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* Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain,
Division of Life Sciences and
** Institute of Liver Studies, King’s College Hospital, London, UK
3To whom correspondence should be addressed. E-mail: mmitjavila{at}ub.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol acetate. We also measured nonheme iron, -tocopherol and retinol concentrations, and superoxide (SOD) and catalase activity in these organs. Rats fed FO were compared to rats fed a corn oil (CO)–rich diet with 100 mg/kg all-rac--tocopherol acetate. The activity and expression of IRP1 in both the liver and the spleen of rats fed FO diets were greater than in those fed the CO diet. FO-fed rats also had lower nonheme iron concentrations in these organs. Hepatic -tocopherol and retinol concentrations and SOD activity were lower in FO-0–fed rats compared to those fed the CO diet. In the spleen, -tocopherol and retinal concentrations were not altered but SOD activity was lower in FO-0– fed rats, whereas catalase activity was greater than in rats fed CO. The results indicate that there is an increase in oxidative stress in the liver and in the spleen of rats fed FO diets. These changes, together with the reduction of nonheme iron concentrations in both FO-0– and FO-1–fed rats, may explain the increase in activity and expression of IRP1. Therefore, the ingestion of FO-rich diets should be monitored under close supervision.
KEY WORDS: • antioxidants • corn oil • fish oil • iron • IRP1 • rats
The health benefits of fish oil (FO) have been extensively studied since the observation that the incidence of cardiovascular disease was low among populations that consume large amounts of FO (1 ,2 ). Although the therapeutic effects of FO have been well described, their impact on iron metabolism remains unclear.
In a previous study (3
), we showed that rats fed a FO diet had an increase in iron absorption compared to rats fed a control diet. We demonstrated that this increase was related to changes in oxidative stress (4
). We also observed (4
) that in rats fed a FO diet with even 200 mg/kg of vitamin E (as all-rac--tocopherol acetate), antioxidant levels (particularly, -tocopherol) were reduced and lipid peroxidation products were increased in both plasma and erythrocytes. The ingestion of FO also decreased nonheme iron concentrations in liver and spleen (3
,4
). However, no studies have been conducted to examine the impact of FO and oxidative stress on iron metabolism in these organs.
The iron regulatory proteins (IRP) are responsible for the regulation of the iron homeostasis by coordinating the use of the mRNA coding for proteins involved in iron metabolism. IRP1 is a bifunctional protein with mutually exclusive functions as an mRNA binding protein that specifically binds the iron-responsive element (IRE) or as the cytoplasmatic isoform of aconitase (c-aconitase) (5 ). IRP1 is sensitive not only to intracellular iron levels but also to oxidative stress, particularly to nitric oxide and hydrogen peroxide (6 ). However, no correlation has been established between FO-rich diets and IRP1 that can explain how FO diets modulate iron metabolism.
The aim of this work was to study the activity and expression of IRP1 in the liver and the spleen of rats fed FO diets with 0 or 100 mg/kg of all-rac--tocopherol acetate. We also determined nonheme iron, -tocopherol and retinol concentrations and superoxide dismutase (SOD, EC 1.15.1.1) and catalase (EC 1.11.1.6) activities in these organs. Rats fed FO were compared to rats fed a corn oil (CO) diet with 100 mg/kg of all-rac--tocopherol acetate.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Sprague-Dawley rats were purchased from Harlan Interfauna Ibérica (Barcelona, Spain) and housed in the Department of Animal Care at the Faculty of Biology at the University of Barcelona. The experimental protocols were reviewed and approved by the Ethical Committee of the Faculty of Biology in accordance with the European Community guidelines.
After weaning, rats were fed isoenergetic semipurified diets for 10 wk (Table 1
) that contained 5% lipids, either CO, rich in (n-6) PUFA, or FO as menhaden oil, rich in (n-3) PUFA. The fatty acid composition of the diets was determined according to Haan et al. (7
) (Table 2
). The diets also had 50 mg of iron/kg. Oils used in these experiments provided between 2.3 and 2.5 mg of -tocopherol/kg diet. The CO diet contained 100 mg/kg all-rac--tocopherol acetate (equivalent to 67 IU -tocopherol/kg) and the FO diets contained 0 (FO-0) or 100 (FO-1) mg/kg all-rac--tocopherol acetate. No other antioxidants were present in the oils or diets. Food was provided daily and any food remaining was also removed daily. Diets were manufactured weekly and stored at -20°C under vacuum for 7 d to prevent oxidation. No significant increase in the peroxidation index of the diets was observed during the storage period. The peroxide value of FO diets was < 5 meq O2/kg. Rats were kept at a constant temperature of 21– 23°C and relative humidity of 50–60% with a 12-h light/dark cycle.
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At the end of the feeding period, food was removed and rats were exsanguinated by cardiac puncture. The liver was perfused through the subhepatic vein with isotonic saline (0.15 mol/L NaCl) to eliminate the blood. The liver and the spleen were then excised, washed with saline, weighed and stored at -80°C until analysis. The hemoglobin concentration and the hematocrit (8 ) were measured in fresh blood samples. Plasma was separated by centrifugation at 1770 x g for 10 min at 4°C and stored at -80°C until analysis.
IRP1 activity and expression.
The spleen and liver samples were homogenized in 3 volumes of HDGC buffer (20 mmol/L Hepes, pH 7.5; 1 mmol/L dithiothreitol; 10% glycerol; and 2 mmol/L trisodium citrate). Citrate was included because it stabilizes the iron–sulfur cluster of aconitases (9 ). The homogenates were centrifuged at 10,000 x g for 30 min at 4°C. The supernatant was recentrifuged at 90,000 x g for 75 min at 4°C and the cytosolic extract was recovered. Protein concentration was determined by the Bradford assay (10 ) using BSA as a standard. The final protein concentration for the liver samples was 5 g/L and 12.5 g/L for the spleen samples.
RNA binding activity was performed by gel shift analysis using a [32P]-labeled RNA of the 59 nucleotides of the IRE of the human ferritin. Synthesis of the radiolabeled IRE-RNA was done in 20 µL containing 1.85 mBq [32P]CTP (370 MBq/mmol); 0.5 mmol/L each of GTP, UTP and ATP; 12 µmol/L CTP; transcription buffer (125 mmol/L potassium-Hepes, pH 7.6; 750 mmol/L CH3COOK; 7.5 mmol/L MgCL2; and 25% glycerol); 35 U ribonuclease inhibitor; 20 U T7 RNA polymerase; and 0.5 µg pTZ19-IRE/EcoRI plasmid. This mix was incubated for 60 min at 37°C. The reaction was stopped by heating 10 min at 75°C, followed by chloroform precipitation. The RNA was resuspended in RNA-free water, aliquoted and stored at -20°C until use.
For the gel retardation binding assay, the cytosolic extract and the binding buffer [100 mmol/L Hepes, pH 7.6; 375 mmol/L MgCl2; 25% glycerol; and 5 mmol/L (CH3COO)2Mg·4H2O] were incubated together for 30 min at room temperature. The amount of IRP1 present in active and inactive (i.e., c-aconitase) forms in each sample was assessed using 2-mercaptoethanol at a final concentration of 2%. After this incubation, the 32P-RNA probe was added and incubated for an additional 30 min at room temperature. The separation of bound RNA from free RNA was accomplished using electrophoresis through a nondenaturing 4% acrylamide (60:1 acrylamide:N,N'-methylbisacrylamide) gel. The gel was fixed in an equal mix of 20% ethanol and 10% acetic acid and dried for 60 min at 70°C. The radioactivity was detected using a phosphorimager (Fuji, Tokyo, Japan).
As a positive gel shift control, 0.5 µg of active recombinant human IRP1 (99 kDa) was added to the [32P]-labeled IRE-RNA probe in every experiment. This allowed us to quantify the amount of IRP1 present in the rest of the samples.
Iron stores.
Nonheme iron concentrations in liver and spleen were determined by the method of Torrance and Bothwell (11 ). Briefly, samples were homogenized in 0.15 mol/L NaCl and then digested in an acid solution (13.5% w/v trichloroacetic acid and 40% v/v HCl 37.5%) for 20 h at 65°C. Iron was measured in the digested supernatant using bathophenanthroline. Iron tritisol was used as standard.
Antioxidants.
In the plasma, -tocopherol and retinol were extracted with methanol:hexane (3:1 v/v). In the liver, 
1 g of tissue was homogenized in 10 volumes of phosphate buffer and stored at -20°C in the presence of butylated hydroxytoluene and EDTA at a final concentration of 0.9 mmol/L and 1.7 µmol/L, respectively. The samples were extracted once with ethanol:chloroform (1:2 v/v) and then with methanol:hexane (1:3 v/v). For the spleen, 0.2 g of tissue was homogenized in 10 volumes of phosphate buffer, and butylated hydroxytoluene and EDTA were also added. The spleen samples were extracted with only methanol:hexane (3:1 v/v). Phospholipids in the liver and the spleen were measured by use of a phospholipid kit. Plasma and tissue -tocopherol and retinol were measured by HPLC (12
).
SOD activity was measured in 1 g of liver and 0.2 g of spleen according to the technique described by Marklund (13
), which is based on the inhibition of pyrogallol autooxidation in the presence of SOD. Protein concentration of the extracts was determined by the Bradford assay (10
) using 
-globulin as a standard.
Catalase activity was measured at room temperature by following the decomposition of hydrogen peroxide at 240 nm (14
). The rate constant k [k = 107 L/(mol·s)] for the first 30 s was calculated. Therefore, 1 g of liver or 0.2 g of spleen was homogenized in 10 volumes of phosphate buffer. Protein concentration of the extracts was determined by the Bradford assay (10
) using -globulin as a standard.
Reagents.
The plasmid pTZ19-IRE/EcoRI, T7 RNA polymerase, ribonuclease inhibitor, binding buffer and human recombinant IRP1 were obtained from MBI Fermentas (Vilnius, Lithuania). The transcription buffer, nucleotides and the ribonuclease inhibitor were obtained from Promega (Southampton, UK). The [32P]CTP was purchased from ICN (Costa Mesa, CA). Acrylamide, Bradford reagent and BSA were obtained from BioRad (Hercules, CA). Iron tritisol and HPLC reagents (HPLC grade) were obtained from Merck (Darmstadt, Germany). The phospholipid kit was obtained from Roche Diagnostics (Mannheim, Germany). All other reagents, including the oils, were obtained from Sigma Chemicals (St. Louis, MO). The peroxide values of CO and FO were < 5 and < 25 meq O2/L, respectively.
Statistical analysis.
The results are expressed as means ± SEM of 7 rats. Statistical analysis of the data was performed using InStat statistical software (v. 2.04a; GraphPad Software, San Diego, CA). Data were analyzed by one-way ANOVA (P < 0.05). The Student– Newman– Keuls multiple-comparison test was used to detect differences among the groups (P < 0.05).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After 10 wk of diet treatment, there were no differences in the body, liver or spleen weights (Table 3 ). Hemoglobin concentration and hematocrit also did not differ among the groups (for the CO group, the hemoglobin concentration was 168 ± 4 g/L and the hematocrit was 0.45 ± 0.01).
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IRP1 activity (2-mercaptoethanol–induced RNA binding activity of cytosolic IRP1) in the liver of rats fed the CO diet was 16.9 ± 1.9% (Fig. 1A and B ). In rats fed the FO-0 and FO-1 diets, this activity was 57 and 61% higher, respectively (P < 0.05; Fig. 1 B). The activity in the spleen of rats fed the CO diet was 13.8 ± 1.3% activity and it was 93 and 73% greater in rats fed the FO-0 and FO-1 diets, respectively (P < 0.05; Fig. 1 A and B).
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Iron stores.
Rats fed the CO diet had 4.7 ± 0.2 µmol/g wet liver of nonheme iron (Table 3) . Rats fed the FO-0 and FO-1 diets had 25 and 30% lower concentrations, respectively. In the spleen, rats fed the CO diet had 21.8 ± 1.6 µmol/g of nonheme iron, whereas FO-0– and FO-1–fed rats had 45 and 29% lower concentrations, respectively.
Antioxidants.
Rats fed the FO-0 and FO-1 diets had lower plasma -tocopherol concentrations than the CO group (Fig. 2A
), even when the amount of all-rac--tocopherol acetate administered was the same as in the CO diet. Retinol in plasma followed a similar pattern (Fig. 2
B). Liver -tocopherol and retinol concentrations in FO-0– fed rats were 23 and 30% lower, respectively, than in rats fed CO (Fig. 2
A and B). The concentrations in rats fed the FO-1 diet did not differ from those of the CO-fed rats. Spleen -tocopherol and retinol did not differ among the groups (Fig. 2
A and B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol contents of membranes and increase oxidative stress (3
,4
,23
–25
). We previously showed (3
,4
) that FO diets reduce nonheme iron stores in rat liver and spleen. Nevertheless, no correlation has been established between oxidative stress induced by FO-rich diets and iron metabolism in these organs. We observed an increase in IRP1 activity and expression in the liver and the spleen of rats fed FO diets. These results were associated with a decrease in nonheme iron stores in these organs. IRP1 activity and expression are mainly modulated by the intracellular levels of iron (26 ). Therefore, a decrease in intracellular iron switches between the c-aconitase activity and the RNA binding activity of IRP1.
IRP1 can also be affected by oxidative stress (6
). In the liver of rats fed the FO-0 diet we observed a reduction in antioxidant defenses: both -tocopherol and retinol concentrations and SOD activity were lower than in rats fed the CO diet. Such reductions in the antioxidant defenses indicate an increase in oxidative stress.
In the spleen, rats fed the FO-0 diet showed a reduction in SOD activity and an increase in catalase activity. Miyasaka et al. (27
) observed no changes in SOD and catalase activity in rats fed a 0.4% FO diet (by gavage) for 1 mo compared to rats fed soybean oil or cocoa butter. However, spleen -tocopherol and retinol were not affected in the present study. This seems contradictory, particularly the results in the plasma and the liver, where significant reductions occurred. These results can be compared to those obtained by McGuire et al. (25
), who observed that after feeding rats diets rich in FO for 4 wk, -tocopherol in the splenocytes did not differ from that in rats fed lard. However, -tocopherol in plasma was significantly lower in rats fed FO. The reason that FO diets can maintain -tocopherol concentrations in the spleen is not clear, particularly when plasma -tocopherol decreases. McGuire et al. (25
) suggested that -tocopherol could be redistributed through the lipoproteins or that cells could adapt to oxidative stress, although there is another explanation. We previously showed that the FO diet increases reticulocyte production (3
,4
), probably because of the destruction of oxidatively aged erythrocytes. It has been demonstrated that the erythrocytes from rats fed a low vitamin E diet behave like aged erythrocytes (28
). Aged erythrocytes are removed from the circulation by splenic macrophages. An increase in erythrocyte removal might lead to an accumulation of -tocopherol and retinol in the erythrocyte membrane.
The reduction of SOD activity in both liver and spleen could be explained by the increase in oxidative stress. Fuji and Taniguchi (29 ) showed a decrease in SOD expression mediated by free radicals. This again indicates an increase in oxidative stress. Catalase activity was affected by the diet in the spleen but not in the liver. Ibrahim et al. (30 ) showed that catalase activity in the liver did not differ in rats fed FO diets with different levels of vitamin E. The liver contains very high levels of catalase, which might be enough to prevent the formation of hydrogen peroxide. However, the levels in the spleen are much lower and increasing them might be necessary to avoid oxidative stress damage.
There is an increase in oxidative stress in the liver of rats fed the FO-0 diet that, together with the reduction in nonheme iron stores in these organs, can explain the increase in activity and expression of IRP1 in these rats. However, the IRP1 in the spleen was responsive to FO feeding and iron stores. The administration of even 100 mg/kg of all-rac--tocopherol acetate in the FO diets does not counterbalance such effects. Therefore, the ingestion of FO-rich diets should be monitored under close supervision.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Abbreviations used: CO, corn oil; FO, fish oil; IRE, iron-responsive element; IRP, iron regulatory protein; SOD, superoxide dismutase.
Manuscript received 25 October 2002. Initial review completed 10 December 2002. Revision accepted 6 January 2003.
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LITERATURE CITED |
|---|
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Bang, H. O. & Dyerberg, J. (1972) Plasma lipids and lipoproteins in Greenlandic west coast Eskimos. Acta Med. Scand. 192:85-94.[Medline]
2. Dyerberg, J. & Bang, H. O. (1979) Lipid metabolism, atherogenesis, and haemostasis in Eskimos: the role of the prostaglandin-3 family. Haemostasis 8:227-233.[Medline]
3. Rodriguez, M. C., Saiz, M. P., Muntane, J. & Mitjavila, M. T. (1996) Fatty acid composition of erythrocyte membranes affects iron absorption in rats. J. Nutr. 126:3109-3117.
4. Miret, S., Sáiz, M. P. & Mitjavila, M. T. (2003) Effects of fish oil- and olive oil-rich diets on iron metabolism and oxidative stress in the rat. Br. J. Nutr. 89:11-18.[Medline]
5. Eisenstein, R. S. (2000) Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu. Rev. Nutr. 20:627-662.[Medline]
6. Hentze, M. W. & Kuhn, L. C. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 93:8175-8182.
7. Haan, G. J., van der Heide, S. & Wolthers, B. G. (1979) Analysis of fatty acids from human lipids by gas chromatography. J. Chromatogr. 162:261-271.[Medline]
8. Drabkin, D. L. & Austin, J. H. (1935) Spectrophotometric studies II. Preparation from washed blood cells: nitric oxide hemoglobin and sulphemoglobin. J. Biol. Chem. 112:51-64.
9. Kennedy, M. C., Mende-Mueller, L., Blondin, G. A. & Beinert, H. (1992) Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc. Natl. Acad. Sci. U.S.A. 89:11730-11734.
10. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[Medline]
11. Torrance, J. D. & Bothwell, T. H. (1968) A simple technique for measuring storage iron concentrations in formalinised liver samples. S. Afr. J. Med. Sci. 33:9-11.[Medline]
12. Bieri, J. G., Tolliver, T. J. & Catignani, G. L. (1979) Simultaneous determination of alpha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am. J. Clin. Nutr. 32:2143-2149.
13. Marklund, S. (1985) Pyrogallol autooxidation. Greenwald, R. A. eds. Handbook of Methods for Oxygen Radical Research 1985:243-247 CRC Press Boca Raton, FL. .
14. Aebi, H. (1984) Catalase in vitro. Methods Enzymol. 105:121-126.[Medline]
15. Connor, W. E. (2000) Importance of n-3 fatty acids in health and disease. Am. J. Clin. Nutr. 71:171S-175S.
16. Engler, M. B. (1994) Vascular effects of omega-3 fatty acids: possible therapeutic mechanisms in cardiovascular disease. J. Cardiovasc. Nurs. 8:53-67.[Medline]
17. Singer, P., Wirth, M., Berger, I., Heinrich, B., Godicke, W., Voigt, S., Taube, C., Jaross, W. & Gehrisch, S. (1992) Long-chain omega 3 fatty acids are the most effective polyunsaturated fatty acids for dietary prevention and treatment of cardiovascular risk factors. Conclusions from clinical studies. World Rev. Nutr. Diet. 69:74-112.[Medline]
18. Uauy, R. & Valenzuela, A. (2000) Marine oils: the health benefits of n-3 fatty acids. Nutrition 16:680-684.[Medline]
19. Drevon, C. A. (1992) Marine oils and their effects. Nutr. Rev. 50:38-45.[Medline]
20. Lands, W. E. (1992) Biochemistry and physiology of n-3 fatty acids. FASEB J. 6:2530-2536.[Abstract]
21. Perona, J. S. & Ruiz-Gutierrez, V. (2000) Effect of two high-oleic oils on the liver lipid composition of spontaneously hypertensive rats. Life Sci. 66:521-531.[Medline]
22. van der Tempel, H., Tulleken, J. E., Limburg, P. C., Muskiet, F. A. & van Rijswijk, M. H. (1990) Effects of fish oil supplementation in rheumatoid arthritis. Ann. Rheum. Dis. 49:76-80.
23. Alexander, D. W., McGuire, S. O., Cassity, N. A. & Fritsche, K. L. (1995) Fish oils lower rat plasma and hepatic, but not immune cell alpha-tocopherol concentration. J. Nutr. 125:2640-2649.
24. Kubo, K., Saito, M., Tadokoro, T. & Maekawa, A. (1997) Changes in susceptibility of tissues to lipid peroxidation after ingestion of various levels of docosahexaenoic acid and vitamin E. Br. J. Nutr. 78:655-669.[Medline]
25. McGuire, S. O., Alexander, D. W. & Fritsche, K. L. (1997) Fish oil source differentially affects rat immune cell alpha-tocopherol concentration. J. Nutr. 127:1388-1394.
26. Kuhn, L. C. (1991) mRNA– protein interactions regulate critical pathways in cellular iron metabolism. Br. J. Haematol. 79:1-5.[Medline]
27. Miyasaka, C. K., de Souza, J. A., Torres, R. P., Filho, J. M., Lajolo, F. M. & Curi, R. (1998) Effect of the administration of fish oil by gavage on activities of antioxidant enzymes of rat lymphoid organs. Gen. Pharmacol. 30:759-762.[Medline]
28. Kay, M. M., Bosman, G. J., Shapiro, S. S., Bendich, A. & Bassel, P. S. (1986) Oxidation as a possible mechanism of cellular aging: vitamin E deficiency causes premature aging and IgG binding to erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 83:2463-2467.
29. Fujii, J. & Taniguchi, N. (1999) Downregulation of superoxide dismutases and glutathione peroxidase by reactive oxygen and nitrogen species. Free Radic. Res. 31:301-308.[Medline]
30. Ibrahim, W., Lee, U. S., Yeh, C. C., Szabo, J., Bruckner, G. & Chow, C. K. (1997) Oxidative stress and antioxidant status in mouse liver: effects of dietary lipid, vitamin E and iron. J. Nutr. 127:1401-1406.
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) and 2-mercaptoethanol–induced (
) RNA binding activity of cytosolic liver IRP1. (B) RNA binding activity of IRP1, expressed as a percentage of 2-mercaptoethanol–induced activity. (C) IRP1 expression (mg/g protein) in liver and spleen. Values are means ± SEM, n = 7. Means for an organ with different letters differ, P < 0.05.