Note: Descriptions are shown in the official language in which they were submitted.
'J 7 ~ 7
METHOD FOR DETECTION OF
THIOT. COMPOUNDS IN A FLUTn SAMPT.F.
Field of the Invention
This invention relates to an analytical aaent
and method for detecting thiol compounds in a fluid
system, including thiol compounds created temporarily
during a reaction step. More specifically, the
analytical agent and method of this invention is
directed to the colorimetric determination of thiol
compounds and can be used in determining the presence
of reagents or enzymes relating to, or causing, the
presence of a thiol compound.
Background of the Invention
Thiol compounds exist in nature and can also be
created by chemical reaction. Such reactions include
disulphide reduction reactions and biochemical
reactions such as:
MS-1476
,
,
- 2 - J~ ~ 3 t~ 7 ~ 7
Liponamide
dehydrogenase
a) ~ CONH2 CONH2
~ SH SH
NADH+H NAD
CH3 o
10 ll e Cholines~erase
b) CH3-l-cH2cH2-s-c-cH3 J
CH3
IC~H3
CH3-N-CH2CH2-5H J + CH3COOH
CH3
Numerous methods are known for the detection of
compounds containing thiol groups. A particularly
useful and frequently quoted method is described by
G. K. Ellman in Arch. Biochem. Biophys., 82, 70-77
(1959). This method is based upon a thiol reaction
with 3,3'-dithio-bis-6-nitrobenzoic acid (Ellman's
reagent) which produces yellow anion of 3-mercapto-
6-nitro-benzoic acid.
One disadvantage of the Ellman method however is
that only shades of yellow are created by the ~educed
disulphides. Color shades in the red or blue region
are generally considered much more desirable and
therefore would be better adapted to the visual
determination of thiols.
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Consequently, it is an objection of this inven-
tion to provide a reliable, accurate, and inexpensive
analytical agent and method for colorimetrically
determining thiol compounds, wherebv the colors
provided by the agent and method are in the red or
blue region.
Other objects and features of this invention
will become apparent to those skilled in the art upon
reading the specification and claims herein.
Detailed Description of the Preferred Embodiment
The agent and method of this invention is
directed to the following reaction:
2 R-SH + 2Fe ~ R-S-S-R + 2Fe
In this reaction, the thiol compound interacts with
iron in the +3 oxidation state, causing the ion to be
reduced to a +2 oxidation state and causing the thiol
compound to oxidize to form a disulphide. The
resulting Fe 2 ion can be complexed with a suitable
ligand to create a color change. In this way, the
molar extinction of Fe 3 and the resulting color
change due to the color-inducing ligand Fe 2 complex
relates directly to the amount of thiol compound
initially present in the system.
Numerous methods are known for detecting Fe
ion by means of suitable complex ligands as colored
complexes. Examples of suitable ligands for this
purpose include compounds from the group comprising
ferroins, cuproins and terroins. Particularly
suitable complex ligands include hydrazones and their
tautomeric azoforms, tetrazolylpyridines,
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1~3~'7~7
pyridyl~uinazolines, bis-isoquinolines, imines,
phenanthrolir,es, bipyridines, terpyridines,
bidiazines, pyridyldiazines, pyridylbenzimidazoles,
diazyltriazines, o-nitroanilines, phenols, tetrazines,
triazines, pyridines, phenazine, auinoxalines,
benzimidazoles, oximes of substituted methyl or
phenyl-2-pyridyl ketones (Smith, 26 Analyt. Chem.,
1534-1538 (1954); Schilt et. al, Talanta 15 (1968)
47S-478; Schilt et. al, 15 Talanta 1055-1058 (1968);
and Schilt et. al, 16 Talanta 448-452 (1969~. A
description of other complex ligands can be found in
Blandamer et. al, J. Chem. Soc., Dalton 1001-1008
(1978), Case, 31 J. Org. Chem, 31 2398-2400 (1966)
and also in British Patent Application 701,843
published Jan 6,54. Of cour~e, complex ligands
other than those mentioned here can al~o be u~ed.
If an embodiment of this invention is used which
incorporates complex ligands in a test strip format,
it may be advantageous for the complex ligands to
also carry hydrophobic groups or ion exchange
functional groups. This will improve bonding of the
complex ligands to the matrix, thereby preventing the
test strip from "bleeding." Examples of hydrophobic
groups which can be used include long-chain alkyl or
aralkyl groups. Polymer bondinq of the complex
ligands is also conceivable.
According to the invention, the test agent can
be used for detecting thiols such as liponic acid
amide, thiocholine, glutathione or coenzvme A, or of
thioglycosides. Furthermore, a thiol precursor can
also be detected such as when a thioester, thioether,
disulphide or thioacetal is formed in a test system.
The test agent of this ir.vention is suitable for
1~ 3g7~7
-- 5 --
detecting enzymes which cleave thio compounds, such
as thioether hydrolases, the thioesterases or thio-
glycosidases. Furthermore, esterases such as
cholinesterase (CHE) can also be detected. Although
the natural substrate of CHE is acetylcholine, this
enzyme is also capable of cleaving acetyl- and
buturylthiocholine, and the test agent can then
detect the free thiol group formed by the cleavage.
This invention can also be used with biochemical
reactions in which free thiol groups are formed on
the basis of redox reactions, such as the reduction
of lipoic acid catalyzed by lipoamide dehydrogenase
in the presence of reduced nicotinamide adenine
dinucleotide (NADH).
Therefore the test agent of this invention can
be used in the analysis of reactions which depend
upon NADH. Typical representatives of NADH-dependent
enzymes include: lactate dehydrogenase, alcohol
dehydrogenase, glucose dehydrogenase, glycerol-
aldehyde dehydrogenase, glycerol phosphate dehydro-
genase, malate dehydrogenase and the like. The NADH
can also be the end product of multi-stage enzymatic
reactions, such as in analyzing glutamate oxalacetate
transaminase (EC 2.6.11), glutamate pyruvate trans-
aminase (EC 2.6.12) or creatinine kinase (EC 2.7.32).
NADH can also be the reaction product in analyz-
ing substrates such as lactate, glucose, malate, urea
and the like. By using the test agent of this
invention in the analysis of NADH-dependent reactions,
a substantial improvement in sensitivity is achieved,
because two moles of Fe2+ are formed per mole of
NADH, and the colored complexes in some cases have
very high extinction coefficients.
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It has proven to be advantageous to supply the
Fe3 complex bonded to the thiol substrate, and
possible complexing agents are EDTA, HEDTA, citric
acid, malic acid, lactic acid, amino acids such as,
for example, alanine, glycine and glutamine, crown
ethers such as, for example, 18-crown-6,phenylaza-
15-crown-5, benzo-15-crown-5 and dibenzopyridino-
15-crown-5, or triazinophanes and kryptates.
The concentration ratio of thiol:Fe3 should be
at least 1:1 and preferably about 1:5. For the
fastest possible reaction, the ratio of 1:20 is
particularly preferred, although the ratio of 1:10 is
also favorable.
The test agent or test system in the context of
the present invention can be used to measure analytes
in a cell. In addition to the Fe3 ions and the
complex ligands, the test agents can also contain all
the reagents necessary for the particular analysis
such as enzymes, substrates, coenzymes, effectors,
antigens, antibodies and the like. These test agents
can contain substances which do not react, such as
buffers, wetting agents and stabilizers.
As described above, enzymes can be determined
which form thiol groups with the aid of NADH and
NADPH as the coenzyme, including liponamide dehy
drogenase and gluthathione reductase. ~eagent
combinations can be prepared from the enzymes,
reagents and substances already mentioned and can be
mixed as a solution or as a powder or in the form of
tablets or a lyophylisate. The reagent combination,
if not already in solution, can be dissolved in water
or another suitable solvent, and a reagent solution
is thereby prepared. If the reagent combination
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consists of individual components, these are mixed
with one another. After the sample (for example
substrate solution, enzyme solution, blood serum,
plasma or urine) has been mixed with an aliquot
portion of the reagent mixture, the resulting color
is measured on a photometer. The particular concen-
tration or substrate concentration is then calculated
via the molar extinction coefficient and the volumes
of reagent or sample added. Both kinetic and end
point measurements are possible.
The Fe system/ligand can also be impregnated
onto absorbent reagent carriers (such as papers,
fleeces and the like) with:
1. one or more reagents or other enzymes
necessary for the particular parameter
detection,
2. a buffer system,
3. wetting agents
4. activators and
5. other auxiliaries.
For this, one or more impregnating solutions can be
prepared in the form of aqueous, organic or mixed
solutions, depending on how the reagents or auxil-
iaries dissolve.
Absorbent or swellable carriers, preferably
filter paper or absorbent fleece of glass or plastic,
are impregnated or sprayed with these solutions and
then dried. The reagent carriers thus prepared can
be used either as rapid diagnostics for directly
determining the contents of a liquid sample, i.e.
body fluids such as blood, urine or saliva, or
foodstuffs such as fruit juices, milk and the like.
The liquid is thereby applied directly to the reagent
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carrier or is immersed briefly in the liquid.
Semiquantitative determination is possible by allocat-
ing a comparison color to the color thus formed.
Quantitative evaluation can be carried out by reflec-
tance photometry.
It is also possible to introduce the test agent
according to the invention into carrier matrices
prepared from casting solutions. Examples which may
be mentioned here are cellulose, cellulose deriva-
tives, gelatin, gelatin derivatives or plastics, suchas polyurethanes and acrylamide. It is advantageous
here if the test agent and, if appropriate, the other
necessary reagents are added directly to the casting
solution. It is thereby possible to produce the test
device, consisting of the carrier and reagents, in
one operation.
By eluting the above-mentioned reagents with
water or buffer or serum from the absorhent carrier,
a reagent solution can be prepared. Substrates or
enzymes can then be determined in a photometer cell
as described above.
Suitable buffers for the test agents mentioned
are phosphate, citrate, borate and GOOD buffers with
alkali metal or ammonium counterions. However, other
systems can also be used. The pH values to be aimed
for are 6 to 10, although 6.5 to 7.5 is preferred.
Wetting agents are, in particular, anionic and
cationic wetting agents which undergo ionic inter-
actions with the compounds according to the inven-
tion. Nonionic wetting agents which activate theenzymes can also be used, and sodium laurylsulphate,
sodium dioctylsulphosuccinate and alkylaryl polyether
alcohols are preferred.
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g
Effectors which can be used are those known for
the particular enzymatic reaction.
Other auxiliaries which may be appropriate are
the customary thickeners, solubilizing agents,
emulsifiers, optical brighteners, contrast media and
the like, such as are known in corresponding tests
with other chromogens.
EXAMPLE
Thiol Detection with FeCl3 and Complex Ligands
To detect NADH in the test svstem described, the
following reagent constituents were introduced into a
cell:
Concentration
in the test
1740 ~l of 0.1 M/l acetate buffer,
pH = 5, 87 mmol/l
100 ~1 of lipoamide 2.5 mmol/l
100 ~l of FeC13 solution 1 mmol/l
60 ~l of dipyridyl 3 mmol/l
20 20 ul of lipoamide dehydrogenase 12 kV/l
After measurement of the reagent blank values,
the reaction was started by addition of 20 ~l of NADH
solution. The extinction maximum measured is at 515
nm. Kinetic investigations showed a stable end point
(change in extinction within 20 minutes = 1%) after a
reaction time of only 1 minutes.
To test the functional capacity and linearity,
NADH concentrations in the range of 1 to 10 mmol/l
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were measured in the test batch. The differences in
extinction measured at 515 nm are summarized in Table
1.
TABLE 1
NADH 515
(mmol/l)
1 0.124
2 0.219
3 0.327
4 0.429
0.513
6 0.633
7 0.735
8 0.822
9 0.964
1.098
The colors obtainable with the various complex
ligands and the corresponding extinction maxima are
summarized in Table 2.
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TABLE 2
Formula Colour ~max
H O
~<~ ~H? red 540
N--N
~ .
~N~ ,N~
2 --N N=NJ \-~/ b l u e 615
3 N~ red 508
- ~ r e d 5 10
NOH
OH
~@~N~N = ~3
S~2
I violet 556
NH2 OCH
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TABLE 2 (continued)
Formula Colour ~max
N-OH
0~0
6 blue 650
H ~ H
H3C CH~
~> ,.
7 N==~ red 522
Q~
N--<~
N ~
8 violet 552
SO ~Na
~>
.~N=N\~SO ~Na
9 violet 560
OC8H17
HO~S ~
N~ ~ ~ violet 583
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i ~ .3 ~ 7 ~ 7
TABLE 2 ~continued)
Formula Colour ~max (nm)
11 CH3 CH3 yello~ 441
I
H2N-N=C C=N-NH2
H1 5C1 7 IC7Hl 5
H2N-N=C C=N-NH2
12 yel low 443
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