Methylene blue increased ATP from substrate-level phosphorylation in isolated brain mitochondria
Mark Kemp · 9 October 2026
In isolated brain mitochondria, methylene blue increased ATP made by substrate-level phosphorylation through succinyl-CoA ligase, even with complex I and ATP synthase blocked. A plain summary of Komlódi & Tretter, Neuropharmacology 2017.
In isolated guinea-pig cortical mitochondria, methylthioninium chloride (methylene blue) stimulated respiration on every substrate tested when ATP synthase was blocked, but increased ATP synthesis only on α-ketoglutarate and glutamate — the substrates that feed succinyl-CoA ligase (Komlódi & Tretter, Neuropharmacology 2017). That identifies substrate-level phosphorylation in the citric acid cycle, not oxidative phosphorylation, as the source of the extra ATP. The effect persisted with complex I and ATP synthase inhibited together, and methylene blue restored membrane potential under those conditions.
Citation: Komlódi T, Tretter L. Neuropharmacology 2017;123:287–298. PMID 28495375.
What the study did
The authors isolated mitochondria from guinea-pig cerebral cortex and fed them one substrate at a time — α-ketoglutarate, glutamate, malate or succinate — while measuring ATP production, oxygen consumption, membrane potential, NAD(P)H and the redox state of methylene blue itself.
The question was specific. Mitochondria make most ATP by oxidative phosphorylation, through ATP synthase. But one step of the citric acid cycle — succinyl-CoA ligase — makes ATP (or GTP) directly, without ATP synthase. That is substrate-level phosphorylation. The authors wanted to know whether methylene blue could drive it.
To isolate it, they blocked ATP synthase with oligomycin and blocked adenylate kinase, so any ATP that appeared had to come from substrate-level phosphorylation.
What it found
With ATP synthase blocked, methylene blue stimulated oxygen consumption on all four substrates. But it raised ATP output only on α-ketoglutarate and glutamate — the two substrates that generate succinyl-CoA, the input to succinyl-CoA ligase. On malate and succinate, which do not, ATP did not rise.
When complex I was inhibited as well as ATP synthase, methylene blue still stimulated substrate-level phosphorylation and restored mitochondrial membrane potential.
The authors proposed the same mechanism Wen and colleagues had described in cells: methylene blue takes electrons from reducing equivalents and hands them to cytochrome c, bypassing complexes I and III. Keeping the electron chain moving keeps the citric acid cycle turning, and succinyl-CoA ligase keeps making ATP.
| Model | Isolated mitochondria from guinea-pig cerebral cortex |
| Compound | Methylthioninium chloride |
| Conditions | ATP synthase blocked (oligomycin); adenylate kinase blocked; complex I blocked in some runs |
| Measured | ATP synthesis; oxygen consumption; membrane potential; NAD(P)H; methylene blue redox state |
| Result | Respiration up on all substrates; ATP up only on α-ketoglutarate and glutamate; membrane potential restored under complex I + ATP synthase inhibition |
| Not tested | Whole cells; animals; humans; oral administration; any concentration beyond those assayed |
What it means, and what it doesn't
This is a biochemistry paper on isolated organelles. It shows that methylene blue can support a second, ATP-synthase-independent route to ATP when the main route is blocked, and that the effect runs through one specific enzyme. The substrate specificity is what makes the finding clean: if it were a general stimulation, all four substrates would have responded.
It does not show anything about intact cells, animals or people. Isolated mitochondria in a chamber with pharmacological blockers are not a model of anything a person experiences. The pathway was isolated using enzyme inhibitors; whether it contributes meaningfully in intact tissue was not tested. The paper is cited here because it describes how the molecule interacts with the electron transport chain and the citric acid cycle, not because it demonstrates an effect of taking it.
Read the paper
Komlódi T, Tretter L. Methylene blue stimulates substrate-level phosphorylation catalysed by succinyl-CoA ligase in the citric acid cycle. Neuropharmacology 2017;123:287–298.
This summarises a laboratory study on isolated mitochondria. It is not evidence of an effect in humans and is not a claim about any product.