The effect of posture and exercise on blood CO kinetics during the optimized carbon monoxide rebreathing procedure

Abstract An indispensable precondition for the determination of hemoglobin mass (Hbmass) and blood volume by CO rebreathing is complete mixing of CO in the blood. The aim of this study was to demonstrate the kinetics of CO in capillary and venous blood in different body positions and during moderate exercise. Six young subjects (4 male, 2 female) performed three 2-min CO rebreathing tests in seated (SEA) & supine (SUP) positions as well as during moderate exercise (EX) on a bicycle ergometer. Before, during, and until 15 min after CO rebreathing cubital venous and capillary blood samples were collected simultaneously and COHb% was determined. COHb% kinetics were significantly slower in SEA than in SUP or EX. Identical COHb% in capillary and venous blood were reached in SEA after 5.0 ± 2.3 min, in SUP after 3.2 ± 1.3 min and in EX after 1.9 ± 1.2 min (EX vs. SEA p < .01, SUP vs. SEA p < .05). After 7th min, Hbmass did not differ between the resting positions (capillary: SEA 766 ± 217 g, SUP 761 ± 227 g; venous: SEA 759 ± 224 g, SUP 744 ± 207 g). Under exercise, however, a higher Hbmass (p < .05) was determined (capillary: 823 ± 221 g, venous: 804 ± 226 g). In blood, the CO mixing time in the supine position is significantly shorter than in the seated position. By the 6th minute complete mixing is achieved in either position giving similar Hbmass determinations. CO-rebreathing under exercise conditions, however, leads to ∼7% higher Hbmass values.


Introduction
Intravascular mixing plays an important role in the transport and action of hormones, drugs, and tracer substances. An empirical parameter for intravascular mixing kinetics is the mixing time (tm), where 95% mixing is generally assumed to be complete [1]. The mixing time is of particular importance for fast-acting drugs such as intravenous anesthetics [2] and for tracers (i.e. radioactive markers and carbon monoxide) for the determination of total hemoglobin as well as blood, red cell and plasma volumes [1]. Values for complete mixing reported in the literature range from three [3] to approximately 15 min [4], and are longer in certain patient groups than in healthy individuals.
The optimized carbon monoxide (CO) rebreathing (oCOR) procedure has become a widely used tool for the determination of hemoglobin mass and for the estimation of blood, plasma and red cell volumes. A crucial prerequisite for this method is complete mixing of the administered CO within the vascular compartment. Without complete mixing, Hbmass and blood volumes would be underestimated when using arterial blood and overestimated when using venous blood. In a recent study by Kellenberger and colleagues [5], it was suggested that complete mixing of carbon monoxide in the vascular compartment does not occur during the oCOR procedure. This was related to the use of the seated position for the determination of Hbmass. It was suggested that pooling of blood in the lower extremities resulted in hemoglobin molecules in that part of the circulation not being tagged with CO.
Biological factors that determine mixing time include cardiac output, tissue perfusion, and venous return to the heart, which are influenced by blood viscosity, body position, and physical activity status, among other factors. When CO rebreathing is performed in a seated position versus a supine position mixing time is likely to be longer due to pooling of blood in the lower extremities. On the other hand, if CO rebreathing were performed while physically active, mixing time is likely to be shortened compared with when rebreathing is performed at rest because of the higher cardiac output and active muscle pump.
In the oCOR method, a CO-bolus is inhaled while the participant is normally in the seated position and CO-rebreathing is limited to 2 min [6,7]. For the measurement of the increase in COHb%, blood is taken before and 7 min after the start of CO inhalation. While very good validity and reliability of the method was consistently determined in healthy subjects [8,9], subjects with very high hemoglobin concentration (>21 g/dL) showed a significantly prolonged mixing time being approximately 14 min [4]. To date, no comparative measurements of the CO mixing time using the oCOR procedure in different body positions exist.
Therefore, the aim of the study was to compare the mixing time of CO in the vascular compartment following a CO bolus administration in the supine versus seated positions. In addition, the influence of physical exercise and the associated higher cardiac output and forced venous return on the mixing time was evaluated. The results of this investigation were then used to evaluate the timing of blood sampling during the oCOR procedure.

Subjects
The study was conducted with six healthy, moderately endurance trained, normal-weight subjects, four men and two women. All subjects were informed about the procedure and possible risks before participation and gave written informed consent. The study was approved by the ethics committee of the University of Bayreuth/Germany (No. O1305/1-GB). Characteristics of the subjects are shown in Table 1.

Study design
In randomized order, three CO rebreathing tests were performed. Rebreathing was performed in the seated (SEA), the supine position (SUP), and during light cycle ergometer exercise (EX, 100 Watts for men, 50 Watts for women). Additionally, each of the three tests was repeated to determine the reliability of each test setup.
Hb-mass was determined using the CO-rebreathing method (oCOR) as described and modified by Schmidt and Prommer [6,7]. Briefly, a bolus of 99.97% carbon monoxide (CO; 0.8 and 1.0 mL CO per kg body mass in females and males, respectively) was administered to subjects and rebreathed along with 3 liters of 100% O 2 for two minutes. Capillary and venous blood samples were taken from an hyperemized earlobe and from a cubital vein via an indwelling catheter. Samples were taken simultaneously every 10 sec in the first minute, every minute until the 10th min, and in min 12 and 15 after the CO-bolus inhalation. COHb% in the capillary and venous blood was determined immediately in triplicate using a CO-oximeter (OSM3, Radiometer, Denmark). End-tidal CO concentration was assessed before as well as 2 min and 6 min after the rebreathing procedure under both resting conditions (SEA and SUP) using a portable CO detector (Draeger Pac7000, Germany).
The test setup on the cycle ergometer was modified to shorten the CO rebreathing to one minute and to increase the volume of oxygen rebreathed with the CO from three liters to 4 liters. During the series of measurements on the cycle ergometer, after being disconnected from the spirometer, the subject exercised for an additional 14 min while inhaling ambient air and exhaling it via a three-way valve towards a flow meter (GMT GmbH; Groß-Gerau, Germany) to determine the exhaled volume. The exhaled air then entered a collection container containing a CO meter (Dräger Pac 7000; Lübeck, Germany), measuring the CO concentration on a minute-by-minute basis.
To accurately determine the volume of CO that has passed into the blood, the amount of CO remaining in the spirometer and in the residual volume is subtracted from the administered CO bolus, as is the CO diffusing to myoglobin and the CO exhaled via respiration (for detailed description see [6,7].

Test specifics
For the test in seated position, the subject sat on a chair with the upper body upright and the legs forming a 90° angle. The arm from which venous blood sampling was performed was almost extended on a table. Blood flow in the hand on the collection side was assisted with a heat pad sealed airtight around the hand. In the supine position, the legs were placed at a 90° angle against a wall. Blood was collected from an arm that was stretched out to the side and positioned at the same level as the upper body. Again, the hand was kept warm using the above method. The measurements on the cycle ergometer were performed with a 100-Watt resistance for the male subjects and with 50 Watts for the female subjects over the entire period of blood collection. The cycle ergometer was specially adjusted for each subject, who sat upright so that he/she could grip the handlebars with an almost extended arm. While the subject was able to support him/herself with one arm, the blood sample was taken from the other, whereby the blood flow was also supported by warming the hand.
Before the rebreathing procedure, there was a ten-minute 'preparation phase' . Here the test subject was already in the position of the subsequent test in seated or supine position and exercised in the third test on the ergometer (Excalibur, Lode, Netherlands) with the load in which the subsequent measurement was performed.

Calculations
For capillary and venous blood samples, COHb% was determined in triplicate with two blood samples collected at rest and one blood sample collected at each post rest time point. The mean of these triplicates was calculated at each sampling period and utilized for further calculation. To compare the change in COHb% during the different testing protocols, the difference in COHb% at each time point from the baseline values was calculated.
Hbmass was calculated as described previously [7] for each time point of blood sampling (formula (1)). CO exhalation was determined from the time point of disconnecting the subject from the spirometer, and diffusion of CO to myoglobin was calculated with a fixed factor of 0.3%/min of the inhaled CO volume [7].
The complete mixing time of blood with CO was estimated in two ways: i. The time when capillary and venous ΔCOHb% were less than 0.1% for the first time. ii. The time when the calculated Hbmass deviated by <2% from the plateau Hbmass values had reached between the 7th and 15th minutes.

Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics (version 25 for Windows, Armonk, NY, USA). Values are presented as the mean ± standard deviation (SD), unless otherwise stated. This was a feasibility study, and a formal power calculation was therefore not required. A specific approach to compute reliability statistics to compare test-retest performance expressed as the typical error of measurement (TEM = standard deviation of the difference score divided by √2) was used (see [11]). When comparing COHb% in capillary blood during test-re-test, TE was below 0.2% in all three experimental setups from the 5th min after CO bolus inhalation (see supplemental material). An analysis of variance (mixed model) was performed comparing the following time courses: (i) COHb% in capillary and venous blood, (ii) COHb% when comparing the two resting positions and the exercise test, and (iii) Hbmass in capillary and venous blood in the three experimental approaches. Paired t-tests were used in case of significant results of the ANOVA. To minimize the risk for type I errors, a correction for multiple measurements according to Benjamini & Hochberg [12] was performed. All tests were 2-sided, and statistical significance was set at p < .05.
Comparison of COHb% from identical time points of both tests under resting conditions (SEA and SUP) was visualized using Bland-Altman plots [13].

Results
All three experimental approaches showed highly significant differences in COHb% between capillary and venous blood (group, time and interaction: each p < .001). COHb% in capillary blood reached a maximum level within a few seconds, whereas COHb% in venous blood increased over several minutes (Figure 1(A-C)). Venous COHb% increased significantly faster in SUP and EX than in SEA and reached capillary values in EX after 1.9 ± 1.2 min, in SUP after 3.2 ± 1.3 min, and in SEA after 5.0 ± 2.3 min (Table 2 and Figure 1; EX vs. SEA p < .01, SUP vs. SEA p < .05). After the venous values reached the capillary values, there was an identical further decreasing of values over time.
In capillary as well as in venous blood significant differences in COHb% between the three test approaches were observed (group and time p < .001; interaction p < .01 in venous blood). As demonstrated in Figure 2(A) for capillary blood COHb% tended to drop faster in the first seconds in SUP than in SEA; from min 6 it was very close in both conditions. COHb% under exercise was significantly lower than the values during the resting conditions. Venous values increased significantly faster in SUP than in SEA, but were almost identical from min 5 (Figure 2(B)). The values under exercise were significantly lower than those in SUP and SEA from min 5 (SUP) and 6 (SEA), respectively. Bland-Altman plots for capillary and venous COHb% determined in SEA and SUP showed no differences between min 5 and min 15 ( Figure 3(A and B)).
Hbmass calculated in capillary and venous blood reached a plateau at different time points for the three experimental conditions (SEA 5.0 ± 1.3 min, SUP 3.8 ± 1.0 min, EX 2.1 ± 1.9 min; SEA vs. SUP and vs EX p < .05 each, Table 2). Capillary and venous values did not differ from each other between min 5 and min 15 for all experimental conditions. In EX, the calculated Hbmass was significantly higher than the values of SUP and SEA ( Figure 4(A and B)). Table 3 shows the Hbmass values determined in the 7 th min after bolus inhalation, which do not differ between SUP and SEA, but are significantly higher in EX.

Discussion
This protocol uniquely demonstrates that intravascular CO mixing time is influenced by resting body position and moderate exercise with the fastest complete mixing time under exercise and slowest complete mixing time in the seated position at rest. From the 6th minute after CO bolus inhalation, there were no differences between Hbmass calculated from capillary and venous blood after CO rebreathing in seated and supine positions. This confirms that the currently recommended carboxyhemoglobin sampling times for the oCOR procedure provide the same results for Hbmass regardless of body position during the procedure. For CO rebreathing under exercise, the calculated Hbmass was 7% higher when compared to Hb mass determined during rest regardless of position.

CO-mixing time
In the literature, CO mixing times in the cardiovascular system have been mostly determined in connection with protocols determining blood volume or its partial volumes. Although the influence of body position on mixing time has not been explicitly studied, shorter times appear to be present in the supine position. Thus, mixing times of 3 min [3] and 5 min [14] have been reported in the supine position, whereas in the seated position the times range from 6 to 10 min have been reported [7,15]. These values apply to healthy adult subjects under resting conditions, whereas significantly longer mixing times (14 min) have been found in polycythemia patients [4].
To our knowledge, the present study is the first to systematically investigate mixing times in different resting body positions and during exercise. Because the cubital venous COHb% values may not exactly match the values in the leg veins [16] and possibly the mixed venous values, in addition to comparing COHb% in cubital venous and capillary blood, we also took as a marker of the mixing time the time point after which the calculated Hbmass yielded a constant value. Since the results of both determination methods deviate only slightly from each other (Table 2), we assume a valid determination of the complete mixing time for each approach. This view is reinforced by the fact that the differences in COHb% between the tests in sitting and supine positions      are no greater than between the tests-retests in identical positions (see data in supplemental material). The reason for the lower mixing time in the supine position compared to the seated position is due in particular to a considerably increased venous return from the legs in the supine position, as is known from situations in weightlessness, bed rest and immersion studies. Thus, after only one minute in weightlessness, a volume shift of 450 mL occurs from the legs predominantly to the thoracic and to a lesser extent to the intracranial region [17]. When changing position from standing to supine, about 11% of the blood volume is redistributed from the legs to the central blood volume and 75% of it to the lungs [18,19]. Therefore, in the supine position, inhaled CO can be mixed much more effectively and results in a short mixing time.
In addition to the role of increased central blood volume, an improved ventilation perfusion ratio might also facilitate a faster mixing time in the supine position. A change in posture from standing to supine causes a redistribution of blood flow in the direction of gravity, resulting in a more even distribution of blood flow in the lungs. Thereby, blood flow in the apical parts of the lung increases and blood flow in the basal parts of the lung decreases [20]. As a result, more areas of the lung participate in gas exchange, which should lead to better diffusion of CO from the lungs to the blood. However, in this study, higher COHb% cannot be detected in arterialized blood in the supine position, so this effect is unlikely to be important for CO rebreathing.
To our knowledge, there are no data in the literature on the mixing time of a marker during physical activity. Our results show a significantly faster mixing time than with the seated position at rest and a tendency to a faster time than the supine position. The causes are primarily a faster circulatory transit time, which is reflected in a higher cardiac output, which increases ~2.5-fold at 100 Watts [21]. Furthermore, the activation of the muscle pump leads to an unloading of blood from the legs, so that, as with the supine position, there is no delay in the mixing time in the leg veins. Like in supine position, during exercise, the uneven distribution of lung blood flow, which is present in a seated position is less pronounced. The decrease of the perfusion gradient depends on the intensity of the work performed [22] probably augmenting the diffusion of CO into the blood, but this cannot be demonstrated here.

Hbmass
Calculation of Hbmass using ΔCOHb% requires accurate determination of the amount of CO diffused into the blood and complete mixing of this amount in the vascular compartment. In addition, diffusion of CO from the blood to the myoglobin must be taken into account, and in the method used here, CO exhalation must also be quantified. Therefore, when making comparisons of Hbmass across time points we adjusted for the variation in CO diffusion to myoglobin and for CO exhalation over time.
From the 5th min after CO bolus breathing, nearly identical Hbmass values are calculated in capillary and venous blood in both sitting and supine positions, so that from this time until the end of the observation period after 15 min, valid Hbmass data are collected with each of these approaches (Figure 4). The importance of the correction for CO lost from blood is evident in the fact that identical Hbmass values (Figure 4) are determined over time despite decreasing COHb% (Figure 3).
If we look at Hbmass values calculated before complete mixing occurred i.e. prior to the 5 th minute, the extent of error due to possible incomplete mixing time is evident (see Table 3 in the supplemental material). If capillary blood is analyzed, an excessively low Hbmass would be measured, and if venous blood is analyzed, an excessively high Hbmass would be measured.
To date, no comparative CO rebreathing tests have been performed in the seated and supine positions. Two studies examined the effect of different postures before a CO rebreathing protocol on the calculation of Hbmass, or COHb%. No posture effect on Hbmass [23], and only a marginal effect on COHb% (0.09%) was found [24]. Our data show that in both positions at the time of the second recommended blood draw for the oCOR procedure, i.e. 7 min after bolus inhalation, complete mixing of CO has occurred in healthy subjects and this measurement protocol can thus be routinely used for Hbmass determination. Interestingly, Breenfeldt-Andersen et al. [25] have recently recommended that the 10-minute rebreathing protocol of Siebenmann et al. [26] be shortened to six minutes based upon complete mixing occurring at this time point. The results of our study provide support for this approach. Table 3. Hbmass (g) calculated from the difference in coHb% in capillary and venous blood obtained before and 7 min after inhaling a co-bolus (mean of min 6, 7 and 8).
Our view is also supported by data from Gore et al. [8] and Garvican et al. [15] showing a 1% increase in Hbmass when the 2nd blood draw during the oCOR occurs at the 10th min compared to the 7th min, which is within the accuracy of the method, which is characterized by a typical error of 1.0-2.0%. Ahlgrim et al. [27] applied CO rebreathing with the identical protocol in patients with impaired ejection fraction and found a 1% increase in mixing time compared to healthy subjects, resulting in a 0.9% lower calculated Hbmass. From these results, they concluded that the mixing time was quite sufficient in these patients and that the method could be used without any restriction. Nevertheless, in clinical investigations of polycythemia patients [4], a possible extension of the mixing time should be considered and verified before starting a scientific study.
When CO rebreathing is performed during physical activity, the calculated Hbmass values are increased by approximately 7% compared to the tests under resting conditions from the 5th minute onwards in both capillary and venous blood. Few comparable data exist in the literature. Keiser et al. [16] found 3% increased values immediately after a 4-min exercise (1 Watt per kg body weight) on the cycle ergometer compared to a CO rebreathing test while completely seated under resting conditions and attributed the difference to incomplete CO mixing in the blood at rest. On the other hand, Gough et al. [28] also described 3% increased Hbmass values when the CO rebreathing test was performed 1-3 h after an ultra-endurance race. Consistent with the argumentation of Gough et al. in our study the increased Hbmass values could be due to altered kinetics of COHb% in blood due to two mechanisms: i. The spleen contains approximately 200mL of blood with a hemoglobin content of approximately 65g under resting conditions, of which up to 40% is released into the circulation during intense exercise [29], which could explain an increase in Hbmass of up to 3.5%. Even if identical Hbmass values were obtained before and after partial splenic depletion [30], different labeling of splenic hemoglobin at rest and during or after exercise cannot be excluded. ii. Increased muscle blood flow and greater capillary surface area during exercise both increase the rate of CO diffusion to intramuscular myoglobin [31] and, because it is not considered, lead to higher calculated Hbmass values.
Consequently, the higher Hbmass in the exercise-test can be attributed in part to a real increase in circulating hemoglobin and in part to an overestimation due to a calculation that is not completely correct. The influence of each of the two factors warrant further investigation.

Limitations
The present study was performed on a relatively small number of subjects. However, the clarity of the differences in mixing time and calculated Hbmass between the conditions under exercise and rest allow clear statistical conclusions [32]. For the rebreathing test under exercise, the procedure had to be adapted. The rebreathing lasted only one instead of two minutes and less CO diffused into the blood (residual CO volume in the spirometer after rebreathing: SEA 1.2 ± 0.3 mL, SUP 0.9 ± 0.3 mL, EX 3.6 ± 1.3 mL). In addition, significantly more CO was exhaled (SEA 0.9 ± 0.1 mL, SUP 0.9 ± 0.1 mL, EX 3.0 ± 2.3 mL). However, although there was less CO in the vascular system at the respective blood sampling times, this is unlikely to have led to an error, as Hbmass determination is not affected by the administration of different amounts of CO between 0.6 mL/kg and 1.0 mL/ kg [33]. The reasons for the higher Hbmass values during CO rebreathing under exercise conditions cannot be clearly elucidated here. Future studies should investigate whether this is a methodological problem or an actual increase in circulating hemoglobin during exercise.

Conclusions
The CO mixing time in the blood after the inhalation of a CO bolus was significantly shorter in the supine position compared to the seated position. Complete mixing, however, was achieved under both conditions around the 6th minute, so that the Hbmass can be calculated equally with both approaches from this time point onwards without differences between venous and capillary blood. When CO rebreathing was performed under exercise, the mixing time was significantly reduced. The calculated Hbmass was increased by 7% compared to resting conditions, which can possibly be attributed to a real increase in circulating hemoglobin and/or to exercise-specific calculation inaccuracies.
The presented results are valid for young healthy subjects; in certain groups of patients, especially in the presence of circulatory disorders, the mixing time should always be checked and taken into account when calculating Hbmass.

Disclosure statement
WFJS. is a managing partner of the company Blood tec GmbH, but he is unaware of any direct or indirect conflict of interest with the contents of this paper. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding
This study was financially supported by regular funds from the University of Bayreuth.