Measurement of radiolabeled lactate production using lactate monooxygenase.
Measurement of radiolabeled lactate production using lactate monooxygenase.
复制标题
使用乳酸单加氧酶测量放射性标记的乳酸产量。
DOI：
10.1006/abio.1993.1069
复制
发表时间：
1993
影响因子：
2.9
通讯作者：
Piomelli,S
中科院分区：
文献类型：
--
作者：
Tilton,WM;Seaman,C;Piomelli,S
在体外代谢研究中，乳酸的产生反映了糖酵解的有序流动。通常很难通过常用的分光光度技术准确测量乳酸的产生，因为细胞环境中存在大量乳酸和丙酮酸，并且在几种异常情况下这些物质可能会进一步增加（1，2）。本报告中描述的系统提供了监测乳酸盐生产的方法，无论最终产品或中间体的背景如何。该技术使用乳酸单加氧酶（LMO）将产生的乳酸盐转化为二氧化碳和乙酸盐，从而测量放射性标记葡萄糖的乳酸盐产量。由于在孵育开始时加入放射性标记的葡萄糖，因此形成的任何放射性标记的CO仅反映新产生的乳酸。用葡萄糖孵育以产生乳酸盐。首先在产生放射性标记乳酸盐的最佳条件下，用均匀放射性标记的葡萄糖对组织进行代谢孵育。在我们的实验室里，我们将这项技术应用于红细胞的研究。由于在全血中，相比之下，白色细胞和血小板对葡萄糖代谢的贡献相对可忽略，因此所有活性基本上反映了红细胞的活性。任何细胞操作都会导致代谢中间产物发生实质性变化;因此，为了获得可靠的结果，必须在采集后立即使用全血。将从正常健康志愿者获得的阻碍物在pH 7.4下缓冲;将25 ml血液添加到0.5ml缓冲液中，所述缓冲液设计成在血液悬浮液中得到以下终浓度：Tes，20 mM;葡萄糖，2.5mM; NaCl，（0.14M; KCl，4 mM; KH，PO，0.7mM; MgSO，1.7mM。(The缓冲液中所含的浓度是所示最终浓度的六倍。）[U-然后加入葡萄糖（2 μ Ci），并将混合物转移到锥形瓶中的封闭孵育系统或透析袋中的开放孵育系统中，如前所述（3）。将细胞悬浮液在Dubnoff代谢培养箱中于37 ℃振荡，通常振荡3小时.
In studies of metabolism in vitro, the production of lactate reflects the orderly flow of glycolysis. It is often difficult to measure accurately the production of lactate by commonly used spectrophotometric techniques, as large amounts of lactate and pyruvate are present in the cellular environment, and these may be further in-creased in several abnormal situations (1, 2). The system described in this report offers the means to monitor lactate production, regardless of the background of endproducts or intermediates. The technique measures lactate production from radiolabeled glucose using the enzyme lactate monooxygenase (LMO) that converts the lactate produced to carbon dioxide and acetate. Since the radiolabeled glucose is added at the beginning of the incubation, any radiolabeled CO, formed reflects exclusively the newly produced lactate. Incubation uith glucose for lactate production. Metabolic incubation of tissue with uniformly radiolabeled glucose under optimal conditions for the production of radiolabeled lactate is first conducted. In our labora-tory, we apply this technique to the study of red blood cells. Since, in whole blood, the contribution of white cells and platelets to glucose metabolism is comparatively negligible in comparison, all activity reflects essentially that of red blood cells. Substantial changes in metabolic intermediates take place with any cell manipulation; thus, in order to obtain reliable results, whole blood must be used immediately upon collection. Hlood obtained from normal healthy volunteers was buffered at pH 7.4; 25 ml of blood was added to 0.5 ml of a buffer designed to give the following final concentrations in the blood suspension: Tes, 20 mM; glucose, 2.5 mM; NaCl,(0.14 M; KCl, 4 mM; KH, PO,, 0.7 mM; MgSO,, 1.7 mM.(The buffer contained six times the final concen-trations shown.)[U-“C] Glucose (2 uCi) was then added, and the mixture was transferred either to a closed incu-bation system in an Erlenmeyer flask or to an open incubation system in a dialysis bag, as described previ-ously (3). The cell suspensions were shaken in a Dubnoff metabolic incubator at 37 C, usually for 3 h.