Carbon monoxide inhalation as a potential aid to enhance aerobic endurance

One of the main aims of sports science is the development of methods to improve athletes' physical performance. As the intensity of sports increases, there is a constant need to find new ergogenic aids and methods that are not prohibited, cause hypoxia, which has been proven to improve performance and, accordingly, can potentially positively affect aerobic performance. One such aid is carbon monoxide (CO) gas, which is regularly encountered by anyone exposed to atmospheric air and cigarette smoke.[1] CO is an odorless, colorless or tasteless gas that can have a negative effect on human health[2] at high doses. Moreover, according to some studies, low doses of this herb can have a positive effect on humans due to its influence on various biological processes. As a signaling molecule, CO is produced endogenously at low concentrations and exerts various important biological effects. At normal and slightly elevated physiological levels, it modulates inflammation and oxidative stress[3] and regulates mitochondrial biogenesis[4] and angiogenesis.[5] The potential efficacy of CO has led to an increase in the number of studies proving its antioxidant,[6] anti-inflammatory,[3] and antithrombotic[7] effects. Researchers are also actively using the simultaneous administration of low doses of CO to assess physiological parameters such as lung diffusion capacity and hemoglobin mass.[8] This procedure has become more frequently utilized in exercise research protocols, and the effects of altitude on human performance have been well documented; however, developing an optimized rebreathing procedure for estimating hemoglobin mass has recently received some attention. A vast quantity of research has examined the effects of CO on the human body, and the main issues are its effects on the human body following acute and chronic exposure to various doses. The determination of a safe concentration of carboxyhemoglobin (HbCO) with respect to health and physical performance is also warranted. Given the availability and proven safety of CO when administered at low doses, it is of great practical interest to search, analyze and review all studies that have investigated CO as a potentially ergogenic aid among healthy physically active people. Eligible studies were searched for in the PubMed, Scopus, Web of Science, Cochrane Library and PROSPERO databases from inception to December 2023 to identify studies on the effects of CO inhalation on performance in healthy physically active individuals. Full texts not available in English were excluded, although no language restrictions were applied in the search. The search terms and algorithms used were as follows: (("carbon monoxide" OR "carbon monoxide inhal*" OR "low- dose carbon monoxide" OR "low-dose CO Inhal*" OR "carbon monoxide rebreathing" OR "carbon compounds" OR "inorganic compound") AND (performance OR "maximal aerobic power" OR "physiological functions" OR "physical indicators" OR "comparative physiology" OR loads OR athletes OR "altitude training" OR endurance OR strength OR speed OR coordination OR recovery OR doping OR "peak load" OR "spacer device*" OR exercise) AND (hemoglobin OR carboxyhemoglobin OR erythropoietin)) OR acizol). The search results were downloaded and filtered in Mendeley Reference Manager (v2.64.0; Mendeley Ltd., London, UK) systematic review software. The PICOS design framework was used to screen the titles and abstracts of the retrieved articles by an independent author. We used the PICOS strategy with the following inclusion criteria to assess the eligibility of the retrieved articles: (a) "P" (population), studies with healthy, physically active human participants with no restrictions to sex or age, nonsmokers; (b) "I" (intervention), participants in the intervention group who received any kind of CO intervention where various physiological parameters and changes in any physical quality were analyzed; (c) "C" (comparator), comparison between individuals who received CO to those who did not receive CO (placebo); (d) "O" (outcome), timing, frequency, dose, and duration of CO intervention; and (e) "S" (study design), randomized controlled trials performed in humans. Studies were excluded from the analysis if (a) CO inhalation was a one-time, nonrepeatable event; (b) multiple supplements were administered; or (c) theses, dissertations, conference papers or others were also excluded. In addition, further articles were selected based on a detailed review of studies where the following criteria were met: (a) the intervention was carried out on animals; (b) the determination of CO in the blood of healthy volunteers was studied; (c) hemoglobin mass levels were determined using the CO rebreathing method; (d) the intervention was performed under normobaric and/or hypobaric hypoxia conditions; and (e) the side effects of CO inhalation and the consequences of CO poisoning were described. A manual search helped identify other suitable articles among the eligible full-text articles to be included in the systematic review. A consensus was reached on the final studies included. Data collection process and data items: Following the completion of the search, the results were compared among the three researchers to ensure that the same number of articles was found. Microsoft Excel Spreadsheet adapted from an example provided by the Introduction to Systematic Reviews Course at York University (UK) was then utilized to extract and tabulate the data. The collected data included the following: (a) authors and year of publication; (b) study design (e.g., double-blind, parallel, or crossover); (c) participants' characteristics (e.g., age); (d) intervention protocol (timing, frequency, dose, and duration); (e) exercise protocol (type, duration, repetitions, and sets); (f) sample size; and (g) relevant outcome measures. The outcomes included changes in carboxyhemoglobin levels, maximal oxygen consumption (VO2max) values, hemoglobin mass levels and erythropoietin (EPO) levels and qualitative or quantitative changes in mitochondrial biogenesis markers and skeletal muscle antioxidant enzymes. The time points extracted started from postintervention until the cessation of measurement per individual study. For each outcome measure, the data are presented as the mean ± standard deviation (SD). If the data were unavailable, they were extrapolated from figures using WebPlotDigitzer (v4.6; Rohatgi, Austin, TX, USA) (n = 1 study). Research has shown that this software has high intercoder reliability and validity. Study risk of bias assessment: All identified studies were assessed for risk of bias by employing the revised Cochrane Risk of Bias Tool for Randomized Trials (RoB 2; Cochrane, Bristol, UK). Disagreements in the assessment of risk of bias between researchers were resolved by discussion or consultation with a senior investigator. Results: Based on keywords and any combinations, 1821 articles were selected from the analyzed databases, 112 of which were excluded (111 duplicates and 1 article were removed due to a lack of data in the found article file). Following analysis of the titles and abstracts, an additional 1663 publications were excluded from the remaining 1709 articles because they did not meet the main criteria and themes of this systematic review. Of the remaining 46 articles, 41 articles were removed, 31 of which used CO as an intervention for healthy subjects, although the effects on performance enhancement were not examined. One study did not administer CO by inhalation, two studies used CO to study the effects of indoor CO doses on human health, four studies utilized elevated CO concentrations to simulate altitude training, and three studies recruited smokers as healthy volunteers. Thus, five English-language studies involving healthy subjects that evaluated the effects of CO inhalation on aerobic endurance were identified (Additional Table 1). In addition, the reference lists of each of the five articles were examined, as well as all coauthors' other publications, to minimize the risk of missing suitable articles. Discussion: This study investigated whether CO inhalation can be considered a potential ergogenic aid to improve aerobic performance. The obtained data demonstrate that CO inhalation can be considered a potentially ergogenic aid in relation to aerobic endurance, causing a statistically significant increase in VO2max, similar to hypoxic exercise.[9] The present work results imply that CO inhalation has practical value as a fitness aid, especially when aiming to improve aerobic performance, which is in agreement with the study hypothesis. However, it is difficult to draw a definitive conclusion, as the research within this field is still in its infancy. The potential performance benefits of CO inhalation, similar to those of hypoxic exercise,[9] are evident; however, this effect has only been examined and developed with prolonged use (at least 5 days per week for four weeks, lasting at least 2 minutes per day in doses of at least 1 mL/kg). Short-term exposure did not yield such an effect (10 days with 1.2 mL/kg bolus,[10] 5 days for 60 minutes with 100 ppm[11] and 5 days for 60 minutes with 200 ppm[12]). Regarding EPO levels, which were measured in only two of the five studies included in this systematic review, a single inhalation of a CO to 2 mixture (total 4 L) for 2 minutes using a special mask (CO dose of 1 mL/kg) was the protocol with a statistically significant (P < 0.05) increase in EPO levels (peaking 4 hours after inhalation).[13] It is worth noting that during the CO inhalation protocol (0.8-1.0 mL/kg) with a bolus every 4 hours, five times a day, for 3 weeks, the values of aerobic endurance (P < 0.1) and the EPO level changed from 8.5 mU/mL to 13 mU/mL 4 hours after CO inhalation (P = 0.07). The authors reported an increasing trend that was not statistically significant, which is most likely due to the small sample size, as the EPO level was only measured in three volunteers.[9] These CO effects may be based on the blocking of oxygen binding sites in the hemoglobin molecule, which leads to a decrease in the oxygen concentration in the renal cortex and an increase in EPO secretion due to erythropoiesis stimulation by myeloid stem progenitor cells.[14] The increase in EPO levels demonstrated in a recent study[13] (EPO before = approximately 1.9 mU/mL and EPO after 4 hours = 2.7 mU/mL) was comparable to its increase after exposure to chronic high-altitude hypoxia in the study of Wachsmuth et al.[15] (EPO before = approximately 10.5 mU/mL and EPO after = 17.9 mU/mL). Notably, other mechanisms may underlie the potential positive effects on performance, as described in the studies of Rhodes et al.[11] and Pecorella et al.[12] These mechanisms may include activation of mitochondrial biogenesis in muscles, increased capillarity and myoglobin and glucose transporter type 4 content in muscles, vasodilation and, as a consequence, increased blood flow to muscles, hence improved muscle trophism, and increased oxygen delivery. The mechanism of vasodilatation development during CO inhalation is currently unclear, but it is believed that CO causes vasodilation similarly to nitric oxide due to guanylate cyclase activation.[16] Furthermore, it should be noted that all detected studies were carried out within the last 15 years, and all can be considered high-methodological studies (Table 1). Th at is, interest in such a seemingly long-known substrate, CO inhalation, as a potentially ergogenic aid has increased relatively recently, suggesting that further research on this topic is warranted.Table 1: Risk of bias in the included studiesUndoubtedly, when assessing the impact of any aid on humans, it is necessary to consider its safety. Analyses of studies involving humans exposed to various doses of inhaled CO have revealed either no effect or an insignificant negative short­term effect on parameters potentially important for athletes, such as visual contrast and hearing threshold,[17] visual field width,[18] operator's functions,[19] ability to adapt to a new response set,[20] O2 diffusion,[21] myocardial perfusion[22] and vigilance.[23] At the same time, even when exposed to relatively high doses, the concentration of carboxyhemoglobin in blood does not exceed 10%, which is the safe threshold for humans and health.[13] Future studies should aim to investigate the effects of different doses of CO on various physical performance parameters among elite endurance-trained athletes, as it is clear that previous studies have involved low-level athletes with VO2max levels ranging from 24.79 ± 5.52 to 58.3 ± 2.3 mL/kg/min. Limitations: Several limitations within this review have been identified. First, it should also be noted that some data points had to be extracted from the presented figures using online software, which increases the risk of error. Furthermore, in the scientific community, it is often preferable to publish studies that have demonstrated statistically significant results, even though studies with results that do not demonstrate statistical significance may be clinically relevant and therefore important to the results of a systematic review. To minimize the occurrence of these selection errors, the current researchers searched the PROSPERO database, a registry of ongoing and completed studies. However, this search did not yield any results. Conclusions: In summary, the present review highlighs that CO inhalation, at least 5 days per week for 4 weeks, lasting at least 2 minutes per day at a dose of at least 1 mL/kg, may be considered a potentially safe and effective ergogenic aid in relation to aerobic endurance (VO2 max) and EPO levels, at least in nonathletes. Additional file: Additional Table 1:Studies investigating the effect of CO inhalation on aerobic endurance in healthy physically active individuals.