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: Cell -> erythrocyte

Citations 4

"Assay Of Purine Nucleoside Phosphorylase In Erythrocytes By Flow Injection Analysis With Fluorescence Detection"
Chem. Pharm. Bull. 1987 Volume 35, Issue 11 Pages 4574-4578
Hayashi Y, Zaitsu K, Ohkura Y

Abstract: The substrate solution consisting of 1 mM inosine in 50 mM phosphate buffer (pH 6.0) was pre-incubated at 37°C for ~5 min before addition of erythrocyte lysate (50 µL). Following incubation for 10 min, the reaction was stopped with 4 M HClO4. The resulting solution was mixed with 2 M K2CO3 (to remove KClO4) and centrifuged and a portion of the supernatant solution was merged (at 0.25 mL min-1) in the flow injection analysis sytem with 0.1 M Tris - HCl buffer (pH 8) as carrier solution. The resulting stream was mixed in the coil with reagent solution [as for the carrier solution but containing 5 mM 3-(4-hydroxyphenyl)propionic acid] and the hypoxanthine present in the stream was degraded to H2O2 on columns of immobilized xanthine oxidase, urate oxidase and horse-radish peroxidase in series. The fluorescence intensity of the final solution was measured at 405 nm (excitation at 305 nm). The limit of determination for hypoxanthine was 0.3 pmol in a 20 µL injection. The method permits the assay of purine-nucleoside phosphorylase in 10 nl of erythrocytes.
Enzyme, purine nucleoside phosphorylase Fluorescence Immobilized enzyme Reactor

"Determination Of Thiamine And Thiamine Phosphate Esters In Blood By Liquid Chromatography With Post-column Derivatization"
Clin. Chem. 1983 Volume 29, Issue 12 Pages 2073-2075
M Kimura and Y Itokawa

Abstract: A modification to the method of Kimura et al. (cf. Anal. Abstr., 1983, 44, 2E36) is described.Blood, erythrocytes or plasma (0.2 ml) and 0.2 mL of aqueous 10% trichloroacetic acid were mixed, and the mixture was centrifuged at 35,000 g.A 100 µL portion of supernatant solution was analyzed by HPLC on a column (25 cm x 4 mm) of µBondapak C18, with 0.2 M NaH2PO4 in aqueous 0.3% acetonitrile as mobile phase (1.0 mL min-1).The eluate was mixed with 0.01% K3Fe(CN)6 in 15% NaOH solution to form thiochrome phosphate esters, and the fluorescence was measured at 450 nm (excitation at 375 nm).Thiamine(I), I mono-, I tri-, and I poly-phosphates were eluted and measured as single peaks.The limit of determination of I or its esters was 30 fmol.
Thiamine Thiamine triphosphate Thiamine monophosphate Clinical analysis HPLC Fluorescence Post-column derivatization

"Automated Determination Of Cholinesterase Activity In Plasma And Erythrocytes By Flow Injection Analysis, And Application To Identify Subjects Sensitive To Succinylcholine"
Clin. Chem. 1989 Volume 35, Issue 1 Pages 77-80
P Laine-Cessac, A Turcant and P Allain

Abstract: The cited determination was carried out with use of a flow injection system incorporating a robotic sample preparation and injection unit, a spectrophotometer and an Apple computer. Plasma or haemolysate (5 µL) was mixed with 0.1 M phosphate buffer (pH 7.5) and 10 µL of substrate solution (succinylcholine, butyrylcholine or acetylcholine) and the mixture was heated at 30°C for 20 min. The reaction was stopped by the addition of 10 µL of physostigmine. The choline formed was determined by addition of a color reagent, containing choline oxidase (forming H2O2), peroxidase, phenol and 4-aminoantipyrine and, after further heating, the absorbance was measured at 500 nm. The calibration graph was rectilinear for 0.5 to 4 mM choline. The results using the three substrates correlated well (r 0.94), and the between-day coefficient of variation for plasma analysis was 7% (n = 12). The preferred substrate is succinylcholine. The use of the flow injection system decreases the volume of reagent and sample solution required by a factor of ten.
Enzyme, cholinesterase Succinylcholine Spectrophotometry Clinical analysis Automation Computer Buffer Calibration Robot

"Continuous-flow Quantification Of Total Mercury In Whole Blood, Plasma, Erythrocytes And Urine By Inductively Coupled Plasma Atomic-emission Spectroscopy"
J. Anal. Toxicol. 1992 Volume 16, Issue 2 Pages 99-101
Buneaux F, Buisine A, Bourdon S, Bourdon R.

Abstract: For the determination of Hg, blood and urine underwent sample preparation (details given), before dilution with aqueous NH3 buffer and reduction by sodium borohydride at pH 9.5. The reduced mixture was analyzed by ICP-AES; the test material sample was not nebulized into the torch, but the Hg vapor was collected in a reactor vial and swept into the plasma by the carrier gas (Ar) using a glass apparatus (described). An oxidative mineralization was not required. Optimization of the experimental conditions is discussed. The separation of liquid and gaseous phases decreased background noise and increased the sensitivity of the method; the detection limit was 4 µg l-1. The coefficient of variation (n = 9) was 9% for 35 µg L-1 of Hg. Recoveries were quantitative. The method can be used for routine determinations but it is recommended that a specific standardization is first performed. Mercury determination in blood and urine can be performed by inductively coupled plasma atomic emission spectroscopy after dilution in an ammonia buffer and reduction by sodium borohydride. The proposed method does not need an oxidative mineralization. The sample is not nebulized into the torch, but the mercury vapor, after collection in a reactor vial, is swept into the plasma by the argon carrier gas using the described glass app.
Mercury Spectrophotometry Optimization Volatile generation Phase separator Volatile generation