Metabolism
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How is NADH from glycolysis normally re-oxidised?
We have seen in the exercise on Breathless after sprinting that under anaerobic conditions the NADH that is formed in glycolysis is re-oxidised at the expense of pyruvate being reduced to lactate. However, we have also seen in the exercise on Life-threatening acidosis in an alcoholic - and in a hunger striker given intravenous glucose that under aerobic conditions pyruvate is oxidised to acetyl CoA, and then undergoes complete oxidation to carbon dioxide and water.
If you incubate isolated mitochondria in an oxygen electrode with NADH you do not observe any consumption of oxygen, regardless of how much ADP is added.
(See the exercise on Overheating after overdosing on E - and slimming by taking dinitrophenol for more information on the oxygen electrode).
What conclusion can you draw from this observation?
This suggests that NADH cannot cross the mitochondrial membrane.
If NADH does cannot cross the mitochondrial membrane, it cannot be re-oxidised via the electron transport chain. This means that we are left with the problem of how NADH produced in the cytosol can be re-oxidised.
In skeletal muscle lactate dehydrogenase acts to reduce pyruvate to lactate, in order to re-oxidise NADH. However, the form of lactate dehydrogenase in heart muscle acts preferentially in the opposite direction, oxidising lactate to pyruvate. Heart takes up lactate from the bloodstream and uses it as a metabolic fuel.
In the following experiments, isolated cardiomyocytes (heart muscle cells) were used to study the metabolism of lactate under various conditions.
Fluoropyruvate
is a potent inhibitor of pyruvate dehydrogenase. While it might also be expected
to inhibit lactate dehydrogenase acting in the direction of reduction of pyruvate
to lactate, it would not be expected to inhibit the oxidation of lactate to
pyruvate, especially in cardiomyocytes, whose lactate dehydrogenase has a very
much higher affinity for lactate than for pyruvate.
Cardiomyocytes were incubated with 100 mmol /L lactate and 1 mmol /L fluoropyruvate in an oxygen electrode. There was consumption of oxygen that was dependent on the addition of ADP, and a P:O ratio of ~2.5 was observed.
After 30 minutes the reaction mixture was pipetted out into a vial and frozen in liquid nitrogen. The cells were disrupted by repeated cycles of thawing and freezing, then centrifuged to remove mitochondria.
There was a significant accumulation of pyruvate in the cytosol.
What conclusions can you draw from these results?
These results suggest that lactate has been oxidised to pyruvate, with the reduction of NAD to NADH, and that the resultant NADH has been re-oxidised in the mitochondria. However, we know that NADH does not cross the mitochondrial membrane.
In further studies with cardiomyocytes incubated with lactate and fluoropyruvate, a variety of inhibitors of electron transport or oxidative phosphorylation were used. The following results were obtained:
final saturation with
oxygen |
pyruvate |
|
| control | 20% |
5 mmol / L |
| + potassium cyanide | 98% |
0 |
| + rotenone | 98% |
0 |
| + antimycin A | 98% |
0 |
| + oligomycin | 95% |
0 |