RAPID ANALYSIS OF ETHANOL IN BODY FLUIDS

Abstract: Novel solid compositions are provided for assaying the levels of lower alcohols in body fluids. A carrier is impreg­ nated with two or more enzymes, the first of which reduces a reducible agent in the presence of the alcohol. A chromogen is provided which reacts with either the reducer or the oxidized compound in the presence of a second enzyme or reagent, depending on the system, to produce a clinically significant indication of the alcohol present. A quantitative determination may be made by the provision of a competi­ tive inhibitor to the first enzyme to provide a graduated response in colour according to the concentration of alcohol. Acetaldehyde trapping agents, buffers and enzyme stabiliz­ ers are provided as required.

Description: "kA'ID ANALYSIS OF ETHANOL IN DONY FLUIDS The present invention relates to the quantitative and qualitative determination of alcohol in biological fluids such as urine, saliva, serum or whole blood. The effect of alcohol on human beings is a significant problem from both a social and medical viewpoint. The consequences of the impaired behaviour of vehicle operators and of persons who consume excessive amounts of alcohol is increasingly being recognized as antisocial behaviour. Both for. medical and social purposes it is important to obtain a fast accurate assay of the body alcohol level in a subject. It is also important to identify the alcohol which causes the impairment. Alcohols other than ethanol, unless treated promptly, may cause permanent damage. At the present time various techniques are available for this purpose but such Lechniques necessitate the use of equipment which is expensive, cumbersome, not sufficiently accurate, or take an unacceptable amount of time to deter mine the alcohol concentration. It is therefore an object of this invention to provide an inexpensive, quick and reliable means of determining accurately the alcohol concentration in a biological flui and a clinically significant identity of the alcohol. In accordance witl.:one aspect of the invention, a quantitative determination of the alcohol level inbody finids is provided, in another aepecta qualitative deiormination Or t)i'. alcohol level in bc body fluids i? provided; and in yet another anpecta simultanoous quantitative and qualitative determination of the alcohol levels in the body fluide may be provided. In accordance with the present invention, a solid carrier io impregnated with the alcohol detecting reactants which, when immersed in the body fluid, gives a visual indication of the information to be determined. In a first embodiment the carrier is impregnated with a reducible compound which reacts with ethanol in the pretence of a first enzyme to produce the corresponding acetaldehyde and tbe reduced compound: the first enzyme; a competitive inhibitor for the first enzyme a chromogen, a colour changing compound which, in the presence of the reduced compound anc. a second enzyme, or with phenazine or thiazine, produces a colour change which represents the con centration of alcohol present in the body fluid; the second enzyme or phenazine or thiazine; an acetaldehyde trapping agent; a buffer to maintain the desired pH : and dithioerythritol or an equivalent sulphydryl group, to protect the enzyme. This system will react with ethanol but not with methanol so as to permit a differential determination between methanol and t11e other lower alcohols. In accordance with a second embodiment, the carrier may be impregnated with an enzyme which catalyzes a reaction between the alcohol and the free oxygen in the body fluid to produce an oxidized compound and hydrogen peroxide, a chrom ogen which is oxidized by the hydrogen peroxide in the presence of a second enzyme to give a colour indicative of the presence of alcohol. To provide quantitative determinations, competitive inhibitor or the first enzymes are employed. ln accordance with a further embodiment, the carrier may he impregnated with a constituent which, with water; produces hydrogen peroxide, a first enzyme catalyst by which the othanol isoxidized by hydrogen perxide to the corressponding acctaldehyde, a nuclactide; a socond enzyme to reduce the nucleotide in the presence of acetaldehyde a chromogen, a colour-changing compound which, in the prosunce of the reduced nuclsotide and o= a third enzyme, or with phenazine or thiazine, produces a colour change which repre sents the concentration of alcohol prescnt in the body fluid; the thrd enzyme and a buffer to maintain desired pH. If the enzymes are not covalently immobilized then an inert protein or proteins such q gelatin or albumin may be added to stabilize tile enzymes while serving to enhance the colours produced. It ill also be appreciated that surfactants such as polyoxy-ethylene-23-laurylether known by the trademark BRIJ35, or its equivalent, may be employed The solid carrier may, for example, be a strip of paper or may be an inert powder material such as silica gel, kieselguhr or microcrystalline cellulose. The inert powder may be compounded with the active ingredients and compressed by known methods into tablet fonn or may be placed in an open-ended tube depending upon the application. It will be apparent to those skilled in the art that the solid carrier may he coated to enhance the optical qualities of the final colour formed. =A samipermeable coating may also be provided on the impregnated carrier over the impregnant, Such coatings may be forned in a conventional manner from materials such as cellophane, cellulose acetate, cellulose butyrate, ethyl cellulose or methyl cellulose so as to permit the alcohol and water to pass through and react with the impregnant while screening or preventing the red cells and larger molecules such as proteins and haemoglobin from passing through. EXAMPLE 1 Solid compositions intended for determining ethanol concentrations in urine or saliva were prepared as follows: 1. Proparation of 125 mM TRIS Goffer p11 0.2containing 0.84g/100 mlgemicarbHzide. To 1.514g of TRIS, 0.04g of cemicorbazide were added and diluted to 100ml with double distilled water (adjusting pH to 8.2 with HCI). At this point gelatin may be added. If golatin is added the solution in; brought to 37 C and 1.5g of gelatin added. 2. Preparakion of stock solution of 25 mt4 pyrazole. 0.17g of pyrazols ware dissolved in 100ml double distilled watcr. 3. Preparation of reagents to be freeze dried: (a) The following were weighed in a lyophilyzing bottle wrapped with aluminum foil to protect from light: 1. Diaphorase 0.170 g (766 Units) 2. NAH0 477 g 3, ADH (5250 Units) 4. INT 0.200 g 5. Albumin 0.14 g 6. BRIJ-35 0.10 g 7. Dithioerythritol 85 ul of 25 mM Stock Solution (b) To this lyophilizing bottle, 14ml of Tris Buffer and O.37ml of 25 mM pyrazole were added. (c) The solution was gently mixed. After 15 minmutes, 120 strips of Whatman filter paper &num;3 were added to the solution in dark room. The size of the strip of filter paper was 5mm x 55mm. (d) The strips were allowed to absorb the reagent for about S minutes and were shaken gently. Any excess fluid was removed to be further used in the next batch. (e) The lyophilizing bottle containing the filter strips was placed in the freezer for at least 3 hours and was freeze dried overnight. Preparation and storage of freeze dried strips: (a) The freeze dried strip (5mm x 55mm) was attached to a semi-rigid plastic strip (5inm x 60mm) by a double sided adhesive tape. (b) Thestrips waro out to give a total of 11 amall strips (5 x 60mm). At the end of onch amall strip there was consequontly a lyophilized pad of 5mm x 5mm which contained all the reagonts. (c) The small strips were stored in dark bottles under a dessicant in a freezer (-15'C) or r4efrigerator. EXAMPLE 2 Solid compositions intended for determining ethanol concentration in serum or plasma were prepared as follows: 1. Preparation of 75 mM TRI5 Buffer ph 8.2 containing 0.84 g/100ml semicarbazide, To 0.908g of TRIS, 0.84g of samicarbazide were added and diluted to 100ml with double distilled water (adjusting pH to 8.2 with HCL). Gelatin may be added as in Step 1 of Example 1. Steps 2 to z were the same as Example 1. EXAMPLE 3 Solid compositions intended for determining ethanol concentration in saliva were prepared as follows: 1. Preparation of 138 Mm TRIS Buffer pH 8.0 containing 0.84 g/100ml sernicarbazide. To 1.671g of TRIS, 0,84 g of semicarbazide were aded and diluted to 100ml with double distilled watex (adjusting pH to 8.0 with HCL). Gelatin may be added as in Step 1 of Example it Steps 2 to 4 were the same as in Example 1. EXAMPLE 4 Solid composition for determin-ation of ethanol concentration in whole blood. Steps 1 to 3 were same as Example 2. Then, the freeze dried strips were dipped into &alpha; 1.2% ethyl cellulose solution in acetone. Acetone is then removed by drying in vacuum. step 4 is same as in Example 1. EXAMPLE 5 Solid compositions intended for determining ethanol presence in urine, serum or saliva when quantitation was not required were prepared as follows : The steps of Example 1 were carried out, except that the pyraolc was oniitd. Those skilled in the art will recognize that the sennitivity Qf detoction of different alcohol levels can ne veried depending on the specific application desired by altering the relative concentrations of pyrazole and alcohol dehydrogenase without departing from the spirit of the invent ion. In use, a solid composition in accordance with the invention and prepared for example as described in the tore- going Examples is dipped for 1-2 seconds in a fluid suspetted of containing ethanol. The intensity of the pink to red colour that develops in the solid composition is proportional to the concentration of ethanol present in the fluid. After I minute, the colour developed is compared to a colour chart. While the colour continues to develop in time, it can be fixed by briefly placing the solid composition on top of a filter paper pad that has previously been soaked in 1.OM pyrazole and dried or dipped in EICl. The solid composition can be used for precise quantitation of ethanol using spectrophotometric instruments. In order to study the accuracy of the present inven tion, ethanol concentration was determined in sample fluids by two methods: 1. A total of 920 urine samples from various populations with different alcohol comsumption habits were collected over a period of 8 weeks, and their ethanol concentration tested both by a solid composition prepared in-accordance with Example 1 and by spectrophotometric analysis. (T.P, Hadjiioannou, S.I. Hadjiioannou, et al. Automated Enzymic Determination of Ethanol in Blood, Serum, and Urine with Miniature Centrifugal Analyzer. Clinical Chemistry, Vol 22(6), 802-805 (1976)). The spectrophotometric results were then conerted to the solid composition scale as follows: > 1600 mg/L 8 6 1200 - 1000 mg/L = 5 800 - 1200 mg/L - 4 400 - 800 mg/L - 3 200 - 400 Ing/L ; 2 - 200mg/L = 1 < 50 mg/L = 0,5 0 = 0 Correlation statistics for the solid composition and the epectrophotolnetric method were calculated on the total samples and were as follows: : 920 is the number of X and Y values. (X) = ay 4 b) ean of all solid composition values (X) = 1.1353 Mean of all Spectrophotometric values (Y) = 1.1951 Slope (a) - 1.0248 Intercept (b) = 0.0315 Correlation Coefficient (r) - 0.9831 2. A total of 580 serum samples of patients who were suspected of having taken drugs, including alcohol, were tested by a solid composition prepared in accordance with Example 2 and by gas chromatography (Barbare R. Manno., Joseph E. Manno. A Simple Approach to Gas Chromotographic Microanalysis of Alcohols in Blood and Urine by a Direct Injection Technique. Journal of Analytical Toxicology. Vol. 2, 257-261, Nov-Dec. (1978)). The gas chromatographio (GC) results were converted to the solid composition scale as follows: ffi\ > 1400 mg/L = 5 1000 - 1400 mg/L = 4 600 - 1000 mg/L = 3 300 - 600 mg/L = 2 50 - 300 mg/L = l1 5- mg/L = 0.5 0 = 0 The data was then correlated and the results were as follows: : 580 is the number X and Y values Mean of all solid composition values (X) = 1.928 Mean of all GC values (Y) = 2.050 Slopn (a) = 1.037 Intercept (b) 1 0.054 Correlation Coefficient (r) = 0.9875 These correlation coefficiente (0.9831 and 0.9875) and alopae (1.0748 and 1.037) indicate that the solid composition in accordance with the invention gives ethanol concentrations that are nearly identical to those given by the use of complex spectrophotometric or gas chromatographic methods. INTERFERENCE STUDIES Interference studies were carried out with the solid composition as prepared in Example 1. TABLE 1 Druge in-vitro Acetone Hydrochlorothiazide Amitriptyline Imipramine Amobarabital Isopropanol Amphetamine Methadone Ascorbic Acid Methamphetamine Butalbital Methanol Chloridiazspoxide Methyprylon Chlorpromazine Morphine.. Cimethidine Naloxone Cocaine Nortriptyline Codeine Oxazepam Diazepam PCP Digokin Phenobarbital Dimenhydrinate Phenylpropanolamine Diphenhydramine Propoxyphane Ephedrine Propranolol Ethchlorvynol Quinine Flurazopam Salicylate Gecoberbital Drups Added in-vitre To pooled urine previously shown to contain no ethan ol by gat chromatographic procodures, drugs listed in Table 1 and ethanol were added auch that the final concentrations of both the drug and ethanol in urine were 1 mg/ml. In the case of methanol, isopropanol and ocetone, their final concentration was 3mg/ml. The urine sample were thstad with the solid composition as prepared in Example 1 in the following manner : (1) after the drug was added and (2) after ethanol was added to asmple containing the drug. The viacal colour change with each testing was recorded. A urine sample frorn the same pooi, containing lmg/ml of ethanol was used as a reference. TABLE 2 Drugs in-vivo Amitriptyline Ludiomil Amoxapine Meprobiline Amphetamine Methadone Barbiturate Methamphetamine Benzodiazepine Morphine Carbamazepine Nortriptyline Chlorpromazine Oxycodone Cimetidine Pentazocine Codeine Imipramine Desimipramine Perphenazine Diphenhydramine Phencyolidine Doxapin Pheniramine Ephedrine Phenothiazine Ethchlorvynol Phenylpropanolamine Flurazwpam Phenytoin Halopyridol Propoxyphen Hydrocodone Quinidine Quinine Drugs in-vivo 166 urine samples from various drug using populations were tested in tie following manner: 1. Drug scrocening was done using thin layor chromolographic procederes to establish the drugs content of the orine. Table 2 lists various drugs found either individually or in various combinations, Most druge usually, oppear with tholrmetabolites (not listed in the table), 2. urine was tested with the solid composition as prepered in Example 1.Visuelcolour change at 1 minute was recorded. (a) If there was a positive reading then the urine was further analyzed with gas chromatographic procedures to establish the urine ethanol leval. (b) If the solid composition did not change colour thcn ethanol was added te the urine such that the final concent ration was 100 mg/100 ml and retested with the solid composition. The visual colour change was recorded at 1 minute. For comparison, a blank urine sample containing 100 mg/ 100 ml of Ethanol was used as reference. All samples were analyzed within the day of their receipt in the laboratory. RESULTS Of INTERFERENCE STUDIES Drugs added in-Vitro Of the 37 different drugs tested none interfered with the colour, as measured visually, or gave positive ethanol readings if ethanol was absent, Urine containing methanol, isopropanol, or acetone did not give a positive reaction in the presence of ethanol. Drugs in-vivo Most of the urine samples had more than two drugs and their metabolite present. 42 urine samples had ethanol as one of the components present. In 124 samples ethanol had to be added. Samples containing large amounts of drugs or many drugs (3 or Inore) generally gave a negative bias of 1+, i.e. 6+ by gas chromatography read 5+ visually. Invariably the endogeneous levels of ethanol in these urines levels wers grester than 100 mg/100 ml. The reducible compound may comprise the co-enzyme nicotinamideadenino-dinuclectide (NAD) and the first enzyme cotalyst may comprise alcohol dehydrogonaso (ADH). The colour-changing compound may comprise a tetrazolium salt and the second enzyms cetalyst may comprise diaphorase. The tetrazolium salt may comprise iodonitroletrazolium chloride (INT), thinzolyl blue tetrazolium bromide (MTT)or nitroblue tetrazolium chloride (NBT). Alternatively, the colourchanging compound may comprise 2-.6-dichlorophenol indophenol, methylene blue or other suitable dyes or combinations thereof. The acetaldehyde trapping agent may comprise semlcar- bazide, hydroxylamine, hydrazille, or sulfhydryl reagents such as penicillamine or cysteine. The buffer may comprise phosphate, pyrophosphate buffer of tris (hydroxymethyl) aminomethane commonly known as tris buffer, The solid composition takes advantage of competitive inhibitors of alcohol denydrosenase to enable higher ethanol concentrations to be determined than would. be possible in the absence of a competitive inhibitor. The competitive inhibitor may be pyrazole, a halogenated pyrazole, methyl, ethyl, propyl, butyl or pentyl pyrazole, or a suitable steroid. In its natural Etate, alcohol dehydrogenase has a very high affinity for alcohol such that the enzyme reacts with alcohol at a maximal constant velocity even at very low concentrations of alcohol, Quantitation by this method is not possible if the concentration of ethanol exceeds 50 mg/l. since quantitation in the range of 50-1600 mg/l or higher is required it is necessary to reduce the affinity of alcohol dehydrogenase for ethanol. In the presence of a suitable concentration of the competitive inhibitor of alcohol dahydrogenase the rate at which the enzymes oxidizes alcohol, and thus it yields the colour reaction, is proportional to the concentration of. alcohol in the said ranges. Pyrazoles substituted in position 4 are more active competi tive inhibi.ors, such that the concentration required in the support system are: pyraole > halogonated pyrazole > methyl pyrazole othyl pyrazole propyl pyrazole > butyl pyrazole pentyl pyrazole. The nonceutrations of compotitive inhibitore other than pyrazole aro calculated by multiplying the concontration of pyrazole uned by the ratios K1(inhibitor)/ K1(pyrazole), where Ki's pro the inhibitor constants for the aloohol dehydrogenase reaction, In accordence with an alternate embodiment the oxygen in the body fluid may be reduced. In such a system, alcohol oxidase is employed as the catalyst to produce the corre sponding aldehyde, The chromogen employed may be chosen front auiacol, o-tolidine, or a benzidine derivative such as tetramethyl benzidine. if a quantitative indication of the alcohol is required then an inhibitor such as urea may be employed in appropriate quantities. And to effect the colour range, a second enzyme such as peroxidase is em- played. This system may be employed for the detection of lower alcohols such as methanol or ethylene glycol. The carrier may be impregnated in two discrete areas with two impregnants, one from this embodiment and from the prior embodiment so that when tested such that both areas give an indicated colour change, ethanol is present. If only one turns colour the indication will be that methanol or other alcohols is present. The second system nay be more fully understood from the following examples: It will be understood flat the alcohols which may be detected by these compositions will be methyl, ethyl or ethylene glycol. EXAMPLE 6 An impregnant for determining alcohol in serum, plasma, saliva or urine using alcohol oxidase. 1. 75 mM TRIS buffer at pH 7.5 was prepared by dissolving 0.900g of tris (hydroxymethyl) aminomethane in 100 ml of distill water. The pE was adjusted with NH4OH, 2. 0,12 g of gelatin was added to 10 ml of the above TRIS buffer, Gointin was dissolved by warming the buffer solution in a warm water bath. 3. Preparation of the reagente to be freoxo dried, (a) The following were weighed in a lyophilyzing bottle wrapped with oluminum foil. 1'. Brij-35 0.0714 gni 2. Albumin 0.1 gm 3. Peroxidase 0.0869 gm 94000 unite) To the above reagents 10 ml of the TRIS buffer containing gelatin was added and mixed gently. It was allowed to stand for 15 minutes. (b) To the above reagent mixture 300 ul (1000 units) of alcohol oxidase was added. The solution was gently mixed. (c) After 15 xninutes 100 strips of Whatman filter paper #3 were added to the solution. The size of the strip filter paper was 5 mm x 50 mm. (d) The strips were allowed to absorb the reagent for about 5 minutes and shaken gently. Any excess fluid was removed to be further used in the next batch. (e) The lyophilizing bottle containing the filter strips was placed in a freezer for at least 3 hours and subsequently freeze dried overnight. 4. Colour changing compound, Depending on the colour change desired any one or combination of the following chemicals can be made in 10 ml of acetone. Colour changing compound Concentration Final Colour a) Guiacol 10 nM 0.012g/lOml brown b) Tetramethylbenzidine 7 nM 0.018g/10ml blue green c) syringaldazille 12 n 0.045g/10ml violet d) o-Tolidine 10 nM 0.021g/lCml green e) Diaminofluorine 5 nM 0.0098g/10m1 blue 5. Inhibitor, 0.159 of urea (0.25 mM) is dissolved in minimal amount of water and added to the acetone solution(4) containing the colour changing compound The lyophilized stripn as prepared in 3e were soaked in the c@@touesolution(5) and wacean solution was docanted off, The strips were dried in a struam of dry nitrogen, 7. Draporation and storage of thc freeze dried strips. a) The atrip (5mm x 50mm) was attached to a semi rigid plestic, strip (50mm x 60mm) by a double sided adhesive tape. b) The strip was then cut to give a total of 10 smal) strips (5mm x 60m). At the end of each small strip there was consequently a lyophilized pad of 5mm x Smm which contained all the reagents. c) The small strips were stored in dark bottles under a dessicant in b refrigerator or a freezer. EXAMPLE 7 A solid composition for determining alcohol in blood using alcohol oxidase Steps 1-3 same as in Example 6. In step 4, in addition to the colour changing compound, ethyl cellulose is added to give a final concentration of 1.2% in acetone. Steps 5-7 same as in example 5. EXAMPLE 8 Solid composition for determining ethanol using alcohol Oxidase when quantitation is not reguired. step 5, the addition of urea, is omitted from Example 6 or 7. The solid composition as described in Examples 6 and reacts with both ethanol as well as methanol and ethylene glycol. When urea is used as the inhibitor at the above concentrations . of methanol and ethanol linearity between 0-1600 mg/l is achieved. Those skilled in the art will recognize that the sensitivity of detection of different alcohol levels can be varied depending on the specific application desired by altering the relative amounts of urea and alcohol oxidase without departing from the spirit of this invention. In use, the solid composition ii accordance with the invention at prepared for oxample an described in the foregoing Examples 5 and 6 is dipped for 1-2 seconde in the fluid suspected of containing the alcohol. After 1 minute the colour developed in the solid composition is compared to a colour chart. 5'he intensity of the color that devciops in the solid composition is proporticnal to the concentration of alcohol present in the fluid. In case of the fluid being blood the solid composition is either washed with a fcw drops of water or wiped with a tissue before comparing to th colour chart. As a further alternative a further system included barium oxide or barium hydroxide or a suitable agent for generating hydrogen peroxide in association with water; the enzyme catalase to oxidize the alcohol to the corresponding aldehyde; nicotinamide-adenine dinucleotide; a second enzyme, such as acetaldehyde dehydrogenase, to reduce the nucleotide in the presence of the acetaldehyde, a chromogen, such as a tetrazolium salt: and a third enzyme, such as dia phorase to reduce the tetrazolium salt in the presence of the reduced nucleotide to give a colour indicative of the amount of reduced nucleotide present. Sodium formate may be added as a competitive substrate (inhibitor) with regard to ethanol thus resulting in quantitation of ethanol on any desired range of concentrations. It will of course be understood as with the prior embodiments, suitable coatings, surfactants, stabilizers and buffers may be employed. This composition is used to impregnate the carrier as formerly and is used in the same manner with the reactants being suitably proportioned to produce the desired results. In the embodiments described freeze drying has been employed to ensure the stability of the composition. It will of course be understood that other forms of drying such as accelerated convection, at suitable temperatures, may be employed or a combination of such techniques may be em ployed. It will aino be understood that other examples or equivalente will be apparent to those skilled in the art without doparting from the spirit of the invention an defined in the appended claims.

Claims: CLAIMS 1. A solid composition for determining the presence of a lower alcohol in a biological fluid such as urine, saliva or blood comprising a solid carrier having the following constituents in effective amounts: a first enzyme for effecting oxidation of said alcohol to produce the corresponding aldehyde and a reduction product, a chromogen reactable with said aldehyde or said reduced product in the presence of second or third agents to effect a colour change indicative of an alcohol presence in the body fluid; an enzyme chosen from the group of said second or third agents or both; and a buffer to maintain a predetermined pfl. 2. A solid composition as claimed in claim 1 further including a reducible compound reactable with ethanol in the presence of said first enzyme. 3 A solid composition as claimed in claim 2 wherein said first enzyme is alcohol dehydrogenese, 4. A solid composition as claimed in claim 3 wherein said reducible compound is nicotinamide adenine dinuclcotide. 5. A solid composition as claimed in claim 1 wherein said composition further includes an inhibitor competitive of said first enzyme whereby said colour change is indicative of the quantity of said reduced compound. 6. A solid composition as claimed in claim 3 further including an acetaldehyde trapping agent. 7. A solid composition as claimed in claim 5 wherein said first enzyme j alcohol dehydrogenase and said inhibitor is chosen from the group comprising a pyrazole, a halogenated pyrazole, an alkyl pyrazole or a steroid. 8. h solid composition as claimed in claim 5 or 7 wherein said second agent is chosen from the group cornprising diaphorase, phenazine or thiazine and said chromagen is chosen from the group comprising a tetrazolium salt, 2-6-dichlorophenol, indophenol or methylene blue. 9. A solid composition as claimed in claim 1 wherein said first enzyme is alcohol oxidase and includes a competitive inhibitor for said first enzyme. 10. A solid composition as claimed in claim 1 further including a reagent chosen from the group comprising barium oxide or barium hydroxide which produces hydrogen peroxide with water, and a nucleotide reactable with said aldehyde to produce a reduced product reactable with said chromogen in the presence of one of said further agents. 11. A composition as claimed in claim 10 wherein said third agent is chosen from the group comprising diaphorase, phenazine or thiazine. 12. A composition as claimed in claim 10 or 11 further including a sulphydryl reducing agent. 13. A composition as claimed in any of claims 9 to 12 including an inert protein. 1. Ar inert carrier including a first discrete area impregnated with a first alcohol determining impregnant and a second discrete area impregnated with a second alechol datermining impregnant said first impregnant being chosen from the group including in effective amounts, nicotinamide adenine dinucleotide, alcohol dehydrogenase, a chromagen reactive it the presence of a second agent chosen from the group comprising diaphorase, phenazine or tiliazine; ; one of diaphorase, phenazine or thiazine, an acetaldohyde trapping agent, a buffer to -maintain a predctermined pH and means for stabilizing said enzymes; and a second alcohol determining impregnant including, in effective amounts; alcohol oxidase; a chromogen reactive in the presence of peroxidase; peroxidase; a buffer to maintain a predetermined pH and means to stabilize said enzymes. 15. An inert carrier as claimed in claim 1,4 wherein said first composition further includes inhibitors, competitive with said alcohol dehydrogenase or said alcohol oxidase; a chromogen reactive in the presence of peroxidase; peroxidase; a buffer to maintain a predetermined pH and means to stabilize said enzymes.

Inventors: ISRAEL, YEDY, KAPUR, BHUSHAN MADHODASS
Applicants: ALCOHOLISM AND DRUG ADDICTION RESEARCH FOUNDATION
Application year: 1984
Main classification: C12Q1
IPC codes: C12Q1