WO2016199928A1 - 腎臓病の病態バイオマーカー - Google Patents
腎臓病の病態バイオマーカー Download PDFInfo
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- WO2016199928A1 WO2016199928A1 PCT/JP2016/067459 JP2016067459W WO2016199928A1 WO 2016199928 A1 WO2016199928 A1 WO 2016199928A1 JP 2016067459 W JP2016067459 W JP 2016067459W WO 2016199928 A1 WO2016199928 A1 WO 2016199928A1
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- amino acid
- renal disorder
- amount
- index value
- renal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Definitions
- the present invention outputs an analysis method including a step of calculating a disease state index value of renal disorder based on the amount of amino acids of D-form and L-form from stool or intestinal contents, a test method for renal disorder, and pathological information about renal disorder
- the present invention relates to a sample analysis system.
- the kidney is an organ whose main role is to maintain the homeostasis of body fluids by filtering waste products and excess water from the blood and discharging them as urine.
- the kidneys are damaged by factors such as immune system abnormalities, allergies to drugs, high blood pressure, diabetes, bleeding and rapid blood pressure drop, infections, dehydration associated with burns, and kidney function decreases.
- AKI acute renal failure
- CKD chronic renal failure
- Acute renal failure refers to renal failure that takes several hours to several weeks as the time to onset.
- Acute renal failure is a condition in which renal function is rapidly reduced due to causes such as ischemia, drugs, and endotoxin shock, increased blood concentrations of urea metabolites and creatinine, metabolic abnormalities, and acid-cis.
- acute renal failure is diagnosed by a rapid rise in serum creatinine level. Since acute renal failure is expected to be recovered by treatment, development of a diagnostic marker that can distinguish earlier acute renal failure is desired.
- the serum creatinine concentration generally used varies depending on conditions such as age, sex, muscle mass, and medicines to be taken, so it cannot be said to be a specific diagnostic marker (Non-patent Document 1).
- NGAL Sphere gelatinase-related lipocalin
- IL-18 interleukin-18
- KIM-1 kidney injury molecule
- FABPs hepatic fatty acid binding proteins
- cystatin C cystatin C and other proteins
- homovanillic acid sulfate trimethylamine-N-
- Chronic renal insufficiency is caused by various renal insufficiencies that cause a decrease in renal function expressed by glomerular filtration rate, or chronic insufficiency (3 months or more) suggesting renal insufficiency Say.
- Chronic renal failure is a disease that affects 13.3 million people, equivalent to about 13% of the adult population in Japan, has a high risk of reaching end-stage renal failure (ESKD), and threatens the health of the people.
- EKD end-stage renal failure
- There is no effective treatment for chronic renal failure and if chronic renal failure progresses and kidney function declines, there is a risk of uremia, necessitating artificial dialysis and kidney transplantation, which imposes a heavy burden on the medical economy (non-patented) Reference 2).
- Non-patent document 3 Non-patent document 4, Non-patent document 5, Non-patent document 6).
- kidney disease kidney disease
- body fluid such as blood, plasma, urine, feces
- body tissues such as sweat, nasal discharge, body hair, nails, skin tissue, and visceral tissues are mentioned, but there is no indication that the D-amino acid in feces was actually used as a diagnostic marker. Absent.
- a technique for identifying and analyzing diagnostic markers for renal failure that vary from earlier diagnostic markers for existing renal failure, such as glomerular filtration rate and serum creatinine concentration, and thereby accurately determining, testing or detecting early renal failure Development of diagnostic technology is desired.
- the present inventors searched for an early diagnostic marker for renal failure using an experimental animal model of early renal injury that can reproduce early renal failure. Specifically, renal artery / vein clipping was performed to induce ischemic kidney injury in mice. While it can be confirmed that inflammation has occurred in the kidney tissue of this ischemic nephropathy-induced mouse (FIGS. 1 and 2), serum creatinine concentration, KIM-1, An animal model of mild kidney injury in which NGAL does not change was created. In such a mild kidney injury animal model, it was found that the D-amino acid in feces was changed (FIGS. 3 and 4). It was confirmed that the D-amino acid change in stool found in this way was associated with renal damage, and the present invention was achieved.
- the present inventors measured the D-form and the L-form for the amino acids in stool in the chronic renal failure patient group and the healthy subject group.
- the present inventors found that such chiral amino acid varies significantly, and led to the present invention.
- the present invention is a method for analyzing feces or intestinal contents, Measuring the amount of D-form and / or L-form of at least one amino acid; Calculating a disease state index value of renal disorder based on the amount of D-form, L-form, or D-form and L-form of at least one amino acid; It is related with the analysis method containing.
- a method for examining renal injury using stool or intestinal contents as a sample Measuring the amount of D-form and / or L-form of at least one amino acid; Calculating a disease state index value of renal disorder based on the amount of D-form, L-form, or D-form and L-form of at least one amino acid;
- the test method further includes a step of associating a pathological index value of renal disorder with a pathological condition of renal disorder.
- Such an inspection method can determine the pathological condition of renal disorder based on a predetermined threshold value of the pathological condition index value.
- the present invention relates to a sample analysis system capable of performing the analysis method of the present invention.
- a sample analysis system includes a storage unit, an input unit, an analysis measurement unit, a data processing unit, and a pathological information output unit, analyzes a sample of stool or intestinal contents, and stores pathological information. Can be output.
- the present invention relates to a program that can be installed in the sample analysis system of the present invention and a storage medium that stores the program.
- the amount of D-amino acid in the sample, the amount of L-form, or the pathological index value calculated based on the amount of D-amino acid and L-amino acid is the value of known kidneys such as blood creatinine Renal failure
- the failure marker there is a possibility that it can be used as a diagnostic marker capable of detecting renal failure earlier than known markers such as blood creatinine.
- feces or intestinal contents used as a sample can be easily acquired non-invasively.
- it since it is a diagnostic marker that can detect early renal failure and it is a sample that can be easily obtained, by adopting the analysis method of the present invention in diagnosis, early diagnosis of renal failure and / or kidney damage Discovery becomes possible.
- FIG. 1A is a photograph showing necrotic cells stained by PAM staining of kidneys of mice in the renal injury induction group.
- FIG. 1B is a graph in which the proportion of necrotic cells is measured in the diagram of FIG. 1A and compared with the control group.
- FIG. 2A is a photograph showing stained inflammatory cells (macrophages and monocytes) by F4 / 80 staining of the kidneys of mice in the renal injury induction group.
- FIG. 2B is a graph comparing the ratio of inflammatory cells in the diagram of FIG. 2A with the control group.
- FIG. 3A is a graph showing changes in D amino acid + L amino acid in stool collected from mice in the control group (Sham) and mice on day 2 and day 10 after induction of renal injury.
- FIG. 1A is a photograph showing necrotic cells stained by PAM staining of kidneys of mice in the renal injury induction group.
- FIG. 1B is a graph in which the proportion of necrotic cells is measured in
- FIG. 3B is a graph showing changes in D amino acids in feces collected from mice in the control group and mice on day 2 and day 10 after induction of renal injury.
- FIG. 3C is a graph showing changes in L amino acids in stool collected from mice in the control group (Sham) and mice on day 2 and day 10 after induction of renal injury.
- FIG. 3D is a graph showing changes in the ratio of D amino acid / L amino acid in feces collected from mice in the control group and mice on day 2 and day 10 after induction of renal injury.
- FIG. 3E is a graph showing changes in the ratio of D amino acid / (D amino acid + L amino acid) in feces collected from mice in the control group (Sham) and mice on day 2 and day 10 after induction of renal injury. is there.
- FIG. 3F shows the evaluation results of statistical significance between the data shown in FIGS. 3A to 3E.
- “ ⁇ LOQ” indicates that it is below the limit of quantification.
- FIG. 4 shows (A) D amino acids for serine, arginine, and alanine in feces collected from mice in the control group (Sham) and mice on day 2 and day 10 after induction of renal injury. It is a graph which shows the fluctuation
- FIG. 5A is a photograph showing a comparison of necrotic cells by performing PAM staining on the kidneys of wild group, sterile group, and inoculated group mice in kidney injury-induced mice.
- FIG. 5B is a graph in which the proportion of necrotic cells was measured and compared in the diagram of FIG. 5A.
- FIG. 6A is a photograph showing comparison of inflammatory cells (macrophages and monocytes) by F4 / 80 staining of kidneys of wild group, sterile group, and inoculated group in kidney-induced mice. It is.
- FIG. 6B is a graph comparing the ratio of inflammatory cells measured in the diagram of FIG. 6A.
- FIG. 7A is a diagram obtained by subjecting the gut microbiota DNA on day 2 and day 10 of mice in the renal injury induction group to electrophoresis after fragmentation.
- FIG. 7B is a diagram comparing the types and number of enteric bacteria on the 2nd and 10th day after surgery of mice in the renal injury induction group.
- FIG. 8 is a configuration diagram of the sample analysis system of the present invention.
- 9A and 9B are flowcharts showing an example of an operation for determining a disease state index value.
- FIGS. 9C to 9D are flowcharts showing an example of an operation for determining a disease state index value and / or disease state information.
- FIG. 10A is a graph showing the amount of D amino acids + L amino acids (total amino acids) in feces collected from healthy subjects and patients with chronic kidney injury.
- FIG. 10B is a graph showing the amount of L amino acids in feces collected from healthy subjects and patients with chronic kidney injury.
- FIG. 10C is a graph showing the amount of D amino acid in stool collected from healthy subjects and patients with chronic kidney injury.
- FIG. 10D is a graph showing the ratio of D amino acid / L amino acid in stool collected from healthy subjects and patients with chronic kidney injury.
- FIG. 11 is a table showing the correlation between the estimated glomerular filtration rate (eGFR) and the amount of each amino acid in stool in patients with chronic kidney injury.
- eGFR estimated glomerular filtration rate
- FIG. 12 is a graph showing the amount of L-Lys in chronic kidney injury patients and healthy subjects (A), a graph showing ROC curves in chronic kidney injury patients and healthy subjects (B), and estimation in chronic kidney injury patients.
- a scatter diagram (C) of glomerular filtration rate (eGFR) and L-Lys amount and a scatter diagram (D) of serum creatinine and L-Lys amount in patients with chronic kidney injury are shown.
- FIG. 13 is a graph showing the amount of D-Lys in chronic kidney injury patients and healthy subjects (A), a graph showing ROC curves in chronic kidney injury patients and healthy subjects (B), and estimation in chronic kidney injury patients.
- a scatter diagram (G) of glomerular filtration rate (eGFR) and D-Lys amount, and a scatter diagram (D) of serum creatinine and D-Lys amount in patients with chronic kidney injury are shown.
- A is a graph showing the amount of D-Lys / L-Lys ratio in chronic kidney injury patients and healthy subjects
- B a graph showing ROC curves in chronic kidney injury patients and healthy subjects
- C Scatter plot of estimated glomerular filtration rate and D-Lys / L-Lys ratio in impaired patients
- C scatter plot of serum creatinine and D-Lys / L-Lys ratio in chronic renal disorder patients
- the method for analyzing stool or intestinal contents includes the following: Measuring the amount of D-form and / or L-form of at least one amino acid; Calculating a disease state index value of renal disorder based on the amount of D-form, L-form, or D-form and L-form of at least one amino acid; including.
- the analysis method can provide data for a doctor to make a diagnosis, and can also be called a preliminary method of diagnosis.
- This analysis method may further include a step of associating a pathological index value of a renal disorder with a pathological condition of the renal disorder.
- Such an analysis method may be performed by an analysis company or an analysis engineer to provide a result associated with a pathological condition of renal disorder.
- the sample analyzed in the present invention is feces or intestinal contents. Therefore, it may be excreted feces, or may be feces staying in the intestines.
- the intestinal contents can be collected by an intestinal catheter.
- intestinal bacterial flora may be directly collected.
- stool collected by stool it is preferable to use stool collected by stool as a sample.
- the collected sample may be used for analysis immediately after being dissolved or mixed in a solvent such as water, methanol, ethanol, acetonitrile, sodium borate aqueous solution, trifluoroacetic acid, or stored at room temperature or under refrigeration. It may be used later for analysis.
- the sample After dissolving the sample in the solvent, the sample may be subjected to analysis after removing insoluble matter by performing filtration and / or precipitation one or more times.
- the amount of amino acids per unit sample mass can be measured.
- the mass of stool or intestinal contents provided to the sample cannot be measured, after dissolving the sample in the solvent, further dilute by adding a solvent so that the amount of the sample becomes constant based on absorbance etc. be able to.
- the amount of amino acid in the present invention may be expressed by mass or mole, or may be expressed by concentration.
- the amino acid used as a renal failure marker is an amino acid having a stereoisomer. More preferred are ⁇ -amino acids, and even more preferred are protein-constituting amino acids. Such amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine.
- stereoisomers of L-isoleucine include D-isoleucine, D-allo-isoleucine, and L-allo-isoleucine.
- stereoisomers of L-threonine include D-threonine, D-allo-threonine, and L-allo-threonine.
- D-form uses at least one amino acid selected from D-amino acid, D-aromatic amino acid, and L-allo-amino acid in addition to D-amino acid.
- the amount of D-form amino acid, the amount of L-form amino acid, or the amount of D-form and L-form amino acids is measured, and the pathological condition index value of renal disorder is calculated based on the measured value.
- amino acids selected from the group consisting of The above-mentioned amino acids are amino acids selected using an experimental animal model of early kidney injury, and can be a marker for early kidney injury in humans.
- histidine, asparagine, arginine, aspartic acid, arosleonine, alanine, proline, methionine, valine, alloisoleucine it is preferable to use at least one amino acid selected from the group consisting of isoleucine, leucine, phenylalanine, lysine, and tyrosine.
- Histidine, asparagine, arginine, aspartic acid, arosleonine, alanine, proline, valine, alloisoleucine, leucine, phenylalanine, and lysine are more effective from the viewpoint of obtaining highly sensitive pathologic index values in human renal disorders, particularly chronic renal disorders.
- histidine, arosleonine, and lysine are used.
- Serine, aspartic acid, glutamic acid, alanine, arginine, lysine and proline are used from the viewpoint of obtaining a pathological index value of renal damage caused by acute stage of renal damage or ischemia.
- D + L-amino acid means the sum of D-form and L-form of amino acid
- D / L-amino acid means the ratio of D-form to L-form.
- the measurement of the amount of D-amino acid in a sample in the present invention may be performed using any method known to those skilled in the art.
- o-phthalaldehyde OPA
- Boc-L-Cys N-tert-butyloxycarbonyl-L-cysteine
- a method of separating a mixture of 100 mM acetate buffer (pH 6.0) and acetonitrile by gradient elution using an analytical column such as ODS-80TsQA is a method for simultaneous measurement of D-form and L-form of amino acids. Can be used.
- D- and L-amino acids are derivatized with a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in advance, and then ODS-80TsQA, Mightysil RP.
- a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in advance, and then ODS-80TsQA, Mightysil RP.
- NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole
- ODS-80TsQA Mightysil RP.
- the optical resolution column system in the present specification refers to a separation analysis system using at least an optical resolution column, and may include a separation analysis using an analysis column other than the optical resolution column. More specifically, passing a sample containing components having optical isomers along with a first liquid as a mobile phase through a first column filler as a stationary phase to separate the components of the sample; Individually holding each of the components of the sample in a multi-loop unit, each of the components of the sample individually held in the multi-loop unit as a stationary phase together with a second liquid as a mobile phase Supplying the second column packing material having an optically active center through a flow path to split the optical isomers contained in each of the sample components, and the optical isomerism contained in each of the sample components
- the D- / L-amino acid concentration in the sample can be measured by using an optical isomer analysis method characterized by including a step of detecting a body ( Patent No.
- D-amino acids can be quantified by immunological techniques using monoclonal antibodies that identify optical isomers of amino acids, such as monoclonal antibodies that specifically bind to D-leucine, D-aspartic acid, etc. (Japanese Patent Application No. 2008-27650).
- renal disorder refers to all disorders that occur in the kidney, and includes not only renal failure but also changes that have not led to renal failure.
- Renal failure is a state in which renal function is lower than normal, and includes all kidney disorders that are used in the normal sense.
- renal failure refers to a state in which renal function falls below 30% of normal, and is roughly classified into acute renal failure and chronic renal failure.
- causes of decreased renal function include multiple factors such as immune system abnormalities, allergies to drugs, high blood pressure, diabetes, bleeding, rapid blood pressure decrease, infection, dehydration associated with burns.
- Acute renal failure (AKI) has been proposed for stage classification such as RIFLE classification, AKIN classification, and KDIGO classification.
- Acute renal failure is classified as Risk (stage 1), Injury (stage 2), Failure (stage 3), Furthermore, it classified into Loss and End stage kidney disease according to the duration. All of these classifications are based on the amount of serum creatinine and the amount of urine. For example, in Risk (stage 1), serum creatinine increases 1.5 to 2.0 times from the baseline or 0.5 ml. urine volume of less than 6 hours / kg / hour is 6 hours or more.
- stage 2 serum creatinine increases 2.0 to 3.0 times from baseline or less than 0.5 ml / kg / hour Urine volume is 12 hours or more, and in Failure (Stage 3), serum creatinine increases 3.0 times or more from baseline, or urine volume of less than 0.3 ml / kg / hour is 24 hours or more. It is based on judgment. On the other hand, these classifications make it possible to classify acute renal failure more accurately by using other indicators, for example, the amount of change in GFR together.
- Chronic renal failure is a diagnostic criterion for stage 1 (with renal impairment but normal renal function, eGFR ⁇ 90) to stage 5 (renal failure, eGFR ⁇ 15) according to the Japanese Society of Nephrology guidelines (2009) It is shown.
- the estimated glomerular filtration rate (eGFR) which is an index here, is calculated from the serum creatinine level, age, and sex, and indicates the ability of the kidneys to discharge waste products into the urine.
- eGFR which is an index here, is calculated from the serum creatinine level, age, and sex, and indicates the ability of the kidneys to discharge waste products into the urine.
- amino acids selected using an experimental animal model of early kidney injury can be a marker for earlier kidney injury in humans.
- the pathological index value of renal disorder is calculated based on the amount of amino acid D-form and / or L-form.
- the disease state index value can be calculated based on the amounts of D-form and L-form amino acids.
- the pathological condition index value may be the amount of each individual biomarker molecule or a numerical value that can be calculated based on the amount of a plurality of biomarkers.
- the pathological index value used in the present specification may be the amount of D form of a certain amino acid, the amount of L form, or the ratio of the amount of a certain amino acid and its stereoisomer, for example, The ratio of the amount of D-form to the amount of L-form of a certain amino acid, the ratio of the amount of L-form to the amount of D-form of a certain amino acid, and the ratio of the amount of D-form to the sum of the amount of D-form and L-form There may be.
- pathological index values can be calculated by adding, subtracting, or integrating any constant or any variable that can affect the amount of chiral amino acids, such as age, weight, sex, BMI, and eGFR And / or division.
- a pathological index value of a subject, and a renal disorder can be associated with
- the disease state index reference value may be a threshold value. If it is an expert, a threshold value can be suitably set from the pathological condition index value of a healthy subject group or a renal failure patient group.
- a threshold value for example, an average value, a median value, and an X percentile value of a healthy person group or a renal failure patient group can be used, but the threshold value is not limited thereto.
- the threshold may be one, type of renal failure (acute, chronic) and cause (for example, drug nephropathy, diabetic nephropathy, IgA nephropathy, membranous nephropathy, nephrosclerosis, etc.), A plurality of conditions can be set according to the state (early, intermediate, late), the amino acid to be used or a combination thereof, and the pathological condition can be classified according to the severity.
- the threshold cutoff value
- the threshold can be determined by performing ROC analysis between a group of healthy subjects and a group of patients with renal impairment at each stage.
- the present invention relates to a method for examining renal injury using stool or intestinal contents as a sample.
- This inspection method includes the following steps: Measuring the amount of D-form and / or L-form of at least one amino acid of protein constituent amino acids, Calculating a disease state index value of renal disorder based on the amount of D-form, L-form, or D-form and L-form of at least one amino acid;
- the method further includes the step of associating a pathological condition index value of renal disorder with a pathological condition of renal disorder.
- the test method of the present invention may be performed by an analysis company or an analysis engineer, and a result associated with a pathological condition of renal disorder may be provided.
- the sample used in the test method, the pathology of the associated renal disorder, and each step performed may be the same as defined in the analytical method.
- FIG. 8 is a configuration diagram of the sample analysis system of the present invention.
- the sample analysis system 10 of the present invention shown in FIG. 8 is configured so that the analysis method and the inspection method of the present invention can be performed.
- Such a sample analysis system 10 includes a storage unit 11, an input unit 12, an analysis measurement unit 13, a data processing unit 14, and an output unit 15, and analyzes a sample of stool or intestinal contents, Pathological information can be output.
- the storage unit 11 stores a threshold value of a disease state index value for determining a renal disorder input from the input unit 12,
- the analytical measurement unit 13 separates and quantifies an amino acid stereoisomer of at least one amino acid of protein constituent amino acids in the feces or intestinal contents of the subject,
- the data processing unit 14 calculates a disease state index value of renal disorder based on the amount of D-form, L-form, or D-form and L-form of the at least one amino acid,
- the data processing unit 14 determines the pathological information of the renal disorder by comparing with the threshold value stored in the storage unit 11,
- the output unit 15 can output pathological information regarding the renal disorder of the subject.
- the storage unit 11 includes a memory device such as a RAM, a ROM, and a flash memory, a fixed disk device such as a hard disk drive, or a portable storage device such as a flexible disk and an optical disk.
- the storage unit stores data measured by the analysis measurement unit, data and instructions input from the input unit, calculation processing results performed by the data processing unit, etc., as well as computer programs and databases used for various processes of the information processing apparatus.
- the computer program may be installed via a computer-readable recording medium such as a CD-ROM or DVD-ROM, or via the Internet.
- the computer program is installed in the storage unit using a known setup program or the like.
- the input unit 12 is an interface or the like, and includes operation units such as a keyboard and a mouse. As a result, the input unit can input data measured by the analysis measurement unit 13, instructions for calculation processing performed by the data processing unit 14, and the like. For example, when the analysis measurement unit 13 is outside, the input unit 12 may include an interface unit that can input measured data or the like via a network or a storage medium, separately from the operation unit.
- the analytical measurement unit 13 performs a process of measuring the amounts of D-form and L-form of amino acids in a sample of feces or intestinal contents suspended in a solvent. Therefore, the analytical measurement unit 13 has a configuration that enables separation and measurement of D-form and L-form of amino acids.
- Amino acids may be analyzed one by one, but some or all types of amino acids can be analyzed together.
- the analytical measurement unit 13 is not intended to be limited to the following, but may be a high performance liquid chromatography system including a sample introduction unit, an optical resolution column, and a detection unit, for example.
- the analysis measurement unit 13 may be configured separately from the sample analysis system, and the measured data or the like may be input via the input unit 12 using a network or a storage medium.
- the data processing unit 14 calculates the pathological index value of the renal disorder from the measured D-form of each amino acid, or the amount of D-form and L-form, and compares it with the threshold value stored in the storage unit 11. Configured to determine renal impairment.
- the data processing unit 14 performs various arithmetic processes on the data measured by the analysis measurement unit 13 and stored in the storage unit 11 in accordance with a program stored in the storage unit. Arithmetic processing is performed by a CPU included in the data processing unit.
- the CPU includes a functional module that controls the analysis measurement unit 13, the input unit 12, the storage unit 11, and the output unit 15, and can perform various controls. Each of these units may be configured by an independent integrated circuit, a microprocessor, firmware, and the like.
- the output unit 15 is configured to output a pathological condition index value and / or pathological condition information, which is a result of performing arithmetic processing in the data processing unit.
- the output unit 15 may be a display device such as a liquid crystal display that directly displays the result of the arithmetic processing, an output unit such as a printer, or an interface unit for outputting to an external storage device or via a network. There may be.
- FIGS. 9A and 9B are flowcharts showing an example of an operation for determining a pathological condition index value according to the program of the present invention.
- FIGS. It is a flowchart which shows the example of this operation
- the program of the present invention is a program that causes an information processing apparatus including an input unit, an output unit, a data processing unit, and a storage unit to determine a pathological condition index value and / or pathological condition information.
- the program of the present invention is as follows:
- the storage unit stores the value of the D-form amount, the L-form amount, or the D-form and L-form amounts of at least one amino acid input from the input unit, Based on the value stored in the storage unit, the data processing unit calculates a disease state index value, A command for causing the information processing apparatus to store the calculated pathologic index value in the storage unit and cause the output unit to output the stored pathologic index value;
- the program of the present invention may be stored in a storage medium or provided via an electric communication line such as the Internet or a LAN.
- the analysis measurement unit may include a command for causing the information processing apparatus to measure the value from the stool sample and store the value in the storage unit.
- a command for causing the information processing apparatus to store the determined disease state information in the storage unit and cause the output unit to output the stored disease state information may be included.
- subjects are not limited to humans, and may include laboratory animals such as mice, rats, rabbits, dogs, monkeys, and the like.
- the subject may be represented as a subject.
- the analytical method of the present invention can be used to collect preliminary data for a diagnostic method for chronic and / or acute renal failure.
- a doctor can diagnose chronic and / or acute renal failure using such preliminary data, but such an analysis method may be performed by a medical assistant who is not a doctor, or by an analysis organization or the like. You can also. Therefore, it can be said that the analysis method of the present invention is a preliminary method of diagnosis.
- a treatment policy can be further determined.
- further treatment such as lifestyle improvement, dietary guidance, blood pressure management, blood glucose level management, lipid management is necessary It becomes.
- lifestyle improvement such as lifestyle improvement, dietary guidance, blood pressure management, blood glucose level management, lipid management
- smoking cessation or exercise for lowering the BMI level is recommended as an improvement in lifestyle habits.
- dietary guidance a reduction in salt intake is recommended for the purpose of improving hypertension.
- blood pressure management administration of an ACE inhibitor or ARB is recommended for the purpose of improving hypertension.
- insulin administration is recommended for the purpose of lowering HbA1c.
- administration of hyperlipidemic drugs is recommended for the purpose of lowering LDL cholesterol.
- the present invention relates to a method for treating a renal disorder, comprising performing the analysis method and the test method of the present invention and further treating the renal disorder.
- FIG. 3B shows the analysis result of the change in D-amino acid content in stool.
- Serine, aspartic acid, glutamic acid, alanine and proline showed significant changes in the D-amino acid content in the renal injury induction group as compared with the control group (Sham).
- serine and proline were higher on both the second day and the tenth day after the operation than on the control group (Sham), and higher on the tenth day than on the second day.
- Aspartic acid and alanine decreased on the second day after the operation compared to the control group (Sham), but were higher than the control group on the tenth day.
- Glutamic acid increased on the 10th day, although no change was seen on the 2nd day.
- serine, aspartic acid, alanine, and proline differ on the second day of the renal failure extreme phase, and can function as detection markers for mild renal injury.
- serine and proline show an increase in D-form on the second day, so that when these D-amino acids are higher than the threshold in feces or intestinal contents, mild renal disorder and It becomes possible to judge.
- aspartic acid and alanine decreased on the 2nd day of the renal failure extreme phase, so when these D-amino acids in stool or intestinal contents were lower than the threshold, it was judged as mild renal injury. It becomes possible to do.
- FIG. 3D shows the analysis result of the change in D -amino acid / L-amino acid ratio in stool.
- Serine, arginine, and aspartic acid showed significant changes in the D-amino acid / L-amino acid ratio in the renal injury induction group as compared to the control group (Sham). Of these, serine and arginine were higher on both the second and tenth days after surgery than on the control group, and higher on the tenth day than on the second day.
- aspartic acid was lower on both the second day and the tenth day after the operation compared to the control group (Sham), and there was almost no difference between the second day and the tenth day.
- Serine and Arginine the D-form / L-form ratio increased on the second day. If it is high, it can be determined as mild kidney damage.
- aspartic acid decreased on the second day of the kidney damage extreme phase, and when these D-amino acids are lower than the threshold in feces or intestinal contents, it is determined that the kidney damage is mild. Is possible. However, there is a possibility that the presence or absence of a significant difference may be changed by increasing the number of parameters.
- FIG. 3E shows the analysis result of the change in the D -amino acid / (D-amino acid + L-amino acid) ratio in feces.
- FIGS. 3A-3E were evaluated. The results are shown in the table of FIG. 3F.
- FIG. 4A shows the results of analysis of changes in the amount of D-amino acid, D / L amino acid ratio, and D / (D + L) amino acid ratio in stool for serine, arginine, and alanine, which are considered to be particularly useful as markers. Shown in (C). For reference, FIG. 4 (D) shows changes in D form in blood for these amino acids.
- mice subjected to ischemia reperfusion are a wild group bred under normal conditions, a sterile group bred under aseptic conditions, and After being raised under aseptic conditions, it was divided into inoculated groups. Mice raised under normal conditions and subjected to sham surgery were used as a control group. Two days after induction of ischemic nephropathy, mice were sacrificed and kidney tissue was collected. The collected kidney tissue was fixed. Sections were obtained from the fixed kidney tissue and subjected to PAS staining. Another section was F4 / 80 stained. The results of PAS staining are shown in FIG.
- the proportion of necrotic cells was evaluated based on the staining result of FIG. 5A. The result is shown in FIG. The results of F4 / 80 staining are shown in the graph of FIG. The proportion of necrotic cells was evaluated based on the staining result in FIG. The result is shown in the graph of FIG.
- FIG. 7A shows the result of electrophoresis performed on the band after PCR.
- the type and number of bacteria contained were determined by culturing the intestinal flora obtained on the 2nd and 10th day after the operation (FIG. 7B).
- Example 2 Materials and methods Research Ethics The present inventors obtained fecal samples and blood samples of 8 patients with chronic renal failure and 3 healthy individuals at Kanazawa University Hospital. For these subjects, informed consent was obtained, and experiments were conducted according to a test approved by the Kanazawa University Medical Ethics Review Committee.
- Amino acid stereoisomers obtained by dissolving feces collected from 8 patients with chronic renal failure and 3 healthy individuals in methanol and using the D, L-amino acid simultaneous high-sensitivity analysis system (Patent No. 4291628) developed by Zaitsu et al. It used for the analysis system. Details of the analysis conditions for each amino acid are described in Miyoshi Y. et al. , Et al. Chromatogr. B, 879: 3184 (2011) and Sasabe, J. et al. Et al., Proc. Natl. Acad. Sci. U. S. A. 109: 627 (2012).
- fecal amino acids were derivatized with NBD-F (4-fluoro-7-nitro-2,1,3-benzoxadiazole, Tokyo Chemical Industry Co., Ltd.) and HPLC system (NANOSPACE SI- 2. See supplementary information of Shiseido Co., Ltd.).
- NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole
- HPLC system NANOSPACE SI- 2. See supplementary information of Shiseido Co., Ltd.
- an in-house manufactured monolithic ODS column (inner diameter 0.53 mm ⁇ 500 mm) was used as the analytical column for reverse phase separation. Fluorescence detection was performed with an excitation wavelength of 470 nm and a detection wavelength of 530 nm. After reverse phase separation, it was transferred to an optical resolution column.
- the estimated glomerular filtration rate (eGFR) was determined based on the measured serum creatinine value and age.
- the formula for determining eGFR is as follows: ⁇ Wherein the unit of age is years, the unit of SCr is mg / dL, and the unit of glomerular filtration rate (GFR) is mL / min / 1.73 m 2 body surface ⁇ . For female patients, a correction factor of 0.739 was applied to the calculated value of the mathematical formula.
- the correlation coefficient between the subject's eGFR value and the total amount of D-amino acid and L-amino acid, L-amino acid amount, D-amino acid amount, and D amino acid / L amino acid ratio was measured for each of the 20 types of amino acids. . The result is shown in FIG.
- D amino acid / L amino acid ratio a high correlation was observed in histidine and lysine (
- L-lysine amount (L-Lys), D-lysine amount (D-Lys), and D-lysine / L-lysine (D / L-Lys) ratio are plotted separately for healthy subjects and patients with chronic renal failure.
- ROC analysis was performed based on the L-lysine amount, D-lysine amount, and D-lysine / L-lysine ratio in healthy subjects and patients with chronic renal failure, and ROC curves were obtained (FIGS. 12B to 14B).
- the cut-off value can be calculated based on the ROC curve. For L-Lys, 781.4 nmol / g, for D-Lys, 24.2 nmol / g, for D / L-Lys, cut 24.2
- nmol / g for D / L-Lys
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Abstract
Description
少なくとも1のアミノ酸のD体及び/又はL体の量を測定する工程、
前記少なくとも1種類のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出する工程、
を含む、分析方法に関する。
少なくとも1のアミノ酸のD体及び/又はL体の量を測定する工程、
前記少なくとも1種類のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出する工程、
腎障害の病態指標値と、腎障害の病態とを関連づける工程
をさらに含む、前記検査方法に関する。かかる検査方法は、予め決められた病態指標値の閾値に基づいて腎障害の病態を決定することができる。
少なくとも1のアミノ酸のD体及び/又はL体の量を測定する工程、
前記少なくとも1種類のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出する工程、
を含む。当該分析方法は、医師が診断を行うためのデータの提供を行うことができ、診断の予備的方法ということもできる。この分析方法は、腎障害の病態指標値と、腎障害の病態とを関連づける工程をさらに含んでいてもよい。このような分析方法は、分析会社や分析技術者により行われて、腎障害の病態と関連づけられた結果が提供されてもよい。
タンパク質構成アミノ酸のうちの少なくとも1のアミノ酸のD体及び/又はL体の量を測定する工程、
前記少なくとも1種類のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出する工程、
腎障害の病態指標値と、腎障害の病態とを関連づける工程をさらに含む。本発明の検査方法は、分析会社や分析技術者により行われて、腎障害の病態と関連づけられた結果が提供されてもよい。検査方法において用いられる試料、関連づけられる腎障害の病態、行われる各工程は、分析方法において定義されたものと同じであってよい。
記憶部11は、入力部12から入力された腎障害を判別するための病態指標値の閾値を記憶し、
分析測定部13は、前記被検体の糞便又は腸内容物中のタンパク質構成アミノ酸のうちの少なくとも1のアミノ酸のアミノ酸立体異性体を分離定量し、
データ処理部14は、前記少なくとも1のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出し、
データ処理部14は、記憶部11に記憶された閾値と比較することにより、腎障害の病態情報を判別し、
出力部15が被検体の腎障害についての病態情報を出力することができる。
具体的に、本発明のプログラムは、入力部、出力部、データ処理部、記憶部とを含む情報処理装置に病態指標値及び/又は病態情報を決定させるプログラムである。本発明のプログラムは、以下の:
入力部から入力された少なくとも1のアミノ酸のD体の量、L体の量、又はD体及びL体の量の値を記憶部に記憶させ、
記憶部に記憶された値に基づきデータ処理部に病態指標値を算出させ、
算出された病態指標値を記憶部に記憶させ、そして
記憶された病態指標値を出力部に出力させる
ことを前記情報処理装置に実行させるための指令を含む。本発明のプログラムは、記憶媒体に格納されてもよいし、インターネット又はLAN等の電気通信回線を介して提供されてもよい。
記憶された病態指標値と、予め記憶部に記憶された閾値とをデータ処理部に比較させることで、腎障害の病態を判別させ、
判別された病態情報を記憶部に記憶させ、そして
記憶された病態情報を出力部に出力させる
ことを情報処理装置に実行させるための指令を含んでもよい。
研究倫理
全ての実験は施設のガイドラインに従って実施され、該施設の動物実験委員会の承認を得て行われた。
アミノ酸のエナンチオマー及びHPLC級のアセトニトリルはナカライテスク(京都)から購入された。HPLC級のメタノール、トリフルオロ酢酸、ホウ酸等は和光純薬(大阪)から購入された。水はMill-QグラジエントA10システムを用いて精製された。
6週齢のC57BL/6JJic雄マウス(日本クレア)に、麻酔下で皮膚及び腹膜を切開した。腎動脈及び静脈クリッピングを40分間行い、その間37℃のヒートプレート上で保温した。クリッピングを外し、腹膜及び皮膚を縫合した。腎動脈及び静脈クリッピングにより、虚血性腎障害が誘導された。虚血性腎障害の誘導に供した腎障害誘導群と、偽手術のみを行った対照群を実験に用いた。
虚血性腎障害の誘導後2日目に、マウスを屠殺し、腎臓組織を回収した。回収した腎臓組織に対し、固定処理を行った。固定された腎臓組織から切片を取得し、PAS染色を行った。さらに別の切片に対しF4/80染色を行った。PAS染色結果を図1(A)に示す。図1Aの染色結果に基づいて壊死細胞の割合を評価した。その結果を図1(B)のグラフに示す。F4/80染色結果を図2(A)に示す。図2(A)の染色結果に基づいて壊死細胞の割合を評価した。その結果を図2(B)のグラフに示す。
虚血性腎障害の誘導後2日目に、マウスから採血及び採尿を行った。採血された血液試料をベクトン・ディッキンソン(BD)マイクロテーナー中で1500×g、10分間遠心により血清を分離した。血清クレアチニンは、FujiDRI-CHEM4000システム(富士フィルム、東京)を用いて測定された。次に、尿中のKIM-1及びNGALを、R&Dシステムズ社製のマウスELISAキットを用いて定量した。
虚血性腎障害の誘導後2日目、及び10日目の対照群(Sham)及び腎障害誘導群のマウスの大腸から採取された糞便をメタノールに溶解し、財津らが開発したD、L-アミノ酸一斉高感度分析システム(特許第4291628号)によるアミノ酸立体異性体分析系に供した。各アミノ酸の分析条件の詳細は、MiyoshiY.、ら、J.Chromatogr.B, 879:3184(2011)及びSasabe,J.ら、Proc.Natl.Acad.Sci.U.S.A.、109:627(2012)に説明される。簡潔には、糞便中のアミノ酸は、NBD-F(4-フルオロ-7-ニトロ-2,1,3-ベンゾオキサジアゾール、東京化成工業株式会社)で誘導体化され、HPLCシステム(NANOSPACE SI-2、株式会社資生堂、の補足情報を参照せよ。)に供された。簡潔には、逆相分離用分析カラムは、自社製のモノリシックODSカラム(内径0.53mm×500mm)が用いられた。蛍光検出は、励起波長470nm、検出波長530nmで実行された。逆相分離の後、光学分割カラムに移された。エナンチオマー分離には、キラルセレクターとして(S)-ナフチルグリシンを用いるスミキラルOA-2500Sカラム(1.5mm×250mm、自家充填、材料は株式会社住化分析センター製)が使用された。各アミノ酸についての、総アミノ酸量、D-アミノ酸量、L-アミノ酸量、D/Lアミノ酸比、D/(D+L)アミノ酸比を、それぞれ図3A~Eに示す。
本明細書及び図面に記載の全ての数値は、平均±標本平均の標準誤差(SEM)で表示される。実験の統計学的解析には、ステューデントのt両側検定、一元配置分散分析(one way ANOVA)、テューキーの多重比較検定(Tukey’s multiple comparison test)等の統計学的手法が利用された。また、これらの検定におけるP値が0.05未満のとき、有意性があると評価された。全ての解析はPrism5(GraphPad Software、カリフォルニア州、ラホヤ)が用いられた。
D-アミノ酸量の変化
糞便におけるD-アミノ酸量の変化についての分析結果を図3Bに示す。対照群(Sham)に比較して、腎障害誘導群でD-アミノ酸含量の有意な変化が見られたのは、セリン、アスパラギン酸、グルタミン酸、アラニン及びプロリンであった。このうち、セリンとプロリンでは、対照群(Sham)と比較して、術後2日目及び10日目の両方で高く、2日目より10日目の方が高かった。アスパラギン酸とアラニンでは、対照群(Sham)と比較して、術後2日目で低下するものの、10日目では対照群よりも高かった。グルタミン酸では、2日目では変化が見られないものの、10日目で増加した。変化のパターンは一定ではないものの、セリン、アスパラギン酸、アラニン、プロリンについては、腎障害極期の2日目で差が出ており、軽度の腎障害の検出マーカーとして機能しうる。より詳細に解析すると、セリン及びプロリンでは、2日目でD体が増加していることから、糞便又は腸内容物においてこれらのD-アミノ酸が、閾値よりも高い場合に、軽度の腎障害と判定することが可能になる。また、アスパラギン酸及びアラニンでは、腎障害極期の2日目で減少していることから、糞便又は腸内容物においてこれらのD-アミノ酸が、閾値よりも低い場合に、軽度の腎障害と判定することが可能になる。しかしながら、母数を増やすことで、有意差の有無が変化する可能性があり、その場合、本発明において軽度の腎障害の検出に用いることができるため、必ずしも上で列挙したアミノ酸に限定されることを意図するものではない。
糞便におけるD-アミノ酸/L-アミノ酸比の変化についての分析結果を図3Dに示す。対照群(Sham)に比較して、腎障害誘導群でD-アミノ酸/L-アミノ酸比の有意な変化が見られたのは、セリン、アルギニン、及びアスパラギン酸であった。このうち、セリンとアルギニンでは、対照群と比較して、術後2日目及び10日目の両方で高く、2日目より10日目の方が高かった。一方、アスパラギン酸では、対照群(Sham)と比較して、術後2日目及び10日目の両方で低く、2日目と10日目にはほとんど差がなかった。より詳細に解析すると、セリン及びアルギニンでは、2日目でD体/L体比が増加していることから、糞便又は腸内容物においてこれらのアミノ酸のD体/L体比が、閾値よりも高い場合に、軽度の腎障害と判定することが可能になる。また、アスパラギン酸では、腎障害極期の2日目で減少していることから、糞便又は腸内容物においてこれらのD-アミノ酸が、閾値よりも低い場合に、軽度の腎障害と判定することが可能になる。しかしながら、母数を増やすことで、有意差の有無が変化する可能性があり、その場合、本発明において軽度の腎障害の検出に用いることができるため、必ずしも上で列挙したアミノ酸に限定されることを意図するものではない。特にD体/L体比では、有意差はでていないものの、ヒスチジン、グルタミン、グルタミン酸、バリン、ロイシンにおいて、腎障害極期の2日目で対照とは異なる動きをしていることから、軽度腎障害の診断に有用でありうる。
糞便におけるD-アミノ酸/(D-アミノ酸+L-アミノ酸)比の変化についての分析結果を図3Eに示す。また、図3A~3Eに示したデータ間の統計的有意差を評価した。その結果を図3Fの表に示す。
上述の軽度急性腎障害動物モデルにおいて、虚血再還流に供するマウスを、通常の条件下で飼育された野生群、無菌下での飼育された無菌群、及び無菌下で飼育された後に、植菌された植菌群に分けた。通常の条件下で飼育し、偽手術を行ったマウスを対照群とした。虚血性腎障害の誘導後2日目に、マウスを屠殺し、腎臓組織を回収した。回収した腎臓組織に対し、固定処理を行った。固定された腎臓組織から切片を取得し、PAS染色を行った。さらに別の切片に対しF4/80染色を行った。PAS染色結果を図5(A)に示す。図5Aの染色結果に基づいて壊死細胞の割合を評価した。その結果を図5(B)に示す。F4/80染色結果を図6(A)のグラフに示す。図6(A)の染色結果に基づいて壊死細胞の割合を評価した。その結果を図6(B)のグラフに示す。
対照群(Sham)及び腎障害誘導群において、術後2日目及び10日目に腸内細菌叢を取得し、RAPD-PCRを行った。PCR後のバンドについて電気泳動を行った結果を図7Aに示す。また、術後2日目及び10日目で取得した腸内細菌叢を、培養することにより、含まれる細菌の種類及び数を決定した(図7B)。
図5及び6の結果により、無菌群では、軽度腎障害に対して、炎症を誘導しやすくなる一方で、腸内に植菌することで炎症性を低下させることができた。したがって、腸内細菌と、腎障害における炎症とが関係性を有することが示された。
図7では、虚血再還流を行った野生群と、偽手術を行った対照群とでは、RAPD-PCRのバンドパターンが変化しており(図7A)、含まれる細菌数及び/又は種類に変化が生じたことが示された。また、図7Bの結果から、術後2日目において、一部の細菌について、有意に数が変化しており、10日目では、有意差はないものの、その細菌の数の違いが現れることが示された。
これにより、軽度の腎障害であっても、腸内細菌の種類や数を変化させることが示された。理論により制限されることを意図するものではないが、糞便中のDアミノ酸は、腸内細菌により産生されるものと考えられることから、腎障害が、腸内細菌の数や種類を変化させ、その結果としてD-アミノ酸の量が変化すると考えられる。これにより糞便中のD-アミノ酸の変化を調べることで、腎障害を鋭敏に感知できると考えられる。
材料及び方法
研究倫理
本発明者らは、金沢大学附属病院において、8名の慢性腎不全患者及び3名健常者の糞便試料及び血液試料を取得した。これらの被験者に対し、インフォームドコンセントを取得し、金沢大学医学倫理審査委員会により承認された試験に従って実験を行った。
アミノ酸のエナンチオマー及びHPLC級のアセトニトリルはナカライテスク(京都)から購入された。HPLC級のメタノール、トリフルオロ酢酸、ホウ酸等は和光純薬(大阪)から購入された。水はMill-QグラジエントA10システムを用いて精製された。
女性患者には、数式の計算値に補正係数0.739をかけた。
被験者のeGFR値と、20種類の各アミノ酸についてD-アミノ酸及びL-アミノ酸の合計量、L-アミノ酸量、D-アミノ酸量、及びDアミノ酸/Lアミノ酸比との間の相関係数を測定した。その結果を図11に示す。
Claims (23)
- 糞便又は腸内容物の分析方法であって、アミノ酸のうちの少なくとも1のアミノ酸のD体及び/又はL体の量を測定する工程、
前記少なくとも1種類のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出する工程、
を含む、分析方法。 - 前記腎障害の病態指標値が、前記少なくとも1のアミノ酸のD体の量、L体の量、又はD体及びL体の合計量である、請求項1に記載の分析方法。
- 前記腎障害の病態指標値が、前記少なくとも1のアミノ酸のD体及びL体の量の和に対するD体の量の比、D体の量に対するL体の量の比、又はL体の量に対するD体の量の比である、請求項1に記載の分析方法。
- 前記少なくとも1のアミノ酸が、セリン、グルタミン酸、ヒスチジン、アスパラギン、アルギニン、アスパラギン酸、アロスレオニン、アラニン、プロリン、メチオニン、バリン、アロイソロイシン、イソロイシン、ロイシン、フェニルアラニン、リジン、及びチロシンからなる群から選ばれる、請求項1~3のいずれか一項に記載の分析方法。
- 前記分析方法が、腎障害の病態指標値と、腎障害の病態とを関連づける工程をさらに含む、請求項1~4のいずれか一項に記載の分析方法。
- 前記少なくとも1のアミノ酸が、ヒスチジン、アスパラギン、アルギニン、アスパラギン酸、アロスレオニン、アラニン、プロリン、メチオニン、バリン、アロイソロイシン、イソロイシン、ロイシン、フェニルアラニン、リジン、及びチロシンからなる群から選ばれる場合、腎障害が慢性腎障害である、請求項5に記載の分析方法。
- 前記少なくとも1のアミノ酸が、ヒスチジン、アロスレオニン、及びリジンからなる群から選ばれる、請求項6に記載の分析方法。
- 前記少なくとも1のアミノ酸が、セリン、アスパラギン酸、グルタミン酸、アラニン、リジン、アルギニン及びプロリンからなる群から選ばれる場合、腎障害が腎障害急性期又は虚血を原因とする腎障害である、請求項5に記載の分析方法。
- 腎障害の病態指標値と、腎障害の病態とを関連づける工程が、健常者群の病態指標値及び腎障害の患者群の病態指標値とから決定される病態指標基準値に基づき決定される、請求項5~8のいずれか一項に記載の分析方法。
- 腎障害の病態指標値と、腎障害の病態とを関連づける工程が、対象の病態指標値を、カットオフ値と比較することにより決定される、請求項5~8のいずれか一項に記載の分析方法。
- 前記カットオフ値が、ROC曲線分析により予め決定される、請求項10に記載の分析方法。
- 糞便又は腸内容物を試料とする腎障害の検査方法であって、
アミノ酸のうちの少なくとも1のアミノ酸のD体及び/又はL体の量を測定する工程、
前記少なくとも1種類のアミノ酸のD体の量、L体の量、D体及びL体の量に基づき腎障害の病態指標値を算出する工程、
腎障害の病態指標値と、腎障害の病態とを関連づける工程をさらに含む、前記検査方法。 - 前記腎障害の病態指標値が、前記少なくとも1のアミノ酸のD体の量、L体の量、又はD体及びL体の合計量である、請求項10に記載の検査方法。
- 前記腎障害の病態指標値が、前記少なくとも1のアミノ酸のD体及びL体の量の和に対するD体の量の比、D体の量に対するL体の量の比、又はL体の量に対するD体の量の比である、請求項10に記載の検査方法。
- 記憶部と、入力部と、分析測定部と、データ処理部と、病態情報出力部とを含む、被検体の糞便又は腸内容物の試料分析システムであって、
前記記憶部は、入力部から入力された腎障害と判別する病態指標値の閾値を記憶し、
前記分析測定部は、前記被検体の糞便又は腸内容物中のアミノ酸のうちの少なくとも1のアミノ酸のアミノ酸立体異性体を分離し定量し、
前記データ処理部は、前記少なくとも1のアミノ酸のD体の量、L体の量、又はD体及びL体の量に基づき腎障害の病態指標値を算出し、
前記データ処理部は、記憶部に記憶された閾値と比較することにより、腎障害を判別し、
前記病態情報出力部が被検体の腎障害についての病態情報を出力する、前記試料分析システム。 - 前記腎障害の病態指標値が、前記少なくとも1のアミノ酸のD体の量、L体の量、又はD体及びL体の合計量である、請求項15に記載の試料分析システム。
- 前記腎障害の病態指標値が、前記少なくとも1のアミノ酸のD体及びL体の量の和に対するD体の量の比、D体の量に対するL体の量の比、又はL体の量に対するD体の量の比である、請求項15に記載の試料分析システム。
- 前記少なくとも1のアミノ酸が、セリン、グルタミン酸、ヒスチジン、アスパラギン、アルギニン、アスパラギン酸、アロスレオニン、アラニン、プロリン、メチオニン、バリン、アロイソロイシン、イソロイシン、ロイシン、フェニルアラニン、リジン、及びチロシンからなる群から選ばれる、請求項13~15のいずれか一項に記載の試料分析システム。
- 前記少なくとも1のアミノ酸が、ヒスチジン、アスパラギン、アルギニン、アスパラギン酸、アロスレオニン、アラニン、プロリン、メチオニン、バリン、アロイソロイシン、イソロイシン、ロイシン、フェニルアラニン、リジン、及びチロシンからなる群から選ばれる場合、腎障害が慢性腎障害である、請求項18に記載の試料分析システム。
- 前記少なくとも1のアミノ酸が、ヒスチジン、アロスレオニン、及びリジンからなる群から選ばれる、請求項19に記載の試料分析システム。
- 前記少なくとも1のアミノ酸が、セリン、アスパラギン酸、グルタミン酸、アラニン、アルギニン、リジン及びプロリンからなる群から選ばれる場合、腎障害が腎障害急性期又は虚血を原因とする腎障害である、請求項18に記載の試料分析システム。
- 前記少なくとも1のアミノ酸が、ヒスチジン、アロスレオニン、及びリジンからなる群から選ばれる、請求項14に記載の試料分析システム。
- 病態指標値の閾値が、ROC曲線分析により予め決定される、請求項13~15の何れか一項に記載の分析方法。
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