Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
122~L0(~8
The present invention relates to the quantitative and
qualitative determination of alcohol in biological fluids such
as urine, saliva, serwrl or whole blood.
The effect of alcohol on human beings is a significant
problem from both a social and medical viewpoint. The conse-
quences of the impaired behaviour of vehicle operators and of
persons who consume excessive amounts of alcohol is increasing-
ly 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, rnay cause permanent damage.
At the present time various techniques are available for
this purpose but such techniques necessitate the use of equip-
ment which is e.Ypensive, cumbersome, not sufficiently accurate,
or take an unacceptable amount of time to determine the alcohol
concentration.
It is therefore an object o,f this invention to provide
an inexpensive, quick and reliable means of determining accur-
ately the alcohol concentration in a biological fluid and a
clinically significant identity of the alcohol.
In accordance with one asnect of the invention, a quan-
titative determination Or the alcohol level in body fluids is
provided; in anotller as~ect a qualitative deterlnination of the
alcohol level in the body fluids is provided; and in yet an-
other aspect a simultalleous quantitative and qualitative deter-
mination of the alcohol levels in the body fluids may be pro-
vided.
In accordance with tlle present invention, a solid car-
~,
rier is impregnated with the alcoh~ c~ng 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
presence of a first enzyme to produce the corresponding acetal-
dehyde and the reduced compound; the first enzyme; a competi-
tive inhibitor for the first enzyme; a chromogen, a colour
changing compound which, in the presence of the reduced com-
pound and a second enzyme, or with phenazine or thiazine, pro-
duces a colour change which represents the concentration of
alcohol present in the body fluid; the second enzyme or phena-
zine or thiazineJ 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 re-
act with ethanol but not with methanol so as to permit a dif-
ferential determination between methanol and the other lower
alcohols.
In accordance with a second embodiment, the carrier may
be impregnated with an enzyme which catalyzes a reaction be-
tween the alcohol and the free oxygen in the body fluid to pro-
duce an oxidized compound and hydrogen peroxide; a chromogen
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 in-
hibitors for the first enzymes are employed.
In accordance with a further embodiment, the carrier may
be impregnated with a constituent which, with water, produces
hydrogen peroxide; a first enzyme catalyst by which the ethanol
is oxidized by hydrogen peroxide to the corresponding acetalde-
hyde; a nucleotide; a second enzyme to reduce the nucleotide in
the presence of acetaldehyde; a chromogen, a colour-changing
~L2~
compound which, in the presence of the reduced nucleotide and
of A third enzyme, or with phenazine or thiazine, produces a
colour change which represents the concentration of alcohol
present in the body fluid; the third enzyme and a buffer to
maintaine desired pH.
If the enzymes are not covalently immobilized then an
inert protein or proteins such as gelatin or albumin may be
added to stabilize the enzymes while serving to enhance the
colours produced.
It will also be appreciated that surfactants such a
polyoxy-ethylene-23-laurylether known by the trademark BRIJ-35,
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, kiesel-
guhr or microcrystalline cellulose. The inert powder may be
compounded with the active ingredients and compressed by known
methods into tablet form 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 be coated to enhance the optical qualities of
the final colour formed.
A semipermeable coating may also be provided on the im-
pregnated carrier over the impregnant. Such coatings may be
formed in a conventional manner from materials such as cello-
phane, 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 con-
centrations in urine or saliva were prepared as follows:
1. Preparation of 125 mM TRIS Buffer pH 8.2 containing
~2Zl~
0.84g/100 ml semicarbazide. To 1.514g of TRIS, 0.84g of semi~
carbazine were added and diluted to lOOml with double distilled
water (adjusting pH to A.2 with ~ICl). At this point gelatin
may be added. If gelatin is added the solution is brought to
37C and 1.5g of gelatin added.
2. Preparation of stock solution of 25 mM pyrazole. 0.17g
of pyrazole were dissolved in lOOml double distilled water.
3. Preparation of reagents to be freeze dried:
(a) The following were weighed in a lyophilyzing hottle
10 wrapped with aluminum foil to protect from light:
1. Diaphorase 0.170 g (766 Units)
2. NAH 0.477 g
3. AD~ (5250 Units3
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 0.37ml of 25 mM pyrazole were added.
(c) The solution was gently mixed. After 15 minutes,
120 strips of Whatman filter paper #3 were added to the solu-
tion in a 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 5 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.
4. Preparation and storage of freeze dried strips:
(a) The freeze dried strip (5mm x 55mm) was attached to
a semi-rigid plastic strip (55mm x 60mm) by a double sided ad-
hesive tape.
-- 4 -
~22~8
(b) The strips were cut to give a total of 11 small
s-trips (5 x 60mm). A~ the end of each small strip there was
consequently a lyophilized pad of 5mm x 5mm which contained all
the reagents.
(c3 The small strips were stored in dark bottles under
a dessicant in a freezer (-15C) or refrigerator.
EXAMPLE 2
Solid compositions intended for determining ethanol con-
centration in serum or plasma were prepared as follows:
1. Preparation of 75 mM TRIS Buffer pH 8.2 containing 0.84
g/lOOml semicarbazide. To 0.908g of TRIS, 0.84g of semicarba-
zide were added and diluted to lOOml 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 4 were the same as Example 1.
EXAMPLE 3
Solid compositions intended for determining ethanol con-
centration in saliva were prepared as follows:
1. Preparation of 138 Mm TRIS Buffer pH 8.0 containing 0.84
g/lOOml semicarbazide. To 1.671g of TRIS, 0.84 g of semicarba-
zide were added and diluted to lOOml with double distilled
water (adjusting pH to 8.0 with HCL). Gelatin may be added as
in Step 1 of Example 1.
Steps 2 to 4 were the same as in Example 1.
EXAMPLE 4
Solid composition for determination of ethanol concen-
tration in whole blood.
Steps 1 to 3 were same as Example 2.
Then, the freeze dried strips were dipped into a 1.2~
ethyl cellulose solution in acetone. Acetone is then removed
by drying in vacuum.
Step 4 is same as in Example 1.
~2~0~3
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
pyrazole was omitted.
Those skilled in the art will recognize that the sensi-
tivity of detection of different alcohol levels can be varied
depending on the specific application desired by altering the
relative concentrations of pyrazole and alcohol dehydrogenase
without departing from the spirit of the invention.
In use, a solid composition in accordance with the in-
vention and prepared for example as described in the foregoing
Examples is dipped for 1-2 seconds in a fluid suspected of con-
taining ethanol. The intensity of the pink to red colour that
develops in the solid composition is proportional to the con-
centration of ethanol present in the fluid. After 1 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 l.OM pyrazole and dried or dipped
in HCl. The solid composition can be used for precise quanti-
tation of ethanol using spectrophotometric instruments.
In order to study the accuracy of the present invention,
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
008
Ethanol in Blood, Serum, and Urine with Miniature Centrifugal
Analyzer. Clinical Chemistry, Vol 22(6), 802-805 (1976)). The
spectrophotometric results were then converted to the solid
composition scale as follows:
>1600 mg/L = 6
1200 -1600 mg/L = 5
800 -1200 mg/L = 4
400 -800 mg/L = 3
200 -400 mg/L = 2
10 50 -200 mg/L = 1
< 50 mg/L = 0.5
O = O
Correlation statistics for the solid composition and the spec-
trophotometric method were calculated on the total samples and
were as follows:
920 is the number of X and Y values. (X) = ay + b)
Mean 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 sus-
pected of having taken drugs, including alcohol, were tested by
a solid composition prepared in accordance with Example 2 and
by gas chromatography (Barbara R. Manno., Joseph E. Manno. A
Simple Approach to Gas Chromotographic Microanalysis of Alco-
hols in Blood and Urine by a Direct-Injection Technique.
Journal of Analytical Toxicology. Vol. 2, 257-261, Nov-Dec.
(1978)).
The gas chromatographic (GC) results were converted to
the solid composition scale as follows:
>1400 mg/L = 5
1000 - 1400 mg/L = 4
-- 7 --
600 ~mg~ 3
300 -600 mg/L = 2
50 -300 mg/L = 1
< 50 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 o~ all solid composition values (X) - 1.928
Mean of all GC values (Y) = 2.050
Slope (a) = 1.037
Intercept (b) = 0~054
Correlation Coefficient (r) = 0.9875
These correlation coefficients (0.9831 and 0.9875) and
slopes (1.0248 amd 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
Drugs in-vitro
Acetone Hydrochlorothiazide
Amitriptyline Imipramine
Amobarabital Isopropanol
Amphetamine Methadone
Ascorbic Acid Methamphetamine
Butalbital Methanol
Chlordiazepoxide Methyprylon
Chlorpromazine Morphine
Cimethidine Naloxone
Cocaine Nortriptyline
~XZl~(:)8
Codeine Oxazepam
Diazepam PCP
Digoxin Phenobarbital
Dimenhydrinate Phenylpropanolamine
Diphenhydramine Propoxyphene
Ephedrine Propranolol
Ethchlorvynol Quinine
Flurazepa~ Salicylate
Secobarbital
Drugs Added in-vitro
To pooled urine previously shown to contain no ethanol
by gas chromatographic procedures, drugs listed in Table 1 and
ethanol were added such that the final concentrations of both
the drug and ethanol in urine were 1 mg/ml. In the case of
methanol, isopropanol and acetone, their final concentration
was 3mg/ml. The urine sample were tested with the solid compo-
sition as prepared in Example 1 in the following manner: (1)
after the drug was added and (2) after ethanol was added to
sample containing the drug. The visual colour change with each
testing was recorded. A urine sample from the same pool, con-
taining lmg/ml of ethanol was used as a reference.
TA~LE 2
Drugs in-vivo
Amitriptyline Ludiomil
Amoxapine Meprotiline
Amphetamine Methadone
Barbiturate Methamphetamine
Benzodiazepine Morphine
Carbamazepine Nortriptyline
Chlorpromazine Oxycodone
Cimetidine Pentazocine
Codeine Imipramine
Desimipramine Perphenazine
Diphenhydramine Phencyclidine
_ g _
Doxepin Pheniramine
Ephedrine Pheno-thia~ine
Ethchlorvynol Phenylpropanolamine
Flurazepam Phenytoin
Haloperidol Propoxyphen
Hydrocodone Quinidine
Quinine
Drugs in-vivo
166 urine samples from various drug using populations
were tested in the following manner:
1. Drug screening was done using thin layer chromatographic
procedures to establish the drugs content of the urine. Table
2 lists various drugs found either individually or in various
combinations. Most drugs usually appear with their metabolites
(not listed in the table).
2. Urine was tested with the solid composition as prepared
in Example 1. Visual colour change at 1 minute was recorded.
(a) If there was a positive reading then the urine was
further analyzed with gas chromotographic procedures to estab-
lish the urine ethanol level.
(b) If the solid composition did not change colour then
ethanol was added to the urine such that the final concentra-
tion 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.
-- 10 --
12~
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 cornponents present. In 124 samples ethanol had to be
added. Samples containing large amounts o~ drugs or many drugs
(3 or more) generally gave a negative bias of 1+, i.e. 6+ by
gas chromatography read 5+ visually. Invariably the endogen-
eous levels of ethanol in these urines levels were greater than
100 mg/100 ml.
The reducible compound may comprise the co enzyme nico-
tinamideadenine~dinucleotide (NAD) and the first enzyme cata-
lyst may comprise alcohol dehydrogenase (ADH). The colour-
changing compound may comprise a tetrazolium salt and the
second enzyme catalyst may comprise diaphorase. The tetrazo-
lium salt may comprise iodonitrotetrazolium chloride (INT),
thiazolyl blue tetrazolium bromide (MTT) or nitroblue tetrazo-
lium chloride (NBT). Alternatively, the colour-changing com-
pound may comprise 2-6-dichlorophenol indophenol, methylene
blue or other suitable dyes or combinations thereof.
The acetaldehyde trapping agent may comprise semicarba-
zide, hydroxylamine, hydrazine, or sulfhydryl reagents such as
penicillamine or cysteine. The buffer may comprise phosphate,
pyrophosphate buffer of tris (hydroxymethyl) aminomethane com-
monly known as tris buffer.
The solid composition takes advantage of competitive
inhibitors of alcohol dehydrogenase 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 pen~yl pyrazole, or a suitable steroid.
In its natural state, alcohol dehydrogenase has a very
high affinity for alcohol such that the enzyme reacts with al-
cohol at a maximal cons-tant velocity even at very low concen-
)8
trations of alcohol. Quantitation by this method is not possi-
ble 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 inhibi-tor of alcohol dehydrogenase the rate at
which the enzymes oxidizes alcohol, and thus it yields the
colour reaction, is proportional to the concentration of alco-
hol in the said ranges. Pyrazoles substituted in position ~
are more active competitive inhibitors, such that the concen-
tration required in the support system are: pyrazole > halogen-
ated pyrazole > methyl pyrazole > ethyl pyrazole > propyl pyra-
zole > butyl pyrazole > pentyl pyrazole. The concentrations of
competitive inhibitors other than pyrazo]e are calculated by
multiplying the concentration of pyrazole used by the ratios
Ki(inhibitor)/Ki(pyrazole), where Ki's are the inhibitor con-
stants ~or the alcohol dehydrogenase reaction.
In accordance with an alternate embodiment the oxygen in
the body fluid may be reduced. In such a system, alcohol oxi-
dase is employed as the catalyst to produce the correspondingaldehyde. The chromogen employed may be chosen from guiacol,
o-tolidine, or a benzidine derivative such as tetramethyl benz-
idine. 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 change, a second enzyme
such as pero~idase is employed. This system may be employed
for the detection of lower alcohols such as methanol or ethy-
lene 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.
08
The second system may be more fully understood from the
following examples:
It will be understood that the alcohols which may be de-
tected 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.
l. 75 mM TRIS buffer at pH 7.5 was prepared by dissolving
0.908g of tris (hydroxymethyl) aminomethane in l00 ml of dis-
tilled water. The pH was adjusted with NH~OH.
2. 0.12 g of gelatin was added to 10 ml of the above TRIS
buffer. Gelatin was dissolved by warming the buffer solution
in a warm water bath.
3. Preparation of the reagents to be freeze dried.
(a) The following were weighed in a lyophilyzing
bottle wrapped with aluminum foil.
l. Brij-35 0.0714 gm
2. Albumin 0.l gm
3. Peroxidase 0.0869 gm (4000 units)
To the above reagents l0 ml of the TRIS buffer containing gel-
atin was added and mixed gently. It was allowed to stand for
15 minutes.
(b) To the above reagent mixture 300 ul (l000 units)
of alcohol oxidase was added. The solution was gently mixed.
(c) After 15 minutes l00 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 re-
moved 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 subse-
- 13 -
~22~
quently freeze dried overnight.
4~ Colour changing compound. ~epending on the colour
change desired any one or combination of the following chemi-
cals can be made in 10 ml of acetone.
Colour changing compound Concentration Final Colour
a) Guiacol 10 nM 0.012g/lOml brown
b) Tetramethylbenzdine 7 nM ~.018g/lOml blue green
c) Syringaldazine 12 nM 0.045g/lOml violet
d) o-Tolidine 10 nM 0.021g/lOml green
e) Diaminofluorine 5 nM 0.00~8g/lOml blue
5. Inhibitor. 0.15g of urea (0.25 mM) is dissolved in min-
imal amount of water and added to the acetone solution(~) con-
taining the colour changing compound
6. The lyophilized strips as prepared in 3e were soaked in
the acetone solution(5) and excess solu-tion was decanted off.
The strips wers dried on a stream of dry nitrogen.
7. Preparation and storage of the freeze dried strips.
a) The strip (5mm x 50mm) was attached to a semi rigid
plastic strip (50mm x 60mm) b~ a double sided adhesive tape.
b) The strip was then cut to give a total 10 small
strips (5mm x 60m). At the end of each small strip there was
consequently a lyophilized pad of 5mm x 5mm which contained all
the reagents.
c) The small strips were stored in dark bottles under a
dessican-t in a 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.
- 14 ~
~.;22~ B
EXAMPLE 8
Solid composition for determining ethanol using alcohol
Oxidase when quan-titation is not required.
Step 5, the addition of urea, is omitted from Example 6
or 7.
The solid composition as described in Examples 6 and 7
reacts with both ethanol as well as methanol and ethylene gly-
col. ~hen urea is used as the inhibitor at the above concen-
trations of methanol and ethanol linearity between 0-1600 mg/l
is achieved.
Those sXilled in the art will recognize that the sensi-
tivity of detection o~ di~ferent 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 in accordance with the in-
vention and prepared for example as described in theforegoing
Examples 5 and 6 is dipped for 1-2 seconds in the fluid sus-
pected of containing the alcohol. After 1 minute the colour
developed in the solid composition is compared to a colour
chart. The intensity of the color that develops in thé solid
composition is proportional to the concentration of alcohol
present in the fluid. In case of the fluid being blood the
solid composition is either washed with a few drops of water or
wiped with a tissue before comparing to the colour chart.
As a further alternative a further system included bar-
ium oxide or barium hydroxide or a suitable agent for generat-
ing hydrogen peroxide in association with water; the enzyme
catalase to oxidize the alcohol to the corresponding aldehyde;
nicotinanide-adenine dinucleotide; a second enzyme, such as
acetaldehyde dehydrogenase, to reduce the nucleotide in the
presence of the acetaldehyde; a chromagen, such as a tetrazo-
lium salt; and a third enzyme, such a.s diaphorase to reduce the
i22~
tetrazolium salt in the presence of the reduced nucleotide to
give a colour indicative of the amount reduced nucleotide pres-
ent. Sodium formate may be added as a competitive substrate
(inhibitor) with regard to ethanol thus resulting in quantita-
tion of ethanol on any desired range of concentrations.
It will of course be understood as with the priorembodiments, suitable coatings, surfactants, stabilizers and
buffers may be employed. This composition is used to im~reg-
nate 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 em-
ployed to ensure the stability of the composition. It will of
course be understood that other forms of drying such as accel-
erated convection, at suitable temperatures, may be employed ora combination of such techniques may be employed.
It will also be understood that other examples or equiv-
alents will be apparent to those skilled in the art without
departing from the spirit of the invention as defined in the
appended claims.
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