Original Article
Determination of putrefactive amine and ammonia concentrations around decomposed corpses
2025 Volume 50 Issue 2 Pages 75-81
Details
2025 Volume 50 Issue 2 Pages 75-81
The surface of a rotting corpse is covered with liquid decomposition products that have flowed out of the body that include putrefactive amines produced via putrefaction and decarboxylation reactions of proteins. Ammonia generated by deamination is also present around the corpse as a liquid or gas. As these putrefactive substances are toxic to humans, we attempted to measure the concentration of putrefactive substances in decomposed corpses in this study. Liquid putrefaction products were collected from the surface of a corpse, and the concentrations of putrefactive amines such as histamine, tyramine, phenethylamine, and tryptamine were analyzed by LC-MS/MS. Ammonia in the liquid and air around the corpse was also measured. Putrefactive amines and ammonia were present on all corpse surfaces. The highest concentrations and postmortem days in parentheses were as follows: histamine 2.26 mg/g (15 days), tyramine 1.77 mg/g (16 days), phenethylamine 4.90 mg/g (24 days), tryptamine 1.58 mg/g (17 days) and ammonia 25.6 mg/g (24 days postmortem). The highest concentration of ammonia in the air was 1310 ppm at 24 days postmortem. The ammonia level in the air around a corpse is toxic to humans. Inhalation of putrefactive amines and ammonia can cause chemical irritation to the respiratory tract and the skin and damage the mucous membrane of the eye. Oral ingestion can also cause poisoning symptoms such as blood pressure changes and headaches. Adequate protection against putrefactive substances is required when in contact with decaying corpses.
The skin of a corpse forms blisters containing liquid putrefactive materials over time. The putrefactive substances produced in the cadaver also flow out of the body through the nostrils, mouth, urethra, and anus (Knight and Saukko, 2016; Carter et al., 2007). Liquid putrefactive substances contain putrefactive amines and ammonia, which adhere to the skin surface of decomposed corpses (Yu et al., 2021). Tissue destruction occurs through the actions of microorganisms and self-enzymes (Dettmeyer, 2018). When proteins in the human body are degraded, they are decarboxylated by microbial proteases to produce putrefactive amines (Jin et al., 2019; Masson et al., 1996; ten Brink et al., 1990) (Fig. 1A). In addition, the air around the corpse is enriched with ammonia gas generated by decomposition (Statheropoulos et al., 2005; Vass et al., 2002). Ammonia is produced when proteins in the human body are degraded by microbial proteases and amino acid oxidase (Fig. 1B). These substances are toxic to humans (Jin et al., 2019; Rice et al., 1976).
Pathways of putrefactive amines and ammonia production. Putrefactive amines are produced from precursor amino acids produced by protein degradation in the human body and further decarboxylated by bacteria (A). Ammonia is produced from precursor amino acids in the human body and bacterial amino acid oxidase (B).
A catastrophe can result in many decomposing corpses. As organic matter always decomposes, these putrefactive amines and ammonia can spread through the surrounding area and cause health hazards to workers in handling a corpse. These putrefactive amines and ammonia have not been investigated yet in detail.
In this study, we measured the concentration of putrefactive amines and ammonia in liquid putrefactive substances on the skin surface of decomposed corpses and ammonia in the air surrounding the corpses. Clarifying the risk of exposure to decomposed corpses can help disease prevention and health management for those who handle corpses.
The corpses in this study were autopsied from October 2020 to October 2022, with mean temperatures above 20°C from death to the viewing of the bodies. We identified their dates and causes of death based on their medical history and autopsy.
Bodies with high concentrations of medicinal drugs or infectious diseases were excluded from the study. The time from death to autopsy was less than 48 days.
Sample collectionApproximately 2.0 g of liquid decomposed samples were taken from the surface of the face, neck, and upper thoracic regions of the corpses using clean disposable spoons during autopsy (Fig. 2A). The air at 50 cm above the chest of the corpse was aspirated with a gas detector tube (Fig. 2B).
Sample collection. Liquid decomposition samples were taken from the surfaces of the corpse with a clean disposable spoon at the autopsy unit (A). Air was aspirated with a detector tube at ~50 cm above the chest of the corpse (B).
Histamine, tyramine, phenethylamine, and tryptamine in the samples were quantified by LC-MS/MS analysis. The liquid putrefactive samples were stored at −20°C until analysis. The sample was accurately weighed and diluted with ultrapure water to obtain an appropriate concentration for the analysis. The diluted sample of 1.5 mL was placed in a 5 mL plastic tube (TM-657, Tomy Kogyo Co., Ltd., Tokyo, Japan) and the following were added: two stainless steel beads (φ4.8 mm, SUB-50, Tomy Kogyo), 0.5 μg of d5-Diazepam (Cat. No.99056107, Hayashi Pure Chemical Ind., Ltd., Tokyo, Japan) as internal standard, 1.5 mL of acetonitrile (LC/MS grade, Cat No.012-19851, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 0.5 g of AOAC powder (Cat. No.5982-0755, Agilent) according to the QuEChERS methods. The mixture was vigorously shaken at 2,500 rpm for 30 sec with a bead-type cell disruption device (MS-100, Tomy Kogyo) at 4°C, and centrifuged at 3,000 rpm for 15 min.
30 μL of the supernatant was injected into the LC-MS/MS apparatus (3200 Q TRAP®, SCIEX) using multiple reaction monitoring (MRM) at positive mode. Allure PFP Propyl column (5.0 μm, 50×2.1 mm, Cat. No.9169552, Restek) was used for LC. Mobile phase A for LC consisted of 0.2% 1M ammonium formate (Cat. No. 1743, Honeywell-Fluka™), 0.2% formic acid (Cat. No.067-04531, FUJIFILM Wako) in purified water. Mobile phase B consisted of 0.2% 1M ammonium formate and 0.2% formic acid in acetonitrile. Mobile phase A was converted to phase B over 16 min at 40°C and a flow rate of 0.5 mL per min. The respective precursor and product ions (Q1/Q3) were as follows: histamine 112.09/95.1, tyramine 138.19/77.1, phenethylamine 122.15/105.1, tryptamine 161.10/144.1 and d5-Diazepam 290.01/154.1. Quantification was performed using the standard addition method. The Pearson’s correlation coefficient of each calibration curve was above 0.99. The quantitative value was calculated by multiplying by the dilution factor.
Measurement of ammoniaAmmonia in the liquid sample was analyzed by the indophenol method. One gram of the collected liquid decomposed samples was diluted 10-fold with ultrapure water. The precipitate was removed by centrifugation, and the supernatant was supplemented with phenol and sodium nitroprusside and then made alkaline. Then, sodium hypochlorite was added for oxidation, and the absorbance at 630 nm of the generated indophenol blue was measured spectrophotometrically to quantify the ammonia concentration. Ammonia gas was measured using a gas detector tube (No.105SE or No.105SB manufactured by Komyo Rikagaku Kogyo Co., Ltd. Kanagawa, Japan). No.105SE was used in the range of 1 ppm to 200 ppm, and No.105SB was used in the range of 50 ppm to 900 ppm of ammonia gas. A gas sampling pump (AP-20, manufactured by Komyo Rikagaku Kogyo Co., Ltd., Kanagawa, Japan) was connected to the base of the detector tube. The tip of the detector tube was fixed at a position 50 cm above the chest. A gas sampling pump drew 100 mL of sampling air through the detector tube. The ammonia concentration was obtained by reading the color change range of the detector tube. In the case of high concentrations, collect 50 mL of sampling air and double the measurement value as the ammonia gas concentration. These measurements were performed twice, and the average value of the concentrations was used as the data.
StatisticsCorrelations between the content of each putrefactive amine or ammonia and postmortem days were analyzed statistically. A p-value less than 0.05 (5%) was considered significant.
Table 1 shows the counts of the investigated cases by sex, place of discovery, cause of death, and days to autopsy. There were 32 cases with ages ranging from 41 to 91 years; the average was 65.3 ± 11.7 years old (mean + standard deviation). All the indoor death cases were dead from internal diseases shown as natural death. Four outdoor death cases had external causes such as suffocation, heat stroke, and drowning. The mean time from death to autopsy was 22.2 ± 12.0 days.
| Total (n=32) | n |
|---|---|
| Sex | |
| Men | 26 |
| Women | 6 |
| Place of Death | |
| Indoor | 28 |
| Outdoor | 4 |
| Cause of Death | |
| Natural Death | 28 |
| Suffocation | 1 |
| Heat Stroke | 2 |
| Drowning | 1 |
| Time from Death to Autopsy | |
| 4–9 days | 5 |
| 10–19 days | 11 |
| 20–29 days | 5 |
| 30–39 days | 8 |
| 40–48 days | 3 |
Putrefactive amines are mainly produced by microbial decarboxylase (Fig. 1A). Concentrations of the four putrefactive amines in the liquid spoilage products appeared to increase with postmortem time until the mid-term, but the variability was so great that no significant changes were observed (Fig. 3A–3D). The maximum concentrations and days for each amine were as follows: histamine 2.26 mg/g on day 15 indoor natural death (Fig. 3A), tyramine 1.77 mg/g on day 16 indoor natural death (Fig. 3B), phenethylamine 4.90 mg/g on day 24 indoor natural death (Fig. 3C), and tryptamine 1.58 mg/g on day 17 indoor natural death (Fig. 3D).
Putrefactive amine concentrations. Concentrations of histamine (A), tyramine (B), phenethylamine (C), and tryptamine (D) in liquid spoilage were plotted against the number of postmortem days. See the text for details.
Ammonia is produced by microbial amino acid oxidase (Fig. 1B). Ammonia concentrations in the liquid putrefactive products had weak positive correlation with postmortem time, reaching a maximum of 25.6 mg/g at 24 days postmortem (Fig. 4A). Similarly, the concentration of ammonia in the air was highly variable but had weak positive correlation with postmortem time, reaching a maximum of 1310 ppm at 24 days postmortem (Fig. 4B). Two cases with maximum ammonia concentrations (liquid and air) were both indoor natural deaths.
Ammonia concentrations. Ammonia concentrations in the liquid putrefactive products (A) and in the air (B) were plotted against the number of postmortem days. See the text for details. Both had weak positive correlation with postmortem time.
Different putrefactive amines are produced from each amino acid. For example, histamine and phenethylamine are produced by decarboxylation of histidine and phenylalanine, respectively (Masson et al., 1996; ten Brink et al., 1990). Different amines are produced by the decarboxylase present in each microorganism. Escherichia coli and Pseudomonas have histidine decarboxylase and tyrosine decarboxylase that produce histamine and tyramine (Silla Santos, 1996). Enterococcus and Clostridium have aromatic amino acid decarboxylase and specifically produce tyramine, phenethylamine, and tryptamine (Silla Santos, 1996). These bacteria are residents on the skin surface and in the intestines of the human body. Although the present study did not examine the microbes in corpse exudates, many of the microbes involved in the early post-mortem period have been reported to be derived from indigenous bacteria that largely originate in the intestines and lungs (Knight and Saukko, 2016). In this study, we measured histamine, tyramine, phenethylamine, and tryptamine as putrefactive amines. Based on our experience, these putrefactive amines are most frequently detected in rotting cadaver. The bias in putrefactive amines produced in liquid putrefactive substances may be related to the types and numbers of putrefactive microorganisms. In this study, we targeted corpses whose average daily temperature was above 20°C from death to discovery. Not only temperature but also factors such as humidity, pH, and nutritional status affect the growth of microorganisms, which in turn affects productivity and activity of decarboxylases in the corpses (Silla Santos, 1996). Therefore, the amounts of putrefactive amines produced in the corpse can be affected by its environment. Putrefactive amines in the corpses were highly variable and their amounts had no significant correlation with postmortem days in this study. It is presumed that the variation is related to the number of microorganisms grown in the corpse body. Only temperature was used as a study condition here. It is possible that nutritional status and environmental factors such as humidity and airflow may be involved in time matter, which may be worth investigating.
Putrefactive amines are toxic to living organisms. The toxicity has been extensively studied in fermented foods (ten Brink et al., 1990; Rice et al., 1976; Silla Santos, 1996). Histamine is a vasodilator amine, the ingestion of 22 mg or more of which causes hypotension, headache, facial flushing, and urticaria (Rice et al., 1976; Taylor, 1986). Excessive intake of tyramine, phenethylamine, and tryptamine releases noradrenaline into the synaptic cleft and excites sympathetic nerves, resulting in hypertension and headache (Rice et al., 1976; Blackwell and Mabbitt, 1965). In addition, many putrefactive amines cause injury to the skin, ocular mucosa, and airway mucosa (Ibe, 2004). This study found up to 4.9 mg/g of phenethylamine in liquid putrefactive products. The ingestion of even 3 mg of phenethylamine has been reported to be toxic to the body; thus, the intake of even small amounts of liquid putrefactive products can be dangerous (Ibe, 2004). In contrast, histamine (maximum concentration 2.23 mg/g), tyramine (maximum concentration 1.77 mg/g), and tryptamine (maximum concentration 1.58 mg/g) are low in concentration and considered unlikely to be toxic to humans (ten Brink et al., 1990). Of note, the toxicity of putrefactive amines should not be attributed to only one amine. A total amount of 1000 mg/kg of putrefactive amines is indicated as the toxicity limit (ten Brink et al., 1990; Silla Santos, 1996). There is concern about the synergistic and additive toxicity due to the coexistence of different putrefactive amines (Stratton et al., 1991; Hui and Taylor, 1985). Increased absorption of histamine and therefore, greater toxicity has been reported when histamine coexists with other putrefactive amines (Chu and Bjeldanes, 1982). Further, sensitive individuals may be adversely affected even at low concentrations (Blackwell et al., 1969). The toxicity of putrefactive amines can be enhanced by drugs taken by the individuals. Monoamine oxidase (MAO) is involved in the degradation and excretion of tyramine (Voigt and Eitenmiller, 1978). Taking a MAO inhibitor (MAOI) for Parkinson's disease or tuberculosis accumulates tyramine in the body, increases norepinephrine, and leads to symptoms such as hypertensive crisis (Blackwell et al., 1969; Isaac et al., 1977). Thus, the oral ingestion of 6 mg of tyramine is toxic to the body when taking MAOIs (Ibe, 2004).
We detected a higher production of ammonia than that of putrefactive amines in this study. Many species of microorganisms have amino acid oxidase activity. Amino acid oxidase degrades all kinds of amino acids and putrefactive amines to produce ammonia (Rice et al., 1976). Each putrefactive amine, on the other hand, is formed by decarboxylation reactions from specific types of amino acids. Therefore, it is considered that a larger amount of ammonia is produced than putrefactive amines in this study. Both ammonia in liquids and air were detected in all corpses and tended to increase with time after death, probably due to the increase in putrefactive microorganisms. Ammonia is highly volatile and much of probably evaporates quickly into the air surrounding the corpse. In this study also, we detected a high concentration of ammonia in the air around the corpse. Liquid ammonia causes injury to the skin and mucous membranes (Amshel et al., 2000; Arwood et al., 1985). Ammonia gas reacts with moisture on the surface of eyes, skin, and respiratory tract mucosa to form ammonium hydroxide, a strong alkali causing lytic necrosis. A concentration of 50 ppm causes odor, 408 ppm irritates the throat mucosa, 698 ppm causes eye damage, 1500 ppm causes laryngospasm and pulmonary edema, and 2500-4500 ppm causes death within 30 min due to respiratory failure (Amshel et al., 2000; Henderson and Haggard, 1943; Latenser and Lucktong, 2000). In this study, 12 of the 32 cases, or 40% of cases, had air ammonia above 408 ppm. The highest gas concentration in this study, 1310 ppm, can cause injuries to the mucous membranes of throat and eyes. Individuals with sensitive airway mucosa may be adversely affected even at low concentrations. If the corpse is in an enclosed space, the concentration in the air is thought to be even higher, increasing the danger to living organisms. In addition, if a large-scale disaster leaves many bodies, it is expected that large amounts of decomposed materials will be generated in the surrounding area. When coming into contact with decomposed carcasses, it is necessary to prevent skin and mucous membrane damage from putrefactive amines and ammonia, toxicity from ingestion, and gas inhalation.
In conclusion, various putrefactive amines and ammonia were detected on the skin surface of all corpses more than 4 days after death. Putrefactive amines can cause toxicity to the human body in a synergistic manner even at low concentration. In addition, there is a high concentration of ammonia gas around decomposing corpses, which may cause respiratory mucosal injury. Persons who come into contact with decomposing corpses require adequate protection against putrefactive substances.
The authors thank the research team in the Department of Forensic Medicine, University of Occupational and Environmental Health Japan.
Conflict of interestThe authors declare that there is no conflict of interest.
