With approval of the Human Studies Committee at the University of Louisville and written informed consent, we studied 10 healthy volunteers. None was obese, taking medication, or had a history of thyroid disease, dysautonomia, or Raynaud’s syndrome.
Each volunteer was studied on two randomly assigned days: 1) Control — no drug —and 2) ondansetron hydrochloride (GlaxoSmithKline, Research Triangle Park, NC) at a target plasma concentration of 250 ng mL−1. Volunteers were blinded as to which solution they received on each study day. The volunteers fasted 8 hours before the start of each study day. They dressed minimally and rested supine on a standard operating room table. Ambient temperature was maintained near 21°C and humidity at 25%.
A 14-gauge catheter was inserted in a right antecubital vein for blood sampling and an 18-gauge catheter was inserted in a left forearm vein for ondansetron or placebo administration. Blood for ondansetron analysis was sampled from the catheter inserted in right antecubital vein before drug administration (blank sample) and at each threshold. Blood was centrifuged, and the plasma was removed and stored at −40°C until analysis.
Ondansetron was infused using a target-controlled infusion with a Stanpump program based on pharmacokinetic data from Colthup et al.14
and de Alwis et al.15
at a target plasma concentration of 250 ng mL−1
. This corresponds to ≈50 mg of ondansetron over the approximately 2.5-hour study period. A similar volume of normal saline was infused on the control day. The infusion began at the start of thermal manipulation and continued until shivering was detected.
Skin and core temperatures were gradually increased with an Allon circulating-water garment (MTRE, North Industrial Park, Israel) and a whole-body convective-air warming coverlet and air heater-blower unit (Bair Hugger Model 250, Augustine Medical, Inc., Eden Prairie, MN) until sweating occurred. Subsequently, skin and core temperature were decreased with an Allon circulation-water garment and convective-air cooling coverlet (PolarAir, Augustine Medical Inc., Eden Prairie, MN) until shivering occurred. The volunteer’s body was insulated with the warming and cooling devices mentioned except for the head, and right forearm and hand, which were used for fingertip blood flow measurement.
Core temperature was measured at the tympanic membrane using Mon-a-therm thermocouples (Tyco-Mallinckrodt Anesthesiology Products, Inc., St. Louis, MO). The aural probes were inserted by the volunteers until they felt the thermocouple touch the tympanic membrane; appropriate placement was confirmed when volunteers easily detected a gentle rubbing of the attached wire. The aural canal was occluded with cotton and taped in place.
Mean skin-surface temperature and cutaneous heat transfer were calculated from measurements at 15 area-weighted sites. Temperatures were recorded at 1-minute intervals from thermocouples connected to calibrated Iso-Thermex® thermometers having an accuracy of 0.1°C and a precision of 0.01°C (Columbus Instruments, Corp., Columbus, OH).
Sweating was continuously quantified on the left upper chest using a ventilated capsule.4
We considered a sweating rate > 40 g·m−2
for at least 5 minutes to be significant.16
Absolute right middle fingertip blood flow was quantified using venous-occlusion volume plethysmography at 5-minute intervals.17
The vasoconstriction threshold was determined post hoc
by an observer blinded to treatment and core temperature.
As in previous similar studies,16
we used systemic oxygen consumption to quantify shivering. A Deltatrac metabolic monitor (SensorMedics Corp., Yorba Linda, CA) was used in canopy mode. Initiation of the shivering threshold was determined post hoc
by an observer blinded to treatment and core temperature. The same observer determined the shivering threshold for all subjects. End-tidal PCO2
was sampled from a catheter inserted into one nostril; gas removed from the catheter was returned to the canopy of the metabolic monitor.
Heart rate, end-tidal PCO2, and oxyhaemoglobin saturation (SpO2) were measured continuously using pulse oximetry, and blood pressure was determined oscillometrically at 5-minute intervals at the left ankle.
An investigator blinded to core temperature and treatment evaluated sedation using the responsiveness component of the Observer’s Assessment of Alertness/Sedation (OAA/S) score ()18
at several times: 1) 15 minutes before starting drug administration, 2) before thermal manipulation started, and 3) at each threshold.
Responsive Component of the Observer’s Assessment of Alertness/Sedation Scale.
Prior to extraction, plasma samples for ondansetron analysis were allowed to thaw at room temperature and vortexed. To 1 mL of plasma, 1 mL 0.1 M sodium phosphate buffer, pH 6.0 and internal standard (prazosine hydrochloride, Sigma-Aldrich, USA) was added and vortexed. Clean Screen solid phase extraction columns (CSDAU-203, World Wide Monitoring, United Chemical Technologies, Inc., Bristol, PA, USA) were preconditioned with 3 mL of methanol, 3 mL of deionised water and 3 mL of 0.1 M sodium phosphate buffer before the plasma samples were loaded (about 0.5 mL min−1). The cartridges were rinsed with 3 mL of deionised water, 2 mL of 1 M acetic acid, 3 mL of methanol and then dried under vacuum for at least 5 min. The extracts were then eluted from the cartridge with 3 mL of freshly prepared dichloromethane — isoproranol — ammonium hydroxide 25% (39:10:1; vol:vol:vol, prepared with sonication) by gravity filtration.
The eluate was evaporated to dryness under nitrogen stream in a water bath at about 40°C. The residues were redissolved in 200 μL mobile phase mixture, briefly mixed in an ultrasonic bath, and loaded into auto-sampler vials. Samples were analyzed by HPLC (Merck-Hitachi, L6200A and AS-2000A, Hitachi, Ltd., Tokyo, Japan) with UV detection (Merck-Hitachi, DAD L-4500) at 250 nm. Aliquots of 20 μL were injected onto a Nucleodur 100-5 CN-RP (Macherey-Nagel, CH), 250mm, 4 mm ID column with an 8 mm precolumn. The mobile phase consisted of 40% acetonitrile, 60% 20 mM bisodiumphosphate buffer, pH 5.4. The flow rate was 1.5 mL min−1 and operating temperatures were 35°C. The data were processed with proprietary HPLC control software (Merck-Hitachi, Model D-7000, Hitachi Instruments, Inc., San Jose, CA, USA).
Retention times of the internal standard and ondansetron (Zofran, GlaxoSmithKline plc, Middlesex, UK) were 2.96 and 3.36 min, respectively. Calibration curves with spiked and extracted plasma passed through the origin and were linear in the calibration range of 150 to 400 ng mL−1 with correlation coefficient values of r2 ≥ 0.99. The intraday and interday coefficients of variation were 2.5% and 3% for quality control samples containing 250 ng mL−1 ondansetron, respectively. For ondansetron the limit of quantification (S/N = 10) was 15 ng mL−1 and the recovery was ≥ 95%.
The cutaneous contribution to the thermoregulatory responses — sweating,19
vasoconstriction, and shivering — is linear.20
We thus used measured skin and core temperatures in °C at each threshold to calculate the core-temperature threshold that would have been observed had skin been maintained at a single designated temperature. For this purpose, we used Equation 1 that corrects core temperature for cutaneous temperature, providing response thresholds that would have been observed at a designated core temperature.
We have previously described the derivation and validation of this equation.16
We used a ß of 0.1 for sweating19
and a ß of 0.2 for vasoconstriction and shivering.20
The designated skin temperature was set at 34°C, a typical intraoperative value.
We determined that a sample size of 10 would detect a 0.5°C difference in shivering thresholds between control and drug days with 90% power. Based on a similar study conducted with the same design in our laboratory with doxapram,21
we assumed that the difference in shivering thresholds on the two study days would have a standard deviation of 0.44 and correlation of 0.75.
Oxygen consumption and calculated respiratory quotient were averaged during baseline (before commencement of drug infusion), during the cooling phase before shivering, and during shivering, respectively. The averaged values were then compared with two-way ANOVA (two factors; drug, period) and post-hoc Bonferroni/Dunn tests.
Ambient temperature, humidity, mean arterial pressure, heart rate, SpO2, and end-tidal PCO2 on each study day were averaged within each volunteer across the warming and cooling periods; the resulting values were then averaged among volunteers. Results for each study day were compared using Wilcoxon signed-rank tests or paired t-tests. Plasma concentrations of ondansetron at each threshold were compared using one-way ANOVA. All results are presented as means ( SD) or median [range], as appropriate; P < 0.05 was considered statistically significant.