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#17023154   2007/02/05 Save this To Up

Development and analytical application of an uric acid biosensor using an uricase-immobilized eggshell membrane.

An uric acid biosensor fabricated from a uricase-immobilized eggshell membrane and an oxygen electrode was presented. The detection schemes involve the enzymatic reactions of the uricase leading to the depletion of dissolved oxygen level upon exposure to uric acid solution. The decrease in oxygen level was monitored and related to the uric acid concentration. The scanning electron micrographs show the microstructure of the eggshell membrane within which the uricase is successfully immobilized. The effects of enzyme loading, pH, temperature, and phosphate buffer concentration on the response of the biosensor were investigated in detail. The uric acid biosensor has a linear response range of 4.0-640 microM with a detection limit of 2.0 microM (S/N=3). The response time was less than 100 s. The biosensor exhibited good repeatable response to a 0.10mM uric acid solution with a relative standard deviation of 3.1% (n=7). The reproducibility of fabrication of the biosensors using four different membranes was good with a R.S.D. of 3.2%. The biosensor showed extremely good stability with a shelf-life of at least 3 months. Some common potential interferents in samples such as glucose, urea, ascorbic acid, lactic acid, glycine, DL-alpha-alanine, DL-cysteine, KCl, NaCl, CaCl2, MgSO4, and NH4Cl showed no interferences on the response of the uric acid biosensor. The biosensor was successfully applied to determine the uric acid level in some human serum and urine samples, and the results agreed well with those obtained by a commercial colorimetric assay kit.

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#16365061   2005/12/20 Save this To Up

Dietary fructooligosaccharides affect intestinal barrier function in healthy men.

In contrast to most expectations, we showed previously that dietary fructooligosaccharides (FOS) stimulate intestinal colonization and translocation of invasive Salmonella enteritidis in rats. Even before infection, FOS increased the cytotoxicity of fecal water, mucin excretion, and intestinal permeability. In the present study, we tested whether FOS has these effects in humans. A double-blind, placebo-controlled, crossover study of 2 x 2 wk, with a washout period of 2 wk, was performed with 34 healthy men. Each day, subjects consumed lemonade containing either 20 g FOS or placebo and the intestinal permeability marker chromium EDTA (CrEDTA). On the last 2 d of each supplement period, subjects scored their gastrointestinal complaints on a visual analog scale and collected feces and urine for 24 h. Fecal lactic acid was measured using a colorimetric enzymatic kit. The cytotoxicity of fecal water was determined with an in vitro bioassay, fecal mucins were quantified fluorimetrically, and intestinal permeability was determined by measuring urinary CrEDTA excretion. In agreement with our animal studies, FOS fermentation increased fecal wet weight, bifidobacteria, lactobacilli, and lactic acid. Consumption of FOS increased flatulence and intestinal bloating. In addition, FOS consumption doubled fecal mucin excretion, indicating mucosal irritation. However, FOS did not affect the cytotoxicity of fecal water and intestinal permeability. The FOS-induced increase in mucin excretion in our human study suggests mucosal irritation in humans, but the overall effects are more moderate than those in rats.

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#23923563   2013/08/08 Save this To Up

Determination of serum lactate with alkylamine glass bound lactate oxidase.

Commercial lactate oxidase (Lactate:O2; oxidoreductase EC 1.1.3.2) from Pediococcus species was immobilized on to alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling with a conjugation yield of 3.2 mg/g support and 105% retention of initial activity. Immobilized enzyme showed maximum activity at pH 6.5, when incubated at 40 degrees C for 12 min and was used for determination of lactic acid in serum. The H2O2 generated from serum lactate by immobilized enzyme was measured colorimetrically at 565 nm by its oxidative coupling with 4-aminoantipyrine and N,N'-dimethyaniline catalyzed by horseradish peroxidase. A linear relationship was observed between A565 and lactic acid concentration ranging from 0.075 mM to 10 mM. The minimum detection limit of the method was 0.075 mM, which was better than that of enzymic colorimetric method employing free enzyme (0.2 mM). Within day and between day coefficient of variations were < 8.0% and < 19%, respectively. Serum lactic acid values determined by the present method were in good correlation (r = 0.99) with the currently used enzymic colorimetric method. The cost of lactate determination for 100 serum samples was less, as compared with Sigma kit method.

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