Agric. Biol. Chem., 47 (7), 1577- 1582, 1983 1577
Antioxidative Effects of a-Tocopherol and Riboflavin-butyrate in Rats Dosed with Methyl Linoleate Hydroperoxide Teruo Miyazawa, Chiharu Sato and Takashi Kaneda Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Sendai 980, Japan Received January 6, 1983
The antioxidative effects of dietary a-tocopherol (TOC) and riboftavin-tetrabutyrate (RTB)against tissue lipoperoxidation caused by the long-term administration of methyl linole- ate hydroperoxide (HPO) to rats were investigated by measuring the spontaneous chemilumi- nescence (CL) intensities and thiobarbituric acid (TBA) reactants of the liver, lung and heart. TOCsupplementation resulted in the effective decrease of CLintensities and TBAreac- tants in the three organs, while RTBsupplementation led to the definitive decay of both indices of the lung and a significant decrease in the CL intensity of the heart, as compared with those of rats dosed with HPOand not given any supplemental antioxidant. Although the activities of glutathione peroxidase and glutathione reductase in the three organs obtained from rats dosed with HPOfor 29 days were clearly lower than those of the control rats, both enzyme activities in rats dosed with antioxidants other than HPOwere gener- ally maintained at levels almost equal to those of the control rats. Glutathione peroxidase of the liver and heart and glutathione reductase of the liver were shownto be further activated by the simultaneous supplementation of TOCand RTB. The results indicated that both TOCand RTBeffectively act as antioxidants in rats dosed with HPOover a long-term. Oneof the antioxidative actions of both agents is ascribed to pro- tection of the glutathione peroxidase system in organs undergoing lipoperoxidation caused by the long-term treatment of dietary hydroperoxides.
An oxidized oil intake has resulted in stimu- be elevated by the short-term dosage of HPO lation of lipid peroxidation in several animal irrespective of any antioxidant supplementa- tissues.1"^ This action has the typical toxic tion.1) effect of deteriorating edible oils, which causes As an extension of the previous work, we several pathological changes such as bleeding have investigated the effects of TOCand RTB and degeneration of the organs and tissues of on tissue lipid peroxidation caused by long- animals.4) The degree of tissue lipoperoxidation term feeding of HPOto rats, for 29 days, and induced by the administration of lipoperoxi- have described the effect of antioxidants on des to rats can be estimated by a chemilumi- the glutathione peroxidase system. nescent assay of tissue homogenates.1>2) The effect of antioxidants on tissue lipoperoxida- MATERIALS AND METHODS tion caused by dietary hydroperoxides should be interesting because it elucidates the bio- Methyl linoleate hydroperoxide (HPO). Methyl lino- leate derived from safflower oil was allowed to autoxi- antioxidative functions in animals. dize, and HPOwas obtained as described previously.1} In a previous work we have demonstrated that antioxidants, i.e. d-a-tocopherol (TOC) Feedingprocedures. Male Wistar rats, weighing about and riboflavin-tetrabutyrate (RTB), inhibit 100 g, were purchased from Nihon Rat Co. As shown in tissue lipid peroxidation induced by a short- Table I, the given amounts of HPO, TOC(Eisai Co.) and RTB (Tokyo Tanabe Co.) diluted with fresh methyl term administration of methyl linoleate hydro- linoleate (ML) were orally administered respectively at peroxide (HPO) to rats.1} And the activities of 48 hr-intervals to rats using a stomach tube for a period hepatic glutathione peroxidase were found to of 29 days. The rats of group I (basal diet rats) were 1578 T. Miyazawa, C. Sato and T. Kaneda dosed only with MLand not given the administration components. During the experimentation period, a basal diet (about 20 g/rat/day, Type F-2 pellet rations from Funabashi Farm Co.) was given to the rats of each group. The vitamin E content was 50mg/kg of ll--*' diet and the selenium content was 0.42 ppm.The vita- min B2 content was lOmg/kg diet. At the 30th day after the start of administration, five animals of each à"-à"ML(group I) group were killed by exsanguination via heart puncture. x--x HPO(group IE) 0-o HPO+TOC(group HI) The liver, heart and lung were then excised. A AHPO+RTB(group IV) Q__DHPO+TOC+RTB (group V) Thiobarbituric acid (TBA) assay. TBAreactants, as ® Control a convenient index for tissue lipoperoxide level, were V measured by the method of Ohkawaet al.5) as has been 10 20 described in a previous paper.3) The TBAvalue was DAYS OF FEEDING expressed as /^mol of malondialdehyde (MDA)/g of 30 wet tissue. Fig. 1. Body Weight Gains of Rats Dosed with Methyl Linoleate Hydroperoxide and Antioxidants. Measurement of tissue chemiluminescence (CL). As Control rats were given neither MLnor supplemental has been described previously,1} the CL intensity was components, and the vertical bar represents the stan- measured using a 5 ml sample of 10%(w/v) tissue- dard deviation, For abbreviations see the legend in physiological saline homogenate in an Chemilumines- Table I. The mean values of body weight gain (n=5) cence Analyzer OX-7C (Tohoku Electronic Industries were plotted. Co.). The CLintensity was expressed in terms of the average count/minute for the 200-sec measurementand during the 29 day feeding; however the HPO corrected for background counts. rats (group II) revealed lower body weight Estimation of a-tocopherol content. The tocopherol gains. content of each tissue was determined by the procedure The CL intensities, TBA values and a- of Abe et al.6) using high performance liquid chroma- tocopherol contents of the liver, lung and tography as has been mentioned previously.3) heart of rats in the five groups are shown in Tables II, III and IV. Assay of glutathione peroxidase system. For the enzymeassay, rats bred in the same condition as de- Liver As shown in Table II, the CL intensi- scribed in Table I (groups II~V) were used, and con- ties and TBA values of liver homogenates trol rats, which were fed a basal diet and not dosed with obtained from the rats of group III and group either MLor other administration components, were employed in place of the MLgroup. The glutathione V (which had been administered with supple- peroxidase activity of the supernatant fraction, ob- mental TOCother than HPO)were significantly tained by centrifugation of the homogenate at 105,000 lower than those of the other three groups x g, was assayed by the method described previously^ which had not been given the supplemental using cumene hydroperoxide as a peroxide donor. The glutathione reductase activity was determined by the Table I. Regimens of Orally method of Racker.7) The specific activities, hereafter Administered Components called activities, of glutathione peroxidase and gluta- thione reductase were expressed as /umol NADPH Administration components 6 (mg/rat/2 days) oxidized/10 min/mg of protein. Protein was assayed by Rat groupa å the method of Miller.8) ML HPO TOC RTB I ML 200 - - ~ II HPO 200 5 - - RESULTS III HPO+TOC 200 5 5 - IV HPO+RTB 200 5 - 0.1 Antioxidative effects of TOCand RTBin rats V HOP+TOC+RTB 200 5 5 0.1 dosed with HPO for 29 days a Each group consists offive rats. Figure 1 shows the growth curve. The body 6 ML, methyl linoleate; HPO, methyl linoleate weight gains of animals in five groups (groups hydroperoxide; TOC, d-a-tocopherol; RTB, I~V) compared favorably with each other riboflavin-2', 3 ',4', 5 '-tetrabutyrate. Antioxidative Effect of V. E and V. B2-Butyrate 1579
Table II. Liver Chemiluminescence, TBAValue and Tocopherol Content in Rats Dosed with Methyl Linoleate Hydroperoxide for 29 Days Chemiluminescent TBAValue a-Tocopherol Rat group (cpm)intensity g wet(nmolwt.)MDA/(/^g/g contentwet wt.) I ML 528dzl56 704±77 9.3±0.7 II HPO 690±198 693±22 8.5±0.7 III HPO+TOC 396± 78* 556±19* 16.5±3.2* IV HPO+RTB 504± 66 638±66 12.2zbl.9* V HPO+TOC+RTB 354± 72* 572±36* 18.9±1.7* Abbreviations are the same as described in Table I. * Significantly different from group II (HPO rat) atiXO.05. Table III. Lung Chemiluminescence, TBAValue and Tocopherol Content in Rats Dosed with Methyl Linoleate Hydroperoxide for 29 Days Chemiluminescent TBAValue a-Tocopherol Rat group (cpm)intensity g (nmolwet wt.)MDA/ Og/g contentwet wt.) I ML 3348d= 678* 437±33 17.8± 4.0 II HPO 5304±1154 443± 8 18.1± 0.5 III HPO+TOC 2628d= 162* 366± 6* 56.0±13.1* IV HPO+RTB 2658± 444* 374±17* 29.7±12.3 V HPO+TOC+RTB 2952± 600* 341±14* 47.8±25.3* Descriptions are the same as in legend of Table II. Table IV. Heart Chemiluminescence, TBAValue and Tocopherol Content in Rats Dosed with Methyl Linoleate Hydroperoxide for 29 Days
Chemiluminescent intensity Rat group (nmol MDA/TBAValue (cpm) gwetwt.) a-Tocopherol Og/gwet wt.) content I ML II HPO III HPO+TOC IV HPO+RTB
1668=b7692±22922172±2250± 348*84*546*
2070± 378* 754±94666±173435±30*481±25470±39 18.5±1.220.6±l.l27.1±1.8*21.3±2.027.9±2.4* V HPO+TOC+RTB Descriptions are the same as in legend of Table II. TOC, The a-tocopherol contents of the livers (groups III, IV and V) were less than those of of rats in group III and group V were higher HPOrats (group II) and of MLrats (group I). than those of HPOrats (group II) and of ML The a-tocopherol contents of the lung were rats (group I). Supplementation of RTB highly elevated in rats supplemented with elevated the a-tocopherol contents as revealed TOC (groups III and V). in the results for group IV. Although the ML Heart The CL intensities of the heart were rats (group I) were not given HPO,the TBA markedly increased in HPOrats (group II), value was almost at the same level as that of and the CLintensities of MLrats (group I) the HPO-dosed rats (group II). and of rats supplemented with antioxidants Lung The CL intensities of the lung of ML (groups III, IV and V) were significantly lower rats (group I) and of rats supplemented with than those of HPOrats (Table IV). The TBA antioxidants (groups III, IV and V) were values of the heart of rats in group III, which noticeably lower than those of HPO rats were given TOC,were clearly lower than those (group II), as shown in Table III. The TBA of the other four groups. The a-tocopherol reactants of the lung of rats given antioxidants contents of the heart were increased in rats 1580 T. Miyazawa, C. Sato and T. Kaneda
Table V. Glutathione Peroxidase Activities of Rats Dosed with Methyl Linoleate Hydroperoxide for 29 Days Glutathione peroxidase Omol NADPHoxidized/10 min/mg protein) Rat group Liver Lung Heart I Control* 279.2±10.5* 242.4±3.1 248.7± 6.4 II HPO 192.7±20.3 229.7±8.3 229.5±18.0 III HPO+TOC 292.2± 5.2* 231.0±7.7 235.8±15.7 IV HPO+RTB 274.0± 3.8* 256.3±3.8* 255.1± 4.7* V HPO+TOC+RTB 311.6± 7.0* 261.7±7.7* 299.5±12.1* Descriptions are the same as in Table II. Control rats were fed a standard basal diet (Type F-2 from Funabashi Farms Co.) and given neither of the supplemental components.
Table VI. Glutathione Reductase Activities of Rats Dosed with Methyl Linoleate Hydroperoxide for 29 Days Ratgroup Omol NADPHoxidized/10Glutathione min/mgreductaseprotein) Lung Liver Heart III HPOControl III HPO+TOC IV HPO+RTB V HPO+TOC+RTB 218.5± 7.9* 161.5±10.0 238.8± 5.9* 226.6± 9.0* 272.4±12.9* 191.6±10.9 188.0± 8.6 188.0± 7.1 223.4± 7.2* 244.3± 3.1* 235.3± 5.9* 192.9±4.8 187.6±12.8 232.8± 9.1* 229.9±11.3* Descriptions are the same as in legend of Table V. given supplemental TOC(groups III and V), animals supplemented with RTB(groups IV similar to those observed for the liver and lung. and V) were increased (Table V). In the heart, the glutathione peroxidase activities were Changes of the glutathione peroxidase system elevated in rats given RTB(groups IV and V), in liver, lung and heart and it was also noted that the glutathione pero- Changes in the activities of glutathione pero- xidase activities were higher in group V which xidase and glutathione reductase in the three had been supplemented with both TOCand organs of rats dosed with HPOand supple- RTB (Table V). mental antioxidants for 29 days are shown in The glutathione reductase activities of the Table V and VI. liver of rats in group II which had been dosed The glutathione peroxidase activities of the with HPO and not any antioxidants were liver of rats in group II, which had been dosed lower than those of the control rats (group I) with HPO and not given any antioxidants, as shown in Table VI. The activities of hepatic were significantly lower than those of the con- glutathione reductase of animals supplemented trol rats (group I) as shown in Table V. The with either TOCor RTB(groups III and IV) hepatic glutathione peroxidase activities of were substantially equal to the level of the animals supplemented with TOC or RTB control rats. By the simultaneous supple- (groups III and IV) were held at almost the mentation of both TOCand RTB(group V), same levels as those of the control rats. Simul- the hepatic glutathione reductase activities taneous supplementation of TOC and RTB were further elevated. In the lung, the reductase (group V) resulted in further elevation of the activities of animals supplemented with RTB hepatic glutathione peroxidase activities. In (groups IV and V) were increased, and these the lung, the glutathione peroxidase activities of activities were higher in group V which had Antioxidative Effect of Y. E and V. B2-Butyrate 1581 been supplemented with both TOCand RTB HPO. On the other hand, RTB is an effective (Table VI). In the heart, the glutathione re- agent, especially in the liver, lung and heart, ductase activities of rats supplemented with at acute intoxication and RTBis also effective RTB(groups IV and V) were maintained at in the lung and heart against chronic intoxica- the same levels as those of the control rats tion with dietary HPO. It is also suggested that (Table VI). the antioxidative functions of dietary TOCare not necessarily the same amongall of the or- DISCUSSION gans in animals, and that RTBhas the added effect of protecting organs which are not effec- The ultraweak CL of systems undergoing tively receiving the antioxidative actions of lipid peroxidation reflects oxidation reactions TOC during acute intoxication with HPO. involving molecular oxygen, and especially Although the functions and mechanisms for the generation of short-lived free radicals and tocopherol as bio-antioxidants have been well the excited states derived from the free radical studied,13} little is known about the suscepti- processes of lipid peroxidation.9~U) The CL bility of the protective effects on individual technique permits monitoring of lipoperoxida- organs against HPO-intoxication. The protec- tion reactions occurring in tissue homogenates tive effect of a-tocopherol is generally regarded and has been shownto have an advantage over as being due to its ability to quench free radi- the detection of lipid peroxides by TBA as- cals as well as lipid peroxy radicals in bio- say.9 1^ During the course of our investiga- membranes.1^ It has been stated that a- tion,1'25 the CL technique has been employed tocopherol, in the process of quenching free to elucidate the antioxidative properties of radicals, is first oxidized to the tocopheryl radi- TOCand RTB, in addition to the TBAassay. cal and subsequently to the tocopheryl quinone, As revealed before, supplementation of TOC and then the tocopheryl radical is recycled to and/or RTBbrought about significant decays a-tocopherol by the action of various thiols of CL intensities and TBA reactants of rat such as reduced glutathione which has been organs as compared with those of rats dosed provided by glutathione reductase.15) The with HPO and not given the supplemental higher tocopherol level observed in the liver of antioxidants (Tables II, III and IV). This shows rats dosed with HPOand supplemented with that both TOCand RTBare demonstrated to RTB (Table II) suggests the protective action be effective antioxidants against tissue lipo- of RTBagainst the consumption of tissue
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