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Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Spain.
Address correspondence to Dr. Julio J. Ochoa, Institute of Nutrition and Food Technology, C/Ramón y Cajal, 4, 18071 Granada, Spain. E-mail: jjoh{at}ugr.es
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Abstract |
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-tocopherol, coenzyme Q, catalase and glutathione peroxidase activities, and cytochromes a+a3, b, c+c1 and cytochrome c oxidase activity were measured. Coenzyme Q10-supplemented animals showed lower hydroperoxide levels; higher content and/or activity of -tocopherol, coenzyme Q, and catalase; and a slightly lower decrease in mitochondrial function. According to that, previously reported positive effects of coenzyme Q supplementation on the life span of rats fed a PUFA-rich diet might be a consequence, at least in part, of a lower oxidative stress level and perhaps, to a minor extent, of a smaller decrease in mitochondrial function.
Heart is a largely postmitotic tissue and one of the organs in which the potential effects of oxidative damage are most readily detectable due to its high dependence on oxidative phosphorylation to derive energy (5). In this sense, there is a link between age-associated changes in heart and clinical cardiac diseases, which are major causes of death in industrialized societies (6–8).
The relationship between dietary polyunsaturated fatty acids (PUFAs) and cardiac diseases has been widely recognized, showing different beneficial effects besides improving the lipid profile. For example, myocardial vulnerability to arrhythmia has been showed to be influenced by dietary fat with a potential benefit of dietary modification to include higher proportion of PUFA in reducing the risk of sudden cardiac death (6,9,10). The major problem of these fatty acids is that they are highly susceptible to the reactive oxygen species attack (11), subsequently increasing the oxidative damage in the organism, especially during aging (12,13). However, supplementation with antioxidants would preserve the advantages of PUFA on health while preventing their deleterious aspects.
The mitochondrial component coenzyme Q10 (CoQ10) or ubiquinone has been used for many years as a dietary supplement intended to promote good health by trapping free radicals. Interest in ubiquinone comes from the fact that it has a pivotal role as a redox link between flavoproteins and cytochromes in the mitochondrial respiratory chain, where it also has important antioxidant properties under lipophilic conditions (14,15). Previous studies (5,14,16,17) have suggested the possible beneficial role of coenzyme Q in aging. In addition, this molecule is commonly used for treating cardiomyopathy, and there is substantial evidence that heart function improves after ubiquinone administration (15). However, no studies have explored the combination of CoQ10 and PUFA-rich diet on oxidative damage of rat heart during aging. According to the above-mentioned premise, the aim of the present study was to investigate, for the first time, changes during aging in lipid peroxidation and functionality of heart mitochondria, depending on supplementation or not with CoQ10, in rats fed a lifelong diet rich in PUFAs.
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METHODS |
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Chromatography was performed using a 60-meter-long capillary column (32 mm inside diameter, 20 mm thickness) impregnated with Sp 2330 FS (Supelco, Inc., Bellefonte, PA). The injector and the detector were maintained at 250°C and 275°C, respectively; nitrogen was used as the carrier gas, and the split ratio was 29:1. Temperature programming (for a total time of 40 minutes) was as follows: initial temperature, 160°C for 5 minutes, 6°C/minute to 195°C, 4°C/minute to 220°C, 2°C/minute to 230°C, hold 12 minutes, 14°C/minute to 160°C.
The mitochondrial membrane hydroperoxide content was determined following the procedure of Jiang and colleagues (21). Tert-butyl hydroperoxide was used to construct a standard curve. When the rats were 24-months old, a second hydroperoxide determination was performed after in vitro oxidation of the samples using 2.5 mM of AAPH (2,2'-zobis (2-amidinopropane) hydrochloride), a potent generator of free radicals.
CoQ10, coenzyme Q9 (CoQ9), and -tocopherol in mitochondrial membrane were assayed by high-performance liquid chromatography, after extraction with ethanol/petroleum ether (60:40) using the method of Kröger (22). The high-performance liquid chromatography system consisted of an apparatus equipped with a diode-array detector (model 168; Beckman Instruments, Inc., Fullerton, CA), and the column was a reverse-phase C18 Spherisorb ODS 1 (25 x 0.46 cm). Coenzymes and -tocopherol were identified by predetermining the retention times of individual standards.
Catalase activity was determined following the method described by Aebi (23), based on monitoring at 240 nm the H2O2 decomposition, as a consequence of the catalytic activity of catalase. The activity was calculated from the first-order rate constant K (s–1). For glutathione peroxidase, the technique of Flohé and Wolfgang (24) was used. Finally, the concentration of mitochondrial cytochromes b, c+c1, and a+a3 was evaluated by measuring the reduced minus oxidized difference spectra with a 
16-Perkin Elmer double-beam spectrophotometer, as previously described (25). Cytochrome c oxidase (CCO) activity was assayed at 25°C using cytochrome c reduced by sodium dithionite, monitoring the absorbance decrease of cytochrome c upon oxidation at 417–409 nm every 10 seconds for 2 minutes using an extinction coefficient for cytochrome c of 40.7 mM–1 cm–1, as previously described (25).
Statistical Analysis
Results are presented as means ± standard error of the mean (SEM) (n = 20 rats). One-way analysis of variance was used to test time-dependent changes, and the Bonferroni test was performed post hoc to evaluate differences among groups. Differences between dietary treatments for a same period of time were evaluated by the Student t test. A p value less than 0.05 was considered significant. Data were analyzed using SPSS statistical software (SPSS for Windows, version 11.0.1; SPSS, Inc., Chicago, IL).
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RESULTS |
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-Tocopherol, CoQ9, and CoQ10-tocopherol than at 6 and 12 months (Table 3) and only the PUFA+CoQ10 group showed higher values at 12 months than at 6 months. Animals supplemented with CoQ10 showed higher values than did the nonsupplemented group at 6 and 12 months. CoQ9 (Table 3) was higher in supplemented animals at 12 and 24 months. Regarding the aging effect, the PUFA group showed lesser values at 12 months compared to 6 months, and higher values at 24 months compared to 12 months. The PUFA+CoQ10 group showed only higher values at 24 months than at 6 months. CoQ10 (Table 3) was higher in the supplemented rats at 12 and 24 months. The aging effect in the PUFA group was observed only at 24 months, registering higher values than those at 6 and 12 months. For the supplemented group, higher values were found at 12 months compared to 6 months, and at 24 months compared to 6 and 12 months.
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CCO Activity
CCO activity is show in Figure 4. In both groups, the activity of this enzyme showed a gradual decrease with age, with lesser values at 12 months than at 6 months and at 24 months than at 6 and 12 months. For all study periods, the CoQ10-supplemented animals showed higher values (p <.05) at 6, 12, and 24 months, respectively, than did their nonsupplemented counterparts.
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DISCUSSION |
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Prior to the analysis of possible effects of CoQ10 supplementation, it was necessary to assess adaptation of the rats to such supplementation. In the present study, we found that supplementation led to higher CoQ10 levels at 12 and 24 months of age in heart mitochondria. Considering that, besides plasma, liver, and spleen, most of the tissues are resistant to increased amounts of coenzyme Q from exogenous sources (26), we can state that our lifelong supplementation schedule based on a low CoQ10 concentration (0.7 mg/kg/day) led to a good adaptation pattern.
The analysis of hydroperoxide levels as an indicator of lipid peroxidation reveals that, regardless of the dietary manipulation, cardiac mitochondrial lipid peroxidation was higher at 12 and 24 months than at 6 months of life. This result agrees with previously reported data (12,27,28), and is consistent with the free radical theory of aging of Harman (1). However, there were important differences between experimental groups with respect to this parameter. Animals fed a PUFA-rich diet and supplemented throughout life with CoQ10 (0.7 mg/kg/day) showed lower values for all the periods studied. In addition, although both groups reached the highest hydroperoxide value at 12 months, the nonsupplemented animals maintained this value until 24 months of age, whereas the supplemented animals were able to decrease it significantly. Thus, at 24 months of age, the supplemented animals reached a hydroperoxide value close to that of nonsupplemented animals at 6 months of life.
There are different ways through which CoQ10 could be able to cause these differences in hydroperoxide levels, such as changes in the mitochondrial fatty-acid profile and therefore its susceptibility to lipid peroxidation, increasing antioxidant defenses or acting on the free radicals sources. We have studied, partially, all these ways.
It has been shown that modifications of the mitochondrial fatty-acid profile can modulate the susceptibility of the mitochondrial membranes to lipid peroxidation during aging (12). In this sense, administration of CoQ10 has been shown capable of modifying the phospholipid fatty-acid composition in monocytes and granulocytes (15). However, under our experimental conditions, the study of the mitochondrial fatty-acid profile shows that lifelong CoQ10 supplementation was not able to modify this parameter significantly. Both experimental groups registered higher levels of saturated fatty acids associated with age and lower monounsaturated fatty acids. This result agrees with previous data reported by our research group (12) for heart in aged rats fed a PUFA-rich diet.
The enzymatic and nonenzymatic components of the antioxidant system studied followed a similar response in both groups with higher levels related to age (except coenzyme Q levels in nonsupplemented animals). Previously, increased cardiac antioxidant levels associated with age have been reported (12,29,30). It has been postulated that a compensatory antioxidant defense system exists to counteract oxidative stress associated with aging, and therefore some antioxidants would be expected to accumulate, such as -tocopherol, at the sites in which it is needed (30).
Despite the similar pattern followed by both experimental groups with respect to the antioxidant system, there were important differences between them. For the nonenzymatic antioxidants, -tocopherol levels were higher, at least at 6 and 12 months of age, in the supplemented animals, and this group also showed a gradual increase associated with age in coenzyme Q content (both CoQ9 and CoQ10), whereas the nonsupplemented group showed no age-associated changes in coenzyme Q content. Results from the present study agree with those of other groups. For example, Kwong and colleagues (26) reported that supplementation with CoQ10 (150 mg/kg/day) for 13 weeks increased the level of this coenzyme and its homologue, CoQ9. Also, Lass and colleagues (31), working with mice, demonstrated that a similar supplementation (123 mg/kg/day) resulted in a concomitant increase in the level of -tocopherol in heart mitochondria. It is of interest to note that, although our study did not use high CoQ10 dosages, as other studies (26,31), we found the mentioned increase in -tocopherol and CoQ9 concentration. The aforementioned suggest that both acute supplementation of high dosages and chronic administration of low dosages of oQ10 are able to induce levels of -tocopherol and CoQ9.
It is noteworthy that the higher levels of -tocopherol shown in nonsupplemented 24-month-old animals were not accompanied by a similar pattern in the coenzyme Q contents, and several results indicate that these two lipid-soluble antioxidants are in fact more efficient when acting together (14). Thus, it seems to indicate that old mitochondrial membrane from supplemented animals could be more prepared against oxidative injury than is that from nonsupplemented animals. To test this hypothesis, we have induced in vitro oxidative stress against these old membranes using the potent free radical generator AAPH. The results indicate that, despite the similar PUFA content of these membranes (and hence a theoretically similar susceptibility to oxidative damage), mitochondrial membranes from old supplemented animals registered lower AAPH-induced hydroperoxide values and therefore proved to be more resistant against oxidative damage. This result is important because one of the problems with aged cardiac mitochondria is that they are more susceptible to oxidative damage and therefore predispose the heart to greater injury during oxidative insults (3).
With respect to enzymatic antioxidants, both showed the highest activities at 24 months, which we believe is due to an increase in free radical generation at this age, because it has been suggested to be the most important factor for the induction of the activities of these enzymatic antioxidants (2). Increased catalase activity has been associated with greater resistance to oxidative damage (2), and it has been suggested that catalase can function as a major pathway for detoxifying H2O2 in cardiac tissue (32). With our data alone, it is difficult to ascertain the mechanisms by which CoQ10 supplementation can increase the activity of this antioxidant enzyme, and thus this issue needs further study. Nevertheless, it has been showed that CoQ10 administration was able to increase in mice heart the gene expression of glutathione S-transferase, another element of the antioxidant system family (5). We can not rule out that a similar interaction could exist between CoQ10 and catalase.
The third possible way by which coenzyme Q could modulate lipid peroxidation levels is by acting on the free radical source, the mitochondrial electron transport chain (METC). Aging has been associated with decay in mitochondrial respiratory chain activity (28) and an increased rate of mitochondrial O2.–/H2O2 generation (33). In addition, it has been observed that CCO activity decreases with age (16), and this decrease has been correlated with the concomitant increase in the flux of mitochondrial 
and H2O2 generation (33). Thus, it has been suggested that there could be an obstruction or partial blockage of electron flow through some respiratory complexes associated with age; therefore, a greater number of free radicals could be generated at these sites along the METC (12,33). Results related to cytochrome content and CCO activity obtained in our study agree with the aforementioned. We have detected higher levels of cytochrome (at least in cytochrome b and c+c1) associated with age in parallel to lower CCO activity. As has been suggested, this situation could give rise to a possible partial blockage of electron flow and therefore higher free radical production. In fact, we have found a good inverse correlation (r = –561; p <.01 for nonsupplemented animals and r = –0.399; p <.01 for supplemented animals) between hydroperoxide content and CCO activity. Thus, taking together the fact that the antioxidant enzymes activity increase at 24 months compared with 12 months and the fact that CCO activity was the lowest at the oldest age, despite of the similar hydroperoxides values between the two ages, we can suggest that at 24 months of life the generation of free radicals was higher than at 12 months and therefore the oxidative insult.
In contrast, CoQ10 supplementation appears to improve these age-associated alterations or changes in the METC. Besides a slight effect on cytochromes content, mostly at 12 months of life, CoQ10 supplementation can increase CCO activity, with respect to that in nonsupplemented animals, throughout life. In addition, CoQ10 supplementation led to a higher content of this molecule and its homologue (CoQ9) at the mitochondrial membrane level (nonsupplemented animals did not show this effect). This should be important for the METC function, because, as has been widely reported, under physiological conditions, the mitochondrial concentration of coenzyme Q does not saturate the enzymes that use it as a substrate (34). It should also be noted that CoQ10 supplementation could modulate the CCO activity either by its effects on lipid peroxidation (allowing a better environment for this activity), and/or by its effect on the inducement of several genes encoding some subunits of this METC complex (5).
In conclusion, on the basis of these results we can suggest that previously reported positive effects of CoQ10 supplementation on mean and maximal life span of rats fed a PUFA-rich diet might be a consequence, at least in part, of a lower oxidative stress level and perhaps, to a minor extent, to a smaller decrease in mitochondrial function. In addition, these results could lead to a better understanding of the beneficial effects on heart function after administration of CoQ10, although investigation of different diets and further studies in humans are still needed to elucidate this protective role of CoQ10.
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Acknowledgments |
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Footnotes |
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Received December 29, 2004
Accepted March 21, 2005
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References |
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-lipoic acid, coenzyme Q10, and caloric restriction on life span and gene expression patterns in mice. Free Radic Biol Med. 2004;27:273-277.
-tocopherol administration on their tissue levels in the mouse: elevation of mitochondrial a-tocopherol by coenzyme Q10. Free Radic Biol Med. 1999;26:1375-1382.[Medline]
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