Mihalik S J, Rainville A M, Watkins P A
Kennedy Krieger Research Institute, John Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Eur J Biochem. 1995 Sep 1;232(2):545-51. doi: 10.1111/j.1432-1033.1995.545zz.x.
Patients with generalized peroxisomal disorders, rhizomelic chondrodysplasia punctata, and Refsum disease are all unable to alpha-oxidize 3,7,11,15-tetramethylhexadecanoic (phytanic) acid. The exact cause of the oxidation defect in these patients is not well characterized, in part because there is only limited knowledge of the biochemical pathway. In 1969, the alpha-oxidation of phytanic acid was reported [Tsai, S.-C., Avigan, J. & Steinberg, D. (1969) Studies on the alpha-oxidation of phytanic acid by rat liver mitochondria, J. Biol. Chem. 244, 2682-2692] to involve the formation of an alpha-hydroxyphytanic acid intermediate prior to removal of the alpha carbon. Subsequently, most researchers have had difficulty detecting this intermediate. In the present study, cofactors known to form hydroxy intermediates by both monooxygenase and dioxygenase reaction mechanisms were incubated with purified rat liver peroxisomes and either [2,3-3H]phytanic acid or [1-14C]phytanic acid. Reaction products were separated by reverse-phase HPLC. A single reaction product, identified as alpha-hydroxyphytanoyl-CoA rather than the free fatty acid, was detected when 2-oxoglutarate/Fe+2/ascorbate, cofactors associated with a dioxygenase reaction mechanism, were present. Concomitant with alpha-hydroxyphytanoyl-CoA production, there was an increased accumulation of formate and CO2. This increase in alpha-oxidation products is evidence that alpha-hydroxyphytanoyl-CoA is a true pathway intermediate and that the entire pathway functions in peroxisomes. In contrast, alpha-hydroxyphytanoyl-CoA was not formed in any quantity in mitochondria. These studies suggest that the alpha-hydroxylation step of phytanic acid oxidation, which has been shown to be defective in Refsum disease, is located in peroxisomes.
患有全身性过氧化物酶体疾病、肢根型点状软骨发育不良和雷夫叙姆病的患者均无法对3,7,11,15-四甲基十六烷酸（植烷酸）进行α-氧化。这些患者氧化缺陷的确切原因尚未完全明确，部分原因是对该生化途径的了解有限。1969年，有报道称植烷酸的α-氧化过程[蔡思聪、阿维根、斯坦伯格（1969年）大鼠肝脏线粒体对植烷酸α-氧化的研究，《生物化学杂志》244卷，2682 - 2692页]涉及在去除α碳之前形成α-羟基植烷酸中间体。然而，随后大多数研究人员难以检测到这种中间体。在本研究中，将已知通过单加氧酶和双加氧酶反应机制形成羟基中间体的辅因子与纯化的大鼠肝脏过氧化物酶体以及[2,3 - 3H]植烷酸或[1 - 14C]植烷酸一起孵育。反应产物通过反相高效液相色谱法分离。当存在与双加氧酶反应机制相关的辅因子2-氧代戊二酸/Fe +2/抗坏血酸时，检测到一种单一反应产物，鉴定为α-羟基植烷酰辅酶A而非游离脂肪酸。伴随着α-羟基植烷酰辅酶A的产生，甲酸盐和二氧化碳的积累增加。α-氧化产物的这种增加证明α-羟基植烷酰辅酶A是真正的途径中间体，并且整个途径在过氧化物酶体中起作用。相比之下，线粒体中未形成任何量的α-羟基植烷酰辅酶A。这些研究表明，植烷酸氧化的α-羟基化步骤（已证明在雷夫叙姆病中存在缺陷）位于过氧化物酶体中。
