Quantification of carbon monoxide (CO) in postmortem human brain tissues after CO poisoning

Abstract

In this study, we aimed to quantify carbon monoxide (CO) in human brain tissue to better understand the toxic mechanisms of CO poisoning. Currently, conventional CO measurement methods are limited; however, the hemoCD assay has proven to be a simple and rapid method for quantifying CO in human tissues. Using this method, CO concentrations were measured in various brain regions, revealing significantly higher CO concentrations in the CO-exposed group (approximately 30–50 pmol/mg) compared to the non-exposed group (approximately 20–30 pmol/mg). However, the absence of region-specific elevation suggests that CO inhalation is not selectively associated with brain areas with high CO affinity or those that typically show abnormal MRI signals during CO intoxication. The observed difference of 10–20 pmol/mg between the CO-exposed and non-exposed groups suggests that an additional 10–20 pmol/mg of external CO may represent a lethal dose, potentially causing death. The results of this study are expected to contribute to the elucidation of the pathogenesis of CO poisoning and ultimately aid in the development of effective treatment strategies.

Carbon monoxide (CO) poisoning is one of the most common forms of poisoning in modern society. According to a report by the National Research Institute of Police Science, CO poisoning accounts for approximately 70% of all poisoning-related deaths in Japan, with several thousand fatalities reported annually¹. Most cases of CO poisoning result from fires or suicides involving charcoal briquette combustion. In forensic practice, it is often necessary to assess the presence and severity of CO poisoning, its physiological effects, and its role in the cause of death. Beyond its toxicity, CO has physiological effects such as vasodilation, neurotransmitter release, and inhibition of inflammatory responses^2–5. A feedback mechanism has also been suggested, where endogenous CO is resynthesized to levels comparable to those prior to its removal, even after being eliminated⁶. Because CO is a potentially lethal substance, understanding its toxicity mechanisms is critical for developing future treatment strategies.

Although CO toxicity is often explained by hypoxic injury and cellular damage, many aspects remain unclear⁷. To clarify the toxic mechanism of any drug or poison, it is necessary to determine the absolute amount and concentration that accumulates in tissues. However, only a few studies have addressed this in relation to CO. Clinically, brain dysfunction is the primary consequence of acute CO poisoning, and delayed neurological damage may also occur⁸. MRI scans in the acute phase often show abnormalities in specific brain regions^9–11. Prognosis for patients with late-onset encephalopathy varies widely, from complete recovery to prolonged unconsciousness or death. The reasons for these differences are not yet clear⁸, but they may relate to differences in CO concentrations across different brain regions. Therefore, quantifying CO levels in various brain regions is crucial for understanding these variations.

In forensic practice, CO toxicity is commonly assessed by measuring CO-hemoglobin (Hb) saturation in the blood, which serves as key evidence for determining whether death resulted from CO poisoning¹². The relationship between CO-Hb saturation and clinical symptoms has been established, and a lethal CO-Hb saturation level is generally considered to be at least 50%¹³. However, this method does not allow for the measurement of absolute CO amounts or concentrations in tissues. Gas chromatography (GC) has also been used to quantify CO^12,14,15. However, it is not a quick or simple method and lacks versatility due to the complexity of sample pretreatment, limited detection sensitivity, and the need for specialized columns and detectors. Furthermore, while high-sensitivity detection methods such as hollow-core antiresonant fiber (HC-ARF) and fluorescent probes have been explored, they remain impractical due to complex experimental procedures and detection sensitivity limitations^16,17.

In this study, we investigated a rapid and simple method for CO quantification using hemoCD assay, as originally reported by Kitagishi et al.^18,19 (Fig. 1). Among these methods, hemoCD-P has approximately 100 times greater affinity for CO than normal Hb, a property with potential therapeutic applications²⁰. The hemoCD assay allows for CO quantification using a standard, inexpensive spectrophotometer, requiring only minimal equipment and simple sample pretreatment. It also eliminates the need for complex calibration curves. Mao et al. previously used this method to measure ultra-trace levels of endogenous CO in various rat tissues, and have proposed the mechanism that external CO, once accumulated in tissues, is transferred to Hb in the blood due to its high CO-binding affinity. The CO storage capacity of tissues likely depends on their heme content, which serves as CO-binding sites¹⁹. If the hemoCD assay can be successfully applied to human tissues, it will facilitate a rapid and simple quantification of CO in human tissues and contribute to the elucidation of CO poisoning mechanisms. Therefore, in this study, we applied the hemoCD assay to postmortem human brain tissue to quantify CO levels in various brain regions and compare them in cases suspected to have died from CO poisoning.

Fig. 1.

Structures of deoxy-hemoCD and CO-hemoCD. Structures of deoxy-hemoCD and CO-hemoCD complexes are shown. hemoCD is composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron (II)(Fe^IITPPS) and a per-O-methyl-β-cyclodextrin dimer with a pyridine linker(Py3CD).

Adapted from Refs.¹⁹.

Results

Application of the HemoCD assay to postmortem human tissues

Using the hemoCD assay, we investigated the feasibility of quantifying endogenous CO in postmortem human brain tissue. A 10 mg sample was used for each analysis, following the hemoCD assay protocol described by Mao et al.¹⁹. A portion of the cadaveric brain tissue was homogenized in phosphate-buffered saline (PBS) and hemoCD-P was added. The homogenate was further sonicated (Fig. 2). After sonication, samples were centrifuged for 15 min to obtain a clear supernatant, which was then filtered through a 0.45 μm filter. Following filtration, 4.5 mg of sodium hydrosulfite (Na₂S₂O₄) was added to the sample, and spectrophotometric analysis was performed. As a result, absorption spectra with two characteristic absorption maxima were observed for deoxy-hemoCD-P (434 nm) and CO-hemoCD-P (422 nm) (Fig. 3), which was consistent with previous results from rat tissues¹⁹. These findings indicate that the assay is adaptable for use in human tissues.

Fig. 2.

Protocol for the hemoCD assay in human brain tissue. Experimental procedure outlining the steps of the hemoCD assay used to measure CO in human brain tissue.

Fig. 3.

Absorbance spectrum of the hemoCD assay in human brain tissue. Absorbance spectrum(422 nm: CO-hemoCD-P, 434 nm: deoxy-hemoCD-P)observed in human brain tissue.

Quantification of endogenous CO in brain regions of the non-CO-exposed group

Next, we quantified CO levels in various regions of the postmortem human brain in cases where no CO exposure had occurred (control group: Ctrl group). As shown in Fig. 4A, CO concentrations in each brain region were as follows: 21.3 ± 4.0 pmol/mg in the cortex of the frontal lobe, 24.5 ± 5.3 pmol/mg in the medulla of the frontal lobe, 25.1 ± 7.2 pmol/mg in the putamen, 20.8 ± 4.4 pmol/mg in the globus pallidus, 21.0 ± 5.4 pmol/mg in the internal capsule, 25.1 ± 7.2 pmol/mg in the caudate nucleus, 24.0 ± 4.3 pmol/mg in the cortex of the temporal lobe, and 23.6 ± 6.9 pmol/mg in the medulla of the temporal lobe. There were no statistically significant differences in CO concentrations among the brain regions. Furthermore, no specific regional accumulation of CO was observed in any individual sample.

Fig. 4.

Comparison of carbon monoxide (CO) concentrations in different brain regions between the control group (A) and the CO-exposed group (B). Box plots showing carbon monoxide (CO) concentrations in different brain regions for the control group (A) and the CO-exposed group (B). Asterisks indicate mean values. One-way ANOVA was conducted for each group, but no significant difference was found. A p-value of < 0.05 was considered statistically significant.

Quantification of CO in brain regions of the CO-exposed group

We then quantified CO levels in the brain regions of individuals exposed to CO (CO group). As shown in Fig. 4B, CO concentrations in each brain region were as follows: 34.6 ± 9.3 pmol/mg in the cortex of the frontal lobe, 35.5 ± 5.6 pmol/mg in the medulla of the frontal lobe, 44.4 ± 7.9 pmol/mg in the putamen, 37.0 ± 9.8 pmol/mg in the globus pallidus, 35.2 ± 10.0 pmol/mg in the internal capsule, 40.0 ± 7.9 pmol/mg in the caudate nucleus, 36.5 ± 8.0 pmol/mg in the cortex of the temporal lobe, and 40.2 ± 5.8 pmol/mg in the medulla of the temporal lobe. Compared to the Ctrl group, CO concentrations were significantly higher in all brain regions (Fig. 5). However, there were no statistically significant differences between individual brain regions within the CO group. Furthermore, CO concentrations in brain tissue did not increase proportionally with blood CO-Hb saturation. No correlation between brain CO concentrations and blood CO-Hb saturation was observed in this study.

Fig. 5.

Comparison of CO concentrations in various brain regions between Ctrl and CO-exposed groups. An independent t-test was performed to compare the Ctrl group and the CO-exposed group. Data are presented as mean ± standard deviation (SD). A p-value of < 0.05 was considered statistically significant.

Discussion

Previously, the hemoCD assay was shown to quantify CO in various tissues, including the rat brain. This study demonstrates for the first time that the method can be applied to human brain tissue without complex additional processing.

Mao et al. used the hemoCD assay to analyze whole rat brains that had not been exposed to CO and found that 26.4 ± 6.2 pmol/mg of endogenous CO had accumulated¹⁹. Our results also indicate that CO concentrations in all examined brain regions of the Ctrl group ranged from 20 to 30 pmol/mg. Interestingly, the amounts of endogenous CO were quite comparable between rat and human brains, as revealed for the first time in this study. As reported, endogenous CO is continuously generated at an average rate of approximately 0.4 mL/h in human adults²¹. It is also estimated that 70% of CO production in living organisms is associated with the breakdown of heme, with 80–90% of this heme originating from Hb. Since Hb levels in rats are comparable to those in humans, and the most endogenous CO production results from heme breakdown^22,23, the similar CO concentrations observed in rats and humans are not contradictory.

In all brain regions of the Ctrl group, CO was present at approximately 20–30 pmol/mg. This suggests that such concentrations are physiologically normal and non-toxic to the human body. These findings suggest that CO concentrations at the levels observed in this study play an essential role throughout the brain. According to the literature, heme oxygenase (HO)-2 is highly expressed in the mammalian brain, where it catalyzes constant heme degradation catalyzed to generate CO^24,25. Unlike cortisol, which is secreted urgently in response to stress, CO appears to exert physiological effects as part of daily biological activities²⁶. Mao et al. also compared CO levels in various rat organs between groups with and without CO exposure and showed that the CO-exposed group had significantly higher CO accumulated than the non-exposed group¹⁹. Our results also showed CO concentrations of approximately 30–50 pmol/mg in the CO-exposed group, which was significantly higher than those observed across all brain regions in the Ctrl group. The concentrations were also similar to those reported by Vreman et al.; however, it is unclear which part of the human brain they analyzed²⁷. Consequently, direct comparisons between our results and those of other studies are challenging due to the lack of region-specific measurements.

Most importantly, these findings suggest that CO inhalation does not result in selective CO accumulation in specific regions of the brain. MRI and other imaging studies have reported that abnormal signals are typically found in the bilateral globus pallidus in acute CO poisoning cases, with additional abnormalities observed in the putamen, caudate nucleus, thalamus, cerebral cortex, hippocampus, cerebellum, etc. in some cases^9–11. Histological studies of postmortem brains from patients who survived long after CO exposure have shown heterogeneous destruction of basic structures in the cerebral cortex and basal ganglia, as well as glial cell damage in the globus pallidus and cerebral medulla²⁸. These findings suggest region-specific CO localization. However, our results did not show elevated CO concentrations corresponding to the sites of abnormal signal development identified by MRI. This strongly suggests that there is no selective uptake of CO by inhalation. The brain contains several heme proteins, such as cytochrome c oxidase, neuroglobin, cystathionine β-synthase. CO may inhibit their functions by coordinating directly to the heme of these enzymes^25,29,30. The cell types composing each brain region analyzed in this study vary, suggesting that different areas of the brain may have different susceptibilities to CO. Our results support this possibility and may contribute to a better understanding of CO toxicity mechanisms. Furthermore, these results could help explain the individual differences in the onset of sequelae often observed in survivors of CO poisoning.

Furthermore, brain CO concentrations in the CO-exposed group, with blood CO-Hb saturation of 50% or higher, remained at approximately 30–50 pmol/mg, regardless of the degree of blood CO-Hb saturation. This suggests that these CO concentrations may represent the maximum amount of CO the brain can accumulate, that is, the saturation threshold. Alternatively, the CO concentration in the brain may have remained at 30–50 pmol/mg due to fatal organ damage, even if the brain’s capacity to store CO exceeds this range. Therefore, the difference in CO concentrations observed between the CO-exposed and Ctrl groups may reflect an additive effect of endogenous CO, potentially leading to fatal outcomes²⁹. In other words, these levels may be lethal and provide critical insights into the toxicity of CO.

It has been pointed out that the reported relationship between blood CO-Hb saturation and clinical symptoms is based on observations made during the process of CO inhalation and gradual increase in blood CO-Hb levels. Therefore, in clinical practice, the severity of acute CO poisoning does not necessarily correlate with blood CO-Hb saturation at the time of presentation^31,32. In other words, CO-Hb saturation alone does not provide a comprehensive understanding of the CO toxicity mechanism. Previous studies have also shown that measurement of CO-Hb saturation through spectrophotometry might not be the best marker for CO poisoning, and that direct measurement of CO may provide more information regarding the severity of CO poisoning, especially since the repartition of CO between blood and hemoglobin-bound compartments is not yet well understood³³. In summary, although the sample size in this study is limited, our findings provide novel insights into the toxicokinetics of the human brain and should be further explored and validated in future studies. This study demonstrated that the hemoCD assay enables direct and reliable quantification of CO in postmortem human brain tissue, and revealed that CO accumulates uniformly across the brain in fatal CO poisoning cases, without region-specific localization.

Methods

Cardiac blood CO-Hb saturation measurement

CO-Hb saturation was measured using cardiac blood collected from cadavers. Each sample was diluted approximately 1:200 with 0.1% sodium carbonate solution. Subsequently, 4.5 mg of sodium hydrosulfite was added to the solution and mixed. After adding 0.2 mL of 1 N sodium hydroxide solution and allowing it to stand, absorbance was measured at (a) 530 nm and (b) 558 nm using a spectrophotometer. CO-Hb saturation was calculated using the formula (2.21 − b/a) × 79, as previously described. The CO-Hb saturation levels for each cadaver are shown in Table 1. This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Clinical Research Review Committee of Osaka Medical and Pharmaceutical University (2023-078-1). As the tissue samples were obtained through medico-legal autopsies conducted under legal authority, obtaining informed consent from the deceased’s families was not institutionally or practically feasible. Therefore, in accordance with national regulations and international ethical standards, an opt-out approach was employed: study information was made publicly available on the Department of Forensic Medicine website (URL: https://www.ompu.ac.jp/u-deps/leg/contact/index.html), allowing families the opportunity to decline participation.

Table 1.

Background information of samples used in this study.

No.	Age	Sex	Cause of death	CO-Hb saturation (%)	Time since death	Situation
1	68	M	Acute myocardial infarction	0.6	3 days	Found in supine position at home
2	35	F	Cardiac sudden death	0.7	2 days	Found in supine position at work
3	82	M	Lethal arrhythmia	0.7	2 days	Found in lateral decubitus position at home.
4	78	M	Hypothermia	2.0	2 days	Found in supine position at home
5	19	F	Exsanguination	5.0	2 days	Murder case
6	25	M	Carbon monoxide poisoning	51.6	3 days	Suicide by charcoal briquettes
7	76	M	Fatal thermal injuries	52.2	19 h	Fire at home
8	74	M	Fatal thermal injuries	55.5	13 h	Fire at home
9	51	M	Carbon monoxide poisoning	66.7	3 days	Suicide by charcoal briquettes
10	75	M	Fatal thermal injuries	72.5	3 days	Suicide by charcoal briquettes
11	25	F	Carbon monoxide poisoning	72.5	2 days	Suicide by charcoal briquettes
12	26	M	Carbon monoxide poisoning	72.7	3 days	Suicide by charcoal briquettes

Samples

A total of 12 cadavers were available for analysis. Details of the cadavers are summarized in Table 1. These cadavers were classified into two groups based on blood CO-Hb saturation levels and findings from the investigative agency. Cadavers with CO-Hb saturation below 10% were classified as the Ctrl group (n = 5), indicating no CO exposure. Cadavers with CO-Hb saturation above 50% were assigned to the CO exposure (CO) group (n = 7). The brain was removed from each cadaver for analysis. Since CO can be produced in decomposed drowned bodies^34,35, any cadavers with suspected decomposition-related CO production, based on external findings at autopsy or autopsy results, were excluded from sample collection.

Sample preparation

Each brain was sectioned into 2-cm-thick slices in the coronal plane, and the following regions were excised, as shown in Fig. 6: cortex of the frontal lobe, medulla of the frontal lobe, putamen, globus pallidus, internal capsule, caudate nucleus, cortex of the temporal lobe, cortex of the temporal lobe, and medulla of the temporal lobe. The excised tissues were placed in 2 mL tubes, rapidly frozen, and stored at − 80 °C.

Fig. 6.

Brain regions used for analysis. The brain regions shown are as follows: 1: cortex of the frontal lobe, 2: medulla of the frontal lobe, 3: putamen, 4: globus pallidus, 5: internal capsule, 6: caudate nucleus, 7: cortex of the temporal lobe, 8: medulla of the temporal lobe.

CO determination in brain tissue by HemoCD assay

The overall protocol for the hemoCD assay is illustrated in Fig. 2. Sample (10 mg) thawed at room temperature was weighed and homogenized using an Automill (Tokken Inc. Japan) in PBS (0.5 mL). After homogenization, hemoCD-P (3 µL) with Na₂S₂O₄ (4 mg) in PBS was added, followed by sonication on ice (10 s×2, amplitude: 30; QSO-NICA). Samples were then centrifuged (14,000×g, 15 min), and the supernatant was filtered through a 0.45 μm pore filter (TecholabSC; LTD. Japan). The filtrate was again treated with Na₂S₂O₄ (4 mg) before absorbance measurement using a UV–Vis-NIR Spectrometer (V-730, Japan Spectroscopy Co., Ltd. Japan). All analyses were processed using JASCO Spectrum Manager Ver. 2 (Japan Spectroscopy Co., Ltd. Japan). Absorbance was measured at 434 nm for deoxy-hemoCD-P and 422 nm for CO-hemoCD-P. CO concentrations (pmol/mg) were calculated using the equation reported by Mao et al.¹⁹.

Statistics and reproducibility

Statistical analyses were performed using GraphPad Prism, version 9.5.1 (GraphPad Software). All data are presented as means ± standard error from at least three independent experiments and were analyzed by one-way analysis of variance (ANOVA) and Student’s t-tests. Differences with P values less than 0.05 were considered statistically significant.

Author contributions

K.M. and M.K. conceived the study. Material preparation conducted by F.M. and T.S. Data collection and analysis were performed by K.M. and J.Y. The first draft of the manuscript was written by K.M. hemoCD was provided by H.K. All authors commented on previous versions of the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Data availability

All relevant data are in the manuscript.

Declarations

Competing interests

The authors declare no competing interests.