U.S. patent number 3,653,841 [Application Number 04/886,538] was granted by the patent office on 1972-04-04 for methods and compositions for determining glucose in blood.
This patent grant is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Bernard Klein.
United States Patent |
3,653,841 |
Klein |
April 4, 1972 |
METHODS AND COMPOSITIONS FOR DETERMINING GLUCOSE IN BLOOD
Abstract
Colorimetric methods and compositions for quantitatively
determining the glucose content of blood plasma or serum by heating
a deproteinized sample of blood plasma or serum with an alkaline
ferricyanide solution, followed by the addition of ferric ions and
a 5-(2-pyridyl)-2H-1,4-benzodiazepine or water soluble salts
thereof to produce a brilliant purple colored solution which can be
quantitated by standard colorimetric means.
Inventors: |
Klein; Bernard (New Hyde Park,
NY) |
Assignee: |
Hoffmann-La Roche Inc. (Nutley,
NJ)
|
Family
ID: |
25389219 |
Appl.
No.: |
04/886,538 |
Filed: |
December 19, 1969 |
Current U.S.
Class: |
436/95;
436/53 |
Current CPC
Class: |
G01N
33/66 (20130101); Y10T 436/118339 (20150115); Y10T
436/144444 (20150115) |
Current International
Class: |
G01N
33/66 (20060101); C09k 003/00 (); G01n 031/22 ();
G01n 033/16 () |
Field of
Search: |
;23/230,23B ;260/239.3D
;252/408 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Hawk et al., Practical Physiological Chemistry, 13th ed.,
McGraw-Hill Co., QP514H4, 1954, p. 567, 575 .
Aloe Scientific Co., Catalog 103, p. 1011, 1041, 1065,
1073.
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Reese; R. M.
Claims
I claim:
1. A process for quantitating the glucose content of blood serum or
plasma comprising:
a. treating a deproteinized sample of blood serum or plasma with an
aqueous alkaline solution containing a source of ferricyanide
ions;
b. heating the mixture from step (a) to a temperature of from about
90.degree. C. to about 100.degree. C.;
c. adding to the mixture from step (b) a source of ferric iron
ions;
d. reacting the mixture of step (c) with a benzodiazepine compound
selected from the group of the compounds of the formula
wherein A is selected from the group consisting of
and
; B is selected from the group of
and --CH.sub.2 --; R.sub.1 is selected from the group consisting of
halogen, hydrogen, trifluoromethyl, nitro, and amino; R.sub.2 is
selected from the group consisting of
, hydrogen, lower alkyl and
; n is an integer from 2 to 7; R.sub.3 is selected from the group
consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and
lower alkanoyloxy; R.sub.4 is 2-pyridyl; R.sub.5 is selected from
the group consisting of lower alkyl, hydrogen,
and
; and R.sub.5 and R.sub.6, where taken together with their attached
nitrogen atom, from a radical selected from the group consisting of
piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl,
lower alkyl substituted pyrrolidinyl, piperidinyl, and lower alkyl
substituted piperidinyl; R.sub.7 is lower alkyl; and R.sub.8 is
selected from the group consisting of lower alkyl and hydrogen, and
water soluble acid addition salts thereof; and
e. colorimetrically quantitating the glucose present by means of
said color.
2. The process in accordance with claim 1 wherein said source of
ferric ions consists essentially of an aqueous solution of ferric
chloride buffered to a pH of between from about 4.0 to about
5.0.
3. The process in accordance with claim 1 wherein said
benzodiazepine compound is added as an aqueous solution buffered to
a pH of from about 4.0 to about 5.0.
4. The process in accordance with claim 1 wherein said
benzodiazepine compound is selected from the group consisting of
7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodi
azepin-2-one and water soluble acid addition salts thereof.
5. A process for quantitating the glucose content of blood serum or
plasma comprising:
a. treating a deproteinized sample of blood serum or plasma with an
aqueous alkaline solution containing a source of ferricyanide
ions;
b. heating the mixture from step (a) to a temperature of from about
90.degree. C. to about 100.degree.C.;
c. adding to the mixture of step (b) an aqueous solution containing
ferric chloride and a buffer;
d. adding to the mixture of step (c) an aqueous solution containing
a buffer and a benzodiazepine compound selected from the group
consisting of compounds of the formula
wherein A is selected from the group consisting of
and
B is selected from the group consisting of
and --CH.sub.2 --; R.sub.1 is selected from the group consisting of
halogen, hydrogen, trifluoromethyl, nitro, and amino; R.sub.2 is
selected from the group consisting of
, hydrogen, lower alkyl and
; n is an integer from 2 to 7; R.sub.3 is selected from the group
consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and
lower alkanoyloxy; R.sub.4 is 2-pyridyl; R.sub.5 is selected from
the group consisting of lower alkyl; hydrogen,
and
; and R.sub.5 and R.sub.6, where taken together with their attached
nitrogen atom, form a radical selected from the group consisting of
piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl,
lower alkyl substituted pyrrolidinyl, piperidinyl, and lower alkyl
substituted piperidinyl; R.sub.7 is lower alkyl; and R.sub.8 is
selected from the group consisting of lower alkyl and hydrogen and
water soluble acid addition salts thereof; and
e. colorimetrically quantitating the glucose present by means of
said color.
6. The process in accordance with claim 5 wherein said source of
ferricyanide ions is potassium ferricyanide.
7. The process in accordance with claim 5 wherein said buffer
present in the aqueous solution containing ferric chloride and the
aqueous solution containing the benzodiazepine compound is a buffer
pair comprising a water soluble salt of acetic acid and acetic
acid.
8. A method for the quantitative analysis of the glucose content of
blood plasma or serum consisting essentially of providing in
continuous flow, the sequential steps comprising:
a. combining in continuous flow a measured specimen of plasma or
serum with an isotonic solution of sodium chloride;
b. passing said mixture through a separating zone, thereby
separating by dialysis in said zone from said mixture a clear
aqueous solution;
c. mixing said clear aqueous solution with a reagent comprising an
alkaline aqueous solution of a water soluble ferricyanide salt;
d. passing said aqueous mixture through heating means thereby
raising the temperature thereof to from about 95.degree. C. to
about 100.degree. C.;
e. mixing said heated aqueous mixture by concurrent flow with a
first reagent comprising a buffered aqueous solution of a ferric
iron salt and a second reagent comprising a buffered, aqueous
solution of a color-forming compound selected from the group
consisting of compounds of the formula
wherein A is selected from the group consisting of
and
; B is selected from the group consisting of
and --CH.sub.2 --; R.sub.1 is selected from the group consisting of
halogen, hydrogen, trifluoromethyl, nitro, and amino; R.sub.2 is
selected from the group consisting of
, hydrogen, lower alkyl and
; n is an integer from 2 to 7; R.sub.3 is selected from the group
consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and
lower alkanoyloxy; R.sub.4 is 2-pyridyl; R.sub.5 is selected from
the group consisting of lower alkyl; hydrogen,
and
; and R.sub.5 and R.sub.6, where taken together with their attached
nitrogen atom, form a radical selected from the group consisting of
piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl,
lower alkyl substituted pyrrolidinyl, piperidinyl, and lower alkyl
substituted piperidinyl; R.sub.7 is lower alkyl; and R.sub.6 is
selected from the group consisting of lower alkyl and hydrogen
thereof and water soluble acid addition salts thereof thereby
forming a colored solution; and
f. flowing said colored solution to an analyzing zone and
photometrically determining quantitatively the amount of glucose
present during the flow of said colored solution through said
analyzing zone.
9. The method in accordance with claim 8 wherein said first reagent
and said second reagent are buffered to a pH of from about 4.0 to
about 5.0 with a buffer pair comprising a water soluble salt of
acetic acid and acetic acid.
10. The method in accordance with claim 8 wherein said colorforming
compound is selected from the group consisting of
7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodi
azepin-2-one and water soluble acid addition salts thereof.
11. The method in accordance with claim 8 wherein said water
soluble ferricyanide salt is potassium ferricyanide and said ferric
iron salt is ferric chloride.
Description
BACKGROUND OF THE INVENTION
The need for a quantitatively accurate method for the determination
of glucose in blood, e.g. plasma and serum, using small amounts of
specimen, yet which is simple enough to be effectively utilized in
the clinical situation and sufficiently economical for mass
screening has long been felt. In addition, it has been considered
most desirable that such a method be readily adaptable to an
automated sequential or continuous flow system in order that a
great many samples may be processed rapidly and with the highest
possible accuracy. There is a need for such an automated sequential
or continuous flow of system which is capable of highly accurate
results before the diagnostic testing of large numbers of persons
for the incidence of diabetes among them. A simple accurate test,
which is both rapid and reliable, is of great value as an aid in
the detection and treatment of diabetes and as an adjunct to
routine screening operations in clinics and for periodic screening
of patients in hospitals, nursing homes and similar
institutions.
Many techniques have been developed for quantitatively determining
the glucose content of blood, plasma or serum. One such technique
utilizes the enzyme glucose oxidase which catalyzes the oxidation
of glucose to gluconic acid. In the more common test, this enzyme
is combined with a substance having a peroxidative activity which
induces the oxidation of an indicator such as o-toluidine in the
presence of hydrogen peroxide formed by the glucose oxidase. This
method, though specific, has proved to be too complex, expensive
and time consuming for general use.
Other further metric techniques which are adaptable to automated
procedures have been found to be not sufficiently sensitive for
today's standards or undesirable in that they require comparatively
large volume of specimen.
The diagnostic compositions and methods of the present invention
provide a reliable, convenient test for the quantitating of glucose
in the blood as well as affording a method whereby the quantitative
determination may be carried out in a continuous sequential or flow
system. Further, the diagnostic compositions and methods of the
present invention overcome many of the disadvantages of the prior
art methods of determining glucose in blood by not requiring a high
degree of laboratory skill and technology using a small specimen
volume, yet being highly accurate in the clinical situation.
BRIEF SUMMARY OF THE INVENTION
In accordance with the invention, a
5-(2-pyridyl)-2H-1,4-benzodiazepine or water soluble salt thereof
preferably in combination with a buffer, is added with an aqueous
solution of ferric chloride to deproteinized plasma or serum which
has been treated with an aqueous alkaline ferricyanide solution,
whereby a purple solution is obtained which can be quantitated as
to its glucose content by standard colorimetric means.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention a compound selected from the group
consisting of compounds of the formula
wherein A is selected from the group consisting of
and
; B is selected from
and --CH.sub.2 --; R.sub.1 is selected from the group consisting of
halogen, hydrogen, trifluoromethyl, nitro and amino; R.sub.2 is
selected from the group consisting of
, hydrogen, lower alkyl and
; n is an integer from 2 to 7; R.sub.3 is selected from the group
consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and
lower alkanoyloxy; R.sub.4 is 2-pyridyl; R.sub.5 is selected from
the group consisting of lower alkyl and hydrogen; R.sub.6 is
selected from the group consisting of lower alkyl, hydrogen,
and
; and R.sub.5 and R.sub.6 where taken together with their attached
nitrogen atom form a radical selected from the group consisting of
piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl,
lower alkyl substituted pyrrolidinyl, piperidinyl and lower alkyl
substituted piperidinyl; R.sub.7 is lower alkyl; and R.sub.8 is
selected from the group consisting of lower alkyl and hydrogen and
water soluble salts thereof, preferably in combination with a
buffer, is added with an aqueous solution of ferric chloride to
deproteinized blood plasma or serum which has been treated with an
aqueous alkaline ferricyanide solution, whereby a purple solution
is obtained which can be quantitated by standard colorimetric
means.
Examples of benzodiazepine compounds of formula I above which are
particularly suitable as the color-forming reagent in the process
of this invention include the following:
7-bromo-1,3-dihydro-1-[4-(4-methyl-1-piperazinyl)butyl]-5-(2-pyridal)-2H-1,
4 -benzodiazepin-2-one;
7-amino-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one;
1-methyl-1-[3-(7-bromo-5-(2-pyridyl)-2
H-1,4-benzodiazepin-2-one;
1-methyl-1-[3-(7-bromo-5-(2-pyridyl)-1,3-dihydro-2-oxo-2H-1,4-benzodiazepin
e-1-yl)propyl]urea whose preparation is disclosed in U.S. Pat. No.
3,464,978 issued Sept. 2, 1969;
7-bromo-1,3-dyhydro-5-(2-pyridyl)-2H-1,4-benzodiazepine;
7-amino-1,3-dihydro-1-methyl-5-(2-pyridyl)-1H-1,4-benzodiazepine;
7-bromo-1,3-dihydro-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodiaze
pin-2-one;
7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one-4-oxide;
7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one;
7-bromo-1,3-dihydro-1-(.beta.-hydroxypropyl)-2-(2-pyridyl)-2H-1,4-benzodiaz
epin-2-one; and
7-bromo-5-(2-pyridyl)-1,3-dihydro-1-[3-(N-cyanomethylamino)propyl]-2H-1,4-b
enzodiazepin-2-one whose preparation is disclosed in U.S. Pat. No.
3,464,978.
The term "lower alkyl" as used throughout this specification
includes both straight and branched chain alkyl groups having from
one to seven carbon atoms such as methyl, ethyl, propyl, isopropyl
and the like. The term "lower alkanoyloxy" refers to both straight
chain and branched chain aliphatic carboxylic acid moieties such as
acetoxy, propionyloxy, butyryloxy and the like. The term "halogen"
includes bromine, chlorine, fluorine and iodine. Also included
within the purview of the present invention are the water soluble
acid addition salts of the compounds of formula I above. Any
conventional water soluble acid addition salts of the compounds of
formula I above may be utilized in the process of this invention to
quantitatively determine the iron content of aqueous solutions.
Among the acid addition salts which can be utilized in accordance
with this invention, includes salts of compounds of the formula I
with organic or inorganic acids such as hydrochloric acid,
hydrobromic acid, nitric acid, sulfuric acid, acetic acid, formic
acid, succinic acid, maleic acid, p-toluenesulfonic acid and the
like.
The color differentiation with varying concentrations of ferrous
ions produced by the compound of formula I above is such that the
concentration of ferrous ions produced by the instant diagnostic
reagent composition in situ can easily be determined by standard
colorimetric instruments. Furthermore, the compounds of formula I
are not sensitive to extraneous sources and therefore are not
affected by trace contaminants. The method of this invention
provides a simple colorimetric means for quantitatively determining
the glucose content of blood plasma and serum.
In accordance with the present invention the glucose content of
blood plasma or serum can be determined by first heating a
deproteinized sample with an aqueous solution containing
ferricyanide ions to form an aqueous solution containing gluconic
acid and ferrocyanide ions, cooling the solution and adding an
aqueous solution containing ferric ions and a compound of formula I
above wherein ferricyanide and ferrous ions are formed and the
ferrous ions thus produced react with the compound of formula I,
preferably in the presence of a buffer, to produce a brilliant deep
purple color and colorimetrically quantitating the amount of
glucose present in the sample. This procedure provides a simple and
quick method for quantitatively determining the glucose content of
a blood sample which is ideally suited for routine diagnostic
use.
In accordance with the present invention, the specimen to be tested
is initially treated with a conventional neutral deproteinizing
agent such as, for example, an aqueous solution of either sodium or
barium hydroxide and zinc sulfate, or an acidic deproteinizing
agent such as, for example, tungstic acid or trichloroacetic acid.
Of these, tungstic acid or an aqueous solution of barium hydroxide
and zinc sulfate are preferred. The specimen is well mixed with the
deproteinizing agent and centrifuged at high speed to obtain a
clear supernate. A 0.1 ml. aliquot of the supernate is then heated
to from 90.degree. C. to about 100.degree. C. preferably about
95.degree. C. with 2.0 ml. of an aqueous alkaline solution
containing ferricyanide ions. The mixture is rapidly cooled after
about 5 minutes heating and treated with 2.0 ml. of an aqueous
solution containing ferric ions such as, for example, ferric
chloride, and 2.0 ml. of an aqueous solution of a compound of the
formula I. The solutions are mixed and the absorbance of the violet
blue color which develops over about 10 minutes is measured at 580
nm against both a standard glucose solution similarly treated and a
reagent blank.
The solution containing ferricyanide ions can be made from any
water soluble ferricyanide salt which does not otherwise interfere
with the reaction such as, for example, potassium ferricyanide and
sodium ferricyanide. Potassium ferricyanide is preferred in the
practice of the present invention. This reagent may be made in
quantity if so desired and used as needed. The appropriate amount
of potassium ferricyanide is dissolved in an aqueous alkaline
medium such as, for example, a 2 percent sodium carbonate solution.
The quantity of ferricyanide salt utilized in preparing the reagent
is variable. However, a sufficient quantity must be utilized to
react with all the glucose in the specimen to furnish a positive
indication of elevated glucose blood levels when the diagnostic
method of the present invention is being utilized as a diagnostic
or a mass screening tool.
Generally, it is preferred that for each ml. of blood plasma or
serum tested, the reagent solution contain from about 1.8 .times.
10.sup.-.sup.5 moles to about 7.0 .times. 10.sup.-.sup.5 moles of
ferricyanide salt, preferably from about 3.5 .times. 10.sup.-.sup.5
moles to about 5.0 .times. 10.sup.-.sup.5 moles per ml. of plasma
or serum utilized.
The quantity of ferric ions added to the sample -- ferricyanide ion
mixture is again variable. However, it is preferred to utilize a
quantity of ferric ion slightly in excess of the molar quantity of
ferricyanide ions added to the sample. The utilization of such an
excess insures that there will be sufficient ferric ions present to
react with the ferrocyanide ions generated by the initial reaction
between the ferricyanide ions and the glucose in the sample. The
ferric ions may be supplied as any water soluble ferric salt which
does not interfere with the diagnostic determination such as, for
example, ferric chloride, ferric nitrate, ferric sulfate and the
like. Of these, ferric chloride is preferred.
The quantity of the compound of formula I which is added to the
aqueous mixture is variable. In all instances, however, there must
be a sufficient quantity of the compound of formula I present to
react with all of the ferrous ions generated by the reaction
between the ferric ions and the ferrocyanide ions. This quantity is
most conveniently determined by equating the quantity of the
compound of formula I with that of the ferric ions to insure the
stoichiometry of the chelation reaction.
It is preferred to maintain the test medium at a pH of about 4.0 to
about 5.0, preferably about 4.5. This can most easily be
accomplished by adding suitable buffers to the ferric ion reagent
and the reagent containing the compound of formula I. Buffering
these reagents also makes them stable in aqueous solution when they
are made up in quantity for large scale laboratory testing.
In general, any recognized buffer pair suitable for the maintenance
of such a pH range as described above can be utilized. Preferably,
there can be utilized as a buffer pair a water soluble salt of
acetic acid and acetic acid. Of the water soluble salts of acetic
acid sodium acetate is preferred. However, ammonium acetate,
potassium acetate or other water soluble salt of acetic acid can be
used, if desired. Although the quantities of the buffer pair
comprising a water soluble acetic acid salt and acetic acid are
variable, the present invention contemplates the use of a
sufficient quantity of the acid component, e.g. acetic acid, to
provide a final test sample having a pH in the range of from about
4.5 to about 5.5. By final test sample is meant a solution
containing the ferricyanide ions, the ferric ions and the
benzodiazepine color reagent. In general, there is contemplated the
preparation of a solution of both the ferric ions and the
benzodiazepine color former which contains per liter about 1.0 mole
of a water soluble salt of acetic acid to about 1.0 to about 2.0
moles of acetic acid.
From the foregoing description it is evident that the compositions
of the present invention may be utilized or handled as prepared
aqueous stock solutions, aqueous concentrates or in a dry powder
form. In either the concentrate or the powder form, sufficient
buffering agents are added to stabilize the compositions when the
working dilutions are made and maintain the pH of the reaction
mixture at between 4.5 and 5.5 preferably about 4.8.
In utilizing the compositions of the present invention, the
addition of the compound of formula I to the test system
immediately produces the desired purple coloration. The color
deepens as the reaction proceeds to completion. The reaction
mixture ceases to undergo any color changes discernible to the
naked eye after it has been allowed to stand for a short time at
room temperature. Accordingly, in order to insure uniform coloring,
the aqueous solution should be allowed to stand until its color
appears to have become constant. In general, it has been found that
the full development of the purple color will occur over a period
of from about 5 to 15 minutes after the addition of the compound of
formula I. In most cases 10 minutes is a sufficient period of time
to allow for full color development.
The quantitation of the glucose in the colored sample can be
carried out by any conventional colorimetric method utilizing
standard spectrophotometers such as a Beckman Spectrophotometer,
Coleman Spectrophotometer and the like.
The principle of the diagnostic method according to the present
invention is based on a series of coupled reactions. Initially,
glucose present in the sample undergoing analysis reduces the
ferricyanide ion in the added first reagent to ferrocyanide ions,
in turn form ferricyanide ions and ferrous ions with the addition
of the second reagent which comprises a source of ferric ions such
as, for example, ferric chloride, a buffer and a compound of the
formula I. The ferrous ions thus generated react with the compound
of the formula I to produce a brilliant deep purple color. The
purple color is thereafter colorimetrically measured and the
glucose content of the sample quantitatively determined.
The quantitative determination of the glucose content in a specimen
is carried out as follows: the optical density of the purple color
developed in the sample by the method of the present invention is
measured against a reagent blank at 580 nm utilizing a standard
spectrophotometer such as, for example, a Coleman
Spectrophotometer, employing a cuvette with a 19 mm. light path.
The quantity of glucose in the specimen is determined in the
conventional manner from the absorbance of the specimen with
reference to the absorbance of the color produced by a glucose
standard similarly treated. The glucose content of the specimen is
calculated in accordance with the following formula:
As indicated heretofore, the present invention provides an
extremely important diagnostic tool. In addition, the method of the
present invention affords a rapid and accurate determination of the
glucose content of body fluids such as plasma or serum with results
that are characterized by a high degree of reproducibility.
In another aspect of the present invention, the analytical
compositions as described are utilized in a method of analyzing the
glucose content of body fluids automatically by discrete sequential
sampling or by continuous flow apparatus. The latter method
consists essentially of mixing specimens in continuous flow with
normal saline, dialyzing the mixture to produce an aqueous
protein-free solution containing the glucose, mixing the aqueous
solution with an aqueous alkaline solution containing ferricyanide
ions, passing the resulting mixture through a heating bath to raise
the temperature thereof to about95.degree. C., mixing the heated
aqueous solution with an aqueous solution of a ferric salt and a
compound of the formula I at a constant pH of from about 4.5 to
about 5.5 and passing the resulting solution through an apparatus
which quantitatively determines the glucose content thereof
photometrically.
FIG. 1 is a schematic flow diagram illustrating a continuous flow
automated system for analyzing glucose in biological fluids
utilizing the diagnostic composition of the present invention.
FIG. 2 is a recording of the photometric response obtained when
utilizing the automated system of FIG. 1.
FIG. 3 is a plot in terms of absorbance of the photometric response
illustrated in FIG. 2.
In FIG. 1, a continuous flow automated testing system is shown
schematically wherein a specimen sample to be tested, i.e. serum or
plasma, is drawn up in sequence from separate sample cups in the
sample plate which rotates at a constant speed to provide the
system with 20-60 specimen samples with a 2:1 wash ratio per hour.
A sample, so drawn, is mixed in flow with normal saline and passed
through a glass mixing coil of conventional design. After the
mixture has passed through the mixing coil, it is next pumped
through a dialyzer module that is provided with a cellophane
membrane or the like through which the glucose passes in aqueous
solution by dialysis. The dialyzer module is maintained at a
constant temperature of 37.degree. C. The residual, non-diffusable
portion of the sample is discarded. As the aqueous glucose solution
passes through the dialyzer module membrane it is admixed with an
aqueous alkaline solution containing ferricyanide ions, preferably
in the form of potassium ferricyanide, the glucose and the
ferricyanide ions are passed in solution through a heating bath
which raises the temperature of the mixture to 95.degree. C. As
this passage takes place the glucose and ferricyanide ions are
reacting to form gluconic acid and ferrocyanide ions. The heated
aqueous stream is then mixed in continuous flow with an aqueous
solution containing ferric ions, preferably in the form of ferric
chloride, and a reagent stream comprising the
5-(2-pyridyl)-2H-1,4-benzodiazepine color reagent of formula I. The
color reagent, preferably
7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodi
azepine-2-one is maintained at a pH of about 4.5 to 5.5, preferably
at about 5.0. The mixture is then passed through a second mixing
coil. As the mixture is in transit through this coil, the ferric
ions and ferrocyanide ions react to form ferricyanide ions and
ferrous ions which in turn react with the benzodiazepine color
reagent to form a brilliant purple coloration. Photometric
measurements are then performed at 580 nm in a 15 mm. flow-cell
colorimeter, i.e., the absorbance of the solution to be tested is
measured at 580 nm in a flow-cell colorimeter using a 580 nm
filter. The results of the colorimetric readings are recorded on a
conventional recording mechanism.
The continuous flow system illustrated in FIG. 1 aspirates at a
rate of 20 to 60 specimens/hour. The rate of flow in ml./min. of
the materials entering the system according to a preferred
technique is illustrated in FIG. 1. The materials entering the
system are pumped into it by any suitable pumping means adjusted to
maintain the rate of flow illustrated in FIG. 1. The mechanism for
the system of the present invention can be conveniently provided by
a manifold assembly prepared in accordance with the system
illustrated in FIG. 1 adaptable to the Technicon Autoanalyzer.
In FIG. 2 the absorbance of solutions containing graduated amounts
of glucose, e.g. 50 mg./100 ml., 100 mg./100 ml. etc. are plotted
as a graph against concentration.
In FIG. 3 the photometric response of solutions containing
different concentrations of glucose is demonstrated. The drawing
illustrates four separate experiments, each of which represents
passage through the automated system of FIG. 1 of a sequence of at
least three solutions having glucose concentrations in the order of
low to high to low, such as, for example, 50 mg. per 100 ml. to 400
mg. per 100 ml. to 50 mg. per 100 ml. These experiments were
conducted to illustrate the sensitivity of the automated system.
The difference in the response curve for similar concentration
sequences represents a variance in the speed with which they were
passed through the system.
The reagents utilized in connection with the automated procedure of
glucose determination comprise aqueous solutions of a ferricyanide
reagent, a ferric ion containing reagent and and the buffered color
forming reagent. The ferricyanide reagent comprises sufficient
ferricyanide to react with all the glucose in the sample, for
example, 0.115 g. potassium ferricyanide dissolved in 1 liter of
0.05 percent sodium hydroxide and 0.9 percent sodium chloride. The
ferric ion containing solutions comprises sufficient ferric ions to
react with all the ferrocyanide ions formed in the initial
reaction, for example, 0.27 g. ferric chloride dissolved in 1 liter
of distilled water and buffered to a pH of about 4.5 with a sodium
acetate/acetic acid buffer couple. The color-forming reagent
comprises sufficient color-forming compound to react with the
ferrous in the ions formed by the reaction of the ferric ions and
the ferrocyanide ions, for example, 2.0 g. of a compound of formula
I, 82.0 g. of ammonium acetate and approximately 60.0 ml. of
glacial acetic acid in a liter of distilled water. The pH of the
solution is maintained between about 4.4 and 4.6.
In the practice of the invention according to the automated
procedure, iron-free distilled water is pumped through the system
for 10 minutes. The system is then switched to reagent and the
pumping is continued until a steady base line is obtained on the
recorder chart. The base line is set to 0.01A (95 percent
transmission).
The standards in the sample tray are aspirated at a rate of 20 to
60 (2:1 wash ratio) samples per hour. The specimens to be analyzed
are then sampled, with a standard glucose specimen which is
aspirated intermittently to insure qualitative control.
The glucose content of each specimen is determined by reference to
a calibration curve prepared by plotting the corrected absorbances
of the glucose standards against concentrations in mg./100 ml.
Table I sets forth a comparison of results obtained when 10
randomly selected plasma specimens were analyzed utilizing the
automated and manual glucose procedures of the present invention.
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TABLE I
Comparison of Manual and Automated Glucose Analysis
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(mg./100 ml.)
Specimen No. Manual Automated Difference
__________________________________________________________________________
1 105 101 -4 2 74 75 +1 3 89 91 +2 4 80 87 +7 5 78 76 -2 6 108 104
-4 7 126 118 -8 8 151 160 +9 9 96 94 -2 10 77 75 -2
__________________________________________________________________________
In Table II, the recovery of glucose added to pooled serum aliquots
is given. An average recovery of 99.3 percent (range 94.2-103.5
percent was realized.) ##SPC1##
For a fuller understanding of the nature and objects of the present
invention, reference may be had to the following examples which are
given merely as further illustrations of the invention and are not
to be construed in a limiting sense.
EXAMPLE 1
To a stirred solution off 22.0 g. of
7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one in
55.0 ml. of dry N,N-dimethylformamide was treated with 11.0 ml. of
a methanolic solution of sodium methoxide (0.0835 mole of
NaOCH.sub.3) and stirred for 30 minutes. After "30 minutes" 15.0
ml. of a toluene solution containing 0.0174 mole of
.gamma.-dimethylaminopropyl chloride was thereafter added, and the
mixture stirred at 75.degree. C. for 5.5 hours. Solvents were
removed under reduced pressure and the residual oil was dissolved
in 100 ml. of dichloromethane. The resultant solution was washed
with water, dried and evaporated. The oil was next dissolved in 100
ml. of ethyl acetate and filtered over 100 g. of activated neutral
alumina (Grade I). Using ethyl acetate as the eluent,
7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodi
azepin-2-one was recovered from the column.
EXAMPLE 2
The
7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodi
azepin-2-one formed in Example 1 was dissolved in sufficient
methanol to provide a 10 percent solution. This solution was then
saturated with hydrogen chloride. A sufficient amount of ether was
added to cause turbidity. The resultant mixture was allowed to cool
for several hours.
7-Bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodi
azepin-2-one dihydrochloride precipitated out on standing and was
separated by filtration. The salt was recrystallized from a
methanol-ether mixture as pale yellow prisms, M.P.
181.degree.-183.degree. dec.
EXAMPLE 3
This example demonstrates the applicability of the test method to
either blood serum or plasma.
In the method, aliquots of plasma or serum were added in 0.1 ml.
quantities to 2.4 ml. of a tungstic acid solution prepared by
mixing one volume 10 percent sodium tungstate and 8 volumes N/12
sulfuric acid. Each mixture was well mixed and then centrifuged at
2,500 r.p.m. for 15 minutes. A 0.1 ml. aliquot of the clear
supernatant liquid was treated with 2.0 ml. of a ferricyanide
solution which had been prepared by dissolving 0.115 g. of
potassium ferricyanide in one liter of a 2 percent aqueous solution
of sodium carbonate. The deproteinized fluid ferricyanide mixture
was heated for 5 minutes in a boiling water bath, rapidly cooled to
about 25.degree. C. and treated with 2.0 ml. of a ferric ion
reagent prepared by dissolving 0.27 g. ferric chloride hexahydrate
in one liter of an acetate buffer comprising 272.0 g. sodium
acetate and 294.0 ml. glacial acetic acid, and 2.0 ml. of a
solution prepared by dissolving 2.0 g. of the compound produced in
Example 2 in one liter of 1M acetate buffer. The solutions were
thoroughly mixed and the absorbance of the violet blue color that
develops was measured after about 10 minutes against a reagent
blank at 580 nm in a Coleman Spectrophotometer using a cuvette with
a 19 mm. light path.
The glucose content of the specimens was obtained by reference to a
calibration curve prepared by plotting the absorbances (A) given by
standard glucose solutions treated in the same manner against
concentration or by the Beer-Lambert formula. Utilizing 4.0 mcg.
glucose/0.1 ml. as a standard, the concentration of the specimen
was calculated according to the formula:
(Absorbance of Specimen/Absorbance of Standard) x 100 = mg.
glucose/100 ml.
For comparative purposes, glucose analyses were also conducted on a
like number of samples utilizing a modified Folin-Wu procedure as
described by B. D. Tonks in American Journal of Clinical Pathology,
22:1009, (1952).
The results obtained utilizing the aforesaid two techniques are set
forth in the following table: ##SPC2##
The comparative data obtained utilizing plasma specimens was
completely analogous to that with serum.
Utilizing similar reagents and quantities as were employed in
Example 3, tests were conducted utilizing the compound prepared in
Example 2 and comparing the results with the glucose content
deproteinized plasma prepared as described in B. Klein in Clinical
Chemistry, 5; 62, (1959) and designated Somogyi filtrates.
The results obtained are set forth in the following table:
##SPC3##
EXAMPLE 5
In an analogous manner to that employed in Examples 3 and 4, serum
specimens from 20 randomly selected blood samples obtained from
healthy individuals and hospitalized patients were tested for
glucose content. For comparative purposes, the glucose content of
the same blood sample was also determined utilizing a standard
glucose oxidase procedure. The glucose oxidase procedure employed
herein is described in detail by R. Richterich and J. P. Colombo in
Klin. Woch., 40, 1208, (1962) and A. Saifer and S. Gerstenfeld in
J. Lab. Clin. Med., 51, 448, (1958).
The results obtained utilizing the two techniques are set forth in
the following table:
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Comparison of Glucose Analyses
---------------------------------------------------------------------------
(milligrams glucose/100 ml.)
Specimen Benzodiazepine Glucose Oxidase Difference No. Test Test in
mg. 1 82 80 +2 2 98 98 +0 3 105 102 +3 4 86 83 +3 5 82 86 -4 6 105
112 -7 7 82 76 +6 8 68 70 -2 9 135 129 +6 10 68 70 -2 11 85 80 +5
12 289 288 +1 13 89 84 +5 14 69 70 -1 15 95 98 -3 16 95 95 +0 17 89
84 +5 18 66 67 -1 19 69 75 -6 20 92 98 -6
* * * * *