Abstract
The purpose of this study was to assess the influence of incorrect determinations of the initial alveolar partial pressure of carbon monoxide (CO) at the beginning of breath holding (Pia _{CO}) on the pulmonary CO diffusing capacity of the lung (Dl _{CO}). Singlebreath maneuvers were performed on 14 anesthetized and artificially ventilated rabbits, using 0.2% CO in nitrogen as the indicator gas mixture. Inflation and deflation procedures were carried out in an identical manner on each animal, with inflation always starting from residual volume. Endtidal partial pressure of CO was determined by respiratory mass spectrometry and was used to calculate Dl _{CO} values with the application of the threeequation (method 1), as well as the conventional (method 2), solution. In each rabbit,method 2 caused Dl _{CO} values to be overestimated when compared withmethod 1, and this overestimation decreased with increasing time intervals of CO uptake. Because we were able to recalculate this deviation using Pia _{CO}values that were obtained by taking the diffusive removal of CO during inflation into account, we concluded that errors in estimating Pia _{CO} by applying method 2 significantly contribute to the discrepancy between both methods.
 conventional solution of breath holding equation
 rabbit
 threeequation methodology
 breath holding
 pulmonary CO diffusing capacity of the lung
conventional methods of analyzing the carbon monoxide (CO) singlebreath diffusing capacity of the lung (Dl _{CO}) are susceptible to functional inhomogeneities and to errors caused by variabilities when the singlebreath maneuver is performed by the subject (10). By using three equations to calculate Dl _{CO}, one each for inhalation, breath holding, and exhalation, Graham et al. (3,4, 5) as well as Cotton et al. (2) have highlighted that by accounting for changes in CO uptake during these three phases rather than by only considering CO uptake during breath holding, values of Dl _{CO} were minimally affected by variations in the way the singlebreath experiment was performed (4) and were independent of changes in breath holding times (5) and of ventilation inhomogeneities in normal subjects (2). Although it has been emphasized by Graham et al. (4) that the conventional solution of the breathholding equation leads to a miscalculation of the true interval of pulmonary CO uptake and the effective alveolar volume, they did not study the influence of a false determination of the initial alveolar partial pressure of CO on Dl _{CO} in detail.
We therefore reexamined the dependence of values in the singlebreath diffusing capacity of CO using the two different methods of calculating Dl _{CO} by applying both the conventional solution of the breath holding equation originated with Krogh (7) as well as the threeequation methodology to data obtained from singlebreath experiments that we performed on anesthetized and mechanically ventilated rabbits.
MATERIALS AND METHODS
Animals.
Fourteen rabbits (Chinchilla crossbreed; mean body wt 3.5 kg, range 2.8–5.6 kg) were used. The rabbits were anesthetized using pentobarbital sodium (19 mg ⋅ kg^{−1} ⋅ h^{−1}iv), paralyzed by alcuronium (0.1 mg ⋅ kg^{−1} ⋅ h^{−1}iv), intubated by a cuffed endotracheal tube, and artificially ventilated using a selfmade, computerized ventilatory servo system.
Experimental procedure.
After the onset of anesthesia, pressurevolume curves were recorded in each animal by inflating and deflating the rabbit lungs using specific steps in volume. The airway pressure was measured during short breath holds using a differential pressure transducer which was connected to the endotracheal tube. Residual volume (V_{R}) was defined as lung volume attained at −20 cmH_{2}O of airway pressure and was calculated from the argon (Ar) dilution induced by inflating the lungs with nitrogen (N_{2}). Anatomical and apparatus dead space was determined by recording expirograms for CO (mean ± SD value: 7.0 ± 0.6 ml) and were used to calculate the effective inflation and deflation times. Starting from V_{R}, the rabbit lungs were inflated using an indicator gas mixture containing 0.2% CO in N_{2} and, after executing the breath holding, were deflated back down to V_{R}. Inflation and deflation procedures were performed identically in each rabbit. Two animal groups were investigated. As can be seen from Table1, the rabbits ofgroup A were inflated and deflated with the application of a flow rate of 55 ml/s, whereas in animalgroup B we used 83 ml/s. Limited by the technical features of our ventilatory servo system, tidal volumes of up to 55 ml or flow rates of up to 83 ml/s were applicable.
Mass spectrometry.
During the singlebreath maneuvers, the rabbit lungs were in each case deflated via a spiral stainless steel tube (3.5 mm ID, length 5 m), where the alveolar gas sample was dried by freezing. We used a respiratory magnetic sector mass spectrometer (M3, Varian MAT, Bremen, Germany) to analyze the alveolar partial pressure of CO within the endtidal portion of the gas sample (stored within the spiral tube) that was continuously sucked into the inlet system of the mass spectrometer (2m heated steel inlet capillary, 5 ml/min) after the deflation had been completed. The relevant gases CO and Ar were detected at the following masstocharge ratios: C^{+}, 12 (C^{+} ions represent a 5% fraction of the total CO); Ar^{+}, 40. We avoided drift errors and cross talk effects by repeatedly comparing the dried alveolar gas sample with a reference gas that only differed in its CO content (11). The signaltonoise ratio was 530:1 at 0.2% CO.
Calculations of Dl_{CO}: Method 1.
The first method of calculating Dl
_{CO} is based on three differential equations defining pulmonary CO transfer during inflation, breath holding, and deflation. As has already been stressed by Graham et al. (3), this approach contrasts with the conventional analysis in which CO uptake is reduced to diffusive removal during breath holding. The solutions of the three equations are
Calculations of Dl_{CO}: Method 2.
In method 2
Analysis.
For t
_{1},t
_{3} → 0, the three equations simply converge at the conventional solution as
Statistics.
Data are expressed as means ± SD. In each animal, mean values of Dl _{CO}(method 2)/Dl _{CO}(method 1) were subjected to Student’s onetailed ttest to check difference from unity. Differences were considered significant whenP < 0.05.
RESULTS
The results of calculating Dl _{CO} frommethods 1 and2 and the corresponding ratios are listed in Table 2. The results show that in each rabbit mean values of Dl _{CO}(method 2)/Dl _{CO}(method 1) are significantly greater than unity. In animal group A (inflation volume = deflation volume = 55 ml; inflation time = deflation time = 1 s;V˙i = V˙e = 55 ml/s), the mean ratio amounted to Dl _{CO}(method 2)/Dl _{CO}(method 1) = 1.1 ± 0.01. In animal group B (inflation volume = deflation volume = 50 ml; inflation time = deflation time = 0.6 s;V˙i = V˙e = 83 ml/s), the mean ratio of Dl _{CO} values according to both methods was 1.09 ± 0.004.
Furthermore, we found a nonlinear dependence of this ratio on the time interval of CO uptake in both groups but which was more pronounced ingroup A. However, in both animal groups, none of the mean ratios Dl _{CO} ^{recalc}/Dl _{CO}(method 1) were different from unity (maximal deviation 0.005) (see Fig. 1).
DISCUSSION
The main result of the present study is that the conventional solution for calculating the singlebreath diffusing capacity causes Dl _{CO} values to be overestimated compared with the threeequation methodology. This finding has already been stated by Graham et al. (4). The authors confined themselves to showing that neglecting inhalation (or inflation) leads to such an overestimation, which is increasingly pronounced with decreasing flow rates. This is fully corroborated by our own results, because we found that Dl _{CO} values obtained using method 1 were more distinctly overestimated when using method 2 in animal group A (V˙i = 55 ml/s) than in group B (V˙i = 83 ml/s). Going beyond their interpretations, we were able to recalculate the deviation between both methods by correcting the initial Pia _{CO}. However, with respect to the small variations in the gas flows between both animal groups, but particularly with respect toV˙i = V˙e, as well as due to the fact that the singlebreath tests were executed automatically, we should emphasize that the present recalculation does not help to exclude errors stemming from a false determination of alveolar volume or varying inspiratory and expiratory flows during other experimental and human settings. In addition, it is certainly not possible to assess the role of functional inhomogeneities in affecting values of Dl _{CO} when considering singlebreath maneuvers that have been performed on anesthetized animals in an identical manner. Nonetheless, the procedure presented may provide an opportunity to later improve the accuracy and precision of Dl _{CO} values determined by using the conventional method.
The present study offers a novel approach to calculating the CO singlebreath diffusing capacity with improved accuracy, requiring only a few additional technical measures (measurement ofV˙i andt
_{1} for determining
In Fig. 2 we show that calculating Pia _{CO} on the basis of Eq. 1 indeed leads to decreasing values of corrected Pia _{CO} and thus to decreasing values of Dl _{CO}. Because Eq. 1 includest _{1} as the main parameter, we found a dependence of this decrease on the time interval of inflation in both animal groups.
Perspectives
By correcting the Pa _{CO} at the beginning of breath holding, we were able to bring values of the singlebreath diffusing capacity of CO, as determined using the conventional method, into line with those obtained by applying the threeequation methodology. However, in this connection it must be emphasized that we used the method for calculating the time interval of CO uptake (conventional method) as proposed by Ogilvie et al. (8). This method may rarely be used in current laboratories today because, according to the recommendation of the American Thoracic Society (1), the method of Jones and Meade (6) appears to be the preferentially applied one. In their theoretical and experimental analysis, the authors made corrective adjustments for the influences of a false determination of the true time interval of CO uptake on the calculation of Dl _{CO}values by simply subtracting 3/10 of the inspiratory time. This procedure nonetheless produces an additional uncertainty compared with the threeequation methodology. Here, the true inspiration and expiration times are exactly calculated using values of anatomical and apparatus dead spaces (please refer toExperimental procedure) where diffusive CO uptake can be neglected. Thus the method of Jones and Meade (6) provides only a rough estimate of the effective time interval of CO uptake. Although Graham et al. (3) did already stress that their method ameliorates the accuracy of Dl _{CO}determinations even in nonuniformly diffusiondistributed lungs, the effect of inaccurate calculations of the Ar dilution on the calculation of the initial Pa _{CO}remains to be evaluated (e.g., in patients with airway obstructions).
Acknowledgments
We thank Barbara Schreiber, Christa Pusch, and Bernd Eixmann for expert technical assistance.
Footnotes

Address reprint requests to H. Heller, Dept. of Physiology, University of Bonn, 53115 Bonn, Nussallee 11, Germany.
 Copyright © 1998 the American Physiological Society