Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus C, Denmark; Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.
Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark.
Biochim Biophys Acta Gen Subj. 2023 Apr;1867(4):130315. doi: 10.1016/j.bbagen.2023.130315. Epub 2023 Feb 4.
Vitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5'-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the "inactive" vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of "harsh" extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms.
We designed a "mild" extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays.
A "mild" extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual "harsh" protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl.
Our procedure reveals a high content of MeCbl in rat liver.
This result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.
维生素 B12（钴胺素，Cbl）由几种分子变体组成，这些变体的区别在于与钴离子配位的可交换配体 X（XCbl）。最典型的 XCbl 形式是氰钴胺素（CNCbl）、羟钴胺素（HOCbl）、甲钴胺素（MeCbl）和 5'-脱氧脱氧腺苷钴胺素（AdoCbl）。细胞将“非活性”维生素 CNCbl 和 HOCbl 转化为两种至关重要的辅酶 AdoCbl 或 MeCbl。令人惊讶的是，与 AdoCbl 和 HOCbl 相比，组织样本中通常很少或没有发现 MeCbl。我们假设 MeCbl 水平低是“苛刻”提取的人为产物，导致 MeCbl 降解和/或转化为其他 XCbl 形式。
我们设计了一种“温和”的提取方案，包括在乙酸铵（pH 4.6）中匀浆大鼠肝脏，用乙醇（最终 60%）稀释，在 70°C 下加热 10 分钟。通过 HPLC 分离 XCbl 并通过同位素稀释测定法定量。
“温和”提取显示 Cbl 的组成如下：37%的 AdoCbl、35%的 MeCbl、15%的 HOCbl 和 13%的 CNCbl。通常的“苛刻”方案（pH 7，80°C 下 20 分钟）分别将这种平衡改变为 33%、5%、43%和 17%。模型测定表明，MeCbl 在 pH 3 和 pH>7 时与硫醇一起加热会发生脱甲基和转化为 HOCbl。其他变化包括 CNCbl 的脱氰和 HOCbl 的破坏。
我们的程序揭示了大鼠肝脏中 MeCbl 的高含量。
这一结果挑战了先前的数据，并指出需要新的研究来描述健康和疾病中内源性 Cbl 形式。
