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Chronic Exposure to Low-Dose Carbon Monoxide Alters Hemoglobin Mass and V˙O2max

SCHMIDT, WALTER F. J.1; HOFFMEISTER, TORBEN1; HAUPT, SANDRA1; SCHWENKE, DIRK2; WACHSMUTH, NADINE B.1; BYRNES, WILLIAM C.3

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Medicine & Science in Sports & Exercise: September 2020 - Volume 52 - Issue 9 - p 1879-1887
doi: 10.1249/MSS.0000000000002330
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Abstract

Carbon monoxide (CO) is best known for its deleterious health effects at high doses. However, in low doses, it seems to have therapeutic and diagnostic value. Along with nitric oxide (NO) and hydrogen sulfide (H2S), CO is one of three known biologically active gaseous signaling molecules (1). As a signaling molecule, CO is produced endogenously in low concentrations and has various important biological effects. At normal physiological levels (and possibly at slightly elevated pharmacological levels), CO has been reported to modulate inflammation and oxidative stresses (2) as well as regulate mitochondrial biogenesis and angiogenesis (3). The potential efficacy of CO has prompted an increase in research focusing on its antioxidative, anti-inflammatory, antithrombotic, and antiapoptotic effects (4). Physiologists and health professionals have also used low-dose CO administrations to assess physiological parameters such as lung diffusion capacity and hemoglobin mass (Hbmass). Since the development of the optimized CO rebreathing procedure for the assessment of Hbmass (5,6), there has been an increased use of this procedure in the exercise and altitude research protocols.

Because of its potential as a signaling molecule that alters physiological functions, it is important to know if the low-dose CO utilized for determining parameters such as Hbmass alters other physiological parameters. Because of its competitive binding with hemoglobin, acute low-dose administrations of CO reduce oxygen-carrying capacity and decrease an individual’s V˙O2max (5,7). This decrease in V˙O2max results in an increased relative exercise intensity at fixed submaximal exercise tasks as reflected by increased heart rate and blood lactate responses (8). Because CO is cleared from the system within 12 h (5), these responses are relatively short-lived. Recently, we have conducted a series of studies designed to examine whether low-dose CO administration has other physiological effects. In our first study, we demonstrated that acute low-dose CO did not influence exercise economy (8). Our second study examined the influence of daily single low doses of CO over the course of 12 d on aerobic performance predictors and peak-power exercise tolerance. Using a single blind parallel-groups design, we demonstrated that there were no significant differences between the placebo and CO groups in Hbmass, V˙O2max, lactate threshold, economy, and peak-power exercise tolerance (9).

Although acute or intermittent exposure does not stimulate erythropoietic processes, chronic continuous exposure to low-dose CO might provide a stronger stimulus and the chronically lower arterial oxygen content might stimulate erythropoietic processes. According to Wenger and Hoogewijs (10), the oxygen-sensing and erythropoietin (EPO)-producing cells of the kidney depend largely on the regional blood oxygen content to determine alterations in oxygen levels. Because regional blood oxygen content is reduced when hemoglobin is ligated with CO, an increase in EPO production might be expected. Support for this concept can be found in the well-known higher hemoglobin concentration and hematocrit (Hct) values in heavy smokers compared with nonsmokers (11). Therefore, we examined the effect of exposure to low-dose CO on Hbmass, erythropoietic activity, and blood volume compartments and as well as V˙O2max. We hypothesized that there would be an increase in Hbmass and subsequently V˙O2max as a result of chronic continuous exposure to low-dose CO inhalation.

METHODS

Subjects

Twenty-two healthy and moderately trained male subjects volunteered for the study. All subjects provided written informed consent, which included the aim and possible risks of the study. The study was approved by the local ethics committee of the University of Bayreuth.

The subjects were randomly distributed to the intervention group (n = 11) inhaling CO or to the placebo group (n = 11) inhaling air instead. Table 1 provides the anthropometric and performance data for all subjects.

T1
TABLE 1:
Anthropometric and performance data of the subjects.

Design of the study

The study was performed using a blinded, placebo-controlled design consisting of a 1-wk baseline period, a 3-wk intervention period, and a 3-wk posttreatment period. The aim of the study was to evaluate the effects of an increase in blood CO–hemoglobin concentration (COHb) by approximately 5% on Hbmass and performance. For this purpose, subjects inhaled a predetermined CO bolus five times per day, starting at 8 am and then every 4 h until midnight. Subjects were allowed to sleep from midnight to 8 am without taking a CO bolus. The placebo group inhaled an ambient air bolus instead of CO from identical syringes on the same time schedule. In order to detect possible hypoxic side effects, the Lake Louise Score, which was originally developed for acute stays at high altitude, was determined daily.

Hbmass was determined using the optimized CO rebreathing method (5) twice before and every week during the inhalation period as well as 2, 7, and 21 d after this period. Two cubital venous blood samples were collected in heparinized (2 mL) and serum separator (5 mL) vacutainer tubes. These samples were taken before and every week during the inhalation period as well as 2, 7, 14, and 21 d after this period. These samples were used for the determination of basic hematological values as well as reticulocytes, ferritin, serum EPO, hepcidin, and serum transferrin receptor concentration. V˙O2max was determined before and at the end of the intervention period and 1 wk thereafter.

Inhalation of CO and calculation of the inhaled CO volume

The CO bolus was administered via a 100-mL syringe. The CO volume was directly inhaled from the syringe, and subjects were instructed to hold their breath for 30 s followed by a rebreathing period of 1.5 min from an anesthetic bag (3.5 L) containing ambient air. The first bolus of the CO volume was calculated from the baseline Hbmass measurements where 0.8–1.0 mL·kg−1 body mass (70.4 ± 4.9 mL, depending on the training state of the subject) increased COHb by 4.7% ± 0.4%. The target COHb concentration was 7% in the seventh minute after the bolus inhalation. Because the COHb concentration did not return to baseline until the following CO administration, COHb was measured in capillarized blood immediately before the further CO inhalations, and the respective inhaled CO volume was calculated according to formula 1.

where CO-Voladm is the CO volume to administer; Δ[COHb]t, target COHb concentration (difference from [COHb]i); [COHb]act, actual COHb concentration before inhaling the CO bolus; [COHb]i, baseline COHb concentration at the initial Hbmass test; CO-Voli, CO volume administered at the initial Hbmass test; and ΔCOHbi, difference in COHb concentration at the initial Hbmass test.

[COHb] was determined every 4 h during the first day of the application period and in random order during the remaining intervention period. On the first day, the [COHb] was between 3.6% ± 0.8% before and 6.8% ± 0.9% after the bolus inhalations (Fig. 1B), and it was very similar the following days.

F1
FIGURE 1:
Changes in COHb concentration after a single CO bolus administration (A) and on the first day of CO administration (B). The target [COHb] 7 min after inhalation was 7%, and the CO bolus was calculated according to formula 1 (see text). The placebo group (B) received ambient air instead.

Sample transport and storage

The whole procedure, including blood sampling, sample transport, sample storage, and sample analyses, was performed according to current World Anti-Doping Agency (WADA) guidelines (12). The blood samples were taken in the morning between 8 am and 9 am after leaving the subject for at least 15 min in a seated position. Within 24 h, the samples were transported to the WADA-accredited Institute of Doping Analysis and Sports Biochemistry in Dresden, Germany, in a refrigerated state monitored by a temperature data logger.

Blood analytical procedures

The Sysmex XT2000i hematological analyzer (Sysmex, Norderstedt, Germany) was used for the determination of hemoglobin concentration ([Hb]), Hct, percentage of reticulocytes (Ret%), immature reticulocyte fraction (IRF), red cell indices, and other routine hematological parameters. The OFF-score as a parameter of suppressed erythropoiesis after ceasing an erythropoiesis boosting measure was calculated as ratio between [Hb] and Ret% (OFF-score = ([Hb] (g·L−1) − 60 × Ret%) (13).

In serum, the following parameters were determined: ferritin (LKFE1, ELISA, and Immulite 1000 [Siemens Healthcare Diagnostics GmbH, Eschborn, Germany]), EPO (LKEP1, ELISA, and Immulite 1000 [Siemens Healthcare Diagnostics GmbH]), soluble transferrin receptor (Quantikine IVD (DTFTR1) Assay (R&D Systems, bio-techne, Wiesbaden, Germany) and Infinite 200 PRO [Tecan, Männedorf, Switzerland]), hepcidin (Quantikine ELISA [DHP250, bio-techne, Wiesbaden, Germany]), and C-reactive protein (high sensitive—LKCRP1, ELISA, and Immulite 1000 [Siemens Healthcare Diagnostics GmbH]).

Hbmass determination

Hbmass was determined by using the optimized CO rebreathing method as described and modified by Schmidt and Prommer (5,6). Briefly, a bolus of 99.97% CO (0.8–1.0 mL CO·kg−1 body mass) was administered to subjects and rebreathed along with 3 L of 100% O2 for 2 min. Arterialized capillary blood samples were taken from a hyperemized earlobe before and 7 min after the rebreathing procedure and analyzed in sextuplicate using an OSM3 hemoximeter (Radiometer, Copenhagen, Denmark). End tidal [CO] was assessed before and 2 min after the rebreathing procedure using a portable CO detector (Draeger Pac7000, Lübeck, Germany). Hbmass was assessed in duplicate before the intervention; there was no significant difference between the two baseline tests (difference, 5.2 ± 15.0 g; n = 22) so the mean of both tests was used as the subject’s baseline value. The typical error for Hbmass measurements determined from these duplicate tests was 1.2%.

Additional tests

Three subjects performed additional nonblinded tests to determine the effects of CO breathing on the position of the oxygen dissociation curve (ODC) and on the circadian behavior of plasma EPO concentration. These tests were not included into the main study to avoid any influences of additional blood loss on Hbmass and the other hematological parameters. In heparinized cubital venous blood samples (2 mL) taken before and until 7.5 h after the inhalation of a CO bolus as described previously, acid base status and the oxygen half-saturation pressure (P50) for the prevailing condition, that is, for actual pH and PCO2, were determined using the ABL 80 blood gas analyzer (Radiometer).

To determine plasma EPO concentration, further venous blood samples were taken for three consecutive days when CO was administered as described previously. Sampling was before inhaling the first CO bolus at 8 am, at 3 pm, and at 11 pm, all for 3 d, as well as on the morning of the fourth day at 8 am. On a separate three consecutive days, blood samples were taken under control conditions.

Aerobic performance

Aerobic performance was determined on a cycle ergometer (Lode, Groningen, the Netherlands) by starting with 100 W (3 min) and increasing the load by 50 W every 3 min (this occurred stepwise every minute by steps of 17, 17, and 16 W) until subjective exhaustion. Key dependent variables were V˙O2max (METALYZER® 3B; Cortex, Leipzig, Germany) and power at exhaustion. Blood samples for lactic acid determination were taken from a hyperemized earlobe before exercise, every 3 min during exercise plus immediately, and 1, 3, 5, and 7 min after exhaustion. The typical error for determination of V˙O2max in our laboratory is 2.5%.

Statistics

Data are presented as the mean ± SD. To determine the sample size for a 3% change in Hbmass due to the CO inhalation intervention, a power analysis was performed assuming the reliability of the CO rebreathing method expressed as typical error as 1.4%. A 90% power was achieved with the sample size n = 10 in both groups (14). An ANOVA with repeated measurements was carried out to detect main effects for group and time as well as group–time interactions. Significance level was set at P < 0.05. In case of significant outcome of the ANOVA (interaction of time and treatment), unpaired t-tests were used to compare mean values for both groups at identical time points, and paired t-tests were used to compare the mean values of identical individuals at different time points. To minimize the risk for type I errors, a correction for multiple measurements was performed. Only in case of significant outcome of the ANOVA, changes of the investigated parameters are described in the Results section. Linear regression analysis was performed to detect any relationship between change in V˙O2max and change in Hbmass.

RESULTS

CO Kinetics and P50

After a single CO inhalation, [COHb] immediately increased in arterialized blood by ~8% and dropped within the following 4 h to ~4% (Fig. 1A). To adjust and control the individual CO administration, [COHb] was measured within 5 min before and exactly 7 min after the CO bolus inhalation. Over the whole day, we determined almost identical oscillations for [COHb] being ~7% 7 min after CO inhalation and ~4% immediately before the next CO inhalation (Fig. 1B). In blood samples randomly obtained during the whole study, [COHb] was always very close to these values.

In the supplemental tests, 30 min after inhaling the CO bolus, the ODC was shifted to the left side (P50 from 28.9 ± 2.7 to 23.8 ± 1.4 mm Hg) and had almost returned to baseline after 4 h (P50 after 1.5 h, 26.2 ± 1.2 mm Hg; after 3.5 h, 27.9 ± 1.9 mm Hg; after 5.5 h, 28.8 ± 0.7 mm Hg; and after 7.5 h, 29.0 ± 1.8 mm Hg).

Changes in hematological parameters

Hbmass significantly increased until the third week of treatment (+43.7 ± 32.0 g) and remained at the higher level for 3 wk after the completion of the intervention (Fig. 2A). In the control group, no change was observed over the measurement period.

F2
FIGURE 2:
Changes in parameters of erythropoiesis during and until 3 wk after chronic CO administration. Hbmass (A), plasma ferritin concentration (B), and plasma EPO concentration (C). Statistic information: for treatment–time interaction: #P < 0.05, ##P < 0.01; post hoc tests vs baseline values: *P < 0.05, **P < 0.01, ***P < 0.001; post hoc tests between both groups at identical time points: +P < 0.05, ++P < 0.01.

Ferritin significantly decreased from the first week until the third week of treatment (−33.9 ± 21.0 ng·mL−1) and completely recovered during the following 3 wk (Fig. 2B). Plasma EPO concentration was not significantly affected during the treatment period, but showed significantly lower values thereafter (Fig. 2C). The additional testing that we performed on a subset of three individuals provided insight into the EPO changes during the 3 d after CO inhalation. EPO tended to increase over time, with low values observed in the morning before inhaling the first CO bolus of the day and increasing values observed during the course of the day (Fig. 3).

F3
FIGURE 3:
Changes in serum EPO concentration during a 3-d continuous CO administration; n = 3.

[Hb] and Hct increased in the second week of the intervention (Table 2), and both the reticulocyte number and the IRF showed significantly higher values after the first intervention week (Table 2). The OFF-score only tended to higher values during the postperiod (Table 2).

T2
TABLE 2:
Hematological data during and after the CO administration period.

The erythrocyte volume was similarly affected to Hbmass, whereas blood volume and plasma volume did not change (Table 2). No change was observed for serum concentrations of hepcidin or transferrin receptor (Table 2).

Performance

Maximum power slightly increased immediately and 1 wk after the intervention from 342 ± 35 to 349 ± 39 W (P < 0.05) and 350 ± 35 W (P < 0.05). V˙O2max did not show significant changes, but there was a tendency to higher values at the end of the intervention (from 4230 ± 275 to 4348 ± 355 mL·min−1; P < 0.1) and 1 wk after the intervention period (4314 ± 347 mL·min−1). There was a significant relationship (P < 0.001) between the individual change in Hbmass and the individual change in V˙O2max (Fig. 4).

F4
FIGURE 4:
Relationship between changes in Hbmass and V˙O2max immediately and 1 wk after a 3-wk continuous CO administration.

DISCUSSION

The most important result was that continuously elevated COHb concentrations enhance erythropoietic processes resulting in increased Hbmass, which was correlated to improvement in V˙O2max.

CO kinetics in blood

As demonstrated in Figure 1B, we were able to adjust [COHb] in blood to an elevated level decreasing the oxygen transport capacity like a stay at moderate altitude (e.g., 2600 m; [15]). The regular CO administration guaranteed [COHb] between 4% and 7% over the whole day for a period of 3 wk. The control of [COHb] furthermore allowed us to adjust the COHb level when CO exhalation had varied, for example, after intensive training units or after very inactive periods like sleeping. The magnitude of [COHb] increase was similar to that observed in blood of moderate smokers, but lower than in heavy smokers where COHb concentrations between 10% and 15% are described (16).

The effect of the intervention on the well-being of our subjects was regularly monitored by the Lake Louise score, but no systematic effect was found. In two cases, light headaches were reported, one in the CO group and one in the placebo group.

Unlike the effects of low inspiratory oxygen content occurring at altitude, CO inhalation not only reduces the oxygen transport capacity but also increases the hemoglobin oxygen affinity (17). After inhaling the CO bolus, the ODC was shifted to the left side as is indicated by the reduction of the P50 by 4 mm Hg. This effect aggravates the hypoxic situation because increased affinity of hemoglobin can hinder oxygen unloading and thus tissue oxygenation. This may induce higher erythropoietic activity than that occurring under comparable inspiratory hypoxic conditions when the ODC is shifted to the right (18,19).

Hbmass and erythropoiesis

Hbmass continuously increased during the 3-wk CO administration period in the mean by 44 g corresponding to 4.8% relative increase with an individual variation ranging from 7 to 123 g. The time course and magnitude of gain in Hbmass are similar to those observed during classic altitude training camps (20) or when applying live high–train low protocols (LH–TL) (21), and correspond to the effects of ~480 h at ~2500 m (22). This effect was expected because CO-induced hypoxia is a well-known stimulant of renal EPO production (23) and the mean increase in [COHb] during the CO administration period corresponded to the decrease in hemoglobin oxygen saturation observed in chronic hypoxia at 2600 m (15). In addition to the decrease in Hb-O2–carrying capacity, also the left-shifted ODC might have contributed to the augmented Hbmass, which can be assumed from the data of Hebbel et al. (24) and Shepherd et al. (19), who described markedly increased [Hb] and Hct in subjects with left-shifted ODC.

Chronic CO effects on the hemoglobin concentration are also proved for heavy smokers (11) who, especially when living at altitude, are characterized by markedly higher [Hb] and Hct values than nonsmokers from the identical altitude (25). There are, to our knowledge, however, no data available comparing Hbmass in smokers and nonsmokers.

In this study, we kept the COHb level high over a period of 3 wk. Shorter CO exposure periods, that is, the administration of a single bout (8) or of one daily CO bout for 10 consecutive days (9), and periods with 40 bouts of CO inhalation within 100 d (26) have not shown any effect on Hbmass. In a recently published article, however, Wang et al. (27) studied the effects of a treadmill-training five times per week after inhaling a CO bolus (1 mL·kg−1 body mass), which increased [COHb] by 4.4% before each training session. They demonstrated a significant increase in Hbmass by 34 g (3.7%; effect size, 0.76) and referred it to the CO-induced hypoxia. However, these authors also report that their matched control group had an increase in Hbmass of 25 g (2.8%; effect size, 0.93). It seems strange that the larger effect size for the control group did not result in significance. We would note that Wang and colleagues did not provide their readers with the significance of the main effects and interaction terms of their statistical analyses for Hbmass or any of their other measured dependent variables, making it difficult to confirm their stated statistical outcomes.

In addition, we would note that Hoppeler and colleagues (28) summarized the existing data on training under hypoxic conditions and did not find any performance-enhancing effects. Also, Hbmass did not increase after a 4-wk 3-h lasting daily exposure to hypobaric hypoxia above simulated 4000 m (29), and LH–TL shows an effective erythropoiesis just when the daily time in hypoxia was greater than 10 h (30). For a CO-induced effective erythropoiesis, only a chronic stimulation has been proved by this study. The effects of other protocols using CO inhalation, for example, similar to the LH–TL concept, remain to be determined.

Concerning the blood volumes, red cell volume showed identical changes to those reported with altitude exposure. In contrast to the altitude effects, however, we did not observe any significant change in plasma and blood volume, which is a well-known fast adaptation increasing [Hb] and O2 transport capacity within the first days in hypoxia. Assuming the hypoxic situation in our study being equivalent to ~2500 m, one would expect a decrease in plasma volume by 13% after 1 wk corresponding to 520 mL (31). There was, however, just a tendency by 120 mL after 2 wk pointing to different effects on electrolyte and water regulating hormones in both kinds of hypoxia.

The increase in Hbmass in this study was accompanied by several markers of increased erythropoiesis. At the beginning of the CO administration period, [EPO] markedly increased showing a circadian time course, as is demonstrated in our additional tests. We found low values in the morning and higher values in the afternoon and during the early night, which has already been described under chronic hypoxic conditions (15) and under regular cobalt administration (32). The low EPO concentration in the morning of the second and third days is likely also due to the time lapse since the previous CO administration (at 12 pm), which occurred approx. 8–9 h before blood sampling (between 8 am and 9 am). After 1 and 2 wk, there was only a tendency for elevated plasma [EPO] left, and after cessation, [EPO] significantly dropped. This behavior is also very well known from altitude studies showing an increase during the first 3 d after ascent and a drop slightly above baseline thereafter (20). After finishing the regular CO administration, erythropoiesis is suppressed like after descent from altitude (33) to adjust Hbmass to the level matching the demands of living in normoxia.

During the inhalation period, ferritin concentration dropped by 34 ng·mL−1, which is in the same magnitude as observed during altitude training (20) and cobalt administration (34). The decrease in ferritin completely compensates for the iron incorporation into the hemoglobin molecules (35), that is, that most of the iron for the additionally produced hemoglobin is taken from the iron stores and not via increased iron absorption. This assumption is confirmed by the unchanged hepcidin concentration during and after the CO inhalation period, which is in contrast to our expectations of a hypoxia-mediated suppression of hepcidin (36). In case of high EPO concentrations, EPO-controlled erythroferrone that is expressed in erythroid precursor cells acts in the liver to reduce the expression of hepcidin (36). Consequently, suppression of hepcidin allows for elevated iron release from storage organs and enhanced absorption of dietary iron by enterocytes. A possible reason for the unchanged hepcidin concentration may therefore be the relatively low EPO concentration during the treatment period.

The reticulocyte count and the IRF significantly increased after 1 wk of CO administration. The increasing IRF indicates the beginning of an erythropoietic stimulation as known from clinical monitoring of the recovery period from bone marrow transplantation (37) or after injections of rhEPO (38). In the latter study, an increase in IRF is evident 36 h after a single dose of rhEPO, reaching a peak after 3 to 4 d. IRF is therefore one of the parameters determined in the hematological module of the athletes’ biological passport and is used for the judgment of suspicious blood profiles (39).

Performance

In this study, we found a close relationship between the individual change in Hbmass and the individual change in V˙O2max (r = 0.70, y = 4.1x − 73.4). The slope of the regression line of ~4 indicates that a change in Hbmass by 1 g results in a change of V˙O2max by 4 mL·min−1, which closely agrees with literature data (40).

The mean change in Hbmass of ∼40 g in this study should therefore result in an increase in V˙O2max of ∼120 mL·min−1, corresponding to 3%. Because of higher methodological noise for V˙O2max determination, however (the typical error using incremental cycle ergometer tests is 2.5% in our laboratory), it is difficult to prove such relative small changes by statistical means. The significant relationship between individual changes in Hbmass and the corresponding changes in V˙O2max after the CO administration period, however, confirms the performance-enhancing effect of increased Hbmass, which is as high as is found after hypoxic training measures. We would note that in a recent meta-analysis of 145 elite athletes, Saunders et al. (41) described a 3% increase in Hbmass and similar increase in V˙O2max after hypoxic exposures (“live high–train high” and “live high–train low” protocols), which is very similar to our results.

Ethical considerations

Our data clearly prove chronic continuous exposure to low-dose CO to increase Hbmass and affect aerobic performance in the same magnitude as do the hypoxic training measures LH–TH and LH–TL. It is therefore not surprising that athletes might have used this method as an ergogenic aid for several years (42) and that this method may be used by a greater number of athletes in the future. This raises the question whether regular CO administration has to be considered as a normal hypoxic training measure like living and sleeping in hypoxic chambers or houses or whether it should be considered as blood manipulation, which has to be banned from WADA.

WADA has to decide whether an athlete may be allowed to inhale a poisonous gas, which in high concentrations is undoubtedly harmful. The health effects associated with exposure to CO range from slight cardiovascular and neurobehavioral effects at low concentrations to unconsciousness and death after acute or chronic exposure to higher concentrations of CO. The symptoms, signs, and prognosis of acute CO poisoning are often individual and frequently correlate poorly with the level of [COHb] (43). On the other hand, in low concentrations, CO is a beneficial gas acting as a messenger molecule like NO, is endogenously produced, and is used for medical diagnostics. Workers are allowed to be exposed for a 40 h·wk−1 or 8 h·d−1 to 35 ppm until 50 ppm CO (43,44), which increase [COHb] until 5% (45), which is similar to the data in our study.

At the moment, it does not seem possible to set a safe upper limit for the continuous use of CO. In addition, like in the early days of EPO abuse, when a series of deaths occurred (46), athletes could use very high doses of CO, which could result in serious health problems or death. We are aware that this study may have opened a “Pandora’s box,” but we are convinced that it will enable us to better control or regulate the handling of CO that is probably already in place.

CONCLUSIONS

Chronic continuous exposure to low-dose CO increasing [COHb] by ~5% significantly increased erythropoietic activity and showed a positive effect on performance. This procedure might therefore be used by athletes, such as altitude training or instead of altitude training, and WADA has to discuss whether it can be accepted as a new training method or has to be banned as a new kind of blood manipulation.

The authors are grateful to those who assisted with the data collection: Daria Schulz, Lisa-Marie Krehl, and Alina Wolf. They also would like to thank all the subjects for participating in the study.

The results of this study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. Results of this study do not constitute endorsement of the American College of Sports Medicine. This study was financially supported by regular funds of the University of Bayreuth. W. F. J. S. 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 article. All other authors declare no conflicts of interest.

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Keywords:

HYPOXIA; ALTITUDE TRAINING; ERYTHROPOIESIS; BLOOD MANIPULATION; POISONOUS GAS

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