Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Street, Sherbrooke, Quebec J1M 0C8, Canada.
BMC Biochem. 2013 Oct 16;14:25. doi: 10.1186/1471-2091-14-25.
Methylmalonyl-CoA mutase (MCM) is an adenosylcobalamin-dependent enzyme that catalyses the interconversion of (2R)-methylmalonyl-CoA to succinyl-CoA. In humans, a deficit in activity of MCM, due to an impairment of intracellular formation of adenosylcobalamin and methylcobalamin results in a wide spectrum of clinical manifestations ranging from moderate to fatal. Consequently, MCM is the subject of abundant literature. However, there is a lack of consensus on the reliable method to monitor its activity. This metabolic pathway is highly solicited in ruminants because it is essential for the utilization of propionate formed during ruminal fermentation. In lactating dairy cows, propionate is the major substrate for glucose formation. In present study, a reversed-phase high performance liquid chromatography (RP-HPLC) was optimized and validated to evaluate MCM activity in bovine liver. The major aim of the study was to describe the conditions to optimize reproducibility of the method and to determine stability of the enzyme and its product during storage and processing of samples.
Specificity of the method was good, as there was no interfering peak from liver extract at the retention times corresponding to methylmalonyl-CoA or succinyl-CoA. Repeatability of the method was improved as compared to previous RP-HPLC published data. Using 66 μg of protein, intra-assay coefficient of variation (CV) of specific activities, ranged from 0.90 to 8.05% and the CV inter-day was 7.40%. Storage and processing conditions (frozen homogenate of fresh tissue vs. fresh homogenate of tissue snapped in liquid nitrogen) did not alter the enzyme activity. The analyte was also stable in liver crude extract for three frozen/thawed cycles when stored at -20°C and thawed to room temperature.
The improved method provides a way for studying the effects of stages of lactation, diet composition, and physiology in cattle on MCM activity over long periods of time, such as a complete lactation period. Interestingly, this sensitive and accurate method could benefit the study of the cobalamin status in experimental studies and clinical cases.
甲基丙二酰辅酶 A 变位酶（MCM）是一种依赖于腺苷钴胺素的酶，可催化（2R）-甲基丙二酰辅酶 A 与琥珀酰辅酶 A 之间的转化。在人类中，由于细胞内腺苷钴胺素和甲基钴胺素形成受损导致 MCM 活性不足，会导致从中度到致命的广泛临床表现。因此，MCM 是大量文献的主题。然而，对于监测其活性的可靠方法尚未达成共识。这个代谢途径在反刍动物中需求量很大，因为它对于利用瘤胃发酵过程中形成的丙酸至关重要。在泌乳奶牛中，丙酸是形成葡萄糖的主要底物。在本研究中，优化并验证了反相高效液相色谱（RP-HPLC）以评估牛肝中的 MCM 活性。该研究的主要目的是描述优化方法重现性的条件，并确定在样品储存和处理过程中酶及其产物的稳定性。
该方法的特异性良好，因为在与甲基丙二酰辅酶 A 或琥珀酰辅酶 A 对应的保留时间处，肝提取物中没有干扰峰。与以前发表的 RP-HPLC 数据相比，该方法的重复性得到了提高。使用 66 μg 蛋白质，特定活性的日内变异系数（CV）范围为 0.90%至 8.05%，日间 CV 为 7.40%。储存和处理条件（新鲜组织的冷冻匀浆与液氮中组织的新鲜匀浆）不会改变酶活性。当在-20°C 下储存并解冻至室温时，分析物在肝脏粗提物中也稳定，可进行三个冷冻/解冻循环。
改进后的方法为研究奶牛泌乳阶段、饮食组成和生理状态对 MCM 活性的影响提供了一种方法，可以进行整个泌乳期等长时间的研究。有趣的是，这种敏感而准确的方法可能有助于钴胺素状态的研究在实验研究和临床病例中。
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