Methods for predicting and stratifying the risk of sarcopenia and NAD deficiency

Abstract

To provide a method for determining whether a subject is having sarcopenia or a subject is having a high risk of developing sarcopenia in a field of a muscle disease, and to provide a method for predicting how responsive the subject is to a nutritional therapy.SOLUTION: A method for supporting a determination as to whether a subject is having sarcopenia includes: the step (a) of measuring the level of trigonelline in a sample derived from blood of a subject; and the step (b) of comparing the level of trigonelline in the sample with a reference value. Reduction of the level of the trigonelline in the sample compared to the reference level is the index of the sarcopenia of the subject.SELECTED DRAWING: None

Description

[Background technology]

[0001] Age-related loss of muscle mass and function is inevitable in all individuals, but its progression varies greatly depending on genetic and environmental factors such as physical activity and nutritional intake.

[0002] Sarcopenia is defined as a condition in which age-related decline in muscle mass and function worsens and impacts quality of life. In contrast, frailty is another classification of age-related decline in physical function, characterized by loss of muscle strength and function, but not muscle mass. Sarcopenia is clinically defined by loss of muscle mass and function, using cutoffs that stratify the aging population for individuals with mobility pathology. Sarcopenia predicts future disability and mortality, and was assigned an official ICD-10 disease code in 2016 (Anker et al., 2016).

[0003] Sarcopenia is common in both men and women over the age of 65, with an estimated global prevalence of 3-30% according to implemented operational definitions. Sarcopenia is associated with numerous adverse physical and metabolic outcomes, including frailty, disability, obesity, diabetes, and osteoporosis. As a result, there are significant healthcare costs attributable to sarcopenia. Although age, sex, body size, heritability, and physical activity are identified adult influences, there remains unexplained variation in muscle mass and strength among older individuals.

[0004] Sarcopenia is a multifactorial syndrome associated with pathophysiological changes such as impaired neuromuscular transmission, altered excitation-contraction coupling, reduced regenerative capacity associated with stem cell depletion, impaired mitochondrial and energy metabolism in muscle fibers, and ultimately marbled fatty and fibrotic skeletal muscle. Thus, the pathogenesis of this syndrome is complex and not fully understood, but it is strongly related to reduced physical activity, hormonal reduction of anabolic hormones (androgens, IGF-1) and malnutrition / malnutrition.

[0005] The MEMOSA (Multi-Ethnic Molecular Determinants of Human Sarcopenia) study was designed to determine the molecular and nutritional changes associated with sarcopenia in humans.

[0006] The inventors of the present application have discovered that the levels of anthranilic acid and trigonelline in the body, and the ratio of trigonelline levels:anthranilic acid levels, are indicative of sarcopenia, or an increased risk of developing sarcopenia, in a subject. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows that serum trigonelline levels are different in sarcopenic patients and are positively correlated with lean muscle mass, grip strength, and gait. [Diagram 2] FIG. 1 shows that serum anthranilic acid levels are different in sarcopenic patients and are negatively correlated with lean muscle mass, grip strength, and gait. [Diagram 3] FIG. 1 shows that serum trigonelline:anthranilic acid ratios differ in sarcopenic patients and are positively correlated with lean muscle mass, grip strength, and ambulation. [Figure 4A] Gene set enrichment analysis (GSEA) of sarcopenic muscles versus control muscles. [Figure 4B] GSEA enrichment plot of selected oxidative phosphorylation gene sets from Figure 4 A. RNA-seq of human sarcopenic skeletal muscle reveals that mitochondrial dysfunction is the major alteration in transcription under sarcopenia. [Figure 5A] Gene set enrichment analysis of sarcopenic muscles versus control muscles. [Figure 5B]GSEA enrichment plot of the oxidative phosphorylation gene set selected from Figure 5A, showing a positive correlation between skeletal muscle mitochondrial functional gene expression and serum trigonelline levels. [Figure 6A] Gene set enrichment analysis of sarcopenic muscles versus control muscles. [Figure 6B] FIG. 6B is a GSEA enrichment plot of the selected oxidative phosphorylation gene set from FIG. 6A, showing a negative correlation between skeletal muscle mitochondrial functional gene expression and serum anthranilic acid levels. [Figure 7A] FIG. 1. Gene set enrichment analysis of sarcopenic versus control muscles. [Figure 7B] FIG. 7B is a GSEA enrichment plot of the oxidative phosphorylation gene set selected from FIG. 7A, showing a positive correlation between expression of skeletal muscle mitochondrial functional genes and the ratio of serum trigonelline:anthranilic acid levels. [Figure 8] FIG. 1 shows that expression of genes such as NMAT1, NAMPT, and PNP is reduced in human sarcopenic muscle. [Figure 9] NAD+ levels in muscle biopsies from control and sarcopenic participants. [Figure 10] FIG. 1 shows that the ratio of serum trigonelline:anthranilic acid levels is positively correlated with lean muscle mass index. Detailed Description of the Invention

[0008] The present invention provides a method for determining whether a subject has sarcopenia or is at high risk of developing sarcopenia, comprising: (a) measuring the level of anthranilic acid in a sample obtained from a subject; (b) comparing the level of anthranilic acid in the sample with a reference value; wherein an increased level of anthranilic acid in the sample compared to a reference level is indicative of sarcopenia or the risk of developing sarcopenia.

[0009] In one embodiment, the method comprises: (a) measuring the level of trigonelline in a sample obtained from a subject; (b) comparing the level of trigonelline in the sample with a reference value; and wherein a decrease in the level of trigonelline in the sample compared to the reference value is indicative of sarcopenia or the risk of developing sarcopenia in the subject.

[0010] The present invention also provides a method for determining whether a subject has sarcopenia or is at high risk of developing sarcopenia, comprising: (a) measuring levels of trigonelline and anthranilic acid in a sample obtained from a subject; (b) determining the ratio of trigonelline:anthranilic acid in the sample; (c) comparing the ratio of trigonelline:anthranilic acid in the sample with a reference value; wherein a comparison of the ratio of trigonelline:anthranilic acid in the sample with a reference value is indicative of sarcopenia or risk of developing sarcopenia in the subject.

[0011] In one embodiment, a decrease in the ratio of trigonelline:anthranilic acid in a sample obtained from a subject compared to the reference value is an indication of sarcopenia or an increased risk of developing sarcopenia in the subject.

[0012] The present invention also provides a method for determining whether a subject has sarcopenia or is at high risk of developing sarcopenia, comprising: (a) measuring levels of trigonelline and anthranilic acid in a sample obtained from a subject; (b) determining the ratio of anthranilic acid:trigonelline in the sample; (c) The ratio of anthranilic acid:trigonelline in the sample comparing with a reference value; wherein a comparison of the ratio of anthranilic acid:trigonelline in the sample with a reference value is indicative of sarcopenia or risk of developing sarcopenia in the subject.

[0013] In one embodiment, an increased ratio of anthranilic acid:trigonelline in a sample obtained from a subject compared to the reference value is indicative of sarcopenia or an increased risk of developing sarcopenia in the subject.

[0014] The present invention also provides a method for determining whether a subject has sarcopenia or is at high risk of developing sarcopenia, comprising: (a) measuring levels of trigonelline and anthranilic acid in a sample obtained from a subject; (b) determining the relative amount or combination of anthranilic acid and trigonelline in the sample; (c) comparing the relative amount or combination of anthranilic acid and trigonelline in the sample with a reference value; wherein a comparison of the relative amount or combination of anthranilic acid and trigonelline in the sample with a reference value indicates sarcopenia or risk of developing sarcopenia in the subject; The above combination is n * f(A)+m * g(B), where A and B are the concentrations of trigonelline and anthranilic acid, f() and g() represent mathematical transformations, and n and m are numerical coefficients that can be positive or negative.

[0015] For example, the combination is 1 * log2(A)+(-1) * log2(B) = log2(A / B) or 1 * It can be defined as log2(A)-2log2(B)=log2(A / (B)^2).

[0016] In one embodiment, the mathematical transformation includes a logarithmic transformation.

[0017] In one embodiment, an increase in the relative amount of anthranilic acid to trigonelline in a sample obtained from the subject compared to the reference value is indicative of sarcopenia or an increased risk of developing sarcopenia in the subject.

[0018] In one embodiment, the present invention relates to a method for determining whether a subject has sarcopenia.

[0019] In one embodiment, the present invention relates to a method for determining whether a subject is at increased risk of developing sarcopenia.

[0020] Sarcopenia is evaluated by one or more of muscle mass loss, muscle strength loss, and physical performance loss. More preferably, sarcopenia is evaluated by two or more of muscle mass loss, muscle strength loss, and physical performance loss. Most preferably, sarcopenia is evaluated by muscle mass loss, muscle strength loss, and physical performance loss. All of these can be measured by methods well known to those skilled in the art.

[0021] Muscle mass can be measured by CT (computed tomography), DXA (dual energy x-ray absorptiometry), MRI (magnetic resonance imaging) or D3 creatine dilution.

[0022] Muscle strength can be measured by grip strength (eg, using handheld dynamometry) or knee extension strength (eg, using quadriceps torque measurements).

[0023] Physical performance can be measured by walking speed, SPPB, 400m walk test, TUG (time up and go) test, or stair climbing test.

[0024] In one embodiment, the levels of trigonelline and anthranilic acid are measured by mass spectrometry. Preferably, the levels of trigonelline and anthranilic acid are measured by liquid chromatography followed by mass spectrometry.

[0025] In one embodiment, the subject is a human subject.

[0026] In one embodiment, the human subject is elderly. In one embodiment, the human subject is elderly.

[0027] In one embodiment, the subject is a companion animal, preferably a dog.

[0028] The present invention provides a method for determining whether a subject has a muscle wasting condition or a muscle disorder, or is at high risk of developing a muscle wasting condition or a muscle disorder, comprising: (a) measuring the level of anthranilic acid and / or trigonelline in a sample obtained from a subject; (b) comparing the levels of anthranilic acid and / or trigonelline in the sample with a reference value; wherein an increased level of anthranilic acid in the sample compared to a reference value or a decreased level of trigonelline in the sample compared to a reference value is indicative of a muscle wasting condition or disease, or a risk of developing a muscle wasting condition or disease.

[0029] The present invention also provides a method for determining whether a subject has a muscle wasting condition or a muscle disorder, or is at increased risk of developing a muscle wasting condition or a muscle disorder, comprising the steps of: (a) measuring levels of trigonelline and anthranilic acid in a sample obtained from a subject; (b) determining the ratio of trigonelline:anthranilic acid in the sample; (c) comparing the ratio of trigonelline:anthranilic acid in the sample with a reference value; wherein comparison of the ratio of trigonelline:anthranilic acid in the sample with a reference value is indicative of a muscle wasting condition or disease, or an increased risk of developing a muscle wasting condition or disease in the subject. In one embodiment, a decrease in the ratio of trigonelline:anthranilic acid in a sample from a subject compared to the reference value is an indication of a muscle wasting condition or disease, or an increased risk of developing a muscle wasting condition or disease in the subject.

[0030] Muscle wasting can be caused by disuse, immobility, prolonged bed rest, or stays in an ICU (intensive care unit).

[0031] The muscle disease may be sarcopenia, cachexia (caused by chronic diseases such as cancer, COPD, heart failure, renal disease, etc.), genetic muscle disorders, and dystrophies such as Duchenne muscular dystrophy, myotoxicity or rhabdomyolysis induced by drugs such as corticosteroids or statins.

[0032] Preferably, the muscle disease is sarcopenia.

[0033] The following embodiments relate to one or more of the above-mentioned methods of the invention.

[0034] In one embodiment, the reference value is determined from samples obtained from the same subject or from samples obtained from a group of subjects.

[0035] In one embodiment, the reference value is determined in a sample from the same subject.

[0036] In one embodiment, the baseline value is measured in the same subject prior to nutritional therapy, for example with a nutritional composition, and compared to the measurement value following nutritional therapy.

[0037] In one embodiment, the reference value is determined in samples obtained from a group of subjects, for example a group of subjects of similar age.

[0038] In one embodiment, the subject is an elderly person.

[0039] In one embodiment, the subject is elderly.

[0040] In one embodiment, the sample obtained from the subject is a plasma sample.

[0041] In one embodiment, the sample obtained from the subject is a serum sample.

[0042] In one embodiment, the sample obtained from the subject is a urine sample.

[0043] The present invention also relates to a nutritional composition for use in the treatment or prevention of sarcopenia in a subject.

[0044] In one embodiment, the subject has been identified as having sarcopenia or is at high risk of developing sarcopenia.

[0045] In one embodiment, the subject has been identified as having sarcopenia or at high risk of developing sarcopenia by a method according to the invention.

[0046] In one embodiment, the nutritional composition comprises a NAD precursor. In one embodiment, the nutritional composition comprises trigonelline. In one embodiment, the nutritional composition comprises a precursor of trigonelline. In one embodiment, the nutritional composition comprises a derivative of trigonelline. Trigonelline is an alkaloid having the chemical formula C7H7NO2 and CAS number 535-83-1.

[0047] In one embodiment, the subject is an elderly person.

[0048] In one embodiment, the subject is elderly.

[0049] In one embodiment, the subject is identified as having sarcopenia by a method according to the invention, the nutritional composition comprises an NAD precursor, and the subject is elderly.

[0050] In one embodiment, the subject is identified as having sarcopenia by a method according to the present invention, the nutritional composition comprises trigonelline and the subject is elderly.

[0051] The present invention also relates to a method of treating or preventing sarcopenia in a subject.

[0052] In one embodiment, the subject has been identified as having sarcopenia or is at high risk of developing sarcopenia.

[0053] In one embodiment, the subject has been identified as having sarcopenia or as being at high risk of developing sarcopenia by a method according to the invention.

[0054] In one embodiment, the method comprises improving the lifestyle of the subject.

[0055] In one embodiment, the lifestyle modification includes physical activity, for example weight-bearing exercise, resistance exercise or endurance exercise.

[0056] In one embodiment, improving the subject's lifestyle comprises modifying diet.

[0057] In one embodiment, the dietary modification comprises administering to the subject at least one nutritional composition as part of a diet to treat or prevent sarcopenia.

[0058] In one embodiment, the nutritional composition comprises an NAD precursor.

[0059] In one embodiment, the subject is an elderly person.

[0060] In one embodiment, the subject is elderly.

[0061] The present invention also relates to methods for predicting the responsiveness of a subject having muscle wasting or a muscle disease, or at high risk of developing muscle wasting or a muscle disease, to a nutritional composition.

[0062] In one embodiment, the nutritional composition comprises an NAD precursor, the subject is elderly, and the muscle disease is sarcopenia.

[0063] The present invention also relates to methods for predicting the responsiveness of a subject with sarcopenia, or at high risk of developing sarcopenia, to a nutritional composition.

[0064] In one embodiment, the nutritional composition comprises an NAD precursor and the subject is elderly.

[0065] In one embodiment, the method of the invention comprises the steps of: (a) detecting the level of one or more biomarkers in a sample obtained from a subject; (b) comparing the level of one or more biomarkers in the sample to a reference value; (c) predicting the subject's responsiveness to the nutritional composition based on the results of step (b); The one or more biomarkers are anthranilic acid and / or trigonelline.

[0066] A subject is predicted to be responsive to the nutritional composition or nutritional therapy if the level of anthranilic acid in the sample is increased compared to the reference value.A subject is predicted to be responsive to the nutritional composition or nutritional therapy if the level of trigonelline in the sample is decreased compared to the reference value.

[0067] In one embodiment, the biomarker is anthranilic acid.

[0068] In one embodiment, the biomarker is trigonelline.

[0069] In one embodiment, the biomarker is a combination of anthranilic acid and trigonelline.

[0070] In one embodiment, the biomarker is anthranilic acid, trigonelline, 3-hydroxy-anthranilic acid, tryptophan, kynurenine, or a combination of these metabolites.

[0071] In one embodiment, the nutritional composition is vitamin B3.

[0072] In one embodiment, the nutritional composition is an NAD precursor, such as nicotinamide riboside, nicotinamide mononucleotide, and nicotinamide.

[0073] In one embodiment, the nutritional composition is tryptophan, kynurenine, and their metabolites.

[0074] In one embodiment, the nutritional composition is trigonelline.

[0075] In one embodiment, the nutritional composition is derived from coffee, such as a coffee bean extract or a coffee beverage, hi one embodiment, the coffee extract comprises one or more of caffeic acid, trigonelline, quinolinic acid, and precursors of quinolinic acid.

[0076] subject The term "subject" refers to any animal, including humans and companion animals. Generally, the subject is a human, or an avian, bovine, canine, equine, feline, caprine, murine, ovine or porcine animal. The subject may be a horse or a companion animal, such as a cat or a dog. Preferably, the subject is a human. The term "elderly" refers to an age of at least 60 years, more preferably greater than 64 years, and most preferably greater than 68 years, in relation to humans. The term "elderly" refers to an age of at least 45 years, preferably greater than 50 years, and more preferably greater than 55 years, including aged controls.

[0077] Sarcopenia Sarcopenia can be assessed by one or more of the following: loss of muscle mass, loss of muscle strength, and impaired physical performance.

[0078] Sarcopenia can be diagnosed in subjects based on the definition of the Asian Working Group for Sarcopenia in Older People (AWGSOP), e.g., as described in Chen et al., 2014. Muscle loss is generally characterized by a low appendicular lean mass normalized to height squared (ALM index), specifically, a low ALM index of 7.00 kg / m2 or lower for men. 2 less than 5.40 kg / m 2Poor physical performance can generally be based on reduced walking speed, specifically a walking speed of <0.8 m / s. Poor muscle strength can generally be based on reduced grip strength, specifically a grip strength of less than 26 kg for men and less than 18 kg for women.

[0079] Sarcopenia in subjects can be diagnosed based on the definition of the European Working Group for Sarcopenia in Older People (EWGSOP), e.g., as described in Cruz-Jentoft et al., 2010. Muscle loss is generally characterized by a low appendicular lean mass normalized to the square of height (ALM index), specifically, a low ALM index of 7.23 kg / m2 for men. 2 less than 5.67 kg / m 2 Poor physical performance can generally be based on reduced walking speed, specifically a walking speed of <0.8 m / s. Poor muscle strength can generally be based on reduced grip strength, specifically a grip strength of less than 30 kg for men and less than 20 kg for women.

[0080] In subjects, sarcopenia can be diagnosed based on the FNIH (Foundation for the National Institutes of Health) definition, for example as described in Studenski et al., 2014. Muscle mass loss can generally be based on low appendicular lean mass (ALM) normalized to body mass index (BMI; kg / m2), specifically ALM-to-BMI of less than 0.789 for men and less than 0.512 for women. Physical performance loss can generally be based on walking speed, specifically a walking speed of <0.8 m / s. Muscle strength loss can generally be based on reduced grip strength, specifically a grip strength of less than 26 kg for men and less than 18 kg for women. Muscle strength loss can also generally be based on reduced grip strength-to-body mass index, specifically a grip strength-to-body mass index of less than 1.00 for men and less than 0.56 for women.

[0081] The D3 creatine dilution method is an alternative approach to measure muscle mass. This method is more widely accepted as a robust standard and a potential future alternative to DXA. The D3 creatine dilution method has been previously described by Clark et al. (2014) and Stimpson et al. (2013).

[0082] The method includes measuring the level of trigonelline, the level of anthranilic acid, and / or the ratio of these levels in a sample obtained from a subject, which may also be referred to as a "test sample." The method is thus typically performed outside the human or animal body, for example on a body fluid sample previously obtained from the subject to be tested.

[0083] The sample may be, for example, a serum, plasma or urine sample. The sample may be obtained from blood, i.e. the sample may comprise whole blood or a blood fraction. The sample may comprise plasma or serum. Preferably, the sample is a serum sample.

[0084] The techniques of collecting blood samples and separating blood fractions are well known in the art.For example, venous blood samples can be collected from patients using needles and stored in plastic tubes.Collection tubes can contain, for example, spray-coated silica and polymer gel for serum separation.Serum can be separated by centrifugation at 1300RCF for 10 minutes at room temperature and stored at -80°C in small plastic tubes.

[0085] Comparison with standard values The method involves comparing the levels of anthranilic acid and trigonelline to baseline levels of anthranilic acid and trigonelline. The term "baseline" is synonymous with "control value" and broadly includes data used by those skilled in the art to facilitate accurate interpretation of technical data.

[0086] The anthranilic acid level, trigonelline level, and / or ratio of trigonelline:anthranilic acid levels in a sample obtained from a subject can be compared with the levels and / or ratios in the same subject, or with the levels and / or ratios in one or more cohorts of control subjects.

[0087] Reference values ​​can be established by analyzing samples from the same subject, for example by assaying repeated samples. Reference values ​​can be established by analyzing samples from individuals in the general population or selected populations, and using a statistical model such as a predictive value method to select positive criteria or a receiver operating characteristic curve that defines optimal specificity (highest true negative rate) and sensitivity (highest true positive rate), as described in Knapp, RG, and Miller, MC (1992). Clinical Epidemiology and Biostatistics. William and Wilkins, Harual Publishing Co. Malvern, Pa. (this document is incorporated herein by reference).

[0088] The reference value may be established by analyzing at least one individual, more preferably at least 5 individuals, more preferably at least 10 individuals, more preferably at least 15 individuals, and most preferably at least 20 individuals, all or a substantial number of said individuals preferably not having sarcopenia.

[0089] How to assign correct reference values ​​is known to those skilled in the art, and these may vary depending on, for example, gender, race, genetic heritage, health status or age.

[0090] Nutritional composition The nutritional composition according to the present invention may be a dietary supplement composition, a functional food, a functional nutritional product, a medical food regulated as a drug, a medical nutritional product, or a dietary supplement.

[0091] The term "nutraceutical" combines the words "nutrition" and "pharmaceutical." Nutraceuticals are foods or food products that provide health and medical benefits, including the prevention and treatment of disease. Nutraceuticals are commonly sold in the form of pharmaceuticals that are not normally associated with food, and are products isolated or purified from food. Nutraceuticals have been demonstrated to have physiological effects or provide protection against chronic diseases. Such products can range from isolated nutrients, dietary supplements, and special diets to genetically engineered foods, herbal products, and processed foods such as cereals, soups, and beverages.

[0092] As used herein, the term "nutraceutical" means useful in both nutritional and pharmaceutical fields.Therefore, the nutraceutical composition can be used as a nutritional supplement to food and beverages, and as a pharmaceutical preparation for enteral or parenteral application, which can be a solid preparation such as a capsule or tablet, or a liquid preparation such as a solution or suspension.

[0093] The nutritional composition according to the invention may comprise trigonelline, a precursor of trigonelline, and / or a derivative of trigonelline. The precursor of trigonelline may be, for example, quinolinic acid, nicotinic acid, and / or nicotinamide.

[0094] Preferably, the nutritional composition according to the present invention comprises NAD precursors, such as nicotinamide riboside, nicotinamide mononucleotide, and nicotinamide and / or nicotinic acid.

[0095] The nutritional compositions according to the present invention may further comprise protective hydrocolloids (gums, proteins, modified starches etc.), binders, film formers, encapsulants / encapsulants, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavoring agents, bulking agents, jellifying agents, gel forming agents, antioxidants and antimicrobial agents.

[0096] Additionally, multivitamin and mineral supplements can be added to the nutritional compositions of the present invention to provide adequate amounts of essential nutrients that are lacking in some diets. Multivitamin and mineral supplements can also be useful in disease prevention and compensation for nutritional loss and malnutrition due to lifestyle patterns.

[0097] The nutritional composition of the invention may be in any galenic form suitable for administration to the body, in particular in any form conventionally used for oral administration, for example in solid forms such as food or feed (additives / supplements therefor), food or feed premixes, fortified food or feed, tablets, pills, granules, dragees, capsules, and effervescent preparations such as powders and tablets, or in liquid forms such as solutions, emulsions or suspensions (for example as beverages, pastes, and oily suspensions). Pastes may be incorporated into hard or soft shell capsules, whereby the capsules are characterized, for example, by a matrix of (fish, porcine, poultry, bovine) gelatin, plant proteins, or lignin sulfonates. Examples of other application forms are forms for transdermal, parenteral, or injectable administration. The nutritional composition may be in the form of a controlled (delayed) release formulation.

[0098] The nutritional composition may comprise a beverage. The term "beverage" includes non-alcoholic and alcoholic beverages as well as liquid preparations added to drinking water and liquid food. Non-alcoholic beverages are, for example, soft drinks, sports drinks, fruit juices, tea, and milk-based drinks. Liquid food may include soups and dairy products. The nutritional supplement composition comprising the compound of the present invention may be added to soft drinks, energy bars, or candies.

[0099] If the nutritional composition is a nutraceutical formulation and further contains a pharma- ceutically acceptable excipient, diluent or adjuvant, the formulation may use standard techniques, such as those disclosed in Remington's Pharmaceutical Sciences, 20th edition Williams & Wilkins, PA, USA. For oral administration, tablets and capsules are preferably used, containing a suitable binder (e.g., gelatin or polyvinylpyrrolidone), a suitable filler (e.g., lactose or starch), a suitable lubricant (e.g., magnesium stearate), and optionally additional additives.

[0100] "Functional foods", "functional nutritional products", "medical foods" and "medical nutritional products" refer to any health food that claims to have health-promoting or disease-preventing properties beyond its basic function of providing nutrients. The general category of functional foods includes processed foods or foods that have been enriched with health-promoting additives, such as "vitamin-fortified" products.

[0101] Dietary supplements, also known as food supplements or dietary supplements, are preparations intended to supplement the diet and to supply nutrients, such as vitamins, minerals, fiber, fatty acids, or amino acids, that may be missing or not consumed in sufficient amounts in an individual's diet. Some countries define dietary supplements as foods, while others define them as drugs or natural health products. Dietary supplements containing vitamins or dietary minerals are included as a category of food in the Codex Alimentarius, a collection of internationally recognized standards, codes of practice, guidelines, and other recommendations on food, food production, and food safety. Codex Alimentarius standards are developed by the Codex Alimentarius Commission, an organization sponsored by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO).

[0102] Prevention or treatment of disease "Prevention" includes reducing the risk and / or severity of a condition or disease. The terms "treatment", "treat", "alleviate" and "alleviate" include both preventative or prophylactic treatment (preventing and / or delaying the onset of the targeted condition or disorder) and curative, therapeutic or disease modifying treatment, including, for example, therapeutic measures to cure, delay, alleviate symptoms, and / or halt the progression of a diagnosed condition or disorder, as well as treatment of patients at risk of or suspected of having a disease, and patients who are unwell or diagnosed as suffering from a disease or medical condition. This term does not necessarily mean that a subject is treated until cured. All of these terms also refer to the maintenance and / or promotion of health of a subject who is not suffering from a disease but is susceptible to an unhealthy condition. These terms are also intended to include synergism or enhancement of one or more primary preventative or therapeutic measures. The terms "treatment," "treat," "alleviate," and "alleviate" are further intended to include treatment of a disease or condition or for the prophylaxis or prevention of a disease or condition. Treatment may be patient-related or physician-related.

[0103] The present invention is further illustrated by the following examples, which are meant to aid one of ordinary skill in the art in practicing the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0104] Example 1 Measurement of metabolites in serum Twenty participants, primarily male Chinese aged 65–79 years, and 20 age-matched control subjects without a diagnosis of sarcopenia were recruited from two studies of healthy community-dwelling older adults in Singapore (the Singapore Sarcopenia Group and the Ageing in a Community Environment Study [ACES]). Sarcopenia was diagnosed according to a standardized operational definition of sarcopenia (Cruz-Jentoft et al., 2010), using the definition from the Asian Working Group for Sarcopenia in Older People (Chen et al., 2014).

[0105] The NHG DSRB (National Healthcare Group Domain Specific Research Board) approved this study (reference number 2014 / 01304) and each participant provided written informed consent. Weight and height were measured to the nearest 0.1 kg or 1 cm. Total lean mass was measured by dual-energy X-ray absorptiometry (DXA) scanning (APEX Software version 4.0.1, Discovery Wi DXA system). Isometric handgrip was measured with a Jamar handheld dynamometer using a standardized protocol, and the average of three trials of the dominant hand was used as the final measurement. Physical performance was assessed by measuring gait speed in a normally paced 6-m walk test. The diagnosis of sarcopenia was based on either the AWGSOP definition, defined as total appendicular lean mass normalized for height < 7.00 kg / m2, evidence of reduced physical performance based on a gait speed < 0.8 m / s, or reduced muscle strength based on a grip strength < 26 kg. Anthropometric parameters in the control and sarcopenic groups were as follows: [Table 1]

[0106] Fasting blood samples were collected from the antecubital fossa using evacuated tubes and an indwelling 20-gauge butterfly needle. Blood was centrifuged at 3000 rpm for 10 min at 4°C, then serum was aliquoted and frozen at -80°C until further analysis. Serum anthranilic acid and trigonelline levels were measured by liquid chromatography followed by mass spectrometry (LC-MS / MS) at BEVITAL laboratories (Bergen, Norway). Semi-open muscle biopsies of the vastus lateralis were taken from 20 male participants and 20 age-matched controls using a BioPince™ (Angiotech) 16G full-core biopsy needle with three adjustable stroke lengths (13mm, 23mm, 33mm), flash frozen in liquid nitrogen, and stored at -80°C until further analysis.

[0107] Differences between sarcopenic cases and controls in serum trigonelline and anthranilic acid levels and their ratio (trigonelline:anthranilic acid) were tested using Wilcoxon test and log2-transformed Student's t-test. We compared these samples with the continuous clinical variables used to assess sarcopenia (appendicular lean muscle mass index (ALMi; kg / m 2 Correlations between serum analyte concentrations (p < 0.05), grip strength and walking speed (p < 0.05), were examined by applying both Spearman's rank correlation and Pearson's correlation after log2 transformation of serum analyte concentrations.

[0108] Figures 1-3 show that serum trigonelline levels, anthranilic acid levels and the trigonelline:anthranilic acid ratio are different in sarcopenic patients and positively correlate with lean muscle mass, grip strength, and gait. Trigonelline (Figure 1), anthranilic acid (Figure 2), and the trigonelline / anthranilic acid ratio (Figure 3) were measured in serum of a human cohort of 20 sarcopenic patients and 20 age-matched controls (all over 65 years old). The left panel represents the log2 values ​​of the analytes in serum of sarcopenic and control patients. The right panel represents the correlation of the analytes in serum with muscle mass, grip strength, and appendicular lean mass index (ALMi) measured by DXA as a proxy for gait speed. The orange line shows the fitted regression line of the clinical variables to the analytes, and the grey area shows the 95% confidence interval of the fitted model. Reported p-values ​​were calculated using Student's T statistic (case-controls) or Pearson correlation (correlation analysis) on Log2-transformed values.

[0109] Example 2 Analysis of muscle gene expression To analyze muscle gene expression, total RNA was extracted from muscle biopsies using a Qiagen kit according to the manufacturer's instructions. RNA quantity was measured with Ribogreen (Life Technologies) and RNA quality was confirmed with a fragment analyzer (Advanced Analytical Technologies) using a standard sensitivity RNA analysis kit. All RNA samples were homogenous and passed quality control with a 260 / 280 nm ratio >1.8 and RIN score >7. For RNA sequencing, 250 ng of total RNA from each sample was used as starting material for library preparation, and ribosomal RNA was removed using a Ribo-Zero magnetic Kit (Illumina). Sequencing libraries were prepared using TruSeq Stranded RNA HT and Ribo-Zero Gold kits (Illumina), followed by 13 cycles of PCR amplification using KAPA HiFi HotStart ReadyMix (Kapa BioSystems). Libraries were quantified with Picogreen (Life Technologies) and size patterns were controlled on a LabChip GX (Perkin Elmer) using a DNA High Sensitivity Reagent Kit. Libraries were then pooled in equimolar ratios and clustered in a paired-end sequencing flow cell (Illumina) at a concentration of 7 pmol. Sequencing was performed on a HiSeq 2500 (Illumina) using V3 chemistry for 2 × 101 cycles. The generated data was demultiplexed using Casava. Reads were aligned to the human genome (hs_GRCh38.p2) using STAR (Dobin et al., 2013) and the number of reads mapping within genes was quantified by HTSeq (Anders et al., 2015) (version HTSeq-0.6.1p1, mode union, strand reverse, quality alignment > 10). Samples had a sequencing depth of 75–104 million reads per sample, of which 34–77 million reads mapped to only one locus.

[0110] Differentially expressed genes between control and sarcopenic samples were defined by using the limma package (Smyth, 2004). Briefly, after excluding genes with mean expression less than 20 reads, data were normalized by the trimmed mean of M value (TMM) method implemented in the edgeR function calcNormFactors (Robinson et al., 2010), and the voomWithQualityWeights function was applied to model mean-variance relationships and estimate sample-specific quality weights (Liu et al., 2015). The Benjamini Hoechberg method was used to correct p-values ​​for multiple testing. When assessing correlations between gene expression and continuous or categorical parameters (ALMi, grip strength, and walking speed), the same procedure used to define sarcopenia was applied. Gene set enrichment analysis was performed using CAMERA (Wu and Smyth, 2012). This analysis is a competitive gene set test that queries whether a set of genes annotated in the Molecular Signatures Database (MSigDB) (Subramanian et al., 2005) is enriched in differentially expressed genes. Pathway analysis was performed using H (hallmark gene set), C2 (curated gene set), and C5 (GO gene set) from the MSigDB v5.2 collection.

[0111] In Figures 4A-4B, RNA sequencing of human sarcopenic skeletal muscle reveals that mitochondrial dysfunction is the major transcriptional change during sarcopenia. (4A) Gene set enrichment analysis of sarcopenic vs. control muscles using a set collection of hallmark genes from CAMERA and MSigDB. Red bars represent gene sets that were increased in sarcopenic muscle, and blue bars represent gene sets that were decreased in sarcopenic muscle. Gene sets are ordered by significance of enrichment. (4B) GSEA enrichment plot of selected oxidative phosphorylation gene sets from A.

[0112] Spearman rank correlations were calculated between TMM-normalized muscle gene expression and each serum analyte of interest (trigonelline, anthranilic acid, and trigonelline:anthranilic acid ratio), and this Spearman rank correlation was used to perform gene set enrichment analysis using a collection of biological hallmark gene sets.

[0113] Legend to Figure 4A: 1. Oxidative phosphorylation, 2. Fatty acid metabolism, 3. MYC target V1, 4. Allograft rejection, 5. MTORC1 signaling, 6. Peroxisomes, 7. Adipogenesis, 8. Epithelial-mesenchymal transition, 9. Apical junction, 10. G2M checkpoint, 11. Bile acid metabolism, 12. Unfolded protein response, 13. Reactive oxygen species pathway, 14. Spindle, 15. Increased UV response, 16. Decreased UV response, 17. PI3K / Akt / mTOR signaling, 18. MYC target V2, 19. Decreased KRAS signaling, 20. Hedgehog signaling, 21. E2F target, 22. Interferon-gamma response, 23. Apical surface, 24. TGF-β signaling, 25. Protein secretion, 26. IL2-STAT5 signaling.

[0114] Figures 5A-5B, 6A-6B, and 7A-7B show the correlation of skeletal muscle gene expression with serum trigonelline levels, anthranilic acid levels, and trigonelline:anthranilic acid ratios. Gene set enrichment analysis (GSEA) of muscle gene expression with serum trigonelline levels (Figures 5A-5B), anthranilic acid levels (Figures 6A-6B), and trigonelline / anthranilic acid ratios (Figures 7A-7B). GSEA was performed using mean-rank gene set tests and a biological hallmark gene set collection from MSigDB. Red bars represent gene sets whose expression in skeletal muscle is positively correlated with serum levels of anthranilic acid in the specimen, and blue bars represent gene sets whose expression in skeletal muscle is negatively correlated with serum levels of anthranilic acid in the specimen. Gene sets are ordered by enrichment significance, and only gene sets with FDR < 1% are shown. The panel on the left represents the GSEA enrichment plot of the oxidative phosphorylation gene set.

[0115] Legend for Figure 5A: 1. Oxidative phosphorylation, 2. MTORC1 signaling, 3. Protein secretion, 4. Adipogenesis, 5. Fatty acid metabolism, 6. MYC target V1, 7. Peroxisomes, 8. DNA repair, 9. Glycolysis, 10. Spindle, 11. Myogenesis, 12. Interferon gamma response, 13. PI3K / Akt / mTOR signaling, 14. Wnt / β-catenin signaling, 15. Interferon alpha response, 16. Allograft rejection, 17. Bile acid metabolism, 18. Androgen response, 19. Cholesterol homeostasis.

[0116] Legend to Figure 6A: 1. Oxidative phosphorylation, 2. Fatty acid metabolism, 3. Adipogenesis, 4. G2M checkpoint, 5. Bile acid metabolism, 6. Peroxisomes, 7. Myogenesis.

[0117] Legend to Figure 7A: 1. Oxidative phosphorylation, 2. Fatty acid metabolism, 3. Adipogenesis, 4. MTORC1 signaling, 5. Peroxisomes, 6. Protein secretion, 7. Myogenesis, 8. G2M checkpoint, 9. MYC target V1, 10. Bile acid metabolism, 11. DNA repair, 12. Cholesterol homeostasis, 13. KRAS signaling down, 14. Glycolysis.

[0118] Figure 8 shows that human sarcopenic muscle has lower expression of genes such as NMAT1, NAMPT, and PNP, which control NAD biosynthesis and NAD salvage from NAD precursors or diet. Gene expression in muscle was measured by high-coverage RNA sequencing. NMNAT: nicotinamide nucleotide adenylyltransferase 1; NAMPT: nicotinamide phosphoribosyltransferase; PNP: purine nucleoside phosphorylase.

[0119] Example 3 Measuring NAD+ levels in muscle biopsies To determine whether NAD levels may be an upstream trigger of the mitochondrial signature in sarcopenic muscle, we measured NAD in aged human muscle biopsies. NAD+ levels were measured in human muscle biopsies as described by Dall et al., 2018. Briefly, 5 mg of muscle tissue from the remaining biopsy was dissolved in 200 μL of 0.6 M perchloric acid, and the supernatant was diluted 250-fold with 100 mM Na2HPO4 (pH 8.0). 100 μL of the diluted sample was combined with 100 μL of reaction mixture (100 mM Na2HPO4 (pH 8), 2% ethanol, 90 U / mL alcohol dehydrogenase, 130 mU / mL diaphorase, 10 μM resazurin, 10 μM flavin mononucleotide, 10 mM nicotinamide) and the fluorescence increase (Ex540nm / Em580) was measured over 10 min. NAD+ content was calculated from the standard curve and normalized to tissue weight.

[0120] Figure 9 shows that there was a significant (32%) decrease in NAD levels in human skeletal muscle of elderly sarcopenic patients. This large decrease in NAD+ levels in skeletal muscle of sarcopenic patients is the first to link low NAD+ levels to age-related pathology in humans and demonstrates NAD deficiency in human skeletal muscle. This result, combined with the preclinical efficacy of NAD precursors such as NR and NMN on aged muscle in mice, extends the clinical relevance of NAD biology in aging and guides clinical translation of sarcopenia and age-related reduced mobility.

[0121] Example 4 Measurement of metabolites in serum of different ethnicities, including women In this example, 70 predominantly Caucasian participants (52 women and 18 men, median age 78 years (interquartile range 76, 81)) were studied.

[0122] These 70 participants were part of the Hertfordshire Sarcopenia Study (HSS), a substudy of the UK Hertfordshire Cohort Study (HCS) designed to investigate lifelong influences on muscle morphology, muscle mass and strength in community-dwelling older adults, or the extension phase (HSSe) of that study. Skeletal muscle characterization performed in this study included body composition and lean mass as determined by dual-energy X-ray absorptiometry (DXA) scanning.

[0123] Fasting blood samples were collected from the antecubital fossa using vacutainer tubes and an indwelling 20-gauge butterfly needle cannula. Blood was centrifuged at 3000 rpm for 10 min at 4°C, and serum was then aliquoted and frozen at -80°C until further analysis. Serum anthranilic acid and trigonelline levels were measured by liquid chromatography followed by mass spectrometry (LC-MS / MS) at BEVITAL laboratories (Bergen, Norway).

[0124] Trigonelline:anthranilic acid ratio and appendicular lean muscle mass index (ALMi; kg / m2 ) was estimated by applying regression corrected for age after log2 transformation of analyte serum concentrations.

[0125] FIG. 10 shows that the ratio of serum levels of trigonelline:anthranilic acid correlates with lean muscle mass as assessed by appendicular lean mass index. Trigonelline and anthranilic acid levels were measured in serum from a human cohort of 70 subjects (52 women and 18 men, all aged 65 years or older). The graph shows the correlation between the log2 value of the ratio of analytes (trigonelline:anthranilic acid) in serum (x-axis) and the appendicular lean mass index (ALMi) (y-axis) measured by DXA as a surrogate for muscle mass. The line shows the regression line fitted to ALMi versus log2(trigonelline:anthranilic acid). This simple linear regression yielded a Pearson correlation coefficient of 0.23, p-value=0.031, and a 51% degree of freedom adjusted R for the coefficients for the analyte ratios based on the regression corrected for age. 2 and p-value = 0.024 was obtained.

[0126] References Anders, S., Pyl, PT, and Huber, W. (2015). HTSeq--a Pythonframework to work with high-throughput sequencing data. Bioinformatics 31,166-169. Anker, SD, Morley, JE, and von Haehling, S. (2016). Welcome to the ICD-10 code for sarcopenia. Journal of cachexia, sarcopenia and muscle 7,512-514. Chen, L.K., Liu, L.K., Woo, J., Assantachai, P., Auyeung, T.W.,Bahyah, K.S., Chou, M.Y., Chen, L.Y., Hsu, P.S., Krairit, O., et al. (2014).Sarcopenia in Asia: consensus report of the Asian Working Group for Sarcopenia.Journal of the American Medical Directors Association 15, 95-101. Clark RV, Walker AC, O'Connor-Semmes RL, Leonard MS, Miller RR,Stimpson SA, Turner SM, Ravussin E, Cefalu WT, Hellerstein MK, Evans WJ. Totalbody skeletal muscle mass: estimation by creatine (methyl-d3) dilution inhumans. J Appl Physiol (1985). 2014 Jun 15;116(12):1605-13. Cruz-Jentoft, A.J., Baeyens, J.P., Bauer, J.M., Boirie, Y.,Cederholm, T., Landi, F., Martin, F.C., Michel, J.P., Rolland, Y., Schneider,S.M., et al. (2010). Sarcopenia: European consensus on definition anddiagnosis: Report of the European Working Group on Sarcopenia in Older People.Age Ageing 39, 412-423. Dall, M., Penke, M., Sulek, K., Matz-Soja, M., Holst, B., Garten,A., Kiess, W., and Treebak, JT. Hepatic NAD(+) levels and NAMPT abundance areunaffected during prolonged high-fat diet consumption in C57BL / 6JBomTac mice.Molecular and Cellular Endocrinology (2018) S0303-7207 (18) 30048. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C.,Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafastuniversal RNA-seq aligner. Bioinformatics 29, 15-21. Liu, R., Holik, A.Z., Su, S., Jansz, N., Chen, K., Leong, H.S.,Blewitt, M.E., Asselin-Labat, M.L., Smyth, G.K., and Ritchie, M.E. (2015). Whyweight? Modelling sample and observational level variability improves power inRNA-seq analyses. Nucleic acids research 43, e97. Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: aBioconductor package for differential expression analysis of digital geneexpression data. Bioinformatics 26, 139-140. Smyth, G.K. (2004). Linear models and empirical bayes methods forassessing differential expression in microarray experiments. Statisticalapplications in genetics and molecular biology 3, Article3. Studenski SA, Peters KW, Alley DE, Cawthon PM, McLean RR, Harris TB,Ferrucci L, Guralnik JM, Fragala MS, Kenny AM, Kiel DP, Kritchevsky SB,Shardell MD, Dam TT, Vassileva MT (2014). The FNIH sarcopenia project:rationale, study description, conference recommendations, and final estimates.J Gerontol A Biol Sci Med Sci. 69(5), 547-558. Stimpson SA, Leonard MS, Clifton LG, Poole JC, Turner SM, ShearerTW, Remlinger KS, Clark RV, Hellerstein MK, Evans WJ. (2013) Longitudinalchanges in total body creatine pool size and skeletal muscle mass using theD3-creatine dilution method. J Cachexia SarcopeniaMuscle. Jun 25. Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert,B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S.,et al. (2005). Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proceedings of the NationalAcademy of Sciences of the United States of America 102, 15545-15550. Wu, D., and Smyth, G.K. (2012). Camera: a competitive gene set testaccounting for inter-gene correlation. Nucleic acids research 40, e133.

Claims

1. A method for assisting in determining whether a subject has sarcopenia, comprising: (a) measuring the level of trigonelline in a blood-derived sample obtained from said subject; (b) comparing the level of trigonelline in the sample with a reference value; wherein a decreased level of trigonelline in the sample compared to the reference value is indicative of sarcopenia in the subject.

2. The method of claim 1 , wherein the blood is fasting blood.

3. 3. The method according to claim 1 or 2, (c) measuring the level of anthranilic acid in a blood-derived sample obtained from the subject; (d) determining the ratio of trigonelline and anthranilic acid in the sample; (e) comparing the ratio of trigonelline to anthranilic acid in the sample with a standard value; wherein a decrease in the ratio of trigonelline to anthranilic acid in the sample compared to the reference value is an additional indication of sarcopenia in the subject.

4. The method according to any one of claims 1 to 3, wherein sarcopenia is assessed by one or more of muscle mass loss, muscle strength loss, and reduced physical performance.

5. 5. The method of claim 4, wherein (i) muscle mass is measured by DXA, MRI or D3 creatine dilution method, (ii) muscle strength is measured by grip strength or knee extension strength, and (iii) physical performance is measured by walking speed, SPPB, 400m walk test, TUG test, or stair climbing test.

6. The method according to any one of claims 1 to 5, wherein the levels of trigonelline and / or anthranilic acid are measured by mass spectrometry.

7. The method of any one of claims 1 to 6, wherein the subject is a human subject.

8. The method of any one of claims 1 to 7, wherein the subject is elderly or geriatric.

Images