Equine insulin dysregulation

Abstract

Described herein are methods of diagnosing the presence or absence of insulin dysregulation in equine animals. The methods comprise (a) performing an assay to determine the level of one or more biomarkers selected from the group consisting of propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid, and urea cycle related metabolites such as argininosuccinate and arginine, in a sample obtained from an equine animal; and (b) comparing the level of the one or more biomarkers determined in step (a) to a reference result; wherein a difference between the level of the one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of insulin dysregulation. Also disclosed are methods of diagnosing the presence or absence of susceptibility to a condition associated with insulin dysregulation in equine animals, methods of monitoring the level of one or more biomarkers in equine animals, and related methods of assessing the effectiveness of a treatment for insulin dysregulation in equine animals.

Description

Background of the Invention

[0001] One of the major endocrine disorders affecting equines is equine metabolic syndrome (EMS). An original definition of the syndrome, associated with insulin resistance, obesity, and susceptibility to laminitis, was developed in the early 2000s (Johnson 2002, Hoffman et al. 2003, Treiber et al. 2005, Treiber et al. 2006). Laminitis is a common and very painful condition in equines that involves the destruction of the lamellar tissue connecting the bone and hoof wall. This can cause the coffin bone (also known as the third phalanx in horses) to sink or rotate away from the hoof wall, potentially leading to euthanasia. Evidence confirming a direct link between insulin and laminitis comes from Australian researchers who showed that maintaining a sustained high plasma insulin concentration of approximately 1000 μIU / mL for an extended period can induce laminitis in ponies and horses (Asplin et al. 2007, de Laat et al. 2010). This has led to the widespread use of the term "horse metabolic syndrome" to describe the phenotype of animals with obesity and insulin resistance that are susceptible to laminitis (Frank et al. 2010).

[0002] Initially, this label was applied to equines exhibiting the following characteristics: (i) increased obesity, whether localized (regional obesity) or generalized (obesity); (ii) insulin resistance, characterized by hyperinsulinemia, or abnormal blood glucose and insulin responses to oral or intravenous glucose and / or insulin loading tests; and (iii) clinical or subclinical laminitis occurring without a recognized cause, such as cereal overeating, colic, colitis, or retained placenta. However, recent acceptance has increased that lean animals may have (even if they have never been obese) or retain (if they were previously obese) the EMS phenotype, and therefore have an increased risk of laminitis. Therefore, obesity and regional obesity have now been excluded from the essential components of EMS. In short, obese animals may or may not be EMS animals, and lean animals may or may not be EMS animals. A recent consensus statement from the European College of Equine Internal Medicine (Durham et al. 2019) clarifies this—the key consistent feature of EMS is the presence of insulin dysregulation (ID). The presence of obesity or other metabolic changes is more variable in EMS animals.

[0003] Therefore, insulin dysregulation is a collective term for perturbations in the balance between plasma concentrations of insulin, glucose, and lipids (Durham et al., 2019). This includes tissue insulin resistance, as well as basal and postprandial hyperinsulinemia. Insulin dysregulation plays a significant role in endometrial dysregulation (EMS), which is associated with an increased risk of laminitis. In fact, laminitis is a major clinical outcome of EMS (Durham et al., 2019). Recent work has confirmed that the severity of insulin dysregulation is associated with any degree of risk of laminitis independent of physical condition scores (Knowles et al., 2023a, 2023b).

[0004] Insulin dysregulation is also associated with middle pituitary dysfunction (PPID), with more than one-third of horses with PPID having insulin dysregulation, and horses with PPID being 2.7 times more likely to have hyperinsulinemia than age-matched controls (McGowan 2018). PPID, also known as Cushing's disease, is the most common disease in older equines. The condition is characterized by the loss of inhibition of the pituitary gland, particularly the middle pituitary, leading to an overproduction of pituitary hormones, including adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (α-MSH), β-endorphin, and corticotropin-like peptide-1 (CLIP) (McGowan 2018). PPID is associated with a wide range of clinical signs, such as increased coat length (hypertrichosis).

[0005] Hypertrichosis and abnormal hair loss patterns, laminitis, muscle wasting, abnormal fat distribution, increased water intake and urination (polydipsia and polyuria), increased susceptibility to infection, and infertility.

[0006] Although insulin dysregulation is associated with a variety of endocrine disorders in equines, it can be difficult to diagnose reliably. As mentioned above, physical condition cannot be used as a reliable indicator of ID status. Insulin dysregulation in equines may also be associated with aging. In a given animal, one or more different components of ID may exist, and different tests are recommended to assess these different ID components. Recent work on ID in ponies suggests that the insulin response to oral sugar may be one of the most important predictors of laminitis risk (Meier et al. 2019). Frequent sampling IV glucose tolerance test (FSIGTT) and normoglycemic insulin clamp are primarily used to assess tissue insulin resistance, but these tests are costly and not practical for routine use. Oral irritation test assesses an animal's response to oral ingestion of hydrolyzable carbohydrates (sugars and starches), which may be associated with laminitis risk. One such test, the oral sugar test (OST), is a practical test that clinicians and owners can easily perform on-site, although the recommended Karo Light Corn Syrup is not available in all countries. Furthermore, diagnostic methods that rely on basal insulin concentrations can often lead to misdiagnosis because different insulin diagnostic tests require different reference ranges, and insulin concentrations can be affected by various factors, including background diet, season, etc., which means that the interpretation of test results can be challenging.

[0007] Therefore, there is a need for more reliable and potentially cheaper and easier-to-implement methods for diagnosing and monitoring ID (as well as susceptibility to ID-related conditions such as EMS, laminitis, diabetes, obesity, and PPID).

[0008] The inventors have developed a specific set of biomarkers, each capable of consistently distinguishing equines with insulin dysregulation from those without, unaffected by diet or other potential confounding factors. Therefore, this invention provides a method for diagnosing the presence or absence of insulin dysregulation in equines based on biomarkers present in samples obtained from animals.

[0009] In one implementation method, the method for diagnosing the presence or absence of insulin dysregulation in equines includes: (a) Assays were performed to determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide riboside, allantoic acid, hexadecatrienoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; (b) Compare the levels of one or more biomarkers identified in step (a) with reference results; The difference between the levels of one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of insulin dysregulation.

[0010] In one implementation, step (a) includes determining the ratio of the level of one or more biomarkers in a sample obtained from equines to the level of a standard biomarker, or determining the ratio of the levels of two or more biomarkers in a sample obtained from equines.

[0011] Because insulin dysregulation is associated with many other conditions, these biomarkers also indicate an increased risk or susceptibility to one or more conditions associated with insulin dysregulation. Therefore, the present invention also provides a method for diagnosing the presence or absence of susceptibility to conditions related to insulin dysregulation in equines based on biomarkers present in samples obtained from animals.

[0012] In one implementation, methods for diagnosing the presence or absence of susceptibility to insulin dysregulation-related conditions in equines include: (a) To determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; (b) Compare the levels of one or more biomarkers identified in step (a) with reference results; The difference between the levels of one or more biomarkers identified in step (a) and the reference result is used to determine the presence or absence of susceptibility to insulin dysregulation-related conditions.

[0013] In one implementation, step (a) includes determining the ratio of the level of one or more biomarkers in a sample obtained from equines to the level of a standard biomarker, or determining the ratio of the levels of two or more biomarkers in a sample obtained from equines.

[0014] The present invention also provides a method for monitoring the levels of one or more biomarkers in equines over time.

[0015] In one implementation, a method for monitoring the levels of one or more biomarkers in equines includes: (a) Assays are performed to determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; and (b) Repeat step (a) at two or more separate time points.

[0016] In one implementation, step (a) includes determining the ratio of the level of one or more biomarkers in a sample obtained from equines to the level of a standard biomarker, or determining the ratio of the levels of two or more biomarkers in a sample obtained from equines.

[0017] The present invention also provides methods for evaluating the effectiveness of treatment for insulin dysregulation in equines and methods for evaluating the effectiveness of treatment for insulin dysregulation-related diseases in equines.

[0018] In one implementation scheme, methods for assessing the effectiveness of treatment for insulin dysregulation include: (a) Assays are performed to determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; and (b) Repeat step (a) at two or more separate time points, at least one of which is after the equine has begun treatment.

[0019] In one implementation, step (a) includes determining the ratio of the level of one or more biomarkers in a sample obtained from equines to the level of a standard biomarker, or determining the ratio of the levels of two or more biomarkers in a sample obtained from equines.

[0020] Similarly, in one implementation method, methods for evaluating the effectiveness of treatment for conditions related to insulin dysregulation include: (a) Assays were performed to determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate; and (b) Repeat step (a) at two or more separate time points, at least one of which is after the equine has begun treatment.

[0021] In one implementation, step (a) includes determining the ratio of the level of one or more biomarkers in a sample obtained from equines to the level of a standard biomarker, or determining the ratio of the levels of two or more biomarkers in a sample obtained from equines.

[0022] Treatment for insulin dysregulation or insulin dysregulation-related conditions may aim to alter the levels of one or more biomarkers of the present invention in equines to make them consistent with or closer to the levels of such biomarkers typical of healthy equines. Therefore, the effectiveness of a treatment can be determined by assessing its ability to achieve the desired change in the levels of one or more biomarkers of the present invention in samples obtained from equines. If a treatment achieves the desired change in the levels of one or more biomarkers of the present invention in equines, for example, if the treatment increases the level of propionylglycine in equines or decreases the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in equines, then the treatment can be determined to be effective. Conversely, if the treatment does not achieve the desired change in the levels of one or more biomarkers of the present invention in equines, for example, if the treatment does not increase the level of propionylglycine in equines or decrease the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in equines, then the treatment can be determined to be ineffective.

[0023] Figure 1 shows propionylglycine (Pyroylglycine) in samples obtained from ID and non-ID animals. Figure 1A ), allantoin ( Figure 1B ), Nicotinamide nucleoside ( Figure 1C ), allantoin ( Figure 1Dhexadecatrienoic acid (HCA) Figure 1E ), argininate ( Figure 1F ), argininosuccinate ( Figure 1G ), creatinine ( Figure 1H ), phenylalanine ( Figure 1I ), tryptophan ( Figure 1J ), Leucine ( Figure 1K ), acetylcarnitine ( Figure 1L ) and 1-palmitoyl-2-oleoyl-GPC (1-palmitoyl-2-oleoyl-GPC) Figure 1M The results of metabolomics analysis of the levels of [specific metabolites] were presented. Animals were fed three different treatment diets, and blood samples were collected before feeding (T1) and at 30 (T2), 60 (T3), and 90 (T4) minutes after feeding. The three groups in each figure correspond to the three diets, and the four time points correspond to four different blood samples (T1–T4). P1 represents ID animals, and P2 represents non-ID animals. The level of each biomarker in each animal was measured relative to the median level of that metabolite in the overall sample population (i.e., all animals at all sampling times), which was set to a value of 1.0.

[0024] The inventors have developed a set of biomarkers present at different levels and / or ratios in equines with insulin dysregulation (ID animals) compared to equines without insulin dysregulation (non-ID animals). This set was developed by collecting blood samples from both ID and ID animals and identifying the levels of different biomarkers in these samples. Blood samples were collected from multiple ID and non-ID animals before and after administration of various control diets, and certain biomarkers were consistently identified as differing in level between the two groups, indicating that the levels of these biomarkers indicate insulin dysregulation in a manner independent of the diet received by the animals and the time point at which the samples were obtained. Therefore, these biomarkers can be used to distinguish between ID and non-ID animals.

[0025] Biomarkers with varying levels between ID and non-ID animals include propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate. Therefore, as used herein, the term "biomarker of the invention" refers to the group of biomarkers comprising propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate. Nicotinamide nucleoside is a precursor to nicotinamide adenine dinucleotide (NAD) and is an important metabolic intermediate. Allantoin and allantoic acid form part of the purine degradation pathway and are intermediates in the adenine partial degradation of NAD. The urea cycle is responsible for processing ammonia produced from the catabolism of amino acids. The catabolism of certain amino acids, such as methionine, threonine, isoleucine, and valine, also produces propionyl-CoA, which can be converted to propionylglycine. The term "urea cycle-related metabolites" includes any metabolites involved in or associated with the urea cycle or its secondary pathways. Specific urea cycle-related metabolites include arginine, argininosuccinate, argininate, ornithine, 3-amino-2-piperidinone, 2-oxoarginine, citrulline, homoarginine, homocitrulline, proline, dimethylarginine, N-acetylarginine, N-δ-acetylornithine, trans-4-hydroxyproline, N-methylproline, N,N,N-trimethyl-alanylproline, betaine, prolylhydroxyproline, aspartic acid, fumaric acid, and urea. In a particular embodiment, the urea cycle-related metabolites are argininosuccinate and argininate. The term "argininate" includes both the protonated and deprotonated forms of arginine acid. Similarly, the term "hexadecatrienoic acid" includes both the protonated and deprotonated forms of the acid (commonly referred to as hexadecatrienoate).

[0026] These biomarkers can be used to diagnose the presence or absence of insulin dysregulation in equines, or the presence or absence of susceptibility to insulin dysregulation-related conditions. Furthermore, these biomarkers can also be used to diagnose the presence or absence of susceptibility to insulin dysregulation-related conditions in equines. As used herein, the term "insulin dysregulation-related conditions" includes, but is not limited to, laminitis, equine metabolic syndrome, diabetes, obesity, and pituitary midline dysfunction (PPID).

[0027] The biomarkers of this invention can also be used to monitor the development of insulin dysregulation and related conditions in equines that have not yet been diagnosed with these conditions. Once equines are diagnosed with insulin dysregulation or related conditions, the biomarkers of this invention can be used to monitor the progression of these conditions and / or assess the effectiveness of any treatments already administered.

[0028] Determination of biomarkers

[0029] This invention provides methods for diagnosing the presence or absence of insulin dysregulation, methods for diagnosing the presence or absence of susceptibility to insulin dysregulation-related conditions in equines, methods for monitoring the levels of one or more biomarkers in equines, and methods for assessing the effectiveness of treatments for insulin dysregulation or insulin dysregulation-related conditions.

[0030] The method of the present invention includes the step of performing assays to determine the levels of one or more biomarkers of the present invention in a sample obtained from an equine animal (also referred to as "equine animal to be diagnosed"). In one aspect, the assay step may include determining the levels of two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve biomarkers of the present invention in the sample.

[0031] As used herein, the term "level" in the context of the level of one or more biomarkers in a sample can refer to an absolute or relative value indicating the level of one or more biomarkers. For example, the level of one or more biomarkers in a sample can be expressed as the concentration of one or more biomarkers in the sample or the total amount of one or more biomarkers in the sample. In one embodiment, the sample obtained from an equine animal may be a dried bloodspot. In this case, the level of a biomarker in the sample can be expressed as the concentration of the biomarker (if the volume of the bloodspot is known) or the total amount of the biomarker in the dried bloodspot. The level of one or more biomarkers in a sample can be expressed in any suitable unit, depending on the assay used to determine the level.

[0032] In one embodiment, the assay step may include determining the concentration of one or more biomarkers in the sample. For example, the assay step may include measuring the concentration of one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve) biomarkers of the present invention in the sample. In one embodiment, the assay step may include determining the total amount of one or more biomarkers in the sample.

[0033] As explained in more detail below, the level of one or more biomarkers in a sample can be expressed as the ratio of the level of one or more biomarkers in the sample to the level of another biomarker in the sample. Therefore, the assay procedure may include determining the ratio of the levels of two or more biomarkers in a sample obtained from an equine animal.

[0034] Alternatively, the level of one or more biomarkers in a sample may be expressed as a ratio of the level of one or more biomarkers in the sample to the level of a standard biomarker in the sample. In one aspect, the assay step may include determining the level of one or more biomarkers in the sample relative to the level of one or more standard biomarkers. For example, the assay step may include determining the ratio of the level of one or more biomarkers in the sample to the level of one or more standard biomarkers. Therefore, the method of the present invention may also include the step of assaying one or more standard biomarkers in a sample obtained from equines.

[0035] A standard biomarker can be any biomarker present in the sample that can be used as a comparison point for the levels of one or more biomarkers. For example, a standard biomarker may be selected as one that is usually or always present in the sample, is easy to measure, and / or exists in the sample at a consistent level, i.e., a biomarker whose level remains unchanged based on the presence or absence of insulin dysregulation. The use of a specific standard biomarker may require collecting samples from animals that are fasting or have not recently been fed and / or supplemented with food.

[0036] The standard biomarker may be an amino acid or a fatty acid. In one embodiment, the standard biomarker may be selected from bicarbonate, bilirubin, calcium, chloride, cholesterol, creatinine, glucose, magnesium, phosphorus, potassium, sodium, urea, phenylalanine, tryptophan, leucine, acetylcarnitine, and 1-palmitoyl-2-oleoyl-GPC. In another embodiment, the standard biomarker may be a protein selected from albumin, transferrin, fibrinogen, haptoglobulin, globulin, alanine transaminase, amylase, alkaline phosphatase, aspartate transaminase, creatine kinase, gamma-glutamyl transferase, lactate dehydrogenase, and sorbitol dehydrogenase, or may be total protein present in the sample. In a preferred embodiment, the standard biomarker is glucose or creatinine. Figure 1H -M indicates that the levels of creatinine, phenylalanine, tryptophan, leucine, acetylcarnitine, and 1-palmitoyl-2-oleoyl-GPC in samples obtained from equines were relatively consistent between ID and non-ID animals. Therefore, these markers represent potentially suitable standard markers.

[0037] In one aspect, the assay step may include determining the relative levels of two or more biomarkers in a sample. For example, the assay step may include determining the ratio of the levels of two or more biomarkers in a sample. In one embodiment, the assay step may include determining the ratio of the level of propionylglycine in the sample to the level of allantoin, allantoic acid, hexadecanoic acid, nicotinamide nucleoside, or urea cycle-related metabolites such as argininosuccinate or argininate.

[0038] Suitable methods for measuring the levels of biomarkers or standard markers in a sample are well known in the art, and any suitable technique may be used in such methods. For example, the steps for determining one or more biomarkers of the present invention may include chromatographic steps, particularly liquid chromatography (LC) steps, such as high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC). Chromatographic steps may include separating and quantifying the biomarker by applying the sample to a chromatographic column coupled to a detector. Suitable columns are well known in the art and include hydrophobically interacting liquid chromatography (HILIC) columns and reversed-phase liquid chromatography (RPLC) columns. In some aspects, the steps for determining one or more biomarkers may include mass spectrometry (MS) steps. Mass spectrometry steps may be combined with chromatographic steps, for example, in the form of HPLC-MS, LC-MS, or LC-MS / MS methods. Nuclear magnetic resonance (NMR) based techniques may also be used to determine the levels of specific biomarkers or standard markers in a sample.

[0039] The inventors have demonstrated that the differences in the levels of the biomarkers of the present invention between samples obtained from ID and non-ID equines are independent of the time of sample acquisition and the diet given to the equines. In the embodiments, the animals were fed three different diets, and samples were obtained at four different time points, including before and 30, 60, and 90 minutes after feeding. In all cases, significant differences in the levels of the biomarkers of the present invention were observed between samples obtained from ID and non-ID animals.

[0040] Therefore, the samples used for the assay step can be obtained from the equine to be diagnosed at any time point. In some embodiments, the samples are obtained before or 30, 60, or 90 minutes after feeding. In some embodiments, the method of the present invention may include the step of performing an assay to determine the levels of one or more biomarkers of the present invention in multiple samples obtained from the equine to be diagnosed at one or more different time points. For example, the samples may be obtained from the equine to be diagnosed before feeding, 30 minutes after feeding, 60 minutes after feeding, and / or 90 minutes after feeding. If the assay step involves determining the levels of one or more biomarkers in multiple samples obtained from the equine, the method of the present invention may include comparing the levels of one or more biomarkers determined in each sample with a reference result. Alternatively, the method of the present invention may include the step of taking the average (e.g., mean) level of one or more biomarkers determined in each sample and comparing the average (e.g., mean) level with a reference result.

[0041] The equine animal to be diagnosed can be given any suitable diet during the period prior to sample acquisition. In some embodiments, the sample can be obtained from an equine animal that received a control diet during the period prior to sample acquisition. The control diet can be a low-non-structural carbohydrate (LNSC) diet, such as a diet containing less than 20% non-structural carbohydrates (typically defined as monosaccharides and starches). Suitable LNSC diets are well known in the art. The control diet can be given to the equine animal to be diagnosed for at least one week prior to sample acquisition, such as at least two weeks, at least three weeks, or at least one month prior to sample acquisition.

[0042] Reference Results

[0043] The diagnostic method of the present invention includes a further step of comparing the levels of one or more biomarkers identified in the assay step with a reference result. The reference result is intended to represent the levels of one or more of the present invention's biomarkers in control equines. The term "level" in this context has the same meaning as defined above with respect to the assay step. Therefore, any discrepancies identified between the levels of one or more biomarkers identified in the assay step and the reference result can be used to determine the presence or absence of susceptibility to ID and / or ID-related conditions.

[0044] As used herein, the term "control equine" refers to an equine that can be compared to the equine to be diagnosed. Specifically, the step of comparing the levels of one or more biomarkers determined in the assay step with a reference result may include comparing the levels of one or more biomarkers of the present invention in a sample obtained from the equine to be diagnosed with the levels of the biomarker in a sample obtained from the control equine.

[0045] In some implementations, the control equine may be a healthy equine. As used herein, the term "healthy equine" or "healthy animal" means an equine that does not have, has not been diagnosed with, or has been confirmed to not have insulin disorders, laminitis, equine metabolic syndrome, diabetes, obesity, or middle pituitary dysfunction. If the control equine is a healthy equine, any differences identified between the levels of one or more biomarkers determined in the assay procedure and the reference result can be used to determine the presence of susceptibility to ID and / or ID-related conditions.

[0046] In some implementations, the control equine may be an unhealthy equine. As used herein, the term "unhealthy equine" or "unhealthy animal" refers to an equine that suffers from, has been diagnosed with, or has been confirmed to have insulin dysfunction, laminitis, equine metabolic syndrome, diabetes, obesity, or pituitary midline dysfunction. If the control equine is an unhealthy equine, any differences identified between the levels of one or more biomarkers determined in the assay procedure and the reference result can be used to determine the absence of susceptibility to ID and / or ID-related conditions.

[0047] The reference result may be a measurement result determining the level of one or more biomarkers in a sample obtained from a control equine animal. The measurement performed on the sample obtained from the control equine animal may correspond to the measurement steps described above performed on the sample obtained from the equine animal to be diagnosed. Similarly, the level of one or more biomarkers in the sample obtained from the control equine animal may be expressed in the same manner as the level of one or more biomarkers in the sample obtained from the equine animal to be diagnosed. Therefore, in one embodiment, the reference result may include a value for the concentration of one or more biomarkers in the sample obtained from the control equine animal or a value for the total amount of one or more biomarkers in the sample obtained from the control equine animal.

[0048] In one embodiment, the reference result may include the ratio of the level of one or more biomarkers of the present invention to the level of a standard biomarker in samples obtained from control equines. The standard biomarker used in the context of the reference result may be any suitable standard biomarker outlined above. Preferably, the standard biomarker used in the context of the reference result is the same as the standard biomarker used in the context of the assay step, i.e., the standard biomarker used in the assay of samples obtained from control equines is the same as the standard biomarker used in the assay of samples obtained from equines to be diagnosed.

[0049] In one embodiment, the reference results may include the ratio of the levels of two or more biomarkers of the present invention in samples obtained from control equines. For example, the reference results may include the ratio of the level of propionylglycine in samples obtained from control equines to the levels of allantoin, allantoic acid, hexadecanoic acid, nicotinamide nucleoside, or urea cycle-related metabolites such as argininosuccinate or argininate.

[0050] In one embodiment, reference results may include a set of reference results. For example, reference results may include the results of one or more assays determining the levels of one or more biomarkers of the invention in samples obtained from two or more control equines, such as samples obtained from 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more control equines. In some embodiments, the set of reference results may include the results of any assay determining the levels of one or more biomarkers of the invention in two or more samples obtained from the same control equine at different time points, such as samples obtained from 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more control equines. Similarly, the levels of one or more biomarkers in samples obtained from (one or more) control equines may be expressed in the same manner as the levels of one or more biomarkers in samples obtained from the equine to be diagnosed. As described above, the set of reference results may include values ​​for the concentrations of one or more biomarkers in samples obtained from (one or more) control equines or values ​​for the total amount of one or more biomarkers in samples obtained from (one or more) control equines. In one embodiment, the set of reference results may include the ratio of the levels of one or more biomarkers of the present invention to the levels of a standard biomarker in samples obtained from one or more control equines. In one embodiment, the set of reference results may include the ratio of the levels of two or more biomarkers of the present invention in samples obtained from one or more control equines. If one or more measurements are performed on samples obtained from multiple control equines to determine the levels of one or more biomarkers of the present invention, the reference results may include the average level of each biomarker in the samples obtained from the control equines. For example, the reference results may include the median level of each biomarker in the samples obtained from the control equines, or the mean level of each biomarker in the samples obtained from the control equines.

[0051] If the control equines include one or more healthy equines and one or more unhealthy equines, the reference results may include the mean level of each biomarker in samples obtained from healthy control equines and the mean level of each biomarker in samples obtained from unhealthy control equines. For example, the reference results may include the median level of each biomarker in samples obtained from healthy control equines and the median level of each biomarker in samples obtained from unhealthy control equines, or the mean level of each biomarker in samples obtained from healthy control equines and the mean level of each biomarker in samples obtained from unhealthy control equines.

[0052] The control equines (one or more) can be of the same class and / or the same age or life stage as the equine to be diagnosed. Animals from the same class can be from the same taxonomic family, genus, or species and / or from the same breed. Equines can be classified into one of the following life stages: foal (0-6 months), weaning (6-12 months), yearling (1-2 years), juvenile (2-4 years), adult (4-20 years), and senior (20 years and beyond). For example, if the equine to be diagnosed is an adult horse, the set of reference results can include determinations of the levels of one or more biomarkers of the present invention obtained from samples obtained from one or more control adult horses.

[0053] In some embodiments, the method of the present invention may include a further step of generating reference results. For example, the method of the present invention may include the step of performing one or more assays to determine the levels of one or more biomarkers of the present invention in samples(s) obtained from one or more control equines. If samples were obtained from control equines, or if multiple assays were performed, for example, multiple assays of samples obtained from the same control equine at different time points, the results may be aggregated into a set of reference results. In the above-described assay steps performed on samples obtained from equines to be diagnosed, the step of measuring samples obtained from one or more control equines may include measuring the concentration of one or more biomarkers of the present invention; determining the level of one or more biomarkers of the present invention relative to the level of a standard biomarker; and / or determining the relative levels of two or more biomarkers.

[0054] Reference results do not need to be prepared every time the method of the present invention is performed. Instead, those skilled in the art can rely on established reference results.

[0055] In one embodiment, the reference results may include predetermined thresholds for one or more biomarkers of the present invention. The predetermined threshold for each biomarker may be a concentration considered healthy (i.e., not indicating ID or related disease) or typical for healthy equines, or a concentration considered unhealthy (i.e., indicating ID or related disease) or typical for unhealthy equines. Similarly, any differences identified between the levels of one or more biomarkers determined in samples obtained from equines to be diagnosed and the predetermined thresholds can be used to determine the presence or absence of susceptibility to ID and / or ID-related diseases.

[0056] In some embodiments, a predetermined threshold for each biomarker may be based on the level of that biomarker in one or more control equines, for example, one or more control equines of the same class and / or the same age or life stage as the equine to be diagnosed. The predetermined threshold for each biomarker can be determined by assessing the level of that biomarker in one or more control equines of the same class and optionally the same life stage as the equine to be diagnosed and taking the average (e.g., mean) value. For example, if the equine to be diagnosed is an adult horse, the predetermined threshold for each biomarker may be the average (e.g., mean) level of that biomarker in healthy or unhealthy adult horses. The predetermined thresholds do not need to be determined each time the method of the invention is performed. Instead, those skilled in the art can rely on thresholds that have already been established.

[0057] In some implementations, the predetermined threshold can be a fixed value, i.e., a single point, or a fixed range of values.

[0058] The predetermined threshold can vary between different genera or species of equines and / or between equines at different life stages. Those skilled in the art will understand that different genera or species or different life stages of equines can typically have higher or lower levels of biomarkers, and therefore will be able to make any necessary adjustments to the predetermined threshold based on the levels of the corresponding biomarkers in equines of a particular species and life stage.

[0059] diagnosis

[0060] The inventors have demonstrated that the biomarkers of this invention can be used to distinguish between ID (infected) and non-ID (infected) equines. Based on this, they have developed a method for diagnosing the presence or absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions in equines.

[0061] The biomarkers of the present invention exhibit different levels between ID (infected) and non-ID (infected) equines. Therefore, the step of comparing the levels of one or more biomarkers determined in the assay step (i.e., the levels of one or more biomarkers determined in assays performed on samples obtained from the equine to be diagnosed) with a reference result allows for the diagnosis of the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to conditions related to insulin dysregulation. In some embodiments, the diagnosis of the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to conditions related to insulin dysregulation can be based on the levels of two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve biomarkers in samples of the present invention.

[0062] The difference between the levels of one or more biomarkers identified in the assay procedure and a reference result can be used to determine the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to insulin dysregulation-related conditions. In other words, the diagnosis of the presence or absence of insulin dysregulation and / or the presence or absence of susceptibility to insulin dysregulation-related conditions can be based on the indication that one or more biomarkers of the present invention are present in a sample obtained from the equine animal to be diagnosed, at levels lower or higher than a reference result.

[0063] Propionylglycine is present at lower levels in ID equines compared to non-ID equines. Therefore, if the control equines are healthy equines—that is, if the reference results include measurements from samples obtained from healthy equines to determine the levels of one or more biomarkers—the lower levels of propionylglycine in samples obtained from the equine to be diagnosed, relative to the reference results, indicate the presence of insulin dysregulation and / or susceptibility to conditions related to insulin dysregulation. This lower level of propionylglycine may be a lower level relative to measurements from samples obtained from one or more control equines, or a lower level relative to a predetermined threshold for propionylglycine.

[0064] In this document, the term "lower" means that the propionylglycine level in a sample obtained from an equine animal to be diagnosed is at least a certain extent lower than a reference result, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% lower. In other words, a propionylglycine level in a sample obtained from an equine animal to be diagnosed that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% lower than a reference result (obtained from a healthy equine animal) indicates the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some embodiments, a statistically significant difference in the propionylglycine level in a sample obtained from an equine animal to be diagnosed compared to a reference result (obtained from a healthy equine animal) indicates the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0065] If the control equine is a healthy equine, i.e., if the reference result includes measurements of the levels of one or more biomarkers in samples obtained from healthy equines, then a propionylglycine level in the sample obtained from the equine to be diagnosed that is not at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than the reference result indicates the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some embodiments, a propionylglycine level in the sample obtained from the equine to be diagnosed that is not statistically significantly lower than the reference result (obtained from a healthy equine) indicates the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In one embodiment, a propionylglycine level in the sample obtained from the equine to be diagnosed that is within 50% of the reference result (obtained from a healthy equine), i.e., differing from the reference result by no more than 50% in any direction, indicates the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. A propionylglycine level in a sample obtained from an equine animal to be diagnosed that is within 45% of the reference result (obtained from a healthy equine animal), such as within 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, indicates the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0066] Conversely, if the control equine is an unhealthy equine—that is, if the reference result includes measurements of levels of one or more biomarkers from samples obtained from unhealthy equines—then the propionylglycine level in the sample obtained from the equine to be diagnosed is not at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher than the reference result, indicating the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some embodiments, the propionylglycine level in the sample obtained from the equine to be diagnosed is not statistically significantly higher than the reference result (obtained from unhealthy equines), indicating the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In one implementation, a propionylglycine level in a sample obtained from the equine animal to be diagnosed that is within 50% of a reference result (obtained from an unhealthy equine animal), i.e., differing from the reference result by no more than 50% in any direction, indicates the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. A propionylglycine level in a sample obtained from the equine animal to be diagnosed that is within 45% of a reference result (obtained from an unhealthy equine animal), such as within 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, indicates the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0067] Compared to non-ID equines, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate are present at higher levels in ID equines. Therefore, if the control equines are healthy equines—that is, if the reference results include measurements of the levels of one or more biomarkers from samples obtained from healthy equines—then higher levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed, relative to the reference results, indicate the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. Similarly, the higher levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate can be higher than the levels measured in samples obtained from one or more control equines, or higher than the predetermined threshold of the biomarker of the present invention.

[0068] In this document, the term "higher" means that the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed are at least a certain extent higher than the reference results of the biomarker of the present invention, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. In other words, levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed, which are at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the reference result of the biomarker of the present invention, indicate the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some embodiments, levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed, which are statistically significantly higher than the reference result of the biomarker of the present invention (obtained from healthy equines), indicate the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0069] If the control equine is a healthy equine, i.e., if the reference results include determinations of the levels of one or more biomarkers in samples obtained from healthy equines, then the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in the samples obtained from the equine to be diagnosed are not higher than by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, indicating the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some embodiments, the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed are not statistically significantly higher than the reference result of the biomarker of the present invention (obtained from healthy equines), indicating the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In one embodiment, the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed are within 50% of the reference result of the biomarker of the present invention (obtained from healthy equines), i.e., differing from the reference result by no more than 50% in any direction, indicating the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. The levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed, within 45% of the reference results (obtained from healthy equines) of the biomarker of the present invention, such as within 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, indicate the absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0070] Conversely, if the control equine is an unhealthy equine, i.e., if the reference results include determinations of the levels of one or more biomarkers in samples obtained from unhealthy equines, then the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in the samples obtained from the equine to be diagnosed are not lower than at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, indicating the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some embodiments, the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed are not statistically significantly lower than the reference result of the biomarker of the present invention (obtained from unhealthy equines), indicating the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In one embodiment, the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed are within 50% of the reference result of the biomarker of the present invention (obtained from unhealthy equines), i.e., differing from the reference result by no more than 50% in any direction, indicating the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. The levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained from equines to be diagnosed, within 45% of the reference results for this biomarker of the present invention (obtained from unhealthy equines), such as within 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, indicate the presence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0071] If the equine animal is indicated to have insulin dysregulation or susceptibility to insulin dysregulation-related conditions, the method of the present invention may further include the step of providing recommendations for intervention.

[0072] In some embodiments, the intervention may be a visit to a veterinary professional. The recommendation may therefore be the need for a visit to a veterinary professional. In some embodiments, the intervention may be notification that the animal is at risk of developing an insulin-related condition such as laminitis and should be monitored. The recommendation may therefore be to repeat the determination of one or more biomarkers of the invention in samples obtained from equines within a recommended timeframe, such as six months, four months, three months, two months, or one month. In some embodiments, the intervention may be a treatment regimen for insulin dysregulation or an insulin-related condition. The recommendation may therefore be the adoption of a treatment regimen. This treatment regimen may include dietary management, exercise, and / or appropriate medications, as well as core management changes. Core management changes may be designed to treat underlying conditions and / or remove stimulating factors. For example, this may include removing feed that may already be involved in the development of ID or ID-related conditions, and feed that may exacerbate the severity of ID or ID-related conditions. The animal may be removed from pasture and provided with a low-non-structural carbohydrate (NSC) diet. Core management changes may also include treatments to reduce blood insulin levels, treatments to minimize lamellar damage, the provision of analgesia, treatments to promote weight loss, and one or more of general animal husbandry and hoof trimming.

[0073] In some embodiments, the method of the present invention also includes recommendations to implement interventions, namely taking the equine to a veterinary professional or initiating treatment for insulin disorders or conditions related to insulin disorders.

[0074] Monitoring methods

[0075] The present invention also provides a method for monitoring the levels of one or more biomarkers of the present invention over time in equines.

[0076] Methods for monitoring the levels of one or more biomarkers in equines include: (a) To determine the levels of one or more biomarkers in samples obtained from equines, wherein said biomarkers are selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate; and (b) Repeat step (a) at two or more separate time points.

[0077] In this monitoring method, two or more individual time points may be spaced at least one week apart, such as at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least six months, at least nine months, at least twelve months, or more. The method is not limited to repeating the determination of step (a) at only two time points. The determination may be repeated at three or more time points, such as four, five, six, seven, eight, nine, ten, or more time points.

[0078] The monitoring method will provide two or more measurements of the levels of one or more biomarkers of the present invention in samples obtained from equines, wherein each measurement corresponds to the level of a given biomarker in samples obtained from equines at different time points. In some embodiments, the method may further include the step of comparing at least two measurements of the levels of one or more biomarkers in samples obtained from equines.

[0079] If equines do not have, have not been diagnosed with, or have been confirmed to not have insulin dysregulation and / or insulin dysregulation-related conditions, then the method of monitoring the levels of one or more biomarkers of the present invention in equines can also be considered as a method of monitoring the development of insulin dysregulation and / or insulin dysregulation-related conditions in equines. Comparisons between different measurements of the levels of one or more biomarkers of the present invention in samples obtained from equines can indicate whether the equine is developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions.

[0080] As described above, propionylglycine is present at lower levels in ID equines compared to non-ID equines, and allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate are present at higher levels in ID equines compared to non-ID equines. Therefore, in some embodiments, if the propionylglycine level is lower in a sample obtained at a later time point than in a sample obtained at an earlier time point, the decrease in propionylglycine level can indicate that the animal is developing insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. Conversely, the absence of a decrease in propionylglycine level between measurements of two samples obtained from equines can indicate that the animal is not developing insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions.

[0081] Similarly, between measurements of two samples obtained from equines, elevated levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate—that is, when the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate in samples obtained at a later time point are higher than the levels of the biomarkers of the present invention in samples obtained at an earlier time point—indicate that the animal is developing insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. The absence of elevated levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate between measurements from two samples obtained from equines indicates that the animal is not developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions.

[0082] In this context, the term "reduction" refers to a decrease in the level of one or more biomarkers of the present invention compared to the level in samples obtained at an earlier time point (e.g., a lower level in samples obtained at a second or subsequent time point compared to the level in samples obtained at a first time point). Between measurements in two samples obtained from equines (e.g., between consecutive measurements), this reduction can be at least by a specific magnitude, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. Propionylglycine is present at lower levels in ID equines compared to non-ID equines. Therefore, a reduction in propionylglycine levels by at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, between measurements in two samples obtained from equines can indicate that the animal is developing insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions. In some implementations, a statistically significant decrease in propionylglycine levels between measurements of two samples obtained from an equine animal can indicate that the animal is developing insulin dysregulation and / or is susceptible to conditions related to insulin dysregulation.

[0083] Conversely, a decrease of at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, in propionylglycine levels between measurements from two equines would indicate that the animal is not developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions. Similarly, a statistically significant decrease in propionylglycine levels between measurements from two equines would indicate that the animal is not developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions.

[0084] In this context, the term "elevation" refers to an increase in the level of one or more biomarkers of the present invention compared to levels in samples obtained at an earlier time point (e.g., a higher level in samples obtained at a second or subsequent time point compared to levels in samples obtained at a first time point). Between measurements in, for example, two samples obtained from equines (e.g., between consecutive measurements), this increase can be at least a specific magnitude, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. Allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate or argininate are present at higher levels in ID equines compared to non-ID equines. Therefore, an increase of at least 5%, for example at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, in the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate or argininate between measurements in two samples obtained from equines, indicates that the animal is developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions. In some embodiments, a statistically significant increase in the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate or argininate between measurements in two samples obtained from equines indicates that the animal is developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions.

[0085] Conversely, the absence of a statistically significant increase in the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate or argininate between measurements in two samples obtained from equines indicates that the animal is not developing insulin dysregulation and / or is susceptible to insulin dysregulation-related conditions.

[0086] If an equine has been diagnosed with insulin dysregulation or an insulin dysregulation-related condition, the method of monitoring the levels of one or more biomarkers of the present invention can be considered as a method of monitoring the progression of insulin dysregulation or an insulin dysregulation-related condition in the equine. Comparisons between different measurements of the levels of one or more biomarkers of the present invention in samples obtained from equines can indicate whether the insulin dysregulation and / or insulin dysregulation-related condition is progressing, stabilizing, or regressing.

[0087] In this document, the term "progressing" refers to a clinical condition that develops or worsens over time, such as when symptoms displayed in equines become more severe, more frequent, or last longer. The term "stable" refers to a clinical condition that does not change significantly over time, i.e., neither worsening nor improving. This can be indicated by consistent or unchanged symptoms. The term "regressing" refers to a clinical condition that improves over time, such as when symptoms displayed in equines may become less severe, less frequent, or last for a shorter period. Those skilled in the art will recognize these different clinical conditions and will understand their importance in monitoring equines.

[0088] For example, in some implementations, a decrease in propionylglycine levels between measurements of two samples from equines (e.g., equines with insulin dysregulation or insulin dysregulation-related conditions) can indicate progression of insulin dysregulation or insulin dysregulation-related conditions. Similarly, an increase in levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, or urea cycle-related metabolites such as argininosuccinate or argininate between measurements of two samples from equines (e.g., equines with insulin dysregulation or insulin dysregulation-related conditions) can indicate progression of insulin dysregulation or insulin dysregulation-related conditions.

[0089] Conversely, between measurements of two samples from equines (e.g., equines with insulin dysregulation or insulin dysregulation-related conditions), an increase in propionylglycine levels can indicate a resolution of insulin dysregulation or insulin dysregulation-related conditions. Similarly, between measurements of samples from equines (e.g., equines with insulin dysregulation or insulin dysregulation-related conditions), a decrease in levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, or urea cycle-related metabolites such as argininosuccinate or argininate can indicate a resolution of insulin dysregulation or insulin dysregulation-related conditions.

[0090] If, between measurements from two samples obtained from equines (e.g., equines with insulin dysregulation or insulin dysregulation-related conditions), the level of one or more biomarkers of the present invention in the equine samples neither increases nor decreases, this indicates that the insulin dysregulation or insulin dysregulation-related conditions is stable. The terms "increase" and "decrease" are described in detail in the context of the monitoring methods described above. If the above definitions of increase and decrease are not met, it can be determined that the level of one or more biomarkers of the present invention does not increase or decrease between measurements from two samples obtained from equines. For example, if the level of one or more biomarkers of the present invention varies by less than 50% in any direction, such as less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% in any direction, it can be determined that the level does not increase or decrease between measurements from two samples obtained from equines. In one embodiment, if the level does not change by a statistically significant amount, it can be determined that the level of one or more biomarkers of the present invention neither increases nor decreases between measurements from two samples obtained from equines.

[0091] Monitoring of the levels of one or more biomarkers of the present invention in equines can be continuous. For example, the monitoring method may include repeating the determinations of steps (a) and (b) approximately weekly, bi-weekly, monthly, six-weekly, two-monthly, three-monthly, four-monthly, six-monthly, nine-monthly, or annually (e.g., over a period of six months, one year, two years, three years, four years, or five years).

[0092] Such continuous monitoring is useful when treating insulin disorders or insulin disorder-related conditions in equines. Therefore, this invention also provides a method for assessing the effectiveness of treatments for insulin disorders and related conditions in equines.

[0093] Insulin dysregulation and insulin dysregulation-related conditions are typically treated with dietary management and exercise and / or medications, as well as core management modifications. Potential core management modifications are defined above. Dietary therapy for insulin dysregulation and insulin dysregulation-related conditions may involve calorie restriction and / or non-structural carbohydrate (NSC) restriction. Medications that can be administered to treat insulin dysregulation or insulin dysregulation-related conditions include levothyroxine; SLGT-2 inhibitors such as velagliflozin, canagliflozin, and ertugliflozin; biguanides such as metformin; GLP-1 analogs such as exenatide; GLP-1 inhibitors and equivalents (Meier et al. 2019, de Laat 2023).

[0094] Methods for evaluating the effectiveness of treatments for insulin dysregulation in equines include: (a) To determine the level of one or more biomarkers in a sample obtained from equines, wherein the biomarkers are selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as argininosuccinate and argininate. (b) Repeat step (a) at two or more separate time points, at least one of which is after the equine has begun treatment.

[0095] Similarly, methods for evaluating the effectiveness of treatments for insulin dysregulation-related conditions in equines include: (a) To determine the level of one or more biomarkers in a sample obtained from equines, wherein the biomarkers are selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as argininosuccinate and argininate. (b) Repeat step (a) at two or more separate time points, at least one of which is after the equine has begun treatment.

[0096] Therefore, this method involves performing assays to determine the levels of one or more biomarkers of the present invention in samples obtained from equines at at least two time points. The first time point may be before the animal begins treatment, so as to provide a baseline assessment of the levels of the biomarkers of the present invention prior to the start of treatment. Similarly, the time points may be spaced at least one week apart, such as at least two weeks, at least three weeks, at least one month, at least two months, or longer (e.g., over a period of up to six months, one year, two years, three years, four years, or five years).

[0097] At least one time point must be after the equine has begun treatment. This time point can be chosen so that there is sufficient time after the start of treatment for it to have an impact on the level of the biomarker of the present invention. For example, the time point after the start of treatment in the equine can be at least one week after the start of treatment, such as at least two, three, four, five, six, seven, or eight weeks after the start of treatment.

[0098] Similarly, this method is not limited to repeating the determination of step (a) at only two time points. These steps can be repeated at three or more time points, such as four, five, six, seven, eight, nine, ten, or more time points. Monitoring the effectiveness of treatment can continue throughout the duration of treatment administration. For example, the effectiveness of treatment can be monitored regularly, such as every two weeks, once a month, once every six weeks, once every two months, once every four months, once every six months, or once a year.

[0099] As outlined in the monitoring methods above, the method for assessing the effectiveness of treatment for insulin dysregulation or insulin dysregulation-related conditions will provide two or more measurements of the levels of one or more biomarkers of the present invention in samples obtained from equines, wherein each measurement corresponds to the level of a given biomarker in samples obtained from equines at different time points. In some embodiments, the method may further include the step of comparing at least two measurements of the levels of one or more biomarkers of the present invention in samples obtained from equines. The comparison results between different measurements of the levels of one or more biomarkers of the present invention in samples obtained from equines can indicate whether the treatment is effective or ineffective.

[0100] Treatment of insulin dysregulation or insulin dysregulation-related conditions may aim to alter the levels of one or more biomarkers of the present invention in equines to make them consistent with or closer to the levels typical of healthy equines. For example, propionylglycine is present at reduced levels in ID equines relative to non-ID equines, so treatment of insulin dysregulation or insulin dysregulation-related conditions may aim to increase propionylglycine levels in equines. Similarly, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate are present at elevated levels in ID equines relative to non-ID equines, so treatment of insulin dysregulation or insulin dysregulation-related conditions may aim to decrease the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, or urea cycle-related metabolites such as argininosuccinate and argininate in equines.

[0101] Therefore, the effectiveness of a treatment can be determined by assessing its ability to achieve the desired change in the levels of one or more biomarkers of the present invention in equines. If the treatment achieves the desired change in the levels of one or more biomarkers of the present invention in equines, for example, if the treatment increases the level of propionylglycine in equines or decreases the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in equines, then the treatment can be determined to be effective. Conversely, if the treatment does not achieve the desired change in the levels of one or more biomarkers of the present invention in equines, for example, if the treatment does not increase the level of propionylglycine in equines or decrease the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in equines, then the treatment can be determined to be ineffective.

[0102] In one implementation, treatment is deemed effective if: (i) the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in samples obtained from equines at a given time point are lower than the levels of the biomarker of the present invention in samples obtained from equines at an earlier time point; and / or (ii) the level of propionylglycine in samples obtained from equines at a given time point is higher than the level of propionylglycine in samples obtained from equines at an earlier time point. Conversely, if: (i) the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in samples obtained from equines at a given time point do not decrease relative to the levels of the biomarker of the present invention in samples obtained from equines at an earlier time point; and / or (ii) the level of propionylglycine in samples obtained from equines at a given time point does not increase relative to the level of propionylglycine in samples obtained from equines at an earlier time point, then the treatment can be determined to be ineffective. The terms “increase” and “decrease” are defined in detail in the context of methods for monitoring the levels of one or more biomarkers in equines, and these definitions also apply herein.

[0103] In one embodiment, the method for assessing the effectiveness of treatment for insulin dysregulation or insulin dysregulation-related conditions may further include a step of comparing the levels of one or more biomarkers identified from samples obtained from equines with a reference result. The term "reference result" is defined in detail above regarding diagnostic methods, and the same definition applies here. In short, a reference result represents the level of one or more of the biomarkers of the present invention in control equines. Therefore, this comparison step indicates whether a given treatment reduces the difference between the level of one or more of the biomarkers of the present invention in samples obtained from equines and the typical level of that biomarker of the present invention in control equines. If the control equines are healthy equines, the treatment can be determined to be effective if the level of one or more of the biomarkers of the present invention in samples obtained from equines at a given time point is closer to the reference result than the level of the biomarkers of the present invention in samples obtained from equines at an earlier time point. Conversely, the treatment can be determined to be ineffective if the level of one or more of the biomarkers of the present invention in samples obtained from equines at a given time point is not closer to the reference result than the level of the biomarkers of the present invention in samples obtained from equines at an earlier time point.

[0104] If the control equines are unhealthy equines, then the treatment can be determined to be effective if the levels of one or more of the present invention's biomarkers in samples obtained from the equines at a given time point are not closer to a reference result than the levels of the present invention's biomarkers in samples obtained from the equines at an earlier time point. Conversely, the treatment can be determined to be ineffective if the levels of one or more of the present invention's biomarkers in samples obtained from the equines at a given time point are closer to a reference result than the levels of the present invention's biomarkers in samples obtained from the equines at an earlier time point.

[0105] If treatment for insulin dysregulation or insulin dysregulation-related conditions is determined to be ineffective, the method of the present invention may further include the step of providing a recommendation for intervention. In some embodiments, this recommendation may be to adjust an existing treatment regimen to change the dosage of the treatment received by the equine. Such a change in dosage may be an increase or a decrease in the dosage of the treatment. For example, if the equine is being treated for insulin dysregulation and it has been determined that the treatment is ineffective in achieving the desired change in the levels of one or more biomarkers of the present invention, the recommendation may be to adjust the existing treatment regimen to increase the dosage of the treatment received by the equine.

[0106] Similarly, in some embodiments, the method of the present invention also includes recommendations for implementing interventions, namely adjusting the treatment regimen to increase or decrease the dosage of treatment received by the equine.

[0107] sample

[0108] The method of this invention refers to the determination of one or more biomarkers in a sample obtained from an equine animal. The sample may be a bodily fluid obtained from an equine animal, such as whole blood, plasma, serum, saliva, or urine, or it may be a dried blood spot.

[0109] In some embodiments, the sample is provided in the form of dried blood spots. Dried blood spot sampling can be used to evaluate a range of biomarkers, including those of the present invention. Dried blood spot samples can be prepared by contacting a small amount of blood with a protein saver card and allowing the sample to air dry. Drying the sample halts many enzymatic reactions that could lead to the degradation of metabolites in liquid samples. Suitable protein saver cards are well known in the art and include, for example, the Whatman 903 protein saver card. Blood can be obtained by puncturing the skin of an equine animal using, for example, a catheter, needle, or lancet, or by any other approved means. Many specific tools are available to help provide blood samples containing an accurate and consistent volume of blood, including the Captainer® B and Mitra® systems. To extract a sample from the protein saver card for the determination of the biomarkers of the present invention, the card can be contacted with a suitable solvent, such as water, methanol, acetonitrile, and / or isopropanol, and mixed to dissolve the sample. Exemplary protocols for measuring metabolites using dried blood spot cards are well known in the art (McCulloch et al., 2021).

[0110] Using dried blood for metabolomics analysis offers numerous advantages. For example, it requires a smaller volume of whole blood compared to methods involving plasma or serum samples. The equipment and expertise required for sample collection are also significantly reduced, as it eliminates the need for trained phlebotomists to use hypodermic needles. Furthermore, once collected, samples are cheaper and easier to store (Allaway et al., 2022).

[0111] If the sample is a whole blood sample, it can be processed by rapid freezing as soon as possible after collection to prevent any in vitro metabolic reactions that may affect the results of the biomarker assays of this invention. In some embodiments, whole blood samples can be preserved using one or more anticoagulants such as EDTA or heparin. Processing whole blood samples may include fractionating the sample to obtain plasma and / or serum fractions. This fractionation can be performed before freezing the sample. In some embodiments, the fractionation of the sample can be performed at a temperature of 10°C or lower, or at a temperature of 5°C or lower.

[0112] In some embodiments, the sample may be obtained from equines that have been fasted or have not recently been fed and / or supplemented with food. For example, in some embodiments, the sample may be obtained from equines that have not been fed for at least 2, 3, 4, or 5 hours, such as at least 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours. In some embodiments, the sample may be obtained from equines at predetermined time points after the provision of feed and / or supplemental feeding. For example, the sample may be obtained from equines at the time of provision of feed and / or supplemental feeding, or 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours after provision of feed and / or supplemental feeding. In some embodiments, the method of the present invention can be applied to a series of samples obtained from equines at different time points. For example, the sample may be obtained from equines at the time of provision of feed and / or supplemental feeding, and 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after provision of feed and / or supplemental feeding.

[0113] The method of the present invention may optionally include the step of obtaining a sample from an equine animal. Suitable samples for use in the method of the present invention are as defined above.

[0114] Horses

[0115] The method of this invention can be applied to a range of equines. In particular, the method of this invention can be used to diagnose diseases in the genus *Equus*. Equus The presence or absence of insulin dysregulation and / or susceptibility to insulin dysregulation-related conditions in animals.

[0116] This genus belongs to the Equidae family ( Equidae The genus *Equus* is part of the family *Equus*, which includes many extant and extinct species. Species in the genus *Equus* include horses (*Equus equine*). Equus caballus ), mule ( Equus mulus ), donkeys, mules, wild horses ( Equus ferus Mountain zebra ( Equus zebra ), African wild ass ( Equus africanus ), domestic donkey ( Equus africanus asinus Fine-striped zebra ( Equus grevyi ), Asian wild ass ( Equus hemionus ), Tibetan wild ass ( Equus kiang ) and ordinary zebra ( Equus quagga Preferably, the animal is the species horse (Horse). Equus caballus ), including horses and ponies. Species: Horse ( Equus caballusAnimals can be of any age, sex, or castrated state, and therefore the method of the present invention can be applied to (but is not limited to) foals, weaning foals, yearlings, foals, fillies, mares, stallions, castrated stallions, or cryptorchidists. The method of the present invention can be used to evaluate genus Equines. Equus Animals, preferably the horse ( Equus caballus All varieties of ).

[0117] General Instructions

[0118] Unless otherwise stated, the present invention will be practiced using conventional chemical, biochemical, molecular biological, immunological, and pharmacological methods within the scope of the art. These techniques are well explained in the literature.

[0119] The term "comprising" encompasses both "including" and "consisting of". For example, a composition that "comprising" X may consist of only X, or it may include additional things, such as X+Y.

[0120] The term “about” associated with the numerical value x is optional and means, for example, x ± 10%.

[0121] The word "substantially" does not exclude "completely". For example, when necessary, a composition that is "substantially free" of Y can be completely free of Y. The word "substantially" can be omitted from the definition of this invention.

[0122] Unless otherwise specified, a process or method comprising multiple steps may include additional steps at the beginning or end of the method, or may include additional intermediate steps. Furthermore, steps may be combined, omitted, or performed in an alternative order, where appropriate.

[0123] This document describes various embodiments of the invention. It should be understood that specific features in each embodiment can be combined with other specific features to provide further embodiments. In particular, embodiments emphasized herein as suitable, typical, or preferred can be combined with each other (except when they are mutually exclusive).

[0124] The invention will be further understood by referring to the following embodiments, which are provided as illustrative purposes and are not intended to be limiting.

[0125] Example

[0126] Example 1

[0127] A study was conducted involving four horses—a 16-year-old female Appaloosa, a 16-year-old quarter horse, a 16-year-old male Tennessee walking horse, and a 21-year-old female quarter horse.

[0128] Two weeks prior to sample collection, at the start of spring, all horses underwent an oral glucose test (OST) at a dose of 0.15 mL / kg body weight. Using published criteria, horses were identified as having insulin dysregulation (ID) or not (non-ID). Of the four horses tested, two were ID and two were non-ID. Additionally, the horses were evaluated to confirm they had no indications of middle pituitary dysfunction (PPID) and their non-declining adrenocorticotropic hormone (ACTH) concentrations were <30 pg / mL.

[0129] Horses were fed three different diets in random order, with at least one week between each diet. The three diets were: (1) Control low non-structural carbohydrate (LNSC) fiber-based pellets (Buckeye™ Nutrition, USA); (2) Control diet plus pure glucose source (D-(+)-glucose, dextrose, (Sigma Aldrich)) (3) Control diet plus a 50:50 mixture of oat starch powder and waxy corn starch (True Nutrition, California, USA).

[0130] The nutritional information for the three diets is shown in the table below (CP = crude protein, WSC = water-soluble carbohydrates, ESC = ethanol-soluble carbohydrates, NSC = non-structural carbohydrates).

[0131]

[0132] On the morning of the sampling day (07:00), horses were placed in their individual enclosures, and a 16-gauge x 14.6 cm Covetrus catheter was inserted under local anesthesia using aseptic techniques. In their individual enclosures, horses had access to water but no other feed. After catheter placement, horses underwent a 30-minute rest period, followed by blood sampling (10 mL serum tube; Covetrus) and feeding. All horses consumed their food within approximately 10 minutes. Blood was collected before feeding and at 30, 60, and 90 minutes after complete consumption of the appropriate treated diet. Remaining blood volumes were applied to Whatman protein preservation cards. After drying, samples were stored with desiccant at -80°C. Samples were then sent to Metabolon for broad-spectrum metabolomics analysis.

[0133] Between sample collections, horses were placed in their family paddocks where they had access to limited pasture feed (semi-dry pasture), hay (11.85% CP, 0.6% starch, 5.9% WSC, 3.75% ESC and 6.5% NSC on a dry matter basis) and water.

[0134] Metabolomics analysis was performed on dried blood samples. Seven metabolites showed consistent differences between ID and non-ID animals at all four time points and under all three dietary conditions. These metabolites were propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, argininosuccinate, argininate, and hexadecanoic acid. The results of this metabolomics analysis are shown in Figure 1. Three groups are shown for each biomarker, corresponding to the three treatment diets. Within each group, time points T1, T2, T3, and T4 correspond to samples collected before feeding and at 30, 60, and 90 minutes after feeding, respectively. P1 represents ID animals, and P2 represents non-ID animals.

[0135] References

[0136] Allaway and others, Front Vet Sci. (Frontiers in Veterinary Science) (2022) 9:887163

[0137] Asplin et al. Vet J. (Veterinary Journal) (2007) 174(3):530-535

[0138] de Laat et al. Equine Vet J. (Equine Veterinary Journal) (2010) 42(2):129-135

[0139] de Laat, “Pathophysiology of Insulin Dysregulation (ID) in Horses and Therapeutic Targets” ACVIM Forum (American College of Veterinary Medicine Forum) (2023)

[0140] Durham et al. J Vet Intern Med. (Journal of Veterinary Internal Medicine) (2019) 33:335-349

[0141] Frank et al. J Vet Intern Med. (Journal of Veterinary Internal Medicine) (2010) 24:467-475

[0142] Hoffman et al. J Anim Sci. (Journal of Animal Science) (2003) 81:2333-2342

[0143] Johnson, Vet Clin North Am Equine Pract. (North American Veterinary Clinical Practice: Equine Clinical Practice) (2002) 18(2):271-293

[0144] Knowles et al. Equine Vet J. (Equine Veterinary Journal) (2023a) 55:12-23

[0145] Knowles et al., “Factors associated with insulin responses to oral sugars in a mixed-breed cohort of ponies”. Equine Vet J. (Equine Veterinary Journal), accepted, (2023b)

[0146] McCulloch et al., “Stability of metabolite profiles using dried bloodspot cards” Metabolon (Metbrolon / Metaboloms) (2021)

[0147] McGowan, “The Intersection of PPID and Laminitis” 64th Annual Convention of the American Association of Equine Practitioners(64th Annual Meeting of the American Equine Physicians Association) (2018)

[0148] Meier et al. BMC Veterinary Research (BMC Veterinary Research) (2019) 15:65

[0149] Treiber et al. J Anim Sci. (Journal of Animal Science) (2005) 83:2357-2364

[0150] Treiber et al. J Nutr. (Journal of Nutrition) (2006) 136(7 Suppl):2094S-2098

Claims

1. A method for diagnosing the presence or absence of insulin dysregulation in equines, the method comprising: (a) To determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as arginine succinate and arginine in samples obtained from equines. (b) Compare the levels of one or more biomarkers identified in step (a) with reference results; The difference between the levels of one or more biomarkers determined in step (a) and the reference result is used to determine the presence or absence of insulin dysregulation.

2. A method for diagnosing the presence or absence of susceptibility to insulin dysregulation-related conditions in equines, the method comprising: (a) To determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; (b) Compare the levels of one or more biomarkers identified in step (a) with reference results; The difference between the levels of one or more biomarkers identified in step (a) and the reference result is used to determine the presence or absence of susceptibility to insulin dysregulation-related conditions.

3. The method of claim 1 or 2, wherein the reference results comprise one or more results from determinations of the levels of one or more biomarkers in a sample obtained from control equines that do not have, have not been diagnosed with, or have been confirmed to be free from insulin dysregulation, laminitis, equine metabolic syndrome, diabetes, obesity, or pituitary midline dysfunction (PPID).

4. The method of claim 3, wherein a lower level of propionylglycine in the sample obtained from the equine animal, relative to the reference result, indicates the presence of insulin dysregulation or susceptibility to conditions related to insulin dysregulation.

5. The method according to claim 3 or 4, wherein higher levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and / or urea cycle-related metabolites such as argininosuccinate or argininate in samples obtained from the equine animals, relative to the reference results, indicate the presence of insulin dysregulation or susceptibility to conditions related to insulin dysregulation.

6. The method of claim 1 or 2, wherein the reference results comprise one or more results from determinations of the levels of one or more biomarkers in a sample obtained from a control equine animal that has been diagnosed or confirmed to have insulin dysregulation, laminitis, equine metabolic syndrome, diabetes, obesity, or pituitary midline dysfunction (PPID).

7. The method of claim 6, wherein the propionylglycine level in the sample obtained from the equine is not higher than the reference result, indicating the presence of insulin dysregulation or susceptibility to conditions related to insulin dysregulation.

8. The method according to claim 6 or 7, wherein the levels of allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and / or urea cycle-related metabolites such as argininosuccinate or argininate in the sample obtained from the equine animal are not lower than the reference result, indicating the presence of insulin dysregulation or susceptibility to insulin dysregulation-related conditions.

9. The method according to any one of claims 3 to 8, wherein the reference result includes the concentration values ​​of one or more biomarkers in a sample obtained from a control equine animal.

10. The method according to any one of claims 3 to 9, wherein the reference result comprises the ratio of the level of one or more biomarkers in a sample obtained from a control equine animal to the level of a standard biomarker.

11. The method according to any one of claims 3 to 10, wherein the reference result comprises the ratio of the levels of two or more biomarkers in samples obtained from control equines.

12. The method according to any one of claims 3 to 11, wherein the method further comprises the step of generating the reference result.

13. The method according to any one of claims 1 to 8, wherein the reference result comprises a predetermined threshold concentration of one or more biomarkers.

14. A method for monitoring the levels of one or more biomarkers in equines, the method comprising: (a) Assays are performed to determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid, and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; and (b) Repeat step (a) at two or more separate time points.

15. A method for evaluating the effectiveness of treatment for insulin dysregulation in equines, the method comprising: (a) To determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; (b) Repeat step (a) at two or more separate time points, at least one of which is after the equine has begun treatment.

16. A method for evaluating the effectiveness of treatment for insulin dysregulation-related conditions in equines, the method comprising: (a) To determine the levels of one or more biomarkers selected from the group consisting of: propionylglycine, allantoin, nicotinamide nucleoside, allantoic acid, hexadecanoic acid and urea cycle-related metabolites such as argininosuccinate and argininate, in samples obtained from equines; (b) Repeat step (a) at two or more separate time points, at least one of which is after the equine has begun treatment.

17. The method according to any one of claims 2 to 13 or 16, wherein the insulin dysregulation-related condition is laminitis, equine metabolic syndrome, diabetes, obesity, or pituitary midline dysfunction (PPID).

18. The method according to any one of claims 1 to 17, wherein step (a) comprises determining the concentration of one or more biomarkers in a sample obtained from an equine animal.

19. The method according to any one of claims 1 to 18, wherein step (a) comprises determining the ratio of the level of one or more biomarkers in a sample obtained from equines to the level of a standard biomarker.

20. The method according to claim 10 or 19, wherein the standard marker is a fatty acid, an amino acid, or selected from phenylalanine, tryptophan, leucine, acetylcarnitine, 1-palmitoyl-2-oleoyl-GPC, creatinine, and glucose.

21. The method according to any one of claims 1 to 20, wherein step (a) comprises determining the ratio of the levels of two or more biomarkers in a sample obtained from a control equine animal.

22. The method according to any one of claims 1-21, wherein the sample is a bodily fluid obtained from an equine animal, preferably whole blood, saliva, plasma, serum or urine, or wherein the sample is a dried blood spot or fecal sample.

23. The method according to any one of claims 1-22, wherein the equine is a horse, pony, mule or donkey.

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