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.
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.
Response to Letter to the Editor:
Kemp GJ. Lactate accumulation, proton buffering, and pH change in ischemically exercising muscle.
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)
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.
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.
We are obviously totally opposed to the wording of this title.
Source of Increasing Pi in Exercising Muscle
As stated in our manuscript, and in every textbook of metabolic biochemistry, the creatine kinase reaction is as follows. (1) Böning and colleagues argue that the creatine kinase reaction is also coupled to ATP hydrolysis. However, where is the evidence for this assumption? Böning et al. (4) present none. We clearly argued against this coupling in our reply to Dr. Kemp on the issue of the fictitious “Lohmann reaction” (see section: is it valid to couple the atpase and creatine kinase reactions together into the ‘lohmann reaction’?). To comprehend the error of this view and all that it implies, read the later section of this response: a böning-kemp view of metabolic biochemistry.
Amount of Proton Release During Glycolysis
It is incorrect to label our presentation of glycolysis as wrong or incomplete. To reinforce this issue, we present the main and summary reactions of intermediary metabolism in contracting skeletal muscle to clearly show that our presentation was valid. We also present these reactions, as the derivation of the main summary reactions of carbohydrate and lipid catabolism is a useful exercise for the student, scientist, and academic alike and, as such, is a great test of the mastery of metabolic biochemistry. (2) (3) (4) (5) (6) (7) (8) (9)
For Glucose Ending in Lactate
(10) (11) (12) (13) (14) Note, as explained in the text, we disagree with the coupling of the ATP hydrolysis of muscle contraction to glycolysis as not all ATP, ADP, and Pi from catabolism cycles 100% between muscle contraction and glycolysis.
When Glycogen Is The Substrate
(15) (16) (17) (18) (19) (20) (21) (22) (23)
For Glycogen Ending in Lactate
(24) (25) (26) (27) (28) (29) Note, as explained in the text, we disagree with the coupling of the ATP hydrolysis of muscle contraction to glycolysis, as not all ATP, ADP, and Pi from catabolism cycles 100% between muscle contraction and glycolysis.
What is “most problematic” with our presentation of these reactions in the minds of Böning et al. is that we did not focus on our Eq. 5 and 6 (9; p. R508), which show glucose or glycogen, “coupled” to ATP hydrolysis. Yes, when you “couple” glycolysis to ATP hydrolysis, you do end up with the production of 2 lactate and 2 H+ (Eqs. 14 and 29). However, the equality of the glycogen vs. glucose source proton release depends on whether you keep ATP turnover the same for both substrates. Our Eq. 6 was based on the fact that when muscle glycogen is used to fuel glycolysis, there is no involvement of the hexokinase reaction (which releases a proton and consumes an ATP). Thus, for every 2 lactate produced and if the 2 ATP gain from glycolysis remains your reference, there is one less proton released when catabolizing glycogen vs. glucose. In other words, for a given rate of ATP turnover, there are less protons released from glycolysis when starting with glycogen (protons:ATP = 2:3) vs. glucose (2:2). This important contrast is overseen when interpreting the resulting balanced reaction of glycolysis coupled to ATP hydrolysis.
Unlike Böning et al., who state that the summary reaction products of 2 lactate + 2 H+ is lactic acid, we view this interpretation to be incorrect. This interpretation is not based on biochemical fact, scientific principles, or research-based evidence, but rather on a convenient explanation construed over the decades since the work of Hill and Meyerhoff (see Ref. 9) to provide a simple explanation of metabolic acidosis. This simple and convenient explanation is based on two disturbing assumptions: 1) that all the ADP, Pi, and H+ from ATP hydrolysis are only used to reform ATP from glycolysis and most disturbingly, 2) that you can take products from completely different reactions, combine them together to form a new end product, and interpret this end product to be causal to altered cellular and system conditions of acid-base balance. It is for these reasons that we chose not to emphasize Eqs. 14 and 29 in our manuscript.
Why do Dr. Kemp and Böning et al. believe in a lactic acidosis? We do not know for sure but speculate that is what they were taught, that is what they presumably teach their students, and that is the foundation upon which they conduct and publish research. Such a publication track record is also clear for Dr. Böning, as revealed below.
“During intense exercise a large amount of lactic acid (La) is produced in the body and may disturb homeostasis, leading to an increase in hydrogen ion (H+) activity.” (2)
“… lactic acid is practically completely dissociated into La− and H+ because of its low pK (3.0). ” (3)
“The extracellular pH defense against the lactic acidosis resulting from exercise can be estimated from the ratios Δ[La]·ΔpH−1 (where Δ[La] is the change in lactic acid concentration and ΔpH is the change in pH) and Δ[HCO3−]·ΔpH−1 (where Δ[HCO3−] is the change in bicarbonate concentration) in blood plasma. The difference between Δ[La]·ΔpH−1 and Δ[HCO3−]·ΔpH−1 yields the capacity of nonbicarbonate buffers (mainly hemoglobin).” (3)
We know of no research evidence or biochemical principles that validate the direct coupling of ATP hydrolysis to the creatine kinase reaction or glycolysis under any muscle contractile condition. As we explained in our response to Dr. Kemp, our Figs. 11 and 14 present the reality of the complexity and interactions of substrates and products across multiple metabolic pathways and isolated reactions and in interaction with the compartmentalization of mitochondria within contracting skeletal muscle.
Exercise-Induced Metabolic Acidosis Without Production of Lactic Acid
We do not find this argument to be logical or clearly in support of your view. Individuals who suffer from McArdle's disease are not only unable to produce lactate, but they are unable to rely on glycolysis for ATP turnover during muscle contraction. As such, McArdle's disease causes a severe reduction in the capacity of nonmitochondrial ATP turnover. Furthermore, the remaining metabolic source of nonmitochondrial ATP turnover is the creatine kinase reaction (Eq. 1), which consumes a proton and the adenylate kinase reaction. Thus McArdle's disease does not disprove the nonmitochondrial ATP turnover cause of metabolic acidosis. If you severely diminish the capacity for nonmitochondrial ATP turnover, you prevent the ability to sustain intense muscle contractions and consequently prevent metabolic acidosis.
Lactate and Proton Release From Muscle to Blood
We addressed this issue in our response to Dr. Kemp in the section are estimates of muscle buffer capacity inaccurate if they assume lactate production contributes to the muscle proton load? However, it needs to be stressed yet again that if you compute a nonbicarbonate buffer capacity based on the assumption that lactate production is the source of protons, you will obviously get a stoichiometry between proton release and lactate release. As we have shown in an earlier quote from Dr. Böning's work (2, 3), he assumes that quantifying lactate quantifies metabolic proton release and that the change in blood lactate quantifies blood proton gain. These assumptions are obviously wrong. Is this a harsh assessment of this work? Maybe. But we once made the same errors but have recognized the errors of our past assumptions and changed the way we teach and conduct research on the topic of acidosis. Many other academics and scientists have done the same. Since the publication of our manuscript (6), there is no longer the excuse of “but that is how we have always interpreted acidosis.”
What is your point here? Yes, the research on muscle buffer capacity is fraught with inconsistencies, and as we explained in our response to Dr. Kemp, fundamental flaws. As most research estimating total or metabolic buffer capacities has used lactate to estimate muscle proton release, which includes research from Dr. Kemp (5) and Dr. Böning (2), this research needs to either be redone or reassessed with revised computations that treat lactate production as a proton consumer and nonmitochondrial ATP turnover as the source of protons.
Calculation of Nonmitochondrial ATP Production
Thank you for detecting this typographical error.
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.
- Copyright © 2005 the American Physiological Society