Note: Descriptions are shown in the official language in which they were submitted.
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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 `
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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
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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
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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
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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
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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
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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.
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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. :
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