San Diego DUI lawyer article

Joyce Chang, x Ph.D. and S. Elliot Kollman, 2 B.A.
The Effect of Temperature on the Formation of
Ethanol by Candida Albicans in Blood
REFERENCE: Chang, J. and Kollman, S. E., "The Effect of Temperature on the Formation of
Ethanol by Candida Albicans in Blood," Journal of Forensic Sciences, JFSCA, Vol. 34, No. 1,
Jan. 1989, pp. 105-109.
ABSTRACT: The effect of temperature on microbial fermentation in blood was studied. Specimens
of human blood from a blood bank were inoculated with Candida albicans, an organism
capable of causing fermentation. A preservative was added to a portion of the inoculated specimens.
These inoculated specimens, as well as uninoculated blood, were stored under various
temperature conditions. Production of ethyl alcohol was monitored over a period of six months.
Fermentation was found to be highly temperature dependent, with refrigeration proving to be
most effective at inhibiting ethanol formation.
KEYWORDS: forensic science, blood, Candida albicans, alcohol, temperature, ethanol
It has been shown that several microorganisms occasionally found in blood specimens are
capable of producing ethyl alcohol [1,2]. Although Blume and Lakatua [1] found that sodium
fluoride effectively inhibited alcohol production from a variety of microorganisms,
one--Candida albicans--appeared to be unaffected by the addition of fluoride. C. albicans
is commonly found in man, usually in the oral cavity and digestive tract, and less commonly
in the vaginal tract of women. Though generally harmless, it can manifest itself as a pathogen.
The organism has been called the most common and most serious pathogen of man [3].
The legal ramifications of this are obvious. If an organism common to man is capable of
producing ethyl alcohol in stored blood, the question arises: Are the results of alcohol analysis
reflective of an individual's level of intoxication or of post-sampling fermentation? With
this in mind, we embarked upon a study of temperature versus ethanol production.
Method
Four plastic collection bags of human blood of 4S0-mL capacity were obtained from the
Peninsula Memorial Blood Bank of Burlingame, California. Each bag contained dextrose
(2.0 g), sodium citrate (1.66 g), citric acid (206 rag), monobasic sodium phosphate (140 mg),
and adenine (17.3 mg). The blood was pooled and half of the pool was inoculated with C.
albicans (Strain ATCC 14056). The inoculum was prepared to produce a final concentration
of approximately 10 000 organisms per millilitre. This concentration was selected from a
prior series of studies in which varying concentrations of this organism were cultured to assess
optimum growth.
Received for publication 27 July 1987; revised manuscript received 20 Jan. 1988; accepted for publication
21 March 1988.
IToxicologist. PharmChem Laboratories, Menlo Park, CA.
2Criminalist, Forensic Laboratory, San Mateo County Sheriff's Department, San Mateo, CA.
105
ASTM International
106 JOURNAL OF FORENSIC SCIENCES
The inoculated and uninoculated blood was divided among 112 10-mL Venoject~ tubes.
Of these tubes, 56 contained 100 mg of sodium fluoride and 20 mg of potassium oxalate. The
other half contained no additives. The tubes were filled under nonsterile conditions with the
uninoculated tubes serving as an experimental control over this study design. Representative
sets of samples (A -- uninoculated, --fluoride; B = inoculated, --fluoride; C = uninoculated,
+fluoride; and D = inoculated, +fluoride) were then divided into temperature storage
sets: refrigerated (6~ room temperature (22~ and body temperature (37~ Specimens
were analyzed after periods of 1 day, 2 days, 3 days, 5 days, 10 days, 35 days, and 6
months.
Analysis was performed by direct injection into a Hewlett-Packard 5880A gas chromatograph
equipped with a flame ionization detector and a 6-ft by Vs-in. (1.8-m by 3-mm)outside
diameter (O.D.) stainless steel column packed with 0.2% Carbowax 1500 on 60/80
Carbopack C. Column temperature was 115~ The injection port and detector temperatures
were 180~
An 0.8-mL specimen of each sample was diluted with 3 mL of deionized water containing
about 0.5% v/v of methyl ethyl ketone internal standard. Secondary alcohol standards of
0.220, 0.122, and 0.340% w/v ethyl alcohol were used to calibrate the instrument. Additionally,
quality control samples (0.177% w/v ethyl alcohol) were analyzed at the beginning,
end, and in the middle of each run. The minimum detectable concentration was determined
to be 0.003% w/v ethyl alcohol.
Results
Duplicate analyses of the two pools, both inoculated and uninoculated, showed that no
ethyl alcohol could be detected at time zero. Therefore, we could assume, with reasonable
certainty, that any ethanol found during the period of study would be the product of microbial
fermentation.
37~
Two representative sets of bank blood were kept at body temperature and analyzed after
periods of 28 h (one day) and 69 h (three days). Figure 1 shows that after 28 h at 37~ only
two of the four specimens in Subset B (inoculated, --fluoride) produced ethyl alcohol in
concentrations of 0.007 and 0.006% w/v. After approximately three days, Subset B showed
A '(Untreated Blood) B (candida Albicans C (Preservative D (Preservative and
only) only) Candl da Albicans )
0 .0~7 ~ &9
28 hrs ~] .0~6 ~ 0
0 0 0
~mmmsm$$1 . 053 o o
FIG. l--Ethanol production in blood at 37~ after periods of 28 and 69 h. Values are in % w/v.
CHANG AND KOLLMAN - TEMPERATURE EFFECT ON ETHANOL FORMATION 107
alcohol production in all four specimens: 0.028, 0.019, 0.041, and 0.053% w/v. Subset A,
which had not been inoculated and contained no sodium fluoride, produced only a trace of
alcohol after 69 h in two of four specimens. Both specimens contained 0.005% w/v ethyl
alcohol. Specimens containing preservative, both inoculated and uninoculated, showed no
detectable production of alcohol after three days at 37~
22~
The room temperature sets of blood were analyzed after periods of 1, 2, 5, 10, 35, and 182
days. The results are illustrated in Fig. 2. As this figure indicates, the production of ethyl
alcohol, once started after 5 days of incubation, was not affected by the presence of sodium
fluoride. However, only the specimens inoculated with Candida albicans showed significant
alcohol production. As with the 37~ sets of specimens, Subset A (uninoculated, --fluoride)
showed slight (0.014 and 0.016% w/v) alcohol production in two of the four specimens. The
uninoculated specimens that contained sodium fluoride (Subset C) showed no alcohol production
even after 35 days of room temperature storage.
6~
Five sets of specimens were kept under refrigeration and analyzed at 1, 5, 10, 35, and 182
days. No evidence of fermentation was found during the first 35 days. After 182 days, only a
trace (0.004% w/v) of ethanol was found in 1 of the 4 uninoculated specimens that contained
no sodium fluoride preservative. Of the 4 specimens inoculated with C. albicans that also
contained no sodium fluoride, only 2 showed slight (0.008 and 0.015% w/v) alcohol production
after 182 days of refrigerated storage. None of the preserved specimens, inoculated or
uninoculated, showed any alcohol production after 182 days at 6~
A (Untreated)
I day
: 2 days
5 days 0 .014
i0 days
35 days (not analyzed)
,~22
.~ d~s %6
.021
0 (Candida only) C (Pr~aervative
only)
F 0
0
E ~ "D~9 0
~ i ~ . 069 8
~lm~ , 062 0
(not analyzed)
0
m~l .~74 (not: analyzed)
~ .839
.066
O (Preservative--
and Candida)
i oo
o
0
i oo
o
.071
| .059
,065
.069
.037
.~3B
(not analyzed)
FIG. 2--Ethanol production in blood at 22~ after periods of 1, 2, 5, I0, 35, and 182 days. Values are
in % w/v.
108 JOURNAL OF FORENSIC SCIENCES
Discussion
We studied the preservation and storage of blood specimens used for alcohol analysis,
with emphasis on the issue of the loss or gain of ethanol over time. Winek and Paul [4] found
no significant variation in alcohol content when blood specimens were analyzed within 14
days of collection regardless of the conditions of storage and preservatives present. This is in
keeping with Glendening and Waugh's findings [5]. However, long-term storage has generally
resulted in a loss of alcohol with time [5-7]. Also, it has been conceded that ethanol can
be produced in blood specimens under certain conditions [1,2].
The issue of ethanol loss during long-term storage has been addressed in length in many
publications. The preservation of blood with sodium fluoride has been shown to prevent
effectively alcohol loss for up to two months when the specimens were stored at room temperature
[5]. For longer periods of time, refrigeration was found to be necessary [5, 8]. Meyer et
al. [8] found the freezing of blood specimens to be most effective in preventing ethanol loss.
The issue of alcohol gain in blood specimens taken from living subjects has received less
scrutiny. The instances of neoformation of alcohol are less common. Nonetheless, the issue,
both legal and scientific, remains.
Tests on postmortem blood specimens, which are more likely to exhibit neoformation of
ethanol, have shown that sodium fluoride is generally sufficient to preserve the integrity of
th,e specimens [1, 9]. However, Blume and Lakatua [1] found that sodium fluoride was ineffective
in preventing ethanol production by C. albicans. Our study generally supports their
conclusions. Although we detected no alcohol in a preserved group of specimens incubated
at 37~ for 69 h, specimens that had been inoculated with C. albicans and stored at room
temperature for more than five days showed significant alcohol formation. Furthermore,
ethanol formation, once started in these inoculated specimens, generally increased although
the absolute amount of ethanol formed appeared to be reaching a plateau concentration at
approximately 0.08% w/v. On the basis that ethanol formation in blood would arise predominantly
by the metabolic conversion of glucose, we calculated the maximum amount of
ethanol that could be created by glucose fermentation. Using a blood glucose concentration
of 95 rag/100 mL of blood, we calculated a first approximation value based upon the complete
conversion of glucose to ethanol via the anaerobic glycotic pathway in which 1 mole of
glucose converts to 2 moles of ethanol. This calculated value is 0.05% w/v. The amount of
ethanol formed in our study exceeded this value. We subsequently discovered that the blood
from the Peninsula Memorial Blood Bank is treated not only with the addition of citrate but
also 2.0 g of glucose per unit of blood. Therefore, our hypothesis of the maximum fermentation
yield could not be assessed.
Room temperature storage of all specimens gave negligible or no ethanol formation until
Day 5, and even under these conditions specimens that were uninoculated and contained
fluoride formed no alcohol over a period of 35 days. Under refrigerated storage, none of the
specimens showed any evidence of fermentation during the first 35 days, and only traces of
alcohol were found after 6 months.
It appears that fermentation proceeds readily only by direct inoculation or contamination
with C. albicans. Under such conditions the formation of ethanol is not inhibited by sodium
fluoride. We have also found that the amount of alcohol formed over time is highly dependent
upon the temperature of storage. Storage for approximately i day (28 h) at 37~ 2 days
at 22~ and 35 days at 6~ produced no alcohol in specimens that were uninoculated and
contained sodium fluoride as a preservative. Under these same storage temperatures and
storage periods, the maximum amount of ethanol formation would be expected in inoculated
and unpreserved specimens. Even in such specimens, the highest concentration of ethanol
attained was 0.007% w/v.
Our study further showed that even when specimens were purposely inoculated with C.
albicans, no alcohol formation took place for 69 h at 37~ if sodium fluoride at 10 mg/mL of
blood was used as a preservative.
CHANG AND KOLLMAN 9 TEMPERATURE EFFECT ON ETHANOL FORMATION 109
Therefore, it appears that legal questions regarding the issue of the neoformation of ethyl
alcohol should be rendered moot if preservatives and short transport times are routinely used
in bringing specimens to the laboratory and refrigeration is used in specimen storage.
Acknowledgments
We would like to express our thanks to Leticia Ruperto for her word processing assistance
and to Kenneth Mark, supervisor of the San Mateo County Toxicology Laboratory, under
whom this study was undertaken.
We would also like to thank Michael Nachtigall, M.S., and the San Mateo County Public
Health Laboratory for providing the C. albicans culture for this study.
References
[1] Blume, P. and Lakatua, D. J., "The Effect of Microbial Contamination of the Blood Sample on the
Determination of Ethanol Levels in Serum," American Journal of Clinical Pathology, Vol. 60, Nov.
1973, pp. 700-702.
[2] Corry, J. E. L., "Methods of Assessing the Effect of Microbes in Blood and Urine on Ethanol Levels,"
paper presented at the Eighth International Conference on Alcohol, Drugs and Traffic Safety,
Stockholm, Sweden, June 1980.
[3] Shepard, M. G., Poulter, R. T. M., and Sullivan, P. A., "Candida Albicans: Biology, Genetics, and
Pathogenicity," Annual Review of Microbiology, Vol. 39, 1985, pp. 579-614.
[4] Winek, C. L. and Louette, J. P., "Effect of Short-Term Storage Conditions on Alcohol Concentrations
in Blood from Living Human Subjects," Clinical Chemistry. Voi. 29, No. 11, 1983, pp. 1959-
1600.
[5l Glendening, B. L. and Waugh, T. C., "The Stability of Ordinary Blood Alcohol Samples Held
Various Periods of Time Under Different Conditions," Journal of Forensic Sciences, Vol. 10, No. 2,
April 1965, pp. 192-200.
[6] Stone, H. M., Muirhead, J. M., and Thompson, H. R., "Preservation and Storage of Blood Samples
Containing Alcohol," in Alcohol, Drugs and the New Zealand Driver, H. M. Stone, Ed., New
Zealand Department of Scientific and Industrial Research, Wellington, 1982, pp. 29-36.
[7] Chang, R. B., Smith, W. A., Walkin, E., and Reynolds, P. C., "The Stability of Ethyl Alcohol in
Forensic Blood Specimens," Journal of Analytical Toxicology. Vol. 8, March/April 1984, pp.
66-67.
[8] Meyer, T., Monge, P. K., and Sakshaug, J., "Storage of BJood Samples Containing Alcohol," Acta
Pharmacotogica et Toxicologica. Vol. 45, 1979, pp. 282-286.
[91 Blackmore, D. J., "The Bacterial Production of Ethyl Alcohol," Journal qfForensic Sciences, Vol.
8, No. 4, Oct. 1968, pp. 73-78.
Address requests for reprints or additional information to
S. Elliot Kollman
Forensic Laboratory
San Mateo County Sheriff's Department
31 Tower Rd.
San Mateo, CA 94402



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