Note: Descriptions are shown in the official language in which they were submitted.
:
This invention relates to a stable, colored reference standard
suitable for use in the determination of enzymatic reactions capable of re-
ducing the colorless 2-(p^iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium
chloride to the red-colored l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenyl-
formazan.
Methods for the quantitative determination of certain enzymes are
based on the production of NADH (reduced nicotinamide adenine dinucleotide) -~
or NADPH (reduced nicotinamide adenine dinucleotide phosphate) as a result
of the enzymatic reaction on a substrate; the NADH or NADPH in turn retuces
colorless 2-tp-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoliwm chloride
~ ~commonly known as INT) in the presence of an electron carrier such as phen-
¦ azine methosulfate, to l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan
I tcommonly known as INT formazan), which is bright red and can be measured
spectrophotometrically. Concentrations of enzyme are tetermined by simul-
taneously running the test reaction on a reference sample containing known
quantities of the enzyme and comparing spectrophotometric readings of the
unknown against the reference sample.
Represcntative enzymatic determinations in which the above-
described general methot is used include the following: Babson, A.L. and
Phillips, G.E., "A Rapid Colorimetric Assay for Serum Lactic DehYdro~enase", i`
Clin. Chim, Acta 12, 210-215 (19653; Van Der Veen et al., "Isoenzvmes of
Creatine Phosphokinase in Tissue Extracts and in Normal ant Patholo~ical
Sera", Clin. Chim, Acta 13, 312-316(1966); and Nachlas, M.M., et al., "The
Determination of Lactic Dehydrogenase with a Tetrazolium Salt", Anal.
Biochem., 1, 3I7-326 (1960). ;
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The accuracy of results using aforementioned methods for deter-
mining enzymatic activities depends, to a large extent, on the preparation ~j
ànd stability of the~reference sample containing the known quantity of
~ enzyme against which results with the test sample are compared. Furthermore, -
; 30 these enzymatic test methods involve double work, since in every instance,
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the test itself must be run on the reference sample, as well as on the un-
known sample. Such dupli ~tion requires extensive checking in order to insure
that inaccuracies have not occurred.
Thus, the need for and the advantages of a stable colored reference
standart for use in enzymatic determinations without simultanéous running of
a control are obvious. However, such a product is not commercially available.
According to the present invention, there is provided a colored
reference standard for use in diagnostic determinations involving enzymatic
reactions in which l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is
produced, which comprises an aqueous solution of:
A. From about 0.001% to about 0.020% by weight, based on the weight of
the total solution, of l-tp-iodophenyl)-5-~p-nitrophenyl)-3-phenylformazan;
B. From about 0.01% to about 0.10% by weight of serum albumin;
C. Prom about 2% to about 10% by weight of solvent selected from the
group consisting of N,N' dimethylformamide and dimethylsulfoxide; and
D. From about 2% to about 10% by weight of isopropyl alcohol;
said aqueous color standard solution having an absorbence maximum at S00 ~-
nanometers,
From 5% to 20% by weight of an inert bulking agent may be added to
the aqueous colored reference standard solution, which may then be lyophilized,
The lyophilized product is easily reconstituted with water. The ~queous
colored rePerence standard solution has an absorbence maximum at 500 nano-
meters and is suitable for use in the determination of certain enzyme
activities, such as serum lactate dehydrogenase, creatine phorphokinase, glu-
cose-6-phosphate dehydrogenase and like.
The lyophilized form of the colored reference standard of this
invention is stable for at least six months at 4C. and 24C. Furthermore,
the lyophilized form of the reference standard of this invention may be
reconstituted with water to form a clear solution which can be utilized,
without difficulty, as a reference standard in enzymatic determinations.
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The l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan in the
colored reference standard of this invention (INT formazan) is commercially
available (National Biochemicals Corp., Cleveland, Ohio) and has the follow-
ing formula:
I ~ N=N-C=N-NH ~ No2
~'
.
INT formazan is frequently encountered in microbiological identification
techniques, particularly for the examination of foods and pathological
specimens where bacteria, if present, reduce the colorless 2-(p-iodophenyl)-
3-(p-nitrophenyl)-5-phenyltetrazolium chloride ~INT) which has the formula:
N ~ ~ I
N N
~3 N02
to the brightly colored INT formazan. Additionally, certain enzymatic re-
actions will cause the reduction of INT to INT formazan and it is in the
determination of these enzymes that the colored reference standard of the
invention finds utility,
The solvents which have been found to improve the solubility
characteristics of INT formazan and to permit easy reconstitution with water
after lyophilization are N,N'-dimethylformamide and dimethylsulfoxide. The
addition of isopropyl alcohol also aids in this solubilizing process. It is
of considerable importance that these solvents do not interfere with the
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absorbence maximum of the aqueous colored reference standard.
The stable, colored reference standard of this invention also con-
tains serum albumin. Bovine serum albumin is preferred. This material is
believed to aid in stabilizing bcth the lyophilized and aqueous forms of the
reference standard.
Suitable inert bulking agents for use in the reference standard
of this invention include natural and synthetic gums such as gum arabic,
sodium alginate, extract of Irish moss, carboxymethyl cellulose, polyvinyl
pyrrolidinone and the like; sugars such as sorbitol, mannitol, sucrose and
the like; and carbohydrates such as starch, dextran and the like. The pre-
ferred bulking agent is a dextran having a molecular weight of approximately
10,000.
A preferred colored reference standard may be prepared according
to the practice of this invention by forming an aqueous solution, suitable
for lyophilization, containing 0.005% to 0.015% by weight, based on the
weight of the total solution of l-tp-iodophenyl)-5-(p-nitrophenyl)-3-
phenylformazan; from 0.02% to 0.075% by weight of bovine serum albumin; from
8% to 14% by weight of dextran having a molecular weight of approximately
2~ 10,000; 2% to 10% by weight of N,N'-dimethylformamide; and from 2% to 10%
by weight of isopropyl alcohol. ;
A particularly preferred colored reference standard, according to i
the teaching of this invention, contains, in an aqueous solution, 0.0123%
by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-
(p-nitrophenyl)-3-phenylformazan; 0.0563% by weight of bovine serum albumin;
9% by weight dextran having a molecular weight of approximately 10,000; 2.5%
by weight of N,N',dimethylformamide and 7.5% by weight of isopropyl alcohol.
The absorbence of the aqueous colored reference standard of this solution
at 500 nanometers is approximately 1.34.
3~ The lyophilized form of the colored reference standard of this
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invention which, as stated a6OYe, has-been found to be stable for at least
6 months at temperatures of 4C and 24C, when reconstituted with water o~
0.1 N hydrochloric acid, is easily handled ~or dilutions which may be
required to construct standard curves for fixed-point colorimetric enzy-
matic assays. Reconstituted colored reference standard solutions are stable
for eight hours at room temperature, and for at least 48 hours at 4C.
In order to prepare the stable colored reference standard of this
invention, the various ingredients named above must be added one to the other
in a particular fashion to obtain a product having the necessary stability
and solubility to permit reconstitution after lyophilization. Such charac-
teristics are achieved by a process which comprises:
A. Completely dissolving from about 0.001% to about 0.020% by weight,
based on the weight of the total solution, l-(p-iodophenyl~-5-(p-nitrophenyl~-
3-phenylformazan, in from about 2% to about 10% by weight of solvent selected
from the group consisting of N,N'-dimethylformamide and dimethylsulfoxide;
B. Adding from about 2% to about 10% by weight of isopropyl alcohol
to (A.) slowly, and mixing thoroughly;
CO Dissolving from about 0.01% to about 0.10% by weig~t of serum
albumin in water;
D. Adding solution ~B.~ to solution ~C.) slowly, with stirring. In the
preferred embodiment of this invention, where lyophilization is desired, the
inert bulking agent is dissolved in water along with the serum albumin. The
final solution obtained in dispensed in aliquots and subjected to the usual
lyophilization procedures.
For use in enzymatic determinations, the a6Ove described 1YG-
philized form of the colored reference standard is reconstituted with water
or O.lN hydrochloric acid. The colored reference standard solution serves
as a standard of known activity against which the unknown test samples are
read in order to determine the activity of enzyme in the unknown. The
colored reference standard s~ e invention may be diluted, if desir-
ed, for construction of curves for the fixed point colorimetric enzymatic
assays.
Thus, there is provided a colored reference standard which has -
excellent stability in the lyophilized state and which, upon reconstitution,
is easily handled in currently used enzymatic determinations.
To further illustrate the present invention, the following
examples are given:
EXAMPLE I
Preparation of the Stable Colored Reference Standard Solution
A. 12.3 mg of INT formazan is dissolved in 2.5 ml of N,N'-dimethyl-
formamide. To this solution 7.5 ml of reagent grade isopropanol is added and
mixed thoroughly.
B. 9.0 g of dextran having a molecular weight of approximately 10,000
and 56.3 mg of bovine serum albumin are dissolved in 90 ml of purified water.
C. Solution A is added slowly to Solution B with stirring. The
combined solution obtained is dispensed in 4.0 ml aliquots into 10 ml vials, `
frozen and lyophilized for 36 hours with a 30C. set-point. The vials are
capped under dry nitrogen. Each vial is reconstituted with 10 ml of 0.1 N -~
hydrochloric acid and the absorbence at 500 nanometers is 1.340 ~ 0.010 which `~-~
represents 540 International Units of lactate dehydrogenase activity at 37C;
or 420 International Units of creatine phosphokinase activity at 30C.
EXAMPLE II
Colorimetric Assay for Serum Lactate Dehydrogenase
Reagents for the assay are prepared as follows:
Color Reagent: 50 mg of INT is dissolved in about 15 ml of water
withprolonged agitation until complete~dissolution is obtained. 125 mg of
nicotinamide adenine dinucleotîde is added and dissolved, follo~ed by the
addition of 12.5 mg phenazine methosulfate. The solution is transferred with
washings immediately to a low actinic 25-ml volumetric flask and diluted with
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water to the mark.
A Buffer Reagent: 1.0 g of ethoxylated oleyl alcohol (Lipal 10-OA,
....
Drew Chemical Co., Boonton, N.J.) is dissolved in 10 ml of water by heating
to about 95C. The solution is diluted with water to 50 ml and 12.1 g of Tris
is added. The pH is adjusted to 8.2 with 3 N HCL and then diluted to lOO ml.
Substrate Reagent: (0.1 M L~+)lactate) 5.0 ml of a 20% solution
of L(+) lactic acid is added to about 50 ml of water. The pH is adjusted to
5.5 with 1 N NaOH and diluted to 120 ml with water. The solution is saturated
wi~h a few drops of chloroform.
Control Reagent: 0.2 g of potassium oxalate and 0.2 g of ethy-
lenediaminetetraacetic acid, disodium dihydrate, are dissolved in 100 ml of
water. The above preparations are described in Babson et al. Clin Chim~ Acta
2: 210-215 (1965).
Procedure: 0.1 ml of serum and 0.2 ml of buffer reagent are
.
pipetted in each of two tubes. 0.5 ml of substrate is added to one tube and
0.5 ml of control reagent is added to the other tube. Both tubes are mixed
and warmed to 37C. At precisely timed intervals, 0.2 ml of color reagent is
added to both tubes, and the contents are mixed and immediately returned to
the water bath. Exactly five minutes after adding color reagent, 5 ml of
0.1 N HCl is added to both tubes and the contents mixed. The difference in
absorbence between the control tube and the serum sample tube, determined at
500 nm within 20 minutes is 0.67. This absorbence is compared with the
colored reference standard of Example I which has an absorbence of 1.340 +
0.010 equivalent to 540 International Units of lactate dehydrogenase activity.
The lactate dehydrogenase activity in the serum is calculated to be 270 Inter-
national Units at 37C.
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