Methods and kits for the detection and quantification of 3-hydroxylated fatty acids and lipopolysaccharides

Abstract

The present invention relates to methods for the detection and / or quantification of 3-hydroxylated fatty acids (3-OH HFA or 3-hydroxylated HFA) in a sample; in particular a biological sample. The present invention also relates to methods for the detection and / or quantification of lipopolysaccharides (LPS) in a sample. The present invention further relates to such methods, for diagnosing and / or prognosing a disorder in a subject; and / or for monitoring the evolution of a disorder in a subject, or risk thereof; and / or for determining the efficacy of a treatment or prevention of said disorder in a subject in need thereof.

Description

METHODS AND KITS FOR THE DETECTION AND QUANTIFICATION OF 3-HYDROXYLATED FATTY ACIDS AND LIPOPOLYSACCHARIDESFIELD OF INVENTION

[0001] The present invention relates to methods for the detection and / or quantification of 3-hydroxylated fatty acids (3-OH HFA or 3 -hydroxylated HFA) in a sample; in particular a biological sample.

[0002] The present invention also relates to methods for the detection and / or quantification of lipopolysaccharides (LPS) in a sample.

[0003] The present invention further relates to such methods, for diagnosing and / or prognosing a disorder in a subject; and / or for monitoring the evolution of a disorder in a subject, or risk thereof; and / or for determining the efficacy of a treatment or prevention of said disorder in a subject in need thereof.

[0004] The present invention further relates to kits specifically adapted to such methods.BACKGROUND OF INVENTION

[0005] Inflammation is the immune system reaction to an internal attack (such as cancer), or an external attack (such as an infection, allergy, trauma) on tissues. Inflammation is often caused by infections from Gram-positive or Gram-negative bacteria. Gram-negative bacteria are characterized by the presence of specific molecules in their outer membrane: endotoxins, also known as lipopolysaccharides or LPS.

[0006] LPS are recognized by specific receptors of the immune system, and trigger fever following the release of cytokines. The structure which is the most recognized by the immune system to trigger inflammation is the hexa-acylated structure of lipid A from Escherichia coli. Lipid A is the biologically active part of LPS on which polysaccharidesare attached. Lipid A structure with six fatty acids adapts perfectly to the Toll-like receptor 4 (TLR-4) expressed by immune cells such as monocytes, macrophages, B lymphocytes, T lymphocytes, dendritic cells, and polymorphonuclear.

[0007] Inflammation can be controlled and can lead to healing, or on the contrary, it can spread and lead to toxic shock, or lead to a selection of disorders, including in a non- exhaustive manner those related to inflammation, auto-immune disorders and proliferative disorders; for example cancer, diabetes and arthritis.

[0008] Accordingly, the determination of lipopolysaccharides (LPS) in biological samples is particularly indicative of the inflammatory state and / or health status of a given subject, or population of subjects, when compared to healthy individuals.

[0009] The detection of such LPSs, and the elucidation of their structure is known in the Art; and mass spectrometry now appears to be a method of choice for their detection and quantification; as detailed in Dardelle et al. (“Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification”; Int. J. Mol. Sci. 2024, 25, 3927).

[0010] Methods for detecting lipopolysaccharides by mass spectrometry are reported in WO2014 / 161613, and in Barros et al. (“Quantitative lipopolysaccharide analysis using HPLC / MS / MS and its combination with the limulus amebocyte lysate assay”; Journal of Lipid Research, Vol. 56, 2015).

[0011] Mass spectrometry -based methods for detecting 2-hydroxylated fatty acids and 3 -hydroxylated fatty acids are also reported in Li et al. (“Strategy for Global Profiling and Identification of 2- and 3-Hydroxy Fatty Acids in Plasma by UPLC-MS / MS”; Anal. Chem. 2020, 92, 5143-5151) and Godon et al. (“Pneumococcal pneumonia and endotoxemia: An experimental and clinical reappraisal”; Eur. J Clin. Invest. 2024; 54:el4077).

[0012] Unfortunately, the available methods for detecting such fatty acid markers do not necessarily meet the sensitivity and / or specificity which can be expected in clinical care and / or in the pharma and food sectors; in particular for assessing the contamination ofsuch samples with lipopolysaccharides, and for determining whether the level of lipopolysaccharides is correlated to an inflammation, or a related disorder.

[0013] For example, the Limulus amebocyte lysate (LAL) test / assay is not well adapted to the detection of 3 -hydroxylated fatty acids in complex samples, such as feces. Indeed, LAL is based on an enzymatic cascade in which the proteins are subject to various physicochemical parameters. Temperature, pH, and ions can directly inhibit the LAL assay. Also, a whole blood assay still poses significant challenges, primarily due to a higher degree of interference. Another effect of interference is endotoxin masking, resulting in issues such as low endotoxin recovery (LER), which is caused by the dissociation of the supramolecule assembly of LPS in the presence of surfactants and chelating agents. The Monocyte Activation test (MAT) also lacks endotoxin specificity.

[0014] Hence, there remains a need for methods for detecting and quantifying bound (i.e. esterified and amide-linked) and free (i.e. unesterified and non-amide-linked) 3- hydroxylated fatty acids (3-OH HFA), which remain sensitive and reproducible for a wide range of samples; in particular biological samples and environmental samples.

[0015] In particular, there remains a need for methods which are applicable to biological samples, and / or which are applicable to the clinical practice and for diagnosis or reglementary purposes.

[0016] The invention has for purpose to meet the above-mentioned needs.SUMMARY

[0017] According to a first main embodiment, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, said method comprising steps of: a) determining a ratio between at least one bound form of 3 -hydroxylated fatty acid(s) and at least one free form of the 3 -hydroxylated fatty acid(s) in a sample from the subject, b) comparing the ratio determined at step a) with a reference value,c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0018] According to a second main embodiment, the invention relates to an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of at least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid.

[0019] According to a third main embodiment, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of: a) determining a level of at least one bound form of 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof, wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said lipopolysaccharides in the sample.

[0020] According to a fourth main embodiment, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) toxicity in a sample, said method comprising steps of: a) determining a level of lipopolysaccharides (LPS) in a sample; b) detecting or quantifying LPS toxicity in the sample, from the level of at least one bound form of 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof, and from the level of lipopolysaccharides in the sample determined at step a).

[0021] According to a fifth main embodiment, the invention relates to a kit for the in vitro diagnosis or prognosis of a disorder, and / or for detecting or quantifying 3 -hydroxylated fatty acids (3-OH- HFA) or lipopolysaccharides or lipopolysaccharides toxicity, comprising: means for determining a level of at least one bound form of 3 -hydroxylated fatty acid(s) in a sample; and means for determining a level of at least one free form of said 3-hydroxylated fatty acid(s) in a sample.DEFINITIONS

[0022] In the present invention, the following terms have the following meanings:

[0023] “A”, “an”, and “the” include plural forms unless the context clearly dictates otherwise.

[0024] As used herein, the term “comprising”, or “comprises” is open-ended and may encompass embodiments in which it is substituted by the terms “consisting” or “consists of’.

[0025] “Diagnosis” or “diagnosing” refers to medical diagnosis, i.e., the process of identifying or determining a pathological state, disease or condition in a subject.

[0026] “Prognosis” refers to the likelihood or expected progression of a disease or disorder, including whether the signs and symptoms will improve or worsen (and how quickly) or remain stable over time; expectations of quality of life, such as the ability to carry out daily activities; the potential for complications and associated health issues; and the likelihood of survival (including life expectancy). Accordingly, a “good prognosis” or “positive prognosis” refers to a beneficial clinical outcome such as long-term survival; and a “bad prognosis” or “negative prognosis” refers to a negative clinical outcome such as worsening of the disease or death.

[0027] “Hydroxy fatty acids’’ or ‘ ‘HFA” refer to a class of fatty acids that contain one or more hydroxyl groups (-OH) attached to the carbon chain.

[0028] “3-hydroxylated fatty acids ”, “P -hydroxylated fatty acids’’ or “3-OH HFA’’ are terms used herein interchangeably, and refer to hydroxy fatty acids characterized by the presence of a hydroxyl group (-OH) attached to the third carbon atom of a molecule's carbon chain. In the context of fatty acids or other organic compounds, the carbon atoms in the chain are conventionally numbered starting from the carboxyl end (-COOH). Accordingly, the term 3-OH HFA may thus refer to odd-chain 3-hydroxylated fatty acids or even-chain 3-hydroxylated fatty acids; for example selected from the group consisting of: odd-chain esterified 3-hydroxylated fatty acids, odd-chain unesterified 3-hydroxylated fatty acids, even-chain amide-linked 3-hydroxylated fatty acids, and even-chain non- amide-linked 3-hydroxylated fatty acids. The 3-hydroxylated fatty acids considered in the present disclosure may include saturated 3-hydroxylated fatty acids and unsaturated 3- hydroxylated fatty acids. Such fatty acids are common components of lipopolysaccharides (LPS), specifically of the lipid A portion of LPS molecules in Enterobacteria, like the commonly cited E. coli lipid A. Such 3-hydroxylated fatty acids may also comprise or consist of fatty acids characterized by a linear or branched aliphatic chain, including iso-3-hydroxylated fatty acids and anteiso-3-hydroxylated fatty acids, which are susceptible to be part of naturally-occurring LPS molecules. When part of such LPS molecules, these fatty acids are attached to the lipid A backbone of LPS molecules and are used as diagnostic markers for determining the presence of endotoxins in biological samples. Unless stated otherwise, the term 3-OH HLA may thus refer to either “bound” 3-hydroxylated fatty acids or “free” 3-hydroxylated fatty acids.

[0029] The “esterified 3-hydroxylated fatty acids” and “amide-linked 3- hydroxylated fatty acids” refer to the “bound” fraction of 3-hydroxylated fatty acids associated to lipopolysaccharides. Such lipopolysaccharides (LPS) may thus include intact, naturally-occurring LPS, and partially degraded LPS retaining one or more fatty acids attached to the lipid A backbone.

[0030] The “unesterified 3-hydroxylated fatty acids” and “non-amide-linked 3- hydroxylated fatty acids” refer to the “free” fraction of 3-hydroxylated fatty acids; i.e.those 3 -hydroxylated fatty acids which are not associated to lipopolysaccharides. As used herein, the term “free 3-hydroxylated fatty acid” thus does not encompass intact LPS and / or LPS molecules lacking one or more of the 3-hydroxylated fatty acids which are constitutive of the lipid A portion of LPS molecules.

[0031] “Level” refers to any measured or measurable amount, level, quantity or concentration, whether relative or absolute, of a given marker (e.g. a 3-hydroxylated fatty acid) in a sample. The level of a marker can be determined relative to a control molecule in a sample, or relative to the level of the same marker in a reference population (i.e., relative to a reference level).

[0032] The term “ratio”, as in “ratio between X and Y” may encompass [X / Y] or [Y / X]; wherein X and Y refer to levels of, respectively, bound forms and free forms of a given 3-hydroxylated fatty acid, or of a plurality of 3-hydroxylated fatty acids. When the determination of a ratio relies on the determination of a level of a plurality of 3- hydroxylated fatty acids, each individual level may be added in order to determine, respectively, the total amount of bound and of free forms of said 3-hydroxylated fatty acids. For example, a ratio between a plurality of bound forms of 3-OH fatty acids consisting of CIO, C12, C14, C16 and C18-chain 3-OH fatty acids, and a plurality of free forms of said 3-OH fatty acids may consist of a ratio between the sum of said 3-OH fatty acids (e.g. the sum of CIO, C12, C14, C16 and C18) in a bound form, and said 3-OH fatty acids (e.g. the sum of CIO, C12, C14, C16 and C18) in a free form. The ratio may thus correspond to a molar ratio.

[0033] “Lipopolysaccharides” or “LPS” are terms used herein interchangeably and refer to the glycolipids which are the main constituents of most Gram-negative bacterial outer membranes. LPS of Smooth-type bacteria are large molecules which, in their “intact” form, generally consist of three parts covalently linked: (i) a long-chain polysaccharide made of repetitive O-chain units termed the “O-antigen”, (ii) a core oligosaccharide which generally consists of about 10 monosaccharides, and (iii) a bisphosphorylated glucosamine disaccharide carrying ester- and / or amide-linked 3- hydroxylated fatty acids structure termed “lipid A”. Unless stated otherwise, the term may also encompass those LPS (e.g. from Rough-type bacteria) which do not comprisethe O-chain polysaccharide moiety. In general LPS according to the present disclosure are characterized by molecular weights ranging from about 2 to about 20 kDa.

[0034] LPS molecules are highly immunogenic and their immunogenicity is largely due to the antigenic O-chain structures, the inflammatory activity being due to the classical hexa-acylated structure termed lipid A. The hexa-acylated structure is mostly present in the Enterobacteriaceae family, which model is generally related to Escherichia coli.

[0035] “Measuring” or “measurement”, or alternatively “detecting” or “detection”, mean assessing the presence, absence, level, quantity, or amount of a given substance, e.g., 3-hydroxylated fatty acids (3-OH HFA) specific of lipopolysaccharides (LPS). “Measuring” or “measurement”, or alternatively “detecting” or “detection” as used herein include the derivation of the qualitative or quantitative concentration of said substance, in particular of the 3-hydroxylated fatty acids (3-OH HFA) which are specific of lipopolysaccharides (LPS).

[0036] “Reference level” refers to the amount, level, quantity or concentration of a biomarker in a sample from a reference subject or to the mean or median level of a biomarker in samples from several subjects in a reference population. A reference level can be a normal reference level or a disease-state reference level. A normal reference level is the level of a biomarker in a substantially healthy subject or in several substantially healthy subjects in a reference population, such as a subject (or several subjects) who is / are not suffering from or otherwise was / were not diagnosed with, a disease or disorder. A disease-state reference level is the level of a biomarker in a diseased subject or in several diseased subjects in a reference population, such as a subject (or several subjects) who is / are suffering from or otherwise was / were diagnosed with, a disease or disorder. A reference level may also be determined in sample(s) from the same patient or patient population, under different conditions and / or time (for example for providing a prognosis on a disorder in a treated population when compared to an untreated population).

[0037] A “Sample” refers to any sample which is obtained directly or indirectly from a starting material, such as an environmental source, a fluid from a cell culture, a microbe colony, a plant, or any combinations thereof.

[0038] A “biological sample” refers to any material obtained directly or indirectly from a biological source, and via suitable methods known to the person skilled in the art from a subject. The sample may be collected in a clinically acceptable manner, e.g., in a way that cells, nucleic acids (such as DNA and RNA), and proteins are preserved. A “biological sample” may be a body tissue and / or a bodily fluid. Examples of biological sample include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, saliva, tears, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat, alveolar macrophages, dental plaque, bone marrow, aqueous humour, perilymph, gastric acid, pancreatic juice, rheum, serous fluid, earwax, rumen.

[0039] “Healthy”, with reference to a subject or a population of subjects, means that said subject (or subjects in the population) is / are not suffering from or otherwise was / were not diagnosed with, a disease or disorder; in particular a LPS-associated disorder.

[0040] “Treating” or “treatment” refers to a therapeutic treatment, to a prophylactic (or preventive) treatment, or to both a therapeutic treatment and a prophylactic (or preventive) treatment; wherein the object is to prevent or slow down or lessen a disease or disorder, its associated symptoms and / or complications. A subject may be successfully "treated" if, after receiving treatment as described herein, including adapted care as described herein, the subject shows at least one of the following: relief to some extent of one or more of the symptoms and / or complications associated with the disease or disorder, and / or improvement in quality-of-life issues. The above parameters for assessing successful treatment and improvement in the symptoms and / or complications are readily measurable by routine procedures familiar to a physician.DETAILED DESCRIPTION

[0041] The 3 -hydroxylated fatty acids (3-OH HFA) are markers specific to lipopolysaccharide (LPS) molecules, which are not found in other biological molecules. Conversely, free fatty acids released from intact or substantially intact LPS molecules can also be measured in a sample, but those free fatty acids are not necessarily related to LPS molecules.

[0042] Surprisingly, it has been found that the detection or quantification in samples of, both, bound (for example esterified) and free (for example unesterified) forms of 3- hydroxylated fatty acids is informative toward the accurate detection and quantification of lipopolysaccharides as a whole, and the detection or quantification of LPS-associated toxicity.

[0043] Interestingly, the detection or quantification of odd-chain 3 -hydroxylated fatty acids is also particularly informative with respect to the detection or quantification of LPS, as it allows for a more accurate and complete determination of novel lipopolysaccharides, including those which may be related to the occurrence of novel Gram-negative bacteria structures.

[0044] Without wishing to be bound by the theory, the inventors are of the opinion that such 3 -hydroxylated fatty acids (3-OH HFA), including odd-chain 3-OH HFAs and evenchain 3-OH HFAs, can advantageously be determined in order to improve the range of lipopolysaccharides (LPS) to be found within samples of interest; including biological and environmental samples which may contain non-conventional Gram-negative bacteria and / or non-conventional lipopolysaccharides and / or unsaturated fatty acids.

[0045] Also, it has now been found that the determination in a sample, in particular a biological sample, of a ratio between fatty acids bound to a LPS molecule and free fatty acids (in particular odd-chain 3 -hydroxylated fatty acids, but also even-chain 3- hydroxylated fatty acids) is particularly indicative of detoxification, and of the inflammatory state of the subject tested. Advantageously, it may further complement available methods for detecting or quantifying 3 -hydroxylated fatty acids and LPS, for abroad range of fatty acid extraction protocols and a wider range of samples, such as complex biological samples and environmental samples;

[0046] Without wishing to be bound by the theory, the inventors are of the opinion that a modulation of the level of bound (z.e. esterified and / or amide-linked) vs. free (i.e. unesterified and / or non-amide-linked) 3-OH fatty acids within a sample correlates well with a modification of the degradation of such lipopolysaccharides (LPS); in particular in vivo. More precisely, the inventors are of the opinion that an imbalance in the degradation of such LPS, as reflected for example by a modulation of a ratio between such bound 3- OH fatty acids and free forms thereof, is a good marker of an imbalance in enzymatic detoxification mechanisms associated to the regulation of inflammation / toxicity which can be associated to LPS.

[0047] Recent reports have further shown that the intestinal microbiome, the gut microbiota and the integrity of the intestinal barrier are closely related to the outcome of many disorders, including metabolic diseases, cardiovascular diseases, nervous system diseases or neurodegenerative disorders, as described in Li et al. (“The intestinal microbiome and Alzheimer’s disease: A review. Animal Model Exp Med. 2018; 1(3): 180- 188) or in Zhang-Sun et al. (“Structure function relationships in three lipid A from the Ralstonia genus rising in obese patients”. Biochimie. 2019, Vol. 159, pp72-80).

[0048] In view of the link between the occurrence of LPS and inflammation, it now appears that the relative increase of free (i.e. unesterified and / or non-amide-linked) 3-OH fatty acids now represents a better and more reliable marker of inflammation in the subject, which can thus serve as a basis for the identification of a disorder in a subject.

[0049] In other word, modulation of the level / amount of free 3-OH fatty acids, when compared to bound 3-OH fatty acids, is associated to modulation of inflammation in said subject. In particular, relative increase of free 3-OH fatty acids when compared to bound 3-OH fatty acid is generally associated to a decrease in inflammation or an inflammation- related disorder. Conversely, relative increase of bound 3-OH fatty acids when compared to free 3-OH fatty acid is generally associated to an increase in inflammation or an inflammation-related disorder.

[0050] For example, the increase of the bound / free 3-OH fatty acid ratio in a biological sample is associated to the occurrence of inflammation or an inflammation-related disorder in the corresponding subject / patient.

[0051] This is consistent with the fact that the modulation of the ratio between the bound and free forms of such 3-OH fatty acids is observed in biological samples, in particular serum and other blood-derived samples, from distinct patient populations, and for distinct conditions.

[0052] Consequently, the detection or quantification of 3 -hydroxylated fatty acids (3- OH HFA) by the in vitro methods described herein can advantageously provide a better insight into the health status of a given subject, and / or the occurrence of an inflammation or inflammation-related disorder, when compared to other available techniques in the Art.

[0053] Alternatively, the method can also serve as a second validation, which may complement the available methods relying on a Limulus amebocyte lysate (LAL) test / assay, which is still widely used for the detection and quantification of bacterial endotoxins. A Limulus amebocyte lysate (LAL) test / assay may comprise or consist of any assay which makes use of LAL or of an amebocyte lysate from of horseshoe crabs of another species such as Tachypleus gigas, to detect LPS or use of recombinant proteins, such as recombinant Factor C (rFC), involved in the LAL clotting cascade (see for example LAL Update October 2005, Volume 22, No.3 or International Food Research Journal 19(2): 423-425 (2012)).

[0054] Alternatively, the method can serve as an alternative to the Limulus amebocyte lysate (LAL) test / assay or other tests like the MAT in complex media. According to some embodiments, the methods for the detection or quantification of 3 -hydroxylated fatty acids or lipopolysaccharides (LPS) do not comprise a step of contacting the sample, or a fraction thereof, with a reagent comprising a limulus amebocyte lysate (LAL).

[0055] Accordingly, the proposed methods for detecting 3 -hydroxylated fatty acids (3- OH HFA), either alone or in combination with other known techniques, are applicable to the diagnosis of disorders correlated to the presence of partially degradedlipopolysaccharides, and / or free (i.e. unesterified or non-amide-linked) 3 -hydroxylated fatty acids (HFA), in such samples.

[0056] Accordingly, the inventors provide herein protocols which are applicable to a plurality of samples, especially biological samples. Advantageously, the proposed methods are also compatible with mass spectrometry quantification, and in particular the quantification of fatty acids in such samples by isotopically labeled fatty acid markers.

[0057] Advantageously, the methods for detection and quantification described herein can be adapted to methods for diagnosing or prognosing a disorder in a subject.

[0058] Accordingly, the inventors also provide experimental evidence that the modulation of a ratio of one or more bound forms of 3-hydroxylated fatty acids relatively to the free forms of said 3-hydroxylated fatty acids, in a biological sample, can be associated to the occurrence of a plurality of disorders, including arthritis, Ulcerative Colitis, Crohn’s Disease and Multiple Sclerosis, in particular inflammatory disorders and other disorders which can be associated to inflammation.

[0059] In a non-exhaustive manner, the disorders which are considered by the present disclosure thus include, in particular, those selected from the group consisting of: inflammatory disorders, autoimmune disorders, proliferative disorders and neurological disorders; for example arthritis, cancer or diabetes.

[0060] In particular, the disorders which are considered by the present disclosure include those which are associated with inflammation, which are associated with lipopolysaccharide (LPS) -containing material, such as Gram-negative bacteria, which are associated with an altered gut microbiome or integrity of the intestinal barrier.

[0061] Hence, the disorders which are considered by the present disclosure include dysbiosis or a dysbiosis-related condition.

[0062] The inflammatory disorder may be selected from the group consisting of: an eye disorder, such as ocular allergy, conjunctivitis, keratoconjunctivitis sicca, uveitis and vernal conjunctivitis; a nose disorder, such as allergic rhinitis; an autoimmune disorder, such as a hematological disorder (e.g. hemolytic anemia, aplastic anemia, pure red cellanemia and idiopathic thrombocytopenia), systemic lupus erythematosus, arthritis such as rheumatoid arthritis, polychondritis, scleroderma, Wegener granulamatosis, dermatomyositis, chronic hepatitis, myasthenia gravis, Steven- Johnson syndrome, idiopathic sprue, an autoimmune or inflammatory bowel disease (e.g. ulcerative colitis and Crohn's disease), irritable bowel syndrome, celiac disease, periodontitis, hyaline membrane disease, a kidney disease such as glomerular disease, alcoholic liver disease, multiple sclerosis, endocrine opthalmopathy, Grave's disease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis, multiple sclerosis, primary biliary cirrhosis, uveitis (anterior and posterior), Sjogren’s syndrome, keratoconjunctivitis sicca, uveitis, and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis, systemic juvenile idiopathic arthritis, cryopyrin-associated periodic syndrome, Muckle- Wells syndrome, nephritis, vasculitis, diverticulitis, interstitial cystitis, glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minal change nephropathy), chronic granulomatous disease, endometriosis, leptospiriosis renal disease, glaucoma, retinal disease, ageing, headache, pain, complex regional pain syndrome, cardiac hypertrophy, musclewasting, catabolic disorders, obesity, fetal growth retardation, intestinal failure, hyperchlolesterolemia, heart disease, chronic heart failure, mesothelioma, anhidrotic ecodermal dysplasia, Behcet’ s disease, incontinentia pigmenti, Paget’s disease, acute or chronic pancreatitis, hereditary periodic fever syndrome, asthma (allergic and non-allergic, mild, moderate, severe, bronchitic, and exercise-induced), acute lung injury, acute respiratory distress syndrome, eosinophilia, hypersensitivities, anaphylaxis, nasal sinusitis, ocular allergy, silica induced diseases, COPD (reduction of damage, airways inflammation, bronchial hyperreactivity, remodeling or disease progression), pulmonary disease, cystic fibrosis, acid-induced lung injury, pulmonary hypertension, polyneuropathy, cataracts, muscle inflammation in conjunction with systemic sclerosis, inclusion body myositis, myasthenia gravis, thyroiditis, Addison’s disease, lichen planus, Type 1 diabetes, or Type 2 diabetes, appendicitis, atopic dermatitis, asthma, allergy, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, chronic graft rejection, colitis, conjunctivitis, cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, Henoch-Schonlein purpura, hepatitis, hidradenitis suppurativa,immunoglobulin A nephropathy, interstitial lung disease, laryngitis, mastitis, meningitis, myelitis myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis, pneumonia, polymyositis, proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, ulcerative colitis, uveitis, vaginitis, vasculitis, or vulvitis.

[0063] In some embodiments, the inflammatory disorder is associated to acute or chronic graft rejection in kidney, liver, heart, pulmonary transplantation, or graft versus-host disease in bone marrow graft. In some embodiments, the inflammatory disorder is a skin inflammatory disorder, for example selected from contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforma, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity angiitis, urticaria, bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, epidermolysis bullosa acquisita, and other inflammatory or allergic conditions of the skin. In some embodiments, the inflammatory disorder is selected from acute and chronic gout, chronic gouty arthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, Juvenile rheumatoid arthritis, Systemic jubenile idiopathic arthritis (SJIA), Cryopyrin Associated Periodic Syndrome (CAPS), Muckle- Wells syndrome, and osteoarthritis. In some embodiments, the inflammatory disorder is selected from Sjogren’s syndrome, allergic disorders, osteoarthritis, conditions of the eye such as ocular allergy, conjunctivitis, keratoconjunctivitis sicca and vernal conjunctivitis, and diseases affecting the nose such as allergic rhinitis. In some embodiments, the inflammatory disorder is associated with transplantation, in particular organ transplantation, organ transplant rejection, and / or graft versus host disease. In some embodiments, the inflammatory disorder is an autoimmune disorder, for example selected from: type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, psoriasis, Behcet’s disease, POEMS syndrome, Crohn's disease, ulcerative colitis, ankylosing spondylitis, axial spondyloarthritis, primary biliary cirrhosis, autoimmune hepatitis, or inflammatory bowel disease.

[0064] In some embodiments, the disorder is a proliferative disorder; in particular cancer, for example selected from blood cancer, liquid and solid cancers.

[0065] In some embodiments, the disorder is a metabolic disease. In some embodiments, the disorder is a cardiovascular disease.

[0066] In some embodiments, the disorder is a neurological disorder; for example a neurodegenerative disorder; for example selected from the group consisting of: Guillain- Barre syndrome, Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis and other demyelinating diseases.

[0067] In some embodiments, the disorder is selected from the group consisting of: Crohn’s Disease, Ulcerative Colitis, Multiple Sclerosis, arthritis, cancer and diabetes.

[0068] In particular, the disorder may be an inflammatory disease, such as arthritis.

[0069] In particular, the disorder may be an autoimmune and / or inflammatory gut disease, such as Crohn’s Disease or Ulcerative Colitis.

[0070] In particular, the disorder may be a neurodegenerative disorder, such as Multiple Sclerosis.

[0071] In some embodiments, the disorder is dysbiosis, sepsis, and / or a disorder associated with a Gram-negative bacterium.Methods & Uses

[0072] According to one main embodiment, the invention relates to an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of at least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample;wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain or even-chain 3 -hydroxylated fatty acid.

[0073] The invention also relates to / describes an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of at least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid.

[0074] The invention also relates to / describes an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of at least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one even-chain 3 -hydroxylated fatty acid.

[0075] The invention also relates to an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of at least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample;wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise at least one oddchain 3 -hydroxylated fatty acid and at least one even-chain 3 -hydroxylated fatty acid.

[0076] Advantageously it is possible to determine the level or ratio of lipopolysaccharides (LPS) in a sample from the level or ratio of a given 3 -hydroxylated fatty acid, or combination thereof, based on the structure of a reference LPS, and in particular based on the structure of the lipid A moiety which is part of said reference LPS. Each bacteria, in particular each Gram-negative bacteria, can be distinguished by a specific LPS profile, as shown in Caroff & Novikov (“Lipopolysaccharides: structure, function and bacterial identification”; OCL 2020, 27, 31).

[0077] A level or ratio of one or more lipopolysaccharides (LPS) may be derived from the level or ratio of the detected / quantified 3 -hydroxylated fatty acids based on:The level of free (z.e. unesterified or non-amide linked) forms of said 3- hydroxylated fatty acid(s);The number of bound (z.e. esterified or amide-linked) forms of 3 -hydroxylated fatty acid(s) covalently linked to the lipid A moiety of a reference lipopolysaccharide (LPS) to be detected / quantified;The molecular mass of the reference lipopolysaccharide (LPS) to be detected / quantified.

[0078] Advantageously, one or more reference lipopolysaccharide (LPS), as defined above, may correspond to a signature of the occurrence of one or more reference bacteria; in particular of one or more reference Gram-negative bacteria.

[0079] Hence, according to another main embodiment, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of: a) detecting or quantifying a level of 3 -hydroxylated fatty acids (3-OH HFA) in said sample according to the method described above, by determining a level ofat least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said lipopolysaccharides in the sample.

[0080] According to some particular embodiments, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of: a) detecting or quantifying a level of at least one bound (i.e. esterified or amide- linked) form of 3 -hydroxylated fatty acid(s) and of at least one free (i.e. unesterified or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof according to the method described above; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said lipopolysaccharides in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid.

[0081] According to some particular embodiments, the invention relates to / describes an in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of: a) detecting or quantifying a level of at least one bound (i.e. esterified or amide- linked) form of 3 -hydroxylated fatty acid(s) and of at least one free (i.e. unesterified or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof according to the method described above; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said lipopolysaccharides in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one even-chain 3 -hydroxylated fatty acid.

[0082] According to some particular embodiments, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of:a) detecting or quantifying a level of at least one bound (i.e. esterified or amide- linked) form of 3 -hydroxylated fatty acid(s) and of at least one free (i.e. unesterified or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof according to the method described above; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said lipopolysaccharides in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise at least one oddchain 3 -hydroxylated fatty acid, and at least one even-chain 3 -hydroxylated fatty acid.

[0083] The methods for detecting or quantifying lipopolysaccharides (LPS) may advantageously be further applied to the detection or quantification of LPS-associated toxicity; for example in biological samples and / or environmental samples.

[0084] Hence, according to another main embodiment, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) toxicity in a sample, said method comprising steps of: a) determining a level of lipopolysaccharides (LPS) in a sample; b) detecting or quantifying LPS toxicity in the sample, from a level of at least one bound form of 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof, and from the level of lipopolysaccharides in the sample determined at step a).

[0085] Without wishing to be bound by the theory, when the LPS molecule contains fewer fatty acids, it has been observed that it is less inflammatory, and can even lead to a structure which blocks inflammation. In vivo, there are enzymatic detoxification mechanisms leading to the release of fatty acids and / or phosphate groups from the LPS molecule, which makes it less or not at all inflammatory. These phenomena also lead to the release of free fatty acid forms in the medium (i.e. unesterified and / or non-amide- linked 3 -hydroxylated fatty acids). Penta- or under-acetylated forms which have lost one fatty acid, or hepta- and over-acetylated forms on which an enzyme adds fatty acids during late steps of the biosynthesis are significantly less active than the canonical hexaacetylated structure.

[0086] Hence, in particular, determination (i.e. detection or quantification) of LPS toxicity may be achieved by detecting or quantifying under- acetylated forms of LPS (i.e. which have lost one or more than one fatty acid), or hepta- and over- acetylated forms which are less active (i.e. toxic and associated to inflammation) than a reference hexaacetylated structure.

[0087] The methods and uses disclosed herein encompass the use of 3 -hydroxylated fatty acids (3-OH HFA) as biomarkers for diagnosis or prognosis purposes. In particular, the methods and uses disclosed herein encompass the use of a ratio between bound (i.e. esterified or amide-linked) forms of 3 -hydroxylated fatty acids (3-OH HFA) and free (i.e. unesterified or non-amide-linked) forms of said 3 -hydroxylated fatty acids (3-OH HFA), as a biomarker for the diagnosis or prognosis of a disorder in a subject. In a non- exhaustive manner, examples are further provided with respect to the diagnosis and prognosis of an inflammatory disorder.

[0088] According to a main embodiment, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, said method comprising steps of: a) determining a level of at least one bound (i.e. esterified or amide-linked) form of 3-hydroxylated fatty acid(s) and of at least one free (i.e. unesterified or non- amide-linked) form of the 3-hydroxylated fatty acid(s) in a sample from the subject, or a ratio thereof, b) comparing the levels or ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0089] In a non-exhaustive manner, and in the context of a method for diagnosis or prognosis of a disorder, a reference value (i.e. a reference level or reference ratio) may thus consist of a physical or temporal endpoint.

[0090] For example, the reference value can be from the same subject from whom a sample is assessed, or the reference value can be from a representative population of samples (e.g., samples from normal subjects not exhibiting a symptom of a given disorder). Therefore, a reference value can provide a threshold measurement which iscompared to a subject sample’s readout in a given sample. Such reference values may be set according to data pooled from groups of sample corresponding to a particular cohort, including but not limited to age (e.g., newborns, infants, adolescents, young, middle-aged adults, seniors and adults of varied ages), racial / ethnic groups, normal versus diseased subjects, smoker v. non-smoker, subject receiving therapy versus untreated subject, different time points of treatment for a particular individual or group of subjects similarly diagnosed or treated or combinations thereof. A reference level or ratio may be based on samples assessed from the same subject to provide individualized tracking. Reference levels or ratios can be established for unaffected individuals (of varying ages, ethnic backgrounds and sexes) without a particular phenotype. For example, a reference value for a reference population can be used as a baseline for the diagnosis or prognosis of a given disorder in a test subject. If a sample from a subject has a level or ratio that is similar to the reference, the subject can be identified to not have the disease, or of having a low likelihood of developing the disorder. Alternatively, reference levels or ratio can be established for individuals with a particular phenotype (z.e. with a particular disorder or symptom thereof). A subject’s value can be compared to the index and a diagnosis or prognosis of the disorder can be determined, such as a disease stage or progression. Alternatively, an index of values can be generated for therapeutic efficacies. The levels and / or ratios can be used to generate values of which is a subject’s value is compared, and a treatment or therapy can be selected for the individual, e.g., by predicting from the levels whether the subject is likely to be a responder or non-responder for a treatment. Reference values can also be established for disease recurrence monitoring (or exacerbation phase in MS), for therapeutic response monitoring, or for predicting responder / non-responder status.

[0091] The value can be a quantitative or qualitative value.

[0092] According to some embodiments, the method comprises a step of determining a level of at least one esterified form of said 3 -hydroxylated fatty acids (3-OH HFA) and of at least one unesterified form of said 3 -hydroxylated fatty acids (3-OH HFA) in said sample; or a ratio thereof.

[0093] According to some embodiments, the method comprises a step of determining a level of at least one amide-linked form of said 3 -hydroxylated fatty acids (3-OH HFA) and of at least one non-amide linked form of said 3 -hydroxylated fatty acids (3-OH HFA) in said sample; or a ratio thereof.

[0094] According to some embodiments, the method comprises a step of determining a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of said 3 -hydroxylated fatty acids (3-OH HFA) and of a plurality of free (i.e. unesterified and / or amide-linked) forms of said 3 -hydroxylated fatty acids (3-OH HFA) in said sample; or a ratio thereof.

[0095] According to some embodiments, the method comprises a step of determining a level of a plurality of esterified forms of said 3 -hydroxylated fatty acids (3-OH HFA) and of a plurality of unesterified forms of said 3-hydroxylated fatty acids (3-OH HFA) in said sample; or a ratio thereof.

[0096] According to some embodiments, the method comprises a step of determining a level of a plurality of amide-linked forms of said 3-hydroxylated fatty acids (3-OH HFA) and of a plurality of non-amide linked forms of said 3-hydroxylated fatty acids (3-OH HFA) in said sample; or a ratio thereof.

[0097] According to some embodiments, the method comprises a step of determining a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of odd-chain 3-hydroxylated fatty acids (3-OH HFA) and of a plurality of free (i.e. unesterified and / or amide-linked) forms of odd-chain 3-hydroxylated fatty acids (3-OH HFA) in said sample; or a ratio thereof.

[0098] Hence, according to some embodiments, the invention relates to an in vitro method for detecting or quantifying 3-hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of the 3-hydroxylated fatty acids and of a plurality of free (i.e. unesterified and / or non-amide-linked) forms of the 3-hydroxylated fatty acids in said sample, or a ratio thereof;b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid.

[0099] Hence, according to some embodiments, the invention relates to an in vitro method for detecting or quantifying odd-chain 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of the odd-chain 3 -hydroxylated fatty acids and of a plurality of free (i.e. unesterified and / or amide-linked) forms of the odd-chain 3 -hydroxylated fatty acids in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said odd-chain 3 -hydroxylated fatty acids (3-OH HFA) in the sample.

[0100] According to some embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, said method comprising steps of: a) determining a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids and of a plurality of free (i.e. unesterified and / or non-amide-linked) forms of the 3 -hydroxylated fatty acids in a sample from the subject, or a ratio thereof; b) comparing the levels or ratio determined at step a) with a reference value; c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0101] According to some embodiments, the methods described herein comprise a step of determining a ratio between a level of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids and a level of free (i.e. unesterified and / or non-amide- linked) forms of the 3 -hydroxylated fatty acids.

[0102] According to some embodiments, the invention relates to an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a ratio between at least one bound (i.e. esterified and / or amide- linked) form of the 3 -hydroxylated fatty acid(s) and at least one free (i.e. unesterified and / or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample; b) detecting or quantifying, from the ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid.

[0103] According to some embodiments, the invention relates to an in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH HFA) in a sample, said method comprising steps of: a) determining a ratio between at least one bound (i.e. esterified and / or amide- linked) form of the 3 -hydroxylated fatty acid(s) and at least one free (i.e. unesterified and / or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample; b) detecting or quantifying, from the ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one even-chain 3 -hydroxylated fatty acid.

[0104] According to some embodiments, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of: a) determining a ratio between at least one bound (i.e. esterified or amide-linked) form of 3 -hydroxylated fatty acid(s) and at least one free (i.e. unesterified or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample; b) detecting or quantifying, from the ratio determined at step a), the occurrence of said lipopolysaccharides in the sample.

[0105] According to some embodiments, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) toxicity in a sample, said method comprising steps of: a) determining a level of lipopolysaccharides (LPS) in a sample; b) detecting or quantifying LPS toxicity in the sample, from a ratio between at least one bound (i.e. esterified or amide-linked) form of 3 -hydroxylated fatty acid(s) and at least one free (i.e. unesterified or non-amide linked) form of the 3 -hydroxylated fatty acid(s) in said sample, and from the level of lipopolysaccharides in the sample determined at step a).

[0106] According to some embodiments, the invention relates to an in vitro method for detecting or quantifying lipopolysaccharides (LPS) toxicity in a sample, said method comprising steps of: a) determining a level of lipopolysaccharides (LPS) in a sample; b) detecting or quantifying LPS toxicity in the sample, from a ratio between a plurality of bound (i.e. esterified or amide-linked) forms of 3 -hydroxylated fatty acid(s) and a plurality of free (i.e. unesterified or non-amide linked) forms of the 3 -hydroxylated fatty acid(s) in said sample, and from the level of lipopolysaccharides in the sample determined at step a).

[0107] According to some embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, said method comprising steps of: a) determining a ratio between at least one bound (i.e. esterified and / or amide- linked) form of 3 -hydroxylated fatty acid(s) and at least one free (i.e. unesterified and / or non-amide-linked) form of the 3 -hydroxylated fatty acid(s) in a sample from the subject; b) comparing the ratio determined at step a) with a reference value; c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0108] According to some embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, said method comprising steps of:a) determining a ratio between a plurality of bound (i.e. esterified and / or amide- linked) forms of 3 -hydroxylated fatty acid(s) and a plurality of free (i.e. unesterified and / or non-amide-linked) forms of the 3 -hydroxylated fatty acid(s) in a sample from the subject; b) comparing the ratio determined at step a) with a reference value; c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0109] According to some embodiments, the methods described herein comprise a step of determining a ratio between a level of a plurality of esterified forms of 3 -hydroxylated fatty acids and a level of a plurality of unesterified forms of the 3 -hydroxylated fatty acids.

[0110] According to some embodiments, the methods described herein comprise a step of determining a ratio between a level of a plurality of amide-linked forms of 3 -hydroxylated fatty acids and a level of a plurality of non-amide-linked forms of the 3 -hydroxylated fatty acids.

[0111] Accordingly the methods described herein may comprise a step of determining: a ratio of a level of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids to a level of free (i.e. unesterified and / or amide-linked) forms of the 3 -hydroxylated fatty acids; or a ratio of a level of free (i.e. unesterified and / or non-amide linked) forms of the 3 -hydroxylated fatty acids to a level of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids.

[0112] Accordingly, the methods described herein may comprise a step of determining: a ratio of a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids to a level of a plurality of free (i.e. unesterified and / or non-amide-linked) forms of the 3 -hydroxylated fatty acids; ora ratio of a level of a plurality of free (i.e. unesterified and / or non-amide linked) forms of the 3 -hydroxylated fatty acids to a level of a plurality of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids.

[0113] A modulation of a ratio between the bound (i.e. esterified and / or amide-linked) and the free (i.e. unesterified and / or non-amide linked) forms of one or more 3 -hydroxylated fatty acids is indicative of the occurrence of a disorder.

[0114] In particular, an increase of the ratio of a level of bound (i.e. esterified and / or amide-linked) forms of 3 -hydroxylated fatty acids to a level of free (i.e. unesterified and / or non-amide-linked) forms of the 3 -hydroxylated fatty acids, when compared to a reference such as the same ratio as measured in healthy individuals, is indicative of the occurrence of a disorder.

[0115] According to some embodiments, the modulation, when compared to healthy individuals may be of about two-fold, three-fold, four-fold, five-fold, ten-fold, or more.

[0116] The modulation of a ratio between the bound (i.e. esterified and / or amide-linked) and the free (i.e. unesterified and / or non-amide-linked) forms of one or more 3 -hydroxylated fatty acids, when compared to a sample from an individual with a known therapeutic treatment is also indicative of the efficacy of said treatment.

[0117] For example, the decrease of the ratio in a subject / patient undergoing therapy, when compared to a subject / patient that is not undergoing the same therapy, may be indicative of the efficacy of said therapy toward the disorder.

[0118] According to some embodiments, the sample may be selected from a group consisting of: a biological sample, a food sample (e.g. a diet supplement), a vaccine sample, a water sample, a beverage sample, a cosmetic sample, an environmental sample.

[0119] The methods described herein may comprise a step of providing a sample; for example a biological sample from a subject.

[0120] The provided methods are applicable to clinical use, but also to veterinary and reglementary purposes.

[0121] Accordingly, the “Subject” may refer to an animal, in particular a human or nonhuman mammal, preferably a human. The subject may be an animal, preferably a mammal, more preferably a primate. The subject may be a human. The subject may be a male. The subject may be a female. The subject may be a child, an adolescent or an adult. The subject may be a patient, i.e., a recipient of health care services.

[0122] The biological sample may be selected from blood, plasma, serum, cerebrospinal fluid, saliva, tears, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat, alveolar macrophages, and dental plaque.

[0123] According to a particular embodiment, said biological sample is selected from the group consisting of; blood, serum, plasma, saliva and urine; preferably blood, serum or plasma.

[0124] The sample may have been previously taken from the subject, i.e., the method as described herein may not comprise an active step of recovering a sample from the subject. Consequently, the methods as described herein may be non-invasive methods, i.e., the methods as described herein may be in vitro methods.

[0125] According to some embodiments, the sample may comprise or consist of bacterial cells or a supernatant thereof, or a fraction thereof; for example Gram-negative bacteria or a supernatant thereof, or a fraction thereof.

[0126] Examples of Gram-negative bacteria which may be associated to the presence of lipopolysaccharides may include those belong to a genus selected from the group consisting of: Escherichia, Bacteroides, Bordetella, Agrobacterium, Yersinia, Prevotella, Porphyromonas, Fusobacterium, Neisseria, Haemophilus, Rhizobia, Vibrio, Coxiella, Legionella, Salmonella, Pseudomonas, Klebsilla, Acinetobacter, Enterobacter, Ralstonia, Akkermansia, Acidovorax, Comamonas, Vitreoscilla, Veillonella, Alistipes,Parabacteroides, Dialister, Oscillibacter, Sutterella, Odoribacter, Citrobacter, Leptospira, Morganella, Aggregatibacter, Cardiobacterium, Eikenella, Desulfovibrio, Yersinia, Burkholderia, Chlamydia, Proteus, Shigella, Helicobacter, Serratia, Campylobacter, Magneto spirillum, Stenotrophomonas, Acinetobacter, Rouxiella, Pantoea, Leclercia, Pasteurella, Capnocytophaga, Flavobacterium and combinations thereof.

[0127] In some embodiments, the sample is a food sample, in particular, intended for animal or human consumption and containing an ingredient such as a cereal, e.g., wheat, barley, oats; a legume, e.g., lentils; meat, e.g., beef, veal, mutton, lamb, pork, rabbit, poultry; a fish, e.g., cod, trout, salmon; seafood, e.g., oysters, mussels, shrimp, lobster; a vegetable or a fruit, e.g., apricot, eggplant, banana, carrot, cherry, cabbage, zucchini, strawberry, green bean, kiwi, lettuce, pear, apple, potato, recombinant protein preparations. In some embodiments, the food sample can be from a manufactured food product, such as, e.g., a ready-made meal, a tin can, cheese samples, milk components, a fermented product, a frozen product.

[0128] In some embodiments, the sample comprises or consists of animal and / or plant cells, or fractions thereof.

[0129] In some embodiment the sample is selected from the non-exhaustive list comprising or consisting of: foliage of a plant, bark of a plant, fruit of a plant, flowers of a plant, seed of a plant, roots of a plant, a cutting of a plant, a graft of a plant, callus tissue of a plant; soil or growth medium surrounding a plant; soil or growth medium before sowing seed of a plant in the soil or growth medium; or soil or growth medium before planting the plant, the plant cutting, the plant graft, or the plant callus tissue in the soil or growth medium.

[0130] In some embodiments, the animal may be a companion animal, or else, a domestic animal, livestock or else, such as those selected from the non-exhaustive list comprising or consisting of: horse, goat, lama, cow, sheep, chicken, duck, goose, pig, donkey, turkey, dog, cat, rabbit, guinea pig, hamster, fish, ferret, bird, parrot, rat, mouse, or else.

[0131] In some embodiments, the animal cell is selected from the non-exhaustive list comprising or consisting of: an adipocyte; a fibroblast; an endothelial cell; an epithelial cell; a bone cell, e.g., an osteoblast, an osteocyte, an osteoclast; a muscle cell, e.g., a myoblast, a myocyte; a blood cell, e.g., a red blood cell, a lymphocyte, a polynuclear; a hepatocyte; a keratinocyte; a nerve cell; and any combination thereof.In some embodiments, the sample is a sample which may contain a microorganism, such as an alga, an archaea, a bacterium, a fungus, a protozoan, a virus. In one embodiment, the microorganism is a pathogen for plants, for animals, and in particular for humans. In certain embodiments, the microorganism is referred to as a contaminant. In some embodiments, the sample comprises nucleic acids from one or more microorganism(s), in particular one or more contaminating microorganism(s), more particularly one or more pathogenic microorganism(s).Fatty acid extraction

[0132] The methods described herein may comprise a step of providing a fatty acid enriched fraction (z.e. isolating a fatty acid-enriched fraction); for example a bound (z.e. esterified and / or amide-linked) fatty acid enriched fraction and / or a free (z.e. unesterified and / or non-amide-linked) fatty acid enriched fraction. The methods described herein may comprise a step of providing a lipopolysaccharide / endotoxin enriched fraction.

[0133] Methods for isolating lipopolysaccharides from a sample may be as disclosed in W02004 / 062690 Al.

[0134] The methods described herein may comprise a step of providing a 3 -hydroxylated fatty acids (3-OH HFA) enriched fraction; for example an esterified 3 -hydroxylated fatty acids (3-OH HFA) enriched fraction and / or an unesterified 3 -hydroxylated fatty acids (3- OH HFA) enriched fraction.

[0135] According to some embodiments, total 3 -hydroxylated fatty acids may be recovered from the sample after an hydrolysis step, thereby recovering an hydrolyzed fraction; and an optional extraction step of the hydrolyzed fraction, thereby recoveringthe total 3 -hydroxylated fatty acids (3-OH HFA) enriched fraction. The hydrolysis step may be achieved in the presence of HC1 in a final concentration ranging from about 5 M to about 8 M; for example 4, 5, 6, 7 or 8M. The hydrolysis step may include an heating step, for example at a temperature ranging from about 80°C to about 100°; for example at about 90°C for 2 to 4 hours.

[0136] According to some embodiments, free (z.e. unesterified and / or non-amide-linked) 3 -hydroxylated fatty acids may be recovered from the sample without an hydrolysis step.

[0137] The methods described herein may comprise a further step of derivatization of a 3 -hydroxylated fatty acids (3-OH HFA) enriched fraction; for example the total 3- hydroxylated fatty acids (3-OH HFA) enriched fraction and / or the free fatty acids (3-OH HFA) enriched fraction.

[0138] The 3-OH HFA enriched fraction may be derivatized through methylation and / or silanization. The 3 -hydroxylated fatty acids can be methylated using, for example, methanolysis, diazomethane or trifluoroborane. The 3 -hydroxylated fatty acids can be derivatized with a chlorosilisane compound.3-hydroxylated fatty acid (3-OH HFA)

[0139] The following 3-OH HFAs, and combinations thereof, are applicable to all the embodiments and sub-embodiments reported herein, including (i) the methods for detecting or quantifying 3-hydroxylated fatty acids (3-OH HFA), to (ii) the methods for detecting or quantifying lipopolysaccharides (LPS), (iii) the methods for diagnosing or prognosing a disorder in a subject, and (iv) the methods for detecting or quantifying LPS toxicity.

[0140] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound (z.e. esterified and / or amide-linked) form of 3-OH HFA and of at least one free (z.e. unesterified and / or non-amide linked) form of said 3-OH HFA selected from the group consisting of: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-30H), 3-hydroxy-decanoic acid (10:0-30H), 3-hydroxy-undecanoic acid (l l:0-3OH), 3- hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3- hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3- hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3- hydroxy-octadecanoic acid (18:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy-heneicosanoic acid (22:0-3OH), 3-hydroxy-tricosanoic acid (23:0-3OH), 3- hydroxy-tetracosanoic acid (24:0-3OH), and combinations thereof.

[0141] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-OH HFA and of at least one free form of said 3-OH HFA selected from the group consisting of: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy-tricosanoic acid (23:0-3OH), and combinations thereof.

[0142] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-heptadecanoic acid (7:0- 3OH) and of at least one free form of 3-hydroxy-heptadecanoic acid (7:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-octadecanoic acid (8:0-3OH) and of at least one free form of 3-hydroxy-octadecanoic acid (8:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-nonadecanoic acid (9:0-3OH) and of at least one free form of 3-hydroxy-nonadecanoic acid (9:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-decanoic acid (10:0-3OH) and of at least one free form of 3-hydroxy- decanoic acid (10:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy- undecanoic acid (l l:0-3OH) and of at least one free form of 3-hydroxy-undecanoic acid(l l:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-dodecanoic acid (12:0-3OH) and of at least one free form of 3-hydroxy-dodecanoic acid (12:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-tridecanoic acid (13:0-3OH) and of at least one free form of 3-hydroxy-tridecanoic acid (13:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-tetradecanoic acid, (14:0-3OH) and of at least one free form of 3-hydroxy-tetradecanoic acid, (14:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-pentadecanoic acid (15:0-3OH) and of at least one free form of 3-hydroxy-pentadecanoic acid (15:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-hexadecanoic acid (16:0-3OH) and of at least one free form of 3-hydroxy-hexadecanoic acid (16:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy -heptadecanoic acid (17:0-3OH) and of at least one free form of 3- hydroxy-heptadecanoic acid (17:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3- hydroxy-octadecanoic acid (18:0-3OH) and of at least one free form of 3-hydroxy- octadecanoic acid (18:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy- nonadecanoic acid (19:0-3OH) and of at least one free form of 3-hydroxy-nonadecanoic acid (19:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-eicosanoic acid (20:0-30H) and of at least one free form of 3-hydroxy-eicosanoic acid (20:0-30H). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-doeicosanoic acid (21:0-3OH) and of at least one free form of 3-hydroxy-doeicosanoic acid (21:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-heneicosanoic acid (22:0-3OH) and of at least one free form of 3-hydroxy-heneicosanoic acid (22:0-3OH). According to someembodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-tricosanoic acid (23:0-3OH) and of at least one free form of 3-hydroxy-tricosanoic acid (23:0-3OH). According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-hydroxy-tetracosanoic acid (24:0-3OH) and of at least one free form of 3- hydroxy-tetracosanoic acid (24: 0-3 OH).

[0143] According to such particular embodiments, the method may comprise a step of determining a level, or ratio thereof, of a single (one) bound form of the 3 -hydroxylated fatty acid and of a single (one) free form of the same 3 -hydroxylated fatty acid. Alternatively, the method may comprise a step of determining a level, or ratio thereof, of a plurality of bound forms of 3 -hydroxylated fatty acids and of a plurality of free forms of the same 3 -hydroxylated fatty acid.

[0144] According to some embodiments, a ratio between a plurality of bound forms of 3-OH fatty acids, and a plurality of free forms of said 3-OH fatty acids may consist of a ratio between the sum of said 3-OH fatty acids in a bound form, and of the same said 3- OH fatty acids in a free form.

[0145] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy- heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy- nonadecanoic acid (9:0-3OH), 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy- undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy- tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy- pentadecanoic acid (15:0-3OH), 3 -hydroxy -hexadecanoic acid (16:0-3OH), 3-hydroxy- heptadecanoic acid (17:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH), 3-hydroxy- nonadecanoic acid (19:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3-hydroxy- doeicosanoic acid (21:0-3OH), 3-hydroxy-heneicosanoic acid (22:0-3OH), 3-hydroxy- tricosanoic acid (23:0-3OH), 3-hydroxy-tetracosanoic acid (24:0-3OH), and combinations thereof.

[0146] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy -heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3- hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy- dodecanoic acid (12:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy- tetradecanoic acid, (14:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy- hexadecanoic acid (16:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy- octadecanoic acid (18:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy- eicosanoic acid (20:0-30H), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy- heneicosanoic acid (22:0-3OH), 3-hydroxy-tricosanoic acid (23:0-3OH), 3-hydroxy- tetracosanoic acid (24:0-3OH).

[0147] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-OH HFA and of at least one free form of said 3-OH HFA, selected from the group consisting of: 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), and combinations thereof.

[0148] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH).

[0149] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA, and a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy- heptadecanoic acid (7:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy- undecanoic acid (l l:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy- nonadecanoic acid (19:0-3OH), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy- tricosanoic acid (23:0-3OH).

[0150] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3- hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3- hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3- hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy-tricosanoic acid (23:0-3OH).

[0151] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of at least one bound form of 3-OH HFA and of at least one free form of said 3-OH HFA, selected from the group consisting of: 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-dodecanoic acid (12:0- 3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0- 3OH), 3-hydroxy-octadecanoic acid (18:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3-hydroxy-heneicosanoic acid (22: 0-3 OH), 3-hydroxy-tetracosanoic acid (24: 0-3 OH), and combinations thereof.

[0152] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and of a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy- octadecanoic acid (8:0-3OH), 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy- dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy- hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH), 3-hydroxy- eicosanoic acid (20: 0-3 OH), 3-hydroxy-heneicosanoic acid (22: 0-3 OH), 3-hydroxy- tetracosanoic acid (24:0-3OH).

[0153] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy-octadecanoic acid (8:0-3OH),3-hydroxy-decanoic acid (10:0-30H), 3-hydroxy-dodecanoic acid (12:0-3OH), 3- hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3- hydroxy-octadecanoic acid (18:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3-hydroxy-heneicosanoic acid (22:0-3OH), 3-hydroxy-tetracosanoic acid (24:0-3OH).

[0154] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and of a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH).

[0155] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy-decanoic acid (10:0-3OH), 3- hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3- hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH).

[0156] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and of a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH).

[0157] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy-decanoic acid (10:0-3OH), 3- hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH).

[0158] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and of a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy- dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy- hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH).

[0159] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and of a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy- dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy- hexadecanoic acid (16:0-3OH).

[0160] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH).

[0161] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and of a plurality of free forms of said 3-OH HFA, selected from the group consisting of: 3-hydroxy- tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy- octadecanoic acid (18:0-3OH).

[0162] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of a plurality of bound forms of 3-OH HFA and a plurality of free forms of said 3-OH HFA, comprising at least: 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH).

[0163] According to some embodiments, the method comprises a step of determining a level, or ratio thereof, of branched 3-OH fatty acids, including iso-3-hydroxylated fatty acids and anteiso-3-hydroxylated fatty acids. According to such embodiments, the method may comprise a step of determining a level, or ratio thereof, of one or more branched 3-OH fatty acids, for example of bound and free forms of 3-OH fatty acids, of which at least one is a branched 3-OH fatty acid.

[0164] According to some embodiments, the step of determining (e.g. detecting or quantifying) a level or ratio thereof of a given 3-OH HFA can be achieved by comparing the detected level to an internal reference, which may consist of an internal standard compound, such as a labeled fatty acid reference compound (e.g. a labeled bound fatty acid or a labeled free fatty acid), in particular a labeled 3-OH fatty acid referencecompound. Examples of internal standard compounds may include isotopically labeled reference compounds, and / or Stable Isotopically Labeled (SIL) reference compounds.

[0165] Internal standard compounds suitable for the present detection and quantification methods, including those applied to the diagnosis or prognosis of disorder, may consist of any compound(s) which may serve as a reference for determining an amount of a give species, or combination thereof. Such internal standard compounds may, for example, comprise or consist of one or more fatty acid reference compounds, or fragments thereof, and / or any compound susceptible to bind to the said species (e.g. the fatty acid(s) to be detected / quantified or a fragment thereof), such as an antigen-binding domain and / or an antibody.

[0166] Accordingly, an internal reference may comprise or consist of one or more isotopically labeled 3-OH fatty acid reference compounds; for example SIL 3-OH fatty acid reference compounds.

[0167] According to some embodiments, the level of 3-OH HEA, or ratio thereof, is determined by mass spectrometry.

[0168] According to some embodiments, the level of 3-OH HEA, or ratio thereof, is determined by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS / MS), Gas Chromatography coupled to Mass Spectrometry (GC-MS), Capillary Electrophoresis MS (CE-MS), Ion Mobility (IM-MS); preferably LC-MS / MS.

[0169] Hence, according to some embodiments, the detection or quantification of one or more 3-OH HFAs of interest can be obtained by (i) determining in the sample a spectrometry signal attributable to said 3-OH HFA, and (ii) determining in the sample a spectrometry signal attributable to said internal reference compound (e.g. an isotopically labeled form of the 3-OH HFA); and then comparing the spectrometry signals determined at (i) and (ii), wherein the comparison is indicative of the presence or level of the one or more 3-OH HFAs in the sample.

[0170] According to a particular embodiment, the invention relates to an in vitro method for diagnosing or prognosing an inflammatory disorder in a subject, in particular arthritis, said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy -heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy-decanoic acid (10:0- 3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3- hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3- hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH), 3- hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3- hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy-heneicosanoic acid (22:0-3OH), 3- hydroxy-tricosanoic acid (23:0-3OH), 3-hydroxy-tetracosanoic acid (24:0-3OH) ; b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0171] According to a particular embodiment, the invention relates to an in vitro method for diagnosing or prognosing an inflammatory disorder in a subject, in particular arthritis, said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-dodecanoic acid (12:0- 3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0- 3OH), 3-hydroxy-octadecanoic acid (18:0-3OH); b) comparing the ratio determined at step a) with a reference value,c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0172] According to a particular embodiment, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, in particular a disorder selected from the group consisting of: an inflammatory disorder, an autoimmune or inflammatory bowel disease, a neurological disorder; said method comprising steps of: a) determining a ratio between one or more bound form(s) of 3 hydroxylated fatty acid(s) and one or more free form(s) of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (11:0- 3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0- 3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0- 3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0173] According to a particular embodiment, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, in particular a disorder selected from the group consisting of: an inflammatory disorder, an autoimmune or inflammatory bowel disease, a neurological disorder; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free forms of the 3 hydroxylated fatty acids in a sample from the subject, wherein the 3 hydroxylated fatty acids are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (11:0- 3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0- 3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0- 3OH); b) comparing the ratio determined at step a) with a reference value,c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0174] According to particular embodiments, the disorder is selected from the group consisting of: arthritis, Crohn’s disease, Ulcerative Colitis, Multiple Sclerosis.

[0175] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being an autoimmune or inflammatory bowel disease, in particular Crohn’s disease or Ulcerative Colitis; said method comprising steps of: a) determining a ratio between one or more of bound form(s) of 3 hydroxylated fatty acid(s) and one or more free form(s) of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0176] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being an autoimmune or inflammatory bowel disease, in particular Crohn’s disease or Ulcerative Colitis; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (11:0- 3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0- 3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0177] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being Crohn’s disease; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (11:0- 3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0- 3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0178] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being Crohn’s disease; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH); b) comparing the ratio determined at step a) with a reference value,c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0179] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being Ulcerative Colitis; said method comprising steps of: a) determining a ratio between one or more bound forms of 3 hydroxylated fatty acid(s) and one or more free form(s) of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0180] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being Ulcerative Colitis; said method comprising steps of: a) determining a ratio between one or more bound forms of 3 hydroxylated fatty acid(s) and one or more free form(s) of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) is or comprises 3-hydroxy- tetradecanoic acid, (14:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0181] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being Ulcerative Colitis; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0182] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being Ulcerative Colitis; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-undecanoic acid (1 l:0-3OH), 3-hydroxy-dodecanoic acid (12:0- 3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0183] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being a neurological disorder, in particular being Multiple Sclerosis; said method comprising steps of:a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (11:0- 3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0- 3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0- 3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0184] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being a neurological disorder, in particular being Multiple Sclerosis; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a sample from the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0185] According to particular embodiments, the invention relates to an in vitro method for diagnosing or prognosing a disorder in a subject, the disorder being a neurological disorder, in particular being Multiple Sclerosis; said method comprising steps of: a) determining a ratio between a plurality of bound forms of 3 hydroxylated fatty acid(s) and a plurality of free form of the 3 hydroxylated fatty acid(s) in a samplefrom the subject, wherein the 3 hydroxylated fatty acid(s) are selected from the group consisting of: 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH); b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

[0186] According to another embodiment, the invention relates to kits for implementing the above-mentioned methods, optionally comprising instructions for carrying out said methods. Hence, the invention also relates to kits comprising one or more of the above- mentioned 3 -hydroxylated fatty acids; in particular in the form of isotopically labeled (e.g. stable isotopically labeled) 3 -hydroxylated fatty acid internal standards.

[0187] In particular, the kits for implementing the above-mentioned methods may comprise a plurality of isotopically labeled 3 -hydroxylated fatty acids; such as isotopically labeled even-chain 3 -hydroxylated fatty acids and / or odd-chain 3- hydroxylated fatty acids.

[0188] In particular, the kits for implementing the above-mentioned methods may comprise a plurality of 3 -hydroxylated fatty acids selected from the group consisting of: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3- hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy-decanoic acid (10:0-3OH), 3- hy droxy -undec anoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3- hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3- hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3- hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH), 3- hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3- hydroxy-doeicos anoic acid (21:0-3OH), 3-hydroxy-heneicosanoic acid (22:0-3OH), 3- hydroxy-tricosanoic acid (23:0-3OH), 3-hydroxy-tetracosanoic acid (24:0-3OH).

[0189] In particular, the kits for implementing the above-mentioned methods may comprise a plurality of isotopically labeled 3 -hydroxylated fatty acids selected from thegroup consisting of: 3-hydroxy -heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0- 3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0- 3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-hexadecanoic acid (16:0- 3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy-octadecanoic acid (18:0- 3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-eicosanoic acid (20:0- 3OH), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy-heneicosanoic acid (22:0- 3OH), 3-hydroxy-tricosanoic acid (23:0-3OH), 3-hydroxy-tetracosanoic acid (24:0- 3OH).

[0190] For example, the kits for implementing the above-mentioned methods may comprise a plurality of 3 -hydroxylated fatty acids, for example isotopically labeled 3- hydroxylated fatty acids, for example stable isotopically labeled 3-hydroxylated fatty acids, the said plurality consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy- dodecanoic acid (12:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy- hexadecanoic acid (16:0-3OH), 3-hydroxy-octadecanoic acid (18:0-3OH).

[0191] For example, the kits for implementing the above-mentioned methods may comprise a plurality of 3-hydroxylated fatty acids, consisting of: 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0- 3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-hexadecanoic acid (16:0- 3OH), 3-hydroxy-octadecanoic acid (18:0-3OH); either in branched or linear forms.

[0192] According to particular embodiments, the kits for implementing the above- mentioned methods, for example for determining the occurrence of a disorder from a biological sample of a patient, may comprise, or consist of: means for determining a level of at least one bound form of 3-hydroxylated fatty acid(s) in a sample; and means for determining a level of at least one free form of said 3-hydroxylated fatty acid(s) in a sample.

[0193] For example, such kits may comprise a first part comprising or consisting of means suitable for determining a level of at least one bound form of 3 -hydroxylated fatty acid(s); and a second part comprising or consisting of means suitable for determining a level of at least one free form of 3 -hydroxylated fatty acid(s).

[0194] Such means may comprise or consist of standards, and / or isotopic standards. Means suitable for determining a bound form of 3 -hydroxylated fatty acid(s) in a sample may comprise or consist of lipopolysaccharides (LPS) or substituted lipid A molecules, in particular substituted lipid A molecules characterized in that at least one bisphosphorylated glucosamine disaccharide is substituted by an ester- and / or amide-linked 3 -hydroxylated fatty acids structure, as internal standard.

[0195] Comparatively, means suitable for determining a level of at least one free form of said 3 -hydroxylated fatty acid(s) in a sample may comprise or consist of corresponding 3 -hydroxylated fatty acid(s) which are not ester- and / or amide-linked to a corresponding LPS or substituted lipid A molecule as defined previously.

[0196] Advantageously, kits according to the invention may further comprise a note of instructions for applying the above-mentioned methods, such as calibration curve and / or a list of defined steps.

[0197] Hence, according to a particular embodiment, the invention relates to a kit for the in vitro diagnosis or prognosis of a disorder, and / or for detecting or quantifying 3 -hydroxylated fatty acids (3-OH- HFA) or lipopolysaccharides or lipopolysaccharides toxicity, comprising: means for determining a level of at least one bound form of 3 -hydroxylated fatty acid(s) in a sample; and means for determining a level of at least one free form of said 3 -hydroxylated fatty acid(s) in a sample.

[0198] According to a particular embodiment, the kit may comprise:a first part comprising lipopolysaccharides (LPS) or substituted lipid A molecules, characterized in that at least one bis-phosphorylated glucosamine disaccharide is substituted by ester- and / or amide-linked 3-hydroxylated fatty acid(s); a second part comprising one or more of the 3-hydroxylated fatty acid(s) in free form.

[0199] In particular, the 3-hydroxylated fatty acids which may be part of said kits can thus be selected from the group consisting of: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3- hydroxy-decanoic acid (10:0-3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy- dodecanoic acid (12:0-3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy- tetradecanoic acid, (14:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy- hexadecanoic acid (16:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy- octadecanoic acid (18:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy- eicosanoic acid (20:0-30H), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy- heneicosanoic acid (22:0-3OH), 3-hydroxy-tricosanoic acid (23:0-3OH), 3-hydroxy- tetracosanoic acid (24:0-3OH); in bound and free form(s).BRIEF DESCRIPTION OF THE DRAWINGS

[0200] Figure 1. A modulation of the bound vs. free 3-OH fatty acid ratio is indicative of the occurrence of arthritis. 1A. Bound / free ratio of a combination of five different 3-OH HF s in healthy vs. arthritic patients. The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of five different 3-OH HFAs, consisting of ClO-chain, C12-chain, C14-chain, C-16 chain and C-18 chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with arthritis. IB. Bound / free ratio of a combination of three different 3-OH HF s in healthy vs. arthritic patients. The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of three different 3-OH HFAs, consisting of ClO-chain, C12-chain, C14-chain, 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with arthritis. . 1C. Bound / free ratio of acombination of two different 3-OH HFAs in healthy vs. arthritic patients. The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of three different 3-OH HFAs, consisting of C12-chain, C14-chain, C-16 chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with arthritis.

[0201] Figure 2. A modulation of the bound vs. free 3-OH fatty acid ratio is indicative of the occurrence of Crohn’s disease. 2A. Bound / free ratio of a combination of four different 3-OH HFAs in healthy vs. Crohn patients The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of four different 3- OH HFAs, consisting of C12-chain, C14-chain, C-16 chain and C-l l chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with Crohn’s Disease. 2B. Bound / free ratio of a combination of two different 3-OH HFAs in healthy vs. Crohn patients The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of two different 3-OH HFAs, consisting of Cl l- chain and C-16 chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with Crohn’s Disease.

[0202] Figure 3. A modulation of the bound vs. free 3-OH fatty acid ratio is indicative of the occurrence of Ulcerative Colitis (UC). 3A. Bound / free ratio of a combination of four different 3-OH HFAs in healthy vs. Ulcerative Colitis patients The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of four different 3-OH HFAs, consisting of C12-chain, C14-chain, C-16 chain and C-18 chain 3- OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y- axis) for patients with UC Disease. 3B. Bound / free ratio of a combination of three different 3-OH HFAs in healthy vs. Ulcerative Colitis patients The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of three different 3- OH HFAs, consisting of Cl l-chain, C12-chain and C14-chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with UC Disease. 3C. Bound / free ratio of a single 3-OH HFAs in healthy vs. Ulcerative Colitis patients The left panel represents the bound / free ratio (in y-axis)corresponding to C14-chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with UC Disease.

[0203] Figure 4. A modulation of the bound vs. free 3-OH fatty acid ratio is indicative of the occurrence of Multiple Sclerosis (MS). 4A. Bound / free ratio of a combination of four different 3-OH HFAs in healthy vs. MS patients The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of four different 3- OH HFAs, consisting of C12-chain, C14-chain, C-16 chain and C-18 chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with MS Disease. 4B. Bound / free ratio of a combination of two different 3- OH HFAs in healthy vs. MS patients The left panel represents the bound / free ratio (in y-axis) corresponding to the sum of two different 3-OH HFAs, consisting of C14-chain and C-16 chain 3-OH fatty acids, for healthy patients. The right panel represents the bound / free ratio (in y-axis) for patients with MS Disease.EXAMPLES

[0204] The present invention is further illustrated by the following examples.Example 1Materials and MethodsStandards

[0205] The standards consist of 3 -hydroxylated fatty acids, including 3-hydroxy- heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0-3OH), 3-hydroxy- nonadecanoic acid (9:0-3OH), 3-hydroxy-decanoic acid (10:0-3OH), 3-hydroxy- undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0-3OH), 3 hydroxytridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3 hydroxypentadecanoic acid (15:0-3OH), 3 -hydroxy -hexadecanoic acid (16:0-3OH), 3 hydroxyheptadecanoic acid (17:0-3OH), 3 hydroxy-octadecanoic acid (18:0-3OH), 3 hydroxynonadecanoic acid (19:0-3OH), 3 hydroxy-eicosanoic acid (20:0-30H), 3 hydroxy-doeicosanoic acid (21:0-3OH), 3 hydroxy-heneicosanoic acid (22:0-3OH), 3-hydroxy- tricosanoic acid (23:0-3OH), 3 hydroxy-tetracosanoic acid (24:0-3OH).Quantification of 3-hydroxylated fatty acids by LC-MS / MSUnesterified and non-Amide-linked 3-hydroxylated fatty acids extraction

[0206] Unesterified and non-Amide-linked 3-hydroxylated fatty acids in their free form are extracted with appropriate solvents such as hexane, chloroform, ethyl acetate (or a mixture of solvents) before evaporation. The method can be applied to complex samples including biological fluids (e.g. blood, synovial fluids, saliva, feces, urine, etc.) organs, tissues, from humans or animals.

[0207] A sample is divided into two parts. One part will be used to quantify the free 3- hydroxylated fatty acids while the other part will be used to quantify the total 3- hydroxylated fatty acids.

[0208] Before any 3-hydroxylated fatty acids extraction, the internal standard is mixed with the product to be analyzed. The internal standard consists of one or a mixture of 3- hydroxylated fatty acids.

[0209] A set of internal standards containing 3-hydroxylated fatty acids with an odd number of carbons (e.g. l l:0-3OH, 13:0-3OH; 15:0-3OH; 17:0-3OH) is used to quantify 3-hydroxylated fatty acids with an even number of carbons. On the contrary, 3- hydroxylated fatty acids with an even number of carbons (e.g. 8:0-3OH, 10:0-3OH; 12:0- 3OH; 14:0-3OH) are used to quantify 3-hydroxylated fatty acids with an odd number of carbons.

[0210] Alternatively, it is possible to use a 3-hydroxylated isotopic fatty acid as the sole internal standard.Total 3 -hydroxylated fatty acids extraction

[0211] In order to quantify free and bound (total) 3 -hydroxylated fatty acids together, a volume of fuming HC1 is added to reach a final concentration in the range of 5 to 8 M, preferably 6 M. The solution is heated preferably at 90 °C for 2 to 4 h.

[0212] In this way, 3 -hydroxylated fatty acids are released from LPSs and are extracted with appropriate solvents such as hexane, chloroform, ethyl acetate (or a mixture of solvents) before evaporation.Quantification of 3 -hydroxylated fatty acids by LC-MS / MS

[0213] The two extracts containing free and total 3 -hydroxylated fatty acids are solubilized in a mixture of 40 % ammonium acetate 5 mM, pH 5 and 60 % acetonitrile / ammonium acetate 5 mM, pH 7,3.

[0214] The solution is injected into an HPLC system. After chromatographic separation, hydroxylated fatty acids are detected with a mass spectrometer.

[0215] The quantification of bound 3 -hydroxylated fatty acids results from the subtraction of free 3 -hydroxylated fatty acids to the total 3 -hydroxylated fatty acids. The targeted 3 -hydroxylated fatty acid contents, from 8:0-3OH to 24:0-3OH, are estimated based on internal standards.Quantification of 3 -hydroxylated fatty acids by GC-MS

[0216] Similarly to the LC-MS2, free and total 3 -hydroxylated fatty acids are extracted. Then, extracted 3-hydroxylated fatty acids are derivatized to allow their volatility. For derivatization, 3-hydroxylated fatty acids can be methylated using, for example, methanolysis, diazomethane or trifluoroborane. Additionally, the 3-hydroxylated fatty acids can be derivatized with a chlorosilisane compound.Determination of the bound / free ratio of the 3 -hydroxylated fatty acids (3 -OH HF A)

[0217] Quantitative values of all observed 3 -hydroxylated fatty acids estimated thanks to the internal standard can be used to determine the bound / free ratio of the 3- hydroxylated fatty acids, also called esterified and amide-linked / unesterified and non- amide-linked ratio.

[0218] The values from several bound and free 3 -hydroxylated fatty acids can be summed before applying the ratio.

[0219] For instance, in patients suffering from arthritis, a bound / free ratio has been estimated by summing the values from 5 different 3 -hydroxylated fatty acids (HFA).

[0220] Values show a difference between healthy (ratio = 6.4) and arthritis patients (ratio = 11). It is not necessary to use the values from all HFA quantified. The number of HFA can be reduced.

[0221] Following the same example and disease, a ratio has been calculated with 3 different HFA or 2 different HFAs: values show a difference between healthy (ratio = 0.1 [3 HFA] or 0.6 [2 HFA]) and arthritis patients (ratio = 0.7 [3 HFA] or 1.3 [2 HFA]).

[0222] The lipidic moiety of the LPS, called Lipid A, is first separated from the LPS molecule by a mild acidic hydrolysis using acidic compounds. Methods for lipid A moiety extraction are known in the Art. See for example W02004 / 062690 Al.Results

[0223] Figure 1 demonstrates that the values from several bound and free 3 -hydroxylated fatty acids can be summed before applying the ratio bound / free. Overall, figures 1A, IB and 1C demonstrate that the said ratio is modulated in patients with an inflammatory disorder such as arthritis. In particular, it is shown herein that the ratio bound / free is increased in arthritis patients. When considering the sum of five different 3 -hydroxylatedfatty acids values, figure 1A shows a difference between healthy (ratio = 6.4) and arthritis patients (ratio = 11). The tested 3-OH fatty acids, as biomarkers in combination, correspond to ClO-chain, C12-chain, C14-chain, C-16 chain and C-18 chain 3-OH fatty acids.

[0224] It is not necessary to use the values from all 3-OH HFA quantified. The number of HFA can be reduced. Following the same example and disease, a ratio has been calculated with 3 different 3-OH HFA (consisting of CIO, C12 and C-143-OH fatty acids) or 2 different 3-OH HFAs (consisting of C12, C14 and C16-3-OH fatty acids), respectively, in figures IB and 1C. Values show a difference between healthy (ratio = 0.1 [3 HFA] or 0.6 [2 HFA]) and arthritis patients (ratio = 0.7 [3 HFA] or 1.3 [2 HFA]).

[0225] Overall, the determination of bound / free ratios is indicative of the health status of the tested population samples. Interestingly, the determination of such ratios based on a plurality of distinct 3-OH HFAs increases the predictability of the test.Example 2 - Application to the in vitro diagnosis of Ulcerative Colitis, Crohn’s Disease and Multiple Sclerosis.Materials and Methods

[0226] The Material & Method is as previously defined in Example 1. A 3 -hydroxylated isotopic fatty acid is used as the sole internal standard.

[0227] The tested samples were obtained from the following patient populations:

[0228] Table 1: tested samples for which ratios have been determined, and compared to a reference value.Results

[0229] Figures 2 to 4 demonstrate that the determination of a ratio from bound and free 3-OH hydroxylated fatty acid(s) in a serum sample is indicative of the occurrence of an inflammatory gut disorder such as Crohn’s disease of Ulcerative Colitis and of a neurological disorder with an inflammatory component such as Multiple Sclerosis. The in vitro detection of such disorders, from serum samples, is also possible when the ratio is calculated from a single 3-OH fatty acid species such as C4-long chain fatty acids.

[0230] Accordingly, the following values can be obtained, for each patient population, based on three distinct serum samples:

[0231] Table 2: comparative values for Crohn Disease (CD), Ulcerative Colitis (UC) and Multiple Sclerosis (MS), based on figures 2 to 4.

[0232] In view of the above, the following observations can be made. It is found that the modulation of the ratio between the sum of bound and the sum of free forms of 3-OH fatty acids is found in all the tested serum samples, which is thus evidence that this modulation can be considered as a biomarker for inflammatory gut disorders and neurological disorders associated to inflammation.

[0233] Also, it is noted that, for all the tested samples, the occurrence of the corresponding disorder is associated to an increase of the bound / free ratio when compared to samples pooled from healthy individuals. This observation applies not only to those ratios which are determined from a single 3-OH fatty acid species, but also to those which are determined from a plurality of 3-OH fatty acids, and for distinct selections of 3-OH fatty acids.

[0234] It can be further noted that the modulation of such ratios is further observed in the presence of a mixture of odd-chain and even-chain fatty acids, including branched 3- OH fatty acids such as 1 l:0-3OH iso and ante-iso fatty acids.

[0235] Overall those data confirm the in vitro use of such biomarkers for the detection of 3-OH fatty acids in biological samples.

Claims

CLAIMS1. An in vitro method for diagnosing or prognosing a disorder in a subject, said method comprising steps of: a) determining a ratio between at least one bound form of 3 -hydroxylated fatty acid(s) and at least one free form of the 3 -hydroxylated fatty acid(s) in a sample from the subject; b) comparing the ratio determined at step a) with a reference value, c) determining, from the comparison of step b), the diagnosis or prognosis of the disorder in said subject.

2. An in vitro method for detecting or quantifying 3 -hydroxylated fatty acids (3-OH- HFA) in a sample, said method comprising steps of: a) determining a level of at least one bound form of the 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said 3 -hydroxylated fatty acids (3-OH HFA) in the sample; wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid.

3. An in vitro method for detecting or quantifying lipopolysaccharides (LPS) in a sample, said method comprising steps of: a) determining a level of at least one bound form of 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof, wherein the 3 -hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3 -hydroxylated fatty acid; b) detecting or quantifying, from the levels or ratio determined at step a), the occurrence of said lipopolysaccharides in the sample.

4. An in vitro method for detecting or quantifying lipopolysaccharides (LPS) toxicity in a sample, said method comprising steps of:a) determining a level of lipopolysaccharides (LPS) in a sample; b) detecting or quantifying LPS toxicity in the sample, from the level of at least one bound form of 3 -hydroxylated fatty acid(s) and of at least one free form of the 3 -hydroxylated fatty acid(s) in said sample, or a ratio thereof, and from the level of lipopolysaccharides in the sample determined at step a).

5. The method according to any one of the preceding claims, wherein step a) comprises determining a ratio between at least one bound form of the 3 -hydroxylated fatty acid(s) and at least one free form of the 3 -hydroxylated fatty acid(s) in said sample.

6. The method according to any of the preceding claims, wherein the 3-hydroxylated fatty acids (3-OH HFA) comprise or consist of at least one odd-chain 3-hydroxylated fatty acid and at least one even-chain 3-hydroxylated fatty acid.

7. The method according to any of the preceding claims, wherein the sample is a biological sample selected from the group consisting of: blood, plasma, serum, cerebrospinal fluid, saliva, tears, lymph, ascetic fluid, cystic fluid, urine, bile, nipple exudate, vomitus, breast milk, wound drainage, feces, vaginal secretions, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, pleural fluid, pericardial fluid, semen, sweat, alveolar macrophages, mucus, pus, dental plaque, bone marrow, aqueous humour, perilymph, gastric acid, pancreatic juice, rheum, serous fluid, earwax, rumen.

8. The method according claim 1, wherein an increase of a ratio of at least one bound form of 3-hydroxylated fatty acid(s) to at least one free form of the 3-hydroxylated fatty acid(s), compared to a reference value, is indicative of the occurrence of said disorder.

9. The method according to claim 1, wherein the disorder is an inflammatory disorder and / or an auto-immune disorder and / or a proliferative disorder and / or a neurological disorder; in particular selected from the group consisting of: Crohn’s Disease, Ulcerative Colitis, Multiple Sclerosis, arthritis, cancer and diabetes.

10. The method according to claim 9, wherein the disorder is an inflammatory disorder; in particular selected from arthritis.

11. The method according to any one of claims 1 to 10, which comprises a step of determining a ratio between a plurality of bound forms of 3 -hydroxylated fatty acids (3-OH HFA) and a plurality of free forms of 3 -hydroxylated fatty acids (3-OH HFA) in said sample.

12. The method according to any one of claims 1 to 10, which comprises a step of determining a ratio between a plurality of bound forms of odd-chain 3 -hydroxylated fatty acids (3-OH HFA) and a plurality of free forms of odd-chain 3 -hydroxylated fatty acids (3-OH HFA) in said sample.

13. The method according to any one of claims 1 to 12, wherein the bound and free 3- OH HFA comprise at least one 3-OH HFA selected from the group consisting of: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-octadecanoic acid (8:0- 3OH), 3-hydroxy-nonadecanoic acid (9:0-3OH), 3-hydroxy-decanoic acid (10:0- 3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-dodecanoic acid (12:0- 3OH), 3-hydroxy-tridecanoic acid (13:0-3OH), 3-hydroxy-tetradecanoic acid, (14:0-3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-hexadecanoic acid (16:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy- octadecanoic acid (18:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-eicosanoic acid (20:0-30H), 3-hydroxy-doeicosanoic acid (21:0-3OH), 3-hydroxy-heneicosanoic acid (22:0-3OH), 3-hydroxy-tricosanoic acid (23:0- 3OH), 3-hydroxy-tetracosanoic acid (24:0-3OH), and combinations thereof.

14. The method according to any one of claims 1 to 13, wherein the bound and free 3- OH HFA comprise at least one 3-OH HFA selected from the group consisting of: 3-hydroxy-heptadecanoic acid (7:0-3OH), 3-hydroxy-nonadecanoic acid (9:0- 3OH), 3-hydroxy-undecanoic acid (l l:0-3OH), 3-hydroxy-tridecanoic acid (13:0- 3OH), 3-hydroxy-pentadecanoic acid (15:0-3OH), 3-hydroxy-heptadecanoic acid (17:0-3OH), 3-hydroxy-nonadecanoic acid (19:0-3OH), 3-hydroxy-doeicosanoicacid (21:0-3OH), 3-hydroxy-tricosanoic acid (23:0-3OH), and combinations thereof.

15. The method according to any one of claims 1 to 14, wherein a level of 3-OH HFA, or ratio thereof, is determined by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS / MS), or Gas Chromatography coupled to Mass Spectrometry (GC-MS), Capillary Electrophoresis MS (CE-MS), Ion Mobility (IM-MS); preferably LC-MS / MS.

16. A kit for the in vitro diagnosis or prognosis of a disorder, and / or for detecting or quantifying 3 -hydroxylated fatty acids (3-OH- HFA) or lipopolysaccharides or lipopolysaccharides toxicity, comprising: means for determining a level of at least one bound form of 3 -hydroxylated fatty acid(s) in a sample; and means for determining a level of at least one free form of said 3-hydroxylated fatty acid(s) in a sample.

17. The kit according to the preceding claim, comprising: a first part comprising lipopolysaccharides (LPS) or substituted lipid A molecules, characterized in that at least one bis-phosphorylated glucosamine disaccharide is substituted by ester- and / or amide-linked 3- hydroxylated fatty acid(s); a second part comprising one or more of the 3-hydroxylated fatty acid(s) in free form.

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