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Low Fatty Acid Unsaturation

A Mechanism for Lowered Lipoperoxidative Modification of Tissue Proteins in Mammalian Species With Long Life Spans

^a Department of Basic Medical Science, University of Lleida, Spain
^b Department of Chemistry and Biochemistry, University of South Carolina, Columbia
^c Department of Animal Biology II (Animal Physiology), Complutense University, Madrid, Spain

Gustavo Barja, Departamento de Biolog\|[iacute]\|a Animal II, Facultad de Biolog\|[iacute]\|a, Universidad Complutense, Madrid 28040, Spain.

Decision Editor: Jay Roberts, PhD

	Abstract

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Carbonyl compounds generated by the nonenzymatic oxidation of polyunsaturated fatty acids react with nucleophilic groups in proteins, leading to their modification. It has not been tested whether fatty acid unsaturation is related to steady-state levels of lipoxidation-derived protein modification in vivo. A low fatty acid unsaturation, hence a low protein lipoxidation, in tissues of longevous animals would be consistent with the free radical theory of aging, because membrane lipids increase their sensitivity to oxidative damage as a function of their degree of unsaturation. To evaluate the relationship between fatty acid composition, protein lipoxidation, and maximum life span (MLSP), we analyzed liver fatty acids and proteins from seven mammalian species, ranging in MLSP from 3.5 to 46 years. The results show that the peroxidizability index of fatty acids and the sensitivity to in vitro lipid peroxidation are negatively correlated with the MLSP. Based on gas chromatography and mass spectroscopy analyses, liver proteins of all these species contain malondialdehyde-lysine and N-carboxymethyllysine adducts, two biomarkers of protein lipoxidation. The steady-state levels of malondialdehyde-lysine and N-carboxymethyl lysine are directly related to the peroxidizability index and inversely related to the MLSP. We propose that a low degree of fatty acid unsaturation may have been selected in longevous mammals to protect their tissue lipids and proteins against oxidative damage while maintaining an appropriate environment for membrane function.

AMONG tissue macromolecules, polyunsaturated fatty acids (PUFA) are the most sensitive to free radical damage, with the sensitivity increasing as a power function of the number of double bonds per fatty acid ⁽¹⁾⁽²⁾. Oxidation of PUFA leads to the formation of hydroperoxides and endoperoxides, which undergo fragmentation to yield a broad range of reactive intermediates, including alkanals, alkenals, hydroxyalkenals, glyoxal, and malondialdehyde (MDA) ⁽³⁾. These carbonyl compounds, and possibly their peroxide precursors ⁽⁴⁾, react with nucleophilic groups in proteins, resulting in chemical modification of the protein ⁽⁵⁾⁽⁶⁾. The modification of amino acids in proteins by products of lipid peroxidation results in the chemical, nonenzymatic formation of a variety of adducts, including malondialdehyde-lysine (MDA-lys) and N-carboxymethyllysine (CML), which thus may serve as indicators of protein oxidative stress in vivo.

Several studies have evaluated the relationship between oxidative stress and maximum life span (MLSP) in different vertebrate species (for a review see Ref. 7). Available research indicates that there are at least two main characteristics of longevous species: a high rate of DNA repair and a low rate of free radical production. Although the latter characteristic is consistent with the free radical theory of aging ⁽⁸⁾⁽⁹⁾, additional factors can also lead to a low level of oxidative damage in longevous animals. Thus, previous studies have shown that the degree of fatty acid unsaturation of all the main phospholipid fractions of liver mitochondria is lower in pigeons (MLSP = 35 years) than in rats (MLSP = 4 years), and is also lower in humans (MLSP = 122 years) than in pigeons ⁽¹⁰⁾. This finding has been extended also to mitochondrial lipids of long-lived compared with short-lived mammalian species ⁽¹¹⁾.

The relationship between fatty acid unsaturation with the steady-state levels of tissue protein modification by products of PUFA oxidation has not been explored in animals showing different MLSPs. We hypothesized that a low fatty acid unsaturation could be advantageous to longevous animals by limiting lipoxidative stress-derived damage, because it would decrease the sensitivity of their tissues to lipid peroxidation and protein modification. Therefore, in the present study we examined the fatty acid composition and the sensitivity to lipid peroxidation, as well as the presence and steady-state levels of MDA-lys and CML adducts in liver proteins of different mammalian species with MLSPs ranging from 3.5 to 46 years.

	Methods

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Animals and Diets
Animals, namely mouse (Mus musculus, n = 6), rat (Rattus norvergicus, n = 8), guinea pig (Cavia porcellus, n = 6), dog (Canis familiaris, n = 4), pig (Sus scrofa, n = 7), cow (Bos taurus, n = 7), and horse (Equus caballus, n = 5), whose MLSPs varied from 3.5 to 46 years, were adult specimens selected at an age within 15–30% of their maximum life span. All animals appeared to be healthy; no animal was obese or scraggy. The recorded values of maximum longevity (in years) were as follows: mouse, 3.5; rat, 4; guinea pig, 8; dog, 20; pig, 27; cow, 30; and horse, 46 ⁽¹²⁾. The animal care protocols were approved by the University of Lleida Animal Experimentation Ethics Committee. Mice, rats, and guinea pigs were euthanized by decapitation. Dogs were euthanized in the Lleida city's dog pound by a single succinylcholine injection. Pigs, cows and horses (farm animals) were euthanized at the abattoir. Diets administered during the adult life of the animals were obtained when the animals were euthanized.

Tissue samples were taken randomly from the main hepatic lobe and were immediately cut into small pieces, washed in 50 mM of phosphate buffer (pH 7.4) to remove all traces of blood, and then stored at -80°C until further analysis. Liver samples (250 mg) were homogenized in 2.5 ml of 50-mM phosphate buffer (pH 7.4) by using a Polytron homogenizer (Brinkmann, Switzerland) for 15 seconds at a setting of 8, and then they were centrifuged at 750 x g for 10 minutes at 4°C to eliminate tissue debris.

Chemicals
Reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified. [²H₈]lysine was purchased from MSD Isotopes (Rahway, NJ). CML and [²H₄]CML, and MDA-lys and [²H₈]MDA-lys, were prepared as previously described ^(6)(13).

Fatty Acid Analyses
Lipids from liver and diets were extracted into chloroform:methanol 2:1 (vol/vol) by the method of Folch and colleagues ⁽¹⁴⁾, in the presence of 0.01% butylated hydroxytoluene. The chloroform phase was separated and then evaporated under N₂. Lipids were transesterified in 2.5 ml of 5% methanolic HCl at 75°C for 90 minutes. The resulting methyl esters were extracted by adding 2.5 ml of n-pentane and 1 ml of saturated NaCl. The n-pentane phase was separated and evaporated under N₂, and the fatty acid methyl esters were redissolved in 100 µl of carbon disulfide. One microliter was submitted to gas chromatography and mass spectrometry (GC/MS) analysis. GC separation was performed in a SP2330 capillary column (30 m x 0.25 mm x 0.20 µm) in a Hewlett-Packard (Palo Alto, CA) 5890 Series II gas chromatograph. A Hewlett-Packard 5989A mass spectrometer was used as detector in the electron-impact ionization mode. GC/MS conditions were as follows: injector and detector port temperatures, 220°C and 250°C, respectively; column temperature ranged from 100°C with an increase of 10°C/min, from 200°C to 240°C at 5°C/min, and a final hold of 12 minutes. Identification of methyl esters was made by comparison with the retention time and mass spectra of authentic standards.

Sensitivity to Lipid Peroxidation
In order to estimate the sensitivity of liver lipids to free radical damage, lipid peroxidation was stimulated in vitro by incubating liver homogenates in the presence of 0.4 mM of ascorbate and 0.05 mM FeSO₄ at 37°C for 6 hours. Preliminary experiments showed that the lipid peroxidation process evaluated by the generation of thiobarbituric acid reactive substances had reached completion by 6 hours. At the end of the incubation, the thiobarbituric acid assay was performed in the presence of 0.07 mM of butylated hydroxytoluene ⁽¹⁵⁾. Malondialdehydebis(dimethylacetal) (Merck, Munich, Germany) was used as standard. The results were expressed as nanomoles of MDA per gram of tissue.

Measurement of CML and MDA-lys in Tissue Proteins
MDA-lys and CML were analyzed in samples from the same animals in which fatty acid analyses and lipid peroxidation assays were performed, except in the case of the mouse, because of the small size of its liver. The liver homogenates were delipidated by using 10 ml of methanol:diethyl ether (1:10 vol/vol), and precipitated proteins were dried under a stream of N₂. MDA-lys and CML were measured in protein samples (1–2 mg) after overnight reduction with a final concentration of 500 mM NaBH₄ in 0.2 M borate buffer, pH 9.2, containing 1 drop of hexanol as an antifoam reagent. Proteins were precipitated with 10% trichloroacetic acid and subsequent centrifugation. Isotopically labeled internal standards were added, and the samples were hydrolyzed at 110°C for 24 hours in 6 N HCl and then dried in vacuo. Residues were rehydrated in 1 ml of 1% trifluoroacetic acid and applied to a 1-ml C-18 solid extraction column (Supelco, Bellefonte, PA), equilibrated with the same solvent. The first milliliter of the flowthrough and an additional 2 ml of 1% trifluoroacetic acid were all pooled and dried in vacuo. The N,O-trifluoroacetyl methyl ester derivatives of the protein hydrolysate were prepared as previously described ⁽¹³⁾. GC/MS analyses were carried out on a Hewlett-Packard Model 6890 gas chromatograph equipped with a 30-m HP-5MS capillary column coupled to a Hewlett-Packard Model 6890 mass selective detector. The injection port was set at 275°C, and the temperature program was 150–225°C at 5°C/min; 225–300°C at 25°C/min, and a final hold of 5 minutes. Quantification was performed by external standardization by using standard curves constructed from mixtures of deuterated and natural abundance standards. Analyses were carried out by selected ion-monitoring GC/MS. The ions used were lysine and [²H₈]lysine, m/z 180 and 187, respectively; CML and [²H₄]CML, m/z 392 and 396, respectively; and MDA-lysine and [²H₈]MDA-lys, m/z 474 and 482, respectively. The amount of products is expressed as the ratio of millimoles of CML or MDA-lys per mole of lysine. The presence of oxidative products in liver proteins was demonstrated by comparing the retention time and characteristic m/z ions with those of authentic standards.

Statistics
Data processing and statistical analyses were performed with the SPSS program for Windows (SPSS Inc., Chicago). The results are expressed as means ± SD. Variable normal distribution was tested by the Kolmogorov–Smirnov test. Interespecies differences between variables were analyzed by using a one-way analysis of variance test. Correlations between variables in the different mammalian species were studied by regression analyses. The .05 level was selected as the point of minimal statistical significance throughout.

	Results

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The fatty acid composition of the diets given to the different mammalian species was analyzed (results not shown). The degree of fatty acid unsaturation (double bond index, DBI) of the diets was not correlated to the MLSP of the different species ( p > .05). The fatty acid composition of liver lipids in the seven mammalian species studied is shown in Table 1 . All fatty acids showed significant differences between species ( p < .0001). 18:2n-6 was the unsaturated fatty acid demonstrating the greatest absolute differences between extremes of MLSP (from 14% in the mouse to 53% in the horse), followed by changes in 22:6n-3 and 20:4n-6 (8–12% decreases in absolute terms and 87–97% relative decreases from rodents to horse). The average chain length (ACL), and the content in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), unsaturated fatty acid (UFA), PUFA, and PUFA of the n-3 and n-6 series, as well as the DBI and peroxidizability index (PI, which takes into account that the sensitivity to peroxidation increases as a function of the number of double bonds per fatty acid) ^(16)(17), showed significant differences among species ( p < .0001, Table 2 ).

View larger version (13K): [in this window] [in a new window]   Figure 1. Relationship between sensitivity to in vitro lipid peroxidation and maximum life span (MLSP) in the livers of mammalian species. Values of lipid peroxidation were plotted as a function of MLSP on double logarithmic axes. After logarithmic transformation of the two variables, the resulting data were fitted to a straight line by linear regression (r = -.74, p < .05). This fit corresponds to the power function y = 2,070 x-0.29. Values are means ± standard error of the mean. Error bars are not visible when they are smaller than the dot. The number of animals in each species is given in Table 2 . MDA, malondialdehyde.

View larger version (15K): [in this window] [in a new window]   Figure 2. Relationship between malondialdehyde-lysine (MDA-lys) adducts in liver proteins of mammals and maximum life span (MLSP). Values of MDA-lys were plotted as a function of MLSP, and data were fitted to a straight line by linear regression, y = 259 - 3.53 x, r = -.93, p < .007. Values are means ± standard error of the mean. Error bars are not visible when they are smaller than the dot. The number of animals in each species is given in Table 2 .

View larger version (15K): [in this window] [in a new window]   Figure 3. Relationship between Nepsilon carboxymethyl lysine adducts in liver proteins of mammals and maximum life span (MLSP). Values of malondialdehyde lysine were plotted as a function of MLSP, and data were fitted to a straight line by linear regression, y = 1,556 - 12 x, r = -.91, p < .01. Values are means ± standard error of the mean. The number of animals in each species is given in Table 2 .

View larger version (15K): [in this window] [in a new window]   Figure 4. Relationship between malondialdehyde-lysine (MDA-lys) protein adducts and the peroxidizability index (PI) of fatty acids in the liver of mammals. Values of MDA-lys were plotted as a function of those of the PI, and data were fitted to a straight line by linear regression, y = 28.17 + 1.57 x, r = .82, p < .04. Values are means ± standard error of the mean. Error bars are not visible when they are smaller than the dot. The number of animals in each species is given in Table 2 .

	Discussion

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The susceptibility of fatty acids to free radical damage increases exponentially as a function of the number of double bonds per fatty acid molecule, with 18:2n-6 being relatively stable to oxidative stress in relation to the highly unsaturated 22:6n-3. In fact, many studies have shown that free radical damage and lipid peroxidation increase as a function of the degree of unsaturation of the fatty acid substrates present in the tissues in vivo ^(1)(23). Increases in PI and decreases in 18:2n-6 have been described in rodent aging ^(17)(18), and the senescent accelerated prone mouse has a somewhat higher 22:6n-3 and PI and a lower 18:2n-6 in the brain than that of their senescent accelerated-resistant counterparts ⁽²⁴⁾. It is reasonable to think that the lower degree of fatty acid unsaturation of longevous animals will protect their tissues against oxidative damage. The low PI of longevous animals can thus be responsible in part for their relatively higher resistance to lipid peroxidation and low content of MDA-lys and CML protein adducts. A previous study ⁽²⁵⁾ has shown that rates and maximum spontaneous—noncatalyzed—in vitro lipid peroxidation of kidney and brain homogenates are negatively correlated with MLSP in 24 (brain) or 9 (kidney) species of mammals from mouse to man. Even though fatty acid composition was not analyzed, it was suggested that for most species the major factor determining the lipid peroxidation potential of tissues was the concentration of peroxidizable substrate and that a change in the composition of lipid membranes occurred during the evolution of increased MLSP in mammals, resulting in a lower susceptibility of tissues to oxygen radical-initiated peroxidation reactions ⁽²⁵⁾. Our study fully supports this last suggestion for the liver, and a previous study ⁽¹⁹⁾ is consistent with the same happening in the kidney, heart, and skeletal muscle. Because lipid peroxidation is known to increase levels of protein carbonyls in nearby proteins ⁽²⁶⁾, the low degree of fatty acid unsaturation of longevous animals can also be an important underlying factor for the negative relationship recently observed between MLSP and the susceptibility of tissue proteins to in vitro oxidative damage (protein carbonyls) in homeothermic vertebrates ⁽²⁷⁾.

Aldehydes generated during the peroxidation of PUFA can result in protein modification by the formation of adducts with epsilon-amino groups of lysine (among others) residues. Several aldehydes are formed by decomposition of lipid hydroperoxides during autoxidation of polyunsaturated lipids, including alkanals and activated aldehydes with -hydroxy, -ß-unsaturated, and 4-hydroxy unsaturated functional groups ^{(3)(28)(29)(30)}. These aldehydes vary in chain length, depending on the orientation of the oxidation site and on the location of the double bonds in the starting lipid, ranging from 9-carbon compounds, such as 4-hydroxynonenal, to small 2- and 3-carbon reactive compounds, such as malondialdehyde, acrolein, and glyoxal. Each of these compounds may react with amino groups of proteins ^(6)(31). Although hexanal is quantitatively (40%) the major aldehyde formed during in vitro lipid peroxidation, Schiff bases (imines) formed between aliphatic aldehydes and amines are relatively labile, and hexanal-lysine adducts dissociate reversibly to the component aldehyde and amine. In contrast, MDA, the aldehyde formed at next highest abundance, and glyoxal, a precursor of CML, yield chemically stable adducts to lysine residues ⁽⁵⁾⁽⁶⁾. For this reason, in this work we have evaluated the MDA-lys and CML adducts occurring in tissue proteins. The GC/MS method described herein provides a means for the specific quantification of MDA-lys Schiff-base and CML adducts in proteins. Both MDA-lys and CML were detected in tissue proteins.

The results indicate that the steady-state levels of these two oxidative products are related to the PI of the fatty acids; that is, a low PI is related to a low degree of protein modification. Furthermore, both a low PI and a low level of protein modification are present in longevous animals. Finally, although CML was originally described as a product of carbohydrate autoxidation ^(32)(33)(34), as the blood glucose concentrations of the species in this study are all 5 mM, we attribute differences among CML levels to differences in formation from lipid peroxidation.

In summary, a low degree of fatty acid unsaturation and a low susceptibility to lipid peroxidation and protein modification, together with a low free radical production ^(7)(35)(36) and high rate of DNA repair ⁽³⁷⁾, can be considered biochemical traits of longevous animals.

	Acknowledgments

 
This work was supported by grants from the Ministerio de Sanidad (FISss, Ref. 98/0752), from the Generalitat de Catalunya (SGR, Ref. 1997SGR00436) and from La Paeria (Ref. X0075) to the Metabolic Pathophysiology Research Group; by grants from the Ministerio de Sanidad (FISss, Ref. 96/1253) to Dr. Barja; by a grant for short-term fellowships from Commissió Interdepartamental de Recerca i Tecnologia (Ref. 1997BEAI400131) to Dr. Pamplona; and by a United States Public Health Services grant (AG11472) to Dr. Thorpe.

Received June 28, 1999

Accepted November 5, 1999

	References

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1. North JA, Spector AA, Buettner GR, 1994. Cell fatty acid composition affects free radical formation during lipid peroxidation. Am J Physiol. 267:C177-C188. [Abstract/Free Full Text]
2. Visioli F, Colombo C, Galli C, 1998. Oxidation of individual fatty acids yields different profiles of oxidation markers. Biochem Biophys Res Commun 245:487-489. [Medline]
3. Esterbauer H, Schauer RJ, Zollner H, 1991. Chemistry and biochemistry of 4 hydroxynonenal, malondialdehyde and related aldehydes. Free Rad Biol Med. 11:81-128. [Medline]
4. Fruebis J, Parthasarathy S, Steinberg D, 1992. Evidence for a concerted reaction between lipid hydroperoxides and polypeptides. Proc Natl Acad Sci USA. 89:10588-10592. [Abstract/Free Full Text]
5. Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe SR, 1996. The advanced glycation end product, Ne-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 271:9982-9986. [Abstract/Free Full Text]
6. Requena JR, Fu MX, Ahmed MU, et al. 1997. Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein. Biochem J. 322:317-325.
7. Perez-Campo R, López-Torres M, Cadenas S, Rojas C, Barja G, 1998. The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach. J Comp Physiol B. 168:149-158. [Medline]
8. Harman D, 1994. Free radical theory of aging. Increasing the functional life span. Ann NY Acad Sci. 717:1-15. [Medline]
9. Yu BP, Yang R, 1996. Critical evaluation of the free radical theory of aging. A proposal for the oxidative stress hypothesis. Ann NY Acad Sci. 786:1-11. [Medline]
10. Pamplona R, Prat J, Cadenas S, et al. 1996. Low fatty acid unsaturation protects against lipid peroxidation in liver mitochondria from longevous species: the pigeon and human case. Mech Ageing Dev. 86:53-66. [Medline]
11. Pamplona R, Portero-Otín M, Riba D, et al. 1998. Mitochondrial membrane peroxidizability index is inversely related to maximum life-span in mammals. J Lipid Res. 39:1989-1994. [Abstract/Free Full Text]
12. Altman PL, Dittmer DS, 1972. Biology Data Book.. Bethesda: Federation of American Societies of Experimental Biology. 1:229-230.
13. Knecht KJ, Dunn JA, McFarland KF, et al. 1991. Effects of diabetes and aging on N^e-(carboxymethyl)lysine levels in human urine. Diabetes. 40:190-196. [Abstract]
14. Folch J, Lees M, Sloane-Stanley GM, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 226:497-509. [Free Full Text]
15. Uchiyama M, Mihara M, 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem. 86:271-278. [Medline]
16. Holman TR, 1954. Autoxidation of fats and related substances. Holman RT, Lundberg WO, Malkin T, , ed.Progress in the Chemistry of Fats and other Lipids 2Academic Press, New York.
17. Laganiere S, Yu BP, 1993. Modulation of membrane phospholipid fatty acid composition by age and food restriction. Gerontology. 39:7-18. [Medline]
18. Gudbjarnason S, 1989. Dynamics of n-3 and n-6 fatty acids in phospholipids of heart muscle. J Intern Med. 225:117-128. [Medline]
19. Couture P, Hulbert AJ, 1995. Membrane fatty acid composition of tissues is related to body mass of mammals. J Membr Biol. 148:27-39. [Medline]
20. Brand MD, Couture P, Hulbert AJ, 1994. Liposomes from mammalian liver mitochondria are more polyunsaturated and leakier to protons than those from reptiles. Comp Biochem Physiol. 108B:181-188.
21. Brenner RR, 1984. Effect of unsaturated fatty acids on membrane structure and enzyme kinetics. Progr Lipid Res. 23:69-96. [Medline]
22. Stubbs CD, Smith AD, 1984. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta. 779:89-137. [Medline]
23. Bondy SC, Marwah S, 1995. Stimulation of synaptosomal free radical production by fatty acids: relation to esterification and to degree of unsaturation. FEBS Lett. 375:53-55. [Medline]
24. Choi JH, Kim J, Moon YS, Chung HY, Yu BP, 1996. Analysis of lipid composition and hydroxyl radicals in brain membranes of senescent-accelerated mice. Age. 19:1-5.
25. Cutler RG, 1985. Peroxide-producing potential of tissues: inverse correlation with longevity of mammalian species. Proc Natl Acad Sci USA. 82:4798-4802. [Abstract/Free Full Text]
26. Forsmark-Andrée P, Dallner G, Ernster L, 1995. Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles. Free Rad Biol Med. 19:749-757. [Medline]
27. Agarwall S, Sohal RS, 1996. Relationship between susceptibility to protein peroxidation, aging, and maximum life span potential of different species. Exp Gerontol. 31:387-392. [Medline]
28. Janero DR, 1990. Malondialdehyde and thiobarbituric acid reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Rad Biol Med. 9:515-541. [Medline]
29. Loidl-Stahlhofen A, Spiteller G, 1994. -Hydroxyaldehydes, products of lipid peroxidation. Biochim Biophys Acta. 1211:156-160. [Medline]
30. Mlakar A, Spiteller G, 1994. Reinvestigation of lipid peroxidation of linolenic acid. Biochim Biophys Acta. 1214:209-230. [Medline]
31. Uchida K, Kanematsu M, Sakai K, et al. 1998. Protein-bound acrolein: potential markers for oxidative stress. Proc Natl Acad Sci USA. 95:4882-4887. [Abstract/Free Full Text]
32. Ahmed MU, Thorpe SR, Baynes JW, 1986. Identification of carboxymethyllysine as a degradation product of fructose-lysine in glycosylated protein. J Biol Chem. 261:4889-4994. [Abstract/Free Full Text]
33. Wells-Knecht KJ, Zyzak DV, Litchfield JE, Thorpe SR, Baynes JW, 1995. Mechanism of autoxidative glycosylation. Identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose. Biochemistry. 34:3702-3709. [Medline]
34. Onorato JM, Thorpe SR, Baynes JW, 1998. Immunohistochemical and ELISA assays for biomarkers of oxidative stress in aging and disease. Ann NY Acad Sci. 854:277-290. [Medline]
35. Barja G, 1998. Mitochondrial free radical production and aging in mammals and birds. Ann NY Acad Sci. 854:224-238. [Medline]
36. Barja G, 1999. Mitochondrial free radical generation: sites of production in states 4 and 3, organ specificity and relationship with aging rate. J Bioenerg Biomembr. 31:347-366. [Medline]
37. Cortopassi GA, Wang E, 1996. There is a substantial agreement among interspecies estimates of DNA repair activity. Mech Ageing Dev. 91:211-218. [Medline]

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