![Figure 2. Glucose metabolism in SSRBCs is dysregulated with EMP bias and resilience to oxidative loading is restored by EMP blockade. HMP flux as a function of O2 content was determined using 1H-NMR spectroscopy to monitor positional 13C-enrichment in lactate isotopomers generated by RBCs incubated with [2-13C]-glucose (± MB to stimulate the HMP). (A) Representative spectrum from normal, unstimulated RBCs is shown (supplemental Figure 5 illustrates spectra for all conditions studied). Per sample, 256 acquisitions were recorded, resulting in a lactate S/N exceeding 1000/1. During acquisition, continuous-wave 13C-decoupling of lactate C1 was performed using Bayesian algorithms.50 Lactate methyl isotopomer signals were deconvoluted and fit to: a doublet D (3-bond 1H-1H coupling only) arising from [12C]-lactate, a doublet of doublets Q3 (3-bond 1H-1H coupling and one bond 1H-13C coupling) arising from HMP-generated [3-13C]-lactate, and a doublet of doublets Q2 (3-bond 1H-1H coupling and 2 bond 1H-13C coupling) arising from EMP-generated [2-13C]-lactate. This approach yielded signal amplitude estimates with a 12% SD for the lowest magnitude (Q3) to ≤ 2% for the highest magnitude (D) peaks. Proportional HMP flux was calculated from peak areas attributable to the pathway-specific lactate isotopomers (eg, Q2→EMP, Q3→HMP, see insets and Table 1). (B) Glucose uptake rate plotted against lactate generation for all conditions studied; note grouping among normal and SSRBC populations, which demonstrate differing lactate/glucose relationships. Both glucose uptake and lactate generation accelerated with MB stimulation. (C) In normal RBCs, HMP flux increased significantly during MB stimulation and deoxygenation blunted this increase. SSRBCs demonstrated similar O2 content–dependent variation in HMP flux, but the HMP increase with MB stimulation was significantly less robust than in normal RBCs. HMP/EMP bias was rebalanced in SSRBCs by incubation with the selective GAPDH inhibitor KA (please refer to Figure 7 for pathway schema); RBCs were then presented with an oxidative challenge. (D) This manipulation stabilized GSH-reducing power in SSRBCs during oxidant loading (mean ± SEM; n = 3-4). *P < .05, reported as Ehc, see supplemental Figure 3 for Ehc components. (E) KA treatment also restored membrane thiol protection in SSRBCs under oxidant loading (n = 3-8; mean ± SEM). *P < .05.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/9/10.1182_blood-2012-02-414037/4/zh89991302620002.jpeg?Expires=1735536673&Signature=1gp9CtsQGEN3AYfMgcGZNMUPmLxB8NQ1o2ASLXZJ96D813sLi-Ic0A-fSm2RQjlpLe3hpLwkscegeb3aHYFNfBMZGQWsZNTcAAs8-ak1L8S10uT7Bc5cYeI9UMPDnDQ9X8fP5QYkoKyS0xl9iPJbSEebbf8fEZF5byHInTIE0k6iR2IasH18XPgzYwq2t~nMYqfvJ4vFqpIf6w~UI5bAxzl7pMG8HipKh3KjxS126qq8ktRhCCRp5QJQPvm25FjsnnQbxmb0oJQZovkjs~vweaTVXMMTiMZNGTHqYpAPlu6sTulNlBM~4jHySEVHcnCED6xKdnt5YR7vICpDB~GOHQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Glucose metabolism in SSRBCs is dysregulated with EMP bias and resilience to oxidative loading is restored by EMP blockade. HMP flux as a function of O2 content was determined using 1H-NMR spectroscopy to monitor positional 13C-enrichment in lactate isotopomers generated by RBCs incubated with [2-13C]-glucose (± MB to stimulate the HMP). (A) Representative spectrum from normal, unstimulated RBCs is shown (supplemental Figure 5 illustrates spectra for all conditions studied). Per sample, 256 acquisitions were recorded, resulting in a lactate S/N exceeding 1000/1. During acquisition, continuous-wave 13C-decoupling of lactate C1 was performed using Bayesian algorithms.50 Lactate methyl isotopomer signals were deconvoluted and fit to: a doublet D (3-bond 1H-1H coupling only) arising from [12C]-lactate, a doublet of doublets Q3 (3-bond 1H-1H coupling and one bond 1H-13C coupling) arising from HMP-generated [3-13C]-lactate, and a doublet of doublets Q2 (3-bond 1H-1H coupling and 2 bond 1H-13C coupling) arising from EMP-generated [2-13C]-lactate. This approach yielded signal amplitude estimates with a 12% SD for the lowest magnitude (Q3) to ≤ 2% for the highest magnitude (D) peaks. Proportional HMP flux was calculated from peak areas attributable to the pathway-specific lactate isotopomers (eg, Q2→EMP, Q3→HMP, see insets and Table 1). (B) Glucose uptake rate plotted against lactate generation for all conditions studied; note grouping among normal and SSRBC populations, which demonstrate differing lactate/glucose relationships. Both glucose uptake and lactate generation accelerated with MB stimulation. (C) In normal RBCs, HMP flux increased significantly during MB stimulation and deoxygenation blunted this increase. SSRBCs demonstrated similar O2 content–dependent variation in HMP flux, but the HMP increase with MB stimulation was significantly less robust than in normal RBCs. HMP/EMP bias was rebalanced in SSRBCs by incubation with the selective GAPDH inhibitor KA (please refer to Figure 7 for pathway schema); RBCs were then presented with an oxidative challenge. (D) This manipulation stabilized GSH-reducing power in SSRBCs during oxidant loading (mean ± SEM; n = 3-4). *P < .05, reported as Ehc, see supplemental Figure 3 for Ehc components. (E) KA treatment also restored membrane thiol protection in SSRBCs under oxidant loading (n = 3-8; mean ± SEM). *P < .05.
