- Email: [email protected]
Nociceptin and urotensin-II concentrations in critically ill patients with sepsis†
British Journal of Anaesthesia 100 (6): 810–14 (2008)
doi:10.1093/bja/aen093 Advance Access publication April 22, 2008
Nociceptin and urotensin-II concentrations in critically ill patients with sepsis† J. P. Williams, J. P. Thompson, S. P. Young, S. J. Gold, J. McDonald, D. J. Rowbotham and D. G. Lambert*
*Corresponding author. E-mail: [email protected] Background. The systemic inflammatory response to infection (sepsis) involves widespread organ dysfunction, including changes in immune modulation, cardiovascular derangements, and neural activation. Two neuropeptide/receptor systems, nociceptin/orphanin FQ (N/OFQ) which acts at the non-classical opioid receptor NOP and urotensin-II (U-II) which acts at the urotensin receptor (UT), have been implicated in neural, immune, and cardiovascular system function. In this study, we make measurements of these peptides in critically ill patients. Methods. Plasma samples from 21 critically ill patients with sepsis were collected over four consecutive days. Plasma N/OFQ and U-II concentrations were determined by radioimmunoassay and compared with biochemical and clinical markers of illness severity, including serum creatinine, bilirubin, platelet and white cell counts, admission APACHE II and serial SOFA scores. Results. Median (inter-quartile range) admission plasma N/OFQ concentrations in sepsis were higher in patients who died within 30 days (n¼4) compared with survivors (n¼17); 3.0 (2.5 – 5.0) vs 1.0 (1.0– 2.5) pg ml21 (P¼0.028). Plasma N/OFQ concentrations were increased in a subgroup of five patients who had undergone major gastrointestinal surgery. There were no significant changes in plasma U-II concentrations. There were no correlations between plasma U-II and N/OFQ concentrations and markers of illness severity and organ system dysfunction. Conclusions. Plasma N/OFQ concentrations were increased in critically ill patients with sepsis who had undergone major gastrointestinal surgery and in patients who subsequently died. Further work is required to clarify the significance of plasma N/OFQ concentrations in sepsis. Br J Anaesth 2008; 100: 810–14 Keywords: complications, sepsis; critical care; inflammatory response; nociceptin/orphanin FQ; urotensin-II Accepted for publication: March 11, 2008
The nociceptin receptor (NOP) is a non-classical naloxone-insensitive opioid receptor. It has similar postreceptor signalling properties to the three classical opioid receptors (MOP, DOP, and KOP), although the responses to agonists and antagonists are unique. The neuropeptide nociceptin/orphanin F/Q (N/OFQ) is the endogenous ligand for NOP.1 2 The nociceptin system is involved in control of pain pathways. Spinal injection of N/OFQ in rodents has anti-nociceptive (analgesic) effects; conversely intracerebroventricular injection has a pro-nociceptive effect.3 Non-nociceptive roles for the nociceptin system have been suggested by various investigators. In small rodents, i.v. N/OFQ causes decreased cardiac output,
vasodilatation, and hypotension.4 – 7 This may be due to effects on the autonomic nervous system and/or release of local vasoactive substances. Intradermal injection of N/ OFQ in rats has been found to cause an increase in vascular permeability, inhibited by a histamine H1 receptor antagonist; the same group went on to show that histamine was released in a dose-dependent manner from a ratperitoneal mast cell preparation in response to N/OFQ.8 †
Presented in part to the Anaesthetic Research Society, London, November 2006 (Williams JP, Gold SJ, Young SP, Thompson JP, Lambert DG. Plasma nociceptin concentrations in SIRS. Br J Anaesth 2007; 98: 290P).
# The Board of Management and Trustees of the British Journal of Anaesthesia 2008. All rights reserved. For Permissions, please e-mail: [email protected]
Downloaded from http://bja.oxfordjournals.org/ at UCSF Library and Center for Knowledge Management on March 9, 2015
Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester LE1 5WW, UK
Nociceptin and urotensin-II concentrations
Methods
second, third, and fourth days where possible. Each sample was collected on ice in an S-Monovettew EDTA container (Sarstedt, Germany), giving a final concentration of 1.6 mg ml21 EDTA, then immediately mixed with 100 ml of the protease inhibitor aprotonin, to a final concentration of 0.6 TIU ml21. Plasma was obtained by centrifugation (3000g, 10 min, 48C) and stored at 2708C for subsequent batch analysis. Plasma N/OFQ and U-II concentrations were determined by commercial radioimmunoassays (Phoenix Pharmaceuticals Inc., CA, USA), performed as described previously,10 19 according to the manufacturer’s instructions. Results below the level of detection were assigned a value of 1.0 pg per sample, the lowest limit of detection of the RIA.
Data description and analysis Descriptive and analytical statistics were performed using PRISM 5.00 for Windows XP (Graphpad Software Inc., CA, USA). Numerical data are presented as medians (inter-quartile range) unless otherwise stated. Comparisons between the groups were made using the Mann – Whitney test. Tests of correlation were made using the two-tailed Spearman rank test. A P-value of ,0.05 was considered to be statistically significant.
Results
Patients With local research ethics committee approval and written informed consent, or assent from the next of kin where appropriate, 21 critically ill patients admitted to our Adult Intensive Care Unit between May 2004 and April 2005 with a diagnosis of sepsis (as defined by the ACCP/SCCM consensus conference)16 were enrolled into this observational pilot study. Physiological data were collected on a daily basis to allow calculation of the Sepsis-related Organ Failure Assessment (SOFA) score.17 Microbiological, biochemical, and haematological data were collected from case note review and computerized laboratory reporting systems. The presumed source of sepsis was diagnosed for each patient by joint consultation with the critical care team and a consultant medical microbiologist, based upon the clinical presentation and the organism(s) isolated. Clinical management was at the discretion of the critical care team, in accordance with the Surviving Sepsis Campaign guidelines.18 Our standard sedation regime consisted of continuous i.v. morphine and midazolam infusions, with daily sedation holds where appropriate.
Peptide extraction and analysis Five millilitres of blood was obtained from an indwelling arterial line at the following time points—within the first 24 h of sepsis being diagnosed (‘admission’), then on the
Four out of the 21 patients died within 30 days of admission to ICU (Table 1). Patients were analysed in two groups: survival or non-survival at 30 days (Table 1). Survivors and non-survivors were equally matched for age, APACHE II score, and length of ICU stay (Table 2). Plasma N/OFQ concentrations sampled on the first day (Fig. 1) were significantly higher in non-survivors compared with survivors; 3.0 (2.5 – 5.0) vs 1.0 (1.0 – 2.5) pg ml21 (P¼0.028). On subsequent days, there were no significant differences in N/OFQ concentrations (Table 3). There were no significant differences in U-II concentrations in survivors and non-survivors at any time point (Fig. 1; Table 3). In the five patients admitted to ICU after surgery (two after a perforated upper gastrointestinal viscus, two after perforated lower gastrointestinal viscus, and one after Ivor-Lewis oesophagogastrectomy) admission plasma Table 1 Patient outcome. Patients who died in hospital within 30 days of admission to the ICU from likely sepsis-related causes. MOFS, multiple organ failure syndrome; PTE, pulmonary thromboembolism Patient
Initial diagnosis
Time from ICU admission to death
Mode of death
1 2 3 4
Faecal peritonitis Faecal peritonitis Pneumonia/urosepsis Pneumonia
1 day 5 days 7 days 14 days
MOFS MOFS MOFS PTE
811
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Human urotensin-II (U-II) is an 11 amino acid neuropeptide which acts on a specific G-protein coupled receptor, the urotensin receptor (UT). The U-II – UT system is widely distributed in cardiovascular tissue, the nervous system (in particular cardiovascular control centres), the kidney, and the respiratory tract.9 Vascular responses to U-II are varied, with some arteries demonstrating intense vasoconstriction, whereas others exhibit (nitric oxide-dependent) vasodilatation. Plasma U-II concentrations are increased in a number of chronic cardiovascular and inflammatory diseases, including congestive cardiac failure, primary hypertension, diabetes mellitus, pre-eclampsia, and chronic kidney disease.10 – 13 The inflammatory response is associated with the release of a number of mediators, including vasoactive neuropeptides; examples include calcitonin gene-related peptide, neuropeptide Y, and substance P.14 15 N/OFQ and U-II may be involved in the inflammatory response given their presence in cardiovascular, immune, and neural tissue. The primary aim was to determine the concentrations of N/OFQ and U-II in the plasma of patients with sepsis. Secondary aims were to assess whether there were associations of these peptides with severity of illness, organ dysfunction, or outcome.
Williams et al.
Table 2 Patient characteristics. Age, APACHE II, ICU stay, and SOFA scores are quoted as median (range) or number (n). There were no significant differences between survivors and non-survivors in terms of age (P¼0.23), APACHE II (P¼0.21), or length of stay (P¼0.48) Non-Survivors
Survivors
21 47 (20– 75) 12:9 18 (7– 30) 7 (1 –30)
4 64 (40 –67) 3:1 20 (18 –26) 6 (1– 10)
17 43 (20– 75) 9:8 18 (7– 30) 7 (1 –30)
5 2 12 1 2
2 1 2 0 0
3 1 10 1 2
7 (1 –17) 5.5 (0– 16) 5 (0 –13) 6 (0 –16)
6 7 6 7
(3– 13) (5– 10) (5– 8) (6– 8)
7.5 (1– 17) 5.5 (0– 16) 5 (0 –13) 6 (0 –16)
Fig 2 Plasma N/OFQ and U-II concentrations in critically ill patients on admission to ICU, comparing those who had major gastrointestinal surgery (n¼5) with those who did not have surgery (n¼16). The data medians are indicated. NS, non-significant.
in plasma U-II concentration (Fig. 2). Plasma N/OFQ and U-II concentrations did not correlate with markers of organ dysfunction and severity of illness, nor with SOFA scores. There were no correlations between concentrations of the two neuropeptides.
Discussion
Fig 1 Plasma N/OFQ and U-II concentrations in critically ill patients with sepsis on admission to ICU. n¼21 for each peptide measured (four non-survivors, 17 survivors). The data medians are indicated. NS, non-significant.
Table 3 Plasma concentrations of N/OFQ and U-II measured serially in critically ill patients with sepsis. Values are reported as median (IQR) pg ml21. The lowest limit of detection of both radioimmunoassays was 1 pg per sample Day
1 2 3 4
N/OFQ
U-II
Non-survivors
Survivors
Non-survivors
Survivors
3.0 (2.5 –5.0) 1.3 (1.0 –3.3) 1.4 (1.0 –2.7) 2.8 (1.5 –3.2)
1.0 (1.0 –2.5) 1.6 (1.0 –3.0) 1.0 (1.0 –3.4) 1.5 (1.0 –3.0)
1.0 (1.0 –1.3) 1.5 (1.0 –1.6) 1.3 (1.3 –1.6) 1.4 (1.3 –2.0)
1.2 (1.1 – 1.4) 1.1 (1.0 – 1.5) 1.3 (1.1 – 1.8) 1.3 (1.0 – 1.8)
N/OFQ concentrations were significantly higher compared with non-surgical patients; 2.9 (2.3 – 4.4) vs 1.0 (1.0 – 2.4) pg ml21 (P¼0.014). There were no significant differences
In this study we found that on admission to ICU plasma N/OFQ concentrations were significantly higher in patients with sepsis who subsequently died within 30 days, compared with those who survived. Of the four patients who died, two were also postoperative patients and had higher N/OFQ concentrations. This could reflect a greater systemic inflammatory response to both sepsis (faecal peritonitis) and surgical trauma. U-II concentrations were not significantly different between these groups. Various studies have shown a consistent range of normal plasma N/OFQ concentrations, ranging from means of 2.5– 10.7 pg ml21 measured by RIA.20 Our group measured plasma concentrations of N/OFQ by RIA in pregnant women and found a range of 7.6– 13.7 pg ml21.21 In the current study, plasma N/OFQ concentrations ranged from less than 1.0 (the lowest level of detection of the RIA) to 7.8 pg ml21, apparently lower than some quoted ‘normal’ values. The significance of this is uncertain. In contrast, U-II has an over 6000-fold range of quoted ‘normal’ healthy plasma concentrations, from 0.6 pg ml21 to 3.6 ng ml21. 22 23 This wide disparity can be at least partly accounted for by differing techniques of measurement.24 25 Plasma U-II concentrations in the current study ranged from less than 1.0 (the lowest level of detection) to 4.2 pg ml21. Importantly, these samples were all processed in the same manner and subjected to a single
812
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n Age (yr) Gender M:F APACHE II ICU stay (days) Sepsis source (n) Abdominal Urosepsis Respiratory CNS Unknown SOFA score Day 1 Day 2 Day 3 Day 4
All
Nociceptin and urotensin-II concentrations
or U-II systems play an important role in localized and systemic inflammatory processes, and to assess the effects of drugs and anaesthesia on these neuropeptide systems.
Funding We would like to thank the British Journal of Anaesthesia/Royal College of Anaesthetists for project grant support.
References 1 Meunier J-C, Mollereau C, Toll L, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 1995; 377: 532– 5 2 Reinsheid RK, Nothacker H-P, Bourson A, et al. Orphanin FQ: a neuropeptide that activates an opioid like G-protein-coupled receptor. Science 1995; 270: 792 – 4 3 Zeilhofer HU, Calo G. Nociceptin/orphanin FQ and its receptor—potential targets for pain therapy. J Pharmacol Exp Ther 2003; 306: 423– 9 4 Champion HC, Czapla MA, Kadowitz PJ. Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. Peptides 1997; 18: 729 – 32 5 Champion HC, Pierce RL, Kadowitz PJ. Nociceptin, a novel endogenous ligand for the ORL1 receptor, dilates isolated resistance arteries from the rat. Regul Pept 1998; 78: 69 – 74 6 Chen Y, Chang M, Wang ZZ, et al. [Nphe1] nociceptin (1-13)-NH2 antagonizes nociceptin-induced hypotension, bradycardia, and hindquarters vasodilatation in the anesthetized rat. Can J Physiol Pharmacol 2002; 80: 31 – 5 7 Malinowska B, Godlewski G, Schlicker E. Function of nociceptin and opioid OP4 receptors in the regulation of the cardiovascular system. J Physiol Pharmacol 2002; 53: 301 – 4 8 Kimura T, Kitaichi K, Hiramatsu K, et al. Intradermal application of nociceptin increases vascular permeability in rats: the possible involvement of histamine release from mast cells. Eur J Pharmacol 2000; 407: 327– 32 9 Lambert DG. Urotensin II: from osmoregulation in fish to cardiovascular regulation in man. Br J Anaesth 2007; 98: 557 – 9 10 Thompson JP, Watt JP, Sanghavi S, Strupish JW, Lambert DG. A comparison of cerebrospinal fluid and plasma urotensin II concentrations in normotensive and hypertensive patients undergoing urological surgery during spinal anesthesia: a pilot study. Anesth Analg 1999; 97: 1501– 3 11 Totsune K, Takahashi K, Arihara Z, et al. Role of urotensin II in patients on dialysis. Lancet 2001; 358: 810 – 1 12 Ng LL, Loke I, O’Brien RJ, Squire IB, Davies JE. Plasma urotensin in human systolic heart failure. Circulation 2002; 106: 2877 – 80 13 Cowley E, Thompson JP, Sharpe P, et al. Effects of pre-eclampsia on maternal plasma, cerebrospinal fluid, and umbilical cord urotensin II concentrations: a pilot study. Br J Anaesth 2005; 95: 495 – 9 14 Arnalich F, Sanchez JF, Martinez M, et al. Changes in plasma concentrations of vasoactive neuropeptides in patients with sepsis and septic shock. Life Sci 1995; 56: 75 – 81 15 Beer S, Weighardt H, Emmanuilidis K, et al. Systemic neuropeptide levels as predictive indicators for lethal outcome in patients with postoperative sepsis. Crit Care Med 2002; 30: 1794 – 8
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batch radioimmunoassay for each peptide, thus between sample comparison is valid. The lack of variation in U-II peptide in patients with sepsis may be related to the effects this peptide displays in health and chronic disease. It has been suggested that the U-II peptide/receptor system is functionally silent in health.9 This suggestion is supported by the findings that gene knock-out mice for the urotensin system appear physiologically normal and that i.v. and intra-arterial U-II given to healthy volunteers results in no systemic cardiovascular changes.26 27 However, when administered by iontophoresis, U-II causes microvascular vasoconstriction in patients with chronic cardiac failure and essential hypertension, whereas normal control groups demonstrate microvascular vasodilatation.28 29 The urotensin system may therefore assume more importance in chronic disease states, rather than in health and acute haemodynamic modulation, as seen in sepsis. In contrast to U-II peptide, concentrations of plasma N/OFQ were raised in individuals with sepsis who subsequently died, and in postoperative patients with sepsis. Previously, Stein and colleagues have described a neuroimmune axis for the classical endogenous opioid peptides and their receptors (MOP, DOP, and KOP). In this model, leucocytes attracted to sites of inflammation deliver opioid peptides to sensory ( pain) nerve endings. Opioid receptors newly synthesized in the dorsal root ganglia in response to inflammation are trafficked to the periphery where they are presented at the neuronal membrane as targets for these peptides.30 In addition, both human peripheral blood mononuclear cells (PBMC) and polymorphonulcear leucocytes express mRNA transcripts encoding for both pre-pro-N/OFQ31 and the NOP receptor (but not MOP, DOP, or KOP receptors).32 Moreover N/OFQ peptide is released on PBMC degranulation while polymorphs demonstrate chemotaxis and recruitment in response to N/OFQ.33 34 N/OFQ also induces the release of lysozyme, a bactericidal enzyme, from human neutrophils,35 although it fails to cause release of reactive oxygen species.34 35 In a toxin-induced colitis model, NOP gene knock-out mice developed significantly less inflammation of the colon than wild-type mice; the authors suggest that N/OFQ acts to stimulate inflammatory cell migration and activation.36 Thus, in a manner similar to the classical opioid systems, leucocytes may be involved in delivering N/OFQ to the nervous tissues at sites of inflammation, experiencing paracrine immunomodulation and forming a ‘nociceptin neuroimmune axis’. Clearly, results from a pilot observational study of this type should be interpreted with caution, but the finding that initial N/OFQ concentrations were higher in nonsurvivors of sepsis would be consistent with a more intense inflammatory response. Exaggerated release of N/OFQ might contribute to reduced myocardial performance and vascular tone, and increased vessel permeability. Further work is needed to ascertain whether the nociceptin
Williams et al.
26 Affolter JT, Newby DE, Wilkinson IB, et al. No effect on central or peripheral blood pressure of systemic urotensin II infusion in humans. Br J Clin Pharmacol 2002; 54: 617 – 21 27 Wilkinson IB, Affolter JT, de Haas SL, et al. High plasma concentrations of urotensin-II do not alter local or systemic hemodynamics in man. Cardiovasc Res 2002; 53: 341 – 7 28 Lim M, Honisett S, Sparkes CD, et al. Differential effect of urotensin II on vascular tone in normal subjects and patients with chronic heart failure. Circulation 2004; 109: 1212 – 4 29 Sondermeijer B, Kompa A, Komesaroff P, Krum H. Effect of exogenous urotensin-II on vascular tone in skin microcirculation of patients with essential hypertension. Am J Hypertens 2005; 18: 1195– 9 30 Stein C, Schafer M, Machelska H. Attacking pain at its source: new perspectives on opioids. Nat Med 2003; 9: 1003 – 8 31 Williams JP, McDonald J, Thompson JP, Rowbotham DJ, Lambert DG. Human peripheral blood mononuclear cells express the mRNA encoding for the nociceptin peptide and its receptor. Br J Anaesth 2006; 96: 260P 32 Williams JP, Thompson JP, McDonald J, et al. Human peripheral blood mononuclear cells express nociceptin/orphanin FQ but not m, d or k opioid receptors. Anesth Analg 2007; 105: 998 – 1005 33 Fiset ME, Gilbert C, Poubelle PE, Pouliot M. Human neutrophils as a source of nociceptin: a novel link between pain and inflammation. Biochemistry 2003; 42: 10498– 505 34 Serhan CN, Fierro IM, Chiang N, Pouliot M. Cutting edge: nociceptin stimulates neutrophil chemotaxis and recruitment: inhibition by aspirin-triggered 15-epi-lipoxin A4. J Immunol 2001; 166: 3650– 4 35 Trombella S, Vergura R, Falzarano S, et al. Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation. Peptides 2005; 26: 1497– 502 36 Kato S, Tsuzuki Y, Hokari R, et al. Role of nociceptin/orphanin FQ (Noc/oFQ) in murine experimental colitis. J Neuroimmunol 2005; 161: 21 – 8
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16 Bone RC, Balk RA, Cerra FB, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992; 101: 1644 – 55 17 Vincent JL, Moreno R, Takala J, et al. The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/ failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med 1996; 22: 707 – 10 18 Dellinger RP, Carlet JM, Masur H, et al. Surviving sepsis campaign guidelines for the management of severe sepsis and septic shock. Crit Care Med 2004; 32: 858– 73 19 Kumar N, Smart D, Mason S, et al. Neither nociceptin nor its receptor are present in human synovial fluid or tissue. Br J Anaesth 1999; 83: 470 – 1 20 Barnes TA, Lambert DG. Nociceptin/orphanin FQ peptide-receptor system: are we any nearer the clinic? Br J Anaesth 2004; 93: 626 – 8 21 Brooks H, Elton CD, Smart D, et al. Identification of nociceptin in human cerebrospinal fluid: comparison of levels in pain and non-pain states. Pain 1998; 78: 71 –3 22 Khan SQ, Bhandari SS, Quinn P, Davies JE, Ng LL. Urotensin II is raised in acute myocardial infarction and low levels predict risk of adverse clinical outcome in humans. Int J Cardiol 2007; 117: 323– 8 23 Heller J, Schepke M, Neef M, et al. Increased urotensin II plasma levels in patients with cirrhosis and portal hypertension. J Hepatol 2002; 37: 767 – 72 24 Gold SJ, Thompson JP, Williams JP, et al. Does smoking increase plasma urotensin II concentrations? Eur J Clin Pharmacol 2007; 63: 253– 7 25 Aiyar N, Guida B, Ao Z, et al. Differential levels of ‘urotensin-II-like’ activity determined by radio-receptor and radioimmuno-assays. Peptides 2004; 25: 1339– 47
doi:10.1093/bja/aen093 Advance Access publication April 22, 2008
Nociceptin and urotensin-II concentrations in critically ill patients with sepsis† J. P. Williams, J. P. Thompson, S. P. Young, S. J. Gold, J. McDonald, D. J. Rowbotham and D. G. Lambert*
*Corresponding author. E-mail: [email protected] Background. The systemic inflammatory response to infection (sepsis) involves widespread organ dysfunction, including changes in immune modulation, cardiovascular derangements, and neural activation. Two neuropeptide/receptor systems, nociceptin/orphanin FQ (N/OFQ) which acts at the non-classical opioid receptor NOP and urotensin-II (U-II) which acts at the urotensin receptor (UT), have been implicated in neural, immune, and cardiovascular system function. In this study, we make measurements of these peptides in critically ill patients. Methods. Plasma samples from 21 critically ill patients with sepsis were collected over four consecutive days. Plasma N/OFQ and U-II concentrations were determined by radioimmunoassay and compared with biochemical and clinical markers of illness severity, including serum creatinine, bilirubin, platelet and white cell counts, admission APACHE II and serial SOFA scores. Results. Median (inter-quartile range) admission plasma N/OFQ concentrations in sepsis were higher in patients who died within 30 days (n¼4) compared with survivors (n¼17); 3.0 (2.5 – 5.0) vs 1.0 (1.0– 2.5) pg ml21 (P¼0.028). Plasma N/OFQ concentrations were increased in a subgroup of five patients who had undergone major gastrointestinal surgery. There were no significant changes in plasma U-II concentrations. There were no correlations between plasma U-II and N/OFQ concentrations and markers of illness severity and organ system dysfunction. Conclusions. Plasma N/OFQ concentrations were increased in critically ill patients with sepsis who had undergone major gastrointestinal surgery and in patients who subsequently died. Further work is required to clarify the significance of plasma N/OFQ concentrations in sepsis. Br J Anaesth 2008; 100: 810–14 Keywords: complications, sepsis; critical care; inflammatory response; nociceptin/orphanin FQ; urotensin-II Accepted for publication: March 11, 2008
The nociceptin receptor (NOP) is a non-classical naloxone-insensitive opioid receptor. It has similar postreceptor signalling properties to the three classical opioid receptors (MOP, DOP, and KOP), although the responses to agonists and antagonists are unique. The neuropeptide nociceptin/orphanin F/Q (N/OFQ) is the endogenous ligand for NOP.1 2 The nociceptin system is involved in control of pain pathways. Spinal injection of N/OFQ in rodents has anti-nociceptive (analgesic) effects; conversely intracerebroventricular injection has a pro-nociceptive effect.3 Non-nociceptive roles for the nociceptin system have been suggested by various investigators. In small rodents, i.v. N/OFQ causes decreased cardiac output,
vasodilatation, and hypotension.4 – 7 This may be due to effects on the autonomic nervous system and/or release of local vasoactive substances. Intradermal injection of N/ OFQ in rats has been found to cause an increase in vascular permeability, inhibited by a histamine H1 receptor antagonist; the same group went on to show that histamine was released in a dose-dependent manner from a ratperitoneal mast cell preparation in response to N/OFQ.8 †
Presented in part to the Anaesthetic Research Society, London, November 2006 (Williams JP, Gold SJ, Young SP, Thompson JP, Lambert DG. Plasma nociceptin concentrations in SIRS. Br J Anaesth 2007; 98: 290P).
# The Board of Management and Trustees of the British Journal of Anaesthesia 2008. All rights reserved. For Permissions, please e-mail: [email protected]
Downloaded from http://bja.oxfordjournals.org/ at UCSF Library and Center for Knowledge Management on March 9, 2015
Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester LE1 5WW, UK
Nociceptin and urotensin-II concentrations
Methods
second, third, and fourth days where possible. Each sample was collected on ice in an S-Monovettew EDTA container (Sarstedt, Germany), giving a final concentration of 1.6 mg ml21 EDTA, then immediately mixed with 100 ml of the protease inhibitor aprotonin, to a final concentration of 0.6 TIU ml21. Plasma was obtained by centrifugation (3000g, 10 min, 48C) and stored at 2708C for subsequent batch analysis. Plasma N/OFQ and U-II concentrations were determined by commercial radioimmunoassays (Phoenix Pharmaceuticals Inc., CA, USA), performed as described previously,10 19 according to the manufacturer’s instructions. Results below the level of detection were assigned a value of 1.0 pg per sample, the lowest limit of detection of the RIA.
Data description and analysis Descriptive and analytical statistics were performed using PRISM 5.00 for Windows XP (Graphpad Software Inc., CA, USA). Numerical data are presented as medians (inter-quartile range) unless otherwise stated. Comparisons between the groups were made using the Mann – Whitney test. Tests of correlation were made using the two-tailed Spearman rank test. A P-value of ,0.05 was considered to be statistically significant.
Results
Patients With local research ethics committee approval and written informed consent, or assent from the next of kin where appropriate, 21 critically ill patients admitted to our Adult Intensive Care Unit between May 2004 and April 2005 with a diagnosis of sepsis (as defined by the ACCP/SCCM consensus conference)16 were enrolled into this observational pilot study. Physiological data were collected on a daily basis to allow calculation of the Sepsis-related Organ Failure Assessment (SOFA) score.17 Microbiological, biochemical, and haematological data were collected from case note review and computerized laboratory reporting systems. The presumed source of sepsis was diagnosed for each patient by joint consultation with the critical care team and a consultant medical microbiologist, based upon the clinical presentation and the organism(s) isolated. Clinical management was at the discretion of the critical care team, in accordance with the Surviving Sepsis Campaign guidelines.18 Our standard sedation regime consisted of continuous i.v. morphine and midazolam infusions, with daily sedation holds where appropriate.
Peptide extraction and analysis Five millilitres of blood was obtained from an indwelling arterial line at the following time points—within the first 24 h of sepsis being diagnosed (‘admission’), then on the
Four out of the 21 patients died within 30 days of admission to ICU (Table 1). Patients were analysed in two groups: survival or non-survival at 30 days (Table 1). Survivors and non-survivors were equally matched for age, APACHE II score, and length of ICU stay (Table 2). Plasma N/OFQ concentrations sampled on the first day (Fig. 1) were significantly higher in non-survivors compared with survivors; 3.0 (2.5 – 5.0) vs 1.0 (1.0 – 2.5) pg ml21 (P¼0.028). On subsequent days, there were no significant differences in N/OFQ concentrations (Table 3). There were no significant differences in U-II concentrations in survivors and non-survivors at any time point (Fig. 1; Table 3). In the five patients admitted to ICU after surgery (two after a perforated upper gastrointestinal viscus, two after perforated lower gastrointestinal viscus, and one after Ivor-Lewis oesophagogastrectomy) admission plasma Table 1 Patient outcome. Patients who died in hospital within 30 days of admission to the ICU from likely sepsis-related causes. MOFS, multiple organ failure syndrome; PTE, pulmonary thromboembolism Patient
Initial diagnosis
Time from ICU admission to death
Mode of death
1 2 3 4
Faecal peritonitis Faecal peritonitis Pneumonia/urosepsis Pneumonia
1 day 5 days 7 days 14 days
MOFS MOFS MOFS PTE
811
Downloaded from http://bja.oxfordjournals.org/ at UCSF Library and Center for Knowledge Management on March 9, 2015
Human urotensin-II (U-II) is an 11 amino acid neuropeptide which acts on a specific G-protein coupled receptor, the urotensin receptor (UT). The U-II – UT system is widely distributed in cardiovascular tissue, the nervous system (in particular cardiovascular control centres), the kidney, and the respiratory tract.9 Vascular responses to U-II are varied, with some arteries demonstrating intense vasoconstriction, whereas others exhibit (nitric oxide-dependent) vasodilatation. Plasma U-II concentrations are increased in a number of chronic cardiovascular and inflammatory diseases, including congestive cardiac failure, primary hypertension, diabetes mellitus, pre-eclampsia, and chronic kidney disease.10 – 13 The inflammatory response is associated with the release of a number of mediators, including vasoactive neuropeptides; examples include calcitonin gene-related peptide, neuropeptide Y, and substance P.14 15 N/OFQ and U-II may be involved in the inflammatory response given their presence in cardiovascular, immune, and neural tissue. The primary aim was to determine the concentrations of N/OFQ and U-II in the plasma of patients with sepsis. Secondary aims were to assess whether there were associations of these peptides with severity of illness, organ dysfunction, or outcome.
Williams et al.
Table 2 Patient characteristics. Age, APACHE II, ICU stay, and SOFA scores are quoted as median (range) or number (n). There were no significant differences between survivors and non-survivors in terms of age (P¼0.23), APACHE II (P¼0.21), or length of stay (P¼0.48) Non-Survivors
Survivors
21 47 (20– 75) 12:9 18 (7– 30) 7 (1 –30)
4 64 (40 –67) 3:1 20 (18 –26) 6 (1– 10)
17 43 (20– 75) 9:8 18 (7– 30) 7 (1 –30)
5 2 12 1 2
2 1 2 0 0
3 1 10 1 2
7 (1 –17) 5.5 (0– 16) 5 (0 –13) 6 (0 –16)
6 7 6 7
(3– 13) (5– 10) (5– 8) (6– 8)
7.5 (1– 17) 5.5 (0– 16) 5 (0 –13) 6 (0 –16)
Fig 2 Plasma N/OFQ and U-II concentrations in critically ill patients on admission to ICU, comparing those who had major gastrointestinal surgery (n¼5) with those who did not have surgery (n¼16). The data medians are indicated. NS, non-significant.
in plasma U-II concentration (Fig. 2). Plasma N/OFQ and U-II concentrations did not correlate with markers of organ dysfunction and severity of illness, nor with SOFA scores. There were no correlations between concentrations of the two neuropeptides.
Discussion
Fig 1 Plasma N/OFQ and U-II concentrations in critically ill patients with sepsis on admission to ICU. n¼21 for each peptide measured (four non-survivors, 17 survivors). The data medians are indicated. NS, non-significant.
Table 3 Plasma concentrations of N/OFQ and U-II measured serially in critically ill patients with sepsis. Values are reported as median (IQR) pg ml21. The lowest limit of detection of both radioimmunoassays was 1 pg per sample Day
1 2 3 4
N/OFQ
U-II
Non-survivors
Survivors
Non-survivors
Survivors
3.0 (2.5 –5.0) 1.3 (1.0 –3.3) 1.4 (1.0 –2.7) 2.8 (1.5 –3.2)
1.0 (1.0 –2.5) 1.6 (1.0 –3.0) 1.0 (1.0 –3.4) 1.5 (1.0 –3.0)
1.0 (1.0 –1.3) 1.5 (1.0 –1.6) 1.3 (1.3 –1.6) 1.4 (1.3 –2.0)
1.2 (1.1 – 1.4) 1.1 (1.0 – 1.5) 1.3 (1.1 – 1.8) 1.3 (1.0 – 1.8)
N/OFQ concentrations were significantly higher compared with non-surgical patients; 2.9 (2.3 – 4.4) vs 1.0 (1.0 – 2.4) pg ml21 (P¼0.014). There were no significant differences
In this study we found that on admission to ICU plasma N/OFQ concentrations were significantly higher in patients with sepsis who subsequently died within 30 days, compared with those who survived. Of the four patients who died, two were also postoperative patients and had higher N/OFQ concentrations. This could reflect a greater systemic inflammatory response to both sepsis (faecal peritonitis) and surgical trauma. U-II concentrations were not significantly different between these groups. Various studies have shown a consistent range of normal plasma N/OFQ concentrations, ranging from means of 2.5– 10.7 pg ml21 measured by RIA.20 Our group measured plasma concentrations of N/OFQ by RIA in pregnant women and found a range of 7.6– 13.7 pg ml21.21 In the current study, plasma N/OFQ concentrations ranged from less than 1.0 (the lowest level of detection of the RIA) to 7.8 pg ml21, apparently lower than some quoted ‘normal’ values. The significance of this is uncertain. In contrast, U-II has an over 6000-fold range of quoted ‘normal’ healthy plasma concentrations, from 0.6 pg ml21 to 3.6 ng ml21. 22 23 This wide disparity can be at least partly accounted for by differing techniques of measurement.24 25 Plasma U-II concentrations in the current study ranged from less than 1.0 (the lowest level of detection) to 4.2 pg ml21. Importantly, these samples were all processed in the same manner and subjected to a single
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n Age (yr) Gender M:F APACHE II ICU stay (days) Sepsis source (n) Abdominal Urosepsis Respiratory CNS Unknown SOFA score Day 1 Day 2 Day 3 Day 4
All
Nociceptin and urotensin-II concentrations
or U-II systems play an important role in localized and systemic inflammatory processes, and to assess the effects of drugs and anaesthesia on these neuropeptide systems.
Funding We would like to thank the British Journal of Anaesthesia/Royal College of Anaesthetists for project grant support.
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batch radioimmunoassay for each peptide, thus between sample comparison is valid. The lack of variation in U-II peptide in patients with sepsis may be related to the effects this peptide displays in health and chronic disease. It has been suggested that the U-II peptide/receptor system is functionally silent in health.9 This suggestion is supported by the findings that gene knock-out mice for the urotensin system appear physiologically normal and that i.v. and intra-arterial U-II given to healthy volunteers results in no systemic cardiovascular changes.26 27 However, when administered by iontophoresis, U-II causes microvascular vasoconstriction in patients with chronic cardiac failure and essential hypertension, whereas normal control groups demonstrate microvascular vasodilatation.28 29 The urotensin system may therefore assume more importance in chronic disease states, rather than in health and acute haemodynamic modulation, as seen in sepsis. In contrast to U-II peptide, concentrations of plasma N/OFQ were raised in individuals with sepsis who subsequently died, and in postoperative patients with sepsis. Previously, Stein and colleagues have described a neuroimmune axis for the classical endogenous opioid peptides and their receptors (MOP, DOP, and KOP). In this model, leucocytes attracted to sites of inflammation deliver opioid peptides to sensory ( pain) nerve endings. Opioid receptors newly synthesized in the dorsal root ganglia in response to inflammation are trafficked to the periphery where they are presented at the neuronal membrane as targets for these peptides.30 In addition, both human peripheral blood mononuclear cells (PBMC) and polymorphonulcear leucocytes express mRNA transcripts encoding for both pre-pro-N/OFQ31 and the NOP receptor (but not MOP, DOP, or KOP receptors).32 Moreover N/OFQ peptide is released on PBMC degranulation while polymorphs demonstrate chemotaxis and recruitment in response to N/OFQ.33 34 N/OFQ also induces the release of lysozyme, a bactericidal enzyme, from human neutrophils,35 although it fails to cause release of reactive oxygen species.34 35 In a toxin-induced colitis model, NOP gene knock-out mice developed significantly less inflammation of the colon than wild-type mice; the authors suggest that N/OFQ acts to stimulate inflammatory cell migration and activation.36 Thus, in a manner similar to the classical opioid systems, leucocytes may be involved in delivering N/OFQ to the nervous tissues at sites of inflammation, experiencing paracrine immunomodulation and forming a ‘nociceptin neuroimmune axis’. Clearly, results from a pilot observational study of this type should be interpreted with caution, but the finding that initial N/OFQ concentrations were higher in nonsurvivors of sepsis would be consistent with a more intense inflammatory response. Exaggerated release of N/OFQ might contribute to reduced myocardial performance and vascular tone, and increased vessel permeability. Further work is needed to ascertain whether the nociceptin
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