Method of testing for renal dysfunction in cats

Abstract

To provide an objective, non-invasive method of testing for renal dysfunction in cats.SOLUTION: A method of testing for renal dysfunction in cats is provided, the method comprising a step of measuring the level(s) of at least one D-amino acid selected from D-tyrosine, D-methionine, D-tryptophan, D-ornithine, and D-histidine.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for testing cats for renal dysfunction 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 drink little water to begin with. Therefore, they are particularly susceptible to cystitis and urinary incontinence 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 is a risk factor for urinary system diseases in young people and kidney diseases in middle-aged and elderly people. It increases the risk of developing heart disease and reduces quality of life (QOL).

[0003] Traditionally, serum creatinine concentration and symmetric dimethylarginine concentration have been used as indicators of impaired renal function in cats. However, serum creatinine (SDMA) levels have not been reported. It is believed that SDMA does not increase until approximately 75% of renal function is lost. It is said that the increase begins when 40% is lost, but abnormal values ​​are detected earlier than creatinine. The reliability of the 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 oxidative stress in mammals, including humans, has become possible. The existence and role of D-amino acids that play important roles in the metabolism have been elucidated, and their specific concentrations in blood and urine have been 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 the effect of serotonin-dependent acetylcholinesterase inhibitors on the function of the kidney is similar to that of serotonin-dependent acetylcholinesterase inhibitors (Patent Documents 1 and 2). D-serine, D-histidine, D-asparagine, D-arginine, D-allose in urine Leonine, D-glutamic acid, D-alanine, D-proline, D-valine, D-alanine The composition ratio of soleucine, D-phenylalanine, and D-lysine decreases, 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 reduced renal function, The amount of specific D-amino acids present in the cat changes, and the level of the D-amino acid is 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: Selected from D-methionine, D-tryptophan, D-ornithine and D-histidine A method for testing for renal dysfunction, comprising the step of measuring the level 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 effectiveness of said intervention, comprising a step of measuring the levels of one or more D-amino acids selected from the group consisting of Valuation method. 3) A method for testing for renal dysfunction in cats, comprising the steps of: The total concentration of all D-amino acids in the urine of the test cat 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 4) A test kit for carrying out the methods of 1) to 3), comprising: A kit for testing for renal dysfunction in cats containing a reagent for measuring renal function. Effect of the Invention

[0010] According to the method of the present invention, the presence or absence and the degree of renal dysfunction in cats can be examined non-invasively, objectively, and simply. This allows for accurate assessment of early changes in declining renal function. [Brief description 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 acids to L-amino acids in cat urine (D / L). [Figure 5-2] The ratio of D-amino acids to L-amino acids 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 an 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 PREFERRED EMBODIMENTS

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

[0013] In the present invention, "renal dysfunction" refers to any abnormality occurring in the kidney that causes impaired renal function. It refers to a condition that occurs when the kidneys become too acidic, but it mainly refers to chronic kidney disease, which cats are prone to. This refers to a condition in which renal function gradually declines, leading to renal failure. Traditionally, renal dysfunction in cats has been diagnosed according to the International Renal Research Institute (IRIS) guidelines, Physical examination (color, urine volume, urine specific gravity, pH) and a comprehensive judgment by a veterinarian based on the interview. In particular, the degree of renal dysfunction is determined according to the IRIS guidelines (2019 revised). They are classified as follows: Healthy: Serum creatinine level is less than 1.6 (mg / dL) or symmetric dimethyl amine SDMA level is less than 14 μg / dL. Stage 1 (early stage): Serum creatinine level is less than 1.6 (mg / dL) or The symmetric dimethylarginine (SDMA) level was 14-17 (μg / dL). Stage 2 (mild): Serum creatinine level is 1.6-2.8 or symmetric dimethicillin Serum arginine (SDMA) level is 18-25. Stage 3 (moderate): Serum creatinine level is 2.9-5.0 or symmetric dimethicone Serum arginine (SDMA) level is 26-38. Stage 4 (severe): Serum creatinine level exceeds 5.0 or symmetric dimethyl amine SDMA level is 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 present invention also includes a method for testing for renal dysfunction, and preferably a method for testing for the presence or absence of renal dysfunction. This naturally includes regular examinations and tests to confirm the effectiveness of preventive or therapeutic interventions. 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 family. Preferably, the cat belongs to the Felinae subfamily, and is exemplified by the domestic cat, gray cat, and jumbo cat. The preferred species are the African wildcat, the sand cat, the black-footed cat, and the 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 under conditions that allowed for the measurement of D-amino acids. The method of collection is not particularly limited, but from the viewpoint of measurement accuracy, fresh urine is preferred. The method of collecting cat urine is preferably natural urine collection (including the use of pet toilets, etc.), cat urine collection, etc. Examples of methods include catheterization, bladder puncture, and urine collection by expressing urine. Natural urine collection is preferable.

[0017] As 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 types (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 gin (D-His) was the same as that of the mouse reported in Patent Document 3. It was found that the levels of ketamine in the kidney disease group increased significantly, and it was confirmed that ketamine is involved in renal dysfunction. Of these, D-tyrosine, D-methionine, and D-trypto Fan and D-ornithine are prominently detected in cat urine, but are also found in other mammals, e.g. It is an amino acid unique to cats and is not detectable in the urine of dogs or mice. Therefore, the amount of D-tyrosine, D-methionine, D-tryptophan and D-o- Lnithine, combined with D-histidine, is a D-amino acid with specific activity for testing renal dysfunction in cats. These are useful marker molecules, and the level of one or more of these can be measured to determine the presence or absence of a specific target molecule. 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, 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, but for example, the concentration of the D-amino acid 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), Ral balance (D-amino acid amount / (D-amino acid amount + L-amino acid amount) × 100) is preferred. A chiral balance is preferable.

[0019] Regarding the chiral balance of D-amino acids in cat urine, at least one selected from the group consisting of 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 are used. In addition to measuring the chiral balance of the acids, we also investigated the chiral balance of D-phenylalanine, D-glutamate, and 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-glycine, 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 in cat urine (D / L) 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-allo-threonine, D-valine and D-leucine The above are preferably D-phenylalanine, D-glutamic acid, D-proline, and 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] In addition, the concentration of D-amino acids in cat urine was - 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 D-phenylalanine, D-valine and D-asparagine, more preferably D-phenylalanine. 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 such as 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, -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-thiol Leucine, 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-asparagine It 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 (total concentration of L-amino acids / total concentration of L-amino acids) and the chiral balance of the total concentration of the D-amino acids (D - Calculate the total concentration of amino acids / (total concentration of D-amino acids + total concentration of L-amino acids) These can also be 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 urine specific gravity, or the urinary clear ratio of the total concentration of the D-amino acids The ratio of D-amino acid concentration to urinary creatinine concentration (total D-amino acid concentration / urinary creatinine concentration) was calculated. These can also be 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 D-amino acids and L-amino acids in urine. As long as the amino acids can be separated and measured, the method is not limited, but generally liquid chromatography is used. LC-MS, LC-MS / M A method for separating and quantifying amino acids 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.) with enzymes, etc. (enzymatic methods) or biosensing methods using enzyme-based biosensors (electrochemical detection, etc.) Alternatively, an enzymatic method can be used, and the enzymatic method is more preferable from the viewpoints of simplicity and rapidity. The enzyme assay using D- or L-amino acid oxidase is used to measure the amount of D- or L-amino acid 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 samples (using a catheter) collected at home or a medical institution should be used. Regarding the use of freezing and thawing, centrifugation, ultrafiltration, gel filtration, and detassium ionization, etc. Pretreatment for analysis, such as amplification, dilution, solid-phase extraction, liquid-liquid partitioning, and derivatization, is appropriately performed. It is preferable that the pretreatment for the enzyme measurement does not contain any substances that inhibit the enzyme reaction. The means of transporting the collected urine is not limited, and may be anything from room temperature to frozen mail.

[0024] As a method for separating chiral amino acids, for example, 4-fluoro-7-nitro-2,1,3 -benzoxadiazole (NBD-F) reagent was used to convert the amino acid into an NBD derivative, A reversed-phase column (first dimension: molecular species separation) and a chiral column with a stationary phase carrying a chiral identifier By two-dimensional liquid chromatography (LC) using a 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), and amino acids were converted to 6-aminoquinolyl-N-hydroxysuccinimidyl carboxylates. The chiral ion was derivatized with bamate (AQC) and subjected to 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) In addition, after the amino group was derivatized with AQC, a weak anion exchange chiral stationary phase was and a second chiral column having a chiral stationary phase of the amphoteric ion exchange type. A method of separating the fluorine-containing compound using liquid chromatography connected to the fluorine-containing compound (Patent No. 6764778) However, it is preferable to use the method described in Japanese Patent No. 6764778.

[0025] Thus, by comparing the measured D-amino acid levels with the reference values, it is possible to determine the D-amino acid status of the subject. Renal dysfunction can be assessed in elephant cats. Here, the reference value is, for example, based on the correlation between the D-amino acid level and renal dysfunction, as follows: It can be set as follows: Renal dysfunction will be evaluated using the IRIS guidelines and other methods. Based on the results, the healthy group consisted of cats that were judged to have no renal dysfunction (healthy) and the control group consisted of cats that had no renal dysfunction (healthy). Cats that are deemed to be at risk of renal dysfunction will be 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 evaluating renal dysfunction were determined. Specifically, the groups were classified based on the results of statistical analysis of the D-amino acid levels in each group. A range of values ​​for the D-amino acid levels that characterize the subjects was determined. This range was determined based on the mean values ​​for each group. The range 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 quartile or third quartile). For example, the level of the D-amino acid of the present invention in urine obtained from a test cat may 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 having a high possibility of having renal dysfunction.

[0026] The reference value may 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 have 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 indicator, 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. The chiral balance of lunitine is 40% or less, and that of D-histidine is 2.2% or less. On the top, 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 is 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 less than 5.5%, and the chiral balance of D-glutamine is 1.2%, D-threonine chiral balance 0.91%, D-serine chiral balance 0.81% 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 D-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 μM or less, and the ratio of the total concentration of the same D-amino acids to the urinary creatinine concentration is 1.84 mmol If the Cre level is less than 10 g, the patient is considered to have "renal dysfunction" or "renal impairment." can be evaluated as having a "high possibility of renal dysfunction."

[0027] Thus, according to the method of the present invention, the presence or absence of renal dysfunction in a 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 appropriate evaluation of renal dysfunction. Other diagnostic indicators for renal dysfunction include, for example, 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. Evaluation method (Patent No. 7084128), urine color (Patent No. 7216529), urine penetration It is also possible to combine it with physical indicators such as blood pressure, fractional excretion rate of various electrolytes, blood pressure and imaging tests. It 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 nitrite, D-tyrosine, D-methionine, and D-tryptophan were detected in the urine of the test cats. The level of one or more D-amino acids selected from D-amino acids such as D-glutamate, 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 carried out in the hope of achieving a preventive or therapeutic effect, and may be a chemical intervention, physical or 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 of such foods include those containing low-salt foods and low-salt modified foods.

[0031] The effectiveness of the intervention was evaluated based on the levels of the relevant 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 evaluated by comparing the levels to a control (absence of the 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 of the present invention. Therefore, the kit includes the tools necessary for collecting cat urine, cat urine or The container is used to store cat urine-containing sand and sheets, and is used to measure the levels of one or more D-amino acids. and a guide or guidance for comparing D-amino acid levels to standards. 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 a cat, comprising: D-methionine, D-tryptophan, D-ornithine and D-histidine. A method for testing for renal dysfunction, comprising the step of measuring the levels of one or more D-amino acids. <2> In addition, D-phenylalanine, D-glutamic acid, D-leucine, and D-threonine D-Proline, D-Serine, D-Alanine, D-Lysine, D-Arginine, D-Glu leucine, 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-amino acids, D-amino acids, 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. <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 kind of amine, preferably D-phenylalanine, D-glutamic acid, D-threonine, D-proline D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, D-phenylalanine, D-glutamic acid, D-alanine chiral amino acids selected from D-amino acids such as leucine, D-allo-isoleucine and D-valine; measuring the weight balance, <3> How to. <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 A step of measuring the ratio of <1> How to. <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 kind, preferably D-phenylalanine, D-glutamic acid, D-proline, D-threonine, D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, D-phenylalanine, D-glutamic acid, D-proline D-alanine, D-allo-isoleucine and D-valine Measuring the ratio of amino acids to L-amino acids; <5> How to. <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. <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 At least one 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-glutamate 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- 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-valinine One or more D-amino acids selected from D-asparagine, D-aspartic acid, and D-aspartic acid are added. measuring the total concentration of six or more D-amino acids obtained by <1> How to. <10> Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids was calculated. The method includes the steps of: <9> How to. <11> The method further comprises the step of calculating the chiral balance of the total concentration of the D-amino acid. <9> How to. <12> The total concentration of six or more D-amino acids was measured to determine the enzyme reaction using D-amino acids as substrates. This is carried out by a quantitative method using the 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-histine The effect of the intervention is determined by measuring the level of one or more D-amino acids selected from the group consisting of ginseng and ginseng. Evaluation method. <14> In addition, D-phenylalanine, D-glutamic acid, D-leucine, and D-threo D-amino acid, D-proline, D-serine, D-alanine, D-lysine, D-arginine, D-glycine Lutein, D-allo-isoleucine, D-allo-threonine, D-valine, D-asparagus measuring the level of one or more D-amino acids selected from D-gin and D-aspartic acid; <13> How to. <15> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D-amino acid, -One or more D-amino acids selected from tryptophan, D-ornithine and D-histidine measuring the chiral balance of the acid; <13> How to. <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 these, preferably D-phenylalanine, D-glutamic acid, D-threonine, D- From proline, D-alanine, D-glutamine, D-allo-isoleucine and D-valine More preferably, D-phenylalanine, D-glutamic acid, D-alanin D-amino acid quinolone, D-allo-isoleucine, and D-valine. Measuring the relative balance of the <15> How to. <17> The step of measuring the level of the D-amino acid includes measuring the level of D-tyrosine, D-tryptophan, L-amino acids selected from one or more of D-amino acids selected from D-ornithine and D-methionine A step of measuring the ratio of the amount of the acid to the amount of the acid. <13> How to. <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, D-phenylalanine, D-glutamic acid, D-proline One or more D-amino acids 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. <19> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D-amino acid, -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. <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-allo-threonine, D-valine, D-asparagine and D-lo At least one 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-glucose Taminic 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. <21> The step of measuring the level of D-amino acids includes measuring the level of D-tyrosine, D-methionine, D-amino acid, -Total 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 D-aspartic acid. measuring the total concentration of the six or more D-amino acids added; <13> How to. <22> Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids was calculated. The method includes the steps of: <21> How to. <23> The method further comprises the step of calculating the chiral balance of the total concentration of the D-amino acid. <21> How to. <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 cat that can be measured by the enzyme measurement method was 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. <26> Furthermore, the ratio of the total concentration of the D-amino acids to the urinary creatinine concentration was calculated. The method includes the steps of: <24> How to. <27> Furthermore, the subject is subjected to an enzyme assay using L-amino acid oxidase. Measuring the total concentration of L-amino acids in cat urine, <24> How to. <28> The method further comprises the step of calculating the chiral balance of the total concentration of the D-amino acid. <27> How to. <29> Furthermore, the ratio of the total concentration of the D-amino acids to the total concentration of the L-amino acids was calculated. The method includes the steps of: <27> How to. <30> <1> ~ <29> A test kit for carrying out the method of the present invention, A kit for testing cats for kidney dysfunction that contains reagents to measure acid levels. <31> Reagents for measuring D-amino acid levels include D-amino acid oxidase , <30> Kit. <32> Further comprising a colorimetric or fluorescent probe and peroxidase, <31> Noki Yat. EXAMPLES

[0034] The present invention will be described more specifically 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 group with reduced renal function (N = 11, hereafter referred to as the renal disease group) (Table 1) The renal function and urinary components were analyzed. IRIS (International Renal Interest Society) Guide urine, physical supplementary tests (color, urine volume, urine specific gravity, pH) and a total urine volume based on a veterinarian interview. The classification was based on the consensus judgment. The classification of the healthy group and the kidney disease group was based on the IRIS guideline. According to the chart, blood (serum) creatinine level is 1.6 (mg / dL) or higher or symmetric Individuals with methylarginine (SDMA) levels of 18 μg / dL or higher were classified as the kidney disease group. (Equivalent to Stage 2).

[0035] [Table 1]

[0036] (2) Preparation of sample solutions and standards Put 10μL of cat urine (natural urine) into a 10mL spitz glass (product name: reinforced hard screw-top test tube) ), mix with 490 μL of methanol:water (9:1, v / v) solution, and then centrifuge (HIT ACHI / CF5RE) for 5 minutes at 2130 x g (3000 rpm) in a refrigerator (4°C). The protein was removed by centrifugation at 400 rpm, 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: The AQC powder was dissolved in ultra-dehydrated acetonitrile at a concentration of 3 mg / 1 mL, i.e., 10 mmol / L. Mix 70μL, 10μL, and 20μL (7:1:2) of each solution in that order, and stir immediately. 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 / allo-Isoleucine, Leu / Leucine, Lys / Lysine, Met / Methionine nine, Phe / Phenylalanine, Pro / Proline, Ser / Serine, Thr / Threonine nine, allo-Thr / allothreonine, 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) were 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) were 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) were connected in series in this order (40 °C) Eluent: methanol:water containing 0.1% (v / v) formic acid and 55 mM ammonium formate ( 90:10, v / v) solution 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 Ionspray 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) Proton ion adduct molecule ([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 expanded into 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 concentration of chiral amino acids was measured in the disease group (D-amino acids (DAA): D-Pr 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 chiral balance of each amino acid (D isomer abundance) was calculated from the urinary chiral amino acid concentration. The ratio of the number of slits in the slits to the number of slits is shown in Figures 3-1 and 3-2.

[0039] 17 types excluding 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-all The chiral balance of o-Thr (aThr), D-Orn, D-Val) was significant in the renal disease group. The chiral balance of D-His was significantly increased in the kidney disease group. It has been confirmed that they are involved in renal function. Among these, D-Tyr, D-Met, D-Trp, 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 / Ls are shown in Figures 5-1 and 5-2. The total concentration 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 renal 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 total concentration ratio (D / L) of DAA and LAA and the chiral balance calculated from the total concentration were also significantly lower. 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 renal 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 healthy subjects versus those with renal disease were compared. ROC (Receiver Error Characteristic) for the ratio of D-amino acids to L-amino acids (D / L) Draw the 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 for each (Table 2). Ral balance, Table 3: Total concentration, Table 4: D-amino acid concentration, Table 5: D-amino acid and L-amino acid concentration The cut off point 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 results showed that the ratio of L-amino acids (D / L) was significant and had diagnostic potential. Many DAA molecular species with AUCs of 0.90 or higher 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) blood test 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 him with 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-ale, D-Val, etc.) and urinary DAA concentration (total concentration) 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 increased by 100% in the kidney and 100% in the peritoneal cavity (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 the enzyme method (1) About the cat test The subjects were cats in the healthy group (N = 14) and the group with impaired renal function (N = 4, hereafter referred to as the renal disease group) (Table 7) We conducted an analysis of the relationship between renal function and urinary components. As in Test Example 1, urine was collected and then analyzed using the current standard IRIS (International Raman Immunosorbent Assay System). Veterinary Renal Interest Society: International Veterinary Nephrology Research Group The classification was based on a comprehensive judgment based on the guidelines and a veterinarian's interview. pH (compact pH meter LAQUAtwin manufactured by Horiba Ltd.), urine Specific gravity (USG) (PAL dog and cat urine specific gravity meter, manufactured by Atago Co., Ltd.), urinary creatinine concentration (CRE) (ARKRAY, Inc., urine chemistry analyzer thinka RT-4010) Measurements of were also carried out.

[0049] [Table 7]

[0050] (2) Total concentration of D-amino acids (DAA) in cat urine Calibration curve creation and quantitative value calculation D-Amino Acid Assay Kit(MET-5136,Colorim Compliant with Cell Biolabs protocol. -Alanine) was prepared at the time of use at 0, 1.56, 3.13, 6.25, 12.5, and 25 A calibration curve was drawn for 100μM, 50μM, and 100μM to measure the total concentration of D-amino acids (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-top test tube). ), mix with 90μL of the kit's included Assay buffer, and then centrifuge ( HITACHI / CF5RE) for 5 minutes at 2130×g (3000r The protein was removed by centrifugation at 400 rpm, and the amino acids in the supernatant were collected. Add 50 μL of the above prepared standard solution and 50 μL of cat urine to a 96-well microplate (No. 6 The mixture was then added to a 100-ml kit (Greiner Bio-One). Built-in enzyme reaction solution (Colorimetric Probe, HRP, D-Amino A Add 50 μL of the mixture (Cid Oxidase mixture), mix immediately in the dark, and then incubate at 37°C for 1 hour. The mixture was then incubated for 1 h (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 Calibration curve creation and quantitative value calculation L-Amino Acid Assay Kit(MET-5054,Colorim Compliant with Cell Biolabs protocol. -Alanine) prepared at the time of use at 0, 62.5, 125, 250, 500, and 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-top test tube). ) and mix with 990μL of the kit's included Assay buffer, then centrifuge. (HITACHI / CF5RE) for 5 minutes at 2130 × g (3000 The protein was removed by centrifugation at 1000 rpm, and the amino acids in the supernatant were collected. Add 50 μL of the above freshly prepared standard solution and 50 μL of cat urine to a 96-well microplate (No. The mixture was then added to a 100 ml flask (Greiner Bio-One Co., Ltd.). 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 (37 °C). The mixture was incubated at ℃ (L-amino acid oxidase (LAO) reaction). Finally, a microplate reader (SH-9000 Lab, Corona Electric Co., Ltd.) was used. ) was used to measure the total LAA concentration in the enzyme reaction solution (wavelength 570 nm).

[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 / (D-form abundance ratio: total DAA concentration 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 the same 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 (ROC) curve, Cutoff point, area under the ROC curve (AUC), sensitivity The specificity, p-value (diagnostic ability) and p-value were calculated. (Table 8). The cut off point is the closest point to the upper left corner, and the null hypothesis is AUC = 0.5. When the value was set to 0, it was confirmed that the feline urinary DAA-related index had diagnostic ability. The total DAA concentration and its related ratios obtained by the method were found to be particularly useful for evaluating renal function. .

[0054] [Table 8]

Claims

1. A method for testing for renal dysfunction in a cat, comprising the step of measuring the level of one or more D-amino acids selected from D-tyrosine, D-methionine, D-tryptophan and D-ornithine in the urine of a test cat.

2. 2. The method of claim 1, further comprising measuring a level of 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-allo-isoleucine, D-allo-threonine, D-valine, D-asparagine and D-aspartic acid.

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

4. The method according to claim 3, further comprising a step of measuring the chiral balance 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-allo-isoleucine, D-allo-threonine, D-valine and D-leucine.

5. 2. The method according to claim 1, wherein the step of measuring the level of a D-amino acid 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 to an L-amino acid.

6. The method according to claim 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-allo-isoleucine, D-allo-threonine, D-valine and D-leucine to the L-amino acid.

7. 2. The method according to claim 1, wherein the step of measuring the level of a D-amino acid 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.

8. The method according to claim 7, further comprising a step of measuring the concentration 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-allo-isoleucine, D-allo-threonine, D-valine, D-asparagine and D-leucine.

9. A test kit for carrying out the method according to any one of claims 1 to 8, which comprises a reagent for measuring D-amino acid levels and is used to test for renal dysfunction in cats.

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