JP4344436B2 - Method for detecting osteoarthritis - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting a joint disease.
[0002]
[Prior art]
Joints are the parts where skeletal components are connected to each other. Normally, each component moves independently of each other, and we play a very important role in our daily lives. Plays. That is, when a disorder is recognized in a joint for some reason, humans cannot perform their original movements, which greatly affects daily life and work.
[0003]
As with other diseases, it is important that early detection and early treatment be performed for joint diseases in which some abnormality is observed in the joint. This is because it is possible to cure the joint disease or delay its progression by identifying the cause of the joint disease and applying appropriate treatment in the early stage.
[0004]
X-ray imaging is known as a representative method for detecting joint diseases.
However, in X-ray imaging, it is difficult to evaluate the degenerative change of cartilage accompanying the progression of the stage, and since it is not a quantitative detection method, there are aspects that are not suitable for detecting the subtle degree of progression of the stage. Furthermore, since the determination of the degree of progression of the stage has many parts based on experience, the conclusion may differ depending on the determiner. Furthermore, it is desirable to avoid radiation (X-ray) exposure as much as possible.
[0005]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method for quantitatively and objectively detecting a joint disease while avoiding the risk of radiation exposure.
[0006]
[Means for Solving the Problems]
The present inventors have found that the amount of a specific oligosaccharide obtained by treating joint fluid with a specific enzyme changes as the stage of joint disease progresses, and the amount of this specific oligosaccharide is determined. By measuring, it was found that the degree of progression of the stage of joint disease can be detected without using X-rays, and the present invention has been completed.
[0007]
That is, this invention provides the detection method (henceforth this invention detection method) of osteoarthritis characterized by including the following processes 1-3.
Step 1: A step of treating joint fluid with an enzyme having a β-N-acetylglucosaminide binding ability of keratan sulfate.
[0008]
Step 2: A step of measuring the amount of an oligosaccharide represented by the following formula (1) contained in the enzyme-treated joint fluid obtained in Step 1.
Gal-GlcNAc (6S) Equation (1)
[Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, (6S) represents that the hydroxyl group at the 6-position is sulfated, and − represents a glycosidic bond (hereinafter the same)] .
[0009]
Step 3: A step of detecting a joint disease by associating a decrease in the amount of Gal-GlcNAc (6S) measured in Step 2 with the onset or exacerbation of osteoarthritis.
The present invention also provides a detection kit for carrying out the detection method of the present invention (hereinafter also referred to as the detection kit of the present invention), which comprises an enzyme having β-N-acetylglucosaminide binding resolution of keratan sulfate as a component. Say).
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below. As described above, the detection target of the detection method of the present invention is osteoarthritis (such as osteoarthritis of the hip, osteoarthritis of the knee).
[0011]
The contents of the detection method of the present invention will be described in the order of the above-described steps.
Process 1 :
This step is a step of treating joint fluid with an enzyme having the ability to bind β-N-acetylglucosaminide of keratan sulfate.
[0012]
Synovial fluid can be used synovial fluid of interest [mammals (including humans)] to detect osteoarthritis. For example, in the case of detecting the human osteoarthritis, we used to collect synovial fluid of joints becomes a problem detection subject, in the case of detecting osteoarthritis in an animal such as a dog or a horse, It is possible to collect and use the joint fluid of the joint which is a problem of the detection target animal.
[0013]
In addition, the joint fluid used in the present invention does not necessarily need to be pretreated before the enzyme treatment, but in order to remove insoluble foreign matters, undegraded products, etc., it is filtered through a filter, an ultrafiltration membrane, It is preferable to perform a solid-liquid separation process such as centrifugation. Also, joint fluid appropriately diluted with physiological saline or the like can be used.
[0014]
The source of keratan sulfate β-N-acetylglucosaminide binding ability (hereinafter also referred to as KS-degrading enzyme) is not particularly limited as long as it has this function. There may be. Further, it may be a living organism in which the ability to produce a KS degrading enzyme is artificially imparted or enhanced by genetic engineering techniques. KS-degrading enzyme is a method according to the source from these sources, for example, if it is a microorganism, KS-degrading enzyme secreted outside or inside the cell under appropriate conditions is obtained by a conventional method. It can be produced by isolation and purification.
[0015]
Specifically, microorganisms belonging to the genus Bacillus can be mentioned as microorganisms used as a source of KS-degrading enzymes. Specifically, Bacillus sp. Ks36 [Bacillus sp. Ks36 (Accession Number: FERM P-10204)], Bacillus Circus KsT202 (Bacillus circulans KsT202 (Accession Number: FERM BP-5285)), and the like. From these microorganisms belonging to the genus Bacillus, endo-β-N-acetylglucosaminidase is produced as a KS-degrading enzyme. The former Bacillus sp. KS-degrading enzyme derived from Ks36 is also called keratanase II and is commercially available from Seikagaku Corporation. The details of the KS-degrading enzyme derived from the latter Bacillus circulans KsT202 are described in WO96 / 16166.
[0016]
Enzymatic treatment of joint fluid with KS-degrading enzyme can be performed by mixing joint fluid and KS-degrading enzyme solution, and incubating this mixed solution. It can also be carried out by bringing the joint fluid into contact with an immobilized enzyme in which a KS-degrading enzyme is bound to a suitable carrier. This enzyme treatment may be carried out by setting the conditions under which this enzyme acts, but it is of course preferable to carry out under the optimum temperature and optimum pH conditions of the enzyme used.
[0017]
When the joint fluid is treated with KS-degrading enzyme, the β-N-acetylglucosaminide bond of keratan sulfate in the joint fluid is degraded to produce the following oligosaccharides.
Gal-GlcNAc (6S) Equation (1)
Gal (6S) -GlcNAc (6S) Equation (2)
[Hereinafter, the oligosaccharide represented by the above formula (1) is referred to as “L2”. The oligosaccharide represented by the above formula (2) is referred to as “L4”. ]
The glycosidic bond between Gal and GlcNAc is preferably a β-1,4 glycosidic bond.
[0018]
As will be described later, although both L2 and L4 are derived from keratan sulfate in joint fluid, it is related to osteoarthritis , particularly “stage progression of osteoarthritis ”. Surprisingly, it is only L2, and there is no significant association with L4. Therefore, in the detection method of the present invention, it is necessary to measure the amount of L2 in joint fluid treated with KS-degrading enzyme (hereinafter also referred to as enzyme-treated joint fluid). It is one of the most characteristic points.
[0019]
Process 2 :
This step is a step of measuring the amount of L2 contained in the enzyme-treated joint fluid obtained in step 1.
[0020]
This measurement method is not limited as long as L2 in the joint fluid treated with the enzyme can be quantitatively measured. For example, a method using liquid chromatography, an electrophoresis method (capillary electrophoresis, a cellulose acetate membrane or the like) is used. Electrophoresis etc. used), immunological measurement methods and the like.
[0021]
An example of a method using liquid chromatography will be specifically described in Examples. In addition, before performing measurement by liquid chromatography, in order to remove insoluble foreign matter and undegraded materials in the joint fluid treated with the enzyme, solid-liquid separation treatment such as filtration by filtration, ultrafiltration, etc. and centrifugation is performed. Preferably it is done.
[0022]
A step of detecting a joint disease by associating a decrease in the amount of Gal-GlcNAc (6S) measured in step 2 with the onset or exacerbation of osteoarthritis.
[0023]
Measured in step 2, related to the amount of L2 in synovial fluid were enzyme treated with osteoarthritis, and the example L2 of becomes osteoarthritis enzyme-treated synovial fluid decreases, osteoarthritis can L2 enzyme-treated synovial fluid with the progress of the stage of the diseases by utilizing the reducing detects osteoarthritis.
[0024]
Incidentally, osteoarthritis and "detection" to grasp wide information related to osteoarthritis, i.e., osteoarthritis types of presence or suffering from, osteoarthritis particular, deformation It means identifying the degree of progression of the stage of osteoarthritis .
[0025]
Thus, step 3 is a step of associating the amount of L2 measured in step 2 with the presence or absence of osteoarthritis, the degree of progression of the stage, and the like. Can be detected.
[0026]
If the amount of L2 in the joint fluid treated with the enzyme is different from normal (specifically, the concentration of L2 in the joint fluid treated with the enzyme is different from normal), it is positive for osteoarthritis. It is possible to correlate with certain, and the presence or absence of osteoarthritis can be detected. Further, the degree of progression of osteoarthritis stage can be identified and detected by the degree of change in the amount of L2 in the enzyme-treated joint fluid. Specifically, the amount of L2 in the joint fluid treated with the enzyme tends to decrease as the stage of osteoarthritis progresses.
[0027]
For example, in the case of administration of medicaments against osteoarthritis patients osteoarthritis, the L2 concentration in synovial fluid was enzyme treated this patient continuously measures, if L2 concentration should close normally, deformation It can be determined that the progression of the stage of osteoarthritis has stopped and is in the direction of improvement. Similarly, if the L2 concentration in the synovial fluid treated with the enzyme does not change, the progression of the stage is stopped and there is no change in the stage, indicating that the L2 concentration in the synovial fluid treated with the enzyme is not normal. For example, it can be determined that the stage is progressing and worsening.
[0028]
Thus, the detection method of the present invention, it is possible to detect osteoarthritis not only understand the condition of the patient osteoarthritis (including osteoarthritis patients first visit) was administered pharmaceutical By evaluating the effect of the drug, extremely useful information including the selection of the drug can be provided regarding the treatment policy.
[0029]
The L2 concentration in the joint fluid treated with the enzyme, which serves as a detection criterion for osteoarthritis, is appropriately determined, and the detection is performed based on the measured L2 concentration in the joint fluid treated with the enzyme. Further, the detection method of the present invention is very preferably used for detection of the degree of progression of the stage.
[0030]
The detection method of the present invention can grasp the state of osteoarthritis by combining with other detection means for joint diseases as necessary. For example, by examining the degree of change in bone and cartilage destruction and proliferation by the X-ray photography described above, and combining this result with the result obtained by the detection method of the present invention, information on osteoarthritis is enriched. be able to.
[0031]
The present invention also provides a detection kit (the detection kit of the present invention) for carrying out the detection method of the present invention. That is, the present invention provides a detection kit containing elements necessary for carrying out the detection method of the present invention to detect osteoarthritis , such as a KS degrading enzyme. The detection kit of the present invention includes other elements that can be contained in a general detection kit necessary for carrying out the detection method of the present invention, such as physiological saline for dilution, purified water, and buffer (joint). Used for dilution of the liquid etc.) may be included as required.
[0032]
Moreover, it is preferable that the detection kit of the present invention includes a standard preparation of L2 to be measured. The standard preparation of L2 is described in, for example, “Shinsei Kagaku Kogaku Koza 3, Carbohydrate II,“ Proteoglycan and Glycosaminoglycan ”, page 62 (1991, Tokyo Kagaku Dojin)” and WO 96/16166. It can be prepared by a method or the like.
[0033]
【Example】
Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.
(1) Detection of the degree of progression of the stage of joint disease by X-ray Detection of the degree of progression of the stage of joint disease by X-ray was performed for 50 patients with osteoarthritis (hereinafter referred to as hip OA) (54.2 ± 16.3 years) ) Was performed by a doctor who has experience in diagnosis of hip OA based on the following Japanese Orthopedic Association criteria (classified as early stage, advanced stage, and late stage).
[0034]
Japan Orthopedic Association Judgment Criteria ( 1) Initial:
i) Joint space / joint joint is incompatible, and partial narrowing is observed.
ii) Bone sclerosis was observed in the acetabular bone.
iii) Mild osteophyte formation is observed for changes in the acetabulum and femoral head.
[0035]
(2) Progression period:
i) Joint space / joint joints There is a joint mismatch and partial subchondral bone contact is observed.
ii) Bone sclerosis and bone capsular capsule Bone sclerosis of the acetabulum, bone capsule of the acetabulum or bone head is observed.
iii) Regarding the changes in the acetabulum and femoral head, osteophyte formation and proliferative changes in the acetabulum are observed.
[0036]
(3) End of term:
i) Joint space / joint joints There is a mismatch of the joint surface, and there is a wide range of disappearance of joint joint space.
ii) Regarding bone sclerosis and bone sac, extensive bone sclerosis and huge bone sac are observed.
iii) Regarding the changes in the acetabulum and femoral head, remarkable osteophyte formation, double image of the acetabulum, and destruction of the acetabulum were observed.
[0037]
(2) Collection of joint fluid The joint fluid was collected from the joint of the patient described in (1) above at the time of surgery or arteriography and stored frozen. The sample was thawed after thawing, and 50 μl or 100 μl was collected in a tube. The sample was weighed and diluted with physiological saline so that the 50 μl sample was 20 times the sample weight and the 100 μl sample was 10 times the sample weight. After dilution, the sample was filtered with a 0.45 μm filter.
[0038]
(3) Preparation of enzyme-treated joint fluid 100 μl of a 20-fold diluted sample was collected in a tube. 20 μl of 0.1 U / ml Keratanase II (Seikagaku Corporation) solution was added, 20 μl of 1M sodium acetate buffer (pH 6.0) was added, and the mixture was lightly stirred and digested at 37 ° C. for 48 hours. For the 10-fold diluted sample, 50 μl of sample, 50 μl of distilled water, 20 μl of keratanase II solution were added, 20 μl of 1M sodium acetate buffer (pH 6.0) was added, and digested in the same manner. Keratan sulfate is decomposed into L2 and L4.
[0039]
After completion of the digestion, the entire amount was placed on a centrifugal ultrafiltration tube (Ultrafree C3GC) having a molecular weight cut off of 30,000 and centrifuged at 12,000 rpm for 15 minutes. L2 and L4 are recovered in the filtrate.
[0040]
(4) Analysis of enzyme-treated joint fluid The ultrafiltered filtrate was transferred to a high performance liquid chromatography (HPLC) vial, and 40 μl thereof was injected into the HPLC. L2 and L4 standard preparations (both were prepared by the method described in "Shinsei Kagaku Kogaku Koza 3, Carbohydrate II," Proteoglycan and Glycosaminoglycan ", p. 62 (1991, Tokyo Kagaku Dojin)). L2 and L4 were analyzed using the elution position of) as an index. The amounts of L2 and L4 were obtained as integral values of this elution curve by monitoring the elution from HPLC with an electrochemical detector (applied voltage: +360 mV).
[0041]
The HPLC conditions for analyzing L2 and L4 are shown below.
1. HPLC conditions for L2 (1) Column: N (CH ₃ ) ₂ -bonded silica gel, φ8 mm × 15 cm (trade name: Senshu Pak., Senshu Science Co., Ltd.)
(2) Elution: 12 mM NaH ₂ PO ₄ /10% acetonitrile (3) Flow rate: 0.65 ml / min
(4) Reaction solution: 50 mM sodium tetraborate containing 1% 2-cyanoacetamide (5) Reaction solution flow rate: 0.5 ml / min
(6) Reaction temperature: 147 ° C
(7) Reaction coil: φ0.4mm × 10m
(8) Detection: electrochemical detection, applied voltage: +360 mV (glassy carbon)
[0042]
2. HPLC conditions for L4 (1) Column: N (CH ₃ ) ₂ -bonded silica gel, φ8 mm × 15 cm (trade name: Senshu Pak., Senshu Science Co., Ltd.)
(2) Elution: 12 mM Na ₂ SO ₄ /10% acetonitrile (3) Flow rate: 0.65 ml / min
(4) Reaction solution: 50 mM sodium tetraborate containing 1% 2-cyanoacetamide (5) Reaction solution flow rate: 0.5 ml / min
(6) Reaction temperature: 147 ° C
(7) Reaction coil: φ0.4mm × 10m
(8) Detection: Electrochemical detection, Applied voltage: +360 mV (The results for glassy carbon L2 are shown in FIG. 1, and the results for L4 are shown in FIG. 2.
[0043]
From this result, it was shown that the L2 concentration in the joint fluid treated with the enzyme significantly decreased as the stage progression of the crotch OA increased. In contrast, there was no significant correlation between the L4 concentration in the synovial fluid treated with the enzyme and the stage of the hip OA.
[0044]
From this result, it was shown that the degree of progression of the stage of joint disease can be detected by measuring the L2 concentration in the joint fluid treated with the enzyme.
[0045]
【The invention's effect】
By using the detection method of the present invention, the degree of progression of the stage of osteoarthritis can be detected objectively and quantitatively while avoiding the risk of radiation exposure. Moreover, this detection method of the present invention can be easily carried out using the detection kit of the present invention.
[Brief description of the drawings]
FIG. 1 is a drawing showing the relationship between the L2 concentration in enzyme-treated synovial fluid and the stage of osteoarthritis of the hip.
FIG. 2 is a drawing showing the relationship between the L4 concentration in enzyme-treated joint fluid and the stage of osteoarthritis of the hip.

Claims (3)

The detection method of osteoarthritis characterized by including the following processes 1-3.
Step 1: A step of treating joint fluid with an enzyme having a β-N-acetylglucosaminide binding ability of keratan sulfate.
Step 2: A step of measuring the amount of an oligosaccharide represented by the following formula (1) contained in the enzyme-treated joint fluid obtained in Step 1.
Gal-GlcNAc (6S) Equation (1)
(Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, (6S) represents that the 6-position hydroxyl group is sulfated, and − represents a glycosidic bond)
Step 3: A step of detecting a joint disease by associating a decrease in the amount of Gal-GlcNAc (6S) measured in Step 2 with the onset or exacerbation of osteoarthritis. The detection method according to claim 1, wherein the enzyme having β-N-acetylglucosaminide binding ability of keratan sulfate is endo-β-N-acetylglucosaminidase derived from a microorganism belonging to the genus Bacillus. The detection kit for performing the detection method according to claim 1 or 2, comprising an enzyme having β-N-acetylglucosaminide binding resolution of keratan sulfate as a component.

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