Detection of glucose-derived D- and L-lactate in cancer cells by the use of a chiral NMR shift reagent.

Abstract

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 1H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1H 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 1H 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.

• Authors: Eul Hyun Suh, Carlos F G C Geraldes, Sara Chirayil, Brandon Faubert, Raul Ayala, Ralph J DeBerardinis, A Dean Sherry
• Journal: Cancer & metabolism (Cancer Metab) Vol. 9 Issue 1 Pg. 38 (Nov 06 2021) ISSN: 2049-3002 [Print] England
• PMID: 34742347 (Publication Type: Journal Article)
• Copyright: © 2021. The Author(s).