Detection of glucose-derived d - and l -lactate in cancer cells by the use of a chiral NMR shift reagent

Background Excessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to l -lactate. Although d -lactate can also be produced from glucose via the methylglyoxal pathway in small amounts, less is known about the amount of d -lactate produced in cancer cells. Since the stereoisomers of lactate cannot be distinguished by conventional 1 H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1 H NMR resonances of d - and l -lactate. Methods The production of l -lactate from glucose and d -lactate from methylglyoxal was first demonstrated in freshly isolated red blood cells using the chiral NMR shift reagent, YbDO3A-trisamide. Then, two different cell lines with high GLO1 expression (H1648 and H 1395) were selected from a panel of over 80 well-characterized human NSCLC cell lines, grown to confluence in standard tissue culture media, washed with phosphate-buffered saline, and exposed to glucose in a buffer for 4 h. After 4 h, a small volume of extracellular fluid was collected and mixed with YbDO3A-trisamide for analysis by 1 H NMR spectroscopy. Results A suspension of freshly isolated red blood cells exposed to 5mM glucose produced l -lactate as expected but very little d -lactate. To evaluate the utility of the chiral NMR shift reagent, methylglyoxal was then added to red cells along with glucose to stimulate the production of d -lactate via the glyoxalate pathway. In this case, both d -lactate and l -lactate were produced and their NMR chemical shifts assigned. NSCLC cell lines with different expression levels of GLO1 produced both l - and d -lactate after incubation with glucose and glutamine alone. A GLO1 -deleted parental cell line (3553T3) showed no production of d -lactate from glucose while re-expression of GLO1 resulted in higher production of d -lactate. Conclusions The shift-reagent-aided NMR technique demonstrates that d -lactate is produced from glucose in NSCLC cells via the methylglyoxal pathway. The biological role of d -lactate is uncertain but a convenient method for monitoring d -lactate production could provide new insights into the biological roles of d - versus l -lactate in cancer metabolism.