CUN.
CHEM. 24/4.
621-626
Enzymatic
Measurement
Frank
S. Cheng1
(1978)
Determination
of Blood
of Rate of Oxygen
Ethanol,
with Amperometric
Depletion
and Gary D. Christian2
A rapid electrochemical
measurement
of blood ethanol
is proposed.
Alcohol is oxidized
by NAD in the presence
of alcohol
dehydrogenase;
and the NADH produced
is
aerobically
oxidized
by horseradish
peroxidase.
The rate
of depletion
of buffer-carried
oxygen,
which is directly
proportional
to the alcohol
concentration
in the sample,
is amperometrically
monitored
with a membrane
oxygen-sensing
electrode.
Only a 5-0 sample
of whole blood
is required,
with no deproteinization,
incubation,
extraction,
or dilution. Results,
obtained
in less than 1 mm, correlate
well with those
obtained
by gas-chromatographic
and
spectrophotometric
methods.
cording
to the
Ethanol
reaction:
+ NAD
dehydrogenase
acetaldehyde
-‘-
+ NADH
NADH
is oxidized
by molecular
of horseradish
peroxidase
(EC
following
reaction.
NADH
+ H
.
.
.
horseradish
coupled
rate
intermethod
.
Several
methods
have been developed
for the assay
of ethanol
concentration
in blood
samples.
Classical
oxidation
methods
for the determination
of blood alcohol,
with use of various
oxidants,
have long been
available
(1-3).
They generally
involve
tedious
procedures for separating
alcohol
from the samples,
and are
therefore
not routinely
used nowadays.
Numerous
modifications
of two original
enzymic
methods
for determination
of ethanol
(4,5)
have been
published
(6-8). Because
of the advantages
of enzymic
over chemical
oxidation
methods,
kits are now sold for
quantitating
blood alcohol
by use of alcohol
dehydrogenase,
some of which have been evaluated
(9).
In 1958, gas chromatography
was proposed
for blood
alcohol
measurement
(10).
Several
improved
procedures, such as the use of the “head space” technique
and
direct
injection
method,
have been recommended.
An
excellent
description
of gas.chromatographic
methods
is available
(11).
Here, we describe
a different
approach
for the enzymic determination
of blood
alcohol.
The method
is
based
on the oxidation
of ethanol
by NAD
in the
presence
of alcohol
dehydrogenase
(EC 1.1.1.1)
acDepartment
of Chemistry,
University
of Washington,
Seattle,
Wash. 98195.
Presented
in part at the 174th national
meeting
of the American
Chemical
Society,
August
28-September
2, 1977, Chicago,
Ill.
‘Present
address:
College of Pharmacy,
The Ohio State University,
Columbus, Ohio 43201.
2Author to whom reprint requests should be addressed.
Received Sept. 9, 1977; accepted Jan. 27, 1978.
(1)
in the presence
as shown in the
+1/202
peroxidase
-
AdditIonal Keyphrases:
alcohol
dehyvi-ogenase
peroxidase
electroanalytical
technique
measurement
Beckman
Glucose Analyzer
comparison
toxicology
oxygen
1.11.1.7)
+ H
NAD
+ H20
(2)
Mn2
The maximum
rate of oxygen
depletion,
which
is
proportional
to the amount
of NADH
produced
in the
ethanol
oxidation,
is monitored
with a Beckman
Glucose Analyzer,
which includes
a Clark oxygen-sensitive
electrode.
The signal is directly
related
to the concentration
of alcohol
in the sample.
Although
reaction
2 has been known
for about
20
years (12), it has never been applied
for analytical
uses.
Our recent
discovery
(13) of conditions
for stable
and
reproducible
measurement
with a linear
relation
between
rate of oxygen
consumption
and NADH
production
makes
possible
this proposed
coupling
with
NAD+dependent
reactions.
Materials
and Methods
Apparatus
We used the Beckman
Glucose
Analyzer
(Beckman
Instruments,
Inc., Fullerton,
Calif. 92634) to measure
the amperometric
current
and electronically
take its
derivative.
Both the “Air Adjust”
and “Glucose
Sensitivity” potentiometers
were adjusted
to maximum
gain,
and the instrument
was operated
in the “U”-mode
to
obtain
maximum
sensitivity.
Measurements
were made
at the thermostated
temperature
of 33.0 ± 0.1 #{176}C.
Both
the direct-current
output
and the derivative
output
were recorded
on a two-pen
strip-chart
recorder
(Linear
Instruments
Corp., Irvine,
Calif. 92714). A Lancer
5-1d
micropipet
(Sherwood
Medical
Industries,
St. Louis,
Mo. 63103) was used to deliver
samples.
Reagents
Tris
pH
8.0.
(hydroxymethyl)methylaminelsuccinate
Prepare
a solution
CLINICAL
buffer,
of 0.5 mol/liter
CHEMISTRY,
Vol.
24,
tris(hydroxNo. 4, 1978
621
ymethyl)methylamine
and 0.17 mol/liter
succinate.
Check and adjust
the pH by adding
small quantities
of
either
tris(hydroxymethyl)methylamine
or succinate.
Horseradish
peroxidase
was obtained
from Worthington
Biochemical
Corp., Freehold,
N.J. 07728.
Alcohol
dehydrogenase
(from yeast) was purchased
from Calbiochem,
San Diego, Calif. 92212.
as the lyophilized
free acid, was purchased
from Boehringer
Mannheim
Biochemicals,
Indianapolis, Ind. 46250.
Buffer/cot
actor
stock
solution.
Dissolve
6.4 mg of
MnCl2”4H20
and 200 mg of 2,4-dichlorophenol
(Aldrich
Chemical
Co., Inc., Milwaukee,
Wis. 53233) in 250 ml
of the buffer.
NAD+/horseradish
peroxidase
stock
solution.
Dissolve 200 mg of NAD
and 1.2 mg of peroxidase
in 100
ml of the buffer/cofactor
stock solution.
This solution
contains,
per liter, 0.13 mmol of Mn2+, 5 mmol of 2,4dichlorophenol,
3 mmol of NAD,
and 10 U of peroxidase.
Ethanol
standards.
Aqueous
standards
were prepared
from
absolute
(i.e., anhydrous)
ethanol
(U.S.
Industrial
Chemicals,
New York,
N.Y. 10016).
Each
aliquot
of ethanol
was measured
by weight.
Alcohol
dehydrogenase
stock
solution.
Dissolve
12
mg of alcohol
dehydrogenase
in 1 ml of distilled,
deionized
water to give an enzyme
activity
of about
25 U
of the enzyme
per 10 l.
The ethanol
“Stat Pack”
(cat. no. 869219)
from Calbiochem
was used for spectrophotometric
measurement
of ethanol
in the correlation
studies
with the present
method.
I
I
I
C
a)
0
150
Cl)
4-
C
>
C
4-
-o
C
200
4-
-D
100’
Procedure
Place
1 ml of the NAD/peroxidase
reagent
in the
reaction
cell of the analyzer
and add 5 l of standard
or
sample.
Allow the oxygen
tension
inside the reagent
to
equilibrate
with atmospheric
oxygen.3
Finally,
add 10 tl of alcohol
dehydrogenase
reagent
to the sample cell to trigger the desired
reaction
between
alcohol
and NAD,
followed
by oxidation
of the resulting
NADH
with oxygen.
The signal,
which represents the concentration
of alcohol present
in the sample,
is obtained
about 20s after adding
alcohol
dehydrogenase.
Results
and Discussion
Analytical
Variables
Reaction
overall
curves.
reaction,
Figure
illustrates
1, a typical
rate curve for the
both
the direct
oxygen
‘ This is necessary
because
peroxidase
reportedly
(14) behaves
as
an oxidase in the aerobic oxidation
of several organic molecules,
such
as dihydroxyfumaric
acid, indoleacetic
acid, and certain dicarboxylic
acids. Therefore,
to avoid these interferences,
we add the blood sample
to the peroxidase
reagent
before adding alcohol dehydrogenase,
and
any organic
compounds
that react with oxygen
in the presence
of
horseradish
peroxidase
are removed
from the sample.
Because
only
trace concentrations
of these compounds
are normally
present
in
blood, these oxidations
usually
use negligible
amounts
of oxygen
carried
in the buffered
NAD/peroxidase
reagent.
622
CLINICAL
CHEMISTRY.
Vol. 24, No. 4, 1978
0
0123
Time
Fig. 1. Representative
Five microliters
of 2 9/liter
reaction
aqueous
mm.
curves
ethanol
was
for the overall
reaction
used for the measurement
current
and the derivative
of the current.
The direct
curve is sigmoidal,
similar
to that observed
with direct
oxidase
reactions
(15). In principle,
the reaction
rate is
maximum
early in the reaction.
After an initial mixing
and instrument
response
period
(15, 16), the recorded
current
goes through
a maximum
rate of change,
generally about
20 s after the reaction
begins,
that is proportional
to the substrate
concentration
(15).
The initial oxygen concentration
in the figure is about
2.4 X 10
mol/liter.
Assuming
complete
reaction
of the
ethanol
and stoichiometric
reaction
with the oxygen,
then 1.1 X 10
mmol of oxygen were consumed
and the
final oxygen concentration
is about 1.3 X 10
mol/liter.
The derivative
recording
is a direct measure
of the slope
of the direct
oxygen
curve
at any given time,
i.e., a
U)
U)
0
0.
>
0
4
3O0
‘C
0
‘C
0
E
E
2c
I0o
200
(N AD’),
Fig. 2. Effect of NAD
Five microliters
of 5 9/liter
0
ethanol
Ethyl
Alcohol
Reagents
of
buffered
:o
reogenf
Fig. 3. Effect of buffer composition
was used for the measurements
measure
of the rate of the reaction.
The maximum
slope
of the direct curve is about 3.0 X 10-6 mmol 02/s, which
corresponds
to the maximum
of the derivative
curve.
Coenzyme
concentration.
Figure
2 shows the effect
of NAD+
concentration
on the maximum
rate of the
coupled
reaction
in
tris(hydroxymethyl)methylamine/succinate
buffer,
in the presence
of 5 ,d of a high
concentration
(6 g/liter)
ethanol
standard.
it is apparent
that
the effect
of coenzyme
concentration
lessens
slightly
beyond
2 g of NAD
per liter (“-‘3 mmol/liter).
Therefore
we used
this
concentration
of NAD+
throughout
the study. This initial concentration
differs
slightly
from those used in the existing
spectrophotometric methods
(6-8),
because
in the present
procedure
NADH
produced
from the alcohol
dehydrogenase
reaction
is recycled
to NAD
in the system;
therefore,
NAD
is not depleted
during
the reaction
and initial
conditions
are more easily maintained.
Buffer
composition.
Pyrophosphate
and phosphate
are the buffers
usually
favored
for the ethanol/dehydrogenase/NAD+
reaction
(17). In the present
procedure,
because
these
buffers
chelate
manganese
ion,
which is essential
for the NADH/peroxidase/02
reaction,
they
or similar
buffers
should
not be used.
Tris(hydroxymethyl)methylamine/succinate
buffer has
been successfully
used in alcohol determinations
(9, 18),
including
a commercial
kit for the enzymatic
determination of ethanol
in biological
fluids.4 This mixed buffer
is suitable
for aerobic
oxidation
of NADH
in the presence of peroxidase
and Mn2+ (13), and so we used it in
the present
study.
Bucolo
(18)
noted
that
the tris(hydroxymethyl)methylamine/succinate
mixture
simultaneously
acts as
a buffer
and a trapping
agent
for the dehydrogenase
reaction.
Therefore
we determined
the effect of buffer
concentration
on the overall
rate of the coupled
reactions. Without
buffer the reaction
does not take place,
and increasing
the buffer
concentration
causes
increased
signals
in the coupled
reaction
(Figure
3). We
used 0.5 mol/liter
tris(hydroxymethyl)methylamine
and
4Calbiochem
o
06
o
Fraction
concentration
aqueous
02
(g/Iiter)
Brochure,
1976.
Different
fractions
of buffered and unbuffered
reagents
were mixed and
for measurements.
The buffered
(pH 8) reagent contained, per liter,
trls(hydroxymethyl)methylamlne,
0.3 mol succlnate, 0.13 mmoI Mn2, 5
DCP, and 10 U horseradish
peroxldase.
The unbuffered
reagent had the
composition
except buffer. Five microliters
of 4 g/lIter ethanol standard
used for assays
used
I mol
mmol
same
was
U)
>..
0
‘C
0
E
2
Horseradish
Fig. 4. Effect of horseradish
Five microliters
0.17
ty.
mol/liter
of 5 g/liter
(U)
peroxidase
aqueous
succinate
Peroxidase
ethanol
was used
to obtain
adequate
sensitivi-
Enzyme
activities.
Figure
4 indicates
the effect of
activity
of horseradish
peroxidase
on the rate of the
overall reaction,
when 5 jl of a 5 g/liter ethanol
solution
is used as a sample.
The optimal
concentration
of peroxidase
for the coupled
reaction
evidently
is near 2.5 U
in the reaction
cell. Because
horseradish
peroxidase
is
relatively
inexpensive,
we used fourfold
that amount
(10
U) throughout
the experiment,
to ensure that the overall
reaction
is not limited
by the rate of the peroxidasecatalyzed
reaction.
Figure
5 illustrates
the rate of the coupled
reaction
as a function
of dehydrogenase
activity.
In the present
procedure
about
25 U of dehydrogenase
is added,
a
compromise
between
sensitivity
of measurement
and
the cost of alcohol
dehydrogenase.
pH.
In the dehydrogenase-catalyzed
reaction
of
equation
1, the equilibrium
of the reaction
lies to the left
at neutral
pH, but may be forced to go completely
to the
right by use of a buffer
to keep the pH alkaline
(19).
Therefore,
most enzymatic
measurements
of ethanol
are made at a pH near 9.0 (9).
CLINICAL
CHEMISTRY,
Vol. 24, No. 4, 1978
623
U,
C
>.
0
.0
0
E
Alcohol
FIg. 5. Effect of alcohol
Five
microliters
of 2.5 g/liter
Dehydrogenose
(U)
dehydrogenase
aqueous
ethanol
was
used
for the measure-
ment
In the present
procedure,
because
Mn2+ is required
for the peroxidase
reaction,
it is not desirable
to have
too high a pH, because
Mn2
is readily
precipitated
in
alkaline
solution.
We studied
the effect of pH for the coupled
reaction
in the present
procedure
by using the tris(hydroxymethyl)methylamine/succinate
buffer
(Figure
6). The
pH profile,
obtained
1 h after adjusting
the pH of the
solution
medium,
indicates
that the rate of the coupled
reaction
is maximum
at pH near 8.5. At this pH, however, Mn2
is not stable, and the signals obtained
for the
coupled
reaction
gradually
decrease
with time. In contrast,
Mn2
is quite stable
near pH 8.0 when tris(hy.
droxymethyl)methylamine/succinate
buffer is used, and
the measurement
of the peroxidase-catalyzed
air oxidation
of NADH
is reproducible
(13). Therefore,
in our
procedure
we used pH 8.0 in the measurement
of the
coupled
reaction,
resulting
in the sensitivity
and precisions
discussed
below.
Trapping
agents.
Not only is an alkaline
pH used to
drive the reaction
of the dehydrogenase-catalyzed
oxidation
of alcohol
to completion,
but several
trapping
agents
have been used in enzymic
alcohol
methods.
Thus, aldehyde
trapping
agents,
such as semicarbazide
hydrochloride
(9), hydrazine
(20), and aminooxyacetic
acid (6, 9), as well as NADH
trapping
agents
such as
tetrazolium
salts (9) have all been applied
to force the
reaction
of the thermodynamically
unfavored
alcohol
oxidation
by ADH.
In the present
procedure,
NADH
generated
from
alcohol
oxidation
is oxidized
to give NAD,
which displaces
the equilibrium
of the dehydrogenase
reaction
in the direction
of NADH
and acetaldehyde,
and it is
therefore
not necessary
to use aldehyde
trapping
agents.
Nevertheless,
we studied
the effect of aldehyde
trapping
agents
on the rate of the coupled
reaction.
On addition
of aminooxyacetic
acid (12 mmol/liter)
or semicarbazide
(15 mmol/liter)
to the reaction
mixture,
the signals
obtained
decreased
with time, perhaps
because
of inhibition
of peroxidase
in the presence
of those
compounds.
On the other
hand,
hydrazine
added
(25
mmol/liter)
to the buffered
reaction
mixture
consumes
oxygen,
and the oxygen
concentration
in the solution
becomes
significantly
less than saturated.
This could
cause nonlinearity
(16).
We therefore
decided
not to
include
any aldehyde
trapping
agents
in the reaction
mixture.
Calibration
Figure
7 shows
signals
recorded
for 5-Ml aqueous
standards.
In each measurement,
5 l of pooled
serum
(with no ethanol
present)
was added to the reaction
cell,
to simulate
clinical
samples.
Because
of the sensitivity
of the present
procedure,
the signals
are read directly
1mm.
‘I
40J
400
C
C,,
3OO
0
>
0
-
20C
x
0
0
E
100l0&
09
TIME
pH
Fig. 7. Recorded
Fig. 6. Effect of pH
Five
microliters
of 3.5
9/liter
ethanol
standard
was used for the meaSure-
ments
624
CLINICAL
CHEMISTRY,
Vol. 24, No. 4, 1978
signals
for aqueous
ethanol
standards
Five microilters
of pooled serum sample was added to the buffered reagent
before addIng the standards. The numbers
on each peak represent
the concentrations
of aqueous ethanol standards
used, In g/llter
0,
/
/
/
/
/
,
0
/
‘a
/0
C
0
y
0.97x
>.
I-
-
0.0438
/
/
0.990
0
,o
I-
/0
C
n 22
‘00
(a
I..
/
0
0
y
‘C
0.96 x - 0.0163
0
E
0.992
fl
‘0
0
22
l.a
2
0
‘0
Lu
1.00
0
1.00
2.00
EtOH
Fig. 8. Calibration
graph
for aqueous
EtOH
(g/liter)
1. AnalytIcal
Recovery
Pooled
a
Human
Difference
ethanol
of Ethanol
Serum
Recovery,
Added to
Added
Found
g/llter
0.50
0.47
-0.03
94
1.00
1.50
2.00
1.03
1.52
1.92
+0.03
+0.02
-0.08
103
101
96
2.50
3.00
2.42
2.90
-0.08
-0.10
97
97
4.00
3.85
-0.15
96
5.00
5.12
+0.12
#{192}y.
a
Mean of three
replicates.
3.00
---GC
Method
-UV
Method
4.00
Fig. 9. Correlation
of results by the present method with those
by gas-chromatography
and an ultraviolet-enzymatic
kit
method
from the digital
panel meter,
with the selection
mode
switched
in the U-mode.
As Figure
6 shows, each measurement
is completed
in 20 s.
The calibration
graph
for the signals
(Figure
8) is
linear from 0 to about
5 g of ethanol
per liter. The detection
limit for ethanol,
when a 5-tl sample
is used, is
about 0.2 g/liter,
but it can be lowered
by using a larger
volume
of sample.
The upper
limit of blood
alcohol
concentration
normally
encountered
in routine
analysis
is about
5 g/liter
(21). When
higher
concentrations
of
alcohol
samples
must be measured,
the volume
of reagent
is doubled
(i.e., place 2 ml of buffered
enzyme
reagent
in the measuring
cell of the analyzer),
thus
doubling
the working
range.
A small blank signal is observed
in the absence
of alcohol (see Figure
7). The same is true if alcohol
dehydrogenase
is added before the sample to remove
possible
traces of alcohol
in the reagents
(from, e.g., recrystallization
in alcohol).
This suggests
that the blank signal
Table
2.00
(g/Iiter)
%
102
98.3
Gas.chromatographic
data supplied
by Washington
State
Toxicology
Labora-
tory
results
from introducing
a sample
that has an oxygen
tension
different
from that in the buffer
solution.
Because a relatively
high buffer concentration
is used, such
a difference
might
be expected
(16).
While the blank
signal can be reliably
recorded,
it is frequently
too small
to be read digitally,
and in such cases it is evaluated
by
extrapolating
the calibration
curve
to zero ethanol
concentration,
as in Figure
8. Of course,
such a calibration
curve can be used directly
without
subtracting
out the blank.
We recommend
that a calibration
graph
with three
different
standards
be prepared
daily. A one-point
recalibration
each hour is sufficient
for successive
runs
during
the day.
Analytical
Recovery
Various
amounts
of aqueous
solution
of ethanol
were
added
to aliquots
of pooled
serum
to produce
concentrations
of 0.5 to 5 g of ethanol
per liter. These samples
were then assayed
(Table
).
The per cent recovery
for
the entire
series averaged
98.3%.
Table
2. Effect
Ethanol
of Isopropanol on Results
by the Present Procedure
lsopropanol
Ethanol
Relative
signal
2.1
0
0.5
1.0
0
1.0
1.0
1.0
0
59
57
45
1.7
1.0
48
g/Ilter
CLINICAL
CHEMISTRY,
Vol.
24, No. 4, 1978
for
625
Precision
method
applied
has high
to routine
We determined
within-day
precision
of the present
procedure
by measuring
a pooled serum sample
fortified
to contain
1.50 g of ethanol
per liter. For 14 assays,
the
results
ranged
within
±6.7%
of a mean
of 1.49; the
standard
deviation
was ±0.055,
with a coefficient
of
variation
±3.6%. Analyses
of blood samples
containing
0.5 to 5 g of ethanol,
on successive
days showed
average
differences
of 0.5%, relative.
State
Correlation
2. Harger,
Studies
Specificity
626
CLINICAL
CHEMISTRY,
Vol.
24,
No. 4, 1978
be successfully
of only 5 zl.
(Washington
samples
that had
Predmore
blood
References
1. Lundquist,
F., The determination
of ethyl alcohol
in blood and
tissues.
In Methods
of Biochemical
Analysis,
7, D. Glick, Ed., Interscience
Publishers,
Inc., New York, N.Y., 1959, p 217.
R. N., Ethyl alcohol.
In Toxicology:
Mechanisms
and
2, C. P. Stewart
and A. Stolman,
Eds., Academic
York, N.Y.,
1961,
p 85.
Press,
Methods,
New
3. Smith,
H. W., Methods
for determining
alcohol.
In Methods
of
Forensic
Science,
4, A. S. Curry, Ed., Interscience
Publishers,
New
York, N.Y.,
1965,
p 1.
4. Bonnichsen,
R., and Theorell,
H., An enzymatic
method
for the
microdetermination
of ethanol.
Scand.
.1. Clin. Lab. Invest.
3, 58
(1951).
5. BUcher,
Bestimmung
Wochenschr.
6.
T., and Redetzki,
von Athylalkohol
29,
Jones,
D.,
7. Rosalki,
termination.
W., A rapid enzymatic
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16, 402
S. B., A new colorimetric
procedure
Anal. Lett. 4,819 (1971).
for blood
method
(1970).
alcohol
de-
T. P., Hadjiioannou,
S. I,, Avery, J., and Malmstadt,
enzymatic
determination
of ethanol
in blood,
with a miniature
centrifugal
analyzer.
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H. M., and Dees,
determination
photometrische
Wege.
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(1951).
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ethanol
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8. Hadjiioannou,
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serum, and urine
22, 802 (1976).
zymatic
615
H., Eine spezifische
auf fermentativem
Gerber,
for estimating
9. Redetzki,
The two enzymes
we used in the present
procedure
are popular
for routine
clinical
analysis.
Horseradish
peroxidase
is used for coupled
measurement
of numerous
oxidase
reactions
(20),
and no serious
interferences
with such coupling
is generally
reported.
Although
alcohol
dehydrogenase
is not fully specific
for
ethanol,
the oxidation
of related
aliphatic
alcohols
is
generally
considered
to be small at the concentrations
in which
they ordinarily
are present
in blood
(6, 22).
However,
isopropanol
or methanol
intoxication
is
sometimes
encountered
with clinical
or forensic
blood
specimens,
and so we evaluated
the effects of these and
other
agents
on our procedure.
Table
2 summarizes
measurements
observed
for isopropanol
in the absence
and presence
of ethanol.
Isopropanol
does not produce
a signal,
and so no false-positive
results
for ethanol
would result. At isopropanol
concentrations
equivalent
to or greater
than that of the ethanol,
there is a slight
inhibition
of the ethanol
signal,
on the order of 20%.
Methanol
at 0.25 g/liter,
acetone
at 1 glliter,
iodoacetic
acid at 2 gfliter, and sodium
fluoride
at 10 g/ liter did not
produce
a signal, and none of these caused
a change
in
the signal for a 1 g/liter
concentration
of ethanol.
Impairment
of the nervous
system
correlates
best
with the arterial
concentration
of alcohol
(23). Mason
and Dubowski
(23) suggest
that arterial
blood be obtained
from a deep cut in the finger tip, which gives
65-85%
arterial
blood in composition.
This technique
could be conveniently
used with the present
procedure
because
of the small sample
requirements.
The present
and can
for samples
We thank Dr. Larry Thomas
and Mr. David
Toxicology
Laboratory)
for providing
been analyzed
by gas chromatography.
Analytical
We compared
results
by the electrochemical
method
with those by gas-chromatography
using an internal
standard,
and an ultraviolet
enzymic
kit method
for
blood-alcohol
determination
(the Calbiochem
Ethyl
Alcohol
Stat Pack, used according
to the supplier’s
instructions).
Twenty-two
whole blood samples,
treated
with 1 g each of sodium
fluoride
and potassium
oxalate
per liter, were obtained
from the Washington
State
Toxicology
Laboratory.
Their
alcohol
concentrations
ranged
between
0.6 and 4 g/liter.
They were analyzed
by the three methods.
Results
are summarized
in Figure
9, along with corresponding
correlation
coefficients
and
regression
equations.
sensitivity
analysis
of
W. L., Comparison
ethanol
in
of four kits for enChem.
22, 83
blood.
Clin.
(1976).
10.
tion
Ann.
11.
gas
12.
Cadman,
W. J., and Johns, T., Gas chromatographic
determinaof ethanol
and other volatiles
from blood. Presented
at the 9th
Conf. on Anal. Chem. and AppI. Spect., Pittsburgh,
Pa., 1958.
Jam, N. C., and Cravey,
R. H., Analysis
of alcohol II. A review of
chromatographic
methods.
J. Chromatogr.
Sci. 10, 263 (1972).
Akazawa,
T., and Conn, E. E., The oxidation
of reduced
pyridine
nucleotides
by peroxidase.
J. Bioi. Chem.
232, 403 (1958).
13. Cheng,
F. S., and Christian,
G. D., Amperometric
of enzyme
reactions
with an oxygen
electrode
using
reduced
nicotinamide
adenine
denucleotide.
Anal.
(1977).
14. Nicholls,
P., Peroxidase
as an oxygenase.
Hayaishi,
Ed., Academic
Press, New York, N.Y.,
air
measurement
oxidation
Chem.
of
49, 1785
In Oxygenase,
1962, p 273.
0.
15. Kadish,
A. H., Little, R. L., and Sternberg,
J. C., A new and rapid
method
for the determination
of glucose by measurement
of rate of
oxygen consumption.
Clin. Chem.
14, 116 (1968).
16. Thomas,
L. C., and Christian,
G. D., The use of amperometric
oxygen electrodes
for measurements
of enzyme reactions.
Anal. Chim.
Acta 89,83 (1977).
17. Sund,
H., and Theorell,
H., Alcohol
dehydrogenases.
In The
Enzymes,
7, 2nd ed., P. D. Boyer,
H. Lardy,
and K. Myrback,
Eds.,
Academic
Press, New York, N.Y., 1963, p 25.
18. Bucolo,
G., Enzymatic
ethanol
assay. U.S. patent
no. 3,926,736,
December
16, 1975.
19. Racker,
E., Crystalline
alcohol dehydrogenase
from bakers’
yeast.
J. Biol. Chem.
184, 313 (1950).
20. Guilbault,
G. G., Handbook
Marcel Dekker,
Inc., New York,
21. Methodology
Press, Cleveland,
of Enzymatic
N.Y., 1976.
for Analytical
Toxicology,
Ohio, 1975, p 148.
22. Mason, M. F., The determination
of blood
chemistry
laboratory.
Lab. Med. 6,28 (1975).
23. Mason,
M. F., and Dubowski,
K. M., Alcohol,
testing in the United States: A Maum#{233}
and some
Clin. Chem.
20, 126 (1974).
Methods
I. Sunshine,
alcohol
of Analysis,
Ed., CRC
in the clinical
traffic,
and chemical
remaining
problems.