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Department of Forensic Medicine & Toxicology, Medical School, University of Ioannina, 45110 Ioaninna, Greece
Department of Microbiology, Medical School, University of Ioannina, 45110 Ioaninna, Greece
c
Department of Medical Physics, Medical School, University of Ioannina, 45110 Ioaninna, Greece
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 30 September 2010
Received in revised form 3 March 2011
Accepted 7 March 2011
Available online 5 April 2011
Ethanol can be produced from all the postmortem available substrates, though with higher rates and
yields from carbohydrates, during the early stages of putrefaction. The so-called higher alcohols (1propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-2-butanol) and 1-butanol could be produced,
from all the available postmortem substrates. However, a quantitative relationship between the
produced ethanol and the potentially produced other alcohols is still missing.
The objective of this study was the development of a simple, mathematical model which could be able
to approximate the microbial produced ethanol in correlation with other produced alcohols. The selected
bacterial species included two Gram+ spore-forming anaerobic bacteria and two (one Gram+ one Gram-)
aerobic/facultative anaerobic bacteria, all being common commensals of the digestive tract and common
colonizers of the corpse. The selected bacterial strains, Escherichia coli, Clostridium perfrigens, Clostridium
sporogenes and Enterococcus faecalis, were cultured separately at 25 8C, for 30 days, under controlled
anaerobic conditions. The produced ethanol and the previously referred alcohols were determined in the
culture medium in 24 h intervals. Using partial least squares (PLS) regression, the estimation of the
relevance score for the available descriptors established the statistical model to assess the ethanol
concentration produced by each studied microbe. E. coli, C. perfrigens, and C. sporogenes produced
different patterns of ethanol and other alcohols, while E. faecalis produced negligible amounts of ethanol
and higher alcohols. In constructing the mathematical models to predict the produced ethanol, 1propanol, 1-butanol, and isobutanol were signicant for C. perfrigens and C. sporogenes, while 1-butanol,
1-propanol, and methyl-butanol were signicant for E. coli. The applicability of these models was tested
in microbial, anaerobic cultures of normal human blood and plasma at 25 8C. The results indicate that
factors such as the type of microbe species, the glucose content and the medium composition apparently
affect the procedure of microbial ethanol, and other alcohols production. However, the models can be
applied with acceptable accuracy and they show potential for application in real postmortem cases.
2011 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Post-mortem blood
Ethanol
Microbial ethanol production
Multivariate statistics
Fermentation
Volatiles
Higher alcohols
Biomarker(s)
1. Introduction
The microbial formation of ethanol is a well recognized
complication in the proper interpretation of postmortem ethanol
analysis results [13]. Many microorganisms potentially present in
a dead body are capable of ethanol production. At least 58 species
of bacteria, 17 species of yeasts and 24 species of molds can
produce ethanol as well as other volatiles through various
biosynthetic pathways [1,4,5].
0379-0738/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2011.03.003
190
anaer incubation system (45 534, BioMerieux) was used to obtain anaerobic
conditions. The inoculated tubes were incubated under anaerobic conditions at
25 8C and 4 8C for 30 days.
2.1.2. Bacterial enumeration
The bacterial counts were performed by the pour plate technique. Decimal
dilutions were performed with the use of tubes containing 9 mL of Maximum
Recovery Dilluent (02-510, Scharlau Chemie, Spain). From each dilution an
inoculum of 1 mL was added into two sterile petri dishes and 15 mL of melted
Plate Count Agar (PCA) (CM0325, Oxoid) at a temperature of 45 8C was poured. After
solidication, 5 mL of plate count agar were overlaid. The plates were inverted and
incubated for 48 h at 30 8C under anaerobic (C. perfrigens and C. sporogenes) or
aerobic (E. coli and E. faecalis) conditions. Plates with 30300 colonies were selected
and colonies were counted under a colony counter (Stuart SC6, Barloworld Scientic
Ltd., UK). The average from the two plates from the appropriate dilution was
calculated. The result was derived from the multiplication of the average colony
count with the dilution factor and was expressed as cfu/mL.
2.1.3. Microbial cultures in human blood and plasma products
Human whole blood from healthy individuals was collected in sterilized blood
tubes containing EDTA (Vacuette EDTAK3, 6 mL), and was centrifuged at 4000 rpm
for 5 min. The supernatant (plasma) was collected and 1 mL portions were placed
into 16 sterilized blood tubes (Vacuette EDTAK3, 3 mL). Each tube was inoculated
with the appropriate volume of bacterial broth of either E. coli, or C. perfrigens or C.
sporogenes in order to obtain an initial concentration of 104 cells/mL (equal to the
culture concentrations used to build the relevant microbial model). Samples were
incubated under anaerobic conditions by using a sterile parane oil overlay and the
GENbag anaer incubation system. At days 0, 1, 2, 3, 5, 7, 11 and 15 two tubes were
removed from the incubation system and alcohols concentrations were determined
by HS-GCFID. Also, two other series of 16 tubes each, containing 1 mL of plasma,
were spiked with the appropriate volume of a concentrated glucose solution (sterile
dextrose 5% (w/v), pyrogen free solution for intravenous infusion, DEMO Ltd.,
Greece) so as to obtain 200 mg/dL nal concentration. The initial glucose
concentration of the spiked plasma samples was chosen to be 200 mg/dL in order
to be the same with the glucose content of the BHI culture medium. Next the
samples were inoculated with E. coli or C. sporogenes and were tested for their
alcohol production capacity as previously described. The aforementioned procedure was also applied to two series of 16 tubes each, containing whole human blood
with EDTA, and the other whole human blood with citric ions, inoculated with C.
perfrigens. All the aforementioned tests in human whole blood and plasma products
were performed in parallel.
2.2. Volatiles determination
2.2.1. Chemicals and solutions
All chemicals were purchased in the highest possible purity and used without
any further purication. Ammonium sulfate, ethanol (99.7%), 1-propanol, 1butanol, isobutanol (methyl-1-propanol), 2-methyl-1-butanol, 3-methyl-2-butanol, and acetonitrile were purchased from Merck in the higher available purity
(Darmstadt, Germany). All aqueous solutions were prepared using double distilled
(DD) water, which was obtained using an Aquatron A400D (Bibby Sterilin,
Staffordshine, UK).
Stock aqueous standard solutions were prepared in concentration 4.00% (w/v) for
ethanol; 0.25% (w/v) for 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl2-butanol; and 0.40% (w/v) for 1-propanol. Acetonitrile was used as internal
standard for the determination of volatiles in aqueous solution of 100 mg/dL. The
above solutions were stored at 4 8C for up to six months. Working solutions were
prepared on a daily basis by mixing the appropriate volumes of the corresponding
stock solutions of each analyte and DD-water. Blood quality control (QC) samples
were prepared from stock solutions in three different concentrations for each
analyte.
Calibration solutions separately for each analyte, were prepared daily, by spiking
aliquots of blank human blood (whole human blood that was certied to be volatile
organic compound free) with the appropriate volume of working solution.
2.2.2. HS-GCFID procedure
GC analyses were performed on a Shimadzu GC 17A gas chromatograph
equipped with a SUPELCOWAXTM10 fused silica capillary column
(30 m 0.25 mm, lm thickness 0.25 mm) and with a FID. The GC was tted with
a Shimadzu AOC-5000 headspace-GC automated sample pretreatment and
injection system. The analyses were performed by slight modications to
previously published methods [1719]. The temperature of the injection port,
the column and the FID was 115 8C, 60 8C and 260 8C, respectively. The carrier gas
was helium with a ow rate of 0.7 mL/min with a constant pressure of 65 kPa. The
injection inlet was set in a split mode with a split ratio of 10:1
The samples were incubated at 50 8C for 8.0 min [18] prior to injection with an
agitation speed of 500 rpm. A 2.5 mL gas tight syringe was used heated at 105 8C. A
head space aliquot of 500 mL was sampled for analysis with a feel speed of 250 mL/s
and a pull up delay 500 ms. The injection speed was 1000 mL/s at a needle
191
Table 1
Validation of the HS-GCFID volatiles determination method.
Volatile
LOD (mg/dL)
LOQ (mg/dL)
R2
Ethanol
1-Propanol
Isobutanol
1-Butanol
3-Methyl-2-butanol
3-Methyl-1-butanol
0.01
0.02
0.005
0.005
0.01
0.01
0.03
0.04
0.015
0.01
0.025
0.025
0.03400
0.0432.0
0.020.08
0.010.08
0.040.32
0.040.32
0.999
0.999
0.989
0.995
0.997
0.999
penetration depth of 20 mm. After injection syringe was ushed with helium for
1.5 min. Acquisition time was 20 min. A standard GCFID chromatogram is
provided as complimentary data (Fig. 1C).
In 10 mL HS vials containing 0.50 g ammonium sulfate were added 500 mL of the
calibration solution or of the culture medium or of blood culture and 500 mL of the
internal standard solution. The vials were then sealed with metal crimp caps tted
with silicon septa and were put to the HS auto sampler for analysis. Ammonium
sulfate was added to increase the ionic strength of the solution. Calibration curves
were constructed for each analyte in six concentration levels within the relevant
working concentration range (Table 1).
1,E+08
1,E+06
1,E+04
1,E+14
1,E+12
1,E+10
1,E+02
10
15
20
25
30
Days
Clostridium sporogenes
Enterococcus faecalis
Clostridium perfringens
Escherichia coli
Fig. 1. Growth curves for each microbe under study showing the number of bacteria
during the incubation period of 30 days at 25 8C under anaerobic conditions.
192
Table 2
Volatiles concentration during the fermentation period of C. perfrigens in BHI culture medium at 25 8C.
Days
Ethanol (g/L)
1-Propanol (mg/dL)
1-Butanol (mg/dL)
Isobutanol (mg/dL)
Methyl-butanol (mg/dL)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0.01
0.01
0.02
0.06
0.05
0.08
0.07
0.11
0.13
0.09
0.12
0.13
0.14
0.12
0.16
0.14
0.13
0.14
0.13
0.14
0.14
0.14
0.15
0.14
0.15
0.14
0.15
0.15
0.15
0.16
0.15
0
0.02
0.04
0.08
0.15
0.2
0.26
0.31
0.32
0.39
0.40
0.46
0.50
0.51
0.52
0.48
0.42
0.55
0.51
0.52
0.53
0.51
0.55
0.63
0.65
0.55
0.64
0.60
0.68
0.69
0.70
0
0.04
0.08
0.16
0.44
0.57
0.78
0.72
0.99
0.72
0.86
1.08
1.35
1.02
1.51
1.18
1.21
1.26
1.23
1.27
1.31
1.25
1.30
1.20
1.16
0.98
1.52
1.47
1.32
1.90
1.33
0
0
0.01
0.01
0.03
0.04
0.05
0.07
0.08
0.07
0.07
0.10
0.11
0.10
0.11
0.11
0.11
0.11
0.10
0.09
0.10
0.11
0.11
0.10
0.13
0.13
0.14
0.13
0.12
0.12
0.15
0
0
0.02
0
0.03
0.03
0.05
0.04
0.06
0.04
0.06
0.07
0.07
0.09
0.09
0.07
0.05
0.04
0.07
0.07
0.06
0.06
0.07
0.08
0.07
0.07
0.08
0.09
0.08
0.10
0.08
Table 3
Volatiles concentration during the fermentation period of C. sporogenes in BHI culture medium at 25 8C.
Days
Ethanol (g/L)
1-Propanol (mg/dL)
1-Butanol (mg/dL)
Isobutanol (mg/dL)
Methyl-butanol (mg/dL)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0.00
0.10
0.73
0.76
0.85
0.87
0.85
0.84
0.78
0.71
0.79
0.81
0.84
0.75
0.77
0.81
0.78
0.81
0.79
0.76
0.77
0.78
0.76
0.66
0.70
0.70
0.73
0.76
0.73
0.67
0.72
0.04
2.42
2.67
4.59
5.07
5.73
5.67
5.82
6.20
5.06
6.55
6.59
6.68
6.67
7.10
8.16
7.50
8.12
7.81
7.62
7.67
8.55
8.21
8.20
8.47
8.64
8.69
8.88
9.36
10.5
10.2
0
0.43
0.70
2.07
2.31
3.56
3.25
3.72
3.81
3.22
4.77
4.81
5.19
6.35
6.45
7.59
6.98
8.85
8.25
8.74
8.00
9.20
8.87
9.35
9.70
10.2
10.5
9.57
11.1
10.2
11.9
0.00
0.70
0.72
1.53
1.76
2.58
2.30
2.56
2.40
2.24
3.03
3.12
3.18
3.36
3.51
4.36
4.57
5.07
4.33
3.99
4.10
4.43
4.20
4.35
4.67
5.38
4.82
4.75
4.90
5.50
6.10
0
0.07
0.10
0.18
0.22
0.24
0.23
0.25
0.21
0.20
0.34
0.32
0.32
0.39
0.38
0.44
0.36
0.57
0.52
0.53
0.52
0.51
0.55
0.52
0.55
0.55
0.55
0.56
0.55
0.55
0.57
193
Table 4
Volatiles concentration during the fermentation period of E. coli in BHI culture medium at 25 8C.
Days
Ethanol (g/L)
1-Propanol (mg/dL)
Isobutanol (mg/dL)
1-Butanol (mg/dL)
Methyl-butanol (mg/dL)
0
0.5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0
0.18
0.36
0.42
0.43
0.44
0.46
0.45
0.44
0.46
0.48
0.47
0.47
0.46
0.50
0.45
0.49
0.49
0.52
0.46
0.46
0.50
0.51
0.56
0.46
0.53
0.49
0.53
0.50
0.52
0.49
0.50
0
0.06
0.09
0.11
0.18
0.24
0.34
0.37
0.38
0.43
0.46
0.45
0.51
0.54
0.62
0.68
0.66
0.72
0.66
0.69
0.76
0.70
0.79
0.81
1.02
0.92
0.92
0.95
0.91
0.86
0.86
0.88
0
0.01
0.02
0.02
0.02
0.02
0.03
0.03
0.03
0.03
0.02
0.03
0.02
0.03
0.03
0.02
0.02
0.02
0.03
0.02
0.03
0.04
0.03
0.02
0.08
0.07
0.07
0.05
0.02
0.07
0.09
0.10
0
0.01
0.04
0.07
0.08
0.09
0.09
0.09
0.10
0.11
0.09
0.10
0.10
0.12
0.09
0.11
0.10
0.12
0.10
0.10
0.10
0.10
0.13
0.16
0.16
0.13
0.11
0.11
0.10
0.10
0.10
0.08
0
0.07
0.06
0.07
0.09
0.09
0.08
0.09
0.11
0.09
0.09
0.11
0.09
0.11
0.08
0.12
0.08
0.12
0.11
0.10
0.10
0.08
0.08
0.08
0.09
0.10
0.09
0.09
0.09
0.09
0.09
0.09
Fig. 2. (A) Four descriptors (4D) model and; (B) one descriptor (1D) model for ethanol production by C. perfrigens.
194
(1)
(2)
(3)
Isobutanol 0:09
Ethanol 0:15 1Propanol 0:14
(4)
(5)
Methyl-butanol 0:15
Ethanol 2:25 1Butanol 0:98
(6)
Fig. 3. (A) Four descriptors model and; (B) two descriptor model for ethanol production by C. sporogenes.
195
Fig. 4. (A) Four descriptors model and; (B) two descriptor model for ethanol production by E. coli.
196
Table 5
Results for the calculated ethanol concentrations after applying each model (Eqs. (1)(6)) in postmortem cases with the standard error (E%) for each case.
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Manner of death
Violent
Natural
Natural
Violent
Natural
Natural
Undetermined
Violent
Undetermined
Undetermined
Undetermined
Natural
Natural
Violent
Natural
Undetermined
Natural
Violent
Undetermined
Undetermined
Undetermined
Undetermined
Violent
Undetermined
Undetermined
Undetermined
Undetermined
Violent
Undetermined
Undetermined
Natural
Violent
Undetermined
Undetermined
Undetermined
Undetermined
Undetermined
Natural
Undetermined
Undetermined
0.10
0.16
0.16
0.19
0.20
0.21
0.21
0.22
0.25
0.29
0.35
0.39
0.42
0.42
0.42
0.47
0.48
0.52
0.58
0.59
0.60
0.62
0.62
0.63
0.64
0.68
0.68
0.69
0.69
0.71
0.72
0.73
0.75
0.81
0.87
0.91
0.94
0.98
1.08
2.58
C.
perfrigens,
Eq. (1)
C. perfrigens,
Eq. (2)
C.
sporogenes,
Eq. (3)
C. sporogenes,
Eq. (4)
Ethanol
(g/L)
E%
Ethanol
(g/L)
E%
Ethanol
(g/L)
E%
Ethanol
(g/L)
E%
Ethanol
(g/L)
E%
Ethanol
(g/L)
E%
0.13
0.15
0.09
0.22
0.06
0.30
0.20
0.27
0.14
0.29
0.36
0.33
0.44
0.54
0.40
0.84
0.72
0.65
0.85
0.29
0.63
1.12
1.02
1.01
0.43
0.43
0.41
0.42
0.25
0.29
0.46
0.67
1.00
0.30
0.95
0.92
1.13
0.43
0.29
1.08
26
8
43
18
70
45
6
23
43
1
2
16
5
28
6
79
50
26
47
52
6
80
64
60
34
36
39
39
64
60
35
8
34
64
9
2
20
56
73
58
0.05
0.05
0.07
0.00
0.09
0.05
0.03
0.03
0.04
0.04
0.06
0.03
0.06
0.19
0.11
0.32
0.29
0.25
0.28
0.04
0.23
0.20
0.45
0.42
0.05
0.11
0.11
0.08
0.02
0.04
0.14
0.26
0.46
0.03
0.42
0.42
0.33
0.10
0.04
0.49
151
129
145
99
146
78
116
87
117
85
82
91
85
56
75
32
39
51
52
93
61
67
27
33
91
84
84
89
98
94
81
64
38
96
52
54
65
90
96
81
0.24
0.71
0.20
0.32
0.18
0.39
0.29
0.36
0.25
0.38
0.43
0.61
0.49
0.59
0.66
0.84
0.75
0.69
0.83
0.37
0.67
0.98
1.01
0.99
1.22
0.50
1.11
0.48
0.34
0.37
1.03
0.71
1.00
0.74
1.56
0.93
1.72
0.49
0.37
1.06
128
344
26
69
12
85
38
64
1
32
23
57
16
41
55
79
57
34
44
37
13
59
63
58
91
27
63
31
51
47
43
3
33
9
80
3
83
50
65
59
0.15
0.80
0.15
0.15
0.15
0.15
0.15
0.15
0.15
0.15
0.15
0.45
0.15
0.15
0.33
0.15
0.15
0.15
0.15
0.15
0.15
0.15
0.15
0.15
0.89
0.15
1.03
0.15
0.15
0.15
0.82
0.15
0.15
0.63
0.99
0.15
0.94
0.15
0.15
0.15
44
399
8
20
25
29
29
31
41
48
57
16
64
64
21
68
69
71
74
75
75
76
76
76
40
78
51
78
78
79
13
79
80
22
13
83
0
85
86
94
0.24
0.24
0.18
0.43
0.11
0.58
0.30
0.52
0.28
0.57
0.63
0.52
0.61
1.05
0.85
1.49
1.42
1.28
1.33
0.55
1.21
0.98
1.95
1.85
0.87
0.80
0.76
0.67
0.48
0.56
0.92
1.30
2.00
0.56
1.84
1.84
1.61
0.74
0.56
2.08
131
53
8
128
45
176
41
139
11
97
81
34
45
151
102
217
196
149
129
6
104
58
214
193
35
17
12
2
31
21
28
78
166
32
112
103
71
24
49
19
0.27
0.29
0.21
0.44
0.15
0.59
0.31
0.53
0.31
0.57
0.63
0.53
0.59
1.03
0.78
1.42
1.37
1.25
1.25
0.56
1.18
0.83
1.86
1.76
0.56
0.78
0.78
0.66
0.49
0.57
0.88
1.26
1.91
0.54
1.76
1.76
1.32
0.73
0.56
1.99
156
82
28
136
27
179
48
144
21
100
80
36
39
145
84
202
186
142
115
5
97
33
200
179
13
15
15
5
30
20
23
73
155
33
103
95
40
26
48
23
The original ethanol concentrations measured for each case are also provided along with the manner of death.
of human plasma (IIIA) the model loses its power probably due to
the fact that ethanol and 1-propanol values are strongly correlated
exhibiting a steady proportion of 1:10.
Our results indicate that the constructed models have the
potential to be applied when different culture media are used,
under controlled experimental conditions. Factors such as the type
of microbial species, the glucose content and the medium
composition apparently affect the procedure of microbial ethanol,
1-butanol and higher alcohols production.
3.6. Use of the models in real postmortem cases
We retrospectively looked our chromatograms archives of the
last six years and we selected 40 chromatograms corresponding to
postmortem cases, having presence of other alcohols during the
original forensic ethanol analysis. The classication of these cases
as resulted from the retrospective review of the forensic pathologists
reports was: 10 cases of natural deaths; 8 cases of violent deaths;
and 22 cases of undetermined cause of death. Furthermore, 39 cases
had marked putrefactive phenomena at autopsy while the other one
had extensive traumatic lesions (Case no 4) making easier the
invasion of microbes. We calculated for these cases the concentrations of higher alcohols and 1-butanol. In Table 5 the original ethanol
concentrations measured for each case and the calculated ethanol
concentrations after applying each model (Eqs. (1)(6)) are provided
along with the standard error for each case.
The models corresponding to C. perfrigens estimated the
microbial produced ethanol with a standard error <40% for 27
out of the 40 cases (68%), 26 of them with marked putrefaction (96%).
Additionally, 21 of these cases (78%) had ethanol lower than 0.7 g/L.
The models corresponding to E. coli estimated the microbial
produced ethanol with a standard error <40% for 25 out of the 40
cases (63%), 24 of them with marked putrefaction (96%). Moreover,
18 of these cases (72%) had ethanol lower than 0.7 g/L.
The models corresponding to C. sporogenes estimated the
microbial produced ethanol with an error <40% for 18 out of the 40
cases (45%) all presented with putrefaction at autopsy, while 12 of
these cases (67%) had ethanol lower than 0.7 g/L.
It is worth mentioning that 28 out of the 29 cases (97%) having
original ethanol concentration lower than 0.7 g/L succeeded a
standard error <40% in predicting the microbially produced
ethanol by at least one of the models.
The cases were selected due to the co-detection of signicant
amounts of volatiles along with ethanol during the original
ethanol analysis a factor making the relevant cases suspicious
for microbial ethanol production, as it was suggested [24]. Then
it was proved that the majority of the selected cases had marked
putrefaction at autopsy another aggravating factor for
microbial activity and ethanol neo-formation [14,15,24,25].
Our results have shown that the models could effectively be
applied in postmortem cases especially when marked putrefaction is present. In addition, the models of C. perfrigens show
better applicability than these of E. coli and than those by C.
sporogenes. Yet, the models are applied more satisfactory when
ethanol concentrations are lower than 0.7 g/L. It is generally
accepted that ethanol concentrations lower than 0.7 g/L is more
possible to be of microbial origin for the majority of cases with
neo-formation of ethanol [2,3].
However, it has to be underlined that so far the study of real
postmortem cases meets serious limitations. As the ethanol
concentrations increase, it is possible part of the detected ethanol
to be of ante mortem origin making the proper interpretation of
postmortem ethanol analysis difcult. In such a case it is extremely
challenging and premature to suggest that one model is more
suitable than another. On the other hand, other microbes might
generate different alcohols patterns resulting to different models.
197
198
[4] J.E.L. Corry, Possible sources of ethanol ante- and postmortem: its relation to the
biochemistry and microbiology of decomposition, J. Appl. Bacteriol. 44 (1978) 156.
[5] V.A. Boumba, K. Ziavrou, T. Vougiouklakis, Biochemical pathways generating
post-mortem volatile compounds co-detected during forensic ethanol analysis,
Forensic Sci. Int. 174 (2008) 133151.
[6] D. Yamima, H. Motani, K. Kamei, Y. Sato, M. Hayakawa, H. Iwase, Ethanol
production by Candida albicans in post-mortem human blood samples: effect
of blood glucose level and dilution, Forensic Sci. Int. 164 (2006) 116121.
[7] J. Chang, S.E. Kollman, The effect of temperature on the formation of ethanol by
Candida albicans in blood, J. Forensic Sci. 34 (1989) 105109.
[8] J.J. Saady, A. Poklis, H.P. Dalton, Production of urinary ethanol after sample
collection, J. Forensic Sci. 38 (1993) 14671471.
[9] H.A. Sulkowski, A.H. Wu, Y.S. McCarter, In-vitro production of ethanol in urine by
fermentation, J. Forensic Sci. 40 (1995) 990993.
[10] B.M. Appenzeller, M. Schuman, R. Wennig, Was a child poisoned by ethanol?
Discrimination between ante-mortem consumption and post-mortem formation,
Int. J. Legal Med. 122 (2008) 429434.
[11] A.C. Gruszecki, C.A. Robinson, S. Kloda, R.M. Brissie, High urine ethanol and
negative blood and vitreous ethanol in a diabetic woman: a case report, retrospective case survey, and review of the literature, Am. J. Forensic Med. Pathol. 26
(2005) 9698.
[12] G.D. Amick, K.H. Habben, Inhibition of ethanol production by Saccharomyces
cerevisiae in human blood by sodium uoride, J. Forensic Sci. 42 (1997) 690692.
[13] R. Nanikawa, K. Ameno, Y. Hashimoto, K. Hamada, Medicolegal studies on alcohol
detected in dead bodies alcohol levels in skeletal muscle, Forensic Sci. Int. 20
(1982) 133140.
[14] S. Felby, E. Nielsen, Congener production in blood samples during preparation and
storage, Blutalkohol 32 (1995) 558.