Prediction of retinal omega-3 polyunsaturated fatty acid content through the measurement of 7 cholesterol ester molecules

Abstract

The present invention relates to a method for determining the omega-3 polyunsaturated fatty acid content of the retina of a subject, comprising determining the content of at least one cholesterol ester in a blood sample of said subject, the omega-3 polyunsaturated fatty acid content of the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate

Description

Title of the invention: Prediction of the content of omega-3 polyunsaturated fatty acids in the retina through the dosage of 7 cholesterol ester molecules Technical field

[0001] The invention relates to the field of ophthalmology and more practically to the prediction of the content of omega-3 polyunsaturated fatty acids in the retina. Prior art

[0002] Age-related macular degeneration (AMD) is a degenerative disease of the central part of the retina, called the macula. It mainly affects older people and is responsible for 50% of blindness in industrialized countries. Approximately 600,000 people are affected by the disease in France. Advanced forms of AMD (called neovascular AMD and atrophic AMD) are associated with vision loss and are usually preceded by early, asymptomatic abnormalities.

[0003] The etiology of AMD is multifactorial, and involves genetic but also environmental factors such as diet. In this context, the role of omega-3 polyunsaturated fatty acids or omega-3 is attracting increasing interest for 3 reasons: - omega-3 polyunsaturated fatty acids, and in particular docosahexaenoic acid (DHA), are major constituents of the retina where they prevent certain pathological processes observed in AMD such as inflammation, neovascularization and cell death; - studies have shown a depletion of the content of omega-3 polyunsaturated fatty acids in the retina of subjects with AMD; - more than 20 epidemiological studies have consistently shown a 40% reduction in the risk of AMD in subjects with a high dietary intake of omega-3 polyunsaturated fatty acids. These data therefore suggest a protective effect of omega-3 polyunsaturated fatty acids against AMD.

[0004] The family of omega-3 polyunsaturated fatty acids includes in particular a precursor fatty acid, α-linoleic acid (or C18:3n-3 or ALA) and three long-chain derivatives, eicosapentaenoic acid (or C20:5n-3 or EPA), docosapentaenoic acid (or C22:5n-3 or DPAn-3) and docosahexaenoic acid (DHA or C22:6n-3). ALA is an essential nutrient for humans, because animal organisms are unable to synthesize it de novo, and therefore depend exclusively on dietary intake. The synthesis of EPA, DPAn-3 and DHA from ALA is possible in humans but very limited. It is therefore recommended to also provide these fatty acids through a diet rich in fish and seafood. Despite these recommendations, the dietary intake of omega-3 polyunsaturated fatty acids in France remains below the recommendations issued by health agencies.

[0005] Knowing the concentrations of omega-3 polyunsaturated fatty acids in the retina therefore appears to be important information for: 1. identifying subjects whose retina is depleted and who could therefore be at risk of AMD, and 2. monitoring the effectiveness of nutritional supplementation based on omega-3 polyunsaturated fatty acids in restoring the physiological levels of these fatty acids in the retina.

[0006] For obvious ethical reasons, however, it is impossible to perform retinal biopsies in vivo in humans for the purpose of biochemical determination of its omega-3 polyunsaturated fatty acid content. Therefore, an indirect measurement is necessary.

[0007] Three studies have attempted to correlate circulating fatty acid content with that of the retina. A first study was limited to investigating associations between retinal omega-3 polyunsaturated fatty acid content and blood levels of various fatty acids, without attempting to predict the omega-3 polyunsaturated fatty acid content of the retina, nor investigating the lipid structures that carry fatty acids in the blood (phospholipids, cholesterol esters, triglycerides, etc.) (Gorusupudi et al. J Lipid Res 2016;57:499-508). The second identified cholesterol esters as a whole as circulating markers of retinal DHA content (Bretillon et al. Exp Eye Res 2008;87:521-528). The third showed that erythrocyte DHA content was not a good circulating marker of retinal DHA content (Acar et al. PLoS ONE 2012;7:e35102).

[0008] None of these studies considered the overall omega-3 polyunsaturated fatty acid content of the retina (they targeted DHA or omega-6 fatty acid / omega-3 fatty acid concentration ratios).

[0009] They also did not allow the construction of a model for estimating the true content of omega-3 polyunsaturated fatty acids as it could be measured through direct analysis.

[0010] To date, two approaches are commonly used to indirectly assess the content of omega-3 polyunsaturated fatty acids in the retina. The first is based on dietary intakes which, through dietary questionnaires, allow a subject to assess the consumption of a particular food rich in omega-3 polyunsaturated fatty acids and to extrapolate this to tissue enrichment. The second consists of an assessment of circulating omega-3 polyunsaturated fatty acid levels and, again, to extrapolate blood enrichment to tissue enrichment. This assessment in the blood compartment can be more or less global and concern whole blood, erythrocytes, whole plasma or different compartments of plasma (phospholipids and cholesterol esters in the majority).

[0011] The use of dietary intakes faces a large number of difficulties, and in particular those linked to dietary assessment methods: length and complexity of dietary questionnaires, memory bias, difficulties in taking into account the great complexity and daily variability of the human diet, inaccuracies in estimating the quantities of food ingested and in the nutrient content of foods, etc.

[0012] Concerning the use of the lipid composition of plasma / of the different compartments of plasma, this is based on the assumption that the fatty acid composition of peripheral tissues (including the retina) follows the same pattern as that of plasma. However, it is known that the relationship between dietary lipids, plasma lipids and tissue lipids is non-linear because it is modulated by several other factors such as metabolism, genetics, age, sex or lifestyle (smoking, alcohol consumption, physical activity). To date, no study has shown the absolute validity or not of this or that compartment in the estimation of the fatty acid content of the retina, apart from the two preliminary studies previously cited showing the invalidity of the use of erythrocytes in adult human subjects and on the contrary the interest of cholesterol esters as a whole. Technical problem

[0013] Thus, and despite major medical interest, it is currently impossible to reliably measure the content of omega-3 polyunsaturated fatty acids in the retina. It is therefore necessary to have a tool for evaluating omega-3 polyunsaturated fatty acids in the retina.

[0014] Advantageously, the invention makes it possible to estimate the content of omega-3 polyunsaturated fatty acids in the retina through the blood measurement of at least one cholesterol ester.

[0015] The inventors have, by refining the use of cholesterol esters as a marker of the content of omega 3 polyunsaturated fatty acids in the retina, used dosage values ​​of one cholesterol ester, preferably seven cholesterol esters, instead of the 25 present in the blood.

[0016] Indeed, the construction and use of a mathematical model advantageously makes it possible to predict the concentration of omega-3 polyunsaturated fatty acids in the retina from a dosage value of a cholesterol ester, preferably 7 cholesterol esters in the blood.

[0017] Thus, the subject of the present invention is a method for determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject comprising the quantification of the content of at least one cholesterol ester in a blood sample of said subject, the content of omega-3 polyunsaturated fatty acids in the retina being correlated with the content of cholesterol esters.

[0018] The invention also relates to a method for preventing a retinal pathology linked to a deficiency in omega-3 polyunsaturated fatty acids by determining the content of omega-3 polyunsaturated fatty acids in the retina according to the determination method according to the invention.

[0019] The invention also relates to a method for monitoring treatment of a pathology linked to a deficiency in omega-3 polyunsaturated fatty acids comprising the steps of administering at least one treatment and determining the content of omega-3 polyunsaturated fatty acids in the retina according to the determination method of the invention.

[0020] The invention also relates to a method for diagnosing omega-3 polyunsaturated fatty acid deficiencies comprising determining the omega-3 polyunsaturated fatty acid content of the retina according to the determination method of the invention. Disclosure of the invention

[0021] Method for determining the content of omega-3 polyunsaturated fatty acids

[0022] The present invention relates to a method for determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject comprising determining the content of at least one cholesterol ester in a blood sample of said subject, the content of omega-3 polyunsaturated fatty acids in the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate.

[0023] Indeed, and advantageously, the inventors of the present invention have demonstrated that the determination of the content of cholesteryl 5,8,11,14,17-eicosapentaenoate (C20:5 co3 methyl ester), preferentially contained in the plasma, makes it possible to predict the content of omega-3 polyunsaturated fatty acids in the retina of a subject.

[0024] The term "omega-3 polyunsaturated fatty acid content of the retina" means the sum in weight percentage of the total fatty acids of all the omega-3 polyunsaturated fatty acids present in the retina, namely, the sum in weight percentage of the total fatty acids of α-linoleic acid (or C18:3n-3 or ALA), eicosapentaenoic acid (or C20:5 co-3 or EPA), docosapentaenoic acid (or C22:5 co-3 or DPA co-3) and docosahexaenoic acid (DHA or C22:6 co-3).

[0025] According to one embodiment of the determination method, the content of at least six additional cholesterol esters is determined.

[0026] The cholesterol esters are chosen from cholesteryl tetradecanoate, cholesteryl pentadecanoate, cholesteryl hexadecanoate, cholesteryl 7-hexadecenoate, le cholesteryl 9-hexadecenoate, le cholesteryl heptadecanoate, le cholesteryl octa-decanoate, le cholesteryl trans-9-octadecenoate, le cholesteryl cis-9-octadecenoate, le cholesteryl 11-octadecenoate, le cholesteryl 9,12-octadecadienoate, le cholesteryl ei-cosanoate, le cholesteryl 6,9,12-octadecatrienoate, le cholesteryl 11-eicosenoate, le cholesteryl 9,12,15-octadecatrienoate, le cholesteryl 11,14-eicosadienoate, le cholesteryl 5,8,11-eicosatrienoate, le cholesteryl 8,11,14-eicosatrienoate, le cholesteryl 5,8,11,14-eicosatetraenoate,, le cholesteryl 15-tetracosenoate, le cholesteryl 7,10,13,16-docosatetraenoate, le cholesteryl 4,7,10,13,16-docosapentaenoate, le cholesteryl 7,10,13,16,19-docosapentaenoate, et le cholesteryl 4,7,10,13,16,19-docosahexaenoate.

[0027] La nomenclature des esters de cholestérol est détaillée dans le tableau 1.

[0028] [Tableaux [tableau 1]] nomenclature biologistes nomenclature chimistes 1 C14:0 cholesteryl ester cholesteryl tetradecanoate 2 C15:0 cholesteryl ester cholesteryl pentadecanoate 3 C16:0 cholesteryl ester cholesteryl hexadecanoate 4 Cl6:1 co-9 cholesteryl ester cholesteryl 7-hexadecenoate 5 C16:l co-7 cholesteryl ester cholesteryl 9-hexadecenoate 6 C17:0 cholesteryl ester cholesteryl heptadecanoate 7 Cl8:0 cholesteryl ester cholesteryl octadecanoate 8 Cl8:1 co-9 trans cholesteryl ester cholesteryl trans-9-octadecenoate 9 Cl8:1 co-9 cholesteryl ester cholesteryl cis-9-octadecenoate 10 Cl8:1 co-7 cholesteryl ester cholesteryl 11-octadecenoate 11 Cl8:2 co-6 cholesteryl ester cholesteryl 9,12-octadecadienoate 12 C20:0 cholesteryl ester cholesteryl eicosanoate 13 Cl8:3 co-6 cholesteryl ester cholesteryl 6,9,12-octadecatrienoate 14 C20:l co-9 cholesteryl ester cholesteryl 11-eicosenoate 15 Cl8:3 co-3 cholesteryl ester cholesteryl 9,12,15-octadecatrienoate 16 C20:2 co-6 cholesteryl ester cholesteryl 11,14-eicosadienoate 17 C20:3 co-9 cholesteryl ester cholesteryl 5,8,11-eicosatrienoate 18 C20:3 co-6 cholesteryl ester cholesteryl 8,11,14-eicosatrienoate 19 C20:4 co-6 cholesteryl ester cholesteryl 5,8,11,14-eicosatetraenoate 20 C20:5 co-3 cholesteryl ester cholesteryl 5,8,11,14,17-eicosapentaenoate 21 C24:l co-9 cholesteryl ester cholesteryl 15-tetracosenoate 22 C22:4 co-6 cholesteryl ester cholesteryl 7,10,13,16-docosatetraenoate 23 C22:5 co-6 cholesteryl ester cholesteryl 4,7,10,13,16-docosapentaenoate 24 C22:5 co-3 cholesteryl ester cholesteryl 7,10,13,16,19-docosapentaenoate 25 C22:6 co-3 cholesteryl ester cholesteryl 4,7,10,13,16,19-docosahexaenoate ,

[0029] Selon un mode de réalisation, les 6 esters de cholestérol supplémentaires sont le cho-lesteryl 9,12,15-octadecatrienoate, le cholesteryl 4,7,10,13,16,19-docosahexaenoate, le cholesteryl 9,12-octadecadienoate, le cholesteryl 6,9,12-octadecatrienoate, le cho- lesteryl 8,11,14-eicosatrienoate, cholesteryl 5,8,11,14-eicosatetraenoate.

[0030] Thus, and according to one embodiment, the content of 7 cholesterol esters is determined.

[0031] The inventors have in fact identified, in addition to cholesteryl 5,8,11,14,17-eicosapentaenoate, six additional cholesterol esters which are cholesteryl 9,12,15-octadecatrienoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate, cholesteryl 9,12-octadecadienoate, cholesteryl 6,9,12-octadecatrienoate, cholesteryl 8,11,14-eicosatrienoate, cholesteryl 5,8,11,14-eicosatetraenoate), among the twenty-five present in the blood that allow for a reliable and precise determination of the content of omega-3 polyunsaturated fatty acids in the retina.

[0032] The term “content of at least one cholesterol ester” means the weight percentage of at least one fatty acid in the cholesterol esters.

[0033] A person skilled in the art may use any known method for determining the cholesterol ester content.

[0034] According to one embodiment and to determine the cholesterol ester content, a method consists of a transesterification step.

[0035] The transesterification step corresponds to an acid catalysis in which the sterol part of the cholesterol ester is exchanged for an alkyl group. The alkyl group is chosen from methyl, propyl, or butyl.

[0036] According to one embodiment, the trans-esterification step is a step of transmethylation of the fatty acids of the cholesterol esters.

[0037] The cholesterol ester content can in fact be determined by the formation of methyl esters of the fatty acids of the cholesterol esters. The methyl esters of the cholesterol esters are obtained after trans-methylation of the fatty acids of the corresponding cholesterol ester.

[0038] Typically, the fatty acid methyl esters of cholesterol esters are formed after transmethylation of fatty acids according to the method of Morrison & Smith (Morrison & Smith J lipid Res 1964;53:600-608).

[0039] The methyl esters of the fatty acids of the cholesterol esters identified according to the present invention are 9,12,15-octadecatrienoic acid methyl ester (C18:3 co-3 methyl ester), 4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6 co-3 methyl ester), 9,12-octadecadienoic acid methyl ester (C18:2 co-6 methyl ester), 6,9,12-octadecatrienoic acid methyl ester (C18:3 co-6 methyl ester), 8,11,14-eicosatrienoic acid methyl ester (C20:3 co-6 methyl ester), 5,8,11,14-eicosatetraenoic acid methyl ester (C20:4 co-6 methyl ester), 5,8,11,14,17-eicosapentaenoate acid methyl ester (C20:5 co-3 methyl ester).

[0040] According to one embodiment, the relative weight content of each of the 25 met- esters The fatty acid content of cholesterol esters is determined.

[0041] The relative weight content of the methyl esters of the fatty acids is analyzed and determined by any method known to those skilled in the art, typically by gas chromatography coupled with flame ionization detection, as described in Acar et al. (Acar et al PLoS One 2012;7(4): e35102).

[0042] The relative proportions of the 25 methyl ester species are calculated and expressed as a percentage of the total cholesterol ester fatty acids. Typically, the proportions are calculated using any data integration software known to those skilled in the art, for example using EZChrom Elite software (Agilent technologies, Massy, ​​France) and are expressed as a percentage of the total cholesterol ester fatty acids.

[0043] This step makes it possible to determine the weight percentage of at least one fatty acid of the cholesterol esters and therefore to determine the content of each cholesterol ester

[0044] The term "correlation" means the calculation of the estimated value of the omega-3 polyunsaturated fatty acid content of the retina from the content of at least one cholesterol ester, preferably from the content of at least 7 cholesterol esters and preferably again from the content of 7 cholesterol esters.

[0045] Typically, the calculation is carried out by any algorithm which makes it possible to predict, for a new sample, the content of omega-3 polyunsaturated fatty acids in the retina from the dosage of at least 1 cholesterol ester, preferably from at least 7 cholesterol esters and preferably again from 7 cholesterol esters.

[0046] As an illustration, and for the determination of the omega-3 polyunsaturated fatty acid content of the retina from a cholesterol ester, the algorithm is based on a linear regression according to the formula:

[0047] [Math.l] (4.1717 x (C20 : 5 w3 EC) where C20:5 co3 EC corresponds to the cholesterol content 5,8,11,14,17-eicosapentaenoate, centered and reduced according to the distribution in the training sample (see Table 2 below).

[0048] Also for illustrative purposes, and for the determination of the omega-3 polyunsaturated fatty acid content of the retina from at least 7 cholesterol esters, the algorithm is based on the sparse group partial least squares (sgPLS) method described by Liquet et al. (Liquet B et al. Bioinformatics. 2016 Jan 1;32(1):35-42).

[0049] The algorithm comprises and preferably consists of 4 steps:

[0050] 1. Several sums and ratios are calculated from the dosage of at least seven esters cholesterol. The measurement of cholesterol ester in plasma is an indirect measurement. Cholesterol esters undergo transesterification which leads to fatty acid methyl esters. The fatty acid methyl esters thus obtained are quantified by known methods, for example by gas chromatography. The relative amount of a fatty acid methyl ester corresponds to a percentage of the total fatty acid methyl esters.

[0051] In one embodiment, the sums and ratios present in the mathematical formula reproduced below (math. 2), are calculated from the dosages of the 7 molecules of interest derived from the 25 fatty acid methyl esters of cholesterol esters.

[0052] The 7 molecules of interest from the 25 fatty acid methyl esters of cholesterol esters are as follows: 9,12,15-octadecatrienoic acid methyl ester (C18:3 co-3 methyl ester), 5,8,11,14,17-eicosapentaenoic acid methyl ester (C20:5 co-3 methyl ester), 4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6 co-3 methyl ester), 9,12-octadecatrienoic acid methyl ester (C18:2 co-6 methyl ester), 6,9,12-octadecatrienoic acid methyl ester (C18:3 co-6 methyl ester), 8,11,14-eicosatrienoic acid methyl ester (C20:3 co-6 methyl ester), 5,8,11,14-eicosatrienoic acid methyl ester (C20:4 co-6 methyl ester).

[0053] The mathematical formula is detailed below

[0054] [Math. 2]: (0.117939545 x C20:5co3_EC) + (0.000429538 x C22:6co3_EC) + (0.127303513 x Tot_co3_EC) - (0.089285375 x ratio_co6_co3_EC) + (0.12438627 x ratio_dietco3_co6_EC) + (0.095117859 x ratio_EPADPAco3DHA_AA_EC) -(0.086291782 x ratio_AA_EPA_EC) + (0.115467043 x tot_co3_LC_EC) in which: - C20:5co3_EC corresponds to the content of cholesteryl 5,8,11,14,17-eicosapentaenoate; - C22:6co3_EC corresponds to the content of cholesteryl 4,7,10,13,16,19-docosahexaenoate - Tot_co3_EC corresponds to the sum of the contents of cholesteryl 9,12,15-octadecatrienoate, cholesteryl 5,8,11,14,17-eicosapentaenoate cholesteryl 7,10,13,16,19-docosapentaenoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate - ratio_co6_co3_EC correspond au ratio de la somme des teneurs en cholesteryl 9,12-octadecadienoate, cholesteryl 6,9,12-octadecatrienoate, cholesteryl 11,14-eicosadienoate, cholesteryl 5,8,11,14-eicosatetraenoate, cholesteryl 7,10,13,16-docosatetraenoate, cholesteryl 4,7,10,13,16-docosapentaenoate sur la somme des teneurs en cholesteryl 9,12,15-octadecatrienoate, cholesteryl 5,8,11,14,17-eicosapentaenoate, cholesteryl 7,10,13,16,19-docosapentaenoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate - ratio_diet co3_co6_EC correspond au ratio de la somme des teneur en cholesteryl 9,12,15-octadecatrienoate, cholesteryl 5,8,11,14,17-eicosapentaenoate et cholesteryl 4,7,10,13,16,19-docosahexaenoate sur la teneur en cholesteryl 9,12-octadecadienoate - ratio_EPADPAco3DHA_AA_EC correspond au ratio de la somme des teneurs en cholesteryl 5,8,11,14,17-eicosapentaenoate, cholesteryl 7,10,13,16,19-docosapentaenoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate on cholesteryl 5,8,11,14-eicosatetraenoate content - ratio_AA_EPA_EC corresponds to the ratio of cholesterol content 5,8,11,14-eicosatetraenoate on the cholesteryl content 5,8,11,14,17-eicosapentaenoate - tot_co3_LC_EC corresponds to the sum of the cholesteryl contents 5,8,11,14,17-eicosapentaenoate, cholesteryl 7,10,13,16,19-docosapentaenoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate.

[0055] 2. All parameters are centered and reduced using the averages and standard deviations of the training sample (the test sample being assumed to come from the same distribution as the training sample). The means and standard deviations of the training sample are provided in the following table:

[0056] [Tables2] Mean Standard deviation C20:5co3_EC 0.70065217 0.43111948 C22:6co3_EC 0.56717391 0.21281879 Tot_co3_EC 1.75413043 0.61243984 ratio_co6_co3_EC 31.3562904 12.3336094 ratio_dietco3_co6_EC 0.04338134 0.01719207 ratio_EPADPAco3DHA_AA_EC 0.20686941 0.11487994 ratio_AA_EPA_EC 11.3806181 4.69275976 tot_co3_LC_EC 1.26782609 0.55703945

[0057] 3. Calculation of the mathematical formula from the centered-reduced test sample.

[0058] 4. In order to obtain the prediction of the concentration of polyunsaturated fatty acids omega-3 in the retina, the result obtained in step 3 is multiplied by the standard deviation of the concentration of omega-3 polyunsaturated fatty acids in the retina of the training sample. The values ​​thus obtained are added to the mean of the concentration of omega-3 polyunsaturated fatty acids in the retina of the training sample.

[0059] The standard deviation and mean of the omega-3 polyunsaturated fatty acid concentration in the retina of the training sample are as follows: - Average: 16.64348 - Standard deviation: 2.915459

[0060] The term “training sample” means the sample of 46 post-mortem donors, to which the sgPLS method was applied in order to obtain the prediction algorithm.

[0061] The term "test sample" means the blood sample from the subject whose retinal omega 3 fatty acid content is to be determined.

[0062] According to the method according to the invention, the blood sample is chosen from whole blood, serum and plasma.

[0063] Preferably, the sample is plasma.

[0064] In one embodiment, the method according to the invention comprises a step of blood collection and plasma isolation. Those skilled in the art may use any known method of plasma isolation. Typically, the plasma may be separated from the red blood cells by centrifugation of a whole blood sample.

[0065] The method according to the invention further comprises a step of extracting total lipids.

[0066] Total lipids may be extracted from a blood sample, by any method known to those skilled in the art. Typically, total lipids from plasma, blood and serum are extracted by the Moilanen & Nikkari method (Moilanen & Nikkari Clin Chhn Acta 1981;114(1):111-116).

[0067] The method according to the invention further comprises a step of isolating cholesterol esters from total lipids.

[0068] Typically, cholesterol esters will be isolated from total lipids, by any method known to those skilled in the art. By way of illustration, cholesterol esters are isolated from total lipids according to a procedure described in Bretillon et al 2008 (Bretillon et al Exp Eye Res 2008;87(6):521-528).

[0069] Method for preventing retinal pathology linked to a deficiency in omega-3 polyunsaturated fatty acids

[0070] The invention relates to a method for preventing a retinal pathology linked to a deficiency in omega-3 polyunsaturated fatty acids comprising the determination of the content of omega-3 polyunsaturated fatty acids in the retina according to the method for determining the content of omega-3 polyunsaturated fatty acids according to the invention.

[0071] The term “retinal pathology linked to a deficiency in omega 3 polyunsaturated fatty acids” means any pathology associated, at least in part, with a reduction in the content of polyunsaturated fatty acids in the retina.

[0072] Indeed, as major components of the human retina, omega-3 polyunsaturated fatty acids, and in particular DHA, are essential for the structure and function of the retina (SanGiovanni, JP & Chew, EY The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina. Prog Retin Eye Res 24, 87-138 (2005)). The highest content of omega-3 polyunsaturated fatty acids is found in the outer membranes of retinal photoreceptor cells (Fliesler, SJ & Anderson, RE Chemistry and metabolism of lipids in the vertebrate retina. Prog Lipid Res 22, 79-131 (1983)), where they exert important biological functions through anti-inflammatory, anti-apoptotic and anti-angiogenic activities. In post-mortem studies, DHA concentration was significantly lower in eyes with retinal pathology than in age-matched controls (Liu, A., Chang, J., Lin, Y., Shen, Z. & Bernstein, P.S. Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration. J Lipid Res 51,3217-3229 (2010)).

[0073] According to one embodiment, the retinal pathology linked to a deficiency in omega 3 polyunsaturated fatty acids is a retinopathy such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity.

[0074] Ocular neovascularization is the most common cause of blindness in all patient populations, regardless of age: retinopathy of prematurity in children, diabetic retinopathy in adults, and age-related macular degeneration in the elderly. An animal model has been developed to study retinopathy (evaluation of the appearance of retinal neovascularization and the growth of these vessels after injury to a mouse eye).

[0075] The influence of omega-3 and omega-6 polyunsaturated fatty acids on vascular growth after injury and the resulting pathological neovascularization were studied in a mouse model with hypoxia-induced retinopathy. It has been shown that increasing the intake of omega-3 polyunsaturated fatty acids reduces pathological angiogenesis. Supplementing with omega-3 polyunsaturated fatty acids would therefore be beneficial in preventing retinopathy (Kip M Connor et al., (2007), Increased dietary intake of co-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nature Medecine; doi:10.1038 / nml591).

[0076] Age-related macular degeneration (AMD)

[0077] According to one embodiment, the pathology is age-related macular degeneration (AMD).

[0078] It has indeed been demonstrated that the content of polyunsaturated fatty acids in the retina of subjects suffering from AMD was reduced (Liu, A., Chang, J., Lin, Y., Shen, Z. & Bernstein, PS Long-chain and very long chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration. J Lipid Res 51, 3217-3229 (2010).

[0079] Numerous publications have also described the reduction in the risk of AMD associated with a high dietary intake of omega-3 polyunsaturated fatty acids (Chong, EW, Kreis, AJ, Wong, .TY, Simpson, JA & Guymer, RH Dietary omega-3 fatty acid and fish intake in the primary prevention of age-related macular degeneration: a systematic review and metaanalysis. Arch Ophthalmol 126, 826-833 (2008)), as well as the reduced risk of AMD associated with high blood concentrations of omega-3 polyunsaturated fatty acids (Merle BM, DElyfer MN, Korobelnik JF, Rougier MB, Malet F, Feart C, Le Goff M, Peuchant E, Letenneur L, Dartigues JF, Colin J, Barberger-Gateau P, Delcourt C. High Concentrations of Plasma n3 Fatty Acids Are Associated with Decreased Risk for Late Age-Related Macular Degeneration. J Nutr 143:505-11 (2013).

[0080] The early and intermediate stages of AMD involve alterations of the pigment epithelium such as hypo- or hyperpigmentation and drusen and are not responsible for significant alteration of visual function.

[0081] Advanced stages (AMD) correspond to exudative forms (wet or neovascular) and atrophic forms (dry), responsible for severe alterations of central vision. According to studies, the prevalence of exudative AMD represents 35 to 65% of advanced stages.

[0082] Early and intermediate AMD:

[0083] Early and intermediate AMD combine one or more of the following elements: - intermediate drusen: small asymptomatic yellowish spots observed in the perimacular region (diameter between 63 pm and 125 pm); - large drusen (also called “serous”): larger drusen (greater than 125 pm), most often asymptomatic but which may be accompanied by a reduction in visual capacity in the dark; - pigmentary alterations such as hypopigmentation or hyperpigmentation of the pigment epithelium. They reflect the death of pigment epithelium cells.

[0084] Exudative AMD

[0085] Exudative AMD is characterized by the proliferation of choroidal neovascularizations that cross Bruch's membrane and develop under the pigment epithelium or in the subretinal space.

[0086] Atrophic AMD

[0087] Atrophic AMD (or geographic atrophy) is characterized by one or more areas of retinal depigmentation, at least 175 pm in diameter, with sharp edges and increased visibility of the choroidal vessels.

[0088] The simplified four-stage classification for the diagnosis and monitoring of AMD can be used as shown in Table 3 (Ferris FL, 3rd, Wilkinson CP, Bird A, Chakravarthy U, Chew E, Csaky K, Sadda SR. Clinical classification of age-related macular degeneration. Ophthalmology 2013;120:844-51).

[0089] [Tables[table 3]] Category 1: No AMD (no abnormalities or abnormalities due to aging) no or few small drusen (diameter less than 63 pm); no pigmentary abnormalities (hypo- and hyper-pigmentation) Category 2: Early AMD Intermediate drusen (between 63 and 125 pm), in the absence of large drusen (greater than 125 pm) Absence of pigmentary abnormalities Category 3: Intermediate AMD Large drusen (greater than 125 pm) And / or pigmentary abnormalities Category 4: Advanced AMD Neovascular or atrophic AMD

[0090] Advanced forms of AMD correspond to category 4 and include neovascular forms and geographic atrophy. These are patients who generally already have a decrease in visual acuity.

[0091] Diabetic retinopathy

[0092] Diabetic retinopathy (DR) is a microvascular complication of diabetes representing the leading cause of blindness in the United States and Europe before the age of 50.

[0093] Excess sugar in the blood weakens the capillary walls. This results in the rupture and then bursting of the retinal vessels. Gradually, large areas of the retina are no longer oxygenated. In response, the retina produces new, even more fragile vessels. The phenomenon increases and extends to the macula where the center of vision is located. The macula thickens, and macular edema occurs, which is then responsible for a drop in visual acuity that can be very significant and only partially reversible (French Federation of Diabetics, diabetic retinopathy and eye diseases).

[0094] Long-chain polyunsaturated fatty acids (LCPUFAs), such as docosahexaenoic acid (DHA, C:22 6n-3) and arachidonic acid (AA, C20:4n-6) play a key role in the pathogenesis of diabetes, along with glucose and lipid metabolism.

[0095] n-3 family VLCFAs have been shown to inhibit many cellular and biochemical processes involved in the pathophysiology of diabetic retinopathy and prevent retinal vascular damage caused by diabetes (Koehrer P, Saab S, Berdeaux O, Isaico R, Grégoire S, et al. (2014) Erythrocyte Phos-pholipid and Polyunsaturated Fatty Acid Composition in Diabetic Retinopathy. PLoS ONE 9(9): el06912. doi:10.1371 / journal.pone.0106912 ).

[0096] Sala-Vila, A. et al. (Dietary Marine co-3 Fatty Acids and Incident Sight-Threatening Retinopathy in Middle-Aged and Older Individuals With Type 2 Diabetes: Prospective Investigation From the PREDIMED Trial. JAMA Ophthalmol 134(10):1142-1149 (2016)), and Sasaki, M. et al. (The Associations of Dietary Intake of Polyun-saturated Fatty Acids With Diabetic Retinopathy in Well-Controlled Diabetes. Invest Ophthalmol Vis Sci 56(12):7473-7479 (2015)) also demonstrated abnormalities in omega-3 polyunsaturated fatty acid metabolism in subjects with diabetic retinopathy.

[0097] Many articles also show prevention of retinal dysfunctions following diabetes in animals (Shen, JH et al. Effect of a-linolenic acid on streptozotocin-induced diabetic retinopathy indices in vivo. Arch Med Res 44(7), 514-520 (2013), Tikhonenko, M. et al. N-3 polyunsaturated fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function. PLoS One 8(l):e55177 (2013), Sapieha, P. et al. Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice. Nutr Diabetes 23;2:e36. doi: 10.1038 / nutd.2012.10 (2012), Tikhonenko, M. et al. Re-modeling of retinal fatty acids in an animal model of diabetes: a decrease in long-chain polyunsaturated fatty acids is associated with a decrease in fatty acid elongases Elovl2 and Elovl4. Diabetes 59(1):219-227 (2010)).

[0098] Retinopathy of Prematurity

[0099] Retinopathy of prematurity (ROP) is a neurovascular disease affecting premature infants. This pathology is induced by a variety of factors that induce a first phase of reduced retinal vessel growth and microvascular degeneration and a second phase of pathological neovascularization, which can lead to retinal detachment.

[0100] Brain and retinal development occurs during the third trimester of pregnancy. Long-chain polyunsaturated fatty acids are selectively transferred from the mother to the fetus. Among these, docosahexaenoic acid (DHA) and arachidonic acid (AA) are the most abundant.

[0101] With routine care, premature infants receive insufficient amounts of DHA and AA as evidenced by low serum levels of these fatty acids.

[0102] Results from in vivo animal studies comparing dietary intake of omega-3 and omega-6 suggest that these essential dietary lipids affect retinal health. Research on long-chain polyunsaturated fatty acids has primarily focused on the role of omega-3 and has demonstrated that long-chain polyunsaturated fatty acids, and particularly DHA, are essential for brain and eye development.

[0103] Low serum levels of arachidonic acid have also been found to be involved in the pathogenesis of retinopathy of prematurity (Chatarina A. Lofqvist, PhD; Svetlana Najm, MD; Gunnel Hellgren, PhD; Eva Engstrom, MD, PhD; Karin Savman, MD, PhD; Anders K. Nilsson, PhD; Mats X. Andersson, PhD; Anna-Lena Hard, MD, PhD; Lois EH Smith, MD, PhD; Ann Hellstrom, MD, PhD, 2018, Association of Retinopathy of Prematurity With Low Levels of Arachidonic Acid, A Secondary Analysis of a Randomized Clinical Trial, JAMA Ophthalmol. doi:10.1001 / jamaophthalmol.2017.6658).

[0104] This highlighting of abnormalities in the metabolism of omega-3 polyunsaturated fatty acids in children with retinopathy of prematurity has also been described by Pallot, C. et al. (Alteration of erythrocyte membrane polyunsaturated fatty acids in preterm newborns with retinopathy of prematurity. Sci Rep 9, 7930 (2019)) and Martin, CR et al. (Decreased postnatal docosahexaenoic and arachidonic acid blood levels in premature infants are associated with neonatal morbidities. J Pediatr 159, 743-749, e741-742 (2011)).

[0105] Two articles also show the effects of omega-3 polyunsaturated fatty acids and their oxygenated derivatives on the retinal phenotype in an animal model of retinopathy of prematurity (Connor, KM et al. Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat Med 13, 868-873 (2007) and Stahl, A. et al. Lipid metabolites in the pathogenesis and treatment of neovascular eye disease. Br J Ophthalmol 95, 1496-1501 (2011)).

[0106] Prevention method

[0107] The terms "prevention" or "prevention method" are not absolute terms and, when applied to a retinal pathology associated with a deficiency in omega 3 polyunsaturated fatty acids such as retinopathy, they designate a procedure or a plan of action designed, even with a low probability of success but which should induce an overall beneficial effect such as the delay in the appearance of the pathology, or the reduction in the severity of one or more symptoms or the stabilization of the pathology.

[0108] Typically, in the case of AMD and diabetic retinopathy, prevention means prevention of the pathology but also prevention of worsening of the pathology.

[0109] Typically, prevention of AMD or diabetic retinopathy means delaying the onset of the pathology.

[0110] Prevention of worsening of AMD or diabetic retinopathy means delaying vision loss or loss of visual acuity in the patient with AMD or diabetic retinopathy.

[0111] Typically, in the case of retinopathy of prematurity, prevention ranges from preventing the onset of the pathology to preventing the severity of the pathology.

[0112] Typically, an increase in the content of omega-3 polyunsaturated fatty acids in the retina is associated with a decrease in the risk of the onset and / or worsening of the pathology and / or its severity.

[0113] Method of treating a retinal pathology linked to an omega-3 deficiency

[0114] The invention also relates to a method for treating a retinal pathology linked to a deficiency in omega-3 polyunsaturated fatty acids comprising: - determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject comprising quantifying the content of at least 1 cholesterol ester in a blood sample of said subject, the content of omega-3 polyunsaturated fatty acids in the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate; - the administration of treatment to the subject needing it.

[0115] Typically, retinal pathology related to omega-3 polyunsaturated fatty acid deficiency is a retinopathy such as age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity.

[0116] Thus, the invention also relates to a method of treating age-related macular degeneration comprising: - determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject comprising quantifying the content of at least 1 cholesterol ester in a blood sample of said subject, the content of omega-3 polyunsaturated fatty acids in the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate; - the administration of treatment to the subject needing it.

[0117] Typically, said treatment may be chosen from a nutritional supplement based on omega-3 polyunsaturated fatty acids, whether or not associated with the administration of vitamins and minerals.

[0118] The invention also relates to a method of treating diabetic retinopathy comprising: - determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject comprising quantifying the content of at least 1 cholesterol ester in a blood sample of said subject, the content of omega-3 polyunsaturated fatty acids in the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate; - the administration of treatment to the subject needing it.

[0119] Typically, said treatment may be chosen from a nutritional supplement based on omega-3 polyunsaturated fatty acids, whether or not associated with the administration of vitamins and minerals.

[0120] The invention also relates to a method of treating retinopathy of the premature including: - determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject comprising quantifying the content of at least 1 cholesterol ester in a blood sample of said subject, the content of omega-3 polyunsaturated fatty acids in the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate; - the administration of treatment to the subject needing it.

[0121] Typically, said treatment may be chosen from a nutritional supplement based on omega-3 polyunsaturated fatty acids, whether or not associated with the administration of vitamins and minerals.

[0122] Method for monitoring treatment of a retinal pathology linked to a deficiency in omega-3 polyunsaturated fatty acids

[0123] The invention relates to a method for monitoring treatment of a pathology linked to a deficiency in omega-3 polyunsaturated fatty acids comprising the steps: - administration of at least one treatment; - determination of the content of omega-3 polyunsaturated fatty acids in the retina according to the determination method according to the invention.

[0124] Typically, the treatment is a nutritional supplement based on omega-3 polyunsaturated fatty acids, whether or not associated with the administration of vitamins and minerals.

[0125] According to one embodiment, the pathology is a retinopathy chosen from age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity.

[0126] Among the emerging preventive strategies, omega-3 polyunsaturated fatty acids can promote normal retinal structure and function, as well as reduce the incidence and slow the progression of AMD (SanGiovanni, JP & Chew, EY The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina. Prog Retin Eye Res 24, 87-138 (2005)) but also prevent all retinopathies (Kip M Connor et al., (2007), Increased dietary intake of co-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nature Medecine ; doi:10.1038 / nml591).

[0127] Typically, an increase in the omega-3 polyunsaturated fatty acid content of the retinal content is synonymous with treatment efficacy.

[0128] Method for diagnosing omega-3 polyunsaturated fatty acid deficiencies

[0129] The invention relates to a method for diagnosing omega-3 polyunsaturated fatty acid deficiencies comprising determining the omega-3 polyunsaturated fatty acid content of the retina according to the determination method according to the invention.

[0130] “Omega-3 polyunsaturated fatty acid deficiency” means an acid deficiency polyunsaturated fats compared to a healthy subject, i.e., a subject not presenting deficiencies in polyunsaturated fatty acids.

[0131] Typically, a decrease in the omega-3 polyunsaturated fatty acid content of the retinal content is synonymous with omega-3 polyunsaturated fatty acid deficiency.

[0132] In one embodiment, the comparison with a control sample is carried out. By "control sample" is meant the distribution of values ​​in healthy subjects, i.e., subjects not presenting deficiencies in polyunsaturated fatty acids. The content of omega-3 polyunsaturated fatty acids in the retina of healthy subjects in the control group is determined by the method according to the invention. Examples

[0133] In the following examples, the inventors determined the circulating lipids having the highest predictive performance in order to determine the content of omega-3 polyunsaturated fatty acids in the retina.

[0134] In these examples, data from eye and blood samples from donors made it possible to establish an algorithm based on the plasma concentration of seven species of cholesterol esters, making it possible to determine the retinal content of omega-3 fatty acids (example 1).

[0135] The inventors have further demonstrated the association between AMD and retinal status in omega-3 polyunsaturated fatty acids (example 2) as well as the impact of dietary supplementation in omega-3 polyunsaturated fatty acids (example 3), thus showing that the combination of these seven cholesterol esters constitutes a biomarker which makes it possible to develop personalized medicine to identify subjects at risk of retinopathy and more particularly of AMD and / or to monitor supplementation in omega-3 polyunsaturated fatty acids. The predictive capacity of only cholesteryl 5,8,11,14,17-eicosapentaenoate has also been demonstrated in example 4.

[0136] Materials and Methods

[0137] In the following examples, the materials and methods detailed below were used.

[0138] Studies on human subjects were conducted in accordance with the guidelines of the Declaration of Helsinki. Written consent was obtained and the protocols were accepted by the local ethics committees (CPP Sud Est I, University Hospital, Saint Etienne, France; CPP Est III, University Hospital, Dijon, France; CPP Sud-Ouest et Outre Mer III, University Hospital, Bordeaux, France).

[0139] Donor Study Human eyeballs, plasma, and erythrocytes were obtained from 46 donors (bodies donated to Science, 30 women and 16 men, median age 86.5 years, interquartile range 76-92 years). Tissues were collected and prepared according to procedures already described and known to those skilled in the art. Samples were stored at -80°C until further analysis.

[0140] Case-control study Subjects were selected from two ongoing studies in Bordeaux (Alienor study) and Dijon (Montrachet study), which use similar methods. Between 2009 and 2011, participants were aged 75 years or older and underwent an eye examination and fasting blood sampling. Eye examinations took place in the ophthalmology departments of the university hospitals of Bordeaux and Dijon and consisted of an ophthalmic history, visual acuity and refraction measurements, and two 45° nonmydriatic retinograms. Retinal photographs were interpreted according to the International Classification and a modification of the grading system used in the Multi-Ethnic Study of Atherosclerosis for drusen size, location, and area. Participants were classified according to their worst eye into the following three exclusive groups: - no AMD, - Early AMD (presence of serous drusen (> 125 microns) and / or reticular drusen and / or pigmentary abnormalities); - Advanced AMD (geographic atrophy or neovascular disease). AMD cases had advanced AMD (geographic atrophy or neovascular disease) and controls were free of any form of early or advanced AMD and had visual acuity better than 20 / 40. Exclusion criteria for both cases and controls were the presence of glaucoma and the presence of diabetes. Thirty-one cases were identified among participants who underwent ophthalmic examination and fasting blood sampling between 2009 and 2011 and controls were matched 1:1 for age (+ / - 2 years), sex and use of lipid-lowering medication. Plasma samples were stored at -80 C until lipid analysis.

[0141] Supplementation study Plasma samples were obtained from 55 subjects participating in the double-blind, randomized clinical trial entitled "Lutein Influence on the Macula of People with AMD Parents (LIMPIA)"-( Korobelnik JF, Rougier MB, Delyfer MN, Bron A, Merle BMJ, Savel H, Chene G, Delcourt C, Creuzot-Garcher C. Effect of Dietary Supplementation With Lutein, Zeaxanthin, and omega-3 on Macular Pigment: A Randomized Clinical Trial. JAMA Ophthalmol 2017;135:1259-66). Healthy adult participants aged 40 to 70 years were recruited. Each participant was randomized in a 1:1 ratio to receive either two capsules per day of a dietary supplement containing lutein, zeaxanthin, vitamin C, vitamin E, zinc, copper, resveratrol, and also fish oil rich in omega-3 polyunsaturated fatty acids (Nutrof Total, Laboratoires Théa) or a placebo containing paraffin. Blood samples were collected at baseline and after 3 and 6 months of supplementation. Whole blood was centrifuged at 3,000 rpm for 10 minutes at 4°C to isolate plasma from red blood cells. Plasma samples were stored at -80°C until further analysis.

[0142] Statistical analyses Donor study: The model with the lowest prediction error was obtained using an extension of the sparse group partial least squares regression method (sgPLS or sparse group PLS) described by Liquet et al (Liquet B et al. Bioinformatics. 2016 Jan 1;32(l):35-42). Case-control study: Differences between cases and controls in predicted retinal omega-3 polyunsaturated fatty acid content, total plasma omega-3 polyunsaturated fatty acids, and total red blood cell omega-3 polyunsaturated fatty acids were assessed by mixed linear regression adjusted for age, body mass index, smoking, omega-3 supplement use, HDL cholesterol, and LDL cholesterol, with a random factor for case-control pairs. Supplementation study: Plasma cholesterol ester concentrations and predicted retinal omega-3 polyunsaturated fatty acid content were compared between supplemented and placebo-treated participants using linear regression models adjusted for age, sex, body mass index (BMI), HDL cholesterol, and LDL cholesterol.

[0143] All p-values ​​less than 0.05 were considered statistically significant. Analyses for the case-control and supplementation studies were performed using SAS software (SAS, version 9.4; SAS Institute Inc., Cary, North Carolina, USA).

[0144] Example 1: Identification of a biomarker of the omega-3 polyunsaturated fatty acid content of the retina

[0145] Rational Blood is a complex fluid composed of cellular components including white and red blood cells, platelets, and a liquid portion called plasma. Blood lipids are found primarily in the membranes of red blood cells and in plasma lipoprotein particles. Red blood cell membranes are composed almost exclusively of phospholipids. Due to a lifespan of approximately 120 days, the fatty acid composition of red blood cell phospholipids is representative of the long-term dietary intake of lipids. In addition, plasma lipoproteins transport biologically important lipid compounds, such as chylomicrons and very low-density lipoproteins (VLDL), which are structures primarily transporting triglycerides. Glycerides formed in the intestine and liver from dietary lipids and carbohydrates, respectively. Their fatty acid compositions are associated with very short-term dietary intake. In contrast, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are involved in the two-way transport of lipids between the liver and peripheral tissues. They are enriched in phospholipids (especially the phosphatidylcholine subclass) and cholesterol esters. The fatty acid compositions of LDL and HDL are considered indices of medium-term dietary fat intake.

[0146] In order to determine whether lipids in one or more of these blood compartments could be reliable indicators of omega-3 polyunsaturated fatty acid content in the retina and omega-3 polyunsaturated fatty acid supplementation, the BLISAR (Biomarkers of LIpid Status And Metabolism in Retinal Ageing) study was conducted between 2015 and 2018.

[0147] Results

[0148] Blood and ocular tissues from 46 human donors from the University of Saint-Etienne (bodies donated to Science) were analyzed to determine their fatty acid composition by lipidomics. In addition to retinal and total plasma lipids, long- and medium-term markers of dietary lipids were studied by isolating and analyzing the fatty acid compositions of plasma (total lipids, phosphatidylcholines, and cholesterol esters) as well as red blood cells. The fatty acid profiles confirm the quantitative importance of omega-3 polyunsaturated fatty acids in the retina (16.8% vs. 3.4% in red blood cells and 2.9% in total plasma lipids).

[0149] By lipidomic analysis, a data set on lipids present in the 4 blood compartments (red blood cells, total plasma, plasma phosphatidylcholines and plasma cholesterol esters), corresponding to 332 different species was generated.

[0150] The model with the lowest prediction error was obtained using an extension of the partial least squares regression method (sgPLS or sparse group PLS) and was characterized by a correlation coefficient of 0.62 between the observed and predicted values ​​of the omega-3 polyunsaturated fatty acid content of the retina, obtained by cross-validation. This predictor of the omega-3 polyunsaturated fatty acid content of the retina is based on an algorithm combining the plasma concentrations of 7 species of cholesterol esters.

[0151] Three of the identified cholesterol esters are from the omega-3 polyunsaturated fatty acid family, i.e., cholesteryl 5,8,11,14,17-eicosapentaenoate, cholesteryl 9,12,15-octadecatrienoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate

[0152] The other four cholesterol esters are from the polyunsaturated fatty acid family omega-6 namely cholesteryl 9,12-octadecadienoate, cholesteryl 6,9,12-octadecatrienoate, cholesteryl 8,11,14-eicosatrienoate, cholesteryl 5,8,11,14-eicosatetraenoate.

[0153] The methyl esters of the above-mentioned cholesterol esters are used to determine and measure the relative amount of fatty acids in cholesterol esters. The method for determining the content of omega-3 polyunsaturated fatty acids is detailed below:

[0154] Method for determining the content of omega-3 polyunsaturated fatty acids in the retina

[0155] The method for determining omega-3 polyunsaturated fatty acids in the retina is broken down into several steps: 1. Blood collection and plasma isolation; 2. Extraction of total lipids from plasma; 3. Isolation of cholesterol esters from total lipids; 4. Formation of methyl esters of fatty acids from cholesterol esters, in other words, methyl esters are formed from the fatty acid resulting from the hydrolysis of the cholesterol ester; 5. Determination of the relative amounts of 25 fatty acid methyl esters of cholesterol esters; 6. Calculation of the estimated value of omega-3 polyunsaturated fatty acid content of the retina from the relative amounts of methyl esters of fatty acids of 7 cholesterol esters.

[0156] Blood collection and plasma isolation A blood sample is collected from a human subject by venipuncture into an EDTA-, citrate-, or heparin-treated tube. The tube containing the whole blood is centrifuged at 3000 rpm for 10 minutes at 4°C to separate the plasma from the red blood cells. The upper phase containing the plasma is removed and stored in a dry tube. Total lipids from the plasma can be extracted immediately, or the plasma sample can be frozen pending further procedures.

[0157] Extraction of total lipids from plasma Total plasma lipids are extracted by the Moilanen & Nikkari method (Moilanen & Nikkari Clin Chim Acta 1981; 114(1): 111-116). In a glass tube, 5 milliliters of a chloroform / methanol mixture (1:1, v:v) are added to a volume of approximately 450 microliters of plasma. The tube is vortexed for 1 minute and then centrifuged at 3000 rpm for 3 minutes. The upper phase is isolated in another glass tube using a Pasteur pipette. Four milliliters of chloroform are added to this second tube along with 3 milliliters of acid sodium chloride (17 mmol / L). The tube is vortexed for 1 minute and then centrifuged at 3000 rpm for 3 minutes. The upper phase is removed using a Pasteur pipette while the lower phase containing the plasma lipid extract is isolated in a glass pillbox. The total lipid extract is first dried under a nitrogen flow and then diluted in chloroform to a concentration of 10 mg / ml. Cholesterol esters can be isolated immediately from the total lipids or frozen pending further procedures.

[0158] Isolation of cholesterol esters from total lipids Cholesterol esters are isolated from total lipids according to a procedure described in Bretillon et al 2008 (Bretillon et al Exp Eye Res 2008;87(6):521-528). A glass plate covered with silica gel is first washed in a migration tank containing 100 mL of ethyl acetate. It is then placed in an oven at 120° for 30 minutes to be activated. The extracted total lipids are deposited on the plate in the form of a band of approximately 3 cm using a glass syringe and then the plate is placed for 25 to 30 minutes in a migration chromatographic tank containing 101 milliliters of a mixture of hexane / ethyl ether / ethyl acetate (80:20:1, v:v:v). The plate is then removed from the tank and left under a hood for a few minutes. It is then exposed to a vaporization of 2',7'-dichlorofluorescein and then observed under ultraviolet light at 366nm.The silica band containing the cholesterol esters located approximately 8 to 9 cm from the deposit is marked with a pencil and then scraped with a razor blade to be collected in a glass tube to which 1 milliliter of toluene is added. The isolated cholesterol esters can be transmethylated immediately for chromatographic analysis or stored at 4°C pending further procedures.

[0159] Formation of methyl esters of fatty acids from cholesterol esters Fatty acid methyl esters of cholesterol esters are formed by transmethylation according to the Morrison & Smith method (Morrison & Smith J Lipid Res 1964;53:600-608). One milliliter of a mixture of Boron Trifluoride / methanol (1:1, v:v) is added to the tube containing the silica gel with the cholesterol esters and toluene. The tube is placed in an oven at 95°C for 2 hours. It is then cooled to room temperature. After adding 5 milliliters of sodium bicarbonate and 5 milliliters of hexane, the tube is vortexed for 1 minute and then centrifuged at 3000 rpm for 3 minutes. The upper phase is isolated in a glass tube using a Pasteur pipette. After drying the methyl esters of fatty acids of cholesterol esters under a nitrogen flow, they are diluted in 1 milliliter of hexane.Fatty acid methyl esters of cholesterol esters can be analyzed immediately by gas chromatography or stored at 4°C pending further procedures.

[0160] Determination of the fatty acid profile of cholesterol esters The fatty acid profile of cholesterol esters was determined by gas chromatography coupled with flame ionization detection according to Acar et al (Acar et al PloS One 2012;7{4): e35102). The fatty acid methyl esters of cholesterol esters were injected into a gas chromatograph equipped with a CPSIL-88 column (100m x 0.25mm inner diameter, 0.20pm film thickness, Varian, Les Ulis, France). Hydrogen was used as the carrier gas at a pressure of 210kPa. The chromatograph oven temperature was 60°C for 5 minutes, then increased to 165°C at 15°C per minute. After being held at 165°C for 1 minute, it was increased to 225°C at 2°C per minute. It is maintained at 225°C for 17 minutes. The injector and detector temperatures are set to 250°C. Fatty acid methyl esters are identified by comparing their retention times to those of commercial standards.The relative proportions of the 25 methyl ester species were calculated using EZChrom Elite software (Agilent Technologies, Massy, ​​France) and expressed as a percentage of total cholesterol ester fatty acids.

[0161] Calculation of the estimated value of the omega-3 polyunsaturated fatty acid content of the retina Use of the relative % of 7 fatty acid methyl esters of cholesterol esters: 9,12,15-octadecatrienoic acid methyl ester (C18:3n-3 methyl ester), 5,8,11,14,17-eicosapentaenoic acid methyl ester (C20:5n-3 methyl ester), 4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6n-3 methyl ester), 9,12-octadecadienoic acid methyl ester (C18:2n-6 methyl ester), 6,9,12-octadecatrienoic acid methyl ester (C18:3n-6 methyl ester), 8,11,14-eicosatrienoic acid methyl ester (C20:3n-6 methyl ester), 5,8,11,14-eicosatetraenoic acid methyl ester (C20:4n-6 methyl ester). The mathematical algorithm predicts, for a new sample (test sample), the omega-3 polyunsaturated fatty acid content in the retina from the dosage of the 7 cholesterol ester molecules in plasma presented above. This algorithm is based on the sparse group partial least squares {sgPLS} method described by Liquet et al (Liquet B et al. Bioinformatics. 2016 Jan 1;32(l):35-42), which was applied to retina, plasma and red blood cell samples collected from 46 human donors (training sample).

[0162] The mathematical algorithm consists of 4 steps: 1. Several sums and ratios are calculated from the dosages of the 7 molecules of interest. 2. All parameters are centered and reduced using the means and standard deviations of the training sample (which assumes that the test sample comes from the same distribution as the training sample). 3. The model obtained by sgPLS is applied to the values ​​obtained in step 2. 4. To obtain the prediction of the concentration of omega-3 polyunsaturated fatty acids in the retina, the result obtained in step 3 is multiplied by the standard deviation of the concentration of omega-3 polyunsaturated fatty acids in the retina of the training sample. The values ​​thus obtained are added to the mean of the concentration of omega-3 polyunsaturated fatty acids in the retina of the training sample.

[0163] Conclusions

[0164] Advantageously, cholesterol ester species of the omega-3 polyunsaturated fatty acid family contributed positively to the estimation of the omega-3 polyunsaturated fatty acid content of the retina while those of the omega-6 polyunsaturated fatty acid family lowered this estimated content, which is consistent with the well-established competitive metabolism of omega-3 and omega-6.

[0165] The identification of a biomarker based on plasma concentrations of 7 specific lipid species is novel and advantageous, as previous studies and attempts, based on the analysis of red blood cells or total plasma lipids, have failed to establish a close correlation between circulating lipids and the content of omega-3 polyunsaturated fatty acids in the retina.

[0166] Advantageously, it is possible to confirm the better robustness of the prediction compared to studies and approaches that have determined the omega-3 polyunsaturated fatty acid contents in red blood cells and whole blood (r = 0.62 versus r = 0.40 for omega-3 fatty acids in total plasma lipids and r = 0.14 for omega-3 fatty acids in red blood cells). This indicates that a more subtle approach focused on a small number of plasma cholesterol esters is more effective for estimating the retinal omega-3 polyunsaturated fatty acid content.

[0167] Furthermore, the identification of cholesterol esters as biomarkers of omega-3 polyunsaturated fatty acid content in the retina reinforces previous observations made by the inventors, suggesting that these lipids are the preferential source of fatty acids for the retina.

[0168] Example 2: Association between AMD and retinal status in omega-3 polyunsaturated fatty acids

[0169] During the second phase of the study, the association between AMD and omega-3 polyunsaturated fatty acid status was investigated.

[0170] First, in a post-mortem case-control study, a lower content of omega-3 polyunsaturated fatty acids was observed in retinas affected by AMD (14.4% ± 1.9%) compared to those of healthy donors (16.8% ± 3.1%), thus confirming previous observations. After adjustment for age, sex, time after death, the difference between the content of polyunsaturated fatty acids omega-3 from AMD-affected retinas and the omega-3 polyunsaturated fatty acid content of control retinas was -2.41% (p = 0.04).

[0171] Then, using the previously described method (measurement of 25 cholesterol esters in plasma, application of the algorithm to these measurements), the predicted content of omega-3 polyunsaturated fatty acids in the retina was compared between 31 subjects with advanced AMD and 31 controls matched for age, sex and use of lipid-lowering drugs. This predicted retinal omega-3 polyunsaturated fatty acid content was lower in cases than in controls (17.8% vs. 18.9%). After adjusting for age, body mass index, smoking, omega-3 supplement use, HDL cholesterol, and LDL cholesterol, the difference in predicted retinal omega-3 polyunsaturated fatty acid content between AMD cases and controls was -1.39% (p = 0.04). In contrast, in red blood cells, the content of omega-3 polyunsaturated fatty acids was similar in cases and controls (6.0% vs. 5.6% p = 0.42, after multivariate adjustment). These observations are in perfect agreement with the pathophysiology of AMD, characterized by the loss of omega-3 polyunsaturated fatty acid-rich metabolism of photoreceptor cells.

[0172] Given the crucial functions played by DHA in retinal structure and function and its depletion in the eyes of patients with AMD, it makes sense to maintain high retinal levels of omega-3 polyunsaturated fatty acids to prevent disease development and / or progression.

[0173] This hypothesis has been consolidated by more than 20 epidemiological studies showing a significantly reduced risk of developing AMD in subjects consuming a lot of omega-3 polyunsaturated fatty acids. However, in both randomized trials (AREDS2 (Lutein + zeaxanthin and omega-3 fatty acids for age-related macular degeneration: the Age-Related Eye Disease Study 2 (AREDS2) randomized clinical trial. JAMA 2013;309:2005-15 and NAT2 Souied EH, Delcourt C, Querques G, Bassols A, Merle B, Zourdani A, Smith T, Benlian P. Oral Docosahexaenoic Acid in the Prevention of Exudative Age-Related Macular Degeneration: The Nutritional AMD Treatment 2 Study. Ophthalmology 2013;120:1619-31.), omega-3 polyunsaturated fatty acid supplementation did not alter disease progression in advanced stages of the disease. A possible explanation for these conflicting data likely lies in the sensitivity of subjects to nutritional supplementation.Indeed, while omega-3 polyunsaturated fatty acid supplementation had no effect on AMD progression when the entire population was considered, it was shown in the NAT2 study that subjects who maintained high and constant blood levels of omega-3 polyunsaturated fatty acids had a . significantly lower risk of developing advanced AMD. This finding highlights the need to monitor the metabolic status of subjects participating in nutritional interventions.

[0174] Example 3: Sensitivity to dietary supplementation with omega-3 polyunsaturated fatty acids of the predicted omega-3 polyunsaturated fatty acid content of the retina

[0175] The sensitivity of the predicted omega-3 polyunsaturated fatty acid content of the retina to dietary supplementation with omega-3 polyunsaturated fatty acids was therefore assessed.

[0176] To this end, blood samples collected from the LIMPIA randomized clinical trial in which healthy middle-aged participants received a daily dose of 676 mg of omega-3 polyunsaturated fatty acids for 6 months, or placebo, were analyzed. The interquartile range of predicted retinal omega-3 polyunsaturated fatty acid content at baseline varied from 17.2% to 19.7% of total fatty acids, with median values ​​of approximately 18.5% for both groups, consistent with previous observations in healthy middle-aged subjects.

[0177] After 3 months of supplementation, the predicted omega-3 polyunsaturated fatty acid content of the retina was significantly increased in the supplemented subjects (median values ​​of 18.8% and 21.6% of total fatty acids in the placebo and supplemented groups, respectively; p < 0.001).

[0178] After 6 months of supplementation, this difference was maintained because the predicted omega-3 polyunsaturated fatty acid content of the retina corresponded perfectly to that calculated at 3 months (median values ​​of 18.9 and 21.7% of total fatty acids in the placebo and supplemented groups, respectively; p < 0.001).

[0179] Thus, and advantageously, a blood biomarker of the omega-3 polyunsaturated fatty acid status of the retina, based on the measurement of 7 plasma lipid species, inversely correlated with the risk of advanced AMD and increased with omega-3 polyunsaturated fatty acid supplementation, has been identified.

[0180] This blood biomarker is thus a reliable and precise tool for the preparation and conduct of clinical trials but also makes it possible to prevent the appearance of retinal pathologies linked to a deficiency in omega-3 polyunsaturated fatty acids as well as to diagnose deficiencies in omega-3 polyunsaturated fatty acids.

[0181] Example 4: Calculation of the predictive capacity of eicosapentaenoic acid (C20:5n-3 or EPA):

[0182] The inventors verified whether it was possible to determine the omega-3 polyunsaturated fatty acid content of the retina from cholesterol alone. 5,8,11,14,17-eicosapentaenoate (C20:5n-3 or EPA) rather than from the 7 cholesterol esters identified by the sgPLS method.

[0183] The result of the linear regression of the concentration of omega-3 polyunsaturated fatty acids in the retina on cholesteryl 5,8,11,14,17-eicosapentaenoate is detailed in Table 4:

[0184] [Tables5] Coefficient p-value C20:5n-3 4.1717 4.98e-06

[0185] The predictive capacity of C20:5n-3 was evaluated by calculating the correlation coefficient between the predicted and observed omega-3 polyunsaturated fatty acid contents in the retina, estimated by cross-validation in the training sample of 46 donors. A correlation coefficient r equal to 0.59 is obtained.

[0186] This result shows that the prediction capacity is almost similar to that obtained by the mathematical formula from the sgPLS method (r=0.62) with only cholesteryl 5,8,11,14,17-eicosapentaenoate (C20:5n-3).

[0187] Advantageously, it is demonstrated here that cholesteryl 5,8,11,14,17-eicosapentaenoate is a blood biomarker of retinal omega-3 polyunsaturated fatty acid status, along with the 7 identified cholesterol esters.

Claims

Claims

1. A method for determining the omega-3 polyunsaturated fatty acid content of the retina of a subject comprising determining the content of at least one cholesterol ester in a blood sample of said subject, the omega-3 polyunsaturated fatty acid content of the retina being correlated with the content of said at least one cholesterol ester, said at least one cholesterol ester being cholesteryl 5,8,11,14,17-eicosapentaenoate.

2. Determination method according to claim 1 characterized in that the content of seven cholesterol esters is determined.

3. A determination method according to claim 2, characterized in that the seven cholesterol esters are cholesteryl 5,8,11,14,17-eicosapentaenoate, cholesteryl 9,12,15-octadecatrienoate, cholesteryl 4,7,10,13,16,19-docosahexaenoate, cholesteryl 9,12-octadecadienoate, cholesteryl 6,9,12-octadecatrienoate, cholesteryl 8,11,14-eicosatrienoate, cholesteryl 5,8,11,14-eicosatetraenoate.

4. Determination method according to any one of claims 1 to 3, characterized in that the blood sample is chosen from whole blood, serum and plasma.

5. A method for preventing retinal pathology related to a deficiency in omega-3 polyunsaturated fatty acids comprising determining the content of omega-3 polyunsaturated fatty acids in the retina according to the determination method according to any one of claims 1 to A.

6. Prevention method according to claim 5 characterized in that the pathology is a retinopathy chosen from age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity.

7. Method for monitoring treatment of a pathology linked to a deficiency in omega-3 polyunsaturated fatty acids comprising the determination of the content of omega-3 polyunsaturated fatty acids in the retina of a patient to whom treatment has been administered, according to the determination method according to any one of claims 1 to 4.

8. Treatment monitoring method according to claim 7 characterized in that said pathology is a retinopathy chosen from degeneration- age-related macular recession, diabetic retinopathy and retinopathy of prematurity.

9. Treatment monitoring method according to claim 7 or 8 characterized in that the pathology is age-related macular degeneration and said treatment is a nutritional supplement based on omega-3 polyunsaturated fatty acids associated or not with the administration of vitamins and minerals.

10. A method for diagnosing omega-3 polyunsaturated fatty acid deficiencies comprising determining the omega-3 polyunsaturated fatty acid content of the retina according to the determination method according to any one of claims 1 to 4.