Eur Respir J 2005; 25: 62–68 DOI: 10.1183/09031936.04.00086104 CopyrightßERS Journals Ltd 2005 Oral L-arginine supplementation in cystic fibrosis patients: a placebo-controlled study H. Grasemann*, C. Grasemann#, F. Kurtz*, G. Tietze-Schillings", U. Vester* and F. Ratjen* ABSTRACT: Exhaled nitric oxide (eNO) is decreased in cystic fibrosis (CF). The effect of oral L- AFFILIATIONS arginine, the precursor of enzymatic nitric oxide (NO) formation, on airway NO in patients with CF *Children’s Hospital #Dept of Human Genetics was studied. "Dept of Pharmacy, University of -1 In a pilot study, oral L-arginine was given in a single dose of 200 mg?kg body weight to eight Duisburg-Essen, Essen, Germany. healthy controls and eight CF patients. Subsequently, the same L-arginine dose was given to 10 patients with CF (five females) t.i.d. for 6 weeks in a randomised double-blind placebo-controlled CORRESPONDENCE H. Grasemann crossover study. Children’s Hospital A single dose of oral L-arginine resulted in a 5.5-fold increase of L-arginine in plasma and a 1.3- University of Essen fold increase of L-arginine in sputum after 2 h. Maximum eNO, within 3 h of L-arginine intake, Hufeland Str. 55 increased significantly in both CF patients (5.4¡2.1 ppb versus 8.3¡3.5 ppb) and controls D-45122 Essen Germany ¡ ¡ L (18.0 8.1 ppb versus 26.4 12.3 ppb). Supplementation of -arginine for 6 weeks resulted in a Fax: 49 2017235983 sustained increase in eNO compared to placebo (9.7¡5.7 ppb versus 6.3¡3.1 ppb). An effect of E-mail: [email protected] L-arginine supplementation on forced expiratory volume in one second was not observed. These data indicate that airway nitric oxide formation in cystic fibrosis patients can be Received: July 21 2004 L augmented with oral -arginine supplementation. Accepted after revision: September 1 2004 KEYWORDS: Cystic fibrosis, L-arginine, lung function, nitric oxide SUPPORT STATEMENT This study was supported by Mukoviszidose e.V. he amino acid L-arginine is the precursor tracheas from mice deficient for the cystic fibrosis for enzymatic nitric oxide (NO) synthesis. transmembrane conductance regulator (CFTR) T NO synthases (NOSs) have been shown to protein but not from wild-type animals [8]. In be involved in regulatory processes in the lung cross-sectional studies in CF patients, a positive including inflammation, host defense and correlation was observed between pulmonary bronchomotor control. The fraction of exhaled function and both eNO [1, 9, 10] and NO NO (eNO) has been found to be reduced in metabolite concentration in sputum [11], suggest- patients with cystic fibrosis (CF) compared to ing that pulmonary function is reduced in CF healthy individuals [1]. The mechanisms that patients with decreased airway NO formation. lead to low airway NO in CF are not completely This view is supported by the recent observation understood but may include mechanical reten- that variants in the neuronal NOS gene (NOS1), tion of NO in airway secretions, conversion of that are associated with low eNO, seem to NO to metabolites, and consumption of NO by predispose to a more rapid decline in pulmonary denitrifying bacteria [2]. Whilst limited evidence function in children with CF [12]. would support all of these concepts, there is also In previous studies, the current authors demon- evidence for reduced enzymatic formation of strated that neither inhalation of NO-containing NO in CF, whereby blood or tissue levels of gas nor infusion of L-arginine had an effect on L-arginine may become rate limiting for NO pulmonary function in CF patients. However, synthesis [3, 4]. a single dose of i.v. L-arginine in a dose of The bronchodilatatory effect of NO (in ppm 500 mg?kg-1 body weight resulted in a significant concentrations) is well known [5–7], and recent increase of eNO in these patients [13, 14]. Oral L- research in animals suggests a causal relationship arginine in doses of 100 mg?kg-1 and 200 mg?kg-1 between airway NOS activity and bronchomotor body weight had been shown to increase eNO in tone in CF. In these experiments, the addition of healthy human subjects [15]. The effect of oral European Respiratory Journal L-arginine resulted in a significant increase of L-arginine supplementation in CF patients has, Print ISSN 0903-1936 electrical field stimulated relaxation of isolated to the present authors’ knowledge, not been Online ISSN 1399-3003 62 VOLUME 25 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL H. GRASEMANN ET AL. ORAL L-ARGININE IN CYSTIC FIBROSIS studied. The current authors speculated that augmentation of corticosteroids at the time of study. All patients included were airway NO formation by oral supplementation of L-arginine in a clinically stable condition after a 2-week treatment period could have a positive effect on airway NO formation and with i.v. antibiotics. pulmonary function in these patients. Therefore, the present authors studied the effect of a single dose of oral L-arginine on Study design eNO and a 6-week supplementation of oral L-arginine in a dose The study was conducted in a randomised double-blind cross- of 200 mg?kg-1 body weight t.i.d. on eNO and pulmonary over design. Patients were either treated with 200 mg?kg-1 function in patients with CF in a randomised double-blind body weight t.i.d. of oral L-arginine for 6 weeks followed by 6 placebo-controlled study. weeks of placebo or vice versa. Measurements of nasal and eNO, as well as pulmonary function, were performed at PATIENTS AND METHODS baseline and after each 6-week treatment period. Study visits The diagnosis of CF in participating patients had been were scheduled in the morning and measurements were confirmed by repeated sweat tests with chloride concentrations performed prior to L-arginine and placebo intake that day, -1 .60 mmol?L , and by mutation analysis of the CFTR gene. respectively. Written informed consent was obtained from each patient, and/or their parents, respectively. The study was approved by Pulmonary function measurements the institutional review board of the University of Essen, Essen, Pulmonary function tests were performed with a bell-spirom- Germany. eter (Volugraph, Mijnhardt, Bunnik, the Netherlands). Results were expressed as percentages of normal reference values Single dose experiments [16, 17]. Subjects Eight patients (five females) with CF were included in this part Nitric oxide measurements ¡ of the study. Mean age was 22 yrs (range 14–37 yrs), mean SD NO measurements were performed according to published ¡ forced vital capacity (FVC) was 68.0 14.5% (range 50–91%) ATS and ERS standards [18, 19]. NO was measured with a and mean forced expiratory volume in one second (FEV1) was chemiluminescence analyser (NOA 280; Sievers, Boulder, CO, ¡ 41.8 21.3% (range 22–75%) of predicted values. Seven patients USA). Nasal NO measurements were performed as recently had chronic colonisation of the airways with Pseudomonas published [18, 20]. Briefly, air from total lung capacity had to aeruginosa and one was colonised with Staphlococcus aureus. be exhaled orally against a flow restrictor to generate a mouth None of the patients received systemic or inhaled corticoster- pressure exceeding 5 cm of H2O for soft palate closure. Air was oids at the time of study. All patients included were in a aspirated from one nostril through a tight fitting nasal olive at clinically stable condition. CF patients were compared to eight a constant sample flow of 50 mL?s-1 generated by an external (five females) healthy nonsmoking controls with a mean age of pump. The other nostril was left open or, at room NO 28 yrs (range 23–44 yrs), who had no history of asthma or concentrations .10 ppb, connected to a reservoir of NO-free atopy, and had no symptoms of upper or lower airway air. The mean of two NO plateau measurements for each infection at the time of study. nostril was used for data analysis. Exhaled NO was measured using single breath on-line measurements for the assessment Study design of lower airway NO [18, 19]. Measurements of eNO were L-arginine (Synopharm, Barsbu¨ ttel, Germany) was diluted with performed at a constant expiratory flow of 3 L?min-1. The indifferent viscous flavouring solution to a 10% L-arginine mean value of three end-expiratory NO levels was calculated solution. This solution was bottled and kept refrigerated until for each subject. it was used. At the time of study each subject ingested the 10% L-arginine solution in a dose of 200 mg of L-arginine per kg Plasma amino acids body weight, diluted with orange juice to a total volume of Plasma samples were deproteinised within 30 min of collec- ,250 mL. tion. Amino acids were determined by ion exchange chroma- Measurements of nasal NO, eNO and blood sampling were tography (IEC) on an amino acid analyser LC 3000 (Eppendorf, performed at baseline as well as 60, 120 and 180 min following Hamburg, Germany) according to the manufacturer’s specifi- the intake of L-arginine. Blood was collected in EDTA cations. Sputum samples were diluted 1:1 with buffer, containing 2.7 mL tubes by venipuncture for the measurement vortexed for 2 min followed by 10 min of centrifugation at of plasma amino acid and urea concentrations in all subjects. 1,000 g for 10 min. Clear supernatant was used for the amino Spontaneously expectorated sputum was collected before and acid IEC. 120 min after L-arginine intake in all but two patients, who were unable to expectorate sputum. Statistical analysis Data were expressed as mean¡SD.