CA1045961A - Enzymatic ethanol assay - Google Patents
Enzymatic ethanol assayInfo
- Publication number
- CA1045961A CA1045961A CA237,298A CA237298A CA1045961A CA 1045961 A CA1045961 A CA 1045961A CA 237298 A CA237298 A CA 237298A CA 1045961 A CA1045961 A CA 1045961A
- Authority
- CA
- Canada
- Prior art keywords
- composition
- acetaldehyde
- trapping agent
- buffer
- acid
- 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.)
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Abstract of the Disclosure A composition useful for determining the ethanol content of body fluids consisting essentially of alcohol dehydrogenase, nicotinamide adenine dinucleotide, a buffer acetaldehyde/trapping agent and an acid.
Description
~O~S9~i~
This invention relates to an improvement in the enzyma~ic assay of ethanol, particularly in body fluids such as saliva, serum an~ urine, wherein a single chemical compound serves the dual function o~ a bufer and a trapping agent by which one of the products of the reaction involved is removed.
A useful method of determining the ethanol content of fluids, and particularly of body fluids when used as a test of recent alcohol ingestion, utilizes the enzymatic reaction whereby ethanol is transformed to acetaldehyde by the action of alcohol ;
dehydrogenase in which nicotinamide adenine di~ucleotide (NAD) serves as a coenzyme and in turn is reduced to its reduced form (NAD~). The course and extent of the reaction are determined by ultraviolet colorimetry at 340 nanometers, at which wave length NAD does not absorb the ultraviolet light but NADH does.
In order for the reaction to be utilized in a practical assay it is necessary for the reaction to go to completion, and this, of course, is not insured by the presence of the enzyme and coenzyme alone. Rather, it is necessary to sequester or to trap in some fashion one of the reaction products, of which the most practical one is the acetaldehyde. Also, as indeed in most enzymatic reactions, the presence of a buffer is necessary to `~
insure that a pH is maintained within the optimum range Eor that particular enzymatic reaction concerned.
A useful trapping agent has been ~ound to be aminooxyacetic acid, described in connection with an ethanol assay in U.S.
Patent No. 3,493,467, of William Drell et al and in Clinical ;
Chemistry 16 402-407 (1970), in an article by Jones, Gerber and Drell.
Subsequent to the appearance of the Drell et al publica-30 tions just noted, it was found that aminooxyacetic acid appar- ~'~
ently underwent a reaction with the NAD, leading to a blank absorbance which was subject to random variations. This `
This invention relates to an improvement in the enzyma~ic assay of ethanol, particularly in body fluids such as saliva, serum an~ urine, wherein a single chemical compound serves the dual function o~ a bufer and a trapping agent by which one of the products of the reaction involved is removed.
A useful method of determining the ethanol content of fluids, and particularly of body fluids when used as a test of recent alcohol ingestion, utilizes the enzymatic reaction whereby ethanol is transformed to acetaldehyde by the action of alcohol ;
dehydrogenase in which nicotinamide adenine di~ucleotide (NAD) serves as a coenzyme and in turn is reduced to its reduced form (NAD~). The course and extent of the reaction are determined by ultraviolet colorimetry at 340 nanometers, at which wave length NAD does not absorb the ultraviolet light but NADH does.
In order for the reaction to be utilized in a practical assay it is necessary for the reaction to go to completion, and this, of course, is not insured by the presence of the enzyme and coenzyme alone. Rather, it is necessary to sequester or to trap in some fashion one of the reaction products, of which the most practical one is the acetaldehyde. Also, as indeed in most enzymatic reactions, the presence of a buffer is necessary to `~
insure that a pH is maintained within the optimum range Eor that particular enzymatic reaction concerned.
A useful trapping agent has been ~ound to be aminooxyacetic acid, described in connection with an ethanol assay in U.S.
Patent No. 3,493,467, of William Drell et al and in Clinical ;
Chemistry 16 402-407 (1970), in an article by Jones, Gerber and Drell.
Subsequent to the appearance of the Drell et al publica-30 tions just noted, it was found that aminooxyacetic acid appar- ~'~
ently underwent a reaction with the NAD, leading to a blank absorbance which was subject to random variations. This `
-2-,1,,,. .. . .......................................... :
.-. . : , :
. . ~ , . ~ ..
~0~596~
difficulty was the subiect of correspondence published in Clin-ical Chemistry 17 Cs~ 458-460 (1971). I have now discovered that two closely related compounds, as well as mix~ures of the two in any proportion, not only furnish the desired and requisite buffer capacity for an assay mixture based upon the enzymatic reaction set forth hereinabove, but enable the complete elimina- ;
tion of the aminooxyacetic acid, these compounds or mixtures of the two quite surprisingly serving as a trapping agent for the - acetaldehyde generated by the reaction. Elimination of the amino-oxyacetic acid leads to a stable assay mixture in which the blank absorbance is substantially constant and predictable and also enables a considerable simplification to be made in formulating the assay mixture since a single substance now serves two separate functions.
The two compounds are 2-amino-2-hydroxymethyl-1,3-propane-diol; and 2-amino-2-methyl-1,3-propanediol. The first of these is commonly known as "Trisamino"; and may also be named as tris (hydroxymethyllaminomethane. It is entered on page 1083 of the Merck Index, 8th edition 1968, under the "generic" name of tro-methamine. The second compound recited appears on page 57 of the Merck Index under the Geneva nomenclature given above. Both of these compounds, which as will be observed, differ only by one hydroxyl radiaal, give a pH somewhat in excess of 10 in diluted a~ueous solution. When partially neutralized with acid, such as hydrochloric, to a pH within the general range of 8.9 to 9.2, then they hehave as buffers with a good buffer capacity, and like-wise exhibit the trapping efect already mentioned.
Any of the usual methods common in this art for putting ;
together the individual components so as to form the inventive combination may be used. For example, all of the components may be put into a single solution, which may conveniently be water, and this may then be freeze dried followed by grinding or sieving , ... . .
~4S96~
so as to produce a dry powdered composition. Alternately, the components m~y be intim~-tely mixed together in their dry state, for which ball milling is convenient. This brings about a homo-geneous composition in the form of a fine powder. Still further, enhanced stability may be given to be combination by supplying it in two moieties. This may conven.iently comprise the NAD as one moiety and the balance of the components as the second~ In order to carry out the assay, preselected quantities of each are dissolved in water and mixed together. The fluid to be assayed for its ethanol content is added in a preselected volume~ and the absorbance of the solution before and after incubation under selected conditions, typically for eight to ten minutes at 30C, is determined. A blank is carried through the procedure in the usual fashion qo that appropriate correction may be automatically made. The details of the actual absorbance procedure ollow the customary sequence, and need not be set forth in detail here. A
typical and recommendable protocol may be found in the Jones et al 1970 article and in the Drell et al patent cited here and :.
above. Of course, when the components are not provided as two moieties, but rather as a single mixture, then a preselected :~
quantity of single mixture is dissolved in water and the assay ~ ~ .
carried out as already described.
Some working examples will now be given.
Example 1.
A dry.blend is made o~ the following substances in the quan- ;
tities named~
2-amino-2-hydroxymethyl-1,3-propanediol842 mg*
Succinic acid 104 mg Ethylene diamine tetra-acetic acid, tetrasodium salt26 mg Alcohol dehydrogenase 216 I.U.**
NAD 17.2 mg * Milligrams ** International Units ?
~, . . .
.. . :
The oregoing lngr2d~ents are Eirs~ ~eparately dried hy desic-cation at room temperature, conveniently over phosphorus pentoxide, and then dry blended to form the inventive composition. For use, 13.5 milliliters (ml) of water are added and the composition is permitted to dissolve. For carryin~ out the assay, a suitable quantity of body fluid such as 0.1 ml of serum, saliva, or the like is diluted with 4.9 ml of water or if preferred of isotonic saline tO.9% aqueous sodium chloride~. This is mixed with the assay ~
solution in the proportion of 0.1 ml of the diluted serum or the ~ ~.
like as already described and 2.6 ml of the assay solution. A
blank ls conveniently carried out in parallel fashion in which 0.1 ml of the saline solu~ion alone is added to 2.6 ml of the assay solution. These operations are conveniently carried out by adding the solutions to a cuvette adapted to photometric measurements.
The two cuvettes are held at 30C for 8 to 10 minutes, whereupon the ultraviolet absorbance of both is determined at 340 nm. The amount of alcohol present in the body fluid may be readily calcu-lated by anyone skilled in the art and indeed the particular appar-atus used may conveniently be calibrated by carrying out a series o tests using diluted alcohol of varying concentrations.
Example 2.
The composition of Example 1 and the procedures set forth therein are carried out as statedl except that in place of 842 mg of 2-amino-2-hydroxymethyl-1,3-propanediol there are used 733 mg o~ 2-amino-2-methyl-1,3-propanediol.
Example 3.
The composition and procedures of Example 1 are carried out as stated except that in place of 842 mg of 2-amino-2-hydroxy-methyl-1,3-propanediol there are used 421 mg of 2-amino-2-hydroxy-methyl 1,3-propanediol together with 369 mg of 2-amino-2-methyl-1,
.-. . : , :
. . ~ , . ~ ..
~0~596~
difficulty was the subiect of correspondence published in Clin-ical Chemistry 17 Cs~ 458-460 (1971). I have now discovered that two closely related compounds, as well as mix~ures of the two in any proportion, not only furnish the desired and requisite buffer capacity for an assay mixture based upon the enzymatic reaction set forth hereinabove, but enable the complete elimina- ;
tion of the aminooxyacetic acid, these compounds or mixtures of the two quite surprisingly serving as a trapping agent for the - acetaldehyde generated by the reaction. Elimination of the amino-oxyacetic acid leads to a stable assay mixture in which the blank absorbance is substantially constant and predictable and also enables a considerable simplification to be made in formulating the assay mixture since a single substance now serves two separate functions.
The two compounds are 2-amino-2-hydroxymethyl-1,3-propane-diol; and 2-amino-2-methyl-1,3-propanediol. The first of these is commonly known as "Trisamino"; and may also be named as tris (hydroxymethyllaminomethane. It is entered on page 1083 of the Merck Index, 8th edition 1968, under the "generic" name of tro-methamine. The second compound recited appears on page 57 of the Merck Index under the Geneva nomenclature given above. Both of these compounds, which as will be observed, differ only by one hydroxyl radiaal, give a pH somewhat in excess of 10 in diluted a~ueous solution. When partially neutralized with acid, such as hydrochloric, to a pH within the general range of 8.9 to 9.2, then they hehave as buffers with a good buffer capacity, and like-wise exhibit the trapping efect already mentioned.
Any of the usual methods common in this art for putting ;
together the individual components so as to form the inventive combination may be used. For example, all of the components may be put into a single solution, which may conveniently be water, and this may then be freeze dried followed by grinding or sieving , ... . .
~4S96~
so as to produce a dry powdered composition. Alternately, the components m~y be intim~-tely mixed together in their dry state, for which ball milling is convenient. This brings about a homo-geneous composition in the form of a fine powder. Still further, enhanced stability may be given to be combination by supplying it in two moieties. This may conven.iently comprise the NAD as one moiety and the balance of the components as the second~ In order to carry out the assay, preselected quantities of each are dissolved in water and mixed together. The fluid to be assayed for its ethanol content is added in a preselected volume~ and the absorbance of the solution before and after incubation under selected conditions, typically for eight to ten minutes at 30C, is determined. A blank is carried through the procedure in the usual fashion qo that appropriate correction may be automatically made. The details of the actual absorbance procedure ollow the customary sequence, and need not be set forth in detail here. A
typical and recommendable protocol may be found in the Jones et al 1970 article and in the Drell et al patent cited here and :.
above. Of course, when the components are not provided as two moieties, but rather as a single mixture, then a preselected :~
quantity of single mixture is dissolved in water and the assay ~ ~ .
carried out as already described.
Some working examples will now be given.
Example 1.
A dry.blend is made o~ the following substances in the quan- ;
tities named~
2-amino-2-hydroxymethyl-1,3-propanediol842 mg*
Succinic acid 104 mg Ethylene diamine tetra-acetic acid, tetrasodium salt26 mg Alcohol dehydrogenase 216 I.U.**
NAD 17.2 mg * Milligrams ** International Units ?
~, . . .
.. . :
The oregoing lngr2d~ents are Eirs~ ~eparately dried hy desic-cation at room temperature, conveniently over phosphorus pentoxide, and then dry blended to form the inventive composition. For use, 13.5 milliliters (ml) of water are added and the composition is permitted to dissolve. For carryin~ out the assay, a suitable quantity of body fluid such as 0.1 ml of serum, saliva, or the like is diluted with 4.9 ml of water or if preferred of isotonic saline tO.9% aqueous sodium chloride~. This is mixed with the assay ~
solution in the proportion of 0.1 ml of the diluted serum or the ~ ~.
like as already described and 2.6 ml of the assay solution. A
blank ls conveniently carried out in parallel fashion in which 0.1 ml of the saline solu~ion alone is added to 2.6 ml of the assay solution. These operations are conveniently carried out by adding the solutions to a cuvette adapted to photometric measurements.
The two cuvettes are held at 30C for 8 to 10 minutes, whereupon the ultraviolet absorbance of both is determined at 340 nm. The amount of alcohol present in the body fluid may be readily calcu-lated by anyone skilled in the art and indeed the particular appar-atus used may conveniently be calibrated by carrying out a series o tests using diluted alcohol of varying concentrations.
Example 2.
The composition of Example 1 and the procedures set forth therein are carried out as statedl except that in place of 842 mg of 2-amino-2-hydroxymethyl-1,3-propanediol there are used 733 mg o~ 2-amino-2-methyl-1,3-propanediol.
Example 3.
The composition and procedures of Example 1 are carried out as stated except that in place of 842 mg of 2-amino-2-hydroxy-methyl-1,3-propanediol there are used 421 mg of 2-amino-2-hydroxy-methyl 1,3-propanediol together with 369 mg of 2-amino-2-methyl-1,
3-propanediol.
Example 4.
The composition and procedures of Example 1 are carried out as stated with the exception that the ethylene diamine tetra-acetic .. ~ . ~ .
... : ;
. . . . .. .. . . .. .
~0~ 6~
acid, tetrasodium salt, is omitted.
Example 5.
The composition and procedures of Example 2 are c~rried out as stated with the exception that the ethylene diamine tetra-acetic acid, tetrasodium salt, is omitted.
Example 6.
The composition and procedures of Example 3 are carried out as stated with the exception that the ethylene diamine tetra-acetic acid, tetrasodium salt, is omitted.
Example 7.
The composition of Example 1 is prepared and dissolved in 13.5 ml of distilled water. The solution is then freeze-dried to yield a dry, homogeneous mixture, which is used for carrying out the assay as already described in Example 1.
Example 8.
The procedure of Example 7 is carried out as stated therein except that the composition of Example 4 instead of the composi-tion of Example 1 is used.
As regards relative proportion of the several components, typical quantities appear in the examples hereinabove. It is con- ;
venient to consider the quantities needed for a single assay, for which for purposes of discussion, one may assume 2.6 ml of assay solution per test, as shown in Example 1. The quantities shown in Example 1 are sufficient for ~ive assays, as will be clear from the detalls given therein. Thus, I prefer the following quantities, per fivP-test batch of dry components.
1) buffer/trapping agent 6.99 millimoles 2) acid - sufficient to give pH in range of 8.8-9.2 3) alcohol dehydrogenase 200-220 I.U. ~ -
Example 4.
The composition and procedures of Example 1 are carried out as stated with the exception that the ethylene diamine tetra-acetic .. ~ . ~ .
... : ;
. . . . .. .. . . .. .
~0~ 6~
acid, tetrasodium salt, is omitted.
Example 5.
The composition and procedures of Example 2 are c~rried out as stated with the exception that the ethylene diamine tetra-acetic acid, tetrasodium salt, is omitted.
Example 6.
The composition and procedures of Example 3 are carried out as stated with the exception that the ethylene diamine tetra-acetic acid, tetrasodium salt, is omitted.
Example 7.
The composition of Example 1 is prepared and dissolved in 13.5 ml of distilled water. The solution is then freeze-dried to yield a dry, homogeneous mixture, which is used for carrying out the assay as already described in Example 1.
Example 8.
The procedure of Example 7 is carried out as stated therein except that the composition of Example 4 instead of the composi-tion of Example 1 is used.
As regards relative proportion of the several components, typical quantities appear in the examples hereinabove. It is con- ;
venient to consider the quantities needed for a single assay, for which for purposes of discussion, one may assume 2.6 ml of assay solution per test, as shown in Example 1. The quantities shown in Example 1 are sufficient for ~ive assays, as will be clear from the detalls given therein. Thus, I prefer the following quantities, per fivP-test batch of dry components.
1) buffer/trapping agent 6.99 millimoles 2) acid - sufficient to give pH in range of 8.8-9.2 3) alcohol dehydrogenase 200-220 I.U. ~ -
4) NAD 16-18 mg
5) heavy metal sequestrant 10-50 mg It will be readily appreciated that when the composition is varried within the limits set forth above then the assay solution ' ~: , . ' ' ~ . .
~04~9~i~
sh~uld be calibrated by the use o~ a known quantity o dilute alcohol.
In any case, in order to achieve eEficient trapping of the acetaldehyde produced during the enzymatic reaction, the buffer/
trapping agent should be present in the assay mixture solution at a concentration of at least 0.3 molar. An excess does no harm, but is of course wasteful. A practical upper limit is 3 molar, although even this may be exceeded without affecting the inventive assay.
As mentioned, both species of the buffer/trapping agent used in accoraance with the invention have a natural alkalinity some-what higher than the optimum pH for the enzymatic reaction con-cerned so that I include an acid in the inventive composition.
Where the composition is prepared by mixing together dry ingred-ients then a solid acid is desirable, particularly one that does not have any water of crystallization in the molecule. Succinic acid is well adapted fo~r this purpose and in general I ind it best. Tartaric acid in any of its isomeric forms may likewise be used as well as other solid acids, such as, for example fumaric, gluconic, citric, malic and the like. Where the assay composition is prepared by a preliminary dissolution and then freeze-dried, the acids already named may be used and non-solid acids as well, such as hydrochloric, sulphuric, phosphoric and the like. The relative proportion9 o acid and buffer/trapping agent axe selected to give a pH in the range already stated when the solid assay mix-ture is dissolved in water in such proportion as to give a molar-ity o at least 0.3 or the buer/trapping agent. Needless to say, the weight proportion will vary depending on the particular acid or mixture of acids selected. A typical weight ratio appears in the examples.
I prefer and find it best to select components which are entirely free o~ any contamination by heavy metal ions. It is i~
.
:~ , lV45g~L
sometimes difficult to ~ind commercial sources of the buffer/
trapping agents disclosed which are sufficien~ly pure in this regard although, of cour5e, they may be purifiea by anyone wish-ing to practice the invention. By way of a safeguard, accord-ingly, I find it convenient to include a heavy metal sequestrant in the inventive composition, even when it is believed that the individual components are sufficiently pure. Indeed, in any case this guards against nickel, chromium or iron ions which might inadvertently be introduced if the user employs water which had been in contact with stainless steel. The heavy metal sequestrant which I prefer and find best is ethylene diamine tetra-acetic acid, which may be added as an acid or as any oE its sodium salts.
The amount used is small in any case, so that whether it is added as the acid or salt form is not critical particularly since any acid contribution therefrom will automatically be compensated for when a determination is made of the quantity of the selected acid needed to achieve a pH in the stated range. Many other heavy metal sequestrants may be employed, such as, or example, diethy-line triamine penta-acetic acid; 8-hydroxyquinoline; orthophen-anthroline; bipyridyl; and many others known to those skilled inthe art of coordination chemistry. The sequestrant may typically be employed in quantities of from about one to five parts per 100 o the buer/trapping agent.
The assay method and composition disclosed herein have util-ity in fields quite diferent rom clinical chemistry. For example, it is often desirable in industrial processes to be able ~o determine the ethanol content o various process liquids, such ;
as spent distillery liquors, and in the reclamation and treatment of water which is possibly subject to fermentative pollution, as may occur in rivers and estuaries receiving assorted industrial wastes. The procedure is thus a manner of manufacture.
~;
4596~
It will be understood that while I have explained the inven-tion with the aid of specific examples, nevertheless consider-able variation i~ possible in choice of materials, proportions, reactions conditions, and the like, with the bxoad scope o~ the invention as set forth in the claims that follow. :
_g_ . : .. :. . .,... :
~04~9~i~
sh~uld be calibrated by the use o~ a known quantity o dilute alcohol.
In any case, in order to achieve eEficient trapping of the acetaldehyde produced during the enzymatic reaction, the buffer/
trapping agent should be present in the assay mixture solution at a concentration of at least 0.3 molar. An excess does no harm, but is of course wasteful. A practical upper limit is 3 molar, although even this may be exceeded without affecting the inventive assay.
As mentioned, both species of the buffer/trapping agent used in accoraance with the invention have a natural alkalinity some-what higher than the optimum pH for the enzymatic reaction con-cerned so that I include an acid in the inventive composition.
Where the composition is prepared by mixing together dry ingred-ients then a solid acid is desirable, particularly one that does not have any water of crystallization in the molecule. Succinic acid is well adapted fo~r this purpose and in general I ind it best. Tartaric acid in any of its isomeric forms may likewise be used as well as other solid acids, such as, for example fumaric, gluconic, citric, malic and the like. Where the assay composition is prepared by a preliminary dissolution and then freeze-dried, the acids already named may be used and non-solid acids as well, such as hydrochloric, sulphuric, phosphoric and the like. The relative proportion9 o acid and buffer/trapping agent axe selected to give a pH in the range already stated when the solid assay mix-ture is dissolved in water in such proportion as to give a molar-ity o at least 0.3 or the buer/trapping agent. Needless to say, the weight proportion will vary depending on the particular acid or mixture of acids selected. A typical weight ratio appears in the examples.
I prefer and find it best to select components which are entirely free o~ any contamination by heavy metal ions. It is i~
.
:~ , lV45g~L
sometimes difficult to ~ind commercial sources of the buffer/
trapping agents disclosed which are sufficien~ly pure in this regard although, of cour5e, they may be purifiea by anyone wish-ing to practice the invention. By way of a safeguard, accord-ingly, I find it convenient to include a heavy metal sequestrant in the inventive composition, even when it is believed that the individual components are sufficiently pure. Indeed, in any case this guards against nickel, chromium or iron ions which might inadvertently be introduced if the user employs water which had been in contact with stainless steel. The heavy metal sequestrant which I prefer and find best is ethylene diamine tetra-acetic acid, which may be added as an acid or as any oE its sodium salts.
The amount used is small in any case, so that whether it is added as the acid or salt form is not critical particularly since any acid contribution therefrom will automatically be compensated for when a determination is made of the quantity of the selected acid needed to achieve a pH in the stated range. Many other heavy metal sequestrants may be employed, such as, or example, diethy-line triamine penta-acetic acid; 8-hydroxyquinoline; orthophen-anthroline; bipyridyl; and many others known to those skilled inthe art of coordination chemistry. The sequestrant may typically be employed in quantities of from about one to five parts per 100 o the buer/trapping agent.
The assay method and composition disclosed herein have util-ity in fields quite diferent rom clinical chemistry. For example, it is often desirable in industrial processes to be able ~o determine the ethanol content o various process liquids, such ;
as spent distillery liquors, and in the reclamation and treatment of water which is possibly subject to fermentative pollution, as may occur in rivers and estuaries receiving assorted industrial wastes. The procedure is thus a manner of manufacture.
~;
4596~
It will be understood that while I have explained the inven-tion with the aid of specific examples, nevertheless consider-able variation i~ possible in choice of materials, proportions, reactions conditions, and the like, with the bxoad scope o~ the invention as set forth in the claims that follow. :
_g_ . : .. :. . .,... :
Claims (7)
1. A composition of matter useful as a reagent in determin-ing the ethanol content of body fluids consisting essentially of:
a) the enzyme alcohol dehydrogenase;
b) the coenzyme nicotinamide adenine dinucleotide;
c) a buffer/acetaldehyde-trapping agent selected from the group consisting of 2-amino-2-hydroxymethyl-1,3-propanediol, 2-amino-2-methyl-1,3-propanediol and mixtures thereof;
d) an acid in quantity sufficient to lower the pH of an aqueous solution of said mixture to within the range of about 8.8 to 9.2;
said mixture being free of any other acetaldehyde trapping agent.
a) the enzyme alcohol dehydrogenase;
b) the coenzyme nicotinamide adenine dinucleotide;
c) a buffer/acetaldehyde-trapping agent selected from the group consisting of 2-amino-2-hydroxymethyl-1,3-propanediol, 2-amino-2-methyl-1,3-propanediol and mixtures thereof;
d) an acid in quantity sufficient to lower the pH of an aqueous solution of said mixture to within the range of about 8.8 to 9.2;
said mixture being free of any other acetaldehyde trapping agent.
2. The composition of Claim 1 wherein said composition is present in the form of two moieties, one containing said coenzyme and the other the balance of the recited components.
3. The composition of Claim 1 which includes a heavy metal sequestering agent.
4. The composition of Claim 3 in which said sequestering agent is ethylene diamine tetra-acetic acid, or a sodium salt thereof.
5. An assay solution useful in the determination of the ethanol content of body fluids consisting essentially of the com-position of Claim 1 dissolved in sufficient water to provide a concentration of said buffer/acetaldehyde-trapping agent of at least 0.3 molar.
6. A composition in accordance with Claim 1 wherein, for each 6.99 millimoles of said buffer/acetaldehyde-trapping agent, 16-18 mg of NAD; and from 200-220 I.U. of alcohol dehydrogenase are present.
7. An assay solution useful in the determination of the ethanol content of body fluids consisting essentially of the com-position of Claim 6 dissolved in sufficient water to provide a concentration of said buffer/acetaldehyde-trapping agent of approximately at least 0.3 molar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA237,298A CA1045961A (en) | 1975-10-08 | 1975-10-08 | Enzymatic ethanol assay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA237,298A CA1045961A (en) | 1975-10-08 | 1975-10-08 | Enzymatic ethanol assay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1045961A true CA1045961A (en) | 1979-01-09 |
Family
ID=4104224
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA237,298A Expired CA1045961A (en) | 1975-10-08 | 1975-10-08 | Enzymatic ethanol assay |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1045961A (en) |
-
1975
- 1975-10-08 CA CA237,298A patent/CA1045961A/en not_active Expired
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