Journal of Analytical Toxicology Advance Access published April 23, 2015 Journal of Analytical Toxicology 2015;1 – 5 doi:10.1093/jat/bkv044
Article
Ethylene Glycol and Metabolite Concentrations in Fatal Ethylene Glycol Poisonings Jenni Viinama¨ki*, Antti Sajantila and Ilkka Ojanpera¨ Department of Forensic Medicine, University of Helsinki, PO Box 40, FI-00014 Helsinki, Finland *Author to whom correspondence should be addressed. Email: jenni.viinamaki@helsinki.fi
Ethylene glycol (EG) is used in antifreeze and other industrial products. It metabolizes to glycolic acid (GA) and oxalic acid (OX) that cause metabolic acidosis and are mainly responsible for the toxicity of EG. During 2010 – 2014, EG or GA was found in 25 postmortem cases in Finland. Of these cases, 21 were classified as fatal EG poisonings and 3 were classified as methanol (MeOH) poisonings. In this study, we report the concentrations of EG and GA in postmortem blood and urine samples of fatal EG or mixed MeOH/EG poisonings. In the fatal EG poisonings, the median EG and GA concentrations were 0.87 and 1.6 g/L in blood and 4.3 and 5.3 g/L in urine. The median urine –blood ratios were 3.8 and 3.1 for EG and GA. These results warrant the use of urine as a primary matrix for screening. In EG positive cases, the quantification of both EG and GA in blood is crucial as GA concentration appears to best indicate a fatal poisoning with an approximate threshold of 1.5 g/L. The measurement of urinary OX does not offer much additional value to toxic alcohol screening as it may originate from varying dietary conditions. Introduction Ethylene glycol (EG) is a colorless, odorless, sweet tasting toxic diol used in antifreeze and other industrial products. It has intoxicating effects similar to those of ethanol, but a dose of 100 mL or 1.4 – 1.6 mg/kg is believed to be fatal to most adults (1 – 3). Poisoning by EG can be deliberate in connection with a suicide attempt or accidental if EG is confused with drinkable alcohol. The metabolism of EG is similar to that of ethanol and methanol (MeOH). EG is first metabolized to glycoaldehyde by the alcohol dehydrogenase (ADH) and further to glycolic acid (GA) by aldehyde dehydrogenase (ALDH). GA is then metabolized to glyoxylic acid and further to oxalic acid (OX) and formic acid (FA). However, unlike ethanol and MeOH, a significant portion of EG is eliminated through kidneys (4). The half-life of EG in poisoning cases is approximately 3 h, but the presence of ethanol increases it at least 5-fold due to ADH’s higher affinity to ethanol (3, 5). In addition, simultaneous ingestion of ethanol may mask the EG intoxication by delaying symptoms (6). The toxicity of EG is mainly caused by its acidic metabolites, particularly GA and OX. Symptoms of EG intoxication include central nervous system depression with seizures, cardiopulmonary complications, acute renal failure and delayed neurological sequelae (7, 8). The impaired organ functions probably result from the metabolic acidosis caused by GA and OX and the precipitation of OX as calcium salt (CaOX) crystals in tissues. Renal injuries have been shown to appear when plasma GA concentration exceeds 0.8–1.0 g/L (9 –11). EG intoxication can be treated with hemodialysis and administration of either 4-methylpyrazole (fomepizole) or ethanol if diagnosed soon after ingestion of EG. Diagnosing EG intoxication may be challenging as methods based on gas chromatography (GC) are available only at larger clinical laboratories. For example in Ireland, only 1 of the 39 acute care hospital laboratories analyzed MeOH or EG in 2010
(12). Enzymatic assays for EG have been presented already in the 1980s (13), but they produce false-positive results due to the less toxic propylene glycol and endogenous compounds (14). An improved enzymatic assay to overcome the issue of false positives was reported in 2011 (15). However, methods used in clinical laboratories seldom include the toxic metabolites of EG even though the severity of the intoxication correlates well with the GA concentration (10). Only recently an enzymatic assay for detection of GA in serum was published, but this method usually requires confirmation and quantification with GC (16). Chromatographic methods that include GA often utilize mass spectrometry (MS), the instrumentation seldom available in hospital laboratories. Furthermore, due to the low incidence of EG intoxication, maintaining such methods is reasonable only in specialized laboratories. Postmortem EG concentrations in various biological matrices have previously been published by several authors (1), but data on metabolite concentrations are scarce. In 2009, Rosano et al. listed EG and GA concentrations in postmortem blood of 12 medical examiner’s cases of fatal EG poisonings (17). However, urinary concentrations of EG and GA in poisoning cases have not been made available. Simultaneous ingestion of MeOH and EG is rare and only one case has been reported previously (18). In this case, however, no autopsy was performed and the cause of death (CoD) determination was based only on the clinical findings and the analysis of the liquid the subject was assumed to have drunk. Neither antemortem nor postmortem blood or urine samples were analyzed to confirm the CoD. Our objective was to add to the scarce literature of toxicologically elaborated investigation of EG fatalities by studying the postmortem blood and urine concentrations of EG and GA in 22 fatal EG poisoning cases using a previously published headspace in-tube extraction GC – MS method (ITEX-GC-MS) (19). In addition, in three mixed MeOH/EG poisonings the respective alcohols and their metabolites were investigated. The usefulness of including urine OX into the method was assessed by analyzing six EG-positive and 100 EG-negative cases for OX. Materials and methods Postmortem samples were collected on a routine basis at medico-legal autopsies carried out by forensic pathologists in Finland. In cases with suspected EG poisoning, EG, GA and FA concentrations in blood and urine samples were analyzed according to a previously published method (19). In addition to EG-positive cases, 100 EG-negative urine samples were analyzed for OX along with EG, GA and FA. OX was purchased from Merck (Darmstad, Germany) and was analyzed as the corresponding dimethyl ester by the ITEX-GC-MS method (19). For quantification, the target ion m/z 59 and qualifier ions m/z 45 and 29 were added to the analysis method.
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Calibration was performed using deuterated GA (2,2-d2-GA) as the internal standard, and calibration plot was created over the concentration range from 0.05 to 5.0 g/L.
Results and discussion During 2010 –2014, the total number of fatalities in Finland was 255,000. Medico-legal autopsy was performed in 52,000 (20%) cases and postmortem toxicological investigation in 33,500 (13%) cases. EG analysis was performed in 1,530 (0.6%) cases and 25 cases, corresponding to 0.01% of all fatalities, were found positive for EG. In 21 of these 25 cases, the underlying CoD was determined as EG poisoning by the forensic pathologist. Three cases were classified as MeOH poisonings, but substantial EG concentrations were present in both blood and urine samples, thus suggesting simultaneous ingestion of EG and MeOH. In one case, CoD was not available. The number of fatal MeOH poisonings during the same period was 68, corresponding to 0.03% of all fatalities. Case data of EG positive cases is presented in Table I, sorted according to the EG concentration in blood. Of the 25 fatal EG or MeOH/EG poisoning cases studied, 19 (76%) were male and 6 (24%) were female. The mean and median ages were 54 and 55 years, respectively, ranging from 38 to 70 years. Previous history of ethanol abuse was reported in 17 (68%) cases and previous history of toxic alcohol abuse in six (24%) cases. Previous intoxications or suicide attempts were reported in 10 (40%) cases. The manner of death (MoD) was accidental in 11 (44%) cases, undetermined in 8 (32%) cases and suicide in 5 (20%) cases. In one case, CoD and MoD were unavailable. Despite the previous history of ethanol abuse of these subjects, ethanol was detected only in two blood samples and one urine sample. In these cases, ethanol levels were low, 0.05 and 0.25 in blood
and 0.27 in urine. The absence of ethanol is consistent with the previous study of MeOH poisonings (20), suggesting faster elimination of ethanol by the ADH or the fact that only EG was ingested. Measurement of ethyl glucuronide would be helpful in determining whether ethanol was used prior to or simultaneously with EG ingestion. Results from EG and GA determinations along with other relevant toxicological findings are presented in Table II. In those 22 cases where no MeOH was detected, the median EG and GA concentrations in blood were 0.87 g/L (range from , LOQ to 5.6 g/L) and 1.6 g/L (range from 0.69 to 2.3 g/L), respectively. These results are comparable to those data published previously (17). The median EG and GA concentrations in urine were 4.3 g/L (range from 1.6 to 18 g/L) and 5.3 g/L (range from 2.6 to 8.3 g/L), respectively. Our study is the first to report urinary EG and GA concentrations in series of fatal EG poisoning cases. In all of these cases, urinary concentrations of EG and GA were higher than those in blood, the median urine – blood ratios being 3.8 and 3.1 for EG and GA, respectively. In all of the fatal EG poisoning cases, both EG and GA were detected in blood and urine samples, though in four cases EG levels in blood were ,0.2 g/L. This warrants the use of urine for screening purposes whenever this sample is available. If the patient is acidotic or suffers from oliguria or anuria, GA should be screened along with EG. There was a significant correlation between the blood and urine EG concentrations at 0.01 level (Pearson correlation, r ¼ 0.947). This finding is analogous with previous studies of ethanol and MeOH poisonings (20 – 22). However, no correlation between the blood and urine GA concentrations was found, being consistent with previous studies of MeOH and its major toxic metabolite FA (20, 22). The lack of correlation is probably due to the uniform level of GA concentrations found in blood, the
Table I Case Information for Fatal Ethylene Glycol and Methanol/Ethylene Glycol Poisonings According to Increasing EG Concentration in Blooda Case no.
Age (years)
Sex
Previous history of alcohol abuse
Previous history of toxic alcohol abuse
Case information
Cause of death
Manner of death
1 2 3 4 5 6
55 43 58 57 59 39
Male Male Male Female Male Male
No No Yes Yes Yes No
No Yes No Yes Yes No
T52.3 na T52.3 T52.3 T52.3 T52.3
Undetermined Na Undetermined Undetermined Undetermined Accidental
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
57 50 67 70 70 39 38 55 58 63 63 38 45 62 53 62 51 50 49
Male Female Male Male Male Male Male Female Male Male Male Female Female Male Male Male Female Male Male
No Yes Yes No Yes Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes
No No Yes No No No Yes No No No No No Yes No No Yes No No No
Taken to hospital unconscious, died 7 days after hospitalization Found dead with a bottle of antifreeze Found dead Found dead Found dead Taken to hospital disorientated and nearly blind, died 2 days after hospitalization Found unconscious, died on the way to the hospital Found dead Found dead with a bottle of windshield washing fluid Found dead Taken to hospital unconscious, died 5 days after hospitalization Found dead Found dead Found dead Found dead with a bottle of antifreeze Found dead with a bottle of antifreeze Found dead with a bottle of antifreeze Found dead Found dead Found dead Found dead Found dead with a bottle of antifreeze Found dead Found dead with a bottle of windshield washing fluid Found dead
T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T51.1 T51.1 T51.1
Accidental Accidental Accidental Accidental Accidental Undetermined Undetermined Undetermined Suicide Suicide Undetermined Accidental Suicide Accidental Suicide Suicide Accidental Accidental Accidental
a
T52.3, toxic effects of glycols; T51.1, toxic effect of methanol; na, data not available.
2 Viinama¨ki et al.
Table II Toxicological Findings in Fatal EG and MeOH/EG Poisoningsa Concentration in blood (g/L)b
Concentration in urine (g/L)b
EG
GA
EG
GA
1
nd/0.08c
nd/1.3c
na
na
2 3
,LOQ 0.10
0.69 1.0
2.0 2.1
7.8 3.3
4
0.13
1.8
1.6
2.9
5 6
0.27 nd/0.44d
1.9 nd/1.4d
2.8 nd
5.4 nd
7 8 9 10 11 12 13
0.48 0.49 0.49 0.56 na/0.83d 0.87 1.0
1.6 1.7 1.7 1.6 na/1.1d 2 1.3
4.3 3.0 3.3 2.7 na 2.9 4.4
7.7 5.7 8.3 5.6 na 4.3 6.1
14
1.1
1.8
3.0
4.4
15 16
1.2 1.8
1.9 1.6
4.8 5.5
5.1 4.9
17
1.8
1.0
6.5
2.6
18 19 20 21 22 23
2.7 3.0 3.7 4.9 5.6 0.22
2.3 1.6 1.9 1.9 1.5 nd
8.9 7.2 7.5 18 18 2.0
8.2 8.0 3.9 5.3 4.0 nd
24
0.25
,LOQ
1.6
0.17
25
0.55
0.43
2.8
2.3
Case no.
Other relevant findings
Blood: sertraline, chlorprotixene, mirtazapine, clonazepam and 7-aminoclonazepam at therapeutic concentrations Blood: cyclizine at therapeutic concentration Blood: acetone 0.2‰ Urine: acetone 0.4‰; isopropanol 0.1‰ Blood: sertraline, desmethyldiazpema, temazepam and diazepam at therapeutic concentrations Blood: desmethyldiazepam and diazepam at therapeutic concentrations Blood: propofol, lorazepam and diazepam at therapeutic concentrations. Drink consumed: ethanol 23% (v/v), acetone 0.7% and EG 120 g/L Blood: lidocaine, propofol and diazepam at therapeutic concentrations Blood: amitriptyline and desmethyldiazepam at therapeutic concentrations Blood: zopiclone and oxazepam at therapeutic concentrations Blood: temazepam, bisoprolol and sildenafil at therapeutic concentrations Blood: warfarin at therapeutic concentration nd Blood: ethanol 0.25‰; desmethyldiazepam at therapeutic concentration Urine: ethanol 0.27‰, Blood: ethanol 0.05‰; zopiclone, diazepam, desmethyldiazepam and haloperidol at therapeutic concentrations Urine: ethyl glucoronide 3.4 mg/L Blood: sertraline, desmethyldiazepam and oxazepam at therapeutic concentrations Blood: desmethyldiazepam, diazepam, temazepam and oxazepam at therapeutic concentrations Blood: high concentration of levomepromazine (3.9 mg/L,25 times therapeutic concentration), desmethyldiazepam and diazepam at therapeutic concentrations Blood: citalopram and norcitalopram at therapeutic concentrations Blood: 7-aminoclonazepam and mirtazapine at therapeutic concentrations nd nd nd Blood: MeOH 5.0‰; FA 0.94 g/L; zopiclone and oxazepam at therapeutic concentrations Urine MeOH 7.0‰; FA 4.1 g/L Blood: MeOH 2.8 ‰; FA 0.83 g/L Urine: MeOH 4.1‰; FA 4.0 g/L Blood: MeOH 1.2‰, FA 0.61 g/L, carbamazepine at therapeutic concentration Urine: MeOH 2.0‰, FA 1.3 g/L.
a
na: not available, nd: not detected, LOQ: limit of quantification. Measured in postmortem samples unless stated otherwise. c EG- and GA-positive results measured in antemortem serum. d EG- and GA-positive results measured in antemortem blood. b
concentrations being similar to those published previously (17). This suggest that GA concentration of 1.5 g/L, twice the concentration causing renal injuries (9 –11), represents a fatal threshold in untreated EG intoxications. However, patients with plasma GA concentrations exceeding 1.5 g/L have recovered, when treated with the combination of hemodialysis and fomepizole (9). As expected, there was no correlation between EG and GA concentrations in neither blood nor urine. Significant negative correlation, however, was found between EG concentration and the GA/ EG-ratio in both blood and urine (r ¼ 20.573 for blood at 0.05 level and r ¼ 20.689 for urine at 0.01 level). In addition, the urinary GA/EG-ratio correlated with the blood EG concentration. In seven cases, EG analysis was not originally requested by forensic pathologists, but a request was sent afterwards, probably due to CaOX found in the microscopy of kidneys. According to the literature, CaOX crystals are present in the kidneys in the majority of fatal EG poisonings (17). However, it has been shown with rats that even at significant GA and OX concentrations in urine CaOX crystals may not be found (23). In the present study, we found one case with considerable EG and GA concentrations both in blood and urine but histological analysis showed
no CaOX crystals (Case 21). This implicates that with large enough EG doses, GA is accumulated and causes death without CaOX crystallization in the kidneys and thus complicates CoD investigation. In 18 (72%) cases, the subjects were found dead. In four cases resuscitation was attempted but found unsuccessful, and in further three cases, the subjects were hospitalized and treated for EG poisoning. In one of the hospitalized cases (Case 1), the admission serum sample tested negative for toxic alcohols in the hospital laboratory. This antemortem serum was re-analyzed together with postmortem blood for EG and GA after the autopsy using the present ITEX-GC-MS method. The postmortem blood sample was negative, but in the antemortem serum concentrations of 0.08 and 1.3 g/L of EG and GA were found, respectively. The initial negative results may have been caused by the low EG concentration in the sample, or the lack of EG in the toxic alcohol screening method. The negative results of postmortem blood are consistent with survival time of 1 week. In Case 6, representing another hospitalized patient, a bottle containing the ingested liquid was analyzed for alcohols and EG. The liquid consisted of ethanol 23% (v/v), acetone 0.7% EG and GA Concentrations in EG Fatal Poisonings 3
(v/v) and EG 120 g/L. The high ethanol concentration probably delayed the symptoms of EG intoxication. The antemortem blood sample was negative for ethanol, but 0.44 g/L of EG and 1.4 g/L of GA were found. The postmortem blood and urine samples were negative for ethanol, EG and GA, which is consistent with the survival time of nearly 3 days. In two of the mixed MeOH/EG poisoning Cases (23,24), the GA concentrations in both blood and urine were very low or not detected at all, whereas the MeOH and FA concentrations were high. This suggests that MeOH was consumed prior to or simultaneously with EG as ADH has a higher affinity to MeOH than EG (4, 24). In the third case (Case 25), MeOH and FA concentrations were substantially lower and GA was present along with EG. In order to assess the value of OX concentrations in the postmortem diagnosis of EG poisoning, six EG positive and 100 EG negative control urine samples were analyzed for OX using the present ITEX-GC-MS method. Of the EG negative urines, 58 were positive for OX, the median concentration being 0.075 g/ L. Despite the small median concentration and the high proportion of negative cases, there were still seven urine samples in which the OX concentration exceeded 0.2 g/L, the highest OX concentration being 0.85 g/L. In EG-positive cases, OX concentrations were generally higher, but in two cases with high urinary EG and GA concentrations, OX concentrations were ,0.1 g/L. These results suggest that analysis of OX does not provide any additional diagnostic value to reveal EG poisonings nor can it be used as a sole substance for verifying EG poisoning. Distribution of urinary OX concentrations is illustrated in Figure 1. The ITEX-GC-MS method has been used for the analysis of EG, GA and FA in the authors’ laboratory for a 5-year period. Over 1,500 postmortem samples have been analyzed, of which
74 blood and 138 urine samples presented notable putrefactive postmortem changes. In these putrefied samples, GA was not detected as a postmortem artefact, whereas FA was positive in 51 (69%) blood and in 53 (38%) urine samples, the median FA concentrations being 0.32 and 0.26 g/L, respectively. In 14 (19%) blood samples, FA concentration exceeded 0.5 g/L, a level generally associated with fatal MeOH poisonings (22, 25). Neither EG nor MeOH was detected in any of these samples, but the abuse of toxic alcohols cannot be entirely discarded. However, in nonputrefied cases such phenomenon of elevated FA has not been observed. Recently, some minor postmortem production of EG has been reported (26). However, the concentration of EG, possibly produced by micro-organisms was negligible compared with that found in EG poisonings and has no influence on the interpretation of toxicological results. Moreover, as we demonstrate in this study, attention in CoD investigation should be paid on the concentration of GA rather than that of EG. Variable OX concentrations are often detected in urine samples, probably originating from varying diets. Conclusions This study reports the postmortem blood and urine concentrations of EG and its major toxic metabolite GA in a series of fatal EG poisoning cases. For screening purposes, urine is preferable to blood as both EG and GA concentrations are considerably higher in urine. When EG intoxication is suspected, the main toxic metabolite GA should be analyzed along with EG, because GA accumulates in the body causing the life-threatening toxic syndrome. In untreated EG intoxications, the blood GA concentration of 1.5 g/L appears to be an approximate threshold fatal concentration. Analysis of the other acidic EG metabolites OX and FA does not provide much additional information in suspected EG intoxication cases as their concentrations are low and cannot be distinguished from non-poisoning cases.
References
Figure 1. Distribution of urinary OX concentration in six fatal EG poisonings and 100 negative control cases.
4 Viinama¨ki et al.
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EG and GA Concentrations in EG Fatal Poisonings 5
Article
Ethylene Glycol and Metabolite Concentrations in Fatal Ethylene Glycol Poisonings Jenni Viinama¨ki*, Antti Sajantila and Ilkka Ojanpera¨ Department of Forensic Medicine, University of Helsinki, PO Box 40, FI-00014 Helsinki, Finland *Author to whom correspondence should be addressed. Email: jenni.viinamaki@helsinki.fi
Ethylene glycol (EG) is used in antifreeze and other industrial products. It metabolizes to glycolic acid (GA) and oxalic acid (OX) that cause metabolic acidosis and are mainly responsible for the toxicity of EG. During 2010 – 2014, EG or GA was found in 25 postmortem cases in Finland. Of these cases, 21 were classified as fatal EG poisonings and 3 were classified as methanol (MeOH) poisonings. In this study, we report the concentrations of EG and GA in postmortem blood and urine samples of fatal EG or mixed MeOH/EG poisonings. In the fatal EG poisonings, the median EG and GA concentrations were 0.87 and 1.6 g/L in blood and 4.3 and 5.3 g/L in urine. The median urine –blood ratios were 3.8 and 3.1 for EG and GA. These results warrant the use of urine as a primary matrix for screening. In EG positive cases, the quantification of both EG and GA in blood is crucial as GA concentration appears to best indicate a fatal poisoning with an approximate threshold of 1.5 g/L. The measurement of urinary OX does not offer much additional value to toxic alcohol screening as it may originate from varying dietary conditions. Introduction Ethylene glycol (EG) is a colorless, odorless, sweet tasting toxic diol used in antifreeze and other industrial products. It has intoxicating effects similar to those of ethanol, but a dose of 100 mL or 1.4 – 1.6 mg/kg is believed to be fatal to most adults (1 – 3). Poisoning by EG can be deliberate in connection with a suicide attempt or accidental if EG is confused with drinkable alcohol. The metabolism of EG is similar to that of ethanol and methanol (MeOH). EG is first metabolized to glycoaldehyde by the alcohol dehydrogenase (ADH) and further to glycolic acid (GA) by aldehyde dehydrogenase (ALDH). GA is then metabolized to glyoxylic acid and further to oxalic acid (OX) and formic acid (FA). However, unlike ethanol and MeOH, a significant portion of EG is eliminated through kidneys (4). The half-life of EG in poisoning cases is approximately 3 h, but the presence of ethanol increases it at least 5-fold due to ADH’s higher affinity to ethanol (3, 5). In addition, simultaneous ingestion of ethanol may mask the EG intoxication by delaying symptoms (6). The toxicity of EG is mainly caused by its acidic metabolites, particularly GA and OX. Symptoms of EG intoxication include central nervous system depression with seizures, cardiopulmonary complications, acute renal failure and delayed neurological sequelae (7, 8). The impaired organ functions probably result from the metabolic acidosis caused by GA and OX and the precipitation of OX as calcium salt (CaOX) crystals in tissues. Renal injuries have been shown to appear when plasma GA concentration exceeds 0.8–1.0 g/L (9 –11). EG intoxication can be treated with hemodialysis and administration of either 4-methylpyrazole (fomepizole) or ethanol if diagnosed soon after ingestion of EG. Diagnosing EG intoxication may be challenging as methods based on gas chromatography (GC) are available only at larger clinical laboratories. For example in Ireland, only 1 of the 39 acute care hospital laboratories analyzed MeOH or EG in 2010
(12). Enzymatic assays for EG have been presented already in the 1980s (13), but they produce false-positive results due to the less toxic propylene glycol and endogenous compounds (14). An improved enzymatic assay to overcome the issue of false positives was reported in 2011 (15). However, methods used in clinical laboratories seldom include the toxic metabolites of EG even though the severity of the intoxication correlates well with the GA concentration (10). Only recently an enzymatic assay for detection of GA in serum was published, but this method usually requires confirmation and quantification with GC (16). Chromatographic methods that include GA often utilize mass spectrometry (MS), the instrumentation seldom available in hospital laboratories. Furthermore, due to the low incidence of EG intoxication, maintaining such methods is reasonable only in specialized laboratories. Postmortem EG concentrations in various biological matrices have previously been published by several authors (1), but data on metabolite concentrations are scarce. In 2009, Rosano et al. listed EG and GA concentrations in postmortem blood of 12 medical examiner’s cases of fatal EG poisonings (17). However, urinary concentrations of EG and GA in poisoning cases have not been made available. Simultaneous ingestion of MeOH and EG is rare and only one case has been reported previously (18). In this case, however, no autopsy was performed and the cause of death (CoD) determination was based only on the clinical findings and the analysis of the liquid the subject was assumed to have drunk. Neither antemortem nor postmortem blood or urine samples were analyzed to confirm the CoD. Our objective was to add to the scarce literature of toxicologically elaborated investigation of EG fatalities by studying the postmortem blood and urine concentrations of EG and GA in 22 fatal EG poisoning cases using a previously published headspace in-tube extraction GC – MS method (ITEX-GC-MS) (19). In addition, in three mixed MeOH/EG poisonings the respective alcohols and their metabolites were investigated. The usefulness of including urine OX into the method was assessed by analyzing six EG-positive and 100 EG-negative cases for OX. Materials and methods Postmortem samples were collected on a routine basis at medico-legal autopsies carried out by forensic pathologists in Finland. In cases with suspected EG poisoning, EG, GA and FA concentrations in blood and urine samples were analyzed according to a previously published method (19). In addition to EG-positive cases, 100 EG-negative urine samples were analyzed for OX along with EG, GA and FA. OX was purchased from Merck (Darmstad, Germany) and was analyzed as the corresponding dimethyl ester by the ITEX-GC-MS method (19). For quantification, the target ion m/z 59 and qualifier ions m/z 45 and 29 were added to the analysis method.
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Calibration was performed using deuterated GA (2,2-d2-GA) as the internal standard, and calibration plot was created over the concentration range from 0.05 to 5.0 g/L.
Results and discussion During 2010 –2014, the total number of fatalities in Finland was 255,000. Medico-legal autopsy was performed in 52,000 (20%) cases and postmortem toxicological investigation in 33,500 (13%) cases. EG analysis was performed in 1,530 (0.6%) cases and 25 cases, corresponding to 0.01% of all fatalities, were found positive for EG. In 21 of these 25 cases, the underlying CoD was determined as EG poisoning by the forensic pathologist. Three cases were classified as MeOH poisonings, but substantial EG concentrations were present in both blood and urine samples, thus suggesting simultaneous ingestion of EG and MeOH. In one case, CoD was not available. The number of fatal MeOH poisonings during the same period was 68, corresponding to 0.03% of all fatalities. Case data of EG positive cases is presented in Table I, sorted according to the EG concentration in blood. Of the 25 fatal EG or MeOH/EG poisoning cases studied, 19 (76%) were male and 6 (24%) were female. The mean and median ages were 54 and 55 years, respectively, ranging from 38 to 70 years. Previous history of ethanol abuse was reported in 17 (68%) cases and previous history of toxic alcohol abuse in six (24%) cases. Previous intoxications or suicide attempts were reported in 10 (40%) cases. The manner of death (MoD) was accidental in 11 (44%) cases, undetermined in 8 (32%) cases and suicide in 5 (20%) cases. In one case, CoD and MoD were unavailable. Despite the previous history of ethanol abuse of these subjects, ethanol was detected only in two blood samples and one urine sample. In these cases, ethanol levels were low, 0.05 and 0.25 in blood
and 0.27 in urine. The absence of ethanol is consistent with the previous study of MeOH poisonings (20), suggesting faster elimination of ethanol by the ADH or the fact that only EG was ingested. Measurement of ethyl glucuronide would be helpful in determining whether ethanol was used prior to or simultaneously with EG ingestion. Results from EG and GA determinations along with other relevant toxicological findings are presented in Table II. In those 22 cases where no MeOH was detected, the median EG and GA concentrations in blood were 0.87 g/L (range from , LOQ to 5.6 g/L) and 1.6 g/L (range from 0.69 to 2.3 g/L), respectively. These results are comparable to those data published previously (17). The median EG and GA concentrations in urine were 4.3 g/L (range from 1.6 to 18 g/L) and 5.3 g/L (range from 2.6 to 8.3 g/L), respectively. Our study is the first to report urinary EG and GA concentrations in series of fatal EG poisoning cases. In all of these cases, urinary concentrations of EG and GA were higher than those in blood, the median urine – blood ratios being 3.8 and 3.1 for EG and GA, respectively. In all of the fatal EG poisoning cases, both EG and GA were detected in blood and urine samples, though in four cases EG levels in blood were ,0.2 g/L. This warrants the use of urine for screening purposes whenever this sample is available. If the patient is acidotic or suffers from oliguria or anuria, GA should be screened along with EG. There was a significant correlation between the blood and urine EG concentrations at 0.01 level (Pearson correlation, r ¼ 0.947). This finding is analogous with previous studies of ethanol and MeOH poisonings (20 – 22). However, no correlation between the blood and urine GA concentrations was found, being consistent with previous studies of MeOH and its major toxic metabolite FA (20, 22). The lack of correlation is probably due to the uniform level of GA concentrations found in blood, the
Table I Case Information for Fatal Ethylene Glycol and Methanol/Ethylene Glycol Poisonings According to Increasing EG Concentration in Blooda Case no.
Age (years)
Sex
Previous history of alcohol abuse
Previous history of toxic alcohol abuse
Case information
Cause of death
Manner of death
1 2 3 4 5 6
55 43 58 57 59 39
Male Male Male Female Male Male
No No Yes Yes Yes No
No Yes No Yes Yes No
T52.3 na T52.3 T52.3 T52.3 T52.3
Undetermined Na Undetermined Undetermined Undetermined Accidental
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
57 50 67 70 70 39 38 55 58 63 63 38 45 62 53 62 51 50 49
Male Female Male Male Male Male Male Female Male Male Male Female Female Male Male Male Female Male Male
No Yes Yes No Yes Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes
No No Yes No No No Yes No No No No No Yes No No Yes No No No
Taken to hospital unconscious, died 7 days after hospitalization Found dead with a bottle of antifreeze Found dead Found dead Found dead Taken to hospital disorientated and nearly blind, died 2 days after hospitalization Found unconscious, died on the way to the hospital Found dead Found dead with a bottle of windshield washing fluid Found dead Taken to hospital unconscious, died 5 days after hospitalization Found dead Found dead Found dead Found dead with a bottle of antifreeze Found dead with a bottle of antifreeze Found dead with a bottle of antifreeze Found dead Found dead Found dead Found dead Found dead with a bottle of antifreeze Found dead Found dead with a bottle of windshield washing fluid Found dead
T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T52.3 T51.1 T51.1 T51.1
Accidental Accidental Accidental Accidental Accidental Undetermined Undetermined Undetermined Suicide Suicide Undetermined Accidental Suicide Accidental Suicide Suicide Accidental Accidental Accidental
a
T52.3, toxic effects of glycols; T51.1, toxic effect of methanol; na, data not available.
2 Viinama¨ki et al.
Table II Toxicological Findings in Fatal EG and MeOH/EG Poisoningsa Concentration in blood (g/L)b
Concentration in urine (g/L)b
EG
GA
EG
GA
1
nd/0.08c
nd/1.3c
na
na
2 3
,LOQ 0.10
0.69 1.0
2.0 2.1
7.8 3.3
4
0.13
1.8
1.6
2.9
5 6
0.27 nd/0.44d
1.9 nd/1.4d
2.8 nd
5.4 nd
7 8 9 10 11 12 13
0.48 0.49 0.49 0.56 na/0.83d 0.87 1.0
1.6 1.7 1.7 1.6 na/1.1d 2 1.3
4.3 3.0 3.3 2.7 na 2.9 4.4
7.7 5.7 8.3 5.6 na 4.3 6.1
14
1.1
1.8
3.0
4.4
15 16
1.2 1.8
1.9 1.6
4.8 5.5
5.1 4.9
17
1.8
1.0
6.5
2.6
18 19 20 21 22 23
2.7 3.0 3.7 4.9 5.6 0.22
2.3 1.6 1.9 1.9 1.5 nd
8.9 7.2 7.5 18 18 2.0
8.2 8.0 3.9 5.3 4.0 nd
24
0.25
,LOQ
1.6
0.17
25
0.55
0.43
2.8
2.3
Case no.
Other relevant findings
Blood: sertraline, chlorprotixene, mirtazapine, clonazepam and 7-aminoclonazepam at therapeutic concentrations Blood: cyclizine at therapeutic concentration Blood: acetone 0.2‰ Urine: acetone 0.4‰; isopropanol 0.1‰ Blood: sertraline, desmethyldiazpema, temazepam and diazepam at therapeutic concentrations Blood: desmethyldiazepam and diazepam at therapeutic concentrations Blood: propofol, lorazepam and diazepam at therapeutic concentrations. Drink consumed: ethanol 23% (v/v), acetone 0.7% and EG 120 g/L Blood: lidocaine, propofol and diazepam at therapeutic concentrations Blood: amitriptyline and desmethyldiazepam at therapeutic concentrations Blood: zopiclone and oxazepam at therapeutic concentrations Blood: temazepam, bisoprolol and sildenafil at therapeutic concentrations Blood: warfarin at therapeutic concentration nd Blood: ethanol 0.25‰; desmethyldiazepam at therapeutic concentration Urine: ethanol 0.27‰, Blood: ethanol 0.05‰; zopiclone, diazepam, desmethyldiazepam and haloperidol at therapeutic concentrations Urine: ethyl glucoronide 3.4 mg/L Blood: sertraline, desmethyldiazepam and oxazepam at therapeutic concentrations Blood: desmethyldiazepam, diazepam, temazepam and oxazepam at therapeutic concentrations Blood: high concentration of levomepromazine (3.9 mg/L,25 times therapeutic concentration), desmethyldiazepam and diazepam at therapeutic concentrations Blood: citalopram and norcitalopram at therapeutic concentrations Blood: 7-aminoclonazepam and mirtazapine at therapeutic concentrations nd nd nd Blood: MeOH 5.0‰; FA 0.94 g/L; zopiclone and oxazepam at therapeutic concentrations Urine MeOH 7.0‰; FA 4.1 g/L Blood: MeOH 2.8 ‰; FA 0.83 g/L Urine: MeOH 4.1‰; FA 4.0 g/L Blood: MeOH 1.2‰, FA 0.61 g/L, carbamazepine at therapeutic concentration Urine: MeOH 2.0‰, FA 1.3 g/L.
a
na: not available, nd: not detected, LOQ: limit of quantification. Measured in postmortem samples unless stated otherwise. c EG- and GA-positive results measured in antemortem serum. d EG- and GA-positive results measured in antemortem blood. b
concentrations being similar to those published previously (17). This suggest that GA concentration of 1.5 g/L, twice the concentration causing renal injuries (9 –11), represents a fatal threshold in untreated EG intoxications. However, patients with plasma GA concentrations exceeding 1.5 g/L have recovered, when treated with the combination of hemodialysis and fomepizole (9). As expected, there was no correlation between EG and GA concentrations in neither blood nor urine. Significant negative correlation, however, was found between EG concentration and the GA/ EG-ratio in both blood and urine (r ¼ 20.573 for blood at 0.05 level and r ¼ 20.689 for urine at 0.01 level). In addition, the urinary GA/EG-ratio correlated with the blood EG concentration. In seven cases, EG analysis was not originally requested by forensic pathologists, but a request was sent afterwards, probably due to CaOX found in the microscopy of kidneys. According to the literature, CaOX crystals are present in the kidneys in the majority of fatal EG poisonings (17). However, it has been shown with rats that even at significant GA and OX concentrations in urine CaOX crystals may not be found (23). In the present study, we found one case with considerable EG and GA concentrations both in blood and urine but histological analysis showed
no CaOX crystals (Case 21). This implicates that with large enough EG doses, GA is accumulated and causes death without CaOX crystallization in the kidneys and thus complicates CoD investigation. In 18 (72%) cases, the subjects were found dead. In four cases resuscitation was attempted but found unsuccessful, and in further three cases, the subjects were hospitalized and treated for EG poisoning. In one of the hospitalized cases (Case 1), the admission serum sample tested negative for toxic alcohols in the hospital laboratory. This antemortem serum was re-analyzed together with postmortem blood for EG and GA after the autopsy using the present ITEX-GC-MS method. The postmortem blood sample was negative, but in the antemortem serum concentrations of 0.08 and 1.3 g/L of EG and GA were found, respectively. The initial negative results may have been caused by the low EG concentration in the sample, or the lack of EG in the toxic alcohol screening method. The negative results of postmortem blood are consistent with survival time of 1 week. In Case 6, representing another hospitalized patient, a bottle containing the ingested liquid was analyzed for alcohols and EG. The liquid consisted of ethanol 23% (v/v), acetone 0.7% EG and GA Concentrations in EG Fatal Poisonings 3
(v/v) and EG 120 g/L. The high ethanol concentration probably delayed the symptoms of EG intoxication. The antemortem blood sample was negative for ethanol, but 0.44 g/L of EG and 1.4 g/L of GA were found. The postmortem blood and urine samples were negative for ethanol, EG and GA, which is consistent with the survival time of nearly 3 days. In two of the mixed MeOH/EG poisoning Cases (23,24), the GA concentrations in both blood and urine were very low or not detected at all, whereas the MeOH and FA concentrations were high. This suggests that MeOH was consumed prior to or simultaneously with EG as ADH has a higher affinity to MeOH than EG (4, 24). In the third case (Case 25), MeOH and FA concentrations were substantially lower and GA was present along with EG. In order to assess the value of OX concentrations in the postmortem diagnosis of EG poisoning, six EG positive and 100 EG negative control urine samples were analyzed for OX using the present ITEX-GC-MS method. Of the EG negative urines, 58 were positive for OX, the median concentration being 0.075 g/ L. Despite the small median concentration and the high proportion of negative cases, there were still seven urine samples in which the OX concentration exceeded 0.2 g/L, the highest OX concentration being 0.85 g/L. In EG-positive cases, OX concentrations were generally higher, but in two cases with high urinary EG and GA concentrations, OX concentrations were ,0.1 g/L. These results suggest that analysis of OX does not provide any additional diagnostic value to reveal EG poisonings nor can it be used as a sole substance for verifying EG poisoning. Distribution of urinary OX concentrations is illustrated in Figure 1. The ITEX-GC-MS method has been used for the analysis of EG, GA and FA in the authors’ laboratory for a 5-year period. Over 1,500 postmortem samples have been analyzed, of which
74 blood and 138 urine samples presented notable putrefactive postmortem changes. In these putrefied samples, GA was not detected as a postmortem artefact, whereas FA was positive in 51 (69%) blood and in 53 (38%) urine samples, the median FA concentrations being 0.32 and 0.26 g/L, respectively. In 14 (19%) blood samples, FA concentration exceeded 0.5 g/L, a level generally associated with fatal MeOH poisonings (22, 25). Neither EG nor MeOH was detected in any of these samples, but the abuse of toxic alcohols cannot be entirely discarded. However, in nonputrefied cases such phenomenon of elevated FA has not been observed. Recently, some minor postmortem production of EG has been reported (26). However, the concentration of EG, possibly produced by micro-organisms was negligible compared with that found in EG poisonings and has no influence on the interpretation of toxicological results. Moreover, as we demonstrate in this study, attention in CoD investigation should be paid on the concentration of GA rather than that of EG. Variable OX concentrations are often detected in urine samples, probably originating from varying diets. Conclusions This study reports the postmortem blood and urine concentrations of EG and its major toxic metabolite GA in a series of fatal EG poisoning cases. For screening purposes, urine is preferable to blood as both EG and GA concentrations are considerably higher in urine. When EG intoxication is suspected, the main toxic metabolite GA should be analyzed along with EG, because GA accumulates in the body causing the life-threatening toxic syndrome. In untreated EG intoxications, the blood GA concentration of 1.5 g/L appears to be an approximate threshold fatal concentration. Analysis of the other acidic EG metabolites OX and FA does not provide much additional information in suspected EG intoxication cases as their concentrations are low and cannot be distinguished from non-poisoning cases.
References
Figure 1. Distribution of urinary OX concentration in six fatal EG poisonings and 100 negative control cases.
4 Viinama¨ki et al.
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EG and GA Concentrations in EG Fatal Poisonings 5
