Use of the o-Phenylenediamine Fluorescence System in the Enzymatic Assay of Serum Uric Acid.

Abstract

A manual enzymatic method is described for sensitive fluorometric determination of uric acid in human serum. This method is based on an enzymatic reaction with uricase to form hydrogen peroxide from uric acid and the following oxidation of o-phenylenediamine with peroxidase and hydrogen peroxide for the production of a fluorescence compound. The specificity and the selectivity in the method are due to the uricase reaction and the fluorometry, respectively. The formed fluorescence in the reaction mixture is measured at 410 nm (an excitation) and 550 nm (an emission). This enzymatic method can determine uric acid at 30-1000 microM, with a between-assay relative standard deviation of 4.35% or less. A good correlation is obtained between the present method and the colorimetric kit method.

Confined Multiple Enzymatic (Cascade) Reactions within Poly(dopamine)-based Capsosomes

“…Abnormal uric acid levels may result from leukemia, toxemia of pregnancy, polycythemia, impaired renal function, or gout disease. − A number of colorimetric assays for the detection of uric acid in biological samples have been developed by coupling the uricase (UR) reaction with the peroxidase-catalyzed oxidation of a chromophore by hydrogen peroxide (H 2 O 2 ). This is a simple, sensitive and specific method, which does not require an expensive apparatus. , UR catalyzes the conversion of uric acid to allantoin, H 2 O 2 , and carbon dioxide (CO 2 ). Next, H 2 O 2 reacts in the presence of horseradish peroxidase (HRP) with Amplex Ultra Red reagent to generate the red-fluorescent oxidation product resorufin, which can be detected by fluorescence spectroscopy at an excitation and an emission wavelength of ∼570 and ∼585 nm, respectively.…”

“…In body fluids, ascorbic acid is one of the main interfering substances due to its strong reducing activity, which disturbs when using oxidase-based chromogenic detection . The enzyme ascorbate oxidase (AO) is commonly added to preserve the chromophore from reduction by elimination of the ascorbic acid, which will be converted to 2- L -dehydroascorbic acid. , We compare capsosomes loaded with equal amounts of L UR , L HRP , and empty liposomes (L) vs capsosomes loaded with equal amounts of L UR , L HRP , and AO loaded liposomes (L AO ) in their ability to convert uric acid into the fluorescent probe via enzymatic conversion in the presence of ascorbic acid. In the latter case, the presence of AO should eliminate the ascorbic acid, leading to more efficient uric acid detection i.e., higher amount of fluorescent product (Figure a).…”

Spectrophotometric Determination of Uric Acid Based on Fading of o-Hydroxyhydroquinonephthalein-palladium(II)-hexadecyltrimethyl-ammonium Complex

“…The relative standard deviation (RSD) for five runs of 0.10 μg ml -1 of UA was 1.5%. This method is about 20-times more sensitive than the conventional methods, 20,21 and gives excellent reproducibilities.…”

“…The sensitivity (ε = 6.5 × 10 5 dm 3 mol -1 cm -1 ) of the proposed method was much greater than that of other spectrophotometric methods. 20,21 In addition, there was a remarkable improvement in the influences of foreign substances, especially ascorbic acid. And the present method, owing to no need for solvent extraction, should be useful for a simple and highly sensitive determination of UA in real samples.…”

“…[17][18][19] While there are many colorimetric assays 20,21 for the determination of UA, most of them are not satisfactory, showing low sensitivity, low reproducibility, time-consuming procedure and interference of foreign substances such as ascorbic acid.…”