|Year : 2016 | Volume
| Issue : 2 | Page : 59-63
Demonstration and quantification of the redistribution and oxidation of carbon monoxide in the human body by tracer analysis
Makoto Sawano M.D., Ph.D.
Department of Emergency Medicine, Saitama Medical Center, Saitama Medical University, Kawagoe-shi, Saitama, Japan
|Date of Web Publication||11-Jul-2016|
Department of Emergency Medicine, Saitama Medical Center, Saitama Medical University, Kawagoe-shi, Saitama
Source of Support: This study was supported by Japanese Grants-in-Aid for Scientific Research, No. 1620902, 21240057, and 19592097., Conflict of Interest: None
Numerous studies have confirmed the role of endogenous carbon monoxide (CO) gas as a signal transmitter. However, CO is considered an intracellular transmitter, as no studies have demonstrated the redistribution of CO from the blood to tissue cells. Tracer analyses of 13 CO 2 production following 13 CO gas inhalation demonstrated that CO is oxidized to carbon dioxide (CO 2 ) in the body and that CO oxidation does not occur in the circulation. However, these results could not clearly demonstrate the redistribution of CO, because oxidation may have occurred in the airway epithelium. The objective of this study, therefore, was to definitively demonstrate and quantify the redistribution and oxidation of CO using time-course analyses of CO and 13 CO 2 production following 13 CO-hemoglobin infusion. The subject was infused with 0.45 L of 13 CO-saturated autologous blood. Exhaled gas was collected intermittently for 36 hours for measurement of minute volumes of CO/CO 2 exhalation and determination of the 13 CO 2 / 12 CO 2 ratio. 13 CO 2 production significantly increased from 3 to 28 hours, peaking at 8 hours. Of the infused CO, 81% was exhaled as CO and 2.6% as 13 CO 2 . Identical time courses of 13 CO 2 production following 13 CO-hemoglobin infusion and 13 CO inhalation refute the hypothesis that CO is oxidized in the airway epithelium and clearly demonstrate the redistribution of CO from the blood to the tissues. Quantitative analyses have revealed that 19% of CO in the circulating blood is redistributed to tissue cells, whereas 2.6% is oxidized there. Overall, these results suggest that CO functions as a systemic signal transmitter.
Keywords: carbon monoxide; redistribution; oxidization; tracer analysis; stable isotope; signal transmitter, quantitative analysis; hemoglobin
|How to cite this article:|
Sawano M. Demonstration and quantification of the redistribution and oxidation of carbon monoxide in the human body by tracer analysis. Med Gas Res 2016;6:59-63
|How to cite this URL:|
Sawano M. Demonstration and quantification of the redistribution and oxidation of carbon monoxide in the human body by tracer analysis. Med Gas Res [serial online] 2016 [cited 2022 Jul 4];6:59-63. Available from: https://www.medgasres.com/text.asp?2016/6/2/59/184598
MS exclusively contributed to the study, read and approved the final version of this paper.
Conflicts of interest
| Introduction|| |
Numerous studies have shown that endogenous carbon monoxide (CO) acts as a signal transmitter (Wu and Wang, 2005). However, CO is thought to act only intracellularly, as no studies have demonstrated redistribution of CO from the blood to tissues (Halperin et al., 1959; Coburn, 1970; Stewart, 1975; Fujimoto et al., 2004; Wang, 2004). Detection of 13 CO 2 production following the inhalation of 13 CO gas demonstrates the oxidation of CO in human tissues (Sawano and Shimouchi, 2010). However, the possibility of CO oxidation in the airway epithelium precludes definitive demonstration of redistribution. The objective of this study, therefore, was to demonstrate and quantify the redistribution and oxidation of CO in the human body by monitoring CO and 13 CO 2 production following infusion of 13 CO-hemoglobin.
| Subjects and Methods|| |
Ethics statement and subjects
The ethics committee of Saitama Medical University in Japan approved the experimental protocol of this study. The subject, who provided written informed consent before the experiment, was a healthy, non-smoking 50 years old male volunteer.
Baseline exhaled gas measurements and sampling
Throughout the experiment, a physician closely observed the subject and monitored his electrocardiogram and pulse-oximetry to determine arterial carboxy- and oxy-hemoglobin fractions (FCOHb and FO 2 Hb), and blood pressure. The experiment was conducted in an operating room equipped with a forced ventilation system. During the experiment, the subject inhaled synthesized air (21% oxygen gas and 79% nitrogen gas), except for limited interruptions.
At the beginning of the experiment, the subject held his breath for 20 seconds and then exhaled into a 1.3-L gas-sampling bag (pylori exhaled gas sampling bag, Fukuda Denshi Co., Ltd., Tokyo, Japan). The procedure was repeated every 10 minutes, until 25 bags of exhaled gas were collected as baseline gas samples. The subject was then asked to breathe freely for 5 minutes using a device consisting of a respiratory circuit, a ventilator (E100, Newport Medical Instruments Inc., Costa Mesa, CA, USA), a mask, a flow sensor (TF-900P, Nihon Kohden Corp., Tokyo, Japan), an electrochemical sensor (Carbolizer mBA-1000, Taiyo Co., Ltd., Osaka, Japan), and a gas circuit with two one-way valves ([Figure 1]). The E100 generates and regulates a constant 6 L/min flow of synthesized air. The Carbolizer electrochemical sensor is capable of determining the CO concentration in the outflow gas every second, at a resolution of 0.1 ppm (Sawano et al., 2006). We processed simultaneous outputs from the Carbolizer and the TF-900P flow sensor to estimate the minute volume of CO (MVCO) and CO 2 (MVCO 2 ) in exhaled gas, each minute. The device is also capable of determining the end-tidal breath CO (ETCO) concentration with each breath.
|Figure 1: Schematic diagram of the device used to estimate the minute volumes of carbon monoxide and carbon dioxide exhalation each minute and the end-tidal breath carbon monoxide concentration with each breath.|
Click here to view
Infusion of 13 CO-hemoglobin
One week prior to initiation of the experiment, we estimated the circulating blood volume of the subject using the CO-hemoglobin dilution technique (Sawano et al., 2006). We drew 0.45 L (approximately 9% of the estimated circulating blood volume) of venous blood from the subject, sealed it in a sterilized plastic bag with 100 units of heparin and 0.4 L of 100% 13 CO gas (Cambridge Isotope Laboratories, Inc., Andover, MA, USA), and then incubated the bag at 37°C for 30 minutes with gentle shaking to obtain 13 CO-saturated autologous blood. The FCOHb of the blood was determined using a CO-hemoximeter integrated into a blood gas analyzer (ABL-720, Radiometer Copenhagen Co., Ltd., Copenhagen, Denmark), and the 13 CO-saturated blood was infused back into the subject within 60 minutes.
During the infusion, venous blood was sampled every 5 minutes to determine the FCOHb using the ABL-720. The infusion was terminated when the FCOHb reached 9%.
Analysis of CO and 13 CO 2 production
Following infusion, exhaled gas was collected in a 1.3-L bag, and the MVCO was measured every hour for the first 12 hours and every 4 hours thereafter, as was also done for baseline exhaled gas samples and MVCO measurement. Production of 13 CO 2 was monitored by measuring increases in the 13 CO 2 / 12 CO 2 ratio in exhaled gas samples using an infrared spectral analyzer (POCone, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). The POCone analyzer is capable of determining changes in the 13 CO 2 / 12 CO 2 ratio relative to baseline (Δ 13 CO 2 / 12 CO 2 ) at a resolution of a part per thousand. A single measurement required 120 mL of sample gas, and the measurement was repeated 10 times for each sample.
Venous blood was sampled every 4 hours to measure FCOHb, and the ETCO was estimated simultaneously using the device described in [Figure 1]. The experiment was terminated 36 hours after infusion, when the Δ 13 CO 2 / 12 CO 2 , FCOHb, MVCO, and ETCO values had returned to baseline levels.
The infused volume of CO ( 13 CO) was calculated from the FCOHb, solubility of CO, and volume of CO-saturated blood infused. We also calculated the total volumes of CO and CO 2 exhaled in the 37-hour period from the beginning of the infusion step until the end of the experiment by adding the measured MVCO and MVCO 2 values. The volume of endogenous CO exhaled during the experimental period was estimated from the baseline MVCO and then subtracted from the total to obtain the volume of exogenous CO derived from the infusion that was exhaled during the experimental period. From these values, the volume and percentage of the infused CO that was then either exhaled or retained in the body were estimated. The volume and percentage of the infused 13 CO that was oxidized and exhaled as 13 CO 2 were estimated from the MVCO 2 and Δ 13 CO 2 / 12 CO 2 time-course data.
We applied Student's t-test and 5% significance level to assess the significance of increase in 13 CO 2 production.
| Results|| |
Time-course changes of end tidal CO and CO-hemoglobin fraction
[Figure 2] shows the changes in ETCO and FCOHb during the course of the experiment. Following infusion, the ETCO and FCOHb increased to 43.2 ppm and 9.8%, respectively. Thereafter, both parameters exhibited almost identical rates of exponential decay and returned to baseline levels after 24 hours.
|Figure 2: Changes in ETCO and FCOHb after beginning of CO-hemoglobin infusion. |
Note: Closed boxes with solid line show the change in end-tidal breath CO (ETCO, left axis) concentration over time. Closed circles with dotted line show the change in carboxyhemoglobin fraction in the venous blood (FCOHb, right axis) over time. CO: Carbon monoxide; h: hour(s).
Click here to view
Time-course change of exhaled 13 CO 2 / 12 CO 2 ratio relative to the baseline
Values for Δ 13 CO 2 / 12 CO 2 over the course of the experiment are shown in [Figure 3]. The Δ 13 CO 2 / 12 CO 2 increased significantly (P < 0.05, vs. 0 hour) from 3 hours to 28 hours after the initiation of 13 CO-hemoglobin infusion, with a peak at 8 hours.
|Figure 3: Change in the 13 CO 2 / 12 CO 2 ratio relative to baseline (Δ13 CO 2 / 12 CO 2 : per mil) over the course of the experiment.|
Note: Closed boxes and error bars represent means and 95% confidence intervals of 10 repeated measurements per sample. CO: Carbon monoxide; CO 2 : carbon dioxide; h: hour(s).
Click here to view
Time-course changes of MVCO and minute volume of 13 CO 2 (MV 13 CO 2 ) exhalations
Changes in the MVCO and MV 13 CO 2 exhalations from the initiation of infusion until the end of the experiment are shown in [Figure 4].
|Figure 4: Changes in the MVCO and MV 13 CO 2 exhalation from the initiation of infusion.|
Note: Closed boxes and solid line show the change in the minute volume of 13 CO 2 (MV 13 CO 2 ) exhalation derived from 13 CO-hmoglobin infusion (left axis) over time. Closed circles and dotted line show the change in minute volume of CO exhalation (MVCO, right axis) over time. CO: Carbon monoxide; CO 2 : carbon dioxide; h: hour(s).
Click here to view
The fate of CO in the body
The quantitative summary of the production and the fate of CO in the body, beginning at the time of CO infusion, until the end of the experiment, is shown in [Table 1] .
|Table 1: Summary of the fate of CO in the body, beginning at the time of CO infusion|
Click here to view
| Discussion|| |
Previous studies have described the residual effects of CO after carboxyhemoglobin elimination in CO-intoxicated patients (Halperin et al., 1959), the toxic effects of chronic exposure to low-concentration CO (Wang, 2004), the poor correlation between blood carboxyhemoglobin levels and the physiologic effects of CO inhalation (Stewart, 1975), and the protective effect against ischemia-reperfusion injury associated with CO inhalation in rats (Fujimoto et al., 2004). These physiologic effects associated with CO inhalation cannot be explained by carboxyhemoglobin-induced hypoxia alone and instead suggest that CO is redistributed from the blood hemoglobin into tissue cells, where it activates or inhibits various heme protein enzymes (Coburn and Mayers, 1971; Piantadosi, 2002). However, such a redistribution of CO under physiologic conditions has yet to be demonstrated (Wu and Wang, 2005).
A previous study reported the production of 13 CO 2 in a human volunteer following the inhalation of 50 ppm of 13 CO gas (Sawano and Shimouchi, 2010). In that study, human blood was circulated through a cardiopulmonary bypass circuit that simulates human blood circulation and gas exchange, with 50 ppm of 13 CO gas supplied to the oxygenator. However, no 13 CO 2 production was detected. These results demonstrated that under physiologic conditions CO is oxidized within the tissues rather than in the circulating blood (Sawano and Shimouchi, 2010). Due to the possibility that the 13 CO 2 detected could have been derived from the oxidation of CO in the airway epithelium, however, the authors of that study were unable to definitively demonstrate the redistribution of CO from the blood to the tissues.
Another study reported a significant increase in Δ 13 CO 2 / 12 CO 2 between 4 and 31 hours, with a peak at 9 hours after 13 CO inhalation, which exposed the airway epithelium to 50 ppm of 13 CO for 4 hours (Sawano and Shimouchi, 2010). Thus, if the oxidation of CO occurs primarily in the airway epithelium, the increase and peak in 13 CO 2 production following 13 CO inhalation should have appeared 4 hours earlier compared with 13 CO-hemoglobin infusion. However, the time course of 13 CO 2 production following inhalation and infusion were almost identical in this study and the past, Sawano and Shimouchi (2010) suggesting that CO oxidation does not occur in the airway epithelium.
In the present study, quantitative analyses revealed that approximately 20% of the infused hemoglobin-bound CO was not exhaled between the initiation of infusion and termination of the experiment and was instead retained in the body. The ETCO, blood FCOHb, MVCO, MV 13 CO 2 and Δ 13 CO 2 / 12 CO 2 all returned to baseline levels at the end of the experimental period ([Figure 2], [Figure 3], [Figure 4]). Therefore, we conclude that the percentage of the infused CO that was retained in the body had been redistributed from the blood into the tissue cells. Taken together, these results demonstrate that a portion of hemoglobin-bound CO moves from the blood to the tissues, where it is then oxidized. The elucidation of this pathway for the redistribution of CO from the circulating blood to the tissues strongly suggests that the role of endogenous CO is not limited to intracellular signal transmission but may extend to systemic or inter-organ signal transmission.
Previous studies estimated that 80% of the total CO body store is bound to hemoglobin in the red blood cells as carboxyhemoglobin and that 20% is bound to intracellular heme proteins (Coburn, 1967, 1970). Another study suggested that these CO body stores are exchangeable and that CO moves from the blood to the tissues, where it binds to heme proteins (Coburn and Mayers, 1971). It is interesting that the ratio of the redistribution of CO from the blood to the tissues revealed by our quantitative analysis in the present study corresponds well with determinations of the distribution of CO body stores identified in these studies. However, the present study included only one subject; thus, further investigations involving more subjects are needed to derive definite conclusions. Another limitation of our quantitative analysis is that we did not measure CO emission from the skin. However, another study reported that the MVCO emission from the skin is no greater than 0.25% of the exhalation level (Nose and Shimouchi, 2008), suggesting that this omission did not significantly affect the results of the present study.
In conclusion, our study demonstrated that a portion of hemoglobin-bound CO is redistributed from the blood to the tissues, where it is then oxidized. However, the enzyme responsible for the oxidation of CO in the tissues of the human body remains to be identified. Several laboratory studies have demonstrated this enzymatic reaction using cytochrome C oxidase extracted from mitochondria of various animal organs (Tzagoloff and Wharton, 1965; Young and Caughey, 1986; Vijayasarathy et al., 1999), but no study has traced the reaction in live human subjects. The enzyme is localized in mitochondria, which are eliminated from red blood cells during the course of their maturation (Zhang et al., 2009). Our study demonstrated that the oxidation of CO does not occur in the circulating blood and suggests that the enzyme responsible for the reaction is located in the tissues. However, further investigation is needed to test this hypothesis.
A part of this paper was presented at the Breath Summit 2015, the scientific meeting of the International Association for Breath Research, 13-15 Sept. 2016, Vienna, Austria.
| References|| |
Coburn RF (1967) Endogenous carbon monoxide production and body CO stores. Acta Med Scand Suppl 472:269-282.
Coburn RF (1970) The carbon monoxide body stores. Ann N Y Acad Sci 174:11-22.
Coburn RF, Mayers LB (1971) Myoglobin O 2
tension determined from measurement of carboxymyoglobin in skeletal muscle. Am J Physiol 220:66-74.
Fujimoto H, Ohno M, Ayabe S, Kobayashi H, Ishizaka N, Kimura H, Yoshida K, Nagai R (2004) Carbon monoxide protects against cardiac ischemia--reperfusion injury in vivo via MAPK and Akt-eNOS pathways. Arterioscler Thromb Vasc Biol 24:1848-1853.
Halperin MH, Mc FR, Niven JI, Roughton FJ (1959) The time course of the effects of carbon monoxide on visual thresholds. J Physiol 146:583-593.
Nose K, Shimouchi A (2008) Case study on changes in exhalation of carbon monoxide and nitrogen oxide in breath and skin gas during 2-day smoking cessation and restart. J Breath Res 2:037026.
Piantadosi CA (2002) Biological chemistry of carbon monoxide. Antioxid Redox Signal 4:259-270.
Sawano M, Mato T, Tsutsumi H (2006) Bedside red cell volumetry by low-dose carboxyhaemoglobin dilution using expiratory gas analysis. Br J Anaesth 96:186-194.
Sawano M, Shimouchi A (2010) A tracer analysis study on the redistribution and oxidization of endogenous carbon monoxide in the human body. J Clin Biochem Nutr 47:107-110.
Stewart RD (1975) The effect of carbon monoxide on humans. Ann Rev Pharmacol 15:409-423.
Tzagoloff A, Wharton DC (1965) Studies on the electron transfer system. LXII. The reaction of cytochrome oxidase with carbon monoxide. J Biol Chem 240:2628-2633.
Vijayasarathy C, Damle S, Lenka N, Avadhani NG (1999) Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex. Eur J Biochem 266:191-200.
Wang R (2004) The evolvement of gasotransmitter biology and medicine: from atmospheric toxic gases to endogenous gaseous signaling molecules. In: Signal transduction and the gasotransmitters: NO, CO and H 2
S in biology and medicine (Wang R, ed), pp3-32. Humana Press, Totowa.
Wu L, Wang R (2005) Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol Rev 57:585-630.
Young LJ, Caughey WS (1986) Mitochondrial oxygenation of carbon monoxide. Biochem J 239:225-227.
Zhang J, Randall MS, Loyd MR, Dorsey FC, Kundu M, Cleveland JL, Ney PA (2009) Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation. Blood 114:157-164.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
|This article has been cited by|
||A brief history of carbon monoxide and its therapeutic origins
| ||Christopher P. Hopper,Paige N. Zambrana,Ulrich Goebel,Jakob Wollborn |
| ||Nitric Oxide. 2021; 111-112: 45 |
|[Pubmed] | [DOI]|
||The relationship of polluted air and drinking water sources with the prevalence of systemic lupus erythematosus: a provincial population-based study
| ||Jiaqi Chen,Wenqiang Qu,Li Sun,Jiansheng Chen,Wei Kong,Fan Wang,Wenyou Pan,Lin Liu,Min Wu,Fuwan Ding,Huaixia Hu,Xiang Ding,Hua Wei,Yaohong Zou,Xian Qian,Meimei Wang,Jian Wu,Juan Tao,Jun Tan,Zhanyun Da,Miaojia Zhang,Jing Li,Jun Liang,Xuebing Feng,Linyu Geng,Lingyun Sun |
| ||Scientific Reports. 2021; 11(1) |
|[Pubmed] | [DOI]|
||Role of Carbon Monoxide in Host–Gut Microbiome Communication
| ||Christopher P. Hopper,Ladie Kimberly De La Cruz,Kristin V. Lyles,Lauren K. Wareham,Jack A. Gilbert,Zehava Eichenbaum,Marcin Magierowski,Robert K. Poole,Jakob Wollborn,Binghe Wang |
| ||Chemical Reviews. 2020; |
|[Pubmed] | [DOI]|
||Nature’s marvels endowed in gaseous molecules I: carbon monoxide and its physiological and therapeutic roles
| ||Xiaoxiao Yang,Wen Lu,Christopher P. Hopper,Bowen Ke,Binghe Wang |
| ||Acta Pharmaceutica Sinica B. 2020; |
|[Pubmed] | [DOI]|
||ICL-based TDLAS sensor for real-time breath gas analysis of carbon monoxide isotopes
| ||Ramin Ghorbani,Florian M. Schmidt |
| ||Optics Express. 2017; 25(11): 12743 |
|[Pubmed] | [DOI]|