REVIEW ARTICLE Anesthesiology 2010; 113:1460–75 Copyright © 2010, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins David S. Warner, M.D., Editor Nitrate-Nitrite-Nitric Oxide Pathway Implications for Anesthesiology and Intensive Care Eddie Weitzberg, M.D., Ph.D.,* Michael Hezel, Ph.D.,† Jon O. Lundberg, M.D., Ph.D.‡ ABSTRACT pects in health and disease. In this article, we present an The gaseous radical nitric oxide is involved in numerous overview of this field of research with emphasis on relevance physiologic and pathophysiological events important in an- in anesthesiology and intensive care. esthesiology and intensive care. Nitric oxide is endogenously generated from the amino acid L-arginine and molecular ox- ITROGEN (N) is a key component of DNA, RNA, ygen in reactions catalyzed by complex nitric oxide synthases. and proteins, which makes it essential for all living Recently, an alternative pathway for nitric oxide generation N organisms. In the form of nitrogen gas (N2), it is the most was discovered, wherein the inorganic anions nitrate Ϫ Ϫ abundant element in the atmosphere, and thereby the largest (NO3 ) and nitrite (NO2 ), most often considered inert pool of nitrogen on Earth. However, atmospheric nitrogen end products from nitric oxide generation, can be reduced would be of no biologic use if not for the nitrogen cycle. As a back to nitric oxide and other bioactive nitrogen oxide spe- first step in this cycle, atmospheric nitrogen undergoes fixa- cies. This nitrate-nitrite-nitric oxide pathway is regulated dif- tion, a process in which nitrogen gas is converted to ammo- ferently than the classic L-arginine-nitric oxide synthase ni- ϩ nium (NH4 ). Ammonium can then be oxidized to a variety tric oxide pathway, and it is greatly enhanced during hypoxia Ϫ of nitrogen oxides, including nitrite (NO2 ) and nitrate and acidosis. Several lines of research now indicate that the Ϫ (NO3 ). The cycle is completed by the denitrification pro- nitrate-nitrite-nitric oxide pathway is involved in regulation cess where nitrate is serially reduced to nitrite, nitric oxide, of blood flow, cell metabolism, and signaling, as well as in nitrous oxide, and, finally, nitrogen gas (N ), which diffuses tissue protection during hypoxia. The fact that nitrate is 2 back into the atmosphere. Bacteria play an essential role in abundant in our diet gives rise to interesting nutritional as- the nitrogen cycle because they are equipped with metabolic machineries suitable for catalyzing its different steps. In the anaerobic denitrification part of the nitrogen cycle, nitrate, * Professor, † Postdoctoral Researcher, Section of Anesthesiology nitrite, and nitric oxide are substrates for specific bacterial and Intensive Care, ‡ Professor, Section of Pharmacology, Depart- ment of Physiology and Pharmacology, Karolinska Institutet, Stock- reductases, and the bacteria use these nitrogen oxides as ter- holm, Sweden. minal electron acceptors for respiration or for incorporation Received from the Department of Physiology and Pharmacology, in biomass. The description of the nitrogen cycle serves as a Section of Anesthesiology and Intensive Care, Karolinska Institutet, relevant prologue to this review because some steps in this Stockholm, Sweden. Submitted for publication August 21, 2010. Accepted for publication September 2, 2010. Support was provided cycle also occur in mammals, where again, bacteria play a by the Swedish Research Council, Torsten and Ragnar So¨derbegs crucial role. Foundation, Swedish Heart and Lung Foundation, Stockholm City Council, Vinnova (Chronic Inflammation Diagnosis and Theraphy The formation of nitrogen oxides by prokaryotes has been CIDaT), Karolinska Institutet (all in Stockholm, Sweden) and Euro- known for more than a century, but it is only during the last pean Union 7th Framework Program (Flaviola), Brussels, Belgium. decades that it has become clear that generation and metab- Dr. Weitzberg and Dr. Lundberg are named coinventors on patent applications (U.S. patent application nos. 12/528.794 and 12/528.798) olism of nitrogen oxides also occur in eukaryotic cells. In relating to therapeutic use of inorganic nitrate and nitrite salts. The 1916, Mitchell et al. observed that humans excrete more figures and tables in this article were prepared by Annemarie B. nitrate than they ingest, but at that time, they could only Johnson, C.M.I., Medical Illustrator, Wake Forest University School of 1 Medicine Creative Communications, Wake Forest University Medical speculate on the mechanisms. In 1981, Green et al. used Center, Winston-Salem, North Carolina. completely germ-free animals to demonstrate a net produc- Address correspondence to Dr. Weitzberg: Department of Physiology tion of nitrate, independent of bacteria, as solid evidence of and Pharmacology, Section of Anesthesiology and Intensive Care, Karo- mammalian nitrate biosynthesis.2 At approximately the same linska Institutet, S-171 77 Stockholm, Sweden. [email protected]. This article may be accessed for personal use at no charge through the Journal time, a series of seminal studies were published, eventually Web site, www.anesthesiology.org. leading to the identification of nitric oxide as a major secre- 1460 Anesthesiology, V 113 • No 6 • December 2010 Nitrate-Nitrite-Nitric Oxide Pathway tory product of mammalian cells. In 1980, Furchgott and Diet Zawadzki identified an endothelium-derived relaxing factor that was later recognized as nitric oxide.3–5 Specific nitric oxide synthases (NOSs) were described that use the N-gua- Bacterial nitrate reductases - Mammalian nitrate reductases NO3 Oxidation nidino nitrogen of L-arginine with molecular oxygen in a (Xanthineoxidase and other?) complex five-step oxidation process to generate nitric oxide.6 Previously, Ferid Murad et al. described that organic nitrates, - - such as nitroglycerine, induce vasodilation by release of nitric NO2 Oxyhemoglobin NO2 oxide, activating soluble guanylyl cyclase and subsequent cy- clic guanosine monophosphate formation.7 These discover- Deoxyhemoglobin/myoglobin Ceruloplasmin Neuroglobin NO Oxygen ies rendered Robert Furchgott, Louis Ignarro, and Ferid Mu- Xanthine oxidoreductase (XOR) Respiratory chain enzymes rad the Nobel Prize in Physiology or Medicine in 1998. It has Endothelial NO synthase Aldehyde oxidase now been established that nitric oxide regulates a vast num- Carbonic anhydrase Protons ber of physiologic processes ranging from vasodilation to VItamin C, polyphenols NOS memory.6 Nitric oxide signaling is partly regulated by the short half-life (milliseconds) in biologic systems because it is Fig. 1. A schematic presentation of a mammalian nitrogen rapidly oxidized to nitrite and nitrate. For this reason, these cycle. Nitric oxide (NO) is generated by nitric oxide synthases inorganic anions have been considered merely as stable-end (NOS) in most cells of the body and participates in regulation of numerous physiologic functions. The bioactivity of nitric metabolites from nitric oxide production, and the scientific Ϫ oxide is partly regulated by its rapid oxidation to nitrite (NO2 ) interest in these anions has primarily been as markers of NOS Ϫ or, in the presence of oxyhemoglobin, to nitrate (NO3 ). activity. Nitrate is the predominant nitric oxide oxidation product in In 1994, two independent groups demonstrated forma- the circulation. In our bodies, nitrate can undergo reduction 8,9 tion of nitric oxide that was independent of NOS. Nitric to nitrite, and this process is strongly dependent on oral oxide gas was generated in the human stomach at high con- commensal bacteria but also to some extent by xanthineoxi- centrations, and this production was dependent on gastric dureductase and possibly other enzymes in tissues. In blood acidity and involved reduction of salivary-derived nitrite. and tissues, nitrite can be further reduced to nitric oxide and Furthermore, nitric oxide generation was greatly enhanced other bioactive nitrogen oxides. There are several enzymatic 8 and nonenzymatic routes that can catalyze this reduction, after intake of nitrate. An enterosalivary circulation of in- most of which are greatly enhanced under hypoxic condi- gested nitrate exists in which, after absorption in the gastro- tions. This mammalian nitrogen cycle can be fueled by the intestinal tract, circulating nitrate is actively secreted in saliva diet because vegetables contain high amounts of inorganic and oral commensal bacteria reduced nitrate to nitrite.10 A nitrate. Modified with permission from reference 15. year later, Zweier et al. demonstrated NOS-independent ni- tric oxide generation from nitrite in the ischemic heart, including regulation of blood flow,18 platelet function,19 thereby extending the occurrence of nitrite reduction outside pulmonary function,20 nerve transmission,21 host defense,22 the gastrointestinal tract.11 From that time, a nitrate-nitrite- metabolic control,23 anesthetic action,24 and pain.25 This nitric oxide pathway has been established in which serial review will describe the current knowledge of the nitrate- reduction of nitrate and nitrite generates nitric oxide and nitrite-nitric oxide pathway with special focus on relevance other bioactive nitrogen oxides throughout the body (fig. to the anesthesiologist and intensive care physician. 1).12–14 This pathway is suggested to be involved in many important biologic processes, including regulation of blood The Classic L-Arginine-NOS-Nitric Oxide flow, cell signaling, and energetic, as well as