The N-methyl-d-aspartate (NMDA)–nitric oxide (NO)–cyclic guanosine 3′,5′-monophosphate (cGMP) pathway is potentially a central target for general anesthetics (1–3). The second messenger, cGMP, regulates a variety of cGMP-dependent kinases, ion channels, and phosphodiesterases, and hence may interact with other previously identified anesthetic targets.
Most volatile and IV anesthetics have been reported to affect this biochemical pathway, generally resulting in a decrease in cGMP concentrations in animal brains (4–7). Plasma cGMP can be measured in humans, and changes of plasma cGMP concentrations in relation to predicted plasma propofol concentrations have been reported during sedation in healthy adult volunteers (8). However, it is not clear whether this observation applies to different depths of general anesthesia and, particularly, to children.
This prospective, double-blind, clinical trial tests the hypothesis that an increase in measured plasma propofol concentration leads to a reduction in plasma cGMP during general anesthesia in children.
With Research Ethics Board approval and written parental consent, 18 healthy children aged 3–6 yr, ASA I or II were enrolled. All children were scheduled to undergo elective lower body urological or general surgical procedures. After inhaled induction of anesthesia with sevoflurane/nitrous oxide/oxygen, an indwelling cannula was inserted and a bolus of propofol 5 mg/kg and rocuronium 1 mg/kg was given to facilitate tracheal intubation. Intermittent positive pressure ventilation was used in all patients with an oxygen/air mixture (Fio2 = 0.3 with a peak airway pressure of 15 cm H2O limiting tidal volumes to 10 mL/kg) and the respiratory rate was adjusted to achieve an end expiratory CO2 concentration of 38–40 mm Hg. Caudal epidural analgesia was performed with 0.25% plain bupivacaine 1.0 mL/kg. Twenty minutes were allowed for the block to become effective. The block was assumed to be successful if, on skin incision, the heart rate and mean arterial blood pressure did not change more than 5% from baseline values obtained immediately before skin incision. Normovolemia and normothermia were maintained throughout the study period.
Sevoflurane and nitrous oxide were discontinued and anesthesia was maintained with a propofol infusion regimen of 30 mg · kg−1 · h−1 for the first 15 min, followed by 26 mg · kg−1 · h−1 for the next 15 min. The propofol infusion was subsequently reduced to 1 mL/h for 5 min, as IRB restrictions required the maintenance of a continuous infusion throughout the study period and was restarted at a rate of 11 mg · kg−1 · h−1 for 15 min. The propofol infusion was then again reduced to 1 mL/h for 5 min and resumed at a rate of 6 mg · kg−1 · h−1 for 15 min. This administration regimen was aimed at producing an estimated steady-state serum propofol concentration of initially 6, 3, and 1.5 μg/mL at the respective stages (9). Rescue anesthesia for light anesthesia was provided when necessary and resulted in the elimination of the patient from the study.
Samples for plasma cGMP and propofol concentrations were collected in an EDTA tube at the three estimated steady-state concentrations, stored on ice, spun 5 min at 2000g at 4°C within 30 min and stored at −80°C until analysis. Cyclic GMP concentrations were measured using an enzyme immunoassay system (Biotrak, APBiotech, Little Chalfont, UK). Plasma propofol concentrations were analyzed using high-performance liquid chromatography (10). Intra- and interassay variability was <10%.
The commercially available pediatric bispectral index (BIS) sensor (Aspect Medical Systems, Newton, MA) was placed on the patient’s forehead and connected to a BIS A-2000 monitor (Aspect Medical Systems) to obtain additional data for depth of anesthesia. BIS values were recorded at the time of blood sampling at the respective time points.
Data were analyzed using Friedman analysis of variance and Wilcoxon signed ranks test as appropriate. P < 0.05 was accepted as significant. The Pearson correlation coefficient was calculated to determine the relationship between cGMP and measured plasma propofol concentrations.
Eighteen patients were studied. The mean (±sd) age and weight were 55.9 ± 12.9 mo and 19.3 ± 6.5 kg, respectively. Sixteen of the 18 patients completed the whole study protocol; in two patients, BIS recording and plasma sampling could not be completed at the predicted 1.5 μg/mL predicted plasma propofol concentration because of earlier than expected termination of surgery. The BIS monitor malfunctioned in two patients and only plasma propofol and cGMP values were analyzed. Plasma sampling errors occurred (insufficient dead-space withdrawn) on six samples and were excluded from analysis. Plasma cGMP analysis did not work in one sample and was excluded from analysis.
Plasma cGMP and corresponding BIS values for the three predicted plasma propofol concentrations are given in Table 1. The plasma cGMP concentrations increased significantly at each predicted reduction in plasma propofol concentration (P < 0.0001).
Measured plasma propofol concentration and cGMP and BIS were negatively correlated (r = −0.62 and r = −0.80, respectively), and the relationship is illustrated in Figure 1.
There were no complications throughout the study period. All children were pain-free in the immediate postoperative period and no child required a rescue propofol dose.
This study demonstrates that plasma cGMP concentrations and BIS increase with a reduction in predicted and measured plasma propofol concentrations in children undergoing general anesthesia. This is the first report of plasma cGMP concentrations in humans in relation to anesthesia that suggests that plasma cGMP may potentially be used as a marker of depth of propofol anesthesia.
The glutamate–NO–cGMP pathway is potentially an effective site for general anesthetics (1–3), and is interlinked with a host of other intracellular pathways, receptor regulation, and intracellular calcium homeostasis. The excitatory neurotransmitter, glutamate, stimulates NMDA receptors, resulting in an intracellular influx of calcium and subsequent stimulation of type I NO synthase, which catalyzes the conversion of l-arginine to NO and l-citrulline. NO activates soluble guanylyl cyclase resulting in the formation of cGMP from guanosine triphosphate. Phosphodiesterases hydrolyze cGMP to inactive 5′GMP (Fig. 2). cGMP is a ubiquitous second messenger and regulates intracellular calcium via cGMP-dependent kinases, ion channels, and phosphodiesterases, and may influence neuronal activity.
There is controversy concerning whether propofol exerts all its anesthetic actions via γ-aminobutyric acid type A receptors β3 subunits, or involves other pathways such as inhibition of phosphorylation of NMDA NR1 subunits (11–13). However, it is most likely that more than one intracellular pathway is involved in the production of clinical anesthesia.
Volatile and IV anesthetics have been reported in animal studies to affect this biochemical pathway resulting in a decrease in cerebral cGMP (4–7). Very little information is available in humans, and only one study in healthy volunteers undergoing propofol sedation demonstrated a decrease in plasma cGMP concentration at loss of verbal contact when compared to baseline measurements (8). The results of the present study in children are in agreement with these investigations and there appears to be an inverse, dose-dependent correlation between propofol and cGMP plasma concentrations.
A limitation of this study is the lack of true baseline samples of plasma cGMP before the induction of anesthesia in children. Children in the age group studied are rarely sufficiently cooperative to allow stress-free blood sampling before the induction of anesthesia. It was speculated that the level of excitement and stress before anesthesia may lead to an artificially high plasma cGMP concentration in nonpremedicated children, whereas the use of premedication s.a. midazolam or nitrous oxide (a NMDA receptor antagonist) may lead to artificially low plasma cGMP concentrations. The effect of sevoflurane after induction of anesthesia may also compound plasma cGMP, although this has not been investigated in humans (8) and should be the subject of future studies.
In adults, general anesthesia using propofol without concomitant systemic analgesia to reduce the nociceptive response to surgical stimulus is ethically difficult to justify, whereas a combination of regional anesthetic technique and mild sedation is safe and indicated. However, in young children, the use of regional anesthesia block without the concomitant administration of general anesthesia is often impossible for allowing surgery to proceed in a safe and comfortable way (14,15).
The current model used to investigate the effects of propofol anesthesia allows the administration of various depths of anesthesia in children. Recently, concerns about the incidence of awareness during pediatric anesthesia have been published (16). Although the true incidence of awareness during pediatric anesthesia is unknown, it is estimated to be approximately 0.8% in the age group of 5–12 yr undergoing elective noncardiac surgical procedures (17). These results support the use of BIS measurement for the assessment of depth of propofol anesthesia; however, concerns regarding the sensitivity to detect all cases of awareness remain (18–20). It is not known whether plasma cGMP concentrations parallel those in human brains. Results of this study demonstrate that real-time plasma cGMP monitoring could potentially serve as an additional depth of propofol anesthesia monitor in the future. Further studies are required to elucidate the applicability of this technique.
In summary, the current study shows that plasma cGMP and BIS are both negatively correlated with measured plasma propofol concentration. Real-time plasma cGMP concentration measurement may potentially serve as a biochemical marker for depth of propofol anesthesia in children, but this will require future studies.
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