Utilizing Stable Isotope Tracers in Preclinical Models of Obesity

Marshall McCue, PhD.

Case studies using small mammals where 13C-labeled nutrient oxidation is tracked second-by-second in real-time using the newest laser-based stable isotope approaches. This webinar is hosted by InsideScientific.

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Liam: Welcome everyone to the second webinar of obesity 2020, a joint webinar series brought to you by Inside Scientific and the American Physiological Society. This is Liam Sanio from Inside Scientific, and I’m very pleased to be your host for today’s event. Between now and December, we have a number of webinars lined up, all focused on the science and technology advancing obesity research. Today’s webinar is titled “Utilizing Stable Isotope Tracers in Preclinical Models of Obesity,” and will feature Dr. Marshall McCue, Chief Scientific Officer from Sable Systems. Marshall will present case studies using carbon-13 to assess metabolism and nutrient oxidation in real time using the newest laser-based stable isotope approaches. And with that, I’m pleased to welcome Dr. Marshall McCue. Marshall, thanks so much for joining us today, and the floor is yours whenever you’re ready.

Dr. McCue: Thank you, Liam, and thank you all for joining this morning. Thanks also to Sable Systems for sponsoring this presentation. So, I’m going to start this webinar by conceding that obesity is a complex issue. It involves cultural and psychological and physiological processes. But at the center of this story is the concept of energy balance, or in the case of obesity, energy imbalance. And as such, I want to focus this presentation on how we can use emerging experimental approaches to gain new insights into mass and energy balance to help us better equip ourselves to study obesity.

Now every good story begins with the familiar and then moves on into the unknown. So, let me start by reviewing some of what we’ve already learned about the behavior in energetics from studies of obese rodent models. These insights were obtained using phenotyping cages that monitor several parameters coincidentally, including movement, feeding and drinking events, and it does this every second for days or even weeks at a time. Now, these systems also simultaneously measure an animal’s energy expenditure, namely their rates of oxygen consumption and carbon dioxide production in real time.

So, these are some graphs summarizing the data collected on 22-gram control mice and 62 gram obese mice. Now there were 8 mice in each group and I’m just showing the mean responses here. Now note that in these and some of the future graphs, I use gray shading to denote the nighttime periods. We call that SCOTO phase. And in the first graph we can see that the total energy expenditure is higher in the obese mice. Well, this makes sense because they’re larger animals. Obese mice show the same average respiratory exchange ratios as control mice, but we can see that the circadian variance is slightly attenuated in these obese mice. Not surprisingly, the feeding behaviors are higher in these obese mice, but the drinking behavior is about the same between these two groups. Locomotion is greatly reduced in the obese mice, especially at night when the control mice are typically active and running around their cages. Now, note that we did not use running wheels in these trials because it would really be unfair to these obese mice. In fact, they rarely, if ever, run on a wheel voluntarily. Finally, and I think most interestingly, is that obese mice need less food to fuel a given amount of energy expenditure. This suggests that they’re simply more efficient from a metabolic perspective.

So today I’m going to propose that the condition obesity is in part a disorder of carbon flux. And the reason why I say this is because carbon comprises 45% of the dry mass of non-obese humans and rodents. This value can easily exceed 60% in the case of obesity. And the primary reason for this is that lipid molecules are particularly carbon rich, with 70% of their molecular mass coming from carbon atoms. So, it would make sense that an understanding of overall carbon flux could help us better understand obesity. Fortunately, nature has given us two naturally occurring isotopes of carbon that we can use to track carbon fluxes in the body. One is carbon-12, which has six protons and six neutrons, and this is the carbon that we typically think of that makes up 99% of the carbon in our bodies. The second isotope is called carbon-13 because it has six protons but seven neutrons, so it’s just a tiny bit heavier. Now this isotopically heavy carbon naturally makes up about 1% of the carbon in our bodies. Now because the isotopic composition of the exhaled CO2 is dependent on the isotopic composition of the substrates that the body is oxidizing, we can start to explore carbon flux in new ways. Another bonus here is that new types of laser-based mass spectrometers are so sensitive that they can measure the amount of this heavy carbon exhaled by a single mouse in real time, second by second. Now I’m not going to bog you down in the technical details, but this device works by measuring the decay of a laser pulse through a gas sample, and it uses mirrors to create an optical path length that’s actually several kilometers long, but it fits in this box.

Now before I get deeper into this methodology, let me explain the general concept of carbon-13 tracing using a simple example. Now take any old mouse from your laboratory or a young mouse. You’ll see that it has some naturally occurring amount of carbon-13 in its tissues. And I’ll explain the units on this graph in the next slide. But in short, higher values mean more of this carbon-13. Now because the mouse is oxidizing the nutrients in its tissues, that is some mixture of proteins and lipids and carbohydrates, the level or the amounts of carbon-13 in its exhaled breath will reflect that of its tissues that are being oxidized or catabolized. Now take that same mouse and inject it with the carbon-13 labeled nutrient, glucose for example. You’ll see that the carbon-13 in its exhaled breath will rapidly increase as it oxidizes the glucose tracer in circulation. Now the magnitude of this isotopic enrichment is pretty linearly dose-dependent. But the time courses of these curve functions are dependent on the physiology of the individual animal, and we’ll discuss how experimental interventions can alter the oxidative kinetics of these carbon-13 tracers and thus help us explore research questions related to obesity.

So, the terminology that’s used for stable isotopes can be a hurdle for many of those who are new to the field, but it really doesn’t have to be. In short, the amount of carbon-13 in a given sample is expressed in terms of an international standard that we call VPDB. This stands for Vienna Peedee Belemnite, and it’s a synthetic mineral. Now, this VPDB material contains carbon-13 and carbon-12 atoms in a particular molar ratio that is 0.011180. Now, pretty much any research device that measures carbon-13 will present these values in terms of delta-carbon-13 PDB. So, you can get your data and then say something like, well, heck, the delta-carbon-13 of the exhaled CO2 of my mouse is negative 15 per mil, and that’s a totally plausible value depending on what your mouse eats. Now if you’re going to be working with very highly isotope-enriched samples, you might report your carbon-13 values in terms of atom percent. And we abbreviate this At%. Now, a simple example is that carbon-13 delta value equaling 4,000 per mil, this is equivalent to an atom percent of 5.294. Now, the final equation on this slide is useful if you ever need to back-calculate atom percent and convert those to delta values.

So, the stable isotope tracer methods that I’ll show you can generally be divided into two broad categories. The first category, highlighted in orange on this slide, focuses on measuring the oxidation of what we call exogenous carbon. Now this is carbon that enters the body in the diet in the form of lipids, carbohydrates, and proteins. The body can then partition this exogenous carbon in various ways. For example, it can be oxidized to meet immediate energy demands, or it can be stored in the tissues for structural purposes, or even for energy stores for later use. Now, obesity and experimental interventions to treat obesity will no doubt influence how the body partitions these nutrients among various locations and processes. The second category of stable isotope tracer methods involves looking at endogenous carbon. This is carbon that’s already inside the body. Now these carbon pools in this category come from adipose and glycogen stores, labile structural proteins, and nutrients that are circulating among these different nutrient pools. Now as you recall, obese organisms have a great deal of endogenous carbon, and it’s important to know when and the extent to which they’re mobilizing and oxidizing this carbon. I would argue that equally important is the ability to identify which of these endogenous carbon pools are actually supplying the carbon that’s going to be oxidized. For example, it would follow that experimental interventions that favor oxidation of fat could be better than those that induce oxidation of other metabolic fuels.

So, let’s see what these carbon-13 labeled tracers actually look like. Take a simple molecule like glucose. Now a common tracer configuration is to have the glucose molecule contain a single carbon atom, carbon-13 atom, at the number one position. Right next to that, we have what we call a uniformly labeled glucose molecule, which contains carbon-13 atoms at all six carbon positions. Now, this tracer costs about six times as much, but it also gives you a signal that’s six times as large. So, your standard carbon-13-1 glucose can be purchased for less than $100 a gram. But unlike radio-labeled tracers, where I got into the business, these things are safe, they don’t require extra paperwork, and they have a nearly infinite shelf life. And for 15 years, I’ve been using Cambridge Isotope Laboratories as my go-to source for carbon-13 tracers, simply because of their huge selection. But there’s other suppliers.

Carbon-13 leucine is another tracer that’s often used in nutrition and obesity studies. And this is an essential amino acid that actually accounts for about 8% of all of the total amino acid residues in the body. What this means is that it’s a good proxy for observing overall protein dynamics. Leucine is particularly useful for carbon-13 oxidation studies because its number one carbon is immediately lost as carbon-13 dioxide during the very first step of catabolism. Second molecule you see here is carbon-13 palmitic acid, and this is a 16-carbon saturated fatty acid that can account for about 20% of all of the fatty acid residues in the body. Palmitic acid is actually particularly rich in the adipose stores of obese animals. And during beta-oxidation, that is the degradation of amino acids, the number one carbon of this palmitic acid is also liberated as carbon-13 dioxide. Ultimately, what this means is that you can measure precisely when these important nutrients are being used as metabolic fuels by the body.

Now, there’s actually thousands of stable isotope-labeled molecules, including vitamins and hormones, complex molecules like triacyl-glycerides and proteins. And if you have the money, you can even get exotic custom-labeled molecules to your specification. But today, I want to focus on the use of monosaccharides, fatty acids, and amino acids that are representative of the major nutrient pools that are relevant to obesity studies and whole-body carbon flux. So, we all suppose that snacking behavior is linked to obesity. So, how do you know if your organisms are snacking? So, if a mouse, for example, removes a large chunk of food from its hopper and then hides it in its bedding – and we call this behavior caching – well, we really can’t measure exactly when that mouse may be snacking on this cached food. So, we took some 20 milligram food pellets, spiked them with as little as one microgram of carbon-13 bicarbonate, and we mixed them throughout the bedding, and then we introduced a mouse. Now, because exogenous bicarbonate quickly dissolves and mixes with the circulating bicarbonate pool in the blood, this tracer appears in the breath within less than a minute after each ingestion event. And as a result, we can clearly identify exactly when each of these snacking events takes place over a four-hour period in this mouse.

So, how long after feeding or ingestion does your animal begin to oxidize the nutrients in its meal to meet its own energy demands? Well, food that needs to be digested and then assimilated across the gut will have slower oxidative kinetics than the bicarbonate that you saw in the previous slide. Now, for glucose, this takes about five minutes for the carbon-13 to be detected in the breath, and then another 15 minutes for the carbon-13 values to reach a peak value in a mouse. Takes a few minutes longer in humans. Now, subsequent exposures to carbon-13 tracers will give a more complex oxidative kinetic outcome because the animal is actually burning or oxidizing some amount of the tracer from the first meal, as well as some of that tracer that came from the second meal. Now fortunately, in a mouse that’s given an acute dose of pretty much any carbon-13 labeled tracer will be isotopically back to normal the next day. This allows researchers to use repeated measures designed in their experiments, which will give them greater statistical power.

Now if the rates of CO2 production by the animal are constant during or between individual experiments, it makes things very easy for tracer studies, and this is because carbon-13 dioxide values give a good proxy of the actual rates of carbon-13 tracer oxidation. The top graph however shows a mouse that was injected with the glucose tracer and then kept within its thermal neutral zone at 30 degrees C. But the next day it received the same amount of tracer and was exposed to a cold temperature, 10 degrees C. So, it would seem, looking at this graph, that the mouse did not oxidize as much glucose that second time around. That’s true only if we consider the carbon-13 values in its breath. The graph just below that shows that when the mouse is exposed to the cold temperature, it’s VCO2 increased. Not surprisingly, it’s undergoing thermogenesis at this temperature. And it turns out that the actual rates of tracer oxidation are most correctly informed by how isotopically enriched the breath is as well as how much total CO2 is being produced. And we can see this in the green equation here. So, the graph on the lower left actually reveals that the rates of the total amount of glucose oxidized is not nearly as different as we might have initially suspected in this case.

Well, how do your experimental interventions affect the extent to which animals oxidize their ingested nutrients? Well, here’s a mouse given an oral gavage of carbon-13 glucose on two subsequent days. So, this is a repeated measures trial. On the first day, it was given a saline injection as a control treatment, and this is represented by the blue function. On the second day, it received a dose of a mitochondrial decoupler, and this is the red function. Now, the area under each of these curves represents the total amount of tracer oxidized, and in this case, it clearly shows that the experimental intervention greatly increased the oxidation of that dietary glucose, that exogenous glucose. Well, can we track exogenous glucose oxidation in humans? I saw a lot of folks responded that they worked with humans. Well, let’s see. This is data that I collected just a couple of weeks ago for this presentation. And to do this, I spiked my morning coffee with 300 milligrams of carbon-13 glucose. And this is such a small amount, I couldn’t even taste the sweetness of the glucose. And I used a simple flow-through mask to measure, to collect the carbon-13 in my breath in real time. And the data that we’re looking at are great, second by second for the first hour or so. But as the day progressed, I had to start attending these things called meetings. And I’m sure everybody here knows what they are. But in any case, you can see that we get a really nice curve that shows exactly how my body was oxidizing my morning sugar over the course of the entire day. Now, if I get a chance to sit still a little longer, I can’t wait to do this again. Maybe it might have to be a weekend. And also, to compare this with obese volunteers.

Now, no matter how much fat we eat, all of our diets can contain complex mixtures of different fatty acids. Well, does your organism treat all types of dietary fatty acids equally? Well, this graph shows how a small bird differentially oxidizes individual fatty acids that differ in their lengths and their degrees of unsaturation. And we can see that this animal is oxidizing more of the palmitic and the oleic acids that are found in its diet. Well, if steric oxidation in this case is occurring at much lower rates, this is the blue function, we can start to ask more targeted questions that allow us to better understand this unique carbon flux pattern. For example, is the body not particularly good at assimilating stearic acid in the first place? Or is the body preferentially sequestering or storing this stearic acid in specific body tissues?

Now, many obesity researchers, especially those who work with rodent models, understand the important roles that brown and beige adipose tissues have on lipid metabolism as well as whole animal energy budgets. Now, we’ve used stable isotope tracers to examine how hamsters with different amounts of brown adipose tissue may differentially oxidize their dietary lipids. So specifically, we compared a population of cold-acclimated Siberian hamsters, which had upregulated brown adipose tissues, with warm-acclimated hamsters that had limited BAT expression, and the hamsters were orally dosed with carbon 13 palmitic acid and then subjected to an acute cold challenge for three hours. We can see how the animals with the metabolically active brown adipose tissue oxidized substantially more of this dietary fatty acid. Now, I’m not showing the data here, but curiously, such differences were not seen in the way that they oxidized dietary carbohydrates or their amino acids.

So, the obesity literature over the years is replete with indictments of dietary fructose. You know the stuff, high fructose corn syrup. And this issue has been even more aggressively adopted by self-trained nutritionists, unfortunately. Now the carbon-13 fructose tracers actually make it very easy to identify small differences in the oxidative kinetics of fructose versus other simple sugars. Now these data on this bird were collected using a more primitive isotope system that only permitted me to get a new data point every 15 minutes, not continuous real-time measurements like we can do now. So, it’d be very easy to repeat this experiment using other model organisms or humans employing the breath testing approach that I previously showed you with my coffee.

Now the proteins that we eat are essentially mixtures of different amino acids, some of which are ketogenic, some are glucogenic, and others are both. Now the glucogenic amino acids like glycine, alanine, cysteine, and histidine may be the most relevant to studies of obesity given the propensity for this carbon to be converted into lipids. So, how does your animal oxidize these different amino acids that they ingest? Well in this case we see that this small bird oxidized the ketogenic leucine much less extensively than the glucogenic glycine. But birds rarely have an issue with obesity, even migratory birds that do fatten up quite a bit. So, I think there’s lots of opportunities to measure dietary amino acid disposal in obese models and I don’t think it’s been doing it’s being done nearly as much as it should or could be.

So, how do you know when your experimental interventions are actually affecting the oxidation of different classes of nutrients in the body? Well, traditionally researchers calculate RER and then they recklessly assume protein oxidation is either negligible or at least constant. Now in case you’re not familiar with the term respiratory exchange ratio, it refers to the ratio of the rate of CO2 production to the rate of oxygen consumption in an animal. And the problem is that animals are actually oxidizing all three metabolic fuels simultaneously. Now, unless an RER value is at an extreme of perhaps 0.7 or 1.0, I’d argue that it’s nearly impossible to accurately partition the amount of lipid, carbohydrate, and protein that the body is using in real time. Now, this physiological computation is analogous to what the well-known three-body problem in physics. This video you can see shows a center of a triangle where each vertex represents the contribution of a metabolic substrate. And you can see that there are infinite combinations of these three substrates that can yield a given RER value.

So fortunately, stable isotopes allow us to circumvent these specific problems with RER. And they enable us or allow us to characterize small changes in endogenous substrate oxidation. So, we can do this by carbon-13 labeling the different endogenous nutrient pools within the body. Now, in practice, we would raise three identical populations of baby animals spiked with different carbon-13 tracers. And this process creates adult animals whose endogenous nutrient pools are now selectively isotopically enriched. This is isotope targeting here. Consider this first group of mice. They’re fed carbon-13 leucine. It can be put in their food or their water – both work well; I prefer water. Now, at the end, all of their proteins in their body become isotopically enriched, but all of their lipids and their carbohydrates still have normal levels of carbon-13 C. So, how enriched are the proteins in their body? That simply depends on how much tracer you give the animals over the course of their life. Now these carbon-13 protein mice will always have elevated levels of carbon-13 in their breath, proving that these animals are always oxidizing some amount of protein. More importantly, we can attribute the changes in the carbon-13 in their breath directly to changes in rates of endogenous protein oxidation.

So, if you assume that the background rate of protein oxidation is negligible, then it’s really impossible to even attempt to measure it. But carbon-13 protein-labeled animals allow us to do just this. Here’s an 800-gram heterothermic mammal that just received an epinephrine injection. Sorry I can’t be more specific about the animal model, this isn’t published yet, but as you can see it was oxidizing some amount of protein and the rates of oxidation immediately dropped due to the experimental intervention. Then it recovered over the course of the next three hours. Now following the stoichiometry, protein oxidation yields say, an RQ, or respiratory quotient, of around 0.8. Now, this rapid change in RER would not otherwise be noticeable without using this carbon-13 tracer approach.

So, here’s another example. We created a population of quail such that all of their proteins were carbon-13 enriched. We then subjected them to a period of fasting that resulted in a 25% weight reduction over the next couple of days. Now, the red trace shows how they sharply reduce their rates of protein oxidation during the first few hours. Interestingly, these decreases in endogenous protein oxidation were kind of episodic. In fact, they follow a strong circadian cycle throughout the remainder of the experiment. Now, endogenous lipid oxidation, on the other hand, this is represented by the blue trace, it doesn’t show such a diel variance. So, how do we isotopically label the lipids in the bodies of these animals?

So palmitic acid is a relatively inexpensive isotope tracer, like glucose, it costs less than $100 a gram. And as you already might suspect, lipids are not particularly soluble in water. So, I like to use ethanol, and not just as a mechanism to relax after work. In fact, we can take this palmitic acid, dissolve it in ethanol, and then spray it equally, all across the rodent chow. And at about 50 degrees C, the ethanol then readily evaporates, leaving you with palmitic acid infused rodent chow, or quail chow. Now the mice or the quail then consume this chow over the course of their lives, giving you animals whose lipid stores are isotopically enriched. This is again targeted labeling. So, such customized animals serve as excellent tools for studying lipid oxidation in vivo. We’ll see some examples.

So, here’s a mouse whose body lipids were enriched in carbon-13. Then we put her under 16 hours of food restriction in this experiment, but you could use any of your favorite experimental interventions. We switched between measuring carbon-13 in her breath and the background air every 60 seconds. Now, because I included all the data points collected at every second, in this graph, we actually get a data cloud. It’s a little different from the other graphs that I’ve shown. But overall, we can see a gradual increase in lipid oxidation that’s followed by a gradual decrease in lipid oxidation. And what I think is most interesting are these green circles, where we can clearly identify transient periods of increased lipid oxidation. Unfortunately, I moved to a new project before I can investigate the mechanistic physiological drivers or signaling responsible for these events. And there’s just underscores how much we have to learn about this process using these techniques.

So, how does physical activity alter the lipid use in your animals or your obese models? Well, we took a carbon-13 lipid labeled mouse and put her on our respiratory treadmill. This measures her metabolic rates as well as her rates of lipid oxidation because she’s carbon-13 labeled. So, she rested for a while, then she ran for eight minutes at 15 meters per minute. Now, during the first minute, her rate of lipid oxidation increased substantially. But during continued running, lipid oxidation actually decreased. In fact, it fell to rates that were lower than its resting levels. This is quite different from the paradigm that an exercise physiologist would provide you. But, keep in mind, mice are not people. In any case, we see that lipid oxidation returned to pre-exercise levels about six minutes into the post-running recovery period. Now, almost any patient that suffers from obesity has tried a weight loss diet, often countless times. We used endogenous protein and endogenous lipid labeling simultaneously in parallel populations of rats to explore the physiological changes that are caused by repeated dieting events. Specifically, we compared populations of rats exposed to what I call yo-yo dieting to other populations that were naive or had no exposure to weight loss dieting. We then subjected the animals to a period of fasting that resulted in a substantial weight loss, 25% of body mass, and we found no differences in the extent of protein oxidation that they underwent. But we see clear evidence that the yo-yo diet group was very effective at minimizing its lipid oxidation. This offers some interesting clues, but we still don’t know the signaling and control mechanisms for this phenomenon. So again, we have a lot to learn.

Now because we’ve talked about carbon-13 labeling of endogenous proteins and lipid stores, I often get asked, well, how about the carbon-13 labeling of carbohydrates and glycogen stores? This has been attempted in literature. Glycogen is just a polymer of glucose, so it might be simple to do. Unfortunately, the chronic administration of a carbon-13 glucose tracer gets messy really fast. And this is because only a small proportion of glucose, incoming exogenous glucose, is stored as glycogen at any given time. And this tracer quickly seeps into all of the other nutrient pools in the body as these glucose carbons are converted into non-essential amino acids or into non-essential lipids. So, this really causes a mess when it comes to tracing because it gets difficult to tell where the carbon-13 that’s appearing in the breath actually came from recently. Now this 2013 paper shows that it’s theoretically possible to do this, but I wouldn’t recommend this approach. It’s very labor-intensive. I’d much prefer acute tracking of the fates of either endogenous nutrients or exogenous materials by putting it directly into the food.

So, there’s some human users here, and I know that so far I’ve focused on examples using small mammals or small animals. But because these metabolic measurements and the carbon-13 tracers are so minimally invasive, we could pretty much easily translate many of these experiments to humans or even larger animal models. Here are some examples of our Sable Systems metabolic tents that we use to measure energy expenditure in these creatures. These systems are particularly useful because the user doesn’t need to wear a mask or even a canopy over their head. In fact, they have relatively unrestricted movement. They’re also pretty comfortable because in doing this, fresh air is continuously pulled through the tents at rates of up to 400 liters per minute. So, these systems can easily be paired with the portable stable isotope analyzers that I showed you before, and this gives researchers huge amounts of flexibility in their experimental designs.

Now, as I mentioned at the start of this presentation, obesity is no doubt a complex issue, but it’s my position that the use of carbon-13 stable isotope tracers gives us a powerful toolset to explore questions related to energy balance and carbon flux within the body. And I’ve showed you several diverse cases, but this is really only the tip of the iceberg. There’s almost no limit to the number of questions that you can ask using these experimental approaches.

Liam: All right, so we’ve had a number of questions come in already, so let’s jump right in. First question today is, and I know you touched on this a bit, but Marshall, maybe you can, can you clarify, can high doses of carbon-13 or other stable isotopes be harmful?

Dr. McCue: Sure, good question. So yeah, like I said earlier, about 1% of our body, a little bit over 1% of our bodies already contain or comprised of carbon-13. And so, the amount of carbon-13 that would be in a typical tracer dose might increase this from 1.11% to 1.12%, and that would provide a very strong tracer signal that can be measured. Now the technical answer is yes, amounts of carbon-13 that if all you ate was carbon-13, first of all, nobody could afford that, but levels of 5% or more could start to interrupt some biological, biochemical processes and start to be harmful. But at the tracer level, the short answer is absolutely not.

Liam: Excellent. All right. Great answer. Next question here. Why don’t we have to worry about isotopic fractionation issues with these types of tracer studies?

Dr. McCue: It’s a good question. So, if you’re a physiological ecologist who’s interested in very, very small differences, or an isotope ecologist who’s interested in differences in carbon-13 levels that are on the order of one per mil, one unit, there is a concern of fractionation. However, because in most of these isotope studies, we are getting orders of magnitude, to several orders of magnitude enrichments in 100 to 1,000 per mil, the effect, the final effect of isotopic fractionation is dwarfed by anything that would be drowned out really by the signal of the tracer itself. So, isotopic fractionation for these particular types of studies that I mentioned today is not a real issue.

Liam: All right, great answer. Yeah, another good question here. So, you mainly talked about carbon-13, of course, but are there other isotopes that you have used? And if so, why might you use alternative isotopes?

Dr. McCue: So, the isotope analyzer that I showed you in some of the images is also capable of measuring oxygen-18, which is the most common. There’s a couple of stable isotope forms of oxygen, but oxygen-18 is the most common heavy form. And that can also be quantified by our stable isotope analyzer when it’s present in carbon dioxide. So, it would be measuring carbon dioxide, oxygen-18. But I’ve also used nitrogen. And I like using nitrogen because you can run it simultaneously with studies of carbon-13 so that you can track carbon and nitrogen flux simultaneously. And I didn’t discuss that today because I was focusing on the breath testing, looking at carbon dioxide. And unfortunately, but it’s just a law of physics, nitrogen is not occurring in carbon dioxide. So, we cannot measure that in a kind of a breath-by-breath situation. But nitrogen-15 is very powerful; another isotope can be used for tracer studies. Deuterium is also useful for doubly-labeled water, which I did not also mention today.

Liam: All right, perfect. Good question here from Jennifer. Have you ever delivered isotopically labeled tracers with osmotic micropump as an alternative to food, for example?

Dr. McCue: I have not done that but that is again one of those iceberg things that there is a so such an open horizon to applying this technology and osmotic micropumps would be a wonderful tool to deliver constant doses of tracer to the organisms, especially in the case of very small organisms that where cannulation can be difficult.

Liam: Definitely, definitely, yeah, an interesting technology. Yeah, here’s another good question. Can you clarify how you measured the breadth of rodent subjects?

Dr. McCue: Sure, I’ll start with the familiar. If we were measuring the breadth of human subjects, it’s pretty easy because we can put a hood over us and exhale and send that gas into the analyzer. But for rodents, it’s actually even simpler because the rodents live in their home cages, their home turf, and we are rapidly pulling air. Room air is entering the cage and we’re pulling/drawing this air at high flow rates, two liters per minute, into the isotope analyzers, so that fresh air is mixing with the rodent breath, but these these devices are so sensitive that we can measure the rodent breath while the animal is in its own cage, and it doesn’t have to be an airtight cage.

Liam: Right, right. All right, and I think in the interest of time, we’ll make this next question the last one, but Natasha has asked, you mentioned oxidation of glucose, fat, and amino acids. Have you considered measuring ketones as well?

Dr. McCue: Yeah, I have not done that yet. I’ve measured ketones in circulation, but it’s just kind of a limitation of measuring anything in circulation. You’re not getting an idea of the flux of that nutrient. So, when ketone levels during weight loss activities perhaps increase, we really cannot attribute that increase to anything specific. Namely, you can’t tell if an increase in ketone levels is actually driven by an increased production of ketones or a decreased rate of the oxidation of those ketones. So, as far as ketone, carbon-13 labeled ketones, I have not explored that yet. And that would be an exciting area of new research for sure.

Liam: Excellent. Well, thanks so much, Marshall, for all of your fantastic insights today, both in your presentation as well as this Q&A session.