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Патент USA US3072549

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3,072,539
United States Patent 0 ” "ice
Patented Jan. 8, 1963
2
1
802,281, ?led March 27, 1959, by the same applicants, in
that the catalyst of the present invention is water-soluble
3,072,539
and is therefore easily applicable to carriers, such as »
‘ DIAGNOSTIC COMPOSITION FOR
DETECTING GLUCOSE
?lter paper strips, together with the glucose oxidase, the
,OtIs
E.
Fancher
and
Dale
A.
Stautfer,
Elkhart,
Ind.,
as
signm-s to Miles Laboratories’ Inc" Elkhart, Ind” a 5 indicator, and such ‘other ingredients as may be required
.
corporation of Indiana
,
to give the appropriate reaction conditions.
No Drawing. Filed Nov. 6, 1959, Ser, No, 851,241
11 Claims. (Cl. 195-1035)
.
_
_
'
,
_
The metalloporphyrin used in the embodiments of this
invention is a mixture of isomeric urohemins or uroferri
_
,
porphyrin chlorides (C40H36N4 OmFeCl), which are char
_ This invention relates tea new and improved diagnos- 10 acterized by four acetic acid and four propionic acid
tic composition and is particularly concerend with a diaggroups attached to the porphyrin ring. The following
IIOSUC test Whlch is useful fol‘ the qualitative detection
structural formula characterizes one isomeric form of its
and quantitative determination of glucose in biological
complex chemical nature:
?uids, such as urine, and wherein the reagent composition
is incorporated upon a bibulous carrier.
'
15
-
The detection of glucose in urine as well as the deter-
We; _
mination of its concentration therein is of great impor. _
so as to regulate their sugar intake and who must fre-
'
_
HOOCCHZCHZP
cians, this glucose indicator may also be used efficiently
‘
, __'
entiation of glucosuria from other'meliturias, and the
-
.
v
\
V
in diabetes detection screening programs, in the di?er- 25
-
‘.i'
'
in routine urinalyses in hospitals‘and physicians’ o?ices,
-
'
l
testing on known diabetics by both patients and physi- "
Because early diagnosis and continued control are so
important in diabetes, a urine-sugar test, to be of greatest
,
/
hOOCCH-T
quently be guided in this regard by a regular check on 20
urine glucose. But beyond its usefulness in regular urine
s.
cnzcnzcoo'?
l-
tance for diabetic patients who must control their diets
like.
m
HOOLMTZ Ii '
’
HOOCCHZCEIZ!
-
Y
“CHZQOOH
-
As indicated above the catalyst used in the practice of
this invention is a ‘chemically reproducible ‘metallopor
value, must be conveniently rapid, simple enough for any 30 'Phyrin, the ‘mode 0f ‘Preparation being as follows!
patient to learn with ease, accurate enough to serve the
clinician, and sensitive enough to re?ect variations‘in‘the = '
The ‘porphyrins are mad‘? in accordance With the de
scription given by MacDonald, 1- Chem- Soc; (1952),
patient’s condition. Moreover, the reagent composition
4176, 4134, and C811- 1. Chem, 34, 1768 (1956). The
must be adequately stabk, ‘
metallation of the thus-produced porphyrins is carried
'
Procedures for the detection of sugar in urineare well 35 0111; according to the general procedure of Erdrnan and
known in clinical chemistry. One such procedure utilizes
COIWiIl, IACS, 69, 750 (1947)- The metalloporphyrin
Benedict’s copper reduction test, another employs a self- '‘
intel’mediat?s are then hydrolysed as Shown by Fischer
heating alkaline copper reduction test in tablet form (US.
Patent No. 2,387,244), still another involves a test which
and Drills “Die Chemie des Pyffols 1L” L Hdlfte, P- 515
It has been suggested by MacDonald that the reaction
depends solely on the action of enzymes (US. Patent No. ‘10 v‘Product, as mentioned above, may be a mixture of
2,848,308). Although these and other procedures have
urohemins in several isomeric 'forms wherein the arrange
greatly contributed to the art of diagnosing for urine
ment of acetic add aHdVPIOPiORiO acid groups yaIiéS
glucose, they have not entirely satis?ed the above-men- '
Separation of the'isomefs is not f?quifed OI‘ even desira
tioned requirement.
,
ble for the practice of this invention.
We have now found a novel and highly useful glucose- 4,
The urohemin mixture has: su?icient activity to catalyze
detecting means which represents an important improve- '’ the oxidation of sensitive indicators such as 2,7-diamino
ment and a fresh approach to the problem of determin- ,
?llofene WithOllt ‘the addition. of amiHe'd'OIlOTS} HOW
ing, glucose in various materials including body ?uids,
such as urine, by a technique which‘utilize's‘ a‘ diagnostic
ever, amine donors, such'as ll-aminobenzothiazole, nico
tinic acid and pyridine make it possible to decrease "the
composition that rivals even the latest ‘and most com- 50 quantity of urohemin required with sensitive indicators
monly used glucose tests in stability and quantitation. '
‘and greatly decrease the time, required for oxidation'of
, Speci?cally, we have now found that a certain synthetic , _ less sensitive indicators, such as o-tolidine.
'
._
metalloporphyrin compound is surprisingly and unexpect- '
' It is understood that such additives as suitable pro
edly capable of acting as catalyst to induce the, oxidation
ted-1V6 agents and Wetting agents may ‘be incorporated
of indicators by hydrogen peroxide formed when glucose 55 therein as illustrated below.- “Furthermore, this glucose
is aerobically oxidized in the presence of glucose oxidase.
test may contain a suitable inert dye to impart tothe
The principles-underlying these'reactions are well known
composition a uniform background color as well as an
and need not be expounded.
''
-~ "
A diagnostic composition according to the present in-
appropriate butter system to maintain a desired pH range
'(pH 3 to 7 with a pH of 5.4 being‘ preferred).
, ’
vention comprises as essential constituents glucose oxi- 60 Although the test device itself may comprise the re
dase, a synthetic metalloporphyrin and a color-forming
‘agent composition in the form of a tablet, powder, or
substance or indicator, such as 2,7-diamino?uorene, o
‘other, embodiment, we prefer, to insurev ease and sim
tolidine, leucoindophenols, etc.
' jplicity of test procedure, to affix the reagent composition
This invention represents an improvement over previon bibulous base materials or carriers, such as strips or
ously described compositions in that the catalyst is a 65 sticks of ?lter paper, by dissolving the component in‘ a
chemically reproducible, stable compound rather than
suitable solvent, impregnating‘ the bibulous strips with
a naturally occurring unstable enzyme mixture of a highly _, . the resulting solution and drying the ‘impregnated test
variable composition. Moreover, it is surprising and unexpected that very small amounts of the catalysts are
su?icient to produce the desired effect.
strips. As will be obvious from the examples, it is very
.easy'. to prepare the instant glucose indicator due to its
Embodiments of 70 manifest simplicity. '
this invention alsohave advantages over- the hemin-amino
iomplexes disclosed in patent‘ application Serial bio. ,
i
'
'
~ ,
r
'
The invention will now be illustrated in greater detail,
not limited, by the following examples:
'
' ' '
3,072,539
4
EXAMPLE I
prepared and tested as in Example I. The test sticks had
the same order of activity as those prepared according
to Example I.
In summary, this invention pertains to a diagnostic com
Preparation of the lmpregnating Solution
Algin (100 mg.) was stirred with 10 ml. of water until
a clear solution resulted. Then 10 ml. of a pH 5.4 buffer
position for the detection of glucose in body ?uids, and
especially in urine, consisting of a bibulous strip or stick
which has been impregnated with a composition compris
ing glucose oxidase, a metalloporphyrin, namely, an iso
No. 3 (disodium salt of 9-o-carboxylphenyl-6-hydroxy 10 meric mixture of urohemins and a color-forming sub
stance, such as, 2,7-diamino?uorene or o-tolidine which
2,3,4,7-tetraiodo-3-isoxanthone) in 100 ml. of hot water
is
oxidizable by hydrogen peroxide in the presence of the
was prepared and 10 ml. of this solution was added to
metalloporphyrin.
the algin-buifer solution with stirring. Then a solution
What is claimed is:
of 100 mg. of 2,7-diamino?uorene dihydrochloride in 10
11. A diagnostic composition for detecting glucose
solution (prepared by dissolving 352.8 g. of sodium citrate
dihydrate and 25.6 g. of anhydrous citric acid in water
and diluting to 1000 ml.) was added with stirring. A
solution of 2 g. of gelatin and 150 mg. of F. D. & C. Red
ml. of hot 50% ethanol was added.
A solution of 500 15
which comprises glucose oxidase, a compound selected
from the group consisting of a uroferriporphyrin chloride
lyophilyzed) in 5 ml. of water was added. The mixture
having the structural formula
was warmed to 40° and a solution of 10 mg. of the syn
thetic urohemin in 5 ml. of water was added.
mg. of glucose oxidase (28,700 U./g., dialyzed and
Preparation of Diagnostic Strip
20
A bibulous carrier, such as, ?lter paper out into narrow
r-cagcngcoon
strips with a water-impervious barrier of ethyl cellulose
one-half inch from the tip were dipped into the impreg
nating solution so that through the process of submer
sion and capillary attraction the entire one-half inch of
the strip up to the barrier was completely impregnated.
These strips were dried in a hot air oven at 100° C. for
from 30 minutes to 5 hours.
Procedure of Testing
In use an impregnated strip made as described above is
dipped into the liquid specimen to be tested. When con
tacted with urine containing glucose, test strips gave posi
tive reactions in about 10 seconds evidenced by various
30 and compounds having the same ferriporphyrin nucleus
dilfering therefrom only in the arrangement of acetic acid
and propionic acid groups and a color-forming substance
oxidizable by hydrogen peroxide in the presence of said
uroferriporphyrin chloride.
35
shades of blue color as follows:
2. A diagnostic composition for detecting glucose
With urine containing 1 or 2% glucose an intense blue
which comprises glucose oxidase, a compound selected
color developed in 10 seconds, while with urine contain
from the group consisting of a uroferriporphyrin chloride
ing 0.1% glucose a light blue color developed in 10 sec
having the structural formula
onds. With urines containing 0.25% and 0.5% glucose
nooccng _
-CH2CH2COOH
the blue colors which developed in 10 seconds were of 40
intermediate intensities, the urine containing the most
glucose being the darkest blue. When dipped in urine
containing no glucose, the sticks underwent no color
nooccnf
change. A simple color chart based on this phenomenon
may be conveniently prepared for use in estimating the 45
glucose content of the urine being tested.
nooccnzorzEXAMPLE II
The impregnating solution was prepared as in Example
I except that the urohemin (10 mg.) together with 20 50
HOOCCH2CH2-
mg. of 2-aminobenzothiazole was dissolved in 5 ml. of
hot 50% aqueous ethanol and this solution was added to
the rest of the mixture.
The diagnostic strips were prepared and tested as in
Example I. These sticks were slightly more active than 55
those prepared according to Example I.
EXAMPLE III
l-cnzcngcoon
— CHZCOOH
- cnzcoou
and compounds having the same ferriporphyrin nucleus
dilfering therefrom only in the arrangement of acetic acid
and propionic acid groups, and a color-forming substance
taken from the group consisting of 2,7-diamino?uorene
and o-tolidine, oxidizable by hydrogen peroxide in the
presence of said uroferriporphyrin chloride.
3. A diagnostic composition for detecting glucose
which comprises glucose oxidase, a compound selected
The impregnating solution was prepared as in Example
I except that the 2,7-diamino?uorene dihydrochloride was 60 from the group consisting of a uroferriporphyrin chloride
having the structural formula
replaced with o-tolidine dihydrochloride as the indicator
substance and the quantity of urohemin used was dou
nooccnz bled.
The sticks were prepared and tested as in Exam
ple I. When dipped in urine containing 1% glucose, a
blue color of moderate intensity developed in 1 minute.
When dipped in urine containing no glucose or in urine
containing 0.1% glucose, no color change was observed
in 1 minute. When wet with urine containing 0.25%
glucose, a faint blue color developed in 1 minute.
HOOCCHZ-
"
EXAMPLE IV
nooconzcnz-
—CH2CH2COOH
- 011200011
70
The impregnating solution was prepared by adding 200
HOOCCH2CH2~ cnzcoon
warmed to 40° to dissolve the amine. The sticks were 75 and compounds having the same ferriporphyrin nucleus
di?ering therefrom only in thearrangement of acetic acid
mg. of 2-aminobenzothiazole to the solution as prepared
according to Example III. The mixture was stirred and
8,072,589
5
having the structural formula
and propionic acid groups and a color-forming substance
taken from the group consisting of 2,4-diamino?uorene
and o-tolidine, oxidizable by hydrogen peroxide in the
presence of said uroferriporphyrin chloride.
HOOCCHg-
'
‘
nooccnjlm-cnzcnzcoon
’
// ‘ \N '\
4. A diagnostic composition for detecting glucose
which comprises glucose oxidase, a compound selected
from the group consisting of a uroferriporphyrin chloride
having the structural formula
6
from the group consisting of a uroferriporphyrin chloride
groups and ‘a color-forming substance taken from the
I
—- FeCl- —
10
-CI-l2CH2COOH
I
4-CH2CH2COOH
HOOCCHZ’
‘ .
nooccnzmz-
\
,HOOCCH2CH2—
" CHZCQOH
HOOCCHZCH2
‘ -.CH2CH2CO.0H
- CH2COOH
and compounds having the same ferriporphyrin nucleus
di?ering therefrom only in the arrangement of acetic acid
20 and propionic acid groups, a complex-forming compound
selected from the class consisting of Z-aminobenzo
thiazole, nicotinic 'acid and pyridine to activate said
_ cnzcoon
— cnzcoon
uroferriporphyrin chloride and a color-forming substance
and compounds having the same ferriporphyrin nucleus
differing therefrom only in the arrangement of acetic acid
oxidizable by hydrogen peroxide in the presence of the
activated uro'feirrinorphyrin chloride‘.
25
Y‘ 1 '
- "
-¢
from the group consisting of a uroferriporphyrin chloride
having the structural formula
5,. A diagnostic composition ‘for detecting ' glucose
which comprises glucose oxidase, a compound selected
j
8. A diagnostic composition for detecting glucose
which comprises glucose oxidase,/ a compound selected
and'propionic acid groups and 2,7-diamino?uorrene, oxi
dizable by hydrogen peroxide in‘v the presence of said
uroferriporphyrin chloride.
30
from the group consisting of ‘a uroferriporphyrin chloride
having the structural formula
.
nooccnz; j
" I
\
\"V- CHZCQOH
HOOCCH2CH2—
—"’ - CHZCOOH
40
45
and compounds having the same ferriporphyrin nucleus
differing therefrom only in the arrangement of acetic acid
and propionic acid groups and 2,7-diamino?uorene.
and compounds having the same ferriporphyrin nucleus
differing therefrom only in the arrangement of acetic acid
and propionic acid groups, a complex-forming compound
selected from the class consisting of Z-aminobenzothia
zole, nicotinic acid and pyridine to activate said uro
6. A glucose~detecting means consisting of a bibulous 50 ferriporphyrin chloride and a color-forming substance
carrier impregnated with a diagnostic composition com
taken from the group consisting of 2,7-diamino?uorene
prising glucose oxidase, a compound selected from the
group consisting of a uroferriporphyrin chloride having
the structural formula
and o-tolidine, oxidizable by hydrogen peroxide in the
presence of the activated uroferriporphyrin chloride.
9. A diagnostic composition for detecting glucose
55
nooccnz -
nooccnz-
nooccnzc'af
—CH2CH2CO0H
which comprises glucose oxidase, a compound selected
from the group consisting of a urofer-riporphyrin chloride
having the structural formula
60
nooccnz-
'7 cnzcoon
~CH2CH2COOH
65
nooccnjcnznooccn2cn2-
— CH2cooH
—-—— - cnzcoon
and compounds having the same ferriporphyrin nucleus
nooccnzcnge
- cuzcoon
di?ering therefrom only in the arrangement of acetic acid 70
and propionic acid groups and 2,7-diamino?uorene oxi
and compounds having the same ferriporphyrin nucleus
dizable by hydrogen peroxide in the presence of the
differing therefrom only in the arrangement of acetic acid
uroferriporphyrin chloride.
and propionic acid groups, Z-aminobenzothiazole to ac
7. A diagnostic composition for detecting glucose
which comprises glucose oxidase, a compound selected 75 tivate said uroferriporphyrin chloride by forming a com
3,072,539
8
plex therewith and 2,7-diamino?uorene oxidizable by
group consisting of ‘a uroferriporphyrin chloride having
hydrogen peroxide in the presence of the activated uro
the structural formula
ferriporphyrin chloride.
10. A diagnostic composition for detecting glucose
nooccng ~
which comprises glucose oxidase, a compound selected
from the group consisting of a uroferriporphyrin chloride
having the structural formula
N
nooc CHg‘I
l/xf- CHZCHZCOOH
/ _- FeCl- -
nooccnzcnz- --
15
I
_~ CHZCOOH
and propionic acid groups, Z-aminobenzothiazole to acti
vate said uroferriporphylrin chloride by forming a com
20
HOOCCH2CH2:
HOOCCH2CIi2
and compounds having the same ferriporphyrin nucleus
differing therefrom only in the arrangement of acetic acid
— c?zooon '
plex therewith, and 2,7-diamino?uorene oxidizable by
hydrogen peroxide in the presence of the activated uro
ferriporphyrin chloride.
References Cited in the ?le of this patent
and compounds having the same ferriporphyrin nucleus
di?ering therefrom only in the arrangement of acetic acid
and propionic acid groups, Z-aminobenzothiazole and
UNITED STATES PATENTS
25
2,848,308
Free ________________ .._. Aug. 19, 1958
203,451
Australia ____________ __ Sept. 27, 1956
FOREIGN PATENTS
2,7-diamino?uorene.
11. A glucose-detecting means consisting of a bibulous
carrier impregnated with a diagnostic composition com- '
prising glucose oxidase, a compound selected from the
OTHER REFERENCES
Chemical Abstracts, vol. 37 (1943), Par. 54232‘
UNITED STATES PATENT OFFICE
‘CERTIFICATE OF CORRECTION
Patent ‘No. 3,072,539
»
January 8, 1963
Otis E. Fancher et a1.
It is hereby certified that error appears in the above numbered pat
ent requiring correction and that the said Letters Patent should read‘ as
corrected below.
Column 1,, line 11, for "'concerend" read —— concerned -~;
line 44‘, for "requirement" read —— requirements ——; column 30
line 101 for "9-o-carb0xylphenyl-" read -— 9—o—carb0xypheny1-_
———; column 5, line 11 strike out "groups and a c0l0r~forming
substance taken from the".
Signed and sealed this 21st-day of April 1964.
{SEAL}
Attest:
ERNEST W. *SWIDER
.
Attesting Officer
EDWARD J.
BRENNER
Commissioner of Patents
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