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The first part of the research and application of purine and its metabolites uric acid detection methods diazotization - xanthine oxidase-HPLC determination of purine food purine (purine C 5 H 5 N 4 ) is an important part of the nucleic acid, an important material in the organism's metabolism. The main source of the human body purine synthesis in vivo nuclease human tissues and from the food intake. Long-term high-purine foods such as seafood, meat intake, plus some inducing factors can cause abnormal purine metabolism, leading to hyperuricemia and gout purine metabolism of the disease. For these diseases, in addition to medical treatment, but also must limit the intake of high-purine foods. Of food composition tables but no accurate and complete data purine content of foods, some food purine content values ??reported in the literature away, therefore, create accurate, simple and feasible method for detecting food purine content, and each kinds of food in purine content accurately detect and can provide data to supplement and improve China's food composition database to provide guidance based on purine metabolism in patients with disorders such as gout patients healthy diet, for the prevention of the occurrence of hyperuricemia and gout, important public health implications. Method for determination of the purine food for use on food after hydrolysis were measured by high performance liquid chromatography of adenine, guanine, hypoxanthine and xanthine content is then calculated by adding the sum. As the final metabolites of purine uric acid, the study will explore the determination of purine total - the first purine All converted to uric acid, calculated by the determination of uric acid content of the purine food total to provide an accurate and sensitive analytical method for the determination of purine content in food. Methods: adenine and guanine with nitrous role diazonium salt, when an acidic aqueous solution of the diazonium salt when heated, is hydrolyzed to produce phenol, the reaction can be adenine, guanine deaminase, respectively, after conversion to generate hypoxanthine purine and xanthine. Hypoxanthine and xanthine, and thus the role of xanthine oxidase transforming generate uric acid, the use of HPLC for the determination of uric acid conversion product, obtained by the determination of uric acid content of the total amount of purine. Experiment the amount of the diazo reaction reagent, reaction temperature and time, the amount of xanthine oxidase, and the optimum pH and temperature, time, and other experimental conditions were optimized to create a new method for the determination of the purine content, and for determination of purine content in food. Results: 1. Experimental results show that the optimum conditions are as follows: (1) HPLC measurement conditions: Column: Agilent XDB-C 18 (4.6 mm × 250 mm, 5μm); mobile phase: H 3 PO 4 -KH 2 PO 4 buffer (7.35 mmol / L, pH 3.8); flow rate: 1.0 mL / min; column temperature: 25 ° C; injection volume: 20 ul; detection wavelength: 254 nm. (2) a diazonium reaction, guanine, adenine concentrations were 4.0 mg / L, the best conversion conditions are as follows: 55 ° C constant temperature water bath for 30 min, the HCl concentration of 0.144 mol / L, NaNO , 2 concentration of 3.0 g / L; (3) xanthine oxidase role: role in a concentration of 100 U / L, the optimum temperature and time for 30 ° C the thermostat 30 min, and the optimum pH 7.5. best experimental conditions, the total purine in 11.18 ~ 111.76μmol / L range and generate good linearity of the uric acid content of the standard curve regression equation A = 4.1885 c - 10.505, correlation coefficient r = 0.9999, precision (RSD) lt; 3.70%, plus The recovery of 101.7% to 113.4%. 3 were determined by this method on the heart-shaped, chicken gizzards, chicken kidney, chicken skin, chicken lung, compared with the the purine total untransformed measured results, using a paired t-test, t = - 0.190, P gt; 0.05 (P = 0.858), no statistically significant difference between the results measured by the two methods. Conclusion: In this study, diazotization - xanthine oxidase in the purine conversion and transformation products of uric acid were measured using HPLC Determination of uric acid can get the total amount of purine, the method has high sensitivity and good accuracy, with The method purine food conversion and the conversion product of uric acid were measured, the results were satisfactory. The second part of the spectrophotometric determination of uric acid in serum long-term intake of purine-rich foods plus some induced factors can cause abnormal purine metabolism, purine metabolism leading to the end product of uric acid deposition in the body . Normal levels of uric acid as an endogenous antioxidants, the body is beneficial, can scavenge oxygen free radicals, chelation of transition metal ions, to prevent the degradation of extracellular superoxide dismutase, protection of vascular endothelial cells it from damage . But when the blood uric acid concentration than normal uric acid salt crystals can be deposited in the joints, soft tissue, bone and kidney, etc., can cause a range of diseases of gout, uric acid Determination monitoring of these diseases, understanding The progress of the disease is important. : Uric acid as a reducing agent, under the conditions of solution pH 3.6, iron (III) is reduced to iron (II), iron (II) to further generate orange-red complex with phenanthroline reaction by spectrophotometry in at 510 nm was measured to establish the spectrophotometric determination of uric acid method, discussed in detail the various factors affecting the color reaction, and applied to the determination of uric acid in serum. Results: 1. Experimental results show that the optimum conditions are as follows: To determine the wavelength of 510 nm, the equilibrium temperature to 55 ° C, the equilibrium time of 30 min, acetic acid buffer solution of pH 3.6, iron (III) concentration of 4.5 mg / L, o of phenanthroline solution concentration of 0.1 g / L, centrifugal speed of 3000 r / min centrifugation time of 25 min. Selected optimum conditions, the uric acid content of 0.25 to 7.00 mg / L range linear standard curve regression equation was A = 0.0023 0.1316c, the correlation coefficient (r = 0.9997), precision RSD lt; 3.70% , the recovery rate of 84.0% to 101.0%. (3) The determination of 12 blood samples, the results are compared with the enzymatic determination of normality analysis, the difference d to follow a normal distribution, using a paired t-test was t = 0.310, P gt; 0.05 (P = 0.762) , indicating the results measured by the two methods was not significantly different. Conclusion: The use of spectrophotometric determination of serum uric acid content, the relative standard deviation is less than 3.70%, and the recovery in the range of 84.0% to 101.0%, uric acid content of 0.25 to 7.00 mg / L within the scope of good linearity. The method is simple instrument, easy to operate, easy to implement, and serum uric acid were measured using this method, with satisfactory results.
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