TITLE

COMPOSITIONS AND METHODS THAT MODULATE DIGESTIBILITY IN A COMPANION

ANIMAL

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 62/434,548 filed December 15, 2016, the disclosure of which is incorporated in its entirety herein by this reference.

BACKGROUND

[0002] The digestibility of an animal regarding food and components thereof, such as organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility, is important for the health and well-being of the animal. These characteristics of the animal can change in response to age and other factors. Beyond selecting macronutrients and amounts thereof, the ability to modulate organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility is difficult. In this regard, most attempts to modulate digestibility are directed to overall digestibility and use macronutrients such as citrus pulp, wheat, corn, apple and beets but not specific compounds.

[0003] The specific biological compounds which increase or decrease digestibility are difficult to ascertain. In addition, measuring digestibility can be time and labor intensive. To the best knowledge of the inventors, the current literature only describes enhancement of overall digestibility and only uses differences between macronutrients or, with respect to specific compounds, uses omega-3 polyunsaturated fats, medium chain triglycerides, methionine, 2-hydroxy-4-butanoic acid, diacylglycerol, yttrium oxide, chromic oxide, polydextrose, and lengths of cellulose fibers. Furthermore, studies regarding digestibility tend to be focused on improving stool quality and not the actual absorption of nutrients by the animal itself.

SUMMARY

[0004] The present disclosure relates generally to pet food compositions; methods of minimizing costs associated with production of a pet food; methods of enhancing nutritional benefit of a pet food; and methods for modulating digestibility of at least one of organic matter, dry matter, fiber, energy, fat or protein in a companion animal. Specifically, the present disclosure relates to metabolites for modulating at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in a companion animal.

[0005] Overall digestibility does not necessarily involve all of the digestibilities noted above: organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility. Therefore, attempts to enhance overall digestibility may fail to address one or more of these important characteristics of the animal. Moreover, the present inventors believed that it was likely that specific digestibilities could be modulated using approaches beyond those already investigated (omega-3 polyunsaturated fats, methionine, 2-hydroxy-4-butanoic acid, and differences between macronutrients).

[0006] The present inventors have developed a predictive model of specific digestibilities by identifying metabolite compounds which correlate to organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility. A very controlled study was employed to minimize other external factors by using multiple canines all fed the same diet. A validation model was then developed by feeding different levels of the identified compounds (via a dietary change) to a group of canines and measuring changes in the corresponding digestibility.

[0007] Accordingly, in a general embodiment, the present disclosure provides a pet food composition comprising: protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in a companion animal. In one embodiment, the pet food composition can provide at least a 10% increase or at least a 10% decrease in at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in the companion animal. In one aspect, the pet food can provide an increase in a digestibility. In another aspect, the pet food can comprise a decrease in a digestibility.

[0008] The present disclosure also provides methods of minimizing costs associated with production of a pet food; methods of enhancing nutritional benefit of a pet food; and methods for modulating digestibility of at least one of organic matter, dry matter, fiber, energy, fat or protein in a companion animal.

[0009] An advantage of one or more embodiments provided by the present disclosure is to improve a specific type of digestibility in a companion animal by adjusting the diet of the animal to increase the amount of a compound which positively modulates the specific type of digestibility or to decrease the amount of a compound which negatively modulates the specific type of digestibility.

[0010] Another advantage of one or more embodiments provided by the present disclosure is to minimize pet food production costs by utilizing ingredients which are appropriately high or low in the identified compounds or precursors thereof, as generally illustrated by the non-limiting examples discussed herein.

[0011] The cost of organic matter can be minimized by utilizing ingredients which are appropriately high in compounds which positively modulate organic matter digestibility and/or appropriately low in compounds which negatively modulate organic matter digestibility.

[0012] The cost of dry matter can be minimized by utilizing ingredients which are appropriately high in compounds which positively modulate dry matter digestibility and/or appropriately low in compounds which negatively modulate dry matter digestibility.

[0013] The cost of fiber can be minimized by utilizing ingredients which are appropriately high in compounds which positively modulate fiber digestibility and/or appropriately low in compounds which negatively modulate fiber digestibility. [0014] The cost of energy-providing matter (foodstuff) can be minimized by utilizing ingredients which are appropriately high in compounds which positively modulate energy digestibility and/or appropriately low in compounds which negatively modulate energy digestibility.

[0015] The cost of fat can be minimized by utilizing ingredients which are appropriately high in compounds which positively modulate fat digestibility and/or appropriately low in compounds which negatively modulate fat digestibility.

[0016] The cost of protein can be minimized by utilizing ingredients which are appropriately high in compounds which positively modulate protein digestibility and/or appropriately low in compounds which negatively modulate protein digestibility.

[0017] Yet another advantage of one or more embodiments of the present disclosure is to decrease the cost and time associated with determining digestibility or specific types thereof by identifying and using surrogate markers.

[0018] Additional features and advantages are described herein and will be apparent from the following Detailed Description.

DETAILED DESCRIPTION

[0019] Definitions

[0020] As used in this disclosure and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" or "the composition" includes two or more compositions. The term "and/or" used in the context of "X and/or Y" should be interpreted as "X," or "Y," or "X and Y." Where used herein, the terms "example" and "such as," particularly when followed by a listing of terms, are merely exemplary and illustrative, and are not exclusive or comprehensive.

[0021] As used herein, "about" is understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, within -5% to +5% of the referenced number, or in one aspect, within -1% to +1% of the referenced number, and in a specific aspect, within -0.1% to +0.1% of the referenced number. Furthermore, all numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0022] All percentages expressed herein are by weight of the total weight of the food composition unless expressed otherwise. When reference is made to the pH, values correspond to pH measured at 25 °C with standard equipment. An "amount" can be the total amount of the referenced component per serving of the composition or per distinct unit of the composition and/or can be the weight percentage of the referenced component by dry weight. Moreover, an "amount" includes zero; for example, the recitation of an amount of a compound does not necessarily mean that the compound is present, unless followed by a range that excludes zero.

[0023] The terms "food," "food product" and "food composition" mean a product or composition that is intended for ingestion by an animal, including a human, and provides at least one nutrient to the animal. Further in this regard, these terms mean that the product or composition is in a form ready for consumption and is not merely an intermediate from which a consumable product or composition is made, although other food compositions can be added in some embodiments. The term "pet food" means any food composition intended to be consumed by a pet. The term "pet" means any animal which could benefit from or enjoy the compositions provided by the present disclosure. For example, the pet can be an avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal, but the pet can be any suitable animal.

[0024] The term "companion animal" means a dog or a cat. In an embodiment, the compositions and methods disclosed herein involve a senior dog or a senior cat. Dogs are considered senior in the last 25% of their lives. The life span of a dog depends on its size and/or its breed, but for the present disclosure a senior dog is a dog that is at least 5 years of age (e.g., at least 6 years of age, at least 7 years of age, or at least 8 years of age). The life span of a cat also depends on its size and/or its breed, but for the present disclosure a senior cat is a cat that is at least 7 years of age (e.g., at least 8 years of age, at least 9 years of age, or at least 10 years of age).

[0025] As used herein, "metabolite" refers to a compound having biological activity in a companion animal that is an intermediate or product of metabolism, and includes precursors thereof. As used herein, "precursor" refers to any compound that metabolizes to a metabolite during metabolism in a companion animal. For example, if the specific metabolite is cysteine, "the metabolite" comprises a cysteine precursor (e.g., methionine).

[0026] The term "effective amount" means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In one embodiment, the present metabolite or combination of metabolites can be present in an effective amount for modulating organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in a companion animal.

[0027] The dosages expressed herein are in milligrams per kilogram of body weight per day (mg/kg/day) unless expressed otherwise.

[0028] The term "long-term administration" means periods of repeated administration or consumption in excess of one month. Periods of longer than two, three, or four months can be used for certain embodiments. Also, more extended periods can be used that include longer than 5, 6, 7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year can also be used. Longer term use extending over 1, 2, 3, or more years are included in the invention. For certain aging animals, the animal will continue consuming on a regular basis for the remainder of its life. This can also be referred to as consumption for "extended" periods.

[0029] The term "regular basis" or "regular administration" means at least monthly dosing with the compositions or consumption of the compositions, and in one aspect, means at least weekly dosing. More f equent dosing or consumption, such as twice or three times weekly, can be performed in certain embodiments. Still, in other embodiments, regimens can be used that comprise at least once daily consumption. The skilled artisan will appreciate that the blood level of a compound or certain metabolites of that compound or which result after the consumption of that compound, may be a useful tool for assessing or determining dosing frequency. For example, for determining feeding amounts for pet food compositions comprising a certain metabolite, the blood concentration of that metabolite, may provide useful information. A frequency, regardless of whether expressly exemplified herein, that allows maintenance of a desired blood level of the measured compound, such as a metabolite, within acceptable ranges can be useful herein. The skilled artisan will appreciate that feeding amounts will be a function of the composition that is being consumed or administered as well as the animal consuming the food, and some food compositions may require more or less frequent administration to maintain a desired blood level of the measured compound (e.g., a metabolite).

[0030] The relative terms "improve," "increase," "enhance," "decrease" and the like refer to the effects of the composition disclosed herein (a composition comprising a metabolites) relative to a composition having a lower amount or lacking such metabolites, but otherwise identical.

[0031] A "blended" composition merely has at least two components having at least one different characteristic relative to each other. In one aspect, moisture content and water activity can be different in the context of the present disclosure. In this regard, description of a composition as "blended" does not imply that the blended composition has been subjected to processing sometimes referenced as "blending," namely mixing components so that they are indistinguishable from each other, and, in one aspect, such processing is avoided when mixing one component with the other components to form a blended composition (e.g., mixing a dry component with a wet or semi-moist component). Further in this regard, in a blended composition each of the at least two components having at least one different characteristic relative to each other can retain their distinct identity and appearance.

[0032] "Wet food" means a pet food having a moisture content from about 50% to about 90%, and in one aspect, from about 70% to about 90%. "Dry food" means a pet food having a moisture content less than about 20%, and in one aspect, less than about 1 %, and in a specific aspect, less than about 10%. "Semi-moist food" means a pet food having a moisture content from about 20% to about 50%, and in one aspect, from about 25% to about 35%.

[0033] "Kibbles" is used synonymously with "chunks" herein and both terms mean pieces of dry or semi- moist pet food which can have a pellet shape or any other shape and can be made by slicing a food composition into separate pieces. Non-limiting examples of kibbles include particulates; pellets; pieces of pet food, dehydrated meat, meat analog, vegetables, and combinations thereof; and pet snacks, such as meat or vegetable jerky, rawhide, and biscuits. A "meat analog" is a meat emulsion product that resembles pieces of natural meat in appearance, texture, and physical structure.

[0034] The term "dietary supplement" means a product that is intended to be ingested in addition to the normal animal diet. Dietary supplements may be in any form, e.g., solid, liquid, gel, tablets, capsules, powder, and the like. In one aspect, they can be provided in convenient dosage forms. In some embodiments, they can be provided in bulk consumer packages such as bulk powders, liquids, gels, or oils. In other embodiments, supplements can be provided in bulk quantities to be included in other food items such as snacks, treats, supplement bars, beverages and the like.

[0035] The compositions disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term "comprising" includes a disclosure of embodiments "consisting essentially of and "consisting of the components identified. Similarly, the methods disclosed herein may lack any step that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term "comprising" includes a disclosure of embodiments "consisting essentially of and "consisting of the steps identified. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein unless explicitly and directly stated otherwise.

[0036] "Digestibility" is the extent a food or component thereof is absorbed in the animal body as it passes through the digestive tract. It varies with the type of food, the specific food formulation, the type of animal, and the specific animal.

[0037] Accordingly, dry matter digestibility is the percentage of the dry matter in a food that is digested by an animal consuming the food (e.g., not excreted as waste). Organic matter digestibility is the percentage of the organic matter in a food that is digested by an animal consuming the food (e.g., not excreted as waste); the amount of organic matter is the difference between the dry matter weight and the weight of the ash. Energy digestibility is the percentage of the energy in a food (e.g., kcals) that is digested by an animal consuming the food (e.g., not excreted as waste). Fat digestibility is the percentage of the fat matter in a food that is digested by an animal consuming the food (e.g., not excreted as waste). Protein digestibility is the percentage of the protein in a food that is digested by an animal consuming the food (e.g., not excreted as waste).

[0038] The present discussion of embodiments, aspects, examples, etc. are independent in that they can apply to all methods and compositions. For example, a metabolite used in a pet food composition can also be used in the method of modulating or a method of minimizing costs associated with making such a pet food, and vice versa.

Embodiments

[0039] In an aspect of the present disclosure, a pet food composition can comprise protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in a companion animal. In one aspect, the pet food composition can include at least 6 distinct metabolites for individually modulating organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility in a companion animal. In one embodiment, the pet food composition can provide an increase in at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in the companion animal. In one aspect, the companion animal can be a senior dog or a senior cat.

[0040] In another aspect of the present disclosure, a method of modulating digestibility of at least one of organic matter, dry matter, fiber, energy, fat or protein in a companion animal is provided. The method comprises administering to the companion animal a pet food composition comprising protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in the companion animal. In one aspect, the companion animal can be a senior dog or a senior cat.

[0041] Yet another aspect of the present disclosure is a method of minimizing costs associated with production of a pet food having a first formulation designed for consumption by a companion animal, such as a senior dog or a senior cat. A further aspect of the present disclosure is a method of enhancing nutritional benefit of a pet food having a first formulation designed for consumption by a companion animal, such as a senior dog or a senior cat. These methods comprise adjusting the first formulation of the pet food to be a second formulation. At least one of the first and second formulations comprises a metabolite for modulating a specific digestibility that is at least one of organic matter, dry matter, fiber, energy, fat or protein in the companion animal. The adjusting comprises changing an amount of the metabolite in the first formulation to a different amount in the second formulation.

[0042] "Minimizing" costs means that the costs associated with making the second formulation are less than the costs associated with making the first formulation, for example on a per serving basis, per unit weight, per unit volume, per total energy, and the like. "Enhanced" nutritional benefit or specific digestibility means that the nutritional benefit or digestibility of the second formulation is greater than the nutritional benefit or specific digestibility of the first formulation.

[0043] As discussed herein, the pet food compositions and methods can contain a metabolite or multiple metabolites for modulating various digestibilities. In various aspects, the compositions and methods can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 metabolites for any one of the digestibilities, or multiple digestibilities, or for each digestibility. As such, in one embodiment, the composition can comprise at least 12 metabolites, including at least 2 metabolites for individually modulating each one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility in a companion animal. In another embodiment, the composition can comprise at least 18 metabolites, including at least 3 metabolites for individually modulating each one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility in a companion animal. Additionally, in one embodiment, the composition can comprise at least four distinct metabolites that modulate at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility. Further, in one embodiment, the composition can comprise at least five distinct metabolites that modulate at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility. Still, in another embodiment, the method can modulate at least two digestibilities selected from the group consisting of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility and where the composition comprises at least six distinct metabolites, three distinct metabolites for modulating each of the at least two digestibilities. Yet, in another embodiment, the method can modulate at least three digestibilities selected from the group consisting of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility and where the composition comprises at least nine distinct metabolites, three distinct metabolites for modulating each of the at least three digestibilities. As previously noted, the metabolite and digestibility relationships can be used for both method and composition embodiments.

[0044] In some embodiments, the metabolite negatively modulates the specific digestibility, and the changing of the amount of the metabolite comprises decreasing the amount of the metabolite. In other embodiments, the metabolite negatively modulates the specific digestibility, and the changing of the amount of the metabolite comprises increasing the amount of the metabolite. In some embodiments, the metabolite positively modulates the specific digestibility, and the changing of the amount of the metabolite comprises increasing the amount of the metabolite. In other embodiments, the metabolite positively modulates the specific digestibility, and the changing of the amount of the metabolite comprises decreasing the amount of the metabolite. These are not mutually exclusive embodiments; a particular embodiment can comprise decreasing the amount of a metabolite that negatively modulates a first specific digestibility and increasing the amount of a metabolite that positively modulates a second specific digestibility, and the first and second specific digestibilities can be the same or different specific digestibilities (e.g., one or more of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility).

[0045] Decreasing the amount of the metabolite can comprise decreasing the amount of the metabolite directly and/or decreasing the amount of an ingredient which comprises the metabolite. In some embodiments, decreasing the amount of the metabolite can comprise decreasing the amount of a precursor of the metabolite directly and/or decreasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, less of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

[0046] Increasing the amount of the metabolite can comprise increasing the amount of the metabolite directly and/or increasing the amount of an ingredient which comprises the metabolite. In some embodiments, increasing the amount of the metabolite can comprise increasing the amount of a precursor of the metabolite directly and/or increasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, more of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

[0047] Generally, the methods and compositions described herein can provide an increase or a decrease in at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in the companion animal. In some embodiment, the increase or decrease can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50%. In one aspect, the compositions and methods can provide an increase. In another aspect, the compositions and methods can provide a decrease.

[0048] In one embodiment, the increase or decrease can be for at least two of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility. In another embodiment, the increase or decrease can be for at least three of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility. In still another embodiment, the increase or decrease can be for at least four of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility. In yet another embodiment, the increase or decrease can be for at least five of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility. In still another embodiment, the increase or decrease can be for organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility and protein digestibility.

[0049] The cost of organic matter used in the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate organic matter digestibility and/or appropriately low in compounds which negatively modulate organic matter digestibility.

[0050] The cost of dry matter used in the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate dry matter digestibility and/or appropriately low in compounds which negatively modulate dry matter digestibility.

[0051] The cost of fiber used in the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate fiber digestibility and/or appropriately low in compounds which negatively modulate fiber digestibility.

[0052] The cost of energy-providing ingredients used in the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate energy digestibility and/or appropriately low in compounds which negatively modulate energy digestibility. [0053] The cost of fat used in the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate fat digestibility and/or appropriately low in compounds which negatively modulate fat digestibility.

[0054] The cost of protein used in the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate protein digestibility and/or appropriately low in compounds which negatively modulate protein digestibility.

[0055] Ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

[0056] In each of these compositions and methods, the pet food composition can be a wet food, a semi- moist food or a dry food. In an embodiment, the pet food composition is one or more components of a blended composition. In some embodiments, the pet food composition is a kibble, and in some embodiments, the pet food composition is a meat analog. Additionally, in another embodiment, the present composition for modulating a digestibility can be a dietary supplement comprising the metabolites described herein. Further, a method of modulating a digestibility can include administering to the companion animal the dietary supplement.

[0057] Such pet food compositions can be administered to the companion animal in amounts ranging from about 3 g of pet food per 1 lb body weight to about 16 g of pet food per 1 lb body weight of the companion animal. Additionally, the metabolites can be present in amounts from about 0.01 weight% to about 10 weight% of the food composition. In one aspect, the metabolites can be present in concentrations of about 0.01 to about 1,000 mg/kg of food. In another aspect, the metabolites can be present in concentrations from about 1 IU to about 500,000 IU per kilogram of food. In one embodiment, the pet food composition can be administered to the companion animal in amounts sufficient to maintain the health and/or body weight of the animal. In one aspect, the administration can be regular administration.

[0058] As noted above and detailed later in this application, the present inventors identified metabolite compounds which correlate to organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility. Thus, the metabolite in the pet food composition can be one of these compounds. Nevertheless, the metabolite can be any metabolite for modulating at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility in a companion animal, even if the metabolite is not explicitly disclosed herein. For example, the metabolite can be a compound identified using the methods disclosed herein but not itself explicitly disclosed herein. Furthermore, the metabolite can be a compound identified using a method not disclosed herein if the compound is reliably correlated to at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility. [0059] As a non-limiting example, the metabolite can modulate organic matter digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), mannose, arachidonate (20:4n6), dihomo-linoleate (20:2n6), docosapentaenoate (n6 DPA; 22:5n6), pantothenate, X-09789, dihomo-linolenate (20:3n3), dihomo-linolenate (20:3n6), 3-hydroxybutyrate (BHBA), adrenate (22:4n6), docosapentaenoate (n3 DPA; 22:5n3), eicosenoate (20: ln9), eicosenoate (20:lnl 1), X-l 1378, alpha linolenate (18:3n3), gamma linolenate (18:3n6), stachydrine, glycerate, gamma-glutamylphenylalanine, X-16975, X-17185, arabitol, creatine, octanoylcamitine, trans-4-hydroxyproline, X-l 7010, glycolate (hydroxyacetate), N-acetylornithine, campesterol, oxidized glutathione (GSSG), phenylalanine, oleoyltaurine, arabonate, X-12450, palmitoleate (16: ln7), acetylcamitine, palmitate (16:0), erythritol, linoleate (18:2n6), stearate (18:0) and mixtures thereof. In one embodiment, the metabolite can modulate organic matter digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), mannose, arachidonate (20:4n6), dihomo-linoleate (20:2n6), docosapentaenoate (n6 DPA; 22:5n6), pantothenate, dihomo-linolenate (20:3n3), dihomo-linolenate (20:3n6), 3- hydroxybutyrate (BHBA), adrenate (22:4n6), docosapentaenoate (n3 DPA; 22:5n3), eicosenoate (20: ln9), eicosenoate (20: lnl 1), alpha linolenate (18:3n3), gamma linolenate (18:3n6), stachydrine, glycerate, gamma- glutamylphenylalanine, arabitol, creatine, octanoylcamitine, trans-4-hydroxyproline, glycolate (hydroxyacetate), N-acetylornithine, campesterol, oxidized glutathione (GSSG), phenylalanine, oleoyltaurine, arabonate, palmitoleate (16:ln7), acetylcamitine, palmitate (16:0), erythritol, linoleate (18:2n6), stearate (18:0) and mixtures thereof.

[0060] As another non-limiting example, the metabolite can modulate dry matter digestibility and can be selected from the group consisting of mannose, trigonelline (N'-methylnicotinate), arachidonate (20:4n6), dihomo-linoleate (20:2n6), -palmitoyl taurine, docosapentaenoate (n6 DPA; 22:5n6), X- 09789, pantothenate, docosapentaenoate (n3 DPA; 22:5n3), X - 16975, 3-hydroxybutyrate (BHBA), X - 11378, linolenate [alpha or gamma; (18:3n3 or 6)], gamma-glutamylphenylalanine, dihomo-linolenate (20:3n3 or n6), eicosenoate (20:ln9 or 11), arabitol, adrenate (22:4n6), X - 17185, Glycerate, glutathione ( oxidized (GSSG)), mannitol, octanoylcamitine, N-acetylomithine, X - 17010, glycolate (hydroxyacetate), stachydrine, campesterol, palmitoleate (16: ln7), creatine, X— 12450, oleoyltaurine, arabonate, trans-4-hydroxyproline, phenylalanine, erythritol, palmitate (16:0), and mixtures thereof. In one embodiment, the metabolite can modulate dry matter digestibility and can be selected from the group consisting of mannose, trigonelline (N'-methylnicotinate), arachidonate (20:4n6), dihomo-linoleate (20:2n6), N-palmitoyl taurine, docosapentaenoate (n6 DPA; 22:5n6), pantothenate, docosapentaenoate (n3 DPA; 22:5n3), 3-hydroxybutyrate (BHBA), linolenate [alpha or gamma; (18:3n3 or 6)], gamma-glutamylphenylalanine, dihomo-linolenate (20:3n3 or n6), eicosenoate (20:ln9 or 11), arabitol, adrenate (22:4n6), Glycerate, glutathione ( oxidized (GSSG)), mannitol, octanoylcamitine, N- acetylornithine, glycolate (hydroxyacetate), stachydrine, campesterol, palmitoleate (16: ln7), creatine, oleoyltaurine, arabonate, trans-4-hydroxyproline, phenylalanine, erythritol, palmitate (16:0), and mixtures thereof. [0061] As yet another non-limiting example, the metabolite can modulate energy digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), mannose, arachidonate (20:4n6), dihomo-linoleate (20:2n6), docosapentaenoate (n6 DPA; 22:5n6), N-palmitoyl taurine, betaine, X-09789, pantothenate, adrenate (22:4n6), dihomo-linolenate (20:3n3), dihomo-linolenate (20:3n6), X-11378, glycerate, 3- hydroxybutyrate (BHBA), docosapentaenoate (n3 DPA; 22:5n3), stachydrine, X-17185, eicosenoate (20:ln9), eicosenoate (20: lnl l), alpha linolenate (18:3 n3), gamma linolenate (18:3n6), gamma-glutamylphenylalanine, X- 16975, creatine, campesterol, glycolate (hydroxyacetate), X-17010, octanoylcarnitine, N-acetylornithine, trans-4- hydroxyproline, phenylalanine, oxidized glutathione (GSSG), arabitol, mannitol, acetylcarnitine, erythritol, oleoyltaurine, X-12450, palmitoleate (16:ln7), arabonate, palmitate (16:0), docosahexaenoate (DHA; 22:6n3), linoleate (18:2n6), stearate (18:0) and mixtures thereof. In one embodiment, the metabolite can modulate energy digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), mannose, arachidonate (20:4n6), dihomo-linoleate (20:2n6), docosapentaenoate (n6 DPA; 22:5n6), N-palmitoyl taurine, betaine, pantothenate, adrenate (22:4n6), dihomo-linolenate (20:3n3), dihomo-linolenate (20:3n6), glycerate, 3- hydroxybutyrate (BHBA), docosapentaenoate (n3 DPA; 22:5n3), stachydrine, eicosenoate (20: ln9), eicosenoate (20: lnl 1), alpha linolenate (18:3n3), gamma linolenate (18:3n6), gamma-glutamylphenylalanine, creatine, campesterol, glycolate (hydroxyacetate), octanoylcarnitine, N-acetylornithine, trans-4-hydroxyproline, phenylalanine, oxidized glutathione (GSSG), arabitol, mannitol, acetylcarnitine, erythritol, oleoyltaurine, palmitoleate (16:ln7), arabonate, palmitate (16:0), docosahexaenoate (DHA; 22:6n3), linoleate (18:2n6), stearate (18:0) and mixtures thereof.

[0062] As a further non-limiting example, the metabolite can modulate fat digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), X-09789, arachidonate (20:4n6), mannose, betaine, oxidized glutathione (GSSG), X-l 1378, dihomo-linoleate (20:2n6), glycerate, campesterol, docosapentaenoate (n6 DPA; 22:5n6), glycolate (hydroxyacetate), pantothenate, adrenate (22:4n6), X-17185, N- acetylornithine, gamma-glutamylphenylalanine, octanoylcarnitine, stachydrine, dihomo-linolenate (20:3n3), dihomo-linolenate (20:3n6), docosapentaenoate (n3 DPA; 22:5n3), 3-hydroxybutyrate (BHBA), X-17010, X- 16975, 15-methylpalmitate (isobar with 2-methylpalmitate), mannitol, citrulline, alpha linolenate (18:3n3), gamma linolenate (18:3n6), phenylalanine, erythritol, arabitol, eicosenoate (20: ln9), eicosenoate (20: lnl l), cholate, gamma-glutamylalanine, creatine, 3-dehydrocholate, docosahexaenoate (DHA; 22:6n3), trans-4- hydroxyproline, 7-ketodeoxycholate, 12-dehydrocholate, chenodeoxycholate, acetylcarnitine, palmitoleate (16:ln7) and mixtures thereof. In one embodiment, the metabolite can modulate fat digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), arachidonate (20:4n6), mannose, betaine, oxidized glutathione (GSSG), dihomo-linoleate (20:2n6), glycerate, campesterol, docosapentaenoate (n6 DPA; 22:5n6), glycolate (hydroxyacetate), pantothenate, adrenate (22:4n6), N-acetylornithine, gamma- glutamylphenylalanine, octanoylcarnitine, stachydrine, dihomo-linolenate (20:3n3), dihomo-linolenate (20:3n6), docosapentaenoate (n3 DPA; 22:5n3), 3-hydroxybutyrate (BHBA), 15-methylpalmitate (isobar with 2- methylpalmitate), mannitol, citrulline, alpha linolenate (18:3n3), gamma linolenate (18;3n6), phenylalanine, erythritol, arabitol, eicosenoate (20:ln9), eicosenoate (20:lnl l), cholate, gamma-glutamylalanine, creatine, 3- dehydrocholate, docosahexaenoate (DHA; 22:6n3), trans-4-hydroxyproline, 7-ketodeoxycholate, 12- dehydrocholate, chenodeoxycholate, acetylcarnitine, palmitoleate (16:ln7), and mixtures thereof.

[0063] As an additional non-limiting example, the metabolite can modulate protein digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), X-09789, mannose, dihomo-linoleate (20:2n6), docosapentaenoate (n6 DP A; 22:5n6), X-11378, trans-4-hydroxyproline, arachidonate (20:4n6), X- 17185, betaine, X- 15461, N-acetylornithine, creatine, stachydrine, 4-hydroxyphenylacetate, eicosenoate (20:ln9), eicosenoate (20:lnl 1), campesterol, gamma-glutamylphenylalanine, pantothenate, mannitol, docosapentaenoate (n3 DPA; 22:5n3), adrenate (22:4n6), alpha linolenate (18:3n3), gamma linolenate (18:3n6), X-16975, glycerate, phenylalanine, oxidized glutathione (GSSG), palmitoleate (16:ln7), octanoylcarnitine, X- 12450, erythritol, 3-hydroxybutyrate (BHBA), 3-(4-hydroxyphenyl)propionate, 1- heptadecanoylglycerophosphocholine, 3-phenylpropionate (hydrocinnamate), oleoyltaurine and mixtures thereof. In one embodiment, the metabolite can modulate protein digestibility and can be selected from the group consisting of trigonelline (N'-methylnicotinate), mannose, dihomo-linoleate (20:2n6), docosapentaenoate (n6 DPA; 22:5n6), trans-4-hydroxyproline, arachidonate (20:4n6), betaine, X- 15461, N-acetylornithine, creatine, stachydrine, 4-hydroxyphenylacetate, eicosenoate (20:ln9), eicosenoate (20:lnl 1), campesterol, gamma- glutamylphenylalanine, pantothenate, mannitol, docosapentaenoate (n3 DPA; 22:5n3), adrenate (22:4n6), alpha linolenate (18:3n3), gamma linolenate (18:3n6), glycerate, phenylalanine, oxidized glutathione (GSSG), palmitoleate (16:ln7), octanoylcarnitine, erythritol, 3-hydroxybutyrate (BHBA), 3-(4-hydroxyphenyl)propionate, 1-heptadecanoylglycerophosphocholine, 3-phenylpropionate (hydrocinnamate), oleoyltaurine, and mixtures thereof.

[0064] As a further non-limiting example, the metabolite can modulate fiber digestibility and can be selected from the group consisting of 1-heptadecanoylglycerophosphoethanolamine, 1- palmitoylplasmenylethanolamine, 1-palmitoylglycerol (1-monopalmitin), alpha-tocopherol, ribitol and mixtures thereof.

[0065] In yet another aspect of the present disclosure, a method enhances nutritional benefit of a pet food having a first formulation designed for consumption by companion animals, and the method comprises administering the pet food having the first formulation to a first companion animal. The method further comprises measuring in a sample of body fluid from the companion animal (e.g., plasma) an amount of a surrogate marker comprising a metabolite that modulates at least one of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility. The method further comprises adjusting the first formulation of the pet food to be a second formulation having a difference from the first formulation selected from the group consisting of (i) an ingredient is present in the second formulation and is absent in the first formulation, (ii) an ingredient is absent in the second formulation and is present in the first formulation, (iii) an ingredient is present in the first and second formulations but in a different amount, and (iv) combinations thereof. The adjusting is based at least partially on the amount of the surrogate marker measured in the previous step.

[0066] The adjusting can comprise directly decreasing the amount of a metabolite negatively modulating a specific digestibility and/or decreasing the amount of an ingredient which comprises a metabolite negatively modulating a specific digestibility. In some embodiments, decreasing the amount of the metabolite can comprise decreasing the amount of a precursor of the metabolite directly and/or decreasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, less of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

[0067] The adjusting can comprise increasing the amount of the metabolite can comprise directly increasing the amount of a metabolite positively modulating a specific digestibility and/or increasing the amount of an ingredient which comprises a metabolite positively modulating a specific digestibility. In some embodiments, increasing the amount of the metabolite can comprise increasing the amount of a precursor of the metabolite directly and/or increasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, more of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

[0068] As noted above, ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

[0069] The method further comprises producing the pet food in the second formulation. In an embodiment, the method comprises administering the pet food having the second formulation to a second companion animal.

[0070] This method can be used to provide customized nutrition for a specific companion animal. For example, the first and second companion animal can be the same specific companion animal such that the animal who is administered the pet food having the first formulation has one or more of their specific digestibilities of the first formulation assessed. Then this same animal is provided with the resultant second formulation which this animal will more effectively digest with respect to at least one of organic matter, dry matter, fiber, energy, fat or protein. Consequently, a pet owner can compensate for their pet's age-induced changes in one or more of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility or protein digestibility. [0071] Alternatively or additionally, this method can be used to provide customized nutrition for companion animals who share one or more of a gender, an approximate age, an approximate size (e.g., body weight, height, and or length) or a breed. For example, the second companion animal can be a different specific animal than the first companion animal but has a characteristic selected from the group consisting of (i) about the same age as the first companion animal, (ii) about the same size as the first companion animal, (iii) the same breed as the first companion animal, (iv) the same gender as the first companion animal, and (iv) combinations thereof. In one embodiment, the second companion animal can be one of a plurality of companion animals who each share the characteristic with the first companion animal. The method can comprise administering the pet food having the second formulation to the plurality of companion animals. In an embodiment, at least a portion of the plurality of companion animals is located remotely relative to the first companion animal.

[0072] The pet food compositions disclosed herein can be any food formulated for consumption by a pet such as a companion animal. In an embodiment, the pet food composition provides complete nutrition as defined by the Association of American Feed Control Officials (AAFCO) and which depends on the type of animal for which the composition is intended (e.g., dog or cat).

[0073] The pet food composition can comprise meat, such as emulsified meat. Examples of suitable meat include poultry, beef, pork, lamb and fish, especially those types of meats suitable for pets. The meat can include any additional parts of an animal including offal. Some or all of the meat can be provided as one or more meat meals, namely meat that has been dried and ground to form substantially uniform-sized particles and as defined by AAFCO. Additionally or alternatively, vegetable protein can be used, such as pea protein, corn protein (e.g., ground corn or corn gluten), wheat protein (e.g., ground wheat or wheat gluten), soy protein (e.g., soybean meal, soy concentrate, or soy isolate), rice protein (e.g., ground rice or rice gluten) and the like.

[0074] The pet food compositions disclosed herein can comprise vegetable oil, a flavorant, a colorant and water. Suitable vegetable oils include soybean oil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil, safflower oil, and the like. Examples of suitable flavorants include yeast, tallow, rendered animal meals (e.g., poultry, beef, Iamb, pork), flavor extracts or blends (e.g., grilled beef), animal digests, and the like. Suitable colorants include FD&C colors, such as blue no. 1, blue no. 2, green no. 3, red no. 3, red no. 40, yellow no. 5, yellow no. 6, and the like; natural colors, such as caramel coloring, annatto, chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene, elderberry juice, pandan, butterfly pea and the like; titanium dioxide; and any suitable food colorant known to the skilled artisan.

[0075] The pet food compositions disclosed hereiri can optionally include additional ingredients, such as other grains and/or other starches additionally or alternatively to flour, amino acids, fibers, sugars, animal oils, aromas, other oils additionally or alternatively to vegetable oil, humectants, preservatives, polyols, salts, oral care ingredients, antioxidants, vitamins, minerals, probiotic microorganisms, bioactive molecules or combinations thereof. [0076] Suitable starches include a grain such as corn, rice, wheat, barley, oats, soy and the like, and mixtures of these grains, and can be included at least partially in any flour. Suitable humectants include salt, sugars, propylene glycol and polyhydric glycols such as glycerin and sorbitol, and the like. Suitable oral care ingredients include alfalfa nutrient concentrate containing chlorophyll, sodium bicarbonate, phosphates (e.g., tricalcium phosphate, acid pyrophosphates, tetrasodium pyrophosphate, metaphosphates, and orthophosphates), peppermint, cloves, parsley, ginger and the like. Examples of suitable antioxidants include butylated hydroxyanisole ("BHA") and butylated hydroxytoluene ("BHT"), vitamin E (tocopherols), and the like.

[0077] Non-limiting examples of vitamins that can be used include Vitamins A, B-complex (such as B-l, B-2, B-6 and B-12), C, D, E and K, niacin and acid vitamins such as pantothenic acid and folic acid and biotin. Non-limiting examples of suitable minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium, boron and the like.

[0078] Non-limiting examples of suitable preservatives include potassium sorbate, sorbic acid, sodium methyl para-hydroxybenzoate, calcium propionate, propionic acid, and combinations thereof.

[0079] Specific amounts for each additional ingredient in the pet food compositions disclosed herein will depend on a variety of factors such as the ingredient included in the first edible material and any second edible material; the species of animal; the animal's age, body weight, general health, sex, and diet; the animal's consumption rate; the purpose for which the food product is administered to the animal; and the like. Therefore, the components and their amounts may vary widely.

[0080] For example, the amount of any of the above-noted ingredients can be decreased or increased based on the estimated effect on one or more of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, or protein digestibility (e.g., an effect identified by one of the methods disclosed herein). In an embodiment, the amount of one or more of the above-noted ingredients can be increased if such ingredients comprise a metabolite that positively modulates one or more of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, and protein digestibility.

Additionally or alternatively, the amount of one or more of the above-noted ingredients can be decreased if such ingredients comprise a metabolite that negatively modulates one or more of organic matter digestibility, dry matter digestibility, fiber digestibility, energy digestibility, fat digestibility, and protein digestibility.

[0081] As noted above, ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

EXAMPLES

[0082] The following non-limiting examples are illustrative of embodiments of the present disclosure.

Methods [0083] Each of the examples was derived from the following study.

[0084] 83 Canines were all fed Diet A for 5 weeks, followed by a 1 week transition period and then 15 were fed Diet B for 5 weeks as shown in the Table 1 below. Plasma samples were taken after overnight fasting using EDTA vacutainer tubes during the fifth week of feeding of each diet. After centrifugation, plasma was aliquoted into cryovials and f ozen at -80 °C.

Table 1

[0085] Metabolomic analysis was carried out using the following methods by Metabolon Inc. Samples were extracted and split into equal parts for analysis on GC/MS and LC/MS MS platforms. Proprietary software was used to match ions to an in-house library of standards for metabolite identification and for metabolite quantitation by peak area integration by Metabolon Inc. Mass and retention index are provided in the following tables such that each metabolite can be uniquely identified and individually distinguished.

[0086] At the time of analysis, samples were thawed and extracts prepared to remove protein, dislodge small molecules bound to protein or physically trapped in the precipitated protein matrix, and recover a wide range of chemically diverse metabolites. A separate aliquot of each experimental plasma sample was taken then pooled for the creation of "Client Matrix" (CMTRX) samples. These CMTRX samples were injected throughout the platform run and served as technical replicates, allowing variability in the quantitation of all consistently detected biochemicals to be determined and overall process variability and platform performance to be monitored. Extracts of all experimental and CMTRX samples were split for analysis on the GC/MS and LC/MS/MS platforms.

[0087] The CMTRX technical replicate samples were treated independently throughout the process as if they were client study samples. All process samples (CMTRX and Grab test mixtures of organic components used to assess GC column performance, process blanks, etc.) were spaced evenly among the injections for each day and all client samples were randomly distributed throughout each day's run. Data were collected over multiple platform run days and thus 'block normalized' by calculating the median values for each run-day block for each, individual compound. This normalization minimizes any inter-day instrument gain or drift, but does not interfere with intra-day sample variability. Missing values (if any) were assumed to be below the level of detection for that biochemical with the instrumentation used and were imputed with the observed minimum for that particular biochemical.

[0088] A number of internal standards were added to each experimental and process standard sample just prior to injection into the mass spectrometers. A measure of the platform variability (7%) was determined by calculating the median relative standard deviation (RSD) for these internal standards. Because these standards are added to the samples immediately prior to injection into the instrument, this value reflects instrument variation. In addition, the median relative standard deviation (RSD) for the biochemicals that were consistently measured in the CMTRX represents the total variability within the process for the actual experimental samples and the variability in quantitation of the endogenous metabolites within these samples (12%). Results for the CMTRX and internal standards indicated that the platform produced data that met process specifications.

[0089] 589 total metabolites were detected in plasma. This total corresponds to many biochemicals (401) that matched a named structure in the reference library (named compounds). The remaining biochemicals (188) represent distinct chemical entities (that is, they represent a single molecule of discrete molecular formula and structure), but they do not currently match a named biochemical in the reference library (unnamed/unknown compounds).

Example 1 (Organic Matter Digestibility ^" )

[0090] Metabolite correlations with organic matter digestibility were determined based on plasma metabolomics (Table 2). This provided a predictive model of compounds which can influence organic matter digestibility either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 3), and noting changes in organic matter digestibility (Table 4), served as a validation model. The metabolite compositions of the two different diets were determined to identify relative levels of specific compounds. Those validated by the model are shown in Table 5.

Table 2. Metabolite correlations with organic matter digestibility. Correlations with a P value < 0.01 are reported.

arachidonate (20:4n6) 0.5395 1.19E-07 5525 303.4

X - 18559 0.5292 2.28E-07 1864.9 254.1 dihomo-linoleate (20:2n6) 0.5242 3.10E-07 5722 307.3

X - 11949 0.5044 9.96E-07 3830 220.1 cysteine -0.4979 1.45E-06 1560.1 218 docosapentaenoate (n6 DPA; 22:5n6) 0.4971 1.51E-06 5625 329.4

N-palmitoyl taurine 0.4829 3.28E-06 5556.8 362.4

1-heptadecanoylglycerophosphoethanolamine -0.4779 4.28E-06 6029 466.3

X - 12339 0.4706 6.28E-06 1055 174.1

1-oleoylglycerophosphoethanolamine -0.4678 7.23E-06 5928 478.3

X - 11549 0.4657 8.05E-06 5093 339.3 betaine 0.4631 9.19E-06 721 118.2

2-aminobutyrate 0.4586 1.15E-05 758 104.1

X - 12435 0.457 1.25E-05 3174 357.2 pantothenate 0.4543 1.42E-05 2218 220.1

X - 15667 0.4526 1.54E-05 2330 202.2

X - 12216 0.4516 1.62E-05 1701 228.1

X - 13543 -0.4503 1.72E-05 1096 484.1

X - 16009 0.4435 2.39E-05 2191 222

X - 09789 -0.4428 2.46E-05 2613 153.1 arginine 0.44 2.82E-05 728 173.2

X - 18487 -0.4373 3.19E-05 1269.6 273.1 dihomo-linolenate (20:3n3 or n6) 0.4357 3.43E-05 5600 305.4

X - 11334 -0.4343 3.66E-05 982 259.1

3-hydroxybutyrate (BHBA) 0.4249 5.61E-05 1203.5 116.9 adrenate (22:4n6) 0.4225 6.25E-05 5684 331.3 docosapentaenoate (n3 DPA; 22:5n3) 0.4157 8.40E-05 5574 329.4 pipecolate 0.415 8.66E-05 1120 130.1

1-palmitoylplasmenylethanolamine -0.4149 8.71E-05 6153 436.4 eicosenoate (20:ln9 or 11) 0.4051 0.0001 5955 309.4 ribitol -0.4091 0.0001 1692.4 217

X - 11378 0.4111 0.0001 5325 445.4 linolenate [alpha or gamma; (18:3n3 or 6)] 0.3932 0.0002 5450 277.3 stachydrine -0.3927 0.0002 860 144.1 glycerate 0.3968 0.0002 1360.7 189 hydroquinone sulfate 0.3995 0.0002 1383 189 gamma-glutamylphenylalanine -0.3814 0.0003 2846 295.1 glycerol 0.3871 0.0003 1311 205

X - 16975 -0.3819 0.0003 2291.8 255.1

X - 12718 0.3879 0.0003 1750 324.1

X - 17185 -0.3835 0.0003 3069.1 215.2

2-oleoylglycerophosphoethanolamine -0.3761 0.0004 5848 478.3 threonate 0.3788 0.0004 1560.7 292.1 creatinine -0.3741 0.0005 730 114.1

X - 16015 -0.3728 0.0005 3788 268.1 arabitol -0.3609 0.0007 1687.5 307.1 creatine 0.362 0.0007 758 132.1

X - 12189 -0.3635 0.0007 1249 273.1

4-vinylphenol sulfate -0.3613 0.0007 3323 199.1 octanoylcarnitine 0.3604 0.0008 4100 288.3

X - 12822 -0.3504 0.0011 2786 389.1 trans-4-hydroxyproline -0.3484 0.0012 705 132.1

X - 17010 0.3473 0.0012 3169.5 189

2-linoleoylglycerophosphoethanolamine -0.3461 0.0013 5650 476.4 glycolate (hydroxyacetate) 0.3449 0.0013 1119 177

X - 18477 -0.3451 0.0013 1604.6 208.2

N-acetylornithine -0.3362 0.0018 875 175.2 beta-hydroxyisovalerate 0.3342 0.0019 1043 117.1

3 -methyl-2-oxobutyrate 0.3345 0.0019 1489 115.1

X - 12263 -0.3318 0.002 1369 275.1

X - 16869 -0.3309 0.0021 5179.8 330.2 campesterol -0.3283 0.0023 2353 343.4

2-hydroxyisobutyrate 0.3269 0.0024 1107.5 130.9

X - 18570 -0.3266 0.0024 3243.1 207 mannitol -0.3261 0.0025 1839 319.1

X - 11843 -0.3259 0.0025 2710 230.1

4-acetylphenol sulfate -0.3247 0.0026 2399 215

X - 18558 0.3198 0.003 1676.8 380.1 glutathione, oxidized (GSSG) -0.32 0.003 1535 613.1 phenylalanine -0.3188 0.0031 2056 166.1 oleoyltaurine 0.3159 0.0034 5599.9 388.3 arabonate -0.3117 0.0039 1736 292.1

X - 12450 0.3102 0.0041 5397 251.4 palmitoleate (16:1 n7) 0.3097 0.0041 5477 253.3 acetylcarnitine 0.3032 0.0051 1203 204.2 palmitate (16:0) 0.3027 0.0051 5619 255.3

X - 12660 -0.3002 0.0055 5667 442.2 erythritol -0.2978 0.0059 1517.5 217

1,5-anhydroglucitol (1,5-AG) -0.2973 0.006 1788.7 217 pseudouridine -0.2956 0.0063 1104 243.1 valine 0.2945 0.0065 1040 118.1 carnitine -0.2934 0.0068 702 162.2

X - 11437 -0.2927 0.0069 2888 231

1-linoleoylglycerophosphoethanolamine -0.2924 0.007 5725 476.3

X - 12156 -0.2893 0.0076 3238 233.1

X - 17305 -0.2886 0.0078 2157.1 296.2 linoleate (18:2n6) 0.287 0.0081 5533 279.3 stearate (18:0) 0.2845 0.0087 5886 283.4 gamma-glutamylmethionine -0.2839 0.0089 1993 279.2

X - 15375 -0.2816 0.0095 2502 328

Table 3. Compound levels present in diets A and B. Values represent scaled imputed values ( ^" data are scaled such that the median value measured across all samples was set to 1.0 and missing values ( ^" if any) were imputed with the observed minimum for that particular compound ^' ).

X - 18559 0.78 0.78 dihomo-linoleate (20:2n6) 0.33 4.06

X - 11949 0.28 0.28 cysteine 0.21 0.24 docosapentaenoate (n6 DP A; 22:5n6) 0.61 4.77

N-palmitoyl taurine 1 0.94

1-heptadecanoylglycerophosphoethanolamine 0.18 2.28

X - 12339 0.96 0.5

1-oleoylglycerophosphoethanolamine 0.16 5.65

X - 11549 0.13 0.13 betaine 0.61 0.69

2-aminobutyrate 1.58 0.83

X - 12435 0.36 0.36 pantothenate 12.83 19.07

X - 15667 0.16 0.16

X - 12216 0.25 0.25

X - 13543 0.1 0.1

X - 16009 0.2 0.2

X - 09789 5.54 0.2 arginine 1 0.63

X - 18487 0.26 0.26 dihomo-linolenate (20:3n3 or n6) 0.42 5.03

X - 11334 0.19 0.19

3-hydroxybutyrate (BHBA) 0.18 0.34 adrenate (22:4n6) 0.82 5.31 docosapentaenoate (n3 DPA; 22:5n3) 0.14 4.84 pipecolate 7.41 1.56

1-palmitoylplasmenylethanolamine 0.21 3.23 eicosenoate (20:ln9 or 11) 0.32 2.11 ribitol 4 7.04

X - 11378 0.05 0.11 linolenate [alpha or gamma; (18:3n3 or 6)] 0.83 1.66 stachydrine 3.62 0.78 glycerate 3.38 9.94 hydroquinone sulfate 0.03 0.03 gamma-glutamylphenylalanine 14.27 1.36 glycerol 2.61 1.85

X - 16975 1.02 0.44

X - 12718 0.42 0.42

X - 17185 0.12 0.06

2-oleoylglycerophosphoethanolamine 0.43 6.03 threonate 0.61 0.22 creatinine 13.3 27.38

X - 16015 0.02 0.02 arabitol 9.47 0.61 creatine 1.98 10.6

X - 12189 0.23 0.23

4-vinylphenol sulfate 0.02 0.02 octanoylcarnitine 0.41 0.53

X - 12822 0.2 0.2 trans-4-hydroxyproline 5.01 4.88

X - 17010 0.68 2.51

2-linoleoylglycerophosphoethanolamine 0.56 2.58 glycolate (hydroxyacetate) 0.89 0.94

X - 18477 0.25 0.25

N-acetylornithine 0.41 0.21 beta-hydroxyisovalerate 0.48 0.48

3 -methy 1-2-oxobutyrate 0.09 0.09

X - 12263 0.14 0.14

X - 16869 0.29 0.29 campesterol 1.56 0.72

2-hydroxyisobutyrate 0.51 0.51

X - 18570 0.08 0.08 mannitol 12.51 1.72

X - 11843 0.19 0.19

4-acetylphenol sulfate 0.4 0.56

X - 18558 0.55 0.47 glutathione, oxidized (GSSG) 0.35 0.16 phenylalanine 0.75 0.64 oleoyltaurine 0.55 1.08 arabonate 4.02 1.03

X - 12450 0.17 1.43 palmitoleate (16:ln7) 0.1 0.81 acetylcarnitine 0.1 0.29 palmitate (16:0) 0.4 0.88

X - 12660 0.63 2.97 erythritol 3.45 0.32

1,5-anhydroglucitol (1,5-AG) 0.28 0.28 pseudouridine 0.07 0.1 valine 0.73 0.58 carnitine 0.32 0.73

X - 11437 0.05 0.05

1 -linoleoylglycerophosphoethanolamine 0.48 2.77

X - 12156 0.19 0.19

X - 17305 0.42 0.42 linoleate (18:2n6) 0.8 0.92 stearate (18:0) 0.33 1.26 gamma-glutamylmethionine 0.47 0.44

X - 15375 0.41 0.97

Table 4. Organic matter digestibility in response to diet A and Diet B.

Table 5. Compounds validated by dietary change.

arachidonate (20:4n6) 5525 303.4 dihomo-linoleate (20:2n6) 5722 307.3 docosapentaenoate (n6 DP A; 22:5n6) 5625 329.4 pantothenate 2218 220.1

X - 09789 2613 153.1 dihomo-linolenate (20:3n3 or n6) 5600 305.4

3-hydroxybutyrate (BHBA) 1203.5 116.9 adrenate (22:4n6) 5684 331.3 docosapentaenoate (n3 DP A; 22:5n3) 5574 329.4 eicosenoate (20:ln9 or 11) 5955 309.4

X - 11378 5325 445.4 linolenate [alpha or gamma; (18:3n3 or 6)] 5450 277.3 stachydrine 860 144.1 glycerate 1360.7 189 gamma-glutamylphenylalanine 2846 295.1

X - 16975 2291.8 255.1

X - 17185 3069.1 215.2 arabitol 1687.5 307.1 creatine 758 132.1 octanoylcarnitine 4100 288.3 trans-4-hydroxyproline 705 132.1

X - 17010 3169.5 189 glycolate (hydroxyacetate) 1119 177

N-acetylornithine 875 175.2 campesterol 2353 343.4 glutathione, oxidized (GSSG) 1535 613.1 phenylalanine 2056 166.1 oleoyltaurine 5599.9 388.3 arabonate 1736 292.1

X - 12450 5397 251.4 palmitoleate (16:ln7) 5477 253.3 acetylcarnitine 1203 204.2 palmitate (16:0) 5619 255.3 erythritol 1517.5 217 linoleate (18:2n6) 5533 279.3 stearate (18:0) 5886 283.4

Example 2 ( ^" Dry Matter Digestibility)

[0091] Metabolite correlations with dry matter digestibility were determined based on plasma metabolomics (Table 6). This provided a predictive model of compounds which can influence dry matter digestibility either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 7), and noting changes in dry matter digestibility (Table 8), served as a validation model. The metabolite compositions of the two different diets were determined to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 9).

Table 6. Metabolite correlations with dry matter digestibility. Correlations with a P value < 0.01 are reported.

X - 16009 0.414 9.07E-05 2191 222

1-palmitoylplasmenylethanolamine -0.4127 9.58E-05 6153 436.4

X - 09789 -0.4077 0.0001 2613 153.1 ribitol -0.4052 0.0001 1692.4 217

X - 18487 -0.4088 0.0001 1269.6 273.1

X - 15667 0.4056 0.0001 2330 202.2 pantothenate 0.3916 0.0002 2218 220.1

X - 12339 0.4011 0.0002 1055 174.1

X - 12216 0.3973 0.0002 1701 228.1 docosapentaenoate (n3 DPA; 22:5n3) 0.3814 0.0003 5574 329.4

X - 16975 -0.3867 0.0003 2291.8 255.1

X - 12435 0.389 0.0003 3174 357.2

3-hydroxybutyrate (BHBA) 0.3858 0.0003 1203.5 116.9

X - 11378 0.3775 0.0004 5325 445.4 linolenate [alpha or gamma; (18:3n3 or 6)] 0.372 0.0005 5450 277.3 gamma-glutamylphenylalanine -0.3693 0.0005 2846 295.1

X - 12189 -0.3716 0.0005 1249 273.1

2-oleoylglycerophosphoethanolamine -0.3648 0.0006 5848 478.3 dihomo-linolenate (20:3n3 or n6) 0.3649 0.0006 5600 305.4 eicosenoate (20: ln9 or 11) 0.3674 0.0006 5955 309.4 arabitol -0.369 0.0006 1687.5 307.1 pipecolate 0.3661 0.0006 1120 130.1 adrenate (22:4n6) 0.366 0.0006 5684 . 331.3

X - 11334 -0.3681 0.0006 982 259.1 hydroquinone sulfate 0.368 0.0006 1383 189 creatinine -0.3576 0.0008 730 114.1 glycerol 0.3535 0.001 1311 205 threonate 0.3492 0.0011 1560.7 292.1

X - 17185 -0.3507 0.0011 3069.1 215.2 glycerate 0.3423 0.0014 1360.7 189 glutathione, oxidized (GSSG) -0.3433 0.0014 1535 613.1

X - 12718 0.3392 0.0016 1750 324.1 mannitol -0.3368 0.0017 1839 319.1 octanoylcarnitine 0.3264 0.0024 4100 288.3 2-linoleoylglycerophosphoethanolamine -0.3262 0.0025 5650 476.4

N-acetylornithine -0.3195 0.003 875 175.2

X - 17010 0.3172 0.0033 3169.5 189

X - 12822 -0.3166 0.0033 2786 389.1

X - 16015 -0.3172 0.0033 3788 268.1 glycolate (hydroxyacetate) 0.3124 0.0038 1119 177 stachydrine -0.3121 0.0039 860 144.1 campesterol -0.3101 0.0041 2353 343.4

X - 12263 -0.3104 0.0041 1369 275.1

3 -methyl-2-oxobutyrate 0.3102 0.0041 1489 115.1 palmitoleate (16:1 n7) 0.3089 0.0043 5477 253.3 creatine 0.3073 0.0045 758 132.1

4-acetylphenol sulfate -0.3061 0.0046 2399 215

X - 12450 0.3038 0.005 5397 251.4 oleoyltaurine 0.3016 0.0053 5599.9 388.3

4-vinylphenol sulfate -0.3015 0.0053 3323 199.1 arabonate -0.3013 0.0054 1736 292.1 trans-4-hydroxyproline -0.3001 0.0055 705 132.1

X - 16869 -0.2996 0.0056 5179.8 330.2 beta-hydroxyisovalerate 0.2994 0.0057 1043 117.1

2-hydroxyisobutyrate 0.2975 0.006 1107.5 130.9 gamma-glutamylmethionine -0.2929 0.0068 1993 279.2 phenylalanine -0.2926 0.0069 2056 166.1

X - 12660 -0.2908 0.0073 5667 442.2 erythritol -0.2879 0.0079 1517.5 217 palmitate (16:0) 0.2862 0.0083 5619 255.3

X - 18558 0.2846 0.0087 1676.8 380.1

X - 11843 -0.2842 0.0088 2710 230.1

X - 18477 -0.2817 0.0094 1604.6 208.2

Table 7. Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values ("if any ^" ) were imputed with the observed minimum for that particular compound ^' ). ID A B mannose 0.21 1.45 cysteine-glutathione disulfide 0.04 0.04 trigonelline (N'-methylnicotinate) 12.47 1.65

X - 11787 0.04 0.06

X - 12672 0.72 0.72 cysteine 0.21 0.24

X - 18559 0.78 0.78 arachidonate (20:4n6) 0.43 4.48 dihomo-linoleate (20:2n6) 0.33 4.06

X - 11949 0.28 0.28

N-palmitoyl taurine 1 0.94

1-heptadecanoylglycerophosphoethanolamine 0.18 2.28

X - 13543 0.1 0.1

2-aminobutyrate 1.58 0.83

1-oleoylglycerophosphoethanolamine 0.16 5.65 docosapentaenoate (n6 DPA; 22:5n6) 0.61 4.77 betaine 0.61 0.69

X - 11549 0.13 0.13 arginine 1 0.63

X - 16009 0.2 0.2

1-palmitoylplasmenylethanolamine 0.21 3.23

X - 09789 5.54 0.2 ribitol 4 7.04

X - 18487 0.26 0.26

X - 15667 0.16 0.16 pantothenate 12.83 19.07

X - 12339 0.96 0.5

X - 12216 0.25 0.25 docosapentaenoate (n3 DPA; 22:5n3) 0.14 4.84

X - 16975 1.02 0.44

X - 12435 0.36 0.36

3-hydroxybutyrate (BHBA) 0.18 0.34 X - 11378 0.05 0.11 linolenate [alpha or gamma; (18:3n3 or 6)] 0.83 1.66 gamma-glutamylphenylalanine 14.27 1.36

X - 12189 0.23 0.23

2-oleoylglycerophosphoethanolamine 0.43 6.03 dihomo-linolenate (20:3n3 or n6) 0.42 5.03 eicosenoate (20:ln9 or 11) 0.32 2.11 arabitol 9.47 0.61 pipecolate 7.41 1.56 adrenate (22:4n6) 0.82 5.31

X - 11334 0.19 0.19 hydroquinone sulfate 0.03 0.03 creatinine 13.3 27.38 glycerol 2.61 1.85 threonate 0.61 0.22

X - 17185 0.12 0.06 glycerate 3.38 9.94 glutathione, oxidized (GSSG) 0.35 0.16

X - 12718 0.42 0.42 mannitol 12.51 1.72 o ctanoylcarnitine 0.41 0.53

2-linoleoy]glycerophosphoethanolamine 0.56 2.58

N-acetylornithine 0.41 0.21

X - 17010 0.68 2.51

X - 12822 0.2 0.2

X - 16015 0.02 0.02 glycolate (hydroxyacetate) 0.89 0.94 stachydrine 3.62 0.78 campesterol 1.56 0.72

X - 12263 0.14 0.14

3 -methyl-2-oxobutyrate 0.09 0.09 palmitoleate (16: ln7) 0.1 0.81 creatine 1.98 10.6

4-acetylphenol sulfate 0.4 0.56 X - 12450 0.17 1.43 oleoyltaurine 0.55 1.08

4-vinylphenol sulfate 0.02 0.02 arabonate 4.02 1.03 trans-4-hydroxyproline 5.01 4.88

X - 16869 0.29 0.29 beta-hydroxyisovalerate 0.48 0.48

2-hydroxyisobutyrate 0.51 0.51 gamma-glutamylmethionine 0.47 0.44 phenylalanine 0.75 0.64

X - 12660 0.63 2.97 erythritol 3.45 0.32 palmitate (16:0) 0.4 0.88

X - 18558 0.55 0.47

X - 11843 0.19 0.19

X - 18477 0.25 0.25

Table 8. Dry matter digestibility in response to diet A and Diet B.

Table 9. Compounds validated by dietary change.

pantothenate 2218 220.1 docosapentaenoate (n3 DP A; 22:5n3) 5574 329.4

X - 16975 2291.8 255.1

3-hydroxybutyrate (BHBA) 1203.5 116.9

X - 11378 5325 445.4 linolenate [alpha or gamma; (18:3n3 or 6)] 5450 277.3 gamma-glutamylphenylalanine 2846 295.1 dihomo-linolenate (20:3n3 or n6) 5600 305.4 eicosenoate (20:ln9 or 11) 5955 309.4

Arabitol 1687.5 307.1 adrenate (22:4n6) 5684 331.3

X - 17185 3069.1 215.2

Glycerate 1360.7 189 glutathione, oxidized (GSSG) 1535 613.1

Mannitol 1839 319.1 octanoylcarnitine 4100 288.3

N-acetylornithine 875 175.2

X - 17010 3169.5 189 glycolate (hydroxyacetate) 1119 177 stachydrine 860 144.1 campesterol 2353 343.4 palmitoleate (16:ln7) 5477 253.3

Creatine 758 132.1

X - 12450 5397 251.4 oleoyltaurine 5599.9 388.3 arabonate 1736 292.1 trans-4-hydroxyproline 705 132.1 phenylalanine 2056 166.1 erythritol 1517.5 217 palmitate (16:0) 5619 255.3

Example 3 (Fiber Digestibility ^" )

[0092] Metabolite correlations with fiber digestibility were determined based on plasma metabolomics

(Table 10). This provided a predictive model of compounds which can influence fiber digestibility either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 11), and noting changes in fiber digestibility (Table 12), served as a validation model. The metabolite compositions of the two different diets were determined in order to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 13).

Table 10. Metabolite correlations with fiber digestibility. Correlations with a P value < 0.01 are reported.

Table 11. Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any) were imputed with the observed minimum for that particular compound).

Table 12. Fiber digestibility in response to diet A and Diet B.

Table 13. Compounds validated by dietary change.

ID Retention Mass

Index

1-heptadecanoylglycerophosphoethanolamine 6029 466.3

1-palmitoylplasmenylethanolamine 6153 436.4 1-palmitoylglycerol (1-monopalmitin) 2119.5 371.3 alpha-tocopherol 2305.4 502.5 ribitol 1692.4 217

Example 4 ( ^" Energy Digestibility ^' )

[0093] Metabolite correlations with energy digestibility were determined based on plasma metabolomics (Table 14). This provided a predictive model of compounds which can influence energy digestibility either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 15), and noting changes in energy digestibility (Table 16), served as a validation model. The metabolite compositions of the two different diets were determined in order to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 17.

Table 14. Metabolite correlations with energy digestibility. Correlations with a P value < 0.01 are reported.

pantothenate 0.4867 2.68E-06 2218 220.1

X - 13543 -0.4823 3.40E-06 1096 484.1

X - 12216 0.4786 4.13E-06 1701 228.1

1-heptadecanoylglycerophosphoethanolamine -0.4775 4.38E-06 6029 466.3 adrenate (22:4n6) 0.4681 7.11E-06 5684 331.3 dihomo-linolenate (20:3n3 or n6) 0.4667 7.66E-06 5600 305.4

X - 16009 0.4636 8.93E-06 2191 222

X - 11334 -0.4634 9.05E-06 982 259.1

X - 15667 0.4627 9.38E-06 2330 202.2

X - 11378 0.4583 1.17E-05 5325 445.4 glycerate 0.454 1.44E-05 1360.7 189

3-hydroxybutyrate (BHBA) 0.451 1.67E-05 1203.5 116.9

X - 18487 -0.4502 1.74E-05 1269.6 273.1 pipecolate 0.4445 2.27E-05 1120 130.1 docosapentaenoate (n3 DPA; 22:5n3) 0.4433 2.41E-05 5574 329.4 hydroquinone sulfate 0.4416 2.61E-05 1383 189 stachydrine -0.4397 2.86E-05 860 144.1 arginine 0.4393 2.91E-05 728 173.2

X - 16015 -0.4365 3.31E-05 3788 268.1

4-vinylphenol sulfate -0.4272 5.06E-05 3323 199.1

X - 17185 -0.42 6.96E-05 3069.1 215.2 eicosenoate (20:ln9 or 11) 0.4199 7.01E-05 5955 309.4 ribitol -0.4161 8.27E-05 1692.4 217

1-palmitoylplasmenylethanolamine -0.4157 8.41E-05 6153 436.4

2-oleoylglycerophosphoethanolamine -0.4071 0.0001 5848 478.3

Iinolenate [alpha or gamma; (18:3n3 or 6)] 0.4105 0.0001 5450 277.3 gamma-glutamylphenylalanine -0.4113 0.0001 2846 295.1 glycerol 0.4112 0.0001 1311 205

X - 16975 -0.3965 0.0002 2291.8 255.1 threonate 0.3996 0.0002 1560.7 292.1 creatinine -0.3852 0.0003 730 114.1 creatine 0.3824 0.0003 758 132.1 campesterol -0.385 0.0003 2353 343.4 glycolate (hydroxyacetate) 0.3893 0.0003 1119 177 X - 17010 0.3842 0.0003 3169.5 189

X - 12718 0.3812 0.0003 1750 324.1 octanoylcarnitine 0.3805 0.0004 4100 288.3

N-acetylornithine -0.3771 0.0004 875 175.2

X - 18570 -0.3808 0.0004 3243.1 207

4-acetylphenol sulfate -0.3708 0.0005 2399 215

X - 11843 -0.3744 0.0005 2710 230.1

2-linoleoylglycerophosphoethanolamine -0.3659 0.0006 5650 476.4 trans-4-hydroxyproline -0.3639 0.0007 705 132.1

3 -methyl-2-oxobutyrate 0.3621 0.0007 1489 115.1 phenylalanine -0.3584 0.0008 2056 166.1

2-hydroxyisobutyrate 0.3579 0.0008 1107.5 130.9

X - 12822 -0.3582 0.0008 2786 389.1 glutathione, oxidized (GSSG) -0.3543 0.0009 1535 613.1

X - 18477 -0.3551 0.0009 1604.6 208.2 arabitol -0.353 0.001 1687.5 307.1

X - 12189 -0.3502 0.0011 1249 273.1

X - 18558 0.3477 0.0012 1676.8 380.1 beta-hydroxyisovalerate 0.3473 0.0012 1043 117.1

X - 16869 -0.3476 0.0012 5179.8 330.2

X - 11437 -0.3461 0.0013 2888 231 mannitol -0.342 0.0015 1839 319.1

X - 12156 -0.3204 0.003 3238 233.1 acetylcarnitine 0.3187 0.0031 1203 204.2 erythritol -0.3193 0.0031 1517.5 217 oleoyltaurine 0.3178 0.0032 5599.9 388.3

X - 12450 0.3177 0.0032 5397 251.4

1,5-anhydroglucitol (1,5-AG) -0.3163 0.0034 1788.7 217

1-linoleoylglycerophosphoethanolamine -0.3152 0.0035 5725 476.3 palmitoleate (16:ln7) 0.315 0.0035 5477 253.3 gamma-glutamylmethionine -0.3131 0.0037 1993 279.2

X - 12263 -0.312 0.0039 1369 275.1 arabonate -0.3093 0.0042 1736 292.1 valine 0.3084 0.0043 1040 118.1 X - 12660 -0.3077 0.0044 5667 442.2 pseudouridine -0.3079 0.0044 1104 243.1 palmitate (16:0) 0.3061 0.0046 5619 255.3

X - 15375 -0.3036 0.005 2502 328 carnitine -0.3014 0.0053 702 162.2

X - 15636 -0.2941 0.0066 3814 243.1

3-methyl-2-oxovalerate 0.2933 0.0068 2106 129.2 catechol sulfate -0.292 0.007 1928 188.9 docosahexaenoate (DHA; 22:6n3) 0.291 0.0072 5518 327.3 linoleate (18:2n6) 0.291 0.0073 5533 279.3

2-arachidonoylglycerophosphoethanolamine -0.2879 0.0079 5674 500.3 stearate (18:0) 0.2879 0.0079 5886 283.4

X - 17305 -0.2874 0.008 2157.1 296.2

1-heptadecanoylglycerophosphocholine -0.2854 0.0085 5750 510.4 gamma-glutamylleucine -0.2827 0.0092 2744 261.2

Table 15. Compound levels present in diets A and B. Values represent scaled imputed values fdata are scaled such that the median value measured across all samples was set to 1.0 and missing values fif any) were imputed with the observed minimum for that particular compound).

X - 12435 0.36 0.36

N-palmitoyl taurine 1 0.94 betaine 0.61 0.69

1-oleoylglycerophosphoethanolamine 0.16 5.65

2-aminobutyrate 1.58 0.83

X - 09789 5.54 0.2 pantothenate 12.83 19.07

X - 13543 0.1 0.1

X - 12216 0.25 0.25

1-heptadecanoylglycerophosphoethanolamine 0.18 2.28 adrenate (22:4n6) 0.82 5.31 dihomo-linolenate (20:3n3 or n6) 0.42 5.03

X - 16009 0.2 0.2

X - 11334 0.19 0.19

X - 15667 0.16 0.16

X - 11378 0.05 0.11 glycerate 3.38 9.94

3-hydroxybutyrate (BHBA) 0.18 0.34

X - 18487 0.26 0.26 pipecolate 7.41 1.56 docosapentaenoate (n3 DPA; 22:5n3) 0.14 4.84 hydroquinone sulfate 0.03 0.03 stachydrine 3.62 0.78 arginine 1 0.63

X - 16015 0.02 0.02

4-vinylphenol sulfate 0.02 0.02

X - 17185 0.12 0.06 eicosenoate (20:ln9 or 11) 0.32 2.11 ribitol 4 7.04

1-palmitoylplasmenylethanolamine 0.21 3.23

2-oleoylglycerophosphoethanolamine 0.43 6.03 linolenate [alpha or gamma; (18:3n3 or 6)] 0.83 1.66 gamma-glutamylphenylalanine 14.27 1.36 glycerol 2.61 1.85 X - 16975 1.02 0.44 threonate 0.61 0.22 creatinine 13.3 27.38 creatine 1.98 10.6 campesterol 1.56 0.72 glycolate (hydroxyacetate) 0.89 0.94

X - 17010 0.68 2.51

X - 12718 0.42 0.42 octanoylcarnitine 0.41 0.53

N-acetylornithine 0.41 0.21

X - 18570 0.08 0.08

4-acetylphenol sulfate 0.4 0.56

X - 11843 0.19 0.19

2-linoleoylglycerophosphoethanolamine 0.56 2.58 trans-4-hydroxyproline 5.01 4.88

3 -methyl-2-oxobutyrate 0.09 0.09 phenylalanine 0.75 0.64

2-hydroxyisobutyrate 0.51 0.51

X - 12822 0.2 0.2 glutathione, oxidized (GSSG) 0.35 0.16

X - 18477 0.25 0.25 arabitol 9.47 0.61

X - 12189 0.23 0.23

X - 18558 0.55 0.47 beta-hydroxyisovalerate 0.48 0.48

X - 16869 0.29 0.29

X - 11437 0.05 0.05 mannitol 12.51 1.72

X - 12156 0.19 0.19 acetylcarnitine 0.1 0.29 erythritol 3.45 0.32 oleoyltaurine 0.55 1.08

X - 12450 0.17 1.43

1,5-anhydroglucitol (1,5-AG) 0.28 0.28 1-linoleoylglycerophosphoethanolamine 0.48 2.77 palmitoleate (16:ln7) 0.1 0.81 gamma-glutamylmethionine 0.47 0.44

X - 12263 0.14 0.14 arabonate 4.02 1.03 valine 0.73 0.58

X - 12660 0.63 2.97 pseudouridine 0.07 0.1 palmitate (16:0) 0.4 0.88

X - 15375 0.41 0.97 carnitine 0.32 0.73

X - 15636 0.2 0.2

3 -methyl-2-oxovalerate 0.07 0.07 catechol sulfate 0.02 0.02 docosahexaenoate (DHA; 22:6n3) 0.24 3.9 linoleate (18:2n6) 0.8 0.92

2-arachidonoylglycerophosphoethanolamine 0.33 10.33 stearate (18:0) 0.33 1.26

X - 17305 0.42 0.42

1-heptadecanoylglycerophosphocholine 0.13 0.24 gamma-glutamylleucine 0.55 0.69

Table 16. Energy digestibility in response to diet A and Diet B.

Table 17. Compounds validated by dietary change.

dihomo-linoleate (20:2n6) 5722 307.3 docosapentaenoate (n6 DP A; 22:5n6) 5625 329.4

N-palmitoyl taurine 5556.8 362.4

Betaine 721 118.2

X - 09789 2613 153.1

Pantothenate 2218 220.1 adrenate (22:4n6) 5684 331.3 dihomo-linolenate (20:3n3 or n6) 5600 305.4

X - 11378 5325 445.4

Glycerate 1360.7 189

3-hydroxybutyrate (BHBA) 1203.5 116.9 docosapentaenoate (n3 DP A; 22:5n3) 5574 329.4

Stachydrine 860 144.1

X - 17185 3069.1 215.2 eicosenoate (20:ln9 or 11) 5955 309.4 linolenate [alpha or gamma; (18:3n3 or 6)] 5450 277.3 gamma-glutamylphenylalanine 2846 295.1

X - 16975 2291.8 255.1

Creatine 758 132.1

Campesterol 2353 343.4 glycolate (hydroxyacetate) 1119 177

X - 17010 3169.5 189

Octanoylcarnitine 4100 288.3

N-acetylornithine 875 175.2 trans-4-hydroxyproline 705 132.1

Phenylalanine 2056 166.1 glutathione, oxidized (GSSG) 1535 613.1

Arabitol 1687.5 307.1

Mannitol 1839 319.1

Acetylcarnitine 1203 204.2

Erythritol 1517.5 217

Oleoyltaurine 5599.9 388.3

X - 12450 5397 251.4 palmitoleate (16:ln7) 5477 253.3 Arabonate 1736 292.1 palmitate (16:0) 5619 255.3 docosahexaenoate (DHA; 22:6n3) 5518 327.3 linoleate (18:2n6) 5533 279.3 stearate (18:0) 5886 283.4

Example 5 (Tat Digestibility)

[0094] Metabolite correlations with fat digestibility were determined based on plasma metabolomics (Table 18). This provided a predictive model of compounds which can influence fat digestibility either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 19), and noting changes in fat digestibility (Table 20), served as a validation model. The metabolite compositions of the two different diets were determined in order to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 21.

Table 18. Metabolite correlations with fat digestibility. Correlations with a P value < 0.01 are reported.

2-oleoylglycerophosphoethanolamine -0.509 7.67E-07 5848 478.3

X - 11378 0.5085 7.87E-07 5325 445.4

X - 12672 0.5083 7.96E-07 1797 256 dihomo-linoleate (20:2n6) 0.5061 9.05E-07 5722 307.3 glycerate 0.5001 1.28E-06 1360.7 189

2-aminobutyrate 0.4973 1.49E-06 758 104.1

X - 12435 0.497 1.51E-06 3174 357.2 campesterol -0.4893 2.32E-06 2353 343.4

4-vinylphenol sulfate -0.4763 4.66E-06 3323 199.1 docosapentaenoate (n6 DPA; 22:5n6) 0.4751 4.96E-06 5625 329.4 glycolate (hydroxyacetate) 0.4628 9.33E-06 1119 177

X - 12216 0.4538 1.46E-05 1701 228.1

4-acetylphenol sulfate -0.4507 1.69E-05 2399 215

1-heptadecanoylglycerophosphoethanolamine -0.4444 2.29E-05 6029 466.3 pantothenate 0.444 2.33E-05 2218 220.1 adrenate (22:4n6) 0.4431 2.43E-05 5684 331.3

X - 17185 -0.4379 3.10E-05 3069.1 215.2 glycerol 0.4301 4.45E-05 1311 205 hydroquinone sulfate 0.4295 4.56E-05 1383 189 ribitol -0.4293 4.60E-05 1692.4 217 catechol sulfate -0.4282 4.84E-05 1928 188.9

N-acetylornithine -0.4272 5.05E-05 875 175.2 gamma-glutamylphenylalanine -0.425 5.58E-05 2846 295.1

X - 11334 -0.4244 5.74E-05 982 259.1

1-palmitoylplasmenylethanolamine -0.4236 5.94E-05 6153 436.4

X - 16009 0.4223 6.30E-05 2191 222

X - 11843 -0.4206 6.78E-05 2710 230.1 pipecolate 0.4204 6.86E-05 1120 130.1

N-palmitoyl taurine 0.4199 6.99E-05 5556.8 362.4 octanoylcarnitine 0.4185 7.45E-05 4100 288.3 indolepropionate -0.4174 7.81E-05 1925 202.1 stachydrine -0.4162 8.22E-05 860 144.1 dihomo-linolenate (20:3n3 or n6) 0.4152 8.58E-05 5600 305.4 docosapentaenoate (n3 DPA; 22:5n3) 0.4142 8.97E-05 5574 329.4 X - 15667 0.4141 9.00E-05 2330 202.2

X - 18570 -0.4041 0.0001 3243.1 207

3-hydroxybutyrate (BHBA) 0.4116 0.0001 1203.5 116.9

X - 18487 -0.4023 0.0001 1269.6 273.1 gamma-glutamylmethionine -0.4042 0.0001 1993 279.2 equol sulfate -0.3941 0.0002 3625 321.2

X - 12236 -0.397 0.0002 1321 245.1 threonate 0.3942 0.0002 1560.7 292.1

X - 17010 0.402 0.0002 3169.5 189

X - 16975 -0.402 0.0002 2291.8 255.1

3-(3-hydroxyphenyl)propionate -0.3861 0.0003 2000 165.1

X - 15636 -0.3892 0.0003 3814 243.1

15-methylpalmitate (isobar with 2-methylpalmitate) -0.3877 0.0003 5698 269.4

X - 11437 -0.375 0.0004 2888 231 indoleacrylate -0.3805 0.0004 2529 186.1 mannitol -0.3716 0.0005 1839 319.1 citrulline -0.369 0.0006 715 176.1

2-linoleoylglycerophosphoethanolamine -0.366 0.0006 5650 476.4 linolenate [alpha or gamma; (18:3n3 or 6)] 0.368 0.0006 5450 277.3 phenylalanine -0.3626 0.0007 2056 166.1 erythritol -0.3633 0.0007 1517.5 217 creatinine -0.3643 0.0007 730 114.1

3 -methyl-2-oxobutyrate 0.3593 0.0008 1489 115.1

1-linoleoylglycerophosphoethanolamine -0.3602 0.0008 5725 476.3

X - 13866 -0.3549 0.0009 2406 253.1

3-methoxytyrosine -0.3536 0.001 1765 212.1 arabitol -0.3518 0.001 1687.5 307.1 eicosenoate (20:ln9 or 11) 0.3495 0.0011 5955 309.4

X - 12212 -0.3481 0.0012 3607 229.1

X - 18558 0.3478 0.0012 1676.8 380.1 phenylalanyltryptophan -0.3443 0.0013 3349 352.2

2-hydroxyisobutyrate 0.3444 0.0013 1107.5 130.9

X - 02249 0.3403 0.0015 4025 267.2 cholate -0.3387 0.0016 5148 407.4 beta-hydroxyisovalerate 0.3381 0.0017 1043 117.1 arginine 0.3376 0.0017 728 173.2 gamma-glutamylleucine -0.3351 0.0018 2744 261.2 garnma-glutamylalanine -0.335 0.0018 1108 219

X - 12189 -0.3335 0.0019 1249 273.1

X - 12156 -0.3327 0.002 3238 233.1

2-hydroxybutyrate (AHB) 0.3332 0.002 1169.4 130.9 creatine 0.333 0.002 758 132.1

3-dehydrocholate -0.3255 0.0025 5050 405.4 docosahexaenoate (DHA; 22:6n3) 0.3239 0.0026 5518 327.3 carnosine -0.325 0.0026 813 225.2 trans-4-hydroxyproline -0.3229 0.0027 705 132.1

X - 17443 -0.3211 0.0029 3102.3 271.2 deoxycholate -0.3174 0.0033 5268 391.4

7-ketodeoxycholate -0.3175 0.0033 4840 405.4

12-dehydrocholate -0.3145 0.0036 4866 405.4

3-methyl-2-oxovalerate 0.3091 0.0042 2106 129.2 chenodeoxycholate -0.3083 0.0043 5250 391.4

X - 12660 -0.3063 0.0046 5667 442.2

X - 16869 -0.3045 0.0049 5179.8 330.2

1 -oleoylglycerophosphocholine -0.3039 0.0049 5700 522.4 pseudouridine -0.3028 0.0051 1104 243.1

1-arachidonoylglycerophosphoethanolamine -0.3026 0.0051 5731 500.3 acetylcarnitine 0.3016 0.0053 1203 204.2

2-arachidonoylglycerophosphoethanolamine -0.2997 0.0056 5674 500.3

1-heptadecanoylglycerophosphocholine -0.2983 0.0058 5750 510.4

1-myristoylglycerophosphocholine -0.2973 0.006 5481 468.3

2-myristoylglycerophosphocholine -0.2935 0.0067 5450 468.3 palmitoleate (16: ln7) 0.2916 0.0071 5477 253.3

X - 12822 -0.2903 0.0074 2786 389.1 methionine -0.2904 0.0074 1252 150.1

X - 15375 -0.2836 0.0089 2502 328 valine 0.283 0.0091 1040 118.1 pentadecanoate (15:0) -0.281 0.0096 1853.5 299.2 phosphate -0.2812 0.0096 1307.7 298.9

Table 19. Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values fif any) were imputed with the observed minimum for that particular compound).

ID A B

cysteine-glutathione disulfide 0.04 0.04

X - 11787 0.04 0.06

X - 13543 0.1 0.1

trigonelline (N'-methylnicotinate) 12.47 1.65

X - 09789 5.54 0.2

arachidonate (20:4n6) 0.43 4.48

1-oleoylglycerophosphoethanolamine 0.16 5.65 mannose 0.21 1.45

X - 18559 0.78 0.78

X - 11549 0.13 0.13 betaine 0.61 0.69

X - 11949 0.28 0.28

X - 16015 0.02 0.02 cysteine 0.21 0.24 glutathione, oxidized (GSSG) 0.35 0.16

X - 12339 0.96 0.5

2-oleoylglycerophosphoethanolamin 0.43 6.03

X - 11378 0.05 0.11

X - 12672 0.72 0.72 dihomo-linoleate (20:2n6) 0.33 4.06 glycerate 3.38 9.94

2-aminobutyrate 1.58 0.83

X - 12435 0.36 0.36 campesterol 1.56 0.72

4-vinylphenol sulfate 0.02 0.02

docosapentaenoate (n6 DP A; 22:5n6) 0.61 4.77 glycolate (hydroxyacetate) 0.89 0.94

X - 12216 0.25 0.25

4-acetylphenol sulfate 0.4 0.56

1-heptadecanoylglycerophosphoethanolamine 0.18 2.28 pantothenate 12.83 19.07 adrenate (22:4n6) 0.82 5.31

X - 17185 0.12 0.06 glycerol 2.61 1.85 hydroquinone sulfate 0.03 0.03 ribitol 4 7.04 catechol sulfate 0.02 0.02

N-acetylornithine 0.41 0.21 gamma-glutamylphenylalanine 14.27 1.36

X - 11334 0.19 0.19

1-palmitoylplasmenylethanolamine 0.21 3.23

X - 16009 0.2 0.2

X - 11843 0.19 0.19 pipecolate 7.41 1.56

N-palmitoyl taurine 1 0.94 octanoylcarnitine 0.41 0.53 indolepropionate 0.07 0.07 stachydrine 3.62 0.78 dihomo-linolenate (20:3n3 or n6) 0.42 5.03 docosapentaenoate (n3 DPA; 22:5n3) 0.14 4.84

X - 15667 0.16 0.16

X - 18570 0.08 0.08

3-hydroxybutyrate (BHBA) 0.18 0.34

X - 18487 0.26 0.26 gamma-glutamylmethionine 0.47 0.44 equol sulfate 0.08 0.08

X - 12236 0.1 0.1 threonate 0.61 0.22

X - 17010 0.68 2.51

X - 16975 1.02 0.44 3-(3-hydroxyphenyl)propionate 0.1 0.1

X - 15636 0.2 0.2

15-methylpalmitate (isobar with 2- 0.37 0.19 methylpalmitate)

X - 11437 0.05 0.05 indoleacrylate 0.06 0.06 mannitol 12.51 1.72 citrulline 2.35 0.38

2-linoleoylglycerophosphoethanolamine 0.56 2.58 linolenate [alpha or gamma; (18:3n3 or 6)] 0.83 1.66 phenylalanine 0.75 0.64 erythritol 3.45 0.32 creatinine 13.3 27.38

3 -methyl-2-oxobutyrate 0.09 0.09

1-linoleoylglycerophosphoethanolamine 0.48 2.77

X - 13866 0.26 0.26

3-methoxytyrosine 0.48 0.48 arabitol 9.47 0.61 eicosenoate (20:ln9 or 11) 0.32 2.11

X - 12212 0.21 0.21

X - 18558 0.55 0.47 phenylalanyltryptophan 0.29 0.29

2-hydroxyisobutyrate 0.51 0.51

X - 02249 0.48 0.48 cholate 25.83 0.83 beta-hydroxyisovalerate 0.48 0.48 arginine 1 0.63 gamma-glutamylleucine 0.55 0.69 gamma-glutamylalanine 1.56 1.06

X - 12189 0.23 0.23

X - 12156 0.19 0.19

2-hydroxybutyrate (AHB) 0.28 0.1 creatine 1.98 10.6

3-dehydrocholate 0.8 0.39 docosahexaenoate (DHA; 22:6n3) 0.24 3.9 camosine 0.38 1.28 trans-4-hydroxyproline 5.01 4.88

X - 17443 0.21 0.21 deoxycholate 0.19 0.19

7-ketodeoxycholate 2.65 0.38

12-dehydro cholate 0.22 0.17

3 -methyl-2-oxo valerate 0.07 0.07 chenodeoxycholate 7.78 0.79

X - 12660 0.63 2.97

X - 16869 0.29 0.29

1 -oleoylglycerophosphocholine 0.12 0.72 pseudouridine 0.07 0.1

1-arachidonoylglycerophosphoethanolamine 0.14 4.6 acetylcarnitine 0.1 0.29

2-arachidonoylglycerophosphoethanolamine 0.33 10.33

1-heptadecanoylglycerophosphocholine 0.13 0.24

1 -myristoylglycerophosphocholine 0.22 3.8

2-myristoylglycerophosphocholine 0.21 3.63 palmitoleate (16: ln7) 0.1 0.81

X - 12822 0.2 0.2 methionine 0.55 0.51

X - 15375 0.41 0.97 valine 0.73 0.58 pentadecanoate (15:0) 0.34 0.7 phosphate 0.76 0.84

Table 20. Fat digestibility in response to diet A and Diet B.

Table 21. Compounds validated by dietary change. ID Retention Mass

Index trigonelline ( '-methylnicotinate) 757 138.1

X - 09789 2613 153.1 arachidonate (20:4n6) 5525 303.4

Mannose 1753.5 203.9

Betaine 721 118.2 glutathione, oxidized (GSSG) 1535 613.1

X - 11378 5325 445.4 dihomo-linoleate (20:2n6) 5722 307.3

Glycerate 1360.7 189

Campesterol 2353 343.4 docosapentaenoate (n6 DPA; 22:5n6) 5625 329.4 glycolate (hydroxyacetate) 1119 177

Pantothenate 2218 220.1 adrenate (22:4n6) 5684 331.3

X - 17185 3069.1 215.2

N-acetylornithine 875 175.2 gamma-glutamylphenylalanine 2846 295.1

Octanoylcarnitine 4100 288.3

Stachydrine 860 144.1 dihomo-linolenate (20:3n3 or n6) 5600 305.4 docosapentaenoate (n3 DPA; 22:5n3) 5574 329.4

3-hydroxybutyrate (BHBA) 1203.5 116.9

X - 17010 3169.5 189

X - 16975 2291.8 255.1

15-methylpalmitate (isobar with 2-methylpalmitate) 5698 269.4

Mannitol 1839 319.1

Citrulline 715 176.1 linolenate [alpha or gamma; (18:3n3 or 6)] 5450 277.3

Phenylalanine 2056 166.1

Erythritol 1517.5 217

Arabitol 1687.5 307.1 eicosenoate (20:ln9 or 11) 5955 309.4 Cholate 5148 407.4 gamma-glutamylalanine 1108 219

Creatine 758 132.1

3-dehydrochoIate 5050 405.4 docosahexaenoate (DHA; 22:6n3) 5518 327.3 trans-4-hydroxyproline 705 132.1

7-ketodeoxycholate 4840 405.4

12-dehydrocholate 4866 405.4

Chenodeoxycholate 5250 391.4

Acetylcarnitine 1203 204.2 palmitoleate (16:ln7) 5477 253.3

Example 6 (Protein Digestibility^

[0095] Metabolite correlations with protein digestibility were determined based on plasma metabolomics (Table 22). This provided a predictive model of compounds which can influence protein digestibility either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 23), and noting changes in protein digestibility (Table 24), served as a validation model. The metabolite compositions of the two different diets were determined in order to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 25).

Table 22. Metabolite correlations with protein digestibility. Correlations with a P value < 0.01 are reported.

1-oleoylglycerophosphoethanolamine -0.4633 9.07E-06 5928 478.3

X - 11378 0.4626 9.43E-06 5325 445.4

X - 18487 -0.4606 1.04E-05 1269.6 273.1

X - 11949 0.4566 1.27E-05 3830 220.1 trans-4-hydroxyproline -0.4536 1.47E-05 705 132.1 hydroquinone sulfate 0.4533 1.49E-05 1383 189

X - 15667 0.4509 1.68E-05 2330 202.2 arachidonate (20:4n6) 0.4458 2.14E-05 5525 303.4 indoleacetylglutamine -0.4384 3.03E-05 2530 302.2 indoleacetate -0.4354 3.48E-05 3760 176.1

1 -palmitoylplasmenylethanolamine -0.4348 3.59E-05 6153 436.4

X - 12672 0.4312 4.22E-05 1797 256

X - 16015 -0.4269 5.13E-05 3788 268.1 phenylacetylglycine -0.4263 5.28E-05 2377 192.1

X - 17185 -0.4247 5.65E-05 3069.1 215.2 dihomo-Iinolenate (20:3n3 or n6) 0.4223 6.29E-05 5600 305.4

4-acetylphenol sulfate -0.4189 7.33E-05 2399 215

X - 13543 -0.4162 8.22E-05 1096 484.1 arginine 0.4156 8.45E-05 728 173.2

X - 11549 0.4097 0.0001 5093 339.3

X - 17305 -0.4113 0.0001 2157.1 296.2

X - 12339 0.4095 0.0001 1055 174.1

X - 11843 -0.3998 0.0002 721 118.2 betaine 0.3915 0.0002 2125 160.1

X - 18559 0.4013 0.0002 2710 230.1

X - 15461 -0.3977 0.0002 1864.9 254.1

N-palmitoyl taurine 0.395 0.0002 5556.8 362.4

N-acetylornithine -0.383 0.0003 875 175.2

2-aminobutyrate 0.3878 0.0003 758 132.1 creatine 0.3829 0.0003 860 144.1 stachydrine -0.3858 0.0003 1630.6 179

4- ydroxyphenylacetate -0.3839 0.0003 5955 309.4 eicosenoate (20: ln9 or 11) 0.383 0.0003 758 104.1 cysteine -0.3796 0.0004 1560.1 218 4-vinylphenol sulfate -0.3728 0.0005 2353 343.4 campesterol -0.3737 0.0005 3323 199.1 ribitol -0.373 0.0005 1692.4 217 creatinine -0.3691 0.0006 2846 295.1 gamma-glutamylphenylalanine -0.3681 0.0006 2218 220.1 pantothenate 0.3679 0.0006 730 114.1 mannitol -0.3622 0.0007 1839 319.1

2-oleoylglycerophosphoethanolamine -0.3616 0.0007 5574 329.4 docosapentaenoate (n3 DPA; 22:5n3) 0.3621 0.0007 5848 478.3 indolepropionate -0.3585 0.0008 5684 331.3

2-hydroxyisobutyrate 0.3577 0.0008 5450 277.3 adrenate (22:4n6) 0.3586 0.0008 1925 202.1 linolenate [alpha or gamma; (18:3n3 or 6)] 0.358 0.0008 1107.5 130.9

X - 18570 -0.3568 0.0009 3243.1 207

X - 12435 0.3526 0.001 3174 357.2

X - 16975 -0.345 0.0013 2291.8 255.1

3-(3-hydroxyphenyl)propionate -0.3412 0.0015 1360.7 189

X - 18558 0.3411 0.0015 2000 165.1 glycerate 0.3415 0.0015 1676.8 380.1

X - 15375 -0.3328 0.002 2502 328

X - 12822 -0.3317 0.0021 2786 389.1

X - 12216 0.3256 0.0025 1701 228.1 equol sulfate -0.3239 0.0027 3625 321.2 phenylalanine -0.3218 0.0028 2056 166.1 glycerol 0.3195 0.0031 1311 205

X - 12236 -0.3156 0.0035 1321 245.1 glutamine -0.3152 0.0035 684 147.2 glutathione, oxidized (GSSG) -0.3143 0.0036 1535 613.1 palmitoleate (16:ln7) 0.3118 0.0039 5477 253.3 octanoylcarnitine 0.3105 0.0041 4100 288.3 gamma-glutamylmethionine -0.3086 0.0043 1993 279.2

X - 15636 -0.3075 0.0044 3814 243.1

X - 12450 0.3031 0.0051 5397 251.4 pseudouridine -0.3017 0.0053 1104 243.1 3-methyl-2-oxobutyrate 0.3016 0.0053 1489 115.1 erythritol -0.3004 0.0055 1517.5 217 phenylacetylglutamine -0.2963 0.0062 2868 265.2

3-hydroxybutyrate (BHBA) 0.2959 0.0063 1203.5 116.9 pipecolate 0.2955 0.0064 1120 130.1

3-(4-hydroxyphenyl)propionate -0.2925 0.0069 1727.9 179.1

1-heptadecanoylglycerophosphocholine -0.2915 0.0071 5750 510.4 catechol sulfate -0.2909 0.0073 2830 149.1

3-phenylpropionate (hydrocinnamate) -0.2905 0.0073 1928 188.9 beta-hydroxyisovalerate 0.289 0.0077 1043 117.1

X - 12212 -0.2871 0.0081 3607 229.1

2-linoleoylglycerophosphoethanolamine -0.2848 0.0086 5650 476.4

N 1 -methyladenosine -0.2847 0.0087 #N/A #N/A indoleacrylate -0.2844 0.0088 2529 186.1 phenylalanyltryptophan -0.2841 0.0088 3349 352.2 oleoyltaurine 0.284 0.0089 5599.9 388.3 acetylcarnitine 0.2817 0.0094 1203 204.2

Table 23. Compound levels present in diets A and B. Values represent scaled imputed values fdata are scaled such that the median value measured across all samples was set to 1.0 and missing values ( ^" if any-) were imputed with the observed minimum for that particular compound ^' ).

1-oleoylglycerophosphoethanolamine 0.16 5.65

X - 11378 0.05 0.11

X - 18487 0.26 0.26

X - 11949 0.28 0.28 trans-4-hydroxyproline 5.01 4.88 hydroquinone sulfate 0.03 0.03

X - 15667 0.16 0.16 arachidonate (20:4n6) 0.43 4.48 indoleacetylglutamine 0.52 0.52 indoleacetate 1.78 0.46

1 -palmitoylplasmenylethanolamine 0.21 3.23

X - 12672 0.72 0.72

X - 16015 0.02 0.02 phenylacetylglycine 0.09 0.28

X - 17185 0.12 0.06 dihomo-linolenate (20:3n3 or n6) 0.42 5.03

4-acetylphenol sulfate 0.4 0.56

X - 13543 0.1 0.1 arginine 1 0.63

X - 11549 0.13 0.13

X - 17305 0.42 0.42

X - 12339 0.96 0.5

X - 11843 0.19 0.19 betaine 0.61 0.69

X - 18559 0.78 0.78

X - 15461 0.97 0.35

N-palmitoyl taurine 1 0.94

N-acetylornithine 0.41 ^■ 0.21

2-aminobutyrate 1.58 0.83 creatine 1.98 10.6 stachydrine 3.62 0.78

4-hydroxyphenyIacetate 5.04 0.72 eicosenoate (20:ln9 or 11) 0.32 2.11 cysteine 0.21 0.24 4-vinylphenol sulfate 0.02 0.02 campesterol 1.56 0.72 ribitol 4 7.04 creatinine 13.3 27.38 gamma-glutamylphenylalanine 14.27 1.36 pantothenate 12.83 19.07 mannitol 12.51 1.72

2-oleoylglycerophosphoethanolamine 0.43 6.03 docosapentaenoate (n3 DPA; 22:5n3) 0.14 4.84 indolepropionate 0.07 0.07

2-hydroxyisobutyrate 0.51 0.51 adrenate (22:4n6) 0.82 5.31 linolenate [alpha or gamma; (18:3n3 or 6)] 0.83 1.66

X - 18570 0.08 0.08

X - 12435 0.36 0.36

X - 16975 1.02 0.44

3-(3-hydroxyphenyl)propionate 0.1 0.1

X - 18558 0.55 0.47 glycerate 3.38 9.94

X - 15375 0.41 0.97

X - 12822 0.2 0.2

X - 12216 0.25 0.25 equol sulfate 0.08 0.08 phenylalanine 0.75 0.64 glycerol 2.61 1.85

X - 12236 0.1 0.1 glutamine 0.18 0.18 glutathione, oxidized (GSSG) 0.35 0.16 palmitoleate (1 :ln7) 0.1 0.81 octanoylcarnitine 0.41 0.53 gamma-glutamylmethionine 0.47 0.44

X - 15636 0.2 0.2

X - 12450 0.17 1.43 pseudouridine 0.07 0.1 3 -methyl-2-oxobutyrate 0.09 0.09 erythritol 3.45 0.32 phenylacetylglutamine 0.33 0.33

3-hydroxybutyrate (BHBA) 0.18 0.34 pipecolate 7.41 1.56

3 -(4-hydroxyphenyl)propionate 14.94 0.38

1-heptadecanoylglycerophosphocholine 0.13 0.24 catechol sulfate 0.02 0.02

3-phenylpropionate (hydrocinnamate) 18.68 0.13 beta-hydroxyisovalerate 0.48 0.48

X - 12212 0.21 0.21

2-linoleoylglycerophosphoethanolamine 0.56 2.58

N 1 -methyladenosine #N/A #N/A indoleacrylate 0.06 0.06 phenylalanyltryptophan 0.29 0.29 oleoyltaurine 0.55 1.08 acetylcarnitine 0.1 0.29

Table 24. Protein digestibility in response to diet A and Diet B.

Table 25. Compounds validated by dietary change.

arachidonate (20:4n6) 5525 303.4

X - 17185 3069.1 215.2 betaine 721 118.2

X - 15461 2125 160.1

N-acetylornithine 875 175.2 creatine 758 132.1 stachydrine 860 144.1

4-hydroxyphenylacetate 1630.6 179 eicosenoate (20:ln9 or 11) 5955 309.4 campesterol 2353 343.4 gamma-glutamylphenylalanine 2846 295.1 pantothenate 2218 220.1 mannitol 1839 319.1 docosapentaenoate (n3 DPA; 22:5n3) 5574 329.4 adrenate (22:4n6) 5684 331.3 linolenate [alpha or gamma; (18:3n3 or 6)] 5450 277.3

X - 16975 2291.8 255.1 glycerate 1360.7 189 phenylalanine 2056 166.1 glutathione, oxidized (GSSG) 1535 613.1 palmitoleate (16:ln7) 5477 253.3 octanoylcarnitine 4100 288.3

X - 12450 5397 251.4 erythritol 1517.5 217

3-hydroxybutyrate (BHBA) 1203.5 116.9

3-(4-hydroxyphenyl)propionate 1727.9 179.1

1-heptadecanoylglycerophosphocholine 5750 510.4

3-phenylpropionate (hydrocinnamate) 2830 149.1 oleoyltaurine 5599.9 388.3

[0096] It should be understood that various changes and modifications to the presently embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.