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Technical note: A new high-performance liquid chromatography purine assay for quantifying microbial flow¹

S. M. Reynal^*,2 and G. A. Broderick^,3

^* Department of Dairy Science, University of Wisconsin, Madison 53706
Agricultural Research Service, USDA US Dairy Forage Research Center, 1925 Linden Drive West, Madison, Wisconsin 53706

³ Corresponding author: glen.broderick{at}ars.usda.gov

An HPLC method was developed to quantify the purines adenine and guanine and their metabolites xanthine and hypoxanthine in hydrolysates of isolated bacteria and omasal digesta and to assess the effect of using either purines only or purines plus metabolites as microbial markers for estimating microbial flow from the rumen. Individual purines and their metabolites were completely resolved on a C18 column using gradient elution with 2 mobile phases. Intraassay coefficient of variation ranged from 0.6 to 3.1%. Hydrolytic recovery of the 4 purine bases from their corresponding nucleosides averaged 101% (control), 103% (when added to bacterial isolates), and 104% (when added to omasal digesta). Mean concentrations of adenine, guanine, xanthine, and hypoxanthine were, respectively, 53, 58, 2.8, and 3.5 µmol/g of dry matter in omasal bacteria and 10, 12, 7.5, and 7.5 µmol/g of dry matter in omasal digesta, indicating that xanthine plus hypoxanthine represented 5% of total purines in bacterial hydrolysates but 41% of total purines in digesta hydrolysates. A significant negative relationship (R² = 0.53) between the sum of adenine and guanine and the sum of xanthine and hypoxanthine in digesta samples (but not bacterial isolates) indicated that 89% of the adenine and guanine originally present in ruminal microbes were recovered as xanthine and hypoxanthine. These results suggested that, when total purines are used as the microbial marker, both purines and their metabolites should be quantified and used to compute microbial nonammonia N and organic matter flows.

Key Words: microbial flow • purines • high-performance liquid chromatography