New radioisotope-free method for measuring bacterial production using [15N5]-2'-deoxyadenosine and liquid chromatography mass spectrometry (LC-MS) in aquatic environments

This study proposed a non-radioactive quantitative measurement of bacterial production using stable isotope nitrogen-15-labeled deoxyadenosine ([N-15(5)]-2'-deoxyadenosine; N-15-dA) by liquid chromatography mass spectrometry (LC-MS). The method of preparing N-5-dA described in this study included incubation of seawater or lake water with N-15-dA for 5-24 h, filtration onto a membrane filter, DNA extraction, enzymatic hydrolysis of DNA to nucleosides and quantification of N-15-dA by LC-MS. In the DNA extraction, the silica beads method was examined first, but a large amount of salts and enzymatic inhibitors used in the method caused failure of subsequent procedures, such as enzymatic hydrolysis and LC-MS analysis. On the other hand, the magnetic beads method showed much better results for the extraction. The incorporation rate of N-15-dA was significantly positively correlated to that of tritium-labeled thymidine (H-3-TdR) in samples of coastal seawaters and lake waters. The average N-15-dA: H-3-TdR incorporation ratio for the seawater sample was 0.55 with 2.5 and 97.5 % confidence intervals of 0.51 and 0.58, respectively; the average ratio for the lake water sample was 0.28 with 2.5 and 97.5 % confidence intervals of 0.23 and 0.34, respectively. The results suggest that the N-15-dA method can be applied to the measurement of bacterial production in aquatic ecosystems, and that this method can accurately predict the DNA synthesis rates measured by the conventional method.