Saliva cyclic GMP increases during anaesthesia

T. Engelhardt*, H. F. Galley, F. M. MacLennan and N. R. Webster

Academic Unit of Anaesthesia and Intensive Care, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK*Corresponding author

Accepted for publication: May 25, 2001


    Abstract
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. Cyclic GMP (cGMP) has been implicated in modulating the effects of general anaesthesia. Changes in cGMP in humans undergoing anaesthesia have not been reported previously.

Methods. In this pilot study we measured cGMP in the saliva of six healthy volunteers and eight patients undergoing general anaesthesia for minor gynaecological procedures. Samples were obtained using a commercially available sampling device and cGMP was determined with an enzyme immunoassay and results expressed as a cGMP per mg protein.

Results. There was no statistically significant variation in salivary cGMP either day-to-day or between time points in healthy volunteers. Analysis of variance of salivary cGMP of patients undergoing general anaesthesia showed that cGMP increased significantly intraoperatively and returned to preoperative levels after surgery (P=0.03).

Conclusions. This is the first time that real time in vivo changes in salivary cGMP levels during general anaesthesia in humans have been demonstrated and may allow an alternative technique for measuring depth of anaesthesia in the future.

Br J Anaesth 2002; 89: 635–7

Keywords: anaesthesia, depth; anaesthesia, general; metabolism, second messengers; monitoring, depth of anaesthesia


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Cyclic GMP (cGMP) is thought to play a central role in mediating the effects of anaesthesia. Nitric oxide is a potent stimulant of soluble guanylcyclase resulting in the production of cGMP, which in turn regulates a variety of cGMP-dependent kinases, ion channels, and phosphodiesterases. There is a reduction of minimum anaesthetic concentration (MAC) of volatile anaesthetic agents in rodents following acute administration of nitric oxide synthase inhibitors.1 This has been disputed and not been reported in chronic administration and deletion of the type I (neuronal) nitric oxide synthase gene.2 3

cGMP is detectable in saliva, providing an easily accessible sample for measurement during anaesthesia. There are no previous data regarding variation in salivary cGMP in healthy subjects in the absence of anaesthesia, and therefore initial studies assessing any variation were required before undertaking studies in anaesthetized subjects. The aim of this study was to assess whether changes in salivary cGMP occurred during anaesthesia in humans.


    Methods and results
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Following local ethics approval and written informed consent we collected saliva from six healthy volunteers (25–32 yr) on 5 consecutive days at two time points (10:00 and 15:00 h) using a commercially available collection device, OrasureTM (Altrix Healthcare, Birkenhead, Merseyside, UK). We also collected saliva from eight ASA grade I or II patients (24–36 yr) undergoing minor obstetric procedures, immediately prior to induction, 10 min into the procedure and within 60 min after the operation. The anaesthetic regimen was standardized. All patients received induction of anaesthesia with propofol and maintenance with isoflurane/nitrous oxide in oxygen at 1.3 MAC via a facemask without the use of an airway. None of the patients had any form of pre-medication or received intraoperative opioids.

Saliva samples were frozen in liquid nitrogen immediately after collection and stored at –80°C until analysis. For the assay, samples were thawed on ice and microwaved for 8 s to halt enzyme activity.4 5 cGMP concentrations were determined using a sensitive enzyme immunoassay kit (R&D Systems Europe, Abingdon, Oxon, UK) as described previously.5 Protein was measured using Bradford reagent (Sigma-Aldrich, Poole, Dorset, UK) and results were expressed as saliva cGMP pmol (mg protein)–1. Data were analysed using Friedman analysis of variance with Wilcoxon signed ranks post hoc testing as appropriate and Bonferroni correction for multiple comparisons as necessary.

There was no statistically significant variation in salivary cGMP either day-to-day or between time points in healthy volunteers. In patients, analysis of variance revealed a statistically significant change in cGMP (P=0.03) during anaesthesia and surgery with no change in salivary protein concentration. Post hoc analysis showed that cGMP increased significantly intraoperatively (P=0.023) and returned to preoperative levels following surgery (Fig. 1).



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Fig 1 Changes in salivary cGMP in eight patients undergoing elective gynaecological surgery. Box and whisker plots show median, 25th and 75th percentile and range. P values refer to Wilcoxon signed ranks post hoc tests.

 

    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Our data show that concentrations of cGMP in saliva remain constant during normal activity and change during anaesthesia. Confounding variables were reduced to a minimum by choosing a group of patients who were essentially healthy, having minor obstetric surgery—thus all patients were female and of a very narrow age range. In addition, all the operations took place at a similar time of day, with the same anaesthetist, and the duration of the operation was similar (10–15 min). The anaesthetic regimen was standardized, with identical procedures for all patients, no pre-medication drugs and no opioid usage. Thus, changes in cGMP are more reliably related to anaesthesia and not other factors.

Studies in anaesthetized animals have shown decreases in cGMP within the central nervous system after halothane or isoflurane anaesthesia.5 However, in the present study, salivary cGMP increased during anaesthesia compared with concentrations immediately pre-induction. This may be partially explained by nitric oxide synthase activity or haemoxygenase activity during anaesthesia. Both nitric oxide, produced by the action of nitric oxide synthase, and carbon monoxide, the product of haemoxygenase activity, are potent activators of soluble guanyl cyclase.6

The results of this study need to be extended to a larger study of various anaesthetic techniques to establish whether this is a common link for a mechanism of anaesthesia in humans. Also, the possible development of a ‘real time’ cGMP monitor is appealing and may allow an alternative technique for measuring depth of anaesthesia.


    Acknowledgements
 
We acknowledge financial support from The Royal College of Anaesthetists, the Association of Anaesthetists of Great Britain and Ireland and the Chief Scientist Office in Scotland.


    References
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
1 Johns RA, Moscicki JC, DiFazio C. Nitric oxide synthase inhibitor dose-dependently and reversibly reduces the threshold for halothane anesthesia. Anesthesiology 1992; 77: 779–84[ISI][Medline]

2 Adachi T, Kurata J, Nakao S, et al. Nitric oxide synthase inhibitor does not reduce minimum alveolar concentration of halothane in rats. Anesth Analg 1994; 78: 1154–7[Abstract]

3 Ichinose F, Huang PL, Zapol W. Effects of targeted neuronal nitric oxide synthase gene disruption and nitroG-arginine methylester on the threshold for isoflurane. Anesthesiology 1995; 83: 101–8[ISI][Medline]

4 Vulliemoz Y,Versoky M, Lapert M, Triner L. Effect of enflurane on cerebellar cGMP and and on motor activity in the mouse. Br J Anaesth 1983; 55: 250–8

5 Galley HF, Le Cras AE, Logan SD, Webster NR. Differential nitric oxide synthase activity, co-factor availability and cGMP accumulation in the central nervous system during anaesthesia. Br J Anaesth 2001; 86: 388–94[Abstract/Free Full Text]

6 Morisaki H, Katayama T, Kotake Y, et al. Roles of carbon monoxide in leukocyte and platelet dynamics in rat mesenteric during sevoflurane anesthesia. Anesthesiology 2001; 95: 192–9[ISI][Medline]