U.S. patent number 3,732,147 [Application Number 05/181,192] was granted by the patent office on 1973-05-08 for colorimetric determination of dehydrogenases.
This patent grant is currently assigned to Miles Laboratories Inc.. Invention is credited to Alan Phillip Fosker, Patrick James Mill.
United States Patent |
3,732,147 |
Fosker , et al. |
May 8, 1973 |
COLORIMETRIC DETERMINATION OF DEHYDROGENASES
Abstract
Test composition and method for assaying dehydrogenase enzymes
in test fluids which comprises contacting the fluid with a test
reagent comprising a coenzyme, a substrate for the dehydrogenase
enzyme, a terminal acceptor dye and Meldola's Blue as an
intermediary hydrogen carrier. A preferable embodiment of the
present invention comprises incorporating the test composition with
a solid carrier therefor and using the same as a dip and read test
device.
Inventors: |
Fosker; Alan Phillip (High
Wycombe, EN), Mill; Patrick James (Beaconsfield,
EN) |
Assignee: |
Miles Laboratories Inc.
(Elkhart, IN)
|
Family
ID: |
10433592 |
Appl.
No.: |
05/181,192 |
Filed: |
September 16, 1971 |
Foreign Application Priority Data
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|
|
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Sep 17, 1970 [GB] |
|
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44,498/70 |
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Current U.S.
Class: |
435/26; 422/400;
435/805 |
Current CPC
Class: |
C12Q
1/32 (20130101); Y10S 435/805 (20130101) |
Current International
Class: |
C12Q
1/32 (20060101); G01n 031/14 () |
Field of
Search: |
;195/13.5R
;23/253TP |
Other References
anal. Abstracts 17:2541 .
Colswick et al., "Methods in Enzymology," Vol. IV, p. 329-336,
(1957)..
|
Primary Examiner: Tanenholtz; Alvin E.
Assistant Examiner: Hensley; Max D.
Claims
What is claimed is:
1. A composition for the determination of a dehydrogenase enzyme in
a test fluid which comprises a coenzyme selected from the group
consisting of nicotinamide adenine dinucleotide and nicotinamide
adenine dinucleotide phosphate, a corresponding substrate for said
enzyme, a terminal acceptor dye and Meldola's Blue as an
intermediary hydrogen carrier.
2. A composition according to claim 1 wherein the terminal acceptor
dye is selected from the group consisting of
2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride
2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium bromide and
2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium iodide.
3. A composition according to claim 1 wherein the enzyme being
determined is lactic dehydrogenase and the substrate is lactic
acid.
4. A composition according to claim 1 wherein the enzyme being
determined is glucose-6-phosphate dehydrogenase and the substrate
is glucose-6-phosphate.
5. A composition according to claim 1 wherein the enzyme being
determined is 6-phosphogluconate dehydrogenase and the substrate is
6-phosphogluconate.
6. A test device for the determination of a dehydrogenase enzyme
comprising a carrier with which is incorporated the composition of
claim 1.
7. A test device according to claim 6 wherein the carrier is a
bibulous paper.
8. A method for the determination of a dehydrogenase enzyme, which
comprises contacting the fluid containing the enzyme with a
predetermined proportion of a composition comprising a coenzyme
selected from the group consisting of dinucleotide and nicotinamide
adenine dinucleotide phosphate, a corresponding substrate for said
enzyme, a terminal acceptor dye and Meldola's Blue as an
intermediary hydrogen carrier.
Description
BACKGROUND OF THE INVENTION
The determination of certain dehydrogenases linked to the coenzymes
nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) is
of medical importance in the diagnosis of several diseases. For
example, an elevated value of lactic dehydrogenase (LDH) can be
indicative of myocardial infarction, convulsive disorder, carcinoma
of the prostate gland or hepatic disease. A rise in
6-phosphogluconate dehydrogenase (6-PGD) in vaginal fluid can be
indicative of trichomonas vaginitis or gynecologic cancer. A
deficiency of glucose-6-phosphate dehydrogenase (G6-PD) can
indicate hereditary as well as drug induced haemolytic anaemia.
DESCRIPTION OF THE PRIOR ART
The coenzyme-linked dehydrogenases referred to above are known to
transfer hydrogen atoms from their substrates to the coenzymes NAD
and NADP. The reduction of the coenzyme is usually followed by a
change in optical density of 340 millimicrons. However, this
wavelength is in the ultra-violet region so that complex and
expensive apparatus is required to measure such changes. A
colorimetric method uses smaller concentrations of coenzymes but
adds a terminal acceptor system which will be reduced by the
reduced coenzyme. Thus, the coenzyme is restored to its original
oxidized state, ready to accept more hydrogen from the substrate,
and the terminal acceptor system is so chosen that its reduction
produces a color change which is proportional to the enzyme
activity.
No single substance is known which will both couple with reduced
coenzyme and give a good color change on reduction. In the past,
both a terminal acceptor dye which gives the color change and an
intermediary carrier have been used in the assay of various
dehydrogenases. In such methods, the carrier is reduced by the
reduced coenzyme, and in turn reduces the terminal acceptor.
Typical acceptors include 2-(p-iodophenyl)-
3-(p-nitrophenyl)-5-phenyl tetrazolium chloride, (hereinafter
referred to as tetrazolium salt) and the corresponding bromide and
iodide and 2,6-dichloroindophenol. Only phenazine methosulfate has
previously been found suitable as an intermediary hydrogen carrier,
but it is unstable and cannot be used for prepackaged reagent
kits.
SUMMARY OF THE INVENTION
We have now found that Meldola's Blue, which is sufficiently stable
to be used in prepackaged kits, is a suitable intermediary hydrogen
carrier for this purpose. More particularly we have found that the
enzymes LDH, 6-PGD and G6-PD can be assayed colorimetrically using
a buffered aqueous mixture consisting of Meldola's Blue,
tetrazolium salt (as defined above) as a terminal acceptor dye, a
substrate for the enzyme, and NAD or NADP as coenzyme, or by use of
test material comprising a carrier member such as a bibulous paper
strip containing or carrying such a solution. When such a
composition or material comes into contact with one of said
enzymes, a color change occurs which can be followed visually or by
means of a spectrophotometer, and which, when compared with
standards containing known amounts of the enzyme to be determined,
serves as a measure of the enzyme concentration.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The reactions which occur in the present invention can be
illustrated as follows:
1. 6-phospho-d-gluconate + NAPD .revreaction.
6-phospho-2-keto-d-gluconate + NADPH.sub.2 + CO.sub.2
2. d-glucose-6-phosphate + NADP .revreaction. d-glucono-.delta.
-lactone-6-phosphate + NADPH.sub.2
3. lactic acid + NAD .revreaction. pyruvic acid + NADH.sub.2
Meldola's Blue is an oxazine dye known chemically as
9-dimethylaminobenzo-.alpha. -phenazoxonium chloride, first
discovered in 1879 by Meldola, and having the formula ##SPC1##
In one aspect therefore the invention comprises a composition for
use in the colorimetric assay of a dehydrogenase enzyme, which
comprises a substrate for the said enzyme, as coenzyme NAD or NADP,
a terminal acceptor dye, and Meldola's Blue as an intermediary
hydrogen carrier.
The composition may be in the form of a buffered aqueous solution.
Preferably however the composition is deposited on or incorporated
in a carrier, and the product is used as a dip and read test
device. Such a test device can be readily made by impregnating a
bibulous material with a solution of the test composition, and
subsequently drying it, or by impregnating the bibulous material
successively with the various ingredients of the test composition,
either singly or two or more at a time, and drying it after each
such impregnation. Alternatively, a finely divided, dry, intimate
mixture of the ingredients can be adhesively attached to the
surface of the carrier. Another embodiment involves incorporating
the test composition with a polymeric semi-permeable membrane and
either using this membrane as such or attaching the membrane to a
carrier. The carrier material may for example be filter paper, wood
strip, synthetic plastic or non-woven or woven fabric, filter paper
having a thickness of from 0.01 inch to 0.02 inch being generally
preferred. Such test devices constitute a further aspect of the
invention.
While the preferred terminal acceptor dye is a tetrazolium salt,
other compounds can be used, including other tetrazolium dyes, and
2,6-dichloroindophenol. The substrate will of course correspond to
the particular dehydrogenase to be determined; thus for determining
LDH, lactic acid will be used; for determining 6-PGD,
6-phosphogluconate; and for determining G6-PD, glucose-6-phosphate.
The coenzyme is NAD or NADP, depending on the coenzyme specificity
of the enzyme to be measured.
Another aspect the invention consists in a method for the assay of
a dehydrogenase enzyme, which comprises contacting a composition
comprising the said enzyme with a predetermined quantity of a
composition as described above in the form of a buffered aqueous
solution, or preferably with a test material comprising a carrier
impregnated with or carrying on its surface such a composition, and
comparing the color developed in the solution, or on the test
material after it has been removed from contact with the
composition containing the enzyme, with a standard color chart.
The following examples illustrate the invention.
EXAMPLE 1
A mixture for use in the colorimetric assay of G6-PD was prepared
from the following ingredients:
0.3 ml of 0.1 molar aqueous sodium phosphate solution (pH 7.5)
0.2 ml. of 0.1 molar aqueous magnesium chloride solution
0.1 ml. of an aqueous solution containing 1.4 millimoles of
NADP
0.1 ml. of an aqueous solution containing 0.3 percent gelatin
0.2 ml. of a aqueous solution containing 3 mg. of disodium
glucose-6-phosphate per ml. of water
0.3 ml. of an aqueous solution containing 5 mg. of tetrazolium salt
per ml. of water
0.05 ml. of an aqueous solution containing 1 mg. of Meldola's Blue
per ml. of water
1.7 ml. of water
To this mixture was added 0.05 ml. of an aqueous solution
containing 0.03 mg. of G6-PD per ml. of water.
The resulting mixture was placed in a cuvette and the change in
optical density at 500 millimicrons was followed with a
spectrophotometer or colorimeter at 25.degree.C. The rate of change
in optical density was linear for 5 minutes and was taken as a
measure of the enzyme activity.
EXAMPLE 2
A mixture was prepared having the same composition as that
described in Example 1 except that 6-phosphogluconate replaced the
glucose-6-phosphate as a substrate. 6-PGD was determined
colorimetrically by observing the rate of change in optical density
with a spectrophotometer or colorimeter at 500 millimicrons and
comparing the results with a standard obtained by carrying out the
test procedure with solutions containing known amounts of 6-PGD. As
in Example 1, the rate of change in optical density was linear for
about 5 minutes.
EXAMPLE 3
A composition for use in the colorimetric assay of LDH was prepared
by mixing the following components:
1.10 ml. of an aqueous borate buffer solution containing 3.10 g.
boric and 1.67 g. borax per 100 ml. water (pH8.6)
0.25 ml. of 0.5 molar aqueous sodium lactate solution
0.10 ml. of an aqueous solution containing 0.3 percent gelatin
0.50 ml. of an aqueous solution containing 1 mg. of NAD per ml. of
water
0.30 ml. of aqueous solution containing 5 mg. of tetrazolium salt
per ml. of water
0.05 ml. of an aqueous solution containing 1 mg. of Meldola's Blue
per ml. of water
0.50 ml. of water
To this mixture was added 0.20 ml. of human serum.
When the resulting composition was placed in a spectrophotometer, a
linear change of optical density was observed for a period of 10
minutes at a rate of 0.183 optical density units per minute at a
wavelength of 500 millimicrons.
EXAMPLE 4
A strip of Whatman No. 120 filter paper about 1 centimeter wide and
14 centimeters long was impregnated successively with the following
solutions and dried in a stream of warm air between each
application:
1. 0.05 molar sodium phosphate in 50 percent aqueous methanol
buffered to a pH of 7.5
2. 0.05 molar magnesium chloride in 50 percent aqueous methanol
3. an aqueous solution containing 3 mg. of disodium
glucose-6-phosphate per ml. of water.
4. an aqueous solution containing 1.4 millimoles of NADP
5. a methanol solution containing 0.04 mg. of Meldola's Blue and 5
mg. of tetrazolium salt per ml. of methanol.
When 0.05 ml. of aqueous solutions containing respectively 0.005 -
0.10 mg. of G6-PD per ml. of water were applied to the strip thus
prepared, the original pale blue color of the strip changed to a
color ranging from a pinkish red to a deep reddish purple dependent
upon the G6-PD enzyme concentration. The strips can be stored for
up to 18 months in the dark in the presence of silica gel without
loss of activity.
EXAMPLE 5
A strip of filter paper was impregnated with the same solutions as
in Example 4 except that 6-phosphogluconate replaced the
glucose-6-phosphate. When the strip was contacted with an aqueous
solution of 6-PGD, a color change occurred which was indicative of
the amount of 6-PGD enzyme present.
EXAMPLE 6
A paper was impregnated as described in Example 4 with the
following solutions and used to assay LDH:
1. 1.5 molar aqueous sodium borate solution (pH8.8)
2. 1.5 molar aqueous sodium lactate solution
3. an aqueous solution containing 2 mg. of NAD per ml. of water
4. a methanol solution containing 0.04 mg. of Meldola's Blue and 5
mg. of tetrazolium salt per ml. of methanol.
When a paper strip so prepared was contacted with a preparation of
beef heart lactic dehydrogenase, the colors produced by solutions
containing 0, 80, 160, 240, 800 and 3,200 international units of
LDH activity could be readily distinguished.
* * * * *