WO2018042072A1

WO2018042072A1 - Furan fatty acid biomarkers of nutritional status

Info

Publication number: WO2018042072A1
Application number: PCT/FI2016/050613
Authority: WO
Inventors: Kati HANHINEVA; Seppo AURIOLA; Marko Lehtonen; Jussi PAANANEN; Vanessa DE MELLO-LAAKSONEN
Original assignee: Afekta Technologies Oy
Priority date: 2016-09-02
Filing date: 2016-09-02
Publication date: 2018-03-08

Abstract

A method for assessing or aiding in the assessment of a nutritional status or the effectiveness of a nutritional regimen in a subject is disclosed, the method comprising analyzing a biological sample from the subject to determine the level (s) of at least one furan fatty acid in the sample; and comparing the level (s) in the sample to a reference value or to a control sample in order to assess the nutritional status or the effectiveness of the nutritional regimen in the subject.

Description

Furan fatty acid biomarkers of nutritional status

BACKGROUND

Cardiometabolic diseases are a major cause of morbidity and mortality in Western societies. Modification of dietary habits can contribute to healthy ageing and the risk for cardiometabolic diseases. However, 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. There is a need for methods for assessing the 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 a nutritional status or the effectiveness of a nutritional regimen in a subject is dislosed. The method comprises analyzing a biological sample from the subject to determine the level (s) of at least one furan fatty acid in the sample; and comparing the level (s) in the sample to a reference value or to a control sample in order to assess the nutritional status or the effectiveness of the nutritional regimen 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 a correlation heatmap of the changes in metabolites and changes in clinical biomarkers and anthropometric measurements.

Correlations were calculated using Spearman rank correlation and the red and blue colours indicate positive and negative correlation, respectively. Size of the circles describe statistical significance (- loglO transformed P-value) , such that a larger circle indicates a higher statistical significance.

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.

Changes in the levels of furan fatty acids, also often referred to as furanoid fatty acids, have now been found to be associated with changes in circulating lipid and other additional clinical biomarkers. Furan fatty acids may remain in plasma for a prolonged time period, so that the changes may be observed e.g. after fasting. They therefore indicate a potential metabolic role in the maintenance of the metabolic homeostasis of lipid compounds in plasma. Thus furan fatty acids may be mediators of improvements in clinical lipid profiles, such as cholesterol status. They may also be predictive biomarkers of metabolic disorders, such as cardiometabolic disorders. Certain furan fatty acids described in this specification have not earlier been identified in blood or as potential biomarkers.

Furan fatty acids may also be used as biomarkers in personalized nutrition, in particular within dietary regimens or schemes involving high intake of fish or fish oil supplements. 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.

A method for assessing or aiding in the assessment of a nutritional status or the effectiveness of a nutritional regimen in a subject is disclosed, the method comprising:

analyzing a biological sample from the subject to determine the level (s) of at least one furan fatty acid in the sample; and

comparing the level (s) in the sample to a reference value, a reference level or a control sample in order to assess the nutritional status or the effectiveness of the nutritional regimen in the subj ect . The nutritional regimen may refer e.g. to a diet and/ or regimen of intake of at least one dietary supplement or dietary item.

In an embodiment, the method comprises comparing the level (s) in the sample to a reference value or to a control sample in order to detect and/or diagnose a metabolic and/or cardiovascular disease in the subject or to determine whether the subject is at risk to develop a metabolic and/or cardiovascular disease. In other words, the probability that the subject will progress from being normal to having a metabolic and/or cardiovascular disease may be determined in the context of the assessment of the nutritional status or the effectiveness of the nutritional regimen in the subject. It may be thus possible to determine or assess the effectiveness of a nutritional regimen for the prevention, treatment or slowing down the progress of a metabolic and/or cardiovascular disease. Metabolic and/or cardiovascular diseases may jointly be referred to as cardiometabolic diseases.

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

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. Coronary artery disease may include e.g. angina and myocardial infarction .

In a second aspect, a method for determining the level (s) of at least one furan fatty acid in a biological sample from a subject is disclosed, the method comprising determining the level (s) of at least one furan fatty acid in the sample. The features of the embodiments described be^¬ low may be combined with the first aspect as well as with the second aspect.

The term "biomarker" may refer to a compound, such as a metabolite, that is 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. The term "biomarker" may in this specification refer to one or more furan fatty acids and/or to one or more additional biomarkers, such as additional clinical biomarkers .

The term "level" of one or more biomarkers may refer to th absolute or relative amount or concentration of the biomarker in the sample. The level (s) of at least one furan fatty acids may refer to the total level of the at least one furan fatty acid, i.e. one or more furan fatty acids, and/or to individual levels of the one or more of the at least one furan fatty acids .

The subject may be an individual subject. The subject may be any animal, or a mammal, such as a human .

A reference level or reference value may refer to a level or value of a biomarker that is indicative of a particular nutritional status, the effectiveness of a nutritional regimen, and/or a metabolic and/or cardiovascular disease. The reference level or reference value may be obtained from a control sample. The reference level or reference value may be predetermined. For instance, the reference level or reference value of a biomarker may be predetermined as an average level or a target level of the biomarker in a control subject, control group or 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 detecting and/or diagnosing a metabolic and/or cardiovascular disease the reference level or value may be a reference level or value in or obtained from a 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 the same body fluid in a control subject, group or population from which the biological sample of the individual subject is obtained.

In an embodiment, the reference level (s) is/are obtained from a reference subject, reference group or reference population that has been subjected to a nutritional regimen or diet devoid of fish and/or seafood. The reference subject, group or population may have been subjected to the nutritional regimen or diet for a sufficient time, e.g. at least two weeks or at least four weeks, prior to obtaining the biological sample (s) from which the reference level (s) is/are obtained.

A decreased or increased level or levels of the at least one furan fatty acid 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 fish and/or seafood intake, such as fatty fish intake. A decreased level or levels of the at least one furan fatty acid may be indicative of a metabolic and/or cardiovascular disease or of an increased risk of developing a metabolic and/or cardiovascular disease, at least in cases in which the level (s) of one or more additional clinical biomarkers are increased simultaneously. However, a decreased level or levels of the at least one furan fatty acid may also be indicative of a diet devoid of or low in fish and/or seafood and therefore not necessarily always indicative of a metabolic and/or cardiovascular disease.

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

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

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

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

In an embodiment, the at least one furan fatty acid comprises at least one compound, or at least two, or at least three, or at least four, or at least five compounds represented by formula I :

Formula I wherein

Ri is COOH, CH₃ or H;

R₂ is CH₃ or H;

n is 2, 3, 4, 5 or 6; and

m is 2, 4, 6, 7, 8, 8, 10, 11 or 12.

In an embodiment, Ri is COOH or CH₃;

R2 is CH₃ or H;

n is 2, 3 or 4; and

m is 2 , 3 or 4.

In an embodiment, the at least one furan fatty acid comprises a furan fatty acid having a molecular weight of 240.10 Da (C12H1605) . This furan fatty acid has been confirmed based on pure compound as being 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) .

In an embodiment, the at least one furan fatty acid comprises a furan fatty acid having a molecular weight of 268.13 Da (C14H20O5) . This furan fatty acid appears to correspond, based on MSMS fragmentation analysis, to 3-carboxy-4-methyl-5- pentyl-2-furanpropionic acid. This furan fatty acid is referred to in this specification as "MW 268.13".

In an embodiment, the at least one furan fatty acid comprises a furan fatty acid having a molecular weight of 238.16 Da (C14H2203) . This furan fatty acid appears to correspond, based on MSMS fragmentation analysis, to 3, 4-dimethyl-5-pentyl-2- furanpropanoic acid. This furan fatty acid is referred to in this specification as "MW 238.16".

In an embodiment, the at least one furan fatty acid comprises a furan fatty acid having a molecular weight of 226.08 Da (C11H1405) . This furan fatty acid appears to correspond, based on MSMS fragmentation analysis, to 3-carboxy-5-propyl-2- furanpropanoic acid. This furan fatty acid is referred to in this specification as "MW 226.08".

In an embodiment, the at least one furan fatty acid comprises a furan fatty acid having a molecular weight of 252.17 Da (C15H2403) . This furan fatty acid appears to correspond, based on MSMS fragmentation analysis, to 3-methyl-5-pentyl-2- furanpentanoic acid. This furan fatty acid is referred to in this specification as "MW 252.17".

Given the nature of the MSMS fragmentation analysis, the structures of the compounds MW 268.13, MW 238.16, MW 226.08 and MW 252.17 indicated above may be to some extent tentative. In an embodiment, the at least one furan fatty acid comprises at least one of 3-carboxy-4 -methyl-5-propyl-2 -furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

In an embodiment, the at least one furan fatty acid is selected from the group consisting of 3- carboxy-4 -methyl-5-propyl-2 -furanpropanoic acid (CMPF), MW 268.13, MW 238.16, MW 226.08, MW 252.17, and any combinations thereof.

In an embodiment, the at least one furan fatty acid comprises at least two of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

In an embodiment, the at least one furan fatty acid comprises at least two furan fatty acids selected from the group consisting of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, MW 252.17, and any combinations thereof.

In an embodiment, the at least one furan fatty acid comprises at least three of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

In an embodiment, the at least one furan fatty acid comprises at least three furan fatty acids selected from the group consisting of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, MW 252.17, and any combinations thereof.

In an embodiment, the at least one furan fatty acid comprises at least four of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

In an embodiment, the at least one furan fatty acid comprises at least four furan fatty acids selected from the group consisting of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, MW 252.17, and any combinations thereof.

In an embodiment, the at least one furan fatty acid comprises 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid (CMPF), MW 268.13, MW 238.16, MW 226.08, and MW 252.17.

In embodiments in which the levels of at least two, three, four or five of the furan fatty acids are determined, it may be possible to obtain a more accurate determination and/or assessment. Further, the total level (s) of the furan fatty acids may have an effect on their biological activity and/or effect. The effect of the total level (s) may be more marked than the effect of the level (s) of individual furan fatty acid compounds .

In an embodiment, the method further comprises analyzing a biological sample from the individual subject to determine the level (s) of at least one additional clinical biomarker, wherein the at least one additional clinical biomarker comprises at least one of total cholesterol, LDL, HDL, LDL-to- HDL cholesterol ratio, triacylglycerols , apoAl, apoB, IL-6, TNF- , free fatty acids, plasminogen activator inhibitor 1 (PAI-1), glucose, insulin, glycated haemoglobin (HbAlc) , or C-reactive protein (hs-CRP) ; and comparing the level (s) of the at least one additional clinical biomarker in the sample to a reference value or to a control sample. The biological sample from which the at least one additional clinical biomarker is determined may be the same biological sample from which the level (s) of the at least one furan fatty acid is determined, or a different biological sample.

In an embodiment, the method further comprises analyzing a biological sample from the individual subject to determine the level (s) of at least one additional clinical biomarker, wherein the at least one additional clinical biomarker is selected from the group consisting of total cholesterol, LDL, HDL, LDL-to-HDL cholesterol ratio, triacylglycerols, apoAl, apoB, IL-6, TNF-a, free fatty acids, plasminogen activator inhibitor 1 (PAI-1), glucose, insulin, glycated haemoglobin (HbAlc) , C-reactive protein (hs-CRP) , and any combinations thereof; and comparing the level (s) of the at least one additional clinical biomarker in the sample to a reference value or to a control sample. In an embodiment, the at least additional clinical biomarker comprises at least one of total cholesterol, LDL, HDL, LDL-to-HDL cholesterol ratio, triacylglycerols , apoAl, or apoB.

In an embodiment, the at least additional clinical biomarker is selected from the group consisting of total cholesterol, LDL, HDL, LDL-to-HDL cholesterol ratio, triacylglycerols, apoAl, apoB, and any combinations thereof.

The level (s) of the at least one additional clinical biomarker may be determined and compared to a reference level in order to assess the nutritional status or the effectiveness of the diet in the subject; and/or to detect and/or diagnose a metabolic and/or cardiovascular disease in the subject or to determine whether the subject is at risk to develop a metabolic and/or cardiovascular disease. The at least one additional clinical biomarker may provide a more comprehensive view or profile and a more accurate determination and/or assessment of the subject than the furan fatty acid(s) alone.

The method may comprise obtaining a biological sample from the subject prior to analyzing the sample.

The biological sample may be a blood sample, a plasma sample, a serum sample, a fasting blood sample, a fasting plasma sample, a fasting serum sample, or a fraction obtainable therefrom.

The level (s) of at least one furan fatty acid in the sample may be determined using one or more techniques selected from liquid chromatography, mass spectrometry, ¹H-NMR and any combination thereof. The liquid chromatography may be reversed phase liquid chromatography, although other techniques may also be contemplated. The liquid chromatography and mass spectrometry may be used in combination. The term ^{λ 1}Η- NMR" may be understood as referring to proton nuclear magnetic resonance, i.e. nuclear magnetic resonance (NMR) with respect to hydrogen-1 nuclei.

The method may comprise analysing a first sample from the subject, wherein the first sample is obtained from the subject at a first time point, and a second sample from the subject, wherein the second sample is obtained from the subject at a second time point, and comparing the level (s) of the at least one furan fatty acid in the first sample to the level of the at least one furan fatty acid in the second sample in order to monitor the progressive nutritional status or the effectiveness of a nutritional regimen or diet in the subject.

The method may further comprise spiking the biological sample with the at least one furan fatty acid prior to determining the level of the at least one furan fatty acid.

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 fatty fish intake or other nutritional recommendation.

The method may further comprise administering a treatment to the subject to thereby treat the subject in order to improve the nutritional status of the subject or in order to treat the subject at risk to develop a cardiometabolic disease. The treatment may be e.g. a treatment effective to prevent and/or treat a metabolic and/or cardiovascular 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 . A kit comprising at least one furan fatty acid is also disclosed. The kit may further comprise instructions for use and optionally further components, such as a carrier or a solvent. The instructions for use may comprise instructions for assessing or aiding in the assessment of the nutritional status or the effectiveness of a nutritional regimen in a subject; or for determining the level (s) of at least one furan fatty acid in a biological sample from a subject.

The use of the kit or the use of at least one furan fatty acid for assessing or aiding in the assessment of the nutritional status or the effectiveness of a nutritional regimen in a subject; or for determining the level (s) of at least one furan fatty acid in a biological sample from a subject is also disclosed.

Formula I wherein

Ri is COOH, CH₃ or H;

R₂ is CH₃ or H;

n is 2, 3, 4, 5 or 6; and

m is 2, 4, 6, 7, 8, 8, 10, 11 or 12

In an embodiment, Ri is COOH or CH₃;

R2 is CH₃ or H;

n is 2, 3 or 4; and

m is 2 , 3 or 4. In an embodiment, the at least one furan fatty acid comprises at least one of 3-carboxy-4- methyl-5-propyl-2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

In an embodiment, the at least one furan fatty acid comprises 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, and MW 252.17.

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 - Participants and study protocol

Samples from an intervention study (described in detail in Tovar et al., Nutr. Metab. (London) 2012, 9, 29, which is incorporated herein in its entirety) involving a diet named "multifunctional diet" (MFD) were analyzed with a non-targeted LC-MS-based metabolite profiling approach.

Other dietary interventions with similar nutritional guidance involving increased intake of fish are described in Hanhineva et al . , Journal of Nutrition 2015, 145(1), 7-17, and in Bondia-Pons et al., 2015, 59, 711-728.

EXAMPLE 2 - Analyses Routine blood analyses were performed according to routine analysis methods on fasting plasma (total and HDL cholesterol, TAG, apo A-l, apo B, hs-CRP) , serum (insulin) or on total blood samples (HbAlc) . An example of such analyses and their results are also described in Tovar et al . , 2012. Routine blood analyses including on fasting plasma (total and HDL cholesterol, TAG, apo A-l, apo B, hs-CRP), serum (insulin) or on total blood samples (HbAlc) are typically performed in the context of nutritional intervention studies, such as those described in Hanhineva et al . , Journal of Nutrition 2015, 145(1), 7-17, and in Bondia-Pons et al . , 2015, 59, 711-728. Non-targeted LC-MS metabolite profiling analysis

EDTA plasma samples were kept frozen at -40 °C for the LC-MS metabolite profiling analysis.

Plasma samples were analyzed by the UHPLC- qTOF-MS system (Agilent Technologies, Waldbronn, Karlsruhe, Germany) that consisted of a 1290 LC system, a Jetstream electrospray ionization (ESI) source, and a 6540 UHD accurate-mass qTOF spectrometry. The samples were analyzed using two different chromatographic techniques, i.e. reversed phase (RP) and hydrophilic interaction chromatography (HILIC) . In addition, data were acquired in both positive (+) and negative (-) polarity. Sample tray was kept at 4 °C during the analysis. Data acquisition software was MassHunter Acquisition B.04.00 (Agilent Technologies) .

In RP technique, two microliters of the sample solution was injected onto a column (Zorbax Eclipse XDB-C18, 2.1 χ 100mm, 1.8 ym) (Agilent Technologies, Palo Alto, CA, USA) kept at 50 °C. Mobile phases, delivered at 0.4 ml/min, consisted of water (eluent A) and methanol (eluent B) , both containing 0.1 % (v/v) of formic acid. Following gradient profile was employed: 0-10 min: 2 → 100% B, 10-14.5 min: 100% B, 14.5-14.51 min: 100 → 2% B; 14.51-16.5 min: 2% B.

In HILIC technique, two microliters of the sample solution was injected onto a column (Acquity UPLC BEH Amide column, 2.1 χ 100mm, 1.7 μιη) (Waters Corporation, Milford, MA) kept at 45 °C. Mobile phases, delivered at 0.6 ml/min, consisted of 50% (v/v) (eluent A) and 90% (v/v) (eluent B) acetonitrile, respectively, both containing 20 mM ammonium formate (pH 3) . Following gradient profile was as employed: 0- 2.5 min: 100% B, 2.5-10 min: 100% B → 0% B, 10-10.1 min: 0 % B → 100% B; 10.1-12.5 min: 100% B.

A Jetstream ESI source, operated in positive and negative ionization mode, conditions were drying gas temperature 325 °C and flow 10 L/min, sheath gas temperature 350 °C and flow 11 L/min, nebulizer pressure 45 psi, capillary voltage 3500 V, nozzle voltage 1000 V, fragmentor voltage 100 V, and skimmer 45 V. For data acquisition, 2 GHz extended dynamic range mode was used and the instrument was set to acquire over the m/z 20-1600. Data were collected in centroid mode at acquisition rate of 1.67 spectra/s (i.e. 600 ms/spectrum) with an abundance threshold 150. For automatic data dependent MS/MS analyses, precursor isolation width was 1.3 Da, and 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. Precursor scan time was based on ion intensity, ending at 20000 counts or after 300 ms . Product ion scan time was 300 ms . Collision energies were 10, 20, and 40 V in subsequent runs. Metabolites that were not included in the automatic MSMS analysis were subjected to targeted MS/MS analyses with precursor isolation width of 1.3 Da. In the RP analysis the scan rates were in MS and MS/MS measurements 4 spectra/s (i.e. 250 ms/spectrum) and 2 spectra/s (i.e. 500 ms/spectrum), respectively. In the HILIC analysis, we used 3.31 spectra/sec (i.e. 300 ms/spectrum) as scan rate for MS and MS/MS. The fixed collision energies of 10 and 20 V were used in targeted MS/MS analysis. Continuous mass axis calibration was performed by monitoring two reference ions from an infusion solution throughout the runs. The reference ions were m/z 121.050873 and m/z 922.009798 in positive mode and m/z 112.985587 and m/z 966.000725 in negative mode.

Data collection and alignment

Data were collected with "Find by Molecular Feature" algorithm in (MassHunter Qualitative Analysis B.05.00, software (Agilent Technologies, USA). The peak collection extraction algorithm was set to collect peaks with threshold was at 200 counts, and the allowed ion species were limited to [M+H] + and [M+Na]+ in ESI (+) , and [M-H] - and [M+Cl]-, in ESI (-) . Only signals over compound height threshold of 2500 counts containing at least with two ions were included in the compound list. Peak spacing tolerance for isotope grouping was 0.0025 m/z plus 7 ppm, with isotope model for common organic molecules. Data files (. cef-format ) were exported to Mass Profiler Professional (Agilent Technologies) for peak alignment. After the first initial alignment, the data were combined in one .cef file, against which the original raw data was reanalyzed. For this recursive analysis, compound mass tolerance was ±15 ppm, retention time ±0.2 min and symmetric expansion value for chromatograms ±10 ppm. Resulting compounds were re-exported to Mass Profiler Professional software for peak alignment and data cleanup resulting in 3532, 3511, 1347, 1441 metabolite features from RP(+), RP(- ), hilic(+), and hilic(-), respectively, which were subjected for chemometrical and statistical evaluation .

Statistical analysis

To account for non-normal distributions, metabolite data was transformed using rank based normal transformation. Each metabolite was analyzed using linear mixed-effect model with the metabolite as a dependent variable. Included independent fixed- effect terms were diet (MFD, CD) , time point, treatment order and interaction between study group and time point, while subject identifier was included as a random term. Interaction between diet and time point was considered the main outcome. All p-values were adjusted for multiple testing using Benjamini- Hochberg false discovery rate (P-FDR) . P-FDR < 0.05 were considered as statistically significant. Statistical analyses were performed using R (version 3.1.2) and nlme package (version 3.1-118). Additionally, multivariate assessment of the data was performed with supervised clustering algorithm Partial Least-Squares Discriminant Analysis (PLS-DA; Simca-13, Umetrics, Sweden) for the samples collected at the end of the interventions. The data were loglO-transformed, pareto-scaled and the model was validated by the Simca-13 internal cross validation.

All the data with combined results from the statistical and chemometrical analysis were exported in excel (Microsoft Office 2013) for further processing. The most significantly altered metabolite signals were filtered out from the data matrix in each of the four analytical conditions by matching with the following criteria: signal was present in at least 80% of samples in one replicate group; P-FDR<0.05; fold change<0.8 or >1.2; and VIP>1 in PLS-DA.

The differential metabolites were identified based on the MS/MS spectral comparison of pure standard compounds and on the search of the candidate compounds in databases, including the Human Metabolome database, METLIN, ChemSpider and SciFinder, and the results verified with the MS/MS spectral features included in the databases or reported in earlier publications. Spearman correlation coefficients (rs) were used to test the relationship between the changes in the cardiometabolic-related biomarkers and in the significantly differentiated metabolites. For these analyses, a P<0.05 was considered statistically significant .

EXAMPLE 3 - Cardiometabolic disease-related outcomes An example of the results of a nutritional intervention in terms of cardiometabolic risk- associated markers have been reported earlier (Tovar et al . , 2012) and can be summarized as follows: compared to baseline, there was a minor weight reduction with both diets (-0.9 %, CD; -1.8%; MFD, P<0.05) . CD did not modify the metabolic variables measured but MFD promoted significant changes in total serum cholesterol (-2611% vs baseline; P<0.0001), LDL-cholesterol (-3411%; P<0.0001), TAG (-1913%; P=0.0056), LDL/HDL (-2712%; P<0.0001), apoB/apoAl (- 10±2%; P< 0.0001), HbAlc (-210.4%; P = 0.0013), hs-CRP (-2919%; P=0.0497) and systolic blood pressure (- 811%, P=0.0123). The differences remained significant after adjustment for weight change (Tovar et al . , 2012) . After consumption of MFD, both the Framingham cardiovascular risk estimate and the Reynolds cardiovascular risk score, which considers CRP values, were reduced by one third (30 ±4% ; P< 0.0001 and 35±3%; P<0.0001, respectively). Other examples of diets with similar effects are also are described in Hanhineva et al . , Journal of Nutrition 2015, 145(1), 7-17, and in Bondia-Pons et al . , 2015, 59, 711-728. EXAMPLE 4 - Metabolomics analysis of fasting plasma

Non-targeted metabolite profiling of plasma samples and the examination of the MS/MS spectral and chromatographic features showed that differential signals were belonging to different classes of compounds in lipid metabolism, including, furan fatty acids (Table 1, Figure 1) .

Furan fatty acids

The plasma furan fatty acid metabolite 3- carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) was clearly increased after ingestion of the diet rich in fish, whereas a significant decrease was recorded for the control diet.

In addition to CMPF, four other compounds were tentatively identified as furan fatty acids have been identified from samples originating from the dietary intervention involving guidance to increase fish intake (Tovar et al . , 2012) . These included 3- carboxy-4-methyl-5-pentyl-2-furanpropionic acid and three other signals (Table 1) . The compound with molecular weight of 238.158 has elemental composition of C14H2203, which refers to a furan fatty acid 3,4- dimethyl-5-pentyl-2-furanpropanoic acid identified from crayfish (Ishii et al., Lipids 1988, 23, 694- 700), whereas the compound with molecular weight of 226.084 refers to molecular formula C11H1405. This formula returns several furan fatty acid hits from the SciFinder database, among which e.g. 3-carboxy-5- propyl-2-furanpropanoic acid exhibits a chemical structure that would match the neutral loss of 44 amu (COO) twice from the molecular structure as visible in the MS/MS spectrum in analogous manner with CMPF. Likewise, the compound C15H2403 eluting in the same chromatographic region as the other furan fatty acids (M 252.172) refers to furan fatty acid structures, e.g. 3-methyl-5-pentyl-2-furanpentanoic acid. This structure, however, was not confirmed with MS/MS. EXAMPLE 5 - Correlation between changes in metabolite concentration and cardiometabolic disease-related outcomes

After finding that furan fatty acids were significantly increased during the intervention involving increased fish intake, we performed correlation analyses to investigate how the metabolic changes are related to the measured clinical biomarkers (Figure 1) . Changes in most of the differential furan fatty acids were mainly associated with changes in the classic circulating lipid biomarkers, such as cholesterol fractions, Apo B and triglycerides. The strongest correlations were observed for changes in total and LDL cholesterol and the LDL-to-HDL cholesterol ratio with particular metabolites, as strong inverse correlation was observed with changes in furan fatty acids, including CMPF and 3-carboxy-4-methyl-5-pentyl-2-furanpropionic acid

The present study shows that the beneficial effect of a diet rich in fish may be mediated via biological activity of furan fatty acids promoting the decrease in clinical lipid biomarkers. Furan fatty acids levels, particularly CMPF and 3-carboxy-4- methyl-5-pentyl-2-furanpropionic acid, increased during the intervention with a diet rich in fish. This observation may be explained mainly on the basis of the significant supply of oily fish that characterizesthe diet. These observations, together with the higher post- intervention levels of three other compounds classified as furan fatty acid species (Table 1), indicate that the variety of diet-derived furan fatty acids is far more diverse than known so far. Moreover, the inverse correlation observed between the levels of circulating fatty acid- containing furan metabolites and LDL cholesterol and LDL/ HDL cholesterol ratio suggests that furan-derived compounds could be involved in the beneficial action of the diet.

Table 1. Main differential furan fatty acids analyzed in the non-targeted LC-qTOF metabolite profiling analysis*

*Included are the neutral molecular mass of the compound (Mass) , retention time either in hydrophilic interaction (hilic) or reversed phase (RP) chromatography, electrospray ionization mode (ESI), tentative identification based on the evaluation of msms

spectral data (detailed msm spectra included as supporting information 3) , false discovery rate corrected P values from the mixed model analysis, average fold changes (Av FC) between the baseline and end of the diet, variable importance for projection (VIP) value from the Partial Least-Squares Discriminant Analysis (PLS-DA) .

Claims

1. A method for assessing or aiding in the assessment of a nutritional status or the effectiveness of a nutritional regimen in a subject, the method comprising:

comparing the level (s) in the sample to a reference value or to a control sample in order to assess the nutritional status or the effectiveness of the nutritional regimen in the subject.

2. The method according to claim 1, wherein the method comprises comparing the level (s) in the sample to a reference value or to a control sample in order to detect and/or diagnose a metabolic and/or cardiovascular disease in the subject or to determine whether the subject is at risk to develop a metabolic and/or cardiovascular disease.

3. The method according to claim 2, wherein the metabolic disease comprises at least one of type 2 diabetes, metabolic syndrome, insulin resistance or prediabetes .

4. The method according to claim 2 or 3, wherein 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, or venous thrombosis.

5. The method according to any one of claims 1 - 4, wherein the at least one furan fatty acid comprises at least one compound, or at least two, or at least three, or at least four, or at least five compounds represented by formula I

Formula I wherein R_x is COOH or CH₃;

R2 is CH₃ or H;

n is 2, 3 or 4; and

m is 2 , 3 or 4.

6. The method according to any one of claims

1 - 5, wherein the at least one furan fatty acid comprises at least one of 3-carboxy-4-methyl-5-propyl-

2-furanpropanoic acid (CMPF) , MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

7. The method according to any one of claims 1 - 8, wherein the at least one furan fatty acid comprises at least two, at least three, or at least four of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), MW 268.13, MW 238.16, MW 226.08, or MW 252.17.

8. The method according to any one of claims 1 - 7, wherein the method further comprises analyzing a biological sample from the subject to determine the level (s) of at least one additional clinical biomarker, wherein the at least one additional clinical biomarker comprises at least one of total cholesterol, LDL, HDL, LDL-to-HDL cholesterol ratio, triacylglycerols , apoAl, apoB, IL-6, TNF- , free fatty acids, plasminogen activator inhibitor 1 (PAI-1), glucose, insulin, glycated haemoglobin (HbAlc) , or C- reactive protein (hs-CRP) ; and comparing the level (s) of the at least one additional clinical biomarker in the sample to a reference value or to a control sample .

9. The method according to any one of claims 1 - 8, wherein the biological sample is a blood sample, a plasma sample, a serum sample, a fasting blood sample, a fasting plasma sample, a fasting serum sample, or a fraction obtainable therefrom.

10. The method according to any one of claims 1 - 9, wherein the level (s) of at least one furan fatty acid in the sample is determined using one or more techniques selected from liquid chromatography, mass spectrometry, ¹H-NMR and any combinations thereof .

11. The method according to any one of claims

1 - 10, wherein the reference level (s) is/are obtained from a reference subject, reference group or reference population that has been subjected to a nutritional regimen or diet devoid of fish and/or seafood.

12. The method according to any one of claims

1 - 11, wherein the method comprises analysing a first sample from the subject, wherein the first sample is obtained from the subject at a first time point, and a second sample from the subject, wherein the second sample is obtained from the subject at a second time point, and comparing the level (s) of the at least one furan fatty acid in the first sample to the level of the at least one furan fatty acid in the second sample in order to monitor the progressive nutritional status or the effectiveness of a diet in the subject.

13. The method according to any one of claims 1 - 12, wherein the method further comprises spiking the biological sample with the at least one furan fatty acid prior to determining the level of the at least one furan fatty acid.

14. The method according to any one of claims 1 - 13, wherein the method further comprises administering a treatment to the subject to thereby treat the subject in order to improve the nutritional status of the subject or in order to treat the subject at risk to develop a cardiometabolic disease.

15. A kit comprising at least one furan fatty acid and instructions for use.

16. The use of the kit according to claim 15 or the use of at least one furan fatty acid for assessing or aiding in the assessment of the nutritional status or the effectiveness of a nutritional regimen in a subject; for detecting and/or diagnosing a metabolic and/or cardiovascular disease in a subject or determining whether the subject is at risk to develop a metabolic and/or cardiovascular disease; or for determining the level (s) of at least one furan fatty acid in a biological sample from a subj ect .