CA1085279A - Measurement of alcohol levels in body fluids - Google Patents
Measurement of alcohol levels in body fluidsInfo
- Publication number
- CA1085279A CA1085279A CA269,354A CA269354A CA1085279A CA 1085279 A CA1085279 A CA 1085279A CA 269354 A CA269354 A CA 269354A CA 1085279 A CA1085279 A CA 1085279A
- Authority
- CA
- Canada
- Prior art keywords
- buffer
- oxygen
- ethanol
- alcohol oxidase
- body fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
Abstract
ABSTRACT OF THE DISCLOSURE
A method of measuring the level of ethanol in a body fluid includes the step of providing a predetermined volume of body fluid, oxidizing 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 consum-ption, the oxidation taking place in the presence of an agent adapted to suppress the formation of oxygen by peroxide decom-position.
Further, a kit for use in the above method comprises (i) a container containing an alcohol oxidase in a suitable buffer, the activity of the oxidase being in the range 1 to 1000 units (as hereindefined) per ml; and (ii) a container containing the agent adapted to suppress the formation of oxygen by peroxide decomposition in a suitable buffer.
A method of measuring the level of ethanol in a body fluid includes the step of providing a predetermined volume of body fluid, oxidizing 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 consum-ption, the oxidation taking place in the presence of an agent adapted to suppress the formation of oxygen by peroxide decom-position.
Further, a kit for use in the above method comprises (i) a container containing an alcohol oxidase in a suitable buffer, the activity of the oxidase being in the range 1 to 1000 units (as hereindefined) per ml; and (ii) a container containing the agent adapted to suppress the formation of oxygen by peroxide decomposition in a suitable buffer.
Description
~al8S'~79 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 -- ;
, oxidants used are able to react with a variety of volatile ~
.', ', substances other than ethanol.
Extraction of ethanol from deproteinated blood and :~;
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.) ;, ,~, , :, l~S;~g 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-~ .
1~)8S~79 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 , ;
~3~
: ,.. , ~ :,.. , ,,/, , . ;
~085Z79 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 ~085~79 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.
5'~7~
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 -- ;
, oxidants used are able to react with a variety of volatile ~
.', ', substances other than ethanol.
Extraction of ethanol from deproteinated blood and :~;
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.) ;, ,~, , :, l~S;~g 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-~ .
1~)8S~79 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 , ;
~3~
: ,.. , ~ :,.. , ,,/, , . ;
~085Z79 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 ~085~79 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.
5'~7~
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 ;
;, ~ -6-., , :.,. ,:, ~ ;
~C~85~7~
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) , .
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.
' :~
1~5'~79 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.
.
.... . . .. ... .
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 ;
;, ~ -6-., , :.,. ,:, ~ ;
~C~85~7~
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) , .
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.
' :~
1~5'~79 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.
.
.... . . .. ... .
Claims (17)
1. A method of measuring the level of ethanol in a body fluid including 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 per-oxide decomposition.
2. A method according to claim 1 wherein said rate of oxygen consumption is measured using an oxygen electrode.
3. A method according to claim 1 or claim 2 wherein said alcohol oxidase is isolated from a strain of Kloeckera yeast.
4. A method according to claim 1 wherein said buffer has a pH of 7 to 9.
5. A method according to claim 4 wherein said buffer has a pH of 7.8.
6. A method according to claim 1 wherein the buffer is a potassium phosphate buffer having a molarity of 0.01 to 2 and a pH of 7 to 9.
7. A method according to claim 6 wherein the buffer has a molarity of 1 and a pH of 7.8.
8. A method according to any one of claims 1,2 or 4 wherein the oxidation takes place at a temperature in the range 20 to 45°C.
9. A method according to any one of claims 1, 2 or 4 wherein the agent is a peroxidase enzyme and a suitable donor molecule.
10. A kit for use in the method of claim 1 comprising:
(i) a container containing an alcohol oxidase in a suitable buffer, the activity of the oxidase being in the range 1 to 1000 units(as hereindefined) per ml; and (ii) a container containing the agent adapted to sup-press the formation of oxygen by peroxide decomposition in a suitable buffer.
(i) a container containing an alcohol oxidase in a suitable buffer, the activity of the oxidase being in the range 1 to 1000 units(as hereindefined) per ml; and (ii) a container containing the agent adapted to sup-press the formation of oxygen by peroxide decomposition in a suitable buffer.
11. A kit according to claim 10 wherein the buffer of the two containers is the same.
12. A kit according to claim 10 or 11 wherein the buffer of each container has a pH of 7 to 9.
13. A kit according to claim 11 wherein the buffer is a potassium phosphate buffer having a molarity of 0.01 to 2 and a pH of 7 to 9.
14. A kit according to claim 13 wherein the molarity of the buffer is l and the pH of the buffer is 7.8.
15. A kit according to any of claims 10, 11 or 13 wherein the second container contains a peroxidase enzyme of activity 1 to 50 units ( as herein defined) per ml and a suitable donor molecule in a concentration of 0.01 to 2 % (w/v).
16. A kit according to claim 10 wherein at least one of the containers also contains a substance capable of complexing with metal ions which inhibit the alcohol oxidase.
17. A kit according to claim 16 wherein the complexing substance is EDTA-Na2 in an amount of 10 micromolar to 10 millimolar.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ZA76/0233 | 1976-01-15 | ||
| ZA760233A ZA76233B (en) | 1976-01-15 | 1976-01-15 | Measurement of alcohol levels in body fluids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1085279A true CA1085279A (en) | 1980-09-09 |
Family
ID=25569865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA269,354A Expired CA1085279A (en) | 1976-01-15 | 1977-01-07 | Measurement of alcohol levels in body fluids |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS5288394A (en) |
| AU (1) | AU505647B2 (en) |
| BE (1) | BE850383A (en) |
| CA (1) | CA1085279A (en) |
| DE (1) | DE2701168A1 (en) |
| FR (1) | FR2338495A1 (en) |
| GB (1) | GB1507810A (en) |
| IE (1) | IE44476B1 (en) |
| NL (1) | NL7700340A (en) |
| SE (1) | SE7700261L (en) |
| ZA (1) | ZA76233B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1157399A (en) * | 1979-06-05 | 1983-11-22 | Thomas R. Hopkins | Alcohol oxidase from pichia-type yeasts |
| US4540668A (en) * | 1979-06-05 | 1985-09-10 | Phillips Petroleum Company | Alcohol oxidase from Pichia-type yeasts |
| US4401122A (en) * | 1979-08-02 | 1983-08-30 | Children's Hospital Medical Center | Cutaneous methods of measuring body substances |
| DE3477812D1 (en) * | 1983-01-12 | 1989-05-24 | Alcoholism & Drug Addiction | Rapid analysis of ethanol in body fluids |
| JPS59232097A (en) * | 1983-05-16 | 1984-12-26 | Shokuhin Sangyo Center | Method and apparatus for determination of freshness |
| US4734360A (en) * | 1983-07-12 | 1988-03-29 | Lifescan, Inc. | Colorimetric ethanol analysis method and test device |
| EP0214336A1 (en) * | 1985-09-13 | 1987-03-18 | Phillips Petroleum Company | Determination of alcohol content in water immiscible organic systems |
| US4556635A (en) * | 1983-08-29 | 1985-12-03 | Phillips Petroleum Company | Determination of alcohol content in water imiscible organic systems |
| AT390803B (en) * | 1988-08-24 | 1990-07-10 | Avl Verbrennungskraft Messtech | METHOD FOR DETERMINING THE CONCENTRATION OF AN ENZYME SUBSTRATE AND SENSOR FOR IMPLEMENTING THE METHOD |
| US5264106A (en) * | 1988-10-07 | 1993-11-23 | Medisense, Inc. | Enhanced amperometric sensor |
| AU625439B2 (en) * | 1988-10-07 | 1992-07-09 | Medisense Inc. | Enhanced amperometric sensor |
| DE3934299C1 (en) * | 1989-10-13 | 1990-10-25 | Gesellschaft Fuer Biotechnologische Forschung Mbh (Gbf), 3300 Braunschweig, De |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1351547A (en) * | 1971-03-22 | 1974-05-01 | Wothington Biochemical Corp | Determination of the ethanol content of a material |
-
1976
- 1976-01-15 ZA ZA760233A patent/ZA76233B/en unknown
-
1977
- 1977-01-07 CA CA269,354A patent/CA1085279A/en not_active Expired
- 1977-01-11 IE IE48/77A patent/IE44476B1/en unknown
- 1977-01-12 GB GB1070/77A patent/GB1507810A/en not_active Expired
- 1977-01-12 SE SE7700261A patent/SE7700261L/en unknown
- 1977-01-12 AU AU21261/77A patent/AU505647B2/en not_active Expired
- 1977-01-13 DE DE19772701168 patent/DE2701168A1/en not_active Withdrawn
- 1977-01-13 NL NL7700340A patent/NL7700340A/en not_active Application Discontinuation
- 1977-01-14 FR FR7701081A patent/FR2338495A1/en active Pending
- 1977-01-14 BE BE174080A patent/BE850383A/en unknown
- 1977-01-14 JP JP349177A patent/JPS5288394A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| IE44476L (en) | 1977-07-15 |
| NL7700340A (en) | 1977-07-19 |
| BE850383A (en) | 1977-07-14 |
| IE44476B1 (en) | 1981-12-16 |
| SE7700261L (en) | 1977-07-16 |
| DE2701168A1 (en) | 1977-07-21 |
| FR2338495A1 (en) | 1977-08-12 |
| JPS5288394A (en) | 1977-07-23 |
| ZA76233B (en) | 1977-08-31 |
| AU2126177A (en) | 1978-07-20 |
| AU505647B2 (en) | 1979-11-29 |
| GB1507810A (en) | 1978-04-19 |
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