WO2018150077A1

WO2018150077A1 - Betaine biomarkers for nutritional status, nutritional regimen, endogenous metabolism, or risk of cardiovascular or metabolic diseases

Info

Publication number: WO2018150077A1
Application number: PCT/FI2017/050099
Authority: WO
Inventors: Kati HANHINEVA; Jenna PEKKINEN; Olli KÄRKKÄINEN; Jukka LEPPÄNEN; Seppo AURIOLA; Marko Lehtonen
Original assignee: Afekta Technologies Oy
Current assignee: Afekta Technologies Oy
Priority date: 2017-02-16
Filing date: 2017-02-16
Publication date: 2018-08-23
Anticipated expiration: 2019-08-16

Abstract

A method for assessing or aiding in the assessment of nutritional status and/or effectiveness of a nutritional regimen, endogenous metabolism, and/or a risk of developing a cardiovascular and/or metabolic disease in a subject is disclosed. The method comprises analysing a biological sample from the subject to determine the level(s) of one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine in the biological sample. The method may further comprise comparing the level(s) of the one or more betaine compounds in the biological sample to a reference value or to the level(s) of the one or more betaine compounds in a control sample in order to assess the nutritional status and/or the effectiveness of the nutritional regimen the endogenous metabolism, and/or the risk of developing the cardiovascular and/or metabolic disease in the subject.

Description

Betaine biomarkers for nutritional status, nutritional regimen, endogenous metabolism, or risk of cardiovascular or metabolic diseases

BACKGROUND

Mounting epidemiological evidence suggests that a diet rich in whole grains has a beneficial effect on health, as it may reduce the risk of chronic diseases, including cardiometabolic diseases, which are a major cause of morbidity and mortality in Western societies. However, the mechanisms responsible for the protective effect are poorly known. Further, modification of dietary habits can contribute to healthy ageing and the risk for cardiometabolic diseases.

There is a need for methods for assessing the metabolic and nutritional status, effectiveness of nutritional regimens, and/or for diagnosing metabolic and/or cardiovascular diseases in individual subjects.

SUMMARY

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

A method for assessing or aiding in the assessment of nutritional status and/or effectiveness of a nutritional regimen, endogenous metabolism, and/or a risk of developing a cardiovascular and/or metabolic disease in a subject is disclosed. The method comprises analysing a biological sample from the subject to determine the level (s) of one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4- aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine in the biological sample. The method may further comprise comparing the level (s) of the one or more betaine compounds in the biological sample to a reference value or to the level (s) of the one or more betaine compounds in a control sample in order to assess the nutritional status and/or the effectiveness of the nutritional regimen the endogenous metabolism, and/or the risk of developing the cardiovascular and/or metabolic disease in the subject.

Many of the attendant features will be more readily appreciated as the same becomes better understood by reference to the following detailed description considered in connection with the accompanying drawings .

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments of the invention and together with the description help to explain the principles of the invention. In the drawings:

Figure 1 illustrates identification of amino-acid derived betaines in the plasma samples. 20 V MSMS fragmentation patterns and abundance in mouse plasma are shown for: a, 5- aminovaleric acid betaine, b, alanine betaine, c, valine betaine, d, phenylalanine betaine, e, pipecolic acid betaine, and f, glycine betaine. Legend: HF, high-fat controls; Rl/2, rye bran groups 1 and 2; Al/4, aleurone groups 1 and 4.

Figure 2 shows the abundances of betaines in fasting plasma samples from three human dietary intervention trials (a, c, e, g, i, k, m, o, q) and the correlations of the levels of the abundances with glycinde betaine abundance (b, d, f, h, j, 1, n, p, r) . Kuopio HG intervention had three dietary groups: A, healthy diet including whole grains; B, whole grain rich diet; C, control diet. s. Correlations of grain intake with plasma levels of some of the betainized compounds in the intervention trials.

Figure 3 shows a. betaines in plasma of conventional (MPF) and germ free (GF) mice, b. Betaines in the colon contents of the mice fed with bran enriched diets, c. betaines in the in vitro 24-h fermentation model with human microbiota incubated with rye bran fractions, d. betaines in tissue matrices from the intestinal tract of the mouse fed with bran enriched feed. FBL = faecal background, Rl = rye bran, R2 = bioprocessed rye bran. Figure 4 illustrates betaines in tissues and effects of 5-AVAB to the energy metabolism of neonatal mouse cardiomyocytes . a. Betaines in mice heart; BET = glycine betaine. b. Betaines in human heart, c. 5-AVAB dose dependently decreases the ability of neonatal mouse cardiomyocytes to utilize palmitoyl acid as an energy source for mitochondrial respiration, d, Comparison of 100 μΜ concentrations of 5-AVAB, meldonium and glycine betaine showed that 5-AVAB produces similar effect to meldonium (but glycine betaine does not) . e, addition of L-carnitine (2 mM) is capable of recovering the cells ability to utilize palmitoyl acid as an energy source, f, 5-AVAB, meldonium and glycine betaine (100 μΜ) did not have an effect on the use of glucose and pyruvate in mitochondrial respiration. g,h. 5-AVAB (100 μΜ) reduces the cellular levels of L-carnitine and acetyl-L-carnit ine in neonatal mice cardiomyocytes .

DETAILED DESCRIPTION

It is obvious to a person skilled in the art that with the advancement of technology, the basic idea of the invention may be implemented in various ways. The invention and its embodiments are thus not limited to the examples described above; instead they may vary within the scope of the claims.

The levels of certain methylated, or betainized, compounds have now been found to be associated with endogenous metabolism. The fact that these betainized compounds have now been found in human blood and tissues indicates that they may be produced endogenously , e.g. in human cells (or cells of other subjects) or by gut microbiota, rather than simply being obtained from nutritional sources and excreted e.g. in urine. The betainized compounds may remain in plasma and tissues for a prolonged time period, so that the changes may be observed e.g. after fasting, or after a change in lifestyle, including nutrition. They therefore indicate a potential metabolic role in endogenous metabolism in plasma and various tissues. They may also be predictive biomarkers of metabolic disorders, such as cardiometabolic disorders. Certain betainized compounds described in this specification have not earlier been identified in blood or tissues and/or as potential biomarkers in humans.

Betainized compounds may also be used as biomarkers in personalized nutrition, in particular within dietary regimens or schemes involving high intake of whole grains and/or products derived from them. Nutritional regimen may affect their levels; for instance, glycine betaine obtainable from nutritional sources may serve as a precursor for them or may induce their production in humans. Individual subjects may exhibit different responses to a particular diet or to particular components of a diet, and therefore universal dietary recommendations may have limited utility. Based on the method according to one or more aspects or embodiments described in this specification, it may be possible to assess and/or monitor the nutritional status of a subject or the effectiveness of a nutritional regimen and, based on the assessment, to take appropriate action to improve the nutritional status, the nutritional regimen or to treat the subject. The nutritional regimen may include intake of whole grains and/or products derived from them. In particular, the nutritional regimen may include high or low intake of whole grains and/or products derived from them. Additionally or alternatively, the nutritional regimen may include intake of foodstuffs rich in glycine betaine. In particular, the nutritional regimen may include high or low intake of foodstuffs rich in glycine betaine. Examples of foodstuffs rich in glycine include whole grains, beetroot (also known as red beet), and quinoa, but other foodstuffs may also be contemplated.

In a first aspect, a method for assessing or aiding in the assessment of nutritional status and/or effectiveness of a nutritional regimen, endogenous metabolism, and/or a risk of developing a cardiovascular or metabolic disease, in a subject is disclosed, the method comprising:

analysing a biological sample from the subject to determine the level (s) of one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine in the biological sample; and comparing the level (s) of the one or more betaine compounds in the biological sample to a reference value or to the level (s) of the one or more betaine compounds in a control sample in order to assess the endogenous metabolism, the risk of developing the cardiovascular or metabolic disease, the nutritional status or the effectiveness of the nutritional regimen in the subject.

In a second aspect, a method for determining the level (s) of one or more betaine compounds in a biological sample from a subject is disclosed, the method comprising determining the level (s) of the one or more betaine compounds in the biological sample, wherein the one or more betaine compounds is/are selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine.

The features of the embodiments described below may be combined with the first aspect as well as with the second aspect .

In the context of this specification, the term "betaine compound" or "betainized compound" may refer to a compound having a positively charged cationic functional group, for example a quaternary ammonium group, and a carboxylate group. The carboxylate group may or may not be adjacent to the cationic group. The quaternary ammonium group may, in one embodiment, be trimethylated . Betaine compounds may include alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and/or tryptophan betaine.

In the context of this specification, the term

"subject" may refer to any animal, or a mammal, such as a human. It may also refer to a non-human primate, a dog, a cat, a horse, a sheep, a goat, a cow, a rabbit, a pig and/or a rodent, such as a mouse or a rat. The subject may be an individual subject. In an embodiment, the subject is a human subject.

In the context of this specification, the term "sample" or "biological sample" may refer to any biological sample obtained from a subject or a group or population of subjects. In an embodiment, the biological sample may be a fasting sample, i.e. a sample obtainable after a period of fasting, e.g. after a period of at least 8 hours.

The biological sample may comprise or be a blood sample, such as a plasma sample, a serum sample, a fasting blood sample, a fasting plasma sample, a fasting serum sample, or a fraction obtainable therefrom. The biological sample may, additionally or alternatively, comprise or be a sample or one or moreother body fluids or biofluids, for example a urine sample, a saliva sample, a bile sample, a tear sample and/or a spinal fluid sample. The biological sample may comprise or be, additionally or alternatively, a tissue sample, such as a liver sample, a pancreas sample, a heart sample, a muscle sample, a gastrointestinal wall sample, an adipose tissue sample, or a fraction obtainable therefrom. The adipose tissue sample may comprise or be a subcutaneous adipose tissue (SAT), a visceral adipose tissue (VAT) and/or brown adipose tissue (BAT) sample.

The method may comprise obtaining a biological sample from the subject prior to analysing the biological sample. Taking a blood sample or a tissue sample of a subject or patient is a part of normal clinical practice. The collected blood or tissue sample can be prepared and serum or plasma can be separated using techniques well known to a skilled person. Methods for separating one or more fractions from biological samples, such as blood samples or tissue samples, are also available to a skilled person. The term "fraction" may, in the context of this specification, also refer to a portion or a component of the biological sample separated according to one or more physical properties, for instance solubility, hydrophilicity or hydrophobicity, or molecular size.

The term "level" of one or more betaine compounds may, in the context of this specification, refer to the absolute or relative amount or concentration of the betaine compound (s) in the biological sample. The level (s) of one or more betaine compound (s) may refer to the total level of the one or more betaine compound(s), combined, and/or to individual levels of the one or more betaine compound (s) . The wording "compared to a control sample" or "comparing to a control sample" as used in this specification may be understood as including embodiments in which control samples are actually analysed in respect of one or more betaine compounds of interest, i.e. in respect of the level (s) or concentration ( s ) of the one or more betaine compounds of interest as described in this specification in connection with the various aspects and embodiments. It will be appreciated, however, that the above wording may also include embodiments in which the corresponding information on the level (s) or concentration ( s ) of the one or more betaine compounds is merely taken from the literature, or has been previously determined, calculated or extrapolated, or is yet to be determined, calculated or extrapolated.

A reference level or reference value may refer to a level or value that is indicative of a particular status of endogenous metabolism, a cardiovascular and/or metabolic disease, nutritional status, and/or the effectiveness of a nutritional regimen. The reference level or reference value may be obtained from a control sample, e.g. a control sample described above. The reference level or reference value may be predetermined, for instance determined, calculated or extrapolated prior to analysing the level (s) of the one or more betaine compounds in the biological sample. For instance, the reference level or reference value of one or more betaine compound (s) may be predetermined as an average level or a target level of the one or more betaine compound (s) in a control sample, control subject, control group, control population, or one or more samples obtained therefrom. A suitable reference level or reference value may be selected based on various criteria. For instance, for assessing or aiding in the assessment of a nutritional status or the effectiveness of a nutritional regimen, the reference level or value may be a reference level or value in a control subject, group or population that has a suitable or desired nutritional status and/or that has been subjected to a particular nutritional regimen or diet, or in one or more samples obtained therefrom. For assessing or aiding in the assessment of a risk of developing a cardiovascular and/or metabolic disease, i.e. detecting and/or diagnosing a cardiovascular and/or metabolic disease, the reference level or value may be a reference level or value in or obtained from a control sample, control subject, group or population that e.g. has not been (or, alternatively, has been) diagnosed earlier with said disease, or that has been determined earlier to be at risk for developing said disease. The reference value may be determined from a comparative biological sample, such as the same body fluid or the same tissue in a control subject, group or population from which the biological sample of the individual subject is obtained.

In an embodiment, the control sample is from a healthy individual or a generalized population of healthy individuals.

In an embodiment, the method is a method for assessing or aiding in the assessment of endogenous metabolism in a human subject. In the context of this specification, the term "endogenous metabolism" may refer to metabolism originating in or from within the subject (as opposed to metabolites, such as betaine compounds, being merely taken into the subject from outside, e.g. by nutrition), such as the production of metabolites produced by or within the subject. Endogenous metabolites may be produced in the subject, and may include metabolites produced by the colonic microbiota in the subject. In other words, the endogenous metabolism may, alternatively or additionally, comprise or refer to metabolism by colonic microbiota in the subject.

The endogenous metabolism may comprise or refer to at least one of endogenous metabolic homeostasis, methylation homeostasis, energy metabolism, methylation metabolism, cellular respiration, or mitochondrial beta-oxidation.

In an embodiment, the method is a method for assessing or aiding in the assessment of a risk of developing a cardiovascular and/or metabolic disease in a human subject. In other words, the probability that the subject will progress from being normal to having a cardiovascular and/or metabolic disease may be determined, or the assessment may assist in the determination of the risk of developing a cardiovascular and/or metabolic disease. Metabolic and cardiovascular diseases may jointly be referred to as cardiometabolic diseases.

The cardiovascular disease may comprise at least one of a coronary artery disease, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, or venous thrombosis. Coronary artery disease may include e.g. angina and/or myocardial infarction.

In an embodiment, the cardiovascular disease comprises at least one of a coronary artery disease, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, and venous thrombosis.

The metabolic disease may comprise at least one of type 2 diabetes, metabolic syndrome, insulin resistance or prediabetes .

In an embodiment, the metabolic disease comprises at least one of type 2 diabetes, metabolic syndrome, insulin resistance, and prediabetes.

In an embodiment, the method is a method for assessing or aiding in the assessment of nutritional status and/or effectiveness of a nutritional regimen in the subject.

The nutritional regimen may refer e.g. to a diet and/ or regimen of intake of at least one dietary supplement or dietary item. The at least one dietary supplement or dietary item may comprise whole grains and/or products derived from them. Additionally or alternatively, at least one dietary supplement or dietary item may comprise one or more foodstuffs rich in glycine betaine.

The one or more betaine compounds may be elevated in a subject, such as a human, after whole grain rich dietary interventions. The one or more betaine compounds may also be elevated in a subject, such as a human, after glycine betaine supplementation. Dietary betaine compounds, such as glycine betaine, may be a source, e.g. a precursor, for one or more betaine compounds according to one or more embodiments described in this specification. Therefore nutrition may have an effect on their level (s) in the subject. However, it appears that the one or more betaine compounds are not merely taken in but metabolised endogenously in the subject. Dietary betaine compounds, such as glycine betaine, may, alternatively or additionally, induce the production of the one or more betaine compounds according to one or more embodiments described in this specification .

A decreased or increased (i.e. compared to the control sample or to a reference value) level or levels of the one or more betaine compounds in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A decreased or increased level or levels of the one or more betaine compounds in the biological sample from the subject may be indicative of a particular nutritional status or nutritional regimen. An example of such a nutritional status or nutritional regimen may be the amount of whole grain intake, so that an increased level (s) of the one or more betaine compounds in the biological sample may be indicative of high whole grain intake or of a diet high in whole grain. A decreased level or levels of the one or more betaine compounds in the biological sample from the subject may be indicative of a metabolic and/or cardiovascular disease or of an increased risk of developing a cardiovascular and/or metabolic disease. However, a decreased level or levels of the one or more betaine compounds may, in some cases, also be at least partially indicative of a diet devoid of or low in whole grain and therefore not necessarily always indicative of a cardiovascular and/or metabolic disease.

A level or levels that is/are decreased or increased at least 1.1-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 1.1-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 1.1-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen. The indicative difference (decrease or increase in the level (s)) may depend e.g. on the type of the biological sample. For example, differences in the level (s) in fasting plasma samples may be relatively small, or smaller than e.g. in tissue samples.

A level or levels that is/are decreased or increased at least 1.2-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 1.2-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 1.2-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

A level or levels that is/are decreased or increased at least 1.3-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 1.3-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 1.3-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

A level or levels that is/are decreased or increased at least 1.4-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 1.4-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 1.4-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

A level or levels that is/are decreased or increased at least 1.5-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 1.5-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 1.5-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

A level or levels that is/are decreased or increased at least 2-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 2-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 2-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

A level or levels that is/are decreased or increased at least 2.5-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 2.5-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic disease. A level or levels that is/are decreased or increased at least 2.5-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

A level or levels that is/are decreased or increased at least 3-fold in the biological sample from the subject may be indicative of a particular status of endogenous metabolism. A level or levels that is/are decreased at least 3-fold in the biological sample from the subject may be indicative of a cardiovascular and/or metabolic disease or of an increased risk of developing a cardiovascular and/or metabolic. A level or levels that is/are decreased or increased at least 3-fold in the biological sample from the subject may be indicative of a particular nutritional status or the effectiveness of a nutritional regimen.

The one or more betaine compounds may be differentially present (increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype as compared to a subject or a group of subjects having a second phenotype .

In an embodiment, the one or more betaine compounds comprise (s) at least one betaine compound selected from alanine betaine, norvaline betaine, 5-aminovaleric acid betaine, 4- aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine.

In an embodiment, the one or more betaine compounds comprise (s) or is 5-aminovaleric acid betaine.

One of the betaine compounds, 5-amino valeric acid betaine (5-AVAB), may accumulate in particular in the heart tissue and other metabolically active tissues, including muscle and brown adipose tissue. It also has structural similarity with meldonium, a drug used to treat coronary artery disease. 5-AVAB may therefore have an inhibitive effect on mitochondrial beta- oxidation similar to meldonium, for instance via inhibition of carnitine-dependent transportation of fatty acids. Increased (or decreased) levels of 5-AVAB may be indicative of endogenous metabolism, including e.g. a shift in energy homeostasis. 5-AVAB may also have other added utility. For example, it may be relatively abundant in various biological samples.

In an embodiment, the one or more betaine compounds comprise (s) at least one of valine/norvaline betaine or alanine betaine. These betaine compounds may be indicative of a particular nutritional regimen, such as a nutritional regimen high in bran. Valine and norvaline betaine, being very similar chemically and having the same mass/charge ratio, may in some embodiments be detected and determined simultaneously. The term "valine/norvaline betaine" may, in some embodiments, therefore refer to valine betaine, norvaline betaine or to both valine betaine and norvaline betaine. The biological sample may be analysed using at least one of liquid chromatography, mass spectrometry, ¹H-NMR, or any combinations thereof. In an embodiment, the biological sample is analysed using one or more techniques selected from liquid chromatography, mass spectrometry, ¹H-NMR, and any combinations thereof. The liquid chromatography (LC) may be hydrophilic interaction liquid chromatography (HILIC) , which is well suited for the analysis of betainized compounds, although other techniques may also be contemplated. The liquid chromatography and mass spectrometry (MS) may be used in combination, often referred to as LC-MS . The term ^{/ l}H-NMR" may be understood as referring to proton nuclear magnetic resonance, i.e. nuclear magnetic resonance (NMR) with respect to hydrogen-l nuclei.

In an embodiment, the method comprises analysing a first biological sample from the subject, wherein the first biological sample is obtained from the subject at a first time point, and a second biological sample from the subject, wherein the second biological sample is obtained from the subject at a second time point, and comparing the level (s) of the one or more betaine compounds in the first biological sample to the level (s) of the one or more betaine compounds in the second biological sample in order to monitor the progressive endogenous metabolism (or progressive status of endogenous metabolism) , the progressive risk of developing a cardiovascular or metabolic disease, the progressive nutritional status or effectiveness of a nutritional regimen in the subject.

In an embodiment, the method further comprises spiking the biological sample with at least one of (or all of) the one or more betaine compounds prior to determining the level (s) of the one or more betaine compounds.

The method may further comprise administering a treatment to the subject to thereby treat the subject in order to improve the endogenous metabolism of the subject, the nutritional status of the subject and/or in order to treat the subject at risk to develop a cardiovascular and/or a metabolic disease. The treatment may be e.g. a treatment effective to prevent and/or treat a cardiovascular and/or metabolic disease. Examples of such treatments may be e.g. administering to the subject an effective amount of a lipid-lowering, cholesterol lowering or cholesterol balancing medicament. The medicament may be e.g. a statin.

The method may further comprise e.g. providing a lifestyle recommendation to the subject based on the assessment of the nutritional status or the effectiveness of the nutritional regimen in the subject. Such a lifestyle recommendation may be e.g. a nutritional recommendation to increase intake of whole grains and/or products derived therefrom, and/or intake of foodstuffs rich in glycine betaine, and/or other nutritional recommendation. A kit for determining the level (s) of one or more betaine compounds in a sample or for performing the method according to one or more embodiments described in this specification is also disclosed. The kit may comprise one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5- aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine. The kit may further comprise reagents for determining the level (s) or for performing said methods and optionally further components, such as a carrier or a solvent. The kit may further comprise instructions for use. The instructions for use may comprise instructions for assessing or aiding in the assessment of endogenous metabolism, a risk of developing a cardiovascular and/or metabolic disease, nutritional status and/or effectiveness of a nutritional regimen in a subject; or instructions for determining the level (s) of the one or more betaine compounds in a biological sample from a subject.

In an embodiment, the one or more betaine compounds is/are selected from alanine betaine, norvaline betaine, 5- aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine.

In an embodiment, the one or more betaine compounds comprise (s) or is 5-aminovaleric acid betaine (5-AVAB).

In an embodiment, the one or more betaine compounds comprise (s) at least one of valine/norvaline betaine or alanine betaine . Use of the kit according to one or more embodiments described in this specification for assessing or aiding in the assessment of nutritional status or effectiveness of a nutritional regimen, endogenous metabolism, and/or a risk of developing a cardiovascular or metabolic disease, nutritional status or effectiveness of a nutritional regimen in a subject, and/or for determining the level (s) of the one or more betaine compounds in a biological sample from a subject is further disclosed .

Use of one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5- aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine for assessing or aiding in the assessment of endogenous metabolism, a risk of developing a cardiovascular or metabolic disease, nutritional status or effectiveness of a nutritional regimen in a subject and/or for determining the level (s) of the one or more betaine compounds in a biological sample from a subject is further disclosed.

In the context of the uses, the subject may be any subject described in this specification. The biological sample may also be any biological sample described in this specification. The nutritional regimen may also be any nutritional regimen described in this specification.

In an embodiment, the one or more betaine compounds comprise (s) or is 5-aminovaleric acid betaine (5-AVAB) .

In an embodiment, the one or more betaine compounds comprise (s) at least one of valine/norvaline betaine or alanine betaine .

It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benefits and advantages. It will further be understood that reference to 'an' item refers to one or more of those items. The term "comprising" is used in this specification to mean including the feature (s) or act(s) followed thereafter, without excluding the presence of one or more additional features or acts.

EXAMPLES

EXAMPLE 1 - Experimental methods

Animal experiments

The animal experiments were approved by the Institutional Animal Care and Use Committee of the Provincial Government of Finland (license number 041003) . C57BL/6J male mice were obtained from National Laboratory Animal Center (Kuopio, Finland) at the age of 9 weeks. The mice were acclimatized for one week and housed 3-4 animals per cage during the weeks 1 to 12 and in single cages from the week 13 until the end of the study. The environment in the animal facility was regulated: temperature 22 ± 1 °C, relative air humidity 55 ± 15% and 12/12 h light/dark cycle with lights on at 7 am. After one week of acclimatization, the mice were fed ad libitum a commercial high-fat diet (D12451, Research Diets Inc., USA) for 9 weeks to induce obesity. The control group was fed ad libitum a commercial low-fat control diet (D12450B, Research Diets Inc., USA) (n=10) . After 9 weeks of pre-feeding the mice were randomized into study groups (n=9-14) and were fed either with D12451, D12450B, or the rye bran or wheat aleurone containing HF diets for additional 9 weeks.

Rye diets contained either rye bran (Rl) or bioprocessed, enzymatically treated and yeast fermented rye bran (R2) where as aleurone diets contained wheat aleurone (Al) or bioprocessed, enzymatically treated aleurone (A4) . More detailed description of the diets have been included in Rosa et al . , J. Agric. Food Chem. 62, 10101-10109 (2014) and Pekkinen et al . , Mol. Nutr. Food Res. 59, 1550-1562 (2015).

8 weeks after starting the study diets enriched with bran, urine samples were collected directly into microcentrifuge tubes and freezed on dry ice. One sample consists of a pool of urine from 3-4 mice. 9 weeks after rye bran or wheat aleurone feeding, the mice were fasted for 8±0.5 hours and sacrificed by decapitation after made unconscious by CO₂ gas. Blood was collected into EDTA tubes (K2-EDTA Microtainer Tubes with BD Microgard Closure, Beckton Dickinson Oy) and centrifuged for 10 minutes 16 000 g at +22°C. The tissues and intestinal tissue contents were collected. The tissue contents were separated from tissues, stored in microcentrifuge tubes and freezed on dry ice. Tissues were rinsed with physiological saline, wrapped in aluminium foil, and snap frozen in liquid nitrogen. All samples were kept at -80 °C until further processing.

Frozen tissue samples were cryo-ground either in 10 ml grinding steel jars with stainless steel ball for 60 seconds with 15 Hz (liver, subcutaneous adipose tissue), or in 2 ml microcentrifuge tubes with 4- or 7-mm stainless steel beads in a precooled 2 ^χ 24 adapters that was shaken for 45 s at 30 Hz (BAT, muscle, heart) using TissueLyser II (Qiagen Finland, Helsinki, Finland) . Samples containing 100 mg (±2 mg) of tissue powder were cryo-weighted into 1.5 mL microcentrifuge tubes and 90% methanol was added (v/v H₂0, LC-MS Ultra CHROMASOLV®, Fluka) in a ratio of 300 pL solvent/100 mg tissue. The samples were shaken for 20 min. Colon contents were weighed, mixed with 90 % methanol (500 μΐ methanolper 100 mg content) and shaken with 4- mm stainless steel beads for 45 s at 20 Hz. All samples were centrifuged for 10 min at 4°C (13 000 rpm) and supernatants were filtered using 0.2-μιη Acrodisc® Syringe Filters with a PTFE membrane (PALL Corporation) and stored at -20°C until LC-MS analyses .

Preparation of plasma samples

The preparation of plasma samples was done essentially as described in Hanhineva et al . , 2015, J. Nutr. 145(1), 7-17. Fasting EDTA plasma samples were collected for the LC-MS metabolite profiling analysis (106 samples at baseline and 106 samples at the end of the study) . An aliquot of the sample, 100 μL, was mixed with 400 μL of acetonitrile (VWR International), mixed on a vortex at maximum speed 15 s, incubated on an ice bath for 15 min to precipitate the proteins, and centrifuged at 16, 000 g for 10 min to collect the supernatant. The supernatant was filtered through 0.2-μιη polytetrafluoroethylene filters in a 96-well plate format. Aliquots of 2 \i were taken from at least half of the plasma samples, mixed together in 1 tube, and used as the quality control (QC) sample in the analysis; a solvent blank was prepared in the same manner.

Other study types

The levels of various betaines can be measured from human subjects participating in clinical trials involving intervention with supplement products, for example glycine betaine (described e.g. in Schwab et al . , 2006, J. Nutr. 136(1), 34-38), or intervention with whole diets, for example increased intake of whole grains (described e.g. in Lankinen et al . , 2011, PLoS One 2011 ; 6 : e22646 ) . In addition, betaines can be measured from other sample types, for example in vitro colonic fermentation (described e.g. in Hanhineva et al . , 2012, PloS One 7(6) :e39322) .

Mass spectrometry

The samples were analyzed by the ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS ) system (Agilent Technologies, a 1290 LC system, a Jetstream electrospray ionization (ESI) source, and a 6540 UHD accurate-mass qTOF spectrometer) . Hydrophilic interaction (HILIC) chromatography was used. The sample tray was kept at 4°C during the analysis. The data acquisition software was the MassHunter Acquisition B.04.00 (Agilent Technologies) .

3 ]i of the sample solution was injected onto the column (Acquity UPLC BEH Amide column, 2.1 χ 100 mm, 1.7 μιη; Waters Corporation) and maintained at 45°C. The mobile phases, delivered at 0.6 mL/min, consisted of 50% acetonitrile (vol: vol; eluent A) and 90% acetonitrile (vol:vol; eluent B) , respectively, both containing 20 mmol/L ammonium formate, pH 3 ( Sigma-Aldrich) . The following gradient profile was used: 0-2.5 min, 100% B; 2.5-10 min, 100% B→0% B; 10-10.1 min, 0% B→100% B; 10.1-14 min, 100% B.

The MS conditions were: Jetstream ESI source, operated in positive ionization mode, conditions were a drying gas temperature of 325°C and flow of 10 L/min, a sheath gas temperature of 350°C and flow of 11 L/min, a nebulizer pressure of 45 pounds per square inch, capillary voltage of 3500 V, nozzle voltage of 1000 V, fragmentor voltage of 100 V, and a skimmer of 45 V. For data acquisition, a 2-GHz extended dynamic range mode was used, and the instrument was set to acquire over the m/ z 50-1600. Data were collected in the centroid mode at the acquisition rate of 2.5 spectra/s (i.e. 400 ms/spectrum) with an abundance threshold of 150.

QC samples were used for the automatic data-dependent MS/MS analyses. From every precursor scan cycle 4 most abundant ions were selected for fragmentation. These ions were excluded after 2 product ion spectra and released again for fragmentation after a 0.25-min hold. The precursor scan time was based on ion intensity, ending at 20, 000 counts or after 300 ms . The product ion scan time was 300 ms . The collision energies were 10, 20, and 40 V in subsequent assays. The continuous mass axis calibration was performed by monitoring two reference ions from an infusion solution throughout the assays. The reference ions were m/z 121.050873 and m/z 922.009798.

Data analysis

Data processing was done using Profinder (Agilent), which enabled manually picking up mass spectrometry peaks associated with amino acid derived betaines. For statistical analysis, parametric ANOVA comparison between three groups for the samples from mice, and paired parametric t-test for the human samples, were used. The a level was set at 0.05 for all statistical tests. Betaines were identified by comparison of retention times and use of targeted MSMS spectrums, which were compared to spectrums of commercial and synthetized chemical standards . Experimental methods for assessing the biological effect of the betaines

The biological effect of betaines can be analysed by typical/general analytical methods used for measuring metabolism-related phenomena/events in biological samples, such as measuring blood glucose and/or insulin levels, analyzing the expression of genes related to carbohydrate and fat metabolism, or conducting a mitochondrial stress test, utilizing for example Seahorse XF24 Analyzer (Agilent Technologies, CA, USA) using suitable cultured cells, such as cardiomyocytes , according to the manufacturer's instructions. Methods e.g. for analysing gene expression and for measuring blood glucose and/or insulin levels are well known to a skilled person. Further, a number of genes related to carbohydrate and fat metabolism are known. The results from such test(s) can be correlated with the altered levels of betaines and linked thus with the metabolic status and health implications thereafter.

EXAMPLE 2 - Identification of novel betainized compounds in plasma and tissues

A group of compounds tentatively identified as betainized metabolites, i.e. betaine compounds, that were increased in the urine of mice were brought to light in a recent non-targeted metabolite profiling on C57BL/6J mouse model fed with rye bran enriched (high fat) feed (Pekkinen et al . , Mol. Nutr. Food Res. 59, 1550-1562 (2015)). The chemical structure of these compounds was verified via chemical synthesis and LC-MS analysis, and they were concluded to include alanine betaine, valine betaine, tryptophan betaine, phenylalanine betaine, pipecolic acid betaine, and 5-aminovaleric acid betaine (5-AVAB) (Fig. 1A-1E) . The compounds are shown in Table 1 and in Fig. 1.

Table 1. Betaine compounds identified. RT refers to retention time using the LC-MS method described in the examples and m/ z to the mass/charge ratio

Betaine compound RT m/z Structure

A semi-targeted method was set up for the novel betaines and plasma samples from a mouse feeding trial comprising native (Rl) or bioprocessed (R2) rye bran (Pekkinen et al . , 2015), or native (Al) or bioprocessed (A4) wheat aleurone fractions (Rosa et al . , 2014, J. Agric. Food Chem. 62, 10101-10109) were examined. Figure 1 illustrates identification of amino-acid derived betaines in the plasma samples. 20 V MSMS fragmentation patterns and abundance in mouse plasma are shown for: a, 5-aminovaleric acid betaine, b, alanine betaine, c , valine betaine, d, phenylalanine betaine, e , pipecolic acid betaine, and f , glycine betaine. Legend: HF, high-fat controls; Rl/2, rye bran groups 1 and 2; Al/4, aleurone groups 1 and 4. Although lower than glycine betaine (Fig. IF), the plasma levels of all these compounds were significantly higher in the enriched groups when compared to the high fat control group (HF) (Fig. 1A-1E), and pipecolic acid betaine (Fig. IE) and phenylalanine betaine (Fig. ID) were detected solely in the test groups, the latter being found only in rye bran fed groups. These betainized compounds have not been described earlier in any mammalian context. In addition to the novel betaines, also other methylated compounds including trigonelline and proline betaine were increased in the plasma samples of mice fed with rye or wheat bran enriched feed.

EXAMPLE 3 - Betaines are elevated after clinical intervention trials with whole grains or glycine betaine

After identifying the betainized compounds in mouse feeding trial, human dietary intervention studies were focused on. Plasma levels of betainized compounds in Healthgrain studies conducted in Kuopio and Naples (Giacco et al . , 2013, Clin. Nutr. 32, 941-949; Giacco et al . , 2014, Nutr. Metab. Cardiovasc. Dis. 24, 837-844) and in glycine betaine supplementation trial (Schwab et al . , 2002, Am. J. Clin. Nutr. 76, 961-967; Schwab et al . , 2006, J. Nutr. 136, 34-38) were analyzed.

Figure 2 shows the abundances of betaines in fasting plasma samples from the three human dietary intervention trials (a, c, e, g, i, k, m, o, q) and the correlations of the levels of the abundances with glycinde betaine abundance (b, d, f, h, j, 1, n, p, r) . Kuopio HG intervention had three dietary groups: A, healthy diet including whole grains; B, whole grain rich diet; C, control diet.

In the dietary intervention studies, whole grain intake correlated with plasma levels of some of the betainized compounds, most notably with pipecolic acid betaine and 5-AVAB levels in the Kuopio HG study, and with valine betaine levels in the Naples HG study (Fig. 2s) . These same betaines were also increased in the subjects on whole grain rich diets when compared to controls in these studies. Furthermore, pipecolic acid betaine and 5-AVAB levels, along with some other betaines, were elevated in the fasting plasma after the intervention with glycine betaine supplementation (Figs. 2m and 2o) , suggesting that the dietary intake of glycine betaine may be at least one of the factors contributing to levels of various betainized compounds found in circulation. This notion is further supported by positive correlations between plasma levels of glycine betaine and betainized compounds in the clinical trials (Fig. 2) . Differences between studies conducted in Kuopio and Naples could be due to e.g. differences in diets or in composition of microbiota.

In addition to the dietary intervention with whole grains, samples from clinical intervention with glycine betaine were examined in order to find out whether glycine betaine present in whole grain foods is responsible for the metabolic shift observed in plasma after whole grain dietary intervention. Similarly as upon the increased whole grain intake, several betainized compounds were elevated in the fasting plasma after the intervention with glycine betaine supplementation (Fig. 2), suggesting that the dietary glycine betaine may be a source for the increment of various betainized compounds found in circulation .

EXAMPLE 4 - Microbiota contributes to the production of betainized compounds

Whole grains are a rich dietary source of glycine betaine, but the other betainized compounds have never been reported from any whole grain containing food material. Four whole grain wheat and rye breads were examined to find out whether any of the compounds could be originating directly from the bread product, and found out that in addition to glycine betaine only trigonelline, proline betaine, and valine betaine were found in low levels (not shown) .

One potential metabolic source for the betainized compounds is colonic microflora, and therefore conventional and germ free mice plasma samples were examined. Figure 3 shows a. betaines in plasma of conventional (MPF) and germ free (GF) mice, b. betaines in the colon contents of the mice fed with bran enriched diets, c. betaines in the in vitro 24-h fermentation model with human microbiota incubated with rye bran fractions. FBL = faecal background, Rl = rye bran, R2 = bioprocessed rye bran.

All the betainized compounds were found in both samples types, but their levels were lower in the germ free mice (Fig. 3a) . The lower levels of betainized compounds in the plasma samples of the germ free mice suggest that microbiota may be contributing to their production. Therefore, next samples from the colonic contents from the mice fed with bran-enriched diets were examined. It was found that all the betainized compounds were present (Fig. 3b) . Majority of the betainized compounds were found also in the group receiving the basal diet, whereas pipecolic acid betaine, phenylalanine betaine, and tryptophan betaine were found only in the bran-fed groups, the latter two being restricted to the groups receiving the rye bran enriched feed. Glycine betaine levels were similar between the groups, suggesting that the extra glycine betaine coming from the feed enriched with bran fractions may potentially serve as precursor for the production of the betainized compounds (Fig. 3b) . Investigation of in vitro model of human colonic fermentation indicated that also human colonic flora is capable of similar metabolic conversions, as upon addition of native or bioprocessed rye bran fractions (Rl and R2, respectively), the production of the betainized compounds continued during a 24- hour in vitro incubation, whereas the levels of the betainized compounds in the faecal background control (FBL) decreased after 4-6 hours, suggesting components in rye bran are able to keep up the production of betainized compounds when compared to faecal background without added bran (Fig. 3c) . Glycine betaine found in the rye bran may be a key component keeping up the production, and indeed, its level decreased during the 24 hour incubation (Fig. 3c). Additionally, investigation of tissue matrices from the intestinal tract of the mouse fed with bran enriched feed showed that the betainized compounds are found in particular in the lower parts of intestine active with microbial fermentation, caecum and colon, suggesting that they are absorbed efficiently. Again, the level of glycine betaine was at a constant level, but the betainized compounds increased in the groups fed with bran enriched feed (Fig. 3d) .

EXAMPLE 5 - 5-Amino valeric acid betaine accumulates to metabolically active tissues

Samples from liver, pancreas, muscle, heart, SAT, VAT, and BAT from the mouse feeding trial were examined in order to find out whether any of the betainized compounds would be absorbed in tissues. All the examined tissues contained varying amounts of several betaines that accumulated upon ingestion of the bran-enriched feed (as shown in Fig. 4a for mouse heart), but 5-AVAB was the compound that had the highest signal among the betainized compounds. Notably, in heart tissue the level of this metabolite was even higher than glycine betaine. In addition to heart, the level of 5-AVAB was particularly high in other metabolically active organs including muscle and brown adipose tissue, being significantly higher in the mice receiving the bran enriched feed when compared to control (not shown) . The notable accumulation of 5-AVAB in the mouse heart tissue prompted further investigation as to whether these compounds could be present in human heart tissue as well. It was found that 5-AVAB is indeed found in human heart tissue alongside other betainized compounds in similar accumulation pattern as in the case of mice (Fig. 4b) . Notably, the compound that had highest accumulation in the organs, 5-AVAB, was produced clearly also in the control group, whereas valine betaine and alanine betaine were particularly high in the bran-fed groups. There might be further chemical conversion between the betaines once entered circulation and target organs, yet to be explored. Another possibility is that the others are mostly excreted via urine, whereas 5-AVAB is actively taken into the tissues.

The molecular structure of 5-AVAB has similarity with meldonium ( 2- [ 2-Carboxyethyl ] -1 , 1 , 1-trimethylhydrazinium, also known as mildronate, THP and MET-88), drug used to treat coronary artery disease. The mechanism of action of meldonium has been associated with reduction of cellular levels of L- carnitine by inhibition of the type 2 organic cation/carnitine transporter (0CTN2, SLC22A5) and L-carnitine biosynthesis enzyme γ-butyrobetaine hydroxylase (Dambrova et al., Pharmacological effects of meldonium: Biochemical mechanisms and biomarkers of cardiometabolic activity. Pharmacol. Res. 2016). Meldonium has been shown to decrease mitochondrial fatty acid oxidation but on the contrary to induce a shift in beta-oxidation from mitochondria to peroxisomes thereby improving cardiac mitochondrial function after ischemia (Liepinsh et al . 2014). The result that in particular the heart tissue had high levels of 5-AVAB after the bran enriched feed in the mouse trial prompted further examination as to whether 5-AVAB could have any similar biochemical function as meldonium. Cardiomyocytes of adult mice were isolated from the other heart cells and were verified as the main source of 5-AVAB signal in the heart tissue as they showed approximately 5 times larger MS peak for 5-AVAB when compared to the other cells in the mouse heart (not shown) . In the cardiomyocytes isolated from the hearts of neonatal mice, 5-AVAB (100 μΜ concentration, 24 hour exposure) significantly decreased levels of L-carnitine (t-test p-value = 0.004) and acetyl-L-carnitine (p < 0.001) when compared to control cells (Figure 4 ) .

Figure 4 illustrates betaines in tissues and effects of 5-AVAB to the energy metabolism of neonatal mouse cardiomyocytes. a . Betaines in mice heart; BET = glycine betaine; b . Betaines in human heart; c . 5-AVAB dose dependently decreases the ability of neonatal mouse cardiomyocytes to utilize palmitoyl acid as an energy source for mitochondrial respiration, d, Comparison of 100 μΜ concentration of 5-AVAB, meldonium (MELDO) and glycine betaine (GLYB) showed that 5-AVAB produces similar effect to meldonium (but glycine betaine does not) . e , Addition of L-carnitine (2 mM, CARNI) is capable of recovering the cells ability to utilize palmitoyl acid as an energy source, f , 5-AVAB, meldonium and glycine betaine (100 μΜ) did not have an effect on the use of glucose and pyruvate in mitochondrial respiration. g, . 5-AVAB (100 μΜ) reduces the cellular levels of L-carnitine and acetyl-L-carnitine in neonatal mice cardiomyocytes. These results suggest that inhibition of the carnitine transporters OCTN2 (responsible for cellular intake) and possibly the mitochondrial carnitine/acylcarnitine carrier protein (CACT, synonyms CAC and SLC25A20) may be the primary cellular mechanisms by which 5-AVAB decreases L-carnitine and acetyl-L-carnitine levels in the cardiomyocytes .

L-Carnitine and acetyl-L-carnitine are in a key role for use of fatty acids in mitochondrial beta-oxidation and degreased levels lead to reduced utilization of fatty acids in energy metabolism. Decreased use of fatty acids in energy metabolism has been considered to protect cardiomyocytes in low oxygen conditions, because use of fatty acids in production of ATP is more oxygen demanding than use of glucose (Dambrova et al . , 2016) . In order to test if 5-AVAB decreased the use of fatty acids in energy metabolism, oxygen consumption of the neonatal mice cardiomyocytes exposed to different concentrations of 5-AVAB (0-250 μΜ) in a medium containing only palmitate as an energy source was measured. When compared to control cells, 5- AVAB caused a dose-dependent reduction in oxygen consumption in the cardiomyocytes, indicating reduced use of fatty acids in mitochondrial ATP production (Fig. 4) . Moreover, in these conditions, 100 μΜ concentration of 5-AVAB inhibited oxygen consumption in cardiomyocytes in a similar manner when compared to same concentration of meldonium (Fig. 4) . By contrast, 100 μΜ concentration of glycine betaine did not have significant effect on utilization of palmitate as an energy source in neonatal cardiomyocytes (Fig. 4) . Furthermore, when 1 mM concentration of L-carnitine is added to the cells, the ability of 5-AVAB to reduce use of fatty acids in energy consumption disappears in both 5-AVAB and meldonium treated cells (Fig. 4), thus confirming that the effect is L-carnitine dependent. The test showed that inclusion of 5-AVAB or meldonium does not have effect on the utilization of glucose as energy source (Fig. 4) .

The present results suggest that 5-AVAB is a novel betaine compound not previously described in humans and a compound modulating the energy homeostasis via inhibition of carnitine transportation, and promoting beneficial health effects related to whole grain consumption including cardioprotective effect and reduction of type 2 diabetes risk. The reduced efficacy to use lipids as energy source forces the body to prefer glucose as energy source over lipids and these biological effects of 5-AVAB could be one of the molecular mechanisms how whole grain diets precondition the hearts tolerance to ischemic insults.

The present results also demonstrate another group of metabolites produced by the microbiota, which may have a direct involvement with host metabolism. Thus they further highlight the role of microbiota in host metabolism, i.e. endogenous metabolism.

The balance between the beta-oxidation of lipids or utilization of sugar/pyruvate in energy homeostasis is known to be critical in metabolic disorders including type 2 diabetes. The present results suggest that betaines may be involved in the modulation of homeostasis in energy metabolism, as exemplified here by 5-AVAB. Further, various betaines described here have not been previously reported to be found in humans.

Claims

1. A method for assessing or aiding in the assessment of nutritional status and/or effectiveness of a nutritional regimen, endogenous metabolism, and/or a risk of developing a cardiovascular and/or metabolic disease in a subject, the method comprising:

analysing a biological sample from the subject to determine the level (s) of one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5- aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine in the biological sample; and

comparing the level (s) of the one or more betaine compounds in the biological sample to a reference value or to the level (s) of the one or more betaine compounds in a control sample in order to assess the endogenous metabolism, the risk of developing the cardiovascular and/or metabolic disease, the nutritional status and/or the effectiveness of the nutritional regimen in the subject .

2. The method according to claim 1, wherein the biological sample comprises or is a blood sample and/or a tissue sample.

3. The method according to claim 2, wherein the blood sample comprises or is a plasma sample, a serum sample, a fasting blood sample, a fasting plasma sample, a fasting serum sample, or a fraction obtainable therefrom.

4. The method according to claim 2 or 3, wherein the tissue sample comprises or is a liver sample, a pancreas sample, a heart sample, a muscle sample, a gastrointestinal wall sample, or an adipose tissue sample, or a fraction obtainable therefrom.

5. The method according to any one of claims 1 - 4, wherein the subject is a human subject.

6. The method according to any one of claims 1 - 5, wherein the method is a method for assessing or aiding in the assessment of nutritional status and/or effectiveness of a nutritional regimen in the subject.

7. The method according to any one of claims 1 - 6, wherein the method is a method for assessing or aiding in the assessment of a risk of developing a cardiovascular and/or metabolic disease in a human subject, wherein the cardiovascular disease optionally comprises at least one of a coronary artery disease, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, or venous thrombosis; and/or the metabolic disease optionally comprises at least one of type 2 diabetes, metabolic syndrome, insulin resistance, or prediabetes.

8. The method according to any one of claims

1 - 7, wherein the method is a method for assessing or aiding in the assessment of endogenous metabolism in a subject, and the endogenous metabolism optionally comprises at least one of endogenous metabolic homeostasis, methylation homeostasis, energy metabolism, methylation metabolism, cellular respiration, or mitochondrial beta-oxidation.

9. The method according to any one of claims 1 - 8, wherein the one or more betaine compounds comprise (s) at least one betaine compound selected from alanine betaine, norvaline betaine, 5- aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine.

10. The method according to any one of claims

1 - 9, wherein the one or more betaine compounds comprise (s) 5-aminovaleric acid betaine.

11. The method according to any one of claims 1 - 10, wherein the one or more betaine compounds comprise (s) at least one of valine/norvaline betaine or alanine betaine.

12. The method according to any one of claims

1 - 11, wherein the biological sample is analysed using at least one of liquid chromatography, mass spectrometry, ¹H-NMR, or any combinations thereof.

13. The method according to claim 12, wherein the liquid chromatography comprises or is hydrophilic interaction liquid chromatography.

14. The method according to any one of claims 1 - 13, wherein the method comprises analysing a first biological sample from the subject, wherein the first biological sample is obtained from the subject at a first time point; analysing a second biological sample from the subject, wherein the second biological sample is obtained from the subject at a second time point; and comparing the level (s) of the one or more betaine compounds in the first biological sample to the level (s) of the one or more betaine compounds in the second biological sample in order to monitor the progressive endogenous metabolism, the progressive risk of developing a cardiovascular or metabolic disease, the progressive nutritional status or the progressive effectiveness of a nutritional regimen in the subject.

15. The method according to any one of claims 1 - 14, wherein the method further comprises spiking the biological sample with at least one of the one or more betaine compounds prior to determining the level (s) of the one or more betaine compounds.

16. The method according to any one of claims 1 - 15, wherein the method further comprises administering a treatment to the subject to thereby treat the subject in order to improve the endogenous metabolism or the nutritional status of the subject and/or in order to treat the subject at risk to develop a cardiovascular or metabolic disease.

17. A kit for determining the level (s) of one or more betaine compounds in a sample or for performing the method according to any one of claims 1 - 16, wherein the kit comprises one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine, and optionally reagents for determining the level (s) or performing said methods and/or instructions for use.

18. Use of the kit according to claim 17 or use of one or more betaine compounds selected from alanine betaine, pipecolic acid betaine, norvaline betaine, 5-aminovaleric acid betaine, 4-aminovaleric acid betaine, valine betaine, phenylalanine betaine, and tryptophan betaine for assessing or aiding in the assessment of nutritional status or effectiveness of a nutritional regimen in a subject, endogenous metabolism, and/or a risk of developing a cardiovascular or metabolic disease; or for determining the level (s) of the one or more betaine compounds in a biological sample from a subject.