Method for assessing intestinal protein absorption

Abstract

A method for assessing protein uptake from a subject's gut includes: a. providing a blood sample from the subject; b. substantially removing proteins exceeding a size limit of 40 kDa from the sample to provide a deproteinized sample; c. determining the level of free amino acids in a subsample of the deproteinized sample CF by colorimetry or fluorescence; d. hydrolyzing the subsample of the deproteinized sample to obtain a hydrolyzed sample, which converts any remaining polypeptides and peptides into free amino acids; e. determining the level of free amino acids in the hydrolyzed sample CT by colorimetry or fluorescence; f. optionally, calculating the level of peptide-derived amino acids in the blood sample CP, where CP = CT - CF; and g. assessing protein uptake from the subject's gut based on the values ​​of CF and CT, and optionally CP. Related applications include diagnostic and therapeutic methods.

Description

Technical Field

[0001] This invention relates to methods for assessing intestinal protein absorption, methods for diagnosing protein malabsorption itself or in combination with intestinal proteolytic enzyme deficiency (including pancreatic exocrine insufficiency (true and apparent insufficiency)), and methods for treating protein malabsorption and pancreatic exocrine insufficiency. Background Technology

[0002] Dietary protein digestion can be considered to begin under the acidic pH conditions of the stomach, through the action of pepsin. The products of pepsin action are high molecular weight polypeptide fragments, traditionally referred to as proteases and peptones (Kyle et al., 2022).

[0003] In the small intestine, peptone is further digested by an intestinal erapsin-peptidase complex, which includes trypsin (trypsin, chymotrypsin, elastase, carboxypeptidase, and intestinal peptidase), which directly or through protein-like reactions (Northtrop, 1947) process peptone and endogenous proteins into free amino acids and peptides containing 2–8 amino acids. The plasma peptide to free amino acid ratio is estimated to be approximately 4:1. According to recent papers in this field, approximately 80% of dietary protein is absorbed in the form of peptides (Shen and Matsui, 2019).

[0004] Protein malabsorption and malabsorption occur when trypsin fails to be secreted into the intestine for various reasons. Examples include bariatric surgery (Han et al, 2021) -> apparent insufficiency, where the enzyme entering the intestine is out of sync with the arrival of digested products; cystic fibrosis in children and the elderly, where pancreatic exocrine insufficiency is common (Price et al, 1977; Rothenbacher et al, 2005, Löhr et al, 2018) -> true insufficiency, where the pancreas itself does not produce the enzyme. Protein malabsorption (and fat malabsorption) is treated with exogenous pancreatic enzyme replacement therapy (PERT) (Fieker et al, 2011).

[0005] There are methodological challenges in rapidly assessing protein malabsorption and the efficacy of PERT in protein digestion. Historically, measurements of fat and nitrogen uptake coefficients (CFA and CNA, respectively) have been used for regulatory approval of porcine PERT (Gan et al, 2017; Borowitz et al, 2022; Freedman et al, 2023). These methods have known limitations, namely, poor reliability, sensitivity, and the cumbersome requirements of long-term fecal collection. Methods based on HPLC, GC, MS, or combinations thereof for measuring amino acids (hydrolyzed free amino acids and total amino acids) in blood absorbed from diet are time-consuming, expensive, and do not yield consistent results (Bernard L, Chauveau B, Rémond D. Effect of the methodology on peptide amino acid concentrations in blood and plasma of sheep. Arch Tierernahr. 2001;54(4):281-96. doi: 10.1080 / 17450390109381985. PMID: 11921851).

[0006] There has long been a need for improved protein absorption tests, rather than tests for protein excretion in feces, which are inexpensive, rapid, and easy to perform. Therefore, the object of this invention is to provide alternative and / or improved methods for assessing protein absorption from an individual's gut, and applications of such methods for purposes such as treating pancreatic exocrine insufficiency, assessing biological age, evaluating dietary protein levels, and predicting the efficacy and absorption of certain drugs.

[0007] definition

[0008] The term “includes” should be interpreted as including, but not limited to, other things.

[0009] The numerical values ​​in the specification and claims of this application should be understood to include: numerical values ​​that are the same when simplified to the same number of significant digits, and numerical values ​​whose difference from the stated values ​​is less than the experimental error of conventional measurement techniques of the type described in this application in determining the value.

[0010] All ranges disclosed herein include the endpoints and can be combined independently (e.g., the range “2 to 10” includes endpoints 2 and 10 as well as all intermediate values).

[0011] The term "about" can be used for any numerical value that includes a basic function that can vary without changing the value. When used with a range, "about" also discloses a range defined by the absolute values ​​of its two endpoints; for example, "about 2 to about 4" also discloses a range "from 2 to 4". The term "about" can refer to a number that is positive or negative 10%.

[0012] The term "exocrine pancreatic insufficiency" (EPI) refers to a condition in which the amount of pancreatic digestive enzymes secreted by the pancreas into the small intestine is reduced to the point that it affects digestion. In the small intestine, pancreatic digestive enzymes play a major role in the digestion of carbohydrates, proteins, and fats, and may also have hormone-like functions. This reduction may be due to decreased production of pancreatic digestive enzymes, pancreatic duct obstruction preventing pancreatic digestive enzymes from reaching the small intestine, or problems in pancreatic regulation (e.g., asynchronous timing of enzyme secretion with the entry of digested matter into the duodenum). EPI can occur in several health conditions, including pancreatitis, cystic fibrosis, pancreatic cancer, type 1 or type 2 diabetes, inflammatory bowel disease, celiac disease, pancreatic or upper gastrointestinal surgery (including bariatric surgery), Sjögren's syndrome, alcohol abuse, and growth retardation. EPI can also be caused by age-related degenerative processes. Common symptoms of EPI include malnutrition, weight loss or inability to gain weight, and diarrhea.

[0013] Pancreatic enzyme replacement therapy (PERT) is a standard treatment for pancreatic lipase (EPI) and involves the oral administration of a digestive enzyme, such as pancreatic lipase, appropriately formulated (usually encapsulated) to survive passage through the stomach to the small intestine. Brand names containing porcine pancreatic lipase include Creon™ and Pancreon™. As an alternative to porcine pancreatic lipase, microbial-derived pancreatic lipases are marketed as Ultresa™. In the context of this invention, any therapy that provides an alternative to, or even a single type of, pancreatic digestive enzyme is considered PERT. In the context of this invention, a preferred form of PERT involves the administration of a suitable protease.

[0014] In this context, the term postprandial refers to the period 0-8 hours after a meal, preferably 0-4 hours, more preferably 0-3 hours, and most preferably 0-2 hours after a meal. Attached Figure Description

[0015] Figure 1. Levels of free amino acids in porcine plasma. Healthy – intact animal group (n=6), Control EPI – pancreatic exocrine insufficiency group (n=6). AUC data are presented as a line at the mean, and C maxData are presented as median ± IQR, and amino acid levels at each time point are given as mean ± SD. A p-value ≤ 0.05 was considered statistically significant; a p-value ≤ 0.1 was considered trending. P-values ​​are presented along with the results in a bar chart.

[0016] Figure 2. Total amino acid levels in porcine plasma. Healthy – intact animal group (n=6), Control EPI – animal group with pancreatic exocrine insufficiency (n=6). AUC data are presented as a line at the mean, C max Amino acid levels at each time point are given as mean ± SD. A p-value ≤ 0.05 is considered statistically significant; a p-value ≤ 0.1 is considered trending. P-values ​​are presented along with the results in a bar chart.

[0017] Figure 3. Levels of peptide-derived amino acids in porcine plasma. Healthy – intact animal group (n=6), Control EPI – animal group with pancreatic exocrine insufficiency (n=6). AUC data are presented as a line at the mean, C max Amino acid levels at each time point are given as mean ± SD. A p-value ≤ 0.05 is considered statistically significant; a p-value ≤ 0.1 is considered trending. P-values ​​are presented along with the results in a bar chart.

[0018] Figure 4 Experimental design and research treatments comparing the efficacy of PERT. JVC – jugular venous catheterization; RMTT – routine meal tolerance test (in the diagram named MTT); PDL – pancreatic duct ligation; EPI – pancreatic exocrine insufficiency.

[0019] Figure 5. Postprandial changes in plasma levels of total amine groups. Data are presented in routine meal tolerance tests (RMTT) using HFD, HFD+SUB; SUB in control healthy and EPI pigs, and in EPI pigs treated with Creon or amylase. Data are expressed as mean ± standard deviation (±SD).

[0020] Figure 6. Area under the absorption curve (AUC) of total amine groups. Data are presented in routine meal tolerance tests (RMTT) using HFD, HFD+SUB; SUB in control healthy and EPI pigs, and in EPI pigs treated with Creon or amylase. Data are expressed as mean ± standard deviation (±SD). AUC data are baseline adjusted. Differences between results were considered significant when p < 0.05. P values ​​between 0.1 and 0.05 were considered trending. P values ​​are presented along with the results bar chart. Summary of the Invention

[0021] The inventors have developed a high-throughput colorimetric / fluorescent method for assessing protein absorption from the gut (see Examples 1-3). Surprisingly, this method is sensitive enough to detect free amino acids and peptides absorbed from the gut into the bloodstream, performing only a simple molecular weight-based separation of resident plasma proteins.

[0022] Six healthy pigs, before and after EPI development, were used for suitability testing. Large plasma proteins were removed using a centrifugal filter with a molecular weight cutoff of 10 kDa. Free and total amino acids were estimated using the ninhydrin method before and after hydrolysis of the filtrate. Peptide-derived amino acids (primarily from nutritional dipeptides and tripeptides) were quantified by subtracting free amino acids from the total amino acids. Analytical accuracy was close to 100%, precision—expressed as intermediate precision percentage less than 5%, and linearity—r. 2 =0.9914.

[0023] In summary, the method of this invention can serve as a tool for monitoring protein digestion and absorption, and as a superior alternative to indirect, fecal-based nitrogen absorption coefficient (CAN) measurement techniques. It is also cost-effective and does not require advanced instrumentation compared to HPLC, GC, MS, etc. The method of this invention is accurate, reproducible, and readily applicable in clinical or research settings. Data obtained from this method can be used to assess the impact of pancreatic enzymes (or their deficiency) on the amino acid to peptide absorption ratio of dietary protein (see Example 4).

[0024] This invention relates to the following. The subject matter disclosed in the following should be considered as disclosed in the same manner as the subject matter disclosed in the patent claims.

[0025] 1. An in vitro method for assessing protein absorption from a subject's gut, comprising:

[0026] a. Provide blood samples from the subjects;

[0027] b. Essentially remove proteins with sizes exceeding 40 kDa from the sample to provide a deproteinized sample;

[0028] c. Determine the level of free amino acids in a subsample of the deproteinized sample by colorimetric or fluorescence methods; this level is designated as CF.

[0029] d. Hydrolyze a subsample of the deproteinized sample to obtain a hydrolyzed sample, wherein the hydrolysis converts any remaining polypeptides and peptides into free amino acids;

[0030] e. The level of free amino acids in the hydrolyzed sample is determined by colorimetry or fluorescence method, and this level is designated as CT;

[0031] f. Optionally, calculate the level of peptide-derived amino acids in the blood sample, denoted as CP, where CP = CT - CF;

[0032] as well as

[0033] g. Based on the values ​​of CF and CT, and optionally further using CP, assess protein absorption from the subject's gut.

[0034] 2. The method of any of the preceding items, wherein an increase in postprandial CF levels relative to postprandial CT or CP levels compared to a healthy reference is considered an indication of protein malabsorption and / or pancreatic exocrine insufficiency.

[0035] 3. The method of any of the preceding items, wherein a decrease in postprandial CP levels compared with a healthy reference is considered an indication of protein malabsorption and / or pancreatic exocrine insufficiency.

[0036] 4. The method according to item 1 further includes assessing the quality of dietary protein based on results from an assessment of protein absorption, wherein CP is calculated, and wherein:

[0037] a. When the CP (Concentration Proportion) is less than 50% compared to the CP of a satisfactory reference dietary protein, the quality of the tested dietary protein is considered poor; and

[0038] b. The quality of a tested dietary protein is considered satisfactory when its CP is at least 50% of that of a reference dietary protein of satisfactory quality.

[0039] 5. The method according to item 1 further includes estimating the subject's biological age based on the level of protein uptake assessed, optionally based on CP, and further independently and optionally in combination with other physiological indicators of biological age.

[0040] Biological age estimation involves comparing the level of protein uptake being assessed with a reference value for similar quantitative protein uptake in a group of reference subjects representing subjects of a specific age or age range.

[0041] 6. According to the method in item 5, wherein the reference subjects are matched with the subjects in terms of age, sex and / or health status.

[0042] 7. The method according to item 1 further includes assessing the subject's ability to absorb drugs based on an assessment of protein absorption.

[0043] 8. The method according to item 7, wherein the drug absorption being evaluated is peptide transporter (PepT1) dependent absorption.

[0044] 9. The method according to item 7 or 8, which includes calculating the level of peptide-derived amino acids in a blood sample, the level being designated as CP, where CP = CT - CF, and where the drug uptake assessment is performed based on CP.

[0045] 10. The method of any of the preceding items, wherein the determination is a colorimetric method and involves a ninhydrin reaction.

[0046] 11. The method of any of the preceding items, wherein the protein removal in step b results in a reduction of at least 98% of proteins having a molecular weight exceeding the size limit.

[0047] 12. The method of any of the preceding items, wherein the protein removal in step b involves ultrafiltration.

[0048] 13. The method of any of the preceding items, wherein the size is limited to 10 kDa.

[0049] 14. The method of any of the preceding items, wherein the hydrolysis step involves acid hydrolysis.

[0050] 15. The method of any of the preceding claims, wherein the method includes calculating the level of peptide-derived amino acids in a blood sample, the level being designated as CP, wherein CP = CT - CF, and wherein the step of assessing protein absorption from the subject’s gut is based on the values ​​of CF, CT, and CP.

[0051] 16. The method of any of the preceding items, wherein the blood sample is a plasma sample, a serum sample, a whole hemolyzed blood sample or a separated red blood cell sample.

[0052] 17. The method of any of the preceding items, wherein the blood sample is a plasma sample or a serum sample.

[0053] 18. The method of any of the preceding items, wherein the blood sample is a plasma sample.

[0054] 19. The method of item 1 or any of its sub-items, wherein the results of an assessment of protein uptake are used to evaluate the outcome of bariatric surgery.

[0055] 20. According to the method in item 19, the evaluation is performed by comparing preoperative and postoperative values.

[0056] 21. A method for treating pancreatic exocrine insufficiency in a subject with this need, comprising:

[0057] a. Assess protein uptake in subjects suspected of having pancreatic exocrine insufficiency using the method according to item 1 or any of its subordinate items; and

[0058] b. If protein malabsorption is detected, oral pancreatic enzyme replacement therapy (PERT) is administered to the subject.

[0059] 22. A method for treating pancreatic exocrine insufficiency in subjects with this need, comprising:

[0060] a. Assess protein uptake in subjects diagnosed with pancreatic exocrine insufficiency and undergoing PERT using the method according to item 1 or any of its sub-items; and

[0061] b. If protein malabsorption is detected, increase the dose of PERT administered to the subject.

[0062] 23. The method according to item 21 or 22, wherein PERT includes oral administration of a protease to the subject.

[0063] 24. The method according to any one of items 21-23, wherein PERT includes oral administration of pancreatic lipase to the subject.

[0064] 25. The method is based on any one of items 21-24, wherein the subject has already undergone weight loss surgery.

[0065] 26. A method for calculating an individualized dosing regimen for a drug in a subject, comprising:

[0066] a. Assess the ability to absorb drugs from the subject's gut using the method described in item 7 or any of its sub-items; and

[0067] b. Individualized drug dosing regimens calculated for the subjects based on this assessment.

[0068] 27. Methods for optimizing the diet of test subjects, including:

[0069] a. Test the protein intake of the subjects after consuming the candidate food using the method described in item 1 or any of its sub-items;

[0070] b. Compare candidate foods to one another based on relative protein intake among subjects; and

[0071] c. Adjust the subjects' diet to include or increase the proportion of foods with relatively good protein intake observed, and / or avoid or reduce the proportion of foods with relatively poor intake.

[0072] 28. Methods for optimizing subject training protocols, including:

[0073] a. Using the method described in item 1 or any of its sub-items, protein intake of subjects was assessed after different training exercises, and the effects of the training exercises on protein intake were compared with each other; and

[0074] b. Adjust the subject’s training protocol to include or increase the relative amount of the following exercises in the training protocol: the protein intake observed after the exercise is relatively high, and / or avoid or reduce the relative amount of the following exercises in the training protocol: the protein intake observed after the exercise is relatively low.

[0075] 29. Methods for screening productive farm animals for productive individuals, including:

[0076] a. Screening farm animals for protein uptake using the method described in item 1 or any of its sub-items; and

[0077] b. Select individual animals with above-average protein uptake capacity for production, and / or discard individual animals that exhibit poor protein uptake.

[0078] The arrangement of this disclosure into chapters with headings and subheadings is solely for the purpose of improving readability and should not be construed as limiting in any way. In particular, this division does not in any way exclude or restrict the combination of features under different headings and subheadings. All references are incorporated herein by citation. Detailed Implementation

[0079] Methods for assessing protein absorption

[0080] In a first aspect, the present invention provides a method for assessing protein absorption from the gut of a subject, comprising:

[0081] a. Provide blood samples from the subjects;

[0082] b. Essentially remove proteins with sizes exceeding 40 kDa from the sample to provide a deproteinized sample;

[0083] c. Determine the level of free amino acids in the first subsample of the deproteinized sample by colorimetry or fluorescence method; this level is designated as CF.

[0084] d. Hydrolyze the second subsample of the deproteinized sample to obtain a hydrolyzed sample, wherein the hydrolysis converts any remaining polypeptides and peptides into free amino acids;

[0085] e. The level of free amino acids in the hydrolyzed sample is determined by colorimetry or fluorescence method, and this level is designated as CT;

[0086] f. Optionally, calculate the level of peptide-derived amino acids in the blood sample, denoted as CP, where CP = CT - CF;

[0087] as well as

[0088] g. Based on the values ​​of CF and CT, and optionally further using CP, assess protein uptake from the subject's gut.

[0089] Blood samples can be plasma samples, serum samples, whole hemolyzed blood samples, or separated hemolyzed red blood cell samples. Plasma or serum samples are preferred, with plasma samples being the most preferred.

[0090] Preferably, the determination is a colorimetric method, and more preferably involves a ninhydrin reaction, which is a classic method for amino acid quantification. However, other methods known for colorimetric or fluorescent quantification of free amino acids can also be used. Commercial reagents from various chemicals can be used for colorimetric (e.g., using genipin) or fluorescent determination of free amino acids in a sample.

[0091] Ninhydrin is a strong oxidizing agent, and in its presence, amine groups in amino acids, peptides, and proteins undergo oxidative deamination, releasing ammonia, CO2, the corresponding aldehyde, and the reduced form of ninhydrin. The colorimetric determination is based on the subsequent reaction of two molecules of ninhydrin (2,2-dihydroxyindane-1,3-diketone) with free α-amino acids to produce a deep purple or blue color known as Ruhemann's purple (Ruhemann, 1910). In this reaction, ninhydrin acts as an oxidizing agent and, under heating conditions, causes deamination and decarboxylation of the amino acid. This reaction is followed by a condensation reaction between the reduced ninhydrin molecule and the released ammonia, as well as a second molecule of n-reduced ninhydrin. At the end of the reaction, a diketohydroindene complex is formed, which has a deep purple color. For amino acids such as proline and hydroxyproline, the test instead produces an imine salt, which has a yellow-orange color. Similarly, proteins with free amine groups, such as asparagine, react with ninhydrin reagent to form a brown product. Amino acids from hydrolyzed proteins or peptides also react with ninhydrin (Mendel, 2004). Ninhydrin reagent is readily available from commercial sources.

[0092] Due to the presence of the amine group (NH2), the amount of the colored complex formed is proportional to the concentration of the amino acid in the analyte solution. The intensity of the Roman violet color can be measured spectrophotometrically, preferably at a wavelength of about 570 nm. Alternatively, the intensity of the yellow-orange color produced by the reaction with proline / hydroxyproline can be measured at a wavelength of about 440 nm. To simultaneously measure both types of amino acids, a wavelength of 520 nm can be used.

[0093] The hydrolysis step (d) preferably involves acid hydrolysis, typically carried out at elevated temperatures. Exposure to acid at elevated temperatures, such as 1H acetate buffer at 100°C, can be used to release amino acids from the peptide, thereby enabling the measurement of the sum of free amino acids and peptide-derived amino acids (i.e., total amino acids). Of course, many different methods for performing peptide hydrolysis are known in the art.

[0094] Removal of interfering blood proteins

[0095] Most proteins present in blood samples (e.g., serum / plasma / hemolyzed erythrocytes / hemolyzed blood) are much larger than dietary peptides (mostly 2-3 amino acids, occasionally up to 8 (i.e., approximately 200-800 Da)) that are the targets of measurement (e.g., albumin approximately 67 kDa, immunoglobulins approximately 160 kDa, fibrinogen approximately 340 kDa). Therefore, interference from colorimetric / fluorescent (e.g., ninhydrin) assays of these proteins can be eliminated by molecular size-based removal techniques (e.g., ultrafiltration). Other suitable methods for protein removal include selective precipitation (e.g., using denaturing solvents or trichloroacetic acid at concentrations selectively precipitating large proteins), gel filtration, and dialysis.

[0096] Preferably, the size removed is limited to about 30 kDa, more preferably about 20 kDa, even more preferably about 10 kDa, still more preferably about 5 kDa, still more preferably about 3 kDa, and most preferably about 1 kDa.

[0097] The protein removal in step b preferably results in a reduction of at least 98%, preferably at least 99%, and more preferably at least 99.9% of proteins having a molecular weight exceeding the size limit.

[0098] The protein removal in step b preferably involves ultrafiltration, preferably using a filter with desired molecular weight size limitations, such as a 10 kDa cutoff filter. Suitable filters that operate using centrifugal force or vacuum suction are well known in the art.

[0099] Conclusions from the assessment

[0100] An increase in postprandial CF levels relative to (healthy) reference levels, compared to postprandial levels of CT or CP, indicates protein malabsorption and / or pancreatic exocrine insufficiency. This is because in malabsorption, dietary peptides cannot be properly absorbed, and their deficiency leads to the dominance of free amino acids in the circulation.

[0101] Compared to a (healthy) reference, decreased postprandial CP levels indicate malabsorption of protein and / or pancreatic exocrine insufficiency.

[0102] The results can be used to evaluate the effects of weight-loss surgery in patients, preferably by comparing values ​​before and after surgery. Suitably, subjects were given similar protein meal challenges before and after surgery prior to sampling for the measurements of this invention. A decrease in postoperative CT or CP indicates poor absorption.

[0103] This result can also be used to evaluate the effectiveness of exercise and training, and to compare the CP value of different meals at different time points before and after training.

[0104] The reference may be the average of comparable measurements from a set of reference samples from a group of reference subjects who do not have protein malabsorption and / or pancreatic exocrine insufficiency. The group may contain at least 10 reference subjects. The test subjects are preferably matched to the reference subjects in terms of age and / or sex.

[0105] Alternatively, the reference could be comparable measured values ​​from samples taken from the same subject at different time points, such as comparing values ​​before and after weight loss surgery to evaluate the effectiveness of the surgery, or comparing values ​​before and after exercise to evaluate the effectiveness of physical training.

[0106] treat

[0107] In a second aspect, the present invention provides a method for treating pancreatic exocrine insufficiency (including insufficiency in the elderly, neonatal insufficiency, alcohol abuse-related insufficiency, weight loss surgery-related pancreatic enzyme insufficiency, and pepsin insufficiency) in subjects with such insufficiency, comprising:

[0108] a. Assess protein uptake in subjects suspected of having pancreatic exocrine insufficiency using the method of the first aspect; and

[0109] b. If protein malabsorption is detected, administer oral pancreatic enzyme replacement therapy (PERT) to the subject.

[0110] In a third aspect, the present invention provides a method for treating pancreatic exocrine insufficiency in subjects with this need, comprising:

[0111] a. Assess protein uptake in subjects diagnosed with pancreatic exocrine insufficiency and undergoing PERT using the method of the first aspect; and

[0112] b. If protein malabsorption is detected, increase the dose of PERT administered to the subject.

[0113] Subjects in the second or third category may have already undergone weight loss surgery.

[0114] PERT may include oral administration of a protease to a subject. PERT preferably includes oral administration of a pancreatic lipase (which includes a protease) to a subject. The pancreatic lipase may be animal / pig-derived or microbial-derived.

[0115] Assessing the quality of dietary protein by evaluating protein absorption from the gut.

[0116] In a fourth aspect of the invention, a method for assessing the quality of dietary protein by evaluating protein absorption from the gut is provided, comprising:

[0117] a. Feed the dietary protein to be evaluated (test dietary protein) to the test subjects;

[0118] b. Blood samples were provided from subjects after feeding;

[0119] c. Essentially remove proteins with sizes exceeding 40 kDa from the sample to provide a deproteinized sample;

[0120] d. Determine the level of free amino acids in subsamples of the deproteinized sample by colorimetric or fluorescence methods; this level is designated as CF.

[0121] e. Hydrolyze a subsample of the deproteinized sample to obtain a hydrolyzed sample, wherein the hydrolysis converts any remaining polypeptides and peptides into free amino acids;

[0122] f. The level of free amino acids in the hydrolyzed sample is determined by colorimetry or fluorescence method, and this level is designated as CT;

[0123] g. Optionally, calculate the level of peptide-derived amino acids in a blood sample, denoted as CP, where CP = CT - CF;

[0124] h. Based on CF and CT values, and optionally further using CP, assess protein uptake from the subject's gut; and

[0125] i. Assess the quality of dietary protein based on the results of protein absorption assessments.

[0126] Step bh can be performed as described with respect to the first aspect. Blood samples can be taken as described with respect to the first aspect.

[0127] Blood samples are preferably collected at 1 h, 2 h, 3 h, 4 h and / or 6 h after feeding.

[0128] If the CP or CT is lower than that of a reference dietary protein with satisfactory dietary quality measured in a similar manner (respectively), this indicates that the tested dietary protein is difficult to digest and its quality is considered poor. In this context, "low" means less than 50%, or in an increasing preferred order of less than 40%, 30%, 20%, 10%, 5%, or 1%.

[0129] Conversely, if the CP or CT is similar to or higher than that of a reference dietary protein with satisfactory dietary quality measured in a similar manner, this indicates that the tested dietary protein is easily digestible, and its quality is considered satisfactory. CP is preferred, but since CF is relatively constant, CT can also be used directly for evaluation without calculating CP. In this context, "high" means at least 50%, or in an increasing preferred order of at least 60%, 70%, 80%, 85%, 90%, 95%, or 100%.

[0130] The dietary protein to be tested can be from conventional or preferably unconventional sources, such as microorganisms, fungi, insects, or synthetic sources. The reference dietary protein can be any protein source of known quality, including meat protein, serum albumin, egg vitellin, cereal protein, milk protein, or protein from vegetable sources. The protein source to be tested can be tested alone and / or as a component of a mixed meal (containing other macronutrients and micronutrients in addition to the protein being tested).

[0131] Test subjects can be laboratory or farm animals, such as mice, rats, gerbils, guinea pigs, rabbits, chickens, turkeys, pigs, cats, dogs, or humans.

[0132] Determine the biological age of the subjects

[0133] In the fifth aspect, methods for determining the biological age of a subject are provided, including:

[0134] a) Provide blood samples from the subjects;

[0135] b) substantially remove proteins with sizes exceeding 40 kDa from the sample to provide a deproteinized sample;

[0136] c) The level of free amino acids in a subsample of the deproteinized sample is determined by colorimetry or fluorescence method, and this level is designated as CF;

[0137] d) Hydrolyze a subsample of the deproteinized sample to obtain a hydrolyzed sample, wherein the hydrolysis converts any remaining polypeptides and peptides into free amino acids;

[0138] e) The level of free amino acids in the hydrolyzed sample is determined by colorimetry or fluorescence method, and this level is designated as CT;

[0139] f) Optionally, calculate the level of peptide-derived amino acids in the blood sample, which is designated as CP, where CP = CT - CF;

[0140] g) Based on the values ​​of CF and CT, optionally further using CP, assess protein absorption from the subject's gut; and

[0141] h) Estimate the subject’s biological age based on the level of protein uptake assessed, optionally based on CP, and further independently and optionally in combination with other physiological indicators of biological age.

[0142] Step ag can be performed according to the first aspect. Blood samples can be taken as described with respect to the first aspect.

[0143] Biological age estimation involves comparing the level of protein uptake being assessed to a reference value, or a set of such reference values, of similarly assessed protein uptake in a group of reference subjects representing a specific age or age range. Reference subjects may be matched to the subjects in terms of age, sex, and / or health status.

[0144] Reference values ​​can be the mean or median (preferably median) protein uptake assessed according to the first aspect: 1 / 100 individuals aged 6–12 years, 2 / 100 individuals aged 13–21 years, 3 / 100 individuals aged 21–30 years, 4 / 100 individuals aged 31–45 years, 5 / 100 individuals aged 46–60 years, 6 / 100 individuals aged 61–75 years, 7 / 100 individuals aged 76–80 years, and 8 / 100 individuals aged 81 years and older. The protein uptake measured by the subjects can then be compared with reference values ​​obtained from the age group closest to the subjects' chronological age.

[0145] Biological age estimation can be based on measurements of additional indicators of biological age, such as DNA methylation, telomere length, proteomics analysis, glycomics analysis, clinical biomarkers, wearable sensor data, and / or other known markers of biological age.

[0146] In some cases, the CP is calculated, and biological age is estimated based on the CP.

[0147] Evaluation of drug absorption and efficacy

[0148] In the sixth aspect, methods are provided for assessing the ability of a drug to be absorbed from the gut of a subject and / or the efficacy of the drug in the subject, including:

[0149] a) Provide blood samples from the subjects;

[0150] b) substantially remove proteins with sizes exceeding 40 kDa from the sample to provide a deproteinized sample;

[0151] c) The level of free amino acids in a subsample of the deproteinized sample is determined by colorimetry or fluorescence method, and this level is designated as CF;

[0152] d) Hydrolyze a subsample of the deproteinized sample to obtain a hydrolyzed sample, wherein the hydrolysis converts any remaining polypeptides and peptides into free amino acids;

[0153] e) The level of free amino acids in the hydrolyzed sample is determined by colorimetry or fluorescence method, and this level is designated as CT;

[0154] f) Optionally, calculate the level of peptide-derived amino acids in the blood sample, which is designated as CP, where CP = CT - CF;

[0155] g) Based on the values ​​of CF and CT, and optionally further using CP, assess protein absorption from the subject's gut; and

[0156] h) Assess the subject’s ability to absorb drugs and / or the expected efficacy of the drug based on an assessment of protein absorption.

[0157] Step ag can be performed according to the first aspect. Blood samples can be taken as described with respect to the first aspect.

[0158] If protein absorption is below the individual's average, drug absorption is also considered below the individual's average ted. Conversely, if protein absorption is above the individual's average, drug absorption is also considered above the individual's average. Assessment may include comparing the assessed protein absorption to reference values ​​obtained from a reference subject or a group of reference subjects. Reference subjects may be matched to the individual in terms of age, sex, and / or health status. Expected drug efficacy may be estimated based on the assessed drug absorption. Drug dosage to subjects may be adjusted based on the results.

[0159] The drug absorption being assessed can be peptide transporter (PepT1) dependent or amino acid transporter dependent. However, this method is not limited to drugs with specific transport mechanisms, as long as absorption is associated with amino acid absorption and / or dipeptide and tripeptide absorption. Examples of drugs that can be assessed using this method include bestatin, β-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors, valacyclovir, L-DOPA-L-Phe, and levodopa.

[0160] In some cases, CP is calculated, and drug uptake is assessed based on CP. This is advantageous for drugs using the same uptake mechanism as dipeptides and tripeptides. In such cases, free amino acid levels do not provide additional information and may even increase noise.

[0161] In the relevant seventh aspect, individualized drug dosing regimens can be designed or adjusted based on the results obtained from the methods in the sixth aspect. The seventh aspect provides methods for calculating individualized drug dosing regimens for subjects, which include:

[0162] a. Assess the ability to absorb drugs from the subject's gut using the sixth aspect of the method; and

[0163] b. Calculate an individualized drug dosing regimen for the subject based on this assessment.

[0164] The calculated individualized dosing regimens can then be applied by physicians in clinical practice. For individuals with below-average peptide absorption, a dose above average is administered to achieve an effective plasma concentration for treatment. For individuals with above-average peptide absorption, a dose below average is administered to avoid over-exposure and thus minimize side effects.

[0165] The drug is preferably transported by PepT1 or an amino acid transporter. The drug may be an anticancer agent, bestatin, a β-lactam antibiotic, an angiotensin-converting enzyme (ACE) inhibitor, valacyclovir, L-DOPA-L-Phe, or levodopa.

[0166] Additional Applications

[0167] In a further aspect, the present invention relates to a method for optimizing the diet of a subject, which is based on testing the protein intake of the subject after consuming various foods using the method of the first aspect. The various foods are compared with each other based on the relative protein intake among the subjects. The subject's diet is adjusted to include or increase the proportion of foods with relatively good observed protein intake, and to avoid or reduce the proportion of foods with relatively poor intake. Subjects may be, for example, elderly / veterinary patients, patients with protein malabsorption, children with growth retardation, or athletes.

[0168] The present invention also relates to a method for optimizing a training program for a subject, such as an athlete, based on testing the subject's protein intake after different training exercises, comparing the effects of these exercises on protein intake with each other. The subject's training program is then adjusted to include or increase the relative amount of exercises following which resulted in relatively high protein intake, and to avoid or reduce the relative amount of exercises following which resulted in relatively low protein intake.

[0169] This invention also relates to a method for screening productive farm animals for production purposes, which involves screening farm animals for protein uptake capacity using the method of the first aspect. Individual animals with above-average protein uptake capacity can be selected for production, and individual animals exhibiting poor protein uptake can be eliminated.

[0170] Discussion of results in the examples

[0171] Based on results obtained from healthy volunteers and animals, and then from the same animals that developed pancreatic exocrine insufficiency, the inventors unexpectedly demonstrated that the ninhydrin method is sensitive enough to rapidly detect peptide / derived amino acid levels in the blood, and clearly showed the differences in protein uptake patterns between healthy pigs and EPI pigs (Figures 1, 2, and 3) and examples.

[0172] Surprisingly, the levels of free amino acids in the blood of both healthy and EPI pigs did not respond to feeding. Furthermore, compared to healthy animals, the highest levels of free amino acids (C0) in EPI pigs were observed. max The levels of free amino acids were significantly higher (p<0.05). Calculations showed that in healthy pigs and EPI pigs, the levels of free amino acids accounted for approximately 10% and 20% of total amino acids, respectively. Conversely, when compared with EPI animals, the levels of total amino acids (which mainly consist of peptide-derived amino acids) in healthy pigs were significantly higher.

[0173] A possible explanation for these characteristics may involve a protein-like reaction in the gut (Watanabe et al., 1992; Gong et al., 2015). The formation of peptides and polypeptides from amino acids and gastric hydrolysates, catalyzed by enzymes (proteases), i.e., the formation of peptide bonds, is called a protein-like reaction. This reaction, occurring in the intestinal lumen, begins in the duodenum (or may begin in the stomach after physiological reflux of pancreatic enzymes) when peptones produced by pepsin in the stomach appear and mix with pancreatic and intestinal peptidases.

[0174] Therefore, pancreatic enzymes not only cleave proteins but also digest peptides derived from the activity of pepsin (a non-specific peptidase) in the stomach. Pancreatic peptidases modify all the end products of protein digestion by coupling free amino acids to peptides and peptides to peptides in protein-like reactions. These end products are produced by specific endopeptidases, carboxypeptidases, and dipeptidases. The deficiency of pancreatic peptidases, and the resulting slowed intestinal protein-like reactions, can explain the higher levels of free amino acids and lower levels of peptide / derived amino acids in the blood of EPI animals. It is speculated that the same pattern of changes in the ratio of free amino acids to total amino acids and postprandial changes can be observed in elderly patients as pancreatic exocrine function declines with aging (Löhr et al, 2018).

[0175] All of the above raises serious questions about current standards of care and the actual treatment outcomes for cystic fibrosis patients of different ages, as exemplified by the treatment of elderly patients with EPI using the same dose of pancreatic lipase. Currently available PERT drugs primarily aim to improve fat absorption. It appears that the needs of EPI patients may vary considerably, therefore the use of PERT drugs (preparations) should be reconsidered and adapted to factors such as age and disease type. For example, growth in cystic fibrosis patients may depend more on absorbed peptides than fat, and glucose homeostasis and the development of type II / III diabetes depend primarily on circulating α-amylase levels (Pierzynowski et al, 2023). Undoubtedly, further research is needed into the roles of absorbed peptides and amino acids in the disease. Research from the 1990s (Pierzynowski et al, 1997) hypothesized that blood peptides are used for tissue protein synthesis, while absorbed amino acids are used for protein repair.

[0176] Most dietary proteins are absorbed in the form of peptides (Goodman, 2010), rather than free amino acids. In addition to assessing protein absorption, the measurement of peptides, unlike free amino acids, may be an important marker of health. Expectedly absorbed peptides play a crucial role in regulating body metabolism and growth (Pierzynowski et al., 1997). Therefore, in addition to nutrient delivery, peptides can induce a number of physiological effects (Miner-Williams et al., 2014; Shen and Matsui, 2019; Choi et al., 2012; O'Keeffe et al., 2018 2-5; Pellegrini et al., 2004). Peptides are absorbed more efficiently than free amino acids (Webb, 1986). The exact mechanisms by which peptides enter the bloodstream are not fully elucidated. Dipeptides and tripeptides have been well demonstrated to enter the bloodstream directly from the intestinal lumen via a transporter protein (PepT1) found on intestinal epithelial cells (Vang et al., 2017). Furthermore, studies have shown that the quantitative and qualitative composition of pancreatic peptidases affects peptide spectra (quantity and quality) (Pierzynowski et al., 2005). Having high-throughput, inexpensive tools to measure peptide uptake could help advance this research area.

[0177] Current findings indicate that the improved high-throughput ninhydrin method is a reliable approach for measuring free amino acids and dietary-derived peptides before and after meals. Both accuracy and precision reflect how close the measured value is to the actual value. More specifically, accuracy reflects how close the measured value is to a known or acceptable value, while precision reflects the repeatability of the measured value, even if these values ​​deviate from the actual values. The validation results of the improved ninhydrin method shown in Table 1 are satisfactory for the key parameters of the measurement: accuracy, precision (repeatability, and intermediate precision). Linearity refers to the ability to provide laboratory test results proportional to the concentration of the measured substance in the test sample. We have demonstrated that this high-throughput, improved ninhydrin method is highly linear. These factors are essential for testing that can be used in clinical research and general clinical applications. CNA measurements have never been shown to possess these key characteristics.

[0178] In summary, this paper provides a rapid, reliable, and simple method for measuring peptide-derived amino acids, which can be used as direct biomarkers of protein digestion and absorption, and is a superior alternative to indirect, fecal-based CNA measurements. The method of this invention is reproducible and physiologically relevant, and can also be readily used in clinical or research settings, as demonstrated in the EPI pig model.

[0179] References

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[0186] Löhr J-M, PanicN, Vujasinovic M, and Verbeke CS. The ageing pancreas:a systematic review of the evidenceand analysis of the consequences InternMed. 2018;283:446–460. Doi: 10.1111 / joim.12745.

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[0188] Gan C, Chen Y-H, Liu L, Gao J-H, Tong H, Tang Ch-W, and Liu R.Efficacy and safety of pancreatic enzyme replacement therapy on exocrinepancreatic insufficiency: a meta-analysis. Oncotarget. 2017 Nov 7; 8(55):94920–94931.Doi:10.18632 / oncotarget.21659.

[0189] Borowitz D, Aronoff N, Cummings LC, Maqbool A, and Mulberg AE. |Coefficient of Fat Absorption to Measure the Efficacy of Pancreatic EnzymeReplacement Therapy in People With Cystic Fibrosis. Gold Standard or CoalStandard? Pancreas 2022;51: 310–318.DOI: 10.1097 / MPA.0000000000002016.

[0190] Freedman SD, Zaworski K, Pierzynowska, Pierzynowski S, Gallotto R,Sathe M, Borovitz D. Validation of an omega-3 substrate challenge absorptiontest as an indicator of global fat lipolysis. PloS ONE 2023, 18(5): e0284651.https: / /

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[0195] Gong M, Mohan A, Gibson A, Udenigwe ChC. Mechanisms of plasteinformation, and prospective food and nutraceutical applications of the peptideaggregates. Biotechnology Reports 5 (2015) 63–69. Doi.org / 10.1016 / j.btre.2014.12.003.

[0196] Pierzynowski SG, Stier Ch, Pierzynowska K. A hypothesis that Alpha-amylase Evokes Regulatory Mechanisms Originating in the Pancreas, Gut andCirculation, which Govern Glucose / Insulin Homeostasis. World J Diabetes. 2023September 15; 14(9): 1341-1348, DOI: 10.4239 / wjd.v14.i9.1341.

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[0199] Miner-Williams WM, Stevens BR, Moughan PJ. Are intact peptidesabsorbed from the healthy gut in the adult human?. Nutr. Res. Rev. 2014, 27(2). 308-329.

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[0201] O’Keeffe MB, FitzGerald RJ. Whey protein hydrolysate inducedmodulation of endothelial cell gene expression. J. Funct. Foods. 2018.40. 102109.

[0202] Pellegrini A, Andreas J, Hulsmeierb W, Hunziker P, Thomas U.Proteolytic fragments of ovalbumin display antimicrobial activity. Biochimicaet Biophysica Acta 1672: 76– 85. 2004.

[0203] Webb KE, Jr. Amino acid and peptide absorption from thegastrointestinal tract . Fed Proc. 1986 Jul;45(8):2268-71.

[0204] Wang Chun-Yang, Shu Liu, Xiao-Nv Xie, Zhi-Rong Tan. Regulationprofile of the intestinal peptide transporter 1 (PepT1). Drug Design,Development and Therapy:11, 3511–3517, 2017.

[0205] Pierzynowski SG, Kruszewska D, Weström B. -The quality of the dietary protein digestion affects animal performance and regulates gut bacterial growth – hypotheses and facts. In: Biology of the growing Animals, SeriesEditors: Pierzynowski SG, Zabileski R.,Volume Editors: Biology of theNutrition in Growing Animals, Editors: Mosenthin R, Zentek J, Zebrowska T.120-143, Elsevier, 2005.

[0206] Chapman, I.; Oberoi, A.; Giezenaar, C.; Soenen, S. Rational Use of Protein Supplements in the Elderly—Relevance of Gastrointestinal Mechanisms. Nutrients 2021, 13, 1227. Example

[0207] The following examples should not be considered limiting. For further information on experimental details, technical readers can refer to the separate chapter entitled Materials and Methods.

[0208] Example 1: Estimation of free amino acids, total amino acids, and peptide-derived amino acids using the ninhydrin method

[0209] The main improvement over the original ninhydrin method is the development of a high-throughput method on microplates. Free amino acids and peptides are separated from large plasma proteins such as albumin and fibrinogen using a 10 kDa centrifugal filter.

[0210] The level of free amino acids is estimated using a complete osmotic solution, while the total amino acid level is estimated using a hydrolyzed osmotic solution. Peptide-derived amino acids are quantified by subtracting free amino acids from the total amino acid level: Peptide-derived amino acids = Total amino acids - Free amino acids

[0211] Required reagents

[0212] 1. Standard amino acid stock solution (amino acid standard, catalog number AAS18, Supelco, Sigma Aldrich) 260 μL + 740 dH2O.

[0213] 2. 0.2 M acetate buffer (pH=5.5) (0.84 g NaOH + 1.465 ml glacial acetic acid + 100 ml dH2O)

[0214] 3.8% w / v ninhydrin reagent (N4876, Sigma-Aldrich) [Preparation: Weigh 8 g of ninhydrin and dissolve in 100 ml of 99.8% acetone]. Photosensitizing! Should be used within 15 minutes.

[0215] 4.50% v / v ethanol.

[0216] 5. Distilled water.

[0217] Required instruments and glassware

[0218] 1.10 kDa rotating column (Amicon Ultra-0.5 centrifugal filter unit, catalog number UFC5010, Millipore, Sigma Aldrich).

[0219] 2. Test / boiling tube (1.5 ml tube with screw cap).

[0220] 3. Pipettes [micropipettes].

[0221] 4. Flat-bottomed microporous plate

[0222] 5. Water bath or dry heating block.

[0223] 6. Spectrophotometer

[0224] Preparation of standard curve

[0225] Prepare amino acid stock solutions. Transfer different volumes (20-70 µl) of standard amino acid stock solutions into appropriately labeled tubes to obtain six standard concentrations ranging from 30.0 to 105.0 µg / ml.

[0226] Sample preparation

[0227] 350 µl of separated plasma was transferred to a centrifuge column (10 kDa rotary column) and centrifuged at 14,000 g for 30 min at room temperature. No significant differences in amino acid content were observed between the separated serum or plasma samples. Samples with high (105 µg / ml), medium (60 µg / ml), and low (30 µg / ml) amino acid concentrations were prepared for use in validation experiments.

[0228] Ninhydrin determination

[0229] Transfer different volumes (20-70 μL) of standard amino acid stock solutions to their respective labeled test tubes to achieve standard concentrations between 30-105 µg / mL. Then transfer the test samples to test tubes: a) for free amino acids, transfer 30 μL of centrifuged buffer; b) for total amino acids, add 30 μL of acetate buffer to 30 μL of centrifuged buffer, and then boil the liquid in a water bath for 1 hour to hydrolyze. Afterward, take 30 μL of the hydrolysate and transfer it to a new test tube.

[0230] To standardize signal intensity, 15 μL of acetate buffer was added to all standards and free amino acid samples. The volume in each tube was then brought to 1000 μL using distilled water. Tubes labeled "blank" were filled with 15 μL of acetate buffer and 985 μL of distilled water. Finally, 250 μL of ninhydrin reagent was added to all tubes, and the tubes were gently vortexed and placed in a boiling water bath for 15 minutes. After cooling the tubes on ice, 250 μL of 50% ethanol was added to each tube and mixed thoroughly. 300 μL of the reaction mixture from each tube was then transferred to a microplate, and the absorbance was recorded at 570 nm using a spectrophotometer. Results were adjusted for dilution.

[0231] Calculation of free amino acids

[0232] C final free = (CSD*33.3) / 1.1

[0233] Where C final is the final concentration of free amino acids, ug / ml; CSD — the concentration of free amino acids obtained from the standard curve; 33.3 — the dilution factor for 30 ul sample, and 1.1 is the coefficient for compensating for plasma deproteinization.

[0234] Calculation of total amino acids

[0235] C final total = (CSD*2*33.3) / 1.1

[0236] Where C final 1 is the final concentration of total amino acids, ug / ml; CSD - the concentration of free amino acids obtained from the standard curve; 2 - the dilution factor for hydrolysis; 33.3 - the dilution factor for 30 ul hydrolyzed sample, and 1.1 is the coefficient for compensating for plasma deproteinization.

[0237] Calculation of peptide-derived amino acids

[0238] C final peptide derived = C final total – C final free

[0239] Example 2: Validation of the high-throughput ninhydrin method

[0240] After quantification, the following parameters are evaluated: accuracy, precision (repeatability and intermediate precision), and linearity.

[0241] Accuracy

[0242] Accuracy was determined based on the percentage recovery of amino acids in samples with high, medium, and low amino acid concentrations. All samples were run in triplicate. The percentage recovery (%) for each level was determined according to the following equation.

[0243]

[0244] The results of the accuracy analysis are shown in Table 1.

[0245] Target Acceptance Criteria

[0246] Acceptable recovery percentages should be between 90% and 110%. We obtained the following recovery percentages: 106.2%, 96.1%, and 97.7% for low, medium, and high amino acid concentration samples, respectively (Table 1).

[0247] Table 1. Accuracy and Intra-batch Precision (Example)

[0248]

[0249] Accuracy

[0250] Precision refers to the degree of consistency among a series of measurements obtained from multiple samplings of the same homogeneous sample under specified conditions. It is usually expressed as a percentage of relative standard deviation (%RSD).

[0251] Repeatability or intra-batch precision

[0252] Repeatability refers to the precision under the same operating conditions over short time intervals. Nine assays (e.g., three concentrations / two replicates each) covering the range specified by the procedure were evaluated. To calculate repeatability, we used data obtained from three samples with high, medium, and low concentrations of amino acids (Table 1).

[0253] Target Acceptance Criteria

[0254] The RSD (n=3) at each level and the overall RSD should be ≤5%. The average RSD (n=3) of the three amino acid levels is reported, and the repeatability of the three amino acid solution samples is calculated to be 0.72% [(1.37+0.25+0.55) / 3=0.72%] (Table 1).

[0255] intermediate precision

[0256] Intermediate precision represents laboratory variability: different days, different analysts, different equipment, etc. To assess intermediate precision, samples with high, medium, and low amino acid concentrations were reanalyzed on three different days. Samples were analyzed in duplicate.

[0257] Target Acceptance Criteria

[0258] The RSD at each level and the overall RSD should be ≤5%. For low, medium, and high amino acid concentration levels, the intermediate precision of the amino acid solution was 3.91%, 3.00%, and 2.52%, respectively. The overall RSD for all three amino acid levels was 3.14% (Table 2).

[0259] Table 2. Intermediate Precision Data (Example)

[0260]

[0261] Recovery rate

[0262] Plasma samples were spiked with a level of free amino acids (500–2500 µg / ml) prior to centrifugation, and recoveries were calculated by comparing the measured values ​​with the amount of free amino acids in unspiked samples. The average recovery rate was 81%, ranging from 69% to 93%.

[0263] Standard curve fitting

[0264] To obtain a standard curve, a mixture of six amino acid standards and a blank were used. The standard curve (cubic curve) was then plotted as the concentration of the amino acid in each standard (µg / ml) relative to the obtained optical density value measured at 570 nm. The coefficient of determination (r) was then calculated. 2 ).

[0265] Target Acceptance Criteria

[0266] (r) for each standard curve 2 The value should be ≥0.98 (Table 3).

[0267] Table 3. Coefficients of determination (r) of the standard curve for different dates 2 )value

[0268]

[0269] Example 3: Proof-of-Concept Study of the Porcine EPI Model

[0270] Results of free amino acid levels in porcine plasma Figure 1A-C is given. The free amino acid level in healthy animals is approximately 2000 µg / ml, while that in EPI animals is approximately 4000 µg / ml. The observed differences are even reflected in the increased AUC and C. max In both groups of experimental animals, the levels of free amino acids remained stable and showed no significant changes after a meal.

[0271] The results described as peptide-derived + free amino acid levels of total amino acids in porcine plasma are in Figure 2A -C is given, while Figure 3A -C. The initial levels of total amino acids or peptide-derived amino acids in healthy pigs and EPI pigs were similar, approximately 13,000 µg / ml and 10,000 µg / ml, respectively. Both total amino acids and peptide-derived amino acids increased after feeding. Figure 2A and Figure 3A Compared to free amino acids alone, total amino and peptide-derived amino acids were significantly increased postprandially. However, healthy animals reached significantly higher levels of total amino and peptide-derived amino acids up to 3 hours postprandial, with Tmax at 120 minutes post-feeding. Differences between groups were reflected in AUC and C. max In terms of values, the AUC and C of the healthy group max The value was significantly higher than that of the EPI group. Figure 2B C and Figure 3B (C).

[0272] Example 4: Evaluation of the effect of digestive enzyme supplementation on protein intake

[0273] The adaptive ninhydrin method of the present invention (see experimental summary) Figure 4 The effects of exogenous digestive enzyme supplementation on dietary protein digestion and the absorption of nutritional peptides and amino acids were tested in eighteen boars before and after the development of pancreatic exocrine insufficiency (EPI) during routine meal tolerance tests (RMTT) of different types.

[0274] In isoenergetic RMTT test meals, the type of macronutrient used affected the percentage relationship between absorbed dipeptides, tripeptides, and free amino acids. Dipeptide and tripeptide absorption was lower in EPI pigs compared to values ​​obtained before pancreatic duct ligation. Supplementation of the diet with creon or amylase during the RMTT significantly and similarly increased dipeptide and tripeptide absorption. Interestingly, PepT1 expression in the duodenum and jejunum of EPI pigs was lower than in healthy pigs and recovered to healthy values ​​upon enzyme supplementation (creeon or amylase alone).

[0275] In summary, the adaptive ninhydrin approach allows for a good probability estimation of the percentage of nutritional dipeptides, tripeptides, and free amino acids absorbed postprandially from dietary protein, as well as their percentage composition in the blood after digestion. Exogenous microbial amylases and creons substantially and similarly improve postprandial peptide absorption and PepT1 expression. Therefore, age-related loss of PepT1 and limited peptide absorption may be associated with pancreatic exocrine dysfunction in older adults.

[0276] Table 4. Composition of the routine meal tolerance test (RMTT) meal, which constitutes 1% of the daily requirement of a pig weighing approximately 16 kg. This meal is isoenergetic.

[0277]

[0278] Calculation of total (amino acids + dipeptides + tripeptides) quantitative absorption

[0279] The effects of Creon and amylase supplementation on plasma levels of total amine groups during the RMTT are shown in... Figure 5A -C. There was no change in the level of free amine groups in plasma after a meal.

[0280] When HFD was enriched with matrix or administered matrix alone, EPI pigs exhibited a significantly lower range of total amino acid uptake compared to control healthy pigs. Figure 6A -C).

[0281] Total quantitative protein absorption

[0282] The composition of isoenergetic diets, containing different levels of protein and other macronutrients from different sources, influences the proteolysis and absorption of their final products. These results are consistent with previous data obtained in EPI pigs fed the same diet, which confirmed the dependence of carbohydrate absorption on its dietary source. Therefore, we can reveal and highlight hidden differences in protein digestion and further absorption when using dietary protein from different sources. Our current study also confirms the data obtained by Rohm et al. (2020), which showed a clear dependence of peptide absorption on dietary protein source in humans.

[0283] Example 5: Assessing the biological age of subjects

[0284] Peptide uptake was measured in a reference group of 20 healthy individuals using the method of the present invention, with ages i) 20-30, ii) 31-40, iii) 41-50, iv) 51-60, v) 61-70, and vi) 71-80. Similarly, peptide uptake was measured in test subjects using the method of the present invention. The biological age of the test subjects was assessed as falling into the age group closest to the mean by comparison with the mean reference group. When the assessed biological age was lower than the chronological age of the test subjects, the test subjects were considered to be in a better-than-average condition, at least in terms of gut health. The biological age corresponding to the chronological age indicates the average condition of the test subjects. When the assessed biological age was higher than the chronological age of the test subjects, the test subjects were considered to be in a worse-than-average condition, at least in terms of gut health. Appropriate interventions were proposed for the test subjects.

[0285] Example 6: Drug Dosage Adjustment

[0286] For individuals requiring cephalosporin treatment, peptide absorption was measured using the method of this invention. For individuals with below-average peptide absorption, a higher-than-average dose of cephalosporin was administered to achieve an effective plasma concentration. For individuals with above-average peptide absorption, a lower-than-average dose of cephalosporin was administered to avoid overexposure.

[0287] Example 7: Diet optimization for athletes / bodybuilders

[0288] Most athletes, especially bodybuilders, consume large amounts of protein to promote muscle growth. Protein intake can be on the order of 2 grams per kilogram of body weight per day. The biggest question is how much of this protein is actually absorbed? Based on practical methods, it is estimated that about 50% of consumed protein ends up in the bloodstream.

[0289] Digestive proteases are essential for protein absorption. It is generally believed that primarily amino acids are absorbed. Our research (and others) shows that dipeptides and tripeptides are absorbed, and we can measure the absorption of these peptides. The availability of these nutritional peptides is highly dependent on the availability of digestive enzymes.

[0290] A healthy body produces enough enzymes to digest a normal amount of protein (approximately 1 g of protein per kg of body weight); however, enzyme production decreases with age. Additional digestive enzymes—especially proteases—can improve peptide formation and influence training / performance effectiveness. Training and performance themselves stimulate pancreatic secretion and improve peptide (protein) absorption.

[0291] Training to improve protein (dietary peptide) absorption

[0292] Test: Six professional cyclists participated in the following activities:

[0293] 1. On the first day, after a regular breakfast at 08:00, six cyclists (aged 40-50) cycled 100 km (approximately 3 hours without food). Immediately after training, basal-0-hour blood samples were drawn, and a protein-rich meal was provided (eaten 1 hour later).

[0294] 2. The cyclist rests for 2-3 hours and obtains a second blood sample.

[0295] 3. The ninhydrin method of the present invention is used to estimate protein uptake as free amino acids and peptide-bound amino acids.

[0296] 4. Over two days, the same people repeat the same procedures, but instead of cycling, they get free time and a spa treatment (massage, water bath, sauna, light swimming, etc.).

[0297] 5. Set the levels of free amino acids and total (peptide-bound) amino acids after SPA and free time as baselines and specify them as 100%. Five hours after the start of the meal, free amino acid levels increased by 20%, while total (peptide-bound) amino acid levels increased by 60%.

[0298] 6. In the same individuals, following the intensive cycling period, amino acid levels decreased by 10% compared to baseline at free-SPA time, while total (peptide-bound) amino acid levels decreased by 30%. Two to three hours after the start of the meal, free amino acid levels increased by 10% compared to baseline at free time, while total (peptide-bound) amino acid levels increased by 100%.

[0299] The results showed that intensive training depleted the peptide-binding amino acids in the blood, and that protein from meals was absorbed at a higher rate than after rest.

[0300] The results also indicate that monitoring postprandial blood peptide levels (peptide absorption) can serve as a tool for assessing meal effectiveness, dietary supplements, training, and the need for digestive enzyme supplementation.

[0301] Example 8: Protein absorption after bariatric surgery

[0302] Patients who have undergone bariatric surgery often exhibit symptoms of pancreatic exocrine insufficiency.

[0303] test:

[0304] 10 patients underwent bariatric surgery (Roux-en-Y gastric bypass) and received a meal trial 6 months later.

[0305] 2. On the first day, a baseline 0 sample was collected before breakfast, and another blood sample was drawn 2-3 hours later.

[0306] 3. Six days later, the same people were tested again, but in addition to their meals, they also ingested 4 Creon 10000 tablets and 150 mg of pepsin.

[0307] 4. The plasma levels of free amino acids and total amino acids were measured using the ninhydrin method of the present invention.

[0308] The levels of free amino acids and total (peptide-bound) amino acids in the 5.0 (baseline) sample were similar in both tests and were designated as 100%.

[0309] 6. Two to three hours after the meal, the level of free amino acids increased by 10% in the first test, while the level of peptide-bound amino acids increased by 40%.

[0310] 7. In the second test, when Creon 10000 and pepsin were used, free amino acids increased by 25% 2-3 hours postprandial, while peptide-bound amino acids increased significantly by 100% or more.

[0311] In summary, the results indicate that impaired gastric and pancreatic enzyme function can reduce the absorption of proteins in peptide form. The effects can be quantified using the method of this invention.

[0312] Example 9: Optimizing the diet of elderly patients

[0313] Older adults constitute the fastest-growing segment of the population. They are at risk of malnutrition, believed to be caused by reduced food intake or impaired gastrointestinal function. Age-related changes are well-known in organs such as the liver, kidneys, and intestines. The pancreas, representing a metabolically active organ responsible for the uptake and breakdown of essential nutrients, undergoes changes in morphology and function with age.

[0314] During childhood, the pancreas increases in volume, plateaus between the ages of 20 and 60, and then declines. This decline involves the pancreatic parenchyma and is associated with reduced perfusion, fibrosis, and atrophy. Due to these changes, pancreatic exocrine function is impaired in healthy older individuals without any gastrointestinal diseases. 5% of people over 70 years of age and 10% of those over 80 years of age have EPI (exocrine pancreatic inflammatory response), with fecal elastase-1 levels below 200 µg / g. -1 Feces, and 5% of people with severe EPI have fecal elastase-1 levels below 100 µg / g. -1Feces. This can lead to indigestion and malnutrition. Patients may experience only mild symptoms such as steatorrhea, diarrhea, abdominal pain, and weight loss. Malnutrition consists of a deficiency of fat-soluble vitamins and affects patients with EPI and older adults. Secondary consequences may include decreased bone mineral density due to impaired absorption of protein and fat-soluble vitamin D caused by impaired pancreatic exocrine function. An unresolved question is whether this age-related decline in pancreatic function requires treatment. Therapeutic interventions, which may include supplementing older individuals with confirmed pancreatic exocrine insufficiency with pancreatic enzymes and / or vitamins, may contribute to healthy aging.

[0315] As we age, we gradually lose muscle and strength. This process of muscle loss with age is called sarcopenia. One of the potential mechanisms of sarcopenia is a lower anabolic response to protein intake. However, it is not entirely clear why older adults respond poorly to protein. In the study by Chapman et al. (2021), the authors integrated data from all previous studies using endogenous marker proteins. Endogenous marker proteins are a special type of protein produced for research purposes, which allows authors to track them throughout the body to measure their absorption. This allowed the authors to make various interesting comparisons, such as differences in protein digestion and absorption between younger and older adults. In younger adults, 60% of ingested protein ends up in the bloodstream after digestion and absorption. For older adults, this is only 40%. Therefore, less ingested protein is available for muscle tissue in older adults. This may suggest that older adults need to consume slightly more protein to compensate.

[0316] In summary, protein digestion and absorption in the circulation of older adults are generally lower. It remains unclear what percentage of digested proteins are absorbed in the form of tripeptides and dipeptides. This may be important for host protein regeneration, especially in older adults. The transport of dietary peptides from the bloodstream to metabolically active tissues (muscle, brain, liver, pancreas, etc.) via the PepT2 transporter is crucial for understanding the role of absorbed dietary peptides.

[0317] test

[0318] 1. Twelve patients aged between 60 and 80 years consented to the meal test.

[0319] 2. On the first day, a baseline 0 sample was collected before breakfast, and another blood sample was drawn 2-3 hours later.

[0320] 3. Six days later, the same people were tested in the same way, but with an additional intake of 4 Creon 10,000 tablets.

[0321] 4. Measure the plasma levels of free and total amino acids using the described ninhydrin method.

[0322] The levels of free amino acids and total (peptide-bound) amino acids in the 5.0 (baseline) sample were similar in both tests and were designated as 100%.

[0323] 6. Two to three hours after the meal, the level of free amino acids increased by 50% in the first test, while the level of peptide-bound amino acids increased by 20%.

[0324] 7. In the second test, when Creon 10000 was used as a dietary supplement, free amino acids increased by 15% 2-3 hours after a meal, while peptide-bound amino acids increased significantly by 70%.

[0325] In summary, the results indicate that elderly patients suffer from pancreatic exocrine insufficiency, which reduces the absorption of proteins in peptide form. The effects can be quantified using the method of this invention.

[0326] Example 10: Children with growth retardation

[0327] It is hypothesized that growth retardation in children is associated with low levels of nutritional peptide absorption. Meal tests using various protein sources in mixed meals were used to assess the need for optimally absorbable protein and / or protease supplementation. As previously described, tests were performed, with continuous postprandial blood samples collected and CF, CT, and CP measured using the ninhydrin method.

[0328] Example 11: Farm Animals

[0329] It is hypothesized that higher levels of dietary dipeptides and tripeptides than amino acids absorbed early in life may predict better productivity in farm animals during their performance phase. Using the method of this invention, protein or mixed meal tests, combined with consecutive or single post-meal blood sampling, can be used for early identification of optimal farm animal individuals in terms of growth and / or productivity.

[0330] Materials and methods

[0331] animal

[0332] The experiment was conducted on hybrid (Polish Landrace × Yorkshire × Hampshire) pigs (Sus scrofadomesticus). Six mixed-sex pigs, weighing 15 kg at the start of the study, were used. +2.3 The study was conducted on pigs. The same pigs underwent pancreatic duct ligation three weeks after arrival at the animal facility. A jugular venous catheter was inserted after confirmation of EPI (exocrine pancreatic insufficiency). The study was approved by the Second Local Animal Ethics Committee in Warsaw, Poland (Approval No. WAW2 / 025 / 2022). Every effort was made to minimize animal suffering.

[0333] Experimental design and research treatment

[0334] After a 7-day acclimatization period, pigs fed a standard diet were assigned to the study.

[0335] By day 10, pigs were gradually switched to a high-fat diet (HFD). A jugular venous catheter was implanted on day 11. On day 15, a meal tolerance test (MTT) was performed on healthy pigs. On day 16, pancreatic duct ligation (PDL) was performed on the pigs. EPI was allowed to develop for 30 days. On day 47, the same MTT test was performed on the EPI pigs. The MTT contained 160 g of HFD.

[0336] feeding

[0337] During the study period, pigs were fed a high-fat diet (HFD) equal to 4% of their body weight daily (Kcynia, Morawski Plant, Poland), with 1% (160 g) given at breakfast (09:00–10:00 hr) and 3% (480 g) at afternoon (17:00–18:00). Upon arrival, the pigs were fed a standard grain-based pelleted diet, which was gradually switched to HFD on day 5, containing 17.5% crude protein, 3.9% crude fiber, 20% crude fat, and 5.2% ash, as well as 5,000 IE / kg vitamin A, 500 IE / kg vitamin D, and 85 mg / kg vitamin E.

[0338] Blood sampling

[0339] Blood samples were collected via a jugular vein catheter 1 hour before feeding, and then at 60, 120, 180, and 240 minutes after feeding, and transferred to BD Vacutainer® glass aprotinin K3 EDTA tubes (BD Diagnostics, New Jersey, USA). The blood samples were immediately placed on ice and then centrifuged at 3000 xg for 15 minutes at 4°C. Plasma was separated and stored at -80°C until further analysis. The levels of free amino acids, total amino acids, and peptide-derived amino acids in the plasma samples were analyzed using the methods described below.

[0340] Statistical analysis

[0341] Statistical analysis was performed on the data generated in this study using the Student's t-test with Welch correction (for normally distributed datasets) or the Mann-Whitney test (when the data are not normally distributed). The Shapiro-Wilk normality test was used to assess the data distribution. Outliers in the dataset were identified using the regression ROUT method, with Q=0.05%. All analyses were performed using GraphPad Prism 10.0, San Diego, USA. Data were not corrected for multiple comparisons. A p-value ≤ 0.05 was considered significant; a p-value ≤ 0.1 was considered trending. Data with a Gaussian distribution are expressed as mean ± standard deviation (±SD), and data with a non-Gaussian distribution are expressed as median ± interquartile range (±IQR).

Claims

1. An in vitro method for assessing protein absorption from a subject's gut, the method comprising: a. Provide a blood sample from the subject; b. substantially remove proteins with sizes exceeding 40 kDa from the sample to provide a deproteinized sample; c. Determine the level of free amino acids in a subsample of the deproteinized sample by colorimetry or fluorescence method, the level being designated as CF; d. Hydrolyze a subsample of the deproteinized sample to obtain a hydrolyzed sample, wherein the hydrolysis converts any remaining polypeptides and peptides into free amino acids; e. The level of free amino acids in the hydrolyzed sample is determined by colorimetry or fluorescence method, the level being designated as CT; f. Optionally, calculate the level of peptide-derived amino acids in the blood sample, the level being designated as CP, where CP = CT - CF; as well as g. Based on the values ​​of CF and CT, and optionally further using CP, assess protein absorption from the subject's gut.

2. The method of any of the preceding claims, wherein an increase in postprandial CF levels relative to postprandial CT or CP levels compared to a healthy reference is considered an indication of protein malabsorption and / or pancreatic exocrine insufficiency.

3. The method of any of the preceding claims, wherein a decrease in postprandial CP levels compared to a healthy reference is considered an indication of protein malabsorption and / or pancreatic exocrine insufficiency.

4. The method of claim 1, further comprising assessing the quality of dietary protein based on results from an evaluation of protein absorption, wherein a CP is calculated, and wherein: a. When the CP is less than 50% compared to the CP of a satisfactory reference dietary protein, the quality of the tested dietary protein is considered poor; and b. The quality of the tested dietary protein is considered satisfactory when the CP is at least 50% of that of a reference dietary protein of satisfactory quality.

5. The method of claim 1, further comprising estimating the subject's biological age based on the assessed level of protein uptake, optionally based on CP, and further independently and optionally in combination with other physiological indicators of biological age. The biological age estimation involves comparing the level of protein uptake being assessed with a reference value for a similar amount of protein uptake in a group of reference subjects representing subjects of a specific age or age range.

6. The method of claim 5, wherein the reference subject is matched with the subject in terms of age, sex, and / or health status.

7. The method of claim 1, further comprising assessing the subject's drug absorption capacity based on an evaluation of said protein absorption.

8. The method of claim 7, wherein the drug absorption being evaluated is peptide transporter (PepT1)-dependent absorption.

9. The method of claim 7 or 8, further comprising calculating the level of peptide-derived amino acids in the blood sample, the level being designated as CP, where CP = CT - CF, and wherein the drug uptake assessment is performed based on CP.

10. The method of any one of the preceding claims, wherein the determination is a colorimetric method and involves a ninhydrin reaction.

11. The method of any of the preceding claims, wherein the protein removal in step b results in a reduction of at least 98% of proteins having a molecular weight exceeding the size limit.

12. The method of any of the preceding claims, wherein the protein removal in step b involves ultrafiltration.

13. The method of any of the preceding claims, wherein the size is limited to 10 kDa.

14. The method of any one of the preceding claims, wherein the hydrolysis step involves acid hydrolysis.

15. The method of any one of the preceding claims, wherein the method comprises calculating the level of peptide-derived amino acids in the blood sample, the level being designated as CP, wherein CP = CT - CF, and wherein the step of assessing protein absorption from the subject's gut is based on the values ​​of CF, CT, and CP.

16. The method of any one of the preceding claims, wherein the blood sample is a plasma sample, a serum sample, a whole hemolyzed blood sample, or a separated red blood cell sample.

17. The method of any of the preceding claims, wherein the blood sample is a plasma sample or a serum sample.

18. The method of any one of the preceding claims, wherein the blood sample is a plasma sample.

19. The method of claim 1 or any of its dependent claims, wherein the results of an assessment of said protein absorption are used to evaluate the outcome of the weight loss surgery.

20. The method of claim 19, wherein the evaluation is performed by comparing pre-operative and post-operative values.

21. Methods for treating pancreatic exocrine insufficiency in subjects with this need, comprising: a. Assessing protein uptake in subjects suspected of having pancreatic exocrine insufficiency using the method according to claim 1 or any of its dependent claims; and b. If protein malabsorption is detected, oral pancreatic enzyme replacement therapy (PERT) is administered to the subject.

22. Methods for treating pancreatic exocrine insufficiency in subjects with this need, including: a. Assessing protein uptake in subjects diagnosed with pancreatic exocrine insufficiency and undergoing PERT using the method according to claim 1 or any of its dependent claims; and b. If protein malabsorption is detected, increase the dose of PERT administered to the subject.

23. The method of claim 21 or 22, wherein the PERT comprises orally administering a protease to the subject.

24. The method according to any one of claims 21-23, wherein the PERT comprises orally administering pancreatic lipase to the subject.

25. The method of any one of claims 21-24, wherein the subject has undergone weight loss surgery.

26. A method for calculating an individualized dosing regimen for a drug in a subject, comprising: a. Assessing the ability of the subject's intestine to absorb drugs using the method of claim 7 or any of its dependent claims; and b. Calculate an individualized drug dosing regimen for the subject based on the assessment.

27. A method for optimizing the diet of subjects, the method comprising: a. Test the protein intake of the subject after consuming the candidate food using the method of claim 1 or any of its dependent claims; b. Compare the candidate foods to each other based on the relative protein intake of the subjects; and c. Adjust the subjects' diets to include or increase the proportion of foods with relatively good protein intake observed, and / or avoid or reduce the proportion of foods with relatively poor intake.

28. A method for optimizing a subject training program, the method comprising: a. Detecting the protein intake of the subject after different training exercises using the method of claim 1 or any of its dependent claims, wherein the effects of the training exercises on protein intake are compared with each other; and b. Adjust the subject's training program to include or increase the relative amount of the following exercises in the training program: the protein intake observed after the exercise is relatively high, and / or avoid or reduce the relative amount of the following exercises in the training program: the protein intake observed after the exercise is relatively low.

29. A method for screening productive farm animals for productive individuals, the method comprising: a. Screening farm animals for protein uptake capacity using the method of any one of claim 1 or any of its dependent claims; and b. Select individual animals with above-average protein uptake capacity for production, and / or discard individual animals that exhibit poor protein uptake.

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