Method for examining renal dysfunction of cat

Abstract

To provide an objective and noninvasive method for examining renal dysfunction of a cat.SOLUTION: A method for examining renal dysfunction in a cat, comprising a step of measuring a level of at least one D-amino acid selected from the group consisting of D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine in urine of a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a test for renal dysfunction in cats using urinary D-amino acids. [Background technology]

[0002] Cats originally evolved in arid regions and therefore have the ability to highly concentrate urine in their kidneys. Cats are animals that naturally drink little water. Therefore, they are particularly susceptible to cystitis and urinary tract infections caused by renal tubular disorders. It is easy to develop urinary syndromes such as urinary stones and urethral obstruction, as well as kidney and urinary system diseases such as renal failure. Furthermore, with the recent spread of dry food, cats are becoming less likely to drink enough water. Insufficient water intake can lead to urinary system diseases in young people and kidney diseases in middle-aged and elderly people. This increases the risk of developing the condition and reduces quality of life (QOL).

[0003] Traditionally, serum creatinine concentration and symmetric dimethylarginine concentration have been used as indicators of decreased renal function in cats. However, serum creatinine (SDMA) levels have not been measured. SDMA is not thought to increase until approximately 75% of renal function is lost. It is said that the increase begins when 40% of the blood is lost, but abnormal values ​​are detected earlier than creatinine. Its reliability as an indicator is not necessarily clear. Instead of blood (minimally invasive), urine (non-invasive) is easier and allows for on-site monitoring. There is a need to establish indicators for this.

[0004] In addition, all amino acids except glycine have two enantiomers: D and L. With the recent advances in analytical technology, the separation ability and sensitivity have improved, and the detection of leukemia in mammals, including humans, has become possible. The existence and role of D-amino acids have become clear, and specific D-amino acids have been detected in blood and urine. It has also been reported that the D-amino acid content in human blood is associated with decreased renal function. D-serine and D-alanine are markers for determining critical stage renal damage. It has been reported that this can be achieved (Patent Documents 1 and 2). In addition, in mice with reduced renal function, Urinary D-serine, D-histidine, D-asparagine, D-arginine, D-allose Leonine, D-glutamic acid, D-alanine, D-proline, D-valine, D-alloy The proportions of soleusine, D-phenylalanine, and D-lysine decrease, and these are the causes of renal failure. It has been reported that this can be an indicator for early diagnosis (Patent Document 3). It has been reported that urinary D-valine levels in cats are drastically reduced (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 080494 [Patent Document 2] International Publication No. 2020 / 080488 [Patent Document 3] Patent No. 5740523 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Pet Nutrition, 20 (19th Conference Issue), 2017, 47-48, Ikeda et al. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an objective, non-invasive method for testing for renal dysfunction in cats. Regarding. [Means for solving the problem]

[0008] The present inventors have conducted research in light of such problems and have found that in cats with impaired renal function, The amount of specific D-amino acids present in the cat changes, and the level of these D-amino acids can be used as an indicator to assess the renal function of the cat. We found that it is possible to evaluate the presence and degree of renal impairment and monitor renal function.

[0009] That is, the present invention relates to the following 1) to 4). 1) A method for testing for renal dysfunction in cats, comprising: detecting D-tyrosine in the urine of a test cat; Selected from D-methionine, D-tryptophan, D-ornithine and D-histidine A method for testing for renal dysfunction, comprising measuring the levels of one or more D-amino acids. 2) In the presence of preventive or therapeutic intervention for feline renal dysfunction, D -Tyrosine, D-methionine, D-tryptophan, D-ornithine and D-histidine and evaluating the effect of said intervention, comprising measuring the level of one or more D-amino acids selected from the group consisting of: Valuation method. 3) A method for testing renal dysfunction in cats, using an enzyme called D-amino acid oxidase The total concentration of all D-amino acids in the urine of the test cats that can be measured by the measurement method was measured by the method. A method for testing for renal dysfunction, comprising the step of measuring the level of 4) A test kit for carrying out the methods 1) to 3), which is for detecting the level of D-amino acids A kit for testing for renal dysfunction in cats containing reagents for measuring renal function. [Effects of the Invention]

[0010] According to the method of the present invention, the presence or absence and degree of renal dysfunction in cats can be detected non-invasively, objectively, and simply. This allows for accurate assessment of early changes in renal function decline. [Brief explanation of the drawings]

[0011] [Figure 1-1] D-amino acid concentrations in cat urine. [Figure 1-2] D-amino acid concentrations in cat urine. [Figure 2-1] L-amino acid concentrations in cat urine. [Figure 2-2] L-amino acid concentrations in cat urine. [Figure 3-1] Chiral balance of D-amino acids in cat urine. [Figure 3-2] Chiral balance of D-amino acids in cat urine. [Figure 4] Total concentrations of D- and L-amino acids in cat urine. [Figure 5-1] The ratio of D-amino acid content to L-amino acid content in cat urine (D / L). [Figure 5-2] The ratio of D-amino acid content to L-amino acid content in cat urine (D / L). [Figure 5-3] The ratio of total D-amino acid concentration to total L-amino acid concentration in cat urine (D / L). [Figure 5-4] Chiral balance of total D-amino acid concentrations in cat urine. [Figure 6] Total concentration of D-amino acids (Tyr, Met, Trp, Orn, and His) in cat urine. [Figure 7-1] Changes in D-amino acids in cat urine. [Figure 7-2] Changes in D-amino acids in cat urine. [Figure 7-3] Changes in D-amino acids in cat urine. [Figure 8-1] Total D-amino acid concentrations in cat urine measured by enzymatic method. [Figure 8-2] Chiral balance of total D-amino acid concentrations in cat urine measured by enzymatic method. [Figure 8-3]Total D-amino acid concentration in cat urine / urinary creatinine ratio measured by enzymatic method. [Figure 8-4] Total D-amino acid concentration in cat urine / urine specific gravity ratio measured by enzymatic method. DETAILED DESCRIPTION OF THE INVENTION

[0012] The method for testing for renal dysfunction in cats of the present invention is to measure D-tyrosine, D- One selected from methionine, D-tryptophan, D-ornithine, and D-histidine The method includes measuring the above D-amino acid levels.

[0013] In the present invention, "renal dysfunction" refers to any abnormality that occurs in the kidneys, resulting in impaired kidney function. It refers to a condition that occurs when the kidneys become damaged, but it mainly refers to chronic kidney disease, which cats are prone to. This refers to a condition in which the function of the kidneys gradually declines, leading to kidney failure. Traditionally, renal dysfunction in cats has been diagnosed according to the IRIS (International Renal Research Society) guidelines, Physical examination (color, urine volume, urine specific gravity, pH) and comprehensive judgment by a veterinarian based on a medical interview. In particular, the degree of renal dysfunction is determined by the IRIS guidelines (2019 revised). and are classified as follows: Healthy: Serum creatinine level is less than 1.6 (mg / dL) or symmetric dimethyl amine SDMA levels were less than 14 μg / dL. Stage 1 (early stage): Serum creatinine level is less than 1.6 (mg / dL) or Symmetric dimethylarginine (SDMA) levels were 14-17 μg / dL. Stage 2 (mild): Serum creatinine level is 1.6-2.8 or symmetric dimethicone Serum arginine (SDMA) levels are 18-25. Stage 3 (moderate): Serum creatinine level is 2.9-5.0 or symmetric dimethicone SDMA levels are 26-38. Stage 4 (severe): Serum creatinine level exceeds 5.0 or symmetric dimethyl amine SDMA levels are over 38.

[0014] In the present invention, "testing for renal dysfunction" refers to determining the presence or absence of renal dysfunction and the degree of renal dysfunction. The test for renal dysfunction of the present invention includes, preferably, a test for the presence or absence of renal dysfunction. This naturally includes periodic testing and testing to confirm the effectiveness of preventive or therapeutic interventions. It can be enjoyed. Here, "inspection" means "measurement," "detection," "judgment," "evaluation," or "evaluation support." This can also be expressed in other terms.

[0015] In the present invention, the subject cat is not particularly limited, but is preferably a cat suspected of having renal dysfunction. Cats with impaired renal function, cats undergoing preventive or therapeutic intervention for renal dysfunction, etc. In addition, cats are animals of the Felidae family, that is, animals of the Carnivora order of the Mammalia class, Felidae family. Preferably, cats of the genus Felinae are included, including domestic cats, grey cats, and jean cats. The preferred species are the African wildcat, sand cat, black-footed cat, and European wildcat. However, domestic cats (Felis silvestris catus) are more preferable, but the breed is not particularly limited. do not have.

[0016] The cat urine samples used were collected in a manner that allowed for the measurement of D-amino acids. If so, the method of collection is not particularly limited, but fresh urine is preferred from the viewpoint of measurement accuracy. The preferred method for collecting cat urine is natural collection (including the use of pet toilets, etc.). These include catheterization, bladder puncture, and urine collection by expression. However, particularly preferred methods are those that do not cause stress to the cat. Natural urine collection is preferable.

[0017] As will be shown in the Examples below, all 20 D-amino acids were detected in cat urine. Human blood (approximately 4 molecular species) and urine (approximately 10 molecular species), mouse blood and urine (approximately 10 molecular species), This is far more than the amount found in rat blood and urine (approximately six molecular species). That is, D-asparagine (D-Asn) and D-aspartic acid (D-Asp) Excluding 17 other amino acids (D-phenylalanine (D-Phe), D-methionine (D-Met), -Glutamic acid (D-Glu), D-leucine (D-Leu), D-threonine (DT hr), D-tryptophan (D-Trp), D-proline (D-Pro), D-serine (D-Ser), D-alanine (D-Ala), D-lysine (D-Lys), D-arginine D-Arg, D-Tyrosine (D-Tyr), D-Glutamine (D-Gln), D -allo-isoleucine (D-aIle), D-allo-threonine (D-aThr), D- Chiral balance of ornithine (D-Orn) and D-valine (D-Val) and the corresponding D- The ratio of amino acids to their L-amino acids (D / L) was significantly decreased in the kidney disease group, and D-histidine was significantly decreased in the kidney disease group. The chiral balance of the D-His is the same as that of the mouse reported in Patent Document 3. It was found that the level of uric acid in the kidney disease group was significantly increased, and it was confirmed that it is involved in renal dysfunction. Among these, D-tyrosine, D-methionine, D-tryptophan, Although phan and D-ornithine are prominently detected in cat urine, they are also found in other mammals, e.g., humans. It is an amino acid unique to cats that cannot be detected in the urine of dogs or mice. Therefore, the amount of D-tyrosine, D-methionine, D-tryptophan and D-o- D-amino acids, consisting of lunithine and D-histidine, are specifically designed for testing renal dysfunction in cats. These are useful marker molecules, and by measuring the presence level of one or more of these, This allows the evaluation of renal dysfunction in test cats.

[0018] The D-amino acid levels measured in the present invention include D-tyrosine, D-methionine, individual D-amino acids selected from D-tryptophan, D-ornithine, and D-histidine; For each amino acid, the concentration of the amino acid may be used. For example, the concentration of the D-amino acid may be used. The ratio of the amount of D-amino acid to the amount of L-amino acid (amount of D-amino acid / amount of L-amino acid), Amount of D-amino acids / (amount of D-amino acids + amount of L-amino acids) × 100) is preferred. A chiral balance is more preferable.

[0019] Regarding the chiral balance of D-amino acids in cat urine, the D-tyrosine and D-methionine one or more selected from the group consisting of thiamin, 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 D-amino acids selected from D-tyrosine and D-tryptophan. In addition to measuring the chiral balance of the acid, we also investigated the chirality of D-phenylalanine, D-glutamic acid, and D-glutamic acid. D-amino acid, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D- Arginine, D-glutamine, D-allo-isoleucine, D-allo-threonine, D-ba one or more selected from D-phosphorus and D-leucine, preferably D-phenylalanine, D-glucan glutamic acid, D-threonine, D-proline, D-alanine, D-glutamine, D-alanine - one or more selected from isoleucine and D-valine, more preferably D-phenylalanine D-glutamic acid, D-alanine, D-allo-isoleucine and D-valine and measuring the chiral balance of one or more D-amino acids that are can be done.

[0020] In addition, the ratio of D-amino acids to L-amino acids (D / L) in cat urine was one selected from D-tyrosine, D-tryptophan, D-ornithine and D-methionine At least one kind, preferably selected from D-tyrosine, D-tryptophan and D-ornithine more preferably, one or more selected from D-tyrosine and D-tryptophan It is preferable to measure the ratio of D-amino acids to L-amino acids (D / L). In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline , D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-alanine -One selected from isoleucine, D-allothreonine, D-valine and D-leucine The above, preferably D-phenylalanine, D-glutamic acid, D-proline, D-threo D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, D-phenylalanine, D-glutamic acid, and D-proline , D-alanine, D-allo-isoleucine, and D-valine. This can be combined with the measurement of the ratio of amino acids to L-amino acids (D / L).

[0021] Regarding the D-amino acid concentration in cat urine, the D-tyrosine, D-methionine, - one or more selected from tryptophan and D-ornithine, preferably D-tyrosine; Measures the concentration of one or more D-amino acids selected from D-tryptophan and D-ornithine In addition, it is preferable to further include D-phenylalanine, D-glutamic acid, D -Threonine, D-Proline, D-Serine, D-Alanine, D-Lysine, D-Arginine , D-glutamine, D-allo-isoleucine, D-allo-threonine, D-valine, D- One or more selected from asparagine and D-leucine, preferably D-phenylalanine , D-glutamic acid, D-threonine, D-proline, D-serine, D-alanine, D- Lysine, D-arginine, D-glutamine, D-allo-isoleucine, D-allo-threo one or more selected from D-valine and D-asparagine, more preferably D-phenylalanine D-threonine, D-serine, D-alanine, D-lysine, D-glutamic acid, D-threonine, D-serine, D-alanine, D-lysine D-arginine, D-glutamine, D-allo-isoleucine, D-allo-threonine The concentration of one or more D-amino acids selected from D-amino acids, D-valine, and D-asparagine is combined. can be measured together.

[0022] Furthermore, the measurement of D-amino acid levels in the present invention includes D-tyrosine, D-methionine Five D-amino acids consisting of 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-allo-isoleucine, D-allo-threonine, D -One or more D-amino acids selected from valine, D-asparagine, and D-aspartic acid The total concentration of 6 or more D-amino acids including the acid is preferably 20 D-amino acids (D-thiamine). Rosine, 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-allo- Isoleucine, D-allo-threonine, D-valine, D-asparagine, D-asparagi This can also be done by measuring the total concentration of the above six or more D- The total concentration of D-amino acids in cat urine containing amino acids is determined by enzyme reactions using amino acids as substrates. It may be the total concentration of all measurable D-amino acids (all D-amino acids excluding acidic amino acids). stomach. Furthermore, the ratio of the total concentration of the D-amino acid to the total concentration of the L-amino acid (D-amino acid The chiral balance of the total concentration of the D-amino acid (D - Calculate the total concentration of amino acids / (total concentration of D-amino acids + total concentration of L-amino acids) These may also be used as the D-amino acid levels of the present invention. In addition, the total concentration of D-amino acids was further calculated by the urine specific gravity of the total concentration of the D-amino acids. The ratio of the total concentration of D-amino acids to the specific gravity of urine, and the ratio of the total concentration of the D-amino acids to the specific gravity of urine The ratio of the total D-amino acid concentration to the urinary creatinine concentration (total D-amino acid concentration / urinary creatinine concentration) was calculated. These may also be used as the D-amino acid levels of the present invention.

[0023] In the present invention, the measurement of D-amino acids or L-amino acids is carried out by measuring the D-amino acids and L-amino acids in urine. The method is not limited as long as the amino acids can be separated and measured, but generally, liquid chromatography is used. LC-MS, LC-MS / MS, which combines liquid chromatography (LC) and mass spectrometry (MS), Separation and quantification methods using S, etc. can be used. In addition, the total amino acid concentration can be measured using D-amino acid. D-amino acid oxidase uses acid as a substrate, and L-amino acid oxidase uses L-amino acid as a substrate. Quantitative methods using enzyme reactions (colorimetry, fluorescence, chemiluminescence, etc.) using enzymes, etc. (enzyme methods) or biosensing methods using enzyme-based biosensors (electrochemical detection, etc.) The enzymatic method is more preferable from the viewpoint of simplicity and speed. The enzyme assay using D- or L-amino acid oxidase is used to measure D- or L-amino acids. Colorimetric determination of hydrogen peroxide produced during oxidative deamination by L-amino acid oxidase This method detects and measures all amino acids in cat urine except for acidic amino acids using a fluorescent probe. The total D-amino acid concentration or the total L-amino acid concentration can be measured. When measuring amino acids in urine, urine collected at home or in a medical institution (using a catheter) Regarding the use of ureters (e.g., bladder puncture, spontaneous urine collection, etc.), freezing and thawing, centrifugation, ultrafiltration, gel filtration, and detangling Pretreatment for analysis such as cleavage, dilution, solid-phase extraction, liquid-liquid partitioning, derivatization, etc. is appropriately performed. It is desirable that the pretreatment for the enzyme assay does not contain any substances that inhibit the enzyme reaction. The form of transportation of collected urine is not limited, and may be at room temperature or by frozen mail.

[0024] Chiral amino acids can be separated using, for example, 4-fluoro-7-nitro-2,1,3 After converting the amino acid into an NBD derivative using the -benzoxadiazole (NBD-F) reagent, A reversed-phase column (first dimension: molecular species separation) and a chiral column with a stationary phase carrying a chiral identifier Two-dimensional liquid chromatography (LC) using ram (second dimension: chiral separation) Separation Quantification (J Chromatogr A. 2010 Feb 12;1217(7):1056-62. doi: 10.1016 / j.chroma.200 9.09.002. Epub 2009 Sep 6), and one-dimensional L using a single reversed-phase column (ODS column). C method (Anal Chim Acta. 2015 May 22;875:73-82. doi: 10.1016 / j.aca.2015.02.054. Epu b 2015 Feb 23), amino acids were converted to 6-aminoquinolyl-N-hydroxysuccinimidyl carboxylates. The α-methyl-4-benzoate (AQC) was derivatized and analyzed by one-dimensional LC using a single chiral column (J Pharm Bio omed Anal. 2015 Nov 10;115:123-9. doi: 10.1016 / j.jpba.2015.05.024. Epub 2015 Jun 16), after derivatizing the amino group with AQC, a weak anion exchange chiral stationary phase was developed. and a second chiral column having a chiral stationary phase of the amphoteric ion exchange type. A method for separating the compound using a liquid chromatography system connected to the Although the method of Japanese Patent No. 6764778 is known, it is preferable to use the method of Japanese Patent No. 6764778.

[0025] Thus, by comparing the measured D-amino acid levels with the reference values, the subject's Renal dysfunction can be assessed in elephant cats. Here, the reference value is determined based on the correlation between the D-amino acid level and renal dysfunction, for example, as follows: It can be set as follows: Renal dysfunction will be evaluated using the IRIS guidelines and other methods. Based on this, a healthy group was composed of cats that were judged to have no renal dysfunction (healthy) and a control group was composed of cats that had renal dysfunction. Cats that are deemed to be at risk are stratified into a renal dysfunction group. The level of the D-amino acid of the present invention is measured by the method described above, and the results of the evaluation of renal dysfunction are then compared. Based on the correlation with D-amino acid levels, appropriate reference values ​​for assessing renal dysfunction were determined. Specifically, each group was classified based on the results of statistical analysis of the D-amino acid levels in each group. The range of values ​​for the D-amino acid levels that characterize the subjects was determined. This range was determined by subtracting the mean value of each group from the mean value of each group. It is determined by setting a certain range above and below the center. Statistical values ​​such as standard deviation (SD), 1 / 2 SD value, 1 / 3 SD value, etc. may be used. It may also be a certain range above and below the median of the group (such as the first or third quartile). For example, the level of the D-amino acid of the present invention in urine obtained from a test cat can be used to evaluate renal function. If the D-amino acid level falls within the range of the impaired group, the cat is considered to have "renal impairment." The patient can be evaluated as having "renal dysfunction," "having renal dysfunction," or "highly likely to have renal dysfunction."

[0026] The reference value may also be determined based on a cutoff value obtained by statistical analysis. . The cutoff value may be, for example, the median or mean value of the calculated biomarkers, RO Values ​​based on C-curve analysis (e.g., Youden's index, The upper left corner coordinates (0,1) of the ROC curve created using a discriminant that separates groups that do not A cutoff value can be set multiple times. For example, When using the protein balance as an index, use the total D-amino acid concentration (5 types, 20 types) in Table 2 below as an index. If the D-amino acid concentration is used as an index, see Table 3 below. If the D-amino acid concentration is used as an index, see Table 4 below. When the ratio of L-amino acids to L-amino acids (D / L) is used as an index, see Table 5 below, and measure by the enzymatic method. When the total concentration of D-amino acids is used as an index, the ROC curve shown in Table 8 below is used. Cutoff values ​​can be set based on the analysis. For example, the chiral balance of D-tyrosine is 0.55% or less, and the chiral balance of D-methionine is D-tryptophan chiral balance is 0.10% or less, and D-o The chiral balance of lunitine is 40% or less, and the chiral balance of D-histidine is 2.2% or less. Above: The chiral balance of D-glutamic acid is 4.8% or less, and the chiral balance of D-valine is 8.9% or less, D-phenylalanine chiral balance 0.52% or less, D-alloy The chiral balance of soleucine is 9.8% or less, and the chiral balance of D-alanine is 43% or less. Below, the chiral balance of D-proline is 5.5% or less, and the chiral balance of D-glutamine is Chiral balance of D-serine is 1.2% or less, and chiral balance of D-threonine is 0.91% or less. The chiral balance of D-allo-threonine is 71% or less, and the chiral balance of D-allo-threonine is 4.0% or less. The chiral balance of arginine is 3.0% or less, and the chiral balance of D-lysine is 20% or less. If the chiral balance of D-leucine is 0.16% or less, "renal function is impaired." The patient can be evaluated as having "renal dysfunction" or "high possibility of having renal dysfunction." In addition, for example, the total concentration of D-amino acids measured by an enzymatic method is 380.0 μM or less, Chiral balance of total D-amino acid concentration (total D-amino acid concentration measured by enzymatic method / (Total concentration of D-amino acids measured by enzymatic method + L-amino acids measured by enzymatic method) The total concentration of D-amino acids is 0.54% or less, and the ratio of the total concentration of D-amino acids to the specific gravity of urine is 350.0 The ratio of the total concentration of D-amino acids to the urinary creatinine concentration was 1.84 mmol. If the Cre level is below 10g / 10g, the patient is considered to have "renal dysfunction" or "renal impairment." can be evaluated as "highly likely to have renal dysfunction."

[0027] Thus, according to the method of the present invention, the presence or absence of renal dysfunction in the subject cat and the state of renal dysfunction can be determined. The condition and severity can be evaluated non-invasively, easily, and accurately. The test method of the present invention may be used in combination with other diagnostic indicators for renal dysfunction other than those of the present invention. This allows for a more multifaceted and accurate evaluation of renal dysfunction. Other diagnostic indicators for renal dysfunction include blood creatinine (Cre), urinary creatinine (Cre), and Atinine, blood urea nitrogen (BUN), blood symmetric dimethylarginine (SDMA), blood Cystatin C, urinary cystatin C, urinary neutrophil gelatinase-binding lipocalin (NGAL) ), urinary L-type fatty acid binding protein (L-FABP), urine protein, urine protein / creatinine ratio (UP C), urinary albumin, etc. In addition, a simple urine specific gravity test using a specific urine collection container is also available. Valuation method (Patent No. 7084128), urine color (Patent No. 7216529), urine penetration It can also be combined with physical indicators such as blood pressure, fractional excretion rates of various electrolytes, blood pressure, and imaging tests. is.

[0028] The testing method of the present invention can be carried out periodically to monitor changes in the D-amino acid levels. This can be used to detect progression of renal dysfunction in test cats.

[0029] The test method of the present invention is also carried out in the presence of preventive or therapeutic intervention for renal dysfunction in cats. By implementing this, it can be used to evaluate the effectiveness of the intervention. That is, the method for evaluating the intervention effect of the present invention is a method for evaluating the effect of preventive or therapeutic intervention for renal dysfunction in cats. In the presence of α-tocopherol, D-tyrosine, D-methionine, D-tryptophan were measured in the urine of the test cats. The level of one or more D-amino acids selected from D-amino acids, D-ornithine, and D-histidine is The method includes a step of measuring.

[0030] Here, preventive or therapeutic intervention for renal dysfunction in cats means prevention of renal dysfunction in cats. It is an intervention that is carried out in the hope of having a preventive or therapeutic effect, and is a chemical intervention, physical mechanical intervention, or Chemical interventions include the administration of natural substances, synthetic substances, compositions, etc. Physical or mechanical interventions include treatments such as dialysis. Low-calorie food, low-fat food, and supplements with adjusted vitamin and mineral content. Examples include the administration of low-salt and low-salt modified foods.

[0031] The effectiveness of the intervention was evaluated based on the levels of the D-amino acid in the test cats resulting from the intervention. For example, the effect of the D-amino acid on the subject cat in the presence of an intervention can be evaluated. The effects of the intervention can be assessed by comparing the levels with a control (absence of intervention). do.

[0032] The kit for testing for renal dysfunction in cats of the present invention is a kit for testing for renal dysfunction in cats that can be used to perform the method for testing for renal dysfunction in cats of the present invention. Therefore, the kit includes the equipment necessary for collecting cat urine, cat urine or The container is for storing cat urine-containing litter or sheets, and is used to measure the levels of one or more D-amino acids. Reagents for determining D-amino acid levels, indicators or guidance for comparing D-amino acid levels with reference values and the like. Reagents for measuring the level of D-amino acids include those that use D- or L-amino acids as substrates. enzymes (e.g., D- or L-amino acid oxidase), colorimetric or fluorescent probes, and peroxidases Examples include oxidase.

[0033] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A method for testing for renal dysfunction in cats, comprising: detecting D-tyrosine in the urine of a test cat; , D-methionine, D-tryptophan, D-ornithine and D-histidine A method for testing for renal dysfunction, comprising measuring the levels of one or more D-amino acids. <2> In addition, D-phenylalanine, D-glutamic acid, D-leucine, D-threonine D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glucan Lecithin, D-allo-isoleucine, D-allo-threonine, D-valine, D-asparagine The method of <1>, wherein the level of one or more D-amino acids selected from D-amino acids, D-aspartic acid, and D-aspartic acid is measured. <3> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D- One or more D-amino acids selected from tryptophan, D-ornithine, and D-histidine measuring the chiral balance of the acid; <1> How to do it. <4> In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-arginine D-isoleucine, D-allothreonine, D-valine, and D-leucine More than one species, preferably D-phenylalanine, D-glutamic acid, D-threonine, D-proline D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, one or more of D-phenylalanine, D-glutamic acid, D-alanine chirality of one or more D-amino acids selected from the group consisting of leucine, D-allo-isoleucine and D-valine measuring the weight balance, <3> How to do it. <5> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-tryptophan, L-amino acids consisting of one or more D-amino acids selected from D-ornithine and D-methionine measuring the ratio of the amount of <1> How to do it. <6> In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-arginine D-isoleucine, D-allothreonine, D-valine, and D-leucine More than one species, preferably D-phenylalanine, D-glutamic acid, D-proline, D-thiamin mononitrate, D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, one or more of D-phenylalanine, D-glutamic acid, D-proline one or more D-amino acids selected from the group consisting of D-alanine, D-allo-isoleucine, and D-valine; measuring the ratio of amino acids to L-amino acids; <5> How to do it. <7> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D- Measure the concentration of one or more D-amino acids selected from tryptophan and D-ornithine The process is <1> How to do it. <8> In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-arginine D-isoleucine, D-allothreonine, D-valine, D-asparagine and D-leucine 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-allo-isoleucine, D-allo-threonine, D-valine and D- Asparagine, more preferably D-phenylalanine, D-glutamic acid, D-amino acid, D-threonine, D-serine, D-alanine, D-lysine, D-arginine, D -Glutamine, D-allo-isoleucine, D-allo-threonine, D-valine and D-allo-threonine measuring the concentration of one or more D-amino acids selected from the group consisting of sparagines, <7> For those Law. <9> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D- The total concentration of five D-amino acids consisting of tryptophan, D-ornithine, and D-histidine or the five D-amino acids plus D-phenylalanine, D-glutamic acid, D-leucine, D-threonine, D-proline, D-serine, D-alanine, D-lysine, D-amino L-arginine, D-glutamine, D-allo-isoleucine, D-allo-threonine, D-valerian D-amino acids selected from the group consisting of thiamin, D-asparagine, and D-aspartic acid are added. measuring the total concentration of the six or more D-amino acids obtained; <1> How to do it. <10> Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids was calculated. comprising the step of: <9> How to do it. <11> further comprising a step of calculating the chiral balance of the total concentration of the D-amino acids. <9> How to do it. <12> Measurement of the total concentration of six or more D-amino acids is performed using enzyme reactions with D-amino acids as substrates. This is carried out by a quantitative method using a reaction. <9> ~ <11> Either way. <13> In the presence of preventive or therapeutic intervention for feline renal dysfunction, D-tyrosine, D-methionine, D-tryptophan, D-ornithine and D-histidine and measuring the level of one or more D-amino acids selected from the group consisting of d-amino acids, d-amino acids, and d-amino acids. Evaluation method. <14> In addition, D-phenylalanine, D-glutamic acid, D-leucine, and D-threo D-Proline, D-Serine, D-Alanine, D-Lysine, D-Arginine, D-Glutamate glutamin, D-allo-isoleucine, D-allo-threonine, D-valine, D-asparagus measuring the level of one or more D-amino acids selected from D-ginseng and D-aspartic acid; <13> How to do it. <15> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D- -One or more D-amino acids selected from tryptophan, D-ornithine and D-histidine measuring the chiral balance of the acid; <13> How to do it. <16> In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline Phosphorus, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D- Selected from allo-isoleucine, D-allo-threonine, D-valine and D-leucine One or more of, preferably, D-phenylalanine, D-glutamic acid, D-threonine, D- From proline, D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, one or more selected from D-phenylalanine, D-glutamic acid, D-alamin, D-amino acid esters of one or more D-amino acids selected from the group consisting of leucine, D-allo-isoleucine, and D-valine. measuring the relative balance, <15> How to do it. <17> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-tryptophan, , D-ornithine, and D-methionine. measuring the ratio of the amount of the acid to the amount of the acid; <13> How to do it. <18> In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline Phosphorus, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D- Selected from allo-isoleucine, D-allo-threonine, D-valine and D-leucine One or more of these, preferably D-phenylalanine, D-glutamic acid, D-proline, D-succinic acid, From leonine, D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, one or more selected from D-phenylalanine, D-glutamic acid, and D-proline are one or more D selected from phosphorus, D-alanine, D-allo-isoleucine and D-valine - measuring the ratio of the amount of amino acids to L-amino acids, <17> How to do it. <19> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D- -Measure the concentration of one or more D-amino acids selected from tryptophan and D-ornithine This is a process in which <13> How to do it. <20> In addition, D-phenylalanine, D-glutamic acid, D-threonine, D-proline Phosphorus, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D- Allo-isoleucine, D-allothreonine, D-valine, D-asparagine and D-lo 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-allo-isoleucine, D-allo-threonine, D-valine and D -asparagine, more preferably D-phenylalanine, D-glucan Amino acid, D-threonine, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-allo-isoleucine, D-allo-threonine, D-valine and D- measuring the concentration of one or more D-amino acids selected from asparagine; <19> How to do it. <21> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D- -The total amount of five D-amino acids consisting of tryptophan, D-ornithine, and D-histidine Concentration, or the five D-amino acids plus D-phenylalanine, D-glutamic acid, D-lo Isine, D-Threonine, D-Proline, D-Serine, D-Alanine, D-Lysine, D- Arginine, D-glutamine, D-allo-isoleucine, D-allo-threonine, D-ba phosphorus, D-asparagine, and one or more D-amino acids selected from D-aspartic acid measuring the total concentration of the six or more D-amino acids added; <13> How to do it. <22> Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids was calculated. comprising the step of: <21> How to do it. <23> further comprising a step of calculating the chiral balance of the total concentration of the D-amino acids. <21> How to do it. <24> A method for testing for renal dysfunction in cats using D-amino acid oxidase The total concentration of all D-amino acids in the urine of the test cats that can be measured by the enzymatic assay was measured by the method. A method for testing for renal dysfunction, comprising the step of measuring the above. <25> The method further comprises the step of calculating the ratio of the total concentration of the D-amino acids to the specific gravity of urine. , <24> How to do it. <26> Furthermore, the ratio of the total concentration of the D-amino acids to the urinary creatinine concentration was calculated. comprising the step of: <24> How to do it. <27> Furthermore, the subject is subjected to an enzyme assay using L-amino acid oxidase. measuring the total concentration of L-amino acids in the urine of the cat; <24> How to do it. <28> further comprising a step of calculating the chiral balance of the total concentration of the D-amino acids. <27> How to do it. <29> Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids was calculated. comprising the step of: <27> How to do it. <30> <1> ~ <29> A test kit for carrying out the method of the present invention, A kit for testing for kidney dysfunction in cats, containing reagents for measuring acid levels. <31> Reagents for measuring D-amino acid levels include D-amino acid oxidase , <30> Kit. <32> Further containing a colorimetric or fluorescent probe and peroxidase, <31> Noki tt. [Example]

[0034] The present invention will be described in more detail below with reference to examples. Test Example 1: Simultaneous separation and analysis of chiral amino acids in urine from cats with kidney disease (1) About the cat test Cats in the healthy group (N = 17) and the renal dysfunction group (N = 11, hereafter referred to as the renal disease group) (Table 1) The relationship between renal function and urine components was analyzed. IRIS (International Renal Interest Society) Guide urine, auxiliary physical tests (color, urine volume, urine specific gravity, pH) and a total The classification was based on the judgement of the health status and kidney disease status. According to the guidelines, blood (serum) creatinine level is 1.6 (mg / dL) or more or symmetric dilation Individuals with methylarginine (SDMA) levels of 18 μg / dL or higher were considered to have kidney disease. (Equivalent to Stage 2).

[0035] [Table 1]

[0036] (2) Preparation of sample solutions and standards 10 μL of cat urine (naturally collected) was placed in a 10 mL spitz glass (product name: reinforced hard screw-top test tube). ), mixed with 490 μL of methanol:water (9:1, v / v), and then centrifuged (HIT ACHI CF5RE) for 5 minutes at 2130 x g (3000 rpm) in a refrigerator (4°C). The protein was removed by centrifugation at 100°C, and the amino acids in the supernatant were collected. In a mL spitz glass, add 0.2 mol / L borate buffer (pH 8.9), collection solution, and A. ccQ·Tag Ultra derivatization reagent, i.e., AQC solution (AdipoGen: AQC powder was dissolved in ultra-dehydrated acetonitrile at a concentration of 3 mg / 1 mL, i.e., 10 mmol / L. Mix 70 μL, 10 μL, and 20 μL (7:1:2) of each solution in this order, and immediately stir. After stirring, the mixture was heated at 55°C for 10 minutes to prepare a derivatized sample solution. Similarly, 0.2 mol / L borate buffer (pH 8.9), 100 μmol / LD, L-amino acid standard solution (amino acids: Ala / alanine, Arg / arginine, Asn / amino acid) Aspartic acid, Asp, Cysteine, Gln, Glutamine, Gl u / glutamic acid, Gly / glycine, His / histidine, Ile / isoleucine, al L-Ile / Isoleucine, Leu / Leucine, Lys / Lysine, Met / Methionine nine, Phe / Phenylalanine, Pro / Proline, Ser / Serine, Thr / Threonine nine, allo-Thr / allo-Threonine, Trp / Tryptophan, Tyr / Tyrosine Val / Valine, Cit / Citrulline, Orn / Ornithine, 0.2 mol / L boric acid (dissolved in buffer), 70 μL, 10 μL, and 20 μL of the AQC solution in the order of (7:×1:2) are mixed, immediately stirred, and heated at 55 °C for 10 minutes to prepare a derivatized amino acid standard solution .

[0037] (3) LC-MS / MS analysis The derivatized sample solution and the derivatized amino acid standard solution prepared in (2) are subjected to LC-MS / MS analysis under the following conditions ( based on Patent No. 6764778) to separate and detect and quantify various chiral amino acids. (Equipment) Exion LC series (AB SCIEX), mass spectrometer / QTRAP6500 + linear ion trap type (AB SCIEX) (Chromatographic separation) Separation chiral column: CHIRALPAK QN-AX <DAICEL> 2.1 mm inner diameter × 150 mm, particle size 5 μm (first chiral column) and CHIRALP AK ZWIX(+) <DAICEL> 3.0 mm inner diameter × 150 mm, particle size 3 μm (second chiral column) are connected in series in this order (40 °C) Eluent: methanol:water ( 90:10, v / v) solution containing 0.1% (v / v) formic acid and 55 mM ammonium formate Elution method: isocratic Mobile phase flow rate: 0.25 mL / min Injection volume: 5μL (Mass spectrometry) Ionization method: Electrospray ionization (ESI) Polarity: Positive ion Curtain Gas (CUR): 30 psi Ion spray voltage (IS): 4500V Temperature (TEM): 600℃ Ion Source Gas 1 (GS1): 80 psi Ion Source Gas 1 (GS2): 80 psi Collision Gas(CAD):10 (detection mode) The precursor ion is protonated ([M+H] + ), product ion SRM in positive ion mode with AQC fragment ion (m / z = 171) Selected Reaction Monitoring) detection.

[0038] (4) Data analysis 1) Analysis of chiral amino acids The data obtained in (3) was plotted on a chromatogram with two axes of retention time and ion intensity. We developed a method to analyze various chiral amino acids derivatized with AQC in cat urine. The urinary chiral amino acid concentrations (D-amino acids (DAA): D-Pr) were measured in the disease group. o,D-Ser,D-Ala,D-Lys,D-Arg,D-His,D-Tyr,D- Gln,D-allo-Ile(aIle),D-Thr,D-allo-Thr(aT hr),D-Orn,D-Val,D-Phe,D-Met,D-Glu,D-Asp, The measured values ​​of D-Asn, D-Leu, and D-Trp are shown in Figures 1-1 and 1-2. -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,LV al,L-Phe,L-Met,L-Glu,L-Asp,L-Asn,L-Leu,L The measured values ​​of -Trp are shown in Figures 2-1 and 2-2. The individual chiral balances (D-isomer abundance) calculated from the concentrations of these urinary chiral amino acids were The ratio of the total number of ions in the saturation zone (D / (D+L)×100(%)) is shown in Figures 3-1 and 3-2.

[0039] 17 types (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-all The chiral balance of o-Thr (aThr), D-Orn, D-Val was significant in the kidney disease group. The chiral balance of D-His was significantly increased in the kidney disease group. It has been confirmed that these are involved in renal function. D-Orn is an amino acid that is specifically detected in cat urine.

[0040] Furthermore, the total DAA or LAA concentration is shown in Figure 4, and the individual D / L is shown in Figures 5-1 and 5-2. The total concentration of D / L is shown in Figure 5-3, and the chiral balance of the total concentration (D-isomer abundance ratio: D / (D+L) In addition to the individual chiral balances and concentrations mentioned above, the total concentration There was also a significant difference in the ratio of individual DAA to LAA (D / L) between the kidney disease 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) of DAA and LAA and the total concentration was also significantly low. These results suggest that D-Tyr and D-Tyr can be used as indicators of renal dysfunction. The total concentrations of only D-Met, D-Trp, D-Orn, and D-His extracted are also shown in FIG. Similarly, a significant decrease was observed in the kidney disease group.

[0041] 2) Verification of usefulness Excel statistical processing software (BellCurve for Excel, Social Sciences Co., Ltd.) Using the information service, the chiral balance, total concentration, and D-amino acid concentration of the healthy group and the renal disease group were compared. ROC (Receiver Error Characteristic) for the ratio of D-amino acids to L-amino acids (D / L) Operating Characteristic curve and set the cutoff point. Cut off, Area under the ROC curve (AUC), Sensitivity The specificity and p-value (diagnostic ability) were calculated (Table 2). Cal balance, Table 3: Total concentration, Table 4: D-amino acid concentration, Table 5: D-amino acid and L-amino acid concentrations The cutoff is the closest point from the upper left corner, and the null hypothesis is In the test with AUC=0.50, chiral balance, (total) concentration, D-amino acids and The quantitative ratio of L-amino acids (D / L) was found to be significant and diagnostic. Many DAA molecular species with AUC of 0.90 or more were also detected. DAA(D-Tyr,D-Trp, D-Orn, D-Met, D-His);DAA(Total ):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-Va l,D-Phe,D-Met,D-Glu,D-Asp,D-Asn,D-Leu,D- Trp;LAA(Total):L-Pro,L-Ser,L-Ala,L-Lys,LA rg,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) have also been found to be useful for evaluating renal function, and urinary chiral amino acids (especially urinary The usefulness of DAA was confirmed.

[0042] [Table 2]

[0043] [Table 3]

[0044] [Table 4]

[0045] [Table 5]

[0046] Test Example 2: Changes in D-amino acids in cat urine over time For cases 1 to 3 shown in Table 6 below, renal function and urinary component association analysis was performed in the same manner as in Test Example 1. Cat breed (1): Abyssinian (neutered male, 3-6 years old) was examined in September 2019. Cat breed (2): Siamese (male, 3-4 years old) diagnosed with Stage 2 kidney disease in November 2020 A blood test in the month diagnosed Stage 2 kidney disease. Cat breed (3): Bengal (neutered male, 4-7 years old) ) was diagnosed with Stage 2 kidney disease in a blood test in November 2016. Changes over time, i.e., urinary chiral balance ( For example, D-His, D-alle, D-Val, etc.) and urinary DAA concentrations (total concentrations) are The correlation with renal function has been confirmed, making it useful for non-invasive evaluation and monitoring of renal function. It was confirmed that DAA in urine was significantly higher than that in the control group (Fig. 7-1, 7-2, 7-3). This is a renal monitoring index that can precisely evaluate the severity (pathological condition) and very early risk.

[0047] [Table 6]

[0048] Test Example 3: Analysis of D,L-amino acids in cat urine using an enzymatic method (1) About the cat test The subjects were cats in the healthy group (N = 14) and the renal dysfunction group (N = 4, hereafter referred to as the renal disease group) (Table 7) We performed an analysis of the relationship between renal function and urinary components. As in Test Example 1, after urine collection, the current standard IRIS (International Routine Immunosorbent Assay System) was used. Veterinary Renal Interest Society: International Veterinary Kidney Research Group The classification was based on a comprehensive judgment based on the guidelines and a veterinarian's interview. pH (Horiba Ltd., compact pH meter LAQUAtwin), urine Specific gravity (USG) (Atago Co., Ltd., PAL-dog and cat urine specific gravity meter), urinary creatinine concentration (CRE) (ARKRAY, Inc., thinka RT-4010 urine chemistry analyzer) Measurements of were also carried out.

[0049] [Table 7]

[0050] (2) Measurement of total D-amino acid (DAA) concentration in cat urine Creating a calibration curve and calculating quantitative values D-Amino Acid Assay Kit(MET-5136,Colorim Compliant with the protocol for the ETRIC (Cell Biolabs). -Alanine) was prepared at the time of use. A calibration curve was drawn for 100 μM, 50 μM, and 100 μM of D-amino acid total concentration (excluding acidic amino acids) in cat urine. Total D-amino acid measurements were performed. Specifically, 10 μL of cat urine was placed in a 10 mL spitz glass (product name: reinforced hard screw-cap test tube). ), mix with 90 μL of the kit's built-in assay buffer, and then centrifuge ( HITACHI CF5RE) for 5 minutes at 2130 × g (3000 rpm) in a refrigerator (4°C). The protein was removed by centrifugation at 1000 rpm, and the amino acids in the supernatant were collected. 50 μL of the above prepared standard solution and 50 μL of cat urine were each placed in a 96-well microplate (No. 6 55801, manufactured by Greiner Bio-One). Built-in enzyme reaction solution (Colorimetric Probe, HRP, D-Amino A Add 50 μL of the HCl solution (C10 Oxidase mixture), mix immediately in the dark, and then heat at 37°C for 1 hour. The mixture was then incubated for 24 hours (D-amino acid oxidase (DAO) reaction). The D in the enzyme reaction mixture was measured using a meter (SH-9000 Lab, manufactured by Corona Electric Co., Ltd.). The total AA concentration was measured (wavelength 570 nm).

[0051] (3) Measurement of total L-amino acid (LAA) concentration in cat urine Creating a calibration curve and calculating quantitative values L-Amino Acid Assay Kit(MET-5054, Colorim Compliant with the standard kit (L -Alanine) prepared at the time of use: 0, 62.5, 125, 250, 500, 1000 μ A calibration curve for M was drawn, and the total concentration of all L-amino acids in cat urine was measured. Specifically, 10 μL of cat urine was placed in a 10 mL spitz glass (product name: reinforced hard screw-cap test tube). ) and mix with 990 μL of the kit's built-in Assay buffer, then centrifuge. (Hitachi / CF5RE) for 5 minutes at 2130 × g (3000 The mixture was centrifuged at 1000 rpm to remove proteins, and the amino acids in the supernatant were collected. 50 μL of the above freshly prepared standard solution and 50 μL of cat urine were each placed in a 96-well microplate (No. 655801, manufactured by Greiner Bio-One Co., Ltd.) were added to each of the Built-in enzyme reaction solution (Colorimetric Probe, HRP, L-Amino Add 50 μL of the acid oxidase mixture, mix immediately in the dark, and then incubate for 3 hours. The mixture was incubated at ℃ (L-amino acid oxidase (LAO) reaction). Finally, a microplate reader (SH-9000 Lab, manufactured by Corona Electric Co., Ltd.) was used. The total LAA concentration in the enzyme reaction solution was measured (wavelength 570 nm) using a chromatograph.

[0052] (4) Data analysis 1) Analysis of DAA-related indicators in cat urine Total DAA concentration and total concentration chiral balance measured by enzymatic method in healthy and renal disease groups D-form abundance ratio: total DAA concentration measured by enzymatic method / (measured by enzymatic method) DAA total concentration + LAA total concentration measured by enzymatic method) × 100 (%), total concentration ratio (enzymatic (total DAA concentration measured by the method / total LAA concentration measured by the enzymatic method) and correlation The ratio of total DAA concentration measured by the enzymatic method to urinary CRE concentration and D measured by the enzymatic method The total AA concentration / USG ratio is shown (Figures 8-1, 8-2, 8-3, and 8-4).

[0053] 2) Verification of usefulness Excel statistical processing software (BellCurve for Excel, Social Sciences Co., Ltd.) Using the information service, the ROC ( Draw the Receiver Operating Characteristic curve, Cut-off point (Cut off), area under the ROC curve (AUC), sensitivity (Sens The specificity, p-value (diagnostic ability), and p-value were calculated. (Table 8). The cutoff is the closest point from the upper left corner, and the null hypothesis is AUC = 0.5. In the test with the value set at 0, it was confirmed that the DAA-related index in cat urine has diagnostic ability. The total DAA concentration and its related ratios determined by the method were found to be particularly useful for assessing renal function. .

[0054] [Table 8]

Claims

1. A method for testing for renal dysfunction in a cat, comprising a step of measuring the total concentration of 15 D-amino acids in the urine of a test cat, comprising five D-amino acids, D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine, as well as D-phenylalanine, D-leucine, D-threonine, D-serine, D-alanine, D-lysine, D-arginine, D-glutamine, D-valine, and D-asparagine.

2. 2. The method according to claim 1, wherein the step of measuring the total concentration of D-amino acids is an enzymatic assay.

3. 2. The method according to claim 1, wherein the step of measuring the total concentration of D-amino acids is an enzymatic measurement method using D-amino acid oxidase.

4. The method of claim 1, further comprising the step of calculating the ratio of the total concentration of the D-amino acids to the specific gravity of urine.

5. The method of claim 1, further comprising the step of calculating the ratio of the total concentration of the D-amino acids to the urinary creatinine concentration.

6. The method according to claim 1, further comprising a step of measuring the total concentration of the L-amino acid corresponding to the D-amino acid in the urine of the subject cat, which can be measured by an enzymatic assay using L-amino acid oxidase.

7. The method of claim 1, further comprising the step of calculating the chiral balance of the total concentration of the D-amino acids.

8. 2. The method of claim 1, further comprising the step of calculating the ratio of the total concentration of the D-amino acids to the total concentration of the corresponding L-amino acids.

9. A test kit for carrying out the method according to any one of claims 1 to 8, comprising a reagent for measuring the total concentration of D-amino acids, for testing for renal dysfunction in cats.

10. 10. The kit according to claim 9, wherein the reagent for measuring the total concentration of D-amino acids comprises D-amino acid oxidase.

11. The kit of claim 10, further comprising a colorimetric or fluorescent probe and peroxidase.

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