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PubMed1995

Determination by enhanced luminescence technique of liver antioxidant capacity.

A simple approach to quantitative determination of antioxidant capacity of rat liver homogenate is proposed. It consists of measuring chemiluminescence generated by a suitable system "detector" for .OH radicals produced from sodium perborate. The system generating the light signal contained luminol and compounds producing enhancement of light emission, such as sodium benzoate and indophenol. Two different methods, utilizing the same technique of enhanced luminescence, were set up. In a previous work, a parameter b, contained in the equation, which best describes the dependence of the intensity of light emission (E) on liver homogenate concentration (C) (E = a.C/exp(b.C), was found to be related to the level of antioxidants in the homogenate. Therefore, in the first method, the light emission from several dilutions of both liver homogenates, and homogenate and antioxidant mixtures, stressed with sodium perborate, was detected by a luminometer. The best fitting of data to theoretical equation provided b values, which were introduced in a system of equations relating such values to the antioxidant concentration. The solution of above system supplied the antioxidant concentration in the homogenate in terms of the equivalent concentration of the antioxidant used. In the other method, evaluations of the antioxidant capacity of liver homogenates were obtained by the determination of the ability of 10% homogenates to quench the light emission induced by either peroxidase or cytochrome c in comparison to the ability of antioxidant solutions. Both methods are able to evidence the decrease of the antioxidant concentration of liver homogenates after oxidative stress with ter-butylhydroperoxide. The value of both concentration changes and standard errors indicates that the method using a standard curve obtained with peroxidase, such as catalyst of radical reaction, and deferoxamine, such as antioxidant, is to be preferred.

Animals↗
PubMed1979

Colorimetric method for the quantitative determination of acetaminophen in serum.

A colorimetric method for quantitatively determining acetaminophen in serum is presented. The procedure involves preparing a protein-free filtrate by the addition of trichloroacetic acid, hydrolyzing the acetaminophen in the filtrate to p-aminophenol, and subsequent reaction of p-aminophenol with phenol and NH4OH to form an indophenol blue chromogen. The absorbance at 620 nm of this chromogen follows Beer's law up to acetaminophen concentrations of greater than 50 mg/1. The recovery of acetaminophen from serum is essentially 100% when compared with appropriately prepared standards. Serum acetaminophen concentrations as low as 1 mg/1 can be detected. Seventy drugs were tested for possible interference and none was found to interfere with the method. The coefficient of variation (day-to-day) is 4%. This new quantitative method provides the necessary sensitivity to quantitate therapeutic as well as toxic serum concentrations of acetaminophen.

Acetaminophen↗
PubMed1987

Role of the isocitrate dehydrogenases and other Krebs cycle enzymes in lactating bovine mammary gland.

The role of the isocitrate dehydrogenases and other Krebs cycle enzymes in bovine mammary metabolism was studied by investigation of their distribution between cytosol and mitochondria. Citrate synthase was used as a marker for mitochondrial disruption, and distributions were normalized to this enzyme. Aconitase, fumarase, and NAD+:malate dehydrogenase were distributed between the mitochondria and the cytosol; evidence for the possible involvement of an aspartate:malate shuttle was also found. The NADP+:isocitrate dehydrogenase is predominantly cytosolic with a small but significant amount of mitochondrial component. Using the dye dichlorophenol-indophenol, a low level of NAD+:isocitrate dehydrogenase activity was observed in bovine mammary tissues. This assay also allows for detection of the enzyme in fresh mitochondria from a variety of other bovine tissues (heart, liver, kidney, and brain). Activities of the isocitrate dehydrogenases were also examined as a function of gestation and lactation. The NAD+:isocitrate dehydrogenase is apparently depressed during gestation with the NADP+ form of the enzyme (cytosolic) elevated postpartum. These results indicate that a substantial portion of Krebs cycle activity may become extramitochondrial in bovine mammary gland at the onset of lactation.

Animals↗
PubMed1998

[Hydrogen peroxide in the aqueous humor: 1992-1997].

Ideas prevailing in 1991 on hydrogen peroxide in the aqueous humor are outlined. They are critically examined under the light of our finding that the method used to establish aqueous humor levels of peroxide generates itself peroxide during the short time span of the analysis. This is due to the fact that the probe used, dichlorophenol indophenol (DCPIP), spontaneously auto-oxidizes in the presence of oxygen. It was concluded then that the level of hydrogen peroxide in the aqueous humor cannot be higher than about 0.3 microM, the detection limit of the DCPIP method. It was also concluded that the statement commonly made in the literature that aqueous humor hydrogen peroxide derives from the oxidation of ascorbate, an abundant component of that fluid, is based solely on the use of the DCPIP method, and so could easily be due to a methodological artifact. The same applies to the statement that the levels of hydrogen peroxide are very high in human senile cataracts. The surprising resistance to accept the results and conclusions of our 1992 publication is documented. Finally, the content is discussed of an oral presentation made at the 1997 ARVO Annual Meeting in which an important portion of our results and conclusions was confirmed, perhaps signaling a shift towards a wider acceptance of our findings.

2,6-Dichloroindophenol↗
PubMed2000

Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.

Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.

Animals↗
PubMedDate not supplied

Identical flow injection spectrophotometric manifold for determination of protein, phosphorus, calcium, chloride, copper, manganese, iron, and zinc in feeds or premixes.

A simple procedure using an identical manifold was developed for determination of nitrogen (protein) phosphorus, calcium, chloride, copper, manganese, iron, and zinc in feeds and feedstuffs. By changing appropriate reagents and detection wavelength, these 8 elements were determined successively with a simple identical double-line flow injection (FI) manifold. Fl spectrophotometric determinations were made by the blue indophenol reaction for ammonium, the molybdenum blue method for phosphate, the cresolphthalein complexone procedure for calcium, and the mercuric thiocyanate procedure for chloride. The chromogenic reagents for copper, iron, manganese, and zinc determination were bis(cyclohexanone)oxalydihydrazone (Cuprizone), 1,10-phenanthroline, formaldoxime, and xylenol orange, respectively. Sample digestion catalyst, Fl manifold, and some chemical parameters were optimized. The proposed procedure had a sampling rate of 90/h for each analyte. The determination ranges (mg/L) were 10-60 for N, 1-15 for P and Ca, 540 for Cl, and 0.5-15 for Cu, Fe, Mn, and Zn, respectively. Results of the analyses of animal feed and feedstuff samples by this procedure did not differ significantly from those obtained by proven manual methods.

Animal Feed↗
PubMed1975

The purification and some properties of electron transfer flavoprotein and general fatty acyl coenzyme A dehydrogenase from pig liver mitochondria.

Electron-transferring flavoprotein (ETF) and acyl dehydrogenases of pig liver mitochondria have been isolated in good yield by a new procedure. ETF and general acyl dehydrogenase appear homogenous, are free of reciprocal contamination, react with neither pyridine nucleotides not cytochrome c, and are completely dependent upon each other for reduction of dichlorophenol indophenol by acyl-CaA substrates. The properties of the present preparation (some of which differ significantly from those previously described) are presented. Sedimentation of ETF in 0.02 M KP-i yields a M-r for the native ETF of 58,00 plus or minus 3,000, whereas sedimentation of reduced and alkylated ETF in guanidine HCl yields a M-r of 26,000. Electrophoresis on sodium dodecyl sulfate gels in the presence or absence of mercaptoethanol gives a M-r of about 27,000 and flavin analysis gives a minimum molecular weight of about the same figure. Thus, ETF appears to contain one flavin (at least 90% FAD, by chromatographic and fluorescence characteristics) per 26,000 M-r, and therefore may be composed of two subunits with one flavin each. Sodium dodecyl sulfate gel electrophoresis of general acyl dehydrogenase in the absence of mercaptoethanol gives a band corresponding to a M-r of 84,000; in the presence of mercaptoethanol a band corresponding to a M-r of 42,000 is found. The minimum molecular weight based on flavin content is 40,500. These data considered in conjunction with previous reports from other laboratories, suggest a structure of four subunits per mol with one flavin per subunit..

2,6-Dichloroindophenol↗
PubMed2001

Inhibition of succinate-cytochrome C reductase by a ferromacrocyclic complex.

Succinate-cytochrome c reductase (SCR) from mouse liver was inhibited strongly and reversibly by an iron (II) macrocyclic complex 3. The inhibition was observed for the enzyme toward the reduction of both 2,6-dichlorophenol indophenol (DCIP) and cytochrome c (cyt c). The inhibition was a mixed type and noncompetitive with respect to the reduction of DCIP and cyt c, respectively. Values of the inhibition constant ranged from 6.6 to 8.3 microM. The IC50 for the complex 3 was found to be 16.6 +/- 0.8 and 12.1 +/- 0.5 microM for the enzyme toward DCIP and cyt c, respectively. The reduced form of complex 3 also exhibited enzyme inhibition but to a less extent. Complex 3, at a lower level, equal to 25% of its LD50 showed about 50% inhibition of the enzyme through in vivo dose-dependent effect. These findings suggested that the structure of the equatorial benzoquinoid macrocyclic ligand of the Fe(II) complex is involved in the enzyme inhibition.

Administration, Oral↗
PubMed2002

Purification and some properties of a blue copper protein from Methylobacillus sp. strain SK1 DSM 8269.

A blue protein was purified from the Methylobacillus sp. strain SK1 that is grown on methanol in the presence of copper ion. This protein was found to be a monomer with a molecular weight of 13,500. The Isoelectric point of the protein was estimated to be 8.8. The spectrum of the protein that was treated with ferricyanide showed a broad peak around 620 nm, but that of the dithionite-treated protein revealed no peaks. It contained 0.83 mol of EDTA-stable copper per mol protein. Under air, the protein accelerated the inactivation of methanol dehydrogenase (MDH). The protein was reducible by phenazine methosulfate or by active MDH that was prepared from cells that were grown in the absence of added copper, but not by methanol, dichlorophenol indophenol, or inactive MDH that was prepared from cells that were grown in the presence of added copper. It was also reducible by active MDH in the presence of methanol. The absorption peak at 340 nm of the active MDH disappeared after the enzyme was treated with ferricyanide, hydrogen peroxide, or the purified blue protein. The inactive MDH also showed no peak at 340 nm. The 340-nm peak was not recovered after incubation of the inactive MDH and blue protein-treated active MDH with dithionite or methanol. The inactive MDH and blue protein-treated active MDH co-migrated with the active MDH preparation on nondenaturing polyacrylamide gel, and contained two non-identical subunits with molecular weights that were identical to those of the active MDH. The N-terminal amino acid sequence of the protein was Ala-Gly-Cys-Ser-Val-Asp-Val-Glu-Ala-Asn-Asp-Ala-Met-Gln-Phe. An analysis of the amino acid composition revealed that the protein contained no tryptophan. It contained three cysteines per mol protein. The blue protein in Methylobacillus sp. strain SK1 was produced only in the cells that were grown in the copper-supplemented medium.

Alcohol Oxidoreductases↗
PubMed2003

[Separation of hydrophobic NAD(P)H dehydrogenase subcomplexes from cyanobacterium Synechocystis PCC6803].

Many efforts have been paid to the separation of an integrated NA(D)PH dehydrogenase (NDH) complex. Several hydrophilic subcomplexes of NDH have been purified from the cyanobacterium Synechocystis PCC6803. However, no hydrophobic NDH subcomplex has ever been separated from cyanobacteria yet. In this paper, two NDH subcomplexes were separated from n-dodecyl beta-D-maltoside(DM)-treated whole cell extracts of Synechocystis PCC6803 by anion exchange chromatography and gel filtration. Both subcomplexes contained the hydrophobic subunit NdhA, suggesting that they were hydrophobic NDH subcomplexes. Of the two subcomplexes, only one subcomplex contained NdhH. These subcomplexes showed NADPH-nitroblue tetrazolium (NBT) oxidoreductase activity and could specifically oxidize NADPH when several quinone analogues were used as electron acceptors, such as ferricyanide, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichlorophenol indophenol (DCPIP), duroquinone, ubiquinone-0 (UQ-0), etc.

Cyanobacteria↗
PubMed2003

[A rapid determination kit for acetaminophen].

Acetaminophen (APAP: N-Acetyl-p-amino-phenol) is widely used as a nonprescription analgesic and antipyretic drug. Although APAP is usually well tolerated when used at the recommended dose, overdose has been associated with lethal hepatic necrosis. This toxicity has been correlated with elevated serum or plasma APAP levels and/or a halftime of elimination from these fluids exceeding four hours. Therefore a rapid method for determination of APAP is needed to treat a patient with an acute APAP overdose. We developed a simple and rapid method for determination of APAP and prepared a test kit. The test kit was based on the colorimetric method (indophenol method), but not including stinking reagents. The reaction mixture assumed pale blue to dark blue according to APAP concentrations and the determination range was 10-200 micrograms/mL with the naked eye. It took only 15 minutes for this test. In addition, the test kit could be adapted to APAP test in urine. We believe that the new APAP determination kit will be a useful tool for emergency diagnosis.

Acetaminophen↗
PubMed2004

Prevention and control of thalassemia at Saraburi Regional Hospital.

OBJECTIVE: To evaluate the program in prevention and control of thalssemia among pregnant women and their spouses, prevention of new cases by screening tests, confirmatory test, genetic counselling, prenatal diagnosis, and selective abortion. SUBJECTS: The pregnant women, attending antenatal care unit, Saraburi center hospital, as well as their spouses. 1 January 2000-31 December 2001 METHOD: As part of the antenatal care assessment, pregnant women before 16 weeks gestation were screened, with pre- and post-test counselling, by osmotic fragility (OF) and dichorophenol indophenol precipitate (DCIP) tests, and confirmed by complete blood count (CBC), mean corpuscular volume (MCV), hemoglobin typing and polymerase chain reaction for alphathal1 (PCR alphathal1) if any of two screening tests was positive. The husbands of those who were carriers of severe thalassemia were encouraged to have thalassemia screening and confirmation. When both the pregnant women and their husbands were carriers of severe thalassemia, the pregnant women would voluntarily perform the prenatal diagnosis. Termination of pregnancy would be offered when the fetus had severe thalassemia. RESULTS: There were 3,739 from 4,214 women (88.7% of all antenatal women), who participated in the program. OF and/or DCIP were positive in 1,742 of 3,739 subjects (46.5%). Of those, 960 from 1,742 (55.1%), had husbands who were willing to have the testing, and OF and/or DCIP were positive in 443 of 960 cases (46.1%). The confirmatory tests revealed carrier and disease of thalassemia, and hemoglobinopathies in 931 of 1,742 women (53.9%), and 135 of 960 husbands (14.0%). The 20 couples who had the possibility of having severe thalassemic newborns, were strongly advised to have prenatal diagnosis. The 12 risk pregnancies had been performed cordocentesis. Finally 3 of 12 (25.0%) fetuses were documented to have severe thalassemia and all of them decided to have selective abortion. CONCLUSIONS: The screening model for thalassemia carriers by using the combination of OF and DCIP is the easy screening model. It can be done quickly, it is inexpensive, therefore it is suitable for large numbers of population screening. The systematic screening, confirmatory of thalassemia diagnosis and prenatal diagnosis are the measure of thalassemia prevention and control, and aims to decrease the number of newborns with severe thalassemia.

Abortion, Eugenic↗
PubMed2004

Prevention of thalassemia: experiences from Samui Island.

Thalassemia is one of the most important genetic disorders in Thailand. A model for prevention of thalassemia by combining 4 strategies including education, carrier screening, counseling and prenatal diagnosis was developed by a thalassemia task force. A thalassemia work group was formed in order to develop a mechanism to integrate the model for real use on Samui Island where thalassemia is common and a specific disease oriented program is required. 200 health professionals working on Samui Island participated in the thalassemia educational courses. The specific training courses were also provided for obstreticians, medical technologists and counselors. A team of well-trained health professionals was established to manage public education. Information booklets, posters, brochures and mass media including spot radio and newspapers were used as educational materials. For carrier screening, blood samples of pregnant women from all health care organizations on Samui Island were collected and screened for thalassemia carriers by using the osmotic fragility (OF) test/the dichlorophenol indophenol precipitation (DCIP) tests at Samui Hospital. Samples with positive results were sent to the Regional Medical Sciences Center in Surat Thani for thalassemia diagnosis. When a carrier was identified, her spouse was offered testing. For at-risk couples, details of the disorder were included in counseling to help them reach a decision that was right to them, in the context of their unique medical, moral and social situations. Amniotic fluid samples were collected for prenatal diagnosis. All patient information was registered by using computer software. After 1 year of integration by using facilities of the Ministry of Public Health service system, a wide range of problems were identified. They underlined the need for effective health service structure co-operation, adequate education of responsible health professionals, explicit policies and a clear line of responsibility at local, regional and national levels for service development and quality management. It is hoped that all information conducted in the present study will be useful to health authorities to develop an explicit policy and promote the health service structure co-operation in the country that will finally lead to successfully reducing the frequency of severe thalassemia in the future.

Endemic Diseases↗
PubMed1998

[Determination of ascorbic acid and its stability of Fructus Hippophae fruit by the method of high-valence iron reduction].

The high-valence iron reduction method was used to determine the ascorbic acid of fructus hippophae fruit. The reduction method had simple, high sensitivity and accuracy in comparison with fluorospectrophotometry or indophenol titration method. Its limit of detection was 0.01 microg/mL, the relative standard deviation and the average recovery was 0.83% (n = 8) and 95.6% (n = 6) respectively. We also researched the influence of the environment factors (temperature, storage period and condition, pH value) on the stability of ascorbic acid in fructus hippophae fruit. The ascorbic acid in fructus hippophae fruit was more stable than thepure ascorbic acid on the same condition of heating (80 C,1 hour), in pH neutral or storing in the refrigerator.

Ascorbic Acid↗
PubMed1991

Activity of phenacetin O-deethylase in rat liver: effect of inducers and inhibitors in vivo.

A method for the determination of microsomal phenacetin O-deethylase activity in the rat liver was described. Optimum conditions in which phenacetin is enzymatically converted to N-acetyl-p-aminophenol were determined. The product of enzymatic reaction was acid-hydrolyzed while heating. The amount of p-aminophenol formed was determined by means of indophenol reaction. The described method was applied to assess activity of phenacetin O-deethylase in normal rats and rats treated with inducers or inhibitors of microsomal monooxygenases, phenobarbital, 3,4-benzpyrene, SKF 525-A and cobaltous chloride, respectively.

Acetaminophen↗
PubMed1991

EM-field effect upon properties of NADPH-cytochrome P-450 reductase with model substrates.

Artificial substrates, including ferricyanide and dichlorophenol indophenol (IP), are frequently used to model the activity of NADPH-cytochrome P-450 reductase, in the xenobiotic-metabolic pathway catalyzed by the P-450 complex. Here, the two oxidants were compared in a microsomal preparation from chicken liver. Low-energy 9.14 GHz perturbation affected both reactions similarly, though the IP reaction may be more sensitive to extremely low energy levels. The reactions of the two oxidants differed from each other in their response to the prior incubation of the microsomes with carbon monoxide and to the presence of superoxide dismutase. The mechanics of the reduction of ferricyanide and the reduction of IP are not identical and the electron-flow paths may be dissimilar. Microwave effect cannot be attributed a temperature change in the reaction medium; it appears to occur at the level of the electron-flow path across the dual-flavin reductase.

Animals↗
PubMed1977

Sensitive, optimized assay for serum AMP deaminase.

A sensitive, optimized assay for serum AMP deaminase (EC 3.5.4.6) is presented, which is based on a colorimetric indophenol determination of ammonia liberated during the enzyme reaction. An average enzyme activity in serum of 2.7 U/liter (n = 36, range 0-6.2) for healthy adults was established. Both the enzyme and colorimetric reactions were optimized to give an assay sensitivity of 0.2 U/liter of serum, a 98-99% analytical recovery in the normal serum enzyme concentration range, and a CV of 7.9% in-run and 10.6% between-run. This optimization included studies on specific buffer, pH, ionic strength, and potential activators. The Km for the enzyme in serum was determinated to be 1.4 X 10(-3) mol/liter, which agrees well with the value reported for human skeletal muscle enzyme. Above-normal serum AMP deaminase activity may be present in various myopathies and could be useful in detecting carriers of the X-linked dystrophies.

AMP Deaminase↗
PubMed1990

Effect of metal chelators on thermoluminescence peaks of spinach chloroplasts and photosystem II particles: probing the water oxidation cycle with 8-hydroxyquinoline.

Inhibition of photosystem II (PS II) activity by 8-hydroxyquinoline (8-HQ) has been investigated in case of spinach chloroplasts and isolated photosystem II particles using the thermoluminescence technique. In presence of 8-HQ, water to methylviologen (MV) photoreduction in isolated chloroplasts is inhibited while the reduction of dichlorophenol indophenol is inhibited in both chloroplasts as well as in photosystem II particles. The activity can be restored fully by addition of diphenylcarbazide (DPC), suggesting that the donor side of water oxidation complex is affected. The changes in the thermoluminescence peaks indicate that the charge recombination processes involving S2 or S3 states of the Kok's cycle are probably affected by 8-HQ treatment.

Chelating Agents↗