Purine quantification in digests from ruminants by spectrophotometric and HPLC methods
Purine quantification in digests from ruminants by spectrophotometric and HPLC methods
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DOI：
10.1017/s0007114599000227
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发表时间：
1999-02-01
影响因子：
3.6
通讯作者：
Becker, K
中科院分区：
文献类型：
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作者：
Makkar, HPS;Becker, K
Zinn & Owens（1986; Canadian Journal of Animal Science 66，157-166）的方法，基于通过HClO 4释放嘌呤碱，然后用AgNO 3沉淀，用于研究从添加到基质如纤维素、淀粉和中性洗涤剂纤维中的冻干瘤胃微生物或大肠杆菌制剂中回收嘌呤。嘌呤的回收率很低（约50%）。在水解条件下（12 M-HClO 4，90-95度，1小时），使用HPLC方法测量的来自添加到基质中的瘤胃微生物制剂的嘌呤的回收率为95- 102%，这表明Zinn和Owens（1986）的方法中较低的嘌呤回收率不是由于核酸的不完全水解。使用HPLC方法，发现腺嘌呤和别嘌呤醇（内标物）是热不稳定的，因为在121度加热时观察到大量破坏。另一方面，另一种常用的内标物咖啡因在121度下稳定。在90-95 ℃下，用总体积为3 ml的2.5 ml 0.6 M-HClO 4（水解期间为0.5 M-HClO 4）1 h观察到来自瘤胃微生物制剂的核酸完全水解，并且在这些条件下腺嘌呤、鸟嘌呤、别嘌呤醇和咖啡因是稳定的。此外，在这些温和的水解条件下，从瘤胃微生物或E。使用Zinn和Owens（1986）的方法，加入基质中的大肠杆菌制剂范围为92 - 108%。根据实验结果，提出了改变水解条件，用分光光度法或HPLC法准确测定嘌呤碱基的方法。
The method of Zinn & Owens (1986; Canadian Journal of Animal Science 66, 157-166), based on release of purine bases by HClO4 followed by their precipitation with AgNO3, was used to study recovery of purines from lyophilized rumen microbial or Escherichia coli preparations added to matrices such as cellulose, starch and neutral-detergent fibre. The recovery of purines was poor (approximately 50 %). Under the hydrolysis conditions (12 M-HClO4, 90-95 degrees for 1 h) used in the method of Zinn & Owens (1986), the recovery of purines from the rumen microbial preparations added to matrices measured using an HPLC method was 95-102 %, suggesting that the lower recovery of purines in the method of Zinn & Owens (1986) was not due to incomplete hydrolysis of nucleic acids. Using the HPLC method, adenine and allopurinol (an internal standard) were found to be heat-labile as substantial destruction was observed on heating at 121 degrees. On the other hand, another commonly used internal standard, caffeine, was stable at 121 degrees. A complete hydrolysis of nucleic acids from the rumen microbial preparation was observed with 2.5 ml 0.6 M-HClO4 in a total volume of 3 ml (0.5 M-HClO4 during hydrolysis) at 90-95 degrees for 1 h, and under these conditions adenine, guanine, allopurinol and caffeine were stable. Moreover, under these milder hydrolysis conditions, the recovery of purine bases from the rumen microbial or E. coli preparations added to matrices ranged from 92 to 108 % using the method of Zinn & Owens (1986). Based on the results, changes in hydrolysis conditions have been proposed for accurate determination of purine bases using spectrophotometric or HPLC methods.