Role of arginine in superficial wound healing in man I.B.J.G. Debats a , T.G.A.M. Wolfs b , T. Gotoh c , J.P.M. Cleutjens d , C.J. Peutz-Kootstra d , R.R.W.J. van der Hulst a, * a Department of Plastic, Reconstructive and Handsurgery, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands b Department of General Surgery, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands c Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan d Department of Pathology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands article info Article history: Received 11 March 2008 Revised 15 July 2009 Available online xxxx Keywords: Nitric oxide Arginine Wound healing Human Acute wounds Arginase NOS abstract Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies sug- gest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The lat- ter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute sur- gical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin. Ó 2009 Elsevier Inc. All rights reserved. Introduction Wound healing represents a complex process, initiated to re- store tissue damage. The amino acid arginine has been identified as an important mediator in this process [1]. Arginine is the sole precursor of nitric oxide (NO), a signal molecule, among others, in- volved in immune responses, angiogenesis, epithelialization and formation of granulation tissue, all essential aspects accompanying wound healing [2]. Nitric Oxide Synthase (NOS) converts arginine to NO and citrul- line. Three isoforms of NOS exist: neuronal NOS (NOS 1) and endo- thelial NOS (NOS 3) are constitutively expressed by neuronal and endothelial cells, respectively. Inducible NOS (NOS 2), is expressed in response to inflammatory cytokines and endotoxins, such as seen during wound repair. The beneficial effects of arginine supple- mentation on wound healing have been attributed to enhanced synthesis of NO by NOS [3–6]. Former studies with rodents showed that arginine-free diets impair wound healing, with decreased breaking strengths of incisions and collagen deposition in granula- tion tissue) [7,8], while supplementation of arginine increased hydroxyproline concentration, a marker of collagen synthesis [9– 12]. In addition, to its role as precursor of NO, arginine can be metabolized by arginase [13–17]. Two different isoforms of this enzyme have been identified: arginase-1 (ARG1) and arginase-2 (ARG2). The liver type, ARG1, is the cytosolic isoform. ARG2 is the mitochondrial isoform, located in kidney, prostate, small intes- tine and the breast. Arginase catalyses the conversion of arginine to ornithine and urea. Ornithine is an essential precursor for collagen and polyamines synthesis [18], both required for wound healing processes [19–25]. Moreover, arginase seems to influence the im- mune response, another important contributor involved in tissue repair. Activated macrophages and neutrophils (PMN’s) show in- creased NO-production for their anti-bacterial function. This NO- production is subsequently down-regulated by arginase through substrate competition [26–28]. 1089-8603/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2009.07.006 * Corresponding author. Address: University Hospital Maastricht, Department of Plastic and Reconstructive Surgery, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Fax: +31 43 3875485. E-mail address: RVH@spch.azm.nl (R.R.W.J. van der Hulst). Nitric Oxide xxx (2009) xxx–xxx Contents lists available at ScienceDirect Nitric Oxide journal homepage: www.elsevier.com/locate/yniox ARTICLE IN PRESS Please cite this article in press as: I.B.J.G. Debats et al., Role of arginine in superficial wound healing in man, Nitric Oxide (2009), doi:10.1016/ j.niox.2009.07.006