Companion animal supplement comprising free glycine

Abstract

The present invention relates to dietary compositions comprising free glycine, and related methods relating to glutathione in companion animals.

Description

Companion animal supplements containing free glycine Technical Field

[0001] This invention relates to dietary compositions containing free glycine, and related methods for processing glutathione in companion animals. Background Technology

[0002] Aging is associated with increased oxidative stress. This leads to a gradual decline in the ability to manage levels of oxidative damage, resulting in changes in cellular biomolecules, including proteins, lipids, and nucleic acids.

[0003] The ability to resist oxidative damage is determined by the capabilities of several antioxidant defense systems. Glutathione is the most abundant and widespread intracellular antioxidant in mammals. Intracellular glutathione levels have been reported to decline with age in many species. Summary of the Invention

[0004] The present invention provides a dietary composition for companion animals, the composition comprising at least about 0.5% free glycine.

[0005] The present invention also provides a method for treating or preventing diseases in companion animals, the method comprising administering the dietary composition of the present invention to the animal.

[0006] The present invention also provides a method for increasing glutathione and / or GSH in companion animals, the method comprising administering the dietary composition of the present invention.

[0007] The present invention also provides a method for determining the presence of glutathione dysfunction in companion animals, the method comprising: (a) providing a blood sample from the companion animal; (b) measuring the levels of glutathione (GSH) and / or glutathione disulfide (GSSG) in the sample; and (c) determining the presence of glutathione dysfunction if the intracellular GSH:GSSG ratio, circulating GSH:GSSG ratio, circulating GSH concentration, or intracellular GSH concentration is less than a threshold. Attached Figure Description

[0008] Figure 1 shows the mean (a) red blood cell (RBC) and (b) whole blood (WB) total glutathione (µM) levels in age-grouped test cats according to Example 1. Individual data are represented by hollow circles, and the mean is represented by a solid circle with a 95% confidence interval. This indicates that there is a significant difference between groups (p≤0.05).

[0009] Figure 2 shows a schematic diagram of the experiments conducted in (a) Example 2 and (b) Example 3. The time points for blood (triangular arrows) and urine (diamond arrows) sample collection and the relevant sample measures are shown (checkmarks indicate that they have been measured).

[0010] Figure 3 shows the mean red blood cell (RBC) glutathione concentrations in aged cats in the test group (supplemented group) and the control group (unsupplemented group) according to Example 3. RBC glutathione is expressed as (a) total glutathione (GSH + GSSG), (b) reduced glutathione (GSH), (c) oxidized glutathione (GSSG), and (d) GSH:GSSG. Individual data are represented by hollow circles, and the mean is represented by a solid circle, with a confidence interval of 95%. This indicates that there is a significant difference between groups (p≤0.05).

[0011] Figure 4 shows the mean (a) plasma and (b) erythrocyte (RBC) glycine (Gly) levels according to Example 3. Individual data are represented by hollow circles, and the mean is represented by a solid circle with a confidence interval of 95%. This indicates that there is a significant difference between groups (p≤0.05).

[0012] Figure 5 shows the mean total GSH (nM / 50µl) of red blood cells (RBCs) in the test dogs divided by age group according to Example 4. Individual data are represented by hollow circles, and the mean is represented by a solid circle, with a confidence interval of 95%. Detailed Implementation

[0013] definition

[0014] Unless otherwise stated, the present invention will be practiced using conventional chemical, biochemical, and molecular biological methods within the scope of the art. Such techniques are well described in the literature. See, for example, *Methods in Enzymology* (Academic Press, Inc.), *Molecular Cloning: A Laboratory Manual, 4th edition* (Cold SpringHarbor Press), Green & Sambrook (2012), *Short Protocols in Molecular Biology* (5th edition), *Molecular Biology Techniques: An Intensive Laboratory Course* (Ream & Field, 1998, Academic Press), *Principles and Techniques of Biochemistry and Molecular Biology* (Hodmann & Clokie, 2018), and *Basic Molecular Biology & Techniques - Recent Advances: Molecular Biology &...* Its Technique (Singh et al., 2021), etc.

[0015] The use of the term "about" when referring to quantities is optional and means that the quantity can vary depending on the structure or function associated with that quantity as understood by those skilled in the art. For example, "about" 1.5% free glycine refers to an amount of free glycine suitable for achieving the effects of the relevant technology, such as a measurable change in plasma glycine and / or RBC glycine levels when that amount of free glycine is fed to a companion animal (e.g., a cat or dog). "About" can also be defined, for example, as + / - 10% of the quantity.

[0016] When two values ​​are involved, “between” (and variations such as “x to y”, “xy”) includes both values. For example, the range “between” 10 mg and 20 mg specifically includes 10 mg, 15 mg and 20 mg.

[0017] A Body Condition Score (BCS) is a semi-quantitative scoring system that measures the physical condition of companion animals based on visual and tactile findings. The BCS includes a 9-point system developed and validated for dogs and cats. The BCS can be combined with body weight (BW), body fat (BF), body mass index (BMI), and / or chest circumference measurements to more accurately determine the BCS and the companion animal's associated health condition. See, for example, LaFlamme DP. Development and validation of a body condition score system for cats: an clinical tool. *Feline Pract*, 1997; 25(5-6):13-18.

[0018] "Companion animals" refers to animals, such as mammals, including (for example) cats (e.g., adult cats, senior cats), dogs (e.g., adult dogs, senior dogs), horses, cattle, pigs, rabbits, guinea pigs, hamsters, gerbils, ferrets, zoo mammals, fish, birds, etc. Preferred companion animals include cats and dogs, such as senior cats and senior dogs. Cats, especially senior cats, are particularly preferred.

[0019] "Complete and nutritionally balanced" (and variations such as "nutritional complete") refers to a composition containing appropriate amounts and proportions of all known essential nutrients, as recommended by recognized authorities in the field of companion animal nutrition. Such authorities are well-known to those skilled in the art, including, for example, AAFCO (Association of American Feed Control Officials) and FEDIAF's "Nutritional Guidelines for Complete and Complementary Pet Food for Cats and Dogs" (http: / / www.fediaf.org / self-regulation / ). A complete and nutritionally balanced composition can be fed as the sole ration for a companion animal and can sustain life and / or promote reproduction without requiring any additional substances other than water.

[0020] The term “comprising” encompasses both “including” and “consisting of”. For example, a composition that “comprising” X can consist of X alone or may include additional elements (e.g., X+Y).

[0021] Generally, the term "disease" refers to the presence or health condition of a companion animal that can be treated using the methods provided herein. "Treatment" encompasses the complete elimination of the disease and the allergic reduction of its symptoms. The term "disease" is used synonymously with "symptom" and "disorder" in this document. A disease can be clinically recognized in the veterinary community. However, disease also includes other presence or health conditions of a companion animal that can be treated using the methods provided herein. "Prevention" refers to reducing the severity and / or likelihood of a disease before its onset.

[0022] The term "free" in relation to amino acids refers to amino acids as monomers, not as amino acid residues that are part of polymers (such as oligopeptides or proteins). Free amino acids can transiently interact with other substances, such as through hydrogen bonds, but they do not covalently bind with other monomers. Therefore, free glycine can refer to the monomeric glycine.

[0023] Glutathione is a tripeptide consisting of glutamic acid (GLU or Glu), cysteine ​​(CYS or Cys), and glycine (GLY or Gly). Glutathione can be a reduced monomer ("GSH") or oxidized to glutathione disulfide—a dimer bonded to a sulfur atom ("GSSG").

[0024] "Whole blood" refers to blood containing cells, fluid, and clotting factors (e.g., a venous blood sample). Whole blood can be separated into its individual components. "Plasma" refers to the fluid remaining after a whole blood sample has undergone a separation process to remove blood cells (usually involving centrifugation). "Serium" refers to plasma that does not contain clotting factors (e.g., fibrinogen). The method of this invention can be applied to whole blood, venous blood, plasma, or serum.

[0025] Glutathione

[0026] Glutathione is the most abundant intracellular antioxidant in mammals. Glutathione exists in mammalian cycles in both oxidized (GSSG) and reduced (GSH) forms. In its reduced state, GSH can react with cysteine ​​residues within proteins to maintain its reduced form, or react with reactive oxygen species (ROS) to neutralize them. This occurs through the thiol group of GSH, which can donate electrons to other molecules. Thus, GSH itself becomes reactive and forms a disulfide bond with another reactive glutathione to generate oxidized glutathione disulfide (GSSG). In the presence of NADPH, GSH can be regenerated from GSSG by glutathione reductase (GSR).

[0027] Glutathione combats oxidative stress by scavenging reactive oxygen species (ROS) and inhibiting macrophage activation, thereby minimizing associated transcription factor activation and cytokine production. Glutathione also reduces superoxide formation in certain macrophage and neutrophil types.

[0028] Therefore, glutathione (or "total glutathione") includes both GSH and GSSG, and unless otherwise stated, this includes both GSH and GSSG in the measurement. Circulating glutathione is the glutathione (GSH + GSSG) that is detectable in circulation (e.g., in blood). Circulating glutathione includes extracellular glutathione (e.g., in whole blood, plasma, or serum) and intracellular glutathione (i.e., in blood cells (e.g., RBCs) suspended in plasma). Unless otherwise stated, references to "glutathione" refer to "circulating glutathione".

[0029] In some embodiments, circulating glutathione is extracellular glutathione, not intracellular glutathione (i.e., glutathione in whole blood, plasma, or serum, rather than glutathione in blood cells, such as glutathione in plasma). In some embodiments, circulating glutathione is intracellular glutathione, not extracellular glutathione (i.e., glutathione in blood cells (e.g., red blood cells (RBCs) and / or white blood cells (WBCs)) suspended in plasma).

[0030] In some implementations, intracellular glutathione is glutathione in RBCs.

[0031] In some implementations, extracellular glutathione is whole blood glutathione, such as plasma glutathione.

[0032] In other embodiments, glutathione is the glutathione in WBCs. Glutathione has been shown to play an important role in the activation of T-lymphocytes.

[0033] Glutathione dysfunction, aging and disease

[0034] Glutathione dysfunction and measurement

[0035] A decreased GSH / GSSG ratio is considered an indicator of oxidative stress. Therefore, determining the presence of glutathione dysfunction may include assessing whether glutathione, GSH, GSSG, or combinations thereof deviate from thresholds. Example thresholds are provided in this article.

[0036] In companion animals, such as healthy young adult cats (e.g., less than 3 years old), the circulating (e.g., extracellular, such as whole blood) GSH:GSSG ratio is at least about 10, for example about 10 to about 25, for example about 16. In companion animals, such as healthy young adult cats, the intracellular (e.g., RBC) GSH:GSSG ratio is at least about 7, for example about 7 to about 21, for example about 12.

[0037] In companion animals, such as healthy young adult cats (e.g., less than 3 years old), circulating (e.g., extracellular, such as whole blood) glutathione is at least about 450 µM, for example about 450 µM to about 550 µM, for example about 480 µM. In companion animals, such as healthy young adult cats, intracellular (e.g., RBC) glutathione is at least about 850 µM, for example about 850 µM to about 1165 µM, for example about 950 µM.

[0038] In companion animals, such as healthy young adult cats (e.g., less than 3 years old), circulating (e.g., extracellular, such as whole blood) GSH is at least about 370 µM, for example about 370 µM to about 510 µM, for example about 450 µM. In companion animals, such as healthy young adult cats, intracellular (e.g., RBC) GSH is at least about 440 µM, for example about 440 µM to about 1260 µM, for example about 860 µM.

[0039] In companion animals, such as healthy young adult dogs (e.g., less than 3 years old), the circulating (e.g., extracellular, such as whole blood) GSH:GSSG ratio is at least about 21, for example about 21 to about 47, for example about 31. In companion animals, such as healthy young adult dogs, the intracellular (e.g., RBC) GSH:GSSG ratio is at least about 20, for example about 20 to about 38, for example about 29.

[0040] In companion animals, such as healthy young adult dogs (e.g., less than 3 years old), circulating (e.g., extracellular, such as whole blood) glutathione is at least about 0.75 µM / ml, for example about 0.75 µM / ml to about 0.90 µM / ml, for example about 0.83 µM / ml. In companion animals, such as healthy young adult dogs, intracellular (e.g., RBC) glutathione is at least about 1.16 µM / ml, for example about 1.16 µM / ml to about 1.36 µM / ml, for example about 1.26 µM / ml.

[0041] In companion animals, such as healthy young adult dogs (e.g., less than 3 years old), circulating (e.g., extracellular, such as whole blood) GSH is at least about 0.72 µM / ml, for example about 0.72 µM / ml to about 0.88 µM / ml, for example about 0.80 µM / ml. In companion animals, such as healthy young adult dogs, intracellular (e.g., RBC) GSH is at least about 1.09 µM / ml, for example about 1.09 µM / ml to about 1.30 µM / ml, for example about 1.19 µM / ml.

[0042] Methods for measuring GSH and / or GSSG will be readily available to those skilled in the art, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (e.g., Enzo Life Sciences, Farmingdale, NY, USA, catalog number ADI-900-160), or by a standard measurement using radiolabeled GSH and / or GSSG (e.g., 13C isotope-labeled GSH and 13C isotope-labeled GSSG) followed by HPLC-MS. GSH can be calculated as the difference between total glutathione and GSSG. GSH and / or GSSG can be normalized to a biomarker (e.g., hemoglobin), or they can be unnormalized to a biomarker (e.g., hemoglobin).

[0043] Glutathione dysfunction refers to abnormal glutathione metabolism, such as insufficient circulating glutathione (i.e., insufficient combined levels of intracellular and extracellular GSH and GSSG), insufficient circulating GSH, and inappropriate circulating GSH:GSSG ratio.

[0044] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes intracellular glutathione concentrations of less than about 950 µM, less than about 900 µM, less than about 850 µM, less than about 800 µM, less than about 750 µM, less than about 700 µM, less than about 650 µM, less than about 600 µM, or less than about 550 µM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is less than about 950 µM, for example, less than about 850 µM. In a particularly preferred embodiment, this concentration is less than about 700 µM, for example, less than about 550 µM. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0045] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes circulating glutathione concentrations less than about 450 µM, less than about 440 µM, less than about 420 µM, less than about 400 µM, less than about 380 µM, less than about 370 µM, less than about 360 µM, or less than about 350 µM. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is less than about 450 µM, for example, less than about 370 µM. In a particularly preferred embodiment, this concentration is less than about 400 µM, for example, less than about 350 µM. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0046] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes intracellular GSH concentrations less than about 860 µM, less than about 800 µM, less than about 700 µM, less than about 600 µM, less than about 550 µM, less than about 500 µM, less than about 450 µM, less than about 440 µM, less than about 400 µM, less than about 350 µM, less than about 300 µM, less than about 250 µM, or less than about 200 µM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is less than about 860 µM, for example, less than about 440 µM. In a particularly preferred embodiment, this concentration is less than about 300 µM, for example, less than about 200 µM. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0047] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes circulating GSH concentrations less than about 450 µM, less than about 440 µM, less than about 420 µM, less than about 400 µM, less than about 380 µM, less than about 360 µM, less than about 340 µM, less than about 320 µM, less than about 310 µM, less than about 300 µM, less than about 280 µM, less than about 260 µM, less than about 250 µM, or less than about 240 µM. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is less than about 450 µM, for example, less than about 370 µM. In a particularly preferred embodiment, this concentration is less than about 310 µM, for example, less than about 250 µM. These concentrations for glutathione dysfunction can, for example, be non-normalized to hemoglobin and can be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0048] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes intracellular GSH:GSSG ratios less than about 12.0, less than about 10.0, less than about 7.0, less than about 6.0, less than about 5.0, less than about 2.5, less than about 2.0, less than about 1.8, less than about 1.6, less than about 1.4, less than about 1.2, less than about 1.1, less than about 1.0, less than about 0.8, less than about 0.7, or less than about 0.6. In some embodiments, the intracellular ratio is the ratio in RBCs. In a preferred embodiment, the ratio is less than about 12, for example, less than about 7.0. In a particularly preferred embodiment, the ratio is less than about 1.1, for example, less than about 0.7. These glutathione dysfunction concentrations may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0049] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes a circulating GSH:GSSG ratio less than about 16.0, less than about 10.0, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, or less than about 2.5. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, the ratio is less than about 16.0, for example, less than about 10.0. In a particularly preferred embodiment, the ratio is less than about 4.0, for example, less than about 2.5. These glutathione dysfunction concentrations may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0050] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes intracellular glutathione concentrations less than about 0.0039 µM, less than about 0.0038 µM, less than about 0.0037 µM, less than about 0.0036 µM, less than about 0.0035 µM, less than about 0.0034 µM, less than about 0.0033 µM, less than about 0.0032 µM, less than about 0.0031 µM, less than about 0.0030 µM, less than about 0.0029 µM, less than about 0.0028 µM, less than about 0.0027 µM, less than about 0.0024 µM, or less than about 0.0023 µM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is less than about 0.0041 µM, for example, less than about 0.0039 µM. In a particularly preferred embodiment, the concentration is less than about 0.0034 µM, for example less than about 0.0023 µM. These concentrations for glutathione dysfunction can, for example, be normalized to hemoglobin and can be measured, for example, by using radiolabeled GSH and / or GSSG (as reported in Example 3).

[0051] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes intracellular GSH concentrations of less than about 4.0 pg / mL, less than about 3.8 pg / mL, less than about 3.6 pg / mL, less than about 3.4 pg / mL, less than about 3.2 pg / mL, less than about 3.0 pg / mL, less than about 2.8 pg / mL, less than about 2.6 pg / mL, less than about 2.4 pg / mL, or less than about 2.2 pg / mL. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is less than about 3.6 pg / mL, for example, less than about 3.2 pg / mL. In a particularly preferred embodiment, this concentration is less than about 2.8 pg / mL, for example, less than about 2.4 pg / mL. These concentrations for glutathione dysfunction can, for example, be normalized to hemoglobin and can be measured, for example, by using radiolabeled GSH and / or GSSG (as reported in Example 3).

[0052] In some embodiments, such as where the companion animal is a cat, glutathione dysfunction includes an intracellular GSH:GSSG ratio of less than about 8.0, less than about 7.5, less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, or less than about 4.5. In some embodiments, the intracellular ratio is the ratio in RBCs. In a preferred embodiment, the ratio is less than about 7.0, for example, less than about 6.0. In a particularly preferred embodiment, the ratio is less than about 5.0, for example, less than about 4.0. These concentrations for glutathione dysfunction can, for example, be normalized to hemoglobin and can be measured, for example, by using radiolabeled GSH and / or GSSG (as reported in Example 3).

[0053] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes intracellular glutathione concentrations less than about 1.16 µM / ml, less than about 1.14 µM / ml, less than about 1.12 µM / ml, less than about 1.10 µM / ml, less than about 1.08 µM / ml, less than about 1.06 µM / ml, less than about 1.04 µM / ml, less than about 1.02 µM / ml, less than about 1.00 µM / ml, less than about 0.98 µM / ml, less than about 0.96 µM / ml, or less than about 0.94 µM / ml. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is less than about 1.16 µM / ml, for example, less than about 1.14 µM / ml. In a particularly preferred embodiment, this concentration is less than about 1.04 µM / ml, for example, less than about 0.94 µM / ml. These concentrations for glutathione dysfunction can, for example, be non-normalized to hemoglobin and can be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0054] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes circulating glutathione concentrations less than about 0.90 µM / ml, less than about 0.85 µM / ml, less than about 0.80 µM / ml, less than about 0.75 µM / ml, less than about 0.70 µM / ml, or less than about 0.65 µM / ml. In a preferred embodiment, the circulating concentration is the concentration in whole blood. In a preferred embodiment, this concentration is less than about 0.90 µM / ml, for example, less than about 0.81 µM / ml. In a particularly preferred embodiment, this concentration is less than about 0.74 µM / ml, for example, less than about 0.67 µM / ml. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0055] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes intracellular GSH concentrations less than about 1.10 µM / ml, less than about 1.08 µM / ml, less than about 1.06 µM / ml, less than about 1.04 µM / ml, less than about 1.02 µM / ml, less than about 1.00 µM / ml, less than about 0.98 µM / ml, less than about 0.96 µM / ml, less than about 0.94 µM / ml, less than about 0.92 µM / ml, less than about 0.90 µM / ml, less than about 0.88 µM / ml, or less than about 0.86 µM / ml. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is less than about 1.09 µM / ml, for example, less than about 1.06 µM / ml. In a particularly preferred embodiment, this concentration is less than about 0.96 µM / ml, for example, less than about 0.85 µM / ml. These concentrations for glutathione dysfunction can, for example, be non-normalized to hemoglobin and can be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0056] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes circulating GSH concentrations less than about 0.90 µM / ml, less than about 0.85 µM / ml, less than about 0.80 µM / ml, less than about 0.75 µM / ml, less than about 0.70 µM / ml, or less than about 0.65 µM / ml. In a preferred embodiment, the circulating concentration is the concentration in whole blood. In a preferred embodiment, this concentration is less than about 0.90 µM / ml, for example, less than about 0.81 µM / ml. In a particularly preferred embodiment, this concentration is less than about 0.74 µM / ml, for example, less than about 0.67 µM / ml. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0057] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes an intracellular GSH:GSSG ratio of less than about 20.0, less than about 19.0, less than about 18.0, less than about 17.0, or less than about 16.0. In some embodiments, the intracellular ratio is the ratio in RBCs. In a preferred embodiment, the ratio is less than about 25.0, for example, less than about 20.0. In a particularly preferred embodiment, the ratio is less than about 16.5, for example, less than about 16.0. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0058] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes a circulating GSH:GSSG ratio less than about 1.8, less than about 1.6, less than about 1.4, less than about 1.2, less than about 1.0, less than about 0.8, less than about 0.6, less than about 0.4, or less than about 0.2. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, the ratio is less than about 1.8, for example, less than about 1.7. In a particularly preferred embodiment, the ratio is less than about 0.7, for example, less than about 0.1. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0059] In some embodiments, such as where the companion animal is a dog, glutathione dysfunction includes intracellular GSSG concentrations of at least about 0.40 nM / ml, at least about 0.60 nM / ml, at least about 0.80 nM / ml, at least about 1.00 nM / ml, at least about 1.20 nM / ml, at least about 1.40 nM / ml, or at least about 1.60 nM / ml. In some embodiments, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, the concentration is at least about 0.39 nM / ml, for example, at least about 0.52 nM / ml. In a particularly preferred embodiment, the concentration is at least about 0.86 nM / ml, for example, 1.42 nM / ml. These concentrations for glutathione dysfunction may, for example, not be normalized to hemoglobin and may be measured, for example, by enzymatically recovering GSSG to GSH using a glutathione detection ELISA (as reported in Examples 1 and 4).

[0060] Measurements to determine the presence of glutathione dysfunction can be performed before and / or after any method of the present invention. Particularly preferred is the measurement to determine the presence of glutathione dysfunction prior to glycine supplementation according to the present invention. In some embodiments, if glutathione dysfunction is determined, the companion animal may be selectively administered the dietary composition of the present invention, optionally including the step of administering the dietary composition. In some embodiments, the measurement to determine the presence of glutathione dysfunction is performed in vitro, for example using blood samples, such as samples containing whole blood, venous blood, plasma, and / or serum.

[0061] The presence of glutathione dysfunction can be determined based on circulating glutathione (e.g., in whole blood and blood cells), extracellular glutathione (e.g., in plasma or serum), or intracellular glutathione (e.g., in RBCs). Measurement in RBCs is particularly preferred.

[0062] Glutathione and aging

[0063] As shown in Example 1, glutathione dysfunction in cats is age-related. Example 4 reports similar results in dogs.

[0064] Lower glutathione levels may be due to age-related changes in the activity of enzymes involved in glutathione synthesis. Glutamate-cysteine ​​ligase (GCL), the rate-limiting enzyme in GSH synthesis, has a Michaelis constant that increases with age, which can lead to decreased GSH and increased GSSG. Protein digestibility in cats has been reported to decline with age, which may affect intracellular amino acid levels and thus glutathione metabolism.

[0065] Glutathione and Disease

[0066] Glutathione dysfunction can lead to oxidative stress, which refers to the harmful effects of reactive oxygen species (ROS) in the body (when ROS production exceeds the body's ability to remove ROS through antioxidant systems such as the glutathione system). ROS are produced by aerobic metabolism. In healthy cells, ROS are regulated by antioxidant mechanisms such as glutathione. Oxidative stress can be measured using biomarkers such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) and / or F2-isoprostaglandins (F2-IsoPs), such as 8-iso-PGF. 2α PGF 2α ; and / or 2,3-dinor-5,6-dihydro-8-isoPGF 2α .

[0067] The effects of increased oxidative stress (e.g., DNA and RNA damage) have been hypothesized to play a central role in age-related loss of physiological function, including immunosenescence, age-related inflammation, cardiovascular and neurodegenerative diseases, osteoarthritis, and type 2 diabetes. Glutathione dysfunction has been associated with many other chronic diseases, including chronic renal failure and chronic kidney disease in felines. Specifically, in chronic kidney disease, circulating glutathione levels in cats are known to be significantly lower than in clinically normal cats. See, for example, Piyarungsri K and Pusoonthornthum R (2016), “Changes in reduced glutathione, oxidized glutathione, and glutathione peroxidase in cats with naturally occurring chronic kidney disease.” *Comparative Clinical Pathology* 25:655-62.

[0068] Compared with healthy cats and dogs, cats and dogs with necrotizing inflammatory liver disease and hepatic lipidosis also had lower GSH concentrations in their liver tissues. See, for example, Center SA, Warner KL, and Erb HN (2002), Liver glutathione concentrations in dogs and cats with naturally occurring liver disease, *American Journal of Veterinary Research*, 63, 1187-97.

[0069] Therefore, in some embodiments, the present invention relates to diseases associated with glutathione dysfunction, including the diagnosis, treatment, and / or prevention of such diseases and conditions. Treatment and / or prevention of such diseases can be achieved by administering the dietary compositions of the present invention. Diseases of particular interest in the context of the present invention include age-related inflammation and chronic kidney disease.

[0070] However, glutathione deficiency can occur without other signs of disease, such as prior to disease onset. The method of the present invention also includes administering the dietary composition to animals (e.g., in clinically healthy older companion animals) in the absence of disease, such that no treatment and / or prevention of disease is performed. Alternatively, administering the dietary composition of the present invention prior to disease onset can prevent disease without treating it.

[0071] glycine

[0072] Glycine Levels and Aging

[0073] The de novo synthesis of glutathione involves a two-step reaction. In the first step, glutamate and cysteine ​​are converted into a dipeptide of Gln and Cys (glutamylcysteine) via glutamate-cysteine ​​ligase (GCL). -GC”). In the second step -GC and Gly participate in a reaction mediated by GSH synthase to generate glutathione.

[0074] Reports in humans indicate that older individuals have lower intracellular glycine levels due to slower protein turnover or reduced de novo synthesis. Since glycine is a precursor to glutathione, decreased glycine levels could explain the decrease in glutathione levels in humans.

[0075] In Example 1, the inventors demonstrated that plasma Gly levels in older cats were within the normal range and comparable to those in younger cats. The same results were observed in dogs in Example 4. "Circulating glycine" is as defined above for circulating glutathione and has been appropriately modified, and unless otherwise stated, references to "glycine" refer to free glycine.

[0076] glycine and glutathione

[0077] Although normal glycine levels were measured in older cats and dogs, the inventors unexpectedly found that supplementing glycine with, for example, the dietary composition of the present invention increased circulating glycine levels (Example 2), even reversed glutathione dysfunction, and improved associated oxidative stress markers (Example 3), without observing toxicity.

[0078] Not wanting to be bound by theory, the inventors believe that in the early stages of supplementation, excessive Gly drive and -GC glutathione synthesis reaction, thereby increasing the de novo generation of glutathione in the second step of the two-step reaction.

[0079] In some embodiments, glycine supplementation according to the invention enhances antioxidant capacity. This may be particularly useful for older cats and dogs. For example, glycine supplementation according to the invention can increase total circulating glutathione, circulating GSH, or the circulating GSH / GSSG ratio. In a preferred embodiment, glycine supplementation according to the invention increases intracellular total glutathione (i.e., GSH and GSSG), and / or intracellular GSH. In a particularly preferred embodiment, glycine supplementation increases RBC total glutathione (i.e., GSH and GSSG) and / or RBC GSH. For example, this increase can be relative to reference values ​​(e.g., those listed above for glutathione dysfunction), and can include increases listed below. Glycine supplementation according to the invention can reduce markers of oxidative stress.

[0080] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the invention increases the intracellular glutathione concentration to at least about 550 µM, at least about 600 µM, at least about 650 µM, at least about 700 µM, at least about 750 µM, at least about 800 µM, at least about 850 µM, at least about 900 µM, at least about 950 µM, at least about 1000 µM, at least about 1050 µM, at least about 1100 µM, at least about 1150 µM, or at least about 1200 µM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, the concentration is increased to at least about 850 µM, for example, at least about 950 µM. In a particularly preferred embodiment, the concentration is increased to at least about 550 µM, for example, at least about 700 µM.

[0081] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the intracellular glutathione concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, this concentration increases by at least about 20% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0082] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention, upon normalization to hemoglobin, increases the intracellular glutathione concentration to at least about 3.9 nM, at least about 4.0 nM, at least about 4.1 nM, at least about 4.2 nM, at least about 4.3 nM, at least about 4.4 nM, at least about 4.5 nM, or at least about 4.6 nM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, this concentration increases to at least about 4.2 nM upon normalization to hemoglobin.

[0083] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the invention increases the circulating glutathione concentration to at least about 350 µM, at least about 360 µM, at least about 370 µM, at least about 380 µM, at least about 390 µM, at least about 400 µM, or at least about 450 µM. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is increased to at least about 370 µM, for example, at least about 450 µM. In a particularly preferred embodiment, this concentration is increased to at least about 350 µM, for example, at least about 400 µM.

[0084] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating glutathione concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration increases by at least about 10% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0085] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the intracellular GSH concentration to at least about 200 µM, at least about 250 µM, at least about 300 µM, at least about 400 µM, at least about 440 µM, at least about 500 µM, at least about 600 µM, at least about 700 µM, at least about 800 µM, at least about 860 µM, at least about 900 µM, at least about 1000 µM, at least about 1100 µM, or at least about 1200 µM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, the concentration is increased to at least about 440 µM, for example, at least about 860 µM. In a particularly preferred embodiment, the concentration is increased to at least about 350 µM, for example, at least about 450 µM.

[0086] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention, upon normalization to hemoglobin, increases the intracellular GSH concentration to at least about 0.38 pg / mL, 0.40 pg / mL, at least about 0.42 pg / mL, at least about 0.44 pg / mL, at least about 0.46 pg / mL, at least about 0.48 pg / mL, at least about 0.50 pg / mL, or at least about 0.52 pg / mL. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, this concentration increases to at least about 0.52 pg / mL upon normalization to hemoglobin.

[0087] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the intracellular GSH concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, this concentration increases by at least about 20% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0088] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating GSH concentration to at least about 250 µM, at least about 300 µM, at least about 310 µM, at least about 350 µM, at least about 370 µM, at least about 380 µM, at least about 400 µM, at least about 420 µM, at least about 440 µM, at least about 460 µM, at least about 480 µM, at least about 500 µM, or at least about 520 µM. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is increased to at least about 370 µM, for example, at least about 450 µM. In a particularly preferred embodiment, this concentration is increased to at least about 250 µM, for example, at least about 310 µM.

[0089] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating GSH concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration increases by at least about 20% compared to the concentration without glycine supplementation (e.g., compared to the ratio before supplementation, or compared to a control companion animal that did not receive supplementation).

[0090] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the intracellular GSH:GSSG ratio to at least about 0.7, at least about 1.1, 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10.0, at least about 11.0, or at least about 12.0. In a preferred embodiment, the intracellular ratio is the ratio in RBCs. In a preferred embodiment, the ratio is increased to at least about 0.7, for example, at least about 1.1. In a particularly preferred embodiment, the ratio is increased to at least about 7.0, for example, at least about 12.0.

[0091] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the intracellular GSH:GSSG ratio by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 400%, at least about 500%, or at least about 1000% compared to the ratio without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the intracellular ratio is the ratio in RBCs. In a particularly preferred embodiment, this ratio increases by at least about 10% compared to the ratio without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0092] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating GSH:GSSG ratio to at least about 2.5, at least about 3.0, at least about 3.5, at least about 4.0, at least about 5.0, at least about 5.5, at least about 6.0, at least about 10, or at least about 16.0. In a preferred embodiment, the circulating ratio is an extracellular ratio, such as a plasma ratio. In a preferred embodiment, the ratio is increased to at least about 10.0, for example, at least about 16.0. In a particularly preferred embodiment, the ratio is increased to at least about 2.5, for example, at least about 5.0.

[0093] In some embodiments, particularly where the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating GSH:GSSG ratio by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 400%, at least about 500%, or at least about 1000% compared to the concentration without glycine supplementation (e.g., compared to the ratio before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating ratio is an extracellular ratio, such as a plasma ratio. In a particularly preferred embodiment, this ratio increases by at least about 10% compared to the ratio without glycine supplementation (e.g., compared to the ratio before supplementation, or compared to a control companion animal that did not receive supplementation).

[0094] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the invention increases the intracellular glutathione concentration to at least about 1.14 µM / ml, at least about 1.18 µM / ml, at least about 1.20 µM / ml, at least about 1.22 µM / ml, at least about 1.24 µM / ml, at least about 1.26 µM / ml, at least about 1.28 µM / ml, at least about 1.30 µM / ml, at least about 1.32 µM / ml, at least about 1.34 µM / ml, or at least about 1.36 µM / ml. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a preferred embodiment, this concentration is increased to at least about 1.14 µM / ml, for example, at least about 1.16 µM / ml. In a particularly preferred embodiment, this concentration is increased to at least about 1.26 µM / ml, for example, at least about 1.36 µM / ml.

[0095] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the intracellular glutathione concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, this concentration increases by at least about 20% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0096] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the intracellular glutathione concentration to at least about 1.06 µM / ml, at least about 1.08 µM / ml, at least about 1.10 µM / ml, at least about 1.12 µM / ml, 1.14 µM / ml, at least about 1.18 µM / ml, at least about 1.20 µM / ml, at least about 1.22 µM / ml, at least about 1.24 µM / ml, at least about 1.26 µM / ml, at least about 1.28 µM / ml, or at least about 1.30 µM / ml. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, the concentration is increased to at least about 1.06 µM / ml, preferably to at least about 1.09 µM / ml. In a particularly preferred embodiment, the concentration is increased to at least about 1.19 µM / ml, for example, at least about 1.30 µM / ml.

[0097] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the invention increases the circulating glutathione concentration to at least about 0.80 nM / ml, at least about 1.00 nM / ml, at least about 1.20 nM / ml, or at least about 1.40 nM / ml. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is increased to at least about 1.00 nM / ml, for example, at least about 1.20 nM / ml.

[0098] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the circulating glutathione concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration increases by at least about 10% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0099] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the circulating GSH concentration to at least about 0.75 nM / ml, at least about 1.00 nM / ml, at least about 1.25 nM / ml, at least about 1.50 nM / ml, at least about 1.75 nM / ml, or at least about 2.00 nM / ml. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is increased to at least about 0.75 nM / ml, for example, at least about 2.00 nM / ml.

[0100] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the circulating GSH concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 75%, at least about 100%, or at least about 150% compared to the concentration without glycine supplementation (e.g., compared to the pre-supplementation ratio, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration increases by at least about 100% compared to the concentration without glycine supplementation (e.g., compared to the pre-supplementation ratio, or compared to a control companion animal that did not receive supplementation).

[0101] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the intracellular GSH:GSSG ratio to at least about 20.0, at least about 25.0, at least about 30.0, or at least about 35.0. In a preferred embodiment, the intracellular ratio is the ratio in RBCs. In a preferred embodiment, the ratio is increased to at least about 25.0, for example, at least about 30.0. In a particularly preferred embodiment, the ratio is increased to at least about 35.0.

[0102] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the intracellular GSH:GSSG ratio by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 75%, at least about 100%, or at least about 150% compared to the ratio without glycine supplementation (e.g., compared to the pre-supplementation concentration, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the intracellular ratio is the ratio in RBCs. In a particularly preferred embodiment, this ratio increases by at least about 30% compared to the ratio without glycine supplementation (e.g., compared to the pre-supplementation concentration, or compared to a control companion animal that did not receive supplementation).

[0103] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the circulating GSH:GSSG ratio to at least about 1.6, at least about 2.0, at least about 2.4, at least about 2.8, at least about 3.2, at least about 3.6, at least about 4.0, at least about 4.4, at least about 4.8, at least about 5.2, or at least about 5.6. In a preferred embodiment, the circulating ratio is an extracellular ratio, such as a plasma ratio. In a preferred embodiment, the ratio is increased to at least about 1.7, for example, at least about 1.8. In a particularly preferred embodiment, the ratio is increased to at least about 3.4, for example, at least about 5.6.

[0104] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the circulating GSH:GSSG ratio by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 400%, at least about 500%, or at least about 1000% compared to the concentration without glycine supplementation (e.g., compared to the ratio before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating ratio is an extracellular ratio, such as a plasma ratio. In a particularly preferred embodiment, this ratio increases by at least about 100% compared to the ratio without glycine supplementation (e.g., compared to the ratio before supplementation, or compared to a control companion animal that did not receive supplementation).

[0105] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention reduces the circulating GSSG concentration to at least about 0.5 nM / ml, at least about 0.4 nM / ml, at least about 0.3 nM / ml, at least about 0.2 nM / ml, or at least about 0.1 nM / ml. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a preferred embodiment, this concentration is increased to at least about 0.4 nM / ml, for example, at least about 0.2 nM / ml.

[0106] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention reduces the circulating GSH concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 75%, at least about 80%, or at least about 90% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration is reduced by at least about 80% compared to the concentration without glycine supplementation (e.g., compared to the ratio before supplementation, or compared to a control companion animal that did not receive supplementation).

[0107] In some embodiments, glycine supplementation according to the method of the present invention results in immunological or physiological changes, such as increased lymphocytes, increased neutrophils, or panleukocytosis.

[0108] In some embodiments, glycine supplementation according to the method of the present invention reduces the plasma 8-OHdG concentration to less than about 37 ng / mL, less than about 36 ng / mL, less than about 35 ng / mL, less than about 34 ng / mL, less than about 33 ng / mL, less than about 32 ng / mL, less than about 31 ng / mL, less than about 30 ng / mL, less than about 29 ng / mL, less than about 28 ng / mL, less than about 27 ng / mL, less than about 26 ng / mL, less than about 25 ng / mL, less than about 24 ng / mL, less than about 23 ng / mL, less than about 22 ng / mL, or less than about 21 ng / mL. In a particularly preferred embodiment, this concentration is reduced to less than about 27 ng / mg.

[0109] In some embodiments, glycine supplementation according to the method of the present invention reduces 8-iso-PGF in urine. 2α The concentration is reduced to less than about 1.4 ng / mg creatinine (Cr), less than about 1.3 ng / mg Cr, less than about 1.2 ng / mg Cr, less than about 1.1 ng / mg Cr, less than about 1.0 ng / mg Cr, less than about 0.9 ng / mg Cr, or less than about 0.8 ng / mg Cr. In a particularly preferred embodiment, the concentration is reduced to less than about 0.8 ng / mg Cr.

[0110] In some embodiments, glycine supplementation according to the method of the present invention reduces PGF in urine. 2α The concentration is reduced to less than about 5.1 ng / mg Cr, less than about 5.0 ng / mg Cr, less than about 4.9 ng / mg Cr, less than about 4.8 ng / mg Cr, or less than about 4.7 ng / mg Cr. In a particularly preferred embodiment, the concentration is reduced to less than about 4.7 ng / mg Cr.

[0111] In some embodiments, glycine supplementation according to the method of the present invention removes 2,3-dinor-5,6-dihydro-8-iso-PGF from urine. 2αThe concentration is reduced to less than about 8.8 ng / mg Cr, less than about 8.6 ng / mg Cr, less than about 8.4 ng / mg Cr, less than about 8.2 ng / mg Cr, less than about 8.0 ng / mg Cr, less than about 7.8 ng / mg Cr, less than about 7.6 ng / mg Cr, less than about 7.4 ng / mg Cr, less than about 7.2 ng / mg Cr, less than about 7.0 ng / mg Cr, less than about 6.8 ng / mg Cr, or less than about 6.6 ng / mg Cr. In a particularly preferred embodiment, the concentration is reduced to less than about 6.6 ng / mg Cr.

[0112] In some embodiments, glycine supplementation according to the method of the invention reduces the concentration of oxidative stress markers by, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70%, compared to the concentration without glycine supplementation (e.g., the concentration before supplementation, or compared to control companion animals that did not receive supplementation). In some embodiments, this concentration is 8-iso-PGF in urine. 2α The concentration. In a preferred embodiment, this concentration is the concentration of 8-OHdG in plasma and / or PGF in urine. 2α The concentration of 2,3-dinor-5,6-dihydro-8-iso-PGF in urine and / or the concentration of 2,3-dinor-5,6-dihydro-8-iso-PGF 2α The concentration. In a particularly preferred embodiment, the concentration is reduced by at least about 30%.

[0113] In some embodiments, glycine supplementation according to the method of the invention increases the intracellular Gly concentration to at least about 260 µM, at least about 270 µM, at least about 280 µM, at least about 290 µM, at least about 300 µM, at least about 310 µM, at least about 320 µM, at least about 330 µM, at least about 350 µM, at least about 400 µM, or at least about 450 µM. In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, the concentration is increased to at least about 280 µM, for example, at least about 300 µM.

[0114] In some embodiments, glycine supplementation according to the method of the invention increases the intracellular Gly concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation). In a preferred embodiment, the intracellular concentration is the concentration in RBCs. In a particularly preferred embodiment, this concentration increases by at least about 15% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to a control companion animal that did not receive supplementation).

[0115] In some embodiments, particularly when the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating Gly concentration to at least about 360 µM, at least about 370 µM, at least about 380 µM, at least about 390 µM, at least about 400 µM, at least about 410 µM, at least about 420 µM, at least about 430 µM, at least about 440 µM, at least about 450 µM, at least about 500 µM, or at least about 550 µM. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration is increased to at least about 400 µM, for example, at least about 550 µM.

[0116] In some embodiments, particularly when the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating Gly concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to the control companion animal that did not receive supplementation). In a particularly preferred embodiment, this concentration increases by at least about 15% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to the control companion animal that did not receive supplementation).

[0117] In some embodiments, particularly when the companion animal is a cat, glycine supplementation according to the method of the invention increases the circulating Gly concentration to at least about 160 nM / ml, at least about 170 nM / ml, at least about 180 nM / ml, or at least about 190 nM / ml. In a preferred embodiment, the circulating concentration is an extracellular concentration, such as a plasma concentration. In a particularly preferred embodiment, this concentration is increased to at least about 170 nM / ml, for example, at least about 190 nM / ml.

[0118] In some embodiments, particularly where the companion animal is a dog, glycine supplementation according to the method of the invention increases the circulating Gly concentration by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to the control companion animal that did not receive supplementation). In a particularly preferred embodiment, this concentration increases by at least about 10% compared to the concentration without glycine supplementation (e.g., compared to the concentration before supplementation, or compared to the control companion animal that did not receive supplementation).

[0119] Glycine supplementation and disease

[0120] The inventors have demonstrated that glycine supplementation reverses glutathione dysfunction. Therefore, glycine supplementation can treat diseases caused by or related to glutathione dysfunction, such as those involving oxidative stress.

[0121] Therefore, in some embodiments, glycine supplementation according to the present invention is associated with the treatment and / or prevention of disease.

[0122] In some embodiments, the disease is chronic kidney disease, dyslipidemia, cataracts, macular degeneration, glaucoma, immunosenescence, oxidative damage, age-related inflammation, neurodegenerative diseases, type 2 diabetes, osteoarthritis, or liver disease. The disease may include CD21+ cell deficiency, insulin insensitivity, or cholesterol oxidation. Chronic kidney disease is particularly preferred.

[0123] In some implementations, the disease is a neurodegenerative disease, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0124] In some implementations, the disease is a cardiovascular disease, such as hypertension or myocardial infarction.

[0125] In some implementations, the disease is a liver disease, such as hepatic lipidosis.

[0126] In some implementations, the disease is associated with high blood cholesterol.

[0127] Glycine supplementation according to the method of the present invention can also be used to prevent the disease, i.e., it can be carried out before the onset of disease symptoms.

[0128] Glycine supplementation in the absence of disease

[0129] In other embodiments, glycine supplementation according to the invention is not associated with disease treatment. These embodiments can be described as non-therapeutic or cosmetic and may involve altering the body composition of a cat or dog (e.g., + / - 10% ideal BCS) or other non-disease signs of aging in the absence of disease. In some embodiments, the glycine supplementation method according to the invention is performed in the absence of disease to increase circulating glutathione, intracellular (e.g., RBC) glutathione, circulating GSH, and / or intracellular (e.g., RBC) GSH as described above.

[0130] In some implementations, glycine supplementation lowers blood cholesterol in companion animals.

[0131] Companion animals

[0132] This invention relates to companion animals. In a preferred embodiment, the companion animal is an older cat. In some embodiments, the older cat is a domestic cat, such as a domestic shorthair. In other embodiments, the companion animal is an older dog. In still other embodiments, the older dog is a beagle or a Brittany beagle.

[0133] In some embodiments, the age of the elderly cat or dog is at least about 6.5 years, at least about 7.0 years, at least about 7.5 years, at least about 8.0 years, at least about 8.5 years, at least about 9.0 years, at least about 9.5 years, at least about 9.7 years, at least about 10.0 years, at least about 10.5 years, at least about 11.0 years, at least about 11.5 years, at least about 12.0 years, at least about 12.5 years, at least about 13.0 years, at least about 13.5 years, or at least about 14.0 years. In a particularly preferred embodiment, the age of the cat or dog is at least about 9.5 years.

[0134] In a preferred embodiment, the elderly cat is at least about 7.0 years old.

[0135] In a preferred embodiment, the age of the senior dog is at least about 12 years, particularly if the dog is a toy-type senior dog or a small senior dog, as defined by Salt et al., Journal of Gerontol A Biol Sci Med Sci., April 2023; 78(4):579–586. In some embodiments, the age of the senior dog is at least about 10 years, particularly if the dog is a large senior dog, as defined by Salt et al., Journal of Gerontol A Biol Sci Med Sci., April 2023; 78(4):579–586.

[0136] Cats are considered particularly vulnerable to oxidative damage because their hemoglobin contains 8-10 reactive sulfhydryl groups, compared to 4 in dogs and other mammals. Therefore, cat hemoglobin molecules are more susceptible to damage after oxidative attack and may require more GSH to maintain the reduced form of the sulfhydryl groups, which is necessary for normal function.

[0137] In some implementations, the companion animal's BCS is within approximately + / - 10% of the ideal BCS for the companion animal.

[0138] In some embodiments, the cat or dog is healthy, for example, without evidence of disease and not receiving drug treatment. In some embodiments, the cat or dog is healthy but has glutathione dysfunction, for example, putting the cat or dog at risk of disease. In some embodiments, the cat or dog suffers from a disease associated with glutathione dysfunction.

[0139] Dietary composition

[0140] The dietary composition (or simply "composition") of the present invention is suitable for feeding companion animals.

[0141] The dietary composition of the present invention comprises free glycine (Gly). The dietary composition may contain any amount of free glycine sufficient to increase circulating glycine, circulating glutathione, circulating GSH, intracellular glutathione, intracellular GSH and / or improve glutathione dysfunction.

[0142] The dietary compositions of the present invention may contain the typical amino acid profile of dietary compositions known in the art, except for glycine, which is added to the dietary compositions of the present invention.

[0143] The dietary compositions of the present invention may contain exogenous free glycine, for example, they may contain free glycine not derived from any other component present in the dietary composition (such as by hydrolysis of proteins already present in the dietary composition). The free glycine may be synthetic (not directly derived from food sources, for example, synthesized in a laboratory from chemical components). Free glycine may be added in solid form, such as powder.

[0144] The content of an ingredient in a dietary composition, such as the content of free glycine, can be expressed as a percentage of the ingredient's weight as is, relative to the total weight of the composition (unless otherwise stated herein, expressed as "%"). In some embodiments, the dietary composition contains at least about 0.5%, at least about 1.0%, at least about 1.5%, at least about 2.0%, at least about 2.5%, at least about 3.0%, at least about 3.5%, at least about 4.0%, at least about 4.5%, at least about 5.0%, at least about 5.5%, or at least about 6.0% free Gly. In a preferred embodiment, the dietary composition contains at least about 1.5% free Gly. In a particularly preferred embodiment, the dietary composition contains at least about 6.0% Gly.

[0145] In some embodiments, the dietary composition comprises about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.0% free Gly. In a preferred embodiment, the dietary composition comprises about 6.0% Gly. In a particularly preferred embodiment, the dietary composition comprises about 1.5% free Gly.

[0146] In some embodiments, the dietary composition contains up to about 6.5%, up to about 6.0%, up to about 5.5%, up to about 5.0%, up to about 4.5%, up to about 4.0%, up to about 3.5%, up to about 3.0%, up to about 2.5%, up to about 2.0%, or up to about 1.5% free Gly. In a preferred embodiment, the dietary composition contains up to about 6.0% Gly. In a particularly preferred embodiment, the dietary composition contains up to about 1.5% Gly.

[0147] In some embodiments, the dietary composition contains at least about 0.5 mM / kg (companion animal body weight), at least about 0.75 mM / kg (companion animal body weight), at least about 1.00 mM / kg (companion animal body weight), at least about 1.25 mM / kg (companion animal body weight), at least about 1.50 mM / kg (companion animal body weight), at least about 1.75 mM / kg (companion animal body weight), at least about 3.50 mM / kg (companion animal body weight), or at least about 7.00 mM / kg (companion animal body weight). In a preferred embodiment, the dietary composition contains at least about 7.00 mM / kg (companion animal body weight) of free Gly. In a particularly preferred embodiment, the dietary composition contains about 1.75 mM / kg (companion animal body weight) of free Gly.

[0148] In some embodiments, the dietary composition contains about 0.5 mM / kg (companion animal body weight), about 0.75 mM / kg (companion animal body weight), about 1.00 mM / kg (companion animal body weight), about 1.25 mM / kg (companion animal body weight), about 1.50 mM / kg (companion animal body weight), about 1.75 mM / kg (companion animal body weight), about 3.50 mM / kg (companion animal body weight), or about 7.00 mM / kg (companion animal body weight). In a preferred embodiment, the dietary composition contains about 7.00 mM / kg (companion animal body weight) of free Gly. In a particularly preferred embodiment, the dietary composition contains about 1.75 mM / kg (companion animal body weight) of free Gly.

[0149] In some embodiments, the dietary composition contains up to about 0.5 mM / kg (companion animal body weight), up to about 0.75 mM / kg (companion animal body weight), up to about 1.00 mM / kg (companion animal body weight), up to about 1.25 mM / kg (companion animal body weight), up to about 1.50 mM / kg (companion animal body weight), up to about 1.75 mM / kg (companion animal body weight), up to about 3.50 mM / kg (companion animal body weight), or up to about 7.00 mM / kg (companion animal body weight). In a preferred embodiment, the dietary composition contains up to about 7.00 mM / kg (companion animal body weight). In a particularly preferred embodiment, the dietary composition contains about 1.75 mM / kg (companion animal body weight) of free Gly.

[0150] In some embodiments, the dietary composition comprises at least about 20g, at least about 30g, at least about 40g, at least about 50g, at least about 60g, at least about 70g, at least about 80g, at least about 90g, or at least about 100g of substance. In a preferred embodiment, the dietary composition comprises at least about 50g.

[0151] In some embodiments, the dietary composition contains at least about 1.0 (mg free Gly) / kCal, at least about 2.0 (mg free Gly) / kCal, at least about 3.0 (mg free Gly) / kCal, at least about 4.0 (mg free Gly) / kCal, at least about 5.0 (mg free Gly) / kCal, at least about 6.0 (mg free Gly) / kCal, at least about 7.0 (mg free Gly) / kCal, at least about 8.0 (mg free Gly) / kCal, at least about 9.0 (mg free Gly) / kCal, at least about 10.0 (mg free Gly) / kCal, at least about 11.0 (mg free Gly) / kCal, at least about 12.0 (mg free Gly) / kCal, or at least about 13.0 (mg free Gly) / kCal. In a preferred embodiment, the dietary composition contains at least about 1.0 (mg free Gly) / kCal, for example at least about 13.0 (mg free Gly) / kCal.

[0152] In some embodiments, the dietary composition comprises about 1.0 (mg free Gly) / kCal, about 2.0 (mg free Gly) / kCal, about 3.0 (mg free Gly) / kCal, about 4.0 (mg free Gly) / kCal, about 5.0 (mg free Gly) / kCal, about 6.0 (mg free Gly) / kCal, about 7.0 (mg free Gly) / kCal, about 8.0 (mg free Gly) / kCal, about 9.0 (mg free Gly) / kCal, about 10.0 (mg free Gly) / kCal, about 11.0 (mg free Gly) / kCal, about 12.0 (mg free Gly) / kCal, or about 13.0 (mg free Gly) / kCal. In a preferred embodiment, the dietary composition contains about 1.0 (mg free Gly) / kCal, for example about 13.0 (mg free Gly) / kCal.

[0153] In some embodiments, the dietary composition contains up to about 1.0 (mg free Gly) / kCal, up to about 2.0 (mg free Gly) / kCal, up to about 3.0 (mg free Gly) / kCal, up to about 4.0 (mg free Gly) / kCal, up to about 5.0 (mg free Gly) / kCal, up to about 6.0 (mg free Gly) / kCal, up to about 7 The dietary composition contains up to about 1.0 (mg free Gly) / kCal, or at most about 8.0 (mg free Gly) / kCal, up to about 9.0 (mg free Gly) / kCal, up to about 10.0 (mg free Gly) / kCal, up to about 11.0 (mg free Gly) / kCal, up to about 12.0 (mg free Gly) / kCal, or up to about 13.0 (mg free Gly) / kCal. In a preferred embodiment, the dietary composition contains up to about 1.0 (mg free Gly) / kCal, for example, up to about 13.0 (mg free Gly) / kCal.

[0154] In some embodiments, the dietary composition comprises at least about 0.2 g / (g total protein) free Gly, at least about 0.2 g / (g total protein) free Gly, at least about 0.4 g / (g total protein) free Gly, at least about 0.6 g / (g total protein) free Gly, at least about 1.2 g / (g total protein) free Gly, at least about 1.8 g / (g total protein) free Gly, at least about 2.4 g / (g total protein) free Gly, or at least about 3.0 g / (g total protein) free Gly. In a preferred embodiment, the dietary composition comprises at least about 0.6 g / (g total protein) free Gly, for example, at least about 2.4 g / (g total protein) free Gly.

[0155] In some embodiments, the dietary composition comprises about 0.2 g / (g total protein) free Gly, about 0.2 g / (g total protein) free Gly, about 0.4 g / (g total protein) free Gly, about 0.6 g / (g total protein) free Gly, about 1.2 g / (g total protein) free Gly, about 1.8 g / (g total protein) free Gly, about 2.4 g / (g total protein) free Gly, or about 3.0 g / (g total protein) free Gly. In a preferred embodiment, the dietary composition comprises about 0.6 g / (g total protein) free Gly, for example, about 2.4 g / (g total protein) free Gly.

[0156] In some embodiments, the dietary composition contains up to about 0.2 g / (g total protein) free Gly, up to about 0.2 g / (g total protein) free Gly, up to about 0.4 g / (g total protein) free Gly, up to about 0.6 g / (g total protein) free Gly, up to about 1.2 g / (g total protein) free Gly, up to about 1.8 g / (g total protein) free Gly, up to about 2.4 g / (g total protein) free Gly, or up to about 3.0 g / (g total protein) free Gly. In a preferred embodiment, the dietary composition contains up to about 0.6 g / (g total protein) free Gly, for example, up to about 2.4 g / (g total protein) free Gly.

[0157] The composition may be a semi-wet composition (total moisture content of about 16% to 50% by weight) or a wet composition (total moisture content of at least about 50% by weight). However, in a preferred embodiment, the dietary composition is a dry composition having a total moisture content of up to about 16% by weight, up to about 8% by weight, up to about 7% by weight, or up to about 6% by weight—for example, up to about 7% by weight.

[0158] In some embodiments, the dietary composition is nutritionally complete for companion animal species. For example, a dietary composition for cats may meet the nutritional profile for adult cats of the Association of American Feed Control Officials (AAFCO). The dietary composition may also comply with the nutritional guidelines of the Federation of European Pet Food Industries (FEDIAF).

[0159] The dietary composition may be solid, liquid, or a mixture of both. In some embodiments, the dietary composition is selected from the group consisting of: pet food, such as cat food, dog food, jelly, gel, treats, chews, biscuits, gravy, broth, sauce, beverages, soup, meat paste, paste, spread, cream, rehydration water, and combinations thereof. Preferably, the dietary composition is cat food or dog food.

[0160] In some embodiments, the dietary composition is a liquid, such as an aqueous solution containing free glycine.

[0161] In some embodiments, the dietary composition is a non-medical composition, such as a nutritional supplement composition.

[0162] The dietary composition may also contain one or more of protein, fat, crude fiber, ash, cysteine, and water. In some embodiments, the dietary composition also contains free cysteine, for example, less than about 0.5% free cysteine, or about 0.5% free cysteine.

[0163] In some embodiments, the dietary composition of the present invention comprises, on a dry matter basis, about 1% to about 50% crude protein, about 0.5% to about 25% crude fat, and about 1% to about 10% supplemental fiber, all on a dry matter basis. The total moisture content of the composition may be about 1% to about 30% moisture. Alternatively, the composition may comprise, on a dry matter basis, about 5% to about 35% crude protein, about 5% to about 25% crude fat, and about 2% to about 8% supplemental fiber, all on a dry matter basis. The total moisture content of the composition may be about 2% to about 20% moisture. Alternatively, the composition may comprise, on a dry matter basis, a minimum protein level of about 9.5% to about 35%, a minimum fat level of about 8% to about 20%, and a minimum supplemental fiber level of about 3% to about 7%, all on a dry matter basis. The minimum metabolizable energy level of the composition may also be about 3.5 kcal / g, for example, about 3.7 kcal / g. The total moisture content of the composition may be about 3% to about 10%.

[0164] The compositions of the present invention may include additional components. Examples of additional components include animal protein, plant protein, starchy substances, vegetables, fruits, egg-based materials, undenatured proteins, food-grade polymer binders, gels, polyols, starch, gums, flavorings, seasonings, salt, colorings, slow-release compounds, minerals, vitamins, antioxidants, prebiotics, probiotics, aroma modifiers, textured wheat protein, textured soy protein, textured lupin protein, textured plant protein, breading, ground meat, flour, ground pasta, water, and combinations thereof.

[0165] Non-limiting examples of optional ingredients may include at least one vegetable. Non-limiting examples of vegetables include carrots, peas, potatoes, cabbage, celery, beans, corn, tomatoes, broccoli, cauliflower, leeks, and combinations thereof.

[0166] Also an optional component that may be used in this article is a filler. A filler may be solid, liquid, or encapsulated air. A filler may be reversible (e.g., thermally reversible, including gelatin) and / or irreversible (e.g., thermally irreversible, including egg white). Non-limiting examples of fillers include gravy, gel, jelly, aspic, sauce, water, air (e.g., including nitrogen, carbon dioxide, and atmosphere), broth, and combinations thereof.

[0167] The composition may contain a colorant. Non-limiting examples of colorants include, but are not limited to, synthetic or natural colorants and any combination thereof. When a colorant is present, the colorant is present in amounts of about 0.0001% to about 5%, about 0.001% to about 1%, or about 0.005% to about 0.1% on a dry matter basis.

[0168] Probiotics, such as species of Lactobacillus or Bifidobacterium, can be added to the composition.

[0169] Optional ingredients that may also be used in this article are at least one fruit. Non-limiting examples include tomatoes, apples, pears, peaches, cherries, apricots, plums, grapes, oranges, grapefruits, lemons, limes, cranberries, raspberries, blueberries, watermelons, cantaloupes, honeydew melons, strawberries, bananas, and combinations thereof.

[0170] The composition may contain other active agents, such as long-chain fatty acids and zinc. Suitable long-chain fatty acids include alpha-linolenic acid, gamma-linolenic acid, linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Fish oil is a suitable source of EPA and DHA. The DHA level may be at least about 0.05%, or at least about 0.1%, or at least about 0.15% of the animal food composition, all on a dry matter basis. The EPA level may be at least about 0.05%, or at least about 0.1%, or at least about 0.15% of the composition, all on a dry matter basis.

[0171] The compositions of the present invention may also contain a source of carbohydrates. Cereals or grains, such as rice, corn, sorghum, barley, wheat, etc., are exemplary sources.

[0172] The composition may also contain other substances, such as dried whey and other dairy byproducts.

[0173] The present invention also provides a method for preparing the dietary composition of the present invention, for example, by combining a dietary composition deficient in free glycine with free glycine to produce the dietary composition of the present invention. Free glycine and the dietary composition deficient in free glycine can be combined in proportions suitable for producing a dietary composition containing at least about 0.5%, at least about 1.5%, or at least about 6.0% free glycine. Preferably, the produced dietary composition contains at least about 1.5% free glycine, for example, at least about 6.0% free glycine. Free glycine and the dietary composition deficient in free glycine can be combined in proportions suitable for producing a dietary composition containing about 0.5%, about 1.5%, or about 6.0% free glycine. Preferably, the produced dietary composition contains about 1.5% free glycine. Particularly preferred is a dietary composition containing 6.0% free glycine. Free glycine and the dietary composition deficient in free glycine can be combined in proportions suitable for producing a dietary composition containing at most about 0.5%, at most about 1.5%, or at most about 6.0% free glycine. Preferably, the produced dietary composition contains up to about 6.0% free glycine, for example up to about 1.5% free glycine.

[0174] Dietary additives

[0175] A dietary additive containing free glycine is also provided, which can be combined with a dietary composition to increase the free glycine content of the dietary composition (thereby producing the dietary composition of the present invention).

[0176] Additives can be added to any dietary composition, such as those deficient in free glycine. A dietary composition may be considered "deficient" in free glycine if it contains insufficient amounts of free glycine to meet the nutritional requirements of the companion animal, or if it may contain insufficient amounts of free glycine according to nutritional standards (e.g., those established by AAFCO). Alternatively, a dietary composition may be considered "deficient" in free glycine if it contains insufficient amounts of free glycine to increase circulating or plasma free glycine levels in the companion animal. For example, a dietary composition may be considered "deficient" in free glycine if it contains less than about 0.5%, less than about 1.5%, or less than about 6%. Dietary compositions containing less than about 6% free glycine (e.g., less than about 1.5% free glycine) are particularly preferred.

[0177] The dietary additives of the present invention may contain or be composed of exogenous free glycine, for example, they may contain free glycine not derived from any other component present in the dietary additive (such as by hydrolysis of proteins already present in the dietary additive). The free glycine may be synthetic (not directly derived from food sources, for example, synthesized in a laboratory by chemical components). Free glycine may be added to the additive in solid form (e.g., powder).

[0178] The additive can be in the form of a substance placed on top of a dietary composition lacking free glycine (such an additive is conventionally called a "topper"). The additive can be mixed with the dietary composition lacking free glycine. The additive may contain no other nutritional value besides the nutritional value of its free glycine content; for example, besides the kcal of its free glycine content, the additive may contain from about 0 kcal to about 100 kcal, or from about 0 kcal to about 30 kcal, or about 0 kcal. The additive can be solid, liquid, or powder.

[0179] The dietary additive may be in the form of a solid, tablet, powder, liquid, or suspension. The dietary additive may contain at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% free glycine.

[0180] The present invention also provides a method for preparing the dietary composition of the present invention, for example comprising mixing the dietary additive of the present invention with the dietary composition to produce the dietary composition of the present invention. The dietary additive and the dietary composition may be combined in proportions suitable for producing a dietary composition containing at least about 0.5%, at least about 1.5%, or at least about 6.0% free glycine. Preferably, the produced dietary composition contains at least about 1.5% free glycine, for example, at least about 6.0% free glycine. The dietary additive and the dietary composition may be combined in proportions suitable for producing a dietary composition containing about 0.5%, about 1.5%, or about 6.0% free glycine. Preferably, the produced dietary composition contains about 1.5% free glycine. Particularly preferred is the production of a dietary composition containing about 6.0% free glycine. The dietary additive and the dietary composition may be combined in proportions suitable for producing a dietary composition containing at most about 0.5%, at most about 1.5%, or at most about 6.0% free glycine. Preferably, the produced dietary composition contains up to about 6.0% free glycine, for example up to about 1.5% free glycine.

[0181] feeding

[0182] In any embodiment of the invention involving the administration of the dietary composition, administration is oral administration (“feeding,” “ingestion,” or “supplementation”). Companion animals may ingest the dietary composition by eating and / or drinking the composition.

[0183] Feeding can be performed daily for at least approximately 2 weeks, such as at least approximately 4 weeks, at least approximately 8 weeks, or at least approximately 12 weeks. In a particularly preferred embodiment, feeding is performed daily for at least approximately 4 weeks.

[0184] In some implementations, the supplemental diet includes at least one meal per day, at least two meals per day, at least three meals per day, or at least four meals per day, such as one meal per day, two meals per day, three meals per day, or four meals per day. Two meals per day are particularly preferred. The calorie content of each meal may be equal or unequal; for example, each meal may account for 50% of the total calorie intake.

[0185] In some embodiments, companion animals are administered at least about 0.5 mM / kg / day, at least about 0.75 mM / kg / day, at least about 1.00 mM / kg / day, at least about 1.25 mM / kg / day, at least about 1.50 mM / kg / day, at least about 1.75 mM / kg / day, at least about 3.50 mM / kg / day, or at least about 7.00 mM / kg / day of free Gly. In a preferred embodiment, companion animals are administered at least about 7.00 mM / kg / day of free Gly. In a particularly preferred embodiment, companion animals are administered at least about 1.75 mM / kg / day of free Gly.

[0186] In some embodiments, companion animals are administered about 0.5 mM / kg / day, about 0.75 mM / kg / day, about 1.00 mM / kg / day, about 1.25 mM / kg / day, about 1.50 mM / kg / day, about 1.75 mM / kg / day, about 3.50 mM / kg / day, or about 7.00 mM / kg / day of free Gly. In a preferred embodiment, companion animals are administered about 7.00 mM / kg / day of free Gly. In a particularly preferred embodiment, companion animals are administered about 1.75 mM / kg / day of free Gly.

[0187] In some embodiments, companion animals are administered up to about 0.5 mM / kg / day, up to about 0.75 mM / kg / day, up to about 1.00 mM / kg / day, up to about 1.25 mM / kg / day, up to about 1.50 mM / kg / day, up to about 1.75 mM / kg / day, up to about 3.50 mM / kg / day, or up to about 7.00 mM / kg / day of free glycine. In a preferred embodiment, companion animals are administered up to about 7.00 mM / kg / day of free glycine. In a particularly preferred embodiment, companion animals are administered about 1.75 mM / kg / day of free glycine.

[0188] In some embodiments, the companion animal is administered a dietary composition of at least about 20 g, at least about 30 g, at least about 40 g, at least about 50 g, at least about 60 g, at least about 70 g, at least about 80 g, at least about 90 g, or at least about 100 g daily. In a preferred embodiment, the companion animal is administered a dietary composition of at least about 50 g daily.

[0189] In some embodiments, the companion animal is administered about 20 g, about 30 g, about 40 g, about 50 g, about 60 g, about 70 g, about 80 g, about 90 g, or about 100 g of the dietary composition daily. In a preferred embodiment, the companion animal is administered about 50 g of the dietary composition daily.

[0190] In some embodiments, the companion animal is administered a dietary composition of up to about 20 g, up to about 30 g, up to about 40 g, up to about 50 g, up to about 60 g, up to about 70 g, up to about 80 g, up to about 90 g, or up to about 100 g daily. In a preferred embodiment, the companion animal is administered a dietary composition of up to about 50 g daily.

[0191] In some embodiments, the companion animal is administered at least about 0.25 g, at least about 0.5 g, at least about 0.75 g, at least about 1.0 g, at least about 1.5 g, at least about 2.0 g, at least about 2.5 g, at least about 3.0 g, at least about 3.5 g, at least about 4.0 g, at least about 4.5 g, at least about 5.0 g, at least about 5.5 g, or at least about 6.0 g of free Gly daily. In a preferred embodiment, the companion animal is administered about 0.75 g of free Gly daily, for example, at least about 3.0 g of free Gly daily.

[0192] In some embodiments, the companion animal is administered about 0.25 g, about 0.5 g, about 0.75 g, about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 3.0 g, about 3.5 g, about 4.0 g, about 4.5 g, about 5.0 g, about 5.5 g, or about 6.0 g of free Gly daily. In a preferred embodiment, the companion animal is administered about 0.75 g of free Gly daily, and particularly preferably about 3.0 g of free Gly daily.

[0193] In some embodiments, companion animals are administered up to about 0.25 g, about 0.5 g, about 0.75 g, about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 3.0 g, about 3.5 g, about 4.0 g, about 4.5 g, about 5.0 g, about 5.5 g, or about 6.0 g of free Gly daily. In a preferred embodiment, companion animals are administered up to about 3.0 g of free Gly daily, for example, up to about 0.75 g of free Gly daily.

[0194] In some embodiments, the companion animal is administered at least about 0.1 g, about 0.2 g, about 0.3 g, about 0.4 g, about 0.5 g, about 0.6 g, about 0.7 g, about 0.8 g, about 0.9 g, about 1.0 g, about 1.2 g, or about 1.5 g of free Gly per kg of body weight per day. In a preferred embodiment, the companion animal is administered at least about 0.3 g of free Gly per kg of body weight per day, for example, at least about 1.5 g of free Gly per kg of body weight per day.

[0195] In some embodiments, the companion animal is administered about 0.1 g, about 0.2 g, about 0.3 g, about 0.4 g, about 0.5 g, about 0.6 g, about 0.7 g, about 0.8 g, about 0.9 g, about 1.0 g, about 1.2 g, or about 1.5 g of free Gly per kg of body weight daily. In a preferred embodiment, the companion animal is administered about 0.3 g of free Gly per kg of body weight daily, and particularly preferably about 1.5 g of free Gly per kg of body weight daily.

[0196] In some embodiments, companion animals are administered up to about 0.1 g, about 0.2 g, about 0.3 g, about 0.4 g, about 0.5 g, about 0.6 g, about 0.7 g, about 0.8 g, about 0.9 g, about 1.0 g, about 1.2 g, or about 1.5 g of free Gly per kg of body weight per day. In a preferred embodiment, companion animals are administered up to about 1.5 g of free Gly per kg of body weight per day, for example, up to about 3.0 g of free Gly per kg of body weight per day.

[0197] Example

[0198] Example 1 investigated whether the circulating glutathione level in cats is affected by age.

[0199] Example 2 investigated whether dietary supplementation with different doses of free Gly could alter circulating Gly levels in older cats.

[0200] Example 3 investigated whether dietary supplementation with free Gly could alter circulating glutathione levels in aged cats, and if so, whether the adjusted glutathione levels were reflected in oxidative stress markers.

[0201] Example 4 investigated whether the levels of circulating glycine and glutathione in dogs are affected by age.

[0202] General materials and methods are provided in the final section following these embodiments.

[0203] Example 1: Glutathione levels in felines decrease with age.

[0204] This example compares the levels of total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), and plasma glycine in young and old cats.

[0205] method

[0206] Thirty-two healthy adult cats (22 females and 10 males) participated in this cross-sectional study. Sixteen were classified as “young” with a median age of 2.9 years (range 1.3 to 2.9 years), and 16 were classified as “senior” with a median age of 9.7 years (range 9.2 to 13.2 years). Throughout the study, cats in both age groups were fed a commercially available, complete, and balanced dry diet (IAMS Multi-Cat, MarsPetcare, USA) that conformed to the American Association of Feed Control and Supervision (AAFCO) adult cat nutritional guidelines. All animals were within the top 10% of their Ideal Body Condition Score (BCS) and were fed a diet designed to maintain an ideal BCS level throughout the study.

[0207] Two 3 mL blood samples were collected from the medial saphenous vein at one-month intervals to compare the concentrations of total glutathione, RBC GSH, RBC GSSG, and plasma Gly in whole blood (WB) and red blood cells (RBC).

[0208] result

[0209] Of the 32 cats participating in this cross-sectional study, one young adult cat aged 2.8 years was removed for health reasons before any data were recorded. Additionally, three cats (two young cats and one older cat) lacked a measurement of WB glutathione due to difficulties in blood collection. The data are shown in Table 1 and Figure 1.

[0210] RBC and WB glutathione levels decreased with age: compared to younger cats, older cats exhibited significantly lower total glutathione; significantly lower GSH; significantly higher GSSG; and consequently significantly lower GSH:GSSG. These findings were observed in both WB and RBC.

[0211] Plasma Gly profile remained constant with age: no significant differences in Gly levels were observed between age groups. Concentrations were all within the reference range for plasma glycine in healthy adult cats (Heinze CR, Larsen JA, Kass PH et al. (2009), American Journal of Veterinary Research (Am J Vet Res) 70, 1374-82).

[0212] Table 1. Red blood cell (RBC) and whole blood (WB) glutathione concentrations and plasma glycine (GLY) levels in young adult cats and older cats.

[0213] All values ​​are averages, with the 95% confidence interval for the average value in parentheses. GSH stands for reduced glutathione; GSSG stands for oxidized glutathione; GSH:GSSG is the ratio of reduced glutathione to oxidized glutathione. This indicates that there is a significant difference between groups (p≤0.05).

[0214] The results showed that circulating glutathione levels in cats decreased with age.

[0215] Compared to younger adult cats, older cats had lower levels of white blood cell (WB) and red blood cell (RBC) glutathione. In particular, older cats were observed to have a significantly lower GSH:GSSG ratio compared to younger cats, indicating increased oxidative stress.

[0216] No significant age-related differences were observed in plasma Gly levels. This suggests that all animals in the preliminary study were healthy and received adequate, comprehensive, and balanced diets.

[0217] Example 2: Dietary supplementation with free Gly increased circulating Gly levels

[0218] This embodiment measured the effect of dietary supplementation with different doses of free Gly on circulating Gly levels in older cats.

[0219] method

[0220] To test the required dose of Gly to increase blood Gly concentrations, 52 senior cats (33 females and 19 males), with a median age of 12.1 years (range 8.1 to 13.6 years), participated in a 16-week study involving dietary rotations over eight 2-week periods (Figure 2a provides a schematic of the study design). Cats were assigned to one of four groups and alternated between a control dry diet (IAMS Adult Cat Original Chicken, Mars Petcare, USA) or test dry diets supplemented with 0.5%, 1.5%, or 6.0% free Gly. All diets met the AAFCO adult cat nutrient profile (Table 2). All cats started on the control diet and were fed it for 2 weeks. Afterward, cats were randomly assigned to each test diet (supplemented with 0.5%, 1.5%, and 6.0% free Gly) for 2 weeks, interspersed with 2-week washout periods of the control diet.

[0221] Older animals were kept within 10% of the ideal BCS and were fed a diet that maintained the ideal BCS level throughout the study period.

[0222] At the end of each 2-week test cycle, 4 mL of fasting (overnight >18 hours) blood was collected from the medial saphenous vein of each cat into a heparin sodium vacuum blood collection tube (BD Vacutainer®). Heparin-anticoagulated Western blotting was performed to determine the primary measure of glycine formation in plasma and RBC Gly. Blood samples collected at the end of the 2-week elution cycle represent the baseline sample for the subsequent 2-week test feeding cycle. Blood samples collected at the end of the test feeding cycle represent the final endpoint sample for the previous 2-week test feeding cycle.

[0223] result

[0224] Of the 52 older cats that participated in the dosage study, one was removed due to health issues.

[0225] Gly supplementation increased RBC and plasma Gly: data are shown in Table 3 below. All 0.5%, 1.5%, and 6.0% free Gly groups showed a statistically significant increase in RBC Gly. In addition, the 1.5% and 6.0% groups showed a statistically significant increase in plasma Gly (the 0.5% group showed a trend toward increasing plasma Gly, but this was below the significance level in this experiment).

[0226] In this embodiment, glutathione levels were normalized to hemoglobin based on the following assumptions: RBC hemoglobin levels in healthy cats (including older cats) vary very little (Moritz A, Fickenscher Y, Meyer K et al. (2004), Veterinary Clin Pathol, 33, 32-8; and the reference ranges for serum biochemical analysis in the Merck Veterinary Manual (2022), https: / / www.msdvetmanual.com / special-subjects / reference-guides / serum-biochemical-analysis-reference-ranges (accessed February 2023)).

[0227] Table 2: Nutritional composition of the diets studied in Examples 2-3.

[0228]

[0229] CYS, cysteine; GLY, glycine.

[0230] Table 3. Plasma and red blood cell (RBC) glycine (GLY) levels in Example 2.

[0231]

[0232] All values ​​are averages, and the values ​​in parentheses represent the 95% confidence interval of the average. This indicates that there is a significant difference between groups (p≤0.05).

[0233] All levels of free Gly supplementation increased RBC Gly.

[0234] All levels of free Gly resulted in a statistically significant increase in RBC Gly. Based on the plasma and RBC Gly results, 1.5% free Gly was selected as the level for exploratory supplementation in the long-term feeding study (Example 3).

[0235] Example 3: Dietary supplementation with free Gly increased glutathione levels and reduced oxidative stress markers.

[0236] This study measured the effects of dietary supplementation with 1.5% free Gly on circulating glutathione levels and biomarkers of oxidative stress in older cats. Using the average body weight and average intake of the cats, 1.5% free Gly was equivalent to 1.75 mM / kg / day of total free Gly.

[0237] method

[0238] Forty-four senior cats (28 females and 16 males), with a median age of 11.7 years (range 7.7 to 13.8 years), participated in this 16-week study, which included a 4-week adaptation period and a 12-week testing period (Figure 2b provides a schematic diagram of the study design). Of these 44 senior cats, 39 participated in Example 2.

[0239] During the 4-week acclimatization period, cats were fed a control dry diet (IAMS Adult Cat Original Chicken, Mars Petcare, USA).

[0240] Subsequently, during the testing period, the senior cohort was randomly divided into two groups of 22 cats each. One group continued to receive the control dry diet, while the other group was immediately switched to a test dry diet supplemented with 1.5% free Gly (Table 2). All diets were designed to meet the AAFCO adult cat nutritional profile.

[0241] Blood and urine were collected at the end of the adaptation period (from cats that had fasted overnight for ≥18 hours) as baseline for all measurements. Blood was collected from the medial saphenous vein for the following measurements: RBC glutathione profile; WBC GSH; plasma and RBC Gly; mitogen-induced lymphocyte proliferation response; the biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the major products of DNA oxidation and widely used as a biomarker of oxidative stress; and biochemical and hematological parameters. Urine was collected for the measurement of F2-IsoPs to further quantify oxidative damage.

[0242] Blood was then collected every 4 weeks to mirror the parameter measurements taken at the end of the adaptation period, but WBC glutathione measurements were omitted in week 4.

[0243] Subsequent urine collections were conducted during the testing period and at the end of the study (week 12).

[0244] Most blood-based aliquots were obtained from 8 mL heparin-anticoagulated WB (BD Vacutainer® 4 mL heparin tubes), except for samples for biochemistry (1.2 mL; serum separation tube, SST, SARSTEDT, Inc.), hematology (1 mL; EDTA, BD Vacutainer®), and WBC glutathione (1 mL; EDTA, BD Vacutainer®), which were collected in separate blood collection tubes.

[0245] result

[0246] Forty-four senior cats were recruited. During the 16-week trial, ten cats were removed due to poor dietary intake, resulting in weight loss exceeding the study-defined threshold (-10% of ideal BCS). This included two control cats removed during the adaptation period, and three control cats and five test cats removed during the testing period, totaling five control and five test cats. This means that at the end of the feeding study, the test power for the primary metric (RBC total glutathione) was >75%, capable of detecting a 40% difference between groups. Data from cats removed during the feeding study were excluded from the statistical analysis of the study metric.

[0247] Supplementation increased total glutathione and RBC GSH in RBCs: data are shown in Table 4 and Figure 3. At week 4 of the testing period, a statistically significant difference in total glutathione and RBC GSH was observed between the test and control groups. No further statistically significant differences were found at the remaining sampling time points during the testing period, although a trend towards higher total glutathione concentrations in the test group was observed at week 12.

[0248] Increased RBC and plasma Gly levels after supplementation: Differences in Gly levels were found between the test and control groups (Table 4 and Figure 4). At all sampling time points during the test period, plasma Gly levels in the test group were significantly higher than those in the control group (p≤0.009). At the first and last time points of the test period, RBC Gly levels in the test group were significantly higher than those in the control group (p≤0.004).

[0249] Leukocyte glutathione: The effects of 1.5% free GLY supplementation on the absolute number of WBC subsets and the concentration of glutathione within WBC subsets were assessed by flow cytometry in specific cell populations: CD4+ T cells, CD8+ T cells, CD14+ monocytes, CD21+ B cells, and granulocytes (Supplementary Table 2). A statistically significant difference was found: at week 8 of the test period, the absolute number of CD21+ cells (a marker of B cells) in the test group was higher than that in the control group (difference value 463.0, 95% CI (188.0, 738.0) cells / year). L; p < 0.001). No further statistically significant differences in absolute cell count or glutathione concentration were observed, whether measured by cell count per μL per subpopulation or by GSH amount per cell.

[0250] No change in lymphocyte proliferation response after supplementation: Throughout the test period, ConA-induced lymphocyte proliferation activity in cats fed a diet supplemented with 1.5% free Gly was similar to that in each other and was not significantly different from that in the control group (p≥0.35; data not shown).

[0251] Reduced oxidative damage markers after supplementation: Gly dietary supplementation was found to affect 8-OHdG and specific urinary F2-IsoPs as markers of oxidative damage (Table 6). A significant decrease in 8-OHdG was observed at week 8, and PGF was observed at week 12. 2α and 2,3-dinor-5,6-dihydro-8-iso-PGF 2α Significantly reduced. Also at week 12, 8-iso-PGF 2α It showed a decreasing trend, but did not reach statistical significance.

[0252] Biochemistry and Hematology: During the study period, parameters in the complete biochemistry panel for both the control and test groups, including symmetric dimethylarginine (SDMA), Na:K ratio, creatine kinase phosphate level, plasma amino acid levels except glycine, blood urea nitrogen (BUN), basophil parameters, MCV, and MCHC, remained within healthy ranges (data not shown). One exception was cholesterol levels; at weeks 4 and 8, cholesterol levels in the test group were significantly lower than in the control group (p≤0.01).

[0253] Table 4. Erythrocyte (RBC) glutathione and glycine (GLY) concentrations and plasma GLY levels in aged cats in the test group (supplemented group) and control group (unsupplemented group) in Example 3 (continued on next page).

[0254]

[0255] All values ​​are averages, with the 95% confidence interval for the average value in parentheses. GSH stands for reduced glutathione; GSSG stands for oxidized glutathione; GSH:GSSG is the ratio of reduced glutathione to oxidized glutathione. This indicates that there is a significant difference between groups (p≤0.05).

[0256] Table 5: Leukocyte glutathione concentration in aged cats in the test group (supplemented group) and the control group (unsupplemented group).

[0257] (a) Absolute cell count (cells / µL), (b) Average quantification per cell (g x 10⁻⁶) - ¹³)

[0258] All values ​​are averages, and the values ​​in parentheses represent the 95% confidence interval for the average. This indicates that there is a significant difference between groups (p≤0.05).

[0259]

[0260] All values ​​are averages, and the values ​​in parentheses represent the 95% confidence interval for the average.

[0261] Table 6. Oxidative stress concentrations in aged cats in the test group (supplemented group) and control group (unsupplemented group) during the GLY feeding study.

[0262]

[0263] All values ​​are averages, with the 95% confidence interval in parentheses. Cr, creatinine; 8-OHdG, 8-hydroxy-2'-deoxyguanosine. This indicates that there is a significant difference between groups (p≤0.05).

[0264] Glycine supplementation increased glycine and glutathione levels in older cats and reduced markers of oxidative stress.

[0265] Feeding older cats a dry diet supplemented with 1.5% free Gly for 12 weeks significantly increased RBC glutathione levels in the early stages, while reducing markers of oxidative stress. Overall, this suggests that administration of free Gly can address age-related decline in glutathione function in this species.

[0266] Significant differences in RBC Gly levels were observed between the study groups at the first and last sampling time points after supplementation. However, no statistical significance was reached at intermediate sampling time points. These findings are consistent with the observations of total RBC glutathione. High RBC Gly levels were correlated with increased intracellular glutathione synthesis at weeks 4 and 12 of the supplementation period.

[0267] To explore the effects of Gly dietary supplementation on oxidative stress beyond glutathione, several biomarkers of oxidative stress were measured. F2-IsoPs are indicators of oxidative stress in vivo. Because F2-IsoPs are chemically stable due to the lack of artificial self-oxidation, they are typically measured in urine. Therefore, this supplementation study aimed to analyze a range of urinary F2-IsoPs. This study showed that after 12 weeks of supplementation with 1.5% free Gly, two urinary biomarkers of lipid peroxidation, PGF, were significantly reduced in the test group. 2α and 2,3-dinor-5,6-dihydro-8-iso-PGF 2α The levels were significantly lower in the test group than in the control group. Furthermore, although there was no statistically significant difference, the urinary 8-iso-PGF in the test group was significantly lower. 2α It shows a decreasing trend. Prostaglandin F 2α (PGF) 2α ( ) is an eicosanoic acid-like substance produced during periods of oxidative stress, which promotes 8-iso-PGF 2α and 2,3-dinor-5,6-dihydro-8-iso-PGF 2α The formation of these oxidative stress markers was observed. Our findings suggest that these markers were reduced in older cats fed a diet supplemented with free Gly.

[0268] Furthermore, oxidative damage to DNA was specifically investigated by measuring plasma 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG), considered one of the best measures of the mutagenic consequences of oxidative stress. At week 8, this biomarker was statistically significantly lower in the test group compared to the control group, but not at weeks 4 or 12.

[0269] In summary, these results indicate that Gly dietary supplementation reduces oxidative stress markers in older cats, which appears to be dependent on an increase in blood cell glutathione.

[0270] The results indicate that dietary supplementation with free Gly, a precursor to glutathione, provides a feasible approach to alleviate age-related glutathione reduction observed in older cats. Significant increases in plasma and RBC Gly levels were observed in the test group at most time points following supplementation.

[0271] In summary, Examples 1-3 above demonstrate that glutathione levels are significantly lower in older cats compared to younger cats. Supplementing older cats with a dry diet containing 1.5% free Gly for 12 weeks induced a significant increase in total RBC glutathione and GSH in the early stages after supplementation, and it was also found to affect WBC levels and oxidative stress markers. Overall, the results indicate that age-related reductions in RBC GSH in cats can be addressed through dietary free Gly supplementation.

[0272] Example 4: Effects of age on glutathione and glycine levels in dogs

[0273] This example compares the total levels of total glutathione, total levels of reduced glutathione (GSH) and oxidized glutathione (GSSG), plasma levels of RBCs, and plasma Gly levels in young and old dogs.

[0274] method

[0275] Twenty-nine healthy adult Beagles (24 females, 5 males) and three healthy Brittany Beagles (1 female, 2 males) participated in this cross-sectional study. Sixteen were classified as “young,” with a median age of 3.6 years (range 2.7 to 4.7 years), and 16 were classified as “old,” with a median age of 11.4 years (range 9.1 to 14.4 years). Throughout the study, dogs in both age groups were fed a commercially available, complete, and balanced dry diet conforming to the American Association of Feed Control and Supervision (AAFCO) adult dog nutritional profile. All animals were within the top 10% of their Ideal Body Condition Score (BCS) and were fed a diet designed to maintain an ideal BCS level throughout the study.

[0276] Two 3 mL blood samples were collected from the medial saphenous vein at one-month intervals to compare the concentrations of total glutathione, RBC GSH, RBC GSSG, and plasma Gly in whole blood (WB) and red blood cells (RBC).

[0277] result

[0278] Of the 32 dogs participating in this cross-sectional study, one 3.1-year-old dog had no GSH data due to a reduced sample size, and one 3.8-year-old dog had plasma glutathione data only at one sampling site. The data are shown in Table 7 and Figure 5.

[0279] Total glutathione and total GSH in canine RBCs decreased with age: levels in older dogs were significantly lower than in younger dogs (p < 0.05). Older dogs had higher plasma GSSG levels, thus the plasma GSH to GSSG ratio differed significantly.

[0280] The results showed that circulating glutathione levels in canines decreased in an age-related manner.

[0281] Compared to younger dogs, older dogs had lower levels of total glutathione and reduced glutathione in their RBCs. Higher plasma GSSG levels were also measured. These indicate increased oxidative stress; confirm the data for cats given in Example 1; and suggest that the age-related decline in glutathione is a common trend across different species.

[0282] Similar to the case in cats, no significant age-related differences were observed in plasma Gly levels. This likely reflects that all animals in the preliminary study were healthy and received adequate, comprehensive, and balanced diets.

[0283] Table 7. Red blood cell (RBC), whole blood (WB), and plasma glutathione concentrations and plasma glycine (GLY) levels in young and old dogs.

[0284] All values ​​are averages, with the 95% confidence interval for the average value in parentheses. GSH stands for reduced glutathione; GSSG stands for oxidized glutathione; GSH:GSSG is the ratio of reduced glutathione to oxidized glutathione. This indicates that there is a significant difference between groups (p<0.05).

[0285] General Materials and Methods

[0286] The following methods are applied throughout the above embodiments.

[0287] Animals: Domestic shorthaired cats, beagles, and Brittany beagles—all spayed / neutered—were housed at the Pet Health and Nutrition Center (PHNC) in Lewisburg, Ohio, USA. All procedures were approved by the Waltham Animal Welfare and Ethics Review Board and the Institutional Animal Care and Use Committee. At the start of the study, the animals were veterinarily determined to be healthy, with no evidence of any systemic diseases requiring treatment (such as arthritis, diabetes, thyroid disease, liver or kidney damage), and had not been vaccinated or given any medications within two weeks prior to blood collection. Throughout the study, routine feeding, care, and exercise protocols were maintained. Cats and dogs were housed separately in free-roaming environments, with access to indoor / outdoor spaces during the day (weather permitting). The rooms were equipped with environmental enrichment facilities, and all animals had social interaction with humans daily, including grooming and play, for at least 20 minutes each day. Water was always readily available. The overall health and condition of each animal were monitored daily by animal care personnel. RBC Glutathione Measurement: For Examples 1 and 4, heparin-anticoagulated WB was placed on a shaker for at least 1 minute, and then 2 mL aliquots were removed for amino acid analysis. The remaining 1 mL sample was centrifuged at 1,000 xg for 30 minutes at 4°C, and the leukocyte layer was removed from the RBC precipitate. GSH and GSSG assays were performed using a glutathione detection ELISA (EnzoLife Sciences, Farmingdale, NY, USA, catalog number ADI-900-160) according to the manufacturer's instructions, with GSSG enzymatically recovered to GSH, including sample pretreatment with metaphosphate to remove interfering proteins. Absorbance was measured every 1 minute at 405 nm in kinetic mode for 12 minutes using a Cytation 3 multi-functional microplate reader (BioTek, Winooski, VT, USA). Total glutathione, GSH, GSSG, and their ratios were measured in Western blotting (WB) and red blood cells (RBC); GSH was calculated as the difference between total glutathione and GSSG. Based on the assumption of minimal variability in RBC hemoglobin levels in healthy cats, data were not normalized to hemoglobin.

[0288] In Examples 2 and 3, 1 mL of heparin-anticoagulated WB aliquots were centrifuged at 2000 x g for 10 minutes. The leukocyte layer was then removed from the RBC precipitate, and the precipitate was washed three times with PBS. The aliquots were then purged with nitrogen, rapidly frozen in liquid nitrogen, and stored at -80°C. The samples were transported on dry ice to Creative Proteomics (New York, USA) for RBC glutathione quantification. Internal standard (IS) solutions containing 13C-labeled GSH and 13C-labeled GSSG were prepared in antioxidant buffer. Serially diluted IS solutions were also prepared in antioxidant buffer. The RBC precipitate was thawed on ice and diluted 50-fold with antioxidant buffer. 50 µL of each RBC solution or IS calibration solution was mixed with 50 µL of IS solution and 300 µL of 40-mM N-ethylmaleimide-acetonitrile. After vortexing at room temperature (RT) for 10 minutes, the resulting solution was centrifuged, and the supernatant was diluted 5-fold with water. Aliquots of 10 µL of the diluted supernatant were taken and analyzed by UPLC-MRMMS on an Agilent 1290 UHPLC system coupled with an Agilent 6495B QQQ mass spectrometer (positive ion detection). Separation was performed using a 10 cm C18 UPLC column with gradient elution (5% to 75% B over 10 minutes) using ammonium acetate buffer (A) and methanol (B) as the mobile phase, at a column temperature of 40°C and a flow rate of 0.20 mL / min. The concentrations of GSH and GSSG were calculated from the IS calibration by interpolating the constructed linear regression curve based on the analyte-to-IS peak area ratio measured from the sample solution.

[0289] Plasma and RBC Gly quantification: Heparin-anticoagulated white blood cells (WB) were centrifuged at 3000 g for 30 min at 4 ± 2°C (for Examples 1 and 4), or at 2000 g for 10 min (for Examples 2-3) to separate plasma, leukocyte layer, and RBCs. For Examples 1 and 4, plasma was removed and deproteinized with 6% sulfosalicylic acid (1:1) to remove plasma proteins; the sample was centrifuged at 4000 g for 25 min; the supernatant was filtered through a 0.45 mm PTFE filter, the pH was adjusted to 2.2, and the sample was immediately frozen at -80°C. For Examples 2-3, plasma was removed and immediately frozen at -80°C. The RBC precipitate was washed three times with cold phosphate-buffered saline (PBS) and then stored at -80°C. Gly assays of plasma and RBCs were performed at the Amino Acid Laboratory, University of California, Davis. Glycine was quantified using a Biochrom 30 amino acid analyzer (Biochrom Ltd., Cambridge, UK).

[0290] WBC Glutathione Measurement: GSH concentrations in WBC subsets were measured by the Veterinary Clinical Flow Cytometry Service at Ohio State University (Columbus, Ohio, USA). The method used was adapted from Webb et al. (2006) using a Cytek® Northern Lights (Cytek Biosciences Inc.) spectroscopic flow cytometer. WBC subsets were differentiated as part of a single-multiplex assay panel using WBC subset markers CD21 (B cells; clone CA2.1D6, Bio-Rad Laboratories Inc., Hercules, CA, catalog number MCA1781R), CD4 (helper T cells; clone vpg34, Bio-Rad Laboratories Inc., catalog number MCA1346F), CD8 (cytotoxic T cells; clone vpg9, Bio-Rad Laboratories Inc., catalog number MCA1347GA), and CD14 (monocytes; clone TÜK4, Bio-Rad Laboratories Inc., catalog number MCA1568GA) and viability marker (propidium iodide, Sigma-Aldrich, Merck, Burlington, MA, catalog number P4170). In addition to the aforementioned subpopulations, granulocytes (the sum of neutrophils, eosinophils, and basophils) were quantified and differentiated based on characteristic light scattering properties and the absence or absence of CD21, CD4, CD8, or CD14 expression. In short: To assess GSH in cells, a non-fluorescent substrate, monochlorodiamine (mBCl, Thermo Fisher Scientific Inc., Waltham, MA, catalog number M1381MP), was added to the assay panel. This substrate forms a fluorescent adduct with glutathione under the catalysis of glutathione S-transferase. The median fluorescence intensity (MFI) of the mBCl-glutathione complex was recorded for each WBC subpopulation (B cells, CD4+ T cells, CD8+ T cells, monocytes, and granulocytes). Then, MFI was correlated with the amount of GSH per cell using a standard curve generated from serial dilutions of known amounts of feline peripheral blood WBCs, which were simultaneously assessed by flow cytometry and a commercial glutathione assay kit (Cayman Chemical, Ann Arbor, MI).Pairwise comparisons were performed for each WBC subset, taking into account both the number of cells in each subset per μL of blood (absolute cell count) and the amount of GSH per cell within each subset.

[0291] Mitogen-induced lymphocyte proliferation: Heparin-anticoagulated white blood cells (WBCs) were centrifuged at 390 g for 30 min at room temperature. The leukocyte layer was removed, and WBCs were isolated using the Histopaque density gradient separation method (Histopaque®-1077, Merck, catalog number 10771 and Histopaque®-1119, Merck, catalog number 11191). The isolated WBCs were counted using a Beckman Z2 granulocyte counter at 1 x 10⁻⁶ for each stimulation condition. 6 Cells were seeded per well in triplicate. Concanavalin A (Con A; from Jack Bean, Merck, catalog number C5275) was added to the wells at concentrations of 0 mg / mL, 1 mg / mL, or 10 mg / mL. Cells were incubated at 37°C in a 5% CO2 enrichment environment for 96 hours. After incubation, the plates were centrifuged at 350 g for 10 minutes at room temperature and subjected to MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Merck, catalog number M5655) according to the manufacturer's instructions to quantify viable cells as a surrogate indicator of proliferation relative to the control group.

[0292] Oxidative DNA Damage: 8-Hydroxy-2'-deoxyguanosine (8-OHdG) was measured using the OxiSelect™ Oxidative DNA Damage ELISA Kit (Cell Biolabs, Inc., USA, Catalog No. STA-320) according to the manufacturer's instructions. Urine samples collected for the measurement of F2-IsoPs were stored at -80°C before being shipped to the Vanderbilt Eicosanoid Core Laboratory (Nashville, Tennessee, USA) for the determination of F2-IsoPs by gas chromatography-negative ionization chemical ionization-mass spectrometry. 2α (PGF) 2α ), free 8-iso-prostaglandin F 2α (8-iso-PGF) 2α ), 2,3-dinor-5,6-dihydro-15-F 2t Quantification of -IsoP and 5-series F2-IsoP was performed using stable isotope dilutions, as described by Milne et al. (2007) and Milne et al. (2013).

[0293] Biochemistry and hematology: conducted at IDEXX Laboratories in the United States.

[0294] Statistical Methods: The sample sizes for Examples 1 and 4 were determined using variance estimates of total RBC glutathione from an unpublished canine study at the Waltham Center for Pet Care Sciences. To use this prior data to calculate the power of the test for the cat study, it was assumed that the variability in cats was comparable to that in dogs. 1000 datasets were modeled using variance components, with a 25% effect size imposed on the older group. A linear mixed-effects model was fitted to each dataset, with age as a fixed effect and animal species as a random effect. The planned comparisons in this study were between the younger adult group and the older group.

[0295] Gly data from a previously unpublished cat study were used to estimate the variance components to calculate the sample size for Example 2. Variance components for plasma Gly and RBC Gly were estimated and used to simulate 1000 datasets, with fold changes of 23% and 30% respectively applied to plasma Gly and RBC Gly at the final sampling point in one diet group. A linear mixed-effects model was fitted to each dataset for each metric, with diet, sampling time point, and their interaction as fixed effects and individual cats as random variables. The planned comparisons for Example 2 were the changes from baseline at each time point between groups.

[0296] Using data collected from Example 1, the test power of Example 3 was calculated by estimating the variance component (in pmol) of total RBC glutathione and simulating 1000 datasets, applying a 40% fold change at the final sampling point. A linear mixed-effects model was fitted to each dataset for each metric, with diet groups, sampling time points, and their interactions as fixed effects, and individual cats as random effects. The planned comparisons for Example 3 were between different sampling time points within each diet group, and between different diet groups at each sampling time point.

[0297] For all analyses, power was calculated as the percentage of simulated datasets in which all planned comparisons involving fixed effect sizes applied were statistically significant. Sample size was determined as the minimum number of animals required to observe the desired effect size with at least 80% power.

[0298] In Examples 1 and 4, all sampling time points were pooled before fitting a linear mixed-effects model, with age groups as fixed effects and individual animals as random effects. Model residuals were visually examined to assess model assumptions (linearity of predictors, normality of residuals, independence of variables, and homoscedasticity). To better fit the model assumptions, some parameters were log10 transformed before model fitting. Plasma GSH to GSSG ratio data were log10 transformed after adding 1 (the minimum added to remove negative values). Mean values ​​for each measure within each age group and differences between age groups (or fold changes in data after log10 transformation), with 95% confidence intervals (CIs), were estimated.

[0299] For all measures in Examples 2-3, the data were fitted to a linear mixed-effects model (to account for repeated measures), with diet, sampling time points, and their interactions as fixed effects, and individual cats as random effects. The model residuals were visually examined to assess the model's assumptions. Parameters deemed to violate the assumptions were log10 transformed before model fitting. The estimated mean and 95% pedigree confidence interval for each diet at each sampling time point were extracted from the model.

[0300] In Example 2, the changes from baseline to each subsequent time point were compared between different diet groups.

[0301] Using the data from Example 3, baseline values ​​for each animal were included as covariates in the model to account for any potential differences between groups. Comparisons were made between different diet groups at each sampling time point.

[0302] For Examples 1, 3, and 4, the primary metric was total RBC glutathione, while for Example 2, the primary variables were plasma and RBC GLY.

[0303] For all comparisons, reported estimates of differences or fold changes were accompanied by a 95% phylogenetic CI and a single-step corrected p-value. A statistically significant difference was determined when the p-value was ≤ 0.05. Statistical analysis was performed using the nlme, multcomp, lme4, and ggplot2 packages in R v4.1.2.

[0304] It will be understood that the above description of the inventors’ work is by way of example only, and modifications may be made while remaining within the scope and spirit of the invention.

[0305] All of the above references are incorporated into this paper in their entirety through citation.

Claims

1. A dietary composition for companion animals, said composition comprising at least about 0.5% free glycine.

2. The composition according to claim 1, wherein it comprises at least about 1.5% free glycine.

3. The composition according to claim 2, comprising about 1.5% free glycine.

4. The composition according to claim 1, wherein it comprises at least about 6.0% free glycine.

5. The composition according to claim 4, comprising about 6.0% free glycine.

6. The composition according to any one of the preceding claims, wherein the moisture content of the composition is at most about 7.0%.

7. The composition according to any one of the preceding claims, wherein the composition comprises a nutritionally complete meal for the companion animal.

8. The composition according to any one of the preceding claims, wherein the companion animal is a cat or a dog.

9. The composition of claim 8, wherein the companion animal is an elderly cat or an elderly dog.

10. The composition according to any one of the preceding claims, wherein the companion animal is a cat, and optionally wherein the companion animal is an older cat.

11. The composition according to claim 9 or 10, wherein the elderly cat or dog is at least about 7.0 years old, optionally wherein the elderly cat or dog is at least about 9.5 years old.

12. A method for treating or preventing a disease in a companion animal, the method comprising administering to the animal the dietary composition of any one of the preceding claims.

13. The method of claim 12, wherein the disease is associated with glutathione dysfunction.

14. The method of claim 12 or 13, wherein the disease is associated with oxidative stress.

15. A method for increasing glutathione and / or GSH in companion animals, the method comprising administering a dietary composition according to any one of claims 1 to 11.

16. The method according to any one of claims 12 to 15, wherein the method increases intracellular glutathione concentration, circulating glutathione concentration, intracellular GSH concentration and / or circulating GSH concentration.

17. The method according to any one of claims 12 to 16, wherein the concentration is increased to at least about 500 µM.

18. The method of claim 17, wherein the concentration is increased to at least about 600 µM.

19. The method according to any one of claims 12 to 18, wherein the method increases the intracellular glutathione concentration, circulating glutathione concentration, intracellular GSH concentration and / or circulating GSH concentration by at least about 20% compared to the concentration without the method.

20. The method according to any one of claims 12 to 19, wherein administration of the dietary composition results in immunological or physiological changes in the companion animal, such as increased lymphocytes, increased neutrophils, or pancytopenia.

21. A method for determining the presence of glutathione dysfunction in companion animals, the method comprising: (a) Provide a blood sample from the companion animal; (b) Measure the levels of glutathione (GSH) and / or glutathione disulfide (GSSG) in the sample; and (c) Determine the presence of glutathione dysfunction if the intracellular GSH:GSSG ratio, circulating GSH:GSSG ratio, circulating GSH concentration, or intracellular GSH concentration is less than a threshold.

22. The method of claim 21, further comprising determining glutathione dysfunction if the intracellular GSH:GSSG ratio is less than about 2.0, the circulating GSH:GSSG ratio is less than about 6.0, the circulating GSH concentration is less than about 500 µM, or the intracellular GSH concentration is less than about 500 µM.

23. The method of claim 21 or 22, wherein the sample comprises whole blood, and the levels of GSH and / or GSSG include (a) the levels of circulating GSH and / or circulating GSSG, or (b) the levels of intracellular GSH and / or GSSG.

24. The method of claim 23, wherein the intracellular GSH and / or GSSG is erythrocyte GSH and / or GSSG.

25. The method of any one of claims 20 to 24, wherein if glutathione dysfunction is determined to exist in step (c), the method includes selecting a companion animal to which the method of any one of claims 12 to 20 is applicable.

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