Methods Study For Determination And Application Of Purine And Uric Acid Part Ⅰ Methods Study For Determination Of Total Amount Of Purine In Food By Diazotization-xanthine Oxidase-HPLC

Methods study for determination and application of purine and uric acid Partâ… Methods study for determination of total amount of purine in food by diazotization-xanthine oxidase-HPLCPurine (C₅H₅N₄) is an important component of nucleic acids, and one of the important organism metabolitess. Body synthesis, nucleic acid decomposition of human tissue and food intaking are the main sources of purine. While intaking of purine-rich foods long-term along with some induced factors can cause purine metabolism diseases, such as gout. We should pay more attention to limit excessively intaking purine-rich food besides traditional medical treatment.However, there is no accurate data on purine content of food in our food composition table, it is reported that values of purine content of food vary greatly. Therefore, the establishment of accurate, simple and feasible method for the detection of food purine content, and accurate detection of the purine content of various foods can provide datas for supplementing and improving food composition database, as well as can provide the basis for the dietary of patients with gout and other disorders of purine metabolism, which have important public health significance to prevent hyperuricemia and gout from occurring.To determine the content of purine in food, the method of high performance liquid chromatography (HPLC) after the hydrolysis of food is mostly used, in which the content of the four purines are firstly measured respectively and then calculates the total sum. The final metabolite of all purines is uric acid, so we can explore a new method for determination of total purine, which makes all purine turn to uric acid at first and calculates the total amount of purine compounds by measuring the content of uric acid in food. It provides an accurate and sensitive analysis method in the determination of total purine contents in food.Methods:Adenine and guanine can reacts to diazonium salt with nitrous salt in the environment of low temperatures and strong acid solution. When the diazonium salt of the acidic aqueous solution was heated, hydrolysis occurs to produce phenol. Though this reaction, we can turn adenine and guanine to hypoxanthine and xanthine after deamination. Hypoxanthine and xanthine can convert to uric acid at the effect of xanthine oxidase, then used HPLC to determine the content of uric acid. Finally, the content of uric acid can be used to measure the amount of purines.In this experiment, we optimized the reagent mount of diazotization reaction, reaction temperature and time, the amount of xanthine oxidase, and pH experimental conditions etc. We established a new method of determination of purine, and used this method for determination of purines in food.Results:1. The experimental results showed that the optimal experimental conditions were as follows:(1) Conditions of HPLC determination: Column: Agilent XDB-C₁₈ (4.6 mm×250 mm, 5μm); Mobile phase: H₃PO₄-KH₂PO₄ buffer (pH 3.8); Flow rate: 1.0 mL/min; Column temperature: 25℃; Injection volume: 20μL; UV detection wavelength: 254 nm.(2) Diazo reaction, when the guanine, adenine concentration was 4.0 mg/L, best conditions were: temperature was 55℃for 30 minutes, HCl concentration was 0.144 mol/L, NaNO2 concentration was 3.0 g/L.(3) The optimal condition of xanthine oxidase: xanthine oxidase concentration was 100 U/L, the optimum temperature was 30℃with constant temperature for 30 minutes, the optimum pH was 7.5.2. Under optimized conditions, it can make a good linearity with uric acid when the total quantity of purine winthin the range of 11.18 111.76μmol/L. Standard curve regression equation was A = 4.1885c - 10.505. Correlation coefficient r = 0.9999, RSD < 3.70 %. Recoveries were between 101.7 %～113.4 %.3. The total amount of purine bases determined in heart-shaped, chicken gizzards, chicken kidneys, chicken skin, chicken lung, then compared this result with the result measured without transformation, by means of matched t-test, there was no statistically significant difference between the results of the two methods(p>0.5).Conclusion:In this study, we use diazotization - xanthine oxidase in the conversion of purine, and we measure the contents of uric acid with HPLC, and get the total amount of purine through the determination of uric acid. This method is sensitive, accurate. To converse purine and measure the transformation products uric acid by this method, the result is satisfactory. Partâ…¡Methods study for determination of Uric Acid by Spectrophotometry and application in SerumPurine-rich foods are intaked long-term with some factors can cause induction of the purine metabolism, leading to product of purine metabolism of uric acid deposition in vivo in the end. As an endogenous antioxidant, normal level of uric acid is beneficial for the body and can scavenge oxygen free radicals, chelate and transfer metal ion, prevent the degradation of extracellular superoxide dismutase, protect vascular endothelial cells from damaging. But when the blood uric acid concentrations exceed over the normal value, uric acid salt crystals can be deposited in joints, soft tissue, bone and kidney, etc., which can lead to a range of diseases such as gout. Therefore, the detection of uric acid content has an important significance in monitoring of these diseases and understanding the progress of the disease.Methods:As a reducing agent, uric acid can reduce Fe (III) to Fe (II) under the pH 3.6 in solution, Fe (II) can react with phenanthroline generate orange-red complexes, then measured at 510 nm by spectrophotometry, which is a new method of determination of uric acid by spectrophotometry. This paper discusses different factors of the color reaction, and applied into determination of the serum uric acid.Results:1. The experimental results showed that the optimal experimental conditions were as follows:λmax was 510 nm, the equilibrium temperature was 55℃, equilibrium time was 30 minutes, acetate buffer pH was 3.6, Fe (III) solution (4.5mg/L), phenanthroline solution (0.1g/L), centrifugation speed 3000 r/min, centrifugation time was 25 minutes.2. Under the optimized conditions, linearity was obeyed in the range of 0.25～7.00 mg/L of uric acid content , the standard curve regression equation was A = 0.1316c + 0.0023, correlation coefficient r = 0.9997, The precisions (RSD, %) for six repeated determinations at three levels of uric acid were less than 3.70 %. The recoveries were between 84.0 % 101.0 %.3. After the determination of 12 blood samples, compared with the results of enzymatic determination , by means of matched t-test, there was no statistically significant difference between the results of the two methods(p>0.5).Conclusions:In this study, the determination of serum uric acid by spectrophotometry have many advantages such as the relative standard deviation is less than 3.70%, the recoveries are between 84.0 % 101.0 %, and it has a good linearity when uric acid content is in the range of 0.25 7.00 mg/L.The method has high sensitivity, good accuracy, as well as that the instrument is simple which is of convenient operation, easy to implement. The proposed method was applied to the determination of uric acid in human serum sample with satisfactory results.