Promoting lipid-soluble retinol and alpha-tocopherol absorption using resistant starch supplementation

Abstract

We demonstrate that supplementation with resistant potato starch (RPS) influences the circulating levels of retinol, alpha-tocopherol, choline, and the choline-derived phospholipid sphingomyelin in serum. Levels of the toxic microbially transformed choline metabolites trimethylamine and trimethylamine oxide did not increase, suggesting that RPS may be an e ective way to enhance choline uptake without promoting increased toxic metabolite production. Increased hydroxysphingomyelin levels in RPS consuming individuals are particularly intriguing given the role these metabolites play in myelin.

Description

[0001] PROMOTING LIPID-SOLUBLE RETINOL AND ALPHA-TOCOPHEROL ABSORPTION USING RESISTANT STARCH SUPPLEMENTATION

[0002] PRIOR APPLICATION INFORMATION

[0003] The instant application claims the benefit of US Provisional Patent Application US63 / 728.887, filed December 6, 2024 and entitled “PROMOTING LIPID-SOLUBLE RETINOL AND ALPHA-TOCOPHEROL ABSORPTION USING RESISTANT STARCH SUPPLEMENTATION”, the entire contents of which are incorporated herein by reference for all purposes.

[0004] The instant application also claims the benefit of US Provisional Patent Application US63 / 712,780, filed October 28, 2024 and entitled “PROMOTING LIPID-SOLUBLE RETINOL AND ALPHA-TOCOPHEROL ABSORPTION USING RESISTANT STARCH SUPPLEMENTATION”, the entire contents of which are incorporated herein by reference for all purposes. BACKGROUND OF THE INVENTION

[0005] Resistant starch (RS) refers to the sum of starch and starch digestion products that are not absorbed in the small intestine and pass through to the large intestine

[0001] . Five categories of RS have been recognized: RS1 ; starch physically inaccessible to digestive enzymes, RS2; native starch granules, RS3; retrograded starch granules, RS4; chemically cross-linked starch, and RS5; starchlipid complexes that resist digestion [2]. Types 2 and 3 historically contributed significantly to dietary fiber intake, though modern American diets contain approximately 4g per day [3]. Lack of dietary RS is thought to contribute to non-communicable diseases like type 2 diabetes, with a target of 15-20g of RS per day required to have meaningful benefit [3]. While the mechanism of action by which RS promotes health benefits have not been elucidated, it is hypothesized that fermentation by microbiota in the large intestines plays a role. However, changes in beneficial microbiota are not strongly correlated with changes in health improvements in clinical trials examining these relationships (see [4] for example).

[0006] The relationship between prebiotic fermentation in the large intestine and nutrient absorption is an area of active investigation. Resistant potato starch (RPS) does not significantly influence macronutrient intake (Bush and Alfa. Submitted). High amylose maize starch (HAMS) promotes increases in magnesium, calcium, and zinc (mineral) absorption in rats in a dosedependent manner in healthy rats [5]. However, absorption of diet-derived vitamins has not been described.

[0007] We therefore measured the abundance of retinol and alpha-tocopherol, two diet-derived essential vitamins with important antioxidant properties, as well as choline, an essential nutrient. Consumption of RPS but not placebo led to significant, meaningful increases in the serum concentration of both vitamins and choline. The increases in retinol and alpha-tocopherol in people consuming RPS are surprising because they require bile acid-mediated emulsification of dietary fats and previous work demonstrated that RPS decreased bile acid conjugation [6], a bile acid property responsible for enhancing fat absorption. Furthermore, administration of taurocholic acid was shown to enhance retinol absorption in a cannulated rat model [7]. Had levels of conjugated bile acids increased with RPS treatment, it might be expected that lipid absorption would also be enhanced, along with fat soluble vitamins. It was also previously reported in this same population that RPS had no effect on macronutrient intake, so the increases in fat soluble vitamins cannot be explained by enhanced consumption of dietary fats, and increased choline cannot be explained by enhanced consumption of protein (Bush and Alfa. Submitted). Every previously reported benefit associated with RPS consumption has been attributed to prebiotic activity involving the microbiota located primarily in the large intestine [8] [9]

[0010]

[0011]

[0012] [4] [6] (Bush and Alfa. Submitted). Given that fat and protein absorption occurs almost exclusively in the small intestine, preceding contact with the microbiota in the large intestine, it is unlikely that RPS-dependent changes in microbial activity physically influence the absorption of lipid soluble vitamins or choline.

[0008] SUMMARY OF THE INVENTION

[0009] According to an aspect of the invention, there is provided a method of increasing circulating retinol levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0010] In another aspect of the invention, there is provided a method of increasing circulating alpha-tocopherol levels in an individual in need of such treatment comprising administeringto said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0011] In another aspect of the invention, there is provided a method of increasing circulating choline levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0012] In another aspect of the invention, there is provided a method of increasing circulating sphingomyelin levels in an individual in need of such treatment comprising administeringto said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0013] In some embodiments of the invention, the individual is a monogastric or pre-ruminant animal, including humans, swine, calves, dogs, or cats. In some embodiments of the invention, the dosage regimen is administering resistant potato starch daily for a period of 1-12 weeks. In this context, “daily” does not necessarily mean “every day”, but may mean for example 19 out of 20 days, 9 out of 10 days, 17 out of 20 days or 8 out of 10 days.

[0014] As discussed herein, the (daily) effective amount may be, for example, 0.5 to 40g, or 0.5 to 30g, or 0.5 to 7g, or 0.5 to 3.5g of resistant starch.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . (A) Consumption of the placebo had no effect on serum retinol levels, but RPS consumption led to a significant increase in serum retinol levels. (B) Consumption of the placebo had no effect on serum alpha-tocopherol levels, but RPS consumption led to a significant increase in serum alpha-tocopherol levels. (Student’s t-test, error bars = SEM)

[0017] Figure 2. (A) Consumption of the placebo had no effect on serum choline levels, but RPS consumption led to a significant increase in serum choline levels. (B) Serum trimethylamine levels were significantly reduced in both placebo and RPS treatment groups. (C) Serum levels of trimethylamine oxide were not significantly affected by either placebo or RPS treatment. (Student’s t-test, error bars = SEM)

[0018] Figure 3. Levels of d 18:1 Sphingomyelins with even fatty acid chains. Levels of d 18: 1 / 8:0 (A), d18:1 / 16:0 (E), di 8:1 / 18:0 (F), d18:20:0 (G), and d18:1 / 26:0 (J) were not affected by treatment. Levels of d18:1 / 10:0 (B), d18:1 / 12:0 (C), di 8:1 / 14:0 (D), d18:1 / 22:0 (H), and d18:1 / 24:0 (I) were significantly increased in the RPS group after 4 weeks but were unaffected by the placebo. (Student’s t-test, error bars = SEM)

[0019] Figure 4. Levels of d18:1 Sphingomyelins with odd fatty acid chains. Levels of d18:1 / 19:0

[0020] (A), d 18: 1 / 21 :0 (B), d18:1 / 23:0 (C), and d 18:25:0 (D) were not affected by treatment. (Student’s t- test, error bars = SEM)

[0021] Figure 5. Levels of d 18: 1 Sphingomyelins with unsaturated fatty acid chains. Levels of d18:1 / 16:1 (A), d18:1 / 18:1 (B), d18:1 / 18:2 (C), d18:20:1 (D), and d18:1 / 24:1 (E) were not affected by treatment. (Student’s t-test, error bars = SEM)

[0022] Figure 6. Levels of hydroxylated di 8: 1 Sphingomyelins. Levels of d18:1 / 17:0-GH (A) and d18:1 / 21 :0-OH (C) significantly increased in response to RPS supplementation but d18:1 / 19:0-OH

[0023] (B) did not. Placebo had no effect on hydroxylated di 8: 1 sphingomyelins. (Student’s t-test, error bars = SEM) Figure 7. Groups of d 18:1 Sphingomyelin. Levels of all d 18:1 sphingomyelins (A), d 18: 1 sphingomyelins with even length saturated fatty acid tails (C), and d18:1 sphingomyelins with unsaturated fatty acid tails (E) were not affected by treatment. Levels of d18:1 sphingomyelins with both even and odd saturated fatty acid chains (B), d 18: 1 sphingomyelins with odd length saturated fatty acid tails (D), and hydroxylated d18:1 sphingomyelins (F) were significantly increased in the RPS group after 4 weeks but were unaffected by the placebo. (Student’s t-test, error bars = SEM)

[0024] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

[0026] As used herein, “treating” in all its grammatical forms does not necessarily require the “curing” of the disease, disorder, episode, occurrence or incident, but rather refers to at least one of the following: lessening the frequency and / or duration and / or severity of feelings of illness and / or one or more symptoms associated with the disease, disorder, episode, occurrence or incident. For example, in the context of “treating” an individual with retinol levels, the “treatment” will result in an increase in the retinol levels as well as the corresponding health benefits associated therewith, as discussed herein.

[0027] As used herein, “circulating levels” refers to the levels of the compound(s) circulating in the blood of the patient or subject. Preferably, the patient or subject is a human, but may also include monogastric or pre-ruminant animals, such as swine, calves, dogs, or cats.

[0028] We investigated the effects of resistant potato starch (Solnul®) in two separate clinical trials. MSP Starch Products Inc manufactures Solnul®, an unmodified RS type 2 that is a food grade quality potato starch for human and animal food applications. While Solnul®, which contains 60% RS by AOAC 2002.02 methods, is used in the trials and experiments discussed herein, it is important to note that as discussed herein, another suitable unmodified potato starch or resistant potato starch (RS type 2) comprising at least 50% resistant starch or at least 55% resistant starch or at least 60% resistant starch on an ‘as is’ basis and meeting USP criteria for potato starch may be used. As discussed herein, we demonstrate that supplementation with resistant potato starch (RPS) influences the circulating level of retinol, alpha-tocopherol, choline, and sphingomyelin in serum.

[0029] In the clinical trial, participants were generally healthy adult males and females between 18-69 years of age with a body mass index (BMI) of 18.0 to S34.9 kg / m2. Enrolled participants agreed to not use any new vitamin, mineral or dietary supplement product until after the study completion and to not take any vitamins, minerals, or dietary supplements 14 days prior to Visit 2 (Randomization) until the completion of Visit 4, since consumption of these products may bias the results for the test product. Individuals with a BMI over 34.9 kg / m2were excluded as their health and any related metabolic changes may impact the results of this study. For similar health reasons, any individuals with a diagnosis of irritable bowel syndrome, dyspepsia, significant gastrointestinal disorders, or other major diseases were excluded. A formal sample size calculation was not performed. The sample size of 25 subjects per study group is consistent with our previous clinical investigation of RS [9].

[0030] A total of 98 participants from Guelph, ON and the surrounding area were screened for eligibility to obtain the required sample size of 75 participants (25 participants per study arm) who were enrolled in the study, which occurred from Oct 20, 2019 to Jan 6, 2020. The identity of the study intervention was blinded to the study staff and participants. The study included a screening visit from 30 days up to 14 days priorto randomization, a run in-period of 14 to 17 days priorto randomization, a baseline visit (Day 0) during which the randomization was performed, and serum collected, and 2 subsequent study visits at Weeks 1 and 4, respectively, where serum was collected.

[0031] During the baseline visit (Day 0), the participants were randomized to receive one of three study interventions as indicated by the randomization scheme: high dose (7g resistant potato starch (RPS)), low dose (3.5g RPS combined with 3.5g digestible corn starch), or placebo (7g digestible corn starch). The 3.5g RPS dose was combined with 3.5g digestible corn starch in order to keep the amount of carbohydrate consistent among all three treatment arms. It should be noted that digestible corn starch does not contain any dietary fiber [9]. The randomization scheme was generated by Nutrasource using SAS 9.4 PROC PLAN on 22OCT19: 15:44:40 with Seed Number: 1887363180. 75 subjects were randomized into 25 blocks with each block containing 3 subjects. The first dose of study intervention was demonstrated and administered by mixing the product in approximately 125 mL of cool or room temperature water and immediately drinking before the investigational product settled. Participants were instructed to consume the investigational product in the morning. A 31-day supply of the study intervention was provided to the study participants during the baseline visit. At Visit 3 (Week 1 ), unused study interventions / empty packaging were collected, and compliance was calculated. AtVisit 4 (Week 4, the final study visit), unused study products / empty packaging were collected, and compliance was calculated.

[0032] Participants were instructed that for Visits 3 and 4, they should return all sachets they were given and to not throw away any open or unopened sachets. Compliance was calculated based on the amount of study product consumed compared to the total amount of study product expected to have been consumed forthe given duration. Compliance forthis study was considered acceptable if participants consumed an average of s 80% of the study product for the given duration. One participant per treatment arm discontinued the study before Visit 4, and 2 participants in RPS high dose arm were excluded due to non-compliance (< 80% product consumption) and use of study-prohibited medication. Safety profiles were based on the safety analysis set (SAF; n = 75), while the full analysis set (FAS; n = 72) included participants who received at least one dose of study product and had at least one outcome assessment after dosing and the per protocol population (PP; n = 70) included only those who completed the study with overall compliance with study parameters. Serum samples were stored at -80°C (MRM Proteomics, Montreal, QC) before mass spectroscopy analysis (The Metabolomics Innovation Centre / The UVic- Genome BC Proteomics Centre, Victoria, BC).

[0033] Mass spectroscopy analysis of metabolites in serum was completed by The Metabolomics Innovation Centre (Victoria, BC). UPLC-MRM / MS was carried out on an Agilent 1290 UHPLC (Agilent Technologies, Santa Clara, CA) coupled to a Sciex 4000 QTRAP mass spectrometer (AB Sciex, Framingham, MA) operated in the multiple-reaction monitoring mode with negative-ion detection. Internal standard solutions were prepared in MeOH-acetonitrile and serially diluted with water. Serum samples were thawed at room temperature, aliquots mixed with the internal standard solution, vortexed, and sonicated in a water bath, before centrifugal clarification. Aliquots of supernatant were mixed with water to create sample solutions. A pooled sample of 10 randomly selected aliquots was also prepared as the QC sample solution, which was injected after every 20 samples. UPLC-MRM / MS data were acquired with Sciex Analyst software and batch processed with Sciex MultiQuant software (AB Sciex). Linear calibration curves of metabolites were constructed using standard solution concentrations for each analyte with an appropriate concentration range versus analyte-to-internal standard peak area ratios. Concentrations detected in each sample were calculated by interpolating the calibration curves with the analyte-to-internal standard peak area ratios measured from each sample.

[0034] For lipid analysis, authentic sphingomyelin compounds were acquired from Cayman Chem Inc. (Ann Arbor, Michigan, USA) or from Avanti Polar Lipids, LLC. (Alabaster, AL, USA). These lipids were used for optimization of the precursor-to-product ion transitions of multiple-reaction monitoring mass spectrometry (MRM / MS) by direct infusion of a standard solution of each of the lipids into a Sciex QTRAP 4000 mass spectrometer via a solvent delivery syringe pump. These lipids were also used to help construction of the putative MRM / MS ion transitions of their homologues in each class, for which the authentic compounds were commercially unavailable or not acquired, according to the structures of lipids deposited in the LIPID MAPS database (https: / / www.lipidmaps.org) or in the Human Metabolome Database (https: / / hmdb.ca).

[0035] 20 pL of human serum from each sample was aliquoted into a 1 .5-mL Eppendorf tube and mixed with 400 pL of a mixed solvent of methanol-chloroform (3:1 , v / v). The samples were vortexed for 1 min, ultra-sonicated in an icy water bath for 3 min and then centrifuged at 21 ,000 g and 5 °C for 10 min inside an Eppendorf 5425R centrifuge. The clear supernatants were transferred to 1-mL micro-vials and dried under a nitrogen gas flow. The dried residues were added with 200 pL of methanol-chloroform (1 :1 , v / v). After 5-s vortex mixing, 30-s ultra-sonication and 2-min centrifugal clarification at 21 ,000 g and 5 °C, 4-pL aliquots of the clear solutions were injected into a Waters XBridge C8 (2.1*50 mm, 2.5 pm) column to run liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) in the dynamic MRM scanning mode on an Agilent 1290 UHPLC instrument hyphenated via an atmospheric pressure electrospray ion source to an Agilent 6495B triple-quadrupole mass spectrometer. The mass spectrometer was operated in the positive-ion mode for detection of sphingolipids and glycerides. For chromatographic separations, a binarysolvent mobile phase composed of 2-mM ammonium acetate solution (pH adjusted to 4 with acetic acid) (solvent A) and 2-mM ammonium acetate in a mixture of water-acetonitrile- isopropanol (20:490:490, v / v / v) (solvent B) was used for gradient elution at 0.4 mL / min and 55 °C. The elution gradient was 0-6 min, 10% to 60% B; 6-22 min, 60% to 100% B and 22-24 min, 100% B. The chromatographic column was re-equilibrated at 10% B for 3 min between injections. For quality control (QC), aliquots of 20-pL serum were pooled from 30 randomly chosen samples. Thirteen 20-pL aliquots of the pooled serum sample were prepared for the same liquid extraction, along with processing of the batch samples. The resultant QC sample solution was injected periodically at the beginning, in the middle and at the end of the LC-MRM / MS batch runs to monitor the analytical precision. The LC-MRM / MS data were recorded and subsequently processed using the Agilent MassHunter 10.0 software suite. Peak areas of individual lipids detected in serum were integrated and used for relative quantitation and subsequent statistics.

[0036] Accuracy of quantitation by LC-MS is affected by multiple factors, including quality and stability of “standard” substances, the use of isotope-labeled internal standards, sample preparation procedures, LC separation conditions, matrix effects (often ionization suppression), and linearity between MS responses vs. concentrations (optimal linearity is only achieved with a certain concentration range for a specific compound), and in-solution stability of analytes during the batch runs. Forthese reasons, the concentrations reported are often lowerthan theirtrue concentration, and relative changes from baseline are considered most informative.

[0037] This study focused on the within group effects of 3.5g / day RPS administration and placebo administration. Baseline metabolite levels were compared to those at four-week time points using Student’s t-test where p values < 0.05 were considered significant.

[0038] RPS consumption for 4 weeks led to a significant 0.3pM (9%) increase in serum retinol levels (p = 0.03) while consumption of placebo for 4 weeks did not have a significant effect (p = 0.06; Figure 1 A). Similarly, RPS consumption for 4 weeks led to a significant 0.8pM (11 %) increase in serum alpha-tocopherol levels (p = 0.04) while consumption of placebo for 4 weeks did not have a significant effect (p = 0.05; Figure 1 B).

[0039] RPS consumption for 4 weeks led to a significant 0.2pM (15%) increase in serum choline levels (p = 0.02) while consumption of placebo for 4 weeks did not have a significant effect (p = 0.1 ; Figure 2A). RPS (p = 0.0002) and placebo (p = 0.000001) treatment led to significant reductions in trimethylamine (Figure 2B), but neither treatment had a significant effect on trimethylamine oxide.

[0040] Neither treatment affected levels of d 18: 1 / 8:0 (Figure 3A), d18:1 / 16:0 (Figure 3E), d18:1 / 18:0 (Figure 3F), d18:20:0 (Figure 3G), or d18:1 / 26:0 (Figure 3J). RPS treatment led to significant increases in the levels of d18:1 / 10:0 (p = 0.04; Figure 3B), d18:1 / 12:0 (p = 0.004; Figure 3C), di 8:1 / 14:0 (p = 0.03; Figure 3D), d18:1 / 22:0 (p = 0.04; Figure 3H), and d18:1 / 24:0 (p = 0.02; Figure 3I) but levels were unaffected by the placebo.

[0041] Neither treatment affected the levels of d18:1 sphingomyelins with odd fatty acid chains (Figure 4) or di 8: 1 sphingomyelins with unsaturated fatty acid chains (Figure 5). RPS consumption led to significant increases in the levels of d18:1 / 17:0-OH (p = 0.02; Figure 6A) and d18:1 / 21 :0-OH (p = 0.04; Figure 6C) but not d18:1 / 19:0-OH (Figure 6B). Placebo treatment had no effect on hydroxylated d18:1 sphingomyelins. Treatment had no effect on the levels of all d18:1 sphingomyelins (Figure 7A), d 18: 1 sphingomyelins with even length saturated fatty acid tails (Figure 7C), and d 18: 1 sphingomyelins with unsaturated fatty acid tails (Figure 7E). RPS consumption led to increases in the levels of d18:1 sphingomyelins with both even and odd saturated fatty acid chains (p = 0.02; Figure 7B), d18:1 sphingomyelins with odd length saturated fatty acid tails (p = 0.03; Figure 7D), and hydroxylated d18:1 sphingomyelins (p = 0.04; Figure 7F) but the placebo had no effect.

[0042] The absorption of dietary vitamins by the intestines is critical for human health because the body cannot synthesize these substances. Retinol, a form of vitamin A, and alpha-tocopherol, a form of Vitamin E, are absorbed from the diet in combination with dietary fats. Notably, conventional potatoes contain minute levels of retinol, provitamin A, and alpha-tocopherol, and neither potatoes nor potato products are considered sources of vitamin A or vitamin E

[0013] . Similarly, potatoes are not good sources of choline

[0014] . Retinol is derived from the diet via two mechanisms: It can be absorbed directly from animal sources, such as eggs and liver, or it can be converted from plant pigments such as beta-carotene, which are abundant in orange-colored vegetables and dark leafed vegetables, in the intestines. Notably, provitamin A beta-carotene is lipid soluble, like retinol, and requires fat for absorption. In Western diets, 70% of vitamin A comes from preformed retinol and the remaining 30% is derived from plant carotenoids, while greater than 70% of vitamin A needs are provided by carotenoids from fruit and vegetables in developing nations

[0015] . Beta-carotene 15,15’-monoxygenase converts provitamin carotenoids to retinol and polymorphisms in BCMO1 , the gene encodingthis enzyme, are known to influence this conversion and contribute to the high variability in provitamin A conversion observed in people

[0016] .

[0043] Vitamin A is required for various physiological processes, including immune system function, proper vision, epithelial integrity, fetal development, and both male and female reproductive systems

[0017] . Inadequate amounts of Vitamin A are associated with various disorders, including diabetes, obesity, cardiovascular disease, and hepatic disorders, as well as various cancers, eye disorders, and skin disease

[0018]

[0017] . Retinol can be metabolized into 11-cis- retinaldehyde, the chromophore component of the visual pigment rhodopsin or all-trans-retinoic acid, a ligand that exerts transcriptional control via the retinoic acid nuclear receptor

[0017] . Therefore, diets deficient in retinol and / or provitamin carotenoids tend to have pleiotropic effects in seemingly unconnected physiological systems.

[0044] Interventions with beta-carotene-supplemented and preformed retinol-supplemented diets have been evaluated in populations with vitamin A deficiency. In one example, supplementing the diet of vitamin A deficient Indonesian children with fruit, vegetables, or preformed retinol led to serum retinol increases of 0.07, 0.12, and 0.23 pM, respectively

[0019] . RPS supplementation for 4 weeks led to a 0.3 pM increase in serum retinol, which produces a greater increase than any of these dietary interventions, suggesting this method of increasing serum retinol is particularly efficacious. This finding is novel, as there are no reports that consumption of conventional potatoes or potato products leads to elevated levels of provitamin A, vitamin A, or retinol. Furthermore, serum retinol levels tend to be maintained under homeostatic control of the liver, which contains most of the retinol reserves in the body, meaning that the success of interventions can often be difficult to gauge

[0020] .

[0045] Among the various forms of vitamin E, alpha-tocopherol is the only form recognized to alleviate vitamin E deficiency in humans, including the inherited disease ataxia with vitamin E deficiency, as well as neuropathy and hemolytic anemia

[0021] . The antioxidant properties of alphatocopherol underlie some of these important health connections. For example, the excessive formation of reactive oxygen species in people with diabetes is thought to damage neurons, driving neuropathic pain, and the administration of exogenous antioxidants like alpha-lipoic acid are thought to provide benefit

[0022] . The antioxidant activity of alpha-tocopherol involves the termination of free radicals via hydrogen donation from the hydroxyl group of the chromane ring

[0023] . This effect is enhanced by vitamin C, which donates a hydrogen ion to restore alpha-tocopherol to a reduced state

[0023] . Absorption of free radicals by retinol and alpha-tocopherol can reduce the harmful effects of free radicals and mitigate these effects in vulnerable populations, such as diabetics.

[0046] Dietary sources of alpha-tocopherol include nuts, seeds and their oils, as well as spinach, broccoli, and some fruits

[0024] . Fat malabsorption and genetic mutations in TTPA, encoding alphatocopherol transfer protein, can adversely affect vitamin E absorption

[0025] . While vitamin E deficiency in the general population is rare

[0026] and increasing vitamin E levels via supplementation tends to be ineffective in some vulnerable populations

[0027]

[0028] , supplementation provides benefits to people at risk of certain disorders, such as dementia and Alzheimer’s disease

[0029] . RPS supplementation for 4 weeks led to a 0.8 pM increase in serum alpha-tocopherol, which is an 11% increase over baseline levels. This finding is novel, as there are no reports that consumption of conventional potatoes or potato products leads to elevated levels of alpha-tocopherol, other tocopherols, or other forms of vitamin E, and potatoes are known to have very low levels of these vitamins

[0013] . Choline is an essential nutrient for human health and development and is involved in the synthesis of phospholipids and acetylcholine, and for DNA methylation

[0030] . Dietary sources of choline include whole eggs, liver, meat, and whole grains

[0014] . While humans can synthesize some choline, this production is incapable of meeting the body’s needs

[0031] . Normal fasting plasma choline levels are between 7-9.3 pM, and levels tend to rise 25-30% following a light meal

[0032] . Choline levels in blood never decrease to less than 50% of baseline levels, even after a week without eating, suggesting that the hydrolysis of membrane phospholipids and liberation of choline may preserve levels in blood

[0031] . RPS supplementation led to a 15% increase in serum choline levels, suggesting that this method of increasing serum choline is particularly efficacious.

[0047] Dietary deficiency of choline can lead to muscle and liver damage, in some cases progressing into non-alcoholic fatty liver disease (NAFLD)

[0031]

[0033] . Furthermore, certain populations are particularly at risk of choline deficiency, including pregnant women

[0034] , those with polymorphisms in the genes encoding proteins important for choline metabolism

[0035] , people requiring total parenteral nutrition

[0036] , those with neurological conditions like Alzheimer’s disease

[0037] , and people at risk of developing NAFLD

[0038] . Dietary supplementation with choline is recommended among pregnant women and those trying to conceive, but the amount of supplemental choline is often inadequate to meet daily requirements, making a combination of dietary intake and supplementation necessary to meet daily targets in this population

[0039] .

[0048] Choline supplementation can also promote adverse health risks. Dietary choline was identified as a risk factor for cardiovascular disease (CVD) and mouse studies demonstrated that microbial transformation of choline enhanced the abundance of the toxic metabolite trimethylamine oxide (TMAO)

[0040] . Importantly, bacteria act on choline to produce the intermediate metabolite trimethylamine (TMA), which is then converted by the liver into the toxic form TMAO

[0041] . Previously, supplementing diets with high amylose maize starch, a form of RS type 2, was shown to increase levels of TMAO in healthy people

[0042] but had no effect on plasma choline orTMAO levels in chronic kidney disease patients

[0043] . In addition to CVD, TMAO has been implicated in the pathogenesis of different types of cancer, type 2 diabetes, chronic kidney disease, and neurodegenerative disorders

[0044] . It is therefore important that supplementation or other activities meant to improve serum choline levels be balanced against the risk of elevating TMAO, especially in those at risk of developing these diseases.

[0049] Sphingomyelins are a diverse class of phospholipid consisting of a sphingosine base attached to a fatty acid, forming a ceramide, and to phosphocholine

[0045] . Choline is required for the synthesis of sphingomyelins, first via conversion into phosphocholine and incorporation into phosphatidyl choline, before the phosphocholine molecule is transferred to a ceramide molecule. These phospholipids play important roles in maintaining the integrity of cell membranes, particularly in cells like neurons and oligodendrocytes that synthesize lipids important for myelination

[0045] . However, sphingomyelin cannot be obtained from the diet, as dietary supplementation with sphingomyelin does not lead to increased serum sphingomyelin levels

[0046] , requiring the body to synthesize sphingomyelin de novo.

[0050] Because of their role in maintaining cell membrane integrity and function, defects in sphingomyelin metabolism are associated with many pathological conditions. Sphingomyelin levels are lower in women with memory impairment, consistent with the role of this phospholipid in neuronal function

[0047] . These phospholipids also play roles in neurological diseases like Alzheimer’s disease and Parkinson’s disease. Neurons exposed to amyloid-beta, the hallmark of Alzheimer’s disease pathology, die rapidly and this effect can be inhibited by blocking the conversion of sphingomyelin to ceramide

[0048] . To this end, novel sphingomyelinase inhibitors are being trialed in patients with PD and AD

[0049]

[0050] . Outside of the nervous system, levels of sphingomyelin breakdown products like ceramides and sphingosine-1-phostphate are associated with increased osteoclastic activity, osteoblast apoptosis, osteoporosis, and reduced bone turnover, and reductions in sphingomyelin are associated with osteoporosis and reduced bone mineral density, reflecting the role sphingomyelin normally plays in promoted bone mineralization

[0051] -

[0051] It has been known for many years that hydroxylated forms of sphingomyelin are abundant in the myelin sheath, as are long chain sphingomyelins

[0052] . Mice lackingthe enzyme required for sphingomyelin hydroxylation develop normally but experience enhanced degeneration of the myelin sheath

[0053] . Degradation of sphingomyelin due to enhanced activity of sphingomyelinase has been reported in serum from patients with multiple sclerosis

[0054] , suggesting that supporting normal sphingomyelin levels may be beneficial in those affected by this disorder. In such cases, the fraction of sphingomyelin as a percentage of total phospholipid levels tends to be around 15% in plasma

[0054] .

[0052] While not wanting to be bound to a particular theory or hypothesis, it is possible that the same mechanism promotes enhanced production and / or release of pancreatic lipase and / or colipase, which could enhance lipid digestion and promote more efficient lipid soluble vitamin absorption

[0055] . Alternatively, RPS has been shown to promote enhanced barrier function, likely due to improved enterocyte function [4], so it is formally possible that even thought absorption of lipid soluble vitamins occurs in the small intestine, RPS promotes increased uptake of provitamin A, beta-carotene, retinol, and alpha-tocopherol. Enhanced enterocyte function and choline absorption could also explain increased levels of choline in serum. Alternatively, RPS may change the composition of the microbiome, reducing its capacity to produce TMA, thereby preserving higher levels of choline available for intestinal absorption into the blood. Notably, RPS-dependent increases in choline absorption did not result in increases in either microbe-derived TMA orTMAO, the liver-modified metabolite of TMA, but they did increase select forms of d 18: 1 sphingomyelin, including hydroxylated and saturated forms. Given that prebiotic activities of fermentable carbohydrates like RPS occur predominantly in the large intestine, away from the site of retinol, alpha-tocopherol, and choline absorption, the observed increases in serum retinol, alphatocopherol, and choline are unanticipated.

[0053] In summary, RPS supplementation increases serum retinol, alpha-tocopherol, and choline concentrations but the placebo did not have a significant effect. While further research is required to elucidate the mechanisms by which these nutrient changes can be attributed to consumption of RPS, the fact that RPS administration on a dosage regimen increases serum retinol, alphatocopherol, and choline, and the subsequent increase in choline-containing sphingomyelin, has been clearly demonstrated.

[0054] According to an aspect of the invention, there is provided a method of increasing circulating retinol levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0055] As such, in this context, “an individual in need of such treatment” may be an individual who has circulating retinol levels lower than a control individual, for example, lower than or less than 20 mcg / dL

[0056] or lower than 0.7 pM in an individual who is a child, for example, a vitamin A deficient child

[0057] or a very low birth weight infant

[0058] , an individual who is pregnant, postpartum, or nursing

[0059] , an individual with polymorphisms in BCMO1 , night blindness, conjunctival xerosis, Bitot’s spot, corneal xerosis, corneal ulcer, keratomalacia, corneal scarring, orxerophthalmic fundus

[0018] , an individual with epithelial proliferation disorders, such as phrynoderma

[0060] , or otherwise is in need of higher circulating retinol or vitamin A levels. The individual might also be someone with less than <0.07 pmol retinol / g of liver

[0061] . The individual may also be taking vita min A, retinol, provitamin A, or carotenoids, including but not limited to beta-carotene, or may be an individual who has been clinically advised or is otherwise deliberately consuming a diet rich in vitamin A, retinol, provitamin A, or carotenoids, including but not limited to beta-carotene, to increase their levels of vitamin A or retinol.

[0056] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for increasing circulating retinol levels or for reducing the severity of night blindness, xerosis of the eye, or related ocular insults compared to an individual of similar age and condition not administered the dosage regimen (referred to herein elsewhere as a "control individual").

[0057] According to another aspect of the invention, wherein the individual is an infant, a post-partum or nursing woman, or is susceptible to vitamin A or retinol deficiency due to genetic factors or family history, there is provided use of native RPS (RS type 2) for mitigating the severity of vitamin A or retinol deficiency.

[0058] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating the severity of vitamin A or retinol deficiency in an individual wherein the individual is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

[0059] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating skin problems, dry eyes, night blindness, or immune system impairment in an individual wherein the individual is susceptible to vitamin A deficiency due to genetic factors, such as polymorphisms in BCMO1 , or family history., for example, skin problems, dry eyes, night blindness or immune system impairment known in the art as being associated with Vitamin A deficiency.

[0060] In some embodiments of the invention, the resistant potato starch is administered daily for a period of 1-12 weeks, for example, 2-12 weeks, 3-12 weeks, 4-12 weeks, 5-12 weeks, 6-12 weeks, 7-12 weeks, 8-12 weeks, 9-12 weeks, 10-12 weeks, 11-12 weeks or 12 weeks. In this context, "daily" does not necessarily mean "every day" but may mean for example 19 out of 20 days, 9 out of 10 days, 17 out of 20 days or 8 out of 10 days or at least 80% of the total days of the treatment period.

[0061] Alternatively, in some embodiments, the dosage regimen may be continued until the circulating retinol levels of the individual have been increased by a certain percentage, for example, by at least 5%, by at least 10%, by at least 15% or at least 20% compared to an initial, pre-dosage regimen circulating retinol level or until the circulating retinol level is above a threshold level, for example, above 20 mcg / dL in an individual with night blindness or an epithelial proliferation disorder, for example, phrynoderma, or above 0.07 pM in an individual who is a vitamin A deficient child or low birthweight individual. As discussed herein, the (daily) effective amount may be, for example, 0.5 to 40g, or 0.5 to 30g, or 0.5 to 7g, or 0.5 to 3.5g of resistant starch.

[0062] According to another aspect of the invention, there is provided a method of increasing circulating alpha-tocopherol levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0063] As such, in this context, “an individual in need of such treatment” may be an individual who has circulating alpha-tocopherol levels lower than a control individual, for example, lower than 12 pM in a vitamin E deficient individual or less than 30 pM in an individual with sub-optimal vitamin E status

[0062] , less than 9.3 pM in an individual who is an infant

[0063] , less than 7.4 pM in a child or adolescent

[0064] , less than 12.0 pM in a pregnant woman

[0065] , or less than 14.0 pM in someone who is elderly

[0066] . The individual might also be someone with genetic mutations in TTPA, ataxia with vitamin E deficiency (AVEA), spinocerebellar ataxia, cholestatic liver disease, cystic fibrosis, or is otherwise needing higher alpha-tocopherol or vitamin E levels

[0067] . The individual may also be taking vitamin E, alpha-tocopherol, or other tocopherols ortocotrienols, or may be an individual who has been clinically advised or is otherwise deliberately consuming a diet rich in vitamin E, alpha-tocopherol, or other tocopherols or tocotrienols to increase their levels of vitamin E or alphatocopherol.

[0064] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for increasing circulating retinol levels or for reducing the severity of symptoms associated with AVEA, spinocerebellar ataxia, cholestatic liver disease, or cystic fibrosis compared to an individual of similar age and condition not administered the dosage regimen (referred to herein elsewhere as a "control individual").

[0065] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating the severity of vitamin E or alpha-tocopherol deficiency in an individual wherein the individual is an infant, a post-partum or nursing woman, or is susceptible to vitamin E or alphatocopherol deficiency due to genetic factors or family history.

[0066] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating the severity of vitamin E or alpha-tocopherol deficiency in an individual wherein the individual is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite. According to another aspect of the invention, there is provided use of native RPS (RS type 2) for treating or mitigating AVEA, spinocerebellar ataxia, cholestatic liver disease, or cystic fibrosis in an individual wherein the individual is susceptible to vitamin A deficiency due to genetic factors, such as polymorphisms in TTPA, or family history.

[0067] In some embodiments of the invention, the resistant potato starch is administered daily for a period of 1-12 weeks, for example, 2-12 weeks, 3-12 weeks, 4-12 weeks, 5-12 weeks, 6-12 weeks, 7-12 weeks, 8-12 weeks, 9-12 weeks, 10-12 weeks, 11-12 weeks or 12 weeks. In this context, "daily" does not necessarily mean "every day" but may mean for example 19 out of 20 days, 9 out of 10 days, 17 out of 20 days or 8 out of 10 days or at least 80% of the total days of the treatment period.

[0068] Alternatively, in some embodiments, the dosage regimen may be continued until the circulating alpha-tocopherol levels of the individual have been increased by a certain percentage, for example, by at least 5%, by at least 10%, by at least 15% or at least 20% compared to an initial, pre-dosage regimen circulating alpha-tocopherol level or until the circulating alpha-tocopherol level is above a threshold level, for example, above 14.0 pM in an individual with ataxia with vitamin E deficiency, spinocerebellar ataxia, cholestatic liver disease, or cystic fibrosis, or above 9.3 pM in an individual who is a vitamin E deficient child or low birthweight individual.

[0069] As discussed herein, the effective amount may be, for example, 0.5 to 40g, or 0.5 to 30g, or 0.5 to 7g, or 0.5 to 3.5g of resistant starch.

[0070] According to another aspect of the invention, there is provided a method of increasing circulating choline levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

[0071] As such, in this context, “an individual in need of such treatment” may be an individual who has circulating retinol levels lower than a control individual, for example, lower than or less than 7.0 pM

[0032] , a pregnant woman or a woman trying to conceive

[0034] , an individual with one or more polymorphisms in the genes encoding proteins important for choline metabolism

[0035] , an individual requiring total parenteral nutrition

[0036] , someone with a neurological condition like Alzheimer’s disease, for example

[0037] , or a person with NAFLD or who is at risk of developing NAFLD due to hereditary factors

[0038] , or an individual who otherwise is in need of higher circulating choline levels. The individual may also be taking choline supplements or prenatal vitamins containing choline, or may be an individual who has been clinically advised or is otherwise deliberately consuming a diet rich in choline to increase their levels of choline in serum. According to another aspect of the invention, there is provided use of native RPS (RS type 2) for increasing circulating choline levels or for reducing the severity of muscle damage, liver damage, NAFLD, or related liver insults compared to an individual of similar age and condition not administered the dosage regimen (referred to herein elsewhere as a "control individual").

[0072] According to another aspect of the invention, wherein the individual is an infant, a post-partum or nursing woman, or is susceptible to choline deficiency due to genetic factors or family history, there is provided use of native RPS (RS type 2) for mitigating the severity of choline deficiency.

[0073] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating the severity of choline deficiency in an individual wherein the individual is using a glucagon- like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

[0074] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for increasing choline levels without increasing TMA and / or TMAO levels in an individual where serum levels of choline need to be increased but increased TMAO levels would be harmful, including someone with CVD, cancer, type 2 diabetes, chronic kidney disease, or a neurological condition, such as Alzheimer's disease.

[0075] In some embodiments of the invention, the resistant potato starch is administered daily for a period of 1-12 weeks, for example, 2-12 weeks, 3-12 weeks, 4-12 weeks, 5-12 weeks, 6-12 weeks, 7-12 weeks, 8-12 weeks, 9-12 weeks, 10-12 weeks, 11-12 weeks or 12 weeks. In this context, "daily" does not necessarily mean "every day" but may mean for example 19 out of 20 days, 9 out of 10 days, 17 out of 20 days or 8 out of 10 days or at least 80% of the total days of the treatment period.

[0076] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for increasing circulating sphingomyelin levels or for reducing the severity of symptoms associated with Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, or multiple sclerosis compared to an individual of similar age and condition not administered the dosage regimen (referred to herein elsewhere as a "control individual").

[0077] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating the severity of excessive sphingomyelin degradation in an individual wherein the individual is susceptible to Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, or multiple sclerosis due to genetic factors or family history.

[0078] According to another aspect of the invention, there is provided use of native RPS (RS type 2) for mitigating the severity of sphingomyelin deficiency in an individual wherein the individual is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

[0079] In some embodiments of the invention, the resistant potato starch is administered daily for a period of 1-12 weeks, for example, 2-12 weeks, 3-12 weeks, 4-12 weeks, 5-12 weeks, 6-12 weeks, 7-12 weeks, 8-12 weeks, 9-12 weeks, 10-12 weeks, 11-12 weeks or 12 weeks. In this context, "daily" does not necessarily mean "every day" but may mean for example 19 out of 20 days, 9 out of 10 days, 17 out of 20 days or 8 out of 10 days or at least 80% of the total days of the treatment period.

[0080] Alternatively, in some embodiments, the dosage regimen may be continued until the circulating sphingomyelin levels of the individual have been increased by a certain percentage, for example, by at least 5%, by at least 10%, by at least 15% or at least 20% compared to an initial, pre-dosage regimen circulating sphingomyelin level or until the circulating sphingomyelin level is above a threshold level, for example, above 20% of the total phospholipid pool in an individual who has Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, or multiple sclerosis.

[0081] As discussed herein, the effective amount may be, for example, 0.5 to 40g, or 0.5 to 30g, or 0.5 to 7g, or 0.5 to 3.5g of resistant starch.

[0082] As will be appreciated by one of skill in the art, “baseline” as used herein in regards retinol, alpha-tocopherol, choline, and sphingomyelin refers to a measured level of retinol, alphatocopherol, choline, or sphingomyelin present in blood of, for example, in a blood sample taken from, one individual priorto that individual starting supplementation with digestion resistant potato starch. Alternatively, the “baseline” level can refer to the measured level of a control individual who is of similar age, weight and general health as the supplemented individual.

[0083] REFERENCES

[0084]

[0001] H. Englyst, S. Kingman, G. Hudson and J. Cummings, "Measurement of resistant starch in vitro and in vivo," Br J Nutr, vol. 75, no. 5, pp. 749-755, 1996.

[0085] [2] M. Sajilata, R. Singhal and P. Kulkarni, "Resistant Starch - A Review," Comp Rev Food Sci Food Saf, vol. 5, no. 1 , pp. 1 -17, 2006.

[0086] [3] D. Miketinas, K. Shankar and M. P. M. Maiya, "Usual Dietary Intake of Resistant Starch in US Adults from NHANES 2015-2016," J Nutr, vol. 150, no. 10, pp. 2738-3747, 2020.

[0087] [4] J. Bush, J. Han, E. Deehan, S. Harding, M. Maiya, J. Baisley, D. Schibli and D. Goodlett, "Resistant potato starch supplementation reduces serum histamine levels in healthy adults with links to attenuated intestinal permeability," J Funct Foods, vol. 108, p. 105740, 2023.

[0088] [5] M. Nisa, L. Kasankala, F. Khan, F. Al-Asmari, M. Abdul Rahim, I. Hussain, A. Angelov and E. R. J. Bartkiene, "Impact of resistant starch: Absorption of dietary minerals, glycemic index and oxidative stress in healthy rats," Clin Nutr ESPEN, vol. 62, pp. 1-9, 2024.

[0089] [6] J. Bush, I. Iwuamadi, J. Han, D. Schibli, D. Goodlett and E. Deehan, "Resistant Potato Starch Supplementation Reduces Serum Free Fatty Acid Levels and Influences Bile Acid Metabolism," Metabolites, vol. 14, no. 10, p. 536, 2024.

[0090] [7] D. Hollander, "Retinol lymphatic and portal transport: influence of pH, bile, and fatty acids," Am J Physiol Gastrointest Liver Physiol, vol. 239, no. 3, pp. G210-G214, 1980.

[0091] [8] M. Alfa, D. Strang, P. Tappia, M. Graham, G. Van Domselaar, J. Forbes, V. Laminman, N. Olson, P. DeGagne, D. Bray, B.-L. Murray, B. Dufault and L. Lix, "A randomized trial to determine the impact of a digestion resistant starch composition on the gut microbiome in older and mid-age adults," Clin Nutr, vol. 37, no. 3, pp. 797-807, 2018.

[0092] [9] M. Alfa, D. Strang, P. Tappia, N. Olson, P. DeGagne, D. Bray, B. Murray and B. Hiebert, "A Randomized Placebo Controlled Clinical Trial to Determine the Impact of Digestion Resistant Starch MSPrebiotic® on Glucose, Insulin, and Insulin Resistance in Elderly and Mid-Age Adults," Front Med (Lausanne), vol. 4, p. 260, 2018.

[0093]

[0010] J. Bush and M. Alfa, "Decreasing levels of Sporacetigenium correlate with improved diabetic parameters in healthy adults consuming MSPrebiotic® digestion resistant starch," J Aging Res Clin Practice, vol. 7, pp. 176-180, 2018.

[0094]

[0011] J. Bush and M. Alfa, "Increasing levels of Parasutterella in the gut microbiome correlate with improving low-density lipoprotein levels in healthy adults consuming resistant potato starch during a randomised trial," BMC Nutrition, vol. 6, no. 1 , p. 72, 2020.

[0095]

[0012] J. Bush, J. Baisley, S. Harding and M. Alfa, "Consumption of Solnul™ Resistant Potato Starch Produces a Prebiotic Effect in a Randomized, Placebo-Controlled Clinical Trial," Nutrients, vol. 15, no. 7, p. 1582, 2023.

[0013] C. Chitchumroonchokchai, G. Diretto, B. Parisi, G. Giuliano and M. Failla, "Potential of golden potatoes to improve vitamin A and vitamin E status in developing countries," PLoS ONE, vol.

[0096] 12, no. 11 , p. e0187102, 2017.

[0097]

[0014] K. Patterson, S. Bhagwat, J. Williams, J. Howe and J. Holden, "USDA Database for the Choline Content of Common Foods," January 2008. [Online]. Available: https: / / www.ars.usda.gov / ARSUserFiles / 80400525 / data / choline / choln02.pdf. [Accessed 20 11 2024].

[0098]

[0015] G. Tang, "Bioconversion of dietary provitamin A carotenoids to vitamin A in humans," Am J Clin Nutr, vol. 91 , no. 5, pp. 1468S-1473S, 2010.

[0099]

[0016] W. Leung, S. Hessel, C. Meplan, J. Flint, V. Oberhauser, F. Tourniaire, J. Hesketh, J. von Lintig and G. Lietz, "Two common single nucleotide polymorphisms in the gene encoding beta-carotene 15,15'-monoxygenase alter beta-carotene metabolism in female volunteers," FASEB J, vol. 23, no. 4, pp. 1041-1053, 2009.

[0100]

[0017] W. Blaner, "Vitamin A and provitamin A carotenoids," in Present Knowledge in Nutrition, Cambridge, MA, Academic Press, 2020, pp. 73-91 .

[0101]

[0018] C. Gilbert, "The eye signs of vitamin A deficiency," Community Eye Health, vol. 26, no. 84, pp. 66-67, 2013.

[0102]

[0019] S. de Pee, C. West, D. Permaesih, S. Martuti, S. Muhilal and J. Hautvast, "range fruit is more effective than are dark-green, leafy vegetables in increasing serum concentrations of retinol and beta-carotene in schoolchildren in Indonesia," Am J Clin Nutr, vol. 68, no. 5, pp. 1058-1067, 1998.

[0103]

[0020] S. Tanumihardjo, "Vitamin A: biomarkers of nutrition for development," Am J Clin Nutr, vol. 94, no. 2, pp. 658S-665S, 2011 .

[0104]

[0021] M. Traber, "Vitamin E inadequacy in humans: causes and consequences," Adv Nutr, vol. 5, no. 5, pp. 503-514, 2014.

[0105]

[0022] A. Atmaca, A. Ketenci, I. Sahin, I. Sengun, R. Oner, H. Erdem Tilki, M. Adas, H. Soyleli and T. Demir, "Expert opinion on screening, diagnosis and management of diabetic peripheral neuropathy: a multidisciplinary approach," Front Endocrinol (Lausanne), vol. 15, p. 1380929, 2024.

[0106]

[0023] M. Zeece, "Chapter Five - Vitamins and Mineral," in Introduction to Food Chemistry, Cambridge, MA, Academic Press, 2020, pp. 163-212.

[0107]

[0024] National Institutes of Health Office of Dietary Supplements, "Vitamin E: Fact Sheet for Health Professionals," 26032021 . [Online]. Available: https: / / ods.od.nih.gov / factsheets / VitaminE- HealthProfessional / . [Accessed 24102024].

[0108]

[0025] K. Ouahchi, M. Arita, H. Kayden, F. Hentati, M. Ben Hamida, R. Sokol, H. Arai, K. Inoue, J. Mandel and M. Koenig, "Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein," Nat Genet, vol. 9, no. 2, pp. 141-145, 1995.

[0026] I. o. Medicine, Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, Washington, DC: The National Academies Press, 2000.

[0109]

[0027] A.-R. E. D. S. R. Group, "A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8," Arch Ophthalmol, vol. 119, no. 10, pp. 1417- 1436, 2001.

[0110]

[0028] R. Xu, S. Zhang, A. Tao, G. Chen and M. Zhang, "Influence of vitamin E supplementation on glycaemic control: a meta-analysis of randomised controlled trials," PLoS ONE, vol. 9, no. 4, p. e95008, 2014.

[0111]

[0029] R. Zhao, X. Han, H. Zhang, J. Liu, M. Zhang, W. Zhao, S. Jiang, R. Li, H. Cai and H. You, "Association of vitamin E intake in diet and supplements with risk of dementia: A meta-analysis," Front Aging Neurosci, vol. 14, p. 955878, 2022.

[0112]

[0030] Office of Dietary Supplements, "Choline: Fact Sheet for Health Professionals," National Institutes of Health, 2 June 2022. [Online]. Available: https: / / ods.od.nih.gov / factsheets / Choline- HealthProfessional / #:~:text=Choline0 / o20is0 / o20an%20essential%20nutrient,phospholipids%20vital %20for%20cell%20membranes.. [Accessed 20 11 2024].

[0113]

[0031] S. Zeisel, "Choline," in Modern Nutrition in Health and Disease, Baltimore, Lippincott Williams & Wilkins, 2014, pp. 416-426.

[0114]

[0032] P. Holm, P. Ueland, G. Kvalheim and E. Lien, "Determination of choline, betaine, and dimethylglycine in plasma by a high-throughput method based on normal-phase chromatographytandem mass spectrometry," Clin Chem, vol. 49, no. 2, pp. 286-294, 2003.

[0115]

[0033] K. Corbin and S. Zeisel, "Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression," Curr Opin Gastroenterol, vol. 28, no. 2, pp. 159-165, 2018.

[0116]

[0034] K. Brunst, R. Wright, K. DiGioia, M. Enlow, H. Fernandez, R. Wright and S. Kannan, "Racial / ethnic and sociodemographic factors associated with micronutrient intakes and inadequacies among pregnant women in an urban US population," Public Health Nutr, vol. 17, no. 9, pp. 1960-1970, 2014.

[0117]

[0035] K. da Costa, O. Kozyreva, J. Song, J. Galanko, L. Fischer and S. Zeisel, "Common genetic polymorphisms affect the human requirement for the nutrient choline," FASEB J, vol. 20, no. 9, pp. 1336-1344, 2006.

[0118]

[0036] V. Vanek, P. Borum, A. Buchman, T. Fessler, L. Howard, K. Jeejeebhoy, M. Kochevar, A. Shenkin, C. Valentine, N. N. T. F. P. M.-V. a. M.-T. E. W. Group and A. B. o. Directors, "A.S.P.E.N. position paper: recommendations for changes in commercially available parenteral multivitamin and multi-trace element products," Nutr Clin Pract, vol. 27, no. 4, pp. 440-449, 2012.

[0037] E. Nurk, H. Refsum, I. Bjelland, C. Drevon, G. Tell, P. Ueland, S. Vollset, K. Engedal, H.

[0119] Nygaard and D. Smith, "Plasma free choline, betaine and cognitive performance: the Hordaland Health Study," Br J Nutr, vol. 109, no. 3, pp. 511 -519, 2013.

[0120]

[0038] A. Buchman, M. Dubin, A. Moukarzel, D. Jenden, M. Roch, K. Rice, J. Gornbein and M. Ament, "Choline deficiency: a cause of hepatic steatosis during parenteral nutrition that can be reversed with intravenous choline supplementation," Hepatology, vol. 22, no. 5, pp. 1399-1403, 1995.

[0121]

[0039] K. Cochrane, J. Hutcheon and C. Karakochuk, "Supplementation practices among pregnant women and those trying to conceive: a population-representative survey in Vancouver, Canada," Appl Physiol Nutr Metab, vol. 49, no. 11 , pp. 1495-1506, 2024.

[0122]

[0040] Z. Wang, E. Klipfell, B. Bennett, R. Koeth, B. Levison, B. Dugar, A. Feldstein, E. Britt, X. Fu, Y. Chung, Y. Wu, P. Schauer, J. Smith, H. Allayee, W. Tang, J. DiDonato, A. Lusis and H. S. , "Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease," Nature, vol. 472, no. 7341 , pp. 57-63, 2011.

[0123]

[0041] J. Zhen, Z. Zhou, M. He, H. Han, E. Lv, P. Wen, X. Liu, Y. Wang, X. Cai, J. Tian, M. Zhang, L. Xiao and X. Kang, "The gut microbial metabolite trimethylamine N-oxide and cardiovascular diseases," Front Endocrinol (Lausanne), vol. 14, p. 1085041 , 2023.

[0124]

[0042] N. Bergeron, P. Williams, R. Lamendella, N. Faghihnia, A. Grube, X. Li, Z. Wang, R. Knight, J. Jansson, S. Hazen and R. Krauss, "Diets high in resistant starch increase plasma levels of trimethylamine-N-oxide, a gut microbiome metabolite associated with CVD risk," Br J Nutr, vol. 116, no. 12, pp. 2020-2029, 2016.

[0125]

[0043] D. S. H. d. J. H. E. M. d. P. B. A. R. S. P. B. P. L. B. R.-A. M. M. D. Kemp JA, "Resistant Starch Type-2 Supplementation Does Not Decrease Trimethylamine N-Oxide (TMAO) Plasma Level in Hemodialysis Patients," J Am Nutr Assoc, vol. 41 , no. 8, pp. 788-795, 2022.

[0126]

[0044] Y. Zhou, Y. Zhang, S. Jin, J. Lv, M. Li and N. Feng, "The gut microbiota derived metabolite trimethylamine N-oxide: Its important role in cancer and other diseases," Biomed Pharmacother, vol. 177, p. 117031 , 2024.

[0127]

[0045] B. Quinville, N. Deschenes, A. Ryckman and J. Walia, "A Comprehensive Review: Sphingolipid Metabolism and Implications of Disruption in Sphingolipid Homeostasis," Int J Mol Sci, vol. 22, no. 11 , p. 5793, 2021.

[0128]

[0046] C. Li, L. Wu, C. Zhu, H. Du, G. Chen and F. Yang, "The impacts of dietary sphingomyelin supplementation on metabolic parameters of healthy adults: a systematic review and metaanalysis of randomized controlled trials," Front Nutr, vol. 11 , p. 1363077, 2024.

[0129]

[0047] M. Mielke, V. Bandaru, N. Haughey, P. Rabins, C. Lyketsos and M. Carlson, "Serum sphingomyelins and ceramides are early predictors of memory impairment," Neurobiol Aging, vol. 31 , no. 1 , pp. 17-24, 2010.

[0048] R. Cutler, J. Kelly, K. Storie, W. Pedersen, A. Tammara, K. Hatanpaa, J. Troncoso and M. Mattson, "Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease," Proc Natl Acad Sci USA, vol. 101 , no. 7, pp. 2070-2075, 2004.

[0130]

[0049] C. Zhu, T. Bilousova, S. Focht, M. Jun, C. Elias, M. Melnik, S. Chandra, J. Campagna, W. Cohn, A. Hatami, P. Spillman, K. Hoppens Gylys and V. John, "Pharmacological inhibition of nSMase2 reduces brain exosome release and a-synuclein pathology in a Parkinson’s disease model," Mol Brain, vol. 14, p. 70, 2021 .

[0131]

[0050] M. Sala, K. Hollinger, A. Thomas, R. Dash, C. Tallon, V. Veeravalli, L. Lovell, M. Kbgler, H. Hrebabecky, E. Prochazkova, O. Nesuta, A. Donohue, J. Lam, R. Rais, C. Rojas, B. Slusher and R. Nencka, "Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer’s Disease," J Med Chem, vol. 63, no. 11 , pp. 6028-6056, 2021 .

[0132]

[0051] A. Seal, M. Hughes, F. Wei, A. Pugazhendhi, C. Ngo, J. Ruiz, J. Schwartzman and M. Coathup, "Sphingolipid-Induced Bone Regulation and Its Emerging Role in Dysfunction Due to Disease and Infection," Int J Mol Sci, vol. 25, no. 5, p. 3024, 2024.

[0133]

[0052] S. Staellberg-Stenhagen and L. Svennerholm, "Fatty acid composition of human brain sphingomyelins: normal variation with age and changes during myelin disorders," J Lipid Res, vol. 6, pp. 146-155, 1965.

[0134]

[0053] I. Zoller, M. Meixner, D. Hartmann, H. Bussow, R. Meyer, V. Gieselmann and M. Eckhardt, "Absence of 2-hydroxylated sphingolipids is compatible with normal neural development but causes late-onset axon and myelin sheath degeneration," J Neurosci, vol. 28, no. 39, pp. 9741-9754, 2008.

[0135]

[0054] A. Momchilova, R. Pankov, A. Alexandrov, T. Markovska, S. Pankov, P. Krastev, G. Staneva, E. Vassileva, N. Krastev and A. Pinkas, "Sphingolipid Catabolism and Glycerophospholipid Levels Are Altered in Erythrocytes and Plasma from Multiple Sclerosis Patients," Int J Mol Sci, vol. 23, no. 14, p. 7592, 2022.

[0136]

[0055] Y. Pirahanchi and S. Sharma, "Biochemistry, Lipase," in StatPearls, Treasure Island, FL, StatPearls Publishing, 2023.

[0137]

[0056] S. Patil, U. Zamwar and A. Mudey, "Etiology, Epidemiology, Pathophysiology, Signs and Symptoms, Evaluation, and Treatment of Vitamin A (Retinol) Deficiency," Cureus, vol. 15, no. 11 , p. e49011 , 2023.

[0138]

[0057] M. Rahman, W. Wahed, G. Fuchs, A. Baqui and J. Alvarez, "Synergistic effect of zinc and vitamin A on the biochemical indexes of vitamin A nutrition in children," Am J Clin Nutr, vol. 75, no. 1 , pp. 92-98, 2002.

[0058] S. Kositamongkol, U. Suthutvoravut, N. Chongviriyaphan, B. Feungpean and P. Nuntnarumit, "Vitamin A and E status in very low birth weight infants," J Perinatol, vol. 31 , no. 7, pp. 471-476, 2011.

[0139]

[0059] WHO Guideline Review Committee, Guideline: Vitamin A Supplementation in Postpartum Women, Geneva, CH: World Health Organization, 2011 .

[0140]

[0060] M. Maronn, D. Allen and N. Esterly, "Phrynoderma: a manifestation of vitamin A deficiency?... The rest of the story," Pediatr Dermatol, vol. 22, no. 1 , pp. 60-63, 2005.

[0141]

[0061] F. Rosales, K. Chau, M. Haskell and A. Shankar, "Determination of a cut-off value forthe molar ratio of retinol-binding protein to transthyretin (RBP:TTR) in Bangladeshi patients with low hepatic vitamin A stores," J Nutr, vol. 132, no. 12, pp. 3687-3692, 2002.

[0142]

[0062] A. Malik, M. Eggersdorfer and G. Trilok-Kumar, "Vitamin E status in healthy population in Asia: A review of current literature," Int J Vitam Nutr Res, vol. 91 , no. 3-4, pp. 356-369, 2021 .

[0143]

[0063] M. Ulak, R. T.-L. A. Chandyo, S. Henjum, P. Ueland, 0. Midttun, P. Shrestha, W. Fawzi, L. Graybill and T. Strand, "Vitamin Status among Breastfed Infants in Bhaktapur, Nepal," Nutrients, vol. 8, no. 3, p. 149, 2016.

[0144]

[0064] T. Mai, K. Nguyen, M. Kawakami and M. N. C. Kawase, "Micronutrient status of primary school girls in rural and urban areas of South Vietnam," Asia Pac J Clin Nutr, vol. 12, no. 2, pp. 178- 185, 2003.

[0145]

[0065] A. Shamim, A. Kabir, R. Me rill, H. Ali, M. Rashid, K. Schulze, A. Labrique, K. West Jr and P. Christian, "Plasma zinc, vitamin B(12) and a-tocopherol are positively and plasma y-tocopherol is negatively associated with Hb concentration in early pregnancy in north-west Bangladesh," Public Health Nutr, vol. 16, no. 8, pp. 1354-1361 , 2013.

[0146]

[0066] P. Assantachai and S. Lekhakula, "Epidemiological survey of vitamin deficiencies in older Thai adults: implications for national policy planning," Public Health Nutr, vol. 10, no. 1 , pp. 65-70, 2007.

[0147]

[0067] M. Traber, "Vitamin E Inadequacy in Humans: Causes and Consequences," Adv Nutr, vol. 5, no. 5, pp. 503-514, 2014.

Claims

CLAIMS1 . A method of increasing nutrient levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen, wherein the nutrient is selected from the group consisting of Vitamin A, Vitamin D and choline.

2. The method accordingto claim 1 wherein the individual in need of such treatment has baseline serum choline levels less than 7.0 pM.

3. The method accordingto claim 1 wherein the individual in need of such treatment has vitamin A deficiency, is a very low birth weight infant, is pregnant, postpartum, or nursing, has polymorphisms in BCMO1 , night blindness, conjunctival xerosis, Bitot’s spot, corneal xerosis, corneal ulcer, keratomalacia, corneal scarring, xerophthalmic fundus, or epithelial proliferation disorders, such as phrynoderma.

4. A method of increasing serum retinol levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

5. The method according to claim 4 wherein the individual in need of such treatment has baseline serum retinol levels less than 20 mcg / dL.

6. The method according to claim 4 wherein the individual in need of such treatment has vitamin A deficiency, is a very low birth weight infant, is pregnant, postpartum, or nursing, has polymorphisms in BCMO1 , night blindness, conjunctival xerosis, Bitot’s spot, corneal xerosis, corneal ulcer, keratomalacia, corneal scarring, xerophthalmic fundus, or epithelial proliferation disorders, such as phrynoderma.

7. The method according to claim 4 wherein the individual in need of such treatment is supplementing with vitamin A, retinol, provitamin A, or carotenoids, including but not limited to beta-carotene, or has been clinically advised or is otherwise deliberately consuming a diet rich in vitamin A, retinol, provitamin A, or carotenoids, including but not limited to beta-carotene, to increase their levels of vitamin A or retinol.

8. The method according to claim 4 wherein the individual in need of such treatment is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

9. A method of increasing serum alpha-tocopherol levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

10. The method according to claim 9 wherein the individual in need of such treatment has baseline serum alpha-tocopherol levels less than 14.0 pM.11 . The method according to claim 9 wherein the individual in need of such treatment has genetic mutations in TTPA, ataxia with vitamin E deficiency (AVEA), spinocerebellar ataxia, cholestatic liver disease, or cystic fibrosis.

12. The method according to claim 9 wherein the individual in need of such treatment is supplementing with vitamin E, alpha-tocopherol, or other tocopherols or tocotrienols, has been clinically advised or is otherwise deliberately consuming a diet rich in vitamin E, alpha-tocopherol, or other tocopherols or tocotrienols to increase their levels of vitamin E or alpha-tocopherol.

13. The method according to claim 9 wherein the individual in need of such treatment is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

14. A method of increasing serum choline levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

15. The method according to claim 14 wherein the individual in need of such treatment has baseline serum choline levels less than 7.0 pM.

16. The method according to claim 14 wherein the individual in need of such treatment is a pregnant woman or a woman trying to conceive, an individual with one or more polymorphisms in the genes encoding proteins important for choline metabolism, an individual requiring total parenteral nutrition, someone with a neurological condition like Alzheimer’s disease, for example, or a person with NAFLD orwho is at risk of developing NAFLD due to hereditary factors.

17. The method according to claim 14 wherein the individual in need of such treatment is supplementing with choline, lecithin, or glycerophosphocholine, has been clinically advised or is otherwise deliberately consuming a diet rich in choline to increase the levels of choline in their blood.

18. The method according to claim 14 wherein the individual in need of such treatment is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

19. The method according to claim 14 wherein the individual in need of such treatment is vulnerable to the toxic effects of trimethylamine oxide (TMAO), including those with cardiovascular disease, type 2 diabetes, and kidney disease.

20. The method according to claim 14 wherein the individual in need of such treatment is also supplementingwith choline, lecithin, or glycerophosphocholine and is needingto reduce to the toxic effects of trimethylamine oxide (TMAO), including those with cardiovascular disease, type 2 diabetes, and kidney disease.21 . The method according to claim 14 wherein the individual in need of such treatment is a student, athlete, or medial doctor or similar professional requiring enhanced mental focus.

22. A method of increasing serum sphingomyelin levels in an individual in need of such treatment comprising administering to said individual an effective amount of a suitable resistant potato starch on a dosage regimen.

23. The method according to claim 22wherein the individual in need of such treatment has baseline serum sphingomyelin levels less than 20% of the total phospholipid pool.

24. The method according to claim 22 wherein the individual in need of such treatment has a neurological condition like Alzheimer’s disease, Parkinson’s disease, for example, or a person with degenerative motor neuron disease like amyotrophic lateral sclerosis or multiple sclerosis or who is at risk of developing Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, or multiple sclerosis due to hereditary factors.

25. The method according to claim 22 wherein the individual in need of such treatment is supplementingwith choline, has been clinically advised or is otherwise deliberately consuming a diet rich in choline to increase the production of sphingomyelin.

26. The method according to claim 22 wherein the individual in need of such treatment is using a glucagon-like peptide 1 (GLP-1) receptor agonist drug, synthetic GLP-1, or another synthetic incretin, such as peptide YY (PYY) prescribed to reduce appetite.

27. The method according to claim 22 wherein the individual in need of such treatment is vulnerable to the toxic effects of trimethylamine oxide (TMAO), including those with cardiovascular disease, type 2 diabetes, and kidney disease.

28. The method according to claim 22 wherein the individual in need of such treatment is a student, athlete, or medial doctor or similar professional requiring enhanced mental focus.

29. The method according to claim 22 wherein the individual in need of such treatment is a very low birth weight infant, is pregnant, postpartum, or nursing, has polymorphisms in BCMO1 , nightblindness, conjunctival xerosis, Bitot’s spot, corneal xerosis, corneal ulcer, keratomalacia, corneal scarring, xerophthalmic fundus, or epithelial proliferation disorders, such as phrynoderma.

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