JP2011058863A - Serum maker for determining chronic nephropathy, detection method and device for the same - Google Patents

Abstract

It is possible to screen patients at an early stage of chronic kidney disease only by blood tests without performing urine tests.
A healthy person has at least one of glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid contained in a blood sample at an early stage of chronic kidney disease. It can be used as a serum marker for determining chronic kidney disease.
[Selection] Figure 2

Description

  The present invention relates to a serum marker for determination of chronic kidney disease, and a detection method and apparatus thereof, and in particular, can be screened by distinguishing early stages 1 and 2 of chronic kidney disease (CKD) from healthy individuals. The present invention relates to a serum marker for determining chronic kidney disease, and a detection method and apparatus thereof.

  In recent years, the number of ESKD patients who require dialysis and transplantation due to end-stage renal failure (ESKD) is increasing all over the world, which is a major problem in the medical economy. Along with this, CKD is also attracting attention as a reserve army of ESKD. Furthermore, since CKD is a serious risk factor for cardiovascular diseases such as myocardial infarction and stroke, early detection of CKD patients is desired.

CKD refers to a state in which either one of the following 1, 2 or both last for 3 months or more.
1. Presence of renal disorder (1) Presence of proteinuria (2) Abnormalities other than proteinuria
1. Presence of renal disorder is clear by pathology, diagnostic imaging, examination (urinalysis / blood), etc. Glomerular Filtration Rate (GFR) <60 (ml / min / 1.73 m ² )

  CKD is a progressive / irreversible disease and is classified into stages 1 to 5 according to the degree of progression as shown in FIG. 1 (Non-patent Document 1).

  Among these, advanced stage 3-5 CKD patients with GFR59 or lower can be distinguished from healthy individuals using serum markers such as serum BUN (Blood Urea Nitrogen), creatinine, cystatin C, etc. (Non-patented) Reference 2 and non-patent reference 3).

  On the other hand, a comprehensive method for measuring intracellular metabolites by a method for measuring metabolites in a sample using a capillary electrophoresis-mass spectrometer (CE-MS) (see, for example, Non-Patent Document 4) is a human or animal body. A method for qualitatively and / or quantitatively determining a low-molecular compound (metabolite) pattern and / or a peptide pattern of a liquid sample derived from the human or animal body, The metabolite and peptide of the liquid sample are then separated by capillary electrophoresis, then directly ionized and detected on-line via an interface with a connected mass spectrometer. In order to monitor the condition of the human or animal body over time, the reference and sample values indicative of the condition, and the deviations and correspondence derived from the values are automatically stored in a database. When separation and analysis of anionic compounds by combining capillary electrophoresis and mass spectrometry, the electroosmotic flow is reversed using a coated capillary whose inner surface is previously cationically coated. A method for separating and analyzing an anionic compound (for example, see Patent Document 1) is known.

Japanese Patent No. 3341765

Am J Kidney Dis 39: S1-S266,2002 Swan SK.The search continues-an ideal marker of GFR.Clinical Chemistry 43: 913-914, 1997 O'Riordan S, Ouldred E, Brice S, Jackson SHD, Swift CG. Serum cystatin C is not a better marker of creatinine or digoxin clearance than serumcreatinine. Br J Clin Pharmacol 53: 398-402, 2002. Soga, T., Ohashi, Y., Ueno, Y., Naraoka, H., Tomita, M., and Nishioka, T., “Quantitative Metabolome Analysis Using Capillary Electrophoresis Mass Spectrometry”, J. Proteome Res. 2. 488 -494, 2003.

  However, since serum markers such as serum BUN, creatinine, cystatin C and the like used for advanced CKD patients in stages 3 to 5 do not change in early CKD patients in stages 1 and 2 above GFR60, It could not be used as a marker for CKD patients. Currently, there are an estimated 2.32 million people in Japan (2.3% of the adult population) in stages 1 and 2, but serum markers useful for distinguishing these early CKD patients from healthy individuals Did not exist.

  It is possible to discriminate between the early stage CKD patients and the healthy person in stages 1 and 2 by conducting a urine test, but only a blood test is performed without conducting a urinalysis when screening healthy persons and early stage CKD patients. Serum markers that can be discriminated by are required.

  The present invention has been made to solve the above-mentioned conventional problems, and an object of the present invention is to provide a serum marker for chronic kidney disease determination that can be distinguished from a healthy person even in early CKD in stages 1 and 2.

  The present invention has been made based on the measurement results of metabolites in serum by CE-MS as described in Non-Patent Document 4 and Patent Document 1.

  That is, when the CE-MS was used to analyze a blood sample (15 samples) of an early stage CKD patient in stages 1 and 2 and a blood sample (7 samples) of a healthy person, as shown in FIG. There were significant differences between healthy C and early CKD patients P for glutamate, glutamine, ornithine, aspartate, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid. In FIG. 2, * indicates a p-value <0.05 of Mann-Whitney test regarding the difference between the average values of the two groups, and ** indicates a p-value <0.01.

  The positioning of each substance in the amino acid metabolic pathway is shown in FIGS. 3 and 4, the positioning in the nucleic acid metabolic pathway is shown in FIGS. 5 and 6, and the positioning in the carbohydrate metabolic pathway is shown in FIG.

  Among these, increases in taurine and hypotaurine in the amino acid metabolic pathway shown in FIG. 4 indicate that the kidney may be exposed to high oxidative stress.

  In addition, a decrease in adenosine and an increase in hypoxanthine in the nucleic acid metabolic pathway shown in FIG. 6 indicate the possibility of increased purine degradation in CKD patients. In addition, the increase in glutamic acid in the nucleic acid metabolic pathway shown in FIG.

  Moreover, the increase in lactic acid in the carbohydrate metabolic pathway shown in FIG. 7 indicates the possibility of a decrease in aerobic glycolysis and an increase in anaerobic glycolysis in CKD patients. The reason for this is not clear, but there is a possibility that aerobic glycolysis is reduced because the pathway to the synthesis of nucleic acid is enhanced from the middle of the glycolysis system. In addition, aspartic acid and glutamic acid may accumulate and increase due to a decrease in metabolism of the TCA cycle.

  The present invention has been made on the basis of the above findings, and is contained in a blood sample by at least one of glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid. The above-mentioned problem is solved by a serum marker for determining chronic kidney disease that can distinguish an early stage of chronic kidney disease from a healthy person.

  The present invention also provides glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid in blood samples as serum markers that can distinguish the early stage of chronic kidney disease from healthy individuals. It is intended to provide a method for detecting a serum marker for determining chronic kidney disease, characterized by detecting at least one of them.

  Here, the serum marker in the blood sample can be detected using a capillary electrophoresis-mass spectrometer.

  The present invention also provides means for preparing a sample suitable for analysis from a blood sample, and at least one of glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid in the sample. And an analytical means for detecting the early stage of chronic kidney disease as a serum marker that can be distinguished from a healthy person. is there.

  According to the present invention, by measuring the concentration of a serum marker in a blood sample of a subject, it can be used as a screening marker that distinguishes the presence or absence of early CKD in stages 1 and 2 from healthy individuals.

  Therefore, early CKD in stages 1 and 2 can be detected only by a blood test without performing a urine test or the like, and the burden on the subject and labor and cost of the test can be reduced.

Diagram showing the stage classification of CKD The figure which compares and shows the comprehensive metabolome analysis result of the healthy person of the serum marker for chronic kidney disease determination by this invention, and the patient of the early stage of CKD Diagram showing part of amino acid metabolism pathway Diagram showing other parts of the amino acid metabolic pathway Diagram showing part of the nucleic acid metabolism pathway Diagram showing another part of the nucleic acid metabolic pathway Diagram showing carbohydrate metabolism pathway The figure which shows the process sequence in embodiment of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  The processing procedure of this embodiment is shown in FIG.

  The supernatants obtained by centrifuging the blood samples of stage 1 and 2 CKD patients and the blood samples of healthy subjects at 3,000 rpm for 10 minutes were collected as serum samples, respectively. The number of samples is 15 CKD patients and 7 healthy subjects.

1. Extraction of Metabolites from Serum 200 μl of serum was placed in 1.8 ml of methanol containing an internal standard substance (Step 100), stirred to inactivate the enzyme, and then 800 μl of pure water and 2 ml of chloroform were added (Step 110 ) The phospholipid was removed and centrifuged at 5000 rpm for 5 minutes at 4 ° C. (step 120). After standing, 800 μl of the separated water-methanol phase was passed through a centrifugal ultrafiltration filter with a molecular weight cut off of 5 kDa to deproteinize (step 130). The filtrate was freeze-dried (step 140), 50 μl of Milli-Q water was added and dissolved in water (step 150), and it was used for CE-MS (CE-TOFMS) measurement (step 160).

2. Measurement of metabolites in a sample by capillary electrophoresis-time-of-flight mass spectrometer (CE-TOFMS) 1000 masses under conditions of cations and anions using CE-TOFMS
The following metabolites were comprehensively measured.

(1) Cationic Metabolite Measurement Conditions 1) Analysis Conditions for Capillary Electrophoresis (CE) A fused silica capillary (inner diameter 50 μm, outer diameter 350 μm, total length 100 cm) was used as the capillary. As the buffer, 1M formic acid (pH about 1.8) was used. The applied voltage was +30 kV, and the capillary temperature was 20 ° C. The sample was injected for 3 seconds at 50 mbar using the pressure method.
2) Analysis conditions of time-of-flight mass spectrometer (TOFMS) The positive ion mode was used, the ionization voltage was set to 4 kV, the fragmentor voltage was set to 75 V, the skimmer voltage was set to 50 V, and the OctRFV voltage was set to 125 V. Nitrogen was used as the drying gas, and the temperature was set to 300 ° C. and the pressure was set to 10 psig. A 50% methanol solution was used as the sheath liquid, and reserpine (m / z 6099.2807) was mixed at 0.5 μM for mass calibration, and the solution was fed at 10 μ / min. All data obtained using the mass numbers of reserpine (m / z 609.2807) and methanol adduct ion (m / z 83.0703) were auto-calibrated.

(2) Conditions for anionic metabolite measurement 1) Analysis conditions for capillary electrophoresis (CE) A SMILE (+) capillary (inner diameter 50 μm, outer diameter 350 μm, total length 100 cm) was used. As the buffer, 50 mM ammonium acetate (pH 8.5) was used. The applied voltage was measured at −30 kV and the capillary temperature at 20 ° C. The sample was injected for 30 seconds at 50 mbar using the pressure method.
2) Analysis conditions of time-of-flight mass spectrometer (TOFMS) The negative ion mode was used, the ionization voltage was set to 3.5 kV, the fragmentor voltage was set to 100 V, the skimmer voltage was set to 50 V, and the OctRFV voltage was set to 200 V. Nitrogen was used as the drying gas, and the temperature was set to 300 ° C. and the pressure was set to 10 psig. As the sheath solution, a 5 mM ammonium acetate-50% methanol solution was used, 20 μMPIPES and 1 μM reserpine (m / z 609.2807) were added for mass calibration, and the solution was fed at 10 μ / min. All data obtained using reserpine (m / z 609.2807) and PIPES monovalent (m / z 301.0534) and divalent (m / z 150.0230) mass numbers were automatically calibrated.

  As a result, a result as shown in FIG. 2 was obtained.

  In the present embodiment, since CE-TOFMS is used for analysis, data of all markers can be obtained comprehensively, and highly accurate determination can be performed.

  The analysis means is not limited to CE-TOFMS, but CE-MS other than TOF, microchip CE-MS, high-performance liquid chromatography other than CE-MS-mass spectrometer (LC-MS), gas chromatography- Even if using an analytical means for detecting a part of a marker such as a mass spectrometer (GC-MS), a single CE including a microchip type, LC, GC, MS, a nuclear magnetic resonance apparatus (NMR), a colorimetric analyzer, etc. good.

C ... healthy person P ... early stage CKD patient in stage 1-2

Claims (4)

  It is possible to distinguish the early stage of chronic kidney disease from at least one of glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid contained in a blood sample from a healthy person Serum marker for determining chronic kidney disease.   Serum markers that can distinguish the early stage of chronic kidney disease from healthy individuals, and at least one of glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid in blood samples A method for detecting a serum marker for determination of chronic kidney disease, characterized by comprising:   The method for detecting a serum marker for determination of chronic kidney disease according to claim 2, wherein the serum marker in the blood sample is detected using a capillary electrophoresis-mass spectrometer. Means for preparing a sample suitable for analysis from a blood sample;
Detection of at least one of glutamic acid, glutamine, ornithine, aspartic acid, hydroxyproline, taurine, hypotaurine, adenosine, hypoxanthine, and lactic acid in the sample as a serum marker that can distinguish the early stage of chronic kidney disease from healthy individuals Analytical means to
An apparatus for detecting a serum marker for chronic kidney disease determination, comprising:

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