Method for testing renal dysfunction of cats

Abstract

The present invention provides an objective non-invasive method for examining renal dysfunction in cats. This method for testing renal dysfunction is a method for testing renal dysfunction in cats, and comprises a step for measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in urine of a subject.

Description

Technical Field

[0001] The present invention relates to an examination for renal dysfunction in cats using D-amino acids in urine. Background Art

[0002] Cats, having evolved in arid environments, have the ability to highly concentrate urine in their kidneys, making them naturally low-water consumers. Consequently, cats are particularly susceptible to urinary tract symptoms such as cystitis, urinary stones, and urethral obstruction, which are caused by renal tubular damage. This in turn leads to renal and urinary tract diseases such as renal failure. Furthermore, the recent popularity of dry cat food has further increased the tendency for cats to inadequately consume water. Inadequate water intake increases the risk of urinary tract diseases in young children and kidney disease in middle and older age, leading to a decline in their quality of life (QOL).

[0003] Previously, serum creatinine or symmetric dimethylarginine (SDMA) levels were measured as indicators of decreased renal function in cats. However, serum creatinine does not rise until approximately 75% of renal function has been lost. On the other hand, although SDMA begins to rise at 40% renal function loss, it does not necessarily show abnormal values earlier than creatinine, and its reliability as an indicator is not necessarily clear. In addition, it is necessary to establish a simpler and on-site monitoring indicator using urine (non-invasive) rather than blood (low-invasive) which is accompanied by pressure or pain.

[0004] In addition, all amino acids except glycine exist two mirror image isomers of D body and L body. With the separation ability caused by the progress of analytical technology in recent years, the improvement of sensitivity, the existence of D-amino acid in mammals including humans and its effect become clear, and there are also reports that the specific D-amino acid content in blood or urine is relevant to renal function decline. For example, there are reports (patent documentation 1, 2) that the D-serine or D-alanine in human blood can become the mark for judging renal damage in the critical period. In addition, there are reports that in mice with renal function decline, the constituent ratio of D-serine, D-histidine, D-asparagine, D-arginine, D-allothreonine, D-glutamic acid, D-alanine, D-proline, D-valine, D-alloisoleucine, D-phenylalanine, D-lysine in urine is reduced, and these can be used as the pointer (patent documentation 3) of early diagnosis of renal failure. In addition, there are reports that in dogs and cats with chronic kidney disease, the D-valine in urine is extremely reduced (non-patent documentation 1).

[0005] [Patent Document 1] International Publication No. 2020 / 080494

[0006] [Patent Document 2] International Publication No. 2020 / 080488

[0007] [Patent Document 3] Japanese Patent No. 5740523

[0008] [Non-patent document 1] Journal of the Society of Pet Nutrition, 20 (19th Conference Issue), 2017, 47-48, Ikeda et al. Summary of the Invention

[0009] The present invention relates to the following 1) to 4).

[0010] 1) A method for examining renal dysfunction in a cat, comprising measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in the urine of a test cat.

[0011] 2) A method for evaluating the effectiveness of a preventive or therapeutic intervention for renal dysfunction in cats, comprising the step of measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in the urine of a test cat in the presence of the intervention.

[0012] 3) A method for examining renal dysfunction in a cat, comprising measuring the total concentration of all D-amino acids in urine of a test cat by an enzymatic assay using D-amino acid oxidase.

[0013] 4) A kit for testing renal dysfunction in cats, which is a kit for carrying out the methods described in 1) to 3) and contains a reagent for measuring the level of D-amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1-1 is the concentration of D-amino acids in cat urine.

[0015] Figure 1-2 is the concentration of D-amino acids in cat urine.

[0016] Figure 2-1 is the concentration of L-amino acids in cat urine.

[0017] Figure 2-2 is the concentration of L-amino acids in cat urine.

[0018] Figure 3-1 This is the chiral equilibrium of D-amino acids in cat urine.

[0019] Figure 3-2 This is the chiral equilibrium of D-amino acids in cat urine.

[0020] Figure 4 It is the total concentration of D-amino acids and L-amino acids in cat urine.

[0021] Figure 5-1 It is the ratio of the amount of D-amino acids to the amount of L-amino acids in cat urine (D / L).

[0022] Figure 5-2 It is the ratio of the amount of D-amino acids to the amount of L-amino acids in cat urine (D / L).

[0023] Figure 5-3 It is the ratio of the total concentration of D-amino acids to the total concentration of L-amino acids in cat urine (D / L).

[0024] Figure 5-4 is the chiral equilibrium of the total concentration of D-amino acids in cat urine.

[0025] Figure 6 It is the total concentration of D-amino acids (Tyr, Met, Trp, Orn and His) in cat urine.

[0026] Figure 7-1 This is the transition of D-amino acids in cat urine.

[0027] Figure 7-2 This is the transition of D-amino acids in cat urine.

[0028] Figure 7-3 This is the transition of D-amino acids in cat urine.

[0029] Figure 8-1 is the total concentration of D-amino acids in cat urine measured by enzymatic method.

[0030] Figure 8-2 is the chiral equilibrium of the total concentration of D-amino acids in cat urine as determined by enzymatic methods.

[0031] Figure 8-3 It is the ratio of total D-amino acid concentration in cat urine to creatinine in urine measured by enzymatic method.

[0032] Figure 8-4 It is the total concentration of D-amino acids in cat urine / urine specific gravity ratio measured by enzymatic method. DETAILED DESCRIPTION

[0033] The present invention is directed to providing an objective, non-invasive method for detecting renal dysfunction in cats.

[0034] The present inventors conducted research to address this issue and found that the amount of specific D-amino acids in urine changes in cats with decreased renal function. This allows the presence or degree of renal dysfunction in cats to be assessed and renal function to be monitored using the D-amino acid levels as an indicator.

[0035] According to the method of the present invention, the presence or extent of renal dysfunction in cats can be examined non-invasively, objectively, and simply, and early changes in renal function decline can be reliably evaluated.

[0036] The method for examining renal dysfunction in a cat of the present invention comprises measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in the urine of a test cat.

[0037] In the present invention, "renal dysfunction" refers to a condition in which the kidneys are abnormal and their function is impaired, primarily referring to chronic kidney disease, which cats are prone to. Chronic kidney disease means a gradual decline in kidney function, leading to renal failure.

[0038] Until now, renal dysfunction in cats has been diagnosed through comprehensive veterinary assessment based on the IRIS (International Renal Interest Society) guidelines, physical examinations (urine color, volume, specific gravity, pH), and interviews. Specifically, the degree of renal dysfunction is categorized as follows based on the IRIS guidelines (revised in 2019).

[0039] Healthy: Serum creatinine value less than 1.6 (mg / dL), or symmetric dimethylarginine (SDMA) value less than 14 (μg / dL).

[0040] Stage 1 (early stage): Serum creatinine value is less than 1.6 (mg / dL), or symmetric dimethylarginine (SDMA) value is 14-17 (μg / dL).

[0041] Stage 2 (mild): Serum creatinine value is 1.6 to 2.8, or symmetric dimethylarginine (SDMA) value is 18 to 25.

[0042] Stage 3 (moderate): Serum creatinine value is 2.9 to 5.0, or symmetric dimethylarginine (SDMA) value is 26 to 38.

[0043] Stage 4 (severe): Serum creatinine value greater than 5.0 or symmetric dimethylarginine (SDMA) value greater than 38.

[0044] In the present invention, "examination of renal dysfunction" includes the presence or degree of renal dysfunction, preferably the presence or absence of renal dysfunction. Furthermore, the examination of renal dysfunction in the present invention naturally includes regular examinations or examinations to confirm the effectiveness of preventive or therapeutic interventions.

[0045] Here, "inspection" can also be expressed as "measurement", "detection", "determination", "evaluation" or "assessment assistance".

[0046] In the present invention, the cat subject is not particularly limited, and preferably includes cats suspected of having renal dysfunction, cats with decreased renal function, and cats undergoing preventive or therapeutic intervention for renal dysfunction. Furthermore, the cat is a cat of the family Felidae, i.e., a cat of the order Carnivora of the class Mammalia. Preferred examples include cats of the genus Felis of the subfamily Felinae. The breed is not particularly limited, and preferably includes domestic cats, desert cats, jungle cats, Libyan wildcats, sand cats, black-footed cats, and European wildcats, and more preferably, domestic cats (Felissilvestris catus).

[0047] The method for collecting cat urine as a sample is not particularly limited, as long as it is collected in a state where D-amino acids can be measured. Fresh urine is preferred from the perspective of measurement accuracy. Preferred methods for collecting cat urine include natural urine collection (including using a pet litter box, etc.), catheterization, cystocentesis, and pressure urine collection. Natural urine collection, which does not put much pressure on the cat, is particularly preferred.

[0048] As shown in the following examples, all 20 D-amino acids were detected in cat urine, which is much higher than in human blood (approximately 4 molecular species) or urine (approximately 10 molecular species), mouse blood and urine (approximately 10 molecular species), and rat blood and urine (approximately 6 molecular species).

[0049] That is, it was found that 17 species (D-phenylalanine (D-Phe), D-methionine (D-Met), D-glutamic acid (D-Glu), D-leucine (D-Leu), D-threonine (D-Thr), D-tryptophan (D-Trp), D-proline (D-Pro), D-serine (D-Ser), D-alanine (D-Ala), D-lysine (D-Lys), D-arginine (D-Arg)) other than D-asparagine (D-Asn) and D-aspartic acid (D-Asp) were present. The chiral balance of D-amino acids (D-Tyr, D-glutamine (D-Gln), D-alloisoleucine (D-aIle), D-allothreonine (D-aThr), D-ornithine (D-Orn), and D-valine (D-Val)) and the amount ratio (D / L) of these D-amino acids to their L-amino acids were significantly decreased in the renal disease group, while the chiral balance of D-histidine (D-His) was significantly increased in the renal disease group, unlike the situation in mice reported in the above-mentioned Patent Document 3, and was confirmed to be related to renal dysfunction ( Figure 3-1 、 3-2 Among them, D-tyrosine, D-methionine, D-tryptophan, and D-ornithine are cat-specific amino acids that are clearly detected in cat urine but cannot be detected in the urine of other mammals, such as humans or mice.

[0050] Therefore, D-amino acids including D-tyrosine, D-methionine, D-tryptophan, and D-ornithine plus D-histidine in cat urine are specific marker molecules for examining renal dysfunction in cats. By measuring the level of one or more selected from these, renal dysfunction in the test cat can be assessed.

[0051] The level of D-amino acid measured in the present invention may be the concentration of each D-amino acid selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine. For example, the level may be the amount ratio of the D-amino acid to the L-amino acid (D-amino acid amount / L-amino acid amount), the chiral balance of the D-amino acid (D-amino acid amount / (D-amino acid amount + L-amino acid amount) × 100), and more preferably the chiral balance.

[0052] Regarding the chiral balance of D-amino acids in cat urine, in addition to measuring the chiral balance of one or more selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, preferably one or more selected from D-tyrosine, D-tryptophan, and D-ornithine, more preferably one or more selected from D-tyrosine and D-tryptophan, the chiral balance of D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, and D-histidine can be combined. , D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-leucine, preferably one or more selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-alanine, D-glutamine, D-alloisoleucine and D-valine, more preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-alanine, D-alloisoleucine and D-valine.

[0053] In addition, regarding the amount ratio (D / L) of D-amino acids to L-amino acids in cat urine, it is preferred to measure one or more selected from D-tyrosine, D-tryptophan, D-ornithine, and D-methionine, preferably one or more selected from D-tyrosine, D-tryptophan, and D-ornithine, more preferably one or more selected from D-tyrosine and D-tryptophan to L-amino acids. In addition, it is possible to further combine and measure an amount ratio (D / L) of one or more selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D- At least one selected from alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-leucine, preferably at least one selected from D-phenylalanine, D-glutamic acid, D-proline, D-threonine, D-alanine, D-glutamine, D-alloisoleucine and D-valine, more preferably the amount ratio (D / L) of at least one D-amino acid selected from D-phenylalanine, D-glutamic acid, D-proline, D-alanine, D-alloisoleucine and D-valine to the L-amino acid.

[0054] In addition, regarding the concentration of D-amino acids in cat urine, it is preferred to measure the concentration of one or more selected from D-tyrosine, D-methionine, D-tryptophan, and D-ornithine, preferably one or more selected from D-tyrosine, D-tryptophan, and D-ornithine. In addition, it is possible to further measure the concentration of one or more selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-leucine in combination. The concentration of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-asparagine is more preferable, and the concentration of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-asparagine is more preferable.

[0055] Furthermore, the level of D-amino acids in the present invention can be measured by measuring the total concentration of five D-amino acids consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, or the total concentration of the five D-amino acids plus D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, D- The total concentration of six or more D-amino acids, including one or more D-amino acids selected from urinary tract infections (e.g., urinary tract infections, urinary tract infections, and urinary tract infections), is preferably the total concentration of 20 D-amino acids (D-tyrosine, D-methionine, D-tryptophan, D-ornithine, D-histidine, D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid). Alternatively, the total concentration of D-amino acids in cat urine containing the six or more D-amino acids may be the total concentration of all D-amino acids (excluding acidic amino acids) that can be measured by an enzyme reaction using amino acids as substrates.

[0056] Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids (total concentration of D-amino acids / total concentration of L-amino acids) or the chiral balance of the total concentration of the D-amino acids (total concentration of D-amino acids / (total concentration of D-amino acids + total concentration of L-amino acids)) can be calculated and used as the level of the D-amino acids of the present invention.

[0057] In addition, the total concentration of D-amino acids can be further calculated by calculating the ratio of the total concentration of D-amino acids to the urine specific gravity (total concentration of D-amino acids / urine specific gravity) or the ratio of the total concentration of D-amino acids to the urine creatinine concentration (total concentration of D-amino acids / urine creatinine concentration), and these can be used as the D-amino acid levels of the present invention.

[0058] In the present invention, the method for measuring D-amino acids or L-amino acids is not limited, as long as the D-amino acids and L-amino acids in urine can be separated and measured. Generally, a separation and quantification method using a combination of liquid chromatography (LC) and mass spectrometry (MS), such as LC-MS or LC-MS / MS, can be used. In addition, when measuring the total concentration of amino acids, a quantitative method (enzymatic method) using an enzyme reaction (colorimetric, fluorescent, chemiluminescent, etc.) using D-amino acid oxidase or L-amino acid oxidase with D-amino acids as substrates or a biosensor using an enzyme as an element (electrochemical detection, etc.) can also be used. However, the enzymatic method is more preferred in terms of simplicity and rapidity.

[0059] The enzymatic assay method using D- or L-amino acid oxidase is a method in which hydrogen peroxide generated when D- or L-amino acids are oxidatively deaminated by D- or L-amino acid oxidase is detected and measured using a colorimetric / fluorescent probe. This method allows the total concentration of all D-amino acids excluding acidic amino acids or the total concentration of all L-amino acids in cat urine to be measured.

[0060] Furthermore, when measuring amino acids in urine, it is preferred that urine collected at home or in a medical institution (e.g., by catheterization, cystocentesis, or spontaneous urine collection) undergo pretreatment for analysis, such as freeze-thaw, centrifugation, ultrafiltration, gel filtration, protein removal, dilution, solid-phase extraction, liquid-liquid partitioning, and derivatization. It is ideal that the pretreatment for enzyme assays does not contain any substance that inhibits the enzyme reaction. The method for transporting the collected urine ranges from room temperature to frozen mailing, and the method is not limited.

[0061] As a method for separating chiral amino acids, for example, there are known methods of separating and quantifying chiral amino acids by two-dimensional liquid chromatography (LC) using a reversed-phase column (first dimension: molecular species separation) and a chiral column having a stationary phase carrying a chiral identifier (second dimension: chiral separation) after converting the amino acid into an NBD derivative using a 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) reagent (J Chromatogr A. 2010 Feb 12; 1217(7): 1056-62. doi: 10.1016 / j.chroma.2009.09.002. Epub 2009 Sep 6); or one-dimensional LC using a single reversed-phase column (ODS column) (Anal Chim Acta. 2015 May 22; 875: 73-82. doi: 10.1016 / j.aca.2015.02.054. Epub 2015). 2015Feb23); a one-dimensional LC method using a single chiral column in which amino acids are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (J Pharm Biomed Anal. 2015Nov 10;115:123-9. doi:10.1016 / j.jpba.2015.05.024. Epub 2015Jun 16). Alternatively, a method is known in which amino groups are derivatized with AQC and then separated using a liquid chromatograph comprising a first chiral column having a weak anion exchange chiral stationary phase and a second chiral column having an amphoteric ion exchange chiral stationary phase connected thereto (Japanese Patent No. 6764778). The method disclosed in Japanese Patent No. 6764778 is preferred.

[0062] Thus, by comparing the measured D-amino acid level with the baseline value, renal dysfunction of the subject cat can be assessed.

[0063] Here, the reference value can be set as follows based on the correlation between the level of the D-amino acid and renal dysfunction, for example.

[0064] Renal dysfunction is assessed by methods such as the IRIS guidelines. Based on its assessment results, it is divided into a healthy group and a renal dysfunction group, wherein the healthy group is composed of cats judged to have no renal dysfunction (health), and the renal dysfunction group is composed of cats judged to have renal dysfunction. In addition, the level of the D-amino acid of the present invention is measured by the above-mentioned method. Then, based on the correlation between the assessment result of renal dysfunction and the level of the D-amino acid, a reference value suitable for assessing renal dysfunction is determined. Specifically, based on the statistical analysis results of the level of the D-amino acid belonging to each group, the numerical range of the level of the D-amino acid that characterizes each group is determined. This numerical range is determined by being set to a certain range above and below the mean value of each group as the center. Here, "a certain range" can use statistical values such as standard deviation (SD) or 1 / 2SD value, 1 / 3SD value, etc., or can be a certain range (the 1st quartile or the 3rd quartile, etc.) above and below the median value of each group as the center.

[0065] Furthermore, for example, when the level of the D-amino acid of the present invention in urine obtained from a test cat falls within the range of the levels of D-amino acids in the renal dysfunction group, the cat can be evaluated as having "decreased renal function", "having renal dysfunction", or "highly likely to have renal dysfunction".

[0066] Alternatively, the reference value may be determined based on a critical value obtained by statistical analysis.

[0067] Examples of the critical value include the median or average value of the calculated biomarker values, and a value based on ROC curve analysis (e.g., the distance from the upper left corner coordinate (0, 1) in an ROC curve created using the Youden's index or a discriminant formula for distinguishing between a group with renal dysfunction and a group without renal dysfunction). Multiple critical values may be set. For example, when chiral equilibrium is used as an indicator, the critical value obtained by performing ROC curve analysis as shown in Table 2 below is set; when the total concentration of D-amino acids (5 types, 20 types) is used as an indicator, the critical value obtained by performing ROC curve analysis as shown in Table 3 below is set; when the D-amino acid concentration is used as an indicator, the critical value obtained by performing ROC curve analysis as shown in Table 4 below is set; when the amount ratio of D-amino acids to L-amino acids (D / L) is used as an indicator, the critical value obtained by performing ROC curve analysis as shown in Table 5 below is set; when the total concentration of D-amino acids measured by an enzymatic method is used as an indicator, the critical value obtained by performing ROC curve analysis as shown in Table 8 below is set.

[0068] For example, the chiral balance of D-tyrosine is less than 0.55%, the chiral balance of D-methionine is less than 3.9%, the chiral balance of D-tryptophan is less than 0.10%, the chiral balance of D-ornithine is less than 40%, the chiral balance of D-histidine is more than 2.2%, the chiral balance of D-glutamic acid is less than 4.8%, the chiral balance of D-valine is less than 8.9%, the chiral balance of D-phenylalanine is less than 0.52%, the chiral balance of D-alloleucine is less than 9.8%, and the chiral balance of D-alanine is more than 43%. When the chiral balance of D-proline is less than 5.5%, the chiral balance of D-glutamine is less than 1.2%, the chiral balance of D-threonine is less than 0.91%, the chiral balance of D-serine is less than 71%, the chiral balance of D-allothreonine is less than 4.0%, the chiral balance of D-arginine is less than 3.0%, the chiral balance of D-lysine is less than 20%, and the chiral balance of D-leucine is less than 0.16%, the patient can be evaluated as having "decreased renal function", "presence of renal dysfunction", or "high possibility of renal dysfunction".

[0069] Furthermore, for example, when the total D-amino acid concentration measured by an enzymatic method is 380.0 μM or less, the chiral equilibrium of the total D-amino acid concentration (total D-amino acid concentration measured by an enzymatic method / (total D-amino acid concentration measured by an enzymatic method + total L-amino acid concentration measured by an enzymatic method)) is 0.54% or less, the ratio of the total D-amino acid concentration to the urine specific gravity is 350.0 μM or less, and the ratio of the total D-amino acid concentration to the creatinine concentration in urine is Cre or less of 1.84 mmol / 10 g, the patient can be evaluated as having "decreased renal function", "presence of renal dysfunction", or "high possibility of renal dysfunction".

[0070] Thus, according to the method of the present invention, the presence or absence of renal dysfunction, and the state or degree of renal dysfunction in a test cat can be non-invasively, simply, and reliably assessed.

[0071] Furthermore, the examination method of the present invention can be used in combination with other diagnostic indicators of renal dysfunction other than the present invention, thereby enabling a more comprehensive and appropriate assessment of renal dysfunction.

[0072] Other diagnostic indicators for renal dysfunction include, for example, blood creatinine (Cre), urine creatinine, blood urea nitrogen (BUN), blood symmetric dimethylarginine (SDMA), blood cystatin C, urine cystatin C, urine neutrophil gelatinase-associated lipocalin (NGAL), urine L-fatty acid binding protein (L-FABP), urine protein, urine protein / creatinine ratio (UPC), and urine albumin. Furthermore, the method may be combined with a simple urine specific gravity evaluation method using a specific urine collection container (Japanese Patent No. 7084128), urine color (Japanese Patent No. 7216529), urine osmotic pressure, differential excretion rates of various electrolytes, blood pressure, or physical indicators such as imaging examinations.

[0073] The testing method of the present invention can be used to detect the progression of renal dysfunction in a test cat by regularly performing the test and tracking the fluctuation of the D-amino acid level.

[0074] Furthermore, the examination method of the present invention can be used to evaluate the effect of preventive or therapeutic intervention for renal dysfunction in cats by performing the examination in the presence of such intervention.

[0075] That is, the intervention effect evaluation method of the present invention includes the following steps: measuring the level of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine and D-histidine in the urine of a subject cat in the presence of preventive or therapeutic intervention for renal dysfunction in the cat.

[0076] Here, preventive or therapeutic intervention for renal dysfunction in cats is an intervention performed in the expectation of a preventive or therapeutic effect on renal dysfunction in cats, and includes chemical intervention, physical or mechanical intervention, or dietary therapy. Chemical interventions include the administration of natural substances, synthetic substances, and combinations thereof, while physical or mechanical interventions include dialysis and other treatments. Dietary therapy includes the administration of low-calorie foods, low-fat foods, supplements with adjusted vitamin and mineral content, and low-salt adjusted foods.

[0077] The effect of the intervention can be evaluated based on the level of the D-amino acid in the subject cat caused by the intervention. For example, the effect can be evaluated by comparing the level of the D-amino acid in the subject cat in the presence of the intervention with that in the control (in the absence of the intervention).

[0078] The kit for testing feline renal dysfunction of the present invention is a kit for performing the method for testing feline renal dysfunction of the present invention. Therefore, the kit may include tools for collecting cat urine, a container for storing cat urine or cat urine-containing litter or sheet material, reagents for measuring the levels of one or more D-amino acids, and indicators or guides for comparing D-amino acid levels with baseline values.

[0079] Examples of reagents for measuring the level of D-amino acids include enzymes that use D- or L-amino acids as substrates (eg, D- or L-amino acid oxidase), colorimetric or fluorescent probes, and peroxidase.

[0080] Regarding the above-mentioned embodiment, the present invention further discloses the following aspects.

[0081] <1> A method for examining renal dysfunction in a cat, comprising measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in urine of a test cat.

[0082] <2> The method according to <1>, wherein the level of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid is further measured.

[0083] <3> The method according to <1>, wherein the step of measuring the level of D-amino acids is a step of measuring the chiral equilibrium of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine.

[0084] <4> The method according to <3>, further comprising the step of measuring the chiral equilibrium of one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-leucine, preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-alanine, D-glutamine, D-alloisoleucine, and D-valine, and more preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-alloisoleucine, and D-valine.

[0085] <5> The method according to <1>, wherein the step of measuring the level of D-amino acids is a step of measuring the quantitative ratio of one or more D-amino acids selected from D-tyrosine, D-tryptophan, D-ornithine, and D-methionine relative to L-amino acids.

[0086] <6> The method according to <5>, further comprising the step of measuring the quantitative ratio of one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-leucine, preferably one or more D-phenylalanine, D-glutamic acid, D-proline, D-threonine, D-alanine, D-glutamine, D-alloisoleucine, and D-valine, and more preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-proline, D-alanine, D-alloisoleucine, and D-valine to L-amino acids.

[0087] <7> The method according to <1>, wherein the step of measuring the level of D-amino acids is a step of measuring the concentration of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, and D-ornithine.

[0088] <8> The method of <7>, further comprising the step of measuring at least one selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-leucine, preferably selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine , D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-asparagine, more preferably the concentration of one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-asparagine.

[0089] <9> The method of <1>, wherein the step of measuring the level of D-amino acids comprises the step of measuring the total concentration of five D-amino acids consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, or the total concentration of six or more D-amino acids consisting of the above five D-amino acids plus one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid.

[0090] <10> The method according to <9>, further comprising calculating the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids.

[0091] <11> The method according to <9>, further comprising the step of calculating the chiral balance of the total concentration of the D-amino acid.

[0092] <12> The method according to any one of <9> to <11>, wherein the total concentration of six or more D-amino acids is measured by a quantitative method utilizing an enzyme reaction using the D-amino acid as a substrate.

[0093] <13> A method for evaluating the effectiveness of a preventive or therapeutic intervention for renal dysfunction in cats, comprising the step of measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in the urine of a test cat in the presence of the intervention.

[0094] <14> The method of <13>, wherein the level of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid is further measured.

[0095] <15> The method according to <13>, wherein the step of measuring the level of D-amino acids is a step of measuring the chiral equilibrium of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine.

[0096] <16> The method of <15>, further comprising the step of determining the chiral equilibrium of one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-leucine, preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-alanine, D-glutamine, D-alloisoleucine, and D-valine, and more preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-alloisoleucine, and D-valine.

[0097] <17> The method according to <13>, wherein the step of measuring the level of D-amino acids is a step of measuring the quantitative ratio of one or more D-amino acids selected from D-tyrosine, D-tryptophan, D-ornithine, and D-methionine relative to L-amino acids.

[0098] <18> The method of <17>, further comprising the step of measuring the amount ratio of one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine and D-leucine, preferably one or more D-phenylalanine, D-glutamic acid, D-proline, D-threonine, D-alanine, D-glutamine, D-alloisoleucine and D-valine, and more preferably one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-proline, D-alanine, D-alloisoleucine and D-valine to L-amino acids.

[0099] <19> The method according to <13>, wherein the step of measuring the level of D-amino acids is a step of measuring the concentration of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, and D-ornithine.

[0100] <20> The method of <19>, further comprising the step of measuring at least one selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-leucine, preferably selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, The concentration of at least one D-amino acid selected from the group consisting of D-amino acid, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-asparagine is more preferably selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-asparagine.

[0101] <21> The method of <13>, wherein the step of determining the level of D-amino acids comprises the step of determining the total concentration of five D-amino acids consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, or the total concentration of six or more D-amino acids consisting of the above five D-amino acids plus one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid.

[0102] <22> The method according to <21>, further comprising calculating the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids.

[0103] <23> The method according to <21>, further comprising the step of calculating the chiral balance of the total concentration of the D-amino acid.

[0104] <24> A method for examining renal dysfunction in a cat, comprising measuring the total concentration of all D-amino acids in urine of a test cat by an enzymatic assay using D-amino acid oxidase.

[0105] <25> The method according to <24>, further comprising the step of calculating the ratio of the total concentration of the D-amino acids to the urine specific gravity.

[0106] <26> The method according to <24>, further comprising calculating the ratio of the total concentration of the D-amino acids to the concentration of creatinine in urine.

[0107] <27> The method according to <24>, further comprising the step of measuring the total concentration of L-amino acids in the urine of the test cat by an enzymatic assay using L-amino acid oxidase.

[0108] <28> The method according to <27>, further comprising the step of calculating the chiral balance of the total concentration of the D-amino acid.

[0109] <29> The method according to <27>, further comprising calculating the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids.

[0110] <30> A kit for testing renal dysfunction in a cat, the kit being used for carrying out the method according to any one of <1> to <29> and comprising a reagent for measuring the level of D-amino acids.

[0111] <31> The kit according to <30>, wherein the reagent for measuring the level of D-amino acid comprises D-amino acid oxidase.

[0112] <32> The kit according to <31>, further comprising a colorimetric or fluorescent probe and peroxidase.

[0113] [Example]

[0114] Hereinafter, the present invention will be described in more detail with reference to examples.

[0115] Experimental Example 1: Simultaneous Separation and Analysis of Chiral Amino Acids in Urine of Cats with Kidney Disease

[0116] (1) About the cat test

[0117] Cats in the healthy group (N=17) and the group with decreased renal function (N=11, hereinafter referred to as the nephrotic group) (Table 1) were used as subjects for analysis of renal function and urine components. Furthermore, after urine collection, classification was performed based on the comprehensive judgment of the IRIS (International Renal Interest Society: International Veterinary Nephropathy Study Group) guidelines, which are the current standards, physical auxiliary examinations (color, urine volume, urine specific gravity, pH value) and veterinary consultations. Regarding the allocation of the healthy group and the nephrotic group, in particular, according to the IRIS guidelines, individuals with a blood (serum) creatinine value of 1.6 (mg / dL) or more or a symmetric dimethylarginine (SDMA) value of 18 (μg / dL) or more were considered to be in the nephrotic group (equivalent to stage 2).

[0118] [Table 1]

[0119]

[0120] (2) Preparation of sample solution and standard

[0121] 10 μL of cat urine (natural urine) was placed in a 10 mL conical bottom glass tube (Spitch Glass) (trade name: reinforced hard screw-cap test tube), mixed with 490 μL of a methanol:water (9:1, v / v) solution, and then centrifuged at 2130 × g (3000 rpm) for 5 minutes in a centrifuge (HITACHI / CF5RE) under refrigeration (4°C) to remove protein, and the amino acids in the supernatant were collected. Next, in a separate 10 mL conical glass tube, 70 μL, 10 μL, and 20 μL of 0.2 mol / L boric acid buffer (pH 8.9), the collection solution, and the AccQ·Tag Ultra derivatization reagent, i.e., AQC solution (manufactured by AdipoGen: AQC powder dissolved in ultra-dehydrated acetonitrile at a concentration of 3 mg / 1 mL, i.e., 10 mmol / L) were mixed in this order (7:1:2), immediately stirred, and then heated at 55°C for 10 minutes to prepare a derivatized sample solution.

[0122] Similarly, 0.2 mol / L borate buffer (pH 8.9), 100 μmol / L D, L-amino acid standard solution (amino acids: Ala / alanine, Arg / arginine, Asn / asparagine, Asp / aspartic acid, Cys / cysteine, Gln / glutamine, Glu / glutamic acid, Gly / glycine, His / histidine, Ile / isoleucine, allo-Ile / alloisoleucine, Leu / leucine, Lys / lysine, Me t / methionine, Phe / phenylalanine, Pro / proline, Ser / serine, Thr / threonine, allo-Thr / allo-threonine, Trp / tryptophan, Tyr / tyrosine, Val / valine, Cit / citrulline, and Orn / ornithine were dissolved in 0.2 mol / L borate buffer), 70 μL, 10 μL, and 20 μL of AQC solution (7:1:2) were immediately stirred and then heated at 55°C for 10 minutes to prepare derivatized amino acid standard solutions.

[0123] (3) LC-MS / MS analysis

[0124] Under the following conditions (based on Japanese Patent No. 6764778), the derivatized sample solution and the derivatized amino acid standard solution prepared in (2) were subjected to LC-MS / MS analysis to separate, detect and quantify various chiral amino acids.

[0125] (Device)

[0126] Exion LC series (AB SCIEX), mass spectrometer / QTRAP6500 + Linear ion trap type (AB SCIEX)

[0127] (Chromatographic separation)

[0128] Chiral separation columns: 2.1 mm inner diameter × 150 mm, 5 μm particle size CHIRALPAK QN-AX (Daicel) (first chiral column) and 3.0 mm inner diameter × 150 mm, 3 μm particle size CHIRALPAK AK ZWIX (+) (Daicel) (second chiral column) connected in series (40°C)

[0129] Eluent: 0.1% (v / v) formic acid and 55 mM ammonium formate in methanol: water (90:10, v / v) solution

[0130] Elution method: isocratic elution

[0131] Mobile phase flow rate: 0.25 mL / min

[0132] Injection volume: 5 μL

[0133] (Mass spectrometry analysis)

[0134] Ionization method: electrospray ionization (ESI)

[0135] Polarity: positive ion

[0136] Curtain air (CUR): 30psi

[0137] Ion spray voltage (IS): 4500V

[0138] Temperature (TEM): 600°C

[0139] Ion source gas 1 (GS1): 80 psi

[0140] Ion source gas 1 (GS2): 80 psi

[0141] Collision gas (CAD): 10

[0142] (Detection Mode)

[0143] By setting the proton ion attached to the molecule ([M+H] + ) was used as the precursor ion, and SRM (Selected Reaction Monitoring) detection was performed in positive ion mode with the AQC fragment ion (m / z=171) as the product ion.

[0144] (4) Data analysis

[0145] 1) Analysis of chiral amino acids

[0146] The data obtained in (3) were expanded into a chromatogram with two axes of retention time and ion intensity, and various chiral amino acids derived from AQC in cat urine were analyzed. The concentrations of chiral amino acids in urine of healthy and renal disease groups were measured (the measured values of D-amino acids (DAA): D-Pro, D-Ser, D-Ala, D-Lys, D-Arg, D-His, D-Tyr, D-Gln, D-allo-Ile (aIle), D-Thr, D-allo-Thr (aThr), D-Orn, D-Val, D-Phe, D-Met, D-Glu, D-Asp, D-Asn, D-Leu, D-Trp are shown in Figure 1-1 and Figure 1-2 In addition, the measured values of L-amino acids (LAA): L-Pro, L-Ser, L-Ala, L-Lys, L-Arg, L-His, L-Tyr, L-Gln, L-Ile, L-Thr, L-Orn, L-Val, L-Phe, L-Met, L-Glu, L-Asp, L-Asn, L-Leu, L-Trp) are shown in Figure 2-1 and Figure 2-2 ).

[0147] Furthermore, the chiral balances (D-form existence ratio: D / (D+L)×100(%)) calculated from the chiral amino acid concentrations in these urines are shown in FIG. Figure 3-1 and Figure 3-2 .

[0148] It was found that the chiral balance of 17 amino acids other than D-Asn and D-Asp (D-Phe, D-Met, D-Glu, D-Leu, D-Thr, D-Trp, D-Pro, D-Ser, D-Ala, D-Lys, D-Arg, D-Tyr, D-Gln, D-allo-Ile (aIle), D-allo-Thr (aThr), D-Orn, and D-Val) was significantly decreased in the renal disease group, while the chiral balance of D-His was significantly increased in the renal disease group, confirming a correlation with renal function. Among them, D-Tyr, D-Met, D-Trp, and D-Orn are amino acids unique to cat urine that were clearly detected in cat urine.

[0149] Furthermore, the total concentration of DAA or LAA is shown in Figure 4 , each D / L is shown in Figure 5-1 、 5-2 , the total concentration D / L is shown in Figure 5-3 The chiral balance of the total concentration (D-isomer ratio: D / (D+L)×100(%)) is shown in Figure 5-4 . In addition to the above-mentioned chiral balances and concentrations, the total concentration and the amount ratio (D / L) of each DAA to LAA were also confirmed to be significantly different between the nephropathy group and the healthy group (D-Phe, D-Glu, D-Thr, D-Trp, D-Pro, D-Ser, D-Ala, D-Tyr, D-Gln, D-aIle, D-aThr, D-Orn, D-Val). The chiral balance calculated from the total concentration ratio (D / L) and total concentration of DAA to LAA was also confirmed to be significantly reduced, indicating that it can be used as an indicator of renal dysfunction. Furthermore, the total concentration of only D-Tyr, D-Met, D-Trp, D-Orn and D-His is also shown in Figure 6 Similarly, a significant decrease was observed in the renal disease group.

[0150] 2) Verification of usefulness

[0151] Using Excel statistical processing software (BellCurve for Excel, Social Survey Research Information Co., Ltd.), the ROC (Receiver Operating Characteristic) curves of the healthy group and the renal disease group (chiral balance, total concentration, D-amino acid concentration, and the ratio of D-amino acid to L-amino acid (D / L)) were drawn, and the cutoff point (Cut off), area under the ROC curve (AUC), sensitivity (Sensitivity), specificity (Specificity), p-value (diagnostic ability) (Table 2: each chiral balance, Table 3: total concentration, Table 4: D-amino acid concentration, Table 5: the ratio of the amount of D-amino acids to L-amino acids (D / L)). Furthermore, the critical point was set to the point closest to the upper left corner. In the test when the null hypothesis (NullHypothesis) was set to AUC=0.50, it was confirmed that there were significant results in terms of chiral balance, (total) concentration, and the ratio of the amount of D-amino acids to L-amino acids (D / L), and it had diagnostic ability. Many DAA molecular species with an AUC of 0.90 or above were also detected. It was found that the simple total concentration (DAA (D-Tyr, D-Trp, D-Orn, D-Met, D-His); DAA (all): D-Pro, D-Ser, D-Ala, D-Lys, D-Arg, D-His, D-Tyr, D-Gln, D-aIle, D-Thr, D-aThr, D-Orn, D-Val, D-Phe, D-Met, D-Glu, D-Asp, D-Asn, D-Leu, D-Trp; LAA (all): L-Pro, L-Ser, L-Ala, L-Lys, L-Arg, L-His, L-Tyr, L-Gln, L-Ile, L-Thr, L-Orn, L-Val, L-Phe, L-Met, L-Glu, L-Asp, L-Asn, L-Leu, L-Trp) can also be used to assess renal function, and the usefulness of chiral amino acids in urine (especially DAA in urine) has been confirmed.

[0152] [Table 2]

[0153]

[0154] Healthy group (N=17) vs. kidney disease group (N=11)

[0155] [Table 3]

[0156]

[0157] Healthy group (N=17) vs. kidney disease group (N=11)

[0158] [Table 4]

[0159]

[0160] Healthy group (N=17) vs. kidney disease group (N=11)

[0161] [Table 5]

[0162]

[0163] Healthy group (N=17) vs. nephropathy group (N=11) Experimental Example 2: Time course of D-amino acids in cat urine

[0164] For Cases 1 to 3 shown in Table 6 below, the correlation analysis between renal function and urine components was performed in the same manner as in Test Example 1 (Cat species (1): Abyssinian cat (castrated) 3-6 years old) was diagnosed with stage 2 kidney disease in a blood test in September 2019. Cat type (2): Siamese cat ( 3-4 years old) was diagnosed with stage 2 kidney disease in a blood test in November 2020. Cat species (3): Bengal cat (castrated (4-7 years old) was diagnosed with stage 2 kidney disease in a blood test in November 2016.

[0165] In terms of temporal changes, i.e., not only in transverse studies but also in longitudinal studies, it was confirmed that the chiral balance in urine (e.g., D-His, D-aIle, D-Val, etc.) and the DAA concentration (total concentration) in urine were related to renal function, and it was confirmed that it is useful in non-invasive evaluation and non-invasive monitoring of renal function ( Figure 7-1 、 7-2 7-3). Urinary DAA is a renal monitoring indicator that can accurately assess the severity (condition) of renal disease in each individual and the risk of early-stage disease.

[0166] [Table 6]

[0167]

[0168] Test Example 3: Analysis of D,L-amino acids in cat urine by enzymatic method

[0169] (1) About the cat test

[0170] Correlation analysis between renal function and urine components was performed on cats in a healthy group (N=14) and a group with decreased renal function (N=4, hereinafter referred to as the renal disease group) (Table 7).

[0171] As in Experimental Example 1, urine was collected and classified based on the current standard IRIS (International Renal Interest Society) guidelines and comprehensive judgment based on veterinary interviews. As auxiliary tests for cat urine, pH (LAQUAtwin, a small pH meter manufactured by Horiba, Ltd.), urine specific gravity (USG) (PAL-Dog and Cat Urine Specific Gravity Meter manufactured by Atago Co., Ltd.), and urine creatinine concentration (CRE) (thinka RT-4010 urine chemical analyzer manufactured by Arkray Co., Ltd.) were also measured.

[0172] [Table 7]

[0173]

[0174] (2) Determination of total concentration of D-amino acids (DAA) in cat urine

[0175] Preparation of calibration curve and calculation of quantitative values

[0176] The D-amino acid analysis kit (MET-5136, Colorimetric, Cell Biolabs) was used according to the instructions. A calibration curve was prepared using the kit's internal standard (D-alanine) at concentrations of 0, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μM to determine the total D-amino acid concentration (all D-amino acids except acidic amino acids) in cat urine.

[0177] Specifically, 10 μL of cat urine was placed in a 10 mL conical bottom glass tube (trade name: reinforced hard screw cap test tube), mixed with 90 μL of the assay buffer (buffer) built into the kit, and then centrifuged at 2130 × g (3000 rpm) for 5 minutes under refrigeration (4°C) to remove proteins, and the amino acids in the supernatant were collected. Next, 50 μL of the standard solution prepared at the time of use and 50 μL of cat urine were added to a 96-well microplate (No. 655801, manufactured by Greiner Bio-One). Next, 50 μL of the enzyme reaction solution built into the kit (colorimetric probe, HRP, D-amino acid oxidase mixture) was added to each well, immediately mixed under light protection, and then incubated at 37°C for 1 hour (D-amino acid oxidase (DAO) reaction). Finally, the total DAA concentration (wavelength 570 nm) in the enzyme reaction solution was measured using a microplate reader (SH-9000Lab, manufactured by Corona Electric Co., Ltd.).

[0178] (3) Determination of total concentration of L-amino acids (LAA) in cat urine

[0179] Preparation of calibration curve and calculation of quantitative values

[0180] The total concentration of all L-amino acids in cat urine was determined by following the instructions of the L-Amino Acid Analysis Kit (MET-5054, Colorimetric, Cell Biolabs). A calibration curve was prepared using the kit's internal standard (L-alanine) at concentrations of 0, 62.5, 125, 250, 500, and 1000 μM.

[0181] Specifically, 10 μL of cat urine was placed in a 10 mL conical bottom glass tube (trade name: reinforced hard screw cap test tube), mixed with 990 μL of the assay buffer (buffer) included in the kit, and then centrifuged at 2130 × g (3000 rpm) for 5 minutes in a centrifuge (HITACHI / CF5RE) under refrigeration (4°C) to remove protein, and the amino acids in the supernatant were collected. Next, 50 μL of the standard solution prepared at the time of use and 50 μL of cat urine were added to each 96-well microplate (No. 655801, manufactured by Greiner Bio-One). Then, 50 μL of the enzyme reaction solution (colorimetric probe, HRP, L-amino acid oxidase mixture) included in the kit was added to each well, immediately mixed under light protection, and then incubated at 37°C for 3 hours (L-amino acid oxidase (LAO) reaction).

[0182] Finally, the total LAA concentration (wavelength 570 nm) in the enzyme reaction solution was measured using a microplate reader (SH-9000Lab, manufactured by Corona Electric Co., Ltd.).

[0183] (4) Data analysis

[0184] 1) Analysis of DAA-related indicators in cat urine

[0185] The total DAA concentration measured by the enzymatic method, the chiral balance of the total concentration (D-body existence ratio: total DAA concentration measured by the enzymatic method / (total DAA concentration measured by the enzymatic method + total LAA concentration measured by the enzymatic method) × 100 (%), which has the same correlation as the total concentration ratio (total DAA concentration measured by the enzymatic method / total LAA concentration measured by the enzymatic method)), the total DAA concentration measured by the enzymatic method / urinary CRE concentration ratio and the total DAA concentration measured by the enzymatic method / USG ratio ( Figure 8-1 、 8-2 , 8-3, 8-4).

[0186] 2) Verification of usefulness

[0187] Using excel statistical processing software (BellCurve for Excel, Social Survey Research Information Co., Ltd.), the ROC (Receiver Operating Characteristic) curve of the healthy group versus the kidney disease group (DAA-related indicators in cat urine) was drawn, and the cutoff point (Cut off), area under the ROC curve (AUC), sensitivity (Sensitivity), specificity (Specificity), and p-value (diagnostic ability) were calculated (Table 8). Furthermore, the cutoff point was set to the point closest to the upper left corner, and in the test when the null hypothesis was set to AUC = 0.50, it was confirmed that the DAA-related indicators in cat urine had diagnostic ability. It was found that the total DAA concentration obtained by the enzymatic method and its related ratio were particularly useful in the assessment of renal function.

[0188] [Table 8]

[0189]

[0190] Healthy group (N=14) vs. kidney disease group (N=4)

Claims

1. A method for examining renal dysfunction, wherein: A method for examining renal dysfunction in a cat comprises measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in the urine of a test cat.

2. The method according to claim 1, wherein Furthermore, the level of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid is measured.

3. The method according to claim 1, wherein The step of measuring the level of D-amino acids is a step of measuring the chiral equilibrium of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine.

4. The method according to claim 3, wherein: The method further includes the step of measuring the chiral equilibrium of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-leucine.

5. The method according to claim 1, wherein The step of measuring the level of D-amino acids is a step of measuring the quantitative ratio of one or more D-amino acids selected from D-tyrosine, D-tryptophan, D-ornithine, and D-methionine relative to L-amino acids.

6. The method according to claim 5, wherein: The method further includes the step of measuring the quantitative ratio of one or more D-amino acids selected from D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, and D-leucine relative to L-amino acids.

7. The method of claim 1, wherein: The step of measuring the level of D-amino acids is a step of measuring the concentration of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, and D-ornithine.

8. The method of claim 7, wherein: The method further includes the step of measuring the concentration of one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-leucine.

9. The method of claim 1, wherein: The step of measuring the level of D-amino acids includes the step of measuring the total concentration of five D-amino acids consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, or measuring the total concentration of six or more D-amino acids consisting of the five D-amino acids plus one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid.

10. The method of claim 9, wherein: The method further includes calculating the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids.

11. The method of claim 9, wherein: The method further includes calculating the chiral balance of the total concentration of the D-amino acid.

12. A method for evaluating the effect of a preventive or therapeutic intervention for renal dysfunction in cats, wherein: The method comprises the step of measuring the level of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in the urine of a test cat in the presence of the intervention.

13. The method of claim 12, wherein: The step of measuring the level of D-amino acids is a step of measuring the chiral equilibrium of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine.

14. The method of claim 12, wherein: The step of measuring the level of D-amino acids is a step of measuring the quantitative ratio of one or more D-amino acids selected from D-tyrosine, D-tryptophan, D-ornithine, and D-methionine relative to L-amino acids.

15. The method of claim 12, wherein: The step of measuring the level of D-amino acids is a step of measuring the concentration of one or more D-amino acids selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, and D-ornithine.

16. The method of claim 12, wherein: The step of measuring the level of D-amino acids includes the step of measuring the total concentration of five D-amino acids consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, or measuring the total concentration of six or more D-amino acids consisting of the five D-amino acids plus one or more D-amino acids selected from the group consisting of D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alloisoleucine, D-allothreonine, D-valine, D-asparagine, and D-aspartic acid.

17. The method of claim 16, wherein: The method further includes calculating the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids.

18. The method of claim 16, wherein: The method further includes calculating the chiral balance of the total concentration of the D-amino acid.

19. A method for examining renal dysfunction, wherein: The present invention is a method for examining renal dysfunction in cats, comprising the steps of measuring the total concentration of all D-amino acids in urine of a test cat, which can be measured by the enzyme assay method using D-amino acid oxidase.

20. The method of claim 19, wherein: The method further includes calculating the ratio of the total concentration of the D-amino acids to the urine specific gravity.

21. The method of claim 19, wherein: The method further includes calculating the ratio of the total concentration of the D-amino acids to the concentration of creatinine in urine.

22. The method of claim 19, wherein: The method further includes the step of measuring the total concentration of all L-amino acids in the urine of the test cat, which can be measured by an enzymatic assay using L-amino acid oxidase.

23. The method of claim 22, wherein: The method further includes calculating the chiral balance of the total concentration of the D-amino acid.

24. The method of claim 22, wherein: The method further includes calculating the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids.

25. A kit for examining renal dysfunction in cats, wherein: A test kit for carrying out the method according to any one of claims 1 to 24, comprising a reagent for measuring D-amino acid levels.

26. The kit according to claim 25, wherein The reagent for measuring the level of D-amino acids contains D-amino acid oxidase.

27. The kit according to claim 26, wherein It further contains a colorimetric probe or a fluorescent probe, and peroxidase.

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