Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to an improved method for the
measurement of alcohol level~, i.e. ethanol levels, in body
fluids and to reagents for use in the method. The measurement
of ethanol in body fluids, especiaLly blood, is a well estab-
lished routine test performed for medical and legal purposes
' throughout the world.
Many chemical methods have been used for the determina-
tion of ethanol in blood, most of which involve the oxidation
of ethanol and determination of the amount of oxidant required,
either volumetrically or colorimetrically. (Landquist, F.,
Methods of Bio-chemical Analysis, Vol. 7, 217, 1959.) All these
methods have in common a serious lack of specificity as all the -- ;
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oxidants used are able to react with a variety of volatile ~
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substances other than ethanol.
Extraction of ethanol from deproteinated blood and
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detection by gas liquid chromatography is highly specific and ~ --
accurate, but very time consuming. The enzymic detection of
ethanol using alcohol dehydrogenase is used extensively in
many laboratories because of the high specificity and sensitivity
of the method (Bonnichsen, R., Theorellj H., Scand. J. Clin.
Lab. and Invest.j 3 58 1951). However, as one is measuring NADH2
formation at 340 nm a spectrophotometer is required, and also
the analysis cannot be perfomed on whole blood.
Guilbault tGuilbault, G.G. and Sadar, S.H., Anal.
Lett. 2,41 1969) suggested using an alcohol oxidase from a
Basidiomycete in the fluorometric estimation of ethanol, as this
has a narrower specificity than alcohol dehydrogenase, although
it will detect methanol. An amperometric enzyme electrode using
the same enzyme has also been suggested by Guilbault (Guilbault,
G.G., and ~ubrano, G.J. Analytica Chimica Acta, 69, 189, 1974.)
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This me-thod monl-tors amperometrically the hydrogen peroxide
produced in the enzymic reaction. The alcohol oxidase from the ~ ;r
Basidiomycete utilises ethanol at only 28% of the rate compared
with methanol.
Accordingly it is an object of this invention to seek
to provide an improved method and, associated kit for determining
ethanol in body fluids.
; The accompanying graph (Fig. 1) of this application
will be further explained hereinafter in association with the
following example. According to this invention there is provided
' a method of measuring the level of ethanol in a body fluid inclu-
- ding the step of providing a predetermined volume of body fluid,
oxidising ethanol in the body fluid by the action of an alcohol
oxidase in a suitable buffer in the presence of excess molecular
oxygen and measuring the rate of oxygen consumption, the oxidation
` taking place in the presence of an agent adapted to suppress the
formation of oxygen by peroxide decomposition.
The rate of oxygen consumption is directly proportional
to the concentration of ethanol in the body fluid. Thus, the
concentration of ethanol in the body fluid is readily determinable
by, for example, reading the concentration of ethanol off an
appropriate graph of concentration of ethanol against rate of
oxygen comsumption or by comparing the rate obtained for any
given sample with a standard.
The rate of oxygen consumption is preferably measured
using an oxygen electrode. The use of an oxygen electrode offers
a number of distinct advantages. An oxygen electrode is relative-
ly inexpensive. It enables the oxygen consumption to be deter-
mined rapidly and in highly turbid or coloured solutions. The
oxygen electrode is a polargraphic device for measuring the con-
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centration of oxygen dissolved in a given medium and depends onthe electrolysis of dissolved oxygen at a weakly negative elec-
trode. The oxygen electrode has been known since the early part
of this century. In 1956, Clark improved the electrode consider-
ably by using an oxygen permeable, non-conducting membrane to
isolate the electrolytic cell from the sample under measurement
- Clark, L.C., Trans. Am. Soc. Art. Int. Org. 2,41. 1956. Oxygen
electrodes are commercially available. The oxygen electrode can
be coupled in known manner to a standard recorder, for following
~ 10 and recording the rate of oxygen consumption.
- As is men-tioned above, the oxidation is carried out in
the presence of excess oxygen i.e. the oxygen must not be a rate
limiting reactant. As the range of likely ethanol concentrations
in the body fluids is known, it is a simple matter to ensure that
excess oxygen is present. Usually, the oxidation is carried out
in air saturated solutions and it is, in this case, necessary
; simply to ensure that sufficient air saturated solution is present
to provide an excess of molecular oxygen. This is readily calcul-
able for any given situation.
The invention will in general be used to measure the
ethanol level in blood. However, the invention can be used to
measure the ethanol levels in other body fluids such as plasma ~;
and serum.
The alcohol oxidase must be capable of catalysing the
oxidation of the ethanol. A number of such oxidases are known
in the art. The preferred oxidase is that isolated from a strain
of Kloeckera yeast which was obtained from Prof. K. Ogata,
Department of Agricultural Chemistry, Kyoto university, Japan.
The oxidase was extracted from the yeast and purified to homo-
geneity by standard techniques of protein purfication as will now
be described briefly. A streak of the yeast was obtained from , ;
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Prof. Ogata and grown on methanol as a carbon source. The cells
of the bulk yeast were broken open by ultrasonic vibrations.
The supernatant from this step was subjected to ammonium sulphate
precipitation, ion exchange chromo-tography on DEAE-cellulose and
gel filtration chromotography on G 200 Sephadex (trademark) and
was then in condition for use. The enzyme at this stage had an
activity of between 90 to 100 units per ml. One unit of enzyme
ac-tivity is defined as the amount of enzyme which causes the con-
sumption of one micromole of oxygen per minute at 37C with
ethanol as a substrate.
Another suitable alcohol oxidase is that used by
Guilbault (Guilbault, G G and Lubrano G.J. Anal. Chim. Acta 69
189, 1974) which was isolated from a Basidiomycete.
The buffer must be such as not to inhlbit the oxidation.
~; The preferred buffer is one having a pH of 7 to 9. The preferred
pH is 7.8. Examples of suitable buffers are potassium phosphate
buffer, borate buffer and tris(hydroxymethyl) amino methane
buffer.
The preferred buffer is a potassium phosphate buffer ~ `~
20 having a molarity of 0.01 to 2 and a pH of 7 to 9. The preferred
molarity is 1 and the preferred pH is 7.8.
The oxidation preferably takes place at a temperature
in the range 20 to 45C. The oxidation can conveniently take
place at abou-t 37C.
Hydrogen peroxide is produced during the oxidation
of the ethanol. Hydrogen peroxide decomposes to produce oxygen
and this decomposition is catalysed by impurities which are
sometimes present in -the alcohol oxidase. It is necessary, there-
fore, for there to be present an agent adapted to suppress the
formation of oxygen by peroxide decomposition. This agent is
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preferably a peroxidase enzyme and a suitable donor molecule
i.e. a molecule capable of being oxidised by ~he hydrogen pero-
xide in the presence of the peroxidase enzyme. Suitable donor
molecules are known in the art and examples thereof are o-toli-
; dine (~,4'-daimino-3,3~-dimethyldiphenyl), o-dianisidine and
amino antipyrine. Another suitable agent is a substance which
will inhibit catalase which, as is known, catalyses the decom-
position of hydrogen peroxide. An example of such a substance
is sodium azide.
The invention provides according to another aspect,
i a kit for use in the abov~ method comprising: ;; ;
(i) a container containing an alcohol oxidase in a
suitable buffer as described above, the activity of the
oxidase being in the range l to lO00 units per ml, preferably
about lO0 units per ml; and
(ii) a container containing the agent adapted to suppress
the formation of oxygen by peroxide decomposition in a suitable
buffer, generally the same b.~ffer as in ~
The agent in (ii) is preferably a peroxidase enzyme
of activity l to 50 units per ml, a suitable donor molecule
in a concentration of O.Ol to 2~ (w/v)-, preferably 0.2% (w/v).
The units of activity are defined hereinafter.
Either container may also contain a substance capable
~ of complexing with metal ions which inhibit the alcohol oxidase.
; The complexing substance is preferably EDTA-Na2 in an amount of
lO micromolar to lO millimolar, preferably about lO0 micromolar.
An example of the invention will now be described. In
this example, the following reagents were used:
l. Alcohol oxidase obtained from Kloeckera yeast in the
manner described above.
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2. Horse radish peroxidase - This enzyme (Donor: hvdrogen
peroxide oxidoreductase, E.C. No. 1.11.1.7) was obtained from
Miles-Seravac, Cape Town, with an acitivity of 60 units/mg. One
unit is defined by the manufactuxers as the amount o~ the enzyme
producing 1 mg purpurogallin in 20 seconds at 20C from pyrogal-
lol.
3. Agent adapted to suppress the formation of oxygen by
peroxide decomposition - A 0.2% (w/v) solution of o-tolidine HCl
was made by dissolving the salt in 1 M phosphate buffer, pH 7.8,
containing 4 units of the horse radish peroxidase described above
per ml of solution. The phosphate buffer was prepared by mixing
suitable proportions of aqueous 1.0 M potassium dihydrogen phos-
phate and 1.0 M dipotassium hydrogen phosphate solutions to the
desired pH~
4. Standard Ethanol Solution - A standard aqueous ethanol
solution (10 ~moles/ml) was prepared using absolute ethanol
dried over magnesium.
The rate of oxygen consumption of a number of samples
of ethanol of known concentration was measured using an oxygen
electrode and the reagents mentioned above. The oxygen electrode
was purchased from Clinical Sciences and Manufacturing Laborat-
ories of Johannesburg. The oxygen electrode was connected
to a circulating water bath maintained at 37C. The electrode was
covered by a 0.0005 inch Teflon (registered trademark) membrane
and the cell volume was maintained at about 1.0 ml. The output
signal was recorded by a commercially available recorder.
0.95 ml of the phosphate-donor-peroxidase buffer system,
pH 7.8, was added to the reaction cell of the oxygen electrode
and the contents allowed to reach thermal equilibrium a-t 37~C. 3
100 ~1 (9~3 units) of the alcohol oxidase was added to the
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reaction cell. The reaction was initiated by the addition of
varying amounts ~o to 50 ~1) of the standard ethanol solution.
The initial rate of oxygen consumption was recorded for each
alcohol concentration used. The rate in each case, after deduc-
tion of a substrate blank, was plotted against concentration
of ethanol solution. The resulting graph is shown in Figure 1.
In this graph the initial rate of oxygen consumption in ~ moles/
;; min is plotted along the ordinate and the amount of ethanol
solution plotted along the abscissa.
The endogenous rate of oxygen consumption was measured
in the absence of ethanol solution before the reaction was
initiated. Oxygen concentration in the air saturated solutions
used was calculated by the method of Glasstone (Glasstone S, ~
Elements of Physical Chemistry, 1st Edition pp 343-344, 1946 D. ~;
Van Nostrand Co. Inc. m New York). The recorder was calibrated
using air saturated water.
In another experiment known amounts of ethanol were
added to blood drawn from persons with similar results being
obtained:
~ 10~1 of freshly drawn blood were added to 1.0 ml of
the phosphate-peroxidase-donor buffer, pH 7.8, in the reaction -
cell of the oxygen electrode. 9.3 units of alcohol oxidase were
then added and the system allowed to equilibrate thermally (37C)
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and to oxidise any alcohol possibly already present in the blood.
Varying aliquots (0-50 ~1) of the aqueous ethanol solution were
then added, and -the initial rates of oxygen consumption recorded.
In every case, -the rates obtained were identical to those
recorded in the absence of blood with the same amount of ethanol.
Thus, the presence of 10 ~1 of whole blood has no effect on
the assay system.
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For accurate work using whole blood as sample material,
the blood should be added to the bu:Efer system first and the
reaction initiated with the alcohol oxidase. By this means, any
endogenous oxygen uptake in the blood can be compensated for.
The method of the invention is based on the initial
rate of oxygen consumption and consequently the procedure is very
rapid. It can be carried out in less than one minute. Whole
blood may be used for sample material, and for routine blood
alcohol analyses~ only 5 ~1 of blood are required. A finger-
prick will therefore supply adequate material for analysis.
No separate sample is required for a blank determination as any
endogenous oxygen uptake is accounted for before addition of
alcohol oxidase. The method is also more specific than most
; of the methods now available.
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