Quantitative analysis of nociceptin in blood of patients with acute and chronic pain Myoung-Hwan Ko, Yun-Hee Kim, Ran-Sook Woo 1 and Kee-Won Kim 1,CA Department of Rehabilitation Medicine and Research Institute of Clinical Medicine; 1 Department of Pharmacology, Institute for Medical Sciences, Chonbuk National University Medical School, Keumam-dong, Chonju, Chonbuk 561-180, Republic of Korea CA Corresponding Author Received 18 June 2002; accepted 10 July 2002 This study aimed to de¢ne the change of serum level of nociceptin in pain patients. Seventy pain patients and 20 normal healthy subjects were enrolled. Patients were divided into three groups ac- cording to the duration of pain; (1) acute (within 4 weeks), (2) subacute (4 weeks to 6 months), and (3) chronic (4 6 months) state. Serum concentration of nociceptin was measured by radioimmunoassay. Serum nociceptin level was signi¢cantly higher in the patients with pain than in normal healthy subjects ( p o 0.01). Furthermore, nociceptin level was signi¢cantly higher in the chronic pain group than acute group ( p o 0.05). Ser- um nociceptin level has a relationship with the existence of pain and its duration. NeuroReport 13:1631^1633 c 2002 Lippincott Williams & Wilkins. Key words: Acute pain; Chronic pain; Human blood; Nociceptin; Radioimmunoassay INTRODUCTION Nociceptin (also named orphanin FQ) is an endogenous agonist for the opioid receptor-like 1 receptor (ORL 1 ) [1,2]. The peptide and its receptor are widely distributed in the brain and spinal cord, and may have important roles in numerous physiological func- tions, such as modulation of nociceptin, locomotor activity, attenuation of stress response and stimulation of food intake [3,4]. Nociceptin exerts its broad spectrum of pharmacological actions through ORL 1 by inhibition of adenylyl cyclase, blockade of voltage-sensitive Ca 2þ channels, and enhancement of K þ conductance [5]. Inhibition of various neurotransmitter release, includ- ing pain related peptides, has been demonstrated [6,7]. There are conflicting results concerning the effects of nociceptin on nociception, i.e. hyperalgesia, reversal of opioid analgesia, no effect or analgesia [8]. In chronic pain models, supraspinally administered nociceptin acts as an anti-opioid peptide [9], while intrathecal nociceptin shows an anti-hyperalgesic effect [10]. In clinical studies, plasma concentration of NC was not changed in women with labor pain [11], and was decreased in females with fibromyalgia syndrome [12]. The role of nociceptin and any clinical significance of this peptide on pain processing in human remains to be explored. In the present study, we have determined the serum concentration of nociceptin in human subject and revealed the difference of its concentration between acute and chronic pain subjects. MATERIALS AND METHODS Subjects: Twenty healthy volunteers (10 men, 10 women; mean age 43; range 23–65) and 70 pain patients (mean age 45; range 19–77) were enrolled. Prior to obtaining written consent, the Ethics Committee of Chonbuk National Uni- versity Hospital approved this study. Patients were divided into three groups according to the duration of pain; (1) acute (within 4 weeks, n ¼ 30), (2) subacute (4 weeks to 6 months, n ¼ 20), and (3) chronic (4 6 months, n ¼ 20) state. The causes of pain included musculoskeletal diseases, cholecys- titis, urinary stone, non-specific chest pain. Subjective degree of pain was assessed by visual analog scale (VAS) and McGill pain questionnaire (MPQ). Blood sampling and radioimmunoassay of nociceptin: Three milliliters of whole blood were withdrawn from each subject at 09.00–10.00 h. Blood samples centrifuged at 2000  g for 20 min at 41C and 1 ml serum was obtained. Serum was loaded onto C-18 Sep-Pak cartridges and washed with 0.1% trifluroacetic acid (TFA). Nociceptin was eluted with 3 ml 60% acetonitrile in 0.1% TFA then freeze dried. Radioimmunoassay of nociceptin was per- formed using a commercially available kit (Phoenix Phar- maceuticals, CA, USA). Briefly, the lyophilized samples were reconstituted with assay buffer of 19 mM mono-basic and 81 mM dibasic sodium phosphate (pH 7.4). Rabbit anti- N/OFQ serum (100 ml) was added to each tube, and samples were incubated overnight at 41C. [ 125 I][Tyr 1 ]-N/OFQ (8000– 10 000 c.p.m./100 ml) was added to each and again incubated 0959-4965  c Lippincott Williams & Wilkins Vol 13 No 13 16 September 2002 1631 SOMATOSENSORY SYSTEMS, PAIN NEUROREPORT