Use of unsaturated fatty acids as biogenic ketones and long chain PUFA precursors for increasing energy status

Abstract

The present invention relates to compositions comprising lipids that are rich in unsaturated fatty acids having a plurality of double bonds in the terminal portion, and / or at least one double bond in the central portion of the fatty acid chain and method relating to administration of same. Methods and kits for ketogenic energy generation are also provided herein. The compositions described herein are useful as a ketogenic energy source that, when administered, increases the body performance of an organism, preferably a mammal. The compositions can also improve the condition of the mammalian body through an enhanced energy input following ingestion of the composition. Other exemplary uses of the compositions herein include treating disorders relating to the nervous system and other conditions that can benefit from enhanced fat-derived energy exposure such as hypometabolism.

Description

USE OF UNSATURATED FATTY ACIDS AS BIOGENIC KETONES AND LONG CHAIN PUFA PRECURSORS FOR INCREASING ENERGY STATUSTECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to compositions comprising lipids with unsaturated fatty acids having a plurality of double bonds in the terminal portion, and / or at least one double bond in the central portion of the fatty acid chain, wherein the fatty acids are formulated to provide improved ketone production with the instant formulations. The compositions described herein are useful as an energy source for increasing the body performance of an organism, preferably a mammal, and / or improving the body condition through enhanced energy input following consumption of the composition. Other exemplary uses of the compositions herein include treating disorders relating to the nervous system and other conditions that can benefit from enhanced fat-derived energy exposure.BACKGROUND OF THE INVENTION

[0002] Catabolism of lipids, including fats and oils (collectively referred to herein as "fats"), represent a major source of energy in particular for non-herbivorous animals, including most mammals. Lipid catabolism normally begins with the liberation of fatty acid(s) ("FA(s)") from a fat source, which is typically in the form of a FA ester. FAs may be saturated, for example, lard and most other animal fats, or unsaturated, for example, oils wherein such unsaturated FAs are the dominant component. Depending on their terminal double bond position, polyunsaturated FAs (PUFAs) are primarily classified in two different groups, (i) o-6 PUFAs, or (ii) o-3 PUFAs, which respectively comprise 6 and 3 carbon atoms between the terminal double bond and the FA nonpolar terminus. Notably, the typical contemporary human diet contains 10-50 times more non-o-3 FAs than fatty acids of the o-3 PUFA type. Historically, this ratio was around 1 to 1.

[0003] The main source of FAs in the human diet are triglycerides, which exist in a variety of forms. For instance, medium chain triglyceride(s) ("MCT(s)") contain 6-12 carbon atoms per FA, and are thus relatively capable of passively crossing cell membranes before being catabolised by intracellular mitochondria. Longer chain triglycerides ("LCT") typically347-004P 1 / 34contain 16-18 carbon atoms per FA (less frequently 14 and 20-22 carbon atoms), and hence depend on protein carriers for entering a cell prior to being catabolized, especially if the LCTs are in an ionised form.

[0004] FAs are typically 0-oxidised in a process that is primarily facilitated by a mitochondrial trifunctional protein, except for particularly long FAs (i.e., having an nC > 22, with the term "nC" referring to the number (n) of carbon atoms (C) that are present in a FA), which are first oxidised by intracellular peroxisomes. Incomplete oxidation of FAs can result in ketone formation, which is especially likely during a state of hypoglycaemia.

[0005] Compared to LCTs, MCTs are oxidised more directly and rapidly following ingestion, mainly in the liver, but also at the cellular level. In this context, MCTs are also generally believed to yield a much greater number of ketones than LCTs Normally, around 15% of ingested medium-chain FAs are bio-transformed by a ketogenic pathway, the rest of the medium-chain FAs being oxidised directly in extrahepatic tissues. However, because FA chain shortening quasi-exponentially increases FA water solubility, which typically causes FA-induced irritation / toxicity to the digestive tract, metabolized MCTs are generally relatively poorly tolerated. MCTs must therefore be ingested in small, repeated doses for achieving a therapeutic effect.

[0006] To address the MCT-induced gastrointestinal-irritancy problem, 'fat enriched' modifications of a typical Mediterranean diet have been investigated in the literature. These studies revealed that eating a modified Mediterranean diet including an abundant amount of olive oil containing a low concentration of phenols, compared to the more customary ketogenic MCT-rich oils, resulted in an increase in the level of ketones in the blood of epileptic children, namely to a therapeutic concentration of 4-5 mM after about 6 (0=4-7) days following diet implementation (Guzel, O. et al., EurJ Paediatric Neurol 23(1): 143-151, 2019). Notably, corn, palm, sunflower, hazel- and coconut oils were not present in the study. Moreover, a very high (72-80 weight-%) proportion of fat in the daily Mediterranean diet was used to enrich the ketone, |3-hydroxy-butyric acid ("BHB"), levels in the blood. A less fatty (60-65 weight-%) modified Mediterranean diet implemented over the course of 6 weeks raised the BHB ketone levels in blood from 0.23 (SD=0.27) mM to a mere 1.03 (0=0.63-1.23) mM, a finding that was dependent on the dementia characteristics of each patient tested. In another study, a Mediterranean diet containing 15-20 weight-% of predominantly olive oil fat did not measurably alter BHB levels in the blood from the347-004P 2 / 34subjects tested (Neth, B. et al. (2020) Neurobiology of Aging 86: 54-63). Correspondingly, only a slight (±0.01 mM) change of BHB ketone levels in the blood (baseline 0.05 (CI=0.04- 0.08) mM) was detected in dogs diagnosed with dementia that were fed a Mediterranean diet including 9 weight-% MCT or olive oil over a course of 3 months. It was reported that MCT raised and olive oil lowered BHB concentrations measured in the blood (cf. supplement info to Berk, A. et al. (2022) Front Vet Sci 9: 935430).

[0007] What is therefore needed are improved compositions and methods that can facilitate fatty acid metabolism associated with a typical mammalian diet for enhancing the bioavailability of these molecules as a ketogenic energy source, which may beneficially increase body performance and / or be useful for treating conditions that can benefit from an enhanced fat-derived energy input, such as disorders relating to the nervous system.SUMMARY OF THE INVENTION

[0008] The chain-length, number, and distribution of fatty acids (FAs) can significantly impact the efficiency of metabolic FA oxidation. For instance, C18:3(n-3), or alpha-linolenic acid ("ALA"), is known to be P-oxidized more efficiently than C18:4(n-3), or stearidonic acid ("SDA"). In addition, C18:3(n-6), or linolenic acid ("LNA") is known to be oxidized more efficiently than C18:l(n-9), or oleic acid ("OA"), and also C18:2(n-6), or linoleic acid ("LO").

[0009] Accordingly, one aim of the present invention is to provide a composition for increasing the metabolic use of fatty acids, preferably comprising n-3 FAs that are predominantly ALA FAs, and less preferably SDA fatty acids, as well-tolerated, plant-based sources of ketones that can act as an alternative or supplementary energy source for the mammalian body by inducing ketone production.

[0010] Another aim of the presently disclosed embodiments is to advocate a general use of the fats disclosed herein as a ketogenic energy source. In some embodiments, the FAs the disclosed ketogenic energy compositions predominantly have a carbon number of nC=18, further comprising 1, 2, 3, or 4 double bonds, provided that at least one of the double bonds is positioned at the n-9 position of the FA chain, and that the total dietary fat amount is less than or equal to 60 weight-%.

[0011] In some embodiments, fats having structures of predominantly n-3 chains can be used in a < 80 weight-% range and, in preferred embodiments, the n-3 chains can be used in a < 60 weight-% range. In some embodiments, the fats have predominantly at least one347-004P 3 / 34double bond in the central portion of the fatty acid chain and the fats can be preferably derived from olives. The nC=18 n-3 FAs described herein represent excellent, rich sources of ketones. However, they can also act as valuable precursors of eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA), if they are ingested in a sufficiently high quantity that leaves any non-oxidised residual available for desaturation and elongation into the long chain n-3 PUFAs disclosed herein.

[0012] In certain embodiments, a further objective of the invention is to provide guidance for the consumption of the fatty acid (FA)-based ketogenic energy compositions disclosed herein. According to some embodiments, the invention encompasses a kit for generating ketogenesis in a mammal, the kit comprising an effective amount of a fatty acid-based ketogenic energy composition and detailed instructions specifying optimized dosing protocols and timing regimens for administration. The instructions accompanying the kit direct a subject user in achieving the intended metabolic effects as described herein. The subject is a mammal, preferably a human. In some embodiments, the composition within the kit is packaged in one or more containers, each container having a volume selected from the group consisting of 100 ml, 250 ml, 500 ml, and 1000 ml. The kit may optionally further comprise instructions for administering the composition, an acetone measurement device, and / or a beta-hydroxybutyrate (BHB) blood measurement assay. According to preferred embodiments, the container housing the ketogenic energy composition comprises glass, and more particularly dark-tinted glass that attenuates ultraviolet (UV) light transmission, thereby protecting the ketogenic composition from photodegradation and preserving its stability and biological efficacy throughout storage and handling. The container configuration ensures that the ketogenic composition retains its functional integrity, thus providing a reliable and optimized dosing system as an integral component of the kit.

[0013] Non-exhaustive examples of the ketogenic energy compositions according to the invention typically comprise the n-3 FA-enriched oils described herein.

[0014] The present invention further relates to compositions that provide the following non-exhaustive listing of features attributable to the engineering and development of the fatty acid component comprised therein:• Efficient ketogenesis in vivo can be achieved not only with compositions comprising MCT, but also with FAs having a relatively long, nC=18 chain if the FAs are of the oligounsaturated n-3 type. Less preferred are FAs of the monounsaturated n-9 or of347-004P 4 / 34biunsaturated n-6 type, which may be additional supported with biophenols that are also dissolved in the oil according to the present invention;• Unusually fast and complete n-3 FAs oxidation in vivo leaves fewer ALA or SDA molecules available for other bio-transformation reactions in the mammalian body, including desaturation and elongation that is required for transformation of ALA into EPA, DPA, and possibly DHA;• The residual, systemically available ALA and / or SDA FAs can be efficiently transformed into especially EPA and then DPA, which are generated from C18:3(n-3) or C18:4(n-3) (ALA or SDA) elongation and desaturation in cases where ALA and / or SDA are ingested in a sufficient quantity;• The secondary generation of ketones originating from the aforementioned FAs, following their respective metabolic degradation, does not simply mirror the published bi-phasic and time-shifted CCh-producing 0-oxidation of the n-3 FAs disclosed herein; rather, these metabolic reactions are apparently circadian rhythm dependent and show a delayed response relative to 0-oxidation;• The time-shift, ketogenesis peak delay and the resulting peak area must be taken into account to maximise benefit of ketones metabolically produced from the FAs described herein;• To maximise ketone production from an ingested oil, the concentration of ketogenic FAs in the oil should be as high as possible, thus requiring judicious selection of the starting oil in the disclosed compositions; and• Practical consideration of the aforementioned features ensures not only an elevation of healthy polyunsaturated FAs concentrations in a body, but this is also helpful for maintaining a body once it is properly energised by the compositions disclosed herein.

[0015] The invention also provides embodiments relating to quantitative formulae for identifying and individualising an optimum daily dose of ketogenic FAs in the compositions described herein, including dosage regimens that take into account, for instance, a subject's body status and daily activity level. Exemplary fatty acids in this context include ALA and OA, in addition to the less preferred, but still biologically effective, LO, LNA and SDA types.347-004P 5 / 34BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG 1: An experimental data set illustrating metabolic fat-dependent ketogenesis, measured using a detector of acetone in exhaled breath, as a function of time over several consecutive days during which the test subject was subject to controlled conditions and consumed a standardized diet at a specified time each day. The diet was varied only in the selection of the fat (oil) consumed (40 g). Whereas no significant ketogenesis is observable after ingestion of butter (which has predominantly saturated FAs) on Day 1, the presence of ketogenesis is measurable in the subject after ingestion of MCT-rich coconut oil (Days 2 and 3) or ALA-rich flaxseed oil (Days 4-6) on the respective day of testing. Curves were generated in the graphical representations shown by fitting to the data one bi-exponential (Bateman's) function and one skewed Gaussian curve, because this combination well- represents 0-oxidation curves (not shown). Like in the results of 0-oxidation pathways producing CO2, the intra-experimental variability might be statistically large, but the data clearly indicate that ALA-rich flaxseed oil acts as a similarly effective ketone precursor in vivo compared to the MCT FAs in the coconut oil.

[0017] FIG. 2: Time-dependence of systemic ketone concentration, as reflected in the acetone concentration measured in exhaled air from a human subject, following ingestion of 60 g of n-3 FA-rich flaxseed oil (left panels), of MCT rich coconut oil (upper two right panels), or of pure MCT oil (right-bottom panel) at different times of day (10:00, 18:00, 24:00). The AUC values shown in the individual panels are background corrected and correspond to the relevant peak values (black symbols), but exclude the peak associated with the tested oil-free meal (grey symbols); the MCT curve and the AUC value in the lowest right panel are moreover scaled to allow for 1.85 higher MCT concentration in the pure MCT oil than in coconut oil. All curves in this Figure were determined by fitting the data with one bi-exponential (Bateman's) function (fitted to the irrelevant peak) and another Bateman's function and one skewed Gaussian curve (fitted to the fat derived ketogenesis peak). The results show a clear and consistent influence of circadian rhythm on BHB-ketonegeneration from the ingestion of MCT-rich coconut oil and also from the ingestion of ALA rich flaxseed oil, which both appear to be similarly ketogenic in the subject tested.

[0018] FIG. 3: Time-dependence of ketogenesis reflected in acetone concentration in breath measured following ingestion of three different fats that are rich in n-3 oligo-347-004P 6 / 34unsaturated FAs, namely flax oil, perilla oil, and echium oil, each of which were dispersed in a green foodstuff, i.e., a smoothie (upper panels) or else ingested directly as a pure fat (lower panels). The data, consistent with the aforementioned tested models, reveal a notable similar temporal evolution of ketogenesis of these fats, suggesting that the fat intake in a bulk form is more ketogenic.

[0019] FIG. 4: Comparison of ketogenesis in a human test subject using different fats (in a pure formulation) that are either (i) rich in n-3 oligo-unsaturated FAs (flaxseed oil), or (ii) in a n-9 mono-unsaturated FA (OA in extra virgin olive oil with >500 mg biophenols / L), or (iii) from a 1 / 1 mixture of these two fats. Ketogenesis was measured following ingestion of the MCT-rich CO fat as is shown for additional comparison. The curves in the lower part of FIG. 4 were determined by fitting the data with one bi-exponential (Bateman's) function (fitted to the irrelevant peak) and another Bateman's function and one skewed Gaussian curve (fitted to the fat derived ketogenesis peak). The data confirm that there is a trend of temporal-dependent generation of ketones from all four tested fats, despite the presence of experimental variability.

[0020] FIG. 5 compares the ketogenic potency of three of the tested fats as described herein using the available experimental data. The basis of this comparison is the dosedependence of total ketogenesis expressed as a cumulative production of 0- hydroxibutyrate (BHB) ketones in the test subject, calculated from the acetone concentration measured using the measuring device (i.e., as the area under the BHB concentration vs. time curve, or "AUC"). The results are shown for the different oils, CO, FO, EVOO. The 1 / 1 mixture of the latter two oils stem from measurements performed under standardized experimental and dietary conditions, except when a difference in the tested fat dose and / or its timing required a deviation. The AUC vs. ingested dose (+) and the corresponding linear fit (dashed line) show that flaxseed oil with ~54% ALA is as ketogenic as coconut oil with ~54% medium chain FAs, and appears to be more ketogenic than the extra virgin olive oil with 75% OA and >500 mg biophenols / L, notwithstanding the similar proportion (87+4%) of (n-3 + n-6 + n-9) FAs with nC=18 in the two oils. Additional detailed comparison of these oils is shown in Table 2.347-004P 7 / 34DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have their plain, general meaning as understood to one of ordinary skill in the art in the relevant technical field.

[0022] The term "about", or "around" when used with a numerical value, means a range surrounding the corresponding numerical value, including the typical measuring error associated with a particular experiment. Unless specifically stated, this value is understood to be, e.g., ± 1%, ± 2%, ± 3%, ± 4%, ± 5%, ± 7.5%, ± 10%, ± 12.5%, ± 15%, ± 17.5%, ± 20%, ± 25%, ± 30%, ± 35%, ± 40% or any other percentage of the numerical value. The term "about" or "around" used in connection with a particular numerical value normally means ± 25%, unless specified otherwise. For the imprecisely known or not uniquely defined quantities, this term implies a range of ± 50%.

[0023] The term "alkenoyl" comprises a -C(O)-alkenyl.

[0024] The term "alkenyl" generally refers to a linear or branched monovalent hydrocarbon radical containing one or several carbon-carbon double bonds in either "cis” (more preferred) or "trans” (less preferred) configuration, which can also be denoted as "Z" or "E", respectively. The preference for the c / s-configuration is not generally transferable to other types of molecules, which can thus be used in either of the two configurations, unless stated otherwise. The linear or branched monovalent hydrocarbon radical may be substituted with one or several chemically suitable substituents. The alkenyl is typically a linear monovalent hydrocarbon radical containing, in some embodiments, 2 to 30, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6 or 2 to 4 C-atoms. When branched, the alkenyl typically contains 3 to 30, 3 to 24, 3 to 22, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, or 3 to 8 carbon atoms.

[0025] A mono-alkenyl or alkenoyl contains one carbon-carbon double bond. If not specified as a mono-alkenyl or -alkenoyl, then an alkenyl- or alkenoyl can be a dialkenyl or alkenoyl, and thus contains two carbon-carbon double bonds, or an oligo- or polyalkenyl or alkenoyl (i.e. polyenyl) entity, which thus contains more than two, preferably 3 or 4, carboncarbon double bonds. Monoalkenoyls with longer chains include, but are not limited to 15c- 24:1 = C24:l(n-9) or nervonic, 13c-22:l = C22:l(n-9) or erucic, llc-20:l = C20:l(n-9) or gondoic, 6c-18:l = C18:l(n-12) or petroselinic, 9c-18:l = C18:l(n-9) or oleic, llc-18:l =347-004P 8 / 34C18:l(n-7) or cis-vaccenic, or the less preferred 9t-18:l or elaidic and llt-18:l or vaccenic alkenoyls. Furthermore, 7c-16:l = C16:l(n-9) = cis-7-hexadecenoic, 9c-16:l = C16:l(n-7) or palmitoleic, or the less preferred 3t-18:l = trans-3-hexadecenoic, and finally 9c-14:l = C14:l(n-5) or myristoleic radical. The C22 oligo-alkenoyl radicals C22 include 13c,16c-22:2 = C22:2(n-6) = 13,16-docosadienoic, 13c,16c,19c-22:3 = C22:3(n-3) = 13,16,19-docosatrienoic, 10c,13c,16c-22:3 = C22:3(n-6) = 10,13,16-docosatrienoic, 7c,10c,13c,16c-22:4 = C22:4(n-6) = 7,10,13,16-docosatetra-enoic (or adrenic), 4c,7c,10c,13c,16c,19c-22:5 = C22:6(n-3) = 4,7,10,13,16,19-docosahexaenoic, 4c,7c,10c,13c,16c-22:5 = C22:5(n-6) = 4,7,10,13,16- docosapentaenoic acid.

[0026] The main oligo-alkenoyls with 20-C-atoms described herein are 14c,17c-20:2 = C20:2(n-3) = 14-cis,17-cis-eicosadienoic, llc,14c-20:2 = C20:2(n-6) = ll-cis,14-cis- eicosadienoic, llc,14c,17c-20:3 = C20:3(n-3) or dihomo-a-linolenic, 8c,llc,14c-20:3 = C20:3(n-6) or dihomo-gamma-linolenic, 5c,8c,llc-20:3 = 20:3(n-9) or 'Mead's', 5c,8c,llc,14c-20:4 = C20:4(n-6) or arachidonic, 8c,llc,14c,17c-20:4 = C20:4(n-3) = 8,11,14,17-all-cis-eicosatetraenoic, and 5c,8c,llc,14c,17c-20:5 = C20:5(n-3) = 5,8,11,14,17- all-cis-eicosa pentaenoic acid.

[0027] Exemplary C18 oligo- and poly-alkenoyls according to the invention, include but are not limited to 12c,15c-18:2 = C18:2(n-3) or alpha-linoleic, 10c,12t-18:2 = C18:2(n-6) = trans-10,trans-12-octadecadienoic, 9c,12c-18:2 = C18:2(n-6) or gamma-linoleic or linoleic, 9c,12c,15c-18:3 = C18:3 (n-3) or alpha-linolenic, 6c,9c,12c-18:3 = C18:3(n-6) or gammalinolenic, 9c,llc,13t-18:3 or alpha-eleostearic, 8t,10t,12c-18:3 calendic, 6c,9c,12c,15c-18:4 = C18:4(n-3) or stearidonic, 3c,6c,9c,12c-18:4 = C18:4(n-6) = 3,6,9, 12-octadecatetraenoic, 3c,6c,9c,12c,15c-18:5 = C18:5(n-3) = 3,6,9,12,15-octadecapentaenoic acid. The main oligo- / poly-alkenoyls with C16 are 10c,13c-16:2 = C16:2(n-3) = 10-cis,13-cis-hexadecadienoic, 7c,10c-16:2 = C16:3(n-6) = 7-cis,10-cis-hexadecadienoic, 7c,10c,13c-16:3 = C16:3 (n-3) = 7- cis,10-cis,13-cis-hexadecatrienoic acid. The above listing of exemplary compounds is not exhaustive since alternative double bond combinations are useful according to the present invention.

[0028] Fatty acid chains comprising more than three double bonds per chain are less preferred than the mono-, di- and tri-unsaturated FA chains. Any number of double bonds per chain smaller than the maximum possible number indicates a state of "partial347-004P 9 / 34saturation", but the preferred meaning of this term refers to 1, 2, or 3 double bonds per chain, preferably in the c / s-configuration.

[0029] The term "biophenol", as used herein, refers to natural non-triglyceride components present in the carrier oils described herein. In the case of extra virgin olive oil, the biophenol component includes oleuropein, oleoeuropein acid, 3,4-DHPEA-EA and 3,4- DHPEA-EDA or oleuropein aglycone mono- and dialdehyde, oleacein, oleaceinic acid, 3,4- DHPEA or hydroxytyrosol, ligstroside, p-HPEA-EA and p-HPEA-EDA or ligstroside aglycone mono- and dialdehyde; oleocanthal, oleocanthalic acid, tyrosol, p-HPEA, methyloleoside, nuzhenide, (e)-3-(l-oxobut-2-en-2-yl) glutaric acid, vanillin, elenolic acid, p-salicylic acid, p- coumaric acid, gallic acid, caffeic acid, ferulic acid, protocatechuic acid, vanillic acid, syringic acid, homovanillic acid, sinapic acid, chlorogenic acid, verbascoside, rutin, apigenin, apigetrin, isorhoifolin, glucocyanidin, antirrhinin, luteolin, luteolol, cynaroside, veronicastroside, diosmetin, hesperidin, quercetin, quercitrin, dihydroquercetin, (+)-l- acetoxypinoresinol, (+)-l-pinoresinol, trans-cinnamic acid, gentisic acid, a-resorcylic acid, catechol, hydroquinone, 2-naphthol, o-coumaric acid. Alternative biophenols are present in other oils relevant to the ketogeneic energy compositions according to the present invention. For instance, terpenoids are the dominant 'biophenol' class in Perilla oil, including the abundant, volatile perilla-ketone, perillaldehyde and limonene, and the less prominent perilla alcohol, but Perilla oil also contains flavonoids (luteolin, apigenin), phenolic acids (rosmarinic acid derivatives) and triterpenoids as well as / 7-caryophyllene, farnesenes (Z, E, a), shisofuran, and trans-shisool. Lignans (primarily secoisolariciresino glycoside) and various phenolic acids (e.g. of the caffeic, chlorogenic, coumaric, ferulic, 4- hydroxybenzoic, gallic, sinapic and vanillic variations) represent the dominant 'biophenols' present in flaxseed oil, which also contains nordihydroguaiaretic acid (NDGA) and secoisolariciresinol diglucoside as well as diphyllin, pinoresinol, matairesinol, secoisolariciresinol, and vanillin, amongst other non-triglycerides. Chia oil also contains numerous flavonoids, phenolic acids, in addition to tocopherols.

[0030] "Conditions" or "disorders" that may benefit from consumption of the ketogenic energy compositions according to the invention include, but are not limited to, conditions or disorders of the nervous system. Exemplary conditions or disorders of the nervous system include, but are not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, bipolar depression, amyotrophic lateral sclerosis and multiple sclerosis,347-004P 10 / 34glucose transporter type 1 deficiency syndrome and other related conditions, including epilepsy, alcohol dependency, insulin resistance, systemic lupus erythematosus associated with psychiatric symptoms, anaesthesia induced memory loss (especially if related to Alzheimer's disease), age associated memory impairment, Friedreich's ataxia, Rabson- Mendenhall syndrome, leprechaunism, coronary arterial bypass graft dementia and the like. The terms "conditions" or "disorders" may also refer to deficiencies encountered by a subject when performing high-performance sports and other acts involving extreme physical activity, in addition to the commonly experienced nocturnal glucose trough.

[0031] The term "nC" as used herein refers to the number (n) of carbon atoms (C) that are present in a particular fatty acid. For instance, a FA with greater than 22 carbons can be referred to as an FA having nC > 22.

[0032] The term "effective amount" herein means the amount of an energy source sufficient to cover at least 5% of the treated body, or body part, or energy demand. For instance, when expressed in terms of the non-esterified FAs in a mammal, this often generates in colon (plasma) 5-50 mM (0.25-2.5 mM) of such FAs, and preferably up to 10- 20 mM (0.5 mM) of non-esterified FAs. If expressed in terms of body mass ("BM") and the daily administered dry fatty product mass ("DPM"), the effective daily dose is estimated to be between DPM|OW> 0.5xBM0 713g / kg / day and DPMhigh <10xBM0 713g / kg / day for a mammal that is predominantly sedentary during its wake phase (the latter value being the total estimated daily energy expenditure of the mammal). The preferred value is in the range of 5-25% of DPMhigh, assuming 50% energy input from glucose. For a predominantly active mammal with a similar BM, the preferred dose is up to 50% higher.

[0033] The term "fat" in its broadest meaning as used herein refers to an oil, a fat, or a mixture thereof. This term further refers to triglycerides and / or diglycerides of fatty acids that can be saturated, which is typical of solid fats, such as lard, or mono-, di-, tri-, or polyunsaturated fats, i.e., comprising 1, 2, 3 or > 4 double bonds per chain, for instance, as in fluid oils.

[0034] The term "food" or "foodstuff" are used herein interchangeably and include but are not limited to baked confectionery foods, cakes, cereals, smoothies, salads, any beverage, dressings or mayonnaise, fried cakes or other kinds of fried foods, ice creams, margarine or fat spreads on breads, oil, powders, or a concentration of any of the347-004P 11 / 34aforementioned substances in a fluid or dry form suitable for incorporation into any of the aforementioned food forms.

[0035] The term "glyceride" generally refers to esters formed from glycerol and fatty acids. In triglycerides, each of the three hydroxyl groups on a glycerol are esterified with a FA which can be similar or dissimilar on the individual group. In diglycerides, two similar or dissimilar FA are attached to one glycerol backbone with an ester bond. "Partial glycerides" refer to esters of glycerol with fatty acids, where not all the hydroxyl groups are esterified, and hence includes diglycerides. Since some of their hydroxyl groups are free, partial glycerides are typically polar. A preferred diglyceride is a diglyceride 1,3-isomer.

[0036] The term "hypometabolism" as used herein, if referring to a mammalian body condition, comprises a condition associated with hypometabolism, including but not being limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, bipolar depression, amyotrophic lateral sclerosis and multiple sclerosis, glucose transporter type 1 deficiency syndrome and the related epilepsy, alcohol dependency, insulin resistance, systemic lupus erythematosus associated with psychiatric symptoms, anesthesia induced memory loss (especially if related to Alzheimer's disease), age associated memory impairment, Friedreich's ataxia, Rabson-Mendenhall syndrome, leprechaunism, coronary arterial bypass graft dementia, etc. If relating to an essentially normal status of a mammalian organism, the term "hypometabolism" refers to any state or outcome that can benefit from an extra delivery of energy following consumption of the ketogenic energy compositions herein, for example, energizing a body during strenuous physical activity, during the nocturnal phase of low systemic glucose concentration and the like.

[0037] In the broadest terms, the health benefits, or improved "biological effect", resulting from the regular use of a composition according to the invention, for example, when treating a condition such as hypometabolism, can relate to the following, interconnected indicia of aging and / or disease: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.

[0038] A person skilled in the art will be well-acquainted with conventional measuring, testing, and quantification protocols for assessing each of the aforementioned biological effect metrics to identify an improvement, either as a baseline measurement exercise, or347-004P 12 / 34for evaluating a change in the condition over time. For instance, the starting point will typically provide the measurement baseline value(s), relative to which the change of the metric following administration of the composition use can be readily determined and evaluated.

[0039] For instance, for assessing the condition of dysbiosis or gut-status, various testing protocols can be employed, including but not limited to, specific indices reflecting microbial diversity and composition in the gut microbiome. The most common markers can be used to evaluate inflammation and the closely related oxidative stress (OS), which may include, but are not limited to, assessing acute-phase proteins, e.g., CRP, serum amyloid A, fibrinogen and procalcitonin, and cytokines, predominantly TNFa, interleukins 1|3, 6, 8, 10 and 12 and their receptors and IFNy; notably certain cytokines can be disease-specific. OS biomarkers appear less disease or tissue-specific and include lipid peroxidation products, e.g., F2-isoprostanes and malondialdehyde, DNA breakdown products (e.g., 8-OH-dG), protein adducts (e.g., carbonylated proteins), or antioxidant status. More novel OS markers include also -omics related ones, as well as non-invasive, questionnaire-based measures, such as the dietary inflammatory-index. Inflammation markers can moreover be useful to assess altered intercellular communication; even more telling are measurements of various biomarkers and signaling pathways that indicate changes in cell communication, particularly those related to inflammation and cellular senescence.

[0040] To assess mitochondrial dysfunction, at least three biomarker classes are commonly evaluated (Hubens et al. (2022) Mitochondrion 62: 187-204): functional markers measured in blood cells (including but not limited to measuring OXPHOS function, mitochondrial membrane potential, mtDNA copy number, germline mtDNA mutations and deletions) biochemical markers of serum / plasma (including but not limited to determination of lactate, pyruvate, Creatine and creatine kinase, acylcarnitine, etc), and DNA markers. While no single biomarkers is likely to reveal all underlying mitochondrial dysfunction, combining biomarkers that cover different aspects of mitochondrial impairment addresses this problem. Deregulated mutrient sensing can involve four associated key protein groups, IGF-1, mTOR, sirtuins, and AMPK, and can lead to various age- related conditions, including diabetes, obesity, and cognitive decline. It can be detected and monitored, for example, by measuring various biomarkers, including but not limited to347-004P 13 / 34altered levels of insulin and insulin-like growth factor (IGF-1), changes in mTOR activity, variations in AMP-activated protein kinase (AMPK) signaling, etc.

[0041] Loss of proteostasis is linked to several human conditions involving deterioration of the organism, particularly neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis (ALS). However, loss of proteostasis can also contribute to metabolic disorders, cardiac diseases, and various age-related diseases, due to the accumulation of misfolded proteins. Studying and quantifying a loss of proteostasis requires complex, but established procedures (Lim & Vendrusco (2025) PLoS Comput Biol 21(6): el013155). To measure epigenetic alterations associated with these conditions, numerous commercially assays ("genetic clocks") are available, which individually yield different results (Bergsmal & Rogaeval (2022) Neuroscience Insights 15:1-11) and hence should best be used in combination and repeatedly to gain robust result. Telomere length, and thus attrition, can be measured directly in cells. Genomic instability markers can include specific mutations in DNA repair genes, chromosomal rearrangements, and aneuploidy, suitable for identifying and characterizing, often cancerous, tissues addressed by an intervention.

[0042] In addition, known biomarkers characteristically associated with a certain condition can be measured with readily available techniques. For example, in case of diabetes, such protocols include but are not limited to, monitoring glucose and, in the case of insulin, concentrations in the blood (both highlighting the acute situation) or HBA1C concentration in blood (as a longer term and thus trend indicator) can be readily determined.

[0043] In case of a cardiovascular condition, blood pressure and / or blood lipid measurements can provide valuable assessment of the health status of a subject, and are hence useful for determining the treatment success; less common but also useful for the purpose are pulse wave-velocity measurements.

[0044] Various neurological conditions can be assessed, for example, by using one of the known validated tests that monitor and asses cognition, the shortest of which not taking more than only a few minutes (Zhang et al. (2019) Aging Disease 6:1258-1269); to gain a deeper insight on the health status of a subject, MRI imaging can be used.

[0045] To assess bone density and to monitor its changes, dual wavelength X-ray is useful, for example, but also telling can be studying relative frequency of fractures in a study population.347-004P 14 / 34

[0046] Many conditions are moreover associated with an increased level of inflammation markers that can be readily assessed by the skilled person using a variety of conventional means by selecting a test method suitable for assessing the beneficial biological effect(s) provided by the compositions according to the invention following administration to a mammalian subject, preferably a human.

[0047] Total daily energy requirement calculations and activity level assessment for a mammalian subject can be established using a number of conventional methods. For example, the estimated total daily energy requirement of an individual can be calculated using well-established predictive equations that calculate basal metabolic rate (BMR) based on demographic and anthropometric parameters. The preferred method employs the Mifflin-St Jeor equation, which has demonstrated superior accuracy compared to alternative formulations, correctly predicting resting metabolic rate within 10% of measured values in approximately 82% of individuals with normal body mass index. For male subjects, the BMR is calculated as: (10 x weight in kilograms) + (6.25 x height in centimeters) - (5 x age in years) + 5, while forfemale subjects the calculation is: (10 x weight in kilograms) + (6.25 x height in centimeters) - (5 x age in years) - 161. Alternatively, the revised Harris-Benedict equation may be employed, utilizing the formulations: BMR = 88.362 + (13.397 x weight in kg) + (4.799 x height in cm) - (5.677 x age in years) for males, and BMR = 447.593 + (9.247 x weight in kg) + (1.736 x height in cm) - (4.330 x age in years) for females. The calculated BMR represents the minimum energy expenditure required for basic physiological functions at rest and is subsequently multiplied by a physical activity level (PAL) factor to determine total daily energy expenditure (TDEE). The physical activity level multipliers follow established guidelines: sedentary lifestyle (BMR x 1.2 for minimal physical activity beyond basic daily living activities), lightly active (BMR x 1.375 for light exercise or sports 1-3 days per week), moderately active (BMR x 1.55 for moderate exercise or sports 3-5 days per week), very active (BMR x 1.725 for hard exercise or sports 6-7 days per week), and extremely active (BMR x 1.9 for very hard physical work or training twice daily). The resulting TDEE value represents the total calories required to maintain current body weight under the specified activity conditions.

[0048] There are also well-known methodologies for activity level assessment and related classification of same. The determination of whether an individual is sedentary or active can be immediately evaluated using standardized physical activity assessment protocols that347-004P 15 / 34quantify both the duration and intensity of weekly physical activity engagement. According to established World Health Organization guidelines and validated screening instruments, adults are classified as sedentary / inactive when they perform less than 150 minutes of moderate-intensity physical activity per week or less than 75 minutes of vigorous-intensity activity per week, essentially engaging in no structured physical activity beyond basic movements required for daily living such as household activities, occupational tasks, or personal care. Conversely, individuals are classified as active when they achieve or exceed the minimum recommended physical activity guidelines of 150-300 minutes of moderateintensity aerobic activity per week (such as brisk walking at 3-4 mph, recreational swimming, or doubles tennis), or 75-150 minutes of vigorous-intensity activity per week (such as running, competitive sports, or high-intensity interval training), or an equivalent combination thereof. The assessment can be rapidly conducted using validated screening tools such as the General Practice Physical Activity Questionnaire (GPPAQ), which provides a four-level physical activity index, or the International Physical Activity Questionnaire Short Form (IPAQ-SF), which categorizes individuals based on metabolic equivalent (MET) minutes per week calculations. For immediate clinical assessment, practitioners can employ simple screening questions that evaluate: (1) frequency of moderate-to-vigorous physical activity sessions per week, (2) average duration of each activity session, (3) nature of occupational physical demands, (4) time spent in sedentary behaviors such as sitting or screen time (with >8 hours daily indicating high sedentary behavior regardless of exercise habits), and (5) use of active transportation methods. Individuals reporting fewer than 3 sessions of moderate activity per week lasting less than 30 minutes each, combined with predominantly sedentary occupational and recreational activities, are classified as inactive and would receive a physical activity level factor of 1.2 in energy calculations, while those meeting or exceeding activity recommendations receive correspondingly higher PAL factors of 1.375- 1.9 depending on the intensity and frequency of their reported physical activity engagement.

[0049] This systematic approach to energy requirement calculation and activity level assessment provides a standardized, reproducible method for determining appropriate dosing regimens for ketogenic energy compositions, ensuring that the administered quantities align with individual metabolic demands and activity-adjusted energy347-004P 16 / 34expenditure patterns while maintaining therapeutic efficacy across diverse patient populations with varying physical activity profiles and metabolic requirements.

[0050] The term "lipid" means herein a substance with at least one fatty acid segment, or fatty acid chain. According to the present invention, a lipid may contain at least one hydrophilic, i.e., lipophobic, segment comprising more water- than fat-soluble, and hence polar, molecular part; the resulting lipid is an "amphipat."

[0051] The term "amphipat" or "amphiphat" or "amphiphile" or "amphipatic" are used interchangeably herein and refer to a chemical compound featuring both hydrophilic and lipophilic properties, i.e., an amphipathic molecule. A simple amphipatic lipid as disclosed herein may be represented according to the following generic formula:Xk— Y|— Zm(I) wherein at least one of the three -indices (k, I, m), which refers to the respective number of hydrophilic segments, is non-zero. The other two indices are then positive or zero. By way of example, if X and Y or Y and Z are lipophilic, the lipid is of a double-chain type; otherwise, it is a block-copolymer. A simple lipid has one positive index (e.g., k > 0 for the lipophilic tail and I = m = 0 for the lacking hydrophilic headgroup) and is thus apolar. A lipid having several lipophilic chains (e.g. k > 0 and I > 0) is normally relatively apolar, even if it contains one small hydrophilic group (m > 0). The latter in any case would define an amphiphilic lipid, i.e. an entity that is partly lipophilic and party hydrophilic.

[0052] The term "oligo" means herein 1, 2, 3, 4, 5 or 6 segments to which the term refers, notably herein typically to double bonds, and most frequently 1 to 4 such segments. In the context of FA unsaturation, this term refers to a FA molecule has 2, 3 or 4, preferably adjacent, double bonds.

[0053] The term "partial" used in conjunction with an oligo- or polyester as described herein means that at least one of the hydroxyl groups on the specified molecule is not der- ivatised, i.e. it exists in a free form. The term "partial glyceride" thus means herein, first, a mono- or di-ester of glycerol with fatty acids. A glycerol-monoester (= monoacylglycerol) can be of either a 1-, 2-, or 3-type glycerol-monoester, i.e. having its only fatty acid esterified to the 1st, 2nd, or 3rdcarbon atom of the glycerol backbone, respectively. A glycerol di-ester (=diacylglycerol) may be a 1,2-isomer or 2,3- isomer (= 1,2- or 2,3-DAG), i.e., having either the 3rd(1,2-) or the 1st(2,4-) carbon atom of the glycerol backbone being non-esterified. The347-004P 17 / 341,3-diacylglycerol (1,3-DAG) isomer has the hydroxyl group on the 2ndcarbon atom in a free form. 1,3-DAG is preferable according to the invention, owing to its biologically inert character. A diacylglycerol is moreover preferred over the corresponding monoacylglycerol, which typically has a less desirable taste for consumption purposes. The term "partial oligosaccharide ester of fatty acids", which includes "partial saccharide ester of fatty acids", means herein a fatty acid(s) oligoester of a saccharide or an oligosaccharide, triose, tetraose, pentose, hexose, heptose or any of their homo- or hetero-oligomers. Preferred oligosaccharides correspond to the most common 'sugar' carbohydrates, for example, erythrose, threose, erythrulose among tetroses; arabinose, ribose, xylose, ribulose among pentoses; glucose, mannose, gulose, galactose, fructose, and sorbose among the hexoses.

[0054] The term "predominantly" as used herein refers to at least greater than 50%, more preferably greater than 68% and most preferably greater than 75%.

[0055] The term "preferred chain(s)" herein refers to collectively the preferred medium chain(s) and the preferred long chain(s) FAs as described herein. For example, any preferred chain should optimally be fluid at normal body temperature, but may also exist as a solid during product storage. The skilled person knows how to combine information on melting temperature of chains and product components with information about their viscosity and miscibility in order to design blends with the desirable (chains) fluidity and overall firmness.

[0056] The term "preferred medium chain(s)" ("PMC") as used herein refers to straight (i.e. linear) and fully saturated (i.e. without double bonds) chains, featuring an even number of C-atoms ("nc"), thus being of a hexanoic (i.e. caproic, C6:0), octanoic (i.e. caprylic, C8:0), decanoic (i.e. capric, C10:0) or dodecanoic (i.e. lauric, C12:0) type. Moreover, C8:0 and C10:0 are especially preferred for relatively small polar lipids (such as DAG or TAG) and C8 and / or CIO and / or C12 for the relatively more polar lipids (such as polysorbates). The fully saturated heptanoic (C7:0), nonanoic (C9:0) and undecanoic (Cll:0) FA, with an odd nc are potentially suitable, but less preferred for the ketogenic energy compositions disclosed herein, as are the branched and unsaturated FA (C6:x to C12:x) with a similar total carbon atoms number and x = 1, 2 double bonds or side chains.

[0057] The term "preferred long chain(s)" ("PLC") in relation to a FA refers to alkenyl chains comprising a n-3 configuration, with preferably 18 carbon atoms and 3 or less preferably 4 double bonds, for example, in alpha-linoleic acid (ALA / ALNA) chains or stearidonic acid (SDA), respectively, or with 1 to 2 centrally located double bonds, including347-004P 18 / 34OA, LO, and LNA. Less preferred long chain FAs are PLCs with nc=20 and 4 double bonds, for example, EPA, and nc=22 and 5 or more double bonds, such as DPA and DHA. Preferred sources of n-3 PLC include flaxseeds and chiaseeds as well as a perilla plant for ALA and Echium plantagineum, cyanobacterium Spirulina, and leaves or borage (Borago officinalis) for ALA and SDA. The preferred sources of OA (the most stable PLC) are especially extra virgin olive oil and high OA canola oil, the ordinary canola oil being less preferred in this respect. Oils rich in LO include safflower, corn, hemp, cotton- and poppy-seed, as well as soybean oil, among others.

[0058] The term "range" used in the context of > 2 numerical values means that the numerical value can be any value encompassed within the specified range. For the purposes according to the present invention, "range" also means that, within the broadest range specified, any narrower range may be selected using 50%, 45%, 40%, 36%, 33%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the entire range. By way of example, a range of 1 to 10 is thus sub-divisible and / or limited to 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9 and 9 to 10 or else to 1 to 3.33, 3.33 to 6.66 and 6.66 to 9.99 or 3.33 to 9.999, or from 1 to 4, 4 to 7, 7 to 10, 1 to 7 or 4 to 10; or from 1 to 3.25, from 3.25 to 5.5, from 5.5 to 7.75, from 7.75 to 10, from 1 to 5.5, from 1 to 7.5, from 3.25 to 7.5 from 3.75 to 10, or from 5.5 to 10.

[0059] The term "resistant starch" refers herein to a carbohydrate having resistance to digestion in the intestine, colon, whether of the RSI type (which is a carbohydrate that is physically protected, e.g. in whole grains or seeds, etc.), or of the RS2 type (in form of ungelatinized resistant granules with B-type crystallinity, e.g. in raw potatoes, legumes, high amylose starches, etc.), or of the RS3 type (retrograded starch, e.g. in cooked and cooled potatoes, bread, corn flakes, food products with prolonged and / or repeated moist heat treatment, etc.), or of the RS4 type (chemically modified, e.g. in chemically cross bonded starches, starch ethers, esters, etc.).

[0060] According to the present invention, FA chain-length and number as well as biodistribution of double bonds (especially the terminal double bond location) each respectively impact FA oxidation. Molecules containing fewer C-atoms and up to 3-4 double bonds (herein referred to as "oligounsaturated" FAs) are typically oxidised more efficiently than longer and less or more unsaturated (herein "polysaturated" FAs or "PUFAs"). The oligo-unsaturation form of the FAs herein are preferably of n-3- / omega-3- / alpha type,347-004P 19 / 34but can also take the form of the bi- or mono-unsaturated chains of the n-6 or n-9- type, especially if used in combination with one or more biophenols. The resulting CO2 peak of this FA structure is detectable in humans for various FAs for about 5.5 ± 0.5 hours following ingestion, but can show a pronounced variation in height. Notably, C18:3(n-3) or alphalinolenic acid ("ALA"), and C18:l(n-6) or oleic ("OA", as well as its corresponding trans- version, elaidic acid), are oxidized more efficiently than C18:3(n-6), or gamma-linolenic acid ("LNA") FAs; however, fatty tissues typically accumulate comparatively more OA than ALA.

[0061] FAs that are neither oxidised nor stored in body fats or incorporated into another lipophilic environment in a mammalian body (e.g., incorporated into a membrane or a protein, such as serum albumin) are deployed to other biological processes, for instance, elongation and desaturation roles, which are especially relevant to the health status of a mammal. Two main reasons for this are that (i) C18:3(n-6) elongation generates predominantly proinflammatory arachidonic acid, C20:4(n-6), whereas (ii) C18:3(n-3) elongation and desaturation generate the comparatively beneficial eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA), respectively (i.e., C20:5(n-3) and C22:5(n-3)).

[0062] Fatty acid incorporation and interconversion processes depend on dietary PUFAs intake as well as the age and gender of the mammalian organism. However, elderly humans oxidise and hydrolyse relatively more long-chain n-3 PUFAs during metabolism compared to younger individuals (M Hennebelle (2016) Nutrition 32: 1211-1216), while females generate more long chain PUFAs from ALA than males, especially during pregnancy. In turn, females P-oxidise a lower proportion of ALA. Taken together, PUFA distribution and concentration in humans is apparently gender, age, and tissue dependent, and likely influenced by the hormonal and metabolic status of the organism, in addition to the measurable PUFA half-life.

[0063] Similar rates and quantities of CO2 generation from the metabolism of C18:l(n-9), C18:2(n-6), and C18:3(n-3) through 0-oxidation prompts a similar conclusion for models of ketogenesis. One should therefore measure a similar total quantity of ketones, or their degradation products, in subjects who ingest similar OA, LNA, or ALA quantities independent of the source of these FAs. Surprisingly, the data disclosed herein in FIG. 1 shows that this is not the case. Here, ALA was revealed to be as ketogenic as MCT FAs and, according to the data presented herein, also shown to be more ketogenic than OA, but347-004P 20 / 34subject to additional factors such as microbiome composition, the biophenol concentration in the oil, etc.

[0064] As described herein, a ketogenic, diet, typically encompassing a low-carbohydrate, high-protein, and a high fat focus, was first introduced decades ago to treat refractory epilepsy in children. Milder versions of this treatment regime involving fewer dietary restrictions are currently used to treat epileptic adults in some cases, mostly based on a diet that relies on MCTs as the main fat component, owing to relatively high metabolic yield of ketones from these medium chain FAs.

[0065] Fats rich in omega-3- (encompassing o-3, n-3-, alpha-, a-fatty acids) are included in other diet programs, owing to the perceived benefits of these FAs. For instance, oils rich in n-3-FAs are also a component of health-focussed or anabolic diets. However, unlike the teachings according to the present invention, the motivation of such diets fails to address the suitability of these FAs for advancing ketogenesis, but instead focuses on the balancing of the intake of non-n-3 and n-3 FAs.

[0066] For example, the inclusion of omega-3 rich linseeds has been reported to meaningfully lower energy utilisation of food ingested by chickens, also when using demucilaged seeds (C. Alzueta et al. (2003) British Poultry Science, 44:67-74). Furthermore, human diet fortification with n-3 FAs (i.e., enrichment with 2 g ALA / day), reportedly did not significantly impact the level of fasting ketones during a 6-hour post-meal observation period, and only moderately (26%) increased the postprandial P-hydroxy-butyrate production in young but not in older adults, without an observable difference for acetoacetate. Collectively, these findings imply no appreciable ketogenesis from such n-3 FAs supplementation had occurred, even if plasma ALA doubled in both groups. Notably, in the older group, the measured EPA concentration in plasma was also 40% higher (M. Hennebelle et al. (2016) Nutrition 32:1211-1216).

[0067] Other authors have reported that ALA is a precursor of EPA and DPA (but not significantly of DHA) in mammals. These reports concluded that this finding is due to an inefficiency of elongation and desaturation enzymes when metabolically acting on the ALA molecule.

[0068] In contrast, the present inventor has found that the unusually high degree of metabolic ALA oxidation in vivo leaves only a small concentration of ALA available for the otherwise efficient ALA transformation into EPA, DPA, and potentially DHA, when the347-004P 21 / 34mammalian body requires more of the latter. To generate practically meaningful levels of EPA and DPA concentrations in the mammalian body, an appropriately high daily ALA dose is therefore required. Table 1 herein shows interconversion factors according to the present invention that are useful for calculating the ingested ALA dose required to achieve a desired systemic concentration of EPA or DPA.

[0069] Equivalents to the compositions and methods described herein therefore fall within the contemplated scope of the invention and the appended claims. The contents of all cited references, patents, and patent applications are hereby incorporated by reference. The components, processes, and methods of these cited disclosures can be selected for use and readily adapted into the embodiments of the presently disclosed invention.EXAMPLES

[0070] EXAMPLES 1-6

[0071] To evaluate the suitability of various fats as effective ketone precursors in vivo, according to embodiments of the present invention, standardised test and boundary conditions were applied in 13 different protocols, each protocol lasting for a period of 2-6 days. Acetone concentration in exhaled air from the test subject acted as a proxy for systemic ketone concentration, since acetone is known to be the final educt of FA oxidation and ketone degradation. The device used to quantify such acetone concentration was the ACE KetoScan Mini device, which was recalibrated after every 300 instances of collected data. To derive the systemic BHB ketone concentration (in mM) from the obtained acetone concentration values (in ppm), the published conversion multiplicator 0.397 was used.

[0072] FIG. 1 shows the average of 1-5, most frequently 2-3, of such repeated measurements performed in the second test series corresponding herein to Examples 1-6. The results of the first test series are not presented herein owing to their relatively large standard deviations. However, following the initial training of the experimental models according to the present invention, the acetone concentration could be determined reliably with a single measurement.

[0073] Two test series included continuous blood glucose measurements measured with the commercial sensor Freestyle Libre 2 (in one test series) and Freestyle Libre 3 (in another test series); both sensors were obtained from Abbott Laboratories. This series of glucose347-004P 22 / 34measurements allowed for investigating a possible correlation between glucogenesis and ketogenesis in the test subject.

[0074] The average body weight of the test subject was 70+3 kg and the adjusted average caloric daily energy intake standardized to around 7000 kJ, net (i.e. adjusted, as needed, from day to day by changing the daily carbohydrates intake to allow for the inter-day variability of the subject's physical activity), such intake corresponding to 60+5% or the test subject's customary caloric intake. Except in the first two test series, the timing of food intake was also standardised. For example, the breakfast meal was served at 10:00 am and dinner was served at 18:00 pm. As required to investigate an intake timing effect, the dinner meal was complemented with a tested oil intake at midnight, then including such oil intake into the calculated total dinner caloric intake.

[0075] In the first two test series, one of the two meals included soba noodles, while the other meal included whole grain rye bread as the non-fatty meal components. Unless specified otherwise, the non-test meals moreover included up to 40 g (subsequently reduced to 30 g or 0 g) butter as the only extra fat. From the third test series onward both meals included whole grain rye bread only; this dietary change was prompted by the discovery, based on concomitant continuous measurements of blood glucose, that the soba noodles are 2-3 times more glucogenic than whole grain rye bread although their glycaemic index is only 1.5-2 times different. Overall, the daily diet during most tests was comprised of, on average (relative standard deviation or RSD, min-max), 43% (0.21, 30-56%) fat, 8% (0.13, 6-9%) proteins and 50% (0.16, 38-62%) carbohydrates, plus 41 g (0.15) fibres / day. The main reason for variability in the data set was attributed to the different daily tested fat dose, chosen in the Examples in the 20-60 g / meal range; RSD within a similar dose group was 2-3 times smaller. In the first two test series, the daily breakfast basis, according to these Examples, included a green smoothie. In the subsequent test series, the smoothie was served either with breakfast or with dinner; the smoothie was always made from vegetables and fruits in a 2 / 1 weight ratio.

[0076] Butter was used as a 'negative control' in the present experiments, as butter contains predominantly saturated FAs. The medium chain triglycerides (MCTs) provided by pure, virgin coconut fat, "VCO" or "CO" served as a 'positive control.' Flaxseed, peri Ila, and echium oils were used as alternative sources of n-3 FAs, whereas the particularly selected347-004P 23 / 34extra virgin olive oil, featuring an unusually high content of biophenols (> 500 mg / L), was chosen as the predominantly n-9 monounsaturated OA source.

[0077] Table 2 shows the characteristic fatty acid distribution in aforementioned oils and also of the previously tested oils in the remaining Examples disclosed herein.

[0078] FIG. 1 illustrates the time-dependent detection of acetone in the breath of the subject both before and after ingestion of the different fats, showing a prominent acetone peak (in ppm, black dots) 8-12 hours following ingestion of the test oil along with the green smoothie forming together the daily dinner. Despite variability of these early proof-of- principle establishing experiments, a common result in the ketogenic energy effect attributable to each individual oil source is clearly recognisable.

[0079] EXAMPLES 7-12

[0080] Examples 7-12, the results of which are illustrated in FIG. 2, were conducted in a similar manner to the first series embodied in Examples 1-6, namely under similar experimental conditions, except that the test oil was ingested at different times of day, i.e., at 10:00 am, 18:00 pm or at midnight. The key results observed are shown in FIG. 2 and reveal the measured time dependences of ketogenesis, with the possible and even likely 1 hour delay between BHB and acetone appearing in vivo following ketogenic diet ingestion (Freemantle, E. et al., (2009) J Nutr Health Aging 13(4): 293-298). The results from Examples 7-12 resemble those observed in the previous test series (Examples 1-6) but, given the fine- tuning to the experimental conditions as described above, are less variable and more mutually consistent.

[0081] Similar to the first experimental series, a small peak was typically measured approximately 8 hours after breakfast, reflecting breath acetone generation from ingestion of the relatively digestion resistant starch during breakfast. The initial peak was superimposed on or followed by a significantly more prominent secondary peak, which consistently started to appear 8 hours or 10 hours after the test fat ingestion, the longer delay time consistently corresponding to the test oil consumption in the evening. In turn, the test oils ingestion in the morning appeared to result in a stronger and longer ketogenesis lasting around 16 hours, rather than just around 12 hours, suggesting a circadian rhythm effect on the bioprocessing of the test fats. Both coconut oil, which abounds in MCTs, and347-004P 24 / 34flaxseed oil, which is rich in ALA, were found to be comparably good sources of a substantive quantity of ketones. The negative control was conversely non-ketogenic.

[0082] EXAMPLES 13-17

[0083] Examples 13-17 relate to testing protocols in which the breakfast meal consisted of full grain rye bread and 30-40 g butter, whereas the main nutritional content of the provided food was again served at 18:00 pm with dinners comprised of green smoothies with or without the test fat included. If the fat was not incorporated in the smoothie it was ingested in bulk as pure oil; the consumed fat amounts are specified in FIG. 3, which shows the resulting data. In this series embodied as Examples 13-17, perilla oil (used as an alternative ALA source), echium seed oil (as an SDA source), and olive oil (as an OA source), were tested, but FIG. 3 shows only the results for the former three fats. Notably, these results are consistent with those described above for the meals including the test oils consumption in the bulk, but strongly suggest that fat bioprocessing is fat presentation dependent since the test fats dispersed in an intensely homogenised green smoothie were observed to be markedly less ketogenic.

[0084] EXAMPLES 18-22

[0085] The results from Examples 18-22 are illustrated in FIG. 4. These experiments compared the outcome of dietary intake of pure fats and their corresponding mixtures using oils extracted from flaxseed and from highly phenol rich olives. The orally consumed fat doses are specified in FIG. 4. The experimental results are consistent with previous experimental findings in the previous Examples, but also reveal appreciable variability of ketogenesis resulting from similar fat intake measured on different days.

[0086] Additional tests were conducted to confirm and reproduce the results of the previous experiments. With these extra tests (not described herein), we observed that increasing the daily caloric intake of the test subject to 100% of their typical energetic requirement did not significantly affect the measured ketogenesis monitored by acetone measurements from the exhaled breath.

[0087] To further investigate and address the experimental variability as described herein, and to include certain data observed in connection with the performed experiments, FIG. 5 summarises the overall outcome of the tested fat dependent ketogenesis models, with exception of the first two test series. The basis of FIG. 5 are dose-effect plots relying347-004P 25 / 34on the area under the ketogenesis vs. time curve measured in each of the representative tests. The plots illustrated in FIG. 5 confirm that nC=18 FAs with at least one double bond in the central portion of the FA chain (i.e. at the n-9 position) are decisively ketogenic. This effect is even more pronounced in fatty acids having 3 double bonds in the terminal chain portion of the FA (i.e. of n-3 type). The latter FAs abound, for example, in flaxseed and perilla oils that are rich in ALA and appear to be at least as ketogenic as the MCTs ingested in coconut oil rich in such medium chain FAs. OA, and probably also the less abundant LA and LNA oils, ingested together with ample biophenols in the tested extra virgin olive oil, were also observed to be ketogenic but less than ALA obtained from flaxseed oil or perilla oil sources.

[0088] The plots shown in FIG. 5 and FIG. 2, for example, show for the first time that it is possible to achieve a beneficial BHB ketone level in human blood by including a moderate amount of ketogenic fats as defined herein in the daily diet, especially if such fats are consumed with the morning breakfast meal.

[0089] These novel conclusions are remarkable compared to known models of ketogenesis in the state of the art. For instance, the present consensus in the art is that a ketogenic diet should comprise at least 80% (60%) arbitrary (MCT) fats in order to elicit a practically meaningful ketogenic effect. The few published reports on the use of a 'fatty' modification of Mediterranean diet are in line with this recommendation (Guzel, O. et al., op. cit.; Neth, B. et al., op. cit.). Pertinent literature also does not advocate use of n-3 FA- rich oils for the purpose of ketone generation in vivo and even the rare study of such use found no practically useful ketone generation from flaxseed oil (Hennebelle, M., op. cit.).

[0090] By contrast, the data reported herein using the disclosed ketogenic energy compositions clearly demonstrate that a single ingestion of ketogenic fats according to the ketogenic energy formulations defined herein can achieve a measurable and practically beneficial BHB ketone level in blood already surprisingly following the first consumption, when the ingested dose contributes to a level of 40-60% fatty component to the daily diet. Combining such fats with carbohydrates consumed in form of resistant starch can then even become therapeutically useful. For even greater therapeutic benefit, one can use fats in the present compositions with an increased proportion of ketogenic fatty acids, for example, oil extracted from genetically modified flax seeds having > 70% and up to 75% ketogenic FAs instead of using > 50% and up to 55% or even fractionate or otherwise chemically347-004P 26 / 34manipulate native ALA rich oils for maximizing ALA content (to approach 100%), as is commonly included in conventional therapeutic MCT oil production.

[0091] The main components of oligounsaturated FO and EO in some embodiments of the ketogenic energy compositions herein are fatty acids having 3-4 double bonds, including C18:3(n-3) at ~56% and ~33%, respectively, C18:4(n-3) at ~12%, and C18:3(n-6) at ~11% in EO but not in FO, and C18:2(n-6) at ~15-17% and C18:l(n-9) at ~15-17%. Moreover, the EO contains slightly more palmitic acid than LO, i.e., C16:0 at ~7% vs. ~5%, respectively. Polyunsaturated fatty-acids (PUFAs) are interconverted in vivo but ALA conversion into the longer chain n-3 PUFAs EPA, DPA, and DHA was considered inefficient for biochemical reasons, i.e., to the relative inefficiency of the enzymatic desaturation and elongation of ALA.

[0092] Allowing for unusually efficient ALA bio-oxidation and utilisation for generating ketones as described herein reveals, however, that the non-oxidised systemically available ALA is relatively efficiently converted into the closely related longer chain PUFAs. In a typical case, daily ingestion of 10 g ALA thus corresponds to a 0.9 g EPA and 0.5 g DPA intake, showing that the doses tested in this study with the corresponding results, provide ample EPA and DHA to a mammalian body following consumption.

[0093] Table 1: Interconversion factors for the disclosed compositionsS / ope* Result ALA EPA DPA DHASource C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3ALA 0.156 0.09 0.05 —EPA 0.549 1.585 0.411 0.876DHA — 2.120 0.415 1.341* expressed as % / g source fatty acid / day.347-004P 27 / 34

[0094] Table 2: Characteristic distribution (in weight-%) of fatty acids in the tested oilsSummary of the compositions comprising the tested fats and oilsMilk Peanut Flaxseed Perilla EchiumFatty acid VCO Butter butter EVOO oil oil oil Butyric C4:0 3.4 Caproic C6:0 0.3 2.2 Caprylic C8:0 7.0 1.1 Capric C10:0 6.3 2.3 Lauric C12:0 47.4 3.4 Myristic C14:0 18.2 10.0 0.1 0.1 0.1 Pentadecanoic C15:0 0.0 0.0 Myristoleic C14:l(n-9) 1.1 Palmitic C16:0 8.5 33.1 10.5 9.7 5.0 5.9 7.0 Palmitoleic C16:l(n-7) 4.0 0.1 0.6 0.1 0.1 0.1 Margaric C17:0 0.2 0.1 0.1 Heptadecenoic C17:l 0.3 0.1 0.1 Stearic C18:0 2.9 9.0 4.5 3.0 3.3 2.2 3.9 Oleic C18:l(n-9) 7.2 26.1 49.5 79.8 17.9 16.0 15.3 c-Vaccenic C18:l(n-7) 0.5 0.5 Linoleic C18:2(n-6) 1.7 2.3 28.0 4.9 15.1 14.5 16.5 gamma- Linolenic C18:3(n-6) 0.1 0.6 0.2 0.2 10.8 alfa-Linolenic C18:3(n-3) 0.1 57.5 60.2 33.0 Stearidonic C18:4(n-3) 12.1 Arachidic C20:0 0.1 1.8 1.5 0.5 0.2 0.2 0.1 Gadoleic C20:l(n-9) 0.1 1.4 0.3 0.2 0.2 0.6 Eicosadienoic C20:2 0 0.0 0.0 Behenic C22:0 3.8 0.1 0.1 0.0 0.0 Lignoceric C24:0 0.1 0 0 0.0 Nervonic C24:l(n-9) 0 0.1Sum 100 100 100 100 100 100 100Total lipid 99.98 >80 47 99.9 99.9 99.98 99.98Water 0.02 15 0.01 0.01 0.02 0.02*VCO= virgin coconut oil; EVOO = extra virgin olive oil.Values > 7.5% are underlined.347-004P 28 / 34

Claims

CLAIMS1. An oligounsaturated ketogenic energy composition comprising one or more lipids comprising:(a) fatty acids having a plurality of double bonds in the terminal portion of the fatty acid; and(b) fatty acids having at least one double bond in the central portion of the fatty acid, wherein the composition provides a measurable increase in ketone levels following administration compared to baseline ketone levels upon metabolism in a mammalian subject.

2. An oligounsaturated ketogenic energy composition for use in promoting ketogenesis in a mammal, wherein the composition comprises one or more lipids comprising:(a) fatty acids having a plurality of double bonds in the terminal portion of the fatty acid; and(b) fatty acids having at least one double bond in the central portion of the fatty acid, wherein the composition provides a measurable increase in ketone levels following administration compared to baseline ketone levels upon metabolism in a mammalian subject.

3. The ketogenic energy composition of claims 1 to 2, wherein the oligounsaturated fatty acid is n-3 fatty acid.

4. The ketogenic energy composition according to claims 1 to 3, wherein the terminal portion of the fatty acid comprises 2 to 4 double bonds, preferably 3 double bonds.

5. The ketogenic energy composition according to any preceding claim wherein the total chain length of the fatty acid is 18 carbons (C18) or 20 carbons (C20).

6. A method for generating ketones in a mammalian subject comprising:347-004P 29 / 34(a) administering to the mammalian subject an effective amount of an oligounsaturated ketogenic energy composition according to any of claim 1 to 5;(b) generating ketones by beta-oxidation of the fatty acids upon metabolism by the mammalian subject; and(c) determining the generation of ketones by measuring an increase in ketone concentration in the mammalian subject following administration of the composition.

7. A method for generating ketones in a mammalian subject comprising:(a) administering to the mammalian subject an effective amount of a ketogenic energy composition comprising one or more lipids comprising one or more long chain fatty acids, wherein the fatty acids have a plurality of double bonds in the terminal portion of the fatty acid, and at least one double bond in the central portion of the fatty acid;(b) generating ketones by beta-oxidation of the fatty acids upon metabolism by the mammalian subject; and(c) determining the generation of ketones by measuring an increase in ketone concentration in the mammalian subject following administration of the composition.

8. A method for treating hypometabolism in a mammalian subject comprising:(a) administering to the mammalian subject an effective amount of an oligounsaturated ketogenic energy composition according to any of claims 1 to 5, preferably wherein the oligounsaturated fatty acid is an n-3 fatty acid;(b) generating ketones by beta-oxidation of the fatty acids upon metabolism by the mammalian subject; and(c) determining the generation of ketones by measuring an increase in ketone concentration in the mammalian subject following administration of the composition.

9. A method for treating hypometabolism in a mammalian subject comprising:(a) administering to the mammalian subject an effective amount of a ketogenic energy composition comprising one or more lipids comprising one or more long chain fatty347-004P 30 / 34acids, wherein the fatty acids have a plurality of double bonds in the terminal portion of the fatty acid, and at least one double bond in the central portion of the fatty acid;(b) generating ketones by beta-oxidation of the fatty acids upon metabolism by the mammalian subject;(c) determining the generation of ketones by measuring an increase in ketone concentration in the mammalian subject following administration of the composition; and / or(d) measuring an improvement of a biological effect associated with the hypometabolism condition in the mammalian subject.

10. The method according to claims 8 or 9, wherein the hypometabolism is associated with a disease or condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, bipolar depression, amyotrophic lateral sclerosis (ALS), multiple sclerosis, glucose transporter type 1 deficiency syndrome and related epilepsy, alcohol dependency, insulin resistance, systemic lupus erythematosus associated with psychiatric symptoms, anesthesia induced memory loss, age associated memory impairment, Friedreich's ataxia, Rabson-Mendenhall syndrome, leprechaunism, coronary arterial bypass graft dementia, or condition associated with an energy-depleted mammal following physical activity.

11. The method according any preceding claim, wherein the determining the generation of ketones is by measuring p-hydroxy-butyric acid (BHB) ketone in a blood sample obtained from the mammalian subject or by measuring acetone in exhaled breath of the mammalian subject.

12. The method according to any preceding claim, wherein the number of carbon atoms (nC) of the long chain fatty acids comprised in the ketogenic energy composition is 18 or 20 carbon atoms.

13. The method according to any preceding claim, wherein the ketogenic energy composition is administered orally or parenterally.347-004P 31 / 3414. The method according to claim 13, wherein the orally administered ketogenic energy composition is in the form of a foodstuff or liquid.

15. The method according to any preceding claim, wherein the ketogenic energy composition is administered sufficient to provide up to 60% of the subject's total daily energy expenditure, optionally wherein the total daily energy expenditure is calculated as resting metabolic rate x physical activity level.

16. The method according to any preceding claim, wherein the dosage of the ketogenic energy composition administered corresponds to a normalised daily administered digestible fat dose of between 0.5xBM°-713g / kg / day and lOxBM0713g / kg / day.

17. The method according to claim 16, wherein the administered daily dose of the ketogenic energy composition is selected based on a 10-50% of estimated total daily energy expenditure of the mammal.

18. The method according to any preceding claim, wherein the dosage of the ketogenic energy composition administered yields a systemic peak concentration of at least 1 mM BHB, preferably measured by acetone concentration in exhaled breath of the mammalian subject.

19. The method according to any preceding claim, wherein the administered composition further comprises a carbohydrate.

20. The method according to claim 19, wherein the carbohydrate is a resistant starch.

21. The method according to any preceding claim, wherein the mammalian subject is a human.

22. A kit for generating ketones in a mammalian subject, comprising an effective amount of the ketogenic energy composition according to any of claims 1 to 5, and instructions specifying optimized dosing protocols and timing regimens for administration of the ketogenic energy composition to measure a ketogenic effect in the mammal, optionally further comprising an acetone measuring device, and a BHB blood measurement assay.347-004P 32 / 3423. The kit according to claim 21, wherein ketogenic energy composition is provided in one or more containers with a volume selected from 100 ml, 250 ml, 500 ml, and / or or 1000 ml.347-004P 33 / 34

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