Lucock M D, Daskalakis I, Schorah C J, Levene M I, Hartley R
Academic Unit of Paediatrics and Child Health, Research School of Medicine, University of Leeds, United Kingdom.
Biochem Mol Med. 1996 Jun;58(1):93-112. doi: 10.1006/bmme.1996.0037.
Although the analysis of low plasma concentrations of 5-methyltetrahydrofolate by several specific HPLC methods has been reported, considerably fewer routine chromatographic techniques exist for the analysis of specific folate coenzymes in the erythrocyte where a nonspecific bioassay indicates that the vitamin achieves a level 10 times higher than that in plasma. By using three separate folypolyglutamate deconjugation procedures and combining an extraction technique which adequately preserves all native folate coenzymes with an HPLC technique utilizing fluorescence, diode array, and off-line radioassay detection capable of resolving all crucial native folates in their monoglutamyl forms, we were unable to demonstrate levels of 5-methyltetrahydrofolate in whole blood hemolysate beyond what might be expected from the plasma component. While the exact nature of erythrocyte folate could not be ascertained, we provide evidence that a proportion of it may exist at the formyl level of oxidation. The complex pH and enzymatic interrelationship between folate coenzymes at the formyl oxidation level is discussed in terms of our extraction technique and findings, as well as in a broader biological context. This paper also describes a simple acid precipitation technique for measuring plasma 5-methyltetrahydrofolate, as well as providing comprehensive data on the chromatographic behavior of all the folylmonoglutamates in reversed-phase and weak anion-exchange modes, including useful spectral data for optimizing detection parameters and identifying individual coenzymes. 10-Formyltetrahydrofolate and 5-methyltetrahydrofolate are the two most important one-carbon-substituted folate coenzymes. 10-Formyltetrahydrofolate is unavailable commercially, probably due to its instability. We chart the chemical synthesis of this important coenzyme and show that it and what is thought to be 5,10-hydroxymethylenetetrahydrofolate are actually minor products compared to the parent 5,10-methenyltetrahydrofolate and the ultimate reaction product, 5-formyltetrahydrofolate. Since intraerythrocyte folate binds to a specific hemoglobin site, we ascertained the total number of binding sites on hemoglobin (Bmax) and the equilibrium dissociation constant (Kd) for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and the antimetabolite methotrexate. Binding affinities were consistent with a low-affinity, low-capacity interaction for all three. It was demonstrated that hemoglobin has a greater affinity for 5-methyltetrahydrofolate than for the other folate derivatives (Kd = 1.2 x 10(-3) M), while rather surprisingly, methotrexate had a higher affinity for hemoglobin than did 5-formyltetrahydrofolate (Kd = 2.5 x 10(-3) and 3.7 x 10(-2) M, respectively.
尽管已有报道通过几种特定的高效液相色谱法分析低血浆浓度的5-甲基四氢叶酸，但用于分析红细胞中特定叶酸辅酶的常规色谱技术要少得多。在红细胞中，一种非特异性生物测定表明该维生素的水平比血浆中高10倍。通过使用三种不同的叶酸多聚谷氨酸去共轭程序，并将一种能充分保留所有天然叶酸辅酶的提取技术与一种利用荧光、二极管阵列和离线放射分析检测的高效液相色谱技术相结合，该检测技术能够分辨出所有关键的单谷氨酰基形式的天然叶酸，我们未能证明全血溶血产物中5-甲基四氢叶酸的水平超过血浆成分所预期的水平。虽然无法确定红细胞叶酸的确切性质，但我们提供的证据表明，其中一部分可能以甲酰基氧化水平的形式存在。根据我们的提取技术和研究结果，以及更广泛的生物学背景，讨论了甲酰基氧化水平下叶酸辅酶之间复杂的pH值和酶促相互关系。本文还描述了一种用于测量血浆5-甲基四氢叶酸的简单酸沉淀技术，并提供了所有单谷氨酰基叶酸在反相和弱阴离子交换模式下色谱行为的综合数据，包括用于优化检测参数和识别单个辅酶的有用光谱数据。10-甲酰基四氢叶酸和5-甲基四氢叶酸是两种最重要的一碳取代叶酸辅酶。10-甲酰基四氢叶酸在商业上无法获得，可能是由于其不稳定性。我们绘制了这种重要辅酶的化学合成过程，并表明与母体5,10-亚甲基四氢叶酸和最终反应产物5-甲酰基四氢叶酸相比，它以及被认为是5,10-亚甲基四氢叶酸实际上都是次要产物。由于红细胞内叶酸与特定的血红蛋白位点结合，我们确定了血红蛋白上5-甲基四氢叶酸、5-甲酰基四氢叶酸和抗代谢物甲氨蝶呤的结合位点总数（Bmax）和平衡解离常数（Kd）。所有三种物质的结合亲和力都符合低亲和力、低容量相互作用。结果表明，血红蛋白对5-甲基四氢叶酸的亲和力比对其他叶酸衍生物更高（Kd = 1.2×10⁻³ M），而令人惊讶的是，甲氨蝶呤对血红蛋白的亲和力比对5-甲酰基四氢叶酸更高（Kd分别为2.5×10⁻³和3.7×10⁻² M）。
