JP4735503B2 - Peritoneal function marker, analysis method thereof and use thereof - Google Patents

Description

  The present invention relates to a marker serving as an index for diagnosing peritoneal function, an analysis method thereof, and a use thereof.

  It is known that when peritoneal dialysis, which is a therapy for renal failure, is performed over a long period of time, the function of the patient's peritoneum tends to decrease due to the stimulation. This causes an increase in peritoneal permeability, resulting in a reduced dialysis effect and a risk of developing encapsulating peritoneal sclerosis (EPS).

  For this reason, in the field of dialysis medicine, it is necessary to diagnose peritoneal function decline in dialysis patients at an early stage. Currently, PET (Peritoneal Equilibration Test) is adopted as a test method for peritoneal function (non-peritoneal test). Patent Document 1). PET stores the dialysate in the patient's peritoneum, measures the changes in creatinine concentration and glucose concentration in the dialysate during the storage period (that is, the dialysis drainage after the storage period) and blood, and removes the peritoneal waste. It is a method for evaluating peritoneal function based on the evaluation results. In addition, the fluctuation | variation of a creatinine density | concentration becomes a parameter | index of waste removal ability, and the fluctuation | variation of glucose concentration becomes a parameter | index of water removal ability. The change in creatinine concentration is the ratio of the creatinine concentration in the dialysate (D) to the blood creatinine concentration (P) at any time during dialysis treatment, the value of “D / P creatinine”; It is evaluated by the value of the ratio “D / D0” between the glucose concentration in the dialysate and the glucose concentration in the dialysate immediately after the injection. The relationship between D / P creatinine and D / D0 and each category of PET is disclosed in, for example, Non-Patent Document 2. For example, the higher value of D / P creatinine, that is, the higher permeability Are classified into four categories of “High”, “High Average”, “Low Average”, and “Low”. Therefore, D / P creatinine and D / D0 of dialysis patients are measured and classified into four categories by PET. According to the classification result, adjustment of osmotic pressure of dialysate, dialysis frequency, hemodialysis By determining the combined use and the shift to hemodialysis, for example, it is useful for preventing the complications as described above. The four categories of PET will be described in more detail. “High”, which has a high permeability, has a relatively high waste removal ability, a low water removal ability, and a “High Average”, which has a relatively high permeability. In general, the waste removal ability is normal, the water removal ability is slightly low, and the permeability is slightly low. “Low Average” has a relatively low waste removal ability and the water removal ability is normal, and the permeability is low. “Low” is evaluated as having relatively low waste removal ability and high water removal ability.

However, this PET requires the dialysate to be drained several times after the start of dialysis (for example, 0 hours, 2 hours and 4 hours after the start), and moreover, blood sampling is also required. It becomes. For this reason, a patient is restrained for a long time for the drainage collection | recovery for every predetermined time, Furthermore, there exists a problem that it takes the pain of blood collection, and both a patient and a doctor take effort. In addition, even in the fast PET method, which is a simplified version of the original PET method, the time for which the patient is restrained to collect the dialysate is the same, and at least three types of dialysate drained over time and blood Since it is necessary to measure the glucose concentration and the creatinine concentration for a total of four types, the measurement itself takes time.
Twardowski ZJ, et al. Peritoneal equilibration test. Perit Dial Bull 1987; 7: 138-47. “Fundamentals of peritoneal dialysis”, Tokyo Medical Company, Akiyasu Yamashita, Date of issue October 1, 1998

  Therefore, an object of the present invention is to provide a new marker for peritoneal function (peritoneal permeability) and an analysis method thereof for providing a new method for diagnosing peritoneal function.

  The peritoneal function marker of the present invention is a marker for diagnosis of peritoneal function, and includes at least one protein selected from the group consisting of leucine-rich α2 glycoprotein, fibrinogen gamma, C4a and albumin precursor. And

  The analysis method of the present invention is a method for analyzing a peritoneal function marker, the marker being the marker of the present invention, comprising a step of qualitatively or quantitatively determining the marker contained in the drainage of peritoneal dialysis. Features.

A method for determining a risk of a decrease in peritoneal function of a subject, comprising the following steps (A) and (B).
(A) The step of measuring the peritoneal function marker of the present invention contained in the peritoneal dialysis drainage of the subject by the analysis method of the present invention (B) The present invention contained in the peritoneal dialysis drainage of a patient whose peritoneal function is known A step of determining a risk of a decrease in the peritoneal function of the subject by comparing the reference value with the measured value of the subject in the step (A) using the measured value of the peritoneal function marker of

  As a result of intensive studies, the present inventors have found that the content of a group of proteins in peritoneal dialysis drainage varies depending on the peritoneal function (peritoneal permeability) of a peritoneal dialysis patient. That is, in the peritoneal dialysis effluent of patients with high peritoneal permeability and those with low peritoneal permeability, there was a change in the content of a total of four proteins, leucine-rich α2 glycoprotein, fibrinogen gamma, C4a, and albumin precursor. It was. For this reason, if these proteins are used as indicators of peritoneal function and the presence and concentration in the dialysis drainage obtained by peritoneal dialysis are qualitatively and quantitatively determined, the peritoneal function of a peritoneal dialysis patient can be diagnosed based on this result. Therefore, if the marker of the present invention and the analysis method thereof are used for the examination of peritoneal function, a new diagnostic method is provided. In addition, as described above, PET that has been widely adopted conventionally needs to collect both dialysis drainage and blood as samples, but if the analysis method of the present invention is used, only dialysis drainage is collected. This is enough to reduce patient pain. Furthermore, when the analysis method of the present invention is used, since one sample of dialysis drainage is sufficient for the sample used for the peritoneal function test, it is necessary to collect a plurality of test samples and from PET having a plurality of test items. However, the operation itself is simple. Therefore, if the present invention is used, the labor of both the patient and the doctor can be reduced and the peritoneal function can be easily tested.

  As described above, the marker of the present invention is a marker for diagnosis of peritoneal function, and is at least one protein selected from the group consisting of leucine-rich α2 glycoprotein, fibrinogen gamma, C4a, and albumin precursor. It is characterized by that.

  Among these four types of markers, the protein showing relatively high peritoneal permeability is leucine-rich α2 glycoprotein, and the proteins showing relatively low peritoneal permeability are fibrinogen gamma, C4a and albumin. It is a precursor. Specifically, when the permeability is relatively high, for example, the leucine-rich α2 glycoprotein concentration in the dialysis drainage is higher than the drainage with a relatively low permeability, and the other three types When the permeation capacity is relatively low, for example, the concentration of leucine-rich α2 glycoprotein in the effluent is lower than the drainage with a relatively high permeation capacity. Each of the three protein concentrations increases. Therefore, as described later, by qualitative or quantitative determination of these proteins in dialysis drainage, if leucine-rich α2 glycoprotein is present or has a relatively high concentration, it can be determined that the peritoneal permeability is relatively high, If fibrinogen gamma, C4a and albumin precursor are present or at a relatively high concentration, it can be determined that the peritoneal permeability is relatively low. A specific determination method will be described later.

  Next, as described above, the analysis method of the present invention is a method for analyzing a peritoneal function marker, wherein the marker is the marker of the present invention, and the marker contained in the drainage of peritoneal dialysis is qualitatively or quantitatively determined. Including the step of: In this analysis method, only one kind of marker protein may be qualitatively or quantitatively determined. However, in order to analyze in more detail, leucine-rich α2 glycoprotein showing relatively high peritoneal permeability, It is preferable to qualitatively or quantitate any one (or two or more) of fibrinogen gamma, C4a and albumin precursor, which show that the peritoneal permeability is relatively low.

  Although an example of the analysis method of the present invention is shown below, the present invention is not limited to this.

  First, the dialysate stored in the patient's peritoneum is drained. The storage time is not particularly limited, but it is preferable to keep the conditions between patients and each analysis constant. As a specific example, for example, a sample obtained by draining dialysate previously stored in the abdominal cavity for 2 to 12 hours is used as an analysis sample, and preferably stored for 8 to 12 hours.

  Subsequently, the qualitative or quantitative determination of the marker of the present invention in the obtained analysis sample is performed. The qualitative method and the quantitative method are not particularly limited, and examples thereof include immunological techniques and various electrophoresis methods such as one-dimensional electrophoresis and two-dimensional electrophoresis. Examples of the immunological technique include a conventionally known method using an antigen-antibody reaction. For example, an ELISA (Enzyme-Linked Immuno Sorbent Assay) method, a Western blotting method, or the like can be employed. Among the above-mentioned electrophoresis methods, according to the two-dimensional electrophoresis method, all marker proteins can be separated by one electrophoresis (one-dimensional and two-dimensional electrophoresis). The marker protein can be qualitatively or quantified all at once from the presence or absence of the marker protein spot and the density of the spot. Further, the analysis result of the marker by qualitative or quantitative analysis may be calculated as a protein amount by using a calibration curve prepared in advance, for example, and the intensity of the spot may be converted into a numerical value, and is not particularly limited.

  Next, a method for examining peritoneal function using the analysis method of the present invention will be described.

  The marker of the present invention, as will be shown in the examples described later, is a result of studying dialysis drainage of patients classified by PET into “High”, “High Average”, “Low Average” and “Low”. It is a protein identified by the inventor's finding that the presence or absence and concentration vary depending on the category. Therefore, if the peritoneal function marker in the dialysis drainage is qualitatively or quantified by the analysis method of the present invention described above, for example, the patient's PET category can be determined from the analysis result. That is, by analyzing only the dialysis drainage by a method different from PET, it is possible to evaluate which PET category it corresponds to. PET is only classified into four categories, but if the marker is quantified by the analysis method of the present invention, the level of permeability is evaluated in more detail even for patients classified into the same category. It is also possible.

  An example of an inspection method for peritoneal function is shown below. In addition, it is not limited to the following content except using the analysis method of this invention and using the marker of this invention as a parameter | index.

(First form)
The drained sample to be examined is subjected to two-dimensional electrophoresis, and the gel after electrophoresis is stained. The conditions for two-dimensional electrophoresis, the total protein amount of the sample to be subjected to electrophoresis, and the like are preferably constant between samples. The staining method is not particularly limited, and examples thereof include fluorescent staining such as silver staining, Coomassie brilliant blue (CBB) staining, and SYPRO Ruby staining (trade name).

  And the spot of the marker protein in the electrophoresis gel after dyeing | staining is confirmed. Here, it can be determined that the darker the leucine-rich α2 glycoprotein spot is, the higher the permeation ability, that is, the “High” category of PET, while the darker the spots corresponding to fibrinogen gamma, C4a and albumin precursor are. It can be determined that the patient has low permeability, that is, corresponds to the “Low Average” or “Low” category.

(Second form)
In order to classify into the PET category in more detail using the analysis method of the present invention, the following method can be used.

  First, as a control, drainage of patients who have been classified in the PET category, for example, drainage of four PET category patients, or patients of “High” category and “Low” category (and “Low Average” category) ” The effluent is subjected to two-dimensional electrophoresis, and the electrophoresis gel is stained. On the other hand, two-dimensional electrophoresis and gel staining are performed on the drainage sample to be examined under the same conditions. Then, by comparing the electrophoretic image of the control with the electrophoretic image of the test object and comparing the presence / absence of the marker spot or the density of the staining, it is evaluated that the marker belongs to the PET category exhibiting the same behavior. .

(Third form)
Classification into the PET category can also be performed by measuring the concentration of the marker in the drainage sample to be examined.

  First, as an evaluation criterion, drainage of patients classified into the PET category, for example, drainage of four PET category patients or “High” category and “Low” category (and “Low Average” category) About drainage, the content rate of each marker protein per fixed amount of total protein is measured. On the other hand, the content rate of each marker protein per fixed amount of the total protein is measured in the same manner for the drainage sample to be examined. Then, the content ratio of each marker protein in the drainage sample to be examined is compared with the result of the evaluation standard, and it is evaluated that it belongs to the category of the evaluation standard indicating the same content ratio.

Furthermore, the present invention is a method for determining a risk of a decrease in the peritoneal function of a subject, and includes the following steps (A) and (B).
(A) The step of measuring the peritoneal function marker of the present invention contained in the peritoneal dialysis drainage of the subject by the analysis method of the present invention (B) The present invention contained in the peritoneal dialysis drainage of a patient whose peritoneal function is known A step of determining a risk of a decrease in the peritoneal function of the subject by comparing the reference value with the measured value of the subject in the step (A) using the measured value of the peritoneal function marker of

  Although the reference value in the step (B) is not particularly limited, for example, the PET category is a measurement value of a patient that is at least one category selected from the group consisting of High, High Average, Low Average, and Low. Is preferred. Specific examples include the evaluation criteria described in the above-described peritoneal function testing method.

  In this way, by comparing the reference value of the marker of the present invention indicating each category with the reference value of the subject, it can be determined which category belongs and which category is approaching. For this reason, according to the present invention, it is possible to determine the degree of risk that the peritoneal function of the subject is reduced, and in particular, by periodically analyzing the amount of the marker contained in the dialysis drainage according to the present invention, the fluctuation of the peritoneal function Etc., and can be reflected in changes in the treatment method, for example.

  EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention further more concretely, this invention is not limited to these.

  Dialysate in the abdominal cavity for 5 patients classified into “High” category, 2 patients classified into “Low Average” category, and 3 patients classified into “Low” category by conventional PET examination Was stored for 8 to 12 hours, and then drained. 4 ml of the effluent from peritoneal dialysis was poured into the trade name Amicon Ultra 4 (Millipore) and centrifuged at 2330 g for 20 minutes, and the protein in the effluent was concentrated by ultrafiltration. The protein concentration in the concentrated effluent was measured using a reagent (trade name DC Protein Assay: manufactured by Nippon Bio-Rad Laboratories).

  The concentrated effluent was suspended in 300 μl of a swelling buffer containing 8 M urea, 4 wt% CHAPS, 1 wt% DTT and 2 wt% amphoteric electrolyte so that the protein was 120 μg to prepare a sample. After this sample was added to an isoelectric focusing gel (trade name IPG strip gel: manufactured by Nippon Bio-Rad Laboratories), isoelectric focusing was performed in the first dimension and SDS-PAGE was performed in the second dimension. The gel was silver stained to visualize protein spots. Then, the electrophoretic image visualized in the two-dimensional electrophoresis gel was captured in a computer with a densitometer (trade name GS-800: manufactured by Nippon Bio-Rad Laboratories). FIG. 1 shows an example of an electrophoretic image using high category patient dialysis drainage, and FIG. 2 shows an example of an electrophoretic image using low category patient dialysis drainage.

  The obtained image data was analyzed by image analysis software (trade name PD-Quest: manufactured by Nippon Bio-Rad Laboratories). Then, protein spots that can be regarded as identical from the spot distribution were recognized (matched) between the gels using the samples of each patient. There were 280 spots matched.

  Subsequently, the concentration in the drainage was calculated for each patient for 280 spots. The concentration of the target spot was a value obtained by dividing “the darkness of the target spot” by “the darkness of the entire gel” for each gel. This standardized the density of staining between gels.

  And from the density | concentration of 280 spots in each patient's drainage, the average value of a High patient group and the average value of a Low Average-Low patient group are each calculated, These are compared and 1.5 times or more are calculated. A spot with a difference was selected. Further, among the selected spots, t-test was performed between the High patient group and the Low Average-Low patient group, and nine spots with p <0.05 were selected. The spots selected in two stages in this manner are spots that clearly have different contents in the drainage between the High patient group and the Low Average-Low group, and are considered to be proteins showing differences in the PET category. It is done. In addition, the average value and the result of the t-test are shown in Table 1 below for the proteins whose types are specified as shown below among the nine spots.

  Nine spots finally selected were cut out from the gel, and by a known method (Toshiaki Taniguchi, Miki Kikuchi "Latest Proteome Analysis Protocol (3) In-Gel Digestion with Protease" Cell Engineering 21: 524-534, 2002) The protein in the gel was trypsin-digested, and the obtained peptide fragment was subjected to ESI-Q-TOF type mass spectrometer (trade name Q-STAR: Applied Biosystems). Identification of proteins based on amino acid sequences from computer database search results using spectral data of “daughter ions” obtained by MS / MS method for “parent ions” observed by mass spectrometry (MS) Went. As a result, four of the nine spots were identified as leucine-rich α2 glycoprotein, fibrinogen gamma, C4a, and albumin precursor, respectively.

  Of the two-dimensional electrophoresis images described above, images of only the spot portions of these four types of marker proteins are shown in FIG. FIG. 3 is an image photograph showing four types of marker protein spots for a High category patient (1 sample) and a Low category patient (1 sample). As shown in FIG. 3, fibrinogen gamma, C4a and albumin precursor were confirmed to have a dark spot in the Low Average-Low patient group, and leucine-rich α2 glycoprotein was confirmed to be a dark spot in the High patient group. From this, by confirming the concentration of leucine-rich α2 glycoprotein, fibrinogen gamma, C4a and albumin precursor in the drainage of the peritoneal dialysis of the patient, either the High patient group in the PET category or the Low Average-Low patient group It can be seen that it is possible to evaluate whether it falls under.

  As described above, if the analysis method for qualitatively or quantifying the peritoneal function marker of the present invention is used, the peritoneal function can be examined only by using the drainage sample. Therefore, it is possible to reduce the burden on the patient and to perform the examination more easily than PET, which has been necessary. For this reason, it can be said that the inspection method using the analysis method of the present invention is extremely useful as an alternative method of the conventional PET.

FIG. 1 is a photograph showing the results of two-dimensional electrophoresis on dialysis drainage of a High category patient in an example of the present invention. FIG. 2 is a photograph showing the result of two-dimensional electrophoresis relating to dialysis drainage of a Low category patient in the Example. FIG. 3 is a photograph showing a spot portion of the marker protein of the present invention in two-dimensional electrophoresis relating to dialysis drainage of High category patients and Low category patients in the above-mentioned Examples.

Claims (8)

A marker for diagnosis of peritoneal function, comprising at least one protein selected from the group consisting of leucine-rich α2 glycoprotein , C4a and albumin precursor. The peritoneal function marker of Claim 1 whose marker which shows that peritoneum permeability is relatively high is leucine rich alpha2 glycoprotein. Markers indicating that peritoneal permeability is relatively low, at least one protein selected from the group consisting of C 4a and albumin precursor, peritoneal function marker of claim 1, wherein. A method for analyzing a peritoneal function marker,
The marker is the marker according to any one of claims 1 to 3,
A method for analyzing a peritoneal function marker, comprising a step of qualitatively or quantitatively determining the marker contained in the drainage of peritoneal dialysis. The analysis method according to claim 4, wherein the method for qualitatively or quantitatively determining the marker is a method using at least one of an immunological technique and an electrophoresis method. The analysis method according to claim 4 or 5, wherein the peritoneal dialysis drainage is drainage collected in 8 to 12 hours from the start of peritoneal dialysis. A method of testing the risk of the subject peritoneal function is reduced, the risk test method comprising the following (A) and (B) step.
(A) The step of measuring the peritoneal function marker according to claim 1 contained in the peritoneal dialysis drainage of the subject by the analysis method according to any one of claims 4 to 6 (B) The peritoneal function is known The measurement value of the peritoneal function marker according to claim 1 contained in a patient's peritoneal dialysis drainage is used as a reference value, and the reference value is compared with the measurement value of the subject in the step (A). Process for testing the risk of decreased peritoneal function of the specimen The reference value in the step (B) is a measurement value of a patient whose peritoneal equilibrium test category is at least one selected from the group consisting of High, High Average, Low Average, and Low. Risk test method.

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