Abstract
Accurate interpretation of the blood ethanol (EtOH) concentration at the time of death presents a difficult task since the origin of detected EtOH in postmortem cases (either in corpses or in specimens after sample collection) may vary. Headspace gas chromatography is the choice method for detecting EtOH in blood or other specimens, due to the accuracy and sensitivity it provides. Possible sources of postmortem EtOH have been the ante-mortem ingestion, the ante-mortem endogenous production and the postmortem microbial neo-formation, which has been considered the most critical factor that could complicate the results. It has been reported that EtOH could be formed postmortem in variable and non-predictable amounts, as a function of the type and number of microorganisms present either in corpses or specimens collected at autopsy. The presence of other volatiles—mostly n-propanol—has been correlated to microbial EtOH production, although the quantitative pattern between them and EtOH still remains obscure. The factors most frequently implicated in the mechanism of postmortem EtOH production in corpses have been considered the number and nature of microbes present, the availability of various types of substrates, the temperature and the time. Complication in the interpretation of blood alcohol concentration could arise due to the atypical distribution of EtOH in the body compartments after death. Specimens to blood EtOH ratios reported in the literature are presented. All the aforementioned aspects are discussed in a comprehensive way, providing a deep insight into this essential problem.
Keywords
There are several reviews on ethanol (EtOH) published each year (Caballeria 2003; Swift 2003; Ramaiah, Rivera and Arteel 2004; Crabb et al. 2004; Poschl and Seizt 2004; Suter 2004; Quertemond and Tambour 2004), but reviews concerning the origin of postmortem EtOH and the evaluation of the levels measured in postmortem specimens are strictly limited (Corry 1978; O’Neal and Poklis 1996; Goulle and Deveaux 2003; Scopp 2004). EtOH analysis is the most frequently performed assay in a forensic toxicology laboratory and gas chromatography is the choice method for detecting EtOH in blood or other specimens, due to the accuracy and sensitivity it provides (Tagliaro et al. 1992). Determination of blood alcohol concentration (BAC) and/or evaluation of the toxicological results are often part of the judicial inquiry in forensic cases. Particularly in postmortem cases, EtOH concentration has to be determined as part of the death investigation, since it might be a causal or a contributory factor.
There have been three possible sources for the EtOH detected in postmortem specimens: (a) antemortem ingestion: EtOH ingested by living persons after the consumption of alcoholic beverages, (b) antemortem endogenous production: EtOH produced endogenously in living persons, under certain conditions, probably due to microbial fermentation in the intestine (“auto-brewery” syndrome), and (c) postmortem microbial fermentation, either in corpses or in specimens post-sampling. However, it has been the microbial induced postmortem EtOH formation, as well as, the source and integrity of the collected specimens that could complicate the results. Despite the adversities, as BAC has been regularly used as evidence in criminal and civil litigation, its unequivocal aetiology, exogenous or endogenous, must be determined, presenting a critical task for accurate scientific interpretations, especially in decomposed bodies (Briglia, Bidanset, and Dal Cortivo 1992; Knight 1996; Grellner and Iffland 1997; Videira de Lima and Midio 1999; Goulle and Deveaux 2003; Norberg et al. 2003; Scopp 2004).
Ingested EtOH could be absorbed from all the mucosal surfaces of the gastrointestinal tract at a rate that has been best described by passive diffusion (Wilkinson et al. 1977; Holford 1987). EtOH is distributed rapidly through the body in a degree governed—especially at equilibrium—by the water content of the various tissues or fluids (Marshall et al. 1983; Holford 1987). Small amounts of the ingested EtOH are excreted via the expired air (0.7%), urine (0.3%), sweat (0.1%), feces, milk, and saliva (Holford 1987). However, elimination of EtOH occurs primarily (∼95–98%) through metabolism in the liver by enzymatic oxidation (Ramchandani and Bosron 2001). The major metabolic pathway for EtOH metabolism is a two step enzymatic process that requires nicotinamide adenine dinucleotide (NAD+) as a cofactor (Von Wartburg, Belhune and Vallee 1964; Li and Vallee 1969; Edenberg and Bosron 1997). In the first step EtOH is converted to acetaldehyde by the isoforms of alcohol dehydrogenase (ADH). In the second step, acetaldehyde is metabolised to acetic acid by the isoforms of aldehyde dehydrogenase (ALDH). Finally acetate is rapidly converted to water and carbon dioxide (CO2) in the Krebs cycle.
In addition to the main metabolic pathway, there are two minor oxidative pathways for converting EtOH to acetaldehyde. The pathway through the cytochrome P4502E1 (CYP2E1) can also oxidize EtOH in the liver (Asai et al. 1996; Lieber 1999). CYP2E1 is inducible, thus accounting for the increased rate of alcohol metabolism that is observed in chronic drinkers at high BAC (Ueno et al. 1996). The second minor oxidative pathway involves the enzyme catalase, which in the presence of peroxides oxidize EtOH to acetaldehyde and acetate (Bradford et al. 1993; Lands 1998). This pathway may act in the brain where no other enzyme is known to convert appreciable amounts of EtOH to acetaldehyde. Non-ADH mediated metabolism of EtOH accounts for no more than 10% of total EtOH metabolism (Cornell et al. 1979).
Regarding antemortem endogenous EtOH production, it could take place in man, probably due to microbial fermentation in the intestine (“auto-brewery syndrome”) as it is demonstrated in a relative review (Logan and Jones 2000). BAC of healthy individuals, as well as, those suffering from various metabolic disorders (diabetes, hepatitis, cirrhosis), attributed to endogenous EtOH production have ranged from 0 to 0.0008 g/L, as measured with gas chromatographic methods (Logan and Jones 2000). These concentrations have been considered too low to have any forensic or medical importance. However, EtOH levels as high as 0.8 g/L, produced endogenously, have been reported, in few rare instances, concerning, either Asian subjects, with very serious yeast infections, due to genetic polymorphism of the enzymes involved in EtOH metabolism (Kaji et al. 1984), or to bacterial overgrowth in the gut, due to abnormalities, such as jejunal blind loops after operation for morbid obesity (Mezey et al. 1975). Regarding this matter, it has been suggested that only the overconsumption of alcoholic beverages could lead to forensically significant BAC (above the legal limits of 0.5 or 0.8 g/L) and not the antemortem endogenous EtOH production (Logan and Jones 2000).
It has been well established in the literature that EtOH could be produced postmortem in variable and nonpredictable amounts, an issue that might cause serious legal implications (Zumvalt 1982; Briglia, Bidanset, and Dal Cortivo 1992; Hansen 1994; Knight 1996; Grellner and Iffland 1997; Videira de Lima and Mayes 1999; Scopp 2004). Since this is the case, we are of opinion that a deep insight into this essential problem would be valuable, underlying all the different aspects it might have, in a comprehensive way.
APPROACHES TO POSTMORTEM ETHANOL PRODUCTION
Postmortem EtOH production in corpses was first reported in 1936 (Nicloux 1936; Wagner 1936) and was later well established by many authors. Relative reports involved corpses either routinely autopsied (Corry 1978; Caplan and Levine 1990; Videira de Lima and Midio 1999), or corpses in different stages of putrefaction, that have been found in the open air after aviation fatalities (Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; Johnson et al. 2004), or have been recovered from water (Zumwalt, Bost, and Sunshine 1982; Gilliland and Bost 1993; Hadley and Smith 2003; Moriya and Hashimoto 2004).
Extensive postmortem EtOH production has been reported in cases of aviation fatalities, where bodies could be found several hours to several days after death (Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993). In these studies, 27% (Canfield, Kupiec, and Huffine 1993), 57% (Kuhlman et al. 1991), and 100% (Mayes et al. 1992) of the EtOH positive blood samples, taken from corpses of different stages of putrefaction, have been attributed to postmortem EtOH formation.
In the majority of cases, where postmortem EtOH formation was considered, very low to moderate concentrations (<0.7 g/L), as compared to the legal limits of 0.5 or 0.8 g/L, have been reported (O’Neal and Poklis 1996). However, maximal blood EtOH concentrations (measured by gas chromatography) produced postmortem, ranging from 1.2 g/L to 2.2 g/L, have been reported in some documented cases. These cases concerned decomposed bodies routinely autopsied (one case with BAC 2.2 g/L (Zumwalt, Bost, and Sunshine 1982), one case with BAC 1.2 g/L (Caplan and Levine 1990) and one with BAC 1.6 g/L (Gilliland and Bost 1993)), corpses from aircrafts accidents (two cases with BAC 1.8 g/L (Canfield, Kupiec, and Huffine 1993) and one case with BAC 1.9 g/L (Mayes et al. 1992)) and a case of a drowned person with 1.7 g/L EtOH measured in pleural cavity fluid (Nanikawa and Kotoku 1974). Moreover, in one single case, the extreme postmortem produced BAC of 3.0 g/L, has been reported (Canfield, Kupiec, and Huffine 1993).
Many other volatiles have been identified in specimens either collected from corpses found after aircraft accidents (Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; Johnson et al. 2004), or recovered from water (Nanikawa and Kotoku 1974; Hadley and Smith 2003; Moriya and Hashimoto 2004), or specimens from decomposed bodies routinely autopsied (Zumwalt, Bost, and Sunshine 1982; Caplan and Levine 1990; Gilliland and Bost 1993; Videira de Lima and Midio 1999). Their presence has been included among the criteria that specify the origin of the postmortem EtOH measured.
Volatiles have been considered to be produced postmortem, primarily due to microbial activity, and their identity and quantity depended on the type and number of microorganisms present in corpses at the different stages of putrefaction (Davis, Leffert, and Capt. Rantanen 1972; Corry 1978). The presence of various volatiles has been reported in postmortem specimens concerning chronic alcoholics (Pounder, Stevenson, and Taylor 1998) and diabetics (Pounder, Stevenson, and Taylor 1998; Smialek and Levine 1998), whose death was attributed to alcoholic ketoacidosis. Furthermore, various types of volatiles, in variable concentrations, have been determined in the blood of living subjects, either after the consumption of alcoholic beverages from alcoholics (Haffner et al. 1997a; Zuba et al. 2002) and from non-alcoholics (Haffner et al. 1997b), or as products of metabolic processes (Jones and Andersson 1995; Haffner et al. 1996; Zuba et al. 1998; Kalapos 2003).
The volatile compounds most commonly detected in postmortem specimens have been the following: methanol (Canfield, Kupiec, and Huffine 1993; O’Neal and Poklis 1996; Amick and Habben 1997), acetaldehyde (Davis, Leffert, and Capt. Rantanen 1972; Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; O’Neal and Poklis 1996; Amick and Habben 1997; Skopp 2004; Lewis et al. 2004), n-propanol (Davis, Leffert, and Capt. Rantanen 1972; Nanikawa et al. 1982; Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; Takayasu et al. 1995a; O’Neal and Poklis 1996; Amick and Habben 1997; Grellner and Iffland 1997; Smialek and Levine 1998; Videira de Lima and Midio 1999; Lewis et al. 2004; Skopp 2004; Moriya and Hashimoto 2004), isopropanol (Davis, Leffert, and Capt. Rantanen 1972; Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; O’Neal and Poklis 1996; Amick and Habben 1997; Grellner and Iffland 1997; Smialek and Levine 1998; Videira de Lima and Midio 1999; Lewis et al. 2004; Skopp 2004), acetone (Davis, Leffert, and Capt. Rantanen 1972; Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; O’Neal and Poklis 1996; Amick and Habben 1997; Smialek and Levine 1998; Lewis et al. 2004), propionic acid (Davis, Leffert, and Capt. Rantanen 1972), n-butanol (Kuhlman et al. 1991; Mayes et al. 1992; O’Neal and Poklis 1996; Grellner and Iffland 1997; Skopp 2004), isobutanol (Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; Skopp 2004; Lewis et al. 2004), sec-butanol (CH3CH(OH)CH2CH3) (Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993; Skopp 2004; Lewis et al. 2004), tert-butanol (CH3C(OH)(CH3)2) (Kuhlman et al. 1991; Mayes et al. 1992; Canfield, Kupiec, and Huffine 1993;) and isoamyl alcohol (Skopp 2004).
The volatile, n-propanol has been considered as the volatile most correlated to microbial postmortem EtOH production (Nanikawa et al. 1982; Takayasu et al. 1995a; Lewis et al. 2004; Moriya and Hashimoto 2004). The reported concentrations of n-propanol ranged from 0.001 to 0.076 mg/g (mean values) in the brain of drowned persons (Moriya and Hashimoto 2004). n-Propanol as a product of putrefaction in postmortem blood has been reported to range from 0.03 to 0.07 g/L (O’Neal and Poklis 1996; O’Neal et al. 1996). Furthermore, n-propanol was detected along with EtOH, in the intra-abdominal bloody fluid, urine and gastric content of living persons suffering from peritonitis (Moriya and Ishizu 1994), thus supporting the idea that n-propanol could monitor microbial activity.
The ratio of EtOH to n-propanol concentration has been determined in order to verify the existence of postmortem EtOH production and the quantitative relationship between the postmortem production of EtOH and n-propanol (Nanikawa and Kotoku 1974; Nanikawa et al. 1982; Feldy and Nielsen 1993; Moriya and Hashimoto 2004). However, the calculated ratios have shown significant variability. Although the aforementioned studies support their validity, it appears more possible that no valid estimation of antemortem EtOH concentration can be made from postmortem n-propanol and EtOH levels.
Regarding the other volatiles (minus n-propanol) detected in postmortem specimens, the quantitative pattern between them and EtOH still remains obscure (Lewis et al. 2004). Possible reasons for this could be the highly variable formation rates that they might have in corpses (Skopp 2004), as well as the fact that they might derive from other sources. Accordingly, there has been skepticism whether the presence of other volatiles could be a marker of postmortem EtOH formation, as it might be misleading (Canfield, Kupiec, and Huffine 1993; O’Neal and Poklis 1996; Johnson et al. 2004).
Further attempts to initiate other indicators of postmortem EtOH formation have led to the determination of other biomolecules as well. The 5-hydroxytryptophol to 5-hydroxyindol-eacetic acid ratio determined in urine (postmortem or not) has been well correlated with antemortem EtOH consumption (Helander, Beck, and Jones 1995; Johnson et al. 2004). The determination of phosphatidyl EtOH in postmortem blood (Hansson et al. 2001), ethyl glucuronide (Wurst et al. 2000), fatty acid ethyl esters (Refaai et al. 2002; Best and Laposata 2003), and fatty acid methyl esters (Emrich et al. 1997) in postmortem tissues have been other markers indicative of antemortem EtOH abuse, thus discriminating from postmortem EtOH production.
MECHANISM OF POSTMORTEM ETHANOL PRODUCTION
Rapidly after death, endogenous microorganisms (particularly from the intestine) invade the body fluids and tissues together with microbes that strike the corpse from the environment. The succession of microbes, endogenous and/or exogenous, finally defines the postmortem EtOH concentration measured (increasing or decreasing it compared to the EtOH levels at the time of death). After death, EtOH concentration could decrease through conversion to acetaldehyde (Smalldon and Brown 1973; Chang et al. 1984), or could increase via microbial alcoholic fermentation from carbohydrates (Corry 1978). Endotoxemia, the result of intestinal bacterial overgrowth after EtOH ingestion in the living, could cause a shift increase in EtOH metabolism (Yuki and Thurman 1980; Persson 1991; Nanji et al. 1993; Adachi et al. 1995). However, to our knowledge, it has not been reported whether or not endotoxemia could cause elevated EtOH levels antemortem that could affect the detected postmortem EtOH concentration. The major challenge, however, has been the potential of EtOH production postmortem and the elucidation of the relative mechanism.
In the attempt to clarify the mechanism of EtOH production in corpses, many experimental studies have been performed. It has been commonly accepted that EtOH could originate postmortem only from microbial activity (Davis Leffert, and Capt. Rantanen 1972; Corry 1978). Experiments performed by using postmortem tissues of conventional and germ-free mice indicated that during putrefaction, high levels of EtOH were produced in tissues of intact mice, whereas no EtOH was produced in the organs of germ-free mice (Davis, Leffert, and Capt. Rantanen 1972). The EtOH concentration varied directly with the postmortem interval under fixed conditions of temperature and humidity, exceeding 0.5 g/L in all tissue samples from the intact mice after 5 days of incubation at room temperature (Davis, Leffert, and Capt. Rantanen 1972). Other studies have shown that EtOH present at the time of death was rapidly degraded postmortem, during the first 2 days at 30°C, whereas neo-formation of EtOH occurred sharply during the same period (Takayasu et al. 1995a 1995b). In another study, in blood samples incubated with microbes at 37°C, for 1 day, the maximum recorded EtOH concentration was followed by a gradual decrease, most likely due to substrate depletion and subsequent EtOH breakdown to acetate and finally to CO2 (Amick and Habben 1997).
EtOH production has been recorded in human blood inoculated with saprogens (mixed population of bacteria and yeast), yeast (Nanikawa, Moriya, and Hashimoto 1988), Candida albicans (Chang and Kollman 1989), and Saccharomyces cerevisiae (Amick and Habben 1997). The concentration of EtOH produced was a function of temperature, time, concentration, and type of substrates, number of microbes, alcohol dehydrogenase activity, NAD+ concentration with or without antibiotics (Nanikawa, Moriya, and Hashimoto 1988; Chang and Kollman 1989; Amick and Habben 1997).
Experiments performed in urine, in order to study the potential of EtOH production, have revealed that this could be possible only if yeast and glucose have been present. The process has been time dependent and required more than 12 h to produce detectable EtOH amounts (Saady, Poklis, and Dalton 1993). When urine samples have been inoculated with C. albicans in the presence of sufficient amount of glucose, EtOH has been produced, after 7 days of incubation at 22°C, in concentration as high as 7.88 g/L (Helander, Beck, and Jones 1995). In vitro production of EtOH in urine has also been recorded by using possible urinary tract pathogens, such as certain yeast species (C. albicans, Candida parapsilosis, and other Candida spp. not albicans) and bacteria (Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis) (Sulkowski, Wu, and McCarter 1995). C. albicans has been proven to be the most effective EtOH producer, because it produced the maximum quantity of EtOH, ∼0.52 g/L. Furthermore, glucose has been proven to be the most sufficient substrate compared to other sugars tested (Sulkowski, Wu, and McCarter 1995).
Concerning other postmortem specimens, a recent study performed in muscle and kidney, collected at autopsy from victims of fatal aviation accidents, EtOH production has been reported. There had been suspicion of microbial contamination of the analyzed specimens. The reported production has been a function of time, temperature and preservation as expected (Lewis et al. 2004).
Consequently, it becomes evident that many factors could influence postmortem microbial production of EtOH, such as the number and nature of microbes present, the type and quantity of substrates available, the temperature and time, the humidity, the availability of air (oxygen), the pH values of postmortem specimens, and their preservation (O’Neal and Poklis 1996).
The following bacteria species have been reported as the major colonizers in corpses and, in parallel, the main EtOH producers: Clostridium perfringens and other Clostridium spp., enterobacteria (particularly, Escherichia coli and Proteus spp.), micrococcaeae (mainly Staphylococcus aureus), streptococci, and Bacillus spp. (Corry 1978). The greatest increases in EtOH levels have been usually attributed to enterococci or enteric bacilli (Corry 1978; Vu et al. 2000). Yeast capable of producing EtOH might also be found in decaying corpses but to a lesser extent (C. albicans and other Candida spp., Saccharomyces cerevisiae and Saccharomyces spp., etc). All microbes examined so far to produce EtOH have been found to possess the enzyme ADH (Corry 1978). Microbial EtOH production could be maintained during fermentation by yeast and moulds that use the Embden-Meyerhof-Parnay pathway for glucose degradation and by bacteria species, anaerobically, through the heterofermentative pathway (Gottschalk 1986). Overall reactions for each pathway could be expressed via the equations:
Experiments, performed on postmortem specimens in vitro, have revealed variations in EtOH content during storage, either due to oxidation and/or evaporation or due to microbial action (Chang and Kollman 1989; Nanikawa, Moriya, and Hashimoto 1988; Sulkowski, Wu, and McCarter 1995; Helander, Beck, and Jones 1995; Amick and Habben 1997; Garriott 2003). Collection of specimens should be performed as early as possible after death in order to minimize the possibility of microbial growth at the corpse. The proper handling of the collected samples should include the immediate addition of the proper amount of a preservative, and finally the storage of the samples as close as possible to 0°C (Sulkowski, Wu, and McCarter 1995; Skopp 2004). The most commonly used preservatives have been fluoride salts although, under certain conditions, they have been found inadequate (Chang and Kollman 1989; Helander, Beck, and Jones 1995). Stability of samples during storage has been considered extremely important, as reanalysis may be requested after weeks or months after sample collection (Skopp 2004).
ATYPICAL DISTRIBUTION OF ETHANOL IN POSTMORTEM SPECIMENS
The phenomena during putrefaction might cause discrepancies between EtOH concentration in autopsy specimens and the actual EtOH concentration at the time of death. For the postmortem EtOH determination, blood is the sample of choice, as it reflects best the effect of EtOH on the brain, because both compartments are in equilibrium, and furthermore corresponds best to the antemortem situation of the deseased. However, practical issues have been (i) the attainment of the proper blood sample postmortem, or the best alternative fluid or tissue, if blood has not been available; and (ii) the determination of the EtOH origin (antemortem or postmortem). By performing multispecimen analysis, the atypical distribution of EtOH throughout the different compartments of the corpse could indicate postmortem EtOH generation (Corry 1978; O’Neal and Poklis 1996). The fluid/blood or tissue/blood ratios described in the literature could be used either to estimate the BAC at the time of death, or as potential indicators of postmortem EtOH production (Budd 1982).
Postmortem redistribution of EtOH from solid organs into blood could be a causative reason for variations in EtOH concentration between different specimens. Redistribution might be facilitated by postmortem diffusion of EtOH from the stomach (Plueckhahn, Path, and Ballard 1967), traumatic injuries (Winek, Winek, and Wahba 1995), aspirated vomits (Pounder and Yonemitsu 1991; Pelissier-Alicot et al. 2004), and extensive clotting of blood (Shepherd, Lake, and Kamps 1992), as well as mechanical factors, such as, the movement of blood through the vasculature (Corry 1978) and postural changes during postmortem inspection and carriage of the body (Skopp 2004).
However, EtOH concentration in different parts of the body could vary in cases where death has occurred during the absorptive phase (Briglia, Bidanset, and Dal Cortivo 1992; Sylvester et al. 1998). Differences have been reported between arterial and venous blood or central and peripheral vessels (Martin et al. 1984; Jones, Jönsson, and Joryeldt 1989; Sylvester et al. 1998; Levine and Smialek 2000). Pooled blood taken at autopsy from the pericardial sac or pleural cavities would be most liable to be contaminated by diffusion from the stomach (Plueckhahn, Path, and Ballard 1967). On the other hand, blood samples taken from the intact heart chambers or the femoral blood vessels have shown no contamination (Plueckhahn 1967; Plueckhahn and Ballard 1968).
Urine has been an alternative specimen widely used for postmortem EtOH determination. Many studies have reported an average urine/blood ratio ranging from 1.01 to 1.5, whereas absolute ratio values reported have shown a greater variation, ranging from 0.21 to 3.67 (Jetter 1938; Bavis 1940; Ellenbrook and Van Gaasbeek 1943; Coldwell and Smith 1959; Heise 1967; Kaye and Cardona 1969; Christopoulos, Kirch, and Gearrien 1973; Backer, Pisano, and Sopher 1980; Budd 1982; Stone and Rooney 1984; Levine and Smialek 2000; Jones and Holmgren 2003). The observed discrepancies suggest that urine alcohol concentration would result in uncertainty in estimating the BAC. Nevertheless, the urine/blood distribution ratios could be informative to some extent on whether the absorption or postabsorptive stage of EtOH kinetics had been reached, or indicative on whether postmortem production has taken place (Jones 2000).
Brain has also shown great variability in postmortem EtOH concentration depending on the part of the brain measured. Absolute brain/blood EtOH concentration ratios ranged from 0.31 to 8.00, which has not been surprising in view of the heterologous make-up of the brain (Gettler and Freireich 1931; Ellenbrook and Van Gaasbeek 1943; Hine 1951; Herold and Prokop 1960; Christopoulos, Kirch, and Gearrien 1973; Backer, Pisano, and Sopher 1980; Budd 1982). Cerebrospinal fluid (CSF), on the other hand, could be a close indicator of the EtOH concentration in the central nervous system (CNS). Average CSF/blood EtOH concentration ratios have been reported to range from 0.9 to 1.18 depending on the time after drinking and the phase of elimination (Gettler and Freireich 1931; Harger, Hulpieu and Lamb 1937; Christopoulos, Kirch, and Gearrien 1973; Backer, Pisano, and Sopher 1980; Budd 1982).
Over the years, vitreous humor (VH) has become a popular alternative specimen for EtOH analysis in postmortem cases (Felby, Pharm, and Olsen 1969; Coe and Sherman 1970; Backer, Pisano, and Sopher 1980; Caplan and Levine 1990; Briglia, Bidanset, and Dal Cortivo 1992; Canfield, Kupiec, and Huffine 1993; Sylvester et al. 1998; Videira de Lima and Midio 1999; Johnson et al. 2004). VH has been a peripheral body compartment that has a delay in alcohol uptake and removal in comparison to blood. Moreover, it has been easy to be obtained during autopsy and has been relatively stable postmortem. Relative studies have shown that the average VH/blood ratio ranged from 0.91 to 1.34 (Felby, Pharm, and Olsen 1969; Coe and Sherman 1970; Scott, Root, and Sanborn 1974; Backer, Pisano, and Sopher 1980; Budd 1982; Stone and Rooney 1984; Sylvester et al. 1998).
Few bacteria have been detected in the VH even in moderately decomposed bodies, suggesting that postmortem production of EtOH in this specimen would be negligible (Zumwalt, Bost, and Sunshine 1982). Therefore, a negative EtOH result in VH could mean that no EtOH has been present at the time of death. However, other studies have questioned the reliability of the VH EtOH concentration when correlated to BAC (Chao and Lo 1993; Jones and Holmegren 2001). Wide variations in VH/blood ratios have been reported when the deceased was in the early absorption phase, ranging from 0.27 to 1.40, whereas the spread was 0.78 to 3.13 for the other pharmakokinetic phases (Chao and Lo 1993). In conclusion, these reports call for caution when trying to estimate BAC indirectly using the EtOH concentration in VH.
Synovial fluid (SF) has been correlated sufficiently with the BAC. Relative studies have shown absolute SF/blood EtOH concentration ratios ranging from 1.06 to 1.67. The ratios have been affected by the metabolic phase of EtOH at the time of death (Winek et al. 1993; Ohshima et al. 1997).
Bile has been also used for estimating the BAC, showing an average bile/blood EtOH concentration ratio of 0.99 (range 0.48 to 2.04) (Backer, Pisano, and Sopher 1980). Regarding central body compartments, data in the literature have been restricted. Liver/blood absolute EtOH concentration ratios have ranged from 0.47 to 0.85 (Christopoulos, Kirch, and Gearrien 1973; Jenkins, Levine, and Smialek 1995), whereas kidney/blood EtOH concentration ratios have ranged from 0.57 to 0.76 (Christopoulos, Kirch, and Gearrien 1973).
Average ratios of bone marrow/blood EtOH concentrations have ranged from 0.34 to 0.79 (Isokoski, Alha, and Laiho 1968; Winek and Jones 1980; Winek and Esposito 1981). However, great individual variations in the ratios have been observed, probably, due to differences in the lipid content of the bone marrow of individuals, making the determination of the exact BAC from a given bone marrow alcohol concentration insecure (Isokoski, Alha, and Laiho 1968; Winek and Esposito 1981). Finally, skeletal muscle has been considered a good alternative specimen to blood even if putrefaction has proceeded, because reported muscle/blood EtOH concentration ratios have been ranging from 0.89 to 0.91 (Felby, Pharm, and Olsen 1969; Krauland, Klugn and Toffel 1979; Nanikawa et al. 1982). Tissues to blood EtOH ratios in postmortem cases and relative references reported in the literature are presented in Table 1.
CONCLUDING REMARKS
It should become evident that the evaluation of the postmortem BAC could be a complicated, multifunctional procedure. Much debate has arisen on whether and how the origin of postmortem EtOH could be determined. Main subjects of this debate have been the correlation of n-propanol with the postmortem generated EtOH and the evaluation of the EtOH results received after multispecimen analysis has been performed. However, up to now, although a documented qualitative relationship has been observed between n-propanol and the postmortem EtOH production, still the quantitative pattern remains obscure.
The identification of the EtOH-producing microbes in postmortem specimen has been proposed in order to identify the bacteria that produce EtOH, though its utilization has not been sufficiently established. In this direction, the performance of microbiological cultures has been suggested (Cohle 1994). However, postmortem cultures have been considered too complicated to proceed and could lead to false positive results, e.g., due to contamination (Tsokos and Püschel 2001). Furthermore, it has been suggested that the absence of bacteria capable of producing EtOH would prove that the postmortem EtOH found was from ingestion (Cohle 1994). Recently, the development of an assay identifying commonly encountered EtOH-producing microorganisms, by the polymerase chain reaction, seems promising for the determination of the origin of postmortem EtOH in forensic samples (Kupfer et al. 1999; Vu et al. 2000).
Meanwhile, feasible accuracy in interpreting the results of postmortem EtOH analysis could be achieved to some extent, provided that proper sample handling (collection, preparation, and storage), determination of other volatiles, and multispecimen analysis are performed.