Kasumov Takhar, Martini Wenjun Z, Reszko Aneta E, Bian Fang, Pierce Bradley A, David France, Roe Charles R, Brunengraber Henri
Department and Nutrition, Case Western Reserve University, 11000 Cedar Road, Cleveland, OH 44106-7139, USA.
Anal Biochem. 2002 Jun 1;305(1):90-6. doi: 10.1006/abio.2002.5639.
We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.
我们开发了气相色谱-质谱分析法，用于测定组织中丙酰辅酶A、甲基丙二酰辅酶A和琥珀酰辅酶A的浓度及质量同位素异构体分布。该分析方法包括用高氯酸提取组织，向提取物中加入[(2)H(5)]丙酰辅酶A和[(2)H(4)]琥珀酰辅酶A内标，以及在寡核苷酸纯化柱上分离短链酰基辅酶A组分。丙酰辅酶A与肌氨酸反应，形成的N-丙酰肌氨酸作为其五氟苄基衍生物进行测定。甲基丙二酰辅酶A和琥珀酰辅酶A被水解，相应的酸作为叔丁基二甲基甲硅烷基衍生物进行测定。该分析方法应用于灌注大鼠肝脏中[U-(13)C(3)]丙酸代谢的研究。虽然丙酰辅酶A仅被M3标记，但由于柠檬酸循环中的同位素交换，琥珀酰辅酶A被M3、M2和M1标记。甲基丙二酰辅酶A被M3和M2标记，反映了S-甲基丙二酰辅酶A变位酶的逆向反应。因此，我们的分析方法能够通过丙酰辅酶A的前体，即丙酸、奇数链脂肪酸、异亮氨酸、苏氨酸和缬氨酸，来测量参与柠檬酸循环回补反应的辅酶A衍生物的周转情况。
