FOOD PRODUCT FOR REGULATING LIPID METABOLITES AND METHODS
20220408757 · 2022-12-29
Assignee
Inventors
Cpc classification
A23K20/147
HUMAN NECESSITIES
A23K20/158
HUMAN NECESSITIES
International classification
A23K20/147
HUMAN NECESSITIES
Abstract
The present invention relates to methods of determining the levels of endogenous margaric acid before and after a meal in a companion animal, wherein the meal is a pet foodstuff having a particular protein to fat ratio, which is useful in increasing the endogenous levels of margaric acid post-prandially and having the beneficial effects as described herein. The pet foodstuff comprises a ratio of protein to fat of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis. The invention also relates to methods of determining levels of lipid metabolites, such as palmitic acid, diacylglycerol (DAG), triacylglycerols (TAGs) and ceramides before and after a meal in a companion animal, wherein the meal is a pet foodstuff having a particular protein to fat ratio, which is useful in controlling, regulating and stabilizing lipid metabolites post-prandially to prevent and/or reduce lipotoxicity in the companion animal.
Claims
1. A pet foodstuff comprising: a ratio of protein:fat of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis, wherein, when fed to a companion animal, lipid metabolite levels including one or more of palmitic acid, diacylglycerol (DAG), triacylglycerol (TAGs), or ceramides of the companion animal measured within five hours after eating the foodstuff have a fold change of less than 1.0.
2. The pet foodstuff of claim 1, wherein the lipid metabolite levels of the companion animal having a fold change of less than 1.0 reduces a risk of one or more of insulin resistance, adipose tissue, or skeletal muscle inflammation in the companion animal.
3. The pet foodstuff of claim 1, wherein the lipid metabolite levels of the companion animal having a fold change of less than 1.0 reduces a risk of one or more of impaired neurological function, nerve cell damage, or cell death in the companion animal.
4. The pet foodstuff of claim 1, wherein, when fed to the companion animal, an endogenous level of margaric acid of the companion animal is greater for at least five hours after eating the foodstuff than an endogenous level of margaric acid of the companion animal after eating a diet without a ratio of protein:fat of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis.
5. The pet foodstuff of claim 1, wherein, when fed to the companion animal, fasted plasma levels of lipotoxic acid including one or more of palmitic acid, diacylglycerol (DAG), triacylglycerol (TAGs), or ceramides measured within five hours after eating the foodstuff do not increase post-prandially.
6. The pet foodstuff of claim 1, wherein the foodstuff is a dry product comprising 5-15% moisture, a semi-moist product comprising 15-70% moisture, or a wet product comprising 70-90% moisture.
7. The pet foodstuff of claim 1, wherein the foodstuff is a cooked product.
8. The pet foodstuff of claim 1, comprising one or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline and one or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid.
9. The pet foodstuff of claim 6, wherein a ratio of the one or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline to the one or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid is 1:0.006 to 1:4.5 on a gram:gram as fed or dry matter basis
10. The pet foodstuff of claim 1, comprising two or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline and two or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid at a ratio of 1:0.014 to 1:3.5 on a gram:gram as fed or dry matter basis
11. The pet foodstuff of claim 1, comprising three or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline and three or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid at a ratio of 1:0.025 to 1:2.5 on a gram:gram as fed or dry matter basis.
12. The pet foodstuff of claim 1, wherein the companion animal is a dog or a cat.
13. A method of preventing or reducing lipotoxicity in a companion animal comprising: feeding a pet foodstuff comprising a ratio of protein:fat of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis to a companion animal, wherein lipid metabolite levels including one or more of palmitic acid, diacylglycerol (DAG), triacylglycerol (TAGs), or ceramides of the companion animal measured within five hours after eating the foodstuff have a fold change of less than 1.0.
14. The method of claim 13, wherein the lipid metabolite levels of the companion animal having a fold change of less than 1.0 reduces a risk of one or more of insulin resistance, adipose tissue, or skeletal muscle inflammation in the companion animal.
15. The method of claim 13, wherein the lipid metabolite levels of the companion animal having a fold change of less than 1.0 reduces a risk of one or more of impaired neurological function, nerve cell drainage, or cell death in the companion animal.
16. The method of claim 13, wherein an endogenous level of margaric acid of the companion animal is greater for at least five hours after eating the foodstuff than an endogenous level of margaric acid of the companion animal after eating a diet without a ratio of protein:fat of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis.
17. The method of claim 13, wherein fasted plasma levels of lipotoxic acid including one or more of palmitic acid, diacylglycerol (DAG), triacylglycerol (TAGs), or ceramides measured within five hours after eating the foodstuff do not increase post-prandially.
18. The method of claim 13, wherein the foodstuff is a dry product comprising 5-15% moisture, a semi-moist product comprising 15-70% moisture, or a wet product comprising 70-90% moisture.
19. The method of claim 13, wherein the foodstuff is a cooked product.
20. The method of claim 13, wherein the pet foodstuff comprises one or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline and one or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid.
21. The method of claim 20, wherein a ratio of the one or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline to the one or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid is 1:0.006 to 1:4.5 on a gram:gram as fed or dry matter basis
22. The method of claim 13, comprising two or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline and two or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid at a ratio of 1:0.014 to 1:3.5 on a gram:gram as fed or dry matter basis
23. The method of claim 13, comprising three or more of aspartic acid, serine, glutamic acid, glycine, alanine, or proline and three or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, or linolenic acid at a ratio of 1:0.025 to 1:2.5 on a gram:gram as fed or dry matter basis.
24. The method of claim 13, wherein the companion animal is a dog or a cat.
Description
BRIEF DESCRIPTION OF THE FIGURES
Examples & Figures
[0071] The invention will now be further described by way of reference to the following Examples and Figures, which are provided for the purpose of illustration only and are not to be construed as being limiting on the invention.
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DETAILED DESCRIPTION OF THE DISCLOSURE
[0077] Aim of the study was to determine different benefits from macronutrient diets in cats and to investigate the difference in macronutrient compositions on post-prandial metabolite profiles in cats.
[0078] A study was performed in 19 cats aged between 1 and 2 years to investigate the impact of different nutrient diets on the post-prandial gut hormone levels. Four diets (Diet 1, Diet 2, Diet 3 and Diet 4) were used to investigate the levels of lipid metabolites of palmitic acid, diacylglycerol (DAG), triacylglycerols (TAGs) and endogenous levels of margaric acid. In addition, two more diets (Diet 5 and Diet 6) were also used to investigate the levels of lipid metabolites of palmitic acid, diacylglycerol (DAG), triacylglycerols (TAGs). Six diets (labelled Diet 1, Diet 2, Diet 3, Diet 4, Diet 5 and Diet 6) were manufactured using the same raw materials but in different proportions to provide a range of protein to fat ratios. Diet 4 was manufactured to have a protein:fat ratio within the target range of 1:0.27 to 1:0.63 (P:F of 1:0.37). The other diets were manufactured so that protein to fat ratios fell on either side of the target range, as well as within the target range. As can be seen in Table 1, diets 1-3 fell above the target range, whereas diets 5 and 6 fell below the target range and diet 4 fell within the target range.
[0079] All cats were fed in four or six consecutive phases of 14 days respectively, in a randomised crossover design with each cat being fed each of the diets in turn. All cats were within 5% of ideal body weight at the start of the trial. For the first 13 days of each phase, cats were fed two meals per day in amounts to maintain a stable, healthy bodyweight. On day 14 of each phase, cats had blood samples taken at 5 time points (one prior to feeding as a baseline and then at 15, 60, 120, & 300 mins following the end of the 20 minute meal).
TABLE-US-00002 TABLE 1 Diet compositions and ratio of Protein:Fat Protein Carbohydrate Fat Energy Diet g/100 g g/100 g g/100 g kcal/100 g P:F 1 7.87 2.55 11.47 132 1:1.46 (low) (low) (high) 2 7.47 9.93 7.67 125 1:1.03 (low) (medium) (medium) 3 7.00 12.68 5.30 119 1:0.76 (low) (high) (medium) 4 10.90 2.71 4.00 95 1:0.37 (high) (low) (medium) 5 10.80 4.71 2.37 82 1:0.22 (high) (low) (low) 6 11.60 9.87 1.30 92 1:0.11 (high) (high) (low)
TABLE-US-00003 TABLE 2 Concentrations of certain amino acids and fatty acids present in the diet associated with the beneficial effects (diet 4). Concentration in diet Nutrient (g/100 g as fed) Aspartic Acid 0.74 Serine 0.38 Glutamic Acid 1.14 Glycine 0.9 Alanine 0.6 Proline 0.55 Myristic Acid 0.03 Palmitic Acid 0.58 Stearic Acid 0.25 Palmitoleic Acid 0.13 Oleic Acid 0.90 Linolenic Acid 0.04
Metabolite Profiling
[0080] Two types of mass spectrometry analyses were applied to all samples. GC-MS (gas chromatography-mass spectrometry; Agilent 6890 GC coupled to an Agilent 5973 MS-System, Agilent, Waldbronn, Germany) and LC-MS/MS (liquid chromatography-MS/MS; Agilent 1100 HPLC-System (Agilent, Waldbronn, Germany) coupled to an Applied Biosystems AP14000 MS/MS-System (Applied Biosystems, Darmstadt, Germany)) were used for broad profiling (van Ravenzwaay et al. 2007).
[0081] Proteins were removed from plasma samples (60 pl) by precipitation. Subsequently polar and non-polar fractions were separated for both GC-MS and LC-MS/MS analysis by adding water and a mixture of ethanol and dichloromethane. For GC-MS analyses, the non-polar fraction was treated with methanol under acidic conditions to yield the fatty acid methyl esters derived from both free fatty acids and hydrolyzed complex lipids. The polar and non-polar fractions were further derivatized with 0-methyl-hydroxyamine hydrochloride (20 mg/ml in pyridine, 50 pl) to convert oxo-groups to 0-methyloximes and subsequently with a silylating agent (MSTFA, 50 pl) before GC-MS analysis. For LC-MS/MS analyses, both fractions were reconstituted in appropriate solvent mixtures. High performance LC (HPLC) was performed by gradient elution using methanol/water/formic acid on reversed phase separation columns. Mass spectrometric detection technology was applied as described in the U.S. Pat. No. 7,196,323, which allows targeted and high sensitivity “Multiple Reaction Monitoring” profiling in parallel to a full screen analysis. To account for inter- and intra-instrumental variation in both GC-MS and LC-MS/MS profiling, data were normalised to the median of reference samples derived from a pool formed from aliquots of all samples from that species. Pooled reference samples were run in parallel through the whole process. The limit of detection and the dynamic range of the semi-quantitative measurements were determined by dilution and spiking experiments during method development. Daily, the signal-to-noise (S/N) ratio threshold of 15 was used for a metabolite to be considered “semi-quantitative”.
Data Analysis
[0082] Data analysis included univariate statistics (mixed linear models) and multivariate analyses (principal component analysis (PCA)).
Multivariate Statistics
[0083] All metabolite data were log-transformed (to ensure an approximate normal distribution), centered and scaled to unit variance. Multivariate analysis was performed using the software Simca (version 13; Umetrics AB, Umea, Sweden).
Univariate Statistics
[0084] For the entire dataset and each individual group to be analyzed statistically, the minimum, maximum, mean and median values were determined. Mean and median values were calculated on a logarithmic scale and then back-transformed to non-logarithmic scale. Figures were constructed in JMP with 95% Confidence Intervals.
[0085] The y axis in the figures is a fold change measurement. Fold change is a measure describing how much a quantity changes going from an initial to a final value. For example, an initial value of 30 and a final value of 60 corresponds to a fold change of 1 (or equivalently, a change to 2 times), or in common terms, a one-fold increase.
Results
[0086] Plasma levels of margaric acid were highest in cats on diet 4. As the cats were not fed dairy fat, the levels are not consistent with the amount of fat consumed, it is likely that the elevated margaric acid levels on diet 4 is the result of enhanced endogenous production (
[0087] Margaric acid has been identified as a marker associated with lower risk/incidence of insulin resistance, type 2 diabetes, inflammation and coronary heart disease. Therefore, increasing the endogenous levels of margaric acid as seen with cats fed diet 4 is beneficial for lowering the risk/incidence of insulin resistance, type 2 diabetes, inflammation and coronary heart disease in companion animals.
[0088] Fasted plasma levels of a number of lipotoxic lipids, including palmitic acid, diacylglycerol (DAG), triacylglycerols (TAGs) and ceramides were lowest in cats following consumption of the diet with a protein:fat ratio of 1:0.37 (diet 4) and did not increase post-prandially on this diet (
[0089] Palmitate (palmitic acid) has been shown to increase the expression and secretion of inflammatory cytokines (e.g. IL-6, TNF-a) in adipocytes (Ajuwon & Spurlock 2005, Bradley et al 2008) and muscle cells (Jove et al 2005, Jove et al 2006). Increased inflammatory cytokines cause tissue damage and also contribute to impaired metabolic function (e.g. insulin resistance) in tissues in which they are elevated. Therefore, the fact that plasma palmitic acid levels are lowest in cats fed a diet with a protein:fat ratio of 1:0.37 (diet 4) both in the fasted state and following a meal provides a benefit of reduced inflammation.
[0090] Palmitate has been shown to impair insulin signalling pathways which can lead to insulin resistance in multiple tissues (e.g. skeletal muscle cells, adipocytes, hepatocytes) (Chavez & Summers 2003, Nakamura et al 2009, Reynoso et al 2003, Sinha et al 2004, Xi et al 2007). Impaired insulin signaling and insulin resistance result in an inability to effectively take up glucose into cells leading to elevations in blood glucose levels and ultimately to the development of type 2 diabetes mellitus. The results show that the low levels of palmitate in cats fed diet 4 (with a protein:fat ratio of 1:0.37) compared to the elevated levels of palmitate when fed the other 5 diets would reduce the risk of or prevent the development of insulin resistance and type 2 diabetes.
[0091] Palmitate also has been shown to induce the accumulation of ceramide and diacylglycerol (DAG) in skeletal muscle cells (Chavez & Summers 2003). These two lipid metabolites have been shown to inhibit insulin signaling in cultured cells and to accumulate in insulin resistant tissues contributing to insulin resistance (Holland et al 2011, Itani et al 2002, Summers 2006, Yu et al 2002). The results show that plasma DAG levels do not increase post-prandially when fed a diet with a protein:fat ratio of 1:0.37 (diet 4), whereas there were large post-prandial increases in DAG levels following consumption of the other 5 diets. Fasted and post-prandial plasma ceramides were lowest when fed diet 4. Since levels of both DAG and ceramides inhibit insulin signaling and contribute to insulin resistance the results show that diet 4 is beneficial in preventing accumulation of these lipid mediators and therefore would reduce the risk of developing insulin resistance and type 2 diabetes. Increased accumulation of ceramides in neuronal cells can lead to apoptosis (a form of cell death) contributing to impaired neurological function and neurodegeneration and therefore a diet that minimized the post-prandial formation of these toxic ceramides (e.g. diet 4) would be beneficial in preventing neuronal cell death and the development of neurological impairment.