WO2010126146A1 - Method or apparatus for determination of severity of renal diseases and method for operation of the apparatus - Google Patents

Abstract

Disclosed is (1) a method or an apparatus for determining the severity of a renal disease, or a method for operating the apparatus. Also disclosed is (2) a dialysis apparatus which is intended to be used for a renal disease patient, or a method for operating the dialysis apparatus. In a sample derived from blood collected from a human, the amount of at least one marker selected from the group consisting of choline, O-butanoylcarnitine, N₁-methylinosine, N²,N²,-dimethylguanosine and N₄-acetylcytidine occurring in the sample is evaluated.

Description

Method or apparatus for determining the severity of kidney disease or method for operating the same

The present invention relates to a method or apparatus for determining the severity of renal disease or an operation method thereof. The present invention also relates to a dialysis device used for a renal disease patient and an operating method thereof.

A state in which renal function is reduced due to various renal disorders is sometimes called renal failure, and the terminal symptom is particularly called uremia. It has also been pointed out that uremia results from the failure of the homeostasis maintenance mechanism.

The substance that causes this uremia is called a uremic substance (uremic toxin). It is known that uremic substances appear and accumulate in the body when renal dysfunction progresses and kidney failure occurs. These uremic substances are present at a higher concentration than the healthy subject, particularly in the plasma of uremic patients. Its plasma concentration is said to correlate with the degree of uremic symptoms.

So far, urea, creatinine, parathyroid hormone, hydrogen ions, potassium, etc. have been pointed out as uremic substances.

Dialysis treatment is currently being performed for the purpose of treating these kidney diseases. When dialysis treatment is performed, it is currently determined whether or not the dialysis treatment is necessary using the creatinine concentration in plasma as an index.

Furthermore, the time for performing this dialysis treatment is currently determined by judging its effect using the urea concentration in plasma as an index.

However, it has been pointed out for many years that there is an incomplete aspect in the determination of introduction of dialysis treatment and the determination of dialysis treatment effect performed in this way (Non-patent Document 1). Nevertheless, to date, no uremic substance has been identified that can be an appropriate indicator of judgment.

An object of the present invention is to provide a method or apparatus for determining the severity of renal disease or an operation method thereof. Another object of the present invention is to provide a dialysis apparatus used for a renal disease patient and an operation method thereof.

The inventors of the present invention conducted thorough trial and error, and compared the three of the pre-dialysis plasma and the post-dialysis plasma of a patient undergoing dialysis treatment (dialysis patient) and the plasma of a healthy person with each other. We searched for low molecular weight compounds that exist only in dialysis patients.

Through this difficult work over a long period of time, the present inventors finally found out that a specific low molecular compound exists in a large amount only in dialysis patients. Furthermore, the present inventors have clarified that the plasma abundance of these specific low-molecular compounds can be used as an index for appropriate determination of introduction of dialysis treatment and appropriate determination of dialysis treatment effect, respectively, and thus completed the present invention. It was.

That is, the present invention is as follows:
Item 1. A method for determining the severity of kidney disease,
(A) at least one marker selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived specimen (B) when the abundance of at least one of the markers evaluated in step (A) is greater than or equal to the reference abundance, serious kidney disease in the donor of the blood-derived specimen A method comprising the step of determining that the degree is high.
Item 2. A device for determining the severity of kidney disease comprising:
(A) At least one selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived sample collected from a human Means for evaluating the abundance of a marker of the species; and (B) comparing the abundance of the marker evaluated by means (A) with a reference abundance, wherein the abundance of at least one of the evaluated markers is the reference abundance In such a case, the apparatus comprises means for determining that the severity of renal disease in the blood-derived sample provider is high.
Item 3. The reference abundance of choline is 20-40 μmol / L, the reference abundance of O-butanoylcarnitine is 30-50 μmol / L, and the reference abundance of N ₁ -methylinosine is 0.6-1.4 μmol / L Item 2 wherein the reference abundance of N ² , N ² , -dimethylguanosine is 0.2 to 0.5 μmol / L and the reference abundance of N ₄ -acetylcytidine is 250 to 400 nmol / L. The device described in 1.
Item 4. Item 3. A method of operating the device according to Item 2, comprising:
(A) at least one selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived sample collected from a human Actuating means (A) to assess the abundance of a marker of a species; and (b) comparing the abundance of the marker assessed by step (a) with a reference abundance and determining at least one When the abundance of the marker is greater than the reference abundance, the method comprises the step of operating the means (B) so as to determine that the severity of renal disease in the blood-derived sample provider is high. Actuation method.
Item 5. The reference abundance of choline is 20-40 μmol / L, the reference abundance of O-butanoylcarnitine is 30-50 μmol / L, and the reference abundance of N ₁ -methylinosine is 0.6-1.4 μmol / L Item 4 wherein the reference abundance of N ² , N ² , -dimethylguanosine is 0.2 to 0.5 μmol / L, and the reference abundance of N ₄ -acetylcytidine is 250 to 400 nmol / L. The method described in 1.
Item 6. A method of dialysis against a patient with renal disease:
(I) a step of performing dialysis on a renal disease patient;
(II) a step of collecting a blood-derived specimen from the renal disease patient;
(III) Choline, O-butanoylcarnitine, N ₁ -methylinosine, and N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid in the blood-derived specimen collected by step (II), and Evaluating the abundance of at least one marker selected from the group consisting of N ₄ -acetylcytidine;
(IV) The abundance of the marker evaluated in step (III) is compared with the reference abundance, and if the abundance of all the evaluated markers is less than or equal to the reference abundance, dialysis by means (I) is terminated. A method comprising the step of:
Item 7. A dialysis machine used for patients with kidney disease:
(I) means for dialysis of patients with renal disease;
(II) means for collecting a blood-derived specimen from the renal disease patient;
(III) Choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N in blood-derived specimens collected by means (II) Means for assessing the abundance of at least one marker selected from the group consisting of ₄ -acetylcytidine;
(IV) The abundance of the marker evaluated by the means (III) is compared with the reference abundance, and if the abundance of all the evaluated markers is less than the reference abundance, the dialysis by the means (I) is terminated. An apparatus comprising: means for:
Item 8. The reference abundance of choline is 20-40 μmol / L, the reference abundance of O-butanoylcarnitine is 30-50 μmol / L, and the reference abundance of N ₁ -methylinosine is 0.6-1.4 μmol / L The reference abundance of N ² , N ² , -dimethylguanosine is 0.2 to 0.5 μmol / L, the reference abundance of kynurenine is 2 to 4 μmol / L, and the reference of phenylacetylglutamine The abundance is 20 to 150 nmol / L, the reference abundance of hippuric acid is 24 to 100 μmol / L, and the reference abundance of N ₄ -acetylcytidine is 250 to 400 nmol / L. 8. The apparatus according to 7.
Item 9. The operation method of the dialysis device according to Item 7, wherein:
(I) actuating means (I) to perform dialysis on a renal disease patient;
(Ii) actuating means (II) to collect a blood-derived sample from the patient with renal disease;
(Iii) Choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N in the blood-derived specimen collected in step (ii) Activating means (III) to assess the abundance of at least one marker selected from the group consisting of ₄ -acetylcytidine;
(Iv) Compare the abundance of the marker evaluated in step (iii) with the reference abundance, and end the step (i) if the abundance of all the evaluated markers is equal to or less than the reference abundance. A method of operating the means (IV).
Item 10. The reference abundance of choline is 20-40 μmol / L, the reference abundance of O-butanoylcarnitine is 30-50 μmol / L, and the reference abundance of N ₁ -methylinosine is 0.6-1.4 μmol / L The reference abundance of N ² , N ² , -dimethylguanosine is 0.2 to 0.5 μmol / L, the reference abundance of kynurenine is 2 to 4 μmol / L, and the reference of phenylacetylglutamine The abundance is 20 to 150 nmol / L, the reference abundance of hippuric acid is 24 to 100 μmol / L, and the reference abundance of N ₄ -acetylcytidine is 250 to 400 nmol / L. 9. The method according to 9.
Item 11. A method for determining the severity of kidney disease, comprising a group of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived specimen The abundance of at least one selected marker is evaluated, and if the abundance of at least one of the evaluated markers is greater than or equal to a reference abundance, the severity of renal disease in the blood-derived sample provider Determining that the value is high.
Item 12. A kit for determining the severity of kidney disease, selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine A kit containing the materials necessary to detect at least one marker.
Item 13. A kit for determining the dialysis effect comprising choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine A kit comprising materials necessary for detecting at least one marker selected from the group consisting of:

According to the present invention, by using a novel human plasma-derived marker, it is possible to more appropriately determine the introduction of dialysis treatment and the determination of dialysis treatment effect than before.

In particular, according to the present invention, it is possible to easily determine the introduction of dialysis treatment and the determination of dialysis treatment effect in a short time at a low cost.

In addition, by determining the severity of kidney disease according to the present invention or performing dialysis against a patient with renal disease, both of the patients with renal disease and medical institutions engaged in diagnosis and treatment receive great benefits. It is expected to be possible.

It is a graph of the amount of creatinine before and after dialysis for each patient. It is a graph of the average value of the amount of creatinine of a patient before and after dialysis, and a healthy person. Positive ion measurement [M + H] ⁺ is abundant in dialysis patients and is significantly reduced by dialysis 18 peaks before dialysis, after dialysis, and the amount contained in healthy human plasma samples as relative area It is the graph compared. Negative ion measurement [MH] ^- before dialysis 6 peak significantly reduced by dialysis included in many dialysis patients were compared after dialysis, and the amount contained in a healthy person's plasma sample as a relative area It is a graph. It is drawing which shows the flow of narrowing down a peak. It is drawing which shows the peak by which the difference was recognized by the significance level 1% also in A group. It is the graph which represented the relative area of each peak with the box-and-whisker chart. The center circle indicates the median value, the box indicates the 25% to 75% value, and the error bar indicates the 10% to 90% value. In group A, there is a significant difference between pre-dialysis vs. dialysis and pre-dialysis vs. healthy individuals, and there is a large variation, which is a peak that decreases to normal levels by dialysis. It is drawing which shows the peak by which the difference was recognized by the significance level 5% also in A group. It is the graph which represented the relative area of each peak with the box-and-whisker chart. In group A, there is a significant difference between pre-dialysis vs. dialysis and pre-dialysis vs. healthy individuals, and there is a large variation, which is a peak that decreases to normal levels by dialysis. It is drawing which shows the peak of B group. It is the graph which represented the relative area of each peak with the box-and-whisker chart. In group B, there was a significant difference between all groups of pre-dialysis vs. post-dialysis, post-dialysis vs. healthy people, and pre-dialysis vs. healthy people, and there was a large variation. . It is drawing which shows the peak of C group. It is the graph which represented the relative area of each peak with the box-and-whisker chart. In group C, there is a significant difference between all groups of pre-dialysis vs. post-dialysis, post-dialysis vs. healthy people, and pre-dialysis vs. healthy people, and there is little variation, and the peak does not decrease to the normal level even after dialysis .

1. Method for determining the severity of kidney disease The method for determining the severity of kidney disease according to the present invention comprises (A) choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² in a blood-derived sample. Evaluating the abundance of at least one marker selected from the group consisting of N, N ² , -dimethylguanosine, and N ₄ -acetylcytidine; and (B) at least of the markers evaluated in step (A) When one kind of abundance is greater than or equal to a reference abundance, the method includes a step of determining that the severity of renal disease in the blood-derived sample provider is high.

The method for determining the severity of kidney disease according to the present invention is a method in which a blood-derived sample is a test subject and whether or not the blood-derived sample is derived from a patient having a high severity of kidney disease. It is.

In addition, the method for determining the severity of kidney disease according to the present invention is a method for testing a patient's blood-derived specimen as a test target in order to determine the severity of the patient's kidney disease.

For patients who are not currently undergoing dialysis treatment, the necessity of introduction of dialysis treatment may be determined based on the severity determined by the method for determining the severity of renal disease of the present invention. That is, if it is judged that the severity is high, it is judged that introduction of dialysis treatment is necessary, and if it is judged that the severity is low, it is judged that introduction of dialysis treatment is unnecessary. Also good.

The method for determining the severity of renal disease of the present invention includes a marker abundance evaluation step (A) and a renal disease severity determination step (B). Hereinafter, the present invention will be described in detail for each process.

(A) Marker abundance evaluation step The marker abundance evaluation step comprises choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine in a blood-derived sample collected from a human. And an abundance of at least one marker selected from the group consisting of N ₄ -acetylcytidine.

As the blood-derived specimen, whole blood, serum, or plasma can be used. Among them, serum and plasma are preferable. Further, plasma is more preferable from the viewpoint that the separation time can be shortened or the degradation of the marker to be evaluated is prevented. In order to prevent degradation of the marker to be evaluated, a substance exhibiting an anticoagulant action by chelating action or the like may be added to these blood-derived specimens. Examples of such a substance exhibiting an anticoagulant action include EDTA (Ethylene Diamine-Tetraacetic Acid). Moreover, you may add the substance which shows a protease inhibitory effect for the same purpose. Examples of the substance exhibiting such anticoagulant action include aprotinin.

In addition, when the blood-derived specimen is once cryopreserved after collection, it may be subjected to a pretreatment step in advance if necessary before being used for means (A) for evaluating the abundance of the marker. Examples of the pretreatment step include a step of removing unnecessary materials by centrifugation or the like. For example, when plasma is used as a blood-derived specimen, if necessary, the precipitate may be removed by centrifugation and then provided to the means (A). As the conditions for centrifugation, for example, 3,000 to 10,000 μg and 5 to 10 minutes are preferable. For example, 10,000g, 10 minutes, etc. are mentioned. As a pretreatment process, you may use for a solid-phase extraction process further as needed. For example, the process using a hydrophobic adsorbent is mentioned. In this case, solid phase extraction may be performed using a hydrophobic adsorbent as a solid phase extraction column. Examples of the solid phase extraction column that is a hydrophobic adsorbent include a Sep-Pak® Light® Cartrides® C18 column (Nihon Waters Co., Ltd.). In this case, elution can be performed using 90% methanol, for example.

Evaluating the abundance of at least one marker selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine, The process is not particularly limited as long as it is a process capable of confirming the presence or absence of these substances or a process capable of quantitative measurement.

If the detection limit of the evaluation means is adjusted to be greater than or equal to the reference abundance of a certain marker, by using this evaluation means, it is possible to evaluate whether or not the marker exists in the specimen in excess of the reference abundance. In this case, as the step of confirming the presence or absence, for example, a step of evaluating that the marker is not present if it is below the detection limit of the evaluation means, and a step of evaluating that the marker is present if it is above the detection limit can be mentioned. .

Examples of the quantitative measurement process include a liquid chromatograph-time-of-flight mass spectrometer (LC-TOF MS), high performance liquid chromatography (HPLC), a spectrophotometer, a chemiluminescence measuring instrument, and the like. LC-TOF MS is preferred from the viewpoint of reproducibility or measurement accuracy.

For example, when LC-TOF MS is used as an evaluation step, multiple internal standards are added to a blood-derived sample in advance, then analysis is performed using LC-TOF MS, and the m / z peak corresponding to the marker The value obtained by dividing the area by the average area of the peak area of m / z corresponding to the internal standard is the relative area (hereinafter referred to as “m / z peak relative area”), and this is compared between different blood-derived specimens. It can be mentioned that the abundance is evaluated.

As the internal standard substance, for example, benzyloxycarbonyl-L-tyrosyl-L-glutamic acid (Z-Tyr-Glu), tosyl-L-arginine methyl ester monohydrochloride (Tos-Arg-OMe / HCl), etc. can be used.

The column used for liquid chromatography (LC) is not limited as long as the marker can be analyzed. It may be a reverse phase column or a normal phase column. A reverse phase column is preferred. The filler is not limited as long as the marker can be analyzed. An example is C18 (octadecyl). The particle system and length can be appropriately set.

Considering the measurement error, according to LC-TOF MS, choline is measured by positive ion measurement [M + H] ⁺ with an accurate mass (m / z) of 104.11 ± 10 mDa. O-butanoylcarnitine has a precise mass (m / z) of 232.14 ± 10 mDa measured by positive ion measurement [M + H] ⁺ . N ₁ -methylinosine is measured as positive ion [M + H] ⁺ with an accurate mass (m / z) of 283.10 ± 10 mDa. N ² , N ² , -Dimethylguanosine is measured as positive ion [M + H] ⁺ with a retention time of 6.52 (min) and an accurate mass (m / z) of 312.13 ± 10 mDa.

If quicker or easier determination is required, select from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine It is preferable to evaluate the abundance of one kind of marker. When more reliable determination is required, it is preferable to evaluate the abundance of two or more types of markers selected from these groups. Depending on the degree of certainty required, the abundance of the three types of markers may be evaluated, or the abundance of the four types of markers may be evaluated.

Conventionally, creatinine has been used as an index for introducing dialysis. However, men and young people with a large amount of muscle are considered to have a high amount of creatinine originally, and the dialysis introduction time tends to be earlier. On the other hand, women and elderly people with low muscle mass are said to have a low amount of creatinine from the beginning, and there is a tendency that it is difficult to accurately determine the dialysis introduction time. On the other hand, choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine have not been used as conventional markers. Therefore, by using these as markers, the dialysis introduction time can be set more appropriately.

(B) Renal disease severity determination step In the kidney disease severity determination step, the abundance of at least one of the markers evaluated in (B) step (A) is greater than or equal to the reference abundance. In this case, it is a step of determining that the severity of kidney disease in the blood-derived sample provider is high.

When step (A) is a step of checking only the presence or absence of a marker, “when the amount of marker is greater than or equal to the reference amount” means when the marker is confirmed to be present means.

On the other hand, when the step (A) is a step of quantitatively evaluating the abundance of the marker, a value between the abundance of each marker in the healthy subject and the abundance in the renal disease patient is calculated. It can be used as a reference abundance. As the abundance in healthy individuals and the abundance in patients with renal disease, it is preferable to use the average value of abundances obtained from a plurality of persons. Which value between the abundance in the healthy person and the abundance in the kidney disease patient is used as the reference abundance may vary depending on the degree of “high severity” to be determined.

When LC-TOF MS is used as a means for evaluation and m / z peak relative area is used as the abundance, the average value of m / z peak relative areas of blood-derived specimens of at least one person, preferably multiple healthy subjects, is used as a reference. What is necessary is just to use as an abundance.

When the plasma concentration is used as the abundance, the abundance of choline in healthy individuals is about 10-15 μmol / L in plasma, and the abundance in patients with renal disease is about 15-45 μmol / L, so for example, When the degree of “severity” to be determined is low, the reference abundance can be determined within the range of 15 to 25 μmol / L, more preferably within the range of 15 to 20 μmol / L. In contrast, if the degree of “severity” to be determined is high, the reference abundance is determined within the range of 35 to 45 μmol / L, more preferably within the range of 40 to 45 μmol / L. can do. Further, when the degree of “severity” to be determined is intermediate between the above, it is within the range of 20 to 40 μmol / L, more preferably within the range of 25 to 35 μmol / L, and even more preferably 25 The reference abundance can be determined within the range of ˜30 μmol / L or within the range of 30 to 35 μmol / L.

The abundance of O-butanoylcarnitine in healthy individuals is about 5-50 μmol / L in plasma, and the abundance in patients with renal disease is about 20-80 μmol / L. When the degree of “height” is low, the reference abundance is determined within the range of 5 to 25 μmol / L, more preferably within the range of 10 to 20 μmol / L, and even more preferably within the range of 10 to 15 μmol / L. can do. On the other hand, when the degree of “high severity” to be determined is high, it is within the range of 60 to 80 μmol / L, more preferably within the range of 65 to 75 μmol / L, and even more preferably 70 to The reference abundance can be determined within the range of 75 μmol / L. When the degree of “severity” to be determined is intermediate between the above, it is within the range of 30 to 50 μmol / L, more preferably within the range of 35 to 45 μmol / L, and even more preferably 40 The reference abundance can be determined in the range of ~ 45 μmol / L.

Since the abundance of N ₁ -methylinosine in healthy individuals is about 10 to 150 nmol / L in plasma and the abundance in patients with renal disease is about 500 to 2,500 nmol / L, for example, If the severity is high, the reference abundance is within the range of 10 to 500 nmol / L, more preferably within the range of 150 to 500 nmol / L, and even more preferably within the range of 200 to 300 nmol / L. Can be determined. On the other hand, when the degree of “severity” to be judged is high, it is within the range of 1,500 to 2,500 nmol / L, more preferably within the range of 1,700 to 2,300 nmol / L, and even more preferably. The reference abundance can be determined within the range of 1,900-2,100 nmol / L. Further, when the degree of “severity” to be determined is intermediate between the above, it is within the range of 600 to 1,400 nmol / L, more preferably within the range of 700 to 1,300 nmol / L, and even more preferably. Can determine the reference abundance within the range of 800-1200 nmol / L.

Since the abundance of N ² , N ² , -dimethylguanosine in healthy individuals is about 15-50 nmol / L in plasma and the abundance in patients with renal disease is about 200-800 nmol / L, If the degree of “severity” to be attempted is low, the reference abundance can be determined within the range of 50 to 200 nmol / L, more preferably within the range of 100 to 150 nmol / L. . On the other hand, if the degree of “severity” to be judged is high, the reference abundance is within the range of 600 to 800 nmol / L, more preferably within the range of 700 to 800 nmol / L. Can be determined. In addition, when the degree of “severity” to be judged is intermediate between the above, it exists within the range of 200 to 500 nmol / L, more preferably within the range of 300 to 400 nmol / L. The amount can be determined.

Since the abundance of N ₄ -acetylcytidine in healthy individuals is about 150 to 250 nmol / L in plasma, and the abundance in patients with renal disease is about 300 to 400 nmol / L, for example, When the degree of “severity” is high, it can be in the range of 150 to 250 nmol / L, more preferably in the range of 200 to 250 nmol / L. On the other hand, when the degree of “severity” to be determined is low, it should be within the range of 300 to 400 nmol / L, more preferably within the range of 350 to 400 nmol / L. it can. If the degree of “severity” to be determined is between the above, it should be in the range of 250 to 400 nmol / L, more preferably in the range of 250 to 300 nmol / L. Can do.

2. Apparatus for determining the severity of kidney disease The apparatus for determining the severity of kidney disease according to the present invention is (A) choline, O-butanoylcarnitine, N ₁ -methyl in a blood-derived sample collected from a human. Means for evaluating the abundance of at least one marker selected from the group consisting of inosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine; and (B) evaluated by means (A) Compare the abundance of the marker with the reference abundance, and if the abundance of at least one evaluated marker is greater than or equal to the reference abundance, determine that the severity of renal disease in the donor of the blood-derived sample is high It is an apparatus provided with the means to do.

The apparatus for determining the severity of kidney disease according to the present invention includes a marker abundance evaluation means (A) and a kidney disease severity determination means (B). Hereinafter, the present invention will be described in detail for each means.

(A) Marker abundance evaluation means Marker abundance evaluation means includes choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine in blood-derived samples collected from humans. And an abundance of at least one marker selected from the group consisting of N ₄ -acetylcytidine.

The marker abundance evaluation means is means for carrying out the marker abundance evaluation step described in the method for determining the severity of renal disease of the present invention. Description of the marker abundance evaluation means is omitted because it is the same as that for the marker abundance evaluation step.

(B) Renal disease severity determination means The kidney disease severity determination means compares the abundance of the marker evaluated by the means (A) with the reference abundance and evaluated at least one kind. When the marker abundance is equal to or higher than the reference abundance, it is a means for determining that the severity of renal disease in the plasma sample provider is high.

When the means (A) is a means for confirming only the presence / absence of the marker, the information “present” may be used as the reference existence quantity as the reference existence quantity.

On the other hand, when the step (A) is a step of quantitatively evaluating the abundance of the marker, it has been described in the renal disease severity determination step of the method for determining the severity of renal disease of the present invention. The reference abundance determined in the same manner as described above may be used.

The means for comparing the abundance of the marker evaluated by the means (A) with the reference abundance is a means for comparing the abundance of the marker evaluated by the means (A) with the reference abundance stored in the memory. Also good.

Further, the means for comparing the abundance of the marker evaluated by the means (A) with the reference abundance may be a means for allowing a person operating the apparatus to compare with the reference abundance. For example, there may be provided means for visually or audibly transmitting the marker abundance evaluated by the means (A) to a person who operates the apparatus. As a means for visually transmitting, there is a display unit that displays the abundance of the marker. As a means for audibly transmitting, there is a voice transmitting section that informs the presence of the marker by voice.

The renal disease severity determination means may further include means for indicating the determination result. The means for indicating the determination result is not limited, but may be a means for visually displaying the determination result on a separate display unit, a means for indicating the determination result audibly, or the determination result. Further, it may be a means for transmitting to the outside.

3. Method of operating apparatus for determining the severity of renal disease The method of operating the apparatus for determining the severity of renal disease of the present invention is as follows: (a) Choline and O-butanoyl in blood-derived samples collected from humans Activating means (A) to evaluate the abundance of at least one marker selected from the group consisting of carnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine And (b) comparing the abundance of the marker evaluated in step (a) with a reference abundance, and if the abundance of at least one of the evaluated markers is greater than or equal to the reference abundance, the blood It is an operating method characterized by comprising the step of operating the means (B) so as to determine that the severity of renal disease in the donor of the derived specimen is high.

The description of the blood-derived specimen, the marker, and the step of evaluating the abundance of the marker is omitted because it is the same as the description of the apparatus for determining the severity of renal disease of the present invention.

4). Dialysis device used for renal disease patient or method for dialysis against renal disease patient The dialysis device used for renal disease patient of the present invention is (I) a renal disease patient. (II) means for collecting a blood-derived sample from the patient with the renal disease; (III) choline, O-butanoylcarnitine, N ₁ -methyl in the blood-derived sample collected by means (II) Means for evaluating the abundance of at least one marker selected from the group consisting of inosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine; (IV) The abundance of the marker evaluated by the means (III) is compared with the reference abundance, and if the abundance of all the evaluated markers is less than the reference abundance, the dialysis by the means (I) is terminated. A device characterized in that it comprises means.
The method of dialysis against a renal disease patient of the present invention comprises (I) a step of dialysis against a renal disease patient; (II) a step of collecting a blood-derived specimen from the renal disease patient; (III) step ( II) Choline, O-butanoylcarnitine, N ₁ -methylinosine, and N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine in blood-derived samples collected by II) Evaluating the abundance of at least one marker selected from the group consisting of: (IV) comparing the abundance of the marker assessed in step (III) with a reference abundance, and When the abundance is less than or equal to the reference abundance, the method includes the step of terminating dialysis by means (I). The method of dialysis against a renal disease patient of the present invention is based on the dialysis apparatus used for the renal disease patient of the present invention. Therefore, the method of dialysis against the renal disease patient of the present invention can be carried out according to the following description of the dialysis apparatus used for the renal disease patient of the present invention. For this reason, the description about the method of dialyzing the renal disease patient of this invention is abbreviate | omitted.

The dialysis apparatus used for the renal disease patient of the present invention comprises dialysis means (I), blood-derived specimen collection means (II), marker abundance evaluation means (III), and dialysis end means (IV). Hereinafter, the present invention will be described in detail for each means.

(I) Dialysis means The dialysis means is a means for dialysis against a renal disease patient.

The dialysis means may be one that is usually used as a dialysis means for renal disease patients, and is not limited. For example, a dialysis unit that removes wastes and the like in the blood by dialysis with a dialyzer, an infusion unit that supplies dialysate to the dialysis unit, and a drainage unit that discharges the dewatered liquid removed from the dialysis unit to the outside And mainly consists of

For example, the dialysis unit may be a device having a general configuration, and a blood pump for feeding blood drawn from the blood vessel of a renal disease patient out of the body into the blood circuit, and in the fed blood There may be provided a dialyzer or the like for transferring the waste product or the like to the dialysate side by diffusion and ultrafiltration. Describing the blood flow in this case, the blood sucked out from the blood vessel of the kidney disease patient is sent to the dialyzer by the action of the blood pump, and the blood passing through the dialyzer is returned to the body again. Further, the flow of the dialysate in this case will be explained. The dialysate sent out from the infusion part is first passed through the dialysate circuit, subjected to water removal treatment in the dialyzer, and then sent to the drainage part. Is done.

(II) Blood-derived specimen collecting means The blood-derived specimen collecting means is means for collecting a blood-derived specimen from the renal disease patient.

The blood-derived specimen collecting means may be provided in any section of the blood circuit of the dialysis means from the time the blood is taken out of the body to the time it is sent to the dialyzer, or is provided separately from the dialysis means. May be.

The blood-derived sample collecting means may be a means for collecting a blood-derived sample from the renal disease patient at regular intervals. The fixed time is not limited, but is preferably 0 to 240 hours, more preferably 0 to 120 hours, and still more preferably 0 to 60 hours.

The blood-derived specimen collecting means may be a means for collecting a blood-derived specimen from the renal disease patient every time stored in the memory.

The blood-derived sample collected from a renal disease patient in the blood-derived sample collecting means may be a dialysis waste liquid.

(III) Marker abundance evaluation means The marker abundance evaluation means includes choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² in the blood-derived specimen collected by the blood-derived specimen collection means. This is a means for evaluating the abundance of at least one marker selected from the group consisting of N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine.

In particular, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine are substances that cannot be sufficiently removed by dialysis based on conventional indicators such as creatinine. For this reason, a dialysis method with higher therapeutic effect than before can be provided by performing dialysis using these abundances as an index.

In particular, N ₁ -methylinosine, N ₄ -acetylcytidine, and N ² , N ² , -dimethylguanosine are components that have a small coefficient of variation and are not affected by background such as body weight, sex, and dialysis history. These components can be used as a substitute for creatinine, which is an indicator of renal function, because there is little variation among individuals.

The introduction time of dialysis is determined based on clinical diagnosis, difficulty of daily life, and creatinine amount of 8 mg / dL or more. However, since the amount of creatinine depends on the muscle mass, for example, even if the amount of creatinine in an elderly woman with a body weight of 30 kg is 1.2 mg / dL, which is above the reference value, the renal function may not actually work. There is also sex. On the other hand, if it is a component that does not depend on the patient background, an absolute specific standard can be used as an index for introducing dialysis. Therefore, N ₁ -methylinosine, N ₄ -acetylcytidine, and N ² , N ² , -dimethylguanosine can be used not only as indicators to show dialysis effects, but also as indicators of renal function evaluation when introducing dialysis. Available.

In particular, kynurenine, phenylacetylglutamine, and hippuric acid are components that are reduced to the level of healthy individuals by the current dialysis treatment using Kt / V as an index. If these components are not reduced to the level of healthy people during dialysis treatment, it is an indication that dialysis treatment is insufficient.
Choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine in blood-derived specimens collected by (II) The abundance of at least one marker selected from the group consisting of may be evaluated sequentially after collection.

Description of the marker abundance evaluation means is omitted because it is the same as the invention relating to the apparatus for determining the severity of renal disease of the present invention.

(IV) Dialysis end means The dialysis end means compares the marker abundance evaluated by the marker abundance evaluation means with the reference abundance, and the abundance of all the evaluated markers is the reference abundance. In the following cases, the dialysis means ends the dialysis.

The reference abundance in this case is an abundance that can serve as an indicator of whether or not the marker has been removed by dialysis in a dialysis patient. That is, when the abundance of the evaluated marker is less than or equal to the reference abundance, it can be determined that the marker has been removed by dialysis more than a certain level and a certain dialysis effect has been achieved. It can be said that the termination is desirable from the viewpoint of reducing the burden on the patient and the treatment efficiency.

When the plasma concentration is used as the reference abundance, the abundance of choline in healthy individuals is about 10 to 15 μmol / L 血漿 in plasma, and the abundance in patients with renal disease is about 15 to 45 μmol / L 、. When the degree of dialysis effect to be achieved is high, it can be within the range of 15 to 25 μmol / L, more preferably within the range of 15 to 20 μmol / L. On the other hand, when the degree of dialysis effect to be achieved is low, it can be in the range of 35 to 45 μmol / L, more preferably in the range of 40 to 45 μmol / L. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it is within the range of 20 to 40 μmol / L, more preferably within the range of 25 to 35 μmol / L, and further preferably within the range of 25 to 30 μmol / L. Or within the range of 30 to 35 μmol / L.

The abundance of O-butanoylcarnitine in healthy individuals is about 5-50 μmol / L in plasma, and the abundance in patients with renal disease is around 20-80 μmol / L. When the degree is high, it can be in the range of 5 to 25 μmol / L, more preferably in the range of 10 to 20 μmol / L, and still more preferably in the range of 10 to 15 μmol / L. In contrast, when the degree of dialysis effect to be achieved is low, it is within the range of 60 to 80 μmol / L, more preferably within the range of 65 to 75 μmol / L, and even more preferably within the range of 70 to 75 μmol / L. It can be. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it is within the range of 30 to 50 μmol / L, more preferably within the range of 35 to 45 μmol / L, still more preferably within the range of 40 to 45 μmol / L. Can be inside.

Since the abundance of N ₁ -methylinosine in healthy individuals is about 10 to 150 nmol / L in plasma and the abundance in patients with renal disease is about 500 to 2,500 nmol / L, for example, dialysis to be achieved When the degree of the effect is high, it can be in the range of 10 to 500 nmol / L, more preferably in the range of 150 to 500 nmol / L, still more preferably in the range of 200 to 300 nmol / L. On the other hand, when the degree of dialysis effect to be achieved is low, it is within the range of 1,500 to 2,500 nmol / L, more preferably within the range of 1,700 to 2,300 nmol / L, and even more preferably 1,900 to 2,100 nmol / L. Can be within the range. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it is in the range of 600 to 1,400 nmol / L, more preferably in the range of 700 to 1,300 nmol / L, and still more preferably in the range of 800 to 1,200 nmol / L. L can be within the range.

The amount of N ² , N ² , -dimethylguanosine in healthy subjects is about 15-50 nmol / L in plasma, and the amount in patients with renal disease is about 200-800 nmol / L. When the degree of dialysis effect to be attempted is high, it can be within the range of 50 to 200 nmol / L, more preferably within the range of 100 to 150 nmol / L. On the other hand, when the degree of dialysis effect to be achieved is low, it can be in the range of 600 to 800 nmol / L, more preferably in the range of 700 to 800 nmol / L. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it can be in the range of 200 to 500 nmol / L, more preferably in the range of 300 to 400 nmol / L.

The amount of kynurenine in healthy individuals is about 1.25 to 2.2 μmol / L in plasma, and the amount in kidney disease patients is about 2.2 to 5.5 μmol / L. For example, the degree of dialysis effect to be achieved is If it is high, it can be in the range of 1.25 to 2.2 μmol / L, more preferably in the range of 1.5 to 2.2 μmol / L, and still more preferably in the range of 2 to 2.5 μmol / L. On the other hand, when the degree of dialysis effect to be achieved is low, it is within the range of 3 to 5 μmol / L, more preferably within the range of 4 to 5 μmol / L, and even more preferably 2.5 to 4 μμmol / L. Can be within the range. Further, when the degree of dialysis effect to be achieved is in the middle of the above, it can be in the range of 2 to 4 μmol / L, more preferably in the range of 3 to 4 μmol / L.

Since the abundance of phenylacetylglutamine in healthy subjects is 18 μmol / L or less in plasma and the abundance in patients with renal disease is about 20 to 460 μmol / L, for example, the degree of dialysis effect to be achieved is If it is high, it can be in the range of 18 μmol / L or less, more preferably in the range of 18 to 20 μmol / L. On the other hand, when the degree of dialysis effect to be achieved is low, it is in the range of 200 to 460 nmol / L, more preferably in the range of 300 to 460 nmol / L, still more preferably 400 to 460 nmol / L. Can be within the range. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it can be in the range of 20 to 150 nmol / L, more preferably in the range of 50 to 100 nmol / L.

The abundance of hippuric acid in healthy individuals is less than 5 μmol / L in plasma, and the abundance in patients with renal disease is around 24 to 750 μmol / L. For example, the degree of dialysis effect to be achieved is high In some cases, it can be in the range of 5 μmol / L or less, more preferably in the range of 5 to 15 μmol / L, and still more preferably in the range of 15 to 24 μmol / L. On the other hand, when the degree of dialysis effect to be achieved is low, it is in the range of 500 to 750 μmol / L, more preferably in the range of 600 to 750 μmol / L, still more preferably 700 to 750 μmol / L. Can be within the range. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it can be in the range of 24 to 100 μmol / L, more preferably in the range of 50 to 80 μmol / L.

Since the abundance of N ₄ -acetylcytidine in healthy subjects is about 150 to 250 nmol / L in plasma and the abundance in patients with renal disease is about 300 to 400 nmol / L, for example, the dialysis effect to be achieved In the case where the degree of is high, it can be in the range of 150 to 250 nmol / L, more preferably in the range of 200 to 250 nmol / L. On the other hand, when the degree of the dialysis effect to be achieved is low, it can be in the range of 300 to 400 nmol / L, more preferably in the range of 350 to 400 nmol / L. Further, when the degree of dialysis effect to be achieved is intermediate between the above, it can be in the range of 250 to 400 nmol / L, more preferably in the range of 250 to 300 nmol / L.

The means for comparing the marker abundance evaluated by the marker abundance evaluation means with the reference abundance is a means for comparing the marker abundance evaluated by the marker abundance evaluation means with the reference abundance stored in the memory. There may be.

Further, the means for comparing the marker abundance evaluated by the marker abundance evaluation means with the reference abundance may be a means for allowing a person operating this apparatus to compare with the reference abundance. . For example, there may be provided means for visually or audibly transmitting the marker abundance evaluated by the marker abundance evaluation means to the person who operates the apparatus. As a means for visually transmitting, there is a display unit that displays the abundance of the marker. As a means for audibly transmitting, there is a voice transmitting section that informs the presence of the marker by voice.

Specifically, the means for ending dialysis may be a means for automatically ending dialysis, or a means for issuing a message for instructing the operator of the dialysis device to end dialysis. As a means for issuing such a message, for example, a means for visually displaying a message for instructing the end of dialysis on a separate display unit may be used, or a means for audibly transmitting it. Good.

Other explanation about the dialysis ending means is the same as the explanation about the kidney disease seriousness determination means in the apparatus for determining the renal disease seriousness of the present invention, and therefore will be omitted.

5). Method of operating a dialysis device used for a renal disease patient The method of operating a dialysis device used for a renal disease patient according to the present invention is (i) performing dialysis on a renal disease patient. Ii) activating means (I); (ii) activating means (II) so as to collect a blood-derived specimen from the renal disease patient; (iii) in the blood-derived specimen collected by step (ii) At least one selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, and N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine Actuating means (III) to assess the abundance of the markers of: (iv) comparing the abundance of the marker assessed by step (iii) with the reference abundance and If the abundance of over is less than the reference amount present is an operation method characterized by comprising the step of actuating the means (IV) to terminate the process (i).

The description of the blood-derived specimen and the marker is omitted because it is the same as the description of the apparatus for determining the severity of renal disease of the present invention and the description of the dialysis apparatus used for renal disease patients.

6). Kit for determining the severity of kidney disease <br/> The kit for determining the severity of kidney disease according to the present invention comprises choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine. , And N ₄ -acetylcytidine, a kit containing materials necessary for detecting at least one marker selected from the group consisting of N ₄ -acetylcytidine.

Materials necessary for detecting at least one marker selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine Is not limited as long as these markers can be detected. For example, the substance etc. which react specifically with these markers are mentioned. A marker can be detected by causing such a substance to act on a blood-derived specimen and detecting the presence or absence or concentration of the reaction product. Examples of substances that specifically react with these markers include enzymes that specifically metabolize these markers. For example, a hydrolase is used as an enzyme, and this hydrolase acts specifically on the marker to generate carbon dioxide (CO ₂ ) and water (H ₂ O), thereby reducing the oxygen (O ₂ ) concentration. In such a case, the marker abundance can be quantitatively analyzed by detecting the decrease in the oxygen concentration using, for example, an oxygen electrode.

7). Kit for judging dialysis effect The kit for judging the dialysis effect of the present invention comprises choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine , Hippuric acid, and N ₄ -acetylcytidine. The kit includes a material necessary for detecting at least one marker selected from the group consisting of N ₄ -acetylcytidine.

The explanation of the marker is the same as that in “Dialyzer used for renal disease patients”.

The material necessary for detecting the marker is the same as that described in the kit for determining the severity of kidney disease.

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the following examples.

1. A human blood-derived specimen was used to search for a marker that could be an indicator of the severity of renal disease or dialysis effect.

Plasma samples were used as blood-derived samples. As the plasma samples, 20 plasma samples separated after blood collection from a dialysis patient at a hospital were used. For comparison, 10 samples of dialysis waste liquid obtained from each dialysis patient were used. As a control sample, plasma samples were similarly obtained for 10 healthy individuals.

Dialysis patient information and ID are shown in Table 1.

LC-TOF MS was used as an analysis means. The analysis was performed as follows. The plasma specimen was centrifuged at 10,000 g for 15 minutes to remove the precipitate. This was subjected to solid phase extraction using a solid phase column (Sep-Pak Light Cartridges C18cartridge; Nippon Waters Co., Ltd.) according to the following procedure, and the obtained sample was used as a measurement sample to be subjected to LC-TOF MS.
[Solid phase extraction conditions]
Procedure 1. Condition the solid phase column with 1 mL MetOH.
Procedure 2. Equilibrate the solid phase column with 1 mL ultrapure water.
Procedure 3. Load 500 μL of plasma sample onto a solid phase column.
Procedure 4. Wash with 2 mL of ultrapure water.
Procedure 5. Elute with 90 μM 90% MetOH.
Procedure 6. To the eluted sample, benzyloxycarbonyl-L-tyrosyl-L-glutamic acid (Z-Tyr-Glu) and tosyl-L-arginine methyl ester monohydrochloride (Tos-Arg-OMe.HCl) were added as internal standard substances.

This measurement sample was subjected to LC-TOF MS. The conditions for liquid chromatography (LC) were as follows.
[LC condition]
LC equipment: Agilent 1100 Series (JEOL)
Column: Cadenza C18 2 x 150 mm, 3 mm (Imtakt)
Pre-column: TCI OPTI-GURD Fit ODS (TCI)
Mobile phase A: 0.05% Formic acid mobile phase B: Acetonitrile elution: Mobile phase B linear gradient from 5% to 95% in mobile phase A 0-15 min
Mobile phase B linear gradient from 95% to 100% in mobile phase A 15-20 min
100% mobile phase B retention 20-35 min
Flow rate: 0.2 mL / min
Measurement sample volume: 5μL
The conditions of time-of-flight mass spectrometry (TOF MS) were as follows.
[TOF MS conditions]
Measurement range: m / z 70 to 1,000
Orifice 1 voltage: 10 to 40 V, -10 to (-40) V Swept ion guide voltage: 500 to 2,500 V Sweep detector: 2,800 V
Measuring device: The Accu TOF JMS-T100LC (JEOL)
Two kinds of amino acid derivatives as internal standard substances were mixed and analyzed in plasma samples before and after dialysis, dialysis waste fluid, and plasma samples of healthy individuals, and [M + H] ⁺ (ESI Positive) ions or [MH] ^- (ESI Negative) The value obtained by dividing the peak area of the ion by the average value of the peak areas of the internal standard was compared as a relative area.

In addition, a significant difference test using a multiple test Tukey-Kramer method was performed on these peaks. Significant difference tests were performed for pre-dialysis vs. post-dialysis vs. pre-dialysis vs. healthy subjects and post-dialysis vs. healthy subjects, respectively.

As an index of dialysis effect, the amount of creatinine contained in each dialysis patient's pre-dialysis plasma sample (Pre-HD), post-dialysis plasma sample (Post-HD), and healthy human plasma sample (Healthy Control) is measured. did. The results are shown in FIGS.

As a result, the amount of creatinine in the plasma sample was 9.6 ± 3.11 mg / dL before dialysis and 4.67 ± 2.03 mg / dL after dialysis. From this result, it was found that the amount of creatinine was reduced by about 50% by performing dialysis.

From the plasma sample before dialysis, the 351 peak in the Positive mode and the 214 peak in the Negative mode were picked up, the respective peaks were quantified, and the relative area was calculated. Based on the retention time of HPLC analysis and the exact mass (m / z) (mass-to-charge ratio) of MS analysis, “retention time_mass-to-charge ratio (m / z)” Each peak was named as follows (the value is up to the second decimal place, and the decimal point is abbreviated). Among these peaks, there are 241 peaks detected by positive ion measurement [M + H] ⁺ and 209 peaks detected by negative ion measurement [MH] ⁻ with relative area before dialysis of 0.0005 or more. Each was analyzed.

In order to examine markers specific to dialysis patients, these peaks were subjected to a significant difference test by the multiple test Tukey-Kramer method. The tests were performed before dialysis vs. after dialysis, before dialysis vs. healthy person, and after dialysis vs. healthy person, respectively. The results are shown in Table 2. The test results of positive ion measurement [M + H] ⁺ and negative ion measurement [MH] ⁻ are shown in Tables 3 to 5 and Tables 6 to 8, respectively. In the table, “**” indicates that there is a difference at the 1% level (p <0.01), and “*” indicates that there is a difference at the 5% level (p <0.05). From this result, it was found that about 25% of the components were significantly (p <0.01) different between dialysis patients and healthy individuals.

Furthermore, among the peaks with significant difference (P <0.01) between plasma components before dialysis and healthy subjects, the components that are mostly contained in patients before dialysis are 46 peaks in positive ion measurement [M + H] ⁺ , negative ion measurement [MH ] ^- in was 46 peak. Furthermore, among the peaks that are significantly different from healthy individuals, there are 18 peaks (2.03_10411, 2.26_28612) of positive ion measurement [M + H] ⁺ that are abundant in dialysis patients and decrease significantly by dialysis. , 3.70_22916, 3.80_25712, 5.43_28315, 6.52_31215, 6.87_23217, 8.29_23113, 8.29_49526, 8.32_26513, 8.32_52928, 8.51_18008, 8.64_30415, 9.24_38924, 9.36_52732, 9.87_44725, 9.91_54136, 10.54_0023) And negative ion measurement [MH] ⁻ showed 6 peaks (3.93_19714, 5.55_32728, 6.57_21117, 7.19_41131, 8.25_26322, and 8.32_30924).

5.43_28315 was N ₁ -methylinosine, 6.52_31215 was N ² , N ² , -dimethylguanosine, 2.03_10411 was choline, and 6.87_23217 was O-butanoylcarnitine.

It was estimated that 2.26_28612 is N ₄ -acetylcytidine, 8.32_26513 and 8.25_26322 are phenylacetylglutamine, 8.32_52928 is phenylacetylglutamine dimer, and 8.51_18008 is hippuric acid.

None of the remaining peaks were consistent with previously reported uremic substances. Taking into account measurement equipment errors, etc., 3.70_22916 is positive ion measurement with LC-TOF MS under the above conditions [M + H] ⁺ , retention time is 3.70 ± 0.5 (min), and accurate mass (m / z) is 229.14 It is a peak derived from a substance not known as a urine toxic substance, which is ± 10 mDa (mDa: millidalton 1 mDa = 0.001 amu).

Similarly, 3.80_25712 is a positive ion measurement [M + H] ⁺ with a retention time of 3.80 ± 0.5 (min) and an accurate mass (m / z) of 257.11 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 8.29_23113 is a positive ion measurement [M + H] ⁺ with a retention time of 8.29 ± 0.5 (min) and an accurate mass (m / z) of 231.10 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 8.29_49526 is a positive ion measurement [M + H] ⁺ with a retention time of 8.29 ± 0.5 (min) and an accurate mass (m / z) of 495.21 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 8.64_30415 is a positive ion measurement [M + H] ⁺ with a retention time of 8.64 ± 0.5 (min) and an accurate mass (m / z) of 304.12 ± 10 mDa, not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 9.24_38924 is a positive ion measurement [M + H] ⁺ with a retention time of 9.24 ± 0.5 (min) and an accurate mass (m / z) of 389.21 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 9.36_52732 is a positive ion measurement [M + H] ⁺ with a retention time of 9.36 ± 0.5 (min) and an accurate mass (m / z) of 527.28 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 9.87_44725 is a positive ion measurement [M + H] ⁺ with a retention time of 9.87 ± 0.5 (min) and an accurate mass (m / z) of 447.22 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 9.91_54136 is a positive ion measurement [M + H] ⁺ with a retention time of 9.91 ± 0.5 (min) and an accurate mass (m / z) of 541.31 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 10.54_30023 is a positive ion measurement [M + H] ⁺ with a retention time of 10.54 ± 0.5 (min) and an accurate mass (m / z) of 300.21 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Similarly, 3.93_19714 negative ions measured [MH] ^- retention time is 3.93 ± 0.5 (min) and precise mass (m / z) is 197.06 ± 10 mDa, the materials are not known as uremic substances It is a derived peak.

Similarly, 5.55_32728 is a negative ion measurement [MH] ⁻ with a retention time of 5.55 ± 0.5 (min) and an accurate mass (m / z) of 327.09 ± 10 mDa. It is a derived peak.

Similarly, 6.57_21117 is a negative ion measurement [MH] ⁻ with a retention time of 6.57 ± 0.5 (min) and an accurate mass (m / z) of 211.08 ± 10 mDa. It is a derived peak.

Similarly, 7.19_41131 is a negative ion measurement [MH] ⁻ with a retention time of 7.19 ± 0.5 (min) and an accurate mass (m / z) of 411.13 ± 10 mDa, which is not known as a urine toxic substance. It is a derived peak.

Similarly, 8.32_30924 is a negative ion measurement [MH] ⁻ with a retention time of 8.32 ± 0.5 (min) and an accurate mass (m / z) of 309.10 ± 10 mDa, which is not known as a urine toxic substance It is a peak derived from a substance.

Positive ion measurement [M + H] ⁺ is abundant in dialysis patients and is significantly reduced by dialysis 18 peaks before dialysis, after dialysis, and the amount contained in healthy human plasma samples as relative area As a result of comparison, the three markers 3.80_25712, 5.43_28315 (N ₁ -methylinosine), and 6.52_31215 (N ² , N ² , -dimethylguanosine) were significantly dialyzed after dialysis and in healthy subjects at p <0.01. (Fig. 3). Note that “**” in post-dialysis (Post-HD) and healthy individuals (Healthy Control) is different at a 1% level compared to that before pre-dialysis (Pre-HD) (p <0.01). )It is shown that. “†” indicates that there is a difference of 1% level (p <0.01) between post-dialysis (Post-HD) and healthy subjects (Healthy Control).

This indicates that these three markers are not sufficiently removed. The other 15 peaks do not show a significant difference between dialysis and healthy people, indicating that dialysis is a substance that can be sufficiently removed to the level of healthy people.

The dialysis time can be adjusted more appropriately by performing dialysis using these three markers as indices. In addition, by using these three markers, dialysis treatment can be performed until the component becomes closer to the plasma component of a healthy person.

Negative ion measurement [MH] ^- before dialysis 6 peak significantly reduced by dialysis included in many dialysis patients were compared after dialysis, and the amount contained in a healthy person's plasma sample as a relative area As a result, since there is no significant difference between the dialysis and the healthy person, all of these are substances that can be sufficiently removed to the normal person level by dialysis.

2. Each peak shown in Table 9 was analyzed by another time-of-flight mass spectrometry (TOF MS). The analysis was performed in the same manner as described above except that the analysis was performed under the following conditions.
[LC condition]
LC equipment: Prominence (Shimadzu)
Column: Cadenza C18 2 x 150 mm, 3 mm (Imtakt)
Pre-column: TCI OPTI-GURD Fit ODS (TCI)
Mobile phase A: 0.05% Formic acid mobile phase B: Acetonitrile elution: Mobile phase B linear gradient from 5% to 95% in mobile phase A 0-15 min
Mobile phase B linear gradient from 95% to 100% in mobile phase A 15-20 min
100% mobile phase B retention 20-35 min
Flow rate: 0.2 mL / min
Measurement sample volume: 5μL
[TOF MS conditions]
Measurement range: m / z 100 to 1,000
Probe voltage: 4.5kV
Curved desolvation line: 200 oC
Nebulizer gas flow rate: 1.5 L / min
Measuring device: LCMS-IT-TOF (Shimadzu)
The results are shown in Table 9. As for the italic component, although some of the components detected during the measurement are similar in numerical value, they are considered to be different components. A blank indicates that a component corresponding to the component detected during the above-described measurement was not detected.

PB is plasma and d is dialysis waste liquid as a sample, and the numbers indicate specimen numbers. JEOL shows the accurate mass measured by The Accu TOF T100LC and Shimadzu measured by LCMS-IT-TOF. Since the data measured by JEOL uses two internal standard substances, the data corrected for each internal standard substance are indicated as Tos (342) and Z (444).

3. A new analysis was performed using a different peak narrowing method from that described above.

Investigated 10 dialysis patients and 16 healthy people. The information is shown in Table 10. The average age of dialysis patients was 64.3 ± 13.3 years, and the average age of healthy individuals was 46.7 ± 14.7 years. The averages of Kt / V, urea reduction rate (URR), plasma creatinine concentration, and blood urea nitrogen (BUN) were 1.092 ± 0.169,67 ± 4%, 11.66 ± 2.86 mg / dL, and 68.2 ± 10.7%, respectively. It was. From the results of biochemical tests, no abnormal values were found in 10 patients.

Fig. 5 shows a simplified flow of narrowing down the peaks. LC-MS analysis showed an SN ratio of 20 or more, and there were 350 peaks of ESI-positive and 211 peaks of ESI-negative in the plasma of patients before dialysis. Analysis of variance (ANOVA) test was performed for these peaks, and statistical processing was performed by Tukey-Kramer method for peaks that were significantly different by ANOVA test.

Table 11 shows the results of the Tukey-Kramer method. Approximately 30% of the components differed between pre-dialysis and healthy individuals. Of these peaks, 76 ESI-positive peaks and 92 ESI-negative peaks were present more in dialysis patients than in healthy individuals. Furthermore, among these peaks, 22 ESI-positive peaks and 32 ESI-negative peaks were components that were significantly reduced by dialysis. Table 12 shows the result of each peak of the Tukey-Kramer method and the variation (Coefficient （of variance: CV) within the peak. ** indicates a significant level of 1%, and * indicates a significant level of 5% and a peak with a difference between groups.

The results of comparing the measurement results before and after dialysis of the ESI-positive 22 component and the ESI-negative 32 component of the marker candidate were able to divide these markers into the following three groups. Group A: There are variations among individual patients, and dialysis reduces to the level of healthy individuals. Group B: There are variations among individuals, and even if dialysis is performed, it does not decrease to the level of healthy people. Group C: There is no variation among individuals.

グ ル ー プ Group A had 43 peaks (Positive: 14 peaks, Negative: 29 peaks). These peaks were significantly different from each other before dialysis vs. healthy subjects, before dialysis vs. after dialysis, and there were variations within the peaks. 6 and 7 show peaks in which a difference was recognized at a significance level of 1% among the A groups.

B group had 6 あ っ た peaks (Positive: 3 peaks, Negative: 3 peaks) (Fig. 8). These peaks were significantly different between all groups of pre-dialysis vs. healthy subjects, pre-dialysis vs. dialysis, and post-dialysis vs. normal subjects, and variations were observed within the peaks.

C group had 5 peaks (Positive: 5 peaks) (Fig. 9). These peaks existed at a certain level because of small variations within the peaks. These peaks were significantly different among all groups.

Next, comparison was made with creatinine, BUN, Kt / V, and URR, which are currently widely used as therapeutic markers. Table 13 shows the correlation function between the current general-purpose marker and the marker candidate component found this time. ** indicates a peak at which correlation was observed at a significance level of 1%, and * indicates a correlation at a significance level of 5%. No. 1 (choline), 11 (N ² , N ² , -dimethylguanosine), 18, and 22 showed a correlation with creatinine, and only No. 1 (choline) showed a negative correlation. In particular, No. 11 (N ² , N ² , -dimethylguanosine), 22 was highly correlated with creatinine at p <0.01.

Regarding the correlation with BUN, No. 13 showed a negative correlation and No. 43 showed a positive correlation, but no correlation was observed in the other peaks.

Regarding the correlation with Kt / V, 3, 5, 23 and 53 (N-α-acetyl-L-arginine) showed a negative correlation.

Regarding the correlation with URR, Nos. 21, 30, and 51 showed a positive correlation.

Finally, among these candidate marker components, an investigation was made as to whether they match the uremic substances reported so far. We searched the Human Metabolome Database and Mass Bank for substances that correspond to 90 components of urine toxic substances reported so far from accurate mass, and identified urine toxic substances from product ions. As a result, as shown in Table 14, 11 peaks coincided with 9 components. No. 7 shows m / z 283.0987, and this component was tentatively estimated from HMDB to be l-methylinosine (N ₁ -methylinosine) (metabocardHMDB02721). m / z 151.0632 was obtained from the product ion of m / z 283.0987, and was identified as l-methylinosine because it was consistent with the reported value.

No. 9 indicates m / z 209.0908, and it was estimated from HMDB as kynurenine (metabocardHMDB00684). M / z イ オ ン 192.0681 was obtained from the product ion of No.9, and was identified as Kynurenine because it matched the pattern of Massbank database (www.massbank.jp).

No. 10 shows m / z 286.0951, which was estimated from HMDB to be N ₄ -acetylcytidine (HMDB04824). m / z 154.0608 was obtained from the product ion of m / z 286.0951, and was identified as N ₄ -acetylcytidine because it was in agreement with the reported value.

Nos. 11 and 17 were estimated from HMDB to be N ² , N ² , -dimethylguanosine (HMDB04824) and hippuric acid (HMDB00714), respectively. These substances were identified as N ² , N ² , -dimethylguanosine and hippuric acid, respectively, because they were consistent with the measurement results of the standard substances. Nos. 15, 16, and 31 were estimated as Phenylacetylglutamine (HMDB06344) from HMDB. However, no fragmentation pattern of this material has been reported so far. Similarly, m / z 212.0028, 377.1887, and 215.1223 were obtained for Nos. 32, 51, and 53, respectively, and were estimated to be indoxyl sulfate, pentosidine, and N-α-acetyl-L-arginine.

The remaining 43 peaks did not match any known uremic substances.

Creatinine is known as one of the substances that accumulate in the body due to kidney damage. In fact, creatinine is currently used as an indicator of renal function. It has been reported so far that creatinine levels are significantly reduced by dialysis. As a result of examining the change in creatinine due to dialysis, the present inventors have found that dialysis significantly reduces creatinine after dialysis but does not decrease to the level of healthy individuals. Currently, dialysis is performed using Kt / V as an index, but it can be said that creatinine is not sufficiently removed by this method.

According to the study by the present inventors, as described above, the components of group A are reduced to the level of healthy individuals by the current dialysis treatment using Kt / V as an index. Ingredients cannot be reduced to the level of healthy individuals by current dialysis treatment using Kt / V as an index.
No.9 (kynurenine), No.15, 16, 31 (phenylacetylglutamine), 17 (hippuric acid), No.32 (indoxyl sulfate), No.51 (pentosidine), No.53 (N-α) -Acetyl-L-arginine) belongs to group A, but if the components of group A are not reduced to the level of healthy individuals during dialysis treatment, it is an indication that dialysis treatment is insufficient.
In contrast to group A, group B is a component that does not decrease to the level of a healthy person. Therefore, if it decreases to the level of a healthy person during dialysis treatment, it can be determined that the dialysis treatment is excessive.
No. 7 (l-methylinosine), No. 10 (N ₄ -acetylcytidine), and No. 11 (N ² , N ² , -dimethylguanosine) belong to group C, but their weight, sex, dialysis history, etc. It is a component that is not affected by the background. Group C can be used as a substitute for creatinine, which is an indicator of renal function, because there is little variation among individuals.
As a result, Group C can also determine the timing of dialysis introduction by using it as an index that indicates the rate of decrease due to dialysis, such as URR, or as an index for evaluating renal function, such as creatinine or BUN. In addition, combining Group A, Group B, and Group C provides an optimal dialysis volume index tailored to the individual.

Claims (8)

1. A method for determining the severity of kidney disease,
   (A) at least one marker selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived specimen (B) when the abundance of at least one of the markers evaluated in step (A) is greater than or equal to the reference abundance, serious kidney disease in the donor of the blood-derived specimen A method comprising the step of determining that the degree is high.
2. A device for determining the severity of kidney disease comprising:
   (A) At least one selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived sample collected from a human Means for evaluating the abundance of a marker of the species; and (B) comparing the abundance of the marker evaluated by means (A) with a reference abundance, wherein the abundance of at least one of the evaluated markers is the reference abundance In such a case, the apparatus comprises means for determining that the severity of renal disease in the blood-derived sample provider is high.
3. A method of operating a device according to claim 2, comprising:
   (A) at least one selected from the group consisting of choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine in a blood-derived sample collected from a human Actuating means (A) to assess the abundance of a marker of a species; and (b) comparing the abundance of the marker assessed by step (a) with a reference abundance and determining at least one When the abundance of the marker is greater than the reference abundance, the method comprises the step of operating the means (B) so as to determine that the severity of renal disease in the blood-derived sample provider is high. Actuation method.
4. A method of dialysis against a patient with renal disease:
   (I) a step of performing dialysis on a renal disease patient;
   (II) a step of collecting a blood-derived specimen from the renal disease patient;
   (III) Choline, O-butanoylcarnitine, N ₁ -methylinosine, and N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid in the blood-derived specimen collected by step (II), and Evaluating the abundance of at least one marker selected from the group consisting of N ₄ -acetylcytidine;
   (IV) The abundance of the marker evaluated in step (III) is compared with the reference abundance, and if the abundance of all the evaluated markers is less than or equal to the reference abundance, dialysis by means (I) is terminated. A method comprising the step of:
5. A dialysis machine used for patients with kidney disease:
   (I) means for dialysis of patients with renal disease;
   (II) means for collecting a blood-derived specimen from the renal disease patient;
   (III) Choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N in blood-derived specimens collected by means (II) Means for assessing the abundance of at least one marker selected from the group consisting of ₄ -acetylcytidine;
   (IV) The abundance of the marker evaluated by the means (III) is compared with the reference abundance, and if the abundance of all the evaluated markers is less than the reference abundance, the dialysis by the means (I) is terminated. An apparatus comprising: means for:
6. A method for operating a dialysis device according to claim 5, wherein:
   (I) actuating means (I) to perform dialysis on a renal disease patient;
   (Ii) actuating means (II) to collect a blood-derived sample from the patient with renal disease;
   (Iii) Choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N in the blood-derived specimen collected in step (ii) Activating means (III) to assess the abundance of at least one marker selected from the group consisting of ₄ -acetylcytidine;
   (Iv) Compare the abundance of the marker evaluated in step (iii) with the reference abundance, and end the step (i) if the abundance of all the evaluated markers is equal to or less than the reference abundance. A method of operating the means (IV).
7. A kit for determining the severity of kidney disease, at least selected from the group consisting of choline O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, and N ₄ -acetylcytidine A kit containing the materials necessary to detect one type of marker.
8. A kit for determining the dialysis effect comprising choline, O-butanoylcarnitine, N ₁ -methylinosine, N ² , N ² , -dimethylguanosine, kynurenine, phenylacetylglutamine, hippuric acid, and N ₄ -acetylcytidine A kit comprising materials necessary for detecting at least one marker selected from the group consisting of:

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