Companion animal supplementation comprising free glycine

Abstract

The invention concerns dietary compositions comprising free glycine, and associated methods concerning glutathione in companion animals.

Description

[0001] COMPANION ANIMAL SUPPLEMENTATION COMPRISING FREE GLYCINE

[0002] FIELD OF THE INVENTION

[0003] The invention concerns dietary compositions comprising free glycine, and associated methods concerning glutathione in companion animals.

[0004] BACKGROUND OF THE INVENTION

[0005] Ageing is associated with increased oxidative stress. This entails a progressive decline in the ability to manage levels of oxidative damage, which results in changes to cellular biomolecules including proteins, lipids, and nucleic acids.

[0006] The ability to withstand oxidative damage is determined by the capacity of several antioxidant defence systems. Glutathione is the most prevalent and abundant intracellular antioxidant in mammals. Intracellular glutathione levels have been reported to decline with age in a number of species.

[0007] SUMMARY OF THE INVENTION

[0008] The invention provides dietary composition for a companion animal, the composition comprising at least about 0.5% free glycine.

[0009] The invention further provides a method of treating or preventing a disease in a companion animal, the method comprising administering to the animal a dietary composition of the invention.

[0010] The invention further provides a method of increasing glutathione and / or GSH in a companion animal, the method comprising administering the dietary composition of the invention.

[0011] The invention further provides a method of determining the presence or absence of glutathione dysfunction in a companion animal, the method comprising:

[0012] (a) providing a blood sample from the companion animal;

[0013] (b) measuring the level of glutathione (GSH) and / or glutathione disulphide (GSSG) in the sample; and (c) identifying glutathione dysfunction as present if the intracellular GSH:GSSG ratio, the circulating GSH:GSSG ratio, the circulating GSH concentration, or the intracellular GSH concentration is less than a threshold value.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows mean (a) red blood cell (RBC) and (b) whole blood (WB) total glutathione (pM) in tested cats by age group, per Example 1. Individual data are shown as open circles and means as solid circles with 95% confidence intervals. * indicates significance between groups (p < 0.05).

[0016] Figure 2 shows schematics for the experiments conducted in (a) Example 2 and (b) Example 3. Blood (triangular arrows) and urine (diamond arrows) sample collection time points and associated sample measures are shown (ticked where measured).

[0017] Figure 3 shows mean red blood cell (RBC) glutathione concentrations for test (supplemented) and control (unsupplemented) senior cats per Example 3. RBC glutathione represented as (a) total glutathione (GSH + GSSG), (b) reduced glutathione (GSH), (c) oxidised glutathione (GSSG), and (d) GSH:GSSG. Individual data are shown as open circles and means as solid circles with 95% confidence intervals. * indicates significance between groups (p < 0.05).

[0018] Figure 4 shows mean (a) plasma and (b) red blood cell (RBC) glycine (Gly) levels per Example 3. Individual data are shown as open circles and means as solid circles with 95% confidence intervals. * indicates significance between groups (p < 0.05).

[0019] Figure 5 shows mean red blood cell (RBC) total GSH (nM / 50pl) in tested dogs by age group, per Example 4. Individual data are shown as open circles and means as solid circles with 95% confidence intervals.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Definitions

[0022] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, and molecular biology, within the skill of the art. Such techniques are explained fully in the literature. See e.g. Methods In Enzymology (Academic Press, Inc.), Green & Sambrook (2012) Molecular Cloning: A Laboratory Manual, 4th edition (Cold Spring Harbor Press), Ausubel et al. (eds) Short protocols in molecular biology, 5th edition (Current Protocols), Molecular Biology Techniques: An Intensive Laboratory Course, (Ream & Field, eds., 1998, Academic Press), Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology (Hodmann & Clokie, 2018), Basic Molecular Biology & Techniques - Recent Advances: Molecular Biology & Its Technique (Singh et al., 2021), etc.

[0023] Use of the term “about” when referring to a quantity is optional, and means that the quantity may be varied according to what the skilled person would recognise as the structure or function associated with the quantity. For example, “about” 1.5% free glycine refers to a quantity of free glycine that is suitable to achieve an associated technical effect, e.g. a measurable change in plasma glycine and / or RBC glycine levels when the amount of free glycine is fed to a companion animal, such as a cat or dog. “About” may also, for example, be defined as + / -10% of a stated quantity.

[0024] “Between” (and variations such as “x toy”, “x-y”), with reference to two values, includes those two values e.g. the range “between” 10 mg and 20 mg encompasses inter alia 10, 15, and 20 mg.

[0025] “Body composition score” or “BCS” is a semiquantitative scoring system for measuring body condition in companion animals, based on visual and palpatory findings. BCSs include a 9 point system developed and validated for dogs and cats. BCS may be combined with bodyweight (BW), body fat (BF), body mass index (BMI) and / or thoracic measurements, to more accurately determine BCS and the companion animal health with which BCS is associated. See e.g. LaFlamme DP. Development and validation of a body condition score system for cats: a clinical tool. Feline Pract 1997; 25(5-6): 13-18.

[0026] “Companion animal” means an animal, such as a mammal, including (for example) cats (e.g. adult cats, senior cats), dogs (e.g. adult dogs, senior dogs), horses, cows, pigs, rabbits, guinea pigs, hamsters, gerbils, ferrets, zoo mammals, fish, birds, and the like. Preferred companion animals include cats and dogs, such as senior cats and senior dogs. Cats, and especially senior cats, are particularly preferred.

[0027] “Complete and nutritionally balanced” (and variations such as “nutritionally complete”) refers to a composition having all known required nutrients in proper amounts and proportions based upon the recommendation of recognized authorities in the field of companion animal nutrition. Such authorities will be well known to the skilled person, and include for example AAFCO (Association of American Feed Control Officials), and the FEDIAF nutritional guidelines for complete and complementary pet food for cats and dogs (http: / / www.fediaf.org / self-regulation / ). A complete and nutritionally balanced composition may be fed as the sole ration to a companion animal, and is capable of maintaining life and / or promoting reproduction without any additional substance being consumed, except for water.

[0028] The term “comprising” encompasses “including” as well as “consisting of’, e.g. a composition “comprising” X may consist exclusively of X or may include something additional (e.g. X + Y).

[0029] In general, the term “disease” refers to a state of being or the health status of a companion animal, which is capable of being treated using the methods provided herein. “Treatment” encompasses the complete removal of disease, as well as palliative alleviation of disease symptoms. The term “disease” is used synonymously with “condition” and “disorder” herein. Diseases may be clinically recognised in the veterinary profession. However, diseases also include other states or being or health statuses of companion animals that can be treated using the method provided herein. “Prevention” refers to reduction in the severity and / or likelihood of disease, prior to onset of disease.

[0030] “Free” in relation to an amino acid refers to the amino acid as a monomer, as opposed to the amino acid residue as part of a polymer (e.g. an oligopeptide or protein). Free amino acids may interact transiently with other substances, for example through hydrogen bonding, but are not covalently bound to other monomers. Thus, free glycine may refer to monomeric glycine.

[0031] “Glutathione” comprises a tripeptide of glutamate (GLU or Glu), cysteine (CYS or Cys), and glycine (GLY or Gly). Glutathione may be reduced as a monomer (“GSH”), or oxidised as glutathione disulphide - a dimer bound at the sulphur atoms (“GSSG”). “Whole blood” refers to blood containing cells, liquid, and clotting factors (such as a sample of venous blood). Whole blood may be separated into fractions. “Plasma” refers to the liquid remaining after a sample of whole blood is subjected to a separation process to remove the blood cells, typically involving centrifugation. “Serum” refers to blood plasma without clotting factors such as fibrinogen. Methods of the invention may be applied to whole blood, venous blood, plasma, or serum.

[0032] Glutathione

[0033] Glutathione is the most abundant intracellular antioxidant in mammals. Glutathione is present in the circulation of mammals in oxidised (GSSG) and reduced (GSH) forms. In the reduced state, GSH can react with cysteine residues within proteins to maintain their reduced forms, or with reactive oxygen species (ROS) to neutralise them. This occurs via the thiol group of GSH, which is able to donate electrons to other molecules. Consequently, GSH becomes reactive itself and forms a disulphide bridge with another reactive glutathione to form oxidised glutathione disulphide (GSSG). GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR) in the presence of NADPH.

[0034] Glutathione counteracts oxidative stress, by scavenging reactive oxygen species (ROS) and inhibiting activation of macrophages, which minimizes associated transcription factor activation and cytokine production. Glutathione also reduces superoxide formation in specific macrophages and neutrophil types.

[0035] Thus, glutathione (or ‘total glutathione’) includes both GSH and GSSG, and where amounts are measured, this includes both GSH and GSSG, unless indicated otherwise. Circulating glutathione is glutathione (GSH + GSSG) detectable in the circulation, for example in blood. Circulating glutathione comprises extracellular glutathione (e.g. in whole blood, plasma or serum) and intracellular glutathione (i.e. in the blood cells that are suspended in plasma, such as RBCs). References to ‘glutathione’ mean ‘circulating glutathione’ unless indicated otherwise.

[0036] In some embodiments, circulating glutathione is extracellular glutathione but not intracellular glutathione (i.e. glutathione in whole blood, plasma or serum, but not in blood cells, e.g. glutathione in plasma). In some embodiments, circulating glutathione is intracellular glutathione, but not extracellular glutathione (i.e. glutathione in blood cells that are suspended in plasma, such as red blood cells (RBCs) and / or white blood cells (WBCs)) . In some embodiments, intracellular glutathione is glutathione in RBCs.

[0037] In some embodiments, extracellular glutathione is glutathione in whole blood, for example glutathione in plasma.

[0038] In other embodiments, glutathione is glutathione in WBCs. Glutathione has been shown to play an important role in the activation of T-lymphocytes.

[0039] Glutathione dysfunction, ageing, and disease

[0040] Glutathione dysfunction and measurement

[0041] A decreased GSH to GSSG ratio is considered indicative of oxidative stress. Determining the presence or absence of glutathione dysfunction may therefore comprise assessing whether glutathione, GSH, GSSG, or combinations of these, deviate from threshold values. Example threshold values are provided herein.

[0042] In companion animals such as healthy young adult cats (e.g. less than 3 years old), circulating (e.g. extracellular, for example whole blood) GSH:GSSG is at least about 10, for example from about 10 to about 25, for example about 16. In companion animals such as healthy young adult cats, intracellular (e.g. RBC) GSH:GSSG is at least about 7, for example from about 7 to about 21, for example about 12.

[0043] In companion animals such as healthy young adult cats (e.g. less than 3 years old), circulating (e.g. extracellular, for example whole blood) glutathione is at least about 450 pM, for example from about 450 pM to about 550 pM, for example about 480 pM. In companion animals such as healthy young adult cats, intracellular (e.g. RBC) glutathione is at least about 850 pM, for example from about 850 to about 1165 pM, for example about 950 pM.

[0044] In companion animals such as healthy young adult cats (e.g. less than 3 years old), circulating (e.g. extracellular, for example whole blood) GSH is at least about 370 pM, for example from about 370 pM to about 510 pM, for example about 450 pM. In companion animals such as healthy young adult cats, intracellular (e.g. RBC) GSH is at least about 440 pM, for example from about 440 pM to about 1260 pM, for example about 860 pM.

[0045] In companion animals such as healthy young adult dogs (e.g. less than 3 years old), circulating (e.g. extracellular, for example whole blood) GSH:GSSG is at least about 21, for example from about 21 to about 47, for example about 31. In companion animals such as healthy young adult dogs, intracellular (e.g. RBC) GSH:GSSG is at least about 20, for example from about 20 to about 38, for example about 29.

[0046] In companion animals such as healthy young adult dogs (e.g. less than 3 years old), circulating (e.g. extracellular, for example whole blood) glutathione is at least about 0.75 pM / ml, for example from about 0.75 pM / ml to about 0.90 pM / ml, for example about 0.83 pM / ml. In companion animals such as healthy young adult dogs, intracellular (e.g. RBC) glutathione is at least about 1.16 pM / ml, for example from about 1.16 pM / ml to about 1.36 pM / ml, for example about 1,26 pM / ml.

[0047] In companion animals such as healthy young adult dogs (e.g. less than 3 years old), circulating (e.g. extracellular, for example whole blood) GSH is at least about 0.72 pM / ml, for example from about 0.72 pM / ml to about 0.88 pM / ml, for example about 0.80 pM / ml. In companion animals such as healthy young adult dogs, intracellular (e.g. RBC) GSH is at least about 1.09 pM / ml, for example from about 1.09 pM / ml to about 1.30 pM / ml, for example about 1.19 pM / ml.

[0048] Methods of measuring GSH and / or GSSG will be readily available to the skilled person, for example by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (e.g. Enzo Life Sciences, Farmingdale, NY, USA, Cat No. ADI-900-160), or according to standard measurements performed using radiolabelled GSH and / or GSSG (e.g. 13C isotope-labelled GSH and 13C isotope-labelled GSSG) followed by HPLC-MS. GSH may be calculated as the difference between total glutathione and GSSH. GSH and / or GSSG may be normalised to a biomarker (such as haemoglobin), or may not be normalised to a biomarker (such as haemoglobin).

[0049] Glutathione dysfunction refers to improper functioning of glutathione metabolism, such as inadequate circulating glutathione (i.e. inadequate combined levels of GSH and GSSG, intracellular and extracellular), inadequate circulating GSH, as well as inappropriate circulating GSH:GSSG.

[0050] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises an intracellular glutathione concentration of less than about 950 pM, less than about 900 pM, less than about 850 pM, less than about 800 pM, less than about 750 pM, less than about 700 pM, less than about 650 pM, less than about 600 pM, or less than about 550 pM. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is less than about 950 pM, for example less than about 850 pM. In particularly preferred embodiments, the concentration is less than about 700 pM, for example less than about 550 pM. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0051] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises a circulating glutathione concentration of than about 450 pM, less than about 440 pM, less than about 420 pM, less than about 400 pM, less than about 380 pM, less than about 370 pM, less than about 360 pM, or less than about 350 pM. In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is less than about 450 pM, for example less than about 370 pM. In particularly preferred embodiments, the concentration is less than about 400 pM, for example less than about 350 pM. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0052] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises an intracellular GSH concentration of less than about 860 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 550 pM, less than about 500 pM, less than about 450 pM, less than about 440 pM, less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, or less than about 200 pM. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is less than about 860 pM, for example less than about 440 pM. In particularly preferred embodiments, the concentration is less than about 300 pM, for example less than about 200 pM. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0053] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises a circulating GSH concentration of than about 450 pM, less than about 440 pM, less than about 420 pM, less than about 400 pM, less than about 380 pM, less than about 360 pM, less than about 340 pM, less than about 320 pM, less than about 310 pM, less than about 300 pM, less than about 280 pM, less than about 260 pM, less than about 250 pM, or less than about 240 pM. In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is less than about 450 pM, for example less than about 370 pM. In particularly preferred embodiments, the concentration is less than about 310 pM, for example less than about 250 pM. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0054] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises an intracellular GSELGSSG ratio of 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 preferred embodiments, the ratio is less than about 12, for example less than about 7.0. In particularly preferred embodiments, the ratio is less than about 1.1, for example less than about 0.7. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0055] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises a circulating GSH:GSSG ratio of 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 preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the ratio is less than about 16.0, for example less than about 10.0. In particularly preferred embodiments, the ratio is less than about 4.0, for example less than about 2.5. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0056] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises an intracellular glutathione concentration of less than about 0.0039 pM, less than about 0.0038 pM, less than about 0.0037 pM, less than about 0.0036 pM, less than about 0.0035 pM, less than about 0.0034 pM, less than about 0.0033 pM, less than about 0.0032 pM, less than about 0.0031 pM, less than about 0.0030 pM, less than about 0.0029 pM, less than about 0.0028 pM, less than about 0.0027 pM, less than about 0.0024 pM, or less than about 0.0023 pM. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is less than about 0.0041 pM, for example less than about 0.0039 pM. In particularly preferred embodiments, the concentration is less than about 0.0034 pM, for example less than about 0.0023 pM. These concentrations for glutathione dysfunction may, for example, be normalised to haemoglobin, and may for example be measured by using radiolabelled GSH and / or GSSG (such as is reported for Example 3).

[0057] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises an intracellular GSH concentration 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 preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is less than about 3.6 pg / mL, for example less than about 3.2 pg / mL. In particularly preferred embodiments, the concentration is less than about 2.8 pg / mL, for example less than about 2.4 pg / mL. These concentrations for glutathione dysfunction may, for example, be normalised to haemoglobin, and may for example be measured by using radiolabelled GSH and / or GSSG (such as is reported for Example 3).

[0058] In some embodiments, for instance wherein the companion animal is a cat, glutathione dysfunction comprises 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 preferred embodiments, the ratio is less than about 7.0, for example less than about 6.0. In particularly preferred embodiments, the ratio is less than about 5.0, for example less than about 4.0. These concentrations for glutathione dysfunction may, for example, be normalised to haemoglobin, and may for example be measured by using radiolabelled GSH and / or GSSG (such as is reported for Example 3).

[0059] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises an intracellular glutathione concentration of less than about 1.16 pM / ml, less than about 1.14 pM / ml, less than about 1.12 pM / ml, less than about 1.10 pM / ml, less than about 1.08 pM / ml, less than about 1.06 pM / ml, less than about 1.04 pM / ml, less than about 1.02 pM / ml, less than about 1.00 pM / ml, less than about 0.98 pM / ml, less than about 0.96 pM / ml, or less than about 0.94 pM / ml. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is less than about 1.16 pM / ml, for example less than about 1.14 pM / ml. In particularly preferred embodiments, the concentration is less than about 1.04 pM / ml, for example less than about 0.94 pM / ml. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0060] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises a circulating glutathione concentration of less than about 0.90 pM / ml, less than about 0.85 pM / ml, less than about 0.80 pM / ml, less than about 0.75 pM / ml, less than about 0.70 pM / ml, or less than about 0.65 pM / ml. In preferred embodiments, the circulating concentration is the concentration in whole blood. In preferred embodiments, the concentration is less than about 0.90 pM / ml, for example less than about 0.81 pM / ml. In particularly preferred embodiments, the concentration is less than about 0.74 pM / ml, for example less than about 0.67 pM / ml. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0061] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises an intracellular GSH concentration of less than about 1.10 pM / ml, less than about 1.08 pM / ml, less than about 1.06 pM / ml, less than about 1.04 pM / ml, less than about 1.02 pM / ml, less than about 1.00 pM / ml, less than about 0.98 pM / ml, less than about 0.96 pM / ml, less than about 0.94 pM / ml, less than about 0.92 pM / ml, less than about 0.90 pM / ml, less than about 0.88 pM / ml, or less than about 0.86 pM / ml. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is less than about 1.09 pM / ml, for example less than about 1.06 pM / ml. In particularly preferred embodiments, the concentration is less than about 0.96 pM / ml, for example less than about 0.85 pM / ml. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0062] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises a circulating GSH concentration of less than about 0.90 pM / ml, less than about 0.85 pM / ml, less than about 0.80 pM / ml, less than about 0.75 pM / ml, less than about 0.70 pM / ml, or less than about 0.65 pM / ml. In preferred embodiments, the circulating concentration is the concentration in whole blood. In preferred embodiments, the concentration is less than about 0.90 pM / ml, for example less than about 0.81 pM / ml. In particularly preferred embodiments, the concentration is less than about 0.74 pM / ml, for example less than about 0.67 pM / ml. These concentrations for glutathione dysfunction may, for example, not be normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0063] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises 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 preferred embodiments, the ratio is less than about 25.0, for example less than about 20.0. In particularly preferred embodiments, 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 normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0064] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises a circulating GSH:GSSG ratio of 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 preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the ratio is less than about 1.8, for example less than about 1.7. In particularly preferred embodiments, 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 normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0065] In some embodiments, for instance wherein the companion animal is a dog, glutathione dysfunction comprises an intracellular GSSG concentration 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 preferred embodiments, the concentration is at least about 0.39 nM / ml, for example at least about 0.52 nM / ml. In particularly preferred embodiments, 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 normalised to haemoglobin, and may for example be measured by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA (such as is reported for Examples 1 and 4).

[0066] Any of the methods of the invention may be preceded and / or followed by measurement to determine whether glutathione dysfunction is present. Particularly preferred is glycine supplementation according to the invention, preceded by measurement to determine whether glutathione dysfunction is present. In some embodiments, if glutathione dysfunction is determined as present, the companion animal may be selected for administration of dietary compositions of the invention, optionally including the step of administering the dietary composition. In some embodiments, the measurement to determine whether glutathione dysfunction is present is conducted in vitro, for example using a blood sample, such as a sample comprising whole blood, venous blood, plasma and / or serum.

[0067] Whether glutathione dysfunction is present may be determined according to 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). Particularly preferred is measurement in RBCs. Glutathione and ageing

[0068] As shown in Example 1, glutathione dysfunction is correlated with age in cats. Similar results are reported for dogs in Example 4.

[0069] Lower levels of glutathione may be due to age-related changes in the activity of enzymes involved in glutathione synthesis. An age-related increase in the Michaelis constant of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, may contribute to GSH reduction and an increase in GSSG. Impaired protein digestibility has been reported with age in cats which could affect intracellular amino acid levels, and hence affect glutathione metabolism.

[0070] Glutathione and disease

[0071] Glutathione dysfunction may lead to oxidative stress, which refers collectively to the harmful effects of ROS in vivo (as when the generation of ROS exceeds the body’s ability to scavenge ROS by antioxidant systems, such as the glutathione system). ROS are generated by aerobic metabolism. In healthy cells, ROS are regulated by antioxidant mechanisms such as glutathione. Oxidative stress may be measured according to biomarkers, such as 8-hydroxy -2’ -deoxyguanosine (8-OHdG) and / or F2-isoprostanes (F2-IsoPs) such as 8-iso-PGF2a; PGF2a; and / or 2,3-dinor-5,6-dihydro-8isoPGF2a.

[0072] The effects of increased oxidative stress, such as DNA and RNA damage, have been postulated to play a central role in age-related loss of physiological functions, including immunosenescence, age-related inflammation, cardiovascular and neurodegenerative disease, osteoarthritis, and type 2 diabetes. Glutathione dysfunction has been linked to a number of other chronic diseases including feline chronic renal failure and chronic kidney disease. In particular, in chronic kidney disease, cats are known to have significantly lower circulating glutathione levels compared to clinically normal cats. See e.g. Piyarungsri K & 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.

[0073] Lower concentrations of GSH have also been characterised in the liver tissue of cats and dogs with necro-inflammatory liver disease and hepatic lipidosis compared with healthy cats and dogs, respectively. See e.g. Center SA, Warner KL & Erb HN (2002) Liver glutathione concentrations in dogs and cats with naturally occurring liver disease. Am J Vet Res 63, 1187-97.

[0074] Thus, in some embodiments, the invention concerns diseases associated with glutathione dysfunction, including the diagnosis, treatment and / or prevention of such diseases and conditions. Treatment and / or prevention of such diseases may be performing by administering a dietary composition of the invention. Diseases of particular interest in the context of the present invention include age-related inflammation and chronic kidney disease.

[0075] However, glutathione deficiency may be present without other signs of disease, for example prior to the onset of disease. Methods of the invention also encompass administration of dietary compositions to animals in the absence of disease (for example in clinically healthy senior companion animals), such that treatment and / or prevention of disease does not take place. Alternatively, administration of dietary compositions of the invention prior to the onset of disease may prevent disease without treating disease.

[0076] Glycine

[0077] Glycine levels and ageing

[0078] De novo glutathione synthesis is dependent on a two-step reaction. In a first step, a dipeptide of Gin and Cys (glutamylcysteine, “%-GC”) is synthesised from glutamate and cysteine by glutamate cysteine ligase (GCL). In a second step, %-GC participates with Gly in a reaction mediated by GSH synthase, producing glutathione.

[0079] Reports in humans suggest that older individuals have lower intracellular levels of Gly due to slower body protein turnover or decreased de novo synthesis. As glycine is a precursor to glutathione, reduced glycine levels may explain reduced glutathione levels in humans.

[0080] The inventors show in Example 1 that plasma levels of Gly were within normal range for the senior cats and comparable with young cats. The same was observed for dogs in Example 4. “Circulating glycine” is defined as above for circulating glutathione, mutatis mutandis, and references to “glycine” mean free glycine unless indicated otherwise.

[0081] Glycine and glutathione

[0082] Despite measurement of normal glycine levels in senior cats and senior dogs, the inventors have surprisingly found that supplementation of glycine, using e.g. dietary compositions of the invention, increases circulating glycine levels (Example 2), and even reverses glutathione dysfunction, with improvements in associated markers of oxidative stress (Example 3), without observable toxicity.

[0083] Without wishing to be bound by theory, the inventors consider that early on during supplementation, an excess of Gly drives the glutathione synthesis reaction with %-GC, increasing de novo generation of glutathione in the second step of the two-step reaction.

[0084] In some embodiments, glycine supplementation in accordance with the invention improves antioxidant capacity. This may be of particular use in senior cats and senior dogs. For example, glycine supplementation in accordance with the invention may lead to increased total circulating glutathione, increased circulating GSH, or increased circulating GSH to GSSG ratio. In preferred embodiments, glycine supplementation in accordance with the invention leads to increased intracellular total glutathione (i.e. GSH and GSSG), and / or increased intracellular GSH. In particularly preferred embodiments, glycine supplementation leads to increased RBC total glutathione (i.e. GSH and GSSG) and / or increased RBC GSH. For example, the increase may be measured relative to reference values such as those listed for glutathione dysfunction above, and may comprise an increase listed below. Glycine supplementation in accordance with the invention may reduce markers of oxidative stress.

[0085] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with the invention increases the intracellular glutathione concentration to at least about 550 pM, at least about 600 pM, at least about 650 pM, at least about 700 pM, at least about 750 pM, at least about 800 pM, at least about 850 pM, at least about 900 pM, at least about 950 pM, at least about 1,000 pM, at least about 1,050 pM, at least about 1,100 pM, at least about 1,150 pM, or at least about 1,200 pM. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is increased to at least about 850 pM, for example at least about 950 pM. In particularly preferred embodiments, the concentration is increased to at least about 550 pM, for example at least about 700 pM.

[0086] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased by at least about 20%, compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0087] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods of the invention 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, when normalised to haemoglobin. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased to at least about 4.2 nM when normalised to haemoglobin.

[0088] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with the invention increases the circulating glutathione concentration to at least about 350 pM, at least about 360 pM, at least about 370 pM, at least about 380 pM, at least about 390 pM, at least about 400 pM, or at least about 450 pM. In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is increased to at least about 370 pM, for example at least about 450 pM. In particularly preferred embodiments, the concentration is increased to at least about 350 pM, for example at least about 400 pM.

[0089] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is increased by at least about 10%, compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0090] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods of the invention increases the intracellular GSH concentration to at least about 200 pM, at least about 250 pM, at least about 300 pM, at least about 400 pM, at least about 440 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 860 pM, at least about 900 pM, at least about 1,000 pM, at least about 1,100 pM, or at least about 1,200 pM. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is increased to at least about 440 pM, for example at least about 860 pM. In particularly preferred embodiments, the concentration is increased to at least about 350 pM, for example at least about 450 pM.

[0091] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods of the invention 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, when normalised to haemoglobin. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased to at least about 0.52 pg / mL when normalised to haemoglobin.

[0092] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased by at least about 20%, compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0093] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods of the invention increases the circulating GSH concentration to at least about 250 pM, at least about 300 pM, at least about 310 pM, at least about 350 pM, at least about 370 pM, at least about 380 pM, at least about 400 pM, at least about 420 pM, at least about 440 pM, at least about 460 pM, at least about 480 pM, at least about 500 pM, or at least about 520 pM. In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is increased to at least about 370 pM, for example at least about 450 pM. In particularly preferred embodiments, the concentration is increased to at least about 250 pM, for example at least about 310 pM.

[0094] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is increased by at least about 20%, compared to the concentration in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation).

[0095] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 preferred embodiments, the intracellular ratio is the ratio in RBCs. In preferred embodiments, the ratio is increased to at least about 0.7, for example at least about 1.1. In particularly preferred embodiments, the ratio is increased to at least about 7.0, for example at least about 12.0.

[0096] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 1,000% compared to the ratio in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the intracellular ratio is the ratio in RBCs. In particularly preferred embodiments, the ratio is increased by at least about 10%, compared to the ratio in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0097] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 preferred embodiments, the circulating ratio is the extracellular ratio, for example the plasma ratio. In preferred embodiments, the ratio is increased to at least about 10.0, for example at least about 16.0. In particularly preferred embodiments, the ratio is increased to at least about 2.5, for example at least about 5.0.

[0098] In some embodiments, in particular wherein the companion animal is a cat, glycine supplementation in accordance with methods 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 1,000% compared to the concentration in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating ratio is the extracellular ratio, for example the plasma ratio. In particularly preferred embodiments, the ratio is increased by at least about 10%, compared to the ratio in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation).

[0099] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with the invention increases the intracellular glutathione concentration to at least about 1.14 pM / ml, at least about 1.18 pM / ml, at least about 1.20 pM / ml, at least about 1.22 pM / ml, at least about 1.24 pM / ml, at least about 1.26 pM / ml, at least about 1.28 pM / ml, at least about 1.30 pM / ml, at least about 1.32 pM / ml, at least about 1.34 pM / ml, or at least about 1.36 pM / ml. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In preferred embodiments, the concentration is increased to at least about 1.14 pM / ml, for example at least about 1.16 pM / ml. In particularly preferred embodiments, the concentration is increased to at least about 1.26 pM / ml, for example at least about 1.36 pM / ml.

[0100] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased by at least about 20%, compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0101] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods of the invention increases the intracellular glutathione concentration to at least about 1.06 pM / ml, at least about 1.08 pM / ml, at least about 1.10 pM / ml, at least about 1.12 pM / ml, 1.14 pM / ml, at least about 1.18 pM / ml, at least about 1.20 pM / ml, at least about 1.22 pM / ml, at least about 1.24 pM / ml, at least about 1.26 pM / ml, at least about 1.28 pM / ml, or at least about 1.30 pM / ml. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased to at least about 1.06 pM / ml, preferably to at least about 1.09 pM / ml. In particularly preferred embodiments, the concentration is increased to at least about 1.19 pM / ml, for example at least about 1.30 pM / ml.

[0102] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with 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 preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is increased to at least about 1.00 nM / ml, for example at least about 1.20 nM / ml.

[0103] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is increased by at least about 10%, compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0104] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is increased to at least about 0.75 nM / ml, for example at least about 2.00 nM / ml. In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is increased by at least about 100%, compared to the concentration in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation).

[0105] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 preferred embodiments, the intracellular ratio is the ratio in RBCs. In preferred embodiments, the ratio is increased to at least about 25.0, for example at least about 30.0. In particularly preferred embodiments, the ratio is increased to at least about 35.0.

[0106] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the intracellular ratio is the ratio in RBCs. In particularly preferred embodiments, the ratio is increased by at least about 30%, compared to the ratio in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 preferred embodiments, the circulating ratio is the extracellular ratio, for example the plasma ratio. In preferred embodiments, the ratio is increased to at least about 1.7, for example at least about 1.8. In particularly preferred embodiments, the ratio is increased to at least about 3.4, for example at least about 5.6.

[0107] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 1,000% compared to the concentration in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating ratio is the extracellular ratio, for example the plasma ratio. In particularly preferred embodiments, the ratio is increased by at least about 100%, compared to the ratio in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation).

[0108] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In preferred embodiments, the concentration is increased to at least about 0.4 nM / ml, for example at least about 0.2 nM / ml.

[0109] In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is reduced by at least about 80%, compared to the concentration in the absence of glycine supplementation (for example compared to the ratio prior to supplementation or compared to a control companion animal not receiving supplementation).

[0110] In some embodiments, glycine supplementation in accordance with methods of the invention leads to immunologic or physiologic changes, for example increased lymphocytosis, increased neutrophilia, or panleukocytosis.

[0111] In some embodiments, glycine supplementation in accordance with methods of the invention reduces the plasma concentration of 8-OHdG 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 particularly preferred embodiments, the concentration is reduced to less than about 27 ng / mL.

[0112] In some embodiments, glycine supplementation in accordance with methods of the invention reduces the urine concentration of 8-iso-PGF2a 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 particularly preferred embodiments, the concentration is reduced to less than about 0.8 ng / mg Cr.

[0113] In some embodiments, glycine supplementation in accordance with methods of the invention reduces the urine concentration of PGF2a 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 particularly preferred embodiments, the concentration is reduced to less than about 4.7 ng / mg Cr. In some embodiments, glycine supplementation in accordance with methods of the invention reduces the urine concentration of 2,3-dinor-5,6-dihydro-8isoPGF2a 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

[0114] 8.2 ng / mg Cr, less than about 8.0 ng / mg Cr, less than about 7.8 ng / mg Cr, less than about

[0115] 7.6 ng / mg Cr, less than about 7.4 ng / mg Cr, less than about 7.2 ng / mg Cr, less than about

[0116] 7.0 ng / mg Cr, less than about 6.8 ng / mg Cr, or less than about 6.6 ng / mg Cr. In particularly preferred embodiments, the concentration is reduced to less than about 6.6 ng / mg Cr.

[0117] In some embodiments, glycine supplementation in accordance with methods of the invention reduces the concentration of a marker of oxidative stress, e.g. by 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 in the absence of glycine supplementation (for example prior to supplementation or compared to a control companion animal not receiving supplementation). In some embodiments, the concentration is urine concentration of 8-iso-PGF2a. In preferred embodiments, the concentration is the plasma concentration of 8-OHdG and / or the urine concentration of PGF2aand / or the urine concentration of 2,3-dinor-5,6-dihydro-8isoPGF2a. In particularly preferred embodiments, the concentration is reduced by at least about 30%.

[0118] In some embodiments, glycine supplementation in accordance with methods of the invention increases the intracellular Gly concentration to at least about 260 pM, at least about 270 pM, at least about 280 pM, at least about 290 pM, at least about 300 pM, at least about 310 pM, at least about 320 pM, at least about 330 pM, at least about 350 pM, at least about 400 pM, or at least about 450 pM. In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased to at least about 280 pM, for example at least about 300 pM.

[0119] In some embodiments, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In preferred embodiments, the intracellular concentration is the concentration in RBCs. In particularly preferred embodiments, the concentration is increased by at least about 15%, compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0120] In some embodiments, in particular where the companion animal is a cat, glycine supplementation in accordance with methods of the invention increases the circulating Gly concentration to at least about 360 pM, at least about 370 pM, at least about 380 pM, at least about 390 pM, at least about 400 pM, at least about 410 pM, at least about 420 pM, at least about 430 pM, at least about 440 pM, at least about 450 pM, at least about 500 pM, or at least about 550 pM. In preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is increased to at least about 400 pM, for example at least about 550 pM.

[0121] In some embodiments, in particular where the companion animal is a cat, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In particularly preferred embodiments, the concentration is increased by at least about 15% compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0122] In some embodiments, in particular where the companion animal is a cat, glycine supplementation in accordance with methods 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 preferred embodiments, the circulating concentration is the extracellular concentration, for example the plasma concentration. In particularly preferred embodiments, the concentration is increased to at least about 170 nM / ml, for example at least about 190 nM / ml. In some embodiments, in particular wherein the companion animal is a dog, glycine supplementation in accordance with methods 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 in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation). In particularly preferred embodiments, the concentration is increased by at least about 10% compared to the concentration in the absence of glycine supplementation (for example compared to the concentration prior to supplementation or compared to a control companion animal not receiving supplementation).

[0123] Glycine supplementation and disease

[0124] The inventors have shown that glycine supplementation reverses glutathione dysfunction. Consequently, glycine supplementation is able to treat diseases arising from or associated with glutathione dysfunction, such as diseases involving oxidative stress.

[0125] Therefore, in some embodiments, glycine supplementation according to the invention is associated with treatment and / or prevention of disease.

[0126] In some embodiments, the disease is chronic kidney disease, dyslipidemia, cataracts, macular degeneration, glaucoma, immunosenescence, oxidative damage, age-associated inflammation, neurodegenerative disease, type 2 diabetes, osteoarthritis, or liver disease. The disease may comprise CD21+ cell deficiency, insensitivity to insulin, or cholesterol oxidation. A particularly preferred disease is chronic kidney disease.

[0127] In some embodiments, the disease is a neurodegenerative disease, such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS).

[0128] In some embodiments, the disease is a cardiovascular disease, such as hypertension or myocardial infarction.

[0129] In some embodiments, the disease is liver disease, such as hepatic lipidosis.

[0130] In some embodiments, the disease is associated with high blood cholesterol.

[0131] Glycine supplementation according to methods of the invention may also be performed to prevent the disease, i.e. may be performed prior to onset of disease symptoms. Glycine supplementation in the absence of disease

[0132] In other embodiments, glycine supplementation according to the invention is not associated with treatment of disease. These embodiments may be described as non-therapeutic or cosmetic, and may involve alteration of the body composition (e.g. + / - 10% ideal BCS) of a cat or dog, or other non-disease signs of senescence, in the absence of disease. In some embodiments, methods of glycine supplementation in accordance with the invention are performed to increase circulating glutathione, intracellular (e.g. RBC) glutathione, circulating GSH, and / or intracellular (e.g. RBC) GSH, as described above, in the absence of disease.

[0133] In some embodiments, glycine supplementation reduces blood cholesterol of the companion animal.

[0134] Companion animals

[0135] The invention concerns companion animals. In preferred embodiments, the companion animal is a senior cat. In some embodiments, the senior cat is a domestic cat, such as a domestic short haired cat. In other embodiments, the companion animal is a senior dog. In other embodiments, the senior dog is a Beagle or Brittany beagle.

[0136] In some embodiments, the senior cat or senior dog has an age of at least about 6.5 y, at least about 7.0 y, at least about 7.5 y, at least about 8.0 y, at least about 8.5 y, at least about 9.0 y, at least about 9.5 y, at least about 9.7 y, at least about 10.0 y, at least about 10.5 y, at least about 11.0 y, at least about 11.5 y, at least about 12.0 y, at least about 12.5 y, at least about 13.0 y, at least about 13.5 y, or at least about 14.0 y. In particularly preferred embodiments, the cat or dog has an age of at least about 9.5 y.

[0137] In preferred embodiments, the senior cat has an age of at least about 7.0 y.

[0138] In preferred embodiments, the senior dog has an age of at least about 12 years, particularly if the dog is a toy senior dog or a small senior dog e.g. as defined by Salt et al. J Gerontol A Biol Sci Med Sci. 2023 Apr; 78(4): 579-586. In some embodiments, the senior dog has an age of at least about 10 years, particularly if the dog is a large senior dog e.g. as defined by Salt et al. J Gerontol A Biol Sci Med Sci. 2023 Apr; 78(4): 579-586.

[0139] Cats are thought to be particularly susceptible to oxidative injury because feline haemoglobin contains 8-10 reactive sulfhydryl groups, rather than 4 as in the dog and other mammalian species. Hence, the feline haemoglobin molecule is more susceptible to disruption following oxidative attack and may have a greater requirement for GSH to maintain sulfhydryl groups in the reduced form, as needed for the maintenance of normal function.

[0140] In some embodiments, the companion animal has a BCS within about + / - 10% of the ideal BCS for the companion animal.

[0141] In some embodiments, the cat or dog is healthy, for example has no evidence of disease and not receiving medication. In some embodiments, the cat or dog is healthy but has glutathione dysfunction, for example putting the cat or dog at risk of a disease. In some embodiments, the cat or dog has a disease associated with glutathione dysfunction.

[0142] Dietary compositions

[0143] Dietary compositions (or simply “compositions”) of the present invention are suitable for feeding companion animals.

[0144] Dietary compositions of the present invention comprise free glycine (Gly). Dietary compositions may comprise any amount of free glycine adequate to increase circulating glycine, circulating glutathione, circulating GSH, intracellular glutathione, intracellular GSH, and / or to improve glutathione dysfunction.

[0145] Dietary compositions of the present invention may comprise an amino acid profile typical of dietary compositions known in the art, with the exception of glycine, which is increased in dietary compositions of the present invention.

[0146] Dietary compositions of the present invention may comprise exogenous free glycine, for example they may contain free glycine that is not derived from any other component present in the dietary composition (as by hydrolysis of protein already present in the dietary composition). The free glycine may be synthetic (not directly derived from food sources, for example by synthesis from chemical components in a laboratory). The free glycine may be added in the form of a solid, for example a powder.

[0147] The content of an ingredient in a dietary composition, such as the free glycine content, may be expressed as the percentage weight of the ingredient per the total weight of the composition on an as-is basis (herein ‘%’ unless indicated otherwise). In some embodiments, the dietary compositions comprise 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 preferred embodiments, the dietary compositions comprise at least about 1.5% free Gly. In particularly preferred embodiments, the dietary compositions comprise at least about 6.0% Gly.

[0148] In some embodiments, the dietary compositions comprise 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 preferred embodiments, the dietary compositions comprise about 6.0% Gly. In particularly preferred embodiments, the dietary compositions comprise about 1.5% free Gly.

[0149] In some embodiments, the dietary compositions comprise at most about 6.5%, at most about 6.0%, at most about 5.5%, at most about 5.0%, at most about 4.5%, at most about 4.0%, at most about 3.5%, at most about 3.0%, at most about 2.5%, at most about 2.0%, or at most about 1.5% free Gly. In preferred embodiments, the dietary compositions comprise at most about 6.0% Gly. In particularly preferred embodiments, the dietary compositions comprise at most about 1.5% Gly.

[0150] In some embodiments, the dietary compositions comprise at least about 0.5 mM / kg (companion animal bodyweight), at least about 0.75 mM / kg (companion animal bodyweight), at least about 1.00 mM / kg (companion animal bodyweight), at least about 1.25 mM / kg (companion animal bodyweight), at least about 1.50 mM / kg (companion animal bodyweight), at least about 1.75 mM / kg (companion animal bodyweight), at least about 3.50 mM / kg (companion animal bodyweight), or at least about 7.00 mM / kg (companion animal bodyweight) free Gly. In preferred embodiments, the dietary compositions comprise at least about 7.00 mM / kg (companion animal bodyweight) free Gly. In particularly preferred embodiments, the dietary compositions comprise about 1.75 mM / kg (companion animal bodyweight) free Gly.

[0151] In some embodiments, the dietary compositions comprise about 0.5 mM / kg (companion animal bodyweight), about 0.75 mM / kg (companion animal bodyweight), about 1.00 mM / kg (companion animal bodyweight), about 1.25 mM / kg (companion animal bodyweight), about 1.50 mM / kg (companion animal bodyweight), about 1.75 mM / kg (companion animal bodyweight), about 3.50 mM / kg (companion animal bodyweight), or about 7.00 mM / kg (companion animal bodyweight) free Gly. In preferred embodiments, the dietary compositions comprise about 7.00 mM / kg (companion animal bodyweight) free Gly. In particularly preferred embodiments, the dietary compositions comprise about 1.75 mM / kg (companion animal bodyweight) free Gly.

[0152] In some embodiments, the dietary compositions comprise at most about 0.5 mM / kg (companion animal bodyweight), at most about 0.75 mM / kg (companion animal bodyweight), at most about 1.00 mM / kg (companion animal bodyweight), at most about 1.25 mM / kg (companion animal bodyweight), at most about 1.50 mM / kg (companion animal bodyweight), at most about 1.75 mM / kg (companion animal bodyweight), at most about 3.50 mM / kg (companion animal bodyweight), or at most about 7.00 mM / kg (companion animal bodyweight) free Gly. In preferred embodiments, the dietary compositions comprise at most about 7.00 mM / kg (companion animal bodyweight) free Gly. In particularly preferred embodiments, the dietary compositions comprise about 1.75 mM / kg (companion animal bodyweight) free Gly.

[0153] In some embodiments, the dietary composition comprises 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 matter. In preferred embodiments, the dietary composition comprises at least about 50 g.

[0154] In some embodiments, the dietary composition comprises 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 preferred embodiments the dietary composition comprises at least about 1.0 (mg free Gly) / cal, for example at least about 13.0 (mg free Gly) / cal.

[0155] 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 preferred embodiments the dietary composition comprises about 1.0 (mg free Gly) / cal, for example about 13.0 (mg free Gly) / cal. In some embodiments, the dietary composition comprises at most about 1.0 (mg free Gly) / kCal, at most about 2.0 (mg free Gly) / kCal, at most about 3.0 (mg free Gly) / kCal, at most about 4.0 (mg free Gly) / kCal, at most about 5.0 (mg free Gly) / kCal, at most about 6.0 (mg free Gly) / kCal, at most about 7.0 (mg free Gly) / kCal, at most about 8.0 (mg free Gly) / kCal, at most about 9.0 (mg free Gly) / kCal, at most about 10.0 (mg free Gly) / kCal, at most about 11.0 (mg free Gly) / kCal, at most about 12.0 (mg free Gly) / kCal, or at most about 13.0 (mg free Gly) / kCal . In preferred embodiments the dietary composition comprises at most about 1.0 (mg free Gly) / cal, for example at most about 13.0 (mg free Gly) / cal.

[0156] 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 preferred embodiments, 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.

[0157] 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 preferred embodiments, the dietary composition comprises about 0.6 g / (g total protein) free Gly, for example about 2.4 g / (g total protein) free Gly.

[0158] In some embodiments, the dietary composition comprises at most about 0.2 g / (g total protein) free Gly, at most about 0.2 g / (g total protein) free Gly, at most about 0.4 g / (g total protein) free Gly, at most about 0.6 g / (g total protein) free Gly, at most about 1.2 g / (g total protein) free Gly, at most about 1.8 g / (g total protein) free Gly, at most about 2.4 g / (g total protein) free Gly, or at most about 3.0 g / (g total protein) free Gly. In preferred embodiments, the dietary composition comprises at most about 0.6 g / (g total protein) free Gly, for example at most about 2.4 g / (g total protein) free Gly.

[0159] The composition can be a moist composition (having a total moisture content of about 16% to 50% by weight), or a wet composition (having a total moisture content of at least about 50% by weight). However, in preferred embodiments, the dietary composition is a dry composition having a total moisture content of at most about 16% by weight, at most about 8% by weight, at most about 7% by weight, or at most about 6% by weight - for example at most about 7% by weight.

[0160] In some embodiments, the dietary composition is nutritionally complete for the companion animal species. For example, dietary compositions for cats may meet the Association of American Feed Control Officials (AAFCO) nutrient profile for adult cats. Dietary compositions may meet The European Pet Food Industry (FEDIAF) nutritional guidelines.

[0161] The dietary composition may be a solid, a 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, a jelly, gel, treats, chew, biscuits, gravy, broth, sauce, beverage, soup, pate, paste, spread, cream, supplemental water, and combinations thereof. Preferably, the dietary composition is cat food or dog food.

[0162] In some embodiments, the dietary composition is a liquid, for example an aqueous solution comprising the free glycine.

[0163] In some embodiments, the dietary composition is a non-medical composition, such as a nutraceutical composition.

[0164] The dietary composition may further comprise one or more of protein, fat, crude fiber, ash, cysteine, and water. In some embodiments, the dietary composition further comprises free Cysteine, for example less than about 0.5% free Cysteine, or about 0.5% free Cysteine.

[0165] In some embodiments, dietary compositions of the invention comprise, on a dry matter basis, from about 1% to about 50% crude protein, from about 0.5% to about 25% crude fat, from about 1% to about 10% supplemental fiber, all by weight of the composition. The composition may have a total moisture content from about 1% to about 30% moisture. Alternatively, the composition may comprise on a dry matter basis, from about 5% to about 35% crude protein, from about 5% to about 25% crude fat, from about 2% to about 8% supplemental fiber, all by weight of the composition. The composition may have a total moisture content from about 2% to about 20% moisture. Alternatively, the composition may comprise on a dry matter basis, a minimum protein level of about from about 9.5% to about 35%, a minimum fat level of from about 8% to about 20%, a minimum supplemental fiber level of from about 3% to about 7%, all by weight of the composition. The composition may also have a minimum metabolizable energy level of about 3.5 Kcal / g, for example about 3.7 Kcal / g. The composition may have a total moisture content from about 3% to about 10%.

[0166] The compositions of the invention may comprise additional components. Examples of additional components include animal protein, plant protein, farinaceous matter, vegetables, fruit, egg-based materials, undenatured proteins, food grade polymeric adhesives, gels, polyols, starches, gums, flavorants, seasonings, salts, colorants, time-release compounds, minerals, vitamins, antioxidants, prebiotics, probiotics, aroma modifiers, textured wheat protein, textured soy protein, textured lupin protein, textured vegetable protein, breading, comminuted meat, flour, comminuted pasta, water, and combinations thereof.

[0167] Nonlimiting examples of optional ingredients can include at least one vegetable. Nonlimiting examples of vegetables include carrots, peas, potatoes, cabbage, celery, beans, corn, tomatoes, broccoli, cauliflower, leeks and combinations thereof.

[0168] Also useful herein, as an optional ingredient, is a filler. The filler can be a solid, a liquid or packed air. The filler can be reversible (for example thermo-reversible including gelatin) and / or irreversible (for example thermo-irreversible including egg white). Nonlimiting examples of the filler include gravy, gel, jelly, aspic, sauce, water, air (for example including nitrogen, carbon dioxide, and atmospheric air), broth, and combinations thereof.

[0169] The composition may comprise a colorant. Nonlimiting examples of colorants include, but are not limited to, synthetic or natural colorants, and any combination thereof. When present the colorants are from about 0.0001 % to about 5%, from about 0.001% to about 1%, from about 0.005% to about 0.1%, on a dry matter basis, of said colorant.

[0170] Additionally, probiotic microorganisms, such as Lactobacillus o Bifidobacterium species, for example, may be added to the composition.

[0171] Also useful herein, as an optional ingredient, is at least one fruit. Nonlimiting examples include tomatoes, apples, pears, peaches, cherries, apricots, plums, grapes, oranges, grapefruit, lemons, limes, cranberries, raspberries, blueberries, watermelon, cantelope, mushmellon, honeydew melon, strawberries, banana, and combinations thereof.

[0172] The composition may contain other active agents such as long chain fatty acids and zinc. Suitable long chain fatty acids include alpha-linoleic acid, gamma linolenic acid, linoleic acid, eicosapentanoic acid, and docosahexanoic acid. Fish oils are a suitable source of eicosapentanoic acids (EP A) and docosahexanoic acid (DHA). The DHA level may be at least about 0.05%, alternatively at least about 0.1%, alternatively 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%, alternatively at least about 0.1%, alternatively at least about 0.15% of the composition, all on a dry matter basis.

[0173] The compositions of the present invention may further comprise a source of carbohydrate. Grains or cereals such as rice, com, milo, sorghum, barley, wheat, and the like are illustrative sources.

[0174] The compositions may also contain other materials such as dried whey and other dairy by products.

[0175] The invention further provides a method of making a dietary composition of the invention, for example by combining a dietary composition that is deficient in free glycine, with free glycine, to produce a dietary composition of the invention. The free glycine and dietary composition deficient in free glycine may be combined in proportions suitable to produce a dietary composition comprising at least about 0.5%, at least about 1.5% or at least about 6.0% free glycine. Preferably the dietary composition produced comprises at least about 1.5% free glycine, for example at least about 6.0% free glycine. The free glycine and dietary composition deficient in free glycine may be combined in proportions suitable to produce a dietary composition comprising about 0.5%, about 1.5% or about 6.0% free glycine.

[0176] Preferably the dietary composition produced comprises about 1.5% free glycine. Particularly preferred is a dietary composition comprising 6.0% free glycine. The free glycine and dietary composition deficient in free glycine may be combined in proportions suitable to produce a dietary composition comprising at most about 0.5%, at most about 1.5% or at most about 6.0% free glycine. Preferably the dietary composition produced comprises at most about 6.0% free glycine, for example at most about 1.5% free glycine.

[0177] Dietary additives

[0178] Also provided is a dietary additive comprising free glycine, which may be combined with dietary compositions to increase the free glycine content of the dietary composition (thereby producing a dietary composition of the invention).

[0179] Additives may be added to any dietary composition, such as a dietary composition that is deficient in free glycine. A dietary composition may be “deficient” in free glycine, if the composition comprises an inadequate quantity of free glycine to meet the nutritional requirements of the companion animal concerned, or may contain inadequate free glycine according to nutritional standards (for example those set by the AAFCO). Alternatively, a dietary composition may be “deficient” in free glycine if it contains a quantity of free glycine that is insufficient to elevate the circulating free glycine or plasma free glycine of the companion animal. For example, a dietary composition may be “deficient” in free glycine is the composition comprises less than about 0.5%, less than about 1.5%, or less than about 6% free glycine. Particularly preferred are dietary compositions comprising less than about 6% free glycine, for example less than about 1.5% free glycine.

[0180] Dietary additives of the present invention may comprise or consist of exogenous free glycine, for example they may contain free glycine that is not derived from any other component present in the dietary additive (as by hydrolysis of protein already present in the dietary additive). The free glycine may be synthetic (not directly derived from food sources, for example by synthesis from chemical components in a laboratory). The free glycine may added to the additive in the form of a solid, for example a powder.

[0181] The additive may be in the form of a substance that is placed on top of the dietary composition that is deficient in free glycine (such an additive is conventionally termed a ‘topper’). The additive may be mixed with the free glycine-deficient dietary composition. The additive may comprise no nutritive value except for that of the free glycine it contains, for example, the additive may comprise from about 0 kcal to about 100 kcal, for example from about 0 kcal to about 30 kcal, for example about 0 kcal, except for the kcal of the free glycine it contains. The additive may be a solid, liquid, or powder.

[0182] The dietary additive may be a solid, tablet, powder, liquid, or suspension. The dietary additive may comprise 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.

[0183] The invention further provides a method for making a dietary composition of the invention, for example comprising mixing a dietary additive of the invention with a dietary composition to produce the dietary composition of the invention. The dietary additive and dietary composition may be combined in proportions suitable to produce a dietary composition comprising at least about 0.5%, at least about 1.5% or at least about 6.0% free glycine. Preferably the dietary composition produced comprises at least about 1.5% free glycine, for example at least about 6.0% free glycine. The dietary additive and dietary composition may be combined in proportions suitable to produce a dietary composition comprising about 0.5%, about 1.5% or about 6.0% free glycine. Preferably the dietary composition produced comprises about 1.5% free glycine. Particularly preferred is producing a dietary composition comprising about 6.0% free glycine. The dietary additive and dietary composition may be combined in proportions suitable to produce a dietary composition comprising at most about 0.5%, at most about 1.5% or at most about 6.0% free glycine. Preferably the dietary composition produced comprises at most about 6.0% free glycine, for example at most about 1.5% free glycine.

[0184] Feeding

[0185] In any of the embodiments of the invention involving administration of a dietary composition, the administration is oral administration (“feeding”, “ingesting” or “supplementation”). The companion animal may consume the dietary composition by eating and / or drinking the composition.

[0186] Feeding may occur daily for at least about 2 weeks, for example at least about 4 weeks, at least about 8 weeks, or at least about 12 weeks. In particularly preferred embodiments, the feeding occurs daily for at least about 4 weeks.

[0187] In some embodiments, the supplementation diet comprises at least one meal daily, at least two meals daily, at least three meals daily, or at least four meals daily, for example one meal daily, two meals daily, three meals daily, or four meals daily. Particularly preferred is two meals daily. The meals may be equal in caloric content or unequal, for example two meals may comprise 50% of the total caloric intake each.

[0188] In some embodiments, the companion animal is 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 free Gly. In preferred embodiments, the the companion animal is administered at least about 7.00 mM / kg / day free Gly. In particularly preferred embodiments, the companion animal is administered at least about 1.75 mM / kg / day free Gly. In some embodiments, the companion animal is 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 free Gly. In preferred embodiments, the companion animal is administered about 7.00 mM / kg / day free Gly. In particularly preferred embodiments, the companion animal is administered about 1.75 mM / kg / day free Gly.

[0189] In some embodiments, the companion animal is administered at most about 0.5 mM / kg / day, at most about 0.75 mM / kg / day, at most about 1.00 mM / kg / day, at most about 1.25 mM / kg / day, at most about 1.50 mM / kg / day, at most about 1.75 mM / kg / day, at most about 3.50 mM / kg / day, or at most about 7.00 mM / kg / day free Gly. In preferred embodiments, the companion animal is administered at most about 7.00 mM / kg / day free Gly. In particularly preferred embodiments, the companion animal is administered about 1.75 mM / kg / day free Gly.

[0190] In some embodiments, the companion animal is administered 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 of the dietary composition per day. In preferred embodiments, the companion animal is administered at least about 50 g of the dietary composition per day.

[0191] 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 per day. In preferred embodiments, the companion animal is administered about 50 g of the dietary composition per day.

[0192] In some embodiments, the companion animal is administered at most about 20 g, at most about 30 g, at most about 40 g, at most about 50 g, at most about 60 g, at most about 70 g, at most about 80 g, at most about 90 g, or at most about 100 g of the dietary composition per day. In preferred embodiments, the companion animal is administered at most about 50 g of the dietary composition per day.

[0193] 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 free Gly per day. In preferred embodiments, the companion animal is administered at least about 0.75 g free Gly per day, for example at least about 3.0 g free Gly per day.

[0194] 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 free Gly per day. In preferred embodiments, the companion animal is administered about 0.75 g free Gly per day, with about 3.0 g free Gly per day particularly preferred.

[0195] In some embodiments, the companion animal is administered at most about 0.25 g, at least about 0.5 g, at least about 0.75 g„ at most about 1.0 g, at most about 1.5 g, at most about 2.0 g, at most about 2.5 g, at most about 3.0 g, at most about 3.5 g, at most about 4.0 g, at most about 4.5 g, at most about 5.0 g, at most about 5.5 g, or at most about 6.0 g free Gly per day. In preferred embodiments, the companion animal is administered at most about 3.0 g free Gly per day, for example at most about 0.75 g free Gly per day.

[0196] In some embodiments, the companion animal is administered at least about 0.1 g, at least about 0.2 g, at least about 0.3 g, at least about 0.4 g, at least about 0.5 g, at least about 0.6 g, at least about 0.7 g, at least about 0.8 g, at least about 0.9 g, at least about 1.0 g, at least about 1.2 g, or at least about 1.5 g free Gly per kg bodyweight per day. In preferred embodiments, the companion animal is administered at least about 0.3 g free Gly per kg bodyweight per day, for example at least about 1.5 g free Gly per kg bodyweight per day.

[0197] 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 free Gly per kg bodyweight per day. In preferred embodiments, the companion animal is administered about 0.3 g free Gly per kg bodyweight per day, with about 1.5 g free Gly per kg bodyweight per day particularly preferred.

[0198] In some embodiments, the companion animal is administered at most about 0.1 g, at most about 0.2 g, at most about 0.3 g, at most about 0.4 g, at most about 0.5 g, at most about 0.6 g, at most about 0.7 g, at most about 0.8 g, at most about 0.9 g, at most about 1.0 g, at most about 1.2 g, or at most about 1.5 g free Gly per kg bodyweight per day. In preferred embodiments, the companion animal is administered at most about 1.5 g free Gly per kg bodyweight per day, for example at most about 3.0 g free Gly per kg bodyweight per day. EXAMPLES

[0199] Example 1 investigated whether circulating glutathione levels are impacted by age in cats.

[0200] Example 2 investigated whether dietary supplementation with different doses of free Gly could alter circulating Gly levels in senior cats.

[0201] Example 3 investigated whether dietary supplementation with free Gly could alter circulating glutathione levels in senior cats, and if so, whether the adjusted level of glutathione is reflected in markers of oxidative stress.

[0202] Example 4 investigated whether circulating glycine and glutathione levels are impacted by age in dogs.

[0203] General materials and methods are provided in a final section, after these examples.

[0204] Example 1: Feline levels of glutathione decline with age

[0205] This Example compared levels of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), and plasma glycine, in young and senior cats.

[0206] Methods

[0207] 32 healthy adult cats (22 female, 10 male) took part in this cross-sectional study. 16 were classified as “young”, with a median age of 2.9 y (range 1.3 to 2.9 y) and 16 as “senior”, with a median age of 9.7 y (range 9.2 to 13.2 y). Cats in both age groups were fed a commercially available complete and balanced dry diet (IAMS Multi-Cat, Mars Petcare, USA) meeting the Association of American Feed Control Officials (AAFCO) nutrient profile for adult cats throughout the study. All animals were within 10% of ideal body condition score (BCS) and diets were provided at levels to maintain ideal BCS throughout the study.

[0208] Two 3-mL blood samples were collected from the medial saphenous vein at an interval of 1 month to compare whole blood (WB) and red blood cell (RBC) total glutathione, RBC GSH, RBC GSSG, and plasma Gly concentrations.

[0209] Results

[0210] Of the 32 cats enrolled for this cross-sectional study, 1 of the young adult cats, aged 2.8 y, was removed due to health reasons prior to any data being recorded. In addition, 3 cats (2 young and 1 senior) had one measure missing for WB glutathione due to difficulties in blood sampling. Data are shown in Table 1 and Figure 1. RBC and WB glutathione decrease with age: Senior cats exhibited significantly lower total glutathione; significantly lower GSH; significantly higher GSSG; and, consequently, significantly lower GSH:GSSG, compared to young cats. These findings occurred in both WB and RBCs. Plasma Gly profile remains constant with age: No significant differences between age groups were observed in the levels of Gly. Concentrations were within reference ranges for plasma Glycine in healthy adult cats (Heinze CR, Larsen JA, Kass PH, et al. (2009) Am J Vet Res 70, 1374-82).

[0211] ble 1. Red blood cell (RBC) and whole blood (WB) glutathione concentrations, and plasma glycine (GLY) levels in the young adult senior cats. easure Young Adult (<3 y) Senior (>9 y) Difference in means Fold change in means p value

[0212] (Senior - Young Adult) (Senior / Young Adult) C total glutathione (pM) 946.6(769.1, 1165.0) 672.3 (549.8,821.9) 0.7 (0.6, 0.9) 0.008* C GSH (pM) 858.3 (586.5, 1256.1) 302.7(209.4,437.8) 0.4 (0.2, 0.6) <0.001* C GSSG (pM) 71.4(51.1,99.7) 268.0(193.9,370.3) 3.8 (2.5, 5.7) <0.001* C GSH: GSSG 12.0(6.9,21.1) 1.12(0.65, 1.93) 0.1 (0.1, 0.2) <0.001*B total glutathione (pM) 479.1 (425.6,532.7) 398.1 (345.6,450.5) -81.1 (-7.3, -0.8) 0.016*BGSH(pM) 446.1 (386.3,505.8) 308.7(249.7,367.6) -6.9 (-10.6, -3.2) <0.001*BGSSG(pM) 26.9(19.5,37.3) 75.2(54.5, 103.5) 2.8 (1.9, 4.2) <0.001*B GSH: GSSG 16.0(10.5,24.2) 3.7 (2.4, 5.6) 0.2 (0.1, 0.4) <0.001*asma GLY (pM) 373.8 (352.4,395.1) 377.0(356.3,397.7) 3.2 (-22.9, 29.3) 0.810 values are means and brackets indicate 95% confidence intervals of the mean. GSH, reduced glutathione; GSSG, oxidised glutathione; H:GSSG, reduced to oxidised glutathione ratio. * indicates significance between groups (p < 0.05).

[0213] The results show age-associated decline in feline circulating glutathione

[0214] Senior cats have lower levels of WB and RBC glutathione compared to young adult cats. Particularly, senior cats were observed to have a significantly lower GSH:GSSG ratios compared to their younger counterparts, indicative of greater oxidative stress.

[0215] No significant age-related differences were observed in plasma levels of Gly. This may reflect the fact that all of the animals in the preliminary study were healthy and offered appropriate amounts of complete and balanced diets.

[0216] Example 2: Dietary supplementation with free Gly increases circulating Gly levels

[0217] This Example measured the effects of dietary supplementation of different doses of free Gly on circulating Gly levels in senior cats.

[0218] Methods

[0219] To test what dose of Gly was needed to increase Gly concentrations in blood, 52 senior cats (33 female, 19 male), with a median age of 12.1 y (range 8.1 to 13.6 y), took part in a 16-week study, comprising diet rotations across eight 2-week blocks (Figure 2a provides a study design schematic). 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 diet supplemented with either 0.5, 1.5 or 6.0% free Gly. All diets met the AAFCO nutrient profile for adult cats (Table 2). All cats began on the control diet and were fed with this diet for 2 weeks. Following this, cats were fed each test diet (supplemented with 0.5%, 1.5% and 6.0% free Gly) in a randomised order, for 2 weeks each, with a 2-week washout with the control diet in between.

[0220] The senior animals were within 10% of ideal BCS and the diets provided were at levels to maintain ideal BCS throughout the study.

[0221] Each 2-week test block concluded with a 4 mL fasted (overnight >18 h) blood sample collected from the medial saphenous vein from each cat into a sodium heparin vacutainer (BD Vacutainer®). Heparin-anticoagulated WB was processed to determine plasma and RBC Gly in both components formed the primary measure. Blood samples collected at the end of a 2-week washout block represented the baseline sample for the subsequent 2-week test feeding block. Blood samples collected at the end of a test feeding block represented the final, endpoint sample for the preceding 2-week test feeding block. Results

[0222] Of the 52 senior cats enrolled for the dosing study, 1 was removed due to healthcare concerns.

[0223] Gly supplementation increased RBC and plasma Gly: Data are shown in Table 3 below. All of the 0.5%, 1.5% and 6.0% free Gly groups showed a statistically significant increase in

[0224] RBC Gly. In addition, the 1.5% and 6.0% groups showed a statistically significant increase in plasma Gly (with the 0.5% group trending to an increase in plasma Gly, but below the level of significance in this experiment).

[0225] Levels of glutathione in this example were normalised to haemoglobin based on the assumption that variation in RBC haemoglobin levels in healthy cats, including seniors, is small (Moritz A, Fickenscher Y, Meyer K, et al. (2004) Vet Clin Pathol 33, 32-8; and MSD Veterinary Manual (2022) Serum Biochemical Analysis Reference Ranges, https: / / www.msdvetmanual.com / special-subjects / reference-guides / senim-biochemical- analysis-reference-ranges (accessed February 2023)).

[0226] Table 2: Nutrient composition of study diets in Examples 2-3.

[0227] CYS, cysteine; GLY, glycine.

[0228] Table 3. Plasma and red blood cell (RBC) glycine (GLY) levels from Example 2.

[0229] % Dosing GLY (pM) Baseline Final Difference in means p value

[0230] Free GLY (Final - Baseline)

[0231] 0% Plasma GLY 355 (334,376) 355 (334,376) 0.1 (-22.4, 22.6) 1

[0232] 0% RBC GLY 282 (251,312) 280 (250,311) -1.1 (-39.8, 37.6) 1

[0233] 0.5% Plasma GLY 359 (337,380) 380 (358,401) 20.8 (-1.8, 43.4) 0.086

[0234] 0.5% RBC GLY 253 (222,283) 347 (316,378) 94.2(55.2, 133.0) <0.001*

[0235] 1.5% Plasma GLY 354 (333,376) 401 (380,423) 47.1 (24.4,69.8) <0.001*

[0236] 1.5% RBC GLY 246 (215,277) 332 (301,363) 86.0(46.9, 125.0) <0.001*

[0237] 6.0% Plasma GLY 365 (343,386) 544 (522,565) 179.0(156.0,201.0) <0.001*

[0238] 6.0% RBC GLY 268 (237,298) 442 (411,473) 174.0(136.0,213.0) <0.001*

[0239] All values are means and brackets indicate 95% confidence intervals of the mean. * indicates significance between groups (p < 0.05).

[0240] All levels of free Gly supplementation increase RBC Gly

[0241] All levels of free Gly caused a statistically significant increase in RBC Gly. In view of the results for plasma and RBC Gly, 1.5% free Gly was selected as the level for exploratory supplementation in a longer-term feeding study (Example 3).

[0242] Example 3: Dietary supplementation with free Gly increases glutathione measures and reduces markers of oxidative stress

[0243] This Example measured the effects of dietary supplementation of 1.5% free Gly on circulating glutathione levels, and markers of oxidative stress, in senior cats. Using the average body weight of a cat and average consumption, 1.5% free Gly equated to 1.75 mM / kg / day total free Gly.

[0244] Methods

[0245] 44 senior cats (28 female, 16 male), with a median age of 11.7 y (range 7.7 to 13.8 y), took part in the 16-week study, comprising a 4-week acclimation phase and a 12-week test phase (Figure 2b provides a study design schematic). Of these 44 senior cats, 39 participated in Example 2.

[0246] For the 4-week duration of the acclimation phase, cats were offered a control dry diet (IAMS Adult Cat Original Chicken, Mars Petcare, USA).

[0247] Subsequently, for the test phase, the senior cohort was randomly divided into two groups of 22 cats, one group remained on the control dry diet and the other was immediately transferred to a test dry diet supplemented with 1.5% free Gly (Table 2). All diets fed met the AAFCO nutrient profile for adult cats.

[0248] Blood and urine were collected at the end of the acclimation phase (from cats fasted overnight >18 h) to act as a baseline for all measures. Blood was collected from the medial saphenous vein for the measurement of: RBC glutathione profiles; WBC GSH; plasma and RBC Gly; mitogen -induced lymphoproliferative response; the marker 8-hydroxy-2'- deoxyguanosine (8-OHdG), one of the major products of DNA oxidation widely used as a biomarker of oxidative stress; and biochemistry and haematology parameters. Urine was collected for the measurement of F2-IsoPs for additional quantification of oxidative damage. Subsequently blood was collected every 4 weeks to mirror the measurement of parameters conducted at the end of the acclimation phase, with the exception of WBC glutathione being omitted at week 4.

[0249] Subsequent urine collection was conducted at the end of the test phase and study (week 12).

[0250] Aliquots for the majority of the blood-based measures were obtained from 8 mL heparin-anticoagulated WB (BD Vacutainer® 4 mL Heparin Tubes) with the exception of samples for biochemistry (1.2 mL; serum separator tube, SST, SARSTEDT, Inc.), haematology (1 mL; EDTA, BD Vacutainer®), and WBC glutathione (1 mL; EDTA, BD Vacutainer®), which were collected into individual blood tubes.

[0251] Results

[0252] 44 senior cats were recruited. 10 cats were removed throughout the duration of the 16-week trial, all due to poor consumption of their respective diets, leading to weight loss in excess of the threshold limits defined for the study (-10% ideal BCS). This comprised the removal of 2 control cats during the acclimation phase, and 3 control cats and 5 test cats during the test phase, totalling 5 control and 5 test cats. This meant the feeding study completed with >75% power against the primary measure, RBC total glutathione, to detect a 40% difference between the groups. Data from cats removed during the feeding study were excluded from the statistical analysis of the study measures.

[0253] RBC total glutathione and RBC GSH increased following supplementation: Data are shown in Table 4, and in Figure 3. Statistically significant differences in RBC total glutathione and RBC GSH were observed between test and control groups in week 4 of the test phase. No further statistically significant differences were identified at the remaining test phase sampling timepoints, although a trend was observed for total glutathione, with higher concentrations for the test group compared to the control group, at week 12.

[0254] RBC and plasma Gly levels increase following supplementation: Levels of Gly were found to differ between the test and control groups (Table 4 and Figure 4). Plasma Gly levels were significantly higher in the test group at all of the sampling timepoints in the test phase compared to the control group (p <0.009). RBC Gly was found to be significantly higher for the test group at the first and last test phase timepoints compared to the control group (p <0.004). White blood cell glutathione: The impact of 1.5% free GLY supplementation on absolute numbers of WBC subsets and on glutathione concentration within WBC subsets was assessed in specific cell populations: CD4+ T-cells, CD8+ T-cells, CD14+ monocytes, CD21+ B-cells, and granulocytes, via flow cytometry (Supplementary Table 2). One statistically significant difference was identified; the absolute number of CD21+ cells (a marker for B-cells) was higher in the test group compared to the control group at week 8 of the test phase (difference 463.0, 95% CI (188.0, 738.0) cells / pL; p < 0.001). No further statistically significant differences in absolute cell counts or glutathione concentration were observed, measured either via the number of cells of each subset per pL or the quantity of GSH per cell.

[0255] Lymphoproliferative response unchanged after supplementation: Cats offered the diet supplemented with 1.5% free Gly had similar ConA-induced lymphoproliferative activities amongst themselves, and not significantly different to control group cats, throughout the test phase (p > 0.35; data not shown).

[0256] Oxidative damage markers reduced following supplementation: Dietary supplementation of Gly was found to influence 8-OHdG and specific urine F2-IsoPs measured as markers of oxidative damage (Table 6). A significant reduction in 8-OHdG was observed in week 8, and significant reductions in PGF2aand 2,3-dinor-5,6-dihydro-8isoPGF2a were observed in week 12. Also in week 12, 8-iso-PGF2a trended towards reduction but did not reach statistical significance.

[0257] Biochemistry and haematology: Parameters within the full biochemistry panel, including symmetric dimethylarginine (SDMA), Na:K ratio, creatine kinase phosphorous levels, plasma levels of amino acids other than glycine, blood urea nitrogen (BUN), basophil parameters, MCV, and MCHC remained within healthy levels during the study in the control and test groups (data not shown). One exception was cholesterol levels, which were significantly lower for the test group compared to the control group at weeks 4 and 8 (p < 0.01). able 4. Red blood cell (RBC) glutathione and glycine (GLY) concentrations, and plasma GLY levels in test (supplemented) and controlunsupplemented) senior cats in Example 3 (cont’d overleaf).

[0258] Measure Test Phase Test Control Difference in means Fold change in means p value

[0259] Week (Test - Control) (Test - Control)

[0260] RBC total 4 0.0041 (0.0037, 0.0046) 0.0034 (0.0030, 0.0039) 0.0007 (0.0001, 0.0013) 0.020* glutathione (pM)

[0261] RBC total 8 0.0030 (0.0025, 0.0034) 0.0027 (0.0023, 0.0032) 0.0002 (-0.0004, 0.0008) 0.659 glutathione (pM)

[0262] RBC total 12 0.0035 (0.0030, 0.0039) 0.0029 (0.0025, 0.0034) 0.0006 (0.0000, 0.0011) 0.063 glutathione (pM)

[0263] RBC GSH (ng) 4 0.0035 (0.0030, 0.0041) 0.0029 (0.0025, 0.0033) 1.23 (1.0200, 1.5000) 0.029*

[0264] RBC GSH (ng) 8 0.0025 (0.0021, 0.0029) 0.0022 (0.0019, 0.0026) 1.11 (0.9170, 1.3500) 0.410

[0265] RBC GSH (ng) 12 0.0029 (0.0025, 0.0034) 0.0025 (0.0025, 0.0029) 1.15 (0.954, 1.4000) 0.182

[0266] RBC GSSG (ng) 4 0.0005 (0.0004, 0.0007) 0.0005 (0.0004, 0.0006) 1.0500 (0.7500, 1.4700) 0.982

[0267] RBC GSSG (ng) 8 0.0004 (0.0003, 0.0005) 0.0005 (0.0004, 0.0006) 0.7700 (0.5500, 1.0900) 0.201

[0268] C GSSG (ng) 12 0.0004 (0.0003, 0.0005) 0.0004 (0.0003, 0.0005) 1.1400 (0.8160, 1.5900) 0.720SH:GSSG 0.965 (0.701, 1.33) 0.987SH:GSSG 8 6.73 (5.00, 9.06) 4.75 (3.52, 6.39) 1.15 (0.796, 1.67) 0.689SH:GSSG 12 7.11 (5.32, 9.49) 7.15 (5.35, 9.54) 1.13 (0.781, 1.64) 0.770 C GLY (pM) 4 295 (277, 312) 246 (229, 263) 48.7 (26.5, 70.8) <0.001* C GLY (pM) 8 280 (262, 297) 259 (241, 276) 21.3 (-0.9, 43.5) 0.063 C GLY (pM) 12 283 (266, 300) 248 (231, 265) 35.2 (13.3, 57.1) <0.001*asma GLY 4 399 (373, 424) 345 (319, 371) 53.7 (20.9, 86.6) <0.001*M) asma GLY 407 (380, 433) 358 (332, 384) 48.6 (15.4, 81.8) 0.002*M) asma GLY 12 398 (373, 423) 343 (317, 369) 55.3 (22.8, 87.8) <0.001*M) values are means and brackets indicate 95% confidence intervals of the mean. GSH, reduced glutathione; GSSG, oxidised glutathione; H:GSSG, reduced to oxidised glutathione ratio. * indicates significance between groups (p < 0.05).

[0269] Table 5: White blood cell glutathione concentrations in test (supplemented) and control (unsupplemented) senior cats, (a) Absolute cell counts (cells / pL), (b) Mean quantification per cell (g x 1013)

[0270] (a) WBC cell Test Phase Test Control Difference in means (Test P value population Week - Control)

[0271] CD4+ 8 748 (590,906) 677 (524,831) 70.9 (-126.0, 268.0) 0.616

[0272] CD4+ 12 825 (673,978) 789 (633,945) 36.2 (-159.0, 231.0) 0.872

[0273] CD8+ 8 254 (192,336) 277 (211,363) 0.9 (0.7, 1.3) 0.813

[0274] CD8+ 12 312 (239,407) 328 (248,432) 1.0 (0.7, 1.3) 0.962

[0275] CD14+ 8 634 (384, 1046) 549 (338,891) 1.2 (0.6, 2.2) 0.846

[0276] CD14+ 12 602 (376,962) 618 (374, 1021) 0.97(0.5, 1.8) 0.994

[0277] CD21+ 8 1128 (907, 1349) 666 (452,879) 463 (188,738) <0.001*

[0278] CD21+ 12 1054 (843, 1265) 908 (689, 1127) 146 (-126, 418) 0.384

[0279] Granulocytes 8 8387 (6351, 10444) 8266 (6287, 10246) 131 (-2424, 2686) 0.991

[0280] Granulocytes 12 7775 (5843,9706) 8336 (6296, 10376) -561 (-3081, 1959) 0.849

[0281] All values are means and brackets indicate 95% confidence intervals of the mean. Brackets indicate 95% confidence intervals of the mean. indicates significance between groups (P < 0.05).

[0282] (b) WBC cell Test Phase Test Control Difference in means (Test P value population Week - Control)

[0283] CD4+ 8 3.02(3.00,3.04) 3.02(2.99,3.04) 1 (0.99, 1.01) 0.889

[0284] CD4+ 12 3.01 (2.99,3.03) 3.00(2.98,3.03) 1 (0.99, 1.01) 0.946

[0285] CD8+ 8 2.99(2.97,3.01) 2.99(2.97,3.01) 1 (0.99, 1.01) 0.997

[0286] CD8+ 12 2.97(2.95,2.99) 2.98 (2.96,3.00) 1.00(0.99, 1.01) 0.697

[0287] CD14+ 8 6.53 (6.17,6.89) 6.72(6.37,7.07) -0.19 (-0.64, 0.26) 0.538

[0288] CD14+ 12 6.42(6.07,6.76) 6.45 (6.10,6.81) -0.04 (-0.48, 0.40) 0.973

[0289] CD21+ 8 2.94(2.92,2.96) 2.96(2.94,2.97) -0.02 (-0.04, 0.00) 0.084

[0290] CD21+ 12 2.93 (2.91,2.94) 2.94 (2.93,2.96) -0.02 (-0.04, 0.00) 0.108

[0291] Granulocytes 8 5.62(5.32,5.93) 5.87(5.58,6.17) -0.25 (-0.63, 0.13) 0.262

[0292] Granulocytes 12 5.36 (5.07, 5.64) 5.34 (5.04, 5.65) 0.01 (-0.36, 0.39) 0.996

[0293] All values are means and brackets indicate 95% confidence intervals of the mean. Brackets indicate 95% confidence intervals of the mean.

[0294] able 6. Oxidative stress measure concentrations in the GLY feeding study test (supplemented) and control (unsupplemented) seniorats.

[0295] Measure Test Phase Test Control Difference in means P value

[0296] Week (Test - Control)

[0297] 8-OHdG (nG / mL) 4 30.3 (24.8,37.0) 37.4(30.7,45.6) 0.81 (0.63, 1.05) 0.130

[0298] 8-OHdG (nG / mL) 8 26.6(21.8,32.5) 37.6(30.8,45.8) 0.71 (0.56,0.91) 0.004*

[0299] 8-OHdG (nG / mL) 12 31.4(25.8,38.2) 33.9(27.8,41.3) 0.93 (0.72, 1.19) 0.821

[0300] 8-iso-PGF2a(nG / mG Cr) 12 0.84(0.57, 1.10) 1.16(0.88, 1.43) -0.32 (-0.65, 0.01) 0.058

[0301] PGF2a(nG / mG Cr) 12 4.71 (3.20,6.94) 7.66(5.14, 11.40) 0.62(0.38, 1.00) 0.049*

[0302] 2,3-dinor-5,6-dihydro-8isoPGF2a(nG / mg Cr) 12 6.65 (5.02,8.80) 9.63 (7.21, 12.9) 0.69(0.49,0.98) 0.040* ll values are means and brackets indicate 95% confidence intervals of the mean. Cr, creatinine; 8-OHdG, 8-hydroxy-2'deoxyguanosine. indicates significance between groups (P < 0.05).

[0303] Glycine supplementation increases glycine and glutathione, and reduces markers of oxidative stress, in senior cats

[0304] Feeding senior cats a dry diet supplemented with 1.5% free Gly for 12 weeks significantly elevated early RBC glutathione levels, with reduction in markers of oxidative stress. Overall, this suggests that administration of free Gly has capacity to address age-associated reduction in glutathione function in this species.

[0305] RBC Gly levels were found to significantly differ between the study groups at the first and last sampling timepoint post-supplementation. However, statistical significance was not achieved at the intermediate sampling timepoint. These findings align with the observations for RBC total glutathione. High RBC Gly levels coordinate with elevations in intracellular glutathione synthesis at week 4 and week 12 of the study’s supplementation phase.

[0306] To explore the impact of dietary Gly supplementation beyond glutathione, several markers of oxidative stress were measured. F2-IsoPs are indicators of oxidative stress in vivo.

[0307] Measurement of F2-IsoPs is commonly performed in urine due to their chemical stability brought about by a lack of artificial auto-oxidation. Hence, the supplementation study set out to profile a series of F2-IsoPs in urine. In this study, it was shown that two urinary markers of lipid peroxidation, PGF2a and 2,3-dinor-5,6-dihydro-8iso PGF2a, were significantly lower in the test group compared to the control group following 12-weeks of 1.5% free Gly supplementation. In addition, although not statistically different, there was a trend with urinary 8-iso-PGF2a being lower in the test group. Produced during periods of oxidative stress, prostaglandin F2a (PGF2a) is an eicosanoid, which promotes the formation of 8-iso-PGF2a and 2,3-dinor-5,6-dihydro-8iso PGF2a. Our findings indicate a reduction in these markers of oxidative stress in senior cats fed a diet supplemented with free Gly.

[0308] Further, oxidative damage to DNA specifically was investigated via determination of plasma 8-oxo-7,8-dihydro-2’deoxyguanosine (8-OHdG) concentrations, considered one of the best measures of the mutagenic consequences of oxidative stress. A statistically significant decrease in this biomarker was identified at week 8 in the test group compared to the control group, which was not apparent at week 4 or 12.

[0309] Taken together, these results suggest dietary supplementation of Gly reduces markers of oxidative stress in senior cats, which appears to be dependent on an increase in blood cell glutathione. The results suggest dietary supplementation with free Gly, a precursor of glutathione, offers a viable route to alleviating the age-associated reduction in glutathione observed in senior cats. Plasma and RBC Gly levels were found to be significantly higher in the test group, at the majority of timepoints post-supplementation.

[0310] In summary, Examples 1-3 above demonstrate that levels of glutathione are significantly lower in senior cats compared to young cats. Offering older cats a dry diet supplemented with 1.5% free Gly for 12 weeks induced a significant elevation in RBC total glutathione and GSH early in the post-supplementation phase, and was also found to influence WBC levels and markers of oxidative stress. Overall, the results suggest age-associated reduction in RBC GSH in cats can be addressed by dietary free Gly supplementation.

[0311] Example 4: effect of age on levels of glutathione and glycine in dogs

[0312] This Example compared total, plasma, and RBC levels of total glutathione, reduced glutathione (GSH), and oxidised glutathione (GSSG), and plasma levels of Gly, in young and senior dogs.

[0313] Methods

[0314] 29 healthy adult Beagles (24 female, 5 male) and 3 healthy Brittany beagles (1 female, 2 male) took part in this cross-sectional study. 16 were classified as “young”, with a median age of 3.6 y (range 2.7 to 4.7 y) and 16 as “senior”, with a median age of 11.4 y (range 9.1 to 14.4 y). Dogs in both age groups were fed a commercially available complete and balanced dry diet meeting the Association of American Feed Control Officials (AAFCO) nutrient profile for adult dogsthroughout the study. All animals were within 10% of ideal body condition score (BCS) and diets were provided at levels to maintain ideal BCS throughout the study.

[0315] Two 3-mL blood samples were collected from the medial saphenous vein at an interval of 1 month to compare whole blood (WB) and red blood cell (RBC) total glutathione, RBC GSH, RBC GSSG, and plasma Gly concentrations.

[0316] Results

[0317] Of the 32 dogs enrolled for this cross-sectional study, 1 dog, aged 3.1 y, had no GSH data due to a reduced sample volume obtained, and 1 dog aged 3.8 y had plasma glutathione data for one sampling occasion only. Data are shown in Table 7 and Figure 5. RBC total glutathione and RBC total GSH decrease with age in dogs: Levels were significantly lower ( / ?<0.05) in senior compared to younger dogs. Plasma GSSG was higher in senior dogs and therefore the ratio of GSH to GSGG in plasma was significantly different.

[0318] The results show age-associated decline in canine circulating glutathione Senior dogs have lower levels of RBC total and reduced glutathione compared to young dogs. Higher levels of plasma GSSG were also measured. These are indicative of greater oxidative stress; corroborate the data given for cats in Example 1; and suggest a species-agnostic trend in glutathione reduction with age.

[0319] Also as for cats, no significant age-related differences were observed in plasma levels of Gly. This may reflect the fact that all of the animals in the preliminary study were healthy and offered appropriate amounts of complete and balanced diets.

[0320] Table 7. Red blood cell (RBC), whole blood (WB), and plasma glutathione concentrations, and plasma glycine (GLY) levels, in the young and senior dogs.

[0321] Measure Young Senior p value

[0322] RBC total glutathione (pM / ml) 1.26(1.16, 1.36) 1.04(0.94, 1.14) <0.001*

[0323] RBC GSH (pM / ml) 1.19(1.09, 1.302) 0.96(0.85, 1.06) <0.001*

[0324] RBC GSSG (nM / ml) 53.53 (39.22,73.06) 55.96(41.00,76.37) 0.822

[0325] RBC GSH:GSSG 28.93 (20.13,37.73) 25.41 (16.61,34.21) 0.527

[0326] WB total glutathione (pM / ml) 0.83 (0.75,0.90) 0.74(0.67,0.81) 0.059

[0327] WB GSH (pM / ml) 0.80(0.72,0.88) 0.70(0.62,0.78) 0.054

[0328] WB GSSG (nM / ml) 24.80(17.84,34.50) 29.59(21.78,41.15) 0.398

[0329] WB GSH: GSSG 31.46(21.02,47.09) 22.42 (14.98,33.56) 0.184

[0330] Plasma total glutathione (nM / ml) 0.99(0.72, 1.38) 1.18 (0.85, 1.63) 0.411

[0331] Plasma GSH (nM / ml) 0.75 (-0.54, 2.04) 0.24 (-1.00, 1.48) 0.523

[0332] Plasma GSSG (nM / ml) 0.23 (0.14,0.39) 0.86(0.52, 1.42) <0.001*

[0333] Plasma GSH:GSSG 3.41 (1.87,5.59) 0.71 (0.00, 1.72) 0.001*

[0334] Plasma GLY (nM / ml) 173.59(157.62, 189.57) 168.58 (152.50, 185.65) 0.621

[0335] All values are means and brackets indicate 95% confidence intervals of the mean. GSH, reduced glutathione; GSSG, oxidised glutathione;

[0336] GSH:GSSG, reduced to oxidised glutathione ratio. * indicates significance between groups (p < 0.05).

[0337] General materials and methods

[0338] The following methods were used throughout the above examples.

[0339] Animals'. Domestic short haired cats, Beagles, and Brittany beagles - all neutered - were housed at the Pet Health and Nutrition Center (PHNC), Lewisburg, OH, USA and all procedures were approved by the WALTHAM Animal Welfare and Ethical Review Body and the Institutional Animal Care and Use Committee. Animals were deemed healthy by a veterinarian at the start of the study with no evidence of systemic disease requiring treatment e.g. arthritis, diabetes, thyroid disorder, liver, or renal impairment requiring treatment and had not been vaccinated or prescribed any medication within 2 weeks of blood sampling. Routine housing, husbandry and exercise regimes were maintained throughout the course of the study. Cats and dogs were separately group housed in a free-living environment with indoor / outdoor access during the day (weather permitting). Rooms were fitted with environmental enrichment and all animals had daily social human interactions which included grooming and play with toys for a minimum of 20 mins. Water was provided ad libitum at all times. The general health and overall condition of each animal were monitored daily by the animal care staff .RBC glutathione measurement: For Examples 1 and 4, heparin-anticoagulated WB was placed on a rocker for at least 1 min before a 2 mL aliquot was removed for amino acid analysis. The remaining 1 mL sample was centrifuged at 1,000 x g for 30 min at 4°C and the buffy coat was then removed from the RBC pellet. Determination of GSH and GSSG was carried out by enzymatic recycling of GSSG to GSH using a glutathione detection ELISA according to manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY, USA, Cat No. ADI-900-160) including pre-treatment of samples with metaphosphoric acid to remove interfering proteins. Absorbance measurements were made at 405 nm for 12 min at 1 min intervals with a microplate reader using kinetic mode (Cytation 3 multi-mode, BioTek, Winooski, VT, USA). WB and RBC total glutathione, GSH, GSSG and their ratio were determined; GSH was calculated as the difference between total glutathione and GSSH. Data were not normalised to haemoglobin based on the assumption that variation in RBC haemoglobin levels in healthy cats is minimal.

[0340] In Examples 2 and 3, 1 mL heparin-anticoagulated WB was aliquoted and centrifuged at 2,000 x g for 10 min. The buffy coat was then removed from the RBC pellet and the pellet washed with PBS three times. The pellet aliquot was then flushed with nitrogen gas, snap frozen in liquid nitrogen and stored at -80 °C. Samples were shipped on dry ice to Creative Proteomics (New York, USA) for the quantification of RBC glutathione. An internal standard (IS) solution containing 13C isotope-labelled GSH and 13C isotope-labelled GSSG, were prepared in an anti -oxidation buffer. Serially diluted IS solutions were also prepared in the anti -oxidation buffer. RBC pellets were thawed on ice and diluted 50x with the anti -oxidation buffer. 50 pL of each RBC solution or IS calibration solution were mixed with 50 pL IS solution and 300 pL 40-mM N-ethylmaleimide-acetonitrile. After vortex-mixing for 10 mins at room temperature (RT), the resulting solutions were centrifuged, and the supernatant diluted 5x with water. 10 pL aliquots of the diluted supernatant were analysed by UPLC- MRM MS on an Agilent 1290 UHPLC system coupled to an Agilent 6495B QQQ mass spectrometer with positive-ion detection. Separation was carried out using a 10 cm long Cl 8 UPLC column with an ammonium acetate buffer (A) and methanol (B) as the mobile phase for gradient elution (5% to 75% B in 10 min), at 40°C and 0.20 mL / min. Concentrations of GSH and GSSG were calculated from the IS calibration by interpolating the constructed linear-regression curves, with the analyte to IS peak ratios measured from sample solutions.

[0341] Plasma and RBC Gly quantification'. Heparin-anticoagulated WB was centrifuged at 4±2°C for 30 mins at 3,000 g (for Examples 1 and 4), or 10 mins at 2,000 g (for Examples 2-3) to separate the plasma, buffy coat, and RBCs. For Examples 1 and 4, plasma was removed and deproteinised with 6% sulfosalicylic acid (1 :1) to remove plasma proteins; samples were centrifuged at 4000 g for 25 mins; the supernatant was filtered through a 0.45 mm PTFE filter and pH adjusted to 2.2 and frozen immediately at -80°C. For Examples 2-3, plasma was removed and frozen immediately at -80°C. The RBC pellet was washed three times in cold phosphate buffered saline (PBS) before also being stored at -80°C. Gly determination of plasma and RBCs was performed at the Amino Acid Laboratory, University of California (Davis), USA. Gly quantification was conducted using a Biochrom 30 amino acid analyser (Biochrom Ltd., Cambridge, UK).

[0342] WBC glutathione measurement: Measurement of GSH concentration in WBC subsets was performed by The Ohio State University Veterinary Clinical Flow Cytometry Service (Columbus, OH, USA). Methods performed were adapted from Webb et al. (2006) utilizing a Cytek® Northern Lights (Cytek Biosciences Inc.) spectral flow cytometer. Markers of WBC subsets CD21 (B-cells; clone CA2.1D6, Bio-Rad Laboratories Inc., Hercules, CA, Cat No. MCA1781R), CD4 (helper T-cells; clone vpg34, Bio-Rad Laboratories Inc., Cat No. MCA1346F), CD8 (cytotoxic T-cells; clone vpg9, Bio-Rad Laboratories Inc., Cat No. MCA1347GA), CD14 (monocytes, clone TUK4, Bio-Rad Laboratories Inc., Cat No. MCA1568GA), and a viability marker (propidium iodide, Sigma- Aldrich, Merck, Burlington, MA, Cat No. P4170) were used to segregate WBC subsets as part of a single multiplexed panel. In addition to the above subsets, granulocytes (neutrophils, eosinophils, and basophils together) were quantified and segregated according to characteristic light scatter properties and the absences of expression of CD21, CD4, CD8, or CD14. Briefly: to assess GSH in the cells, a non-fluorescent substrate, monochlorobimane (mBCl, Thermo Fisher Scientific Inc., Waltham, MA, Cat No. Ml 38 IMP) was included in the panel, which forms fluorescent adducts with glutathione when catalyzed by the enzyme glutathione-S-transferase. The median florescence intensity (MFI) of the mBCl -glutathione complex was recorded for each WBC population subset (B-cell, CD4+ T-cells, CD8+ T-cells, monocytes, and granulocytes). MFI was then correlated to the amount of GSH per cell using a standard curve generated by serial dilutions of known quantities of feline peripheral blood WBCs assessed by both flow cytometry and a commercial glutathione assay kit (Cayman Chemical, Ann Arbor, MI). Pairwise contrasts were performed for each of the WBC subsets considered both as the number of each subset per pL of blood (absolute cell counts) and as the amount of GSH per cell within each subset.

[0343] Mitogen-induced lymphoproliferative response'. Heparin-anticoagulated WB was centrifuged at RT for 30 mins at 390 g. The buffy coat was removed and WBCs isolated by Histopaque gradient density separation (Histopaque®-1077, Merck, Cat No. 10771 and Histopaque®-1119, Merck Cat No. 11191). Isolated WBCs were counted using a Beckman Z2 particle cell counter, plated at IxlO6cells / well in triplicate for each stimulation condition. Concanavalin A (Con A; from Jack Bean, Merck, Cat No. C5275) was added to the wells at either 0, 1 or 10 mg / mL. Cells were incubated at 37°C in a 5% CO2 enriched environment for 96 h. After incubation, plates were centrifuged at RT for 10 mins at 350 g and a tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Merck, Cat No. M5655) performed as per manufacturer’s instructions to quantify viable cells as a proxy of proliferation relative to the control.

[0344] Oxidative DNA damage: 8-hydroxy-2' -deoxyguanosine (8-OHdG) was measured using the OxiSelect™ Oxidative DNA Damage ELISA Kit (Cell Biolabs, Inc., USA, Cat No. STA- 320) according to the manufacturer’s instructions. Urine samples collected for the measurement of F2-IsoPs were stored at -80°C prior to shipment to Vanderbilt Eicosanoid Core Laboratory (Nashville, Tennessee, USA) for quantification of F2-IsoPs F2a (PGF2a), free 8-iso-prostaglandin F2a (8-iso-PGF2a), 2,3-dinor-5,6-dihydro-15-F2t-IsoP; and 5-series F2- IsoP by gas chromatography-negative ion chemical ionization-mass spectrometry employing stable isotope dilution as described in Milne et aL (2007) and Milne et aL (2013).

[0345] Biochemistry and haematology: Performed at IDEXX laboratories, USA.

[0346] Statistical Methods: The sample size for Examples 1 and 4 was determined using variance estimates for RBC total glutathione from an unpublished study in dogs at the Waltham Petcare Science Institute. In order to use this previous data to power for the study in cats, variability in cats was assumed to be comparable to dogs. The variance components were used to simulate 1,000 data sets, with the 25% effect size induced on to the senior group. Each data set was fit to a linear mixed effects model, with age as the fixed effect and animal as the random effect. Planned comparisons for this study were between the young adult and senior groups.

[0347] Data on Gly from a previous, unpublished study conducted in cats were used to estimate the variance components for calculating the sample size for Example 2. The variance components for plasma Gly and RBC Gly were estimated and used to simulate 1,000 data sets, with fold changes of 23% and 30% induced on the final sampling occasion in one diet group for plasma Gly and RBC Gly, respectively. Each data set for each measure was fit to a linear mixed effect model with diet, sampling occasion and their interaction as the fixed effects and individual cat as the random variable. Planned comparisons for Example 2 were the change from baseline at each time point compared between groups.

[0348] Data collated from Example 1 was used to power Example 3 by estimating the variance components of RBC total glutathione in pmol and simulating 1,000 data sets, with a 40%-fold change induced on final sampling occasion. Each data set for each measure was fit to a linear mixed effect model with diet group, sample occasion and their interaction as the fixed effects and individual cat as the random effect. Planned comparisons for Example 3 were within each diet between each of the sampling occasions, as well as between the diet groups at each sampling occasion.

[0349] For all analyses, power was calculated as the percentage of simulated data sets where all planned contrasts involving the fixed effect level where the effect size was induced were statistically significant. The sample size was determined as the minimum number of animals required to see the desired effect size with a minimum of 80% power.

[0350] During Examples 1 and 4 all sampling occasions were combined prior to being fit to a linear mixed effects model, with age group as the fixed effects and individual animal as the random effects. The model residuals were examined visually to assess the assumptions of the model (linearity of predictors, normality of residuals, independence of variables and homoscedasticity). To improve agreement with model assumptions, a loglO transformation was applied to some parameters, prior to model -fitting. Plasma GSH to GSSG ratio data was log 10 transformed after the addition of 1; (the minimum value to remove negative values). The mean of each measure for each age group and the difference between age groups (or fold change where data were loglO transformed) were estimated with 95% confidence intervals (CI).

[0351] For all measures in Examples 2-3, the data were fit to a linear mixed effects model (to account for repeated measures), with diet, sampling occasion and their interaction as the fixed effects, and individual cat as the random effect. The model residuals were examined visually to assess the assumptions of the model. Parameters that were deemed to violate the assumptions were loglO transformed prior to model fitting. The estimated means and 95% family-wise CI were extracted from the model for each diet at each sampling occasion.

[0352] In Example 2, the change from baseline to each subsequent time point was compared between the diets.

[0353] With the data from Example 3, the baseline value for each animal was included in the model as a covariate to account for any differences between the groups that may exist. Contrasts were made between diets at each of the sampling occasions.

[0354] For Examples 1, 3 and 4, the primary measure was RBC total glutathione, whilst the primary variables for Example 2 were plasma and RBC GLY.

[0355] For all comparisons, the estimated differences or fold changes were reported alongside the 95% family-wise CI and single-step adjusted P-values and a statistically significant difference was determined when the P-value < 0.05. Statistical analyses were performed in R v4.1.2 using libraries nlme, multcomp, lme4 and ggplot2. It will be understood that the inventors’ work has been described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention.

[0356] All references described above are incorporated by reference in their entirety.

Claims

CLAIMS1. A dietary composition for a companion animal, the composition comprising at least about 0.5% free glycine.

2. The composition of claim 1, comprising at least about 1.5% free glycine.

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

4. The composition of claim 1, comprising at least about 6.0% free glycine.

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

6. The composition of any one of the preceding claims, wherein the composition comprises a moisture content of at most about 7.0%.

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

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

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

10. The composition of any one of the preceding claims, wherein the companion animal is a cat, optionally wherein the companion animal is a senior cat.

11. The composition of claim 9 or claim 10, wherein the senior cat or senior dog is at least about 7.0 years of age, optionally wherein the senior cat or senior dog is at least about 9.5 years of age.

12. A method of 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 claim 13, wherein the disease is associated with oxidative stress.

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

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

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

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

19. The method of any one of claims 12-18, wherein the method increases the intracellular glutathione concentration, the circulating glutathione concentration, the intracellular GSH concentration, and / or the circulating GSH concentration by at least about 20% compared to the concentration in the absence of the method.

20. The method of any one of claims 12-19, wherein administering the dietary composition leads to immunologic or physiologic changes in the companion animal, for example increased lymphocytosis, increased neutrophilia, or panleukocytosis.

21. A method of determining the presence or absence of glutathione dysfunction in a companion animal, the method comprising:(a) providing a blood sample from the companion animal;(b) measuring the level of glutathione (GSH) and / or glutathione disulphide (GSSG) in the sample; and(c) identifying glutathione dysfunction as present if the intracellular GSH:GSSG ratio, the circulating GSH:GSSG ratio, the circulating GSH concentration, or the intracellular GSH concentration is less than a threshold value.

22. The method of claim 21, comprising identifying glutathione dysfunction as present 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 pM, or the intracellular GSH concentration is less than about 500 pM.

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

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

25. The method of any one of claims 20-24, wherein if glutathione dysfunction is identified as present in step (c), the method comprises selecting the companion animal as suitable for a method according to any one of claims 12-20.

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