This Real Science Exchange episode was recorded during a webinar, which was part of a series. Watch all the presentations from this series here: https://balchem.com/animal-nutrition-health/resources-categories/real-science-lecture-series/previous-lectures/page/10/
This Real Science Exchange episode was recorded during a webinar, which was part of a series. Watch all the presentations from this series here: https://balchem.com/animal-nutrition-health/resources-categories/real-science-lecture-series/previous-lectures/page/10/
Early in lactation, the cow is incapable of eating enough to meet her dramatically increased requirements. As the cow’s intake decreases near calving, there are fewer nutrient contributions from dry matter intake and she must alter nutrient partitioning to meet her increased needs by mobilizing fat and muscle stores. (1:18)
Triglycerides from fat stores are broken down into non-esterified fatty acids (NEFA) and glycerol. NEFA has two different fates in the postpartum cow: to the mammary gland as a precursor for milk fat synthesis, or to the liver to be oxidized for energy production. Glycerol enters the gluconeogenic pathway in the liver as a glucose precursor. (4:41)
The capacity for the liver to use NEFA for energy is limited by the capacity of the TCA cycle. When the TCA cycle is at capacity, excess NEFA can either undergo incomplete oxidation to ketones or be repackaged back into triglycerides. If the capacity for other tissues to use ketones for energy is exceeded, then blood concentrations of ketones rise and negative outcomes from subclinical and clinical ketosis can occur. If triglycerides accumulate in the liver, negative outcomes associated with fatty liver can occur. Triglycerides can be transported out of the liver via very low-density lipoprotein (VLDL) export; however, VLDL export does not keep up with triglyceride concentration during the transition period in dairy cows, largely because of a limiting amount of phosphatidylcholine. (5:51)
Dr. White describes a series of experiments in her lab using liver cells in culture to investigate the relationship between choline supplementation and VLDL export. As choline supplementation to the cell culture increased, so did VLDL export from the cells into the media. In addition, increasing choline supplementation to the cell culture also decreased cellular triglyceride content. (10:54)
Using gene expression and radiolabeled tracers over a series of experiments, Dr. White’s group found that as choline supplementation increased, so did complete oxidation of NEFA to energy. This was accompanied by decreased incomplete oxidation to ketone bodies and decreased accumulation of lipids in the liver cells. Glucose and glycogen were also increased with increasing choline supplementation to the cell culture, and a decrease in reactive oxygen species was observed. In addition, choline-supplemented cultures exhibited an increase in metabolic pathways associated with methionine regeneration and methyl donation. (15:29)
Dr. White then details the complexity of the metabolic pathways that intersect between choline and methionine. In similar experiments supplementing cell cultures with increasing amounts of methionine and choline, there were no effects of methionine on lipid export, oxidative pathways, or glucose metabolism. The main benefit of methionine was a marked increase in glutathione production. It’s important to note that no interactions between choline and methionine were observed in this series of experiments. (19:37)
There seems to be a clear biological priority for different sets of pathways for choline and methionine. Choline seems to be influencing lipid, glucose, and oxidative pathways, while methionine is primarily serving its role as an essential amino acid for cellular protein structure and generation, acting as a methyl donor, and impacting inflammation. Importantly, both the choline and methionine results observed in cell culture are paralleled in transition dairy cow studies. (24:14)
Dr. White’s lab further investigated the impact of methionine on inflammation. When cells were challenged with LPS to provoke an inflammatory response, methionine mitigated the inflammatory response. Similar results have been observed in liver tissue samples of transition cows. Methionine mitigated inflammatory markers and increased glutathione but did not influence reactive oxygen species. Conversely, choline decreased reactive oxygen species but did not change glutathione. (27:47)
Choline and methionine are both essential nutrients, there are biological priorities for them as methyl donors, and they are not mutually exchangeable. The lack of interaction between choline and methionine in vivo or in vitro supports the idea of different biological roles for these nutrients. (32:09)
Dr. White takes questions from the webinar audience. (34:53)
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Balchem (00:00):
The following podcast is taken from a webinar presented by Dr. Heather White from the University of Wisconsin, titled “The Dual Essentiality of Choline and Methionine”. To view the full webinar and access the slides referenced during this podcast, visit balchem.com/realscience and use the search bar to jump to this webinar from May 4th, 2020.
Commercial (00:29):
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Dr. Heather White (01:18):
So I am really excited to talk to you today about the dual essentiality of choline and methionine. Certainly a lot of research that you've heard already today has highlighted the aspect of methyl donor being an important part of this discussion. And a lot of our research in the lab over the last few years has focused on this. So there's no doubt that the transition to lactation period is or can be in and of itself a challenging period. And much of those challenges are brought out of the fact that the animal enters into a period of negative energy balance. So the animal, as she approaches calving, which is denoted here in this vertical line as she approaches parturition, what she consumes in the yellow line exceeds her requirement highlighted in blue. And so she's in a positive energy balance prior to, prior to parturition. That however, changes very quickly as she has that calf.
Dr. Heather White (02:21):
And the requirement increases quite drastically right after the time of calving with the onset of lactation. What she consumes again here in yellow is not able to meet her requirement and therefore she enters a period of negative energy balance. We talk about this as being a negative energy balance, but that's really an oversimplified way of thinking of it. It's actually negative energy balance, negative macronutrient balance, negative micronutrient balance. So she has a period of time where she simply cannot eat enough to keep up with her requirements. And as nutritionists, it's our job to provide both the macronutrients and the micronutrients that it takes to make up that difference. So if we think about how this is typically met, then we can think about what our opportunities are during this period to help as she goes through the transition to lactation. If we use glucose here as just one of the nutrients examples, the cow typically is consuming her feed, which is fermented in the rumen, and one of those key volatile fatty acids propagate is absorbed and taken up by the liver where it is used to generate glucose.
Dr. Heather White (03:38):
Again, just highlighting one example here, but that glucose goes to the mammary gland and is the precursor for lactose synthesis within the mammary gland, which as we all know is the typically one of the limiters or the volume regulators of milk production. But as this cow that's approaching calving decreases her feed intake, we enter into a period where we have less contribution from dry matter intake and the cow has to use alternative precursors or change the nutrient partitioning, if we will. One of the most common ways she does that is by mobilizing body stores. Typically fat stores also muscle stores, although we don't talk about them nearly as much when she mobilizes these fat stores, they're providing different nutrients to the body to make up that deficit. Remember that this adipose store is made up of triglycerides, which we heard about already this morning, fatty acids, three of them stored on a glycerol backbone.
Dr. Heather White (04:41):
And it's important for us to recognize that those fatty acids are nonsterile fatty acids. NEFA have a different fate than the glycerol backbone they were stored on. Those fatty acids can have two different direct fates within the body as related to this discussion. One is they can be taken up by the mammary gland and can serve as a precursor for milk fat synthesis. The other is that they can be taken up by the liver and oxidized for energy production, and we know that the liver will take up nonsteroid fatty acid in a direct proportion to how much is presented to the liver. So in result to the blood flow and the concentration of NEFA, the liver, the glycerol backbone has a different fate. The glycerol can actually enter into the gluconeogenic pathway and can also serve as a glucose precursor.
Dr. Heather White (05:36):
Although it's a relatively nominal contribution to the whole body glucose pool, that glucose then, just as glucose derived from other precursors, can go to the mammary gland to support milk lactose production. The capacity for the liver to use nonsteroid fatty acids for energy is limited to a certain extent, and there are alternative fates which involve production of ketone bodies or triglycerides. And so let's take a look at this a little bit closer. As nonsteroid fatty acids go to the liver there oxidized completely to produce energy via the TCA cycle. And this cycle, because it is cyclic, requires a carrier and has a finite capacity based on how much of that carrier is there. So we can think of the capacity of the TCA cycle, much like a carousel that we would take our kids, grandkids, nieces, and nephews to ride. And only as many kids can ride the carousel at one time as there are animals or horses avail available on the carousel.
Dr. Heather White (06:43):
So the carrier of the TCA cycle is OAA or acetate. And so once all of those are occupied, we realize a limitation to the capacity for complete energy oxidation of fatty acids. When that happens, we have to have alternative fates for these fatty acids that are still coming to the liver. Those alternative fates are incomplete oxidation to ketone bodies, and the one we primarily talk about is beta hydroxybutyrate or BHB. Now, ketone bodies get a bad rap, but in and of themselves they're not a bad thing. They are in fact a way for the liver to export energy equivalents to other tissues within the body. And other tissues can readily use these ketone bodies such as the mammary gland and the brain muscle. And so these BHB can go through and other ketone bodies can be circulated through the body and used by other tissues.
Dr. Heather White (07:42):
What we do know though is that if production of ketone bodies by the liver or direct absorption from the rumen exceeds the extent of peripheral tissue use, then blood concentration of these metabolites increases and there are negative outcomes associated with increases in BHV. And that's where we get into subclinical and clinical ketosis. In parallel to this, the other alternative fate is that the fatty acids can be reasterified back into triglyceride. So basically three of those are put back on triglyceride backbone and they can be stored within the liver. Now we heard really good explanations of this from Dr. Mcfadden this morning as he kicked off discussions about fatty acid and VLDL export VLDL is in fact the primary way of which triglycerides could be exported from the liver. And this happens in a much greater extent in other species such as rodents and humans.
Dr. Heather White (08:46):
But what we've come to learn over the years, and again, what you heard highlighted this morning, is that VLDL export does not keep up with triglycerol accumulation during the transition period in dairy cows, largely because of the lack or the limited amount of phosphatidylcholine because of room and degradation of those intermediates. As we have export of VLDL, they can go into circulation and be used for milk fat synthesis or energy as an energy source for other tissues. And again, you saw this earlier from Joe, but if we want to really hone in on the export of VLDL, we have to think about what the key components of it are and understand how we may influence that. Nutritionally, this has been of a lot of interest in the, in the field over the years because it does in fact allow for a way to export the fat from the liver, which could have negative impacts but once exported could have benefits to the rest of the body.
Dr. Heather White (09:48):
So these VLDL are comprised of triglycerides, apo, lipoproteins, cholesterol, and phospholipids, including phosphatidylcholine. This is in fact one of the main early benefits that were noted of choline was the ability for supplementation of a cow with choline to reduce triglyceride accumulation in the liver. What was very challenging for a long time to do, however, was to definitively demonstrate that this was because of an increase in VLDL. The decrease in triglyceride accumulation was speculated or hypothesized to be through this increase VLDL but the technology did not allow for us to quantify it directly at the time, what we were able to do within a cell culture system. So just a a note, a housekeeping note here. In the bottom right corner of your slide, you'll see a cell, I'll try to keep it very clear, which of these data are from cell culture experiments versus cow experiments and those cell culture experiments will have the cell picture here.
Dr. Heather White (10:54):
So through a series of experiments done in our lab, we were able to quantify VLDL secreted from liver cells in culture when we gave those liver cells increasing amounts of choline chloride. Now, another note here, remember, if we're working in culture with liver cells, we can ignore any degradation by the rumen, so we can directly use choline chloride and not have to focus on the rumen degradation aspects. So as we increased amounts of choline chloride supplemented to the cells here on the left panel in black bars, we observed a linear increase in VLDL export from the cell into the media. And this was very exciting because we were also able to show in a parallel set of experiments that across a different range of choline treatments, that there was also a decrease in cellular triglyceride across a range of choline. So again, being able to mimic what we were seeing in the cow with decreased cellular triglyceride, yet being able to demonstrate the mechanism that that was through an increase in VLDL export, we have gone on to do this collaboratively with Joe McFadden as well through the mechanisms or the techniques rather, that he described this morning and have found similar patterns in the second set of experiments on the right.
Dr. Heather White (12:20):
So the question is, does just decreasing liver lipids, is that enough to result in the increase in milk production that we see in the dairy cow when we supplement with RU protected choline? This slide is from a study done at University of Florida and Charlie Staples lab where they supplemented rumen protected choline from three weeks before calving to three weeks after calving. So the classic transition period supplementation and the choline supplemented animals are shown in orange while the control animals are shown in blue. And this figure here demonstrates energy corrected milk, which was significantly increased during the first three weeks after calving. What was really interesting is that all those supplementation stopped at 21 days after calving. They followed these animals through 15 weeks of lactation, and they found a tendency for increase in milk production about five pounds of milk a day to persist across the 15 weeks.
Dr. Heather White (13:24):
So again, as we ask if a decrease in liver lipids is enough to increase milk production, we now have to ask if it's enough to increase in a persistent or sustained manner. So what is it or how is it that we're able to observe an increase in milk production even after supplementation has ended? Dr. McFadden also highlighted this morning that there are three pretty recent meta-analyses of choline supplementation effects. And so I have one of them here to highlight that this isn't just an observation in the study out of Florida when 21 studies were analyzed. So we've got about 66 treatment means and 1300 cows represented here. When we look at the average supplementation rate of choline ion, there's a noted increase in milk yield, 1.6 kilos per day in energy corrected milk, in milk fat yield, milk, protein yield, and dry matter intake.
Dr. Heather White (14:21):
And those were significantly and consistently increased across the studies. I'm gonna note here, and I'll come back to this in a little bit as we start to talk about the intersection with methionine, but at the concentrations, at the average concentrations of choline and methionine as a percent of metabolizable protein prepartum and postpartum, they did not find interactions of these two nutrients rather independent benefit of choline. And so this really raises the question, if choline can significantly influence milk production or production performance, what is the mechanism through which choline supports that benefit or that production gain? Is it just through the classic decrease in liver lipids or fatty liver that we've long talked about, or is there something additional that's happening? And so this is the question we set out to ask, and we largely used a cell culture model because it allows us to specifically look at different pathways in a very controlled setting setting.
Dr. Heather White (15:29):
I won't bore you with all of the details or the pathways. Although it makes grad students cringe when you put five years of their work into one slide. I think this slide really highlights what we found across a series of experiments. So this is a similar setup to what I showed before, where we have mobilized fatty acids, non aster fatty acids coming in and they're going into these different points, either complete oxidation to energy, to ketone body production or to lipid accumulation. And across a series of experiments, what we found using gene expression and radiolabeled tracers were that as we increased choline supplementation, we noted increased complete oxidation of fatty acids to energy. We noted decreased incomplete oxidation to ketone bodies, and we noted decreased accumulation of lipids within the liver cells.
Dr. Heather White (16:32):
Interestingly, we also looked at glucose production. And one thing to note here is that not only is glucose production relying on some of the similar precursors but it also relies heavily on energy generated by the TCA cycle through this complete energy production because it is a very energetically expensive pathway. And what we noted through these experiments was as we increased choline supplementation to the cells, we had increase in the generation of glycogen, which is the short term storage form of glucose within the cell culture system. We have to remember there's no demand from the mammary gland, which would be sending signals to the liver to release glucose. So we have to look at glucose and glucose glycogen together. We also found a decrease in reactive oxygen species. And Ross is one of the many things that we hear is a buzzword right now.
Dr. Heather White (17:33):
What we know about Ross is that oxidation of fatty acids innately produces oxidative stress. So even though we want a cell to oxidize fatty acids, there is a generation of reactive oxygen species or raw that is associated with that. We also know that at some point, accumulation of Ross probably has detrimental effects to the cells, although we're not at the same point here as we are with ketone bodies. There's no well-defined threshold that would tell us what that limit is. But we did notice that there was a decrease in reactive oxygen species as we increase choline supplementation within the cells. So if we put all of this together and we look at it by pathway, either of lipid, export of oxidation, either complete or incomplete in the associated raw glucose production we see some really interesting and consistent effects of choline in the cell culture system.
Dr. Heather White (18:38):
We see increases in glycogen production. And although I didn't show it in the the last slide, no difference in glutathione production, which I'll come back to later. You might be wondering, well, these are just hepatocytes. So is this really indicative of what's happening in the cow? And what's been really insightful for us is that as we look to our collaborative work with Florida and then into the literature as well, all of these areas with check marks are areas where we have indications that the same thing are happening in vivo. So the same pathways are being influenced in the cow. We're just able to look at those pathways much more specifically, and the cell culture system. So one of the pathways that you notice here at the bottom of the slide that I didn't show you in the last is methionine regeneration. And this would be through the pathway you've heard of already from both of our first speakers through methyl donation.
Dr. Heather White (19:37):
And what we observed was when we supplemented cells with additional amounts of choline, we found an increase in the pathways associated with methionine regeneration and methyl donation. And so that's a good point here to dig a little closer into this because it is in fact the way that choline and methionine intersect physiologically. So if we talk about methyl groups and methyl donors, methyl donor is simply a lab biocarbon with three hydrogens. It is a methyl group that can be easily given from one molecule to another. So it's located on the end and it's donatable, if we will. So we can see in these circled highlights. Methionine has a methyl group choline and its downstream metabolite butane have three methyl groups and folic acid the most commonly referred to methyl donor. And human nutrition has one methyl group as well. So these methyl groups, or this methyl donating action of these molecules serve as a way for them to intersect within the physiology of the cell, of the tissue and of the body.
Dr. Heather White (20:46):
So if we, we look at that intersection, let's start by orienting ourselves to the pathway we, we began our discussion with, which is VLDL packaging. VLDL requires phosphatidylcholine and other phospholipids as I mentioned earlier, and those can be derived directly from choline. If it was as simple as this we wouldn't all be so intrigued by these talks today. So fortunately, for those of us doing research in this area or unfortunately for, for trying to piece it all together, it's a lot more complex than this. In fact, phosphatidylcholine can also be formed through methylation of phosphoethanolamine. And we heard Joe talk this morning about the role of different fatty acids and lipids in the composition of these phospholipids. If we orient ourselves back up here to choline and green, and we look at the ability of choline to serve as a methyl donor, the downstream metabolite of choline is beane, and that beane can serve as a methyl donor, which allows a methyl group to be given to regenerate methionine.
Dr. Heather White (21:56):
Now, we're all on the same page here that methionine is an essential amino acid. It cannot be created by from scratch by a mammalian cell, but because it also has a methyl donor, it serves a key role in fueling Sam, which Dr. Hanson did a really good job overviewing that Sam is the universal methyl donor, and that it can in fact donate this CH three here, this methyl group, which leaves us with an SAH, this methyl depleted SAH can go through, continue through this cycle. Again, the cycles that require carriers and regeneration through homocysteine and can be regen used to regenerate the methionine. So the carbon sulfur structure of the amino acid itself is still there. We just have to put an amino acid, or I'm sorry, put a methyl group back on that the methyl group can come from choline and beane, or it can come from folic acid as shown here in the middle of the diagram with the tetrahydrofolate pathway.
Dr. Heather White (23:02):
So if we have a methyl donor available within the cell, we can regenerate that valuable amino acid methionine. Now, if we look back at this SAM donation of methyl donors, there are hundreds, perhaps thousands of trans methylation pathways or methyl donation pathways within the cell that demand this methyl group. One of the many is methylation of phosphoethanolamine to generate phosphatidylcholine, and that requires three methyl groups, so three different methionine to SAM transfers. So this demonstrates the intricate relationship or intersection between these two nutrients. The other thing that I'll highlight in this diagram is in the top left of your slide, you'll see the downstream byproduct of both beane metabolism through glycine and cysteine metabolism is glutathione. We know that homocysteine and cysteine can be toxic if in excessive concentrations in the blood. And so there are, there are some benefits to generating glutathione from those intermediates.
Dr. Heather White (24:14):
There's also a benefit because glutathione serves as an antioxidant within the cell. And so we know that we need glutathione and other antioxidants to combat oxidative stress within the tissue. So what happens when we culture cells with increasing amounts of methionine within these series of experiments, we did that with choline and a factorial design. And I've now listed the methionine effects off to the right here. There were no effects of methionine on the lipid export or oxidative pathways or on glucose metabolism. And I will note that there were no interactions between choline or methionine across the series of experiments. What was a market benefit of methionine was increase in glutathione production, this green arrow here at the bottom and the ability to influence inflammatory response. There was also a benefit because methionine was generate or was provided. We didn't need the methionine regeneration. So this lack of interaction on these pathways highlights a few things to us.
Dr. Heather White (25:25):
First, there seems to be a clear set of pathways that the biological priority is for in terms of choline and methionine, and that there is a difference in the roles, whereas choline seems to be influencing these lipid glucose and oxidative related pathways. Methionine is primarily serving as a methionine as an essential amino acid, both for cellular protein structure and generation, but also as a methyl donor, but then also in inflammation. And just as with the choline response, we can see this paralleled in animal studies in transition dairy cows. So this is a set of although not published yet as a formal meta-analysis. This is a set of studies using postpartum pre and postpartum methionine supplementation that's pulled together by one of our graduate students at the University of Wisconsin. There's a lot on this slide. Really, it just highlights the extent of studies done with either supplementation of rumen protected methionine or ru protected methionine analog.
Dr. Heather White (26:36):
And what we find is that response and milk yield itself is inconsistent across the studies, but what's very consistent or more consistent is a response in milk protein percent. So again, just listing the studies out here and significant effects demonstrated with the red bars showing the extent or the scope of the benefit. And so there's consistently an increase in milk protein percent when we supplement methionine during the transition period. Again, I'm just focusing on transition right now. There's more comprehensive reviews of methionine supplementation through the whole lactation. And so one of the things that we did in a separate set of cell culture experiments was that we challenged cells to try to dig into this inflammatory response. So if there's a benefit of methionine on inflammation we should be able to look at that more closely if we can in fact do an inflammatory challenge in cells, and we can do that by putting in LPS very similar to how we would use an LPS challenge for a mastitis challenge in a dairy cow.
Dr. Heather White (27:47):
We can put that on cells. Let's start in the top left panel. There's a lot of bars here, but there's two things to look at as you move from left, right. We supplemented the cells with more methionine and within each pair of bars, the white bar is no LPS challenge, and the black bar is the same concentration of methionine and lysine with an LPS challenge. So undoubtedly, LPS challenge did in fact cause an inflammatory response within the cells which is good because this is now a very nice cell culture model. But also note that as we supplemented more methionine, so if you look at the third pair of bars in each of these panels we were able to mitigate the inflammatory response by supplementing cells with methionine. This is similar to what has been observed in a transition cow study. These are the same pro-inflammatory cytokine markers that were done in gene expression and liver tissue biopsies.
Dr. Heather White (28:51):
And the tissue samples were taken at minus 10 days relative to calving across the transition period to 29 days after calving. And on the left side here, top and bottom panels cows supplemented with methionine are in the black circles and without methionine in the white circles. And we can see that there are changes in the inflammatory markers when cows were supplemented with methionine. This study is one of the few that looked at factorial design and methionine and choline, and on the right are the main effects of choline, which did not influence the inflammatory markers. This is interesting to note because in this study as well, they, they did not find an interaction of methionine and choline on these pathways. So the the story as we dig into this with inflammation and oxidative stress, and I think that Joe highlighted it really well first thing this morning.
Dr. Heather White (29:47):
This is one of the forefront edges of research in our field right now. And so we don't have as clear of a picture on understanding inflammation and oxidative stress as we do on some of the other pathways. We talk about the inflammation and oxidative stress are certainly related, but they may be more complex than a direct relationship. So when we talk about inflammatory markers, and then we also talk about the Ross or oxidative stress associated with fatty acid oxidation, those may be related, but they may not be in a one-to-one relationship. They may be more complex than that. So in our work and that of others, methionine has mitigated inflammatory markers and has increased glutathione. Conversely, choline has decreased reactive oxygen species, but without a change in glutathione. And so this really highlights an interesting intersection. I'll also note that methionine, or remind you that methionine did not influence reactive oxygen species.
Dr. Heather White (30:52):
So we seem to have some relationship between these pathways, but maybe independent roles of the two nutrients to influence them. We're still learning what these balances mean in regards to inflammation and oxidative stress, specifically during the transition to lactation period. So piecing this all together, supplementation of rumen protected choline resulted in vivo in the transition cow in increased milk yield energy corrected milk, milk protein and fat yield, and increased dry matter intake to support those by using a mechanistic cell culture model, we've been able to explore that increase that these benefits are likely through increased glucose production, complete oxidation increased VLDL export and methyl donation, and through a consistent decrease in lipid accumulation and BHV or ketone body production supplementation with ru protected methionine or rumen protected methionine analog increases milk yield and milk protein percent. And this really highlights that methionine is essential not only for cell protein generation, but for protein production.
Dr. Heather White (32:09):
And within our mechanistic cell culture model, we were able to find that this is through increased glutathione and that methionine ameliorates inflammatory response during both LPS challenge and in vivo. We've seen that it does this during the transition to lactation period. So some take home messages if we want to apply this to ration formulation or nutrition in the field. Both choline and methionine are essential nutrients, and it is becoming more and more clear that there are biological priorities for these different methyl donors and that they're not mutually exchangeable. So it is important whether that methyl donor comes from choline or from methionine and that that leads to different apparent priorities within the liver cells. There's no evidence for choline and methionine interactions at average doses in vivo and transition cows or across the ranges that we studied in cell culture, which again suggests and supports biological priorities, nutrients and vivo. So with that, I'd like to take a second to acknowledge and thank all of the folks that make this possible. Tawny Chandler is now a postdoc at Cornell, and she was the primary person on the cell culture work and gratitude to collaborators, Charlie Staples and Marco Enovia of University of Florida Rick Gruer, and then funding sources for the work. And with that, I'd be happy to take any questions.
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Q&A question (34:53):
So right off the top one of our viewers had a, an interesting observation. It's, it's kind of silly. We continue to talk about using choline or methionine as an either or proposition given their separate roles. And you talked about this a lot in your presentation. So and the analogy that was used as a producer doesn't really make choices between a pre dip and a and a post dip. They just use both. So aside from cost, which we'll have a few questions about cost, but aside from cost, why do you think we continue to have this discussion as though it's an either or, rather than looking about how we should use these products together, is there more research that we need to do? What, what is it that is kind of gonna help us close the loop here between supporting use of these two nutrients together?
Dr. Heather White (35:42):
I think that's an excellent question, and I think it's born out of the fact that there are intersections biochemically. So when nutrients can go into the same pathway, sometimes we falsely assume that they can replace each other. I teach this to our undergraduate students and the graduate students as well. And the phrase that I really like to use is that just because something is biochemically possible does not mean that it's biologically relevant. And I think that that's what a lot of this is born out of. As I demonstrated in that very complex diagram, both choline and methionine can enter into the same biochemical pathway. However, I think it's very unlikely that an animal or a tissue would use a limiting amino acid to serve the role in a pathway that other nutrients can do. And so I think that we have to remember just because two things are going into the same pathway, or because it's possible, it doesn't mean that it's energetically feasible, and it doesn't mean that physiologically it's likely to happen. And so I think a lot of this was born out of us recognizing the potential intersection in the pathways before we had enough research to support what each of the nutrients roles were in those pathways.
Q&A question (37:01):
So now that I've asked you to make the case for using both nutrients, if, if we forced you to make a choice, I got a couple questions along these lines, but if we forced you to make a choice, do you select choline? Do you select methionine and show your work? Yeah. No, I'm kidding.
Dr. Heather White (37:16):
Yes. Right. So show my math on the marker board behind me. So you just told me that a producer doesn't have to pick between pre and post-it, but I recognize the importance of the question. So if I had to pick and a lot of these reasons were highlighted in the studies you saw by our previous two speakers, I would supplement choline, prepartum and methionine postpartum. So you didn't tell me I had to pick, only one just had to pick. I'm gonna go with one at a time if I have to. And the reasons for that is I think that based on some of my other work on liver metabolism that demonstrates the liver is already changed at the day of calving and cows that will subsequently develop a metabolic disorder at day eight or 12, I think that we need to influence the liver before calving happens. Combined with the effects on the offspring which you've heard a little bit of today, not extensively, there are a lot of benefits on the calf of prepartum choline supplementation. So I think that I would put choline, prepartum and then certainly the demands for methionine once lactation begins are very high. And so that would be the strategy I would take if I had to make a choice, although I think that the cow needs both of them during both parts.
Q&A question (38:39):
So are, are there any parody effects on choline and methionine supplementation?
Dr. Heather White (38:46):
Yeah, so the recent meta-analysis out of University of Florida did identify some interactions with parity where multiparous cows respond consistently better to choline supplementation, primiparous animals are still growing, and because we haven't had a lot of primiparous only, or first lactation, cow only studies, I think we still have to learn a little bit more about the role of methionine and choline in those cows.
Q&A question (39:17):
I had a few questions about ROS and is ROS related to utter edema? Do you know anything about that relationship that you can enlighten us?
Dr. Heather White (39:28):
So I don't have a direct answer for that. ROS it can be secreted from the liver cells. So that's actually how we measured it in culture is we measured what was secreted from the liver cell into the media. And so it is possible that Ross generated from the liver could contribute to whole body or systemic oxidative stress. We haven't been able to dig into that in our lab, and I'm not aware of any studies that have really been able to dig into that as a relationship to utter edema or anything specifically like that. One challenge is that antioxidants and reactive oxygen species are very, very reactive, for lack of better word, but labile in the blood. And as soon as we take a blood sample, if it's exposed to oxygen, then we get all of these changes. And so it makes it very challenging to quantify it within a cow situation. So a lot of work still needs to be done there. I think
Q&A question (40:30):
Another related question, could you explain a little why choline reduces Ross and Methionine doesn't? And, and, and if methionine increases glutathione, would you expect a reduction in Ross?
Dr. Heather White (40:44):
Originally we did. I remember a lot of conversations with with Tawny Chandler working on, on this data thinking, well, if one increases, if glutathione increases, then surely Ross would be decreased. But as we started thinking about it more, I think it's more complex than that because we really can get ourselves in a circular discussion where we ask, do we want glutathione to be high in a cell? So bear with me here. A theoretical approach to this, if glutathione's an antioxidant, do we want it to be spent, if you will? Is it currency that can be spent to alleviate the stress within a cell or do we want it to accumulate and is it that we want it to be at a certain level within the cell or a certain content or concentration, but then beyond that, there's no benefit of an increase.
Dr. Heather White (41:37):
So we want it to be spent to neutralize antioxidants. And these are questions we don't know the answer to. What I'd love to be able to tease out is within the choline treatments was Ross decreased because glutathione was made but used to neutralize the Ross. And we didn't capture that initial increase in glutathione. But if that's the case, then one would still argue that an increase in glutathione with methionine should have decreased Ross. So where we're at on that discussion, and we're going to do some, some more work looking at glutathione and Ross across other research projects going on. But where we're at is really thinking it's not a one-to-one relationship which is why I indicated the re the balance there is probably more complex than just a direct increase in one equals a decrease in the other.
Q&A question (42:31):
So another d different subject here. So if I was under the impression that ketones couldn't pass the blood brain barrier in ruminants, did, did, did I miss something or could you, could you explain if I had a misconception here?
Dr. Heather White (42:47):
Yeah. So the central nervous system of all species can use BHB for energy. It's, I don't know that we wanna say it's a direct crossing of the barrier. It is an energy precursor that can be used. And one of the mechanisms for glucose sparing and ruminants is that central nervous system can use more ketones than other animals. So it's one of the ways that ruminant animals can spare glucose just for use by glucose obligatory tissues like the mammary gland and the fetus. So I'd have to go back to the straight physiology textbooks to see if it crosses the blood-brain barrier as the BHB itself or as the energy equivalent, but I know it is an important energy source for central nervous system.
Q&A question (43:40):
Okay, we'll take what I think might be our last question for you here, Dr. White. We, we talk a lot about glutathione, but glutathione can be regenerated, methionine can lead to glutathione, trine synthesis, which is also an antioxidant. So what about trine?
Dr. Heather White (43:57):
So we didn't look directly at trine in any of our work. The, the really definitive intermediate characterization of going through and doing mass spec of all of these intermediates in the methyl transferase pathways is probably still needed. It hasn't been done extensively to my knowledge in ruminants, so in cow either cell culture or in cow studies. So we didn't measure trine in any of our studies. And I don't know that trine has been extensively characterized in any of these methionine or choline studies, at least that I can recall right now. It's, it's probably an intermediate that plays a role, but if it's, you know, I, I can't address specifically if it's something we should look at for more than that.
Q&A question (44:43):
Dr. Heather Wyatt, University of Wisconsin, thank you for a fantastic presentation adding to our fund of knowledge today on this pretty important topic.
Balchem (44:52):
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