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Reversible Effects of Long-Term Caloric Restriction on Protein Oxidative Damage

^a Department of Pharmacology, University of North Texas Health Science Center at Fort Worth
^b Department of Biological Sciences, Southern Methodist University, Dallas, Texas

Michael J. Forster, Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 E-mail: Forsterm{at}hsc.unt.edu.

Jay Roberts, PhD

	Abstract

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The age-associated increase in oxidative damage in ad libitum-fed mice is attenuated in mice fed calorically restricted (CR) diets. The objective of this study was to determine if this effect results from a slowing of age-related accumulation of oxidative damage, or from a reversible decrease of oxidative damage by caloric restriction. To address these possibilities, crossover studies were conducted in C57BL/6 mice aged 15 to 22 months that had been maintained, after 4 months of age, on ad libitum (AL) or a 60% of AL caloric regimen. One half of the mice in these groups were switched to the opposite regimen of caloric intake for periods up to 6 weeks, and protein oxidative damage (measured as carbonyl concentration and loss of sulfhydryl content) was measured in homogenates of brain and heart. In AL-fed mice, the protein carbonyl content increased with age, whereas the sulfhydryl content decreased. Old mice maintained continuously under CR had reduced levels of protein oxidative damage when compared with the old mice fed AL. The effects of chronic CR on the carbonyl content of the whole brain and the sulfhydryl content of the heart were fully reversible within 3–6 weeks following reinstatement of AL feeding. The effect of chronic CR on the sulfhydryl content of the brain cortex was only partially reversible. The introduction of CR for 6 weeks in the old mice resulted in a reduction of protein oxidative damage (as indicated by whole brain carbonyl content and cortex sulfhydryl), although this effect was not equivalent to that of CR from 4 months of age. The introduction of CR did not affect the sulfhydryl content of the heart. Overall, the current findings indicate that changes in the level of caloric intake may reversibly affect the concentration of oxidized proteins and sufhydryl content. In addition, chronic restriction of caloric intake also retards the age-associated accumulation of oxidative damage. The magnitude of the reversible and chronic effects appears to be dependent upon the tissue examined and the nature of the oxidative alteration.

PROGRESSIVE accumulation of molecular oxidative damage has been postulated to be an important cause of aging-related losses in cellular functions and, ultimately, of declines in physiological capacity of the whole organism ^(1)(2)(3). This hypothesis is supported by studies in insects (Drosophila melanogaster) in which retardation of age-associated accumulation of molecular oxidative damage, achieved through overexpression of antioxidant enzymes, resulted in increased longevity and preservation of motor capacity ⁽⁴⁾. In mammalian species, long-term restriction of caloric intake results in a similar pattern of effects. It has been known for decades that feeding regimens that provide essential nutrients but restrict caloric intake by 30–50% from ad libitum result in a prolongation of laboratory rodents' life span. Such restricted caloric intake has also been reported to lower the incidence of certain age-associated diseases and to delay several age-associated biochemical, physiological, and functional changes ^(3)(5)(6)(7). Currently available data accord with the hypothesis that the life-prolonging and beneficial functional effects of caloric restriction can be attributed to a chronic lowering of the steady-state level of oxidative stress, primarily through a decrease in the rate of generation of reactive oxygen species and, perhaps, secondarily via enhancement of antioxidative defenses ^(2)(3)(8)(9).

We have previously reported that C57BL/6 mice maintained for 15 to 23 months on a regimen of 40% caloric restriction (CR) showed lower concentrations of protein and DNA oxidative damage when compared with age-matched ad libitum (AL)-fed controls ^(10)(11). The same dietary regimen resulted in increases in the mean and maximum life span ⁽¹²⁾ and a retardation of age-associated losses of sensorimotor and cognitive performance ^(13)(14). Additional studies have suggested that age-related losses of function in mice can be predicted based upon levels of protein oxidative damage in those regions of the brain associated with cognitive and motor capacity ⁽¹⁵⁾. Overall, the close association of oxidative protein damage with longevity and with functional decline provides strong correlative support for the hypothesis that oxidative damage is a causal agent in the aging process. At a practical level, these attributes establish protein oxidative damage as a potentially valid and reliable biomarker of aging.

In analyzing the mechanism of caloric restriction effects on protein oxidative damage, and in the assessment of oxidative damage as a useful biomarker of aging, an important consideration is the potential for CR to produce reversible effects on the level of oxidative damage. A difference in the level of oxidative damage between AL and CR mice of a given age could result from a deceleration of aging processes underlying the progressive accumulation of oxidative damage or, alternatively, from a reversible effect of CR on the efficiency of cellular functions, resulting in a lowering of oxidative damage. The latter possibility is consistent with previous literature suggesting that caloric restriction regimes initiated during adulthood, midadulthood, or thereafter can have beneficial effects on longevity and/or age-associated pathology ^(16)(17)(18). Thus, if the beneficial effects of CR are attributable to its ability to lower the level of oxidative stress/damage, then it could be predicted that such lowering should occur even when CR is introduced during or after midadulthood. However, there is little indication from previous studies as to how rapidly such modulation would occur following introduction of CR. Some findings suggest that the oxidative damage present during aging can be reversed relatively rapidly. For example, the spin-trapping compound, N-tert-butyl--phenylnitrone (PBN) has been shown to reduce the steady-state levels of protein oxidative damage in various regions of the brain after as little as two weeks of treatment ^(19)(20).

The goal of the current study was to address the possibilities outlined above by performing crossover studies in old mice maintained under AL or CR feeding regimens for up to 18 months. In previous studies, increases in protein oxidation were inferred from increases in protein carbonylation ⁽¹¹⁾, or from a loss of protein sulfhydryls (-SH) in tissue homogenates ⁽¹³⁾. It was demonstrated that in the brains of AL mice, there was an age-related -SH loss of approximately 20% and an increase in carbonyls of nearly 70%, over the period from approximately 8 to 27 months of age. However, older mice maintained from 4 months under CR had higher -SH, and lower protein carbonyl, when compared with the age-matched AL mice. If caloric restriction acts in a cumulative fashion to decelerate the rate of age-associated increase in oxidative damage (-SH loss or carbonyl), then changing of the dietary regimens for a relatively short period (up to 6 weeks) was expected to have little effect. On the other hand, if the effects of CR involve a reversible lowering of oxidative damage, then a decrease in oxidative damage was expected to occur in the AL mice following introduction of the CR regimen, and an increase in oxidative damage was expected in CR mice following introduction of the AL regimen.

	Methods

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Animals
One hundred thirty-three male C57BL/6Nnia mice aged 4 to 22 months were obtained from the National Institute on Aging (NIA) caloric restriction colonies maintained at the National Center for Toxicological Research (NCTR; Jefferson, AR). The chronic feeding regimens were initiated and maintained at the NIA colonies until shipment of the mice to the University of North Texas Health Science Center (UNTHSC) vivarium at the appropriate target age. Following receipt at UNTHSC, the mice were maintained for 2–4 weeks under the same feeding regimens and housing conditions, prior to initiation of the crossover experiments. The mice were housed individually in 28 x 19 x 12.5-cm solid bottom polycarbonate cages with wire tops modified into two mouse units by insertion of a stainless steel divider. The mice were maintained on a 12-hour light/dark cycle, with the light portion beginning at 0600 hour.

Caloric Restriction
Beginning at 4 months of age, some of the mice were maintained on a CR regimen permitting daily access to 60% of the intake of a companion group of mice given AL access to the diet (NIH-31, Purina Feeds). The CR mice were fed a special NIH-31 formulation providing a correction for intake of essential nutrients. Following receipt in the UNTHSC vivarium, the CR mice were placed on a night feeding regimen in which they were fed at 2200 hours, approximately 2-hours prior to the acrophase of the normal circadian feeding cycle of the AL mice. The purpose of this procedure was to provide proper phasing of circadian cycles in the AL and CR groups ⁽²¹⁾. Mortality and body weight analyses for designated cohorts of AL and CR mice maintained by NIA/NCTR were reported previously ^(10)(12).

Following adaptation within the UNTHSC vivarium, approximately half of the old AL and DR mice were switched to the opposite diet condition, and the remaining half were maintained in their long-term diet condition to serve as controls. This procedure resulted in four diet groups based upon the combinations of long-term and short-term diet conditions: ALAL; ALCR; CRCR; CRAL. In experiments examining -SH content of the heart, and carbonyl content of the brain, these conditions were maintained for 1, 3, or 6 weeks following switching of the dietary regimens. The results of the carbonyl experiment were reported previously ⁽¹³⁾. For the experiment on -SH content of brain tissue (cortex), all mice were euthanized 6 weeks following switching of the dietary regimens. To verify the effect of age and CR on carbonyl and -SH content reported in previous studies ^(11)(13), young, nonswitched mice (ALAL, aged 5.5 to 8 months) were compared with the older nonswitched (ALAL, CRCR) groups. Old mice used in the whole brain carbonyl experiments were 15 months of age when the dietary regimens were switched, whereas switched mice in the -SH experiments were 20–22 months of age. These age groups were selected based upon estimates from previous studies of the minimum time periods required for significant age-related changes in protein carbonyl ⁽¹¹⁾ and -SH concentrations ⁽¹³⁾. At the end of the switch experiments, the mice were euthanized by CO₂ inhalation, and tissue samples (brain and heart) were quickly dissected and frozen at –80° until processing.

Determination of Carbonyl Content
Protein carbonyl content was measured in homogenates prepared from whole brain according to Levine and colleagues ⁽²²⁾, using the 2-4-dinitrophenylhydrazine (DNPH) procedure, as reported previously ⁽¹¹⁾. A consideration of sensitivity and reliability for this method has been published ⁽²⁰⁾. A 5% (weight/volume) tissue homogenate was prepared in 5 mmol phosphate buffer (pH 7.5), containing the protease inhibitors, leupeptin (0.5 mg/ml), aprotenin (0.5 mg/ml), and pepstatin (0.7 mg/ml) and 0.1% Triton X, using a Teflon and glass homogenizer. The homogenate was centrifuged at 700 g, and 300-µl aliquots of the resulting supernatant containing 1.6–2.0 mg protein were treated with 300 ml of 10 mmol DNPH dissolved in 2 N HCL or with 2 N HCL alone in the controls. Samples were then incubated for 1 hour at room temperature, stirred every 10 minutes, precipitated with 10% trichloroacetic acid (final conc.), and centrifuged for 3 minutes at 16,000 g. The pellet was washed three times with 1 ml ethanol/ethyl acetate (1:1, v/v) and redissolved in 1 ml 6 mol guanidine in 10 mmol phosphate buffer-trifluoroacetic acid (pH 2.3). The difference in absorbance between the DNPH-treated and the HCL-treated samples was determined at 366 nm, and the results were expressed as nmol carbonyl groups/mg of protein using the extinction coefficient of 22.0 mmol^-1 cm^-1 for aliphatic hydrazones.

Determination of Sulfhydryl Content
Total -SH content (i.e. protein -SH and nonprotein -SH) was determined by the method of Ellman ⁽²³⁾. Tissues were homogenized in 0.02 mol EDTA (disodium salt) with a ratio of 0.1 g tissues to 2 ml of the EDTA solution, and analyzed for protein and sulfhydryl concentration. The total sulfhydryl concentration was determined by incubating 10 µl aliquots of homogenate with 150 µl 0.2 mol Tris (pH 8.2), 40 µl 0.02 mol EDTA (disodium salt), 790 µl methanol, and 10 µl of 0.01 mol DTNB (in methanol) for 15 minutes, followed by centrifugation at 3000 g for 15 minutes. Absorption was measured at 412 nm against sample-free and DTNB-free blanks. Glutathione (GSH) was used as a standard.

Data Analysis
All groups of each experiment were considered as levels of a single factor in a one-way analysis of variance (ANOVA). Planned comparisons between switched and nonswitched groups of old mice, and between young and old nonswitched groups were made using single degree of freedom F tests within this overall analysis. The effect of shifts in level of caloric intake in AL and CR groups were considered using two-way ANOVA with long-term diet (AL or CR) and switch (switched or nonswitched) or switch duration (0, 7, 21, or 42 days) as the factors.

	Results

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Body Weight of Old Mice Following Shifts in Caloric Intake
Switching of the dietary regimes had marked effects on body weights of mice over the subsequent 6 weeks (Fig. 1). When the old AL mice were placed on 40% caloric restriction (ALCR), there was a gradual loss of body weight (approximately 15%) over the course of 6 weeks. In contrast, the difference in body weights of old ALAL mice and nonswitched mice on CR (CRCR) was 37%. When old CR mice were placed on AL feeding (CRAL), the increase in body weight occurred more rapidly than weight loss of the ALCR group, with stable weight occurring within 3 weeks following switching of the dietary regime. The stable weight of the CRAL group was approximately 13% lower than that of the ALAL group. A two-way ANOVA on the body weight data indicated a significant main effect of long-term diet, F(1,26) = 31.3, p < .001, as well as an interaction of long-term diet with switch duration, F(3,26) = 6.7, p = .002. Planned comparisons within the ANOVA verified that significant changes in body weight had occurred by 6 weeks in the ALCR group, and by 3 weeks in the CRAL group (all ps < .033).

View larger version (40K): [in this window] [in a new window]   Figure 1. Effects of short-term and long-term caloric restriction (CR) on body weight (g ± SE) of aged C57BL/6 mice. Mice maintained from 4 months of age on the ad libitum (AL) or CR feeding regimen were switched to the opposite feeding condition at 20 months of age yielding two experimental groups, namely, AL switched to CR (ALCR) (left panel), and CR switched to AL (CRAL) (right panel). Separate groups of 3 to 8 old mice were euthanized 7, 21, or 42 days following the diet switch (hatched bars). Average weights of age-matched controls (nonswitched, ALAL and CRCR) are shown as solid bars (labeled on the abscissa as "0" days following switch). *Indicates a significant difference from ALAL control; **indicates a significant difference from CRCR control (ps < .05, planned individual comparison within ANOVA).

View larger version (42K): [in this window] [in a new window]   Figure 2. Effects of age and short-term and long-term caloric restriction (CR) on brain carbonyl content (nmol carbonyls/mg protein ± SE) of aged C57BL/6 mice. Mice maintained from 4 months on ad libitum (AL) or CR feeding regimen were switched to the opposite feeding condition at 15 months yielding two experimental groups, namely, AL switched to CR (ALCR) (left panel), and CR switched to AL (CRAL) (right panel). Average carbonyl content of age-matched controls (nonswitched, ALAL and CRCR) are shown as solid bars, whereas young controls (nonswitched, ALAL, aged 5.5 months) are shown in the gray bar (controls indicated as "0" days following switch). Separate groups of 4 to 26 old mice were euthanized 7, 21, or 42 days following the diet switch (hatched bars). Values for each sample were the average of from 3 to 6 determinations on the same sample. *Indicates a significant difference from ALAL control; **indicates a significant difference from CRCR control (ps < .05, planned individual comparison within ANOVA). Figure adapted from (13).

View larger version (39K): [in this window] [in a new window]   Figure 3. Effects of age and short-term and long-term caloric restriction (CR) on brain (cortex) sulfhydryl content (nmol SH/mg protein ± SE). Mice maintained from 4 months on the ad libitum (AL) or CR feeding regimen were switched to the opposite feeding condition at 20 months yielding two experimental groups: AL switched to CR (ALCR), and CR switched to AL (CRAL). Average sulfhydryl content of age-matched controls (nonswitched, ALAL and CRCR) are shown as solid bars, whereas young controls (aged 5.5 months) are shown in the gray bar. Separate groups of 7 to 13 old mice were euthanized 42 days following the diet switch (hatched bars). Values for each sample were the average of from 3 to 6 determinations. *Indicates a significant difference from old AL control.

Reversibility of the Effect of CR Upon Heart -SH Content
In order to examine the reversibility of the effect of CR on -SH content of nonbrain tissue, a crossover experiment was also conducted using heart tissue from the 22-month-old mice. The results of this experiment suggested a unidirectional effect (Fig. 4). The one-way ANOVA on these data indicated a significant overall effect, F(8,28) = 2.8, p = .018. Similar to the observations in brain tissue, a 13.5% loss of -SH content with age was suggested by comparison of the young control (ALAL) group (5.5 months) with the old ALAL group (p = .031). The -SH concentration of the old control CR group (CRCR), however, was 97% of the young ALAL group, and was significantly higher when compared with the old ALAL (p = .040). Over the 6 weeks following the switch to CR, there was no indication of increased heart -SH concentration in the ALCR groups (all ps > .650). However, when the CR mice were placed on the AL feeding regime (CRAL), a steady loss of -SH was evident over the 6-week period thereafter, with an 18% loss evident by 3 weeks (p = .013). The -SH content of the ALCR groups at 3 and 6 weeks following the switch was lower than that of the nonswitched AL controls (ALAL). A two-way ANOVA on -SH content for the 22-month-old mice revealed nonsignificant effects of switch duration (p = .059) and of the interaction of long-term diet and switch duration (p = .072), that tended to support the results of the planned individual comparisons.

View larger version (43K): [in this window] [in a new window]   Figure 4. Effects of age and short-term and long-term caloric restriction (CR) on heart sulfhydryl content (nmol sulfhydryl/mg protein ± SE) of aged C57BL/6 mice. Mice maintained from 4 months on the ad libitum (AL) or CR feeding regimen were switched to the opposite feeding condition at 22 months yielding two experimental groups, namely, AL switched to CR (ALCR) (left panel), and CR switched to AL (CRAL) (right panel). Average carbonyl content of age-matched controls (nonswitched, ALAL and CRCR) are shown as solid bars, whereas young controls (nonswitched, ALAL, aged 5.5 months) are shown as gray bars (controls indicated as "0" days following switch). Separate groups of 3 to 8 old mice were euthanized either 7, 21, or 42 days following the diet switch (hatched bars). Values for each sample were the average of 3 to 6 determinations. *Indicates a significant difference from ALAL control; **indicates a significant difference from CRCR control (ps < .05, planned individual comparison within ANOVA).

	Discussion

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Richardson and McCarter ⁽²⁴⁾ have suggested that mechanisms underlying the life-prolonging effects of caloric restriction may be envisioned to follow two alternate models. According to one model, termed the "rate" model, CR slows down the rate of the biological processes underlying aging, whereas the second, termed the "set-point" model, proposes that CR prolongs survival by elevating the set-point of the efficiency of the putative physiological processes that affect life span. Therefore, it is of theoretical significance that the effect of long-term caloric restriction on the level of protein oxidative damage within some tissues can be fully reversed after 3 to 6 weeks of AL feeding. This was the case for oxidative damage measured as protein carbonylation in homogenates of the whole brain, or as loss of -SH in homogenates from heart. A decrease in protein oxidative damage (carbonylation) equivalent to that produced by long-term caloric restriction could also be induced within 3–6 weeks following implementation of CR in old, AL-fed mice. The findings for protein carbonylation in the brain, and for -SH of the heart, clearly indicate that the concentration of protein oxidative damage can be rapidly and reversibly modulated by the level of caloric intake. Such findings fail to conform to the rate model, insofar as there was no indication of any effect of long-term CR on the rate of accrual of oxidative damage with increasing age. Indeed, it appeared that for carbonyl content of the brain, all of the effect of long-term CR was lost within 2–3 weeks following reinstatement of AL feeding. These results appear to be more consistent with the set-point model. If so, the ability of CR to prolong survival and improve function should depend upon maintenance of low caloric intake and the accompanying lowered level of oxidative stress/damage.

The mechanism by which changes in caloric intake directly and reversibly modulate the concentration of oxidative damage is not yet clear. Based upon previous investigations in chronic CR animals, the reversible effects of CR on oxidative damage could involve a decrease in generation of reactive oxygen species (ROS), but probably do not reflect a modulation of antioxidant defenses. Sohal and colleagues ⁽¹¹⁾ reported that rates of mitochondrial O₂^- and H₂O₂ generation in brain, kidney, and heart were dramatically reduced in mice maintained chronically under CR, whereas activities of individual antioxidative enzymes in these tissues did not follow a consistent pattern. It is not known whether or not the effects of CR on ROS generation are rapidly inducible or reversible, although effects of CR on ROS generation are observed within 5 months following implementation of the CR regimen ⁽²⁵⁾.

While the current results establish a direct sensitivity of oxidative damage to the ongoing level of caloric intake, some results of the current study, as well as those published elsewhere (e.g., 26), are not completely consistent with a pure set-point model for the effects of CR. In contrast to the reversible effects of chronic CR on carbonyl content of the brain, the effect of chronic CR on -SH of the brain cortex was only partially reversed following reinstatement of the AL feeding regimen. In this experiment, old mice maintained on long-term CR failed to differ substantially from young controls, whereas old ALAL mice evidenced a 20% decrease in -SH. Switching of the old mice from CR to AL or from AL to CR resulted in a change of 10% in the predicted directions, thus accounting for slightly less than 50% of the difference between old CRCR and ALAL groups. These results indicated that although some of the -SH may be subject to direct modulation by caloric regimens, the higher -SH concentration in old CRCR mice must also reflect an ability of CR to prevent age-associated accumulation of oxidative damage.

A similar interpretation has been applied to results obtained for oxidative damage to mitochondria from skeletal muscle of the mouse ⁽²⁶⁾. In those experiments, CR was observed to dramatically retard or completely prevent the age-associated accumulation of oxidative damage, when such damage was measured as increased protein carbonylation, increased thiobarbituric acid-reactive substance (TBARS), or as decreased -SH. In crossover experiments that were the same as those conducted in the current study, the degree to which such long-term effects of CR could be reversed in old mice was less than 8%. Similarly, imposition of the CR regime for 6 weeks in old AL mice did not significantly lower the level of oxidative damage in skeletal muscle mitochondria.

It should be pointed out that in the experiments involving skeletal muscle mitochondria, as well as the current experiments on sulfhydryl content of brain and heart, the effects of short-term shifts in caloric intake were consistently observed, yet often did not yield statistical significance. It is worth pointing out that, given the relatively small sample sizes employed in these studies (e.g., 7 to 13 samples per group in Fig. 3), the probability of detecting such a small effect is relatively low. Assuming that the data shown in Fig. 3 for CRCR versus CRAL represents a true difference of 15 nmole -SH/mg protein (approximately 10%), a sample size of 30 would have been required to detect this as a significant difference (p < .05, 1-tailed) with a power of 90%. With the sample sizes used in the current study (an average of 10.5), power was 49%.

Taking into account data available to us, it seems that the effects of CR on protein oxidative damage can conform to both the rate and the set point models described above. Therefore, we propose a hypothetical model in which both rate and set point effects may contribute to the beneficial consequences of long-term caloric restriction (Fig. 5). This model is based upon the assumption that reversible oxidative damage, which varies directly with the level of caloric intake, can be differentiated from accumulated damage, which results from irreversible, age-associated loss of physiological efficiency. In an additive model, the steady-state level of oxidative damage measured at any given time would be the sum of the accumulated, irreversible oxidative damage and the reversible damage.

View larger version (26K): [in this window] [in a new window]   Figure 5. A conceptual model of the effects of caloric restriction (CR) on oxidative damage. It is proposed that the steady-state level of oxidative damage at a given age is a sum of the damage resulting from an irreversible decline in physiological efficiency (accumulated damage; gray area) and damage that varies directly with the level of caloric intake (reversible damage). Thus, implementation of CR to individuals maintained under ad libitum (AL) feeding (left) may lower the level of oxidative damage by a certain amount at a given age (indicated by down arrows). In individuals maintained under chronic CR, two different models may apply. In model A, chronic CR results in a low steady-state level of oxidative damage via a reversible action, without affecting the accumulation of oxidative damage with age. Reinstatement of AL caloric intake in the chronic CR individuals results in a return to AL levels of oxidative damage at a given age (indicated by up arrows), and thus the effect of chronic CR is fully reversible. In model B, maintenance of lower steady-state damage by CR retards the decline in physiological efficiency, resulting in slower accumulation of oxidative damage. Reinstatement of AL caloric intake in the chronic CR individuals results in a partial reversal (up arrows) toward AL levels, which is of constant magnitude as a function of age, whereas with time there is an increasingly larger proportion of the effect of CR that is irreversible. This model envisions that CR retards the basic underlying mechanism of aging.

While the present results for the -SH content of the heart were generally consistent with a set point model for the effect of CR (Fig. 5), the results were dissimilar to those obtained for carbonyl in the brain. As appears to be true in all tissues examined thus far, there was higher -SH in old mice maintained continuously under CR (CRCR) when compared with the mice on chronic AL feeding (ALAL). However, in the heart tissue it was possible to rapidly reverse the effect of long-term CR on -SH by reinstatement of the AL feeding regime, but it was not possible to induce an increase in -SH of the old mice by imposing the CR regimen. Without further study, a definitive explanation for the unidirectional effects in this experiment is not possible. However, one explanation would be that the relevant physiological/biochemical systems modulated by caloric intake become unresponsive during aging. Continuous maintenance of mice under the CR regimen could retard such age-related changes, with the result that older CR mice continue to respond rapidly to changes in caloric intake, whereas age-matched mice maintained under the AL regimen become unresponsive to shifts in caloric intake. Based upon this hypothesis, it would be expected that somewhat younger AL mice placed on the CR regimen might show an increase in -SH of the heart. The findings in the brain carbonyl experiment would be consistent with this explanation, given that the mice used in that experiment were 7 months younger than those used in the -SH experiments.

A comparison of the present and previous studies suggests that the nature of the effect of CR on oxidative damage may conform to either of the models in Fig. 5, depending on the tissue examined, as well as the type of oxidative damage under study. Several factors might contribute to these differences. In the current studies, the effect of CR on brain carbonyls was fully reversible, whereas only partial reversibility was evident for brain -SH. The relatively high level of proteolytic activity in the brain may be a factor for the removal of oxidized proteins ^(19)(27), and the ability of caloric intake to modulate proteolytic activity may be a factor in its ability to produce rapid changes in oxidative damage. On the other hand, loss of -SH does not seem to increase susceptibility of proteins to proteolysis, a difference that might explain the contrasting insensitivity of this type of protein damage to short-term changes in caloric intake.

Another factor likely to explain differences in response to CR is the nature and quantity of the specific proteins that are actually present and subject to carbonylation or loss of -SH within a particular tissue. Recent studies have suggested that age-associated protein oxidative damage is targeted to specific proteins and is not necessarily a random phenomenon ^(28)(29). It is thus not surprising that different tissues, containing different concentrations of proteins with differing susceptibility to oxidative modification, may respond in a different fashion to restriction of caloric intake.

There is agreement among many investigators that utility of a biomarker measurement should depend upon its ability to predict, with greater accuracy than chronological age, one or more end points of biological aging as defined in terms of life span or some indicator of health and/or functional capacity during late life ^{(30)(31)(32)(33)(34)}. The hypothesized link between oxidative damage and biological aging processes makes measures of oxidative damage especially attractive as potential biomarkers. Obviously, measurements of protein damage as made in the current study would be too invasive to serve as aging biomarkers. Nevertheless, specific oxidatively modified proteins may yet be identified, which could be quantified noninvasively, that might provide accurate prediction of functional aging effects within specific systems during aging. Unfortunately, the current studies reveal a characteristic of oxidative damage, when measured as total carbonyl or -SH concentration, that tends to detract from its potential use as an aging biomarker. The fact that the level of protein oxidation may vary markedly with the level of caloric intake suggests that oxidative damage could also vary as a function of other physiological conditions. Thus, a single point measurement of oxidative damage is not necessarily an accurate reflection of the level of accumulated oxidative damage, the component that should afford the greatest predictability with respect to longevity and/or functional life span.

	Acknowledgments

 
This research was supported by Grants R01 AG 13563 and R01 AG 7695 from the National Institute on Aging, National Institutes of Health.

Received September 16, 1999

Accepted March 15, 2000

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