Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: acyl-CoA profiles in short-chain fatty acid oxidation defects
Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: acyl-CoA profiles in short-chain fatty acid oxidation defects
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DOI：
10.1016/j.ymgme.2012.10.007
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发表时间：
2012-12-01
影响因子：
3.8
通讯作者：
Bennett, Michael J.
中科院分区：
文献类型：
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作者：
Palladino, Andrew A.;Chen, Jie;Bennett, Michael J.
脂肪酸氧化缺陷的主要积累代谢产物是线粒体内酰基辅酶A。通常，测量次级代谢物如酰基肉毒碱、酰基甘氨酸和二羧酸以研究这些病症。方法尚未适用于主要为酰基辅酶A积累的缺陷中的组织酰基辅酶A测量。我们的目的是开发一种使用流动注射串联质谱法测量脂肪酸氧化缺陷小鼠组织中存在的脂肪酰辅酶A种类的方法。在加入[C-13（2）]乙酰辅酶A、[C-13（8）]辛酰辅酶A和[C-17]十七烷酸辅酶A的内标物后，从组织样品中提取酰基辅酶A并直接注入质谱仪。使用506.9中性丢失扫描和多反应监测（MRM）获得数据。该方法可以鉴定野生型小鼠肝脏中所有长链、中链和短链酰基CoA种类，包括预测的3-羟基酰基CoA种类。我们使用短链酰基辅酶A脱氢酶（SCAD）基因敲除小鼠的肝脏验证了该方法。正如预期的，与野生型相比，SAD -/-小鼠肝脏中[C-4]丁酰-CoA种类显著增加。然后，我们在短链3-羟酰辅酶A脱氢酶（SCHAD）缺陷小鼠的肝脏中测试了该测定法，以确定该模型中酰基辅酶A蓄积的概况。中链物质（包括3-羟酰基-CoA）的积累更适度，这与SCHAD酶的已知链长特异性一致。(C)2012 Elsevier Inc. All rights reserved.
The primary accumulating metabolites in fatty acid oxidation defects are intramitochondrial acyl-CoAs. Typically, secondary metabolites such as acylcarnitines, acylglycines and dicarboxylic acids are measured to study these disorders. Methods have not been adapted for tissue acyl-CoA measurement in defects with primarily acyl-CoA accumulation. Our objective was to develop a method to measure fatty acyl-CoA species that are present in tissues of mice with fatty acid oxidation defects using flow-injection tandem mass spectrometry.Following the addition of internal standards of [C-13(2)] acetyl-CoA, [C-13(8)] octanoyl-CoA, and [C-17] heptadecanoic CoA, acyl-CoA's are extracted from tissue samples and are injected directly into the mass spectrometer. Data is acquired using a 506.9 neutral loss scan and multiple reaction-monitoring (MRM).This method can identify all long, medium and short-chain acyl-CoA species in wild type mouse liver including predicted 3-hydroxyacyl-CoA species. We validated the method using liver of the short-chain-acyl-CoA dehydrogenase (SCAD) knock-out mice. As expected, there is a significant increase in [C-4] butyryl-CoA species in the SAD -/- mouse liver compared to wild type. We then tested the assay in liver from the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficient mice to determine the profile of acyl-CoA accumulation in this less predictable model. There was more modest accumulation of medium chain species including 3-hydroxyacyl-CoA's consistent with the known chain-length specificity of the SCHAD enzyme. (C) 2012 Elsevier Inc. All rights reserved.