Journal of Neuroscience Methods 164 (2007) 155–160 Determination of cerebrospinal fluid concentrations of arginine and dimethylarginines in patients with subarachnoid haemorrhage Jens Martens-Lobenhoffer a,∗ , Endre Sulyok b , Endre Czeiter c , Andras B ¨ uki c , Jana Kohl d , Raimund Firsching d , Uwe Tr¨ oger a , Stefanie M. Bode-B¨ oger a a Institute of Clinical Pharmacology, Otto-von-Guericke University Magdeburg, Leipziger Straße 44, 39120 Magdeburg, Germany b Department of Health Promotion and Family Care, Faculty of Health Sciences, P´ ecs University, Hungary c Department of Neurosurgery, Faculty of Medicine, P´ ecs University, Hungary d Department for Neurosurgery, Otto-von-Guericke University, Magdeburg, Germany Received 1 February 2007; received in revised form 2 April 2007; accepted 6 April 2007 Abstract Elevated cerebrospinal fluid (CSF) concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), are assumed to be related to delayed vasospasm after subarachnoid haemorrhage (SAH). However, data on CSF concentrations of l-arginine, ADMA and its structural isomer symmetric dimethylarginine (SDMA) are very sparse in humans. We here present a new hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS–MS) method for the precise determination of these substances in CSF. The method requires only minimal sample preparation and features isotope labeled internal standards. First data of patients with SAH showed that on the day of admission CSF concentration values of l-arginine and ADMA were not significantly different from controls, but increased markedly during the course of the hospital stay. The decrease of the l-arginine to ADMA ratio points to a progressive impairment of the NO production rate in the brain after SAH which is confirmed by a simultaneous decrease in nitrate and nitrite concentrations in CSF. © 2007 Elsevier B.V. All rights reserved. Keywords: CSF; Traumatic brain injury; Subarachnoid haemorrhage; l-Arginine; ADMA; SDMA 1. Introduction Nitric oxide (NO) is a cell signaling substance of paramount importance in various tissues. In vascular endothelial cells NO is synthesized by the enzyme endothelial nitric oxide synthase (eNOS) and it is essential for the regulation of vascular tone and endothelial function (Cooke and Dzau, 1997). In neurons it is synthesized by the enzyme neuronal nitric oxide syn- thase (nNOS) and is involved in the processes of learning and memory (Fedele and Raiteri, 1999). In contrast to these two constitutive isoenzymes, a further isoform of NOS (iNOS) is inducible by immune responses of the organism (Cooke and Dzau, 1997). In all cases, l-arginine (Arg) is the substrate for the NOS enzyme family, being converted into citrulline and NO. The NOS enzyme family can be competitively inhibited ∗ Corresponding author. Tel.: +49 391 6713068; fax: +49 391 6713062. E-mail address: jens.martens-lobenhoffer@medizin.uni-magdeburg.de (J. Martens-Lobenhoffer). by the Arg metabolite asymmetric dimethylarginine (ADMA) (Bode-B¨ oger et al., 1996). ADMA is eliminated mainly by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) to produce dimethylamine and citrulline (Ogawa et al., 1989). In this way, Arg, ADMA, NOS and DDAH regulate the production rate of NO, whereas elevated ADMA levels lead to deficiency in NO production (MacAllister et al., 1996). In plasma, elevated ADMA concentrations are an established risk marker for a range of vascular diseases (Cooke, 2004). However, reports regarding ADMA concentrations in cerebrospinal fluid (CSF) are sparse. For example, in patients suffering from multiple sclerosis, CSF ADMA levels are elevated (Conte et al., 2005), whereas the uri- nary excretion of ADMA and SDMA in such patients seems to be reduced (Rawal et al., 1995). A main focus of current research is on CSF ADMA levels after subarachnoid haemor- rhage (SAH), a disease pattern that may follow a traumatic brain injury or non-traumatic vascular rupture, for example, in the case of a cerebral aneurysm. A severe complication of SAH is the delayed onset of cerebral vasospasm. It developed in 30–70% of the patients after both non-traumatic (Pluta, 2005) and traumatic 0165-0270/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2007.04.005