(6R)-5,6,7,8-Tetrahydro-L-Biopterin and Its Stereoisomer Prevent Ischemia Reperfusion Injury in Human Forearm Lila Mayahi, Simon Heales, David Owen, Juan P. Casas, Joanne Harris, Raymond J. MacAllister, Aroon D. Hingorani Objective— 6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4) is a cofactor for endothelial nitric oxide synthase but also has antioxidant properties. Its stereo-isomer 6S-5,6,7,8-tetrahydro-L-biopterin (6S-BH4) and structurally similar pterin 6R,S-5,6,7,8-tetrahydro-D-neopterin (NH4) are also antioxidants but have no cofactor function. When endothelial nitric oxide synthase is 6R-BH4 – deplete, it synthesizes superoxide rather than nitric oxide. Reduced nitric oxide bioavailability by interaction with reactive oxygen species is implicated in endothelial dysfunction (ED). 6R-BH4 corrects ED in animal models of ischemia reperfusion injury (IRI) and in patients with cardiovascular risks. It is uncertain whether the effect of exogenous 6R-BH4 on ED is through its cofactor or antioxidant action. Methods and Results—In healthy volunteers, forearm blood flow was measured by venous occlusion plethysmography during intra-arterial infusion of the endothelium-dependent vasodilator acetylcholine, or the endothelium-independent vasodilator glyceryl trinitrate, before and after IRI. IRI reduced plasma total antioxidant status (P0.03) and impaired vasodilatation to acetylcholine (P0.01), but not to glyceryl trinitrate (P0.3). Intra-arterial infusion of 6R-BH4, 6S-BH4 and NH4 at approximately equimolar concentrations prevented IRI. Conclusion—IRI causes ED associated with increased oxidative stress that is prevented by 6R-BH4, 6S-BH4, and NH4, an effect mediated perhaps by an antioxidant rather than cofactor function. Regardless of mechanism, 6R-BH4, 6S-BH4, or NH4 may reduce tissue injury during clinical IRI syndromes. (Arterioscler Thromb Vasc Biol. 2007;27:1334-1339.) Key Words: 6R-5,6,7,8-tetrahydro-L-biopterin  antioxidant  endothelial dysfunction  ischemia  reperfusion 6 R-5,6,7,8-terahydro-L-biopterin (6R-BH4) is an essential cofactor for endothelial nitric oxide synthase (NOS) that catalyzes production of the vasodilator and atheroprotective mediator NO. 1,2 Biosynthesis of 6R-BH4 occurs in the endothelium, promoting NO synthesis by facilitating electron transfer from the reductase domain of NOS to arginine. 6R-BH4 also stabilizes the NOS dimer and exerts an alloste- ric effect, enhancing substrate binding. 3 Changes in 6R-BH4 availability influence NO production. A reduction in 6R-BH4 leads not only to a diminished NO synthesis but also to the generation of NOS-derived superoxide (O 2  ) that has been implicated in the development of endothelial dysfunction. 4 Ischemia reperfusion injury (IRI), such as occurs after thrombolysis or balloon angioplasty, is associated with endo- thelial dysfunction, which may contribute to cellular damage. The mechanism underlying endothelial dysfunction in IRI is incompletely understood but adhesion of activated neutro- phils to endothelial cells, 5 an increase in oxygen radical generation, 6,7 the elaboration of inflammatory cytokines, and a reduction in NO production 8,9 are all believed to play a role. In humans, local delivery of 6R-BH4 by intra-arterial infusion improves endothelium dependent vasodilation in patients with coronary artery disease, 10 type II diabetes mellitus, 11 elevated cholesterol, 12 raised blood pressure, 13 and in smokers, 14 leading to the proposal that an acquired defi- ciency in 6R-BH4 and defective NOS catalysis underlies endothelial dysfunction in these states. Supplementation with 6R-BH4 also reduces IRI in the rat heart 15 and kidney 16 and in the pig heart. 17 In animals, the administration of antioxi- dants such as ascorbic acid or N-acetylcysteine reduces endothelial dysfunction induced by IRI. 18 –20 Therefore, the effect of 6R-BH4 on endothelial function in IRI could also be mediated by an antioxidant action, 21 a property shared with its stereoisomer 6S-5,6,7,8-tetrahydro- L-biopterin (6S-BH4), 22 as well as with 6R,S-5,6,7,8- tetrahydro-D-neopterin (NH4), 14 rather than by correction of defective NOS catalysis. Although approximately equipotent as antioxidants, 14,22 only 6R-BH4 has substantial capacity to support NOS catalysis. 23,24 We therefore examined, for the first time, the effect of 6R-BH4 on endothelial dysfunction during IRI in humans and tested whether this was mediated through correction of 6R-BH4 deficiency and defective NOS catalysis or through an antioxidant action. Original received November 17, 2006; final version accepted March 15, 2007. From Centre for Clinical Pharmacology (L.M., D.O., J.P.C., J.H., R.J.M., A.D.H.), University College London, London, UK; Neurometabolic Unit (S.H.), Hospital for Neurology and Neurosurgery (UCLH Foundation Trust), Queen Square, London, UK. Correspondence to Dr Lila Mayahi, Centre for Clinical Pharmacology, BHF laboratories, Department of Medicine, UCL, 5 University Street, London, UK, WC1E 6JJ. E-mail l.mayahi@ucl.ac.uk © 2007 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/ATVBAHA.107.142257 1334 Downloaded from http://ahajournals.org by on August 15, 2022