Circulating interleukin-18 concentrations and a loss-of-function P2X7 polymorphism in heart failure Guy D. Eslick a,b , Binu V. Thampan a , Marek Nalos c , Anthony S. McLean c , Ronald Sluyter a, ⁎ a Department of Medicine, Nepean Clinical School, The University of Sydney, Nepean Hospital, Penrith, New South Wales, Australia b School of Public Health, The University of Sydney, Sydney, New South Wales, Australia c Department of Intensive Care Medicine, Nepean Hospital, Penrith, New South Wales, Australia Received 24 March 2008; Accepted 9 May 2008 Available online 3 August 2008 Abstract We examined if a loss-of-function polymorphism in the P2X 7 receptor (1513C) corresponded with circulating interleukin(IL)-18 concentrations in heart failure (HF) patients. IL-18 values were significantly elevated in HF subjects compared to healthy control subjects. No association was seen between the polymorphism and IL-18 concentrations in HF patients. In HF patients, IL-18 values had an inverse relationship with ejection fraction, mean arterial pressure and body mass index, while high IL-18 concentrations were associated with increased mortality. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Heart failure; IL-18; Interleukin-18; P2X7; Polymorphism ⁎ Corresponding author. School of Biological Sciences, The University of Wollongong, Wollongong, NSW 2522, Australia. Tel.: +61 2 4221 5508; fax: +61 2 4221 4135. E-mail address: rsluyter@uow.edu.au (R. Sluyter). Heart failure (HF) represents a major health problem in many developed nations including Australia [1]. Circulating interleukin(IL)-18 is elevated in HF patients with concentra- tions directly proportional to disease severity [2–4]. However, in a proportion of HF patients, IL-18 con- centrations are similar to those measured in normal subjects. The reason for this variation remains unknown. We have previously shown that a single nucleotide polymorphism in the human P2RX7 gene (A1513C), which substitutes alanine for glutamic acid at residue 496 in the P2X 7 receptor, when present in homozygous dosage impairs ATP-induced release of IL-18 from monocytes and in whole blood [5]. We hy- pothesized that this polymorphism may reduce circulating IL-18 concentrations and thereby mitigate disease severity in HF patients. Thus, a pilot study was conducted to com- pare circulating IL-18 concentrations, P2RX7 genotype and disease severity in HF patients. We studied 30 patients (mean age 62 years, 21 male) with acute or chronic HF admitted to Nepean Hospital's Intensive Care Unit or Cardiology Ward, and 16 healthy controls from hospital staff (mean age 49 years, 10 male). Patient clinical information was obtained by a retrospective review of medi- cal records. Peripheral blood was collected into serum- and EDTA-containing vacutainer tubes, placed on ice for 30 min, centrifuged at 1700 g for 15 min at 4°C and cell-free super- natants stored at - 80°C. Samples were analysed using an IL-18 ELISA kit (Medical and Biological Laboratories, Nagoya, Japan). Exon 13 of the P2RX7 gene was amplified by PCR [6] and the genotype at nucleotide position 1513 determined by restriction enzyme digestion (Hha I; New England Biolabs, Beverly, MA) or sequencing. Data, presented as means and ranges, was collected in a blinded fashion and analysed using Stata 9 (StataCorp LP, College Station, TX). The alpha level of significance was p b 0.05. Previous studies have demonstrated that serum or plasma IL-18 concentrations in HF patients are elevated compared to healthy controls [2–4]. To determine if IL-18 values alter depending on the sample choice, we compared IL-18 concentrations in serum and EDTA plasma from 6 control and 6 HF subjects. IL-18 concentrations in serum (350 pg/ml, 93–1042 pg/ml) and plasma samples (312 pg/ ml, 87–1003 pg/ml) were similar and not statistically signifi- cant (paired t test, p N 0.05). Moreover, there was a signifi- cant correlation between the serum and plasma IL-18 values (Spearman's bivariate correlation test, r = 0.9231, p b 0.0001). Since IL-18 concentrations were comparable in either sam- ple type and because the largest previous studies used serum [2,3] we compared serum IL-18 concentrations in HF and control subjects. Similar to other studies [2–4], the mean circulating IL-18 concentration for HF patients (517 pg/ml, 179–855 pg/ml) was significantly higher than that of control subjects (217 pg/ml, 134–408 pg/ml) (Mann Whitney U test, p = 0.0054) (Fig. 1A). We then compared the allele frequency of the 1513C polymorphism in control and HF subjects, and determined 81 Letters to the Editor