Hi, today I'm going to be talking to you about how best to use laboratory analysis in equine endocrinological disease. Now, we are going to be focusing particularly on PPID and EMS. I know sometimes they, they're not the most exciting, but they are the most talked about and the most diagnosed.

The other endocrinological diseases in the equine industry really aren't that significant, at this moment in time. So what's the point of today? I think firstly, it's going to be trying to understand exactly what is going on and how the test can be used and how some may fail you and how we can hopefully reduce the risks of that happening.

But why are we worried about it? We know that endocrinological diseases, EMS and and PPID inherently are more common in the older horse, and that they put these horses at risk of laminitis, which can be career, life limiting. Also with insulin dysregulation, we know that's associated with laminitis and primarily PPID exacerbates insulin dysregulation amongst other problems.

And also endocrinopathies can lead to another, a lot of other problems such as loss of muscle mass along the top line, poor performance, and potentially reduce the ability to treat other diseases with steroids because of the risk of laminitis. Now each of those three points I'm going to highlight some recent literature when we get to that stage. Not so much about the testing, but just some stuff that's come out in the last, last 12 months or so.

With the mounting evidence that insulin dysregulation in itself leads to laminitis, it's really important that this highlights how much we have to screen these horses as we start to move into our geriatric population or at risk patients where you're talking about the slightly overweight horse. What we also know is that as horses age, PPID increases, and that this can lead to various diseases that can weaken tissues as well. So making these horses slightly more susceptible to tendon and ligament injuries.

Now PVID therefore not only could be a risk for life threatening disease, but it also might lead to poor performance, and I say stoppers using steroids if we need to in RAO or musculoskeletal diseases. Now I think most of you will be fully aware of what pituitary pars intermediate dysfunction is, PPID. It's a previously, it's a disease of relatively minor importance, but over the last decade has become one of the most encountered diseases in equ practise.

So making sure we know how to test for it and get the most out of our tests is significant. Why are we seeing this? It's driven by a greater knowledge and understanding of endocrinopathic laminitis, and also an increasing geriatric population.

The slight problem that we see is that the more we learn, the more questions we ask, the more questions arise as well. So it seems that rather than becoming a slightly more black and white situation, we're only ever increasing our problem, making it more and more of a grey disease. But what is PPID in itself?

We know that it's a disease that affects the pars intermedia of the anterior pituitary gland, with varying changes seen in that pituitary gland from mild enlargement and proliferation to full out tumour development. And I think at a basic level, it's important to understand the pathophysiology of this disease, so that we can actually start to understand why testing at certain stages in the disease becomes important. As I said before, PPID is a grey area disease with variable changes seen in the past intermediate, which correlates with a highly marked variability in the clinical presentation of these patients.

In 2008, I'm sure anyone who's done the reading but has seen this seminal paper, reviewed the histopathological grading system within the pituitary gland. And they graded it between 1 and 5, with 1 being completely normal, no changes on the histopathology. 2 showing focal and multifocal pituitary intermediate hypertrophy or hyperplasia.

And the clinical interpretation was that this was completely normal for the age. And then we go into grade 3 where we sort out the diffuse adenomatous changes. 4, adenosis hyperplasia with microadenomas, and then finally, adenomas in the pituitary or parts in anterior.

And each of those stages as we go from 3 to 4 to 5, we start to see the ACTH elevations, and this is where we talk about being able to test for that patient. But should we really be thinking about making sure that we get them in that grade 2, grade 3 area where we can hopefully negate the changes and stop it progressing into grade 5? We're looking particularly at how do we test for PPID.

There are a number of options available, which most of you will obviously be aware of, basal ACTH. Which is done in summer or autumn, the TRH stimulation, dexamethasone suppression test, which I hope that nobody's really doing, . And hopefully we'll be able to bring some clarity into that.

What I would like to say is make sure that you don't forget to do assess concurrent insulin dysregulation, because we, as I mentioned before, we know that PPID leads to an increased risk of insulin dysregulation. So it's really significant to make sure when doing this, that you are looking at insulin dysregulation as well. And one conversation I regularly have with people is, if we're not getting the response we want on our ACTHs as we treat them, do we need to chase that number down to the perfect normality for that part of the season?

And sometimes the answer is yes, absolutely, if we're clinically worried about the case. Sometimes though, no, the answer is no, because what are we trying to affect? Are we trying to affect hypertrichosis, so we don't really worry about a little bit of extra hair growth.

Or should we be looking to see what the laminitis risk is? And there's some recent work that's going to come out in the next year or so, it's currently in review. There's actually making a grading system to use looking at incidences, looking at ACTH, looking at clinical parameters, and therefore presenting a risk ratio for those horses for laminitis.

And I think once we've got that, then we can start really considering. Do we need to push that ACTH down to normal, or can we maybe look at an insulin in the blood and say actually this horse isn't at that much risk of laminitis, therefore, let's not chase the the number down to to the normal reference range. When we look at Bayes LazyTHH, the most common and the easiest test, obviously.

First and foremost, with all ACTH testing. There's some particulars that have to be undertaken to make sure that the sample is accurate. Firstly, we have to take it into into EDTA and plastic tube for EDTA as well, and it must be kept chilled.

Ideally you should be separating that sample as soon as possible and ideally again by centrifuge, cos that will allow you to freeze it if there's gonna be any delay. If you are separating by gravity, do not freeze that sample. What we know is that any hemolysis can affect the result leading to a false positive.

We don't know what exactly it is that caused from the hemolysis that leads to a cross reactivity with the emulite test, but it leads to massive increases in your ACTH. So it's important, as with all laboratory tests at this moment, to say, if you don't believe the result, if the result does not fit with the clinical parameters or the, Progression of the disease as you see it, then get it repeated, get a new sample and get it sent back in and hopefully the result will appear more in line with what you're expecting. But when do you take a basal ACTH?

There are two times of year that I'd recommend taking one, and one is in the autumn, so that we're hitting it around late September, September, late September, and one in the spring, so that we're getting that low level. I think it's really important to do both because often you'll have good control during the Nadir through the, you know, sort of late April time, but then you won't have good control as you hit into the September period. And that can lead to an increased risk of laminitis, so sometimes in some cases, we are recommending changes in the dosing strategy depending on the time of year, but that does take a very involved patient and a lot of very involved owner, sorry, and a lot of repeat sampling.

Reference ranges will continue to be a massive debate, the equine Endocrinological group, headed up by Andy and by Hal Shotton and that group, and Nick Frank, sorry, . Have recently released some new information and we're talking now about grey areas rather than a definitive number. And the rationale behind that is that actually, if you're going to give a grey area going above the reference range, you should be giving one going below because the bell shaped curve of all reference values will indicate that some will be normal or abnormal right at either end of it.

So this is the data that Andy's created off about 8, just shy of 80,000 samples, so pretty significant numbers, which is unusual in the equine world. The grey line is the reference value if you were to take it as absolute throughout the year, whereas the yellow and the orange are are 95% confidence intervals. We're now keeping our grey area as between those two lines throughout the year.

So if you have a horse that is coming in, let's say at 25 in April. Then, and it has severe clinical symptoms, then we can be fairly confident that actually it has PPID. Whereas if you flip it on its head, if you're talking about a horse with a 37 at the same time of year, but absolutely no clinical signs, it may be above reference range, but should you really be interpreting that as a PPID case?

And the answer probably is no. So it's taking into into your mind both the reference range but also the clinical symptoms, and I think that's really significant, you know, if you're sending it in, add a history so we can make a sensible comment and guide you guys there. But some of the more recent research that we've been pushing out that Andy and and Julie, our resident have been pushing out, has looked at the breed specificity of, of this disease process.

And what we can see is that there are significant differences between each of the breeds, and therefore we have to be cautious with with some of our interpretation. This is, as I say, it's in review at the moment, hopefully will be publishing, which means we can actually use the data until then it's sort of in our back pocket. But what we can see is that as we go into September time, our reference ranges are about true for the the Connemaras, the thoroughbreds, and even the new forests.

But as you go into your Welsh section A's and your Shetlands and miniature Shetlands, you start to see that actually that ACTH can be dramatically high in what were completely and utterly normal horses. So if you have a Shetlands who doesn't appear to be symp symptomatic, but you've tested ACTH for another reason, maybe owner driven, and it's coming in at 100 in September, take it with a pinch of salt, reconsider the options and maybe say, let's test later in the year, or let's do a TRH once we're back into the Nadir period. So that's some really good new stuff that's coming out that I think will dramatically change how we diagnose diagnose the disease based on the clinical presentation.

But what can lead to a false positive, and I think, firstly, freezing, gravity, freezing gravity separated samples is a significant problem that we do come across on a nearly daily basis. So make sure that you aren't gravity separating, taking the serum off and then freezing. If you're going to gravity separate, just send it in as a chilled sample and it does have a pretty good stability over a number of days, so don't worry too much.

What about pain? It's a, there's a variable amount of research in this, so some papers historically have looked back and said only extreme pain will lead to an elevation in our ACTH. Whereas, a recent study by Lawson in 2020 showed that there's absolutely no correlation between pain and the elevations in ACTH's.

Stress was a very similar situation where we had variable research showing sometimes it leads to an increase, sometimes it doesn't. But what I would say is anecdotally, talking to tens of people pretty much every single day about ACTH's is that both pain and stress do seem to occasionally lead to dramatic elevations, and I don't think there's a rhyme or reason, but we do see some horses with huge numbers. I just remember one case with a referring vet who was adamant it was happening, and so he did his own little trial and that horse had huge spikes up into the thousands following transportation for only 20 minutes.

So, for me, I only test at least 24 hours after a horse has arrived in the hospital, for example, or if I can avoid pain, then I do, otherwise if it's with say a severe laminitis, then I'll take it with a pinch of salt and maybe change my treatments once we're out of the laministic period. Obviously, as I mentioned, ACTH PPID is often one big grey area, so how are we going to deal with those borderline results? Now it's important to note this is a slowly progressive disease and therefore we, it is absolutely inevitable that we are going to get ambiguous results as we do this do this work.

But what can we do to increase our likelihood of an accurate result? One, obviously, as I mentioned, was our autumnal testing with biostimulation, and the other is TRH and now where there are seasonal testing available. Sorry, seasonality just on, on a basic level is that the more we do it in the autumn, the more likely the sensitivity and specificity are better.

So we can see that in spring, summer, and winter, we have a sensitivity of about 80% and specificity of 82%. Whereas in autumn, we're looking at about 100% sensitivity and 95% specificity. Now, this work was done nearly a decade ago by Andy in Victoria and then followed up by Catherine McGowan in 2013.

So there is a lot of new stuff that's coming out as we talk, so those numbers might be a little bit more adjusted, but either way you're gonna have a better result, a better sensitivity and specificity if we're looking at it in the autumnal period. But what about TRH stimulation tests? Why are we gonna run this?

And I think first and foremost, it's for those grey area results where you don't get the result you're expecting, you know, you've got a clinically obvious PPID and the ACTH isn't high or you've got real concern from some other direction and that you just need to double check that this is truly the result. So it has greater sensitivity and specificity than a basal ACTH, but the results need to be interpreted in line with season. So if we look at July and November, so we split it into three sections, July and November, November to July, and then August and October, August to October.

We can see that actually our reference ranges are quite, quite OK, so abnormal is anything above 250, and, and actually the suspicious area is relatively small, 110 to 250. November to July, even better. So that's a really good time to be doing a TRH.

Whereas when we look in August to October, this was the more recent research, we can see that grey area starts to get much, much bigger with a reference range of only anything above 500 being significant. So it opens up just another grey area. If the owners are aware of that and you're getting a TRH test in the autumn, that's absolutely fine, but I think it's important to make sure and educate your owner that those results might be a little bit unhelpful if you're doing it in the autumnal period.

The other thing just to mention is that you can't run a TRH and a Caro light at the same time because they both interact and lead to false positives, so make sure you're doing those on separate days. And the question is, obviously, does, does the TRH stimulation work? Is it actually useful in those grey area results?

So some recent unpublished work that Andy did with the University of Surrey showed that about 25% of equivocal results on the basal ACTH will come back positive on the TRH, so 25% isn't actually too bad. And approximately 24% of equivocal results will come back positive on an autumnal test, so you could actually just wait until your autumnal period. But if there's a reason, there's laminitis or whatever it might be, then doing it earlier that is, is also advantageous.

And what this shows is that the higher the ACTH, and the orange bars of the number of patients that have that basal ACTH, and then the blue is the the same horses with the TRH, is that the higher the ACT basal ACTH, the more likely that you're gonna have a high TRH stim, but in those equivocal ones down in sort of the, the low 20s, 30s, we, we end up being about 25%. As a brief slide, just so it's here, I don't think we need to talk about it. What are our treatment options?

Obviously Pergoli, a couple of options there, cyproheptadine, is sometimes added in our really difficult patients. And then there are anecdotal evidence about the these other three, but I wouldn't be reaching for them in routine. We are starting to see a fair bit of usingerglide BID rather than FID as the label treatment dose, and that's because there is this question about how long the half-life is.

So sometimes it can be quite useful to swap to BID and see if there is an improvement in the clinical or or in the, the values following that. But what is the correct time to start treatment, because that's what we need to know really. Are we worried about subclinical disease, susceptibility to laminitis, white lie disease?

Are we worried about hair growth? Probably not. We can clip the horse out, PUPD, yes, there can be other problems.

All the rest there, so it's significant to think about where in that spectrum of disease do we want to start treating and I think. What we see is that in over 4600 cases, the most likely clinical symptom was laminitis. And if we think about, say, heart disease, for example, I don't want to be treated when I've had a heart attack.

I'd much prefer to be treated when I have high cholesterol and I can reduce the risk. So if we can get on top of this disease process much much earlier in the whole process, then we're doing well and we can actually talk about trying to stop the disease occurring rather than allowing it to progress and occur. And it's a clinical disease common, that's the significant question.

So again, we go back to our clinical symptoms, sorry, our grading, . And then we look at is subclinical PPID a common finding. So in a study of 155 horses, what we found was that those with grade 1, none of those horses had clinical symptoms, but there were some histopathological changes in about 13 of them.

When we go to grade 2, we start to see 2% of one horse had clinical symptoms, but 4949 of them had histopathological changes. As we go into grade 3, we start to see 5%, grade 4, 21%, and grade 5, 94%. But only 27% of the total horses found to have histopathological changes had clinical symptoms, so nearly 75% of those with clinical changes, histopathological changes have no clinical symptoms.

And actually they're the ones that we want to be hitting early on so that we can make sure that they don't have disease later on in their life. Is subclinical disease actually important? We know it exists.

We know those horses are there, they're at risk. Are they really at risk? So in 2013, McGowan looked at all of this and said, is there a risk of laminitis, increased risk of laminitis in PPID versus non-PPIT?

All of the horses were over 15 years of age. In those horses that were PPID negative based on ACTH testing, 3% had laminitis. Of PPID horses that were greater than 15, they were tested again on ACTH.

13% had laminitis. So it's a 4.65% times sorry, risk factor.

So to me, that's a pretty significant change. If you tell me a disease process for myself that I've got a 4.65 times risk factor, I'm gonna want it treated as soon as is humanly possible.

So we've started, we've decided, we've got the disease, we know that we want to crack on. How are we actually going to make sure that the disease is under control and that we are on the correct dose? Firstly, we can never predict what the correct dose of her glide will be for each individual horse.

And efficacy and correct dose could be judged from clinical improvement and maintenance of good clinical health. So hair, attitude, PUPD. But the problem is it's very subjective.

So what we can see in this study, I can't remember any of that was from, sorry, that between day 90 and day 80, actually, it took 180 days for 90% of them to their heritages and to respond to treatment. And actually some of the others didn't even respond anywhere near as well. So actually, That's a long time to wait to decide whether your treatment is appropriate or not.

So for me, clinical signs are not a good way to judge whether it's working. So does ACTH work? Most cases will respond within the first month of treatment.

So we're gonna see that. 91% of cases will be responding to treatment and that's unpublished data from Andy but that's looking at a large number of cases coming through the laboratory. But if there's only a mild improvement, so you know, you've got it some way to being under control by 4 weeks, you could consider a dose increase if they're, if it's still very high, or it might be that you just wait another 4 weeks and retest cos some of them will trend downwards over another 4 weeks.

But most of them, as I say, will be done by 4 and you can really assess where we're at at that stage. And then ideally, as I said before, every 6 months it would be a realistic level at which you'd be wanting to retest. So How well do they respond in sort of the categorical numbers is, is quite a significant question as well.

So 402 horses were involved in this study, all with ACTHs that were greater than 50, ranging all the way up to 1250. And the averages went significantly down over time, but what we can see is that those that started, With values that were relatively low, had much better, you know, sorry, er, sorry, I got a bit confused there. There was a 50%, 100% improvement in 40% of them, so most of them, did very, very well, and, and then 30% of them were completely normal.

There was this category of less than 10% who hadn't less, sorry, about 10% who had less than 10% improvement. So it is important to keep doing our testing because you don't want to miss those few percent that are on the outliers, of, of response. As I mentioned earlier, I just wanted to make sure, mentioned a couple of recent studies.

There's a lot coming out at the moment about testing, but it's sort of quite an esoteric topic at the moment. I don't think there's a lot that's gonna change how we do it clinically. Whereas some of the clinical symptoms are, are interesting.

So this study said that there is muscle atrophy occurs in about 50% of PPID affected horses. And obviously that may have important implications for both performance and quality of life. And what we know is that in humans, a loss of muscle mass results dramatically in decreased strength, aerobic capacity, metabolic rate, and also general wellbeing.

So the more we can ameliorate that in horses, hopefully, the longer and the more comfortable they will be through their geriatric years. So bans in 2000 this year, sorry, showed that there was atrophy of type 2A and type 2B muscles, and that they found Pergoli ameliorated muscle wastage in 36 and 46% of courses at 3 and 6 months, so. Not as good as maybe you would like, but maybe you're battling once you lose that muscle in geriatric, we know in humans it's much, much harder to rebuild your muscle once you've lost it as a geriatric patient, so.

If we can try and stop it occurring early, coming back to that subclinical disease, all the better. But if we have got to that stage, then hopefully, 40, 50% of them will start to rebuild their muscle by about 6 months time. Obviously there was no control, well, not obviously, sorry, there was no control in this study, so we have to take it with a slight pinch of salt.

And in a much older paper, Monica Alleman, looking at this in 2002, showed that there was no improvement in muscle fibre after three months of treatment, so. We've got to question where the the line truly is. So, there was another study that looked at some different markers, muscle ring finger one, great name, to me, which is a marker of proteolysis.

And associated with muscle loss as it degrades, the, the myogenic transcription factors, really, I don't fully understand it if I'm honest, but it's, it's out there, . But they show no improvement in the amount of muscle ring finger, one, with Perlite, again, so harking back to the Aeman paper of 2002 that actually maybe we're not seeing the improvements that we would like. They state in their comment that pole eye treatment did lead to weight loss, er and improved in fasting insulin, but that weight loss was planned, so they had actually put these horses on diets, so it's a slightly naughty answer that they come up with in that one.

And previous studies have been a bit equivocal about muscle loss, so they showed up regulated calpa, which again leads to pro proteolysis. And another paper that showed no difference between PPID and EM and normal horses. So again, just take it all with a pinch of salt.

Common sense says that actually a lot of these horses are losing muscle, but let's try and stop it happening. Inflammation, so this was a 2020 paper and they compared PPID versus non-PPID cases. And we're seeing a relative lympheenmia, although probably not clinically all that significant.

And they also found there was no difference in antioxidant markers and they. And anecdotal evidence. Although I have had disease problems such as, corneal ulcers leading to problems as well.

Is anaemia significant in these patients? So should we be looking at baseline biochemistries and hematologies in them? We know that it occurs in old age, irrespective of PPID status, and it could affect exercise intolerance.

So when looking at the two of them, there is a comparative anaemia in our PPID patients compared to our non-PPID patients. It's not significantly low enough to lead to exercise intolerance or any disease processes such as that. But to me it would be very much worth monitoring to make sure that we don't allow it to progress and become more significant, because, you know, some of these older geriatrics can be sitting at around 21% of the PCV as they get older, which definitely has an effect on their ability to exercise.

There's some potential new therapies, sorry I meant to move this slide, but we'll we'll talk about it here, and because we know that hyperinsulinemia induces laminitis, and it's thought to do so via insulin growth factor one receptors in the in the hoof. So insulin has been shown to affect it, leading to this dephosphorization of the receptors, which exerts an effect on the lamely making them stretch, making them break. And it looks like there might be potential to use antibodies to block IGFR one, therefore reducing the risk that that you're gonna start to see that stretching the damage to the lamellae.

And they have this study, Rhan Ranama, sorry for the pronunciation, in 2020, found that human antibodies could bind to the IGF1R and therefore could be a potential therapy. It's not, nothing's come of it in the last year, so who knows, but it is out there as a potential option. One significant question is, what is the risk of laminitis when treating steroids, and it's always a question that's in the back of my head when I'm gonna start any of these horses on steroids.

And I think it should always be considered seriously, but is there an increased risk? So a study in 2019, they did an infusion of ACTH into these horses, measured various plasma steroid levels throughout that period, and found there was a significant increase in a whole range of steroids. Therefore, could that increasing circulating steroids predispose to the risk of laminitis, and the question is, I don't know.

But my gut feeling is, if you know you have a horse with PPID and EMS or even just one or the other, then we should be seriously considering the use of steroids, whether we reduce the dose, whether we hold until we've got maybe the EMS at least under control. Would, is normally how I approach those cases. Now, if we're in a life threatening disease, it would not stop me using it.

So immune-mediated hemolytic anaemia, the horse's PCB is 12. I would absolutely use steroids and take the risk at that stage. And I don't remember the last horse that I induced laminitis with steroids, in the hospital, so my, my risk factor is, is low in my head.

So hopefully that's PPID, we've got our basal ACTHs, we can do them in autumn and spring, ideally so that we're getting that autumnal peak and therefore we're getting much increased sensitivity specificity. We can do TRH testing, which is gonna increase again, our sensitivity specificity, giving about 25% of patients who are equivocal, a positive answer or a negative, which is also very helpful. So we are now able to give ourselves a much better chance of being able to diagnose the the disease process.

Now moving on to equine metabolic syndrome. EMS is a collection of risk factors increasing the risk of laminitis. These can be grouped into insulin dysregulation, obesity, and adipokine dysregulation.

So, you know, your stereotypical fat cob, sorry for the picture of this one, is an at-risk patient or the PPID patient, who is going to exacerbate any insulin disregulation. But why do we investigate this disease when 70% of horses in one recent study were overweight? So we can pretty much categorically say that a lot of those are gonna have EMS so why don't we just get them all on a diet, partly because we, we struggle with our owners, but what it does is it prevents, provides a laminitis risk factor ratio, and we're hopefully, as I said a moment ago, gonna have a bit more information on exactly how to be able to do that ratio.

It also provides a baseline monitoring, so we can say to the owner, OK, here's your level today, let's get the diet done for a couple of months and then hopefully we're gonna see that number improve and therefore all the hard work that you've done has been worthwhile. But we've got to be aware of testing limitations as we go through. So what are the components of EMS that increase the likelihood of laminitis occurring?

It is the insulin resistance, that is what the problem is, so we're seeing a transient or long-lasting hyperinsulinemia in these horses. And what's interesting is that in horses compared to humans, we see this persistent hyperinsulinemia that is maintained consistently, whereas in humans, once they've reached a hyperinsulinemia peak, normally they drop off as you get er overuse of the pancreas and and and they can't produce any more insulin. And a regional or generalised adiposity is an increased risk of laminitis, as we know.

So what is it that causes it directly, so we know it's a hyperinsulinemia, we know that, we've done studies, we've shown that. And it's thought that the pro-inflammatory status of course of the ID EMS might induce an increased risk of laminitis as well. And the question is, is another one is, is EMS pro-inflammatory, and it has been shown compared to PPID to be inflammatory via metabolone work, and also low levels of carnitine and arginine.

And carnitine is necessary for the transportation of fatty acids into mitochondria for energy production. Therefore, it's been hypothesised that obese individuals with higher plasma fatty acid con er concentrations use more carnitine up. An arginine has been shown to increase oxidation of long chain fatty acids and glucose, but it is also known for its strong vasodilatory effect, mediated by nitric oxide.

Therefore, low arginine concentrations in horses with high insulin response could be associated with some sort of endothelial dysfunction, potentially involved in the pathophysiology of endocrine laminitis. So those two markers, just a recent study in 2020, could be involved in our laminitis. So they did postulate, could we supplement with carnitine or urgently to try and reduce it, there there was no answer to that question, but it's something that might come up in the future.

But what is insulin dysregulation or insulin resistance? And I think it's important to separate the two, insulin dysregulation, often associated with PPID. We don't have, we don't know it's regulated, sorry, resistant per se.

But resistance is a subnormal response to normal insulin res concentrations. With a reduced sensitivity to insulin by the body's insulin dependent processes. And therefore the inability of a known quantity of exogenous or endogenous insulin to increase glucose uptake and in utilisation individual as much as in the normal population.

And finally, a given concentration of insulin produced a less than expected biological effect. In other words, how much insulin is floating around in the blood does not have as much of an effect on the insulin receptors and therefore uptake of glucose from the blood into the cells. And what are the components of insulin dysregulation?

First and foremost, insulin resistance. And it's clear that it plays a central tenant role in the disease process. But it's not always the sole cause of disease process.

Therefore, when looking at EMS patients, we are concerned with untangling if the insulin dysregulation is driven solely by insulin resistance, the pancreatic response or a complete combination of both of them. So we know with insulin resistance, there will be a compensatory pancreatic secretion. In other words, increase leading to hyperinsulinemia, and also there'll be a reduced hepatic insulin clearance.

This hyperinfinemia in itself leads to impaired in insulin signalling and lipogenesis leading to more insulin resistance. Insulin resistance therefore leads to a hyperglycemia because there's gonna be an impaired glucose uptake into the cells and reduce suppression of gluconeogenesis. And this in itself stimulates B cell production and therefore hyperinsulinemia.

There's also gonna be a reduced suppression of lipolysis and hepatic BLDL synthesis leading to a hypertriglyceridemia. So all hand in hand, there's a lot of things going on in this insulin dysregulation. As I mentioned, we know that insulin causes, let's say is central to laminitis, and insulin is normally released into the circulation after eating, and that plays a part in my further testing that I'm gonna talk about in a moment.

As I said, normally moves sugars into the cells, and excessive insulin levels of cells can be harmful to blood flow, to cell division and growth, and either of these could be involved in laminitis. And what we know is that if aquis experience sustained high insulin levels, they develop laminitis. So what about the testing of it?

A group in 2006 compared horses with laminitis and those without that were laminitis, naive, and what they found was that the basal insulin in those that were naive versus those with laminitis prone, so the, the hashed out section, the basal insulin was much, much higher. As was the triglyceride, linked in with the insulin resistance that I just mentioned a moment ago, and the body condition score was just above the non-laminitic group. Well they also found was that this group had a much much more significant response in insulin to feeding at different times of year.

So we can see that again the two greyed out areas where the laminitis prone ones, and we can see the much, much higher response in insulin. And so does insulin differ in laminitis prone horses because that's the crux of the matter. And so there was another study that looked at 12 horses, 12 normal sort of 8 fat ponies, and they get fed one set of food that was just hay and rice bran and one that was with glucose added.

And fat ponies, the 8 fat ponies, showed an excessive insulin response, especially with high sugar diets. So we can see that they are much, much higher in that response. What does that matter?

I mean, high insulin. What we can see is that those horses with a much higher insulin responses, so the going up into the 4 hundreds, had a much higher risk of laminitis occurring, . Within a relatively short time period, so 0% of horses with with an incident of less than 195 had laminitis within a short period, whereas a much, much higher percentage of them had it, in the higher insulin levels.

And also the higher the insulin levels, the sooner the laminitis was likely to occur. So looking at the individual tests themselves, how are we going to, what are we gonna do, which ones are we going to use and and how do we make our decisions? So First and foremost, it's always important to consider are we gonna get false positives, false negatives, because both of them are a problem.

Now false positives in this case probably aren't too significant. We're not talking about COVID, we're talking about EMS. The thing we're gonna recommend is a diet, so if it's a bit overweight, it's not a problem.

False negatives are a problem though, because if we sit on our hands and say, OK, actually that incident was completely normal when that horse was truly at risk, then we are going to miss our opportunity to get that horse treated and make sure everything goes well. So historically we have done fasting insulin, so starve overnight, but what we found is that that test had a really high frequency of false negatives. And therefore we don't ever recommend that test anymore.

Whereas resting, feed as usual. And then test our our insulin about 2 hours later. So fast is never recommended, whereas resting, we would never really recommend as a primary diagnostic because there is still that chance, you know, if they're on an appropriate hay diet, let's say, very low sugar hay, that you're not going to get an elevated insulin, it's not testing every single part of their diet.

But sometimes, as we'll come on to, we are now using that as a really significant way to adjust horses' lifestyle and dietary modification. So what do we use as a primary test, dynamic testing is the way forward. And this this dynamic test assesses the magnitude of your postprandial hyperinsulinemia, so that post eating, how high is the insulin liquids, and it's far more sensitive as a diagnostic test than the resting insulin concentrations.

What we recommend is that we do withhold feed for approximately 6 hours before administering the corn syrup. So normally, you sort of I say to an owner, leave only one flake of hay in after about 10 p.m.

And perform the test first thing in the morning. Or you could theoretically feed as normal in the morning and then fast for 4 to 6 hours before performing in the afternoon. What we're now recommending is about 45 mLs of car light per 100 kg.

That was some work that, the RVC did, a few years ago, and then test between 60 and 90 minutes. So, you, you can do a 60 and a 90 or you can do a shortened at 75 minutes. With the carrilight, we are having a few horses that are, are rejecting it, so you can still do the 1 gramme per kilogramme of, glucose in feed with a little bit of chaff.

But we have a better strike rate with the Caroline. As we're in the slightly colder months, if you just warm it up slightly in the microwave, it makes it far, far easier to administer. It's much more of a liquid rather than a pure syrup.

So that, that can be helpful. The My Group in 2018, showed that the oral glucose test is a predictor of laminitis with those having higher insulins and gluco levels far more likely to be prone to laminitis. So these tests really are a good way of confirming the risk of laminitis.

So for me, the Carali is a first line recommended diagnostic test for insulin dysregulation. And as I say, sometimes, we do see it fail, so we can go to the 1 gramme, of glucose. And sometimes that failure in, in the Caroli, we have a few postulated ideas.

One is, was the horse starved, so actually, was there a lot of food sitting in the stomach and therefore, the glucose didn't go from the stomach into the small intestine, which is essential for the, the, the test to work. Also Caroli is a natural product, so the exact sugar levels might change from from container to container. Now that shouldn't be a significant change, but there is that potential.

And what's really important when we do this test is, oh sorry, is to remember to take an insulin and a glucose when you take that 75 minute sample. And the rationale behind that is that you can't analyse the insulin level unless you know that a high enough glucose peak has been achieved. And we are seeing enough cases where that glucose peak is not reached that we're saying, do another test.

. And also it means that you can compare tests as you go forward, whereas sometimes if you don't know that that glucose level, you wouldn't be able to do that. So make sure you are doing that whenever you do the test. As I mentioned, we generally recommend at least a short period of starvation or reduced food, overnight so that we know what's going on.

Ed Knowles did a study a little while ago and found that in the statistics, it was acceptable to test directly from pasture. The one problem was that results were more variable, so you can see that, you know, that there are some significant differences, as we go through the 60 to 90 minute period, but that they weren't affecting the diagnosis, so they felt that at least you could still most likely get the diagnosis. But given that variability and the poor repeatability of the study, we still recommend that we do starving because then we're gonna get the best out of the test that we possibly can.

Also, a horse is gonna be absolutely fine without food for a few hours overnight. So, we, we can rest assured that we're not doing any harm to the horse. If though, we're still feeling like we're not getting the test results we want, so in other words, we, we look at a horse, we think this is an EMS patient, it's ticking all the boxes, and yet our carolite is normal or we're getting poor glucose absorption, our resting is normal.

What are we gonna do to diagnose this? One final test that we could do is our insulin response test. So this came from the combined glucose insulin test, which is never used in clinical practise.

It's far too involved and, you know, far too many injections, far too much sampling. So, a group in 2013, so a little while ago now, looked at how could we shorten this, how can we make this into a more viable test to do. And they do the, they found the two-step insulin response test.

And what this is is you take a baseline sample, looking at your glucose, so get a red sorry, a a grey top tube, take your glucose beforehand, inject 0.1 international units per kilo of insulin, and it has to be bovine neutral insulin, not the long lasting stuff that, you use in, in small animals. And then in the short test, you take a sample 30 minutes later.

And what you're looking for in a normal horse, it should drop the glucose down to less than 50%, whereas in an insulin resistant horse, you're going to see the baseline not increase, not decrease below 50%. And it's important to note that I am talking purely about insulin resistant horses, rather than insulin disregulated horses, because those that are just purely disregulated, say, due to PPID may not have such a significant response in this blood test. So sometimes this test is recommended if other tests are inconclusive or a definitive answer has not been reached on on the testing so far.

But what's important to note is that there is a risk of hypoglycemia in these patients. So if the horse, and if the if the horse is not insulin dysregulated. So feed these horses straight after the second blood sample, so as soon as you've got that 30 minute sample, give them a nice load of feed.

And if you think they are becoming hypoglycemic, in other words, you're seeing some tremors. I've, I've only ever seen tremors, nothing more significant than that, then you can give them about 100 mLs of 50% glucose IV and monitor them. So, whenever doing this test, take a bag of 50% glucose with you so that you can make sure you can administer it as and when required.

But I've seen it, I think, twice, in all the times I've done it. And what about adiicine? So this is a test that we introduced, Lipoate when a hospital introduced probably about 5 years ago, and what they are are biologically active molecules that work in autocrine and paracrine function throughout the whole body.

Their function is to regulate energy metabolism, cardiovascular function, reproductive status, immune function, they are massively important throughout the body, and this is general adapines rather than anything specific. The study adipokines in the equine population include leptin, adiponectin, angiotensinogen, as part of the, the RAS system and a few others. But what we know is that the abnormal production regulation of adipokines appears to be associated with a number of diseases in humans, dogs, cats, and horses, sorry, and these can include metaboloid syndromes such as type 2 diabetes, atherosclerosis, heart disease, and even cancer in patients.

And it's a relatively new area in veterinary medicine, particularly in the equine field, so we are constantly learning about it and constantly changing. Now leptin initially seemed like a great marker to work with. It increases with obesity, it's sometimes seen increasing luteal phase, it's associated with inflammation and decreased with testosterone.

It also is massively involved in suppressing appetite, increasing energy expenditure and stimulating angiogenesis, and it also is involved in modulation of insulin sensitivity. The problem was that as we did this, or as other people did this, sorry, they found that leptin tells you what you already know, is the horse fat. It doesn't help beyond that, it confirms obesity, it does not confirm an insulin problem.

And also, as I say, it's seen in luteal changes, so in mares, you're gonna get changes normally within the season, so it really isn't all that helpful. So we moved on from leptin fairly rapidly. Adiponectin became the one that was, more interested, interesting, sorry.

It's only produced with mature adipocytes, compared with leptin which is produced in a number of cells. There are multiple forms, trimus, hexas, high molecular weight versions, and initially, the or the thought is that high molecular weight version is the most biologically active. And that was the one that we were testing, but due to problems with the, the reagent that we were buying in, we've now moved back to total adiponectin.

And we did look at our numbers because we obviously wanted to make sure that the total admectin wasn't going to be giving us lots of false positives or negatives. And we found an absolute correlation between our high molecular weight levels and our total ad connecting. So although the reference ranges have changed, the test is still as sensitive in our mind as it was previously.

So there was a lot of studies that looked at this, that said there would be a drop in horses, the drop in out of connecting those horses lose weight. What we're seeing in reality is that sometimes that drop isn't as significant as we want. And I think that harks back to the idea of is there a genetic disease in in play in a lot of these cases that we're never gonna resolve the adipocyte function, metabolic functionality in our Shetlands.

So, in fact, It's not always that helpful unless you're talking about some thoroughbreds. What's really useful though is that it gives you that baseline sample, so you go and see a horse today and you want to say, OK, I want a test for EMS and you can do this test, and you can get a pretty good indication of whether that horse is suffering from EMS. There are some increases seen with some various things in in humans, but we're not really significantly worried about those in horses.

There is a slightly higher levels in females and geldings versus males, but it's not too significant. What we did find, or what has been shown in studies is that chronic exercise leads to a decrease in decrease in adiponectin. So it nicely correlates with losing weight, getting fitter, getting healthier.

And as I mentioned, it's functionally indicator of fat activity, and it works in both the liver and muscle to to increase beta oxidation, glucan decrease the gluconeogenesis, and importantly increase insulin sensitivity. What we know is that increased fat leads to decreased adiponectin, and that's particularly true of visceral fat. And that decreased adiponectin is linked with insulin resistance, type 2 diabetes, hypertension, and ventricular hypertrophy.

That's all in humans, but we now know, we now know that it's associated with insulin dysregulation, resistance, sorry, in horses as well. So as I mentioned, Unru in 2012 did a study looking at horses and saying. Does adiponectin decrease as we lose weight?

And what he found was that prior to a weight loss programme, insulin resistant horses had a markedly high adiponectin, and that after their weight loss programme, they had a much more normal adiponectin. Sadly, as I mentioned, this isn't reality in, sorry, this isn't true in reality, and I think that you know, that, that study was done on a lot of horses that were made to put weight on and then lose it, and they were often slightly less native in native breeds in their phenotype. So how do we test it?

Serum or plasma, can be either works. So, EDG and heparin also appear to be consistent, but, red top tubes is normally the best. Stable for in humans for about 36 hours, and we, our research also found about the same.

And what's important, it doesn't fluctuate with exercise or feed, so it's a very stable primary diagnostic tool. So How do we summarise, one first thing again, just as I mentioned with the ACTH, remember that not all labs that references are the same, so everything I'm putting on here are Lieckman Hospital labs, reference ranges, not another, not another company, so just make sure you interpret it in line with the laboratory you use. But these are our rough, well, not our rough, these are our reference ranges.

As I said, don't use fast, far too high false negatives. Sorry, fast insulin, false negatives, resting insulin can be helpful, but we'll come on to the new testing in a moment. Post glucose insulin and post-Carolite quite useful.

And adiponectin also very useful. So the potential new testing that I've been talking or sort of leading to is. We know that various different foods can induce hyperinsulinemia, but it doesn't seem to be that one type of food is consistent throughout all horses.

So we know that some horses have a prolonged hyperinsalemia, others have spikes, and it might be that actually one type of hay is massively difficult for one horse and might not be for the other. So we've started doing a lot more testing in these cases, looking at which feed is actually acceptable for the patient. And it's really interesting.

So we've had quite a few in the hospital where the hay that the horse, the owner had brought in that was supposedly quite good for horses with laminitis risk for, you know, as per the manufacturer actually was inducing a hyperinsalemia, whereas our soaked or our steamed hay didn't induce a hyperinsulinemia. Also, we're doing it a lot for working out whether a horse can go out to grass or not, because that's often the question the owners want to answer. So doing this test, host weight loss so that you're in a really good place, you know that horse is about as good as it's gonna be.

And then you give them the food whatever they're gonna have, and about 2 hours later, you then take the sample. So following the feed, whatever it is, you should be starving, so they're not topping up that feed with whatever else is given. And sometimes it can be quite good to do it over, you know, let them graze for quite a long time so that you're, you're creating a real situation.

And does weight loss help? So obviously it's significant, it's really important to know if we do all this weight loss, if we put these sources through it, can we retest them to show that everything has improved? And De la Clerk, in 2020 showed that utilising an oral glucose test, comparison, they said that a 5% weight loss leads to a 22% decrease in your insulin response.

Some horses didn't respond at all. But I think it is important. So this is just one of my favourite patients.

This horse was on death or with laminitis, morbidly obese, and through starvation, or not starvation, but through dieting alone, ended up looking like it does six months later, and its insulin response went from horrendously high to complete normality, through that period. So it really does work to get these horses back into the place they should be. So this was, it's a few years old, 3 years old now, but it's becoming something on the market.

I know that there are specialist companies that are producing versions of it. So Velagic flowsin was a studied, drug in 2018. It's a sodium glucose co-transporter 2 inhibitor.

So what this does is it stops the, kidneys reabsorbing glucose, leading to a glucose urea and a decreased blood glucose and insulin. In that study, 49 horses were were tested, and the maximum glucose in the, in the treatment horses was 20% lower versus the ones not in the treatment. And the incidin was 45% lower in the treatment.

So also the really significant part was that the treated group had no laminitis, whereas the control group did have some laminitis. Now it is a drug company funded study, but the data does look genuinely quite good, and I would be, I'm looking forward to that coming out as a, a licenced product, but as I say, there is actually a, a one out on, on the market, as a special currently. It is very important when using these medications though to monitor your triglycerides because they do lead to a hyper tri triglyceridemia, which can obviously have significant effects on liver function.

So don't take these drugs as a mild thing, they, they really do need monitoring if you're going to start using them. So this was just some work that came out in 2020, again just highlighting some of the recent literature. And, and stating is EMS pro-inflammatory, so therefore likely leading to an increased risk of laminitis.

What they found was there was no evidence of increased SAA, but there was an increase in IL-6 of pro-inflammatory cytokine, also active in A, again another inflammatory cytokine. But there were increased IL8 IL 10 as well, so it's, it's a bit of a grey area is the honest answer, and actually when you look at things like exercise, that is pro-inflammatory in itself. So, luxurious out as to whether in itself it's pro-inflammatory.

I don't think that's the cause of laminnis, I think it's the, the changes within the cellular structures associated with insulin growth factor. So To summarise all of that, that was a lot of information about various different things. I think when we look at PVID testing, as I said, we've got our basal or our TRH, they're our main tests nowadays, there are others, but they're not in reality what we're, we're worried about.

Basel remember your seasons, twice a year is what is ideal, otherwise do it annually at the same time so you can truly compare ideally in the autumnal period. TRH stimulation is very good, it increases your sensitivity and specificity, helps to untangle some of those grey area results, and it's now possible year round, but take care in the autumnal period because that grey area on the TRH is so big that you may just lead yourself into another grey area. EMS testing, I said, don't do a fasted, you could do a resting if you want, but probably not far better to do a dynamic testing, carolit or glucose.

Instant testing is possible, but care with hyperglycemia. And what's really important is now our new testing techniques of testing what food is appropriate for each individual horse. So starve, give the feed, starve, retest two hours later and see what your insulin response is.

Thank you very much for listening and obviously if you have any questions, get in contact with me.