Jones Patricia M, Bennett Michael J
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
Biochim Biophys Acta. 2011 Nov;1811(11):657-62. doi: 10.1016/j.bbalip.2011.06.026. Epub 2011 Jun 30.
L-3-Hydroxy fatty acids are unusual metabolites and rarely occur in significant quantities in normal human physiology. Genetic defects of both long-chain and medium-/short-chain mitochondrial L-3 hydroxyacyl coenzyme A dehydrogenases (LCHAD, M/SCHAD) have been identified as significant metabolic diseases in humans often with severe clinical phenotypes and pathophysiology that appears to differ from other defects of straight chain fatty acid oxidation. It is felt that accumulation of these atypical fatty acid species may play a role in this pathology. We have therefore developed an assay to measure these compounds in body fluids, and tissue culture medium to help in the diagnosis of these disorders and to better study the effects of 3-hydroxy fatty acid accumulation.
We have developed a stable isotope dilution, selected ion-monitoring gas chromatography-mass spectrometric assay for the measurement of all 3-hydroxy fatty acids from chain lengths C6 to C18 using 1,2 (13)C-labeled internal standards for all species. Authentic patient samples were utilized to develop reference intervals for control subjects, for those associated with patient samples confirmed at the molecular level to have either LCHAD or M/SCHAD deficiency and for patients who did not have disease but were fasting or on diets high in medium-chain fatty acids. Likewise, skin fibroblasts were obtained from patients with confirmed disease for additional study. Samples were also obtained from the hadh (M/SCHAD) knockout mouse.
The measurement of 3-hydroxy fatty acids in patient plasma is a valuable tool in the identification of defects of both enzymes. Severe starvation, prolonged fasting and increased medium-chain triglycerides in the diet produce a profile that is similar to that seen in M/SCHAD deficiency, making this a more difficult condition to diagnose but these biomarkers provide an important clue to the diagnosis, particularly in non-fasted, diet-controlled patients. Fibroblast studies in LCHAD deficiency demonstrate that long-chain 3-hydroxy fatty acid accumulation can be observed in cultured tissues. Incubation of cultured fibroblasts from LCHAD deficient patients with labeled fatty acids demonstrated a process of chain lengthening that has not previously been recognized.
The measurement of body fluid and cultured cell 3-hydroxy fatty acids provides both diagnostic and pathogenic information regarding these genetic diseases of fatty acid oxidation in the mitochondrion. Presently, the measurement of medium- and short-chain species provides a major metabolic biomarker for the recognition of M/SCHAD deficiency.
L-3-羟基脂肪酸是一类特殊的代谢产物，在正常人体生理过程中很少大量出现。长链和中/短链线粒体L-3-羟基酰基辅酶A脱氢酶（LCHAD、M/SCHAD）的基因缺陷已被确定为人类的重要代谢疾病，通常具有严重的临床表型和病理生理学特征，似乎与直链脂肪酸氧化的其他缺陷不同。人们认为这些非典型脂肪酸种类的积累可能在这种病理过程中起作用。因此，我们开发了一种检测方法，用于测量体液和组织培养基中的这些化合物，以帮助诊断这些疾病，并更好地研究3-羟基脂肪酸积累的影响。
我们开发了一种稳定同位素稀释、选择离子监测气相色谱-质谱分析法，使用所有物种的1,2（13）C标记内标来测量碳链长度从C6到C18的所有3-羟基脂肪酸。利用真实患者样本为对照受试者、在分子水平上确诊为LCHAD或M/SCHAD缺乏的患者样本以及未患疾病但处于禁食状态或食用富含中链脂肪酸饮食的患者建立参考区间。同样，从确诊疾病的患者身上获取皮肤成纤维细胞进行进一步研究。样本也取自hadh（M/SCHAD）基因敲除小鼠。
测量患者血浆中的3-羟基脂肪酸是识别这两种酶缺陷的有价值工具。严重饥饿、长期禁食和饮食中中链甘油三酯增加会产生与M/SCHAD缺乏症相似的特征，这使得这种情况更难诊断，但这些生物标志物为诊断提供了重要线索，特别是在非禁食、饮食控制的患者中。对LCHAD缺乏症的成纤维细胞研究表明，在培养的组织中可以观察到长链3-羟基脂肪酸的积累。用标记脂肪酸培养LCHAD缺乏症患者的成纤维细胞，显示出一个以前未被认识的链延长过程。
测量体液和培养细胞中的3-羟基脂肪酸可提供有关线粒体脂肪酸氧化这些遗传疾病的诊断和致病信息。目前，中链和短链物种的测量为识别M/SCHAD缺乏症提供了主要的代谢生物标志物。
