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Past studies have suggested that some carbon monoxide (CO) moves from blood haemoglobin to tissue cells and that mitochondrial cytochrome c oxidase oxidizes CO to carbon dioxide (CO2). However, no study has demonstrated this redistribution and oxidization of CO under physiological conditions. The objective of this study was to trace the redistribution and oxidization of CO in the human body by detecting 13CO2 production after the inhalation of 13CO. In Experiment 1, we asked a healthy subject to inhale 50 ppm 13CO gas. In Experiment 2, we circulated heparinized human blood in a cardio-pulmonary bypass circuit and supplied 50 ppm 13CO gas to the oxygenator. We sequentially sampled exhaled and output gases and measured the 13CO2/12CO2 ratios. In Experiment 1, the exhaled 13CO2/12CO2 ratio increased significantly between 4 to 31 h of 13CO inhalation. In Experiment 2, the output 13CO2/12CO2 ratio showed no significant increase within 36 h of 13CO input. Experiment 1 demonstrated the oxidization of CO in the human body under physiological conditions. Experiment 2 confirmed that oxidization does not occur in the circulating blood and indicated the redistribution of CO from blood carboxyhaemoglobin to tissue cells.
Since endogenous carbon monoxide (CO) production by heme degradation was first reported in the early 1950s [1, 2], numerous studies have revealed its physiological roles and regulatory mechanisms . However, two pathways in the disposition and catabolism of endogenous CO remain unidentified. One pathway is the redistribution of CO from circulating blood to tissue cells. It is supposed that 80% of endogenously produced CO binds to haemoglobin in the circulating blood and is finally exhaled . Although several studies have suggested that a portion of CO moves from blood haemoglobin to tissue and binds to intracellular heme proteins [5–8], this redistribution of CO has not been demonstrated under physiological conditions . Another pathway is the oxidization of CO to carbon dioxide (CO2) by cytochrome c oxidase in mitochondria. It has been believed that a small metabolic endpoint of endogenous CO is the oxidization to CO2  a process that has been demonstrated in vitro [10–12]. However, in vivo oxidization of CO under physiological conditions has not been shown .
The objective of this study was to trace these unidentified redistribution and oxidization pathways of CO in the human body through detection of 13CO2 production by exhaled gas analysis following the inhalation of 13CO gas. 13C is a stable isotope of 12C with an abundance ratio of 1.1%.
The experimental protocol of this study was approved by the ethics committee of Saitama Medical University.
The subject was a healthy male adult volunteer with no smoking habits who gave written informed consent. First, the subject inhaled the synthesized air and exhaled into a 1.3-L gas sampling bag (Pylori exhaled gas sampling bag, Fukuda Denshi Co., Ltd., Tokyo, Japan) after holding his breath for 20 s. Twenty-five bags of exhaled gas were collected as the background.
Then the subject inhaled synthesized air that contained 50 ppm 13CO gas. During this procedure, a physician closely observed the subject and monitored his electrocardiogram, pulse-oximetry, and blood pressure in an operating room equipped with a forced ventilation system. Exhaled CO and CO2 concentrations were continuously analyzed by an electrochemical sensor (Carbolizer mBA-1000, Taiyo Co., Ltd., Osaka, Japan). This sensor is capable of determining CO and CO2 concentrations every second with resolutions of 0.1 ppm and 0.1%, respectively . The procedure was terminated before the exhaled CO concentration achieved 50 ppm, which is almost equivalent to 10% blood carboxyhaemoglobin concentration (COHb%). Following the inhalation of 13CO gas, exhaled gas was sampled every hour for the first 12 h, every 2 h for the next 12 h, and thereafter every 4 h, by the same procedure as the background. We also sampled venous blood every 6 h to measure COHb% by a CO-hemoximtre integrated into a blood gas analyzer (ABL-720, Radiometer Copenhagen Co., Ltd., Copenhagen, Denmark). In this study, we detected 13CO2 production by measuring the increase of the 13CO2/12CO2 ratio in exhaled gas using an infrared spectral analyzer (POCone, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). This analyzer is capable of determining changes in the 13CO2/12CO2 ratio with a resolution of 0.1 per mil (0/00) relative to the background. A single measurement required 120 mL of sample and the background gases, and the measurement was repeated ten times for each sample. We terminated the experiment 36 h after inhalation, when the elevated 13CO2/12CO2 ratio of the sample had recovered to the background level.
The objective of Experiment 2 was to ensure that the 13CO2 production shown in Experiment 1 was totally derived from the oxidization of 13CO in tissue cells. Fig. 1 shows the experimental device that simulates human blood circulation and gas exchange on a 1/10 scale. We filled a CAPIOX cardiopulmonary bypass circuit (Terumo Co., Tokyo, Japan) with 400 mL of human whole blood, 120 ml of saline, and 1000 units of heparin. A centrifugal pump regulated the circulating blood flow at 0.5 l/min. We infused 100% CO2 gas into the circuit to simulate tissue CO2 production.
First, we supplied synthesized air at 0.5 l/min to the oxygenator as input gas, adjusted the infusion rate of CO2 to maintain the output CO2 concentration between 5% and 6%, and collected the output gas in the sampling bag as the background. Next, we added 50 ppm 13CO to the input gas until the carboxyhaemoglobin concentration of the blood exceeded 10%. Then we collected output gas every 4 h for 36 h.
We measured the 13CO2/12CO2 ratios of the output gas and the carboxyhaemoglobin concentration of the blood by the same procedures as in Experiment 1.
The significance of the increase of the 13CO2/12CO2 ratios was statistically assessed using Student’s t test, with significance level of 5% (p<0.05).
Figs. 2 and and33 show the increase in the exhaled 13CO2/12CO2 ratios relative to the background and the blood carboxyhaemoglobin concentrations plotted against time in Experiments 1 and 2. Error bars of the 13CO2/12CO2 ratios indicate two standard deviations or 95% confidence intervals of ten repeated measurements.
In Experiment 1, the exhaled 13CO2/12CO2 ratio increased significantly between 4 and 31 h after 13CO inhalation (p<0.05). The maximum increase in the 13CO2/12CO2 ratio was +2.10/00 of the background 9 h after inhalation. In Experiment 2, the output 13CO2/12CO2 ratio showed no significant increase for 36 h after the 13CO input.
First, we ensured the subject was not subjected to symptomatic CO intoxication in Experiment 1. The threshold level for the carboxyhaemoglobin concentration to manifest the mildest symptoms of CO intoxication has reported to be 10% [14, 15]. We have developed a system for non-invasive, continuous carboxyhaemoglobin densitometry by expiratory gas analysis that enables continuous real-time monitoring of the carboxyhaemoglobin concentration using the Carbolizer sensor . Using this technique, we controlled the duration of 13CO gas inhalation to limit the maximum carboxyhaemoglobin concentration of the subject to below 10%. In addition to the monitors equipped in the intensive care unit of our hospital, at least two physicians continuously monitored the subject’s consciousness and neurological status for any unexpected manifestations during the experiment.
Over the past decade, numerous studies have clarified the physiological role of endogenous CO as a gas transmitter that activates or inhibits various enzymes by binding to heme proteins within a cell or adjacent cells. However, most researchers are sceptical about the role of CO as a transorganic or systemic signal transmitter, because the redistribution of CO from blood carboxyhaemoglobin to tissue cells has not been shown. It is widely accepted that the only destination for haemoglobin-bound CO is to be exchanged with oxygen in the lung and to be exhaled. Previous studies have shown remnant effects of CO after carboxyhaemoglobin elimination , a poor correlation of blood carboxyhaemoglobin levels with the physiological effects of CO inhalation , and a protective effect of CO inhalation against ischemia-reperfusion injury in rats . These findings suggest that a fraction of CO moves from blood to tissue, but no tracer analysis study has demonstrated this redistribution.
Meanwhile, several laboratories have confirmed the oxidization of CO by mitochondrial cytochrome c oxidase extracted from various animal organs [10–12]. However, those studies detected CO2 production from the purified enzyme gassed with CO in a closed chamber and no tracer analysis study has demonstrated the oxidization of CO in living animal tissue under physiological conditions.
In Experiment 1, we detected 13CO2 production deriving from inhaled 13CO, which demonstrated the oxidization of CO in a living human body under physiological conditions. In Experiment 2, we detected no 13CO2 production, which confirmed that oxidization does not occur in the circulating blood. Taken together, these experiments indicate that a portion of inhaled CO is redistributed from the blood to tissue cells and is oxidized there.
There is still the possibility that CO oxidization occurs in airway epithelium. Inhaled CO contacts airway epithelium before it binds to blood haemoglobin and we may have detected 13CO2 that is derived from the oxidation of CO in the airway epithelium tissue. If the oxidization occurs in the airway, 13CO2 production would increase during 13CO inhalation and decrease soon after the termination of inhalation. However, a significant increase in 13CO2 production started 4 h after the termination of 13CO inhalation and was sustained for 31 h, with a peak at 9 h in Experiment 1. This time delay between CO inhalation and CO2 production may reflect the time required for redistribution and thus refuting the hypothesis of CO oxidization in the airway epithelium. Still, further evidence is required to confirm such CO redistribution, including the detection of 13CO2 production after infusion of 13CO-bound carboxyhaemoglobin (13CO-saturated autologous blood).
This study was supported by Japanese Grants-in-Aid for Scientific Research 1620902，21240057, and 19592097.