Postmortem toxicology of alcohols: A cross-matrix study of ethanol and methanol in 150 cases from New Delhi,India

Abstract

Ethanol is the most commonly abused drug worldwide, and excessive alcohol consumption is a major contributing factor in accidents and violent crimes. Methanol is a toxic alcohol and is less commonly detected but holds significant forensic relevance due to its potential for causing fatal poisoning. The study aims to detect the concentrations of ethanol and methanol in postmortem blood, vitreous humor, urine, and cerebrospinal fluid, and to create a database of the involvement of alcohol in postmortem cases. A total of 150 postmortem cases with different causes of death were enrolled for the study. Gas Chromatography-Headspace was employed for the quantitative determination of ethanol and methanol concentrations in the biological specimens. Out of 150 cases, 108 cases (72%) exhibited positive ethanol concentrations. The high prevalence of positive ethanol concentration, particularly in cases of road traffic accidents and hanging, indicated a significant amount of alcohol use prior to death and underscores the role of alcohol consumption in fatal incidents. Methanol was detected in only those cases that had a high ethanol concentration, which may be due to adulteration or contamination of methanol in the alcoholic beverages that the deceased consumed before death. The study highlighted that an alternative/ complementary specimen to the blood should be considered to assess the alcohol concentration and to differentiate antemortem ingestion of alcohol from postmortem formation. The toxicological analysis of alcohol is important in understanding consumption patterns and in developing protocols and policies for preventing such cases.

Keywords

Ethanol methanol GC-HS forensic toxicology postmortem biological specimens

Introduction

In forensic practice, one of the most significant and challenging jobs for a forensic pathologist is to ascertain the cause of death. The toxicological examination- from the appropriate collection of samples, to their analysis and interpretation often plays a crucial role in this process.¹ Ethanol is a psychoactive substance found in alcoholic beverages.² It is one of the most widely abused substances in the world and is frequently encountered in clinical and forensic toxicology laboratories.³ Even with the rise in the use of illegal drugs, including cocaine, heroin, and phencyclidine, ethanol is still the most often found drug in postmortem toxicological analyses.^3,4

According to the World Health Organization (WHO) report, almost three million individuals die every year globally due to the harmful use of alcohol.⁵ Excessive use of alcohol has been reported as the primary cause of road traffic accidents (RTA), suicides, violent crimes, drowning, and accidental falls. Alcohol consumption holds significant societal implications, influencing public health, safety, and forensic investigations.^4,5 In postmortem examinations, the detection and quantification of alcohol in biological specimens play a pivotal role in elucidating the circumstances surrounding death, determining the cause and manner of death, and informing legal proceedings.^3,6,7 The accurate assessment of ethanol concentration in postmortem blood is crucial for distinguishing between antemortem ingestion and postmortem formation, which can profoundly impact forensic investigations.^8,9 Antemortem ingestion of ethanol may impair judgment and coordination, potentially leading to fatal accidents or altercations. Conversely, the postmortem formation of ethanol through microbial activity or contamination can complicate forensic analyses and the interpretation of ethanol concentrations.^10,11

In postmortem cases, blood is the usual specimen analyzed for determining the alcohol concentration but as blood is suspectable to contamination or postmortem ethanol formation by microbes, and in cases where it is not possible to collect blood samples such as putrefaction, decomposed bodies, and traumatic injuries; so other biological samples which are more resistant to postmortem changes such as vitreous humor, cerebrospinal fluid (CSF) and urine should be collected and analyzed. These alternative specimens should be used alongside blood to provide a more comprehensive assessment and interpretation of alcohol concentrations.^4,8,12,13 Urine is an important biological specimen in postmortem toxicological analysis as it is less susceptible to postmortem changes than blood but there are also studies reporting that a high amount of alcohol can be formed in urine if the deceased is suffering from certain medical conditions, such as diabetes and glycosuria and if Candida albicans (microorganism) is found in urine.^1,8,14 Vitreous humor is an important biological specimen in forensic toxicology analysis, as it is easy to retrieve and the risk of contamination by diffusion of ethanol or microbes is minimal even in decomposed bodies.^15–19 The possibility of using CSF as an alternative biological specimen for toxicological analysis has been recently reviewed, and there has not been yet any extensive research on the use of CSF in forensic toxicology analyses.^20,21 Wachholz et al. reviewed data from 45 autopsy cases to detect and compare concentrations of ethanol in blood and CSF and reported that CSF is less likely to undergo postmortem changes and can be used to determine ethanol concentration at the time of death.²¹

Gas Chromatography-Headspace with Flame Ionization Detector (GC-HS-FID) was used for the determination and analysis of ethanol and methanol in postmortem biological specimens. GC-HS-FID is a widely accepted and validated method for the quantitative determination of volatiles in postmortem biological specimens, as this analytical technique offers high sensitivity, specificity, and reproducibility, making it the gold standard in clinical and forensic toxicology laboratories.²²

Existing research on alcohol analysis in postmortem investigations often focuses on single specimen analysis or lacks sufficient sample size, particularly within the context of the Indian population. The present study aims to fill this gap by conducting analysis in multiple postmortem biological specimens, i.e., blood, urine, vitreous humor, and CSF, and to contribute valuable insights into the complexities of postmortem alcohol analysis to enhance the forensic investigative process and inform forensic pathology practice and medico-legal decision-making.

Material and methods

Study design

150 postmortem cases with different causes of death were enrolled for this study. The following biological samples were collected: blood, vitreous humor, urine, and CSF. The samples were obtained from 150 cases brought for postmortem examination in the mortuary of All India Institute of Medical Sciences (AIIMS), New Delhi, from February 2022 to November 2023. Also, demographic data regarding the victim (age, gender) and brief case history were collected.

Recruitment of subjects

After obtaining informed consent from a legally authorized relative (LAR) of the deceased, the biological samples were collected from subjects during the postmortem examination. Postmortem cases with an alcohol history were recruited in this study. The alcohol history of the deceased was taken from the relative/family of the victim.

Inclusion criteria:

Samples were collected from subjects who were brought to the mortuary for postmortem examination within seven days of death.

Samples were collected from only those cases in which the deceased had a history of alcohol use or consumption.

Exclusion criteria:

Cases in which the deceased had no history of alcohol use were excluded from the study.

Postmortem sample collection

After obtaining informed consent from the LAR, biological specimens were collected from the deceased during the autopsy.

Blood samples were collected mainly from the femoral vein to prevent contamination of the blood with gastrointestinal contents, peritoneal fluid, and other substances due to the increased permeability of vascular walls and biological membranes after death.

Urine samples were collected directly from the bladder by puncturing the pubis region with a 10 ml syringe with a wide-bore needle.

CSF samples were collected either directly from the spinal cord by lumbar puncture; or from the back of the neck by a cisternal needle puncture. The body was kept in the prone position, with support provided to the upper chest region.

Vitreous samples were collected from either eye by puncturing at an oblique angle on the lateral aspect of the eyeball with a thin needle. Following the aspiration of the vitreous, a normal saline solution was injected to preserve the contour of the eyeball in a cosmetic aspect.

Storage of samples

All the samples were collected in 4-mL vacutainers containing Ethylenediamine tetra acetic acid (EDTA) and sodium fluoride (NaF) as preservatives and stored at 4°C, and analysis was carried out within 5 days of collection.

Quantitative analysis

Instrumentation

Instrument

Gas Chromatography system, Model No. 7890A, and headspace sampler, Model No. 7697A with a Flame Ionization Detector from Agilent, USA were used for the detection of ethanol and volatiles in the samples.

The column used was DB-624; 30 m × 530 µm × 3 µm.

ChemStation^® software was used for the data analysis of the signals.

Carrier and detector gas of purity 99.99% were used. Nitrogen was used as carrier gas. Hydrogen and zero air were used for the detector.

Chemicals/reagents

Ethanol (GC grade), n-propanol (GC grade), and methanol (GC grade) were used from Merck, Germany. Ultrapure water was used from Rions, India.

Laboratory ware

Volumetric flasks were used from Merck, India. Twenty-milliliter Headspace vials were used from Agilent, USA.

Miscellaneous

Micropipettes of volume 100–1000 µL and 20–200 µL were used from Corning, USA. Septa - polytetrafluoroethylene (PTFE), aluminum crimp, and crimper for sealing the HS vial were used from Agilent, USA.

Experimental procedure

Six calibration standards of ethanol and methanol were prepared in the concentration range of 1–1000 mg/100 mL for ethanol and 2–500 mg/100 mL for methanol. The prepared calibration standards of ethanol and methanol were sealed in headspace vials with septa and crimp and run on GC-HS to calibrate the method. The temperature of the headspace oven, loop, and transfer line were set at 70°C, 80°C, and 90°C respectively. The carrier gas utilized was nitrogen, with a flow rate of 8 mL/min. Hydrogen and zero air were used with a flow rate of 40 and 400 mL/min, respectively. The gas chromatography oven was heated at 50°C for 5 min, then 35°C/min to 200°C for 1 min. The temperature of the detector was set at 250°C. DB-624 column was used for separation, and quantitation of the signals was performed using Chem Station software. A six-point calibration curve was drawn by plotting six different concentrations of ethanol and methanol according to the peak-area ratios with n-propanol as the internal standard. The method exhibited excellent linearity, with correlation coefficients of 0.9998 for ethanol and 0.9996 for methanol. The limit of detection (LOD) for ethanol and methanol was 0.3 mg/100 mL and 0.5 mg/100 mL, respectively, and the limit of quantitation (LOQ) for ethanol and methanol was 1 mg/100 mL and 2 mg/100 mL, respectively.

To validate the method in terms of recovery and accuracy, standard references of ethanol and methanol were spiked in blood, vitreous humor, urine, and CSF to check if there were any interferences in the biological matrices and to measure the recovery %. Biological matrices can contain various endogenous compounds that may interfere with the analysis of ethanol. Spiking standard references allowed us to evaluate if there were any interferences from the matrix components that could affect the accuracy and reliability of the analytical method. The average recovery percentage of ethanol and methanol in the postmortem biological specimens was in the range of 97–100%. No interferences were detected in any of the specimens that were tested.

The proposed method for GC-HS-FID is simple, fast, and reliable, and several samples can be analyzed in a short period for the quantitative determination of methanol and ethanol in biological samples for clinical and forensic toxicology purposes.

Sample preparation

One milliliter of the postmortem biological specimen and 90 µL internal standard were placed into a 20 mL HS vial. The vials were sealed with PTFE septa and aluminum crimp and then run on GC-HS-FID for quantitative analysis of ethanol and methanol in the biological specimens.

Results

One hundred and fifty postmortem cases were studied during the period from February 2022 to November 2023. The age group of the subjects was 18–70 years. In this study group, 96% of the subjects were male, and 4% of the subjects were female (Table 1).

Table 1.

Demographic characteristics of the study group.

Total number of cases	150
Period	February 2022 to November 2023
Age [mean ± SD]	37.39 ± 11.26 years
Gender	Male: 144 subjects Female: 6 subjects

Cause and manner of death: The study included only those cases in which the deceased had an alcohol history. Deceased had an alcohol history in the following cases: RTA, hanging, brought dead (BD), found dead and fall from height (FFH).

RTA cases include drivers of two-wheeler and four-wheeler vehicles, and pedestrians.

Hanging cases were suicide cases by hanging.

Brought dead cases refer to sudden death cases in which the subject got ill or fainted and was taken to a hospital and was declared dead at the hospital. In these cases, no treatment was given at the hospital as the patient was declared dead on reaching the hospital, and after that, the body was sent for postmortem examination.

Found dead cases refer to those where the subject was found dead at their home by his/her family or on the road by police, and then the body was sent for postmortem examination.

Fall from height cases were where subjects have fallen from a terrace, stairs, or balcony.

Out of 150 postmortem cases, positive ethanol concentration was detected in 108 cases. Among 108 cases with positive ethanol concentration, the highest number of cases was of hanging, followed by RTA, found dead, brought dead and fall from height (Figure 1).

Figure 1.

Distribution of cases with positive alcohol concentration.

The concentration of ethanol detected in the biological samples is shown in Supplementary Table 1.

Table 2 shows the mean postmortem ethanol concentration in blood, vitreous humor, urine, and CSF. It can be observed that in most cases, there was an increase in ethanol concentration as follows: blood < vitreous humor < CSF < urine. Also, it was noted that there was no significant difference in blood and vitreous humor alcohol concentration. These results suggested a high degree of consistency in the relationship between femoral blood alcohol concentration and alcohol concentration in different biological specimens, that is, vitreous humor, urine, and CSF. This consistency strengthens the reliability of using alternative biological specimens, such as vitreous humor and CSF, for postmortem alcohol analysis when blood samples are unavailable or contaminated.

Table 2.

Minimum, maximum, mean values, and standard deviation of ethanol concentration in biological specimens.

Variable	Obs	Mean	SD	Max
Blood	150	116.82	139.96	750.12
Vitreous humor	150	122.80	145.33	745.81
Urine	144	140.35	161.30	780.83
CSF	148	132.80	157.62	772.75

Figures 2, 3, and 4 show the correlation of ethanol in biological specimens using Spearman rho.

Figure 2.

Correlation of ethyl alcohol concentration using Spearman rho in blood and vitreous humor (mg/100 ml).

Figure 3.

Correlation of ethyl alcohol concentration using Spearman rho in blood and CSF (mg/100 ml).

Figure 4.

Correlation of ethyl alcohol concentration using Spearman rho in blood and urine (mg/100 ml).

Methanol

Methanol is a toxic alcohol. Methanol poisoning has resulted in a significant number of fatalities worldwide.^23,24 Methanol intoxication often occurs as a result of consuming methanol in illicit alcoholic drinks or ingesting specific fluids that contain methanol. Methanol toxicity is primarily related to its metabolites- formaldehyde and formic acid.^24–26 Methanol is cheap in comparison to ethanol and is thus used for the adulteration of alcoholic beverages, and this can lead to serious health consequences, including poisoning and death.^25,26 Instances of toxic alcohol intoxication have been reported worldwide.^26–28

In this study, out of 108 cases with positive ethanol concentration, in 33 cases methanol was also detected in the biological samples. Methanol was only detected in cases which have high ethanol concentrations. When ethanol is present at elevated levels, it competes with methanol for the enzyme alcohol dehydrogenase, thereby slowing methanol's conversion into its toxic metabolites, formaldehyde and formic acid. This competitive inhibition preserves unmetabolized methanol in the body for a longer period. Methanol was detected in the majority of blood and vitreous humor samples, with concentrations varying significantly across cases. Methanol was less frequently detected in CSF and urine, with most urine samples showing no methanol. The variation in methanol detection across matrices may be attributed to differential distribution in body fluids, or postmortem redistribution dynamics that vary between biological matrices.

The concentration of methanol detected in the biological samples is shown in Supplementary Table 2.

Figure 5 shows the GC-HS chromatograms of a case with positive methanol and ethanol concentration in biological specimens.

Figure 5.

GC-HS chromatograms of case 108 (a): blood sample; (b) vitreous humor sample; (c) CSF sample; and (d) urine sample.

Discussion

In this study, quantitative analysis of ethanol and methanol was carried out in multiple postmortem biological specimens. The principal reason for analyzing ethanol in alternative biological specimens in postmortem toxicology is to provide corroborating evidence of blood alcohol concentration and strengthen the conclusion that the deceased had ingested an alcoholic beverage prior to death.

Ethanol is the psychoactive constituent of alcoholic beverages, and it is the most often detected drug in postmortem toxicology. Alcohol consumption is a widespread phenomenon and holds significant societal implications, influencing public health, safety, and forensic investigations.^29,30 Research published in The Lancet in 2018 revealed that around one-third of the worldwide population, or 2.4 billion individuals, consume alcohol, and alcohol-related health issues account for the annual deaths of 2.2% of women and 6.8% of men.³¹

Excessive alcohol consumption can lead to a reduction in self-control, deterioration of judgment, heightened likelihood of aggressive behavior and actions, and increase the risk of violent crimes.^32,33 Olds et al. reported that excessive drinking and drunkenness are common causes of many fatal accidents and thus quantitative analysis of ethanol concentrations in postmortem specimens is a crucial component of investigation into an unnatural death.³⁴

In India, alcohol consumption amounted to about five billion Liters in 2020 and was estimated to reach about 6.21 billion Liters by 2024. There are various factors for the rise in alcohol consumption due to a variety of sociocultural practices, different state policies and practices regarding alcohol, a lack of community awareness of alcohol-related issues, misinformation about alcohol use in the media, different drinking habits among alcohol users, and the emergence of social drinking as a habit as a result of the nation's widespread urbanization.^33,35 The regulation of alcohol in India primarily falls under the purview of state governments, as per the Seventh Schedule of the Constitution, which lists “alcoholic liquors” as a state subject. Thus, leading to significant variations in policies and laws across different states. Some states have stringent regulations, including high taxes, restricted availability, and prohibition in certain areas, while others have more lenient policies. The legal drinking age differs among states, with the majority setting it at 21 years.^35,36

In this study, the comprehensive analysis of 150 postmortem cases over a span of nearly two years, representing a wide age range with a predominant male demographic. Among the identified causes of death, RTAs and hanging were the most prevalent. Remarkably, a substantial proportion of cases (72%) exhibited positive ethanol concentration, which was detected in 108 cases, with the majority surpassing 10 mg/100 mL, indicative of significant ethanol ingestion prior to death.

In 18 cases, ethanol was observed to be low, specifically below 10 mg/100 mL, and is solely detected in blood samples without corresponding concentrations in other biological matrices, i.e., vitreous humor, CSF and urine, and it gives rise to the possibility of postmortem ethanol formation due to microbial activity or contamination, emphasizing the importance of rigorous forensic analysis and interpretation.

These findings underscore the critical role of ethanol analysis in postmortem investigations, aiding in the determination of cause and manner of death, particularly in cases where ethanol ingestion may have contributed to or influenced the fatal outcome. For instance, higher ethanol concentrations may be anticipated in cases of RTAs, where impaired judgment or coordination due to alcohol consumption could contribute to the fatal outcome. Out of 36 positive RTA cases, seven cases were of four-wheeler driver and 14 cases were of two-wheeler drivers, and in all these cases, the level of alcohol was found above 30 mg/100 mL (permissible alcohol limit in India). In 2024, Delhi Traffic Police reported the data that cases of drunk driving had increased by 22% from the previous year. Police data from 1 January to 31 March 2024 showed that 6591 drunk drivers have been prosecuted. This was linked to the rise in sales of alcohol. Stringent checking of drivers was suggested by the enforcement agencies.³⁷

Hanging is the second most common method for suicide in India and the United States.³⁸ According to the National Crime Records Bureau (NCRB) data, in 2022, after ‘family problems’ and ‘illness’, drug and alcohol use was the most common cause of suicide in India.³⁹ In a case-control research study conducted by Gururaj on completed suicides in Bangalore, alcohol use was found to be a major risk factor for suicide, with alcohol users having a nearly 25-fold increased risk of committing suicide. The suicide rate rose by almost six times for women who were married to alcohol abusers. According to a study conducted in Chennai by Vijayakumar et al., alcohol users had greater suicide rates than non-users.³³ Zerbini et al. (2012) found a correlation between alcohol use and an increase in suicide rates. They specifically focused on hanging as a prevalent means of suicide, and reported that the high percentage of positive results for alcohol intake suggested that it is a risk factor for suicide, and concluded that it is crucial to cultivate and augment preventative strategies for this risk group.³⁸ In this study, out of 52 hanging cases, 43 cases have positive alcohol concentration in high amount indicating that the deceased had consumed alcohol prior to committing suicide.

In 2023, a study by the Indian Council of Medical Research (ICMR) reported that binge drinking is a major risk factor for unexplained sudden death among young adults in India aged 18–45 years.⁴⁰ In this study, in brought dead (or sudden death) cases and found dead cases, subjects have a history of illness and chronic alcohol use. In three brought dead cases, the deceased had a history of consuming excessive alcohol before death. In forensic and medical examinations of such cases, the presence of a history of illness and chronic alcohol use can provide a critical context for understanding the potential causes of death.

In 2022, the transport department of the Delhi government issued a notification that Forensic investigation under section 135A of the Motor Vehicle Act should be carried out for all road crashes in Delhi, India.⁴¹ Alcohol use is becoming a bigger problem for public health in India. Stringent alcohol policies and education are needed to reduce alcohol consumption.³³ Campaigns and initiatives for health education are crucial in increasing public knowledge of the negative effects of long-term alcohol use. The media, politicians, medical professionals, and the general public should all be the focus of these campaigns..^32,33,42

This study also highlighted the detection of methanol in the postmortem biological specimens. Various studies have reported that methanol is not produced postmortem, and blood concentrations over 10 mg/L may indicate significant exposure to methanol before death. Out of 108 cases with positive ethanol concentration, methanol was detected in the biological samples in 33 cases. Methanol was only detected in cases with high ethanol concentration, suggesting that the presence of methanol in the samples may be attributed to the contamination or adulteration of methanol in the alcohol drunk by the deceased before death. Methanol adulteration in alcoholic beverages poses serious health risks and emphasizes the need for stringent regulatory measures to ensure consumer safety.^43,44

A study conducted by Bonchev et al. in 2016 reported that they found methanol in the blood of patients with acute alcohol intoxication and that this could be due to the presence of methanol in the alcoholic beverages that the patients had consumed. In the field of toxicology, the long-term use of alcoholic beverages containing methanol is linked to the demographic of those who are addicted to alcohol, individuals commonly referred to as habitual drinkers. In this study, they further reported that extended exposure to methanol at sub-intoxicating concentrations poses a risk to chronic drinkers. This is because the increased plasma concentrations of methanol in the bloodstream are directly linked to the pathogenesis of chronic alcoholism and the resulting organ damage.⁴⁵

Overall, the study emphasizes the multifaceted nature of alcohol-related fatalities and the importance of thorough forensic analysis in determining the circumstances surrounding death. By elucidating the prevalence and implications of ethanol and methanol detection, as well as associated challenges, the study contributes to our understanding of alcohol-related mortality and informs public health interventions aimed at reducing the burden of alcohol-related harm.

Limitations

Firstly, the participants for our research study were selected from the cases that were brought to the mortuary of AIIMS, New Delhi, potentially limiting the generalizability of findings to broader populations. Furthermore, 96% of the subjects were male, restricting the applicability of the results across genders. Another limitation of this study is the use of n-propanol as the internal standard, as it is known that n-propanol can potentially form during decomposition or microbial activity in cadavers. Although no such occurrence was observed in our samples and quality control measures ensured reliability. Future studies could use tert-butanol to further enhance reliability, particularly in cases involving advanced decomposition. Furthermore, the method employed in the study required 1 mL of biological specimen for alcohol determination, which, while reliable, may be considered excessive when additional analyses are needed. We recommend careful consideration of sample volume in scenarios where multiple analyses are necessary. Future studies should address these limitations to enhance applicability and efficiency.

Conclusion

In this study, we conducted research that improves the interpretation of postmortem alcohol findings. Since ethanol is a putrefactive product that is formed postmortem, methods are needed to determine if ethanol was ingested before death. Ethanol levels in postmortem blood must be evaluated in relation to whether the individual had consumed alcohol and was intoxicated at the time of death, or if the levels were higher than a certain threshold. This study highlighted that the use of alternative/complementary specimens to the blood is of great relevance for forensic purposes to assess the alcohol concentration, ultimately facilitating more informed and accurate interpretations in medico-legal contexts. The detection of methanol in biological samples underscores the significance of continuous monitoring and regulatory actions to deter the dissemination and use of contaminated alcohol. The results of this study will also increase awareness and can help direct community initiatives, influence policy decisions, and enable people to make educated decisions about their alcohol consumption, all of which can help to mitigate the negative effects of alcohol use.

Supplemental Material

sj-docx-1-msl-10.1177_00258024261418791 - Supplemental material for Postmortem toxicology of alcohols: A cross-matrix study of ethanol and methanol in 150 cases from New Delhi, India

Supplemental material, sj-docx-1-msl-10.1177_00258024261418791 for Postmortem toxicology of alcohols: A cross-matrix study of ethanol and methanol in 150 cases from New Delhi, India by Neha Afaria, A.K. Jaiswal, Sudhir K. Gupta and Kulbhushan Prasad in Medicine, Science and the Law

Footnotes

Abbreviations

ORCID iD

Neha Afaria

Ethics approval and consent to participate

The research was conducted in accordance with the Declaration of Helsinki. The approval for the study was obtained from the ethics committee of the All-India Institute of Medical Sciences, New Delhi, India with Ref. No. IECPG-786/23.12.2021, RT-21/27.01.2022, OT-02/29.09.2022. The biological samples were collected after a legally accepted guardian have signed an informed consent form before the autopsy.

Consent for publication

Obtained.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. Supplementary data related to this article, including two tables, are available and have been submitted alongside the manuscript. These materials can be accessed in the online version of the article as supplementary files.

Supplemental material

Supplemental material for this article is available online.

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