Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

Catalytic photoinduced electron transport across a lipid bilayer mediated by a membrane-soluble electron relay.

Unidirectional photocatalytic electron transfer from a hydrophilic electron donor encapsulated in the interior of a liposome, to a hydrophilic electron acceptor on the other side of the membrane, has been achieved using the simple membrane-soluble electron relay 1-methoxy-N-methylphenazinium (MMP(+)). The total amount of photoproduct (>140 nmol) exceeds the number of moles of MMP(+) present (125 nmol), thus showing that the transport of electrons is catalytic.

Ultrasensitive detection of the reduced form of nicotinamide adenine dinucleotide based on carbon nanotube field effect transistor.

We developed a simple, ultrasensitive, and quantitative detection method for the reduced form of nicotinamide adenine dinucleotide (NADH), based on carbon nanotube field effect transistors (CNTFETs). Following the injection of NADH at different concentrations, we obtained different electrical signals from a semiconductor characterization system mimicking biological catalysis of NADH dehydrogenase (CoI). Here, FET was fabricated via photolithography, attaching silicon wells, as the detection chamber, on the channel area of the single wall carbon nanotube (SWCNT). SWCNTs were functionalized with phenazine derivant, a counterpart of the key functional prosthetic group of CoI enzyme. In the presence of NADH, electrons transferred to phenazine derivant through SWCNT, by analogous means of the electron transport chain formed by a series of iron-sulfur (FeS) clusters in CoI. Using this method, the limit of detection was as low as 1 pM, and the range of linear response was 10 pM to 500 nM. Significantly, this approach possesses great potential for applications in real-time detection of NADH at extremely low concentrations, and rigorous analysis for NADH in electrochemical fields.

Visible wavelength spectrophotometric assays of L-aspartate and D-aspartate using hyperthermophilic enzyme systems.

Methods with which to simply and rapidly assay L-aspartate (L-Asp) and D-aspartate (D-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of L- and D-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an L-aspartate dehydrogenase (L-AspDH) system to colorimetrically assay L-Asp and a system of three hyperthermophilic enzymes--aspartate racemase (AspR), L-AspDH, and L-aspartate oxidase (L-AO)--to assay D-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD(+))-dependent L-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, D-Asp was measured after first removing L-Asp in the sample solution with L-AO. The remaining D-Asp was then changed to L-Asp using racemase, and the newly formed L-Asp was assayed calorimetrically using NAD(+)-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 µM L- and D-Asp in the assay systems. In addition, methods were applicable to the L- and D-Asp determinations in some living cells and foods.

Biosensor assay of neuropathy target esterase in whole blood as a new approach to OPIDN risk assessment: review of progress.

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 microL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area.

Cell surface oxygen consumption: a major contributor to cellular oxygen consumption in glycolytic cancer cell lines.

Oxygen consumption for bioenergetic purposes has long been thought to be the prerogative of mitochondria. Nevertheless, mitochondrial gene knockout (rho(0)) cells that are defective in mitochondrial respiration require oxygen for growth and consume oxygen at the cell surface via trans-plasma membrane electron transport (tPMET). This raises the possibility that cell surface oxygen consumption may support glycolytic energy metabolism by reoxidising cytosolic NADH to facilitate continued glycolysis. In this paper we determined the extent of cell surface oxygen consumption in a panel of 19 cancer cell lines. Non-mitochondrial (myxothiazol-resistant) oxygen consumption was demonstrated to consist of at least two components, cell surface oxygen consumption (inhibited by extracellular NADH) and basal oxygen consumption (insensitive to both myxothiazol and NADH). The extent of cell surface oxygen consumption varied considerably between parental cell lines from 1% to 80% of total oxygen consumption rates. In addition, cell surface oxygen consumption was found to be associated with low levels of superoxide production and to contribute significantly (up to 25%) to extracellular acidification in HL60rho(0) cells. In summary, cell surface oxygen consumption contributes significantly to total cellular oxygen consumption, not only in rho(0) cells but also in mitochondrially competent tumour cell lines with glycolytic metabolism.

Chemiluminescence assay for tetrahydrobiopterin based on the generation of hydrogen peroxide using isoluminol-microperoxidase in the presence of 1-methoxy PMS.

We developed a novel highly sensitive chemiluminescence (CL) method for BH(4). The principle of the proposed method is based on active oxygen formation induced by 1-methoxy-5-methyl phenazinium methyl sulphate (1-methoxy PMS) in the presence of dissolved oxygen. Furthermore, active oxygen is determined by a CL assay involving the luminol reaction with microperoxidase. In this report, we examined the mechanism of formation and identified the reactive oxygen species derived from BH(4) employing 1-methoxy PMS. Additionally, optimum conditions for the CL assay of BH(4) were established.

Electron transfer from NADH bound to horse liver alcohol dehydrogenase (NAD+ dependent dehydrogenase): visualisation of the activity in the enzyme crystals and adsorption of formazan derivatives by these crystals.

The crystals of holoenzyme from native and cross-linked alcohol dehydrogenase exhibit electron transfer from NADH to phenazinium methosulfate (PMS), and then to the tetrazolium salt sodium 3,3'-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonate (XXT). The slow dissociation of the cofactor and/or the conformational change associated can now be bypassed. The reduction product, formazan, did not diffuse out of the crystals in buffer and the crystals turned colored. In the presence of dimethyl sulfoxide or dimethoxyethane, the formazan diffused out to the solution. The reaction rates were found to be, respectively, 18% and 15% of the redox reaction rate of ethanol with cinnamaldehyde, close to the activity determined for the enzyme in solution in the presence of dimethoxyethane. The use of system PMS-tetrazolium salt is a useful tool to visualize the activity of dehydrogenases and other electron transferring systems in the crystalline state. The adsorption of formazan by the alcohol dehydrogenase crystals occurs in solution.

Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases.

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells.

Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ(1)) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ(1), whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40-60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.

An improved, simple screening method for detection of glucose-6-phosphate dehydrogenase deficiency.

We established a new, simple and rapid screening method for detection of glucose-6-phosphate dehydrogenase (G6PD)-deficiency by using a new formazan substrate, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) with a hydrogen carrier of 1-methoxyphenazine methosulfate (1-methoxy PMS), instead of a combination of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and phenazine methosulfate (PMS), as used in many previous formazan methods. WST-8 does not react with haemoglobin, and the formed formazan is highly water-soluble, differing from MTT. Thus, the whole procedure can be performed in aqueous solution in a tube or well without any special equipment other than micropipettes. Within 1 h at room temperature, the strong orange colour of the WST-8 formazan formed in normal blood samples could be distinguished, by naked eye, from G6PD-deficient blood samples with less than 50% residual activity. We also found that reagents in the WST-8/1-methoxy PMS method were more resistant against exposure to sunlight than those in an MTT/PMS method. As the new method is both qualitative and quantitative, it is possible to express G6PD activity as increase of NADPH concentration by reading absorbance at 460 nm after incubation for 30 or 60 min.

A new assay system for guinea pig interferon biological activity.

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.

Bilirubin dehydrogenase, an enzyme in Aspergillus ochraceus IB-3 useful for diagnostic measurement of bilirubin.

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 microM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.

A biomimetic phospholipid/alkanethiolate bilayer immobilizing uricase and an electron mediator on an Au electrode for amperometric determination of uric acid.

A biomimetic bilayer membrane immobilizing uricase (urate oxidase; EC 1.7.3.3) (UOx) and a redox agent of 1-methoxy-5-methylphenazinium (MMP) was fabricated on an Au electrode substrate with use of the Au substrate coated with a self-assembled monolayer of n-octanethiolate (OT/Au) and L-alpha-phosphatidylcholine beta-oleoyl-gamma-palmitoyl (PCOP). The preparation was carried out by successively immersing an Au electrode substrate in an ethanol solution of OT, an MMP aqueous solution, and a suspension of proteoliposome formed by PCOP containing UOx and MMP. The prepared electrode exhibited such fast steady amperometric responses to uric acid as to allow its determination within 20 s after injecting uric acid, indicating that UOx-catalyzed electrochemical oxidation of uric acid was accomplished with assistance of electron mediation by MMP between UOx and the Au substrate. An increase in the response currents with increasing concentration of uric acid was obtained in a concentration range of uric acid found in healthy human blood. Any interference in the current response that is caused by direct anodic oxidation of uric acid or ascorbic acid was not observed at the prepared sensor electrode because the densely packed bilayer effectively blocked the diffusion of these substrates toward the Au surface, making it possible to determine amperometrically uric acid at the electrode with high precision.

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT.

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres.

A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., 'nothing dehydrogenase' activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and 'nothing dehydrogenase') when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.

The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films.

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. I. Optimalization of the staining procedure.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a "fixation" in 0.025% glutaraldehyde (under NADP+ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.