Determination Of Xanthine Oxidase Activity Research Articles

Xanthine Oxidase: Isolation, Assays of Activity, and Inhibition

Xanthine oxidase (XO) is an important enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid which is excreted by kidneys. Excessive production and/or inadequate excretion of uric acid results in hyperuricemia. This paper presents a detailed review of methods of isolation, determination of xanthine oxidase activity, and the effect of plant extracts and their constituents on it. Determining the content and activities of XO can be used for diagnostic purposes. Testing inhibition of XO is important for detection of potentially effective compounds or extracts that can be used to treat diseases that are caused by increased activity of XO.In vitrobioassays are used to examine test material for XO inhibition, as inhibitors of XO may be potentially useful for the treatment of gout or other XO induced diseases. Several authors reported on the XO inhibitory potential of traditionally used medicinal plants.

Chemiluminescent determination of xanthine oxidase activity in milk.

A chemiluminescent method for determining xanthine oxidase (XOD) activity was developed and applied to the assay of milk enzyme activity using a photomultiplier luminometer. Various kinds of milk and cream samples were analysed for XOD content. In pasteurized milk, XOD activity depended on the fat content and in UHT milk it disappeared owing to the heat treatment. Milk sample preparation was very simple, requiring only homogenization at 40 degrees C followed by a 1:10 dilution with UHT ('XOD-free') milk. The assay was carried out at 25 degrees C. The response obtained from XOD standard solutions in milk was linear from 0.1 to 500 enzyme units (U) l-1, but for the actual milk samples values ranged only from 1 to 135 U l-1. The detection limit at 2 SD was 0.1 U l-1 in milk, while in buffer it was 100 times lower. The intra-assay and interassay CV for XOD activity in milk were 6-12%.

42 CHEMILUMINESCENT ASSAYS IN THE STUDY OF PURINE METABOLISM

The measurement of purine nucleosides and bases in biological fluids is a particularly challenging problem because their concentrations are extremely low and very limited amount of the specimen is often available (e.g. paediatric specimens). Abnormal levels of these compounds are associated with inborn errors of purine metabolism, neoplastic diseases and conditions leading to a net ATP degradation. Xanthine oxidase-initiated isoluminol chemiluminescence, in the absence of peroxidase, has been employed in our laboratories for the assay of hypoxanthine/xanthine, guanine, guanosine, inosine and adenosine. Any purines lower in the catabolic sequence than that to be assayed are removed by a preincubation in the presence of appropriate enzymes. The assayed purine is converted to xanthine and/or hypoxanthine. Isoluminol chemiluminescence elicited by xanthine oxidase/hypoxanthine-xanthine system, is enhanced by adding p-iodophenol to the reaction mixture. The assay is rapid, specific, sensitive (1 nmol/liter) and suitable for the assay of purines in deproteinized biological fluids, provided that uric acid is removed by preincubation with uricase and that EDTA is added to the reaction mixture. Internal standardization is required. A very sensitive luminometric assay for the determination of xanthine oxidase activity in biological samples is also described.

Spectrophotometric assay of xanthine oxidase with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as chromogen

A new method for the determination of xanthine oxidase activity, based on the oxidation of 2, 2'-azino-di(3-ethylbenzthiazoline-6-sulphonate) (ABTS) by use of uricase and peroxidase, is described. The absorbance increase of the oxidized form of ABTS, measured after 10 min at 410 nm is proportional to xanthine oxidase activity. The method is sensitive, precise (CV below 8.3%), and linear up to 20 U/l. The analytical recovery of the ABTS-method was quantitative. Comparison with the UV and colorimetric NBT-method gave good correlation ( r ⩾ 0.984). Reference values for serum xanthine oxidase activities determined with the new ABTS-method on 83 healthy persons are 0 to 1.20 U/l.

A new method for the assay of xanthine oxidase activity.

A colorimetric method for the assay of xanthine oxidase activity, based on the production of hydrogen peroxide, is described. [chemical formula] The precision, accuracy, sensitivity and specificity of the method were found to be satisfactory for the rapid and reliable determination of xanthine oxidase activity.

A sensitive method for the determination of xanthine oxidase activity

1. 1. A sensitive radioactive method is described for the determination of xanthine oxidase activity. 2. 2. [8- 14C] xanthine is incubated with the enzyme in phosphate buffer pH 7.4 at 37° for 3 h. The reaction is stopped with TCA and the protein-free filtrate is passed through a Dowex 50 column. The xanthine is quantitatively adsorbed to the resin and the uric acid is entirely recovered in the first 10 ml of 0.1 N HCl eluate and is counted. The method is calibrated against purified xanthine oxidase assayed spectrophotometrically. 3. 3. The method is able to measure 2.10 −7 and is linear up to 5.10 −5 international units of xanthine oxidase and is therefore more sensitive than the standard assays by 3–4 orders of magnitude. 4. 4. The method has been successfully used for the assay of the enzyme in serum, whole blood and tissue extracts.