Quantitative isolation of radiolabeled metabolites without chromatography: measurements of the biosynthesis of purines, pyrimidines, and urea in isolated hepatocytes.
Quantitative isolation of radiolabeled metabolites without chromatography: measurements of the biosynthesis of purines, pyrimidines, and urea in isolated hepatocytes.
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无需色谱法即可定量分离放射性标记代谢物：测量分离的肝细胞中嘌呤、嘧啶和尿素的生物合成。
DOI：
10.1016/0003-2697(80)90606-5
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发表时间：
1980
影响因子：
2.9
通讯作者：
Tremblay,GC
中科院分区：
文献类型：
--
作者：
Wendler,PA;Tremblay,GC
放射性标记的前体掺入尿素，乳清酸，尿苷核苷酸和腺嘌呤核苷酸的测量被用来证明与载体共结晶分离代谢产物的程序的可靠性。用分离的肝细胞悬液将前体掺入产品中。通过在高温下用载体饱和孵育混合物的酸溶性部分的等分试样，并在溶液冷却时使放射性标记代谢物和载体共结晶，基本上实现了各放射性标记产物的直接分离。当[14C] NaHCO3作为前体时，该程序允许从同一孵育混合物中分离放射性标记的尿素、乳清酸和尿苷核苷酸。通过重结晶至恒定比活度以及在共结晶前使用酶去除推定代谢物，获得了与载体分离的放射性标记代谢物与载体具有相同化学特性的证据。改变条件以提供[14C] NaHCO3分别掺入尿素、乳清酸和尿苷核苷酸的8.4倍、180倍和13倍的速率范围，在这些活性范围内以六种不同速率进行的重复测定结果显示，单个测定值与其平均值的差异分别为3.2 ± 1.0、1.1 ± 0.3、1.2 ± 0.3、1.3 ± 0.4、1.3 ± 0.5。和3.3 ± 1.5%（平均值± SE）。与载体共结晶分离的方法已被证明是廉价、可靠的，并且非常适合于多个样品的同时分析。这些程序的优点和局限性进行了讨论。
Measurements of the incorporation of radiolabeled precursors into urea, orotic acid, uridine nucleotides, and adenine nucleotides were used to demonstrate the reliability of procedures for the isolation of metabolites by cocrystallization with carrier. Incorporation of precursor into product was carried out with suspensions of isolated hepatocytes. Direct isolation of each radiolabeled product was accomplished essentially by saturating aliquots of the acid-soluble fraction of the incubation mixture with carrier at elevated temperatures, and allowing the radiolabeled metabolite and carrier to cocrystallize as the solutions cooled. When [14C]NaHCO3was employed as the precursor, the procedure permitted isolation of radiolabeled urea, orotic acid, and uridine nucleotides from the same incubation mixture. Evidence that the radiolabeled metabolite isolated with carrier was of the same chemical identity as the carrier was obtained by recrystallization to constant specific activity and also by the use of enzymes to remove the putative metabolite prior to cocrystallization. Conditions were varied to provide a range of rates of 8.4-fold, 180-fold, and 13-fold for the incorporation of [14C]NaHCO3into urea, orotic acid, and uridine nucleotides, respectively, and the results of duplicate assays at six different rates over these ranges of activity showed individual determinations to differ from their means by 3.2 ± 1.0, 1.1 ± 0.3, and 3.3 ± 1.5% (average ± SE), respectively. The method of isolation by cocrystallization with carrier has proved inexpensive, reliable, and well suited to the simultaneous analysis of multiple samples. The advantages and limitations of these procedures over more conventional methods are discussed.