Katz J, Wals P, Lee W N
Cedars-Sinai Medical Center, Los Angeles, California 90048.
J Biol Chem. 1993 Dec 5;268(34):25509-21.
Fasted rats were intragastrically infused with either [2,3-13C]lactate or [1,2,3-13C]lactate. The infusate also contained 14C-labeled lactate and [3-3H]glucose. Glucose, alanine, glutamate, and glutamine were isolated from liver and blood. There was near complete equilibration of lactate and alanine, and the relative specific activities and relative enrichments were the same in blood and liver. Glucose was cleaved enzymatically to lactate. The compounds were examined by gas chromatography-mass spectroscopy. From the mass isotopomer spectra of the lactate, glutamate, and glutamine and their cleavage fragments the positional isotopomer composition of these compounds was obtained. The enrichment and isotopomer pattern in the lactate from cleaved glucose represents that in phosphoenolpyruvate (PEP). When [1,2,3-13C]lactate was infused the mass isotopomer spectrum of glutamates consisted only of compounds containing either one, two, or three 13C carbons per molecule (m1, m2, and m3). There was little 13C in C-4 and C-5 of glutamate. The rate of pyruvate decarboxylation is low, and 3-4% of the acetyl-CoA flux in the Krebs cycle is contributed by lactate carbon. The major isotopomers in lactate, alanine, and PEP were m3 and m2 with 13C in C-2 and C-3. The predominant isotopomer in PEP from [2,3-13C]lactate was m2 with 13C in C-2 and C-3. There was much more of m1 isotopomer with 13C in C-3 and C-2 than the m1 isotopomer with 13C in C-1. There was very little m3, the isotopomer with 13C in all three carbons. Most of the 13C in C-3 and C-4 of glucose and C-1 of glutamate was introduced via 13CO2 fixation. From the isotopomer distribution and the rate of glucose turnover we deduced, applying the analysis described in the "Appendix," the absolute rates of gluconeogenic pathways, recycling of PEP and the Cori cycle, and flux in the Krebs cycle. The flux from oxaloacetate (OAA)-->PEP was seven times that of OAA-->citrate, and about half of PEP was recycled to pyruvate via pyruvate kinase. The mass isotopomer patterns in glutamate and glutamine were similar but differed from those of lactate and glucose. It appears that the glutamates are derived from alpha-ketoglutarate from a different Krebs cycle pool than PEP. The flux from OAA to PEP in this pool was two to three times that of OAA to citrate.(ABSTRACT TRUNCATED AT 400 WORDS)
对禁食的大鼠进行胃内灌注[2,3-¹³C]乳酸盐或[1,2,3-¹³C]乳酸盐。灌注液中还含有¹⁴C标记的乳酸盐和[3-³H]葡萄糖。从肝脏和血液中分离出葡萄糖、丙氨酸、谷氨酸和谷氨酰胺。乳酸盐和丙氨酸接近完全平衡，血液和肝脏中的相对比活性和相对丰度相同。葡萄糖通过酶促反应裂解为乳酸盐。通过气相色谱-质谱法对这些化合物进行检测。从乳酸盐、谷氨酸和谷氨酰胺的质量同位素异构体谱及其裂解片段中获得这些化合物的位置同位素异构体组成。裂解葡萄糖产生的乳酸盐中的丰度和同位素异构体模式代表磷酸烯醇丙酮酸（PEP）中的情况。当灌注[1,2,3-¹³C]乳酸盐时，谷氨酸的质量同位素异构体谱仅由每分子含有一个、两个或三个¹³C碳原子的化合物（m1、m2和m3）组成。谷氨酸的C-4和C-5中几乎没有¹³C。丙酮酸脱羧的速率较低，三羧酸循环中3-4%的乙酰辅酶A通量由乳酸盐碳贡献。乳酸盐、丙氨酸和PEP中的主要同位素异构体是m3和m2，¹³C位于C-2和C-3。[2,3-¹³C]乳酸盐产生的PEP中的主要同位素异构体是m2，¹³C位于C-2和C-3。C-3和C-2中含有¹³C的m1同位素异构体比C-1中含有¹³C的m1同位素异构体多得多。三个碳原子都含有¹³C的m3同位素异构体非常少。葡萄糖的C-3和C-4以及谷氨酸的C-1中的大部分¹³C是通过¹³CO₂固定引入的。根据同位素异构体分布和葡萄糖周转率，我们应用“附录”中描述的分析方法，推断出糖异生途径的绝对速率、PEP和科里循环的再循环以及三羧酸循环中的通量。草酰乙酸（OAA）→PEP的通量是OAA→柠檬酸通量的7倍，约一半的PEP通过丙酮酸激酶再循环为丙酮酸。谷氨酸和谷氨酰胺中的质量同位素异构体模式相似，但与乳酸盐和葡萄糖的不同。似乎谷氨酸来自与PEP不同的三羧酸循环池中的α-酮戊二酸。该池中从OAA到PEP的通量是从OAA到柠檬酸通量的两到三倍。（摘要截断于400字）
