Lingering construct of lactic acidosis

Lingering construct of lactic acidosis

We would like to thank Dr. Kemp (6) and Böning et al. (4) for their letters to the editor and Dr. Lindinger et al. (8) for their editorial contributions to the topic of the biochemistry of metabolic acidosis in contracting skeletal muscle. As we stated in our original manuscript (9), there have been far too few academic discussions and reviews on this topic, resulting in the incorrect acceptance of the lactic acidosis construct as fact.

To specifically address the comments from each letter, we provide answers to each query separately, below. We provide a short response to the editorial of Dr Lindinger et al. after our responses to the two letters.

Nevertheless, before responding to specific contents of the two letters, we would like to reinforce the clear and simple evidence that we feel is being overlooked in the two letters to the editor. To argue for a lactic acidosis means that both of the phosphoglycerate kinase and lactate dehydrogenase reactions are incorrect.

The fact is that the biochemistry of these two reactions (Figs. 1 and 2) is factual and, as such, irrefutable and not open for debate or discussion. These reactions clearly reveal that the lactic acidosis construct is a fictional concept that serves to reinforce misunderstood muscle biochemistry and preserve decades of errors and misinterpretation. To argue the acceptance of this fiction is unscientific.

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Fig. 1.

The substrates and products of the phosphoglycerate kinase reaction. The product 3-phosphoglycerate is the first “carboxylic acid” formed in glycolysis. The phosphate transfer of this reaction reveals that a proton was never present to be released to the cytosol and alter cellular proton exchange and pH. As such, 3-phosphoglycerate and all of the remaining glycolytic carboxylic acid intermediates do not function as acids as they never have a proton that can be released into solution. Arrow pointing away from a bond represents bond/group removal. Arrow pointing to a bond represents addition of an atom/group.

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Fig. 2.

The substrates and products of the lactate dehydrogenase (LDH) reaction. Two electrons and a proton are removed from NADH, and a proton is consumed from solution, to reduce pyruvate to lactate. Arrow pointing away from a bond represents bond/group removal. Arrows pointing to a bond represent addition of an atom/group.

We entertain this series of letters to the editor because they once again allow us to state the truths about metabolic acidosis, highlight continued misinterpretations, and hopefully eradicate any thought of a lactic acidosis from academia and scientific research.

WHAT WAS THE PURPOSE OF OUR MANUSCRIPT?

We were very clear in what we wanted to accomplish with our manuscript. Our purpose was to show that it is a biochemical impossibility for lactic acid to be produced in skeletal muscle. In addition, we presented evidence based on simple computations and past research that revealed that there is no stoichiometry to lactate and proton production in contracting skeletal muscle, with proton production far in excess of lactate production. Finally, we stated and provided research evidence in support of the cause of metabolic acidosis to be an increased dependence on nonmitochondrial ATP turnover.

It was not our intention to provide definitive numbers for total proton release from muscle metabolism during exercise, and we did not attempt to provide definitive values for a muscle buffer capacity. We also identified that our numbers would not be totally accurate based on the pH dependence of proton release from ATP hydrolysis and proton binding to metabolites such as ADP and inorganic phosphate (Pi) (9; see p. R513, left column, paragraph 1).

WHAT IS THE PURPOSE OF THE LETTER OF DR. KEMP?

We found it difficult to comprehend what the real purpose of the letter of Dr. Kemp is. Is Dr. Kemp attempting to say that he agrees that lactate production does not produce protons, but then argue that when experiencing extreme acidosis, muscle seems to produce a small fraction of protons during ATP hydrolysis and one proton per lactate? Does this imply that lactate production does produce protons during acidic conditions, even if Dr. Kemp previously agreed that lactate production and proton release are unrelated biochemical events? Is Dr. Kemp saying that the decreased ionization of ATP during a developing acidosis means that ATP hydrolysis does not cause acidosis? What does Dr. Kemp view to be the proton releasing and proton consuming reactions of muscle metabolism? Is the main fundamental criticism that we should have tallied true proton release numbers based on partial ionization models?

On the basis of the absence of a clear purpose for Dr. Kemp's criticisms, we read some of his past research (5), and the research of his colleagues (1, 7). This process was extremely helpful, for we found the following quotes from these studies.

“… ‘proton balance’ only involves glycolytic lactate/H⁺ generation and net H⁺ consumption by PCr splitting.” (Ref. 5; p. 901, Abstract point 1)

“Total H⁺ load (lactate load less H⁺ consumption) was used to estimate cytosolic buffer capacity.” (Ref. 5; p. 901, Abstract point 3)

A schematic biochemical summary shows H⁺ production linked to lactate production (Ref. 5; p. 904, Fig. 1).

Dr. Kemp balances the substrates and products of glycolysis (commencing with glycogen) in an ischemic condition (no mitochondrial respiration), then couples glycolytic ATP production to ATP hydrolysis (e.g., muscle contraction) and reveals the following.

Glycogen-_n + H₂O → Glycogen-_n-1 + 2 Lactate + 2 H⁺ (Ref. 5; p. 905)

“In normal cellular functions the creatine kinase reaction is coupled to ATP utilization and resynthesis; the sum of both reactions is classically known as the Lohmann reaction.” (Ref. 7; p. C1741)

“During exercise, the net H⁺ load that results in pH changes is the difference between glycolytic H⁺ production (=change in lactate) and H⁺ consumption by PCr splitting.” (Ref. 1; p. 2392)

We will address the errors or misleading nature of all of the aforementioned quotes in the sections to follow. Clearly, the view of Dr. Kemp and his associates is that lactate production causes H⁺ production, and subsequently, that total proton release (proton load) can be indirectly calculated from the change in lactate accumulation when adjusted for the protons consumed in the creatine kinase reaction. Of course, these beliefs and interpretations of metabolic biochemistry in skeletal muscle are incorrect, as we further explain in this response.

IS IT VALID TO COUPLE THE ATPase AND CREATINE KINASE REACTIONS TOGETHER INTO THE LOHMANN REACTION?

Bendahan et al. (1), Kemp (5, 6), and Kushmerick (7) based their whole presentations of muscle metabolism on the assumption that ATP turnover is tightly coupled to the creatine kinase reaction during intense muscle contractions, or in Kemp's example, ischemic exercise. Kushmerick (7) presents the coupled balance of the Lohmann reaction (it is not a reaction inbiochemical terms, nor is it biochemically coupled) as follows. where α, β, and γ represent ionization coefficients and CK is creatine kinase.

At best, such an approach may only be accurate for the initial reactions of repeated muscle contractions. At worst, the Lohmann reaction concept is an oversimplification of the contribution of the phosphagen system to the biochemistry of skeletal muscle metabolism. In an anoxic environment (no muscle mitochondrial respiration), the ADP produced from ATP hydrolysis is not solely consumed by the creatine kinase reaction, but is also used in glycolysis and the adenylate kinase (myokinase) reaction (ADP + ADP → ATP + AMP). The Pi produced from ATP hydrolysis is also used as a substrate for glycolysis. AMP is converted to IMP and ammonia (AMP deaminase) during acidosis, revealing that the sum of AMP, IMP (or ammonia), and glycolytic flux equals the inaccuracy of the Lohmann reaction in accounting for all ATP regeneration. Because there is no equality or known stoichiometry between ATP hydrolysis and each of glycolysis, the creatine kinase reaction, and the AMP deaminase reaction (they can all be quantified, but their relative proportions can vary depending on the exercise condition) in working muscle, summary reactions of part or all of the phosphagen system are misleading and inaccurate.

Our Eqs. 3 and 4 (9; p. R507), Eqs. 5 and 6 (9; p. R508), Fig. 11 (9; p. R510) and Fig. 14 (9; p. R511) provide a detailed account of the origin and fate of ADP, Pi, and H⁺ during muscle metabolism. We included mitochondrial respiration, for almost all exercise conditions have functioning mitochondria, oxygen provision—and hence substrate consumption (O₂, ADP, Pi, H⁺)—and ATP production from mitochondrial respiration. It is only when presenting the complete metabolic milieu in working muscle that the true cause of acidosis is revealed. Falsely coupling separate reactions is not a valid presentation of metabolic biochemistry and leads to misunderstandings of proton balance.

DOES THE pH DEPENDENCE OF THE PROTON YIELD FROM ATP HYDROLYSIS CHANGE THE BIOCHEMICAL CAUSE OF ACIDOSIS?

We agree that the proton yield of ATP hydrolysis is pH dependent. However, this fact is largely irrelevant to the understanding of the cause of metabolic acidosis. Skeletal muscle fatigue during intense exercise is rapid in onset and unpreventable during “all out” efforts. As we depict in Fig. 3, the ATP turnover of skeletal muscle during such conditions mirrors the muscle force production profile, as researched by Spriet et al. (10). Thus the period of greatest ATP turnover occurs early, during the initial phase of muscle fatigue, and decreases rapidly, resulting in relatively small ATP turnover during what has now become an extreme acidic condition.

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Fig. 3.

Relative change in the force production and estimated rates of ATP turnover and proton release during 64 repeated contractions of the human quadriceps muscles for 1.6 s at 20 Hz electrical stimulation, with occluded blood flow. The darker the intensity of the shading represents greater rates of estimated proton release. Adapted from Spriet et al. (10).

In short, once muscle is acidic, it is also severely compromised in its ability to generate force and thus demand ATP turnover. These are not necessarily cause-and-effect responses. Clearly, the metabolic period of relevance to the development of metabolic acidosis is the condition change from neutral cell pH to moderate acidosis (pH decrease from 7.0 to ∼6.6), conditions where Dr. Kemp seems to agree that decreases in pH are best explained by ATP hydrolysis. Applying our presentation of metabolic biochemistry and applying the decreasing proton release from ATP during acidosis reveal that during severe acidosis, there are major constraints to added proton release (ATP proton gain and decreased ATP demand). The important issue here is not what happens to proton balance when the cell pH approaches 6, but how cell pH got that low to begin with.

Finally, this issue reveals the inconsistency in Dr. Kemp's arguments within his letter (6). Dr. Kemp commences his letter with recognition to the proton release from ATP hydrolysis and that this is likely to cause initial reductions in muscle pH during repeated intense (as well as hypoxic or anoxic) contractions. Dr. Kemp states, “I will argue that while this analysis [that ATP hydrolysis disconnected from mitochondrial respiration causes acidosis] of proton generation is broadly correct at resting pH, it is much less so at low pH values found in exercising muscle.” Dr. Kemp then presents admirable evidence for the decreased proton release from ATP hydrolysis as pH declines, but then states the following, “…at pH 6, most of the ‘glycolytic’ protons actually do come from glycolysis to lactate.” Dr Kemp bases this last statement in part on his Eq. 5b, revealing the decreased ionization of ATP at pH 6, as well as summary reactions of ATP hydrolysis, glycolysis, and lactate production (6; see Table 1). However, what Dr. Kemp fails to realize is that this summary reaction that we detailed in Eqs. 1–6 reveals that the proton product accompanying lactate does not come from lactate production, but from ATP hydrolysis (9; Fig. 11, p. R510). Thus, at a pH near 6, there is really minimal proton release, and his summary reaction (our Eq. 5) should be written with proton release adjusted by an ionization constant close to zero.

ARE ESTIMATES OF MUSCLE BUFFER CAPACITY INACCURATE IF THEY ASSUME LACTATE PRODUCTION CONTRIBUTES TO THE MUSCLE PROTON LOAD?

We do not understand the effort of Dr. Kemp to argue against the logic and factual content of our manuscript on the grounds of muscle buffer capacity. Earlier, we revealed in the quotes from past research of Bendahan et al. (1), Dr. Kemp (5), and Kushmerick (7) that these scientists accept the notion that lactate production is stoichiometric to proton release in contracting skeletal muscle in the absence of mitochondrial respiration. As such, these scientists use lactate production to estimate the proton load (adjusted for the creatine kinase reaction). This is incorrect for all the reasons presented in our original manuscript and which require no further repetition here. You cannot estimate a muscle buffer capacity using lactate production to account for proton release. Lactate production consumes a proton, and thus the estimates of muscle buffer capacity used by Dr. Kemp and Dr. Bendahan must therefore grossly underestimate the true buffer capacity, as they underestimate both proton load (they should use their own data for nonmitochondrial ATP turnover) and metabolic proton removal (they need to add lactate production to the reactions comprising metabolic buffering). It does not matter what sensitive methods are used to quantify ATP, ADP, creatine phosphate and muscle pH, the incorrect estimation of proton loads condemns these data to major error.

Response to Letter to the Editor: Böning D, Benele R, and Maassen N. Lactic acid still remains the real cause of exercise-induced metabolic acidosis.

A BÖNING-KEMP VIEW OF METABOLIC BIOCHEMISTRY

As previously stated, Dr. Kemp and Dr. Böning have made fundamental biochemical errors in their arguments in support of a lactic acidosis. To put such views into their true perspective for metabolism, Dr. Kemp and Böning et al. believe that you can take products of totally different reactions, couple these reactions together, and combine the products into new molecules to provide a valid assessment of muscle metabolism.

The question that we have is if you view this approach to be valid then why stop at the production of this new fictitious molecule called lactic acid? We can continue this principle, seemingly unconstrained by the laws of bioenergetics and Einstein's theories, to bring together whatever end products from different reactions we like, simply to support whatever unscientifically based theory we can dream up.

For example, the proton from ATP hydrolysis can leave the muscle cell where it is buffered by bicarbonate (HCO₃⁻). Thanks to the application of this “logic”, the incomplete oxidation of glucose to lactate can now be changed, as follows. In summary The interpretation of this reaction is that glucose oxidation outside of the mitochondria produces CO₂!

However, we can continue this view of metabolic biochemistry. Lactate leaves the muscle and can be circulated to the liver where it is reduced to pyruvate by a reversal of liver lactate dehydrogenase, and then converted to glucose via liver gluconeogenesis. Thus, applying the views of Dr. Kemp and Böning et al., we could also combine these reactions, as follows. In summary

These new reactions reveal that the incomplete oxidation of glucose uses ATP and produces glucose. Not only that, but the real culprit in the cause of acidosis is not lactic acid at all, but the production of glucose acid (glucose + 2H⁺).

Obviously, these reactions are fictitious, but no more so than the production of lactic acid, or the existence of the Lohmann reaction. We hope that this extension of the principles employed by Dr. Kemp and Böning et al. places the following interpretation of Böning et al., “The resulting metabolite is lactic acid in spite of the last step for proton release being ATP hydrolysis,” (4) into correct perspective—clear fiction. Response to Editorial: Lindinger MI, Kowalchuk JM, Heigenhauser GJF. Applying the laws of physics to muscle acid-base chemistry.

We are indebted to the prior work of Lindinger et al. (8) for teaching us to recognize the importance of the strong ion difference (SID) in understanding proton balance and activity. Nevertheless, we feel that it is important to clarify an underlying message contained in this editorial.

The question crucial to the main purpose of our original manuscript is “Where do the protons come from?” Clearly, protons do not come from the lactate dehydrogenase reaction and therefore the production of lactate. However, the Lindinger et al. (8) presentation of the physical chemistry pertaining to the acid-base balance of aqueous solutions indirectly argues that there is no evidence for proton consumption and release from chemical reactions and that changes in cellular or blood pH are caused predominantly by changes in strong ions (their Eq. 7). We do not agree.

Despite the solid basic science foundation of the SID concept, we feel that it alone does not answer the question of where do the protons come from. If it did, then the answer is water, and this opposes what we know of the organic chemistry of the reactions of intermediary metabolism. Certainly, the SID approach can explain the final aqueous solution pH when given component changes for a given proton release condition of cellular metabolism. Thus we feel that to best comprehend the acid-base changes associated with intense exercise, you need a combination of a stoichiometric approach fine tuned by the SID method. For example, given the metabolic proton release conditions of intense exercise, the SID computations would predict the final muscle pH. Such a scenario is presented in our Fig. 17 (9; p. R513), where the final pH resulting from this imbalance in proton release and consumption would adhere to the SID concept.

What Is the Take-Home Message?

We have learned that despite a wealth of e-mail messages from around the world that congratulated us on our recent publication, there remain numerous physiologists and metabolic biochemists consumed by the inertia of viewing muscle acidosis as a lactate-caused condition.

We encourage Dr. Kemp and Böning et al. to reassess their own data (2, 5), view proton production to be caused by ATP hydrolysis, and recalculate muscle proton loads and buffer capacities from their prior research. We thank Lindinger et al. (8) for their insightful explanation of the SID, and we will include the SID, and our interpretations of its merit and limitations, in our future manuscripts on the topic of the biochemistry of metabolic acidosis.

It has been more than 80 years since the work of Hill and Meyerhoff (9). We have known the organic and acid-base chemistry of the lactate dehydrogenase reaction for more than 50 years. It has been 28 years since the initial publications that criticized the lactic acidosis construct. We are impatient for science to recognize that three is no such thing as a lactic acidosis, thus allowing more valid research and learning of the intricacies of muscle metabolic regulation and proton balance.

We continue to hypothesize that an increased dependence on nonmitochondrial ATP turnover is currently the most valid explanation of the cause of exercise-induced metabolic acidosis.