Food product for reducing muscle breakdown and methods thereof

Abstract

The present invention relates to methods of determining the levels of 3-methylhistidine 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 decreasing the levels of 3-methydlhistidine 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 pet foodstuff described is fed to a companion animal for use in reducing and/or preventing sarcopenia. The present invention also relates to methods of feeding the pet foodstuff described and/or dietary regimes to provide the companion animal with the benefit of reducing and/or preventing sarcopenia.

Claims

1. A method comprising: (a) measuring a level of 3-methylhistidine in a blood sample from a cat, (b) feeding a cat a foodstuff comprising a protein to fat ratio of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis, and further comprising i) one or more of aspartic acid, serine, glutamic acid, glycine, alanine and proline, and ii) one or more of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, and linolenic acid, wherein the ratio of i) to ii) is 1:0.006 to 1:4.5 on a gram:gram as fed or dry matter basis, and (c) measuring the level of 3-methylhistidine in a blood sample from the cat after feeding the foodstuff, wherein a reduction and/or maintenance in 3-methylhistidine level post-prandially is indicative of a foodstuff effective at reducing skeletal muscle breakdown in a cat.

2. The method of claim 1, wherein the cat is above 8 years of age.

3. The method of claim 1, wherein the levels of 3-methylhistidine are measured at least once between an hour or immediately before the foodstuff is fed to the cat and at least once between at least 30 minutes to 5 hours after the foodstuff has been fed to the cat.

4. The method of claim 1, wherein the foodstuff has a protein to fat ratio of about 1:0.33 to 1:0.55 on a gram:gram as fed or dry matter basis.

5. The method of claim 1, wherein the foodstuff has a protein to fat ratio of about 1:0.45 on a gram:gram as fed or dry matter basis.

6. The method of claim 1, wherein the foodstuff has a protein to fat ratio of about 1:0.37 on a gram:gram as fed or dry matter basis.

7. The method of claim 1, wherein the foodstuff is a nutritionally balanced pet foodstuff.

8. The method of claim 1, wherein the foodstuff has a low caloric density.

9. The method of claim 1, further comprising a step of formulating a foodstuff comprising a protein to fat ratio of 1:0.27 to 1:0.63 on a gram:gram as fed or dry matter basis prior to feeding the cat the foodstuff.

Description

EXAMPLES

(1) The invention will now be further described by way of reference to the following Example and FIGURE, which are provided for the purpose of illustration only and are not to be construed as being limiting on the invention.

(2) FIG. 1: Graph showing plasma levels of 3-methylhistidine in cats fed the diets over time. The graph shows that cats fed Diet 4 (having P:F of 1:0.37 gram:gram as fed or dry matter basis) obtained low and stable plasma levels of the muscle breakdown product 3-methylhistidine compared to the other diets. The y axis is a fold change measurement.

(3) 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.

(4) 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.

(5) A study was performed in 19 cats aged between 1 and 2 years to investigate the impact of 5 diets on various aspects of metabolism. Five diets (labelled Diet 1, Diet 2, Diet 3, Diet 4, and Diet 5) were manufactured using the same raw materials but in different proportions to provide a range of protein to fat ratios.

(6) All cats were fed in five consecutive phases of 14 days in a randomised crossover design with each cat being fed each of the 5 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).

(7) 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 11.60  9.87  1.30 92 1:0.11 (high) (high) (low)

(8) 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 (g/100 g Nutrient 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

(9) 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 API4000 MS/MS-System (Applied Biosystems, Darmstadt, Germany)) were used for broad profiling (van Ravenzwaay et al. 2007).

(10) Proteins were removed from plasma samples (60 μl) 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 O-methyl-hydroxyamine hydrochloride (20 mg/ml in pyridine, 50 μl) to convert oxo-groups to O-methyloximes and subsequently with a silylating agent (MSTFA, 50 μl) 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”.

(11) Data Analysis

(12) Data analysis included univariate statistics (mixed linear models) and multivariate analyses (principal component analysis (PCA)).

(13) Multivariate Statistics

(14) 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, Umeå, Sweden).

(15) Univariate Statistics

(16) 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.

(17) Figures were constructed in JMP with 95% Confidence Intervals.

(18) Results

(19) The study surprisingly showed that cats fed diets with protein to fat ratios of 1:0.37 (diet 4) had low fasting levels of 3-methylhistidine (a marker of muscle breakdown) which did not increase following food intake. In contrast, consumption of other diets with different protein:fat ratios resulted in either increased fasting levels of 3-methylhistidine, increased post-prandial levels of 3 methylhistidine or both. A low level of 3-methylhistidine is indicative of minimal muscle breakdown whereas increased levels of 3-methylhistidine are consistent with increased breakdown of muscle protein.

(20) The results therefore show that the ratio of protein to fat in the food can have a marked impact on the breakdown of muscle protein in the cat. Based on these results, diets with a protein to fat ratio the same as diet 4 (i.e. 1:0.37) and within a range either side of that of diet 4 (i.e. 1:0.27 to 1:0.63) would provide a benefit to the cat of reducing muscle breakdown in comparison to the other diets and therefore can lead to minimising muscle turnover and may lead to better metabolic responses to perturbation, healthier longevity and improved outcome with ageing.

(21) At all sample points after meals of Diets 1, 2, 3 and 5, the 3-methylhistidine concentrations were significantly higher than after Diet 4. From 30 minutes to an hour post meal until 325 minutes post meal, levels of GIP after Diets 1, 2, 3 and 5, were significantly higher than after Diet 4. (FIG. 1).

(22) The benefits of reducing/preventing skeletal muscle breakdown by feeding a diet with a protein:fat ratio of 1:0.27 to 1:0.63 as described in the present invention are that normal skeletal muscle mass can be maintained for longer in the lifespan of the animal contributing to the avoidance of weakness, frailty and reduced activity commonly associated with aging in companion animals. Preventing/reducing skeletal muscle breakdown is also beneficial because an adequate skeletal muscle mass is essential to supply required amino acids in order to maintain the protein turnover of vital tissues and organs in the post-absorptive state and potentially to provide substrate for gluconeogenesis in both fed and fasted states.