Original communication| Volume 139, ISSUE 3, P377-384, March 2006

Download started.


Cytoprotective function of tetrahydrobiopterin in rat liver ischemia/reperfusion injury

  • Yuzuru Hara
    Department of Surgery, School of Medicine, and Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
    Search for articles by this author
  • Kenichi Teramoto
    Reprint requests: Kenichi Teramoto, MD, PhD, Department of Hepato-Biliary-Pancreatic Surgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
    Department of Surgery, School of Medicine, and Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
    Search for articles by this author
  • Kozo Ishidate
    Department of Surgery, School of Medicine, and Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
    Search for articles by this author
  • Shigeki Arii
    Department of Surgery, School of Medicine, and Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
    Search for articles by this author


      Tetrahydrobiopterin (BH4) is a key coenzyme of nitric oxide synthase (NOS), which is associated with a cytoprotective function in various ischemia-reperfusion (I/R) injury models. There have been a few reports on the efficacy of BH4 in the treatment of I/R injury in other organs; therefore, the aim of this study was to investigate the effect of BH4 related with NOS reaction in hepatic I/R injury.


      A model of 70% liver I/R injury with a 100-minute ischemic time was created in rats, and the non–ischemic lobes were then resected. The rats were given BH4 (BH4 group) or saline solution (saline group) before reperfusion. The specific inducible NOS blocker 1400W was used to evaluate the effect of endogenous inducible NOS in the I/R hepatic injury. Survival, nitric oxide products (nitrate and nitrite), NOS expression, and nitrotyrosine (ie, the peroxynitrite product) were measured after reperfusion.


      On day 7, the survival rate was 62.5% in the BH4 group, as opposed to 14.3% in the saline group (P = .0004); 1400W administration to the BH4 group decreased the survival rate to 0% (P = .003). BH4 prevented the significant increase in total bilirubin levels (P < .01) after 12-hour reperfusion. The increases in serum alanine transaminase levels (after 3 hours and 12 hours of reperfusion) were significantly (P < .01) attenuated in the BH4 group. BH4 increased the nitrate/nitrite concentrations in liver tissue (P < .05) and reduced nitrotyrosine production, and the protein assay showed that BH4 increased inducible NOS and endothelial NOS expression. Histologic examination of the liver revealed that BH4 mitigated the damage that was caused by liver I/R.


      Exogenous BH4 increased nitric oxide production, which attenuated the hepatic I/R injury.
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Surgery
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Mosher B.
        • Dean R.
        • Harkema J.
        • Remick D.
        • Palma J.
        • Crockett E.
        Inhibition of Kupffer cells reduced CXC chemokine production and liver injury.
        J Surg Res. 2001; 99: 201-210
        • Peralta C.
        • Prats N.
        • Xaus C.
        • Gelpí E.
        • Roselló-Catafau J.
        Protective effect of liver ischemic preconditioning on liver and lung injury induced by hepatic ischemia-reperfusion in the rat.
        Hepatology. 1999; 30: 1481-1489
        • Wanner G.A.
        • Ertel W.
        • Muller P.
        • Hofer Y.
        • Leiderer R.
        • Menger M.D.
        • et al.
        Liver ischemia and reperfusion induces a systemic inflammatory response through Kupffer cell activation.
        Shock. 1996; 5: 34-40
        • Pannen B.H.
        • Al-Adili F.
        • Bauer M.
        • Clemens M.G.
        • Geiger K.K.
        Role of endothelins and nitric oxide in hepatic reperfusion injury in the rat.
        Hepatology. 1998; 27: 755-764
        • Isobe M.
        • Katsuramaki T.
        • Hirata K.
        • Kimura H.
        • Nagayama M.
        • Matsuno T.
        Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver.
        Transplantation. 1999; 68: 803-813
        • Liu P.
        • Xu B.
        • Spokas E.
        • Lai P.S.
        • Wong P.Y.
        Role of endogenous nitric oxide in TNF-alpha and IL-1beta generation in hepatic ischemia-reperfusion.
        Shock. 2000; 13: 217-223
        • Palmer R.M.
        • Ferrige A.G.
        • Moncada S.
        Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
        Nature. 1987; 327: 524-526
        • Radomski M.W.
        • Palmer R.M.
        • Moncada S.
        Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets.
        Br J Pharmacol. 1987; 92: 181-187
        • Furlong B.
        • Henderson A.H.
        • Lewis M.J.
        • Smith J.A.
        Endothelium-derived relaxing factor inhibits in vitro platelet aggregation.
        Br J Pharmacol. 1987; 90: 687-692
        • Lefer A.M.
        • Tsao P.S.
        • Lefer D.J.
        • Ma X.L.
        Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia.
        FASEB J. 1991; 5: 2029-2034
        • Kurose I.
        • Wolf R.
        • Grisham M.B.
        • Granger D.N.
        Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide.
        Circ Res. 1994; 74: 376-382
        • Beckman J.S.
        • Beckman T.W.
        • Chen J.
        • Marshall P.A.
        • Freeman B.A.
        Apparent hydroxyl radical production by peroxynitrite.
        Proc Natl Acad Sci USA. 1990; 87: 1620-1624
        • Haddad I.Y.
        • Pataki G.
        • Hu P.
        • Galliani C.
        • Beckman J.S.
        • Matalon S.
        Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.
        J Clin Invest. 1994; 94: 2407-2413
        • Kinoshita H.
        • Milstien S.
        • Wambi C.
        • Katusic Z.S.
        Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery.
        Am J Physiol. 1997; 273: H718-H724
        • Kaufman S.
        Unsolved problems in diagnosis and therapy of hyperphenylalaninemia caused by defects in tetrahydrobiopterin metabolism.
        J Pediatr. 1986; 109: 572-578
        • Sakai N.
        • Kaufman S.
        • Milstein S.
        Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264).
        Mol Pharmacol. 1993; 43: 6-10
        • Werner-Felmayer G.
        • Werner E.R.
        • Fuchs D.
        • Hausen A.
        • Reibnegger G.
        • Schmidt K.
        • et al.
        Pteridine biosynthesis in human endothelial cells.
        J Biol Chem. 1993; 268: 1842-1846
        • Gross S.S.
        • Levi R.
        Tetrahydrobiopterin synthesis.
        J Biol Chem. 1992; 267: 25722-25729
        • Saura M.
        • Perez-Sala D.
        • Canada F.J.
        • Lamas S.
        Role of tetrahydrobiopterin availability in the regulation of nitric-oxide synthase expression in human mesangial cells.
        J Biol Chem. 1996; 271: 14290-14295
        • Tiefenbacher C.P.
        • Chilian W.M.
        • Mitchell M.
        • DeFily D.V.
        Restoration of endothelium-dependent vasodilation after reperfusion injury by tetrahydrobiopterin.
        Circulation. 1996; 94: 1423-1429
        • Rusche K.M.
        • Spiering M.M.
        • Marletta M.A.
        Reactions catalyzed by tetrahydrobiopterin free nitric oxide synthase.
        Biochemistry. 1998; 44: 15503-15512
        • Gorren A.C.
        • List B.M.
        • Schrammel A.
        • Pitters E.
        • Hemmens B.
        • Werner E.R.
        • et al.
        Tetrahydrobiopterin-free neuronal nitric oxide synthase.
        Biochemistry. 1996; 355: 16735-16745
        • Mayer B.
        • Pitters E.
        • Pfeiffer S.
        • Kukovetz W.R.
        • Schmidt K.
        A synthetic peptide corresponding to the putative dihydrofolate reductase domain of nitric oxide synthase inhibits uncoupled NADPH oxidation.
        Nitric Oxide. 1997; 1: 50-55
        • Stuehr D.J.
        Mammalian nitric oxide synthase.
        Biochim Biophys Acta. 1999; 1411: 217-230
        • Schmid R.A.
        • Hillinger S.
        • Walter R.
        • Zollinger A.
        • Stammberger U.
        • Speich R.
        • et al.
        The nitric oxide synthase cofactor tetrahydrobiopterin reduces allograft ischemia-reperfusion injury after lung transplantation.
        J Thorac Cardiovasc Surg. 1999; 118: 726-732
        • Kakoki M.
        • Hirata Y.
        • Hayakawa H.
        • Suzuki E.
        • Nagata D.
        • Tojo A.
        • et al.
        Effects of tetrahydrobiopterin on endothelial dysfunction in rats with ischemic acute renal failure.
        J Am Soc Nephrol. 2000; 11: 301-309
        • Hillinger S.
        • Sandera P.
        • Carboni G.L.
        • Stammberger U.
        • Zalunardo M.
        • Schoedon G.
        • et al.
        Survival and graft function in a large animal lung transplant model after 30 h preservation and substitution of the nitric oxide pathway.
        Eur J Cardiothorac Surg. 2000; 20: 508-513
        • Ishii M.
        • Shimizu S.
        • Nawata S.
        • Kiuchi Y.
        • Yamamoto T.
        Involvement of reactive oxygen species and nitric oxide in gastric ischemia-reperfusion injury in rats.
        Dig Dis Sci. 2000; 45: 93-98
        • Garvey E.P.
        • Oplinger J.A.
        • Furfine E.S.
        • Kiff R.J.
        • Laszlo F.
        • Whittlei B.J.
        • et al.
        1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo.
        J Biol Chem. 1997; 272: 4959-4963
        • Boer R.
        • Ulrich W.R.
        • Klein T.
        • Mirau B.
        • Haas S.
        • Baur I.
        The inhibitory potency and selectivity of arginine substrate site nitric-oxide syntherase inhibitors is solely determined by their affinity toward the different isoenzymes.
        Mol Pharmacol. 2000; 58: 1026-1034
        • Stuehr D.J.
        Structure function aspects in the nitric oxide synthases.
        Annu Rev Pharmacol Toxicol. 1997; 37: 339-359
        • Pou S.
        • Pou W.S.
        • Bredt D.S.
        • Snyder S.H.
        • Rosen G.M.
        Generation of superoxide by purified brain nitric oxide synthase.
        J Biol Chem. 1992; 267: 24173-24176
        • Mayer B.
        • Klatt P.
        • Werner E.R.
        • Schmidt K.
        Kinetics and mechanism of tetrahydrobiopterin-induced oxidation of nitric oxide.
        J Biol Chem. 1995; 270: 655-659
        • Scott-Burden T.
        Regulation of nitric oxide production by tetrahydrobiopterin.
        Circulation. 1995; 91: 248-250
        • Kimura H.
        • Katsuramaki T.
        • Isobe M.
        • Nagayama M.
        • Meguro M.
        • Kukita K.
        Role of inducible nitric oxide synthase in pig liver transplantation.
        J Surg Res. 2003; 111: 28-37
        • Meguro M.
        • Katsuramaki T.
        • Kimura H.
        • Isobe M.
        • Nagayama M.
        • Kukita K.
        Apoptosis and necrosis after warm ischemia-reperfusion injury of the pig liver and their inhibition by ONO-1714.
        Transplantation. 2003; 75: 703-710
        • Lee V.G.
        • Johnson M.L.
        • Baust J.
        • Laubach V.E.
        • Watkins S.C.
        • Billiar T.R.
        The roles of iNOS in liver ischemia-reperfusion injury.
        Shock. 2001; 16: 355-360
        • Meguro M.
        • Katsuramaki T.
        • Nagayama M.
        • Kimura H.
        • Isobe M.
        • Kimura Y.
        A novel inhibitor of inducible nitric oxide synthase (ONO-1714) prevents critical warm ischemia-reperfusion injury in the pig liver.
        Transplantation. 2002; 73: 1439-1446
        • Liu T.H.
        • Robinson E.K.
        • Helmer K.S.
        • West S.D.
        • Castaneda A.A.
        • Chang L.
        Does upregulation of inducible nitric oxide synthase play a role in hepatic injury?.
        Shock. 2002; 18: 549-554
        • Yagnik G.P.
        • Takahashi Y.
        • Tsoulfas G.
        • Reid K.
        • Murase N.
        • Geller D.A.
        Blockade of the L-arginine/NO synthase pathway worsens hepatic apoptosis and liver transplant preservation injury.
        Hepatology. 2002; 36: 573-581
        • Hsu C.M.
        • Wang J.S.
        • Liu C.H.
        • Chen L.W.
        Kupffer cells protect liver from ischemia-reperfusion injury by an inducible nitric oxide synthase-dependent mechanism.
        Shock. 2002; 17: 280-285
        • Rivera-Chavez F.A.
        • Toledo-Pereyra L.H.
        • Dean R.E.
        • Crouch L.
        • Ward P.A.
        Exogenous and endogenous nitric oxide but not iNOS inhibition improves function and survival of ischemically injured livers.
        J Invest Surg. 2001; 14: 267-273