This Article Abstract Full Text (PDF) All Versions of this Article: 105/14/1635    most recent 01.CIR.0000014986.29834.71v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van der Loo, B. Articles by Lüscher, T. F. Search for Related Content PubMed PubMed Citation Articles by van der Loo, B. Articles by Lüscher, T. F. Related Collections Animal models of human disease

(Circulation. 2002;105:1635.)
© 2002 American Heart Association, Inc.

Brief Rapid Communications

Cardiovascular Aging Is Associated With Vitamin E Increase

Bernd van der Loo, MD; Ralf Labugger, MSc; Claude P. Aebischer, PhD; Jeremy N. Skepper, PhD; Markus Bachschmid, MSc; Volker Spitzer, PhD; Juliane Kilo, MD; Lukas Altwegg, MD; Volker Ullrich, PhD; Thomas F. Lüscher, MD FRCP

From the Division of Cardiology (B.v.d.L., J.K., L.A., T.F.L.), Cardiovascular Centre, University Hospital, Zurich, and the Division of Cardiovascular Research (R.L., T.F.L.), Institute of Physiology, University Zurich-Irchel, Switzerland; the Department of Vitamin Research (C.-P.A., V.S.), F. Hoffmann-La Roche Ltd, Basel, Switzerland; the Multi-Imaging Centre (J.N.S.), University of Cambridge, Cambridge, UK; and the Department of Biology (M.B., V.U.), University of Konstanz, Germany.

Correspondence to Thomas F. Lüscher, MD, FACC, Cardiology, University Hospital, Raemistrasse 100, CH-8091 Zurich, Switzerland. E-mail cardiotfl{at}gmx.com

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Aging is an independent risk factor for the development of cardiovascular disease. Therefore, therapies to delay vascular aging may have enormous medical consequences. In this context, vitamin E is of particular interest, mainly because of its antioxidative properties.

Methods and Results— In 3-year-old rats, which are not susceptible to atherosclerosis, vitamin E levels, as measured by reversed-phase high-performance liquid chromatography, were markedly increased both in plasma and in major organs (P<0.01 to P<0.0001). The highest increase (at least 70-fold) was found in the aortic wall.

Conclusions— This unexpected accumulation of vitamin E appears to be a compensatory mechanism that attempts to counterbalance age-associated oxidative stress and that may represent a self-regulatory protective adaptation.

Key Words: aging • cardiovascular diseases • vitamin E • antioxidative defense

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Cardiovascular diseases become more frequent with increasing age.¹ Therefore, the aging process itself becomes part of pathophysiological mechanisms.² Recently, we have shown a markedly increased production of superoxide and subsequent formation of peroxynitrite with aging.³ Vitamin E is an important electron source for the reduction of peroxynitrite.⁴ Therefore, its therapeutical use in aging may seem particularly attractive.

Vitamin E is usually supplemented as -tocopherol, which is its most biologically active form. The protective effect of additional vitamin E beyond normal dietary provision remains controversial. The Cambridge Heart Antioxidant Study (CHAOS)⁵ suggested that supplementation with -tocopherol may reduce the risk of cardiovascular death, whereas the much larger Heart Outcomes Prevention Evaluation (HOPE) study⁶ indicated no significant reduction in death from cardiovascular causes in patients with vitamin E supplementation.

Although dietary addition of vitamin E is frequently marketed, little is known in regard to the requirement for vitamin E, especially for older people. Studies in other mammals have presented contradictory results on vitamin E–associated antioxidant capacity with increasing age.^7,8 No studies have been performed, to date, that have estimated the concentration of vitamin E in the arterial vessel wall.

Our aim was to clarify whether tissue levels or plasma levels of vitamin E change with increasing age. To this end, we performed measurements of -tocopherol in aorta, heart, liver, and plasma and of -tocopherol in plasma of young adult, middle-aged, and old healthy male rats. Rats are one of the most suitable models for studies of cardiovascular aging in mammals. Their life span is short, they do not develop atherosclerosis, and nutrition can easily be controlled.

We demonstrate an age-related vitamin E increase, in particular within the aortic vessel wall. This may be considered a counter-regulatory mechanism to prevent oxidative tissue damage.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
F1 (F344xBN) healthy male rats, fed ad libitum, were obtained from the National Institute on Aging, Bethesda, Maryland. Animals belonged to three age groups: 4 to 6 months old ("young"), 19 months old ("middle-aged"), and 32 to 35 months old ("old"). Animals were fed a standardized, sterilized diet (NIH-31, Harlan Teklad Laboratory Diets, Indianapolis, Ind) that contained identical concentrations of vitamin E (45 IU/g) in all age groups.

Surgical Procedures
Rats were anesthetized, and, after medial sternotomy, the aorta was cannulated and blood was taken and centrifuged at 4000 rpm at 4°C for 10 minutes. The aorta, cleaned of adhering tissue, was used for vitamin E determination from the beginning of the renal arteries down to the bifurcation. The heart and the liver were excised in situ. Aortic tissue, heart, liver, and plasma were immediately snap-frozen in liquid nitrogen and stored at -70°C until further use for vitamin E determinations (n=11 for young, n=7 for middle-aged, and n=10 for old rats).

Reversed-Phase High-Performance Liquid Chromatography
Reversed-phase high-performance liquid chromatography was performed as described previously.⁹ Data were acquired via a chromatography server (Fission Instruments) and analyzed by a VAX-based multichannel data acquisition system (Multichrom, VG Data Systems Ltd). The separation was performed on a reversed-phase column (C₁₈) at 30°C, and the tocopherols were detected fluorometrically. To guarantee reproducible results, the method has been monitored with internal control samples and by external quality assurance programs (National Institute of Standards and Technology, Gaithersburg, Md).

Electron Microscopy
From 2 additional young and 2 additional old animals, the aorta was isolated, fixed, and embedded for electron microscopy as described previously.³ Thin sections were viewed with a CM100 transmission electron microscope (Philips). Intimal thickness was defined as the distance from the luminal surface of the endothelium to the surface of the first elastic lamellae.

Measurement of Superoxide
·O₂^- concentrations in aortic tissue were determined with a lucigenin-enhanced chemiluminescence method as described previously.³

Calculations and Statistical Analysis
Results were analyzed by ANOVA followed by Bonferroni’s and Dunn’s correction. Data are presented as mean±SEM. Means were considered significantly different at P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
In the plasma of old animals, -tocopherol was increased 2-fold compared with young rats (Figure 1A). Plasma levels of -tocopherol were, given a detection limit of 0.02 mg/L, at least 10-fold higher in old rats (Figure 1B). Because -tocopherol was undetectable in the plasma of young animals, we did not investigate the concentration of tissue -tocopherol. We refrained from adjusting vitamin E plasma levels to cholesterol because vitamin E concentrations in plasma generally do not correlate with tissue levels.

View larger version (16K): [in this window] [in a new window]   Figure 1. Levels of -tocopherol (A) and of -tocopherol (B) in plasma of young, middle-aged, and old animals as measured by reversed-phase high-performance liquid chromatography. Values represent the mean±SEM. The limit of detection is defined as the x-fold signal of the baseline signal in which the standard deviation is still <7% of the mean signal. A, Young vs old, P<0.0001; middle-aged vs old, NS; and young vs middle-aged, P<0.001. B, Young vs old, P<0.0001; and young vs middle-aged and middle-aged vs old, NS.

In the liver, which is considered to be a major site of storage for tocopherols, there was also a significant, 2-fold increase of -tocopherol in parallel to the plasma levels in old rats as compared with young rats. The concentration of -tocopherol in plasma and liver of middle-aged rats was in between that of young and old animals (Figures 1A and 2A). In the heart, levels of -tocopherol were nearly 3-fold increased in old animals as compared with young and middle-aged animals (Figure 2B).

View larger version (32K): [in this window] [in a new window]   Figure 2. Levels of -tocopherol in liver (A), heart (B), and aortic vessel wall (C). For heart and liver, values represent the mean±SEM. For determination of -tocopherol in the aortic vessel wall, aortic segments of corresponding anatomical sites from each animal were pooled for each age group according to weight. Each pool contained 1 g (young animals), 0.8 g (middle-aged animals), and 1.5 g (old animals) of aortic tissue. D, Superoxide production in the aortic vessel wall (n=6 for young, middle-aged, and old animals). Reproduced in part from J Exp Med (2000;182:1731–1743) by copyright permission of The Rockefeller University Press. A, Young vs old, P<0.0001; middle-aged vs old, P<0.01; and young vs middle-aged, NS. B, Young vs old and middle-aged vs old, P<0.01; and young vs middle-aged, NS. D, Young vs old and middle-aged vs old, P<0.0001.

The most astonishing finding was that -tocopherol in the aorta was extremely high (at least 70 times greater) in old animals as compared with young animals in which it was below detection limits (Figure 2C). The aortic wall of old rats contained one-third of the concentration of -tocopherol found in the liver, a fact that underlines the magnitude of -tocopherol levels in the aorta of old animals. This marked increase in -tocopherol was paralleled by a significant increase of ·O₂^--generated chemiluminescence (Figure 2D). Although aortic -tocopherol levels in middle-aged animals were comparably high to those in old animals, oxidative stress was significantly lower (Figure 2, C and D).

Electron microscopy revealed no overt signs of atherosclerosis, such as accumulation of foam cells, smooth muscle cells, or extracellular lipid. Old aortas exhibited a marked increase in intimal thickness, which increased from 2.5±0.9 µm in young aorta (mean of 50 different measurements) to 8.7±3.4 µm in the old aorta (P<0.001; data not shown). This change is similar to that seen in human arteries in which an age-related increase in the intima-media thickness has been reported in the femoral arteries of old but healthy humans.¹⁰ The steep increase of vitamin E content occurs independently of the development of atherosclerosis (although the increase in intimal thickness may possibly be an early precursor)¹¹ and can be considered as primarily dependent on aging.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
With the same model of aging, we have previously shown that vascular aging is closely associated with augmented ·O₂^- generation and peroxynitrite formation that, in turn, exhibits new intracellular messenger functions.³ Vitamin E may reduce oxidative stress by directly scavenging reactive oxygen species.¹² Therefore, our discovery of a specific tissue distribution and, in particular, of a dramatic increase in -tocopherol in the aortic vessel wall of old animals is likely to have distinct pathophysiological significance.

On the basis of these data, we postulate the existence of a compensatory antioxidant defense system to counteract oxidative stress–associated vascular aging. It is reasonable to assume that absorption of vitamin E can increase with age to reduce oxidative damage to vascular tissue. It would be expected to accumulate at the sites in which it is needed most to protect against the development of age-associated changes, such as endothelial dysfunction. Because mammals cannot synthesize vitamin E, intestinal absorption and subsequent transport determines how much of the ingested vitamin enters the circulation. An age-associated increase in the total amount of vitamin E absorbed in the intestine of old rats has already been described¹³ and would support the concept of a self-protective age-dependent adaptation. Under physiological conditions, the metabolism of vitamin E is incompletely understood to date.¹⁴ Therefore, we do not feel able to speculate on a possible decreased catabolism in aging.

Controlled upregulation, dependent on demand, may be determined by age-associated increases in oxidative tissue damage.³ Our concept is further strengthened by the facts that -tocopherol was particularly increased in the vasculature in which oxidant stress was high and is apparently still able to reduce oxidative radicals in the aorta of middle-aged animals, whereas in the extreme age group, it cannot.

In vitro experiments¹⁵ have shown that the -tocopheroxyl radical may exhibit pro-oxidant activities. However, the analytical method we used does not allow the quantification of -tocopherol radicals. It seems unlikely, albeit possible, that vitamin E itself may be involved in the cardiovascular aging process. The pathophysiological relevance of vitamin E as a pro-oxidant under in vivo conditions remains to be determined.¹⁴

The interpretation of vitamin E plasma levels is possibly limited by the fact that they are not adjusted to plasma cholesterol. However, the main body of evidence (-tocopherol in major organs) we present remains unaffected by this.

In conclusion, it seems implicit that mammals are capable of regulating age-related, increased demand for vitamin E. Our results suggest that sufficiently high levels of vitamin E may be built up from a normally balanced diet to counter-balance oxidative stress-associated vascular aging. This counter-regulation eventually remains unsuccessful because, in old age, oxidative damage occurs although the antioxidant defense is dramatically "switched on."

The relevance of our findings to aging in humans is not known. Corresponding studies in humans remain to be performed.

	Acknowledgments

 
This work was supported by grants from the Swiss National Research Foundation; from Aetas, Geneva, Switzerland; and from the Swiss Heart Foundation. The University of Konstanz was supported by the Deutsche Forschungsgemeinschaft. The Multi-Imaging Center was supported by funding from the Wellcome Trust.

Received December 17, 2001; revision received February 7, 2002; accepted February 7, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Lüscher TF, Noll G. The endothelium in coronary vascular control. Heart Dis. 1995; 3: 1–10.
   
2. Lakatta EG. Heart aging. Circ Res. 2001; 88: 984–986.
   
3. van der Loo B, Labugger R, Skepper JN, et al. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med. 2000; 192: 1731–1743.
   
4. Lee J, Hunt JA, Groves JT. Manganese porphyrins as redox-coupled peroxynitrite reductases. J Am Chem Soc. 1998; 120: 6053–6061.
   
5. Stephens NG, Parsons A, Schofield PM, et al. Randomized controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet. 1996; 347: 781–786.
   
6. Yusuf S, Dagenais G, Pogue J, et al. Vitamin E supplementation and cardiovascular events in high risk patients: the Heart Outcomes Prevention Study Investigators. N Engl J Med. 2000; 342: 154–160.
   
7. Matsuo M, Gomi F, Dooley MM. Age-related alterations in antioxidant capacity and lipid peroxidation in brain, liver, and lung homogenates of normal and vitamin E-deficient rats. Mech Aging Dev. 1992; 64: 273–292.
   
8. De AK, Darad R. Age-associated changes in antioxidants and antioxidative enzymes in rats. Mech Aging Dev. 1991; 59: 123–128.
   
9. Aebischer CP, Schierle J, Schuep W. Simultaneous determination of retinol, tocopherols, carotene, lycopene, and xanthophylls in plasma by means of reversed-phase high-performance liquid chromatography. Methods Enzymol. 1999; 299: 348–362.
   
10. Kornet L, Hoeks AP, Lambregts J, et al. In the femoral artery, differences in mean wall shear stress within subjects are associated with different intima-media thicknesses. Arterioscler Thromb Vasc Biol. 1999; 19: 2933–2940.
    
11. Bots ML, Hofman A, Grobbee DE. Increased common carotid intima-media thickness: adaptive response or a reflection of atherosclerosis? Findings from the Rotterdam Study. Stroke. 1997; 28: 2442–2447.
    
12. Carr AC, Zhu BZ, Frei B. Potential antiatherogenic mechanisms of ascorbate (vitamin C) and alpha-tocopherol (vitamin E). Circ Res. 2000; 87: 349–354.
    
13. Hollander D, Dadufalza V. Lymphatic and portal absorption of vitamin E in aging rats. Dig Dis Sci. 1989; 34: 768–772.
    
14. Brigelius-Flohe R, Traber MG. Vitamin E: function and metabolism. FASEB J. 1999; 13: 1145–1155.
    
15. Upston JM, Terentis AC, Stocker R. Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J. 1999; 13: 977–994.
    

This article has been cited by other articles:

				J. J. Ochoa, J. L. Quiles, M. Lopez-Frias, J. R. Huertas, and J. Mataix Effect of Lifelong Coenzyme Q10 Supplementation on Age-Related Oxidative Stress and Mitochondrial Function in Liver and Skeletal Muscle of Rats Fed on a Polyunsaturated Fatty Acid (PUFA)-Rich Diet J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2007; 62(11): 1211 - 1218. [Abstract] [Full Text] [PDF]

				J. L. Quiles, J. J. Ochoa, M. C. Ramirez-Tortosa, J. R. Huertas, and J. Mataix Age-related mitochondrial DNA deletion in rat liver depends on dietary fat unsaturation. J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2006; 61(2): 107 - 114. [Abstract] [Full Text] [PDF]

				J. J. Ochoa, J. L. Quiles, J. R. Huertas, and J. Mataix Coenzyme Q10 Protects From Aging-Related Oxidative Stress and Improves Mitochondrial Function in Heart of Rats Fed a Polyunsaturated Fatty Acid (PUFA)-Rich Diet J. Gerontol. A Biol. Sci. Med. Sci., August 1, 2005; 60(8): 970 - 975. [Abstract] [Full Text] [PDF]

				F. VIOLI, R. CANGEMI, G. SABATINO, and P. PIGNATELLI Vitamin E for the Treatment of Cardiovascular Disease: Is There a Future? Ann. N.Y. Acad. Sci., December 1, 2004; 1031(1): 292 - 304. [Abstract] [Full Text] [PDF]

				J. C. Sullivan, E. D. Loomis, M. Collins, J. D. Imig, E. W. Inscho, and J. S. Pollock Age-related alterations in NOS and oxidative stress in mesenteric arteries from male and female rats J Appl Physiol, October 1, 2004; 97(4): 1268 - 1274. [Abstract] [Full Text] [PDF]

				F Violi, L Loffredo, L Musella, and A Marcoccia Should antioxidant status be considered in interventional trials with antioxidants? Heart, June 1, 2004; 90(6): 598 - 602. [Abstract] [Full Text] [PDF]

				F. Micheletta, S. Natoli, M. Misuraca, E. Sbarigia, U. Diczfalusy, and L. Iuliano Vitamin E Supplementation in Patients With Carotid Atherosclerosis: Reversal of Altered Oxidative Stress Status in Plasma but not in Plaque Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 136 - 140. [Abstract] [Full Text] [PDF]

				F. Violi, F. Micheletta, L. Iuliano, D. Steinberg, and J. L. Witztum How to Select Patient Candidates for Antioxidant Treatment? * Response Circulation, December 10, 2002; 106 (24): e195 - e195. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 105/14/1635    most recent 01.CIR.0000014986.29834.71v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van der Loo, B. Articles by Lüscher, T. F. Search for Related Content PubMed PubMed Citation Articles by van der Loo, B. Articles by Lüscher, T. F. Related Collections Animal models of human disease