A method for assessing lipid absorption by re-esterification assay and its application

Abstract

This invention discloses a method for assessing lipid reesterification, belonging to the field of biochemistry. Based on radioisotope-labeled assisted liquid scintillation counting technology, this invention provides a method for evaluating the reesterification of functional lipids. For the reaction of fatty acids with coenzyme A to form acyl-CoA, coenzyme A is labeled with a radioisotope, and the reesterification rate of fatty acids is quantified by calculating the isotope recovery rate in the product. For the reaction of sterols with acyl-CoA to form sterol fatty acid esters, sterols and acyl-CoA are labeled with two different isotopes respectively. The reduction of acyl-CoA in the reactants and the formation of sterol fatty acid esters in the product are characterized by detecting the intensity of the two isotopes simultaneously carried in the product, thus quantifying the reesterification rate of sterols. This invention uses labeled coenzyme A as an evaluator for the metabolic process, precisely locating the lipid metabolic pathway to the esterification reaction involving coenzyme A, eliminating the influence of unknown metabolic pathways.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a method and application for assessing reesterification of lipid absorption. Background Technology

[0002] Functional lipids are a class of lipids with special physiological functions. They refer to a large class of fat-soluble substances that are needed for human nutrition and health and have a positive effect on the prevention and treatment of some corresponding nutrient deficiencies and endogenous diseases, especially hypertension, heart disease, cancer, and diabetes.

[0003] Functional lipids can be classified according to their function into functional fatty acids, fat-soluble vitamins, sterols, squalene, etc.; other functional lipid derivatives include coenzyme Q10, curcumin, resveratrol, lycopene, lutein, astaxanthin, etc. Fatty acids and phytosterols are widely present in food. Common functional fatty acids in food are mainly medium- and long-chain fatty acids such as DHA, EPA, linoleic acid, linolenic acid, arachidonic acid, and oleic acid. Phytosterols (including sterols and steranols) are tetracyclic triterpenoid natural products, widely found in vegetable oils, grains, vegetables, fruits, and other foods commonly consumed by Chinese residents. The daily intake of phytosterols in my country is 257.7–473.7 mg. Common phytosterols in food include sitosterol, stigmasterol, and campesterol, while their corresponding phytosterols, such as stigmasterol and campesterol, have higher saturation. These functional lipids have alleviating effects on arteriosclerosis and cardiovascular diseases, and significantly regulate the receptors of proteins related to bile acid metabolism. Therefore, how to evaluate the absorption efficiency of dietary lipids by measuring the reesterification rate and thus guide the rational intake of dietary lipids is a key focus of attention.

[0004] The digestibility and absorption capacity of functional lipids reflects their bioavailability. Functional lipids are all fat-soluble substances. Lipids dissolved in mixed micelles undergo intracellular re-esterification, and the esterified lipids are subsequently incorporated into chylomicrons, thereby participating in lymphatic and blood circulation metabolism. The majority of lipids in chylomicrons originate from the acyl-CoA esterified form obtained through intestinal cell re-esterification. By regulating key control points in the esterification process, the esterification rate can be increased, thereby improving bioavailability.

[0005] Bioavailability is a crucial indicator for evaluating the absorption capacity and efficiency of functional lipids; however, current technologies cannot accurately assess this process in vitro. For example, current methods using CaCO₂ cell models evaluate cell membrane permeability based on monolayer membrane permeability, not the overall absorption rate. Furthermore, in vivo pharmacokinetic assessments of absorption generally exhibit poor parallelism compared to in vitro models due to individual animal variability.

[0006] The most crucial step in the absorption phase is the formation of chylomicrons from functional lipids in digestive fluids, along with triglycerides and phospholipids. These chylomicrons then enter the lymphatic circulation and are utilized by the body. Studies have shown that the key factor influencing chylomicron formation is the re-esterification process of lipids. Esterified functional lipids are further absorbed and utilized; therefore, evaluating chylomicron formation capacity requires focusing on the esterification process.

[0007] Patent CN 114736947A discloses a method for determining the bioavailability of triglycerides based on pH-stat combined with a CaCO-2 model. This method combines an in vitro digestion model and a CaCO-2 intestinal absorption model to evaluate the bioavailability of triglycerides. However, this method can only evaluate the portion that is easily absorbed and crosses the intestinal cell membrane. It cannot measure the portion of triglycerides that are utilized by the body, i.e., those used in chylomicrons in the lymphatic circulation (in other words, triglyceride bioavailability).

[0008] Therefore, simplifying and refining the evaluation model of lipid bioavailability in the body is beneficial for evaluating the absorption rate of functional lipids in food, thereby forming a reasonable dietary pattern. Summary of the Invention

[0009] The purpose of this invention is to provide a method for detecting the re-esterification efficiency of functional lipids in intestinal epithelial cells during digestion and absorption, and to use it for evaluating the nutritional value of lipids in food.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention provides a method for assessing the reesterification of lipids based on radioisotope-labeled assisted liquid scintillation counting technology. The lipids are primarily fatty acids and steroidal compounds. When the lipids are fatty acids, the method includes the following steps:

[0012] (1) Add the fatty acid to be tested to a monolayer cultured intestinal epithelial cells and incubate to load the cells with the fatty acid to be tested; then add a radioactive isotope. 14 C-labeled coenzyme a, when incubated, causes fatty acids to undergo a re-esterification reaction with coenzyme a within the cell to produce... 14 C-labeled acyl-CoA;

[0013] (2) After the reaction is terminated, the cell culture medium is centrifuged, the organic layer is collected, and the solvent is removed to obtain the metabolites.

[0014] (3) The metabolites were chromatographically analyzed using thin-layer chromatography. The band corresponding to acyl-CoA was cut off, transferred to a scintillation bottle containing scintillation solution, stirred and incubated, and the isotopes were detected using a liquid scintillation counter. 14 C strength, calculation 14 C recovery rate characterizes the reesterification rate of the fatty acids to be tested;

[0015] When the lipid is a sterol, the method includes the following steps:

[0016] 1) Radioactive isotopes are labeled on the sterols to be tested. 3 H was then added to a monolayer of intestinal epithelial cells for incubation; subsequently, a radioactive isotope was added. 14 C-labeled acyl-CoA, upon incubation, causes a re-esterification reaction between sterols and acyl-CoA within the cell, producing a product that simultaneously possesses... 14 C and 3 H-labeled sterol fatty acid esters;

[0017] 2) After the reaction is terminated, the cell culture medium is centrifuged, the organic layer is collected, and the solvent is removed to obtain the metabolites;

[0018] 3) The metabolites were analyzed by thin-layer chromatography. The bands corresponding to the sterol fatty acid esters were cut out, transferred to a scintillation bottle containing scintillation solution, stirred and incubated, and the isotopes were detected by a liquid scintillation counter. 3 H and 14 C strength, the sterol re-esterification rate to be tested is calculated according to formula (Ⅰ);

[0019]

[0020] The dosage of [14C] acyl-CoA in the reactants was […]. 14 C addition amount, dosage [3H] sterol in reactants 3 H addition amount, single-layer model [14C] coenzyme a in the product 14 C content, [3H] sterol fatty acid esters in the monolayer model are the products 3 H content.

[0021] Furthermore, the fatty acids are mainly medium- and long-chain fatty acids, including but not limited to DHA, EPA, linoleic acid, linolenic acid, arachidonic acid, and oleic acid.

[0022] Furthermore, the steroidal compounds are phytosterols, including but not limited to sitosterol, stigmasterol, campesterol, and brassosterol.

[0023] The principle of the re-esterification assay for assessing lipid absorption provided by this invention is as follows:

[0024] For fatty acid re-esterification, fatty acids react under the action of coenzyme A to generate acyl-CoA and acylglycerol. Coenzyme A is labeled with radioactive isotopes, and the product acyl-CoA will carry the radioactive isotope label. The intensity of radioactive isotopes in the product is detected, and the re-esterification rate of fatty acids is quantified by calculating the radioactive isotope recovery rate.

[0025] For sterol re-esterification, sterols react with acyl-CoA to generate sterol fatty acid esters and CoA. Two different radioisotopes are used to label sterols and acyl-CoA, respectively, generating sterol fatty acid esters simultaneously labeled with both radioisotopes. The reduction in acyl-CoA in the reactants and the formation of sterol fatty acid esters in the products are determined by detecting the intensity of the radioisotopes, thus quantifying the sterol re-esterification rate.

[0026] This invention precisely locates the metabolic pathway to the esterification reaction involving the enzymes by labeling them with radioactive isotopes, effectively eliminating the influence of unknown metabolic pathways.

[0027] Furthermore, the intestinal epithelial cells are CaCO-2 cells, and the CaCO-2 cell model is a classic model for studying nutrient absorption. After the CaCO-2 cells are cultured to a stable state, they are transferred to a Transwell 12-well plate and cultured for a further period to form a monolayer of cells.

[0028] Furthermore, the transmembrane resistance of cells was measured during cell culture. When the transmembrane resistance was greater than 350 Ω, the monolayer membrane model was successfully constructed.

[0029] Furthermore, coenzyme A 14 The C-labeled dose is 0.015-0.06 μCi, preferably, coenzyme a. 14 The C-labeled dose was 0.03 μCi.

[0030] Furthermore, the sterols to be tested 3 H-labeled dose is 0.001-0.004 μCi, acyl-CoA 14 The C-labeled dose is 0.015-0.06 μCi. Preferably, the sterol to be tested... 3 The H-labeled dose was 0.002 μCi, and the acyl-CoA content was... 14 The C-labeled dose was 0.03 μCi.

[0031] Furthermore, the incubation conditions for the fatty acids or sterols to be tested in intestinal epithelial cells were: incubation at 37°C for 5 min.

[0032] Further, in step 1), the sterol is dissolved in a 45% (w / v) aqueous solution of 2-hydroxypropyl β-cyclodextrin, and then added to a monolayer cultured intestinal epithelial cells and incubated at 37°C for 5 min.

[0033] Furthermore, in step 1), after incubation with the sterol to be tested, add... 14 C-labeled acyl-CoA and fatty acid-free bovine serum albumin were incubated to induce a re-esterification reaction. Bovine serum albumin served as a cellular nutrient source to support the esterification process.

[0034] Furthermore, in steps (1) and 1), an equal dose of unlabeled coenzyme A or acyl-CoA is added to the control group. Furthermore, in steps (1) and 1), the re-esterification reaction time is 12–24 h.

[0035] Furthermore, in steps (2) and (2), physiological saline solution with a volume ratio of 2:1 of chloroform:methanol was added to terminate the reaction.

[0036] Furthermore, in steps (2) and (2), the centrifugation conditions are: centrifugation at 800×g for 10 min at 4℃.

[0037] Furthermore, in steps (2) and (2), the solvent is removed by evaporation under a nitrogen stream.

[0038] Furthermore, the metabolites were resuspended and spotted onto a thin-layer silica gel plate. The mobile phase was a mixture of n-hexane, ethyl acetate, and acetic acid with a volume ratio of 80:20:1, and the chromatography time was 45 min.

[0039] Meanwhile, samples containing only acyl-CoA or sterol fatty acid esters were used as controls for the separation and identification of metabolites by spot chromatography.

[0040] Furthermore, in step (3), radioactive acyl-CoA is generated to quantify the reaction and exclude other non-radioactive acyl-CoA. 14 C-labeled CoA pathway: Unlabeled acyl-CoA was added to the resuspension of metabolites, followed by thin-layer chromatography.

[0041] Another object of the present invention is to provide the application of the re-esterification assay method for assessing lipid absorption in evaluating the nutritional value of lipids in food. The application includes: firstly, extracting and identifying specific functional lipid components from the food, and determining metabolites based on the target components; then, using the aforementioned re-esterification assay method to detect the isotope intensity carried on the target metabolites, and subsequently calculating the re-esterification rate of the target components.

[0042] The beneficial effects of this invention are as follows:

[0043] The key factor in the formation of chylomicrons is the re-esterification process of lipids. This invention provides a method for evaluating the re-esterification of functional lipids based on radioisotope-labeled assisted liquid scintillation counting technology. In this method, coenzyme A or acyl-CoA is radioisotope-labeled, and the labeled coenzyme A is used as an evaluator to measure the metabolic process. The intensity of the radioisotope of the products catalyzed by the above enzymes is detected, and the metabolic pathway of functional lipids is accurately located to the esterification reaction involving coenzyme A, eliminating the influence of unknown metabolic pathways.

[0044] Compared to current in vitro experiments that use CaCO-2 cell models to evaluate lipid absorption capacity by assessing cell membrane permeability through monolayer membrane permeability, the method of this invention focuses on the energy metabolism response within intestinal epithelial cells, which is closer to the actual lipid absorption situation and provides a more accurate evaluation.

[0045] Compared to current methods for assessing absorption through pharmacokinetic analysis in in vivo experiments, the method of this invention precisely locates the re-esterification process of lipids, can accurately evaluate lipid absorption, and has good repeatability. Attached Figure Description

[0046] Figure 1 This is a diagram illustrating the mechanism of functional lipid re-esterification.

[0047] Figure 2 Esterification efficiency of different lipids (fatty acids, phytosterols).

[0048] Figure 3 To assess the bioavailability of different lipids (fatty acids, phytosterols).

[0049] Figure 4 Permeability of different lipids (fatty acids, phytosterols) to intestinal cells

[0050] Figure 5 Bioavailability of different lipids (fatty acids, phytosterols).

[0051] The diagram shows oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:4), EPA (C20:5), DHA (C22:6), sitosterol, stigmasterol, campesterol, and brassicasterol. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0054] Example 1: Establishing an evaluation model for intracellular reesterification

[0055] The most crucial step in the absorption phase is the formation of chylomicrons from functional lipids in digestive fluids, involving triglycerides and phospholipids. These chylomicrons then enter the lymphatic circulation and are utilized by the body. Studies have shown that the key factor influencing chylomicron formation is the re-esterification process of lipids. Esterified functional lipids are further absorbed and utilized; therefore, evaluating chylomicron formation capacity requires focusing on the esterification process. Using a CaCO₂ monolayer cell model as a prototype, and employing radioactive isotope labeling of key enzymatic reactants, this study simulates intracellular chemical reactions using the cell model to investigate the metabolites, metabolism, and dynamic processes of functional lipids. This allows for the establishment of a method for measuring intracellular re-esterification, providing insights into evaluating the absorbability of functional lipids in food.

[0056] When the CaCO₂ cells reached approximately 80% cellularity, a single-cell suspension was prepared according to cell culture and passage methods. The cell concentration was calculated, and the cell suspension concentration was adjusted to 3 × 10⁻⁶. 5 Cells / mL. Pipette 0.5 mL of cell suspension into the upper chamber of a (3 μm, 24 mm) Transwell 12-well plate, and add 1.5 mL of fresh cell culture medium to the lower chamber. The control group is not seeded with cells and receives an equal volume of cell culture medium. Change the culture medium in the inner chamber daily and the culture medium in the lower chamber every other day.

[0057] Using a Millicell-ERS transmembrane resistance meter, the transmembrane resistance (TEER) of CaCO-2 monolayer intestinal epithelial cell model cells was measured starting from day 1 after cell seeding.

[0058] TEER=(R-Ro)×A

[0059] In the formula: TEER is the transmembrane resistance value (Ω·cm) 2 Ro and R are the blank resistance and detection resistance, respectively, and A is the cell membrane area (surface area of ​​the Transwell 12-well plate, 1.12 cm²). 2 ).

[0060] When TEER > 350, the monolayer membrane is successfully established.

[0061] Esterification of functional lipids, including medium- and long-chain fatty acids and phytosterols, can be used as... Figure 1 The reaction equation shown indicates that fatty acids react under the action of coenzyme A to produce acetyl-CoA and diacylglycerol, thus allowing them to be released through... 14 The ratio of C-labeled coenzyme A to acyl-CoA is used to quantitatively determine the esterification efficiency of fatty acids via radiometric analysis. Phytosterols can react with acetyl-CoA to produce sterol esters and coenzyme A, which have different structural formulas but can exist equivalently. Therefore, this can be achieved through... 14C-labeled acetyl-CoA supplemented with 3 Radiometric radiometric analysis of H-sterol esters (Acylated-products) using radioisotope ratios to determine the esterification efficiency of sterols.

[0062] Specifically, fatty acid esterification: 14 C-labeled CoA was used to quantify the fatty acid reesterification rate by measuring the generated acyl-CoA.

[0063] Sterol esterification: 3 H-labeled sterols, 14 C-labeled acyl-CoA was measured by determining the decrease in acyl-CoA in the reactants (upper chamber) and the amount in the products (lower chamber). 3 The content of H plant sterol fatty acid esters was calculated using a formula to determine the re-esterification rate.

[0064] The following section uses oleic acid as an example to explain the evaluation method for intracellular reesterification of functional lipids.

[0065] Fatty acid esterification: Add 0.38 nmol to the upper chamber of a Transwell 12-well culture plate. 14 C-labeled CoA (0.03 μCi) was co-incubated with 1 mmol / L oleic acid for 24 h. An equal dose of unlabeled CoA was added to another well, and the product bands were determined by comparing the labeled and unlabeled CoA. The labeled CoA was then separated by thin-layer chromatography to quantify the CoA reaction efficiency.

[0066] The reaction was stopped by adding 4.8 mL of a chloroform:methanol mixture (2:1, v / v) and 1 mL of physiological saline. The mixture was centrifuged at 800 × g for 10 min at 4 °C, and the lower organic layer containing the lipids to be tested was collected and transferred to a new tube. The tube was then evaporated to dryness under a gentle nitrogen stream. Finally, 10 μg of the sample was added to 50 μL of chloroform to prepare the lipids to be tested.

[0067] Add 10 μg of oleyl-CoA to 50 μL of chloroform and thoroughly swirl the tube to mix. This serves as a control for the next step of thin-layer chromatography separation of oleyl-CoA.

[0068] To quantify the radioactive oleoyl-CoA produced in the reaction and exclude other non-radioactive compounds... 14For the C-labeled CoA pathway, 5.47 nmol / L of oleyl-CoA was added to the lipid sample. The lipid mixture was then placed on a thin-layer silica gel plate (Merck, NJ, USA) and developed in hexane:ethyl acetate:acetic acid (80:20:1, v / v / v) for 45 minutes. The oleyl-CoA band was cut and transferred to a scintillation vial. 10 mL of OptiPhaseHiSafe scintillation buffer (Perkin-Elmer) was added to the vial, and the mixture was incubated overnight with stirring. Radioactivity was then measured on an 16500 scintillation counter (Beckman) and determined according to... 14 C recovery rate quantifies fatty acid re-esterification rate.

[0069] Recovery rate = (Number of radioactive elements recovered / Number of radioactive elements to be recovered) × 100% (counting unit: cpm).

[0070] Sterol esterification: Add 45% (w / v) aqueous solution of 2-hydroxypropyl β-cyclodextrin to the inner chamber of a Transwell 12-well culture plate. 3 H-stigmasterol (0.002 μCi) was incubated in a water bath at 37 °C for 5 min. At the start of the reaction, 0.52 nmol of […] was added. 14 [C] Oleyl-CoA (0.03 μCi) and 10 nmol of fatty acid-free bovine serum albumin were incubated for 24 h. In another well, 7.48 nmol of oleyl-CoA and 10 nmol of fatty acid-free bovine serum albumin were added and incubated for 24 h. The product bands were determined by comparing labeled and unlabeled oleyl-CoA. The reaction was stopped by adding 4.8 mL of a chloroform:methanol mixture (2:1, v / v) and 1 mL of physiological saline. The mixture was centrifuged at 800 × g for 10 min at 4 °C, and the lower organic layer was collected, transferred to a new tube, and evaporated to dryness under a gentle nitrogen stream. Then, 10 μg of the sample was added to 50 μL of chloroform to prepare the lipids to be tested.

[0071] Add 10 μg of stigmasterol oleate to 50 μL of chloroform and thoroughly swirl the tube to mix. This serves as a control for the next step of thin-layer chromatography separation of stigmasterol oleate.

[0072] The lipid mixture was then placed on a thin-layer silica gel plate (Merck, NJ, USA) and developed in hexane:ethyl acetate:acetic acid (80:20:1, v / v / v) for 45 minutes. The band corresponding to stigmasterol oleate was cut off and transferred to a scintillation vial. 10 mL of OptiPhaseHiSafe scintillation buffer (Perkin-Elmer) was added to the vial and incubated overnight with stirring.

[0073] Finally, measurements were taken on an LS6500 blink counter (Beckman). 14 C 3H radioactivity, and the data are calculated according to the following formula.

[0074] Reaction calculation formula:

[0075]

[0076] The dosage of [14C] oleoyl-CoA in the reactants was... 14 C addition amount, dosage [3H] stigmasterol is a reactant 3 H addition amount, single-layer model [14C] coenzyme a in the product 14 C content, in the single-layer model, [3H]stigmasterol oleate is the product. 3 H content.

[0077] Example 2: Comparison of re-esterification rates among fatty acids and phytosterols

[0078] 1. Determination of re-esterification rate of different fatty acids

[0079] Following the method described in Example 1, 0.38 nmol was added to the inner chamber of a Transwell 12-well culture plate. 14 C-labeled CoA (0.03 μCi) was co-incubated with 1 mmol C18:1, C18:2, C18:3, C20:4, C20:5, and C22:6 for 24 h. An equal dose of unlabeled CoA was added to another well. The product bands were determined by comparing labeled and unlabeled CoA, and the labeled CoA was then separated by thin-layer chromatography to quantify the CoA reaction efficiency.

[0080] The reaction was stopped by adding 4.8 mL of a chloroform:methanol mixture (2:1, v / v) and 1 mL of physiological saline. The mixture was centrifuged at 800 × g for 10 min at 4 °C, and the lower organic layer was collected and transferred to a new tube. The tube was then evaporated to dryness under a gentle nitrogen stream. Finally, 10 μg of the sample was added to 50 μL of chloroform to prepare the lipids to be tested.

[0081] Add 5.47 nmol / L of acyl-CoA to the lipids to be tested, then place the lipid mixture on a thin-layer silica gel plate (Merck, NJ, USA) and develop the bands in hexane:ethyl acetate:acetic acid (80:20:1, v / v / v) for 45 minutes. Cut off the Acyl-CoA band, add 10 mL of scintillation buffer to a vial, and incubate overnight with stirring. Then measure the radioactivity on a scintillation counter and according to... 14 C recovery rate quantifies fatty acid re-esterification rate.

[0082] 2. Determination of reesterification rate of different phytosterols

[0083] Add water dissolved in 45% (w / v) 2-hydroxypropyl β-cyclodextrin to each well of the Transwell 12-well culture plate. 3 H(0.002μCi) sitosterol, stigmasterol, campesterol, and brassosterol were incubated in a 37°C water bath for 5 min. At the start of the reaction, 0.52 nmol of [ 14 [C]Oleyl-CoA (0.03 μCi) and 10 nmol of fatty acid-free bovine serum albumin were incubated for 24 h; in another well, 7.48 nmol of oleyl-CoA and 10 nmol of fatty acid-free bovine serum albumin were added and incubated for 24 h. The reaction was stopped by adding 4.8 mL of chloroform:methanol mixture (2:1, v / v) and 1 mL of physiological saline. The mixture was centrifuged at 800 × g for 10 min at 4 °C, and the lower organic layer was collected, transferred to a new tube, and evaporated to dryness under a gentle nitrogen stream. Then, 10 μg of the sample was added to 50 μL of chloroform to prepare the lipids to be tested.

[0084] The lipid mixture was then placed on a thin-layer silica gel plate (Merck, NJ, USA), and the bands were developed in hexane:ethyl acetate:acetic acid (80:20:1, v / v / v) for 45 minutes. The band corresponding to oleic acid sterol ester was cut off and transferred to a scintillation vial. 10 mL of OptiPhaseHiSafe scintillation buffer (Perkin-Elmer) was added to the vial, and the mixture was incubated overnight with stirring. Measurements were then performed using an LS6500 scintillation counter (Beckman). 14 C 3 H radioactivity, and the data are calculated according to the following formula.

[0085]

[0086] The dosage of [14C] acyl-CoA in the reactants was […]. 14 C addition amount, dosage [3H] sterol in reactants 3 H addition amount, single-layer model [14C] coenzyme a in the product 14 C content, [3H] sterol fatty acid esters in the monolayer model are the products 3 H content.

[0087] 3. Results Analysis

[0088] The results are as follows Figure 2As shown in the figure, comparing the re-esterification rates of fatty acids and phytosterols, the fatty acid chain length has a significant impact on the re-esterification rate, with the main re-esterification pattern being C18 > C20 > C22. Furthermore, the effect of the number of fatty acid double bonds on the re-esterification rate can also be observed in the figure; the higher the degree of unsaturation, the lower the re-esterification rate, and the re-esterification rate increases with increasing saturation. The re-esterification rate of C18:1 exceeds 70%, while that of C22:6 is around 5%, showing a significant difference in re-esterification capacity.

[0089] Analysis of phytosterols showed that sitosterol had the highest re-esterification rate, but it was much lower than that of fatty acids. Sitosterol had the highest esterification rate, followed by stigmasterol, while the other two were comparable, both <10%.

[0090] Comparative Example 1: In vitro digestion simulation

[0091] 1. Prepare simulated gastric juice (SGF) and simulated intestinal juice (SIF).

[0092] The composition of 1.25× simulated gastric juice (SGF) is 47.2 mM NaCl, 25 mM NaHCO3, 6.9 mM KCl, 0.9 mM KH2PO4, and 0.12 mM MgCl2 (H2O). 20 6. 0.5 mM (NH4)2CO3, 15.6 mM HCl and 0.15 mM CaCl2 (H2O) 20 )2.

[0093] The composition of 1.25× simulated intestinal fluid (SIF) includes: 6.8mM KCl, 8.3mM HCl, 0.8mM KH2PO4, 38.4mM NaCl, 0.33mM MgCl2·6(H2O), 85mM NaHCO3, and 0.6mM CaCl2·2(H2O).

[0094] The two 1.25× digestion solutions were diluted to a 1:1 ratio to achieve a final SGF concentration of 1.5 mM and a final SIF concentration of 0.15 mM.

[0095] 2. Pepsin and trypsin were added to SGF and SIF, and their specific enzyme activities were measured. Then, 1g of functional lipids (C18:1, C18:2, C18:3, C20:4, C20:5, C22:6, sitosterol, stigmasterol, campesterol, and brassosterol) were added to 10mL of SGF, the pH of SGF was adjusted to 3, and digestion was carried out at 37℃ for 2h. The residue was mixed with an equal volume of SIF simulated solution, and the reaction was carried out for 2h. The reaction was terminated by heating at 90℃ for 10min to inhibit enzyme activity.

[0096] After 240 minutes of simulated digestion, the small intestinal residue was collected and centrifuged at 18,000 rpm for 50 minutes at 4°C. The supernatant was collected as the mixed micelles for lipid determination. Bioaccessibility is the percentage of the target lipid in the mixed micelles relative to the total lipids in the digestate.

[0097] 3. Results Analysis

[0098] The results are as follows Figure 3 As shown, (1) the bioavailability of medium- and long-chain fatty acids is much higher than that of phytosterols, consistent with the pattern obtained from the re-esterification model. (2) The bioavailability pattern of phytosterols is consistent with... Figure 2 The bioavailability was consistent, all above 20%, and the differences in bioavailability among different structures were not significant. The order from high to low was slightly different from the re-esterification rate. The bioavailability of rapeseed sterol was higher than that of stigmasterol, which may be related to the absence of double bonds in its ring structure.

[0099] The results above show that, compared with Comparative Example 1, the values ​​of Example 2 are similar to those of Comparative Example 1. However, in vitro digestion can only represent the lipid mixing micelle stage of functional lipids forming digestive juice, and cannot represent the entire absorption process. The re-esterification model focuses on the energy metabolism reaction in intestinal epithelial cells. The strength of this energy-consuming reaction is represented by the re-esterification rate, which represents its absorption. Using the re-esterification rate to represent the absorption of substances is closer to the real lipid absorption and the evaluation is more accurate.

[0100] Comparative Example 2: In vitro digestion simulation + CaCO-2 cell culture

[0101] The digestion products obtained in Comparative Example 1 were cultured in CaCO₂. The method for constructing the CaCO₂ monolayer intestinal epithelial cell model was the same as in Example 1.

[0102] The digest sample was dissolved in a small amount of dimethyl sulfoxide (DMSO) and diluted with HBSS to a working solution of 50 μmol / L, ensuring the DMSO concentration in the final working solution was below 0.5%. The transmembrane transport of the digest products was assessed. 0.5 mL of the digest working solution was gently added to the upper chamber, and 1.5 mL of HBSS (blank control) was added to the lower chamber. Transwell plates were incubated for 120 min in a (37±1)℃, 50 rpm air bath shaker. 200 μL of the receiving solution was then aspirated from the receiving chamber, and the corresponding volume of HBSS was added. The lipid content in the receiving solution was determined by GC-MS.

[0103] The results are as follows Figure 4As shown, (1) the interspecific patterns are similar to those in Comparative Example 1. The fatty acid absorption rate is much higher than that of phytosterols, but the fatty acid absorption ratio (intestinal cell permeability) is reduced to about 40%. At the same time, the differences in bioavailability among different structures are not significant. This may be due to the presence of digestive products and bile acids in the in vitro digestive fluid, which have subsequent effects. In addition, in vitro digestion will cause the loss of some effective substances, resulting in a decrease in intestinal cell permeability. (2) The effect of lipid type on absorption: the bioavailability of medium and long chain fatty acids is much higher than that of phytosterols, which is consistent with the pattern obtained from the re-esterification model.

[0104] The numerical values ​​and trends of Example 2 are similar to those of Comparative Example 2. However, the differences between the samples in Comparative Example 2 are difficult to observe, and the two simulated processes include the stages of lipids mixing into digestive fluids and entering intestinal epithelial cells, which cannot represent the absorption process.

[0105] Comparative Example 3: Bioavailability, i.e., metabolic kinetics

[0106] Sixty male SD rats (300-400g) were selected and acclimatized for 7 days. They were randomly divided into two groups: a control group and a functional lipid group. Before the formal experiment, all rats were fasted for 12 hours. During the experiment, each rat was administered functional lipids (C18:1, C18:2, C18:3, C20:4, C20:5, C22:6, sitosterol, stigmasterol, campesterol, and phytosterol) at 50 mg / kg body weight via gavage. Blood was collected from the eyes of each SD rat at predetermined time intervals (0, 2, 4, 6, 9, 12, 16, 24, 36, and 48 hours). Plasma was collected by centrifugation at 5000 rpm for 10 min and stored at -80℃ for analysis. Rat serum was collected, centrifuged at 2000 rpm for 5 min, and 50 μL of supernatant was added, with 5α-cholesterol as an internal standard. The supernatant was saponified with KOH and CH2Cl2 and washed three times to remove impurities. Finally, the drug was derivatized with 5 μL of 1-MIM and 95 μL of MSHFBA, and determined by gas chromatography-mass spectrometry. Bioavailability was calculated as the plasma concentration versus time integral AUC.

[0107] The results are as follows Figure 5 As shown: (1) There was no significant difference in the in vivo metabolism of fatty acids among C18, while the differences among C20:4, C20:5, and C22:6 were more obvious. (2) The bioavailability of phytosterols was less than 5%, with sitosterol being higher than the other four, but there was no significant difference among the four sterols. This proves that the low solubility of sterols affects their bioavailability, and the long digestion and absorption stage leads to no significant difference in the bioavailability of sterols with different structures.

[0108] Therefore, accurately evaluating absorption capacity before the formation of lipid chylomicrons is crucial. Compared to Comparative Example 3, the evaluation model obtained in Example 1 closely approximates the absorption and metabolism patterns obtained from in vivo experiments. This demonstrates that the re-esterification model in Example 1 can effectively evaluate the absorption of functional lipids (medium- and long-chain fatty acids and phytosterols), and can quantify the absorption efficiency of sterols with different structures that have low absorption rates. The model can quantify more realistic absorption and the influence of structure on absorption.

[0109] Example 3: Evaluation of the absorption efficiency of functional lipids in food

[0110] The extraction of functional lipids involved placing 1 kg of unpeeled Yangbi walnuts into a 5L fully automatic hydraulic oil press, pressing at room temperature with a pressing pressure of 48 MPa for 2 hours to obtain a transparent, bright yellow crude oil.

[0111] Lipids were analyzed by GCMS under the following extraction conditions:

[0112] GC separation conditions: programmed temperature rise, initial temperature 100℃, hold for 1 min, rise at 40℃ / min to 250℃, rise at 2.5℃ / min to 300℃ and hold for 10 min.

[0113] MS detection conditions: Ion source temperature: 250℃; transfer line temperature: 290℃; electron intensity: 70 eV; solvent delay: 10 min; detection mode: SIM mode. Scanning ion range: 50–650 m / z.

[0114] The results showed that the main fatty acids and sterols in walnuts were linoleic acid, linolenic acid, oleic acid, arachidonic acid, DHA, and EPA, while the main phytosterols were sitosterol, stigmasterol, campesterol, and brassosterol.

[0115] The metabolites were determined based on the target components described above; the intensity of the isotopes carried on the target metabolites was detected using the re-esterification assay method described in Example 1, and the re-esterification rate of the target components was then calculated.

[0116] The results showed that the re-esterification rate of functional lipids in walnuts followed the trend of linoleic acid > linolenic acid > oleic acid > arachidonic acid > sitosterol > stigmasterol > campesterol > campesterol.

Claims

1. A re-esterification assay method for assessing lipid absorption, characterized in that, The lipid is a sterol, and the method comprises the following steps: 1) radioactive isotope to be used as a label 3 H, to the monolayer of epithelial cells and incubated; then, radioactive isotope 14 C-labeled acyl-coenzyme A is added and incubated so that the sterol is re-esterified in the cell with the acyl-coenzyme A to form a sterol fatty acid ester 14 C and 3 H-labeled sterol fatty acid ester; 2) After the reaction is terminated, the cell culture solution is centrifuged, the organic layer is collected, and the solvent is removed to obtain a metabolite; 3) The metabolites are chromatographed by thin layer chromatography, the band corresponding to the sterol fatty acid ester is cut off, transferred to a scintillation vial containing scintillation liquid, incubated with stirring, and the isotope is detected by liquid scintillation counting 3 H and 14 C intensity, and the re-esterification rate of the sterol to be tested is calculated according to formula (I); (Ⅰ)， where the administered dose of [14C] fatty acyl CoA is a reactant in 14 C added, administered dose of [3H] sterol is a reactant in 3 H added, monolayer model [14C] CoA is a product in 14 C added, monolayer model [3H] sterol fatty acid ester is a product in 3 H added.

2. The re-esterification assay method for assessing lipid absorption according to claim 1, wherein, The intestinal epithelial cells are CaCO-2 cells; the CaCO-2 cells are transferred to a Transwell 12-hole plate after being cultured to a stable state, and are continuously cultured to form a monolayer of cells.

3. The re-esterification assay method for assessing lipid absorption according to claim 1, wherein, In step 1) the sterol to be tested 3 The H-labeled dose was 0.002 μCi, and the ester acyl-coenzyme A 14 The C-labeled dose was 0.03 μCi.

4. The re-esterification assay method of assessing lipid absorption according to claim 1, wherein, In step 1), the sterol is dissolved in a 45% (mass / volume) 2-hydroxypropyl β-cyclodextrin aqueous solution, and then is added to the monolayer of intestinal epithelial cells and is incubated at 37 DEG C for 5 min.

5. The re-esterification assay method for assessing lipid absorption according to claim 3, wherein, In step 1) after incubation of the sterol to be tested, the following are added 14 C-labeled ester coenzyme A, fatty acid-free bovine serum albumin incubation for re-esterification reaction.

6. The re-esterification assay method of assessing lipid absorption according to claim 1, wherein, In step 3), after the metabolite is resuspended, is spotted on a thin layer silica gel plate, a mobile phase of a mixture of n-hexane: ethyl acetate: acetic acid at a volume ratio of 80:20:1 is used, and the chromatography time is 45 min.

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