Metabolic flux data from 1H-NMR spectroscopy after RBC incubation with [2-13C] D-glucose
| Positional 13C enrichment (lactate) . | Normal . | SS . | ||||||
|---|---|---|---|---|---|---|---|---|
| Oxygenated . | Deoxygenated . | Oxygenated . | Deoxygenated . | |||||
| − MB . | + MB . | − MB . | + MB . | − MB . | + MB . | − MB . | + MB . | |
| Raw data | ||||||||
| Carbon 2 | 0.29 ± 0.01 | 0.16 ± 0.02 | 0.31 ± 0.01 | 0.24 ± 0.02 | 0.31 ± 0.00 | 0.25 ± 0.01§ | 0.31 ± 0.01 | 0.27 ± 0.01 |
| Carbon 3 | 0.03 ± 0.00 | 0.10 ± 0.00 | 0.03 ± 0.00 | 0.06 ± 0.01 | 0.04 ± 0.01 | 0.08 ± 0.00§ | 0.01 ± 0.00§ | 0.05 ± 0.01 |
| Corrected* | ||||||||
| Carbon 2 | 0.29 ± 0.01 | 0.16 ± 0.02 | 0.31 ± 0.01 | 0.24 ± 0.02 | 0.31 ± 0.00 | 0.25 ± 0.01§ | 0.31 ± 0.01 | 0.27 ± 0.01 |
| Carbon 3 | 0.03 ± 0.00 | 0.09 ± 0.00 | 0.02 ± 0.00 | 0.06 ± 0.01 | 0.03 ± 0.01 | 0.07 ± 0.01§ | 0.00 ± 0.00§ | 0.05 ± 0.01 |
| Flux estimates | ||||||||
| Proportional [2-13C] D-glucose flux through HMP† | 0.05 ± 0.00 | 0.76 ± 0.16 | 0.04 ± 0.01 | 0.18 ± 0.06 | 0.06 ± 0.01 | 0.19 ± 0.01§ | 0.00 ± 0.00§ | 0.11 ± 0.02 |
| Maximum NADPH generation per glucose, mol/mol‡ | 0.31 ± 0.03 | 4.54 ± 0.95 | 0.23 ± 0.06 | 1.10 ± 0.38 | 0.36 ± 0.08 | 1.13 ± 0.04§ | 0.03 ± 0.02§ | 0.65 ± 0.10 |
| Positional 13C enrichment (lactate) . | Normal . | SS . | ||||||
|---|---|---|---|---|---|---|---|---|
| Oxygenated . | Deoxygenated . | Oxygenated . | Deoxygenated . | |||||
| − MB . | + MB . | − MB . | + MB . | − MB . | + MB . | − MB . | + MB . | |
| Raw data | ||||||||
| Carbon 2 | 0.29 ± 0.01 | 0.16 ± 0.02 | 0.31 ± 0.01 | 0.24 ± 0.02 | 0.31 ± 0.00 | 0.25 ± 0.01§ | 0.31 ± 0.01 | 0.27 ± 0.01 |
| Carbon 3 | 0.03 ± 0.00 | 0.10 ± 0.00 | 0.03 ± 0.00 | 0.06 ± 0.01 | 0.04 ± 0.01 | 0.08 ± 0.00§ | 0.01 ± 0.00§ | 0.05 ± 0.01 |
| Corrected* | ||||||||
| Carbon 2 | 0.29 ± 0.01 | 0.16 ± 0.02 | 0.31 ± 0.01 | 0.24 ± 0.02 | 0.31 ± 0.00 | 0.25 ± 0.01§ | 0.31 ± 0.01 | 0.27 ± 0.01 |
| Carbon 3 | 0.03 ± 0.00 | 0.09 ± 0.00 | 0.02 ± 0.00 | 0.06 ± 0.01 | 0.03 ± 0.01 | 0.07 ± 0.01§ | 0.00 ± 0.00§ | 0.05 ± 0.01 |
| Flux estimates | ||||||||
| Proportional [2-13C] D-glucose flux through HMP† | 0.05 ± 0.00 | 0.76 ± 0.16 | 0.04 ± 0.01 | 0.18 ± 0.06 | 0.06 ± 0.01 | 0.19 ± 0.01§ | 0.00 ± 0.00§ | 0.11 ± 0.02 |
| Maximum NADPH generation per glucose, mol/mol‡ | 0.31 ± 0.03 | 4.54 ± 0.95 | 0.23 ± 0.06 | 1.10 ± 0.38 | 0.36 ± 0.08 | 1.13 ± 0.04§ | 0.03 ± 0.02§ | 0.65 ± 0.10 |
Values are reported as mean ± SEM; for all groups, n = 3-5.
MB indicates methylene blue; HMP, hexose monophosphate shunt; G6P, glucose 6-phosphate; and NADPH, nicotinamide adenine dinucleotide phosphate.
With a natural abundance of [2-13C] lactate of 1.1% and an assumed random distribution of natural 13C among lactate carbons, the relative intensities of the signals are: [12C] lactate (D-peak): 97.8%; [2-13C] lactate (Q2): 1.1%; and [3-13C] lactate (Q3): 1.1%. Values for carbon 2 and 3 enrichment in the lactate methyl signal, corrected for natural 13C abundance, were therefore calculated as follows25 : Q2corr = Q2 − ((D × 0.011)/0.978)).
The equation for estimating the proportion of [2-13C] D-glucose metabolized by the HMP relative to total (eg, HMP + EMP) was as follows25 : Q3corr/Q2corr = 2(HMP fraction)/(1 + 2[HMP fraction]).
Moles of NADPH that may be generated per mole [2-13C] D-glucose metabolized were determined according to known stoichiometry for NADPH generation from glucose: due to pentose- and hexose-phosphate recycling within the HMP, each glucose molecule may be functionally recycled 3 times through the oxidative HMP branch. Because each molecule of G6P oxidized to ribose-P generates 2 NADPH, the maximum NADPH generation (and glutathione recycling) may reach 6 times the HMP flux estimate.32
Significant difference between normal and sickle cell (P < .05).