Center for Environmental and Systems Biochemistry, University of Kentucky, Lexington, Kentucky 40536, United States.
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, United States.
Anal Chem. 2022 Sep 13;94(36):12286-12291. doi: 10.1021/acs.analchem.2c00490. Epub 2022 Aug 30.
Biologically important 2-hydroxy carboxylates such as lactate, malate, and 2-hydroxyglutarate exist in two enantiomeric forms that cannot be distinguished under achiral conditions. The D and L (or R, S) enantiomers have different biological origins and functions, and therefore, there is a need for a simple method for resolving, identifying, and quantifying these enantiomers. We have adapted and improved a chiral derivatization technique for nuclear magnetic resonance (NMR), which needs no chromatography for enantiomer resolution, with greater than 90% overall recovery. This method was developed for 2-hydroxyglutarate (2HG) to produce diastereomers resolvable by column chromatography. We have applied the method to lactate, malate, and 2HG. The limit of quantification was determined to be about 1 nmol for 2HG with coefficients of variation of less than 5%. We also demonstrated the method on an extract of a renal carcinoma bearing an isocitrate dehydrogenase-2 (IDH2) variant that produces copious quantities of 2HG and showed that it is the D enantiomer that was exclusively produced. We also demonstrated in the same experiment that the lactate produced in the same sample was the L enantiomer.
生物重要的 2-羟基羧酸，如乳酸、苹果酸和 2-羟基戊二酸，存在两种无法在非手性条件下区分的对映异构体形式。D 和 L（或 R、S）对映异构体具有不同的生物学起源和功能，因此需要一种简单的方法来拆分、鉴定和定量这些对映异构体。我们已经对核磁共振（NMR）的手性衍生技术进行了改编和改进，该技术不需要色谱法进行对映异构体拆分，总回收率超过 90%。该方法最初是为 2-羟基戊二酸（2HG）开发的，可产生通过柱色谱法可拆分的非对映异构体。我们已经将该方法应用于乳酸、苹果酸和 2HG。2HG 的定量下限约为 1 nmol，变异系数小于 5%。我们还在携带异柠檬酸脱氢酶-2（IDH2）变体的肾癌细胞提取物上进行了该方法的演示，该变体产生大量的 2HG，并且表明仅产生 D 对映异构体。我们还在同一项实验中证明，在同一样本中产生的乳酸是 L 对映异构体。
