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At least 19 recordsLinked to original sources
PubMed1992

Aqueous humor hydrogen peroxide analysis with dichlorophenol-indophenol.

Hydrogen peroxide is now reported to be a normal aqueous humor component present, in the low microM concentration range, in the animal species which have been studied. This finding was established with the exclusive use of the dichlorophenol-indophenol method of analysis. In this procedure, aqueous humor is added to a blue, oxidized dichlorophenol-indophenol solution. The 605 nm absorbance of this solution immediately decreases in response to the reducing action of ascorbate present in the sample. The extent of reoxidation of the solution upon the addition of peroxidase, as measured by the increase in its 605 nm absorbance, can be quantitatively related to the concentration of H2O2 in the sample. A close examination of this method revealed that reduced dichlorophenol-indophenol spontaneously reoxidizes at a rate of 0.03 nmol min-1 microM-1, with generation of H2O2. H2O2 generation was unequivocally established by analysis of the temporal dependency of the absorbance increase produced by peroxidase in the absence of added H2O2 and by the sensitivity of this phenomenon to catalase. This spontaneous production of H2O2, on the other hand, cannot be attributed to ascorbate auto-oxidation because added ascorbate quantitatively reacts with dichlorophenol-indophenol, provided that an excess of the latter is maintained. This method then has an enormous potential to overestimate H2O2 in any sample. On the other hand, the response of the assay system to a given level of H2O2 depends on the level of reduction previously produced by ascorbate. This results in an artifactual positive correlation between ascorbate and H2O2 levels in samples containing variable amounts of ascorbate. In spite of these serious limitations the method can still be useful to measure H2O2 if appropriate precautions are taken. When using it for the analysis of rabbit aqueous humor H2O2 without correcting for the H2O2 generated during the assay and ignoring differences in the level of ascorbate in the samples, we obtained an average value of 25.3 microM H2O2, which coincides with that reported in the literature for the rabbit, but is obviously incorrect. When analysing aqueous humor there was the additional variable of the aqueous humor itself inhibiting the rate of dichlorophenol-indophenol auto-oxidation and so the final, corrected figure for H2O2 concentration in the aqueous humor became uncertain, since the auto-oxidation of the substrate could not be properly subtracted.(ABSTRACT TRUNCATED AT 400 WORDS)

2,6-Dichloroindophenol↗
PubMed2002

Design of novel electron transfer mediators based on indophenol derivatives for lactate sensor.

Novel indophenol derivatives which have limited solubility in water solution functioned as excellent electron-transfer mediators for lactate oxidase and a lactate sensor, showing good sensor performance, including a high sensitivity and good durability. These less water-soluble mediators were prepared by the O-alkylation or acylation of indophenols in order to prevent the mediator being leached from the sensor. Consequently the characteristic substituent effects of indophenol derivatives were found to be follows, (1) modification of the phenolic OH group improved sensor durability; (2) the presence of chloride groups at the 2,6-positions of the phenol ring improved the sensitivity of the sensor; (3) the introduction of substituents at the quinoide moiety led to a deterioration in durability. A mediator based lactate sensor using these derivatives was found to be nearly independent of oxygen concentration, showed a low level of interference effect, and a quite long durability.

Biosensing Techniques↗
PubMed1985

Visible spectrophotometric determination of cephalosporins and penicillins by indophenol derivatization with and without alkaline degradation to ammonia.

Cephalosporins and penicillins give reproducible yields of ammonia on degradation in 0.5 M sodium hydroxide solution at 100 degrees C: the ammonia formed was determined in the degraded solutions using the indophenol reaction. In another approach the ammonia driven off on refluxing alkaline solutions of the cephalosporin or penicillin was collected in dilute hydrochloric acid solution and determined using the indophenol reaction. For eight of the fourteen cephalosporins and penicillins studied identical yields were recorded using the two procedures: these varied from 29% for penicillin G to 137% for cephalonium based on the production of one ammonia molecule per beta-lactam molecule. For six other cephalosporins the distillation method gave substantially higher yields of ammonia than did the direct determination. Eight cephalosporins and penicillins were found to give substantial indophenol-type reactions without prior hydrolysis of the beta-lactam, but the sensitivities were usually lower than for the hydrolysis method. Manual spectrophotometric procedures for the determination of cephalosporins and penicillins based on these reactions have been developed.

Journal Article↗
PubMedDate not supplied

Determination of ammonia in cerebrospinal fluid using the indophenol direct method.

We have determined ammonia in cerebrospinal fluid (CSF) with the indophenol direct method. The results were compared with an enzymatic method. The method is very simple, and precision (coefficient of variation 1.6%) and linearity (r = 0.9999, p < 0.001) of the method are excellent. The recoveries of the method are very good (within-sample recovery: range 88-93, median 93%; between-sample recovery: 88-93, median 91%). In a population of 23 neurological patients not suffering from liver disease, the reference values ranged from 8 to 26, median 18 microM. Males and females did not differ (p = 0.5). The values obtained with the indophenol method were equal to the enzymatic method (range 9-28, median 18 microM, p = 0.6). On storage in the deep freeze (-20 degrees C), there was no change in CSF ammonia concentration for at least 1 mo. When stored at 4 degrees C (refrigerator), ammonia determinations have to be performed within 2 d. CSF storage at room temperature results in artificially elevated ammonia levels and should be avoided.

Ammonia↗
PubMed1996

Arterial ammonia with Blood Ammonia Checker II and with indophenol reaction to assess presence of hepatic encephalopathy.

Hepatic encephalopathy (HE) is associated with elevated arterial ammonia levels. The relationship is variable, in part due to ammonia methodology. One method, based on the indophenol reaction (IPh), is interfered with a number of amino acids including all aromatic amino acids. We have determined arterial ammonia simultaneously with the Blood Ammonia Checker II (BAC) as reference method and with the IPh method. The difference BAC-IPh, mumol/l, was assumed to express the interference in the indophenol method (IFI) by amino acids. It may be positive or negative. The aim was to establish the value of BAC in comparison with IPh in the diagnosis of liver disease and overt HE and to assess any added value of IFI. Of two reference groups without disturbances, A (n = 39) had not and B (n = 13) had encephalopathy. Group C consisted of 125 liver patients (34 no cirrhosis, 91 cirrhosis) of which 55 had no manifest HE (C:HE-) and 70 had HE (C:HE+). Median BAC ammonia nitrogen (NH3-N), mumol/l: A 21, B 35, C 80, C:HE - 57 and C:HE+ 98 (A < B < C and A < B < C:HE - < C:HE +, P < 0.001). Median IPh NH3-N, mumol/l: A 27, B 30, C 30, C:HE - 25 and C:HE + 35 mumol/l (A = B = C and C:HE - < C:HE+, P < 0.01). IFI medians: A -6, B 3, C 40, C:HE - 29 and C:HE + 58 mumol/l (A < B (P < 0.05) < C (P < 0.0001); A, B < C:HE - and C:HE+; C:HE- < C:HE + (all P < 0.0001)). While BAC correlated weakly with IPh in the (sub)groups C, C:HE-, C:HE+ (r = 0.3, 0.3, 0.4, P < 0.05), it correlated strongly with IFI (r = 0.9, 0.9, 0.8, P < 0.0001). There was no correlation between IPh and IFI. BAC, as well as IFI, could discriminate all liver patients (C) from both reference groups A and B with 100% positive likelihoods. BAC, IPh and IFI could discriminate between HE- and HE+. To differentiate cirrhosis from non-cirrhosis the specificity of IPh was uniformly high and the sensitivity satisfactory, whereas BAC had a high sensitivity but an insufficient specificity. In conclusion, in blood, BAC is the ammonia determination of choice. It differentiates between reference groups (encephalopathic or not) and liver disease and the more so HE. The combination of BAC and IPh (indicating IFI) may eventually be shown useful to rapidly assess the severity of underlying liver disease in HE patients. In other biological fluids, IPh is excellent when the inhibiting influence of non-protein nitrogen substances is absent or can be eliminated.

Adult↗
PubMed1982

Determination of ammonia in saliva using indophenol, an ammonium electrode and an enzymatic method: a comparative investigation.

Three methods for determination of ammonia in saliva are reported. The indophenol method on diluted saliva has the best precision (coefficient of variation 0.8%) and the lowest reagent cost. The ammonium electrode method is the quickest, but it requires simultaneous determination of the potassium content of the specimen. The enzymatic method give the same result as the electrode method, but is more expensive. Deproteinisation proved not to be necessary. In one hour 10, 20 or 40 determinations can be performed with the enzymatic, indophenol- or the electrode method, respectively.

Ammonia↗
PubMed2005

[Direct determination of ammonia in plasma with indophenol method].

A new method for direct determination of ammonia in plasmas with indophenol reagent is described. It is shown that using sodium citrate as anticoagulant in phosphate buffer within pH 10.5-11.2, indophenol reagent reacts sensitively with ammonia in the plasmas to form a stable blue compound with a maximum absorption at 630 nm with low interference. The apparent molor absorptivity is 2.48 x 10(4) L x mol(-1) cm(-1) , and the linear range 0-1500 micromol x L(-1) (r = 0.9999) with a detection limit of 1.7 micromol x L(-1). The relative standard deviation is 1.13% (n=11) and the recoveries are in the range of 93.6%-101.6% with an average recovery of 97. 0%. The proposed method has been applied to detect ammonia in the plasmas from 43 healthy persons and 97 patients who suffered from liver diseases with satisfactory results.

Ammonia↗
PubMed2005

Interference of laccase in determination of cellobiose dehydrogenase activity of Pleurotus ostreatus (Florida) using dichlorophenol indophenol as the electron acceptor.

Pleurotus ostreatus (Florida), ITCC 3308 produces approximately 9.0 U/ml extracellular cellobiose dehydrogenase (CDH) in cellulose medium after 7 days of growth. However, no activity could be detected if the assay was done with cellobiose as the substrate and 2,6-dichlorophenol indophenol (DPIP) as the electron acceptor in absence of any laccase inhibitor. Kinetic study showed that V(max)/K(m) value was very high for rDPIP (reduced 2,6-dichlrophenol indophenol) oxidation by laccase. Oxygen consumption rate of rDPIP oxidation by the enzyme was found to be highest among all the tested substrates. The present study indicated that rDPIP was a good substrate for laccase. Therefore, caution is needed to measure CDH activity by monitoring DPIP reduction in a system where laccase is likely to be present.

2,6-Dichloroindophenol↗
PubMed1983

Analysis of the effect of industrial effluent on pigments, proteins, nucleic acids, and the 2,6-dichlorophenol indophenol Hill reaction of rice seedlings.

The effect of an industrial effluent on cellular macromolecular composition and indophenol photochemical activity of isolated chloroplasts of rice (Orvza sativa L. C. V. Mushoori) seedlings have been investigated. Each experiment consisted of two parts. One was the effect of various concentrations and the other was the time-dependent changes induced by the undiluted effluent. The total pigments, proteins, and nucleic acids of rice seedlings declined with an increase in effluent concentration and the time of incubation. The loss in contents of macromolecules like deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was relatively more marked in the root than in the shoot. RNA and chlorophyll (Chl) contents of the seedlings were found most susceptible to effluent stress. Loss in Hill reaction activity measured as photoreduction of 2,6-dichlorophenol indophenol (DCPIP) of isolated chloroplasts could be correlated in a general way with the loss of pigments, proteins and nucleic acids. Diphenyl carbazide- and Mn2+-induced restoration of loss in DCPIP photoreduction suggests that the damage of oxygen evolving systems is the initial site of action of the effluent.

2,6-Dichloroindophenol↗
PubMed1999

Mechanism of the Gibbs Reaction. Part 4.(1) Indophenol Formation via N-Chlorobenzoquinone Imine Radical Anions. The Aza-S(RN)2 Chain Reaction Mechanism. Chain Initiation with 1,4-Benzoquinones and Cyanide Ion.

The mechanism of the Gibbs reaction, a colorimetric phenol assay that applies N-chlorobenzoquinone imines 1 in an aqueous basic medium, was investigated. It is concluded that N-chloroimine radical anion 7 generated in a single electron transfer (SET) from the anion of phenol 4 to N-chloroimine 1 can produce indophenol dye 3 in three distinct routes. For more reactive reagent-substrate pairs, a route is proposed that involves a fast combination of the radical pair in the solvent cage and, consequently, the total rate of which exhibits a pH-independent second-order kinetics, as does the preceding SET itself. For less reactive reagents, a route is proposed in which the N-chloroimine radical anion 7 escapes from the solvent cage to initiate a chain reaction, evidenced by its characteristic kinetics. It has been found in the kinetic experiments that during propagation the chlorine of the chain carrier N-chloroimine radical anion 7 is substituted by the anion of 4 in a bimolecular rate-determining step. Therefore, the mechanism of the chain reaction is termed S(RN)2. In the case when the anion of 4 is less active, a competitive reaction along a third route can proceed in which the N-haloimine radical anion 7 yields benzoquinone imine 6 by the elimination of halogenide and the abstraction of an H-atom from the medium. Compound 6 is also known to give indophenol 3 with a second-order but pH-dependent rate that is considerably faster than the rate in the first route. On the basis of the different kinetic characteristics outlined above a clear distinction can be made among these three pathways. In this paper, evidence is also presented for the initiating SET. Furthermore, it is of high importance that the N-haloimine radical anion 7 can also be generated from reagent 1 using external electron donors and, independently of its origin, it can be spin trapped with 2,2,6,6-tetramethylpiperidine-N-oxyl.

Journal Article↗
PubMed1997

Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-gamma and aldolase C.

NADH-dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-gamma. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.

Amino Acid Sequence↗
PubMed1973

The linkage of genes for the human interferon-induced antiviral protein and indophenol oxidase-B traits to chromosome G-21.

13 independent mouse-human somatic cell hybrid clones derived from beta-propiolactone-inactivated Sendai stimulated cell fusion of human cells with mouse cells were tested for their sensitivities to human and mouse interferon. All of them were protected by mouse interferon and only six of the clones were protected by both human and mouse interferon. Only the six that were protected by human interferon were shown to express the human dimeric form of indophenol oxidase. Complete chromosomal analysis of the clones indicated human chromosome G-21 to be the only human chromosome in common for the six clones which had both phenotypes present. Nine subclones were derived from one of the clones expressing both phenotypes. Eight of the nine subclones were shown to retain both phenotypes, whereas one subclone lost both. Chromosomal analysis of the subclones indicated the loss of chromosome G-21 from the subclone which lost both phenotypes. It is apparent from these findings that the gene(s) for indophenol oxidase (IPO-B) and the gene(s) for the antiviral protein are syntenic and that they are linked to human chromosome G-21.

Animals↗
PubMed1977

Mitochondrial membrane-linked reactions in carcinogenesis: change in steroselective uncoupling of oxidative phosphorylation by aliphatic dicarbonyls and in the Arrhenius plot of NADH-indophenol reductase.

The previously observed alterations in the energy transducing system of rat liver mitochondria during 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) carcinogenesis were investigated using aliphatic dicarbonyl compounds as molecular probes and the effect of temperature on the membrane-linked NADH-indophenol reductase. The vicinal diketone, diacetyl, uncouples oxidative phosphorylation in normal rat liver mitochondria while the higher diketones, acetylacetone and acetonylacetone, are increasingly less effective in that order; diacetyl totally abolishes respiratory control with substrates the oxidation of which involves the NADH leads to CoQ segment, but only partially with succinate which bypasses this segment. Diacetyl, likewise, uncouples oxidative phosphorylation in liver mitochondria from rats fed 3'-Me-DAB, but the mitochondria are most resistant to this uncoupling (in terms of the P/O ratio) at the time period when the respiratory control index (determined in the absence of diacetyl) is at the dye-induced minmum. This time period is at 3 to 4 weeks of dye administration, representing the cumulative dose for tumorigenesis threshold. At this threshold period of feeding 3'-Me-DAB, discontinuities in the Arrhenius plot of the mitochondrial membrane-localized NADH-indophenol reductase appear, with a return toward the control state (no break) at 8 weeks, only to reappear in the plot of the enzyme from tumor mitochondria, suggesting sequential membrane phase transitions in the mitochondria during azo dye carcinogenesis.

2,6-Dichloroindophenol↗
PubMed2001

In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction.

Two methods for measurement of urease activity are described and demonstrated on extracts from several crop plants. With an in-gel staining method based on the principle of Fishbein's noninhibitory stain for urease as little as 25 microU of jackbean urease can be detected within 2 h following electrophoresis. A comparison with published in-gel staining methods shows that the sensitivity is improved by at least two orders of magnitude. The second method allows quantification of urease activity from small amounts of plant material without the need for special laboratory equipment. It employs the detection of ammonium by the indophenol reaction. To eliminate reducing agents which are often necessary to maintain urease activity during extraction, but which interfere with ammonium detection, a simple spin-column procedure is used. The quantification of less than 5 mU/ml extract is possible.

Electrophoresis↗