WST-5
-A Novel
Reagent for Clinical Assays of Dehydrogenases and Their Related
Substrates-
Munetaka
Ishiyama
Research section in Dojindo Labs
Tetrazolium salts are known to produce formazan dyes under
reducing conditions.
These salts have found applications in clinical assays that
are based on a spectrophotometric measurement of dehydrogenase
activities. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H
tetrazolium chloride (INT) and nitrotetrazolium blue (NTB)
have been used for this purpose over several decades. However,
formazan dyes derived from these tetrazolium salts are insoluble
in water, and these dyes stain tubes and cells of clinical
analyzers. In order to conquer these problems, we recently
developed a novel tetrasulfonated tetrazolium salt, 2,2'-dibenzothiazolyl-5,5'-[4-di(2-sulfoethyl)carbamoylphenyl]
-3,3'-(3,3'-dimethoxy-4,4'-biphenyl) ditetrazolium, disodium
salts (WST-5) which produces a highly water-soluble formazan
dye. A structural formula of WST-5 is shown in Fig. 1.
This tetrazolium salt has two tetrazolium skeletons as well
as NTB, and four sulfonic acid groups were introduced to tetrazolium
skeletons as water-soluble functions. 
Fig. 2 shows absorption spectra of WST-5 formazan at various
pHs. The wavelength of absorption maximum is 580 nm and molar
extinction coefficient is about 27000 in the range of pH 6.2
to pH 9.0. WST-5 was readily reduced by nicotinamide adenine
dinucleotide reduced form (NADH) in the presence of an electron
mediator such as 1-methoxy-5-methylphenazinium methosulfate
(1-methoxy PMS), which provides the basis to spectrophotometric
assays of NADH (Fig. 3).
In the several pH between 6.0 and 9.0, WST-5 shows a good
linear response with correlation coefficient (r) = 0.990 up
to 0.1 mM NADH. The time courses of the reducing reaction
of WST-5 with NADH at verious pH in the presense of 1-methoxy
PMS were shown in Fig. 4.
The reducing reaction of WST-5 completes within 5 minutes,
and resulting formazan was stable over 24 h.
WST-5 was applied to measurement of lactic acid. Lactic acid
is a substrate of lactate dehydrogenase (LDH) and it turns
to pyruvic acid by the enzymatic reaction as shown in Scheme
2.
LDH-mediated reducing reaction of nicotinamide adenine dinucleotide
(NAD) produces NADH at pH over 8.8. In the measurement of
lactic acid, measuring of the resulting NADH at 340 nm (UV
method) has been widely used because of its simplicity. In
the optimized condition, lactic acid was measured by WST-5
and NTB (Fig. 5).
Both WST-5 and NTB exhibited linear responses up to 0.1 mM
of lactic acid. The sensitivities of these tetrazolium salts
were 7-hold higher than that of UV method. Without a detergent
resulting NTB formazan was precipitated, on the other hand,
formazan dye derived from WST-5 was not precipitated. Newly
developed WST-5 will be useful reagent for clinical assays
of dehydrogenases and their related substrates.
For more detail information:
Munetaka Ishiyama
(ishi@dojindo.co.jp)
Dojindo Laboratories
Research Section
Research & Development Department
