On 2016 Sep 30, Paul Brookes commented:

I submitted a response to this opinion piece to the journal (Circ. Res.), but unfortunately was informed that they do not accept or publish correspondence related to this type of article. So, here's my un-published letter, which raises a number of issues with the article...

A recent Circ. Res. viewpointLoscalzo J, 2016 discussed the complex relationships between redox biology and metabolism in the setting of hypoxia, with an emphasis on the use of biochemically correct terminology. While there is broad agreement that the field of redox biology is often confounded by use of inappropriate methods and language Kalyanaraman B, 2012,Forman HJ, 2015, concern is raised regarding some ideas on reductive stress in the latter part of the article.

In discussing the fate of glycolytically-derived NADH in hypoxia, the reader is urged to “Remember that while redirecting glucose metabolism to glycolysis decreases NADH production by the TCA cycle and decreases leaky electron transport chain flux, glycolysis continues to produce NADH". First, glucose undergoes glycolysis regardless of cellular oxygenation status; this simply happens at a faster rate in hypoxia. As such, glucose is not redirected but rather its product pyruvate is. Second, regardless a proposed lower rate of NADH generation by the TCA cycle (which may not actually be the case Chouchani ET, 2014,Hochachka PW, 1975) NADH still accumulates in hypoxic mitochondria because its major consumer, the O2-dependent respiratory chain, is inhibited. It is clear that both NADH consumers and producers can determine the NADH/NAD+ ratio, and in hypoxia the consumption side of the equation cannot be forgotten.

While the field is in broad agreement that NADH accumulates in hypoxia, the piece goes on to claim that “How the cell handles this mounting pool of reducing equivalents remained enigmatic until recently.” This is misleading. The defining characteristic of hypoxia, one that has dominated the literature in the nearly 90 years since Warburg's seminal work Warburg O, 1927, is the generation of lactate by lactate dehydrogenase (LDH), a key NADH consuming reaction that permits glycolysis to continue. Lactate is “How cells handle the mounting pool of reducing equivalents.”

Without mentioning lactate, an alternate fate for hypoxic NADH is proposed, based on the recent discovery that both LDH and malate dehydrogenase (MDH) can use NADH to drive the reduction of 2-oxoglutarate (α-ketoglutarate, α-KG) to the L(S)-enantiomer of 2-hydroxyglutarate (L-2-HG) under hypoxic conditions Oldham WM, 2015,Intlekofer AM, 2015. We also found elevated 2-HG in the ischemic preconditioned heart Nadtochiy SM, 2015, and recently reported that acidic pH – a common feature of hypoxia – can promote 2-HG generation by LDH and MDH Nadtochiy SM, 2016.

While there can be little doubt that the discovery of hypoxic L-2-HG accumulation is an important milestone in understanding hypoxic metabolism and signaling, the claim that L-2-HG is “a reservoir for reducing equivalents and buffers NADH/NAD+” is troublesome on several counts. From a quantitative standpoint, we reported the canonical activities of LDH (pyruvate + NADH --> lactate + NAD+) and of MDH (oxaloacetate + NADH --> malate + NAD+) are at least 3-orders of magnitude greater than the rates at which these enzymes can reduce α-KG to L-2-HG Nadtochiy SM, 2016. This is in agreement with an earlier study reporting a catalytic efficiency ratio of 10⁷ for the canonical vs. L-2-HG generating activities of MDH Rzem R, 2007. Given these constraints, we consider it unlikely that the generation of L-2-HG by these enzymes is a quantitatively important NADH sink, compared to their native reactions. It is also misleading to refer to the α-KG --> L-2-HG reaction as a "reservoir for reducing equivalents", because even though this reaction consumes NADH, it is not clear whether the reverse reaction regenerates NADH. Specifically, the metabolite rescue enzyme L-2-HG-dehydrogenase uses an FAD electron acceptor and is not known to consume NAD+ Nadtochiy SM, 2016,Rzem R, 2007,Weil-Malherbe H, 1937.

Another potentially important sink for reducing equivalents in hypoxia that was not mentioned, is succinate. During hypoxia, NADH oxidation by mitochondrial complex I can drive the reversal of complex II (succinate dehydrogenase) to reduce fumarate to succinate Chouchani ET, 2014. This redox circuit, in which fumarate replaces oxygen as an electron acceptor for respiration, was first hinted at over 50 years ago SANADI DR, 1963. Importantly (and in contrast to L-2-HG as mentioned above), the metabolites recovered upon withdrawal from a fumarate --> succinate "electron bank" are the same as those deposited.

Although recent attention has focused on the pathologic effects of accumulated succinate in driving ROS generation at tissue reperfusion Chouchani ET, 2014,Pell VR, 2016, the physiologic importance of hypoxic complex II reversal as a redox reservoir and as an evolutionarily-conserved survival mechanism Hochachka PW, 1975 should not be overlooked. Quantitatively, the levels of lactate and succinate accumulated during hypoxia are comparable Hochachka PW, 1975, and both are several orders of magnitude greater than reported hypoxic 2-HG levels.

While overall the article makes a number of important points regarding reductive stress and the correct use of terminology in this field, we feel that the currently available data do not support a quantitatively significant role for L-2-HG as a hypoxic reservoir for reducing equivalents. These quantitative limitations do not diminish the potential importance of L-2-HG as a hypoxic signaling molecule Nadtochiy SM, 2016,Su X, 2016,Xu W, 2011.

Paul S. Brookes, PhD.

On 2016 Sep 30, Paul Brookes commented:

I submitted a response to this opinion piece to the journal (Circ. Res.), but unfortunately was informed that they do not accept or publish correspondence related to this type of article. So, here's my un-published letter, which raises a number of issues with the article...

A recent Circ. Res. viewpointLoscalzo J, 2016 discussed the complex relationships between redox biology and metabolism in the setting of hypoxia, with an emphasis on the use of biochemically correct terminology. While there is broad agreement that the field of redox biology is often confounded by use of inappropriate methods and language Kalyanaraman B, 2012,Forman HJ, 2015, concern is raised regarding some ideas on reductive stress in the latter part of the article.

In discussing the fate of glycolytically-derived NADH in hypoxia, the reader is urged to “Remember that while redirecting glucose metabolism to glycolysis decreases NADH production by the TCA cycle and decreases leaky electron transport chain flux, glycolysis continues to produce NADH". First, glucose undergoes glycolysis regardless of cellular oxygenation status; this simply happens at a faster rate in hypoxia. As such, glucose is not redirected but rather its product pyruvate is. Second, regardless a proposed lower rate of NADH generation by the TCA cycle (which may not actually be the case Chouchani ET, 2014,Hochachka PW, 1975) NADH still accumulates in hypoxic mitochondria because its major consumer, the O2-dependent respiratory chain, is inhibited. It is clear that both NADH consumers and producers can determine the NADH/NAD+ ratio, and in hypoxia the consumption side of the equation cannot be forgotten.

While the field is in broad agreement that NADH accumulates in hypoxia, the piece goes on to claim that “How the cell handles this mounting pool of reducing equivalents remained enigmatic until recently.” This is misleading. The defining characteristic of hypoxia, one that has dominated the literature in the nearly 90 years since Warburg's seminal work Warburg O, 1927, is the generation of lactate by lactate dehydrogenase (LDH), a key NADH consuming reaction that permits glycolysis to continue. Lactate is “How cells handle the mounting pool of reducing equivalents.”

Without mentioning lactate, an alternate fate for hypoxic NADH is proposed, based on the recent discovery that both LDH and malate dehydrogenase (MDH) can use NADH to drive the reduction of 2-oxoglutarate (α-ketoglutarate, α-KG) to the L(S)-enantiomer of 2-hydroxyglutarate (L-2-HG) under hypoxic conditions Oldham WM, 2015,Intlekofer AM, 2015. We also found elevated 2-HG in the ischemic preconditioned heart Nadtochiy SM, 2015, and recently reported that acidic pH – a common feature of hypoxia – can promote 2-HG generation by LDH and MDH Nadtochiy SM, 2016.

While there can be little doubt that the discovery of hypoxic L-2-HG accumulation is an important milestone in understanding hypoxic metabolism and signaling, the claim that L-2-HG is “a reservoir for reducing equivalents and buffers NADH/NAD+” is troublesome on several counts. From a quantitative standpoint, we reported the canonical activities of LDH (pyruvate + NADH --> lactate + NAD+) and of MDH (oxaloacetate + NADH --> malate + NAD+) are at least 3-orders of magnitude greater than the rates at which these enzymes can reduce α-KG to L-2-HG Nadtochiy SM, 2016. This is in agreement with an earlier study reporting a catalytic efficiency ratio of 10⁷ for the canonical vs. L-2-HG generating activities of MDH Rzem R, 2007. Given these constraints, we consider it unlikely that the generation of L-2-HG by these enzymes is a quantitatively important NADH sink, compared to their native reactions. It is also misleading to refer to the α-KG --> L-2-HG reaction as a "reservoir for reducing equivalents", because even though this reaction consumes NADH, it is not clear whether the reverse reaction regenerates NADH. Specifically, the metabolite rescue enzyme L-2-HG-dehydrogenase uses an FAD electron acceptor and is not known to consume NAD+ Nadtochiy SM, 2016,Rzem R, 2007,Weil-Malherbe H, 1937.

Another potentially important sink for reducing equivalents in hypoxia that was not mentioned, is succinate. During hypoxia, NADH oxidation by mitochondrial complex I can drive the reversal of complex II (succinate dehydrogenase) to reduce fumarate to succinate Chouchani ET, 2014. This redox circuit, in which fumarate replaces oxygen as an electron acceptor for respiration, was first hinted at over 50 years ago SANADI DR, 1963. Importantly (and in contrast to L-2-HG as mentioned above), the metabolites recovered upon withdrawal from a fumarate --> succinate "electron bank" are the same as those deposited.

Although recent attention has focused on the pathologic effects of accumulated succinate in driving ROS generation at tissue reperfusion Chouchani ET, 2014,Pell VR, 2016, the physiologic importance of hypoxic complex II reversal as a redox reservoir and as an evolutionarily-conserved survival mechanism Hochachka PW, 1975 should not be overlooked. Quantitatively, the levels of lactate and succinate accumulated during hypoxia are comparable Hochachka PW, 1975, and both are several orders of magnitude greater than reported hypoxic 2-HG levels.

While overall the article makes a number of important points regarding reductive stress and the correct use of terminology in this field, we feel that the currently available data do not support a quantitatively significant role for L-2-HG as a hypoxic reservoir for reducing equivalents. These quantitative limitations do not diminish the potential importance of L-2-HG as a hypoxic signaling molecule Nadtochiy SM, 2016,Su X, 2016,Xu W, 2011.

Paul S. Brookes, PhD.