Tikunov Andrey P, Stoskopf Michael K, Macdonald Jeffrey M
Joint Department of Biomedical Engineering, NC State University and UNC Chapel Hill, Chapel Hill, NC 27599, USA.
Metabolites. 2014 Jan 7;4(1):53-70. doi: 10.3390/metabo4010053.
The metabolism of 2-13C/15N-glycine and U-13C-glucose was determined in four tissue blocks (adductor muscle, stomach and digestive gland, mantle, and gills) of the Eastern oyster (Crassostrea virginica) using proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR) spectroscopy. The oysters were treated in aerated seawater with three treatments (5.5 mM U-13C-glucose, 2.7 mM 2-13C/15N-glycine, and 5.5 mM U-13C-glucose plus 2.7 mM 2-13C/15N-glycine) and the relative mass balance and 13C fractional enrichments were determined in the four tissue blocks. In all tissues, glycine was metabolized by the glycine cycle forming serine exclusively in the mitochondria by the glycine cleavage system forming 2,3-13C-serine. In muscle, a minor amount of serine-derived pyruvate entered the Krebs cycle as substantiated by detection of a trace of 2,3-13C-aspartate. In all tissues, U-13C-glucose formed glycogen by glycogen synthesis, alanine by glycolysis, and glutamate and aspartate through the Krebs cycle. Alanine was formed exclusively from glucose via alanine transaminase and not glycine via alanine-glyoxylate transaminase. Based on isotopomer analysis, pyruvate carboxylase and pyruvate dehydrogenase appeared to be equal points for pyruvate entry into the Krebs cycle. In the 5.5 mM U-13C-glucose plus 2.7 mM 2-13C/15N-glycine emergence treatment used to simulate 12 h of "low tide", oysters accumulated more 13C-labeled metabolites, including both anaerobic glycolytic and aerobic Krebs cycle intermediates. The aerobic metabolites could be the biochemical result of the gaping behavior of mollusks during emergence. The change in tissue distribution and mass balance of 13C-labeled nutrients (U-13C-glucose and 2-13C/15N-glycine) provides the basis for a new quantitative fluxomic method for elucidating sub-lethal environmental effects in marine organisms called whole body mass balance phenotyping (WoMBaP).
利用质子（1H）和碳-13（13C）核磁共振（NMR）光谱法，测定了美国东牡蛎（Crassostrea virginica）四个组织块（闭壳肌、胃和消化腺、外套膜和鳃）中2-13C/15N-甘氨酸和U-13C-葡萄糖的代谢情况。将牡蛎置于充气海水中进行三种处理（5.5 mM U-13C-葡萄糖、2.7 mM 2-13C/15N-甘氨酸以及5.5 mM U-13C-葡萄糖加2.7 mM 2-13C/15N-甘氨酸），并测定四个组织块中的相对质量平衡和13C丰度。在所有组织中，甘氨酸通过甘氨酸循环代谢，仅在线粒体中由甘氨酸裂解系统形成丝氨酸，生成2,3-13C-丝氨酸。在肌肉中，少量丝氨酸衍生的丙酮酸进入三羧酸循环，这通过检测到微量的2,3-13C-天冬氨酸得以证实。在所有组织中，U-13C-葡萄糖通过糖原合成形成糖原，通过糖酵解形成丙氨酸，并通过三羧酸循环形成谷氨酸和天冬氨酸。丙氨酸仅由葡萄糖通过丙氨酸转氨酶形成，而非由甘氨酸通过丙氨酸-乙醛酸转氨酶形成。基于同位素异构体分析，丙酮酸羧化酶和丙酮酸脱氢酶似乎是丙酮酸进入三羧酸循环的等效位点。在用于模拟12小时“退潮”的5.5 mM U-13C-葡萄糖加2.7 mM 2-13C/15N-甘氨酸暴露处理中，牡蛎积累了更多的13C标记代谢物，包括无氧糖酵解和有氧三羧酸循环中间体。有氧代谢物可能是软体动物在暴露期间张口行为的生化结果。13C标记营养素（U-13C-葡萄糖和2-13C/15N-甘氨酸）的组织分布和质量平衡变化为一种新的定量通量组学方法提供了基础，该方法用于阐明海洋生物中的亚致死环境效应，称为全身质量平衡表型分析（WoMBaP）。
