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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:847-851 (2005)
© 2005 The Gerontological Society of America

Effects of Age and Caloric Restriction on Lipid Peroxidation: Measurement of Oxidative Stress by F2-Isoprostane Levels

Walter F. Ward1,3,, Wenbo Qi2,3, Holly Van Remmen2,3,4, William E. Zackert5, L. Jackson Roberts, II5 and Arlan Richardson2,3,4

Departments of 1 Physiology
2 Cellular and Structural Biology, and the
3 Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center at San Antonio.
4 Geriatric Research, Education and Clinical Center and Research Service, South Texas Veterans Health Care System, San Antonio.
5 Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee.

Address correspondence to Walter F. Ward, PhD, Department of Physiology–MSC-7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. E-mail: wardw{at}uthscsa.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 REFERENCES
 
The free radical theory of aging proposes that the accumulation of oxidative damage is a key component of the aging process. The discovery of F2-isoprostanes (F2-isoPs) and their establishment as a sensitive and accurate biomarker of lipid peroxidation represents a major advance for measuring the oxidative stress status of an organism. We have shown that plasma free and total (free plus esterified) F2-isoPs increase with age (185% and 66%, respectively), and that these increases are reduced by life-extending caloric restriction (50% and 23%, respectively). In addition, we found that levels of esterified F2-isoPs increase 68% with age in liver, and 76% with age in kidney. Caloric restriction modulated the age-related increase, reducing the esterified F2-isoPs levels 27% in liver and 35% in kidney. These age-related increases in esterified F2-isoPs levels correlate well with DNA oxidation, as measured by 8-oxodeoxyguanosine production demonstrating that F2-isoPs are an excellent biomarker for age-related changes in oxidative damage to membranes.

CURRENTLY, one of the most popular theories of aging is the free radical or oxidative stress theory of aging. This theory is based on the fact that cells exist in a chronic state of oxidative stress resulting from an imbalance between pro-oxidants and antioxidants. Because of this imbalance, which occurs as a consequence of aerobic metabolism, an accumulation of oxidative damage is proposed to occur with age in a variety of macromolecules within the cell. This steady-state accumulation of oxidative damage is thought to be an important mechanism underlying aging and the age-related increase in pathology as well as the progressive decline in the functional efficiency of various cellular processes (1–4). Over the past decade, the oxidative stress hypothesis of aging has gained wide acceptance because numerous studies have shown a strong correlation between increasing age and the accumulation of oxidative damage to cellular macromolecules, and because the increased survival observed with use of caloric restriction (CR) has been correlated with reduced oxidative damage (5–10). It also appears that certain types of pathological lesions that arise with age are associated with increased levels of oxidative damage to cellular macromolecules.

Lipid peroxidation is one type of oxidative damage that has been extensively studied with respect to age. The allylic hydrogens in the polyunsaturated fatty acid components of phospholipids in cellular membranes make membranes extremely sensitive to free radical oxidation. One of the major problems confronting investigators measuring lipid peroxidation is the accuracy of the assays, which is discussed in detail by Rikans and Hornbrook (11). Recently, an alternative method for measuring lipid peroxidation was developed that is more accurate and sensitive than the previous methods of measuring lipid peroxidation by malondialdehyde (MDA) and 4-hydroxy-2-nonenol (4-HNE) levels. The laboratories of Roberts, Morrow, and Janssen (12,13) were the first to describe the nonenzymatic production of a group of prostaglandin-like compounds that arise from free radical attack on membrane phospholipids. Arachidonic acid esterified to membrane phospholipids are converted to compounds isomeric to prostaglandin F2{alpha} and are referred to as F2-isoprostanes (F2-isoPs). Esterified F2-isoPs are released from the membrane (by the action of phospholipase) into the blood stream, and are ultimately excreted in the urine (14). Thus, plasma levels of free F2-isoPs provide a measure of the total endogenous production of F2-isoPs by all tissues of the body plus those derived from oxidation of plasma lipoproteins (15). Data obtained over the past 5 years demonstrate that the formation of F2-isoPs provides an excellent marker of oxidative stress because these compounds can be measured with precision down to the subpicomolar level; they are relatively stable compounds; they do not exhibit diurnal variations; they are present in detectable quantities in all normal biological tissues; and lastly, F2-isoPs levels are modulated by the antioxidant status of the organism but are not affected by the lipid composition of the diet (12,13).

Although F2-isoP levels appear to be an excellent marker of lipid peroxidation, the literature provides only limited information on their usefulness as a biomarker of lipid peroxidation with age. Roberts and Reckelhoff (15) showed that plasma levels of free and esterified F2-isoPs were dramatically increased with age in male Sprague-Dawley rats. In this study, we measured the levels of F2-isoPs in plasma as well as in liver and kidney tissue of male F344 rats fed ad libitum and a CR diet. We found that F2-isoP levels increase with age in the plasma, and in liver and kidney tissue, and that these increases are modulated by CR. Furthermore, the changes in F2-isoP levels in liver and kidney tissue can be directly correlated with oxidative damage to DNA, i.e., the age-related accumulation of 8-oxo-deoxyguanosine (8-oxodG) in DNA (16). Thus, F2-isoP levels appear to be an excellent biomarker of lipid peroxidation for aging studies.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 REFERENCES
 
Animals
Specific pathogen-free male Fischer 344 (F344) rats were obtained from the National Institute on Aging colony and were singly housed under barrier conditions in the Veterans Administration animal facility on a 12-hour light/dark cycle. Acidified water was provided ad libitum, and the animals were maintained on the NIH-31 diet (17). The animals subjected to CR were fed 60% of the caloric intake of their ad libitum-fed litter mates. All procedures for handling the rats were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and the Subcommittee for Animal Studies at the Audie L. Murphy Memorial Veterans Hospital.

Blood (2 ml) was collected from the inferior vena cava of anesthetized animals into prechilled heparin-coated tubes and centrifuged at 1500 x g for 10 minutes at 4°C. The plasma was transferred to 1.5 ml microcentrifuge tubes and rapidly frozen in liquid nitrogen for storage at –80°C. The animal was then euthanized by exsanguination, and the liver and kidney were removed and immediately frozen in liquid nitrogen for storage at –80°C. All samples were analyzed within 6 months of being collected.

Measurement of F2-isoPs
The method utilized for measurement of F2-isoPs follows the procedure exactly as described by Roberts and Morrow (12), which involves extraction, thin layer chromatography (TLC) purification, and quantification by gas chromatography–mass spectrometric analysis as described below.

Lipid Extraction and Base Hydrolysis of Esterified F2-IsoP
Tissue (150–200 mg) was homogenized in 10 ml of ice-cold Folch solution (CHCl3:MeOH, 2:1) containing 5 mg/100 ml butylated hydroxytoluene (BHT), and the tube was flushed with N2 and sealed. The tubes were allowed to stand for 30 minutes at room temperature with vortexing every 10 minutes. The extracted lipids were then mixed with 2 ml of 0.9% NaCl and vortexed for 1 minute followed by centrifugation at 3000 x g for 5 minutes at 4°C, and the aqueous layer was aspirated and discarded. The organic layer was then evaporated to dryness under N2 at 37°C.

Plasma (0.5–1.0 ml) was added to 10 ml of ice-cold Folch solution containing 5 mg/100 ml BHT and 50 mg/100 ml triphenylphosphine (TPP). The tubes were vortexed for 1 minute, and 5 ml of 0.043% MgCl2 was added; the solution was mixed then centrifuged at 3000 x g for 3–5 minutes at 4°C. The upper MgCl2/methanol layer was aspirated, and the underlying CHCl3 layer was transferred to a clean tube, evaporated to dryness, and stored under nitrogen at 37°C until analyzed. The dried organic layer was redissolved in 0.5 ml of MeOH containing 5 mg/100 ml BHT. An equal volume (0.5 ml) of 15% KOH was added, mixed, and incubated at 37°C for 30 minutes. The samples were acidified to pH 3 with 1N HCl.

Measurement of Free 8-Isoprostanes
Using a Hamilton syringe, 20 µl (1 ng) of d4 8-isoPGF2{alpha} (internal standard) was added to 10 ml of deionized water, pH 3.0. One milliliter of untreated plasma (for free isoprostanes) or base hydrolyzed sample (for total isoprostanes) was added, mixed, and centrifuged at 2500 x g for 3 minutes at 4°C. The supernatant was added to a C18 Sep-Pak column (Waters Corp., Milford, MA), which had been prewashed with 5 ml of methanol followed by 7 ml of deionized water, pH 3.0. The loaded column was washed with 10 ml of water, pH 3.0, and 10 ml of heptane. The isoprostane fractions were eluted with 10 ml of heptane/ethyl acetate (1:1) into 20 ml glass scintillation vials. The eluate was dried by adding anhydrous Na2SO4, and the supernatant was rapidly transferred to a Silica Sep-Pak column (Waters Corp.), which had been rinsed with 5 ml of ethyl acetate (EtOAc). The column was washed with 5 ml of EtOAc, and the isoprostane fractions eluted with ethyl acetate/methanol (EtOAc/MeOH; 1:1) into 5 ml borosilicate reactive vials.

Derivatization
The column fractions were dried under N2 at 37°C, and the residues were redissolved in 40 µl of pentafluorobenzyl bromide, which has been completely dried with anhydrous CaSO4, plus 20 ml of 10% N,N-diisopropylethylamine in acetylnitrile. The sample solution was incubated at 37°C for 20 minutes, dried down under N2 at 37°C, and then reconstituted with 50 µl of MeOH/CHCl3 (3:2).

The silica TLC plates were prewashed with 100 ml of EtOAc/EtOH (90:10), oven dried at 90°–100°C for 10 minutes, and stored in a dessicator. Using a lead pencil, a line was drawn 13 cm from the origin, and 50 µl of sample was spotted onto each lane of the TLC plate with 5 µl (5 µg) of 8-iso-PGF2{alpha} methyl ester spotted in a separate lane as a standard. Following drying of the plate, the sample was developed in a TLC tank containing 100 ml of 93% CHCl3/7% EtOH. The plates were removed when the solvent front reached the 13 cm line, and the solvent was allowed to evaporate from the plates. The standard was visualized by spraying 10% phosphomolybdic acid in EtOH onto the plate. A pencil line was then drawn 0.6 cm above and 0.9 cm below the center of the standard band, and the silica within the lines was scraped onto a weighing paper and transferred to a 1.7-ml tube. One milliliter of EtOAc was added to the tube for extraction of the isoprostane compounds, and the supernatant was collected and dried down under N2.

The dried samples were reconstituted with 20 µl of N,O-bistrifluoroacetamide (BSTFA) and 8 µl of dried dimethylformamide (DMF), and were incubated at 37°C for 5 minutes. The samples were again dried down and redissolved in 20 µl of dry undecane. The samples were then analyzed by gas chromatography–mass spectrometric analysis using negative ion chemical ionization (NICI), which was carried out using a DB 1701 fused silica capillary column (Agilent Technology, Palo Alto, CA) (0.25 mm diameter, 0.25 µm film thickness), at temperatures of 190–300°C. The carrier gas is methane at a flow rate of 1 ml/min with an ion source temperature of 250°C, electron energy of 70 eV, and a filament current of 0.25 mA.

Measurement of DNA Oxidation
The levels of 8-oxodG in nuclear DNA, from the same liver and kidney tissue utilized for 8-iso-PGF2{alpha} analysis, were determined as we have previously described (16). Nuclear DNA (100–150 µg) was isolated from 100 mg of tissue using a DNA Extractor WB kit (Waco Chemicals, Richmond, VA) and digested to nucleotides by incubation with nuclease P1 and alkaline phosphatase. The levels 8-oxodG and 2-deoxyguanosine (2-dG) in the deoxynucleoside mixture were determined by high performance liquid chromatography with electrochemical and ultraviolet detection (Model 5200; ESA Inc., Chelmsford, MA) using a reverse phase, isocratic system using a 3 mm x 15 mm x 4.6 mm Supelcosil LC-18-T column (Supelco, Bellefonte, PA) with a sodium acetate mobile phase (25 mM NaAc, pH 5–5.2) containing 5% methanol. The quantities of 8-oxodG and 2-dG eluting from the column were measured, and the data are expressed as the ratio of nmoles of 8-oxodG to 105 nmoles of 2-dG.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 REFERENCES
 
Effect of Age and CR on F2-IsoP Levels in Plasma
Figure 1 shows the effect of age and CR on the levels of free and total (free + esterified) plasma levels of F2-isoPs in F344 rats, comparing 4- to 6- and 22- to 24-month-old animals. Although we observed a significant increase with age in the plasma levels of both free F2-isoPs (0.07 ± 0.01 ng/ml to 0.20 ± 0.06 ng/ml) and total F2-isoPs (0.45 ± 0.09 ng/ml to 0.75 ± 0.14 ng/ml), the increase in free F2-isoPs, as a percentage, was significantly greater than that of total F2-isoPs, 185% as compared to 66%. Figure 1 also shows that plasma levels of F2-isoPs were decreased significantly in the CR rats. Plasma free F2-isoPs decreased from 0.20 ± 0.06 to 0.10 ± 0.01, a decrease of 50%. There was less of a decrease in total plasma F2-isoPs levels in response to CR, 0.75 ± 0.14 to 0.58 ± 0.10 ng/ml, a reduction of approximately 23%.



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  		Figure 1. Effect of age and caloric restriction (CR) on plasma isoprostane (F2-isoP) levels. The levels of free and total (free + esterified) F2-isoP in 4- to 6-month-old (Y-AL) and 22- to 24-month-old rats fed ad libitum (O-AL) or a CR-diet (O-CR). Each bar represents the mean and standard error of the mean of data obtained from five rats. *The age-related increase and the decrease with CR in free and total isoprostane levels were significant at the p <.05 level as determined by t test

 
Effects of Age and CR on Lipid and DNA Oxidation in Liver
Figure 2 presents a comparison of the effects of age and CR on levels of esterified isoprostanes in the livers of F344 rats with another important and well documented oxidative modification, DNA oxidation (8-oxodG). The levels of F2-isoPs in liver increased from 10.34 ± 0.85 ng/g to 17.42 ± 1.24 ng/g, an increase of approximately 68%. This age-related increase was suppressed by CR. The level of F2-isoPs in the liver of the old CR animals averaged 12.76 ± 1.29 ng/g, which represents an age-related increase of slightly more than 23% in the CR animals or, conversely, CR produced approximately a 27% reduction as compared to that in the old rats fed ad libitum. We also found that the changes in F2-isoP levels paralleled the changes in 8-oxodG in nuclear DNA from the liver. In the ad libitum-fed group there was a 94% increase of 8-oxodG levels with age. CR reduced the age-related increase to 36%, or conversely, a decrease of approximately 30% when comparing old CR rats with old ad libitum-fed rats.



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  		Figure 2. Effect of age and caloric restriction (CR) on oxidative damage in liver. The levels of esterified isoprostane (F2-isoP) and 8-oxodeoxyguanosine (8-oxodG) in the liver from 4- to 6-month-old (Y-AL) and 22- to 24-month-old rats fed ad libitum (O-AL) or a CR diet (O-CR). Each bar represents the mean and standard error of the mean of data obtained from five rats for F2-isoP measurements and from six animals for 8-oxodG measurements. *The values for the O-AL rats are significantly higher (p <.05 level) than those for the Y-AL or O-CR rats as determined by t test

 
Effect of Age and CR on Lipid and DNA Oxidation in Kidney
Figure 3 compares the levels of F2-isoPs extracted from the kidney tissue of young and old ad libitum-fed rats and old CR rats. The total F2-isoP levels measured in young animals averaged 5.04 ± 0.49 ng/g. With age, kidney F2-isoP levels increased approximately 76% to an average of 8.84 ± 1.13 ng/g. Again, CR decreased the age-related increase in the old animals to a level of 5.76 ± 0.28 ng/g. This can be viewed as an age-related increase of approximately 14% as compared to the young ad libitum-fed animals, or a reduction of approximately 35% as compared to the old ad libitum-fed animals. The levels of 8-oxodG levels in kidney tissue exhibited a 280% increase with age, and CR reduced this increase to 175% or, conversely, CR produced a 27% reduction when the levels in the old CR rats are compared to those in the old ad libitum-fed rats. Thus, both lipid peroxidation and DNA oxidation increased with age in the kidney of rats fed ad libitum, and these increases were ameliorated by CR.



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  		Figure 3. Effect of age and caloric restriction (CR) on oxidative damage in kidney. The levels of esterified isoprostane (F2-isoP) and 8-oxodeoxyguanosine (8-oxodG) in kidney from 4- to 6-month-old (Y-AL) and 22- to 24-month-old rats fed ad libitum (O-AL) or a CR diet (O-CR). Each bar represents the mean and standard error of the mean of data obtained from five rats. *The values for the O-AL rats are significantly higher (p <.05 level) than those for the Y-AL or O-CR rats as determined by t test

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
REFERENCES
 
The peroxidation of phospholipids in membranes is potentially important to cellular function and is proposed to play a central role in a number of disease processes such as atherosclerosis, Parkinson's disease, Alzheimer's disease, and carcinogenesis (5). The phospholipid in membranes is an important site for the generation of reactive oxygen species in a cell because of its high content of polyunsaturated fatty acids. The allylic hydrogens in the polyunsaturated fatty acids are extremely sensitive to attack by free radicals; this sensitivity leads to lipid peroxidation and the generation of peroxyl radicals. The peroxyl radicals in turn are capable of abstracting allylic hydrogens from other fatty acids to produce more lipid hydroperoxides, thereby generating a chain reaction of lipid peroxidation (18).

Lipid peroxidation is potentially important physically because it decreases membrane fluidity, making it easier for phospholipids to exchange between the two monolayers, thereby increasing the leakiness of the membrane bilayer to substances that do not normally cross the membrane other than through specific channels (19). Lipid peroxidation can also produce cytotoxic compounds such as 4-HNE, which has been shown to inhibit protein and DNA synthesis and is especially toxic to mitochondria. Continued oxidation of fatty acid side-chains and their fragmentation to produce aldehydes and hydrocarbons will eventually lead to loss of membrane integrity. For example, rupture of the membranes of lysosomes can release hydrolytic enzymes into the cell causing an amplification of the damage (18). In addition, lipid peroxidation can cause cross-linking and inactivation of membrane proteins (19).

The majority of published studies measuring lipid peroxidation with age have relied on the measurement of MDA or 4-HNE. For example, increased MDA or 4-HNE has been reported in skeletal muscle (20), liver (21,22), lung, pancreas, and testes (18). However, the thiobarbituric acid assay, which commonly is used to measure MDA, is not specific to MDA; furthermore, MDA is not a specific marker of lipid peroxidation (12). In addition, both MDA and 4-HNE are very reactive and therefore unstable compounds, which can form adducts. In contrast, F2-isoPs are very stable compounds. Because of their stability and because they are derived from complex lipids, which are primarily found in membranes, the F2-isoPs are an excellent biomarker of membrane peroxidation.

Although there are numerous studies on the effect of age and CR on lipid peroxidation using the thiobarbituric acid assay or other assays of MDA or 4-HNE, there is very little information on either the effect of age on membrane peroxidation using the F2-isoP assay, or on the levels of F2-isoPs in rodents fed a CR diet. Therefore, in this study, we examined the effects of age and CR on plasma and tissue levels of free and esterified F2-isoPs in one of the most commonly used animal models for aging studies, the F344 rat. In addition, we compared the changes in F2-isoP levels with another sensitive assay of oxidative damage, the formation of 8-oxodG in DNA. Oxidative damage of DNA, a well established and sensitive measure of oxidative damage, results in the accumulation of 8-oxodG residues within the DNA, the levels of which can be measured with great precision by high performance liquid chromatography coupled with electrochemical detection.

We found that the levels of free F2-isoPs in plasma increased with age in male F344 rats, increasing approximately 185% (Figure 1). We also observed that total plasma F2-isoP levels, which represent both free F2-isoPs and F2-isoPs esterified to plasma lipids, were also increased with age, approximately 66%. Although these data are consistent with the observations of Roberts and Reckelhoff (15) (who used Sprague-Dawley rats of approximately the same age range), the age-related increase in plasma free and total F2-isoP levels that we observed was not as large as reported by them. The difference in the increase in F2-isoP levels with age between the two studies could be due to different strains of rats used (e.g., F344 vs Sprague-Dawley), because Sprague-Dawley rats become very obese with age (23) or because the Sprague-Dawley rats in the study by Roberts and Reckelhoff (15) were not maintained under barrier conditions, i.e., these rats might have been exposed to more stress than were the rats in our study.

Plasma levels of free F2-isoPs provide investigators with an excellent integrated measure of the oxidative stress status of the whole organism, because these F2-isoPs arise from the free radical attack of arachidonic acid moieties associated with membrane phospholipids in the various tissues of the organism. The plasma free F2-isoPs, which are initially esterified to phospholipids in the tissues of the organism, arise in the blood stream after phospholipase action, and are quickly (half life {approx}20 minutes) excreted into the urine (12). Thus, plasma free F2-isoP levels provide one with a measure of the endogenous production of F2-isoPs by the whole organism at a specific time. We were interested in determining the effects of CR on the level of plasma free F2-isoPs because a great deal of research over the past decade has shown that the levels of various types of oxidative damage are reduced by CR in tissues of rodents (3). We observed a significant reduction in plasma free F2-isoP levels in the old CR rats compared to those in the old rats fed ad libitum. In fact, CR abolished the age-related increase in plasma free F2-isoP levels, e.g., the levels of plasma free F2-isoPs were not significantly different for young rats fed ad libitum and for old CR rats. These are the first data showing that CR resulted in a reduction of plasma free F2-isoP levels, and are consistent with the previous data, suggesting that the CR rodents are under reduced oxidative stress compared to rodents fed ad libitum.

The elevation of plasma F2-isoP levels in the old rats fed ad libitum that we observed suggests that the old rats are subject endogenously to an increased level of oxidative stress compared to the young rats fed ad libitum and that CR reduces the level of endogenous oxidative stress to the organism. To test this possibility, we also measured the levels of esterified F2-isoPs in liver and kidney. As predicted from our data on plasma free F2-isoP levels, we observed a significant increase with age in the levels of esterified F2-isoPs in both the liver and kidney of the rats fed ad libitum. These data represent the first study, to our knowledge, to measure F2-isoP levels in liver and kidney tissue with either age or CR. The changes in F2-isoP levels with age and CR were parallel to similar changes in the levels of 8-oxodG, an excellent marker of DNA oxidation (16).

Summary
Our data demonstrated that both free and esterified F2-isoP levels increase with age, firmly establishing that, as an organism ages, it is under greater oxidative stress. Our data provide further support for the beneficial effects of CR in protecting cells against oxidative damage through the observation that CR lowered levels of F2-isoPs. These data also demonstrated a reduction in membrane lipid peroxidation in the tissues of the organism and therefore a reduction in membrane damage. In addition, our study clearly showed that F2-isoPs provide investigators with a sensitive, precise, and highly reproducible biomarker for measuring lipid peroxidation in tissues of a rodent as a function of age. Furthermore, levels of free plasma F2-isoPs provide investigators with a measure of the level of oxidative damage to membranes from all sites in the body, thereby giving an integrated picture of the overall oxidative stress status of the organism.


   Acknowledgments
 
This work was supported by a Merit Review grant (HVR, AR), an Environmental Hazards Center grant from the Department of Veteran Affairs (HVR, AR), and by National Institutes of Health grants R01 AG025362-01 (WW), R37 GM42056 (Merit Award to LJR), and R01 AG23843 (AR).


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received December 27, 2004

Accepted March 21, 2005


   REFERENCES
Top
 Abstract
 Methods
 Results
 Discussion
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Q. Ran, H. Liang, Y. Ikeno, W. Qi, T. A. Prolla, L. J. Roberts II, N. Wolf, H. VanRemmen, and A. Richardson
Reduction in Glutathione Peroxidase 4 Increases Life Span Through Increased Sensitivity to Apoptosis
J. Gerontol. A Biol. Sci. Med. Sci., September 1, 2007; 62(9): 932 - 942.
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 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
G. Cambonie, B. Comte, C. Yzydorczyk, T. Ntimbane, N. Germain, N. L. O. Le, P. Pladys, C. Gauthier, I. Lahaie, D. Abran, et al.
Antenatal antioxidant prevents adult hypertension, vascular dysfunction, and microvascular rarefaction associated with in utero exposure to a low-protein diet
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1236 - R1245.
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 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
K. C. Kregel and H. J. Zhang
An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R18 - R36.
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D. Giustarini, I. Dalle-Donne, S. Lorenzini, A. Milzani, and R. Rossi
Age-related influence on thiol, disulfide, and protein-mixed disulfide levels in human plasma.
J. Gerontol. A Biol. Sci. Med. Sci., October 1, 2006; 61(10): 1030 - 1038.
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