IE44476B1

IE44476B1 - Measurement of alcohol levels in body fluids

Info

Publication number: IE44476B1
Application number: IE48/77A
Authority: IE
Original assignee: Chembro Holdings Pty Ltd
Priority date: 1976-01-15
Filing date: 1977-01-11
Publication date: 1981-12-16
Also published as: FR2338495A1; GB1507810A; AU2126177A; JPS5288394A; DE2701168A1; SE7700261L; ZA76233B; BE850383A; AU505647B2; IE44476L; CA1085279A; NL7700340A

Abstract

1507810 Determination of ethanol in body fluid CHEMBRO HOLDINGS (PTY) Ltd 12 Jan 1977 [15 Jan 1976] 01070/77 Heading G1B Ethanol in a body fluid is determined by a method comprising providing a predetermined volume of body fluid, oxidizing ethanol in the body fluid using an alcohol oxidase in buffer solution in the presence of excess molecular oxygen and measuring the rate of oxygen consumption using for example an oxygen electrode, the oxidation taking place in the presence of an agent operative to suppress the formation of free oxygen by decomosition of peroxide. The agent may be a peroxidase together with o-tolidine, o-dianisidine, aminoantipyrine, or alternatively a catalase inhibitor such as sodium azide. The alcohol oxidase may be obtained from Kloeckera yeast or from a Basidiomycete. The buffer may be a 0À01 to 2 m potassum phosphate buffer pH 7 to 9, and the reaction mixture may contain 10 microns to 10 mm of a metal ion complexing substance such as EDTA-Na.

Description

TATENT APPLICATION BY (71) CHEMBRO HOLDINGS (PROPRIETARY) LIMITED A COMPANY REGISTERED ACCORDING TO THE LAWS OF THE REPUBLIC OF SOUTH AFRICA, OF 105 QUARTZ STREET, HILLBROW, JOHANNESBURG, REPUBLIC OF SOUTH AFRICA.

Pnct 12|p THIS invention relates to an improved method for the measurement of alcohol levels, 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 established routine test performed for medical and legal purposes throughout the world.

Many chemical methods have been used for the determination 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., Theorell, H., Scand. J. Clin. Lab. and Invest., 3 58, 1951). However, as one is measuring NADHg formation at 340 nm. a spectrophotometer is required, and also the analysis cannot be performed on whole blood.

Guilbault (Guilbault, 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 Lubrano, G.J., Analytica Chimica Acta, 69, 189, 1974). This method monitors amperometrically the hydrogen peroxide produced in the enzymic reaction. The alcohol oxidase from the Basidiomycete utilises ethanol at only 28% of the rate compared with methanol.

According to this invention there is provided 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 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 consumption 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 relatively inexpensive. It enables the oxygen consumption to be determined rapidly and in highly turbid or coloured solutions. The oxygen electrode is a polaragraphic device for measuring the concentration of oxygen dissolved in a given medium and depends on the electrolysis of dissolved oxygen at a weakly negative electrode. The oxygen electrode has been known since the early part of this century. In 1956, Clark improved the electrode considerably by using an oxyger. permeable, non-conducting membrane to isolate the electrolytic cell from the sample under measurement - Clark, t.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 such as a Cimatic Cimapot T5 Recorder for following and recording the rate of oxygen consumption.

As is mentioned 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 calculable for any given situation.

, The invention will in general be used to measure the ethano’l 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 homogeneity by standard techniques of protein purification as will now be described briefly. A streak of the yeast was obtained fromProf. 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 chromatography on DEAE-cellulose and gel filtration chromatography on G 200 Sephadex 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 activity is defined as the amount of enzyme which causes the consumption of one micromole of oxygen per minute at 37°C with ethanol as 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 inhibit the oxidation.

The preferred buffer is one having a pH of 7 to S. 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 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 45°C, The oxidation can conveniently take place at about 37°C.

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, therefore, for there to be present an agent adapted to suppress the formation of oxygen by peroxide - 5 44476 decomposition. This agent is preferably a peroxidase enzyme and a suitable donor molecule i.e. a molecule capable of being oxidised by the hydrogen peroxide in the presence of the peroxidase enzyme. Suitable donor molecules are known in the art and examples thereof are o-tolidine (4,41-diamino-3,3'-dimethyldiphenyl), o-dianisidine and amino antipyrine. Another suitable agent is a substance which will inhibit catalase which, as is known,catalyses the decomposition of hydrogen peroxide.

An example of such a substance is sodium azide The invention provides according to another aspect, a kit for use in the above 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 1 to 1000 units per ml, preferably about 100 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 buffer as in (i).

The agent in (ii) is preferably a peroxidase enzyme of activity 1 to 50 units per ml, a suitable donor molecule in a concentration of 0,01 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-Na^ in an amount of 10 micromolar to 10 millimolar, preferably about 100 micromolar. - 6 44476 ρ5·... Λη example of the invention will nov; be described. In this example, the following reagents were used: 1. Alcohol oxidase obtained from Kloeckera yeast in the manner described above. 2. Horse radish peroxidase - This enzyme (Donor: hydrogen peroxide oxidoreductase, E.C. Ho. 1.11.1.7) was obtained from Hi 1es-Seravac, Cape Town, with an activity of 60 units/ mg. One unit is defined by the'manufacturers as the amount of enzyme producing 1 mg purpurogal1in in 20 seconds at °C from pyrogallol. 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 H 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 phosphate and 1,0 H dipotassium hydrogen phosphate solutions to the desired pH. 4. Standard Ethanol Solution - A standard aqueous ethanol solution (10 μmoles/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 nn oxygen electrode and the reagents mentioned above. The - 7 4447® oxygen electrode was purchased from Clinical Sciences and Manufacturing Laboratories of Johannesburg. The oxygen electrode was connected to a circulating water bath maintained at 37°C, by a 0,0005 inch Teflon The electrode was covered (trademark) membrane and the cell volume was maintained at about 1,0 ml. The output signal was recorded by a Cimatic Cimapot T5 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 at 37°C. 100 yl (9,3 units) of the alcohol oxidase was added to the reaction cell. The reaction was initiated by the addition of varying amounts (0 to 50 yl) of the standard ethanol solution. The initial rate of oxygen consumption was recorded for each alcohol concentration used. The rate in each case, after deduction 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 ymoles/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: lOyl of freshly drawn blood were added to 1,0 rol 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 (37°C) and to oxidise any alcohol possibly already present in the blood. Varying aliquots (0-50yl ) of the aqueous ethanol solution were then added, and the initial rates of oxygen consumption recorded. In every case, the tares obtained were identical to those recorded in the absence of blood with the same amount of ethanol. Thus, the presence of lOyl of whole blood has no effect on the assay system.

For accurate work using whole blood as sample material, the blood should be added to the buffer 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 yl 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

CLAIMS:

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 5 ih 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.

2. A method according to claim 1 v/herein the rate of oxygen 10 consumption is measured using an oxygen electrode.

3. A method according to claim 1 or claim 2 v/herein the alcohol oxidase is isolated from a strain of Kloeckera yeast.

4. A method according to any one of the preceding claims 15 wherein the buffer has a pH of 7 to 9.

5. A method according to claim 4 wherein the buffer has a pH of 7,8. 1044476

6. A method according to any one of claims 1 to 3 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 5 molarity of 1 and a pH of 7,8.

8. A method according to any one of the preceding claims wherein the oxidation takes place at a temperature in the range 20 to 45°C.

9. A method according to any one of the preceding claims 10 wherein the agent is a peroxidase enzyme and a suitable donor molecule.

10. A method according to claim 1 substantially as herein described.

11. A kit for use in the method of claim 1 comprising: 15 (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 20 suitable buffer.

12. A kit according to claim 11 wherein the buffer of the two containers is the same.

13. A kit according to claim 11 or claim 12 wherein the buffer of each container has a pH of 7 to 9.

14. 5 A kit according to claim 12 wherein the buffer is a potassium phosphate buffer having a molarity of 0,01 to 2 and a pH of 7 to 9.

15. A kit according to claim 14 wherein the molarity of the buffer is 1 and the pH of the buffer is 7,8.

16. 10 A kit according to any of claims 11 to 15 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).

17. A kit according to any of claims 11 to 16 wherein at 15 least one of the containers also contains a substance capable of complexing with metal ions which inhibit the alcohol oxidase. 12 4 4 4 7 6

18. A kit according to claim 17 wherein the complexing substance is EDTA-Na^ in an amount of 10 micromolar to 10 millimolar.

19. A kit according to claim 11 substantially as herein 5 described.