How Lactate Alkalinizes Your Muscles

Interactive Transcript

spk_0 This is how lactate alkalinizes your muscles.

spk_0 You heard that right, not as sitifies alkalinizes your muscles.

spk_0 I'm Dr. Chris Masterjohn, I have a PhD in nutritional sciences,

spk_0 and you are watching a Masterclass with Masterjohn Energy

spk_0 Metabolism lesson.

spk_0 This is a course where we break down the biochemistry

spk_0 of how we use the energy in food to fuel all the important

spk_0 processes in our body that contribute to our wellness

spk_0 performance and longevity.

spk_0 And in this lesson, we're going to look at the biochemistry

spk_0 of how lactate is alkalinizing.

spk_0 You may have heard that lactate is something that is produced

spk_0 when our muscles make lactic acid when we're exercising,

spk_0 that that acidifies our muscles and contributes to fatigue,

spk_0 failure, and delayed onset muscle soreness,

spk_0 or you may have heard the arguments against this.

spk_0 There are many well-known people who have been spending a long time,

spk_0 big parts of their career, countering this narrative like Andy Galpin.

spk_0 You may have heard, if you're deep in the science,

spk_0 you may have read the work of George Brooks.

spk_0 We're going to take a deep look into the biochemistry.

spk_0 We're going to learn how we do not make lactic acid in our metabolism.

spk_0 We make lactate.

spk_0 When we make lactate is alkalinizing,

spk_0 we're going to look at how some of the presentations

spk_0 in biochemistry textbooks on the one hand

spk_0 give us everything that we need to understand why it is lactate

spk_0 that we make and not lactate gas,

spk_0 that we never make lactate gas in our metabolism.

spk_0 But on the other hand, do not equip us very well

spk_0 to understand what actually is the source of acidity

spk_0 in exercising skeletal muscle or in metabolism in general,

spk_0 because they don't put enough emphasis on acid base balance

spk_0 to bother showing you with completeness

spk_0 where all the protons, which are the cause of acidity,

spk_0 are going in the chemical reactions.

spk_0 So we're going to take a look at the burglary and leninja

spk_0 presentations of glycolysis to see where they go wrong

spk_0 in equipping us with the knowledge we need to understand

spk_0 the acid base balance during exercise.

spk_0 And finally, we're going to end with some practical implications.

spk_0 We'll talk about what actually does cause fatigue

spk_0 in exercise skeletal muscle and exercising skeletal muscle.

spk_0 And we will also look at the research on lactate supplements

spk_0 briefly to see how we can leverage what we learn in this biochemistry

spk_0 towards how we should interpret the human trials

spk_0 with lactate supplements in the context of other ways

spk_0 that we could leverage nutrition and supplementation

spk_0 towards improving exercise performance.

spk_0 So super briefly on the screen is the typical presentation

spk_0 of glycolysis divided into two phases

spk_0 based on investing energy versus generating energy

spk_0 where we invest to ATP, we get as a payoff from that.

spk_0 It's also called the preparatory and payoff phases.

spk_0 As a payoff, we get four ATP, so that's net two ATP gain

spk_0 and we also get two NADH.

spk_0 We're going to now look at it from a different perspective

spk_0 how we could divide it between acidifying phases

spk_0 and alkalizing phases.

spk_0 Before we do that, just to make sure we're all on the same page,

spk_0 I want you to think of a proton as a hydrogen ion

spk_0 as an H plus and as acidity.

spk_0 These are all the same things.

spk_0 The simplest element is hydrogen, one proton, one electron.

spk_0 Take away that electron.

spk_0 You form the positively charged hydrogen ion,

spk_0 which is the same thing as a proton.

spk_0 The accumulation of protons is the cause of acidity.

spk_0 We can measure it and express it as the pH.

spk_0 The P in pH means the negative logarithm of the negative logarithm

spk_0 of the hydrogen ion concentration.

spk_0 Because it's negative, the lower the pH, the more acidic,

spk_0 the higher the pH, the less acidic,

spk_0 because it's logarithmic as we go one unit on the pH scale,

spk_0 we're moving by a factor of 10.

spk_0 So seven is neutral, six is 10 times more acidic,

spk_0 five is 100 times more acidic, four is 1000 times more acidic,

spk_0 and so on.

spk_0 If we divide glycolysis into the acidifying

spk_0 and alkalizing phases, we are going down to the production

spk_0 of two phosphoenol pyruvate.

spk_0 So we're going almost to the end of glycolysis

spk_0 as the acidifying part.

spk_0 And the alkalizing phase is the pyruvate kinase reaction

spk_0 and the lactate dehydrogenase reaction.

spk_0 I'm depicting lactate as the end product of glycolysis,

spk_0 because there's a rich body of evidence

spk_0 that contrary to the presentation of the textbooks,

spk_0 lactate is the dominant, if not the overwhelmingly

spk_0 or nearly exclusive end product of glycolysis.

spk_0 The protons released are fractional,

spk_0 because it's not only the case that we have

spk_0 covalent bonds changing as we change the construction

spk_0 of a molecule that may release a stoichiometric amount

spk_0 of protons or consume them, meaning we can mathematically

spk_0 account that as we go from one lactate to one pyruvate,

spk_0 we must do one or another thing with the reactants

spk_0 and substrates involves that add up into a mathematical formula

spk_0 where everything is expressed in whole numbers.

spk_0 In addition to that, we also have hydrogen ions

spk_0 that are binding to or releasing from the substrates

spk_0 and products in these reactions.

spk_0 And we can express those as probabilities.

spk_0 So we have an average fractional proton yield.

spk_0 We will talk about that, but to simplify things here,

spk_0 I've rounded these all off to whole numbers.

spk_0 And when we do that to make it easier to understand,

spk_0 we are releasing five or six protons in the acidifying phase

spk_0 of glycolysis, five if we come from glycogen,

spk_0 six if we come from glucose, and then during the alkalonizing phase,

spk_0 when we end at lactate, we're consuming four protons.

spk_0 That is netacidic regardless of whether we came from

spk_0 glycogen or free glucose.

spk_0 But if we remove lactate from the cell,

spk_0 we have to remove one proton per lactate molecule

spk_0 because of the way that the transporters designed.

spk_0 And that means that if we break glucose into two lactate

spk_0 and remove two lactate from the cell,

spk_0 we remove two more protons.

spk_0 That is a net clearance of six protons in the alkalonizing phase.

spk_0 And that will allow us to break even if we came from glucose

spk_0 or to be net alkalonizing if we came from glycogen.

spk_0 As brief review, these are the 10 reactions of glycolysis.

spk_0 We phosphorylate glucose to glucose six phosphate, not shown here.

spk_0 We also could have broken glycogen down to glucose one phosphate.

spk_0 And I summarize that to glucose six phosphate.

spk_0 Then we I summarize the glucose six phosphate to fructose six phosphate.

spk_0 We then phosphorylate that to fructose one six bis phosphate.

spk_0 That is with the enzyme phosphofructokinase or PFK.

spk_0 Super important enzyme because it's the first committed step in glycolysis.

spk_0 You could have done other things with this glucose six phosphate.

spk_0 And also because it's highly regulated.

spk_0 So with respect to our discussion, the most important regulation of it is that

spk_0 it is strongly inhibited by acidity.

spk_0 The fructose one six bis phosphate then gets split apart into glyceraldehyde three phosphate

spk_0 and dihydroxyacetone phosphate.

spk_0 If glycolysis is proceeding smoothly forward,

spk_0 the dihydroxyacetone phosphate converts to a second molecule

spk_0 of glyceraldehyde three phosphate.

spk_0 This concludes the energy investment or preparatory phase.

spk_0 It now begins the energy generating or payoff phase.

spk_0 And it also marks the point where we have two of everything now

spk_0 because we've split the molecule in half.

spk_0 So everything here happened once.

spk_0 Everything forward is going to happen twice.

spk_0 So two molecules of glyceraldehyde three phosphate

spk_0 are going to get oxidized by two NAD plus generating two NADH and two protons.

spk_0 And that's going to form one three bis phosphate glycerate.

spk_0 In doing that, we are using the enzyme glyceraldehyde three phosphate dehydrogenase or GAPDH.

spk_0 And that is going to be another super important enzyme because it's an NAD plus sensitive step.

spk_0 The one NAD plus sensitive step of glycolysis.

spk_0 So we're going to need this NADH to be in net oxidized in the respiratory chain in order

spk_0 to allow this step to go forward.

spk_0 And it's also the most acidifying step in glycolysis.

spk_0 We're going to see additional reasons why that's true momentarily.

spk_0 Then one three bis phosphate glycerate, we have two of those.

spk_0 That produces two ATP yields two molecules of three phosphoglycerate.

spk_0 That gets a summarized to two molecules of two phosphoglycerate.

spk_0 That gets rearranged using water to two molecules of phosphoenol pyruvate.

spk_0 That then produces two more ATP for a total production of four ATP

spk_0 and a net production of two ATP yielding two molecules of pyruvate.

spk_0 This then stops as it's typical at pyruvate only we, as I said before, we are always

spk_0 or almost always going to actually continue going on to lactate.

spk_0 But that concludes the typical presentation in a textbook and in most online sources

spk_0 of the 10 steps of glycolysis.

spk_0 All right, so here is the redox-acid-base reaction that occurs with NAD plus and NADH.

spk_0 NAD plus in the upper left we can see that it binds two electrons and two hydrogen ions.

spk_0 One of those hydrogen ions becomes NADH and the other is left in solution.

spk_0 One of those electrons was neutralizing the positive charge on the NAD plus.

spk_0 The other was gluing that first proton to the NADH molecule.

spk_0 That can also be written as NAD plus accepting a hydride ion,

spk_0 which is an H negative instead of an H plus.

spk_0 That's a proton with two electrons and that becomes NADH and there's one proton that's left over.

spk_0 Either way, because we have two electrons with electric neutrality, we have to balance that with

spk_0 two protons. One of those protons is always going to be released into solution.

spk_0 Simply to account for the fact that NAD plus has one electron neutralizing its positive charge

spk_0 and one getting added on as NADH.

spk_0 So when you hydrolyze ATP as hydrolysis hydrolyze implies you're using water

spk_0 and the reason that you're using water is because to break apart the terminal phosphate,

spk_0 you have an oxygen that is essentially shared with the phosphate next to it.

spk_0 And you're going to have to reconstitute the oxygen of the phosphate that you're ripping off

spk_0 if it can no longer share the oxygen with the phosphate next to it.

spk_0 So you take the oxygen from water to do that and one of the hydrogens of the water

spk_0 will tend to stay on the phosphate molecule, but the other will be left into solution as an extra proton.

spk_0 Conversely, if you synthesize ATP in the mitochondrial respiratory chain, you go the opposite way.

spk_0 You do dehydration synthesis and you remove the OH from the phosphate with a proton that you've

spk_0 taken from solution and that will reconstitute the ATP molecule and you will have a removed a

spk_0 proton from solution.

spk_0 So ATP synthesis is alkalizing and ATP hydrolysis is acidifying.

spk_0 However, the substrate level phosphorylation in glycolysis do not use dehydration synthesis.

spk_0 And you can see in the Lenninger textbook that depicted why in the case of the phosphoglycerate

spk_0 kinase reaction, they've shadowed in this pinkish orange color a box around what is moving and

spk_0 it's not actually phosphate, it's phosphite, it's PO3, not PO4.

spk_0 And you do not need to reconstitute it, you do not need to reconstitute its fourth oxygen

spk_0 because you're not generating free phosphate in water.

spk_0 Instead, you're taking PO3 and you're leaving behind the oxygen of the substrate and you're in

spk_0 this case you're allowing it to become a carboxyl group and you are moving PO3 onto the adenosine

spk_0 diphosphate molecule. Now remember, adenosine diphosphate that phosphate on the end, it has its own

spk_0 oxygen. So you don't need to take out OH when you move it there because you're just taking PO3,

spk_0 you're not taking PO4 and you're just putting PO3 where PO3 needs to be, you're not dealing with any

spk_0 extra oxygen. So in the substrate level phosphorylation of glycolysis, there's no fourth oxygen that

spk_0 ever needs to be dealt with, there's no hydrolysis, there's no dehydration synthesis, there's just

spk_0 a transfer of a PO3 and there's no proton exchange. However, in the second substrate phosphorylation

spk_0 reaction, you do happen to absorb a proton for a completely different reason. So here you have

spk_0 the same type of transfer of PO3 from phosphino pyruvate to ATP but this hydrogen ion that you see

spk_0 getting used is not doing anything with the phosphate, it's actually making the last hydrogen

spk_0 on the carbon show up. The way this happens is that as the PO3 leaves, the hydrogen ion from solution

spk_0 comes in and gets added as an OH. This is what's called an enol form of pyruvate and the enol

spk_0 is a double bonded carbon within OH coming off of it. Enol's taught to marise to ketones.

spk_0 ketones are a double bonded oxygen in the middle of a molecule. In general, whenever you have an enol

spk_0 like this, it's going to taught to marise to the ketone form and what we typically think of as

spk_0 pyruvate is the ketone form. And so that hydrogen that came in on the oxygen winds up down at the

spk_0 bottom on the carbon. So that's the reason that a proton is being used up in the pyruvate

spk_0 kinase reaction. Phosphorylation and defosphorylation of sugars within glycolysis do release protons.

spk_0 Although this is not a case of hydrolysis and dehydration synthesis, this is a case of

spk_0 exchanging the proton on the end for the PO3 or phosphite that's going to come off of the ATP

spk_0 molecule. So PO3 jumps off the molecule. Hydrogen jumps off the molecule as a proton. And the

spk_0 PO3 gets put in the proton's place. And so in each phosphorylation reaction at the beginning of

spk_0 glycolysis, you do release one proton. All of this becomes quite a bit more complicated when we

spk_0 think of the fact that both phosphate species as well as sugars, as well as nucleotides, as well

spk_0 as everything else that is capable of ionization has a degree to which they will tend to ionize

spk_0 or deionize. In this case, we're talking about proton exchanges. So the ionization is a deprotonation

spk_0 and the deionization is a protonation. So something is, in this example, we have phosphoric acid,

spk_0 has a neutral charge. It becomes charged or ionized with a negative one charge if it loses one proton.

spk_0 That is the deprotonated state. Phosphoric acid is the protonated state because that proton is

spk_0 added. The relative degree to which they will tend to lose or gain a proton is a matter of

spk_0 probability. And the probability depends on the concentrations of substrates of reactants and

spk_0 products. Because H plus is a reactant or product depending in which way you're going, then the pH,

spk_0 the concentration of hydrogen ions, becomes a critical determinant of the degree to which you would

spk_0 protonate or deprotonate. In general, if it's a very acidic environment, the concentration of

spk_0 hydrogen goes up and that favors protonation. If the concentration of hydrogen ions goes down,

spk_0 it's more alkaline or more basic. It's less acidic. Facilitates a deprotonation. We can express

spk_0 the probability that they'll ionize as the pKa. Technically, the K is the equilibrium constant

spk_0 of a reaction. It's saying, if you have these two things that can interconvert at chemical

spk_0 equilibrium, when they're both going forward and backward in the same at a constant rate,

spk_0 what would you expect to have more of? The thing on the right or the thing on the left? But if it's

spk_0 a K of an acid, we can call it the K sub A, the K of the acid. And we can then convert that to a

spk_0 pKa, the negative logarithm of the Ka. And the pKa happens to be the pH at which we have 50% the thing

spk_0 on the right and 50% the thing on the left. That's how you think of it technically. But you can

spk_0 more intuitively think about it as if you have two convertible chemical species in a protonated

spk_0 and deprotonated state. They can act as a buffer when the pH of the solution they're buffering

spk_0 is close to their pKa. And the pKa is the pH that they're going to defend. So in this particular

spk_0 case, I took the conversion of phosphoric acid to dihydrogen phosphate, hydrogen phosphate and

spk_0 phosphate from the bird biochemistry book. And you can see that their pKa is are 2.12, 7.21 and 12.67.

spk_0 What that means is that you're not really going to see much action between phosphoric acid and

spk_0 dihydrogen phosphate unless you get way more acidic than most of our body tissues would tolerate.

spk_0 And you're not going to see much action between hydrogen phosphate and phosphate until you get

spk_0 way more alkaline than most of our tissues would tolerate. And so what you're primarily going to

spk_0 see as free phosphate and solution is dihydrogen phosphate and hydrogen phosphate. And when they're

spk_0 in solution, they're going to buffer the solution and they're going to defend the pH of 7.21.

spk_0 If you get less than that, then you're going to favor the protonated form. So it will absorb

spk_0 hydrogen ions. And if you get higher than that, you're going to favor the deprotonated form. So

spk_0 the hydrogen ions get released. Now it's also the case that the pKa's are not intrinsic

spk_0 properties of the chemicals, but they depend on the experimental conditions in which they were

spk_0 measured. And that's why if you look at different sources, you're going to see different values

spk_0 for the pKa's. For example, I took the Lenninger biocamistry diagram for the same thing and they

spk_0 show the pKa as 2.14 instead of 2.12, 6.86 instead of 7.21 and 12.4 instead of 12.67.

spk_0 The pKa's that I'm going to follow are taken from a paper that are not the ones on the screen.

spk_0 The ones that I'm primarily going to use in the rest of this presentation are taken from a paper

spk_0 by Robert Roberts that I'll show you later where the most recent pKa's under the conditions that

spk_0 are most relevant to those that prevail in skeletal muscle were compiled from the existing

spk_0 literature. These pKa's that are on the screen are from that paper. What I want to show you now is

spk_0 that the pKa of the phosphate will be radically different depending on what it's bound to.

spk_0 So as free phosphate, the ability of phosphate to bind to one proton is rated at a pKa of 11.59.

spk_0 But when you have glucose 6 phosphate, the pKa of the phosphate group to bind one proton drops

spk_0 down to 6.1. That means the glucose has made that phosphate tremendously more acidic than it was

spk_0 on its own. Why is that? Because the carbohydrate molecule is rich in oxygens and it has an

spk_0 acidifying effect because of the electronic negativity of the oxygens. If you look at ATP,

spk_0 you're going to see considerably higher pKa's but they're still way lower than the phosphate.

spk_0 But they're higher than glucose 6 phosphate. So ATP is 6.48, ADP is 6.38, and NP is 6.2.

spk_0 And what you're seeing here is the primary influence is the acidifying effect of the ribose.

spk_0 But it's slightly mitigated by the basic effect of the nitrogen-rich adenine that's behind it.

spk_0 But then the effect of the ribose is mitigated by the distance of the terminal phosphate

spk_0 from the ribose molecule. Because it's only the terminal phosphate that's going to tend to be

spk_0 able to bind to even one proton. All these other ionization sites are fully ionized in the phosphate

spk_0 moieties that are bound to ATP. So in ATP, the terminal phosphate is fairly far away from the ribose,

spk_0 but it's closer in ADP because you've chopped off one phosphate. And it's a lot closer in NP

spk_0 because you've chopped off both phosphates. And so what you see is the acidifying effect of the

spk_0 ribose becomes stronger and stronger as the terminal phosphate gets closer to it.

spk_0 The reason this is so important is that we're going to see that it's not just about the

spk_0 stoichiometric balance of the covalent changes to the molecules in the pathways. It is also about

spk_0 the relative PKAs of the ionizable groups of all the different compounds involved. So Robert Roberts

spk_0 is, you know, if George Brooks has for decades been the lone voice crying in the wilderness that

spk_0 lactate is not a useless and harmful waste product of metabolism. Robert Roberts from Queensland

spk_0 University and Queensland Australia has been the lone voice crying in the wilderness for the last

spk_0 decades saying that lactate is alkalizing, your acidosis is coming from somewhere else.

spk_0 His papers and what I'm telling you now, in fact, indicate that there's essentially no such thing

spk_0 as lactate acidosis. You can have hyperlactate, you can have hyperlactatemia alongside and in parallel

spk_0 with metabolic acidosis, but the lactate acid is not getting made in the body and it can't cause

spk_0 lactate acidosis. All right. So Robert Roberts has done the best work to date in compiling the

spk_0 relevant factors. And one of the things that he's pointed out, he's not the first to point this out,

spk_0 but building, standing on the shoulders of giants before him outlined the effects of competition

spk_0 between positively charged minerals and hydrogen ions. He's the first to integrate these competitive

spk_0 ion effects with how they would influence all of the different contributions to acidity in glycolytic

spk_0 energy metabolism during high intensity exercise. One of the, and so what you see in this table is

spk_0 he's showing you the different ATP complexes that can form. It can bind to one hydrogen ion,

spk_0 it can bind to two, three or four. It can bind to one magnesium, it can bind to two magnesium,

spk_0 and it can get one proton added with one magnesium or it can bind to potassium or it can bind

spk_0 to sodium. And most of these are very small. If you look over here where you see exponential

spk_0 notation, you're talking about super low quantities. Although if you're talking about exponential

spk_0 notation to the negative two, you're talking about, you know, it's not that small. You're

spk_0 moving a decimal point over two places. But in general, the large, the predominant effects are where

spk_0 you see these single-digit decimals. In the non-competitive column, this is what happens if you just

spk_0 look at the PKA. So the PKA of ATP implies that it should be 23% protonated at pH 7.0. But if you

spk_0 take into account the effects of magnesium, potassium, and sodium, then you come down here and you

spk_0 see that it is 3% protonated at 7.0. Then of course, this changes as you go down in pH. So

spk_0 arresting muscle cells is going to be pH 7 to 7.2. And in exercise, you could easily get down to

spk_0 6.7 and you can get down to 6.2. So as you go down to 6, this is kind of the limit of where you could

spk_0 see high-intensity exercising skeletal muscle. And you see that 75% protonation of ATP is only 24%

spk_0 when you take into account the other cations. But 24% is a lot higher than 3%. So there's still a

spk_0 huge difference with pH. It's just that it's nowhere near as protonated as you would expect from looking

spk_0 at the PKA. Alright, now here we have all the different species that free phosphate can form.

spk_0 And we can see it combined one, two, or three hydrogens. It can bind magnesium, magnesium with one

spk_0 or two hydrogens, potassium, potassium with one or two hydrogens, two potassium and one hydrogen,

spk_0 two potassium, sodium, sodium and hydrogen, sodium and two hydrogen, two sodium, two sodium and one

spk_0 hydrogen. So when you take into account all these different possibilities, it looks at first like

spk_0 phosphate would be 36% protonated at 7.0. But when you look at the net effects, the fact that so many

spk_0 mineral bindings can actually exist in protonated form winds up allowing this to bind an average of

spk_0 1.3 protons. Of course, this goes up quite a bit to 1.8 protons when you go down in acidity.

spk_0 Now the reason this is important is because if you're removing phosphate from a solution,

spk_0 then depending on the balance of cations and the pH and so on, you could be removing 2.6

spk_0 protons, excuse me, you could be removing the phosphate that is binding 2.6 protons.

spk_0 If you remove two phosphates from solution in the GAPDH reaction of glycolysis,

spk_0 where two molecules of glycerol di-3 phosphate and two free phosphate and two NAD wind up producing

spk_0 two NADH and two molecules of 1.3 BIS phosphoglycerate. Now the fact that it can go up to 1.8 at pH

spk_0 6 seems to imply considerable buffering capacity, but the demands of covalent reactions in glycolysis

spk_0 can override that. For example, in the GAPDH reaction, if you need PO4, you're not taking two extra

spk_0 hydrogen ions along for the ride. So it doesn't matter if the phosphate has buffered additional

spk_0 protons if you're going to take that phosphate away, use it in the reaction, and throw away all

spk_0 the protons that are bound to it. So what you see is that on the one hand, the free phosphate has

spk_0 buffering capacity where it gets more protonated in the more acidic state, but on the other hand,

spk_0 certain reactions in glycolysis are going to become more acidifying when they use that phosphate

spk_0 because all the work it did to bind up extra protons gets thrown in the trash when that phosphate gets

spk_0 entered into a chemical reaction. Alright, so now let's go in order. Let's look at the reactions of

spk_0 glycolysis and just put them in order to see how things are stringing together. The two phosphorylation

spk_0 that the beginning are going to each release a proton. That's two protons. This is the bird biochemistry

spk_0 textbook showing the glycerolide-3phosphate dehydrogenase reaction. It shows you that NAD plus

spk_0 becomes NADH and H plus. This happens twice. So it shows you the addition of two more protons. We're

spk_0 at four total now. What it doesn't show you though is the organic chemistry of the phosphate reaction.

spk_0 It totally leaves this unexplained, but that phosphate was coming from what was primarily hydrogen

spk_0 phosphate. A little bit of dehydrogen phosphate, right? An average of 1.3 protons carried by that

spk_0 phosphate. Those protons are not present on the one three bifosphoglycerate molecule. That

spk_0 means they too are released into solution, right? So this is the NAD is stoichiometric. For each NAD

spk_0 plus you're going to get NADH plus H plus. It's going to one-to-one even out like that. But there's a

spk_0 very large non-stochymetric fractional probability-based release of an average of up to 2.6 protons

spk_0 by removing the phosphate from solution. So technically we're not up to four, we're up to 6.6 now.

spk_0 The Lenninger textbook actually shows you the organic chemistry of the phosphate,

spk_0 but it doesn't even mention it in the text or show it in the equation, but it shows you by drawing a

spk_0 box around the PO4 from the HPO4. It leaves the hydrogen out. That box gets added to this carbon.

spk_0 It implies that one of these hydrogens winds up over here and you assume that it's the one from

spk_0 here, but what about the second hydrogen? Where did it go? It's not balanced in this equation.

spk_0 So they're visually showing you that there's a hydrogen and it disappears and they're not telling

spk_0 you where it went. Where it went was another proton that got released into the solution. By

spk_0 the biochemistry textbook does show it consuming a proton, but offers no explanation for where that

spk_0 proton gets used up. You can see that there's a hydrogen here. There's two hydrogen here. There's one

spk_0 hydrogen here and they're all exactly the same. So this H-plus disappears without ever showing where it

spk_0 went and you're left to kind of assume that it got soaked up into the ATP molecule only it didn't

spk_0 because as I showed you before, mechanistically, the substrate level phosphorylations transfer PO3,

spk_0 there's no additional oxygens involved. There's no hydrolysis dehydration synthesis. There's no reason

spk_0 this H-plus should get sucked up into the ATP molecule. In fact, the leningere biochemistry textbook

spk_0 consistent with the other reaction I showed you is more clearly demonstrating the organic chemistry

spk_0 of the phosphate transfer and it shows you that it's PO3 for PO3. There's no hydrogen involved.

spk_0 So the bird biochemistry textbook is actually giving you an extra hydrogen ion not explaining it

spk_0 at all, but by the organic chemistry of the phosphate transfer, it doesn't exist. Now, this is what

spk_0 actually happens for why a proton does get consumed in pyruvate kinase. As I indicated before,

spk_0 that hydrogen is becoming the third hydrogen of the last carbon. You're taking that phosphate away

spk_0 and you're leaving behind the oxygen and that oxygen is going to want to pull in a hydrogen

spk_0 if it can get one because a COH bond is not usually going to ionize. But as it so happens,

spk_0 because of the inaltotron, inaltotron, the ionization that I showed you before,

spk_0 you wind up making that double bond transfer up here. You form a ketone carbonyl and you bring

spk_0 the hydrogen down here to make the terminal methyl group. You're never going to have almost never

spk_0 going to have a carbon-carbon bond having a hydrogen ionize off of it. So that is why you're absorbing

spk_0 the proton the pyruvate kinase reaction. When let-in-ger shows the pyruvate kinase reaction,

spk_0 unlike the Berg biochemistry textbook figure, which states out, I showed you the Berg textbook figure

spk_0 earlier. So the Berg pyruvate kinase reaction is shown up here and it shows you that it doesn't

spk_0 really clearly explain where it goes, but it shows you the proton coming in that winds up making

spk_0 up that methyl group at the end. The let-in-ger textbook does not show you this at all. So it

spk_0 it shows the PO3 transferring and you have a CH2 here, you have a CH3 here and nothing in the text

spk_0 and nothing in the figure explains where that extra hydrogen came from. But it came from a drawing

spk_0 a proton out of solution, which had an alkalizing effect. Any textbook will acknowledge that the

spk_0 pyruvate to lactate conversion converts NADH plus H plus to NAD plus for all the reasons we've

spk_0 already described. The lactate dehydrogenase reaction is intrinsically alkalizing. For the purposes

spk_0 of our lesson here, we're going to consider lactate always the end product glycolysis. These two

spk_0 papers will tell you much more about that. One is called lactate is always the end product glycolysis

spk_0 from 2015, from Rogotsky et al. And one from George Brooks et al. Tracing the lactate shuttle to the

spk_0 mitochondrial reticulum in 2022, compile abundant evidence that lactate is the default end product

spk_0 of glycolysis. So if we look at these textbook descriptions, we see that the Berg biochemistry

spk_0 textbook does show us two protons released in the initial phosphorylation, two released in the

spk_0 reduction of NAD plus, two consumed in the pyruvate kinase reaction, and it wrongly shows us that

spk_0 two are consumed in the phosphoglycerate kinase reaction. So according to the graphics in the

spk_0 Berg biochemistry textbook, there are net zero protons going from glucose to pyruvate.

spk_0 Yet in the stoichiometry, it ignores all of those and only shows a yield of two protons that

spk_0 came from the conversion of two NAD plus to two NADH. It completely ignores all of the other

spk_0 proton transactions that it showed in the graphics of glycolysis. Now, Lenninger shows the exact

spk_0 same stoichiometry, and it's somewhat more self-consistent because it never shows you the proton

spk_0 exchanges in the reactions except for the conversion of NAD plus to NADH. But Lenninger does show you

spk_0 in the graphics that there are phantom magical appearances and disappearances of hydrogens in

spk_0 the molecule and never accounts for how they got there or where they went. And so none of this is

spk_0 to say that the biochemistry textbooks, you know, that the authors aren't good enough to do this work,

spk_0 it seems to me, I mean, I do think there's a mistake in the Berg phosphoglycerate kinase reaction,

spk_0 but it just seems like they don't consider the acid-based balance of the reactions to be important

spk_0 to fully account for the protons. And so they just often leave it out. But that leaves the student in a

spk_0 pretty bad position because you think that's your go-to source when you want to, you know, light bulb

spk_0 goes off in your head and you say, oh, I wonder where the acidity is coming from. And you go to the

spk_0 textbook and they show you some of the protons and they don't show you other protons and they don't

spk_0 tell you what happened to them and they don't put it in the stoichiometry. And so once you want to

spk_0 start understanding a question like where does acidity come from in skeletal muscle glycolysis,

spk_0 you're left needing to go way beyond the textbook to get anywhere when you want to answer a question

spk_0 like that. And I think that's unfortunate because it's not a whole lot of extra effort to show

spk_0 the proton transactions. All right. So that's where we get the net acid-based balance in glycolysis

spk_0 from. And that's why the production of lactate is intrinsically alkalizing. But this is why we know

spk_0 that we do not ever, ever, ever, ever, ever make lactic acid. These figures are from the Berg

spk_0 Biochemistry textbook and they show you on the top the glyceraldehyde-3-phosphate-dirogenase reaction

spk_0 split into two half reactions. And then on the bottom is the phosphoglycerate-chinese reaction.

spk_0 So in this first half reaction, they're depicting this hydrogen shown in blue that is being

spk_0 added to NADH. You then have water which has an OH group that gets added in its place,

spk_0 constructing the carboxyl group, and then the proton that is left over from H2O is the proton

spk_0 that's released into solution. Before that carboxyl group could ever deprotonate or do anything

spk_0 of the sort, we are switching it out for a phosphate group. And that's going to happen through

spk_0 dehydration synthesis. But remember, phosphate is mostly complex as hydrogen phosphate in solution.

spk_0 And so this dehydration synthesis is taking a hydrogen from the phosphate and it's taking OH

spk_0 from that molecule. And that's coming out as water and the phosphate's getting added there.

spk_0 No net transactions of protons, no carboxyl group ionizing. And then in the phosphoglycerate-chinese

spk_0 reaction, we have Berg, you know, wrongly showing us the proton here. But what's most important

spk_0 is the PO3 is coming off being added to ADP to make ATP. And what did you do? You took it off

spk_0 leaving behind the negative charge. So this is the carboxyl group formed in the ionized state,

spk_0 never protonated, never deprotonates to form an acid. You did not form three phosphoglyceric acid,

spk_0 you formed three phosphoglycerate. That three phosphoglycerate became two phosphoglycerate,

spk_0 phosphino pyruvate and pyruvate. And then lactate never became three phosphoglyceric acid

spk_0 or phosphoenol pyruvate acid or pyruvate acid. We're not saying lactate pyruvate glycerate

spk_0 to save syllables. We're saying lactate pyruvate glycerate because these are formed and stay in

spk_0 their anionic negatively charged deprotonated form and never contributed a proton to solution

spk_0 at any point. And you can look at the carboxyl group from the from the point where it is first

spk_0 formed in the phosphoglycerate kinase reaction. And you can carry it through all the other reactions.

spk_0 And you can watch that carboxyl group stay ionized right up until you get to lactate. So the

spk_0 human body does not make lactic acid. It never makes lactic acid. Now what is the fate of that lactate?

spk_0 So as extensively documented by George Brooks, especially in that article tracing the lactate

spk_0 shuttle to the mitochondrial reticulum, we have a cytosolic lactate dehydrogenase that is

spk_0 converting two pyruvate to two lactate and oxidizing two NADH to two NAD+. It's going to travel

spk_0 through the voltage dependent anion channel or VDAC in the outer mitochondrial membrane into the

spk_0 intermembrane space of the mitochondrion. We're kind of in the intermembrane space. We're kind of in

spk_0 the cytosol and that's because the VDAC is very promiscuous about what it transports lactate, pyruvate,

spk_0 NADH, NADH plus hydrogen ions. Any of that stuff gets back and forth through the VDAC, whereas the

spk_0 inner mitochondrial membrane which guards the entry to the matrix, which is the very inside of

spk_0 mitochondrion has very specific channels in its membrane that are very discerning in what they

spk_0 let in and out. And that makes the mitochondrial matrix very different in composition from the

spk_0 intermembrane space or from the cytosol, whereas because of the promiscuous nature of the VDAC,

spk_0 the cytosol and the intermembrane space are going to be fairly consistent in their composition

spk_0 with one another. When the lactate comes in here, it then can be converted back to pyruvate by

spk_0 mitochondrial lactate dehydrogenase. Now, lactate dehydrogenase converting pyruvate to lactate releases

spk_0 energy. Lactate dehydrogenase converting lactate back to pyruvate incorporates energy. It's hard to do

spk_0 that without an energy source. So what you find is that mitochondrial lactate dehydrogenase is

spk_0 physically connected to complex four of the respiratory chain and the heat that is released when oxygen is

spk_0 converted to water in complex four is funneled into the mitochondrial lactate dehydrogenase reaction

spk_0 to convert the lactate back to pyruvate. And then the two pyruvate enter the mitochondrial matrix

spk_0 through the mitochondrial pyruvate carrier where they can be further oxidized in further reactions.

spk_0 Now, when you do that, you convert the NAD back to NADH plus H plus. So you've reversed the

spk_0 alkalonizing effect of the cytosolic LDH reaction. However, you've done two things.

spk_0 One is that NAD plus is needed to keep glycolysis going. You have moved the NADH over here,

spk_0 even though these can go back and forth in the V-dact, now you're way over here. And you've got

spk_0 the take some time to get back over here, right? In the in the approximate vicinity of glycolysis,

spk_0 you've gotten rid of NADH. You've kept NAD plus there. And you've gotten rid of the acidity that's

spk_0 going to inhibit glycolysis. You've moved it over here. More to the point, if the respiratory

spk_0 chain is actively running, that NADH is going to be quickly converted to NAD plus. And then you've

spk_0 preserved the alkalinization. You've preserved the provision of NAD plus. And it doesn't matter that

spk_0 these can get back here because they're going to be very quickly oxidized, retaining the alkalinity

spk_0 and retaining the redox reactivity because of the respiratory chain's activity. If lactate

spk_0 accumulates in muscle, it's going to be because the oxidation of the NADH by the mitochondrial

spk_0 respiratory chain is not keeping up with the degree of glycolysis. But because the mitochondrial

spk_0 respiratory chain is one of your chief alkalinization strategies, then that also means that accumulating

spk_0 lactate is going to be a defense against the rising acidity that would occur under those conditions.

spk_0 An alternative fate for lactate would be to leave the cell. And it can leave the cell to a nearby

spk_0 cell or it can leave the cell into the blood. If it leaves the cell to from a fast twitch muscle

spk_0 fiber to a slow twitch muscle fiber nearby, you're never going to see it rise in the blood. And it's

spk_0 going to get oxidized in the slow twitch muscle fiber. If you exceed the capacity of the slow

spk_0 twitch muscle fibers to do that, you're going to spill it into the blood. It's going to get taken

spk_0 up by the liver that's called the quarry cycle and it's going to be converted into glucose.

spk_0 Either way, you have removed one proton per lactate molecule from the muscle cell because lactate

spk_0 transport goes through a series of transporters in the monocarboxylate transporter family or

spk_0 MCT family, which transport things that have one carboxyl groups such as lactate or pyruvate, etc.

spk_0 And they are H plus simporters. Simport means transport in the same direction. Antiport means

spk_0 transport in opposite directions. These are proton-coupled simporters, which means one for one.

spk_0 Lactate leaves, H plus leaves. So if two lactate accumulate at the end of glycolysis and then leave

spk_0 the cell, they carry two protons with them. So not only did the lactate dehydrogenase reaction

spk_0 remove two protons into the NAD plus formation, the lactate molecule transported two protons,

spk_0 or two lactate molecules transported two protons out of the muscle cell. So you've now doubled

spk_0 the alkalineizing effect of the lactate dehydrogenase reaction and you've tripled your net alkalinization

spk_0 of the alkalineizing phase of glycolysis. This is from a graduate student in Robert Roberts lab.

spk_0 So Roberts was at this 23 years ago. They say the results of our study showed that hexokinase

spk_0 drops pH by 1.52. The glycerol, glycerol-hyde 3-phosphate dehydrogenase reaction

spk_0 coupled to the phosphoglycerate kinase reaction drops at 0.43. And the ATP hydrolysis reaction drops

spk_0 at 0.93. So these are all acidifying reactions. Whereas the pyruvate kinase reaction raises the

spk_0 pH 1.9 and the LDH reaction raises the pH 3.1. Raising the pH 3.1 units is a thousand fold alkalinization.

spk_0 So they conclude down here. These results confirm that lactate production does not cause metabolic

spk_0 acidosis and that biochemical contributors to the development of acidosis include glycolytic flux

spk_0 and NADH plus H plus accumulation and ATP hydrolysis. I mean what an understatement.

spk_0 Lactate production does not cause metabolic acidosis. These results indicate that lactate

spk_0 production makes you 1,000 fold more alkaline. Now granted these are in vitro conditions and

spk_0 Roberts has since done a lot of modeling that includes lots of empirical data in humans with

spk_0 metabolites and in vitro pKa and competitive cation binding where I think the modeling is a lot

spk_0 very superior to the in vitro what happens when I just take this isolated enzyme in solution

spk_0 that was done here. But this provides proof of principle that the early glycolysis reactions

spk_0 are acidifying and pyruvate kinase and LDH are alkalizing. In 2017, Roberts published a paper

spk_0 that in PLOS1 that compiled all the different reactions of non-mitocondrial energy metabolism

spk_0 focusing on glucose metabolism and the phosphogen system. And what you can see here is that the delta H

spk_0 shown on the right is the stoichiometric gain or loss of an electron as a result of the

spk_0 creatine kinase helps mitigate the loss of ATP but that is the same as if you synthesized ATP

spk_0 in the mitochondrial respiratory chain. So the creatine kinase reaction is removing one proton

spk_0 from solution. The AMP-DM and ACE reaction which is what happens when you start to get down to

spk_0 AMP and then you start breaking it down further than that. Ultimately that could go down to uric acid.

spk_0 That also is removing one proton from solution. You're going to be doing a lot more of creatine

spk_0 kinase than AMP-DM-DM-DM-DM-DM-DM-DM-DM. ATP hydrolysis releases one proton, the phosphorylation

spk_0 of glucose and fructose 6-phosphate, each release one proton, the glyceraldehyde 3-phosphate

spk_0 dehydrogenase reaction, gap D-H, it releases one proton. But remember not shown here is the

spk_0 non-stochymetric net effect of removing phosphate from solution, you're removing buffering

spk_0 capacity and you're releasing double the number of protons as you are when you just do the

spk_0 stochomatory. And then the pyruvate kinase reaction is going to remove one proton and looks like

spk_0 this got cut off on the screen but lactate production is also going to remove one proton.

spk_0 This has it all incorporating the pKa's and the competitive cation effects of the relevant

spk_0 reactions and that then adjusts what you would expect based on the stochometry alone.

spk_0 And that means that the AMP-DM-DM-DM-DM-DM reaction is a little bit more alkaline than you would

spk_0 have predicted based on the stochometry. ATP hydrolysis is a little bit less acidifying than you

spk_0 would have predicted. Creatine kinase is basically right on the money at pH 7 but it starts to lose its

spk_0 power slightly at pH 6. And ATP hydrolysis at pH 7 is significantly acidifying but that effect

spk_0 completely disappears by the time you get to pH 6. And that's a result of the fact that as you

spk_0 start hydrolysis and a lot of phosphate the buffering capacity the phosphate starts picking up.

spk_0 Now on the other hand the reaction that uses the free phosphate and removes it from solution

spk_0 because it's removing a buffer from solution it's becoming a lot more acidifying at pH 6 than

spk_0 it is pH 7. So pH 6 is the light bars, pH 7 is the dark bars. So you can see the gap DH reaction

spk_0 is in net you know almost one proton releasing at pH 7 but it's you know more than 1.5 proton

spk_0 releasing when you get to pH 6. Then you can identify a number of other reactions so one that we

spk_0 haven't really talked about is that there's a slight acidifying effect of the phosphorylease

spk_0 reaction that breaks apart glycogen but when you use the phosphorylease reaction you get to glucose 1

spk_0 phosphate and you don't have to use the hexokinase reaction. The hexokinase reaction is much more acidifying

spk_0 in the phosphorylease reaction and that's why going from free glucose through glycolysis instead

spk_0 of coming from glycogen leads to more acidity but that's very mitigated at pH 6. The phosphoryptokinase

spk_0 reaction is considerably acidic at pH 7 it kind of disappears at pH 6 and then when you get to

spk_0 phosphorylease reaction you have an interesting effect here where one three bisphosphoryglycerate

spk_0 which came out of the glycerol that had three phosphate dehydrogenase reaction

spk_0 has a very high ability to bind protons but three phosphoryglycerate which comes out of the three

spk_0 phosphoryglycerate kinase reaction does not and so even though you're not doing a proton transfer

spk_0 in this reaction you are in net changing a substrate from one that binds protons pretty tightly to

spk_0 one that does not bind them tightly and so you do have net acidification here and then you can see

spk_0 pyruvate kinase is substantially alkalizing at pH 7 and it's to lose that effect at pH 6 and

spk_0 lactate dehydrogenase is pretty much always one to one alkalizing so lactate dehydrogenase is always

spk_0 your most alkalizing reaction and it never really changes even as the pH drops to 6 which is

spk_0 beyond the maximum you'd see an exercising skeletal muscle the lactate dehydrogenase stays the

spk_0 most alkalizing reaction when he modeled the effective glucose to pyruvate versus glucose to lactate

spk_0 along pH one thing that you can see is glycogen to lactate is always slightly more alkaline than glucose

spk_0 to lactate glycogen to pyruvate slightly more alkaline than glucose to pyruvate as you go down

spk_0 in pH you unfortunately wind up with glycolysis becoming much more acidifying and so that kind of

spk_0 explains why you know as you approach muscular failure you get closer to it instead of further

spk_0 away right you don't have intrinsic buffering capacity you're burning through more glucose to

spk_0 try to get that last stretch of ATP and as it becomes more acidifying as it becomes more acidic

spk_0 in the muscle that doing more glycolysis gets more acidic you know which which you know it's like

spk_0 the first the closer you get the failure the closer you get and then you fail and then the other

spk_0 thing you notice is that you know when you go to lactate you're way more alkaline than when you go

spk_0 to pyruvate and this reflects the fact that lact making lactate is highly alkalizing in this next paper

spk_0 robergs took data from 25 human exercise trials that provided the time course of metabolite

spk_0 accumulation during exercise at relevant intensities and then the data from biochemical reactions pk a's

spk_0 competitive ion effects etc from the 2017 paper slightly updated and then put these all into a

spk_0 model that said okay if you have a hypothetical exercise session that is totally anoxic and hits

spk_0 failure at the three minute mark what would be the time course effect of the production of

spk_0 acidity from different sources and what you can see here is that at the very beginning the creatine

spk_0 kinase reaction slightly outperforms ATP hydrolysis creatine kinase is alkalizing ATP hydrolysis is

spk_0 acidifying you you'll see that like in the first seconds of muscle contraction the pH goes up

spk_0 a tiny tiny bit and then it starts falling and at first ATP hydrolysis is the powerful acidifier

spk_0 but this gets replaced by glycolysis as soon as glycolysis kicks in and the effective

spk_0 ATP hydrolysis really starts to disappear as you get to the one to one and a half minute mark

spk_0 because ATP hydrolysis becomes net pH neutral at lower pHs due to the

spk_0 due to the proton binding capacity of free phosphate and so what takes over as a source of acidity

spk_0 is early glycolysis but especially the gap pH reaction which is always your most acidic reaction

spk_0 and as you get to failure you know you're not you're not changing the pH anymore once you stop

spk_0 contracting on the top you see the alkalizing factors and as creatine kinase starts to fade away

spk_0 the later glycolysis pyruvate kinase and LDH reactions take over and LDH is always your most

spk_0 alkalizing reaction and it starts to really exceed the pyruvate kinase reaction by a margin as you

spk_0 get deeper into exercise towards the failure point they did not include hexokinase in this model

spk_0 because they were modeling it from glycogen to me when I look at these papers it looks to me like

spk_0 yes glycogen is dominant but you still have a lot of hexokinase going on the limitations that they

spk_0 acknowledged was that this is a specific extreme model of anoxic environment three minutes to failure

spk_0 that's not reflective of a lot of different types of exercise it's one thing to model that this is

spk_0 where H plus is being generated it's another thing to say that this is the cause of muscle pH

spk_0 in order to prove cause and effect you'd have to intervene to stop one of those reactions

spk_0 and show that that alkalizes the muscle or to stop LDH reaction so that it satisfies the muscle

spk_0 so they're not showing the cause and effect behind the pH it's still up for debate they're

spk_0 modeling the different sources of an isolated set of things that can generate protons or remove them

spk_0 they did not model lactate transport which would provide very powerful additional alkalizing effects

spk_0 because they said there's not enough information on the causes of the magnitude the quantitative

spk_0 magnitude of the transport and what causes it to vary under different conditions and the alkalizing

spk_0 effects of the respiratory chain were totally ignored which are the dominant alkalizing effects

spk_0 until your glycolysis starts exceeding its capacity I would add additional limitations that they

spk_0 didn't look at glycerol 3 phosphate I'm going to show you that and I also think you know I

spk_0 so I extracted all 25 of these studies to look for evidence of which where could you see where

spk_0 glycolysis was getting backed up was you know was all of glycolysis running at the same time

spk_0 or were you stopping at a certain point and if you were stopping at a certain point was it

spk_0 at LDH because you ran out of any D plus or was it at PFK because you got too acidic what I found

spk_0 was very few of those studies showed you the difference between those different stopping points in

spk_0 glycolysis you know there's tremendous amount of studies that measure lactate or ATP ADP

spk_0 creatine phosphate there are not many studies that look at fructose 1 6bis phosphate there are

spk_0 you know even I found one study and it wasn't included in the 25 that looked at any representative

spk_0 of lower glycolysis after the gap DH reaction before pyruvate I found one study that was not

spk_0 included in this model so I do think that this model is limited by the very limited number of

spk_0 studies that are actually looking granularly at the different points of glycolysis and so for

spk_0 that reason I did my own analysis just looking at the individual studies to see what additional points

spk_0 they tell us and we'll get to that in a moment but for now I want to take a brief break from

spk_0 the biochemistry of lactate and just say okay what do we know about fatigue and this is a great

spk_0 diagram that was tweeted by Chris Beardley of Strength and Conditioning Research where he listed

spk_0 the mechanisms of fatigue like if lactate gas that doesn't cause fatigue what does right and so

spk_0 he modeled not modeled but he reasoned the effect on ATP in the second column because the point

spk_0 that he wants to make here is that you don't run out of ATP but you have a lot of fatigue mechanisms

spk_0 that prevent you from running out of ATP and so you could regard a lot of these as what is going

spk_0 to happen when the body is in danger of running out of ATP but it needs to make sure it doesn't but

spk_0 as we go through them you'll see that some of them are actually oh damages happening it's kind of

spk_0 an accident that it's conserving ATP and some of them might be purposeful regulation so

spk_0 coordination disruption is when the the ability of the muscle to keep up starts dropping and the

spk_0 brain does not step up its game to compensate and make the muscle keep going. Super spinal CNS

spk_0 fatigue is when discomforting sensations increase the perception of effort and make you want to stop

spk_0 they're eroding your willpower. Spinal CNS fatigue is when motor neurons of the spinal cord

spk_0 desensitize to the amount of the brain telling them what to do and they start to get less sensitive

spk_0 to that so they transmit less signal of muscle. Loss of cell membrane excitability is

spk_0 this is really about like your energy capacity is going down and it's not properly pumping out

spk_0 the ions so your resting potential in a neuron is built on the the ion gradients and if they all

spk_0 get led in through an ion channel to activate the neuron they got to get pumped back out so if

spk_0 you're running into a problem where you're going to run out of ATP if you use enough to keep

spk_0 the neuron excitable by pumping the ions back out then you're between a rock and a hard place

spk_0 in that if you conserve that ATP you are going to be less effective at pump I guess not a rock

spk_0 in a hard place it's just you're at a point where you have to lessen the muscular contraction by

spk_0 one way or another one is you run out of ATP and the other is that you conserve it by making by not

spk_0 pumping the ions out of the neuronal membrane as well and if you don't pump the ions out to conserve

spk_0 your ATP you're going to have the neuron less excitable and it's going to be less stimulatory to the

spk_0 muscular contraction you can have damage from proteases that get activated by calcium ions calcium

spk_0 ions are released as a signal to contract muscle they're released as a signal to release

spk_0 their transmitters and they have to be energetically pumped back into their place and so if you are

spk_0 losing energy to control the calcium ion distribution they can activate proteases that start causing

spk_0 damage to the membranes that are controlling the coupling of neural activity with muscular activity

spk_0 you can lose sensitivity to calcium ions because you are just you know too much too much calcium

spk_0 ion signaling leads to becoming tolerant of it and desensitize to it that makes you contract muscle

spk_0 less and then finally you have accumulation of phosphate and hydrogen ions here's our acidity

spk_0 so it's a major mechanism you know but it's one of seven major mechanisms that he's highlighting

spk_0 and the hydrogen ions are not caused by lactic acid but nevertheless it is a mechanism of fatigue

spk_0 so we do have to understand where it's coming from one of the hypotheses that

spk_0 robergs was not covering that I've seen in the literature and I you know I took this from

spk_0 in a paper called an obsession with CO2 and this is the CO2 hypothesis of acidity and so I

spk_0 looked at some of the papers that were cited in favor of it and this is a paper where they

spk_0 did 30 seconds of maximal exercise on a bike and six people they took muscle biopsies three people

spk_0 they took vein blood samples and they did the exact same protocol and they put them all in one

spk_0 table to compare them so one thing that you can see is that if you look at venous plasma

spk_0 the lactate and mill equivalence per liter is the same as millimoles per liter the lactate goes from

spk_0 one to 13 so it increases 13 fold the muscle lactate goes from six to 47 now on the one hand

spk_0 it's not as great as a fold increase it's 7.8 fold instead of 13 fold but on the other hand the

spk_0 lactate concentration the muscle gets way higher than the concentration the blood and like I

spk_0 said before I don't think or according to robergs we don't know that much about what dictates the

spk_0 quantitative transport we do know that the it goes through the MCT which is a proton

spk_0 simporder but we don't know you know what dictates why the muscle would transfer it at x rate you

spk_0 know you can imagine easy solutions to this right if lactate transport removes protons from the

spk_0 muscle at the expense of acidifying the blood then presumably maintaining blood pH within narrow

spk_0 boundaries that are consistent with life is more important than getting the protons out of the

spk_0 muscle especially when the muscle can tolerate more acidity than the organism can in the blood

spk_0 you can just accumulate high concentrations of lactate and absorb protons that way where the

spk_0 protons are actually not they're actually being removed the total number of protons decreasing

spk_0 when you accumulate lactate it's just moving from one compartment to another when you

spk_0 transport lactate into the blood so you know presumably I would think that this is going to be

spk_0 limited by the fact that you can only transfer protons into the blood at the rate that the blood

spk_0 can tolerate but you can accumulate lactate which has less of an alkalizing effect on the muscle

spk_0 but costs less to the rest of the body and so I think the muscle is going to seek a balance between

spk_0 those two priorities and that's why you have you know considerable increase in the blood and yet

spk_0 really massive accumulation of high levels in the muscle cell to reach 47 millimoles per liter

spk_0 lactate you can also see that muscle CO2 goes from 46 to 106 tour and I try to calculate that out

spk_0 to millimoles per liter and I get that that's 1.41 to 3.25 millimoles per liter and that's

spk_0 increasing 2.3 fold and the hydrogen ions increase 2.5 fold which is pretty close to what the CO2 is

spk_0 doing you know so I can see someone looking at this and I can see them saying well you know the

spk_0 acidity is really following the CO2 CO2 goes up to 2.3 fold hydrogen ions go up 2.5 fold CO2 is

spk_0 mostly the acidity because CO2 is acidic why because CO2 dissolves in solution it becomes hydrated

spk_0 it becomes carbonic acid that dissociates a proton to become bicarbonate well here's the problem I

spk_0 have with that that is that the bicarbonate goes nowhere so the the bicarbonate is going from 9 to

spk_0 7 in the muscle and it's going from 27 to 28 in the blood right so the CO2 is what's acidic

spk_0 how is it becoming acidic if it's not ionizing the bicarbonate right it's and I think you know

spk_0 the explanation is it's pretty simple you absolutely need to go back and forth between CO2

spk_0 and bicarbonate to get CO2 from the muscle cell to the lungs but that happens extremely rapidly

spk_0 with the carbonic and hydrace reaction 13,000 times higher than spontaneous and CO2 rapidly crosses

spk_0 cell membranes and it only takes 5 seconds to get from your muscle cell to your breath so yeah the

spk_0 CO2 partial pressure is increasing but that's just representing a 5 second lag to get out the breath

spk_0 okay on the other hand they publish a study same protocol it's probably the same study is common

spk_0 in when you do small studies you want to get more papers published so you take one study and you make

spk_0 three papers out of it so I think that's what they did but they showed that in another paper that

spk_0 CO2 lagged by minutes right so it's it's immediately immediately it's coming in the breath but it

spk_0 stays high for a few minutes you're not exercising anymore why is that so they reasoned that

spk_0 the CO2 is being stored in the muscle but it wasn't being stored in the muscle is bicarbonate so

spk_0 it must have been stored some other way and they suggested it was forming carbon mate ions on

spk_0 the amino groups of proteins and amino acids and that is a one-to-one release of a proton per CO2 molecule

spk_0 so if carbon mate is forming and is accounting for some of the acidity you got to compare it to the

spk_0 other possible sources so how do you get CO2 it's after you get to lactate you convert it back to

spk_0 pyruvate in the intermembrane space you oxidize the pyruvate with pyruvate dehydrogenase and

spk_0 you enter the citric acid cycle and you run the citric acid cycle and all of that is facilitated by

spk_0 running the respiratory chain by the time lactate is accumulating by definition you are not making CO2

spk_0 out of the lactate now it's not to say that you're not making CO2 anymore it's just to say that the

spk_0 marginal increase in lactate is reflecting the marginal increase of glycolysis that exceeds the

spk_0 capacity for the respiratory chain to meet that marginal increase and the acidity rises when the

spk_0 respiratory chain can't keep up so if the acidity is going up in parallel to the lactate that is

spk_0 reflecting the margin that does not generate CO2 that is acidifying because the respiratory chain

spk_0 could not step up any higher to get more CO2 out of that lactate so you have to realize that by the

spk_0 point that the acidity is happening you it's happening in parallel to you not making

spk_0 CO2 to meet the level of glycolysis so it's pretty unlikely that CO2 is accounting for the

spk_0 increase in acidity that happens when the respiratory chain can't keep up but if we just

spk_0 logic our way through it if you fully oxidize the glucose in the respiratory chain you'd get

spk_0 602 if 602 are 10% forming carbamate that's 0.6 CO2 per glucose molecule by contrast in the first half

spk_0 of glycolysis you can generate six protons so you know that's 10 times the theoretically yield from

spk_0 the acidifying phase of glycolysis as you could get from carbamate even if you're fully oxidizing

spk_0 but again you acidify when you are not fully oxidizing to CO2 so let's look at three individual studies

spk_0 and we're going to focus on this first one eight healthy men performed an isometric contraction on

spk_0 the knee extensor machine at 2 thirds maximal force they took biopsies at rest 20 seconds

spk_0 into the contraction and at the point of fatigue all right so I've put the metabolites that they've

spk_0 measured along with the concentrations at rest at 20 seconds and at fatigue and then I calculated

spk_0 the fold increase at 20 seconds and the fold increase at fatigue and I drew out the pathway here

spk_0 where some of these great out steps indicate that they didn't measure that but it is how you get from

spk_0 one to the to the other that is important to draw attention to the first thing to note is that

spk_0 the lactate is the thing that's most increased the second most increased point is glycerol 3

spk_0 phosphate we have not talked about that yet but glycerol 3 phosphate accumulates as a side

spk_0 reaction when d-hap does not get metabolized through gap dh so if any d is not abundant and the

spk_0 gap dh reaction doesn't go forward then d-hap accumulates but if any dh is abundant and d-hap

spk_0 is abundant then that will drive forward the reaction to form glycerol 3 phosphate I believe the

spk_0 primary drivers of this reaction going forward are going to be the concentration of d-hap

spk_0 and the high n-a-dh to n-a-d plus ratio I do think it's possible that there is some influence

spk_0 of pH because based on the little research that I did it looks like the pH optimums of these

spk_0 enzymes are a little bit different such that acidity is going to favor the production of glycerol 3

spk_0 phosphate but I think it's going to be mostly driven by the n-a-dh to n-a-d plus ratio

spk_0 and you know to some extent also the degree to which the influx is inhibited or not

spk_0 right so the influx into d-hap is high and the n-a-d plus is low and the n-a-dh is high

spk_0 you're going to get a lot of d-hap and a high n-a-dh to n-a-d plus ratio and that's going to drive

spk_0 d-hap into glycerol 3 phosphate instead of down through glycolysis it's also the case that

spk_0 glycerol 3 phosphate is able to deliver electrons to the respiratory chain through what is called

spk_0 the glycerol 3 phosphate shuttle which is using an enzyme called mitochondrial glycerol 3 phosphate

spk_0 dehydrogenase and it does make sense that if the flux through glycolysis expands in exercising

spk_0 skeletal muscle you're going to need to have more glycerol 3 phosphate in order to turn over

spk_0 those electrons because it's not disappearing when it does that it's it's cycling right so you will

spk_0 need some more glycerol 3 phosphate but you only need enough to accommodate the flux to the pathway

spk_0 and if you look at glycerol 3 phosphate being 28.7-fold increased compared to the metabolites on

spk_0 either side of it being 2-3-fold increased then you're around an order of magnitude higher than

spk_0 you need to be to accommodate the flux going through the pathway so clearly this is not a reflection

spk_0 of the use of the glycerol 3 phosphate shuttle primarily it's a tiny bit that and it's mostly

spk_0 a side reaction to generate NAD plus and to get rid of acidity. It also appears that there is any

spk_0 addition of the phosphofructokinase reaction or PFK reaction which converts fructose 6-phosphate to

spk_0 fructose 1-6-bisphosphate why don't I say that because if you look at glucose 6-phosphate and fructose 6-phosphate

spk_0 they're both way more accumulated than fructose 1-6-bisphosphate. In fact I added them up and I

spk_0 took the ratio and there is 6.7-fold greater accumulation of hexos monophosphates that's glucose

spk_0 and fructose 6-phosphates then there is fructose 1-6-bisphosphate. So I would say the third thing that's

spk_0 the first thing that's happening here is lots of lactate production. The second thing that's

spk_0 happening here is lots of glycerol 3-phosphate production and the third thing that's happening here

spk_0 is a considerable amount of PFK inhibition. Like I said earlier PFK is strongly inhibited by acidity

spk_0 and so you're responding to the fact that the gap DH reaction is the most acidic reaction in

spk_0 all of glycolysis. So you don't want to press it forward under conditions of acidity. In fact

spk_0 as Robert says shown it gets more acidic the more acidic you are. So running gap DH is a vicious spiral

spk_0 towards never-ending acidity and you don't want to run it if you're not able to complete the

spk_0 rest of this going forward. In particular when your respiratory chain being limited is the

spk_0 primary driver of acidity. So there are a number of compensations that are happening here

spk_0 but it's pretty clear that lactate production while it's the biggest one is not capable of

spk_0 fully regenerating enough NAD to keep gap DH running forward enough that you wouldn't instead

spk_0 run the glycerol 3-phosphate production. It's also clear that that situation would be a lot worse

spk_0 if PFK weren't inhibited by acidity and so you have basically a trickle through middle glycolysis

spk_0 because you are driving the middle into a side reaction and you're inhibiting the top and you are

spk_0 essentially accumulating acidic exos monophosphates at the expense of risking running the gap DH

spk_0 reaction without being able to get it all down to lactate because that risk would be

spk_0 running the most acidic part of glycolysis but that's you know it's not all driven by acidity

spk_0 inhibition. It's also driven by collateral damage of the acidity is also the low NAD supply

spk_0 and that's really what's driving this side reaction. So when I look at this and I look at the

spk_0 Roberts modeling papers which I think are amazing I do think that missing glycerol 3-phosphate

spk_0 production is a major missing piece of them and I also think that you know PFK inhibition is

spk_0 kind of a wild card where the degree of PFK inhibition might vary in different studies and that's

spk_0 going to be a really big determinant of how much running gap DH is contributing to acidity.

spk_0 And then I also think that we might be underestimating the hexokinase reaction here. That was kind

spk_0 of left out of the Roberts models to my understanding because the assumption is you're coming down from

spk_0 glycogen under these conditions and I think you are you know but you have a lot more G6P

spk_0 than G1P which I think indicates that even though your fold increase in G1P is almost double your

spk_0 even though your fold increase in G1P is considerably higher than your fold increase in free glucose

spk_0 almost double. The increase in glucose 6-phosphates hard to say where it's coming from but it just

spk_0 it just seems like you you could have plenty of hexokinase going on these conditions and so

spk_0 when I look at you know what are the lingering sources of acidity here.

spk_0 Haxos monophosphate accumulation is the most acidic thing that's not being considerably inhibited

spk_0 in all of glycolysis. So I do I think it's possible I'd love to talk to him about it but I do

spk_0 think it's possible that the Roberts model might be underestimating the contribution that you

spk_0 can get from hexokinase under conditions where Haxos phosphates are accumulating. PFK is strongly

spk_0 inhibited and GAPDH is largely driven around. Now I couldn't find any other studies that looked at

spk_0 three phosphoglycerate or any other reflection of the alkalizing phase of glycolysis prior to

spk_0 this study showed that glycerol 3-phosphate goes up 23.6 full during maximal exercise which I

spk_0 think is consistent with the last study showing that glycerol 3-phosphate production is very important

spk_0 and this study showed glycerol 3-phosphate went up 12.8 full during electrical stimulation of

spk_0 contraction. So there is a pretty consistent finding that when these studies look for glycerol 3-phosphate

spk_0 they do find that it is not as significant as lactate but very considerable. So what can we

spk_0 conclude? First of all the primary culprit in acidity is that the alkalizing respiratory chain

spk_0 is lagging behind acidifying glycolysis. That is number one. Number two within that context

spk_0 if you look at the molar sum of glycerol 3-phosphate and lactate when you start to it failure

spk_0 72.4 that is out competing the increase in inorganic phosphate of 52.8 which is fairly close to

spk_0 the accumulation of hexos monophosphates at 41.9. So that indicates that NADH plus H accumulation

spk_0 which is reflected in the glycerol 3-phosphate and lactate accumulation. That is the greatest

spk_0 driver of acidity followed by ATP hydrolysis followed by hexos monophosphate accumulation.

spk_0 I was saying before that the hexos monophosphates look like they are the greatest accumulation

spk_0 that is contributing to acidity. What I mean by that is lactate and glycerol 3-phosphate are

spk_0 fixing the problem of NADH plus H accumulation. The fact that they are so high reflects that the

spk_0 accumulation is the primary driver of acidity but it is also the one that is primarily being fixed

spk_0 by reversing that accumulation in the production of lactate and glycerol 3-phosphate.

spk_0 So what I mean when I say the hexos monophosphates are the ones left standing is that they are the

spk_0 ones that aren't being fixed. They are not being cleared and they are acidic. So I think that is

spk_0 kind of the elephant standing in the room is that quite often they are left as the one thing that is

spk_0 not resolved that is a very acidic. ATP hydrolysis is going to be the driver of acidity that is reflected

spk_0 in the inorganic phosphate accumulation but Robert has shown that ATP hydrolysis stops being acidic

spk_0 as you go forward. So I think looking at the inorganic phosphate left over at fatigue is not really

spk_0 how you can measure it and his modeling indicates that about 25% of the acidity that would be generated

spk_0 across three minutes of anoxic contraction to failure is generated by ATP hydrolysis.

spk_0 Carbomate formation may make some contribution but as lactate rises this is reflecting the margin at

spk_0 which carbohydrate no longer generates additional CO2 and there really is no hard evidence of carbon

spk_0 made accumulation in the muscle. Like I said before it has been hypothesized that the delay

spk_0 and CO2 release is a result of stored CO2 in the muscle but it might just be that once you

spk_0 are not exercising anymore now you are oxidizing that lactate and you could be replenishing glycogen

spk_0 but the point is it is an open question about the degree to which carbomate formation might be a

spk_0 contributor to acidity in exercising skeletal muscle. The primary defenses against acidity are

spk_0 lactate production glycerol three phosphate formation and PFK inhibition. PFK inhibition is

spk_0 slowing the input into the highly acidic gap DH reaction. So what does this mean for lactate

spk_0 supplements? Andy Galpin tweeted out this 2024 August paper on a randomized controlled trial

spk_0 crossover design looking at a lactate supplement and you know the the the TLDR of it is

spk_0 they increased the amount of work done by 4% with the oral lactate supplement and it didn't

spk_0 impact anything else that they measured. If you look at what they did they gave people a calcium

spk_0 magnesium vitamin D3 lactate pill with 372 milligrams of lactate and every 50 pounds of body weight

spk_0 got one more pill so if you weighed 150 pounds you would have gotten they rounded up to the

spk_0 I guess they rounded up to the nearest 50 pounds or not even the nearest but to the next 50 pounds

spk_0 I don't see what the point of measuring this against the placebo is like we all know that adding

spk_0 calories before a workout is going to increase performance usually and so

spk_0 I think it would have been much more sensible to compare this against

spk_0 isocaloric glucose instead of a blank placebo they don't clarify

spk_0 whether it was isocalorically controlled and I think that makes a difference in interpreting

spk_0 whether this 4% increase in work is a reflection of the uniqueness of the lactate supplement

spk_0 versus the coloric supply. If you look at the discussion they they overview the other

spk_0 lactate supplement data so time to exhaustion during constant load was unaffected by the addition

spk_0 of lactate to a carbohydrate sports drink. Bicarbonate and pH were greater during three hours of

spk_0 constant load cycling following following ingestion of a polylactate solution but no mention of a

spk_0 exercise performance benefit. Time to exhaustion during short duration high-intensity

spk_0 treadmill exercise was barely extended less than 2% by high doses of lactate ingestion.

spk_0 Neither 20 nor 40 kilometer time trial performance was influenced by lactate ingestion.

spk_0 Okay so so far every performance related metric is negative in all of these studies. Time to

spk_0 exhaustion during high-intensity cyclurgometer exercise was appreciably extended 17% by high doses

spk_0 of oral lactate so that's the one outlier. More recently other commercially available supplements

spk_0 containing lactate have been shown to have unappreciable effects on skeletal muscle endurance during

spk_0 resistance exercise but interpretation pertaining to lactate supplementation per se was complicated

spk_0 by the addition of other potentially active ingredients. All right so as it stands there's quite a

spk_0 number of studies all negative regarding actual performance one showing a you know a pH effect

spk_0 but this 117% increase in high-intensity cycle or gamma or exercise time to exhaustion is the one

spk_0 outlier. So if we look at that study the reason I the reason I I struggle to take this seriously is

spk_0 the lactate is 20 120 or 220 milligrams of lactate per kilogram of body mass in the form of calcium lactate

spk_0 we're just we're looking at like on the order of like 15 grams of lactate and that's a considerable

spk_0 amount of energy being provided to compare it to blank placebo right the placebo is aspartame in water

spk_0 right so it's you're definitely getting you know a potentially significant caloric source and I

spk_0 just don't think that's the right control to try to fair it out whether there's something unique

spk_0 about lactate and giving you a 17% extra time to exhaustion so I just I honestly don't take these

spk_0 trials all that seriously unless they're isocloric and they're overwhelmingly negative anyway

spk_0 so the way that I view that is as follows everything that we said about lactate so far means that

spk_0 lactate does what it does best when your cells make it from pyruvate if you make lactate from pyruvate

spk_0 it generates NAD plus if you make lactate to pyruvate from pyruvate it alkalinizes you lactate

spk_0 supplied exogenous is not going to alkalize you it it really can't because um you know there

spk_0 could be some varying indirect effects but the pka of lactate is down in the mid-three which means

spk_0 that it's not going to absorb a proton unless your a thousand times more acidic they need

spk_0 ever get in in in a maximally exercised muscle cell and if you oxidize lactate into pyruvate

spk_0 in order to burn it for energy you are generating NADH in the process and you are releasing a proton

spk_0 the process which is going to be acidifying it's not going to generate NAD plus and it's not going

spk_0 to generate it's not going to generate an alkalinizing effect so I just I just don't see how it is

spk_0 going to provide the same benefit that you would get when you make lactate yourself so I think

spk_0 better strategies would be ones that we didn't really cover here for example boosting intercellular

spk_0 carnasean levels with beta alanine supplementation can increase your muscle specific buffering capacity

spk_0 acutely bicarbonate glutamine and organic acids such as citrate alpha-ketoglutarate, malate,

spk_0 fumerate, succinate can all be bicarbonate sparing because they are anaplorotic and they

spk_0 fill the citric acid cycle without needing the pyruvate carboxylase reaction which sucks bicarbonate

spk_0 out of the system and so all of those can be alkalinizing bicarbonate you know bicarbonate you

spk_0 are acute buffering capacity and it makes a lot of sense to also train for increasing your

spk_0 endogenous productions against buffering acidity such as if you have you know an I'm not a trainer

spk_0 and I'm not an exercise physiologist but just reasoning through this if you had a performance event

spk_0 three months from now and it's you know your off season now or six months from now let's say

spk_0 you have a performance event that six months from now you might want to spend three to four months

spk_0 training without the crutches to increase your endogenous buffering capacity and then you might

spk_0 want to load beta alanine to improve your carnasing stores as you are going a couple of months into

spk_0 the performance event and then you might want to supplement strategically around specific training

spk_0 sessions in a periodized manner or the actual performance event to acutely increase your

spk_0 buffering capacity even beyond what you've trained endogenously when the time requires it

spk_0 and I do think it would be very interesting to study lactate more I just don't think that lactate

spk_0 supplements make the most sense in the context of viewing the alkalizing effects of lactate

spk_0 it's producing lactate endogenously that is alkalizing it's transporting it out of the

spk_0 muscle cell not into the muscle cell that's alkalizing and so while lactate is an excellent fuel

spk_0 source and not a metabolic waste product I think most of the benefits you get from lactate come

spk_0 from when you make it out of pyruvate all right this has been masterclass with master john energy

spk_0 metabolism lesson on how lactate alkalizing your muscles if you are not taking the full course

spk_0 and you've only seen this video check out the link the description to the full course and I hope

spk_0 to see you in the next class

How Lactate Alkalinizes Your Muscles

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