Ross B D, Kingsley P B, Ben-Yoseph O
Department of Radiology, School of Medicine, University of Michigan, Ann Arbor 48109-0553.
Biochem J. 1994 Aug 15;302 ( Pt 1)(Pt 1):31-8. doi: 10.1042/bj3020031.
The isotopically substituted molecule D-[1,6-13C2,6,6-2H2]glucose is introduced for measuring the relative activities of the pentose phosphate pathway (PPP) and glycolysis in a single incubation. PPP activity in cultured cells was determined by gas chromatography/mass spectrometric analysis of lactate produced by cells incubated with [1,6-13C2,6,6-2H2]glucose. Two other isotopes, [1,5,6-13C3]glucose and [6-13C,1,6,6-2H3]glucose, were not satisfactory for measurements of this activity. This method has four advantages over the traditional one in which 14CO2 production from [1-14C]glucose and [6-14C]glucose is compared: (1) repeated measurements can be made on a single set of cells, (2) only a single incubation is required, (3) extensive CO2 production by Krebs-cycle activity does not interfere with the measurements and (4) it is not necessary to measure the amount of glucose consumed in order to calculate relative activities of the PPP and glycolysis. Preliminary observation indicates that rat brain PPP activity may be measured in vivo with [1,6-13C2,6,6-2H2]glucose when combined with microdialysis techniques.
引入同位素取代分子D-[1,6-¹³C₂,6,6-²H₂]葡萄糖，用于在单次孵育中测量磷酸戊糖途径（PPP）和糖酵解的相对活性。通过对用[1,6-¹³C₂,6,6-²H₂]葡萄糖孵育的细胞产生的乳酸进行气相色谱/质谱分析，来测定培养细胞中的PPP活性。另外两种同位素，[1,5,6-¹³C₃]葡萄糖和[6-¹³C,1,6,6-²H₃]葡萄糖，不适合用于该活性的测量。与传统方法相比，该方法具有四个优点，传统方法是比较[1-¹⁴C]葡萄糖和[6-¹⁴C]葡萄糖产生¹⁴CO₂的情况：（1）可以对同一组细胞进行重复测量；（2）只需要单次孵育；（3）三羧酸循环活性产生的大量CO₂不会干扰测量；（4）计算PPP和糖酵解的相对活性时，无需测量消耗的葡萄糖量。初步观察表明，当与微透析技术结合使用时，可用[1,6-¹³C₂,6,6-²H₂]葡萄糖在体内测量大鼠脑PPP活性。
