells at advanced age, and its rate of deposition in cardiac myocytes in cell culture is accelerated by growth under hyperoxic conditions. In flies, the rate of accumulation of lipofuscin varies directly with ambient temperature and activity, and inversely with lifespan.
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| Overall, there are a number of chemical modifications, both oxidative and non-oxidative, that accumulate in proteins with age. While much of the research in this area has focused on slow, cumulative chemical modification of long-lived extracellular proteins, there is growing evidence that these reactions also proceed inside the cell where ROS are formed from mitochondria and from reactive metabolic intermediates, such as glyceraldehyde-3-phosphate and methylglyoxal (Chapter 35). The generation of these compounds is also accelerated under pathological conditions, for example, in plaque deposits in the vascular wall in atherosclerosis and in neurodegenerative diseases (see Box).
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