Professor Dom D'Agostino: Finale

They're not fermentable, but I think ketones can be potentially used as a precursor for the biosynthesis of lipids. So I know that acetoacetate plays a big role in synthesizing myelin. So for neuroregeneration, that's why ketones are quite remarkably effective for traumatic brain injury, because they can help repair the myelin sheath. And if you radiolabel acetoacetate, then a lot of it gets incorporated into myelin, which is a fat-like substance or fat-containing substance. And I think maybe other people have shown too, with radiolabeling and tracer studies, that ketone metabolism for energy is marginally sufficient. And I think it's not maybe all or none, but I think generally speaking, ketones require oxidative phosphorylation to generate ATP from that. But I do think they could be directed for biosynthetic processes. And we know that a big part of the Warburg effect, we're talking about metabolism, but a big part of that is to redirect metabolites for the biosynthetic expansion of the tumor biomass.

And that could be achieved in part by ketones feeding into that. I mean, you could say that about anything. You could say that about anything that you eat, amino acids.

Yeah. And I mean, we've published, and Tom Seyfried is on the paper. I think the title of the paper, it's International Journal of Cancer, a good journal, and it said ketone supplementation extends survival and suppresses tumor growth or something like that. It's International Journal of Cancer where we actually used 1,3-butanediol and we used a ketone ester, and that worked in that way. Probably, you know, in hindsight, we would pivot to use some of the other compounds, but we also took the ketone salts, the DL ketone salts, and we grew the cells in culture, and we actually grew them in 25 millimolar of glucose because that's how you actually, so that's, yeah, that's, I mean, that's actually the standard protocol that Tom Seyfried sent us to grow the cancer cells, and it just makes them like super highly aggressive. So in the presence of 25 millimolar of glucose, we added the ketones, and the ketones decreased cell growth and decreased proliferation, and also increased dead cells. So we did ethidium homodimer-1 and calcein to look at live-dead cell analysis, like a standard kind of assay for that. So we saw suppression of tumor growth, of cancer cell growth suppression, and we also saw greater cell death by not changing anything, the glucose in the media, not changing glucose or glutamine, but simply adding ketones. And that study was done by Dr. Angela Poff, and that was part of her PhD dissertation. Prior to her doing those studies, I had a medical student do that, and the title of her poster for her student presentation was that ketones kill cancer. I remember a lot of students are, and I was like, okay, the results seem sometimes like students could be basically picking cells out. So when Angela came into the lab, her thing was to replicate that finding. And I think we might have used a couple different, there were brain cancer cell lines, but she replicated it. And I think others have actually, there was a paper by Skinner, and he was at University of Florida, and I tried to get in touch with him because he used a neuroblastoma cell line, threw on ketones in a dish, and saw more cell death and less proliferation. And then Eugene Fine from Einstein did it with acetoacetate. And then my student did it, and it was like, wow, these seem too good to be true. And then Angela replicated, and then we ended up publishing that.

Nope.

Yeah. We use like a serum that has like glutamine in it, and maybe we supplement it a little bit. And we use like DMEM, and we have like a whole cocktail, and we grow the VM-M3 cells with like, you know, a solution that basically makes them grow as fast as possible. So the idea is make them grow as fast as possible so we can get enough cells, and then we collect them, and then we inject them into the flank of the tumor, and then they just, it's a syngeneic model. So they just like, they just go, the cancer cells go to the liver typically. They pepper the liver with all these micrometastases. It's really gnarly, disgusting look. It's like oozing tumors coming out of the liver. And then the cancer cells also, they go to the kidney, they go to the lungs, and then after about a week to two weeks, they get into the brain and they start causing brain tumors. So that is what's kind of unique and controversial about this model is that you can inject it into the flank, and then it actually causes, you know, brain cancer. So most glioblastoma cells and cell lines, it's very controversial, but most people who study glioblastoma kind of say that, you know, a glioblastoma should not grow outside of the brain, and that's what's kind of unique about this tumor. And Tom would argue that, and he like, you know, I had some pushback about this model, and he was like, it's turned on. I mean, there are, I guess, reports where someone had maybe gotten a liver or something from a brain tumor patient, and then that liver had like the glioblastoma cells. So there is evidence of glioblastoma cells going and causing metastasis to organs of the body.

Yeah, Tom Seyfried has sent you a couple papers that will convince you that it's at least possible. But I do think that there's so many mutations in these GBMs that they're just so glycolytic, and they also have like a phenotype, like, you know, they have a unique phenotype. They're like a tumor-associated macrophage, right? And they're a bit different than, they're like that, you know, this transformed glioblastoma cell that's like a tumor-associated macrophage hybrid kind of thing. But I do think that it's, you know, that model is one of the best models out there, and they're kind of unique to study because I also think they suck up glucose and glutamine, and I think that's relevant to this discussion and also highly relevant to creating therapies to manage this disorder.

Yeah, the senior author, yeah, Thomas. Yep.

Yeah, it was on the figure. I wrote the section. It wasn't super, yeah, it wasn't super, super prominent in the article.

No, I mean, in hindsight, I should have spent more time expanding that, you know, oxidative stress. But I think the important take-home message was that ketone metabolic therapy is putting the patient into a glucose ketone index of one to two. I push back a little bit and say one to four. And it deemphasized anything that was not uniquely metabolic. So it deemphasizes, you know, hence the title of the paper, right? The framework for ketone metabolic therapy for glioblastoma. So I think maybe I wanted to get more of the hyperbaric in. Also, the hyperbaric, we don't know the dosage, the timing, and then the scheduling of that. Also, I have to say, as someone who studied oxygen toxicity seizures, is that when you have a glioblastoma, I am hesitant to tell people to jump into a hyperbaric chamber at the standard, you know, hard shell chamber, shall we say, dosages of 2.5 atmospheres or three atmospheres, which we have published on, right? Because having a tumor makes you much more vulnerable to having a seizure that could be evoked by oxygen toxicity. So my recommendation is to do like, you know, low-dose oxygen, like 1.3, 1.5, do that for a couple weeks, and you have to understand that that's more like the bleeding edge of science. So this idea of creating, you know, to metabolically compromise the tumor and then hit it with an oxidative stress. So I'm almost more likely to recommend something like metformin or recommend some other drugs like IV vitamin C, right? So you give millimolar concentrations of circulating vitamin C functions as a glucose antagonist. Ascorbic acid uses the same transporter as glucose. So it's functioning that way, and also the high dose of vitamin C functions as a pro-oxidant. It drives something called the Fenton reaction, and that you could actually enhance iron-induced oxidative stress. And we talked about the ferroptosis overdrive hypothesis being very prominent, especially in GBM. So driving that. So I'm a little bit more comfortable mentioning that. And with the hyperbarics, I think there's a lot of potential for hyperbarics, but I think it has to, not everybody has access to that too. So that becomes, you know, an accessibility problem for patients that just do not have access.

Well, I gotta say, I mean, like just north of me is the Aviv Clinic, and they have like literally tens of thousands of, you know, older patients that they do hyperbaric oxygen therapy for longevity, and they have greater, you know, cognitive function. They do better reaction times, decision-making. They're using it as a cognitive enhancer. It stimulates the production of stem cells and the mobilization of those stem cells to hone in on sites of injury and inflammation. It has a lot of remarkable properties. The folks in Israel showed it lengthens telomeres. So I'm a little bit less convinced of that, although, or if that's beneficial, not I believe in the results, but maybe the interpretation of the results. So, and you know, I'm in touch with, you know, NFL guys, sports teams, and people. So hyperbaric oxygen, what it does, it enhances wound healing. Like everybody, I mean, it's FDA approved for that. So when you're banged up on the football field or you're like a special operator, you're just like really going at it in the gym, right? And you get inside a hyperbaric chamber, it's really augmenting those healing processes. And it's doing it, it's creating oxidative stress, but the adaptive response to the oxidative stimulus and the repair mechanisms are what happens. Right.

So, and I think that could be different in older patients because their adaptive response to an oxidative stress stimulus can be different. So, I know, I mean, I'm in my 50s now. So when I go and I do like a super brutal squat workout or deadlift workout, it takes me like five days to recover now because I don't have like, you know, the testosterone and all that hormone stuff that I did when I was 20, right? And I recovered like in a day or two. But now, I kind of liken it to that, right? So if you go and do a super hard workout and then take blood work and look at that blood work and say, well, whatever you did, stop doing that because you have all these, you know, inflammatory markers, oxidative stress. The same thing kind of happens with hyperbaric oxygen therapy. But it's the training stimulus and the adaptive response to that stimulus that produces the beneficial effect. So that's, I'm a firm believer that if you're in a state of therapeutic ketosis and you do hyperbaric oxygen therapy, the adaptive therapeutic beneficial effects of that hyperbaric therapy will be greatly augmented, and you'll also decrease the potential for oxygen toxicity. I mean, that's clear, the anti-seizure effect. And it tends to negatively impact the cancer cells and positively impact the healthy cells.

No, like all of our literature is actually without ketogenic. All of our literature that's not from my lab, right? So we're the ones that, but what I can say, you know, there's 14 different FDA-approved applications. So one is radiation necrosis for cancer patients that have, you know, damage from radiation therapy. And it was observed in attending the Undersea and Hyperbaric Medical Society meetings, like they would show these case reports and like, oh, the patient like healed up and somehow, you know, the tumor went into remission and the patients had, so, but they were talking about it, you know, by the way, like here's what hyperbaric oxygen did to reverse this radiation necrosis and enhance, and this cancer patient just seemed to be like, you know, it was one after another, like these patients were doing better. When we do hyperbaric oxygen therapy with the standard diet, we did not see an increase in survival. There was like a little bit of a trend, but it didn't improve survival statistically significant. You know, if we did a cohort of 50, maybe we would have had statistical significance. But what it did do, it enhanced the tumor suppression effect of the ketogenic diet. So there's some unique synergy there. And I think, you know, that if you have cancer and you're undergoing radiation therapy, insurance will cover it. But the more you improve your metabolic health, I could say if your glucose is high and you're in and you're, you know, and you don't have good metabolic health, I worry about the oxygen somehow augmenting maybe, you know, inflammatory processes that could, you know, worsen the situation. But I think each case needs to be, I mean, we are, each individual is like a unique metabolic entity, and I think hyperbaric oxygen needs to be titrated to the individual. And what we don't have are biomarkers that we can look at as people are going through hyperbaric therapy to titrate the dosage, timing, and scheduling of the hyperbaric therapy. So this is like, you know, what we always discuss when we get together as hyperbaric, you know, experts in the field, like, you know, we need to, and there's a couple biomarkers that the best one we have now is HSCRP. So if you have a patient and their HSCRP keeps climbing with hyperbaric oxygen therapy, which I've seen on the five-day-a-week schedule, back that off to three days a week, Monday, Wednesday, Friday, and then you often see, you know, they continue to get benefits, but the HSCRP goes down. That means the oxidative stress load over time, they're adapting to that.

Yeah, that's a huge, that's a big question, and I think it caused a lot of confusion, right? Because people just breathe 100% oxygen, right? You're going to, interestingly, and I've done the experiments back when we used to do like larger animal experiments. If you measure, I'll start off by saying this. If you do an experiment with an animal model experiment or just take a human and you put an oxygen electrode in the human's brain and you measure the partial pressure of oxygen in the brain tissue breathing normal air and then you put a mask on them, which is 100% oxygen, what do you think would happen to brain PO2?

No, it goes down. It goes down, and it goes down for a number of reasons. So when you breathe 100% oxygen, it causes hyperoxic vasoconstriction. So unless you titrate in CO2, and that's called carbogen, right? So that's another thing that we could talk about. Yeah, breathing 100% oxygen actually causes what's known as hyperoxic vasoconstriction. And this, I was not fully aware of that until we were doing the experiments, and I was like, this can't be right. Let's switch the oxygen electrode. And then I was like, oh, okay, yeah. And it's in the books, actually, like in old, you know, physiology books, maybe not like in medical books.

So under normal conditions, a normal healthy person's hemoglobin is saturated, right? And hemoglobin is how we get oxygen into the cells. When you breathe 100% oxygen, hemoglobin is already pretty much saturated. You know, if you're on the sidelines of a football field or you have a patient with COPD and you throw 100% oxygen, you know, you might jump it up a few percentages. So you might increase it a little bit, but for all intents and purposes, you know, hemoglobin is saturated and you're not going to get more oxygen into the tissues, right? You may actually decrease it through vasoconstriction. So what you have to do is you actually have to increase the partial pressure of oxygen. The dose of oxygen is a function of the concentration of oxygen times the atmospheric, the pressure, the barometric pressure. So that will dictate the partial pressure of oxygen in the tissues and in the blood. So by increasing the barometric pressure, you're pushing more oxygen into the liquid phase of blood, and that's called the plasma. If oxygen is at a higher concentration in the plasma phase, then that gets into all the nooks and crannies of a tumor that has erratic, you know, blood vessel growth, right? So a tumor, the core of the tumor becomes hypoxic because the red blood cells kind of get trapped in like the erratic blood vessels and things like that. Hyperbaric oxygen, by virtue of pushing the oxygen into the liquid medium, the plasma, rapidly reverses tumor hypoxia and superoxygenates the tumor by getting the oxygen into the actual liquid phase, right? And when you do that, the mitochondria are all screwed up and damaged. And when those mitochondria, you know, their tissue PO2 goes from, you know, three millimeters of mercury to two thousand millimeters of mercury from that. That causes a massive oxidative burst. That's kind of site-specific, right? Like the healthy cells can handle that, but the tumor cells are used to thriving in a low-oxygen environment, pumping out lots of lactate that like protects them from the immune system and things like that. So you reverse the tumor hypoxia, you hyperoxygenate the tumor, and then that tumor actually starts to die from the high concentration of oxygen making reactive oxygen species. But if you were to then hit that tumor with radiation, then boom, like actually the radiation, when you hit the tumor with radiation, it kills the tumor by generating reactive oxygen species, which is entirely dependent upon the PO2 of the tumor.

Yeah. Yep. So there are a lot of clever ways that you could reverse tumor hypoxia, hyperoxygenate, and use a toolbox of different drugs to increase that oxidative stress five to ten or maybe even a hundred times higher, especially if you knock out like a few key enzymes with relatively non-toxic compounds. And that's the direction I...