Prediction of omega-3 polyunsaturated fatty acid content in the retina by measuring seven cholesterol ester molecules

Abstract

The present invention relates to a method for determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject, the method comprising determining the content of at least one cholesteryl ester in a blood sample from the subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina correlates with the content of said at least one cholesteryl ester, and said at least one cholesteryl ester is cholesteryl 5,8,11,14,17-eicosapentaenoate.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to the field of ophthalmology, and more specifically to predicting the content of omega-3 polyunsaturated fatty acids in the retina.

[0002] [Prior Art] Age-related macular degeneration (AMD) is a degenerative disease of the central part of the retina, known as the macula. It mainly affects older people and accounts for 50% of cases of blindness in industrialized countries. In France, approximately 600,000 people suffer from the disease. Advanced forms of AMD (known as neovascular AMD and atrophic AMD) are associated with vision loss and are generally preceded by early and asymptomatic abnormalities.

[0003] The etiology of AMD is multifactorial, involving genetic and environmental factors such as diet. In this context, the role of omega-3 polyunsaturated fatty acids, or omega-3, has gained increasing interest for three reasons: - omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are key components of the retina that counteract certain pathological processes observed in AMD, such as inflammation, angiogenesis and cell death; - Studies have shown a depletion of omega-3 polyunsaturated fatty acid levels in the retina of subjects suffering from AMD; - Over 20 epidemiological studies have very consistently shown a 40% reduced risk of AMD in subjects consuming a diet high in omega-3 polyunsaturated fatty acids. The data therefore suggest a protective effect of omega-3 polyunsaturated fatty acids against AMD.

[0004] The family of omega-3 polyunsaturated fatty acids particularly encompasses the precursor fatty acid, alpha-linoleic acid (or C18:3n-3 or ALA) and the three longer 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, since animal organisms cannot synthesize it de novo and therefore rely solely on dietary intake. The synthesis of EPA, DPAn-3 and DHA from ALA is possible in humans, but is very limited. It is therefore also recommended to provide these fatty acids through a diet rich in seafood. Despite these recommendations, the dietary intake of omega-3 polyunsaturated fatty acids in France remains below the recommendations issued by health authorities.

[0005] It is therefore essential to be able to determine the content of omega-3 polyunsaturated fatty acids in the retina.

[0006] Knowing the concentration of omega-3 polyunsaturated fatty acids in the retina is indeed important information for: 1. identifying subjects whose retina has deteriorated and who may therefore be at risk for AMD, and 2. monitoring the effectiveness of nutritional supplements based on these fatty acids in restoring the physiological content of omega-3 polyunsaturated fatty acids in the retina.

[0007] However, for obvious ethical reasons, it is not possible to perform retinal biopsies in vivo in humans for the purpose of biochemical assays of omega-3 polyunsaturated fatty acid content, so indirect measurements are required.

[0008] Measurement of omega-3 polyunsaturated fatty acids in blood or plasma is also not sufficient to measure the content of omega-3 polyunsaturated fatty acids in the retina.

[0009] Therefore, it is necessary to have a robust and reliable method for determining the content of omega-3 polyunsaturated fatty acids in the retina.

[0010] Three studies have attempted to correlate the content of circulating fatty acids with that in the retina. The first limited its study to investigating the association between the retinal content of omega-3 polyunsaturated fatty acids and the blood content of different fatty acids, without attempting to predict the content of omega-3 polyunsaturated fatty acids in the retina or studying the lipid structures that carry the fatty acids in the blood (phospholipids, cholesteryl esters, triglycerides, etc.) (Gorusupudi et al., J. Lipid Res., 2016; 57: 499-508). The second identified total cholesteryl esters as a circulating marker of retinal DHA content (Bretillon et al., Exp. Eye Res., 2008; 87: 521-528). A third study showed that red blood cell DHA content is not a good circulating marker of retinal DHA content (Acar et al., PLoS ONE 2012; 7: e35102).

[0011] None of these studies considered the overall content of omega-3 polyunsaturated fatty acids in the retina (they targeted DHA, or the ratio of omega-6 / omega-3 fatty acid concentrations).

[0012] Nor were they able to develop models to estimate the true content of omega-3 polyunsaturated fatty acids, which could then be measured by direct analysis.

[0013] To date, two approaches are commonly used to indirectly assess the retinal content of omega-3 polyunsaturated fatty acids. The first is based on food intake, which allows the assessment of foods rich in omega-3 polyunsaturated fatty acids and the consumption of such foods in the subject through a food questionnaire, from which tissue enrichment can be estimated. The second consists of the assessment of the content of circulating omega-3 polyunsaturated fatty acids, again estimating tissue enrichment from blood enrichment. This assessment in blood compartments is more or less global and can concern both whole blood, red blood cells, whole plasma, or various compartments of plasma (mainly phospholipids and cholesteryl esters).

[0014] The use of dietary intake faces many difficulties, particularly those related to dietary assessment methods: length and complexity of dietary questionnaires, recall bias, difficulties in taking into account the great complexity and day-to-day variability of the human diet, imprecision in estimating the amount of food ingested and the nutrient content of foods.

[0015] Regarding the use of the lipid composition of the various compartments of plasma / 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, since it is modulated by several other factors such as metabolism, genetics, age, sex or lifestyle (smoking, alcohol consumption, physical activity). Apart from the two previously mentioned preliminary studies showing the ineffectiveness of the use of erythrocytes in adult human subjects and, on the contrary, the overall benefit of cholesteryl esters, to date, there have been no studies showing the absolute validity of such compartments in the estimation of the content of fatty acids in the retina or vice versa.

[0016] [Technical issues] Therefore, despite the great medical interest, it is not possible to date to measure the content of omega-3 polyunsaturated fatty acids in the retina in a reliable manner.Therefore, it is necessary to provide a tool for evaluating omega-3 polyunsaturated fatty acids in the retina other than the dietary intake method, which is complex to implement and inaccurate, and other than the methods based on the lipid composition of plasma / various plasma compartments, which do not give reliable results regarding the content of fatty acids in the retina.

[0017] Advantageously, the invention makes it possible to estimate the content of omega-3 polyunsaturated fatty acids in the retina by blood assay of at least one cholesteryl ester.

[0018] The inventors have refined the use of cholesteryl esters as markers of the content of omega-3 polyunsaturated fatty acids in the retina by using the assay values ​​of one cholesteryl ester, preferentially seven cholesteryl esters, instead of the 25 present in blood.

[0019] This method, based on the determination of the cholesteryl ester content, allows the prediction to be more robust, which is compared with studies and approaches that determined the content of omega-3 polyunsaturated fatty acids in red blood cells and whole blood (r=0.62, versus r=0.40 for omega-3 fatty acids of total lipids of plasma and r=0.14 for omega-3 fatty acids of red blood cells).

[0020] Specifically, the construction and use of a mathematical model advantageously enables the prediction of the concentration of omega-3 polyunsaturated fatty acids in the retina from the assay values ​​of one cholesteryl ester, preferably seven cholesteryl esters in the blood.

[0021] This approach is advantageously based on a small number of plasma cholesteryl esters and is more effective and reliable in determining the retinal content of omega-3 polyunsaturated fatty acids.

[0022] Therefore, one 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 quantifying the content of at least one cholesteryl ester in a blood sample from said subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina is correlated with the content of cholesteryl esters.

[0023] Another subject of the present invention is a method for preventing retinal pathologies associated with a deficiency of omega-3 polyunsaturated fatty acids by determining the content of omega-3 polyunsaturated fatty acids in the retina with the determination method of the present invention.

[0024] The present invention also relates to a method for monitoring the treatment of a pathology associated with a deficiency of 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 by the determination method of the present invention.

[0025] The present invention also relates to a method for diagnosing a deficiency of omega-3 polyunsaturated fatty acids, which comprises determining the content of omega-3 polyunsaturated fatty acids in the retina of a patient to whom the treatment has been administered using the determination method of the present invention. Summary of the Invention

[0026] (Method of determining omega-3 polyunsaturated fatty acid content)

[0027] The present invention relates to a method for determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject, the method comprising determining the content of at least one cholesteryl ester in a blood sample from the subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina correlates with the content of said at least one cholesteryl ester, said at least one cholesteryl ester being cholesteryl 5,8,11,14,17-eicosapentaenoate.

[0028] Specifically and advantageously, the inventors of the present invention have demonstrated that determining the content of cholesteryl 5,8,11,14,17-eicosapentaenoic acid (C20:5 ω3 methyl ester), which is preferentially found in plasma, can predict the content of omega-3 polyunsaturated fatty acids in the retina of a subject.

[0029] The expression "content of omega-3 polyunsaturated fatty acids in the retina" is understood to mean the sum of all omega-3 polyunsaturated fatty acids present in the retina, as a percentage by weight of total fatty acids, i.e. the sum of α-linoleic acid (or C18:3n-3 or ALA), eicosapentaenoic acid (or C20:5 ω-3 or EPA), docosapentaenoic acid (or C22:5 ω-3 or DPA ω-3) and docosahexaenoic acid (DHA or C22:6 ω-3), as a percentage by weight of total fatty acids.

[0030] According to one embodiment of the determination method the content of at least six additional cholesteryl esters is determined.

[0031] Cholesteryl esters include cholesteryl tetradecanoate, cholesteryl pentadecanoate, cholesteryl hexadecanoate, cholesteryl 7-hexadecenoate, cholesteryl 9-hexadecenoate, cholesteryl heptadecanoate, cholesteryl octadecanoate, cholesteryl trans-9-octadecenoate, cholesteryl cis-9-octadecenoate, cholesteryl 11-octadecenoate, cholesteryl 9,12-octadecadienoate, cholesteryl eicosanoate, cholesteryl 6,9,12-octadecatrienoate, cholesteryl 11-eicosenoate, cholesteryl 9,12,15-octadecadieno ...11-eicosenoate, cholesteryl 9,12,15-octadecadienoate, cholesteryl 11-eicosenoate, cholesteryl 9,12,15-octadecadienoate, cholesteryl 11-eicosenoate, cholesteryl 9,12,15-octadecadienoate, cholesteryl 11-eicosenoate, cholesteryl 11-octadecadieno The cholesteryl ester is selected from cholesteryl ctadecatrienoate, cholesteryl 11,14-eicosadienoate, cholesteryl 5,8,11-eicosatrienoate, cholesteryl 8,11,14-eicosatrienoate, cholesteryl 5,8,11,14-eicosatetraenoate, cholesteryl 15-tetracosenoate, cholesteryl 7,10,13,16-docosatetraenoate, cholesteryl 4,7,10,13,16-docosapentaenoate, cholesteryl 7,10,13,16,19-docosapentaenoate, and cholesteryl 4,7,10,13,16,19-docosahexaenoate.

[0032] The nomenclature of cholesteryl esters is detailed in Table 1.

[0033] [Table 1]

[0034] According to one embodiment, the six additional cholesteryl esters 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, and cholesteryl 5,8,11,14-eicosatetraenoate.

[0035] Therefore, according to one embodiment, the content of seven cholesteryl esters is determined.

[0036] The inventors have indeed identified, besides cholesteryl 5,8,11,14,17-eicosapentaenoate, six additional cholesteryl esters, namely 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 and cholesteryl 5,8,11,14-eicosatetraenoate, among the 25 present in blood, which makes it possible to obtain a reliable and accurate determination of the content of omega-3 polyunsaturated fatty acids in the retina.

[0037] The expression "content of at least one cholesteryl ester" is understood to mean the percentage by weight of at least one fatty acid of cholesteryl esters.

[0038] A person skilled in the art could use any known method for determining cholesteryl ester content.

[0039] According to one embodiment, to determine the content of cholesteryl esters, a method consists of a transesterification step.

[0040] The transesterification step corresponds to an acid catalyzed reaction in which the sterol moiety of the cholesteryl ester is exchanged with an alkyl group, the alkyl group being selected from methyl, propyl or butyl.

[0041] According to one embodiment, the transesterification step is a step of transmethylation of fatty acids of cholesteryl esters.

[0042] The content of cholesteryl esters can in fact be determined by the formation of methyl esters of the fatty acids of the cholesteryl esters, which are obtained after transmethylation of the fatty acids of the corresponding cholesteryl esters.

[0043] Typically, the methyl esters of fatty acids of cholesteryl esters are formed after transmethylation of fatty acids according to the method of Morrison & Smith (Morrison & Smith, J. Lipid Res., 1964; 53: 600-608).

[0044] The methyl esters of fatty acids of cholesteryl esters identified according to the invention are 9,12,15-octadecatrienoic acid methyl ester (C18:3 ω-3 methyl ester), 4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6 ω-3 methyl ester), 9,12-octadecadienoic acid methyl ester (C18:2 ω-6 methyl ester), 6,9,12-octadecatrienoic acid methyl ester (C18:3 ω-6 methyl ester), 8,11,14-eicosatrienoic acid methyl ester (C20:3 ω-6 methyl ester), 5,8,11,14-eicosatetraenoic acid methyl ester (C20:4 ω-6 methyl ester), 5,8,11,14,17-eicosapentaenoic acid methyl ester (C20:5 ω-3 methyl ester), methyl ester).

[0045] According to one embodiment, the relative weight content of each of the 25 methyl esters of fatty acids of the cholesteryl esters is determined.

[0046] The relative weight content of methyl esters of fatty acids is analyzed and determined by any method known to those skilled in the art, typically gas chromatography coupled to flame ionization detection, as described in Acar et al. (Acar et al. PLoS One 2012; 7(4): e35102).

[0047] The relative ratios of the 25 methyl esters are calculated and expressed as a percentage of the total fatty acids of cholesteryl esters. Typically, said ratios are calculated from any data integration software known to those skilled in the art, for example using EZChrom Elite software (Agilent Technologies, Massy, ​​France), and expressed as a percentage of the total fatty acids of cholesteryl esters.

[0048] This step makes it possible to determine the percentage by weight of at least one fatty acid of the cholesteryl esters and therefore the content of each cholesteryl ester.

[0049] The term "correlation" is understood to mean the calculation of an estimate of the content of omega-3 polyunsaturated fatty acids in the retina from the content of at least one cholesteryl ester, preferably from the content of at least seven cholesteryl esters, more preferably from the content of seven cholesteryl esters.

[0050] Typically, the content of omega-3 polyunsaturated fatty acids in the retina is estimated using an algorithm based on the content of at least one cholesteryl ester, preferably based on the content of at least seven cholesteryl esters, more preferably based on the content of seven cholesteryl esters.

[0051] Typically, the calculation is carried out by any algorithm that makes it possible to predict, for a new sample, the content of omega-3 polyunsaturated fatty acids in the retina from the assay of at least one cholesteryl ester, preferably from at least seven cholesteryl esters, more preferably from seven cholesteryl esters.

[0052] By way of example, and for the determination of the content of omega-3 polyunsaturated fatty acids in the retina from cholesteryl esters, the algorithm is based on linear regression according to the following formula:

[0053]

number

[0054] Also by way of example, and for the determination of the content of omega-3 polyunsaturated fatty acids in the retina from at least seven cholesteryl esters, the algorithm is based on the sparse group partial least squares (sgPLS) method described in Liquet et al. (Liquet B et al., Bioinformatics, 2016 Jan 1; 32(1): 35-42).

[0055] The algorithm includes the following four steps, which are prioritized:

[0056] 1. From the assay of at least seven cholesteryl esters, several sums and ratios are calculated. The assay of cholesteryl esters contained in plasma is an indirect assay. The cholesteryl esters undergo transesterification to produce fatty acid methyl esters. The fatty acid methyl esters thus obtained are quantified by known methods, for example gas chromatography. The relative amount of fatty acid methyl esters corresponds to the proportion of the total fatty acid methyl esters.

[0057] In one embodiment, the sums and ratios present in the formula (equation 2) reproduced below are calculated from assays of seven molecules of interest derived from 25 cholesteryl ester fatty acid methyl esters.

[0058] The seven molecules of interest derived from the 25 cholesteryl ester fatty acid methyl esters are: 9,12,15-octadecatrienoic acid methyl ester (C18:3 ω-3 methyl ester), 5,8,11,14,17-eicosapentaenoic acid methyl ester (C20:5 ω-3 methyl ester), 4,7,10,13,16,19-docosahexaenoic acid methyl ester (C22:6 ω-3 methyl ester), 9,12-octadecatrienoic acid methyl ester (C18:2 ω-6 methyl ester), 6,9,12-octadecatrienoic acid methyl ester (C18:3 ω-6 methyl ester), 8,11,14-eicosatrienoic acid methyl ester (C20:3 ω-6 methyl ester), and 5,8,11,14-eicosatrienoic acid methyl ester (C20:4 ω-3 methyl ester). ω-6 methyl ester).

[0059] The formula is detailed below.

[0060]

number

[0061] 2. All parameters are centered and shrunk using the mean and standard deviation of the training samples (assuming that the test samples come from the same distribution as the training samples). The mean and standard deviation of the training samples are shown in the table below:

[0062] [Table 2]

[0063] 3. Calculation of formula from centering-reduced test sample.

[0064] 4. To obtain a 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 value thus obtained is added to the average concentration of omega-3 polyunsaturated fatty acids in the retina of the training sample.

[0065] The standard deviations and means of the concentrations of omega-3 polyunsaturated fatty acids in the retina of the training samples are as follows: - Average value: 16.64348 - Standard deviation: 2.915459

[0066] The expression "training samples" is understood to mean samples of 46 post-mortem donors, to which the sgPLS method was applied in order to obtain a prediction algorithm.

[0067] The expression "test sample" is understood to mean a blood sample from a subject in which it is desired to determine the retinal content of omega-3 fatty acids.

[0068] According to the method of the present invention, the blood sample is selected from whole blood, serum and plasma.

[0069] Preferably, the sample is plasma.

[0070] In one embodiment, the method according to the invention comprises the steps of blood collection and plasma isolation. A person skilled in the art could use any known method for isolating plasma. Typically, plasma can be separated from red blood cells by centrifuging a sample of whole blood.

[0071] The method according to the invention further comprises the step of extracting total lipids.

[0072] Total lipids can be extracted from blood samples by any method known per se to those skilled in the art. Typically, total lipids from plasma, blood and serum are extracted by the method of Moilanen & Nikkari (Moilanen & Nikkari, Clin. Chim. Acta. 1981; 114(1): 111-116).

[0073] The method of the present invention further comprises the step of isolating the cholesteryl esters from the total lipids.

[0074] Typically, cholesteryl esters are isolated from total lipids by any method known to those skilled in the art. By way of example, cholesteryl esters are isolated from total lipids according to the procedure described in Bretillon et al. 2008 (Bretillon et al., Exp. Eye Res., 2008; 87(6): 521-528).

[0075] (Method of preventing retinal pathology associated with deficiency of omega-3 polyunsaturated fatty acids)

[0076] The present invention relates to a method for preventing retinal pathology associated with a deficiency of omega-3 polyunsaturated fatty acids, comprising determining the content of omega-3 polyunsaturated fatty acids in the retina by the method for determining the content of omega-3 polyunsaturated fatty acids of the present invention.

[0077] The expression "retinal pathology associated with a deficiency of omega-3 polyunsaturated fatty acids" is understood to mean any pathology that is at least partially associated with a decreased content of polyunsaturated fatty acids in the retina.

[0078] Specifically, as the major components of the human retina, omega-3 polyunsaturated fatty acids, especially DHA, are essential for retinal structure and function (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 membrane 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 perform important biological functions through anti-inflammatory, anti-apoptotic and anti-angiogenic activity. In a postmortem study, concentrations of DHA in eyes with retinal pathology were significantly lower than age-matched controls (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] According to one embodiment, the retinal pathology associated with a deficiency of omega-3 polyunsaturated fatty acids is a retinopathic disorder such as age-related macular degeneration, diabetic retinopathy, or retinopathy of prematurity.

[0080] Ocular neovascularization is the most common cause of blindness, which is present in all patient populations regardless of age: retinopathy of prematurity in infants, diabetic retinopathy in adults and age-related macular degeneration in the elderly. An animal model was developed to study retinopathy, assessing the appearance of retinal neovascularization and the growth of these vessels after lesions in mouse eyes.

[0081] Using a mouse model of hypoxia-induced retinopathy, the effect of omega-3 and omega-6 polyunsaturated fatty acids on vascular growth after injury and therefore induced pathological angiogenesis was studied. It was demonstrated that increased intake of omega-3 polyunsaturated fatty acids reduced pathological angiogenesis. Thus, taking omega-3 polyunsaturated fatty acid supplements would be advantageous in preventing retinopathy (Kip M Connor et al., (2007), Increased dietary intake of ω-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis, Nature Medicine; doi:10.1038 / nm1591).

[0082] (Age-related Macular Degeneration (AMD))

[0083] According to one embodiment, the pathology is age-related macular degeneration (AMD).

[0084] Indeed, it has been demonstrated that the content of polyunsaturated fatty acids in the retina of subjects with AMD is 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)).

[0085] Numerous publications have also reported a reduced 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 meta-analysis, Arch. Ophthalmol., 126, 826-833 (2008)) and a 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)).

[0086] The early and intermediate stages of AMD involve hypo- and / or hyperpigmented changes in the pigment epithelium and drusen and do not cause significant changes in visual function.

[0087] Late-stage AMD corresponds to exudative (wet or neovascular) and atrophic (dry) AMD and is responsible for severe deterioration of central vision. Studies show that the prevalence of exudative AMD accounts for 35% to 65% of the late-stage disease.

[0088] Early and intermediate AMD:

[0089] Early and intermediate AMD combine one or more of the following factors: - intermediate drusen: small asymptomatic yellowish spots observed in the perimacular area (diameter 63 μm-125 μm); - Large drusen (also called "serous" drusen): drusen of larger size (greater than 125 μm), usually asymptomatic, but may be associated with impaired night vision; - Hypo- or hyperpigmented changes of the pigment epithelium, reflecting cell death of the pigment epithelium.

[0090] wet AMD

[0091] Wet AMD is characterized by the proliferation of choroidal neovascular vessels that penetrate Bruch's membrane and grow beneath the pigment epithelium or into the subretinal space.

[0092] atrophic AMD

[0093] Dry AMD (or geographic atrophy) is characterized by depigmentation of one or more areas of the retina measuring at least 175 μm in diameter, with sharp edges and increased visibility of the choroidal vessels.

[0094] A simplified four-stage classification for diagnosing and monitoring AMD is sometimes 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).

[0095] [Table 3]

[0096] Late forms of AMD correspond to category 4 and include neovascular forms and geographic atrophy. These are generally patients who already have impaired vision.

[0097] (diabetic retinopathy)

[0098] Diabetic retinopathy (DR) is a microvascular complication of diabetes that represents the leading cause of blindness in people under 50 years of age in the United States and Europe.

[0099] Excess sugar in the blood weakens the walls of the capillaries. This is followed by rupture and then collapse of the retinal blood vessels. Gradually, large areas of the retina are deprived of oxygen. In response, the retina creates new, weaker blood vessels. This phenomenon gradually spreads to the macula, where the visual centre is located. The macula thickens and macular edema occurs, which can cause a loss of vision that is very serious and may only be partially reversible (Fédération Francaise des Diabetiques la retinopathie diabetique et les maladies des yeux [French Federation of Diabetics, diabetic retinopathy and eye diseases]).

[0100] Long-chain polyunsaturated fatty acids (LCPUFAs), such as docosahexaenoic acid (DHA, C:22 6n-3) and arachidonic acid (AA, C20:4n-6), play important roles in the pathogenesis of diabetes, as well as in glucose and lipid metabolism.

[0101] It has been demonstrated that LCPUFAs of the n-3 family inhibit many cellular and biochemical processes involved in the pathobiology of the diabetic retinopathy pathway and prevent retinopathy vascular damage caused by diabetes (Koehrer P, Saab S, Berdeaux O, Isaico R, Gregoire S, et al., (2014) Erythrocyte Phospholipid and Polyunsaturated Fatty Acid Composition in Diabetic Retinopathy, PLoS ONE 9(9): e106912. doi:10.1371 / journal.pone.0106912).

[0102] Sala-Vila, A. et al. (Dietary Marine ω-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 Polyunsaturated Fatty Acids With Diabetic Retinopathy in Well-Controlled Diabetes, Invest. Ophthalmol. Vis. Sci., 56 (12): 7473-7479 (2015)) have also demonstrated abnormalities in the metabolism of omega-3 polyunsaturated fatty acids in subjects with diabetic retinopathy.

[0103] Many papers have also shown prevention of diabetic retinopathy in animals (Shen, JH et al., Effect of α-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 (1): 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., Remodeling 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)).

[0104] (Retinopathy of Prematurity)

[0105] Retinopathy of prematurity (ROP) is a neurovascular disease that affects premature infants. This pathology is triggered by various factors that induce a first phase of reduced retinal vascular growth and microvascular degeneration, and a second phase of pathological neovascularization, which can result in retinal detachment.

[0106] Brain and retina development occurs during the third trimester. 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.

[0107] With standard treatment, premature infants receive insufficient amounts of DHA and AA, as evidenced by the low serum content of these fatty acids.

[0108] Results from in vivo studies in animals comparing nutritional intakes of omega-3 and omega-6 suggest that these essential dietary lipids influence retinal health. Research conducted on long-chain polyunsaturated fatty acids has focused primarily on the role of omega-3, demonstrating that long-chain polyunsaturated fatty acids, and more specifically DHA, are essential for brain and eye development.

[0109] It has also been observed that the pathogenesis of retinopathy of prematurity may involve low serum levels of arachidonic acid (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).

[0110] This evidence of metabolic abnormalities of omega-3 polyunsaturated fatty acids in infants affected by 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)).

[0111] Two papers also show the effect of omega-3 polyunsaturated fatty acids and their oxygenated derivatives on retinal phenotype in animal models of premature retinopathy (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)).

[0112] (Preventive measures)

[0113] The terms "prevention" or "prevention method" are not absolute terms and, when applied to retinal pathology associated with a deficiency of omega-3 polyunsaturated fatty acids, such as retinopathy, refer to a treatment or plan of action designed to induce an overall beneficial effect, such as a delay in the appearance of pathology, or a reduction in the severity of one or more symptoms, or a stabilization of pathology, even if the likelihood of success is low.

[0114] Typically, in the case of AMD and diabetic retinopathy, prevention includes not only preventing pathology, but also preventing the worsening of pathology.

[0115] Prevention of AMD or diabetic retinopathy involves delaying the appearance of pathology.

[0116] Preventing the worsening of AMD or diabetic retinopathy includes slowing the progression of vision loss or decline in patients with AMD or diabetic retinopathy.

[0117] Typically, in the case of retinopathy of prematurity, prevention ranges from preventing the appearance of pathology to preventing the pathology from becoming severe.

[0118] Typically, increased content of omega-3 polyunsaturated fatty acids in the retina is associated with a reduced risk of the appearance and / or worsening of pathology and / or severity.

[0119] The method of prevention according to the invention is an in vitro method of prevention based on the in vitro determination of the content of omega-3 polyunsaturated fatty acids in the retina, in fact, this determination is carried out on a blood sample chosen from whole blood, serum and plasma, preferentially plasma.

[0120] (Method of Treating Retinal Pathology Associated with Omega-3 Deficiency)

[0121] The present invention also relates to a method of treating a retinal pathology associated with a deficiency of 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 one cholesteryl ester in a blood sample from the subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina correlates with the content of the at least one cholesteryl ester, and wherein the at least one cholesteryl ester is cholesteryl 5,8,11,14,17-eicosapentaenoate; - Administration of treatment to a subject in need of treatment.

[0122] Typically, retinal pathologies associated with a deficiency of omega-3 polyunsaturated fatty acids are retinopathies such as age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity.

[0123] Therefore, the present 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 one cholesteryl ester in a blood sample from the subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina correlates with the content of the at least one cholesteryl ester, and wherein the at least one cholesteryl ester is cholesteryl 5,8,11,14,17-eicosapentaenoate; - Administration of treatment to a subject in need of treatment.

[0124] Typically, said treatment may be selected from nutritional supplements based on omega-3 polyunsaturated fatty acids, optionally combined with the administration of vitamins and minerals.

[0125] The present 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 one cholesteryl ester in a blood sample from the subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina correlates with the content of said at least one cholesteryl ester, and wherein said at least one cholesteryl ester is cholesteryl 5,8,11,14,17-eicosapentaenoate; - Administration of treatment to a subject in need of treatment.

[0126] Typically, said treatment may be selected from nutritional supplements based on omega-3 polyunsaturated fatty acids, optionally combined with the administration of vitamins and minerals.

[0127] The present invention also relates to a method of treating retinopathy of prematurity comprising: - determining the content of omega-3 polyunsaturated fatty acids in the retina of a subject, comprising quantifying the content of at least one cholesteryl ester in a blood sample from the subject, wherein the content of omega-3 polyunsaturated fatty acids in the retina correlates with the content of the at least one cholesteryl ester, and wherein the at least one cholesteryl ester is cholesteryl 5,8,11,14,17-eicosapentaenoate; - Administration of treatment to a subject in need of treatment.

[0128] Typically, said treatment may be selected from nutritional supplements based on omega-3 polyunsaturated fatty acids, optionally combined with the administration of vitamins and minerals.

[0129] Methods for monitoring the treatment of retinal pathology associated with a deficiency of omega-3 polyunsaturated fatty acids

[0130] The present invention relates to a method for monitoring the treatment of a pathology associated with a deficiency of omega-3 polyunsaturated fatty acids, comprising the following steps: - administering at least one treatment; - determining the content of omega-3 polyunsaturated fatty acids in the retina according to the determination method of the invention.

[0131] The present invention also relates to a method for monitoring the treatment of a pathology associated with a deficiency of omega-3 polyunsaturated fatty acids, which comprises determining the content of omega-3 polyunsaturated fatty acids in the retina of a patient to whom the treatment has been administered, by means of the determination method of the invention.

[0132] Typically, the treatment is a nutritional supplement based on omega-3 polyunsaturated fatty acids, optionally combined with the administration of vitamins and minerals.

[0133] According to one embodiment, the pathology is a retinopathy selected from age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity.

[0134] Among the new preventive strategies, omega-3 polyunsaturated fatty acids make it possible to promote normal retinal structure and function, and to prevent the occurrence 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)), as well as to prevent all retinopathy (Kip M Connor et al., (2007), Increased dietary intake of ω-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis, Nature Medicine; doi:10.1038 / nm1591)).

[0135] Typically, an increase in the retinal content of omega-3 polyunsaturated fatty acids is synonymous with efficacy of the treatment.

[0136] The monitoring method of the invention is an in vitro monitoring method based on the in vitro determination of the content of omega-3 polyunsaturated fatty acids in the retina, in fact, this determination is carried out on a blood sample chosen from whole blood, serum and plasma, preferentially plasma.

[0137] (Method of diagnosing deficiency of omega-3 polyunsaturated fatty acids)

[0138] The present invention relates to a method for diagnosing a deficiency of omega-3 polyunsaturated fatty acids, comprising determining the content of omega-3 polyunsaturated fatty acids in the retina by the determination method of the present invention.

[0139] The expression "deficiency in omega-3 polyunsaturated fatty acids" is understood to mean a deficiency in polyunsaturated fatty acids compared to healthy subjects, ie subjects that do not have a deficiency in polyunsaturated fatty acids.

[0140] Typically, a decrease in the retinal content of omega-3 polyunsaturated fatty acids is synonymous with a deficiency of omega-3 polyunsaturated fatty acids.

[0141] In one embodiment, comparison is made with a control sample.The expression "control sample" is understood to mean the distribution of values ​​in healthy subjects, i.e. subjects who do not have a deficiency in polyunsaturated fatty acids.The content of omega-3 polyunsaturated fatty acids in the retina of the healthy subjects of the control group is determined by the method of the present invention.

[0142] The diagnostic method according to the invention is an in vitro diagnostic method based on the in vitro determination of the content of omega-3 polyunsaturated fatty acids in the retina, in fact, this determination is carried out on a blood sample chosen from whole blood, serum and plasma, preferentially plasma.

[0143] [Example] In the following examples, we determined the circulating lipids with the best predictive performance for determining the content of omega-3 polyunsaturated fatty acids in the retina.

[0144] In these examples, data derived from the collection of eyes and blood from donors made it possible to establish an algorithm based on the plasma concentrations of seven cholesteryl esters and to determine the retinal content of omega-3 fatty acids (Example 1).

[0145] The inventors further demonstrated the association between AMD and omega-3 polyunsaturated fatty acid retinal status (Example 2) and the effect of omega-3 polyunsaturated fatty acid dietary supplementation (Example 3), and therefore showed that the combination of these seven cholesteryl esters constitutes a biomarker that allows developing personalized medicines and / or monitoring omega-3 polyunsaturated fatty acid supplementation to identify subjects at risk of retinopathy, more particularly AMD.The predictive ability of 5,8,11,14,17-eicosapentaenoic acid cholesteryl alone is also demonstrated in Example 4.

[0146] Materials and Methods

[0147] In the examples that follow, the materials and methods detailed below were used.

[0148] Studies involving human subjects were performed in accordance with the guidelines of the Declaration of Helsinki. Written informed consent was obtained and the protocol was accepted by the local ethical 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).

[0149] (Donor Research) Human eyes, plasma and red blood cells were obtained from 46 donors (cadaveric donors, 30 females and 16 males, median age 86.5 years, interquartile range 76-92 years). Tissues were collected and prepared according to procedures previously described and known to those skilled in the art. Samples were stored at -80 °C until further analysis.

[0150] (Case-Control Study) Subjects were selected from two ongoing studies in Bordeaux (Alienor study) and Dijon (Montrachet study), using similar methods. Between 2009 and 2011, participants were aged 75 years or older and underwent an eye examination and fasting blood sampling. The eye examination was performed in the Ophthalmology Departments of the University Hospitals of Bordeaux and Dijon and consisted of an ophthalmic history, visual acuity and refraction measurements, as well as two 45° non-mydriatic retinal photographs. The retinal photographs were interpreted according to the International Classification and according to a modification of the classification system used in the Multi-Ethnic Study of Atherosclerosis for size, location and surface area of ​​drusen. Participants were classified into three exclusive groups according to their worst eye: - No AMD, - Early AMD (presence of serous drusen (>125 microns) and / or reticular drusen and / or pigmentary abnormalities); - Late stage AMD (geographic atrophy or neovascular disease). AMD case donors had late AMD (geographic atrophy or neovascular disease) and control donors had no forms of early or late AMD and had visual acuity better than 20 / 40. Exclusion criteria for both case and control donors were the presence of glaucoma and the presence of diabetes mellitus. Thirty-one case donors were identified from participants who underwent eye examination and fasting blood sampling between 2009 and 2011, and controls were matched 1:1 for age (+ / - 2 years), sex, and lipid-lowering medication use. Plasma samples were stored at −80°C until lipid analysis.

[0151] (supply research) Plasma samples were obtained from 55 subjects who participated in a double-blind randomized clinical trial entitled "Lutein Influence on Macula of Persons Issued From 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). Adult participants aged 40 to 70 years and in good health 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 fish oil rich in omega-3 polyunsaturated fatty acids (Nutrof Total, Laboratoires Thea), or a placebo containing paraffin. Blood samples were collected at the start of supplementation and after 3 and 6 months. Whole blood was centrifuged at 3000 rpm for 10 minutes at 4°C to isolate plasma from red blood cells. Plasma samples were stored at -80°C until further analysis.

[0152] (statistical analysis) Donor study: The model with the lowest prediction error was obtained using an extension of the partial least squares regression method (sgPLS, i.e. sparse group PLS) described by Liquet et al. (Liquet B et al., Bioinformatics, 2016, Jan 1; 32(1): 35·42). Case-control donation study: Differences between case and control donations in predicted content of omega-3 polyunsaturated fatty acids in the retina, total omega-3 polyunsaturated fatty acids in plasma and erythrocytes were assessed by mixed linear regression adjusted for age, body mass index, smoking, use of omega-3 supplements, HDL cholesterol and LDL cholesterol, with a random factor of case-control donation pairing. Supplementation study: Plasma concentrations of cholesteryl esters and predicted content of omega-3 polyunsaturated fatty acids in the retina were compared between supplemented and placebo participants using linear regression models adjusted as a function of age, sex, body mass index (BMA), and HDL and LDL cholesterol.

[0153] All p values ​​below 0.05 were considered statistically significant. Analyses for the case-control cadaveric donation study and the supplementation study were performed using SAS software (SAS, version 9.4; SAS Institute Inc, Cary, North Carolina, United States).

[0154] Example 1: Identification of biomarkers of omega-3 polyunsaturated fatty acid content in the retina

[0155] (Theoretical basis) Blood is a complex fluid composed by 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. The membranes of red blood cells are composed almost exclusively of phospholipids. Due to a life span of about 120 days, the fatty acid composition of red blood cell phospholipids is representative of long-term dietary intake of lipids. In addition, plasma lipoproteins transport biologically important lipid compounds such as chylomicrons and very low density lipoproteins (VLDL), structures that primarily transport triglycerides formed in the intestine and liver from dietary lipids and carbohydrates, respectively. Their fatty acid composition is related to very short-term food 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 rich in phospholipids (especially the subclass of phosphatidylcholine) and cholesteryl esters. The fatty acid composition of LDL and HDL is considered to be an indicator of medium-term consumption of dietary fat.

[0156] To determine whether lipids from one or more of these blood compartments could constitute reliable indicators of omega-3 polyunsaturated fatty acid content in the retina and of omega-3 polyunsaturated fatty acid supplementation, the BLISAR (Biomarkers of LIpid Status And metabolism in Retinal ageing) study was carried out between 2015 and 2018.

[0157] (result)

[0158] Blood and ocular tissues of 46 human donors from the University of Saint-Etienne were analyzed to determine their fatty acid composition by lipidomics. In addition to retinal lipids and total plasma lipids, long-term and mid-term markers of dietary lipids were studied by isolating and analyzing the fatty acid composition of plasma (total lipids, phosphatidylcholine and cholesteryl esters) and erythrocytes. The fatty acid profile confirmed the quantitative importance of omega-3 polyunsaturated fatty acids in the retina (16.8% versus 3.4% in erythrocytes and 2.9% in total lipids of plasma).

[0159] Lipid analysis generated a set of data on lipids present in four blood compartments (erythrocytes, total plasma, plasma phosphatidylcholine and plasma cholesteryl esters) corresponding to 332 different species.

[0160] The model with the smallest prediction error was obtained using an extension of the partial least squares regression method (sgPLS, i.e. 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 in the retina, obtained by cross-validation. This predictor of the omega-3 polyunsaturated fatty acid content in the retina is based on an algorithm combining the plasma concentrations of seven cholesteryl esters.

[0161] Three of the cholesteryl esters identified are derived from the family of omega-3 polyunsaturated fatty acids, namely cholesteryl 5,8,11,14,17-eicosapentaenoate, cholesteryl 9,12,15-octadecatrienoate and cholesteryl 4,7,10,13,16,19-docosahexaenoate.

[0162] The other four cholesteryl esters are derived from the family of omega-6 polyunsaturated fatty acids, namely cholesteryl 9,12-octadecadienoate, cholesteryl 6,9,12-octadecatrienoate, cholesteryl 8,11,14-eicosatrienoate and cholesteryl 5,8,11,14-eicosatetraenoate.

[0163] The methyl esters of the aforementioned cholesteryl esters are used to determine and measure the relative amounts of fatty acids in the cholesteryl esters. The method for determining the content of omega-3 polyunsaturated fatty acids is detailed below:

[0164] (Method for determining the content of omega-3 polyunsaturated fatty acids in the retina)

[0165] 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 cholesteryl esters from total lipids; 4. Formation of methyl esters of fatty acids of cholesteryl esters, in other words, methyl esters are formed from fatty acids resulting from the hydrolysis of cholesteryl esters; 5. Determination of the relative amounts of 25 methyl esters of fatty acids in cholesteryl esters; 6. Calculation of an estimate of omega-3 polyunsaturated fatty acid content in the retina from the relative amounts of fatty acid methyl esters of seven cholesteryl esters.

[0166] Blood collection and plasma isolation Blood samples are collected from human subjects by venipuncture into tubes treated with EDTA, citrate or heparin. The tubes containing whole blood are centrifuged at 3000 rpm for 10 min 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 of the plasma can be extracted immediately or the plasma samples can be frozen pending the remaining procedures.

[0167] Extraction of total lipids from plasma Total lipids of plasma are extracted by the method of Moilanen & Nikkari (Moilanen & Nikkari, Clin. Chim. Acta, 1981; 114(1): 111·116). Five milliliters of a chloroform / methanol (1:1, v:v) mixture are added to a volume of approximately 450 microliters of plasma in a glass tube. The tube is vortex mixed 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, as well as 3 milliliters of acidified sodium chloride (17 mmol / L). The tube is vortex mixed for 1 minute and then centrifuged at 3000 rpm for 3 minutes. The upper phase is removed using a Pasteur pipette and the lower phase, containing the extract of plasma lipids, is then isolated in a glass sample vial. The total lipid extract is first dried under a stream of nitrogen and then diluted in chloroform to a concentration of 10 mg / ml. Cholesteryl esters can be immediately isolated from the total lipid or frozen pending the remaining procedures.

[0168] Isolation of cholesteryl esters from total lipids Cholesteryl esters are isolated from total lipids according to the 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 pre-cleaned in a transfer tank containing 100 mL of ethyl acetate. It is then placed in an oven at 120 °C for 30 minutes to activate it. The extracted total lipids are deposited on the plate in the form of a band of about 3 cm using a glass syringe, and the plate is then placed for 25-30 minutes in a chromatographic transfer tank containing 101 milliliters of a hexane / ethyl ether / ethyl acetate (80:20:1, v:v:v) mixture. The plate is then removed from the tank and left under a fume hood for a few minutes. It is then exposed to the evaporation of 2',7'-dichlorofluorescein and then observed under 366 nm ultraviolet light. The band of silica containing the cholesteryl esters, located approximately 8-9 cm from the deposit, is marked with a pencil, then scratched with a razor blade and collected in a glass tube to which 1 milliliter of toluene is added. The isolated cholesteryl esters may be immediately transmethylated in preparation for chromatographic analysis or stored at 4 °C pending the remaining procedures.

[0169] Formation of methyl esters of fatty acids from cholesteryl esters The methyl esters of fatty acids of cholesteryl esters are formed by transmethylation according to the method of Morrison & Smith (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 along with the cholesteryl esters and toluene. The tube is placed in an oven at 95°C for 2 hours. It is then cooled to ambient temperature. After the addition of 5 milliliters of sodium bicarbonate and 5 milliliters of hexane, the tube is vortex mixed 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 cholesteryl esters under a stream of nitrogen, they are diluted in 1 milliliter of hexane. The methyl esters of fatty acids of cholesteryl esters may be analyzed immediately by gas chromatography or stored at 4°C pending the remaining procedures.

[0170] Determination of fatty acid profiles of cholesteryl esters. The fatty acid profile of the cholesteryl esters is determined by gas chromatography coupled to flame ionization detection according to Acar et al. (Acar et al. PLoS One 2012; 7(4): e35102). The fatty acid methyl esters of the cholesteryl esters are injected into a gas chromatograph equipped with a CPSIL·88 column (100 m × 0.25 mm internal diameter, 0.20 μm film thickness, Varian, Les Ulis, France). Hydrogen is used as carrier gas at a pressure of 210 kPa. The temperature of the chromatograph oven is 60 °C for 5 min, then increased to 165 °C at 15 °C / min. After holding at 165 °C for 1 min, it is increased to 225 °C at 2 °C / min. It is held at 225 °C for 17 min. The injector and detector temperatures are set at 250 °C. The fatty acid methyl esters are identified by comparing their retention times with those of commercial standards. The relative proportions of the 25 methyl esters were calculated using EZChrom Elite software (Agilent Technologies, Massy, ​​France) and are expressed as cholesteryl esters as a percentage of total fatty acids.

[0171] Calculation of an estimate of omega-3 polyunsaturated fatty acid content in the retina Relative % use of seven methyl esters of fatty acids in cholesteryl 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 makes it possible to predict the content of omega-3 polyunsaturated fatty acids in the retina for a new sample (test sample) from the assay of the seven molecules of cholesteryl esters in plasma presented above. 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), applied to samples of retina, plasma and erythrocytes collected from 46 human donors (training samples).

[0172] The mathematical algorithm consists of four steps: 1. Several sums and ratios are calculated from the assays of the seven molecules of interest. 2. All parameters are centered and shrunk using the mean and standard deviation of the training samples (this assumes that the test samples are drawn from the same distribution as the training samples). 3. The model obtained by sgPLS is applied to the values ​​obtained in step 2. 4. To obtain a 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 value thus obtained is added to the average concentration of omega-3 polyunsaturated fatty acids in the retina of the training sample.

[0173] (Conclusion)

[0174] Advantageously, cholesteryl ester species from the omega-3 polyunsaturated fatty acid family positively contributed to the estimated content of omega-3 polyunsaturated fatty acids in the retina, whereas cholesteryl ester species from the omega-6 polyunsaturated fatty acid family reduced this estimated content, which corresponds to the well-established competitive metabolism of omega-3 and omega-6.

[0175] Previous studies and attempts, based on the analysis of erythrocyte or total plasma lipids, did not allow to establish a close correlation between circulating lipids and the content of omega-3 polyunsaturated fatty acids in the retina, so the identification of biomarkers based on the plasma concentrations of seven specific lipid species is innovative and advantageous.

[0176] Advantageously, it is again possible to see more robust predictions, compared to studies and approaches that determined the content of omega-3 polyunsaturated fatty acids in red blood cells and whole blood (r=0.62, versus r=0.40 for omega-3 fatty acids in plasma total lipids and r=0.14 for omega-3 fatty acids in red blood cells), indicating that a more refined approach centered on a small number of plasma cholesteryl esters is more effective at estimating the retinal content of omega-3 polyunsaturated fatty acids.

[0177] Furthermore, the identification of cholesteryl esters as biomarkers of omega-3 polyunsaturated fatty acid content in the retina strengthens our previous observations and suggests that these lipids are the preferential source of fatty acids for the retina.

[0178] Example 2: Association of omega-3 polyunsaturated fatty acids with AMD and retinal condition

[0179] During the second phase of the study, the association between AMD and omega-3 polyunsaturated fatty acid status was investigated.

[0180] First, the postmortem case-control study confirmed previous observations by showing that the content of omega-3 polyunsaturated fatty acids was lower in retinas affected by AMD (14.4% ± 1.9%) compared to retinas from donors in good health (16.8% ± 3.1%). After adjusting for age, sex, and time since death, the difference between the content of omega-3 polyunsaturated fatty acids in retinas affected by AMD and in control retinas was -2.41% (p = 0.04).

[0181] Next, using the methods described previously (measurement of 25 cholesteryl esters in plasma and application of an algorithm to these measurements), the predicted content of omega-3 polyunsaturated fatty acids in the retina was compared between 31 subjects with late AMD and 31 control donors matched for age, sex, and use of hypolipidemic medications. This predicted content of omega-3 polyunsaturated fatty acids in the retina was lower in case donations than in control donations (17.8% vs. 18.9%). After adjustment as a function of age, body mass index, smoking, use of omega-3 supplements, HDL cholesterol and LDL cholesterol, the difference in predicted content of omega-3 polyunsaturated fatty acids in the retina 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 case and control donors (6.0% vs. 5.6%, p=0.42 after multivariate adjustment). These observations are entirely consistent with the pathobiology of AMD, which is characterized by the loss of omega-3 polyunsaturated fatty acid-rich photoreceptor cell metabolism.

[0182] Given the important role that DHA plays in retinal structure and function, and its depletion in the eyes of patients with AMD, it is logical to maintain high retinal levels of omega-3 polyunsaturated fatty acids to prevent the onset and / or progression of the disease.

[0183] This hypothesis is supported by more than 20 epidemiological studies showing a significant reduction in the risk of developing AMD in subjects with a high intake of omega-3 polyunsaturated fatty acids. However, in two 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.), supplementation with omega-3 polyunsaturated fatty acids did not modify disease progression in later stages of the disease. One possible explanation for this conflicting data is perhaps the sensitivity of the subjects to nutritional supplementation. Specifically, when considering the entire population, supplementation with omega-3 polyunsaturated fatty acids had no effect on the progression of AMD. On the other hand, the NAT2 study showed that subjects who maintained high and consistent blood levels of omega-3 polyunsaturated fatty acids had a significantly lower risk of developing late-stage AMD. This finding highlights the need to monitor the metabolic status of subjects participating in nutritional interventions.

[0184] Example 3: Sensitivity of predicted omega-3 polyunsaturated fatty acid content in the retina to dietary supplementation with omega-3 polyunsaturated fatty acids

[0185] Therefore, the sensitivity of the predicted omega-3 polyunsaturated fatty acid content in the retina to dietary supplementation with omega-3 polyunsaturated fatty acids was evaluated.

[0186] To this end, we analyzed blood samples taken in the LIMPIA randomized clinical trial, in which healthy middle-aged participants received 676 mg of omega-3 polyunsaturated fatty acids daily for 6 months, or in the context of a placebo. The interquartile range of the predicted content of omega-3 polyunsaturated fatty acids in the retina varied from 17.2% to 19.7% of total fatty acids at baseline, with a median value of approximately 18.5% for the two groups, which is in line with previous observations in middle-aged healthy subjects.

[0187] After 3 months of supplementation, predicted omega-3 polyunsaturated fatty acid content in the retina was significantly increased in supplemented subjects (median total fatty acids in placebo and supplemented groups, 18.8% and 21.6%, respectively; p<0.001).

[0188] After 6 months of supplementation, this difference was maintained, as the predicted content of omega-3 polyunsaturated fatty acids in the retina corresponded perfectly with the values ​​calculated at 3 months (median total fatty acids in the placebo and supplemented groups, 18.9% and 21.7%, respectively; p<0.001).

[0189] Advantageously, therefore, a blood biomarker of omega-3 polyunsaturated fatty acid status in the retina has been identified, based on the measurement of seven plasma lipid species, that is inversely correlated with risk of late stage AMD and that is increased with omega-3 polyunsaturated fatty acid supplementation.

[0190] This blood biomarker is therefore a reliable and accurate tool for preparing and conducting clinical trials, making it possible to prevent the appearance of retinal pathologies associated with a deficiency of omega-3 polyunsaturated fatty acids and also to diagnose a deficiency of omega-3 polyunsaturated fatty acids.

[0191] Example 4: Calculation of predictive ability of cholesteryl 5,8,11,14,17-eicosapentaenoate (C20:5 ω3 methyl ester)

[0192] We investigated whether it was possible to determine the content of omega-3 polyunsaturated fatty acids in the retina from a single cholesteryl ester, cholesteryl 5,8,11,14,17-eicosapentaenoate (C20:5 ω3 methyl ester), rather than from the seven cholesteryl esters identified by the sgPLS method.

[0193] The results of the linear regression of retinal omega-3 polyunsaturated fatty acid concentrations against cholesteryl 5,8,11,14,17-eicosapentaenoate are detailed in Table 4:

[0194] [Table 4]

[0195] The predictive ability of 5,8,11,14,17-eicosapentaenoic acid cholesteryl was evaluated by calculating the correlation coefficient between the predicted retinal omega-3 polyunsaturated fatty acid content and the content observed and estimated by cross-validation in a training sample of 46 donors. A correlation coefficient r equal to 0.59 is obtained.

[0196] The results show that the predictive ability is substantially similar to that obtained by the formula derived from the sgPLS method (r=0.62) using only cholesteryl 5,8,11,14,17-eicosapentaenoate.

[0197] Advantageously, cholesteryl 5,8,11,14,17-eicosapentaenoate, as well as the seven cholesteryl esters identified, are again shown to be blood biomarkers of omega-3 polyunsaturated fatty acid status in the retina.

Claims

1. 1. A method for determining the sum of all omega-3 polyunsaturated fatty acids present in a subject's retina as a percentage by weight of total fatty acids, comprising determining the weight percentage of at least one cholesteryl ester in a blood sample from the subject, wherein the sum of all omega-3 polyunsaturated fatty acids present in the retina as a percentage by weight of total fatty acids correlates with the weight percentage of said at least one cholesteryl ester, said at least one cholesteryl ester being cholesteryl 5,8,11,14,17-eicosapentaenoate.

2. 2. The method according to claim 1, characterized in that the weight proportions of seven cholesteryl esters are determined.

3. The method according to claim 2, wherein the seven types of cholesteryl 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, and cholesteryl 5,8,11,14-eicosatetraenoate.

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

5. A method for using at least cholesteryl 5,8,11,14,17-eicosapentaenoate as a biomarker for preventing retinal pathology associated with a deficiency of omega-3 polyunsaturated fatty acids, the method comprising determining the sum of all omega-3 polyunsaturated fatty acids present in the retina as a weight percentage of total fatty acids by the determination method described in any one of claims 1 to 4.

6. 6. The method according to claim 5, wherein the pathology is a retinopathy selected from age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity.

7. A method for monitoring the treatment of a pathology associated with a deficiency of omega-3 polyunsaturated fatty acids, comprising determining the sum of all omega-3 polyunsaturated fatty acids present in the retina of a patient to whom the treatment has been administered, as a percentage by weight of total fatty acids, by the determination method described in any one of claims 1 to 4.

8. 8. The method of monitoring a treatment according to claim 7, characterized in that the pathology is a retinopathy selected from age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity.

9. 9. A method for monitoring a treatment according to claim 7 or 8, characterized in that the pathology is age-related macular degeneration and the treatment is a nutritional supplement based on omega-3 polyunsaturated fatty acids, optionally combined with the administration of vitamins and minerals.

10. A method for using at least cholesteryl 5,8,11,14,17-eicosapentaenoate as a biomarker for diagnosing a deficiency of omega-3 polyunsaturated fatty acids, the method comprising determining the sum of all omega-3 polyunsaturated fatty acids present in the retina as a weight percentage of total fatty acids by the determination method described in any one of claims 1 to 4.

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