Rotenone Stereospecifically Increases (S)-2-Hydroxyglutarate in SH-SY5Y Neuronal Cells

Abstract

The α-ketoglutarate metabolite, 2-hydroxyglutarate (2-HG), has emerged as an important mediator in a subset of cancers and rare inherited inborn errors of metabolism. Because of potential enantiospecific metabolism, chiral analysis is essential for determining the biochemical impacts of altered 2-HG metabolism. We have developed a novel application of chiral liquid chromatography-electron capture/atmospheric pressure chemical ionization/mass spectrometry, which allows for the quantification of both (R)-2-HG (D-2-HG) and (S)-2-HG (L-2-HG) in human cell lines. This method avoids the need for chiral derivatization, which could potentially distort enantiomer ratios through racemization during the derivatization process. The study revealed that the pesticide rotenone (100 nM), a mitochondrial complex I inhibitor, caused a significant almost 3-fold increase in the levels of (S)-2-HG, (91.7 ± 7.5 ng/10(6) cells) when compared with the levels of (R)-2-HG (24.1 ± 1.2 ng/10(6) cells) in the SH-SY5Y neuronal cells, a widely used model of human neurons. Stable isotope tracers and isotopologue analysis revealed that the increased (S)-2-HG was derived primarily from l-glutamine. Accumulation of highly toxic (S)-2-HG occurs in the brains of subjects with reduced L-2-HG dehydrogenase activity that results from mutations in the L2HGDH gene. This suggests that the observed stereospecific increase of (S)-2-HG in neuronal cells is due to rotenone-mediated inhibition of L-2-HG dehydrogenase but not D-2-HG dehydrogenase. The high sensitivity chiral analytical methodology that has been developed in the present study can also be employed for analyzing other disruptions to 2-HG formation and metabolism such as those resulting from mutations in the isocitrate dehydrogenase gene.

Figure 1

Schematic of the TCA cycle. Abbreviations: α-KGD, α-ketoglutarate dehydrogenase; AT, aspartate transaminase; CS, citrate synthase; L2HGDH, L-2-hydroxyglutarate dehydrogenase; MD, malate dehydrogenase; MutIDH, mutated isocitrate dehydrogenase; SCS, succinyl-CoA synthase; SD, succinate dehydrogenase.

Figure 2

Formation of 2-HG-bis-PFB followed by LC-ECAPCI/MS/MS.

Figure 3

Product ions derived from LC-ECAPCI/MS/MS analysis of [M-PFB]⁻ at m/z 327 derived from 2-HG-bis-PFB.

Figure 4

Chromatographic separation of (R)-2-HG from (S)-2-HG using normal-phase chiral LC-ECAPCI/MS.

Figure 5

Calibration curves for (R)-2-HG and (S)-2-HG. (R)-2-HG: y = 0.0021x + 0.0056, r² = 0.995. (S)-2-HG: y = 0.0022x + 0.0046, r² = 0.988.

Figure 6

Separation of (R)-2-HG and (S)-2-HG from SH-SY5Y neuronal cells using normal-phase chiral LC-ECAPCI/MS. The cells were treated with (A) DMSO and (B) 100 nM rotenone.

Figure 7

Rotenone induces stereospecific increases in (S)-2-HG. Student’s two-tailed t test against vehicle control: *p < 0.05, **p < 0.001 (n = 4). Rotenone induced a stereoselective increase in (S)-2-HG when compared with (R)-2-HG. SH-SY5Y cells were treated with 100 nM rotenone or vehicle (DMSO) for 6 h. Student’s two-tailed t test for (S)-2-HG against (R)-2-HG after rotenone treatment: **p = 0.0016 (n = 4).

Figure 8

Glutamine contributes to increased (S)-2-HG production in response to rotenone. SH-SY5Y cells were grown in the presence of 2 mM L-[¹³C₅¹⁵N₂]-glutamine and either 100 nM rotenone or vehicle DMSO for 6 h followed by isotopic enrichment analysis by LC-ECAPCI-MS/MS. Student’s two-tailed t test against vehicle control: ***p < 0.001 (n = 4).