SEARCH

SEARCH BY CITATION

Keywords:

  • Docosahexaenoic acid;
  • Arachidonic acid;
  • Lipid peroxide;
  • Reactive oxygen species;
  • Antioxidative enzymes;
  • Rat brain

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Abstract : Female Wistar rats (100 weeks old) were divided into two groups ; one group was fed a high-cholesterol diet (HC) and the other a high-cholesterol diet plus docosahexaenoic acid (HC-fed DHA rats). Fatty acid concentrations in brain tissues were analyzed by gas chromatography. In the HC-fed DHA rats, brain catalase (CAT), GSH, and glutathione peroxidase (GPx) increased in the cerebrum but not in the brainstem or cerebellum. The rate of increase was 23.0% for CAT, 24.5% for GSH, and 26.3% for GPx compared with that in the HC animals (p < 0.05). In the cerebrum of the HC-fed DHA rats, CAT and GPx increased, with an increase in the ratio of DHA to arachidonic acid. The cerebrum, unlike the other areas of the brain, seems to be more sensitive to DHA in stimulating CAT and GPx. We suggest that DHA plays an important role in inducing an antioxidative defense against active oxygen by enhancing the cerebral activities of CAT, GPx, and GSH.

An increase in docosahexaenoic acid (DHA) level in the brain has been anticipated to have deleterious effects by enhancing lipid peroxidation (Eddy and Harman, 1977), although the regions of the brain where such deleterious effects are exerted remain obscure. The hypothesis that reactive free radicals and lipid peroxides (LPOs) play a pivotal role in the pathogenesis of atherosclerotic disease and ischemic or traumatic brain injuries has been reinforced (Braughler and Hall, 1989). Our observation that DHA in the brain in certain circumstances might act as an antioxidant (Hossain et al., 1998) is of special significance because of the brain's intrinsic potential for free radical generation. The increased presence of polyunsaturated fatty acids in cell membranes renders cells more susceptible to damage by lipid peroxidation (Saito and Nakatsugawa, 1994) ; but the brain is poor in antioxidative enzymes such as catalase (CAT), superoxide dismutase, and glutathione peroxidase (GPx) (Mizuno and Ohta, 1986 ; Cohen, 1988), and the level of antioxidant substances such as GSH is also relatively low (Sinet et al., 1980). The CNS is highly susceptible to oxidative insult (Halliwell, 1989). Therefore, the damaging effects are dependent obviously on the balance between cellular peroxidation processes and the antioxidative defenses.

There has been very little quantitative information regarding the interaction of DHA and the concentrations of reactive oxygen species (ROS), antioxidative enzymes, and antioxidants in different regions of brains of aged rats, with special reference to hypercholesterolemia. In general, increased levels of LPOs (Ando et al., 1990) and cholesterol in brain tissue are encountered with aging (Kelly et al., 1995). Enhancement in vulnerability to oxidative stress seen with aging is in part the result of higher levels of cholesterol (Joseph et al., 1996 ; Urano et al., 1998). Cholesterol-induced brain tissue damage has been directly proved in in vitro isolated brain synaptosomes (Vatassery et al., 1997).

A recent study shows that DHA in aged and hypercholesterolemic rats decreases cerebral LPOs (Hossain et al., 1998). To explain the role of DHA in protecting the brain from oxidation, we examined the effects of DHA on the levels of ROS and GSH, and on the activities of GPx and CAT in the brains of aged and hypercholesterolemic rats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Materials

All standard fatty acids and 2′,7′-dichlorofluorescein were obtained from Sigma (St. Louis, MO, U.S.A.) and 2′,7′-dichlorofluorescin diacetate was from Molecular Probes (Eugene, OR, U.S.A.). All other chemicals were of analytical grade.

Animals and diets

Female Wistar rats (100 weeks old) were randomly divided into two groups. One group was fed a high cholesterol (HC) rat chow pellet diet (a semisynthetic pellet diet without fish oil ; Funabashi Farm Co. Ltd., Chiba, Japan) ; the other group (HC + DHA) was fed the same HC pellet diet supplemented orally with an ethyl ester derivative of all cis-4,7,10,13,16,19-DHA (DHA-95E) (antioxidant vitamin C or E unenriched ; purity > 95% ; Harima Chemicals, Inc., Tokyo, Japan). DHA-95E was gently emulsified in 5% gum arabic solution, as a vehicle, in ice-cold water with an ultrasonic cell homogenizer (Taitec VP-5, Taitec Co. Ltd., Tokyo, Japan) before administration. The HC group was administered an equal volume of the above 5% gum arabic solution without DHA-95E. Both the HC pellet feeding and subsequent DHA supplementation were performed for 12 weeks. Food consumption was monitored throughout the experimental period. The basal HC diet by weight contained 6.0% water, 21.3% protein, 5.1% fat, 3.1% fiber, 5.0% carbohydrate, 57.7% nonnitrogen, 1% cholesterol, 1% cholic acid, and total energy kilocalories per gram 4.2. The fatty acid profile of the fat used in the diet is given in Table 1.

Table 1. Fatty acid composition (mol%) of the basal HC pelletResults are mean ± SE values of quadruplicate determinations. ND, not detected.
Myristic acid, C14:00.09 ± 0.09
Palmitic acid, C16:015.62 ± 0.530
Palmitoleic acid, C16:1 (n-7)ND
Stearic acid, C18:05.41 ± 0.09
Oleic acid, C18:1 (n-9)21.13 ± 0.17
Linoleic acid, C18:2 (n-6)52.43 ± 0.80
Linolenic acid, C18:3 (n-3)4.49 ± 0.13
Arachidic acid, C20:00.14 ± 0.09
Eicosenoic acid, C20:1 (n-9)0.33 ± 0.13
Arachidonic acid, C20:4 (n-6)ND
Eicosapentaenoic acid C20:5 (n-3)0.06 ± 0.06
Docosapentaenoic acid, C22:5 (n-3)ND
Docosahexaenoic acid, C22:6 (n-3)ND
Lignoceric acid, C24:0 (n-3)0.11 ± 0.07

Tissue preparation

After sodium pentobarbital (65 mg/kg of body weight) anesthesia intraperitoneally, the rats were killed in accordance with the procedures outlined in the Guidelines for Animal Experimentation of Shimane Medical University. The brainstem, cerebellum, and cerebrum were separated from the whole brain on ice. Tissues were weighed and homogenized in icecold 0.32 M sucrose buffer (pH 7.4) with a Polytron homogenizer (PCU 2-110, Kinematica GmbH, Steinhofhale, Switzerland). The homogenates were adjusted to a final concentration of 100 mg of tissue/ml of buffer and immediately subjected to the assays described below.

Sample preparation and enzyme assays

Fractionations of brain homogenates were performed as previously described by Roy et al. (1984). After centrifuging the ice-cold homogenates at 1,000 g for 5 min at 4°C to discard unruptured tissues and other cellular debris, the supernatant was used for CAT activity. The remaining supernatant was further centrifuged at 12,500 g for 30 min at 4°C. The resultant supernatant containing microsome and cytosol was used for GPx activity assay.

The activity of CAT (EC 1.11.1.6) was measured by exploiting its peroxidation function at 20°C according to the procedures of Johansson and Borg (1988) and Wheeler et al. (1990). In brief, 300 μl of 0.25 M phosphate buffer (pH 7.0), 60 μl of 44 mM H2O2, 300 μl of 6 M methanol, and 600 μl of diluted sample were mixed and incubated at 20°C for 20 min. The reaction was terminated by the addition of 50 μl of 7.8 M KOH, followed by a rapid addition of 900 μl of 22.8 mM Purpald in 0.48 M HCl. After vortex mixing, the incubation mixtures were left for 20 min at 20°C. Then 300 μl of 0.0652 M potassium periodate in 0.5 M KOH was added ; again after brief vortex mixing, the absorbance of the purple formaldehyde adduct produced was measured at 550 nm. CAT activity was determined by linear least-squares regression of the absorbance of formaldehyde standards.

GPx (EC 1.11.1.0) was assayed according to the method of Paglia and Valentine (1967) with some modifications. Each 3.0-ml assay volume contained 0.2 ml of sample, 0.05 M phosphate buffer (pH 7.0) containing 5 mM EDTA, 0.14 mM NADPH, 3.0 mM sodium azide, 5.0 mM GSH, 1 U glutathione reductase, and 100 μM H2O2. Changes in absorbance were recorded at 340 nm for 5 min with a Hitachi U-3210 spectrophotometer, and the enzyme activity was calculated as micromoles of NADPH oxidized per minute.

GSH assay

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

An appropriate volume of homogenate of the brainstem, cerebellum, or cerebrum region was quickly mixed with perchloric acid (final concentration, 2% vol/vol) containing 1 mM EDTA and centrifuged at 2,000 g for 10 min at 4°C. The protein-free supernatant was neutralized with equimolar potassium bicarbonate (Huang and Philbert, 1995). The concentration of GSH was measured by the nonenzymatic method of Akerboom and Seis (1981).

ROS assay

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

The basal level of ROS was determined by the procedure of Montoliu et al. (1994). An appropriate volume of freshly prepared tissue homogenate was diluted in 100 mM potassium phosphate buffer (pH 7.4) and incubated with a final concentration of 5 μM dichlorofluorescin diacetate in methanol for 15 min at 37°C. The dye-loaded samples were centrifuged at 12,500 g for 10 min at 4°C. The pellet was vortex mixed at ice-cold temperatures in 5 ml of 100 mM phosphate buffer (pH 7.4) and again incubated for 60 min at 37°C. The fluorescence measurements were performed with a Hitachi 850 spectrofluorometer at 488 nm for excitation and 525 nm for emission wavelengths. The cuvette holder was maintained at 37°C. ROS were quantified from the dichlorofluorescein standard curve in methanol (0-100 nM).

Other determinations

Fatty acid composition was determined by the one-step analysis of Lepage and Roy (1986) as previously described (Hashimoto et al., 1998) by using gas chromatography. Protein concentration was estimated by the method of Lowry et al. (1951). The level of LPOs in brain tissues was estimated with an LPO assay kit (Wako Pure Chemical Industries Ltd., Osaka, Japan) by measuring the thiobarbituric acid-reactive substances.

On completion of the study, the levels of polyunsaturated fatty acids and LPOs in brain tissues were reported by Hossain et al. (1998 ; see Table 2).

Table 2. Effect of DHA on LPOs (nanomoles per milligram of protein) in different regions of the brain of HC-fed and HC + DHA groupsaResults are mean ± SEM values (n = 10).
 HCHC + DHA
  1. aThis table is reproduced from Hossain et al. (1998) with the permission of Elsevier Science.

  2. bp < 0.01,

  3. cp < 0.001,

  4. dp < 0.05 vs. cerebrum of the HC group.

Brainstem0.231 ± 0.053 c0.312 ± 0.040
Cerebellum0.305 ± 0.024 cb0.299 ± 0.030
Cerebrum0.539 ± 0.040 bc0.389 ± 0.049d

Statistical analysis

Results are reported as mean ± SEM values. Statistical analysis of the results used Student's t test or one-way ANOVA followed by Fisher's PLSD test for post hoc comparisons. For correlation coefficients, simple regression analysis was performed. A level of p < 0.05 was considered statistically significant.

Effect of DHA on the activities of brain CAT and GPx

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

In the HC group, the activities of brain CAT and GPx were significantly lower in the cerebrum than in the brainstem or the cerebellum (p < 0.05). In the HC + DHA group, the cerebral activities of these enzymes increased significantly in the cerebrum, but not in the brainstem or cerebellum (Figs.1 and 2) ; compared with the HC group, the rate of increase was 23.0% for CAT (p < 0.05) and 26.3% for GPx (p < 0.05).

image

Figure 1. Catalase (CAT) activity in different brain regions of HC-fed rats (□) and HC + DHA rats (▪). Data are mean ± SEM (bars) values with n = 10-13. One unit is defined as micromoles of methanol converted to formaldehyde equivalent to micromoles of H2O2 oxidized per minute. *p < 0.05, compared with HC control (unpaired Student's t test). †p < 0.05, compared with brainstem and cerebellum of HC rats (one-way ANOVA with post hoc Fisher's PLSD test).

Download figure to PowerPoint

image

Figure 2. GPx activity in different brain regions of HC-fed rats (□) and HC + DHA rats (▪). Results are mean ± SEM (bars) values with n = 10-13. One unit is defined as micromoles of NADPH oxidized per minute. *p < 0.05, compared with HC control (unpaired Student's t test). †p < 0.05, compared with brainstem and cerebellum of HC rats (one-way ANOVA with post hoc Fisher's PLSD test).

Download figure to PowerPoint

Effect of DHA on the levels of brain GSH

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

The finding for brain GSH was similar to those of the brain CAT and GPx. In the HC group, the level of brain GSH was significantly lower in the cerebrum than in the brainstem or the cerebellum (p < 0.05) ((Fig. 3). The HC + DHA group showed a 24.5% increase in cerebral GSH (p < 0.05) compared with the HC group, but there was no significant difference in the GSH concentration in the brainstem or the cerebellum.

image

Figure 3. GSH levels in different brain regions of HC-fed rats (□) and HC + DHA rats (▪). Data are mean ± SEM (bars) values with n = 8-10. *p < 0.05, compared with HC control (unpaired Student's t test). †p < 0.05, compared with brainstem and &Dagger;p < 0.05 ~ 0.1, compared with cerebellum of HC rats (one-way ANOVA with post hoc Fisher's PLSD test).

Download figure to PowerPoint

Effect of DHA on the levels of brain ROS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Within the limits of the study, no changes were detected in ROS concentrations in either the HC or the HC + DHA group (Fig. 4). In neither group was brain CAT nor brain GPx related to the respective values of ROS.

image

Figure 4. Effect of long-term administration of DHA on the basal levels of ROS in different brain regions of HC-fed rats (□) and HC + DHA rats (▪). Data are mean ± SEM (bars) values with n = 10-13. No significant differences were found in any region for either diet.

Download figure to PowerPoint

Relationship between values in rat brain

The cerebral concentrations of DHA in the HC and HC + DHA groups were associated with the corresponding values of CAT and GPx ([CAT] = 0.325 [DHA] - 5.82, r = 0.710, p < 0.0005 ; and [GPx] = 0.804 [DHA] - 39.9, r = 0.750, p < 0.0002) (Fig. 5A). The concentration ratios of DHA to arachidonic acid (AA) ([DHA][AA]-1) in brain tissues were also related to the activities of CAT and GPx ([CAT] = 19.5 [DHA][AA]-1 - 0.977, r = 0.870, p < 0.0001 ; and [GPx] = 38.5 [DHA][AA]-1 - 5.68, r = 0.730, p < 0.0002) (Fig. 5B). Neither the brainstem nor the cerebellum played any part in these relationships. The whole brain activity of CAT in the HC and HC + DHA groups ()Fig. 5C) was, however, associated with the respective levels of GPx ([CAT] = 0.216 [GPx] + 17.5, p < 0.039, r = 0.401).

image

Figure 5. Relationships between the values of the rat brain in HC-fed rats (▵, ○, and □) and HC + DHA rats (▴, ○, and ▪). Cerebrum (▵, n = 10 ; ▴, n = 10), brainstem (○, n = 10 ; ▪, n = 10), and cerebellum (□, n = 10 ; ▪, n = 10). A : Relationship between the cerebral levels of GPx or CAT and DHA. B : Relationship between the cerebral level of GPx or CAT and the [DHA][AA]-1 ratio. C : Relationship between the whole brain activities of CAT and GPx. #The levels of cerebral DHA and the cerebral [DHA][AA]-1 ratio are reproduced from Hossain et al. (1998) with the permission of Elsevier Science.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

The present study demonstrated that (1) brain GSH and brain peroxidase, such as CAT and GPx, in the HC + DHA group increased in the cerebrum but not in the brainstem or cerebellum, (2) the cerebral DHA concentrations in the HC and the HC + DHA groups were associated with the CAT and GPx activities, whereas the whole brain CAT activity was related to the respective levels of GPx, and (3) there was no change in the concentrations of ROS in the brains of the HC or the HC + DHA groups.

The most remarkable finding in the present study was the stimulating effect of DHA on the cerebral activities of peroxidase in aged and hypercholesterolemic rats. Regarding these enzymatic functions, CAT and GPx metabolize peroxide, including H2O2, and protect the cellular membranes from lipid peroxidation (Younes and Seigers, 1980 ; McCay et al., 1981). GPx can limit the production of LPOs by directly catalyzing the conversion of peroxidated lipid into lipid alcohol (Christophersen, 1968). We suggest that increased activities of cerebral CAT and GPx in the HC + DHA rats enhance the capacity of the cerebrum to detoxify H2O2 and LPOs ; that is, one might think that long-term administration of DHA might have induced adaptive changes in the anti-oxidative defenses probably to compensate for greater free radical generation in the cerebrum of the HC + DHA rats. Indeed, we found that the DHA supplement used in the HC rats reduced cerebral LPOs (Table 2). This simple interpretation, as an adaptive response to DHA in these rats to combat the oxidative stresses, however, does not appear relevant, because basal levels of ROS did not change significantly by supplementation with DHA. Regarding this contradiction, we postulate that CAT activity, increased by the DHA supplement used, produces an excess amount of O2 from H2O2, and xanthine oxidase, in this case, facilitates the transformation of O2 to oxygen radicals such as superoxide (O2-), which in turn recycles the O2 derived from H2O2 (Fig. 6). If this is true, cerebral DHA would have two actions, i.e., (1) derive O2 from H2O2, leading to increments in ROS, and (2) reduce LPO concentrations, such as ROS components. Thus, the DHA supplement used in this study appears to mask a possible change in the levels of ROS.

image

Figure 6. Recycling of oxygen (O2) derived from hydrogen peroxide (H2O2).

Download figure to PowerPoint

Activities of GPx and superoxide dismutase have been reported as lower in the cortical and hippocampus areas than in the other regions of the brain (Brannan et al., 1980 ; Mizuno and Ohta, 1986). These results are qualitatively consistent with our results, with a general description of a decrease in CAT, GPx, and GSH levels in the cerebral region of HC-fed rats. Thus, it is likely that the decrease in the above antioxidative defenses in the cerebrum of HC-fed rats would predispose the area to oxidation attack. In this case, the higher the basal values of the brain CAT and GPx were elevated, the more the brain sensitivity to DHA diminished (Figs. 1 and 2). Thus, the lowest basal levels of the cerebral CAT and GPx in the HC group would become more sensitive to DHA, requiring more DHA to stimulate them. The present findings further strengthen the reasoning that increased LPO levels in the brain seem to be not merely due to increased levels of precursor polyene fatty acids, but rather to altered protection systems against lipid peroxidation formation (Ando et al., 1990).

The relationship between the activities of CAT and GPx in the whole brain of the HC and HC + DHA groups suggests that CAT acts in concert with GPx. As the basal activities of these two enzymes rose in the order of the cerebrum < the cerebellum < the brainstem, higher levels of GSH and lower levels of LPOs observed in these brainstem and cerebellum regions appear to be harmonized by the control of the regional CAT and GPx activities.

GSH, a substrate of GPx, is a major water-soluble thiol and plays an important role in the maintenance of cellular defense mechanisms in multiple ways, including its participation in direct reactions with free radicals or in the enzymatic reduction of hydroperoxides (Ishikawa et al., 1986). An increase in the GSH level in the cerebrum of the HC + DHA rats suggests contribution of the peroxidatic substrate to neutralize the hydroperoxide radicals in the cerebral region. We did not find any relationship between the values of cerebral DHA and GSH. Thus, the GSH concentrations in the cerebrum of HC-fed rats increased without predication.

We examined the relationship between the level of CAT or GPx and the [DHA][AA]-1 ratio in the brains of the HC and HC + DHA groups, because the cerebral [DHA][AA]-1 ratio was considered to be a marker for host defense capability against damage induced by oxidants (Hossain et al., 1998). In only the cerebrum was the relationship between CAT and the [DHA][AA]-1 ratio or GPx and the [DHA][AA]-1 ratio detected. In this case, we observed an inverse relationship between the levels of DHA and AA, thereby suggesting that dietary DHA facilitates the expulsion of cerebral AA, elevates the [DHA][AA]-1 ratio, and potentiates the CAT and GPx activities. In addition, AA might contribute to an increase in LPOs not only by autoperoxidation, but also by triggering the AA-cascade production of endoperoxides, which themselves have free radical characters (Egan et al., 1976), whereas DHA with its specialized helical conformation (Stubbs, 1992) in the membrane bilayer plays a role as a structural fatty acid (Bazan, 1990 ; Fonlupt et al., 1994) and is believed to turn over at a slower rate (Gazzah et al., 1995). Thus, the inverse relationship between these polyunsaturated fatty acids offers a possible explanation for the protection of the brain from oxidation.

We propose that a low concentration of GSH and low activities of CAT and GPx in the cerebrum would make it particularly susceptible to oxidation stress compared with the brainstem and cerebellum regions. Now, it could be postulated that DHA-induced decrease in AA with a concomitant increase in the [DHA][AA]-1 ratio might provide a line of defense against the peroxidative stress in the cerebrum through activation of the antioxidative enzymes. Further investigations along this line will be needed to clarify this postulation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

We thank Dr. Joe Clark, assistant professor, Department of Biochemistry, Oxford University, for his useful comments regarding the manuscript. It is a pleasure to acknowledge Harima Chemicals, Inc., Tokyo, Japan, for its generous gift of DHA-95E as an ethyl ester derivative of all cis-4,7,10,13,16,19-docosahexaenoic acid. This study was supported, in part, by a Grant-in-Aid(C) from the Ministry of Education, Science and Culture of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. ROS assay
  6. RESULTS
  7. Effect of DHA on the activities of brain CAT and GPx
  8. Effect of DHA on the levels of brain GSH
  9. Effect of DHA on the levels of brain ROS
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES
  • 1
    Akerboom T.P.M. & Seis H. (1981) Assay of glutathione, glutathione disulfide and glutathione mixed disulfides in biological samples, inMethods in Enzymology, Vol. 77 (Jakoby W. B., ed), pp. 373382. Academic Press, New York.
  • 2
    Ando S., Kon K., Aino K. & Totani Y. (1990) Increased levels of lipid peroxides in aged rat brain as revealed by direct assay of peroxide values.Neurosci. Lett. 113,199204.
  • 3
    Bazan N.G. (1990) Supply of n-3 polyunsaturated fatty acids and their significance in the central nervous system, inNutrition and the Brain (Wurtman R. J. and Wurtman J. J., eds), pp. 124. Raven Press, New York.
  • 4
    Brannan T.S., Maker H.S., Weiss C. & Cohen G. (1980) Regional distribution of glutathione peroxidase in the adult rat brain.J. Neurochem.35,10131014.
  • 5
    Braughler J.M. & Hall E.D. (1989) Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation.Free Radic. Biol. Med. 6,289301.
  • 6
    Christophersen B.O. (1968) Formation of monohydroxy-polyenic fatty acids from lipid peroxides by a glutathione peroxidase.Biochim. Biophys. Acta 164,3546.
  • 7
    Cohen G. (1988) Oxygen radicals and Parkinson's disease, inOxygen Radicals and Tissue Injury (Haliwell B., ed), pp. 130135. Federation of American Societies for Experimental Biology, Bethesda, Maryland.
  • 8
    Eddy D.E. & Harman D. (1977) Free radical theory of aging : effect of age, sex and dietary precursors on rat-brain docosahexaenoic acid.J. Am. Geriatr. Soc. 25,220229.
  • 9
    Egan R.W., Paxton J. & Kuehl F.A.J r. (1976) Mechanism for irreversible self-deactivation of prostaglandin synthetase.J. Biol. Chem. 251,73297335.
  • 10
    Fonlupt P., Croset M. & Lagarde M. (1994) Incorporation of arachidonic and docosahexaenoic acids into phospholipids of rat brain membranes.Neurosci. Lett. 171,137141.
  • 11
    Gazzah N., Gharib A., Croset M., Bobillier P., Lagarde M. & Sarda N. (1995) Decrease of brain phospholipid synthesis in free-moving n-3 fatty acid deficient rats.J. Neurochem.64,908918.
  • 12
    Halliwell B. (1989) Oxidants and the central nervous system : some fundamental questions. Is oxidant damage relevant to Parkinson's disease, Alzheimer's disease, traumatic injury or stroke ?Acta Neurol. Scand. 126,2333.
  • 13
    Hashimoto M., Shinozuka K., Hossain M.S., Kwon Y.M., Tanabe Y., Kunitomo M. & Masumura S. (1998) Antihypertensive effect of all-cis-5,8,11,14,17-icosapentaenoate of aged rats is associated with an increase in the release of ATP from the caudal artery.J. Vasc. Res. 35,5562.
  • 14
    Hossain M.S., Hashimoto M. & Masumura S. (1998) Influence of docosahexaenoic acid on cerebral lipid peroxide level in aged rats with and without hypercholesterolemia.Neurosci. Lett. 244,157160.
  • 15
    Huang J. & Philbert M.A. (1995) Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells.Brain Res. 680,1622.
  • 16
    Ishikawa T., Akerboom T.M.P. & Sies H. (1986) Role of key defense systems in target organ toxicity, inTarget Organ Toxicity, Vol. I (Cohen G., ed), pp. 129143. CRC Press, Boca Raton, Florida.
  • 17
    Johansson L.H. & Borg L.A.H. (1988) A spectrophotometric method for determination of catalase activity in small tissue samples.Anal. Biochem. 174,331336.
  • 18
    Joseph J.A., Villalobos-Molina R., Denisova N., Erat S., Culter R. & Strain J. (1996) Age differences in sensitivity to H2O2- or No-induced reductions in K(+)-evoked dopamine release from superfused striatal slices : reversals by PBN or Trolox.Free Radic. Biol. Med. 20,821830.
  • 19
    Kelly J.F., Joseph J.A., Denisova N.A., Erat S., Mason R.P. & Roth G.S. (1995) Dissociation of striatal GTPase and dopamine release responses to muscarinic cholinergic agonists in F344 rats : influence of age and dietary manipulation.J. Neurochem. 64,27552764.
  • 20
    Lepage G. & Roy C.C. (1986) Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 27,114120.
  • 21
    Lowry O.H., Rosebrough N.J., Farr A.L. & Randall R.J. (1951) Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193,265275.
  • 22
    McCay P.B., Gibson D.D. & Hornbrook K.R. (1981) Glutathionedependent inhibition of lipid peroxidation by a soluble, heat-labile factor not glutathione peroxidase.Fed. Proc. 40,199205.
  • 23
    Mizuno Y. & Ohta K. (1986) Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of oxygen free radicals, and some of the related enzymes in adult and aged rat brains.J. Neurochem. 46,13441352.
  • 24
    Montoliu C., Vallés S., Renau-Piqueras J. & Guerri C. (1994) Ethanol-induced oxygen radical formation and lipid peroxidation in rat brain : effect of chronic alcohol consumption.J. Neurochem. 63,18551862.
  • 25
    Paglia D.E. & Valentine W.N. (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase.J. Lab. Clin. Med. 70,158169.
  • 26
    Roy D., Pathak D.N. & Singh R. (1984) Effects of chlorpromazine on the activities of antioxidant enzymes and lipid peroxidation in the various regions of aging rat brain.J. Neurochem. 42,628633.
  • 27
    Saito M. & Nakatsugawa K. (1994) Increased susceptibility of liver to lipid peroxidation after ingestion of a high fish oil diet.Int. J. Vitam. Nutr. Res. 64,144151.
  • 28
    Sinet P.M., Heikkila R.E. & Cohen G. (1980) Hydrogen peroxide production by rat brainin vivo. J. Neurochem. 34,14211428.
  • 29
    Stubbs C.D. (1992) The structure and function of docosahexaenoic acid in membranes, inEssential Fatty Acids and Eicosanoids : 3rd International Congress (Sinclair A. and Gibson R., eds), pp. 116121. American Oil Chemists' Society, Champaign, Illinois.
  • 30
    Urano S., Sato Y., Otonari T., Makabe S., Suzuki S., Ogata M. & Endo T. (1998) Aging and oxidative stress in neurogeneration.Biofactors 7,103112.
  • 31
    Vatassery G.T., Quach H.T., Smith W.E. & Ungar F. (1997) Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains.Lipids 32,879886.
  • 32
    Wheeler C.R., Salzman J.A., Elsayed N.M., Omaye S.T. & Korte D.W.J r. (1990) Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity.Anal. Biochem. 184,193199.
  • 33
    Younes M. & Siegers C.P. (1980) Lipid peroxidation as a consequence of glutathione depletion in rat and mouse liver.Res. Commun. Chem. Pathol. Pharmacol. 27,119128.