General anesthesia in phocids is potentially more complicated than in other mammals because of the high degree of development of anatomic and physiologic adaptations to diving.1–3 Isoflurane, a potent respiratory depressant,4 is frequently used in anesthesia of pinipeds.3,5,6 Calculation of the respiratory anesthetic index (a measure of the therapeutic index of an inhalant anesthetic agent defined as the ratio between the end-tidal concentration causing respiratory arrest and the minimum alveolar concentration) demonstrates a greater degree of respiratory depression associated with isoflurane than halothane.4 As a result, institution of IPPV has been recommended in anesthesia of pinnipeds.3
Intermittent monitoring of arterial blood gas analysis is the gold standard in assessment of ventilatory status, although sample collection is technically challenging to perform and access to arterial sites may be limited.7 This is particularly true of phocids, in which arterial access is limited. Furthermore, sample collection of arterial blood is not a benign procedure. Potential complications include transient or permanent artery occlusion, hemorrhage, hematoma, impaired peripheral circulation, air emboli, and infection.8 A potential alternative to arterial blood sample collection to measure blood gas tensions as a monitor of adequate ventilation in intubated patients is ETCO2 monitoring by capnography. Measurement of ETCO2 has been found to be an acceptable approximation of PaCO2 in people9–11 and domestic animals12,13 in which cardiopulmonary function is normal. In healthy humans, the ETCO2 underestimates PaCO2 by 5 to 10 mm Hg under anesthesia, and this is attributed to the presence of alveolar dead space.14 However, the P(a-ET)CO2 is not equal across species; in horses, it may be up to 30 mm Hg.15 Reference range values for P(a-ET)CO2 have not been reported for pinniped species.
The importance of ETCO2 measurements to provide an assessment of adequate ventilation merits its inclusion as one of the basic standards of monitoring during general anesthesia by the American Society of Anesthesiologists16 and is one of the suggestions made by the American College of Veterinary Anesthesiologists for anesthetic monitoring.17 When orotracheal intubation is used, ETCO2 is easily measured by attaching a capnograph to the proximal end of the endotracheal tube. To our knowledge, reference range values for ETCO2 or P(a-ET)CO2 have not been reported for harp seals. High concentrations of ETCO2 under general anesthesia have been reported for other pinniped species, and this may indicate the development of hypoventilation as a result of sedative and anesthetic effects of administered agents used as well as a natural degree of CO2 tolerance.18,19 The purpose of the study reported here was to assess the ability of ETCO2, as measured with a handheld sidestream microstream capnograph, to accurately predict PaCO2 measurements in harp seals anesthetized with isoflurane with manual IPPV.
Materials and Methods
Animals—This project was performed according to animal use protocols approved by the animal care committees of the institutions involved in this project, both of which operate under the auspices of the Canadian Council on Animal Care. Three 5-month-old harp seal (Phoca groenlandica) pups weighing from 27 to 45 kg were used for this study. They were collected as weaned pups on the ice pack in the Gulf of St Lawrence approximately 4.5 months prior to this experiment. These seals were enrolled as noninfected controls in an experimental study examining the effect of lungworms on the health of seals. As part of that study, they were scheduled to be euthanatized (via sodium pentobarbital, IV) for tissue sample collection. Consequently, no seals were euthanatized for the sake of the study presented here.
Seals were kept in an indoor tank (3.71 × 3.55 × 1.36 m) filled with salt water (flow rate, 50 to 60 L/min; temperature, −1.0° to 10°C; salinity, 24% to 30%) with a haul-out area of 1.82 × 3.55 m. The ambient air temperature was maintained between 8° and 10°C with a complete air exchange every 5 minutes. Seals were fed once a day ad libitum with shrimp, capelin, and herring and given a vitamin-mineral supplement.a
Experimental protocol—Following removal from the tank and a general clinical examination, each seal was premedicated with midazolam (0.2 mg/kg) injected IM into semitendinosus muscle groups. Mask induction of anesthesia was followed by isoflurane at a vaporizer setting of 4%. Isoflurane was delivered with O2 as a carrier gas via a Bain coaxial nonrebreathing system and a flow rate of 200 mL/kg/min. Following apparent loss of muscle tone (assessed in the thoracic limb), endotracheal intubation was attempted with a cuffed endotracheal tube of 7-mm internal diameter with the seal in sternal recumbency. After endotracheal intubation, seals were manually ventilated with a peak inspiratory pressure varying between 15 and 20 cm H2O. Anesthesia was maintained with isoflurane delivered in O2 at a vaporizer concentration ranging from 1% to 2% with a fresh gas flow of 200 mL/kg/min.
Cardiovascular and respiratory variables were monitored with a Doppler ultrasound probeb placed directly on the globe of the eye, a reflectance pulse oximeterc placed on the hard palate for pulsatile O2 saturation of hemoglobin, and a sidestream microstream end-tidal CO2 analyzerd connected to the proximal end of the endotracheal tube. The capnograph used in this study was calibrated prior to experimentation by use of a calibration gas recommended by the manufacturer. Body temperature was monitored with an esophageal thermometer.c To provide direct arterial samples for blood gas tension analysis, an indwelling cathetere was inserted in the median artery and connected to an extension set and 3-way stopcock to allow anaerobic withdrawal of arterial blood samples. Briefly, with the seal in dorsal recumbency, a 5-cm cutaneous incision was made on the medial aspect of the radiocarpal joint, followed by blunt dissection to expose the median artery. The catheter was inserted in the arterial lumen for approximately 15 cm, glued in place, and connected to a transducer, allowing arterial blood pressure monitoringf between arterial blood sample collections. The seal was then returned to sternal recumbency. To achieve a range of values, ventilation was adjusted randomly to ETCO2 values in ranges from 30 to 40, 40 to 50, and 50 to 60 mm Hg. Concentrations of ETCO2 were stabilized within each target concentration for 5 minutes.
An arterial blood sample was withdrawn anaerobically at each ETCO2 concentration by use of a heparinized 1-mL syringe and analyzed immediately with a point-of-care analyzer.g Samples were temperature corrected to esophageal temperature at the time of collection according to human nomograms included with the blood gas analyzer. During the period that the arterial sample was withdrawn (approx 10 seconds), ETCO2 was recorded and the mean of these values calculated. This mean value was used for subsequent data analysis.
Seals were euthanatized at the end of the procedure by IV administration of sodium pentobarbital. The experimental protocol required between 127 and 174 minutes. A necropsy, including histologic examination of tissue specimens, was performed on each seal to detect any disease process that could have affected results.
Statistical analysis—The correlation between ETCO2 measured by the capnograph and the corresponding PaCO2 was examined by linear regression analysis. A mixed multiple regression model with individual as a random factor was used to correlate the gradient between ETCO2 and PaCO2, body weight, arterial blood pressure, heart rate, and isoflurane setting. Data from paired samples were subjected to the Bland-Altman method for assessment of suitability of 2 methods of clinical measurement.20 For a new method to replace a gold-standard method, the limits of agreement must lie with-in an acceptable range, one that would not affect clinical decision-making. A modification of the original Bland-Altman method was used.21,22 In the original Bland-Altman method,20 bias is calculated as the gold-standard method results minus the new method results (ie, P(a-ET)CO2). In the present study, bias was calculated as the new method result minus the gold-standard method result (ie, P(ET-a)CO2).21,22 This provides a more intuitive method of presenting the bias, where an underestimation of the gold-standard method by the new method is presented as a negative value. In addition, a further technique described by Bland and Altman23 was used (regression analysis for nonuniform differences). This allows calculation of the limits of agreement when unequal variances exist at different levels of the independent variable.23,24 Effect of VR on P(ET-a)CO2 was examined graphically. Analyses were performed by use of software programs.h,i Values are reported as mean ± SD unless otherwise stated. Values of P < 0.05 were considered significant.
Results
Of the 25 paired samples (7 to 9 samples/seal), data from 24 paired samples were included in the subsequent analysis. The first sample of 1 seal was discarded because of technical difficulties with the arterial catheter. Including all remaining values (n = 24) in the analyses, a significant (P < 0.001) linear correlation (r = 0.94) was found between PaCO2 and ETCO2 (Figure 1). No significant association was observed between P(ET-a)CO2, body weight (P = 0.79), arterial blood pressure (P = 0.25), heart rate (P = 0.89), and isoflurane setting (P = 0.12). The bias and limits of agreement indicated an underestimation of PaCO2 by ETCO2 (mean bias, −4.01 mm Hg) with wide limits of agreement (−4.01 mm Hg ± 1.96 SD [−13.10 to 5.07 mm Hg]; Figure 2). Visual examination of the Bland-Altman plot revealed an increasing P(ET-a)CO2 as PCO2 increased. Therefore, data were analyzed by use of a second technique described by Bland and Altman23 (regression analysis for nonuniform differences) to account for a change in variances as the independent variable changes (Figure 3).
Scatterplot of PaCO2 (mm Hg) versus ETCO2 (mm Hg; n = 24) in 3 harp seals. Notice the best-fit linear trend line (bold line) and the line of equality for comparison (fine line). Values of ETCO2 below and above the line of equality overestimate and underestimate, respectively, the concomitantly measured values of PaCO2.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1131
Bland-Altman plot of P(ET-a)CO2 versus the mean of ETCO2+ PaCO2 (mm Hg) in 3 harp seals. Notice the limits of agreement (upper and lower dashed lines; ± 1.96 SD). Fewer numbers of points are illustrated than analyzed as a result of replication of points (n = 24).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1131
Bland-Altman regression analysis for data with nonuniform variances, illustrating P(ET-a)CO2 (mm Hg) versus the mean of ETCO2 + PaCO2 (mm Hg) in 3 harp seals. Notice the limits of agreement (upper and lower dashed lines; ± 1.96 SD). Fewer numbers of points are illustrated than analyzed as a result of replication of points (n = 24).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1131
A plot of VR versus P(ET-a)CO2 revealed that the greatest P(ET-a)CO2 was associated with a VR of < 5 (Figure 4). Five of 6 samples taken at a VR of < 5 had a P(ET-a)CO2 of ≥ 9 mm Hg. Values of ETCO2 at VR < 5 and ≥ 5 were 49.4 ± 6.2 mm Hg (range, 39.5 to 58.0 mm Hg) and 43.1 ± 8.8 mm Hg (range, 31.5 to 58 mm Hg), respectively.
Graph of VR (breaths/min) versus the P(ET-a)CO2 (mm Hg; n = 24) in 3 harp seals.
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1131
A final Bland-Altman analysis was performed following removal of data from 6 paired samples taken when the VR was < 5. The bias and limits of agreement indicated an underestimation of PaCO2 by ETCO2 (mean bias, −2.24 mm Hg) with limits of agreement of −7.79 to 3.30 mm Hg (−2.24 mm Hg ± 1.96 SD). Rectal temperature, as measured immediately prior to induction of anesthesia, was 37.0 ± 0.6°C (range, 36.0° to 38.8°C). Esophageal temperature was 32.1 ± 0.41°C (range, 31.7° to 33.2°C) during the sample collection period.
Systolic, diastolic, and mean arterial blood pressures during the sample collection period were 87.7 ± 23.7 mm Hg, 55.5 ± 9.8 mm Hg, and 67.0 ± 11.2 mm Hg, respectively. Heart rate during the sample collection period was 69.1 ± 6.1 beats/min. No noteworthy pathologic processes were detected during necropsy examinations.
Discussion
Examination of the 24 paired samples by the Bland-Altman method revealed an underestimation of PaCO2 by ETCO2 of −4.01 mm Hg. Although this appears to be a small degree of underestimation, the limits of agreement must be examined to assess the usefulness of the new method (ETCO2) in a clinical context. The limits of agreement were wide (range of values, −13.10 to 5.07 mm Hg).
A plot of VR versus the P(ET-a)CO2 revealed an association between a VR of < 5 and a greater P(ET-a)CO2. Consequently, a Bland-Altman plot was redrawn with these low values of VR removed. The outcome was a smaller degree of underestimation (−2.24 mm Hg) and narrower limits of agreement (range of values, −7.79 to 3.30 mm Hg).
Given the occurrence of unequal variances as the concentration of PCO2 changed, an alternative method of analyzing data was performed. This regression analysis for nonuniform differences confirmed the increase in variance as PCO2 increased. The increase in P(ET-a)CO2 as ETCO2 increased has been reported previously and may reflect a decrease in capnograph accuracy as ETCO2 increases.12,13
We have presented 2 methods of data analysis. However, from a clinical perspective, it is simpler to maintain a VR of ≥ 5 with IPPV, resulting in an underestimation of PaCO2 by −2.24 ± 5.54 mm Hg. Such a range of limits of agreement is unlikely to alter clinical decision-making during anesthesia. Although it is true that the mean ETCO2 was increased at a VR of < 5, compared with that at a VR of ≥ 5, the ranges of ETCO2 encountered included those within the reference range. Therefore, it may be more useful to use the arbitrary cutoff value of VR < 5, rather than rely on ETCO2 values.
There are many common sources of methodologic error that may contribute to ETCO2 inaccuracy when sidestream capnographs are used. These include a high sample-collection flow rate (typically 150 mL/min) from the capnograph exceeding the expired gas flow and leading to dilution of the ETCO2 sample, inappropriate positioning of the sample collection line so that dead airspace is sampled, water contamination leading to erroneously high ETCO2 readings, and high respiratory rates exceeding the response time of the apparatus.10,25 Use of a microstream capnograph with a low sample-collection rate (25 to 50 mL/min) will decrease the likelihood of ETCO2 sample dilution and contamination with water vapor. Conversely, microstream capnography may be associated with a slight time delay as a result of the greater transit time associated with low sample-collection flow rates.10 This is relevant when high respiratory rates are encountered, typically > 60 breaths/min.10
Although most ETCO2 readings underestimated PaCO2, overestimation was seen on 3 occasions. This has been attributed to normal capnograph bias or to software or hardware design.13 Other possible, albeit less likely, causes include calibration error, low respiratory rate, or high mixed venous PCO2.10
Limitations in our study include the low esophageal temperature during the sample collection period. As has been noted in harp seals2 and other pinniped species,5,26 body temperature under anesthesia tends to decrease towards ambient temperature. In the study by McDonell,2 ambient temperature was not given, although the body temperature (rectal) of anesthetized harp seals decreased in a similar manner to those reported here, from a preinduction range of 36.0° to 38.8°C to a value ranging from 31.7° to 33.2°C during the sample-collection period. The hypothermia observed is likely to be associated with decreased metabolism, CO2 production,27 and depressed thermoregulation. It has been found that temperature correction of PaCO2 during moderate hypothermia maintains the predictive value of ETCO2 and PaCO228,29 despite a likely decrease in CO2 production. Consequently, although the moderate hypothermia encountered during our study may have decreased the production of CO2, this should not have altered the relationship between ETCO2 and PaCO2.
The wide variation in values of mean arterial blood pressure may reflect a combination of a decrease in cardiac output associated with hypothermia or vasodilation associated with isoflurane anesthesia.
Unfortunately, cardiac output was not measured. However, this variation in mean arterial blood pressure did not adversely affect the P(ET-a)CO2, suggesting a minimal effect on pulmonary blood flow.
Findings of our study indicate that ETCO2 monitoring via sidestream microstream capnography is useful as an estimate of PaCO2 in anesthetized juvenile harp seals. Furthermore, results obtained with this capnograph indicate a P(a-ET)CO2 similar to that reported for humans and dogs. However, the accuracy of this estimate is decreased when VR is < 5.
ABBREVIATIONS
| IPPV | Intermittent positive-pressure ventilation |
| ETCO2 | End-tidal partial pressure of CO2 |
| P(a-ET)CO2 | PaCO2 to ETCO2 difference |
| P(ET-a)CO2 | ETCO2 to PaCO2 difference |
| VR | Ventilation rate per minute |
SeaTab, Pacific Research Laboratories, San Diego, Calif.
Ultrasonic doppler flow detector, model 811-B, Parks Medical Electronics Inc, Aloha, Ore.
Vet Ox Plus 4800 monitor, Heska Corp, Flamborough, ON, Canada.
NPB-75, Nellcor Puritan Bennett, Plesanton, Calif.
Intracath 19GA 8 in, Becton-Dickinson, Infusion Therapy Systems Inc, Sandy, Utah.
Bioview 2F52A, NEC San-ei, Tokyo, Japan.
I-STAT portable clinical analyzer, Sensor Devices Inc, Waukesha, Wis.
SAS Institute, Cary, NC.
Microsoft Excel × for Mac Service Release 1, Microsoft Corp, Redmond, Wash.
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