1. fheor. Biology. (1977) 67, 11 l-120
On the Etiology of Aging RICHARD Department
DELONG
of Biology, Del Mar College, Corpus Christi, Texas 78404, U.S.A. AND LOYD
Department
POPLIN
of Chemistry, Del Mar College, Corpus Christi, Texas 18404, U.S.A.
(Received 6 November
1975, and in revised,fbrm
18 October 1976)
Amino acid racemization is presented as an hypothesis for the cause of aging. Effects on important biomolecules resulting from changes in their three-dimensional conformations and resulting changes in activities and specificities are discussed. Aging is viewed as a natural process associated with attaining thermodynamic equilibrium.
Although a number of hypotheses have been proposed as the cause of aging, none are completely satisfactory and, usually, are difficult to subject to experimentation. Another proposal for the cause of aging is presented here but this hypothesis should lend itself to experimentation very well. A mechanism for aging to be meritorious should possess the following features: (1) it should be accumulative, (2) it should lead solely to abnormal anatomy and physiology, (3) it should be non-genetic, (4) it should lend itself to experimentation. The hypothesis we submit has all these attributes. We propose that aging is caused mainly by the racemization of L-amino acids to D-amino acids as time passes. Within cells and tissues, D-amino acids accumulate causing geometrical changes in important biomolecules such as structural and functional proteins. Accumulative changes in these molecules could lead to the symptom called aging. Biological stereospecificity is now common knowledge. One aspect of biological stereospecificity is the ability of certain molecules to differentiate between substrate enantiomers. This type of differentiation is related to the absolute three-dimensional orientation of the substrates and molecules 111
112
R.
DELONG
AND
L.
POPLIN
involved. Therefore, biological stereospecificity results from an enzymcsubstrate interaction (Phillips, 1966). Should enantiomers be formed in either enzyme or substrate, the interaction could be modified either qualitatively or quantitatively, or both. This would be especially so among large molecules such as proteins. Proteins are polymers, therefore, they are subject to polymeric configurational changes. There are, at least, three main types of polymeric configurations. These are: (1) isotactic polymers in which all chiral centers have the same configurations ; (2) syndiotactic polymers in which successive chiral centers alternate in configuration; (3) atactic polymers in which the configuration of chiral centers is random. It is well known that different physical and chemical properties exist for such polymers. The changes encountered in proteins, because of introduction of enantiomers into the polymeric molecule, could lead to dramatic effects biologically. In the last few years amino acid racemization has been used to determine the age of fossils (Bada, Luyendyk & Maynard, 1970; Wehmiller & Hare, 1971; Bada & Protsch, 1973; Bada, Kvenvolden & Peterson, 1973; Bada, Schroeder, Protsch & Berger, 1974). The ratio of the biologically active L-amino acid enantiomers to the D-form decreases with age and, knowing the rate constants for the racemization reaction, the age of the bone or shell specimen can be calculated. Amino acid racemization occurs not only in free amino acids but, also, in amino acids bound in proteins (Bada & Schroeder, 1975). At first thought, racemization of individual amino acids bound in a polypeptide would seem to require tremendous distortion of the protein molecule or the breakage of the polypeptide chain to yield fragments. However, when the probable mechanism for racemization is considered exaggerated motions do not seem to be necessary. In fact, the rate of racemization for free amino acids and for amino acids bound in peptide chains should not differ greatly. The primary mechanism for racemization is hydrogen abstraction from the chiral carbon atom of the amino acid to form a three-co-ordinate planar carbanion (Neuberger, 1948). With this mechanism, the most important factors in determining the rate of racemization would not be the three-dimensional environment of the amino acid, but the electron-withdrawing and resonancestabilizing capacities of the substituents on the a-carbon atom. The applicability of this mechanism to the environment in which the polypeptides and proteins are maintained should be considered. This reaction might be expected to be operative only under the influence of a strong base in order to abstract the hydrogen ion but experimental evidence has shown that racemization also occurs at physiological pH’s in both free and bound amino acids (Bada & Schroeder, 1975). Within the pH range of 5-8, it has
THE
ETlOLOGY
OF
AGING
113
been determined that the racemization rate is virtually independent of pH. It can be concluded, then, that racemization of amino acids could be occurring in living organisms as well as fossils. Although the half-lives of racemization for even the most reactive amino acids are thousands of years at geologic temperatures, racemization is a temperature-sensitive process and its rate would be expected to increase very rapidly as the temperature increases. Our objective is to examine the available data on geologic samples using their rate constants and half-lives to obtain an estimate of the degree of racemization in a living human being and, then, relate the possible physiological or structural changes that might be expected to occur if all the hormones, enzymes, structural proteins and, in fact, all amino acidcontaining substances in the body were under the influence of racemization. To obtain an idea of the magnitude of racemization that might occur in the body, half-life data from geologic dating can be treated according to the Ahrrenius equation In k = j-$
f In A
where k is the rate constant, E,, the activation energy, R the universal gas constant, T the absolute temperature and A is an empirical frequency factor. Accordingly, a plot of Ink versus the reciprocal of the absolute temperature should yield a straight line. Since the half-lives at different temperatures are proportional to the rate constants at those temperatures, an extrapolation to physiological temperature for humans (37°C) should yield the half-life at that temperature. For example, using the data from Bada (see Table 1) for the racemization of aspratic acid and phenylalanine at various temperatures, the following results are obtained (Fig. 1). From the plot for aspartic acid, where three points are available, a good straight-line fit is observed and extrapolation at 37°C yields a half-life of 490 years. Similarly, for phenylalanine, extrapolation yields a half-life, at 37°C of 302 years. These time periods involve a number of human generations so, perhaps, a more reasonable approach to determine the magnitude of this phenomenon in the human body, would be to take the first order rate expression, calculate the rate constant for racemization at 37°C and use the rate constant to determine the amount of racemization occurring over a 70-year period (average life expectancy for humans). Thus, for phenylalanine, In 4-1 = k(f,-t,) A2 1.“.
114
R.
DELONG
AND
FIG. 1. Arrhenius plot of racemization acid ; P, phenylalanine.
L.
POPLIN
half-lives extrapolated
TABLE
to 37°C. A, aspartic
1
Half-lives (years) for racemization of amino acids at pH 7.6
Aspartic acid Phenylalanine
0°C
20°C
25°C
430,000 160,000
15,000 -
zoo0
3,500
where A, is the L-amino acid at t, (time) and A, the L-amino acid at t,. In the special case of the half-life the equation yields: In hi = k(302-0) k = 2.295 x 10m3 year-‘.
THE
ETIOLOGY
Using the rate constant, the AI/A,
OF
AGING
115
ratio at 70 years can be calculated.
In 2 = (2.295 x low3 year-‘)
(70 years)
2
In $ = O-1607 2
A -! = I.174 A2
A, =0*85 This implies that in 70 years approximately 15 % of the original L-phenylalanine will have racemized at 37°C. For aspartic acid, 9.4% racemization will have occurred in 70 years. A smaller extent of racemization would be observed for other amino acids since they have longer half-lives. An assumption in the above calculation is that all protein molecules are present in the body throughout the entire 70-year period and, obviously, this is not the case. However, when the seemingly trivial differences in overall structure and amino acid sequence that lead to gross changes in activity of structural and functional proteins are considered, probably a much smaller magnitude of racemization could still affect the activity of both these types of proteins and, hence, the entire organism. There is some experimental evidence to support our hypothesis. Helfman & Bada (1975) have published results of aspartic acid racemization in tooth enamel taken from living humans. These results are shown in Table 2. They have found that about 8% of aspartic acid had racemized in tooth enamel after 60 years. Extrapolation and calculation of the geologic data for aspartic acid predict an 8-l % racemization of aspartic acid after 60 years in living humans. This experimental evidence is in exceptional agreement with the theoretical. It should be understood, though, that this evidence, by itself, does not distinguish between cause and effect of aging. However, it does establish a definite relationship between racemization and aging in life. Aging is a complicated process. We do not propose that amino acid racemization would be the only mechanism for aging. However, we do believe that it may be one of the important mechanisms that exists. Since amino acids are part of the most preponderant and important molecules of life, such as enzymes, hormones, ribonucleoproteins and structural proteins, any mechanism that could cause an accumulative change in configuration of these molecules would certainly have a profound effect on the physiology and anatomy of life. Amino acid racemization would accomplish just that.
116
R. DELONG
AND L. POPLIN
TABLE 2 Results of aspartic acid racemization analysis of human tooth enamel proteins Age of enamel? (year) 5 6 8 8 1% IQ 151: 23’ 35 42 48 54 56 58 61 62 66(?) 69 72
Spe of
tooth ___.--~.-Third molar Premolar Deciduous molar Premolar Third molar Third molar Third molar Third molar Third molar Incisor Canine Third (?) molar First molar First (?) molar Second molar Incisor Second molar Second molar Second molar
Age of individual (years) 17 11 8 13 27-a 27-b 27-c 35 47 45 53 66 58 60 67 65 72(?) 75 78
D/L aspartic acid ratio Amino acid Gas analyzer chromatography ..-~ __ -. 0.045 0.042 + 0.045 0.030 0.043 0.038 0.046 0.045 0.045 0.053 0.055 0.068 0,068 0.077 0.078 0.094 0.082 0.088 0.112 + 0.085 0.089 0.085 0.080
t Insufficient aspartic acid was isolated for dipeptide analysis. j. Analyses made on teeth from three different 27-year-old individuals. Cells perform thousands of different reactions and these reactions are involved with structural proteins, hormones, ribonucleoproteins and enzymes. All these molecules possess complex three-dimensional structures
and they must maintain their conformations or a loss of biological activity results. Molecules that began in life having all L-amino acids in their structures will, upon introduction of D-enantiomers by racemization, be changed so that the conformations of these molecules will affect their specificities and activities,
which,
in turn,
could
lead to abnormal
physiology
and
anatomy. Since, in life, many physiological functions are the result of chain reactions involving complicated combinations of any of the aforementioned molecules and racemization could affect all or most of them, biological effects could be greatly magnified by only minute amounts of D-enantiomers being present in any one of them. It should be obvious that racemization’s importance in aging would be in those biomolecules having a slow turnover rate. Architectural proteins,
THE
ETIOLOGY
OF
AGING
117
at least with present knowledge, would best fit this requirement. Central nervous system tissue should be very important in this respect, because it is not renewable and yet it controls the functioning of most of the endocrine and muscular systems. Concerning the architectural proteins of the cell, tissue or body, any accumulative changes occurring in their configurations could lead to abnormal function and architecture. Cellular membranes and organelle membranes which are composed, in part, of proteins perform many very important functions of cellular metabolism and reproduction. Any changes in their architecture could lead to changes in permeability, active transport and many other functions which are vitally important to the proper functioning of a cell. Extracellular architectural proteins of the body would be affected also by any changes in their molecular configurations. For example, collagen might lose its elasticity or supportive strength, or both, because of configurational changes within the molecules. Racemization of proline and/or epimerization of hydroxyproline could result in geometrical changes of the collagen molecules and, thus, cause a loss in its supportive function and/or a loss in elasticity. These two characteristics are symptoms of aging. As stated before, we consider the racemization hypothesis to be the most plausible for the etiology of aging. Nevertheless, one of the more popular hypotheses for aging is the free-radical hypothesis (Pryor, 1971). Arguments for this mechanism lose their impact when it is considered that: (1) free radicals are very important in normal chemical and biological activities within a cell; (2) free radicals are usually not accumulative to any great degree; (3) pertaining to aging the mechanism is difficult to put to experimentation; (4) free radical reactions may not occur in a progressively regulated fashion but may occur intermittently which does not parallel the symptoms of aging. Another popular hypothesis advanced for the cause of aging is the deoxyribonucleic acid hypothesis (Hayflick, 1968). An accumulation of changes in DNA has been postulated as the cause of aging. However, this mechanism is contradictory to reality. If aging were caused by any change in DNA, then aging would be inherited. This does not occur. Each generation of life begins anew. If DNA errors caused aging, each successive generation would start life older than the preceding generation did. After a number of generations had been produced, the latter generations would begin life already quite aged. Obviously, this does not happen. There are numerous other hypotheses, also, for aging. Somatic mutation, error catastrophe, autoimmunity and others have been postulated but all lack certain criteria for the cause of aging which render them questionable.
118
R.
DELONG
AND
L.
POPLIN
Orgel (1963) proposed an hypothesis for aging. He maintained that aging could be caused by increasing errors in translation of proteins which would ultimately lead to catastrophe. Frank (1953), Seelig (1971, 1972), Hoffman (1974) and Gael & Yeas (1975) have presented models and treatments of asymmetric syntheses. These tend to show that the “error catastrophe” hypothesis of Orgel is not correct. Gael & Yeas’ (1975) treatment is far more rigorous than any of the others. The main concern, with all of these authors, is the probable stability and accuracy of the translational apparatus and the chance of an “error catastrophe” occurring which might be related to aging in life. Our main premise is not based on errors in translation but on configurational errors accumulating in little or non-renewable already synthesized molecules, such as, structural proteins of nervous, muscular and connective tissues; both intracellular and extracellular. Throughout our thesis we have presented amino acid racemization and, specifically, amino acid racemization within proteins as being the cause of aging. Although we do think protein racemization to be the most important, we do not mean to imply that only such racemization is responsible for aging. Any amino acid-containing compound and, indeed, any carbohydrate or lipid compound, containing chiral carbon atoms, which possesses a slow turnover rate might contribute to the complex process of aging through racemization. A number of experimental approaches should be available to test the racemization hypothesis: (1) samples of a tissue could be taken from very young animals, or even humans, followed by the same type of tissue samples from progressively older animals and all of them tested for D-enantiomer concentrations, (2) in vitro cell cultures could be established and, as time passes, monitored for D-enantiomer concentration, (3) proper microbial cultures might also be tested, (4) cell cultures could be fed varying concentrations of D-enantiomers and then observed for correlation, if any, between an increase in intracellular D-enantiomers and an increase in ther ate of aging. Correlation between D-enantiomer concentration and age in approaches 1 or 2 above would be suggestive that amino acid racemization may have an important role in the cause of aging in life. Correlation between D enantiomer concentration and the rate of aging in approach 4 above would indicate, quite emphatically, that amino acid racemization has an important role in the etiology of aging in life. If, by feeding cell cultures D-enantiomers, cells were found to absorb Denantiomers and incorporate them into structural and functional molecules, not only would it indicate racemization’s involvement in aging but it could demonstrate that a complementary mechanism to racemization might exist,
THE
as well, for heterotrophic
ETIOLOGY
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AGING
119
It@. Upon ingestion of food, heterotrophs would be consuming some D-enantiomers already present in the food from racemization. Should these D-enantiomers be incorporated into biomolecules, similar effects to those produced by racemization could be expected. The net result should be the same, namely: aging. Accumulation of enantiomers as being the cause of aging in all of the various life-forms, at first, may seem dubious. However, reasonable explanations to account for the different life-spans of life-forms in relation to racemization are available: (1) death should not be used as the prime criterion for aging because death (or conversely, life-span) need not be relevant to aging at all; for example, disease, accidental or predatory death, (2) the racemization threshold needed to cause death could be very different in varying life-forms; for instance, the degree of racemization required to kill a hummingbird might be much lower than that required to kill an elephant because of the differences in heart mass and heart rate between the two life-forms and, thus, the hummingbird would be expected to have a shorter life-span than the elephant would. Yet both deaths could have been due to aging caused by racemization, (3) there exists a definite correlation between the length of the growth period of a life-form and its life-span. During growth and maturation much cellular reproduction and turnover is occurring and the chemistry of life also is turning over rapidly. Enantiomeric accumulation under these conditions probably would be very small, hence, the longer the growth period the longer the lifespan would be, because racemization would only reafly become significant after growth had stopped, (4) differences in biochemical and biophysical pathways among different life-forms could account for varying life-spans and yet racemization could be operating as the cause of aging. Less racemization may be needed in one life-form (to render life incapable) than in another because of these differences, (5) differences in body or cellular temperature among the various life-forms could be expected to produce widely different life-spans and yet racemization could be operating in the aging process. Racemization is very temperature-dependent, so life-forms with lower temperatures would be expected to have longer life-spans than those with higher temperatures. Any one, or any combination, of these explanations might be possible to account for the existence of different life-spans among the various life-forms and, yet, still be compatible with the accumulative enantiomeric hypothesis. An appealing feature of the accumulative enantiomeric hypothesis is that it is a consequence of a natural process. It is not necessary to conjure up a variety of exotic assumptions that must drastically alter some normal mechanism, as so many other aging hypotheses do. Rather, it is a normal, universal manifestation of entities tending to achieve equilibrium.
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YOPLIN
Aging might even be viewed as an exemplification of the second law of thermodynamics. The chemical environment of life begins in a highly nonrandom state. As entropy dictates, all processes in nature proceed toward randomness. Hence, the stereoisomeric non-randomness of organic chemicals at birth must lead to a more random stereoisomeric state. Racemization accomplishes this dictate. REFERENCES L., KVENVOLDEN, K. A. & PETERSON, E. (1973). Nature, Land. 245, 308. L., LIJYENDYK, B. P. & MAYNARD, J. B. (1970). Science, N. Y. 170, 730. L. & PROTSCH, R. (1973). Proc. natn. Acad. Sci. U.S.A. 70, 1331. L. & SCHROEDER, R. A. (1975). Naturwiss. 62, 71. L., SCHROEDER, R. A., PROTXH, R. & BERGER, R. (1974). Pruc. natn. Acad. U.S.A. 71,914. FRANK, J. C. (1953). Biochem. Biophys. Acta 11, 459. GOEL, N. S. & YCAS, M. (1975). J. theor. Biol. 54, 245. HAYFLICK, L. (1968). Sci. Am. 218, 3, 32. HELFMAN, P. & BADA, J. L. (1975). Proc. rzatn. Acad. Sci. U.S.A. 72, 2891. HOFFMAN, G. W. (1974). J. molec. Biol. 86, 349. NEUBERGER, A. (1948). Adv. Protein Chem. 4, 298. ORGEL, L. C. (1963). Proc. natn. Acad. Sci. U.S.A. 49, 5 Il. PHILLIPS, D. C. (1966). Sci. Am. 215, 78. PRYOR, W. A. (1971). Chem. Eng. News49, 34. SEELIG, F. F. (1971). J. theor. Biof. 31,355. SEELIG, F. F. (1971). J. theor. Biul. 32, 93. SEELIG, F. F. (1972). J. theor. Biol. 34, 197. WEHMILLER, J. & HARE, P. E. (1971). Science, N. Y. 173, 907. BADA, BADA, BADA, BADA, BADA,
J. J. J. J. J.
Sci.
On the Etiology of Aging RICHARD Department
DELONG
of Biology, Del Mar College, Corpus Christi, Texas 78404, U.S.A. AND LOYD
Department
POPLIN
of Chemistry, Del Mar College, Corpus Christi, Texas 18404, U.S.A.
(Received 6 November
1975, and in revised,fbrm
18 October 1976)
Amino acid racemization is presented as an hypothesis for the cause of aging. Effects on important biomolecules resulting from changes in their three-dimensional conformations and resulting changes in activities and specificities are discussed. Aging is viewed as a natural process associated with attaining thermodynamic equilibrium.
Although a number of hypotheses have been proposed as the cause of aging, none are completely satisfactory and, usually, are difficult to subject to experimentation. Another proposal for the cause of aging is presented here but this hypothesis should lend itself to experimentation very well. A mechanism for aging to be meritorious should possess the following features: (1) it should be accumulative, (2) it should lead solely to abnormal anatomy and physiology, (3) it should be non-genetic, (4) it should lend itself to experimentation. The hypothesis we submit has all these attributes. We propose that aging is caused mainly by the racemization of L-amino acids to D-amino acids as time passes. Within cells and tissues, D-amino acids accumulate causing geometrical changes in important biomolecules such as structural and functional proteins. Accumulative changes in these molecules could lead to the symptom called aging. Biological stereospecificity is now common knowledge. One aspect of biological stereospecificity is the ability of certain molecules to differentiate between substrate enantiomers. This type of differentiation is related to the absolute three-dimensional orientation of the substrates and molecules 111
112
R.
DELONG
AND
L.
POPLIN
involved. Therefore, biological stereospecificity results from an enzymcsubstrate interaction (Phillips, 1966). Should enantiomers be formed in either enzyme or substrate, the interaction could be modified either qualitatively or quantitatively, or both. This would be especially so among large molecules such as proteins. Proteins are polymers, therefore, they are subject to polymeric configurational changes. There are, at least, three main types of polymeric configurations. These are: (1) isotactic polymers in which all chiral centers have the same configurations ; (2) syndiotactic polymers in which successive chiral centers alternate in configuration; (3) atactic polymers in which the configuration of chiral centers is random. It is well known that different physical and chemical properties exist for such polymers. The changes encountered in proteins, because of introduction of enantiomers into the polymeric molecule, could lead to dramatic effects biologically. In the last few years amino acid racemization has been used to determine the age of fossils (Bada, Luyendyk & Maynard, 1970; Wehmiller & Hare, 1971; Bada & Protsch, 1973; Bada, Kvenvolden & Peterson, 1973; Bada, Schroeder, Protsch & Berger, 1974). The ratio of the biologically active L-amino acid enantiomers to the D-form decreases with age and, knowing the rate constants for the racemization reaction, the age of the bone or shell specimen can be calculated. Amino acid racemization occurs not only in free amino acids but, also, in amino acids bound in proteins (Bada & Schroeder, 1975). At first thought, racemization of individual amino acids bound in a polypeptide would seem to require tremendous distortion of the protein molecule or the breakage of the polypeptide chain to yield fragments. However, when the probable mechanism for racemization is considered exaggerated motions do not seem to be necessary. In fact, the rate of racemization for free amino acids and for amino acids bound in peptide chains should not differ greatly. The primary mechanism for racemization is hydrogen abstraction from the chiral carbon atom of the amino acid to form a three-co-ordinate planar carbanion (Neuberger, 1948). With this mechanism, the most important factors in determining the rate of racemization would not be the three-dimensional environment of the amino acid, but the electron-withdrawing and resonancestabilizing capacities of the substituents on the a-carbon atom. The applicability of this mechanism to the environment in which the polypeptides and proteins are maintained should be considered. This reaction might be expected to be operative only under the influence of a strong base in order to abstract the hydrogen ion but experimental evidence has shown that racemization also occurs at physiological pH’s in both free and bound amino acids (Bada & Schroeder, 1975). Within the pH range of 5-8, it has
THE
ETlOLOGY
OF
AGING
113
been determined that the racemization rate is virtually independent of pH. It can be concluded, then, that racemization of amino acids could be occurring in living organisms as well as fossils. Although the half-lives of racemization for even the most reactive amino acids are thousands of years at geologic temperatures, racemization is a temperature-sensitive process and its rate would be expected to increase very rapidly as the temperature increases. Our objective is to examine the available data on geologic samples using their rate constants and half-lives to obtain an estimate of the degree of racemization in a living human being and, then, relate the possible physiological or structural changes that might be expected to occur if all the hormones, enzymes, structural proteins and, in fact, all amino acidcontaining substances in the body were under the influence of racemization. To obtain an idea of the magnitude of racemization that might occur in the body, half-life data from geologic dating can be treated according to the Ahrrenius equation In k = j-$
f In A
where k is the rate constant, E,, the activation energy, R the universal gas constant, T the absolute temperature and A is an empirical frequency factor. Accordingly, a plot of Ink versus the reciprocal of the absolute temperature should yield a straight line. Since the half-lives at different temperatures are proportional to the rate constants at those temperatures, an extrapolation to physiological temperature for humans (37°C) should yield the half-life at that temperature. For example, using the data from Bada (see Table 1) for the racemization of aspratic acid and phenylalanine at various temperatures, the following results are obtained (Fig. 1). From the plot for aspartic acid, where three points are available, a good straight-line fit is observed and extrapolation at 37°C yields a half-life of 490 years. Similarly, for phenylalanine, extrapolation yields a half-life, at 37°C of 302 years. These time periods involve a number of human generations so, perhaps, a more reasonable approach to determine the magnitude of this phenomenon in the human body, would be to take the first order rate expression, calculate the rate constant for racemization at 37°C and use the rate constant to determine the amount of racemization occurring over a 70-year period (average life expectancy for humans). Thus, for phenylalanine, In 4-1 = k(f,-t,) A2 1.“.
114
R.
DELONG
AND
FIG. 1. Arrhenius plot of racemization acid ; P, phenylalanine.
L.
POPLIN
half-lives extrapolated
TABLE
to 37°C. A, aspartic
1
Half-lives (years) for racemization of amino acids at pH 7.6
Aspartic acid Phenylalanine
0°C
20°C
25°C
430,000 160,000
15,000 -
zoo0
3,500
where A, is the L-amino acid at t, (time) and A, the L-amino acid at t,. In the special case of the half-life the equation yields: In hi = k(302-0) k = 2.295 x 10m3 year-‘.
THE
ETIOLOGY
Using the rate constant, the AI/A,
OF
AGING
115
ratio at 70 years can be calculated.
In 2 = (2.295 x low3 year-‘)
(70 years)
2
In $ = O-1607 2
A -! = I.174 A2
A, =0*85 This implies that in 70 years approximately 15 % of the original L-phenylalanine will have racemized at 37°C. For aspartic acid, 9.4% racemization will have occurred in 70 years. A smaller extent of racemization would be observed for other amino acids since they have longer half-lives. An assumption in the above calculation is that all protein molecules are present in the body throughout the entire 70-year period and, obviously, this is not the case. However, when the seemingly trivial differences in overall structure and amino acid sequence that lead to gross changes in activity of structural and functional proteins are considered, probably a much smaller magnitude of racemization could still affect the activity of both these types of proteins and, hence, the entire organism. There is some experimental evidence to support our hypothesis. Helfman & Bada (1975) have published results of aspartic acid racemization in tooth enamel taken from living humans. These results are shown in Table 2. They have found that about 8% of aspartic acid had racemized in tooth enamel after 60 years. Extrapolation and calculation of the geologic data for aspartic acid predict an 8-l % racemization of aspartic acid after 60 years in living humans. This experimental evidence is in exceptional agreement with the theoretical. It should be understood, though, that this evidence, by itself, does not distinguish between cause and effect of aging. However, it does establish a definite relationship between racemization and aging in life. Aging is a complicated process. We do not propose that amino acid racemization would be the only mechanism for aging. However, we do believe that it may be one of the important mechanisms that exists. Since amino acids are part of the most preponderant and important molecules of life, such as enzymes, hormones, ribonucleoproteins and structural proteins, any mechanism that could cause an accumulative change in configuration of these molecules would certainly have a profound effect on the physiology and anatomy of life. Amino acid racemization would accomplish just that.
116
R. DELONG
AND L. POPLIN
TABLE 2 Results of aspartic acid racemization analysis of human tooth enamel proteins Age of enamel? (year) 5 6 8 8 1% IQ 151: 23’ 35 42 48 54 56 58 61 62 66(?) 69 72
Spe of
tooth ___.--~.-Third molar Premolar Deciduous molar Premolar Third molar Third molar Third molar Third molar Third molar Incisor Canine Third (?) molar First molar First (?) molar Second molar Incisor Second molar Second molar Second molar
Age of individual (years) 17 11 8 13 27-a 27-b 27-c 35 47 45 53 66 58 60 67 65 72(?) 75 78
D/L aspartic acid ratio Amino acid Gas analyzer chromatography ..-~ __ -. 0.045 0.042 + 0.045 0.030 0.043 0.038 0.046 0.045 0.045 0.053 0.055 0.068 0,068 0.077 0.078 0.094 0.082 0.088 0.112 + 0.085 0.089 0.085 0.080
t Insufficient aspartic acid was isolated for dipeptide analysis. j. Analyses made on teeth from three different 27-year-old individuals. Cells perform thousands of different reactions and these reactions are involved with structural proteins, hormones, ribonucleoproteins and enzymes. All these molecules possess complex three-dimensional structures
and they must maintain their conformations or a loss of biological activity results. Molecules that began in life having all L-amino acids in their structures will, upon introduction of D-enantiomers by racemization, be changed so that the conformations of these molecules will affect their specificities and activities,
which,
in turn,
could
lead to abnormal
physiology
and
anatomy. Since, in life, many physiological functions are the result of chain reactions involving complicated combinations of any of the aforementioned molecules and racemization could affect all or most of them, biological effects could be greatly magnified by only minute amounts of D-enantiomers being present in any one of them. It should be obvious that racemization’s importance in aging would be in those biomolecules having a slow turnover rate. Architectural proteins,
THE
ETIOLOGY
OF
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at least with present knowledge, would best fit this requirement. Central nervous system tissue should be very important in this respect, because it is not renewable and yet it controls the functioning of most of the endocrine and muscular systems. Concerning the architectural proteins of the cell, tissue or body, any accumulative changes occurring in their configurations could lead to abnormal function and architecture. Cellular membranes and organelle membranes which are composed, in part, of proteins perform many very important functions of cellular metabolism and reproduction. Any changes in their architecture could lead to changes in permeability, active transport and many other functions which are vitally important to the proper functioning of a cell. Extracellular architectural proteins of the body would be affected also by any changes in their molecular configurations. For example, collagen might lose its elasticity or supportive strength, or both, because of configurational changes within the molecules. Racemization of proline and/or epimerization of hydroxyproline could result in geometrical changes of the collagen molecules and, thus, cause a loss in its supportive function and/or a loss in elasticity. These two characteristics are symptoms of aging. As stated before, we consider the racemization hypothesis to be the most plausible for the etiology of aging. Nevertheless, one of the more popular hypotheses for aging is the free-radical hypothesis (Pryor, 1971). Arguments for this mechanism lose their impact when it is considered that: (1) free radicals are very important in normal chemical and biological activities within a cell; (2) free radicals are usually not accumulative to any great degree; (3) pertaining to aging the mechanism is difficult to put to experimentation; (4) free radical reactions may not occur in a progressively regulated fashion but may occur intermittently which does not parallel the symptoms of aging. Another popular hypothesis advanced for the cause of aging is the deoxyribonucleic acid hypothesis (Hayflick, 1968). An accumulation of changes in DNA has been postulated as the cause of aging. However, this mechanism is contradictory to reality. If aging were caused by any change in DNA, then aging would be inherited. This does not occur. Each generation of life begins anew. If DNA errors caused aging, each successive generation would start life older than the preceding generation did. After a number of generations had been produced, the latter generations would begin life already quite aged. Obviously, this does not happen. There are numerous other hypotheses, also, for aging. Somatic mutation, error catastrophe, autoimmunity and others have been postulated but all lack certain criteria for the cause of aging which render them questionable.
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Orgel (1963) proposed an hypothesis for aging. He maintained that aging could be caused by increasing errors in translation of proteins which would ultimately lead to catastrophe. Frank (1953), Seelig (1971, 1972), Hoffman (1974) and Gael & Yeas (1975) have presented models and treatments of asymmetric syntheses. These tend to show that the “error catastrophe” hypothesis of Orgel is not correct. Gael & Yeas’ (1975) treatment is far more rigorous than any of the others. The main concern, with all of these authors, is the probable stability and accuracy of the translational apparatus and the chance of an “error catastrophe” occurring which might be related to aging in life. Our main premise is not based on errors in translation but on configurational errors accumulating in little or non-renewable already synthesized molecules, such as, structural proteins of nervous, muscular and connective tissues; both intracellular and extracellular. Throughout our thesis we have presented amino acid racemization and, specifically, amino acid racemization within proteins as being the cause of aging. Although we do think protein racemization to be the most important, we do not mean to imply that only such racemization is responsible for aging. Any amino acid-containing compound and, indeed, any carbohydrate or lipid compound, containing chiral carbon atoms, which possesses a slow turnover rate might contribute to the complex process of aging through racemization. A number of experimental approaches should be available to test the racemization hypothesis: (1) samples of a tissue could be taken from very young animals, or even humans, followed by the same type of tissue samples from progressively older animals and all of them tested for D-enantiomer concentrations, (2) in vitro cell cultures could be established and, as time passes, monitored for D-enantiomer concentration, (3) proper microbial cultures might also be tested, (4) cell cultures could be fed varying concentrations of D-enantiomers and then observed for correlation, if any, between an increase in intracellular D-enantiomers and an increase in ther ate of aging. Correlation between D-enantiomer concentration and age in approaches 1 or 2 above would be suggestive that amino acid racemization may have an important role in the cause of aging in life. Correlation between D enantiomer concentration and the rate of aging in approach 4 above would indicate, quite emphatically, that amino acid racemization has an important role in the etiology of aging in life. If, by feeding cell cultures D-enantiomers, cells were found to absorb Denantiomers and incorporate them into structural and functional molecules, not only would it indicate racemization’s involvement in aging but it could demonstrate that a complementary mechanism to racemization might exist,
THE
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It@. Upon ingestion of food, heterotrophs would be consuming some D-enantiomers already present in the food from racemization. Should these D-enantiomers be incorporated into biomolecules, similar effects to those produced by racemization could be expected. The net result should be the same, namely: aging. Accumulation of enantiomers as being the cause of aging in all of the various life-forms, at first, may seem dubious. However, reasonable explanations to account for the different life-spans of life-forms in relation to racemization are available: (1) death should not be used as the prime criterion for aging because death (or conversely, life-span) need not be relevant to aging at all; for example, disease, accidental or predatory death, (2) the racemization threshold needed to cause death could be very different in varying life-forms; for instance, the degree of racemization required to kill a hummingbird might be much lower than that required to kill an elephant because of the differences in heart mass and heart rate between the two life-forms and, thus, the hummingbird would be expected to have a shorter life-span than the elephant would. Yet both deaths could have been due to aging caused by racemization, (3) there exists a definite correlation between the length of the growth period of a life-form and its life-span. During growth and maturation much cellular reproduction and turnover is occurring and the chemistry of life also is turning over rapidly. Enantiomeric accumulation under these conditions probably would be very small, hence, the longer the growth period the longer the lifespan would be, because racemization would only reafly become significant after growth had stopped, (4) differences in biochemical and biophysical pathways among different life-forms could account for varying life-spans and yet racemization could be operating as the cause of aging. Less racemization may be needed in one life-form (to render life incapable) than in another because of these differences, (5) differences in body or cellular temperature among the various life-forms could be expected to produce widely different life-spans and yet racemization could be operating in the aging process. Racemization is very temperature-dependent, so life-forms with lower temperatures would be expected to have longer life-spans than those with higher temperatures. Any one, or any combination, of these explanations might be possible to account for the existence of different life-spans among the various life-forms and, yet, still be compatible with the accumulative enantiomeric hypothesis. An appealing feature of the accumulative enantiomeric hypothesis is that it is a consequence of a natural process. It is not necessary to conjure up a variety of exotic assumptions that must drastically alter some normal mechanism, as so many other aging hypotheses do. Rather, it is a normal, universal manifestation of entities tending to achieve equilibrium.
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Aging might even be viewed as an exemplification of the second law of thermodynamics. The chemical environment of life begins in a highly nonrandom state. As entropy dictates, all processes in nature proceed toward randomness. Hence, the stereoisomeric non-randomness of organic chemicals at birth must lead to a more random stereoisomeric state. Racemization accomplishes this dictate. REFERENCES L., KVENVOLDEN, K. A. & PETERSON, E. (1973). Nature, Land. 245, 308. L., LIJYENDYK, B. P. & MAYNARD, J. B. (1970). Science, N. Y. 170, 730. L. & PROTSCH, R. (1973). Proc. natn. Acad. Sci. U.S.A. 70, 1331. L. & SCHROEDER, R. A. (1975). Naturwiss. 62, 71. L., SCHROEDER, R. A., PROTXH, R. & BERGER, R. (1974). Pruc. natn. Acad. U.S.A. 71,914. FRANK, J. C. (1953). Biochem. Biophys. Acta 11, 459. GOEL, N. S. & YCAS, M. (1975). J. theor. Biol. 54, 245. HAYFLICK, L. (1968). Sci. Am. 218, 3, 32. HELFMAN, P. & BADA, J. L. (1975). Proc. rzatn. Acad. Sci. U.S.A. 72, 2891. HOFFMAN, G. W. (1974). J. molec. Biol. 86, 349. NEUBERGER, A. (1948). Adv. Protein Chem. 4, 298. ORGEL, L. C. (1963). Proc. natn. Acad. Sci. U.S.A. 49, 5 Il. PHILLIPS, D. C. (1966). Sci. Am. 215, 78. PRYOR, W. A. (1971). Chem. Eng. News49, 34. SEELIG, F. F. (1971). J. theor. Biof. 31,355. SEELIG, F. F. (1971). J. theor. Biul. 32, 93. SEELIG, F. F. (1972). J. theor. Biol. 34, 197. WEHMILLER, J. & HARE, P. E. (1971). Science, N. Y. 173, 907. BADA, BADA, BADA, BADA, BADA,
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