University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Classification: Biological tissue -> liver -> rat

Citations 8

"Determination Of Glutathione In Biological Material By Flow Injection Analysis Using An Enzymic Recycling Reaction"
Anal. Biochem. 1988 Volume 174, Issue 2 Pages 489-495
F. A. M. Redegeld*, M. A. J. van Opstal, E. Houdkamp and W. P. van Bennekom

Abstract: Rat hepatocyte preparation or rat liver homogenate was deproteinized with HClO4. For determination of oxidized glutathione(I), N-ethylmaleimide was added; the excess was destroyed by alkaline hydrolysis (pH 11.2; 3 M potassium phosphate buffer) and HClO4 was added to neutralize. After centrifugation, the supernatant solution was injected into a carrier buffer (0.4 M potassium phosphate of pH 6.75) for flow injection analysis. The reagent solution (each at 0.2 mL min-1) were 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) in carrier buffer and 0.5 mM NADPH containing 3 iu mL-1 of glutathione reductase (in carrier buffer). The mixture flowed at 0.6 mL min-1 through a single-bead string reactor for enzymatic recycling before measurement of absorbance at 412 nm. The calibration graphs were rectilinear up to 200 and 400 pmol of oxidized and reduced I, respectively. The detection limit was 1 pmol. Glutathione was determined in isolated rat hepatocytes and results correlated well with those by spectrophotometry (r = 0.977). A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h-1 and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughout, high sensitivity, and good reproducibility.
Glutathione Spectrophotometry Calibration Method comparison Optimization Chromogenic reagent

"Method For The Sensitive Analysis Of Individual Molecular Species Of Phosphatidylcholine By High Performance Liquid Chromatography Using Post-column Fluorescence Detection"
J. Chromatogr. B 1987 Volume 415, Issue 1 Pages 241-251
Anthony D. Postle

Abstract: Total phosphatidylcholine(I) in CHCl3 extracts of rat liver and lung was isolated from other phospholipid classes by HPLC on a column (30 cm x 4.9 mm) of µPorasil (10 µm) with hexane - propan-2-ol - water (60:80:9) as mobile phase (1 mL min-1) and detection at 200 nm. The eluate containing I was evaporated and the residue was dissolved in trifluoroethanol for analysis of individual molecular species by reversed-phase HPLC. Columns (25 cm x 4.6 mm) of µBondapak C18 (10 µm), Spherisorb ODS 1 and ODS 2 (5 µm), and Apex ODS 1 and ODS 2 (5 µm) were evaluated with mobile phases (1 mL min-1) of 40 mM choline chloride in methanol - water - acetonitrile. Initial detection was at 200 nm followed by post-column derivatization at 50°C with 1,6-diphenylhexa-1,3,5-triene and fluorescence detection at 460 nm (excitation at 340 nm). The between-run coefficient of variation (n = 10) were 0.32 to 0.68%. The stationary phase Apex ODS 2 gave the most efficient separation, and both saturated and unsaturated species could be determined.
Phosphatidylcholine HPLC Fluorescence Heated reaction Post-column derivatization

"Analysis Of 6R- And 6S-tetrahydrobiopterin And Other Pterins By Reversed-phase Ion-pair Liquid Chromatography With Fluorimetric Detection By Post-column Sodium Nitrate Oxidation"
J. Chromatogr. B 1993 Volume 617, Issue 2 Pages 249-255
Yoshihiro Tani* and Tomochika Ohno

Abstract: A mixture of D-neopterin, biopterin, pterin, 7,8-dihydropterin, 6R-L-erythro-5,6,7,8-tetrahydrobiopterin and 6S-L-erythro-5,6,7,8-tetrahydrobiopterin was analyzed by HPLC at 40°C on a column (25 cm x 4.6 mm) of Cosmosil 5C18 equipped with a guard column (5 cm x 4.6 mm) of the same packing. Elution (1 ml/min) was effected with 0.1 M phosphate buffer of pH 3 containing 5% methanol, 3 mM sodium octylsulfate, 0.1 mM Na2EDTA and 0.1 mM ascorbic acid. Post-column derivatization was achieved by mixing the eluate with a stream of 5 mM NaNO2 (1 ml/min) before passing through a reaction coil (no dimensions given) at 80°C; fluorescent detection was performed at 440 nm (excitation at 350 nm). The calibration graphs were linear from 0.02-5 ng of each compound injected, with detection limits of 10^-20 pg. Recoveries from tissues ranged from 84.7-99.6% and the within-assay RSD were 0.7-3.7%. The method was applied to the analysis of homogenates of rat liver, kidney and 6 regions of the brain.
d-Neopterin Biopterin Pterin 7,8-Dihydropterin 6R-L-erythro-5,6,7,8-tetrahydrobiopterin 6S-l-erythro-5,6,7,8-tetrahydrobiopterin Fluorescence HPLC Post-column derivatization Heated reaction

"Quantitative Determination Of Unchanged Cisplatin In Rat Kidney And Liver By High Performance Liquid Chromatography"
J. Chromatogr. B 1995 Volume 663, Issue 1 Pages 181-186
Kazuhiko Hanada, Naomi Nagai and Hiroyasu Ogata*

Abstract: A quantitative analytical method for measuring unchanged cisplatin (CDDP) and high- and low-molecular-mass metabolites (fixed and mobile metabolites) in rat kidney and liver was developed. Unchanged CDDP, separated from fixed and mobile metabolites in tissue homogenates by consecutive procedures of fractionation and ultrafiltration, was determined by high performance liquid chromatography (HPLC) with post-column derivatization. Although unchanged CDDP was found to be partly metabolized to fixed metabolites during the preparation of cytosolic ultrafiltrates, the recovery of unchanged CDDP gave a constant value (about 70%), which was independent of tissue type and CDDP concentration (from 1 to 10 µg/ml). The detection limit for unchanged CDDP in the cytosolic ultrafiltrate was 20 ng/ml, corresponding to a concentration detection limit of 65 ng Pt per g of tissue in the kidney and liver. The concentrations of fixed and mobile metabolites were determined as platinum concentrations in the tissue homogenate and in the cytosolic ultrafiltrate using atomic absorption spectrometry after correcting for transformation of unchanged CDDP to fixed metabolites. The distribution of unchanged CDDP, mobile metabolites and fixed metabolites in rat kidney and liver, after bolus injection of CDDP (5 mg/kg), was determined using this method.
Cis-platin Cisplatin, metabolites HPLC Post-column derivatization

"Improved Flow Injection Analysis Method For Determining Selenium In Biological Samples, And The Effect Of Captopril Administration On Selenium Levels And Glutathione Peroxidase Activity In Rat"
Biol. Pharm. Bull. 1993 Volume 16, Issue 11 Pages 1069-1072
Nobuyuki SUZUKI, Eriko FUJITA, Hiroyuki YASUI, Eiji AOYAMA, Hisashi TANAKA, Terumichi NAKAGAWA

Abstract: An improved flow injection analysis (FIA) method has been developed for the determination of trace selenium in biological samples, and this method has been applied to investigate the effect of captopril, an antihypertensive drug having a thiol group, on selenium concentrations in the rat blood, liver and urine. After oral administration of captopril, selenium levels in the blood decreased, while those in the liver increased significantly. However, no pronounced effect was observed on the urinary excretion rate. The glutathione peroxidase activities in the blood and the liver were comparable to the changes in the selenium levels.
Selenium Captopril

"Rapid And Sensitive Determination Of Proteins In Biological Samples By Flow Injection Analysis"
Bunseki Kagaku 1987 Volume 36, Issue 4 Pages 256-260
Shuto, T.;Koga, M.;Tanaka, I.;Akiyama, T.;Igisu, H.

Abstract: The sample was injected into a carrier stream of aqueous potassium hydrogen phosphates of pH 7.4 and then mixed with a reagent stream of 60 µg mL-1 of Coomassie Brilliant Blue G250 (C. I. Acid Blue 90) in aqueous 2.7% HClO4 before passing through the reaction coil (10 cm x 1 mm) followed by absorbance measurement at 600 nm. For determination of bovine serum albumin(I) as test protein, rectilinear calibration was obtained from 40 ng (detection limit) to 1 µg, with coefficient of variation of 0.2% (n = 10) at 1.0 µg of I. Up to 60 samples h-1 could be analyzed. The method was applied to the determination of proteins in rat liver microsomes.
Albumin Proteins Spectrophotometry

"Determination Of Vitamin B6 In Foods And Other Biological Materials By Paired-ion High Performance Liquid Chromatography"
J. Agric. Food Chem. 1985 Volume 33, Issue 3 Pages 359-363
Jesse F. Gregory and Debra Feldstein

Abstract: Food, human plasma or milk, or rat liver or muscle samples, were homogenized in sulfosalicylic acid with 4'-deoxypyridoxine (as internal standard) and CH2Cl2. After centrifugation, the aqueous phase was applied to a column of Bio-Rad AG2-X8 anion-exchange resin (200 to 400 mesh; Cl- form) with 0.1 M HCl as mobile phase. The fraction containing pyridoxine was subjected to HPLC on a column (3 cm x 4.6 mm) of octadecylsilica (3 µm) with gradient elution with 0 to 2.5% of propan-2-ol in 33 mM potassium phosphate - 8 mM octanesulfonic acid (pH 2.2). Post-column derivatization was with NaHSO3 in 1 M sodium phosphate buffer (pH 7.5) and fluorescence detection was at 400 nm (excitation at 330 nm). The coefficient of variation were <2%; recoveries were 78.7 to 103.2%. Good correlation with published data was achieved.
Vitamin B6 Pyridoxine HPLC Ion exchange Fluorescence Post-column derivatization

"Separation Of Ascorbic Acid, Isoascorbic Acid, Dehydroascorbic Acid And Dehydroisoascorbic Acid In Food And Animal Tissue"
J. Micronutr. Anal. 1990 Volume 7, Issue 1 Pages 67-70
Vanderslice, J.T.;Higgs, D.J.

Abstract: Samples (fresh broccoli and rat liver and kidney) were extracted with 8% acetic acid - 3% HPO3, the extracts were washed with hexane, with centrifugation, and the acid layers were analyzed on two PLRP-S (5 µm) columns (15 cm x 4.6 mm and 25 cm x 4.6 mm) in series operated at 4°C with 0.2 M NaH2PO4 (pH 2.14) as mobile phase (0.5 mL min-1). Post-column derivatization was carried out by oxidation with HgCl2 and subsequent reaction with o-phenylenediamine; detection was by fluorescence at 430 nm (excitation at 350 nm). Baseline separation of the cited analytes was achieved for both standards and sample extracts.
Ascorbic acid isoascorbic acid dehydroascorbic acid dehydroisoascorbic acid Fluorescence Column Post-column derivatization