U.S. patent number 3,926,736 [Application Number 05/502,210] was granted by the patent office on 1975-12-16 for enzymatic ethanol assay.
This patent grant is currently assigned to Calbiochem. Invention is credited to Giovanni Bucolo.
United States Patent |
3,926,736 |
Bucolo |
December 16, 1975 |
Enzymatic ethanol assay
Abstract
This invention provides an improved enzymatic assay for ethanol
in body fluids, whereby a single substance acts simultaneously as a
buffer and as a trapping agent for one of the products of the
enzymatic reaction.
Inventors: |
Bucolo; Giovanni (Irvington,
NY) |
Assignee: |
Calbiochem (La Jolla,
CA)
|
Family
ID: |
23996831 |
Appl.
No.: |
05/502,210 |
Filed: |
August 30, 1974 |
Current U.S.
Class: |
435/26 |
Current CPC
Class: |
C12Q
1/32 (20130101) |
Current International
Class: |
C12Q
1/32 (20060101); C12K 001/04 () |
Field of
Search: |
;195/13.5R ;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Fan; C. A.
Attorney, Agent or Firm: Larsen; Delmar H.
Claims
Having disclosed the invention, I claim:
1. A composition of matter useful as a reagent in determining 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.
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 composition of
claim 1 dissolved in sufficient water to provide a concentration of
said buffer/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/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 composition of
claim 6 dissolved in sufficient water to provide a concentration of
said buffer/trapping agent of approximately at least 0.3 molar.
Description
This invention relates to an improvement in the enzymatic assay of
ethanol, particularly in body fluids such as saliva, serum and
urine, wherein a single chemical compound serves the dual function
of a buffer 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 dinucleotide (NAD) serves a a
coenzyme and in turn is reduced to its reduced form (NADH). 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 for that particular
enzymatic reaction concerned.
A useful trapping agent has been found to be aminooxyacetic acid,
described in connection with an ethanol assay in U.S. Pat. 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. publications just
noted, it was found that aminooxyacetic acid apparently underwent a
reaction with the NAD, leading to a blank absorbance which was
subject to random variations. This difficulty was the subject of
correspondence published in Clinical Chemistry 17 [5] 458-460
(1971). I have now discovered that two closely related compounds,
as well as mixtures 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 elimination 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 aminooxyacetic 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 -propanediol;
and 2-amino-2-methyl-1,3-propanediol. The first of these is
commonly known as "Trisamino"; and may also be named as
tris(hydroxymethyl)aminomethane. It is entered on page 1083 of the
Merck Index, 8th edition 1968, under the "generic" name of
tromethamine. 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 radical, give a pH somewhat in excess of 10 in diluted
aqueous solution. When partially neutralized with acid, such as
hydrochloric, to a pH within the general range of 8.9 to 9.2, then
they behave as buffers with a good buffer capacity, and likewise
exhibit the trapping effect 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 so as to produce a
dry powdered composition. Alternately, the components may be
intimately mixed together in their dry state, for which ball
milling is convenient. This brings about a homogeneous composition
in the form of a fine powder. Still further, enhanced stability may
be given to the combination by supplying it in two moieties. This
may conveniently 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 8 to
10 minutes at 30.degree. C, is determined. A blank is carried
through the procedure in the usual fashion so that appropriate
correction may be automatically made. The details of the actual
absorbance procedure follow 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, both of which are included herein by
reference. Of course, when the components are not provided as two
moieties, but rather as a single mixture, then a preselected
quantity of the 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 of the following substances in the quantities
named: 2-amino-2-hydroxymethyl-1,3- propanediol 842 mg.* Succinic
acid 104 mg. Ethylene diamine tetra-acetic acid, tetrasodium salt
26 mg. Alcohol dehydrogenase 216 I.U.** NAD 17.2 mg. *Milligrams?
**International Units?
The foregoing ingredients are first separately dried by desiccation
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 carrying 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 (0.9% 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
is conveniently carried out in parallel fashion in which 0.1 ml of
the saline solution alone is added to 2.6 ml of the assay solution.
These operations are conveniently carried out by adding the
solutions to a cuvet adapted to photometric measurements. The two
cuvets are held at 30.degree. C 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 calculated by
anyone skilled in the art and indeed the particular apparatus used
may conveniently be calibrated by carrying out a series of tests
using diluted alcohol of varying concentrations.
EXAMPLE 2.
The composition of Example 1 and the procedures set forth therein
are carried out as stated, except that in place of 842 mg of
2-amino-2-hydroxymethyl-1,3-propanediol there are used 733 mg of
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-hydroxymethyl-1,3-propanediol there are used 421 mg of
2-amino-2-hydroxymethyl-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
acid, tetrasodium salt, is omitted.
EXAMPLE 5.
The composition and procedures of Example 2 are carried 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 composition of
Example 1 is used.
As regards relative proportion of the several components, typical
quantities appear in the examples hereinabove. It is convenient 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 five assays, as will be clear from the details given
therein. Thus, I prefer the following quantities, per five-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 varied
within the limits set forth above then the assay solution should be
calibrated by the use of a known quantity of dilute alcohol.
In any case, in order to achieve efficient 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
accordance with the invention have a natural alkalinity somewhat
higher than the optimum pH for the enzymatic reaction concerned so
that I include an acid in the inventive composition. Where the
composition is prepared by mixing together dry ingredients then a
solid acid is desirable, particularly one that does not have any
water of crystallization in the molecule. Succinic acid is well
adapted for this purpose and in general I find 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
proportions of acid and buffer/trapping agent are selected to give
a pH in the range already stated when the solid assay mixture is
dissolved in water in such proportion as to give a molarity of at
least 0.3 for the buffer/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 of any contamination by heavy metal ions. It is sometimes
difficult to find commercial sources of the buffer/trapping agents
disclosed which are sufficiently pure in this regard although, of
course, they may be purified by anyone wishing to practise the
invention. By way of a safeguard, accordingly, 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 of 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, for example, diethyline triamine penta-acetic acid;
8-hydroxyquinoline; orthophenanthroline; bipyridyl; and many others
known to those skilled in the art of coordination chemistry. The
sequestrant may typically be employed in quantities of from about 1
to 5 parts per 100 of the buffer/trapping agent.
It will be understood that while I have explained the invention
with the aid of specific examples, nevertheless considerable
variation is possible in choice of materials, proportions, reaction
conditions, and the like, with the broad scope of the invention as
set forth in the claims that follow.
* * * * *