Note: Descriptions are shown in the official language in which they were submitted.
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~OE ~1/8 012
The inver,tion relates to an ac3ent for detecc;ng
glucose in biological fluids, ~hich essentially com~
prises an adsorbent matrix~ a glucose oxidase, a peroxid-
ase and a chromogen as re3gent anci contains a nitrate and,
optionally~ a UV absorber.
Diabetes mellitus is a chronic disease ~hich must
be continuously monitored. It has proved to be reason~
able for the diabetic to monitor his o~ln metabolic status
afte,r ins~ruct;on by ~he phys;cian ;n order to be able,
1~ if necessary, immediately to adjust the necessary insulir
dose ~o suit ~he changing requiremcnts. The check of
metaboLism is carried out sush that the conten~ of
~lucose in the urine or in the blood is routineLy
determined. In general, the diabetic determines his O~il
urinary glucose value semiquantitatively, whilst~ in
contrast, quantitative measuremen~ of blood sugar is
carried out by an expert.
The determination of urinary glucose sl1ould cover
a concentration rancge up to about 5 g of c3lucose/100 ml
~- ~f urine.
hi present, there arè t,to met,hods for rapid semi--
~uantitative C~etermiQatiOn o-F c3lucose:
1. Test ;ablecs accordincJ to the non-specific reduction
r,letllocl o-F ~ EDlKr and
~ Test strips ~hich are irDprec~nated ~ich c~lucoce oxidase,
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which reacts soecifically, a chromogen and a peroxidase.
The glucose oxidase in the test strips catalyzes
the ox;dation of glucose with oxygen to for~, gLuconolact-
one and hydrogen peroxide. In a subsequent non-speçifi~
reaction~ ehe latter oxidizes a chromogen, ~i~h peroxid-
ase catalysis, to g;ve a dyestuff.
The t~o methods ~entioned are associated uith dis-
ad~antages: -
! Th2 reduction method is non-specific, since
other reducing com?ounds, such as, for exanple, galactose,
are also de~ected. Furtherrrlore, i~ is necessary to use
corrosive reagents for this rnethod (G. Friedrich~ "Der
;nformierte Arzt" 8 (9), Off-print, 1~80). In addition,
~he reaction is associa~ed ~ith a not inconsiderable
'l~ evolution ~f heat alld ~iih an interle~ g forr,7atiun of
foam. The performance of ~he tests ~lith tablets and ~est
tubes is elaborate, inconvenient and costly.
The same applies to the second rnethod of deterrnin-
at;on~ S;nce thc semiquantitative test strips for urine
hitherto only comprise a ran~e of indication up to a
axilnum of 2 Q of glucose/100 ml of urine~ they are un-
suitable for day in day-out routine testing, since higher
glucose concentrations should also be de~ected.
In order to get round this a'isadvantage, the use
of a diLuting de~ice has been proposed t~orge and
Schlebusch, "t1edizinische Klinik" 75, 537-591, '1980~.
~;lutir-g devices of this type are disadvan~ageous for
simple routine deterlnination, since man-,pulationc are
expected of the unpractised diabetic, ~lith ~hich he is
~ ~?t --
not con~ersant (pipetting, diluting and evalua-ting). The
hygienic requirements are Yery stri~t and cannot al~lays
be fulfilled~
In contrast, it is intended to provide the dia-
betie with a simple tes~, which is easy to learn, ishygienieally aceeptable ancl ~Ihich eomprises as largD a
eoncentration range as possible.
Thus the objeet of the present invention was an
improved agent for determining glucose in biological
fluids~
It is kno~n, from Bioehem~ Journal 42, 221 (1948),
that glueose oxidase is inhibited as much as 13 % by
sodium nitrate~ This inhibitory effeet is so small for
glucose test strips, in ~hich a large exeess of enzymes
1~ ;s employed, that no use has hilherto Deen made of il.
Our own experinents showed that a eorresponding rapid test
for deterrnining glucose ;s not inhibited by nitrates.
Conversely, it has no~ been found, surprisingly,
that by impregnating glucose test strips ~ith nitra~ces,
their range OT indication is signi-ficantl) enlarged,
partieular~y uhen hydrophobic films (IJ~ ~lishinsky in
Quality Control ;n Clinical Chermistry; Amido, Rosalhi and
~. Kampen, page 425, de Gruyter, Berlin 1975) are used.
By this means the range of indica~:ion of 2 CUstOnlary
semiquant;tative test reg;on, ~Ihich extends to a max;murr,
of a'~out 2 9 o~ glucose/1~0 ;nl of urine, can be extended
to rnore than , g of glucose/lOO rnl of urine. These modi-
fied tesc strips chus comply, in respect of their range
of use, with the rec-uirements placeo on a sirnple dia~nos~:ic
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aid by a diabetir. Furthermore~ a ~est strip of this
type fulfils the hygienic dernands~
Furthermore, it has been found that ~he resist~
ance of these test strips to ligllt can be improved oy the
5 ~ddit;on of UV absorbers.
The object described above has now been achieved
by ~he agent containing, in addition ;o the cus-tomary
components, a nitrate and, optionally, a llV absorber.
Accordingly, the inveneion relates to an agerlt
10 ~or the detection of glucose in biological fluids, essen-
tially composed of an adsorbent matrix~ containing a
chromogen, a glucose oxidase, a peroxidase and, option-
ally, a stabilizer, which contains a nitrate and, optionally,
a UV absorber.
~h. inver,tior~ furthcr rclate~ to the use o-f ~rnmon-
ium nitrate or a nitrate of an element of the first group
of the periodic table, ammonium and sodi~m nitrate being
preferred. Examples of UV absorbers which can be used
are the derivatives of benzophenone, of benzoic acid,
20 oxalic acid or of benzotriazine.
Compounds of this type are described in "IJllrllanns
Enzy~;lopadie der technischen Chemie" ~lJllrnann's Encyclo-
pedia of Industrial Chemistry), ~th edition, volu~e 15
pa~e 259r Verla~ Chemier Weinheim, Federal Republic of
25 Germany. 2,2'-Dihy~roxy-4-methoxybenzophenone (Cyasorb
UV 24, Arnerican Cyanamide)t 2-~ydroxy-4-methoxybenzo~
phenone-5-sulfollic acid (UV absorber HMBS "Riedel",
Riedel-de Haen AG, SeeLze, Federal Rep~blic of Gerrr!any)
or dihydroxybenzGpllenone are preferred.
The agent accordin~ to the invention is thus com-
posed o~ an ~idsorbing support matrix, in i~hich a glucose
oxidase, a peroxidase, a chromogen, a i~ater-soluble
nitrate and, optionally, a UV absorber are impregnat2di.
A(l absorbent plane structures of natural or synthetic
or;~in can be used as the support matrix, such as, for
example, non-uoven fabrics, papers, asbes~os or polymer
f;lms.
The agent is advansageously prepared such that
the chromogen, the buffer and the enzymes, together with
the nitrate, in aqueous solution are appliecl to the test
pciper. The UV absorber can also be dissolved in this irn-
pregnating solution. I~ is also possible to apply the UV
absorber i~ith a iJater repelling agen~c to the support
ma~rix in a further steo. The sequence of imprec~rla~ion
steps is arbitrary; it is also possible to start uith the
application of the i~ater-repel~ing ~igent. A process has
proved effective in uhich initially an indicator base
paper is impregnated ~ith chromogen, buffer, enzymes,
~etting agent, stabilizer, a i~ater soluble nitrate and a
UV absorber. Then a further UV absorber and a ~Jater-
repelling a~ent, for example ethylcellulose, dissolved in
an organic solvent, ~hich is not miscible with Jater,
such a~ toluene, are applied.
The preferreo process for ~he ?repciraiion of an
agen. according to the .nvention ~hus co~priscs t~o steps.
hfter drying~ the reaction support is lar,ina~ecl on~o a
plastic film for easier manipulatiorl.
The amount of nitrate :sed in each case is
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no~ critical; it can be adjusted to sui~ the particular
envisa~ed use, that is to say the range of concentration
to be co~ered. It can be between 0.1 and 7 X Sv:~) rela-
tive to ammonium nitrate. A concentrat;on bet~leen 0.3
S and ~.0 X (v:~3 is preferred.
The amount ~f UV absorber used is also not criti-
cal~ ln general, a larger amount of UV absorber is
emplo~ed for a larger amount of nitrate. The maximum con-
centration of the UV absorber is limited by ;-ts solubility
in the solvent chosen. The cor,centrations can vary between
Or1 and ~.0 % ~:v), however~ a concentration range
betweel- 0.4 and 3.0 X (~:v) is preferred.
In order to use the agent descrihed, it is
br;efly dipped into the ~u;d to be tested. h graded
~5 coLor reaction develops, depending on the content of glu-
cose in the fluid, which can be graded semiquanti~atively
up to 5 9 of glucose/100 ml oF urine.
The advantage of the invention emerged f-rom com-
para~ive investigat;ons w;th rapid diagnostic aids accord-
in~ to the state of the ar~.
Three test strip papers ~ere prepared accordingto Examples 1 to 3, of which 1 a represents the state of
the art. 1 b was prepared using a water-soluble ni~ra-.e~
1 c corresponds to 1 b, but the paper was additionall
;mpregnated ~lith a UV absorber.
The follo~;nrJ tests ~ere carried out ~ith the
f;nished p 2 p ers
Paperc 1 a, 1 b and 1 c were eYposed to sunli~ht~
After about 20 l~inutes of this, 1 b riscolored to such
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an intense green that it gave a false positi~e result on
subsequent ~etting ~/ith a glucose-free solotion. In con-
trast, papers 1 a and 1 c did not change their original
color even after 60 rnin~
In a further series of tests, papers 1 A ancl
1 c uere dipped in samples of urine ~Ihich contained 50,
15n, 500, 1,500~ 3,000 and 5,000 mg of gluGose/100 ~nl oF
urine. The conc~ntration s~eps up to 1~,500 Ing/dl could be
easily clifferentiated with paper 1 a, ~hiLst the two
10 higher glucose concentrations ~ere not distinguished. In
contrast, the entire spectrum from 50 mg of glu~ose/100 ml
oF urine up to 5,000 mg of glucose/100 ml of urine could
be differentiated with paper 1 c.
The invention is illustrated in more detail by
1~ means of the -;ollo~Jing examples.
Example 1 t Paper 1 a
_ .
Solution 1 The following lJere dissolved in 100 ~nl of
0.1 M citrate buffer, pH 5.5:
0.5 cg of gela-tin
20 7.0 mg of tartrazine
0.6 g oF o-tolidinc dihydrochloride
0.2 g of peroxidase
0.~ 9 of glucc)se oxidase
1,0 g o~ polyethylene gLycol 1500.
Paper 2316 of Schle;cher anci Schull, Dassel,
Federal Republic of Gernlany, ~,las inlpregna~ed ~ith this
irnpregnation solution. After drying at 80C in an oven,
the paper uas subjected to a second3ry iMpregnation ~/-ith
Solution 2 containing 0 6 CJ Of ethylcellulose in 1G0 ~nl
: . .
s-j ~Lr~
of toluene.
Exa,-,lple 2 ,t Paper 1 b
A pa~er 1 b ~as prepared in accordance ~ith
Exampl~ 1, but an add;tional 2 9 of ammonium nitrate were
5 dissGlved in solution 1. The secondary impregnation was
h so lu~ i on 2 .
Example 3 / Paper 1 c
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Th e f o l lOh'i ng were additionally di sso lved i n
100 ml of ~he impregnation solution 1 according to
Example 1:
2.0 ~ o~ ammonium nitrate and
1rO ~ OT UV absorber HMBS "Riedel"~
The following lere dissolved in 100 ml of tol~ene
for ~he seconclary impregnation:
0.5 ~, of ethylcellu!ose and
2.0 9 of Cyasorb, Cyanamide.
Papers 1 a, 1 b and 1 c showed ~he propelties
described abuve.
