THE ALTERATIONS IN LIPID AND PROTEINS IN ERYTHROCYTE MEMBRANES OF ENDOTOXIN/DGALACTOSAMINE (LPS/GaIN)-ADMINISTERED RATS

Öz

Objective: To clarify the effects of endotoxin/D-galactosamine (LPS/GaIN) on erythrocyte membranes (ghost) of rats.

Materials and methods: After 18 hours of intraperitoneal coadministration of 200 mg/kg D-galactosamine (GaIN) and 200 µg/kg lipopolysaccharide (LPS), male Wistar rats were decapitated and ghost was prepared by ultracentrifugation. Malondialdehyde (MDA), sulfhydryl (-SH) and protein carbonyl (PC) contents and Na+ ,K+ -ATPase activity were measured in ghost, spectrophotometrically. MDA and PC were measured in also plasma of rats. Furthermore, sodium dodesyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) technique was used for the determination of the modifications in cytoskeletal ghost proteins .

Results: LPS/ GaIN administration resulted in hepatitis in male Wistar rats as evaluated by assessments of serum alanine transaminase (ALT) and aspartate transaminase (AST) activities. While plasma MDA levels were determined to increase, ghost MDA and PC contents were observed unchanged. However, -SH content as well as Na+ ,K+ -ATPase activity of ghost were found decreased. As shown by the electrophoretic gel patterns, significant reduction in the intensities of some cytoskelatal membrane proteins, especially in α spectrin and β–spectrin, was observed in erythroctes of LPS/ GaIN-administered rats.

Conclusion: From these findings, we concluded that reduced Na+ ,K+ -ATPase activity may be associated with the diminished – SH content in ghost of LPS/GaIN-administered rats. Furthermore, alterations seen in cytoskeletal proteins of ghost may participate to LPS/GaIN-induced hepatotoxicity by causing the deformation of cell membrane structure and abnormality of membrane functions, ultimately.

Anahtar Kelimeler

• Lipid/protein oxidation
• cytoskeletal proteins
• LPS/GaIN
• ghost
• rat

ENDOTOKSİN/D-GALAKTOZAMİN (LPS/GaIN) VERİLEN SIÇANLARIN ERİTROSİT ZARLARINDA LİPİT VE PROTEİN DEĞİŞİKLİKLERİ

Öz

Amaç: Endotoksin/D-galaktozamin (LPS/GaIN)’in sıçan eritrosit zarı üzerine etkilerini aydınlatmak.

Gereç ve yöntem: Erkek Wistar sıçanlar, intraperitoneal 200 mg/kg D-galaktozamin (GaIN) ve 200 µg/ kg lipopolisakkarit (LPS) uygulamasından 18 saat sonra kesilerek ultrasantrifüj yöntemi ile eritrosit zarı (ghost) elde edildi. Sıçanların eritrosit zarlarında, malondialdehit (MDA), sülfidril (-SH) ve protein karbonil (PC) içeriği ve Na+ ,K+ -ATPaz aktivitesi spektrofotometrik olarak ölçüldü. MDA ve PC sıçanların plazmalarında da tayin edildi. Bunun yanısıra, eritrosit zar iskeletinin proteinlerindeki değişiklikler sodyum dodesil sülfat gel elektroforezi (SDS-PAGE) tekniği ile saptandı.

Bulgular: LPS/GaIN uygulamasının, erkek Wistar sıçanlarda, serum alanin transaminaz (ALT) ve aspartat transaminaz (AST) ölçümleri sonucu, hepatit oluşturduğu belirlendi. Sıçanların plazma MDA düzeylerinde artış bulunurken, eritrosit zarlarında MDA ve PC düzeylerinin değişmediği gözlendi. Bununla birlikte sıçan eritrosit zarlarının –SH içeriği ve Na+ ,K+ -ATPaz aktivitesinde azalma olduğu saptandı. Eritrosit zar iskeleti proteinlerinin SDS-PAGE örneklerinde ise, proteinlerin bir kısmında, özellikle de, α-spektrin ve β-spektrin band yoğunluklarında azalmalar olduğu gözlendi.

Sonuç: Bulgularımız, Na+ ,K+ -ATPaz aktivitesindeki azalmadan zar –SH içeriğindeki azalmanın sorumlu olabileceğini düşündürmektedir. Bunun yanısıra, eritrosit zar iskeletinin proteinlerinde görülen değişiklikler, hücre zar yapısında ve dolayısıyla fonksiyonlarında anormalliklere neden olarak LPS/GaIN hepatotoksisitesine katkıda bulunabilir.

Anahtar Kelimeler

• Lipit/protein oksidasyonu
• zar iskelet proteinleri
• LPS/GaIN
• eritrosit zarı
• sıçan

Kaynakça

1. Arduini A, Stern A, Storto S, Belfiglio M, Mancinelli G, Scurti R and Federici G. Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes 1989; 273: 112.
2. Basu S and Eriksson M. Oxidative injury and survival during endotoxemia. FEBS Letters 1998; 438: 159.
3. Bidlack WR, Tappel AL. Damage to microsomal membrane by lipid peroxidation. Lipids 1983; 8: 177.
4. Brandes RF, Koddenberg G, Gwinner W, Kim D, Kruse HJ, Busse R, Mugge A. Role of increased production of superoxide anions by NAD(P)H oxidase and xanthine oxidase in prolonged endotoxemia. Hypertension 1999; 33: 1243.
5. Chamulitrat W, Skrepnik NV, and Spitzer JJ. Endotoxin- induced oxidative stress in the rat small intestine. Role of nitric oxide. Shock 1996; 5: 217.
6. Cheng CC, Etoh J, Tanimura T, Egashira Y, Ohta T and Sanada H. Effects of dietary gluten on the hepatotoxic action galactosamine and /or endotoxin in rats. Biosci Biotech Biochem 1996; 60:439.
7. Özdemirler G, Küçük S,Orhan Y, Aykaç-Toker G, Uysal
8. M. Lipid and protein oxidation in erythrocyte membrane

9. of hypercholesterolemic subjects.Clin Biochem 2001; 34:335.
10. Gwozdzinski K, Pieniazek A and Kaca W. Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins. Int J Biochem Cell Biol 2003; 35:333.
11. Ikejima K, Enomoto N, Iımuro Y, Ikejima A F D, Xu J, Forman DT, Brenner DA and Thurman RG. Estrogen increases sensitivity of hepatic Kupffer cells to endotoxin. Am J Phsiol 1998; 274:669.
12. Kadota Y, Kamada T, Yoshimura N, Otsuji S. Changes in erythrocyte membrane fluidity by endotoxin in rats. J Anesth 1992; 6:145.
13. Kitajima H, Yamaguchi T, and Kimoto E. Hemolysis of human erythrocytes under hydrostatic pressure is suppressed by crosslinking of membrane proteins. J Biochem 1990; 108:1057.
14. Kovachich GB and Mishra OP. The effect of dopamine on Na+,K+-ATPase activity in nerve ending membranes are prevented by N-ethylmaleimide. Neurochem Int 1983; 5: 117.
15. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680 .
16. Levine RL, Garland D, Oliver CN, Amıcı A, Climent I, Lenz AG, Ahn BW, Shaltiel S, and Stadtman ER. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990; 186: 464.
17. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265.
18. Moore RB, Hulgan TM, Green JW and Jenkins LD. Increased susceptibility of the sickle cell membrane Ca+2,Mg+2-ATPase to t-butylhyroperoxide: Protective effects of ascorbate and desferal. Blood 1992; 79: 334.
19. Mutlu-Türkoğlu Ü, Tamer-Toptani S, Öztezcan S, Uysal M and Toker G. The effects of N-acetyl cysteine treatment and nitric oxide synthase inhibition on oxidative stress induced by E.coli endotoxin. Biochem Arch 1998; 14:33.
20. Pöschl JMB, Leray C, Ruef P, Cazenave JP, Linderkamp O. Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro. Crit Care Med 2003; 31: 924 .
21. Rice-Evans C, Hochstein P. Alterations in erythrocyte membrane peroxidation of lipids. Biochem Biophys Res Comm 1981; 100:1537.
22. phenylhydrazine induced
23. Sewerynek E, Abe M, Chen L, Ortiz GG and Reiter RJ. Oxidative changes in the liver, brain and lens of lipopolysaccharide-treated rats. Arch Med Res 1995; 2: S109 .
24. Sprong RC, Winkelhuyzen-Janssen AML, Aarsman CJM, Van Oirschot JFLM, Vander Bruggen T and Van Asbeck BS. Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high- dose increases mortality. Am J Respir Crit Care Med 1998; 157: 1283.
25. Uyesaka N, Hasegawa S, Ishioka N, Ishioka R, Shio H and Schechter AN. Effects of superoxide anions on red cell deformability and membrane proteins Biorheol 1992; 29: 217.
26. Vimal V, Devaki T. Herpatoprotective effect of allicin on tissue defense system in galactosamine/endotoxin challenged rats. J Ethnopharmacol 2004; 90: 151.
27. VonRückmann B, Jöns T, Dölle F, Drenckhahn D and Schubert D. Cytoskeleton-membrane connections in the human erythrocyte membrane: Band 4.1 binds to tetrameric band 3 protein. Biochim Biophys Acta 1997; 1325:226 .
28. Wang H, Xu DX, Lv JW, Ning H, Wei W. Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in d-galactosamine-sensitized mice. Toxicol 2007; 237: 49.
29. Yagi K, Nishigaki I, Ohama H. Measurement of serum TBA value. Vitamins (Japan) 1968; 37: 105 .