Kohen E, Kohen C, Hirschberg J G, Wouters A W, Bartick P R, Westerhoff H V, Charyulu K K, Schachtschabel D O
Blood Cells. 1980;6(4):753-65.
Microspectrofluorometric evaluation of coenzyme-linked transient changes in blue fluorescence, triggered by microinjections of metabolic intermediates, allows the definition of dynamic parameters in the characterization of cells. The observed fluorescence transients can be simulated by appropriate equations accounting for NAD(P) reduction-reoxidation, with NAD(P) as rate-limiting or not. From the above, the rate constants K1 and K2 of NAD(P) reduction and reoxidation can be determined. Other useful parameters in the metabolic evaluation of different cell lines, comprising normal and transformed fibroblasts, glia-glioma, melanoma lines, and a mouse embryo clone, can be derived from the relationship between injected dose of substrate and rise or decay rates of NAD(P) in equilibrium or formed from NAD(P)H transients. Reoxidation of NAD(P)H seems to be a useful target for such studies in view of possible impairment in malignant cells and X-irradiated cells. Cells followed by fluorometry are retrieved for subsequent ultrastructural and other analyses. Thus, the metabolic patterns associated with the operation of intracellular pathways or organelle interactions, and their aberrations can be recognized. On this basis eventually a classification of different cell lines according to structure-function should be feasible.
通过微量注射代谢中间体引发的辅酶连接的蓝色荧光瞬态变化的显微分光荧光测定，能够定义细胞特征中的动态参数。观察到的荧光瞬态可以通过考虑NAD(P)还原-再氧化的适当方程进行模拟，NAD(P)可以是限速的，也可以不是。由此，可以确定NAD(P)还原和再氧化的速率常数K1和K2。不同细胞系（包括正常和转化的成纤维细胞、神经胶质-胶质瘤、黑色素瘤细胞系以及小鼠胚胎克隆）代谢评估中的其他有用参数，可以从底物注射剂量与平衡状态下NAD(P)的上升或衰减速率之间的关系中得出，或者从NAD(P)H瞬态形成的关系中得出。鉴于恶性细胞和X射线照射细胞可能存在的损伤，NAD(P)H的再氧化似乎是此类研究的一个有用靶点。通过荧光测定跟踪的细胞被回收用于后续的超微结构和其他分析。因此，可以识别与细胞内途径或细胞器相互作用操作相关的代谢模式及其异常。在此基础上，最终根据结构-功能对不同细胞系进行分类应该是可行的。
