WO2012081508A1

WO2012081508A1 - Method for assaying glycosphingolipid

Info

Publication number: WO2012081508A1
Application number: PCT/JP2011/078512
Authority: WO
Inventors: 徹平戸; 登田中; 秀人森本
Original assignee: 日本ケミカルリサーチ株式会社
Priority date: 2010-12-13
Filing date: 2011-12-09
Publication date: 2012-06-21
Also published as: JP5977174B2; JPWO2012081508A1

Abstract

Disclosed is a simple, economical, and highly sensitive method for assaying a glycosphingolipid. This method for assaying a glycosphingolipid comprises a step for deacylating the glycosphingolipid using sphingolipid-ceramide deacylase to obtain a lyso form, and a step for detecting the generated lyso form by using mass spectrometry.

Description

Determination of glycosphingolipids

The present invention includes a step of deacylating a glycosphingolipid using a sphingolipid ceramide deacylase to form a lysosphingoglycolipid, and detecting the obtained lysosphingoglycolipid using mass spectrometry. The present invention relates to a method for quantifying glycosphingolipid, and particularly to a method for quantifying glucocerebrosid or globotriaosylceramide.

In patients with Gaucher's disease, a type of lysosomal disease, the activity of glucocerebrosidase is absent or decreased due to genetic factors. As a result, glucocerebrosid is accumulated in organs and blood without being decomposed, resulting in hematopoietic organs. Symptoms such as disability are caused. Glucocerebroside (glucosylceramide) is a type of glycosphingolipid in which glucose is β-glycoside-bonded to the hydroxyl group at the 1-position of ceramide, and is a generic name for heterogeneous molecules in which the chain length of the constituent fatty acid is not constant. is there. In mammals including humans, glucocerebroside is metabolized by glucocerebrosidase (glucosylceramidase) through hydrolysis at a β-glycoside bond (FIG. 1).
Therefore, as a treatment method for Gaucher's disease, glucocerebrosidase accumulated in the patient's organs is administered by administering glucocerebrosidase to the patient by intravenous injection or the like and supplementing glucocerebrosidase that is lacking in the patient's body. Enzyme replacement therapy is being performed to break down and improve various symptoms such as hematopoietic disorders.

There are multiple clinical types of Gaucher's disease. Among them, the so-called infant type and younger type are characterized in that they show progressive central nervous symptoms from infancy or early childhood. Therefore, in order to suppress the progression of the disease state, it is desirable to start treatment early. For this purpose, it is necessary to diagnose Gaucher disease at the infancy stage. *

In patients with Fabry disease, a type of lysosomal disease, the activity of α-galactosidase A is absent or decreased due to genetic factors, and as a result, ceramides and trihexosides such as globotriaosylceramide are not degraded. It accumulates in the blood and causes various symptoms such as cardiovascular lesions and kidney damage. Globotriaosylceramide is a type of glycosphingolipid in which three hexoses are linked via the hydroxyl group at the 1-position of ceramide, and its hetero-molecular chain length is not constant. It is a generic name. In mammals including humans, ceramide trihexoside including globotriaosyl ceramide is metabolized by α-galactosidase A at the non-reducing end α-galactoside bond (FIG. 2). .

Therefore, as a treatment method for Fabry disease, α-galactosidase A is administered to a patient by intravenous injection or the like, and α-galactosidase A that is deficient in the patient's body is replenished, so that globo accumulated in the patient's organs. Enzyme replacement therapy is being performed to break down ceramides and trihexosides such as trioosylceramide to improve various symptoms such as cardiovascular lesions. *

Fabry disease is often asymptomatic in early childhood, and limb pain, skin rash, etc. are observed after school age, and severe symptoms such as cardiovascular lesions and kidney damage appear after puberty. Is a progressive disease that becomes. Therefore, it is desirable to start treatment early in order to suppress the progression of the disease state. For that purpose, it is necessary to diagnose Fabry disease at the stage of asymptomatic infants.

In addition to Gaucher's disease and Fabry disease, genetic diseases caused by glycosphingolipid accumulation include Krabbe disease, metachromatic leukodystrophy, G _M1 -gangliosidosis, G _M2 -gangliosidosis and the like. In this case, galactocerebroside, sulfatide, ganglioside G _M1 , ganglioside G _{M2 and the} like accumulate in the patient's body.

As a method for diagnosing these genetic diseases, there is a gene diagnostic method in which the base sequence of a patient's gene is analyzed using a DNA sequencer to detect the presence or absence of a gene abnormality. The gene diagnosis method is a highly versatile diagnosis method that can be used for diagnosis of almost all genetic diseases.
However, the genetic diagnosis method requires a complicated procedure of extracting DNA or RNA from a sample such as blood collected from each patient and then sequentially specifying the base sequence of the DNA or RNA. In addition, even in the same genetic disease, the mutation position on the gene is not constant depending on the patient, so in order to detect the abnormality of the gene, it is necessary to investigate the presence or absence of the abnormality throughout the gene, Gene diagnosis is a time-consuming and expensive diagnosis. Therefore, genetic diagnosis is not always an appropriate method for diagnosing many infants.

In patients with Fabry disease, the concentration of ceramide trihexoside (sphingoglycolipid) such as globotriaosylceramide contained in blood or urine is increased compared to healthy subjects. Therefore, as a comparatively simple method for diagnosing Fabry disease instead of genetic diagnosis, ceramide trihexoside contained in the blood or urine of patients is analyzed by mass spectrometry (electrospray ionization tandem mass spectrometry: ESI / MS). A method of quantifying using this is known (Patent Document 1). However, ceramide trihexoside is a group of heterogeneous molecular species whose constituent fatty acid chain lengths are not constant. Therefore, in order to quantify ceramide trihexoside directly using mass spectrometry, prepare a standard product corresponding to each molecular species with different fatty acid chain lengths, create a calibration curve, and individually prepare each molecular species. It is necessary to quantify and is complicated. Therefore, in the above document, ceramide / trihexoside is measured using, for example, ceramide / trihexoside whose fatty acid is heptadecanoic acid as a standard product. Therefore, the measured value is an approximate value.

A product obtained by removing a fatty acid from a sphingoglycolipid (deacylated form) is called a lysosphingo glycolipid (lyso form). As a method for converting to a lyso form in this way, a method of specifically hydrolyzing an amide bond in a glycosphingolipid (eg, glucocerebroside, globotriaosylceramide, etc.) with a sphingolipid ceramide deacylase (SCDase) There is. (Figure 3). SCDase has been isolated from various organisms so far, including SCDase derived from Pseudomonas (Patent Document 2, Patent Document 3), SCDase derived from Schwannella (Patent Document 3), and G8 derived from a marine bacterium, Shebanera alga. SCDase (patent document 4) derived from a strain, SCDase (patent document 5) derived from a strain (non-fermentative gram-negative bacillus AI-2 (FERM P-16124)) obtained from desquamation of atopic dermatitis patients, Streptomyces A genus-derived SCDase (Patent Document 6) and the like are known.

In Patent Document 2 and Patent Document 3, it is confirmed by detecting the obtained lyso form by mass spectrometry that a glycosphingolipid is deacylated with the above SCDase to obtain a lyso form.

In addition, by utilizing the increase in the concentration of lysosphingoglycolipid in blood in patients with Fabry disease, the lysosphingoglycolipid thus increased is quantified by electrospray tandem mass spectrometry, so that Fabry disease Has been reported (Patent Document 7).

JP 2005-512061 A JP-A-8-84587 JP 10-45792 A WO2002 / 026963 JP-A-10-257784 JP-A-7-1079888 US Patent Publication 2010/0047844

Under the above background, the object of the present invention is to provide a simple and economical glycosphingolipid, particularly glucocerebrosid and globotriosylceramide, which are causative in genetic diseases caused by glycosphingolipid accumulation. It is to provide a more sensitive quantitative method.

In the research for the above purpose, the present inventors hydrolyzed glycosphingolipid with sphingolipid ceramide deacylase (SCDase) to obtain a lyso form, and then analyzed the lyso form by mass spectrometry. The present inventors have found that glycosphingolipids can be quantified with high sensitivity. Furthermore, when hydrolyzing a sphingoglycolipid using SCDase, the present inventors sonicated a solution or suspension containing the sphingoglycolipid, frozen it once, thawed, and then SCDase. When the lysosphingo glycolipid-containing solution thus obtained is subjected to an enzymatic reaction and analyzed by mass spectrometry, the lysosphingo glycolipid-containing solution is subjected to ultrasonic treatment in advance. It was found that the quantitative value of was stabilized and the detection sensitivity was further increased. The present invention has been completed based on these findings.

That is, the present invention provides the following.
[1] A method for quantifying glycosphingolipid,
(A) A specimen containing glycosphingolipid is dissolved or dispersed in an aqueous solution, and a sphingolipid ceramide deacylase is added to the glycosphingolipid-containing liquid thus obtained to deacylate the glycosphingolipid contained therein by hydrolysis. Releasing lysosphingoglycolipid, and
(B) a quantitative method comprising the step of quantifying the glycosphingolipid by measuring the glycosphingolipid by mass spectrometry,
[2] The specimen is obtained from any one selected from the group consisting of kidney, liver and other organ tissues collected from the subject, blood, cerebrospinal fluid and other body fluids, urine, skin, muscle, and interstitial tissue. The quantification method of [1] above,
[3] The quantification of [1] or [2] above, wherein the glycosphingolipid is selected from the group consisting of glucocerebroside, globotriaosylceramide, galactocerebroside, sulfatide, ganglioside G _M1 and ganglioside G _M2. Method,
[4] The quantification method of the above [1] or [2], wherein the glycosphingolipid is glucocerebroside or globotriaosylceramide,
[5] The sphingolipid ceramide deacylase is composed of SCDase derived from Pseudomonas, SCDase derived from Shewanella, SCDase derived from Streptomyces, and SCDase derived from non-fermentative Gram-negative bacilli AI-2 (FERM P-16124) A quantification method according to any one of the above [1] to [4], which is selected from the group;
[6] The quantification method according to any one of the above [1] to [5], wherein the mass spectrometry method is tandem mass spectrometry (MS / MS),
[7] The quantification method according to any one of the above [1] to [6], wherein the mass spectrometry method is high performance liquid chromatograph tandem mass spectrometry (LC / MS / MS),
[8] A method for diagnosing a disease caused by glycosphingolipid accumulation by quantifying the glycosphingolipid using the quantification method according to any one of [1] to [7] above,
[9] disease pathogenesis accumulation of glycosphingolipids, Gaucher's disease, Fabry's disease, Krabbe disease, metachromatic leukodystrophy, G _{M1 -} is selected from the group consisting of gangliosidosis _- gangliosidosis and G _M2 The diagnostic method of [8] above,
[10] The diagnostic method of [8] above, wherein the disease caused by glycosphingolipid accumulation is Gaucher disease or Fabry disease,
[11] A method for preparing a lysosphingoglycolipid-containing liquid,
(A) dissolving or dispersing a specimen containing glycosphingolipid in an aqueous solution, and sonicating the glycosphingolipid-containing liquid thus obtained;
(B) freezing the sonicated liquid;
(C) thawing the frozen solution; and
(D) a step of adding a sphingolipid ceramide deacylase to the melted liquid, deacylating the glycosphingolipid contained in the liquid by hydrolysis, and releasing lysosphingoglycolipid. Preparation method,
[12] The specimen is obtained from any one selected from the group consisting of kidney, liver and other organ tissues collected from the subject, blood, spinal fluid and other body fluids, urine, skin, muscle, and interstitial tissue. The preparation method of [11] above,
[13] Preparation of [11] or [12] above, wherein the glycosphingolipid is selected from the group consisting of glucocerebroside, globotriaosylceramide, galactocerebroside, sulfatide, ganglioside G _M1 and ganglioside G _M2. Method,
[14] The method according to [11] or [12] above, wherein the glycosphingolipid is glucocerebrosid or globotriaosylceramide,
[15] The sphingolipid ceramide deacylase comprises SCDase derived from Pseudomonas, SCDase derived from Shewanella, SCDase derived from Streptomyces, and SCDase derived from non-fermentative Gram-negative bacilli AI-2 (FERM P-16124) A preparation method according to any one of the above [11] to [14], which is selected from the group;
[16] A method for quantifying glycosphingolipid,
(A) sonicating the lysosphingoglycolipid-containing solution prepared in any one of [11] to [15] above;
(B) a quantification method comprising a step of quantifying the glycosphingolipid by measuring lysosphingoglycolipid contained in the ultrasonically treated liquid by mass spectrometry;
[17] The quantification method of [16] above, wherein the mass spectrometry method is tandem mass spectrometry (MS / MS),
[18] The quantification method of the above [16], wherein the mass spectrometry is high performance liquid chromatograph tandem mass spectrometry (LC / MS / MS),
[19] A method for diagnosing a disease caused by glycosphingolipid accumulation by quantifying the glycosphingolipid using the quantification method according to any one of [16] to [18] above,
[20] The disease caused by the accumulation of glycosphingolipid is selected from the group consisting of Gaucher disease, Fabry disease, Krabpe disease, metachromatic leukodystrophy, G _M1 -gangliosidosis and G _M2 -gangliosidosis The diagnostic method of [19] above,
[21] The diagnostic method of the above [19], wherein the disease caused by glycosphingolipid accumulation is Gaucher's disease or Fabry disease.
[22] A method for producing a lysosphingoglycolipid-containing liquid,
(A) dissolving or dispersing a material containing glycosphingolipid in an aqueous solution, and sonicating the glycosphingolipid-containing liquid thus obtained;
(B) freezing the sonicated liquid;
(C) thawing the frozen solution; and
(D) a step of adding a sphingolipid ceramide deacylase to the melted liquid and deacylating the glycosphingolipid contained in the liquid by hydrolysis to release the lysosphingoglycolipid. ,Production method.

According to the present invention, glycosphingolipids, particularly glucocerebrosides and globotriaosylceramides can be quantified simply, economically and with higher sensitivity. Furthermore, according to the present invention, it is possible to provide a lysosphingoglycolipid-containing solution used for the determination.
In particular, the glycosphingolipid diagnostic method according to the present invention is simple and economical, and thus can be used not only for diagnosis of specific subjects but also for large-scale diagnosis, for example, large-scale screening of newborns. It is also possible to grasp the medical condition over time by periodically diagnosing a specific patient. Further, by diagnosing a patient before and after treatment such as enzyme replacement therapy, the degree of the effect of the treatment can be known.

FIG. 1 is a drawing showing glucocerebrosides and locations in the molecule that are hydrolyzed by glucocerebrosidase. An arrow indicates a portion hydrolyzed by glucocerebrosidase, and R indicates an acyl group of a fatty acid. FIG. 2 is a drawing showing globotriaosylceramide and the portion of the molecule that is hydrolyzed by α-galactosidase A. An arrow indicates a portion hydrolyzed by α-galactosidase A, and R indicates an acyl group of a fatty acid. FIG. 3 is a drawing showing hydrolysis of a sphingoglycolipid by a sphingolipid ceramide deacylase (SCDase). An arrow indicates a portion hydrolyzed by SCDase, R1 indicates an acyl group of a fatty acid, and R2 indicates a sugar or a sugar chain. A: Glycosphingolipid, B: Glycosphingolipid FIG. 4 is a drawing showing an ion chromatogram of globotriaosylsphingosine (lyso form). The vertical axis represents ion intensity (cps: counts / second), and the horizontal axis represents retention time (min). FIG. 5 is a drawing showing a calibration curve of globotriaosylceramide. The vertical axis is the peak area (counts) of the ion chromatogram of globotriaosylsphingosine (lyso form), and the horizontal axis is the concentration of globotriaosylceramide (ng / ng) in the glycosphingolipid (globotriaosylceramide) -containing solution. mL).

In the present invention, the glycosphingolipid to be quantified is not particularly limited as long as the glycosphingolipid is hydrolyzed with SCDase to produce a lysosphingoglycolipid. In addition to those derived from organisms such as microorganisms, plants, animals, Any of those synthesized artificially (for example, industrially) may be used.
Preferable specific examples of the glycosphingolipid in the present invention are glucocerebroside, globotriaosylceramide, galactocerebroside, sulfatide, ganglioside G _M1 and ganglioside G _M2 . Patients Glucocerebroside Gaucher disease, patients globotriaosylceramide Fabry disease, galactocerebrosidase patients b Sid Krabbe disease, sulfatide patients metachromatic leukodystrophy, ganglioside G _M1 is G _{M1 -} gangliosidoses patient, And ganglioside G _M2 is a glycosphingolipid known to accumulate in the body of patients with G _M2 -gangliosidosis, respectively. Therefore, by quantifying these glycosphingolipids, it is possible to specify a patient or a carrier of a disease whose pathogenesis is the accumulation of glycosphingolipids. Therefore, determination of glycosphingolipids of the invention are disease pathogenesis accumulation of glycosphingolipids, particularly Gaucher disease, Fabry disease, Krabbe disease, metachromatic leukodystrophy, G _{M1 -} gangliosidosis, G _M2 - It can be used as a diagnostic method for gangliosidosis.

In the present invention, “specimen containing glycosphingolipid” or “material containing glycosphingolipid” means microorganisms, plants, animals (including humans), etc. containing glycosphingolipid, and artificial (for example, All samples containing glycosphingolipids synthesized industrially) are included. Here, animal tissues and the like refer to kidney, liver and other organ tissues, blood, cerebrospinal fluid and other body fluids, urine, skin, muscle, and interstitial tissues.
Diagnosis of diseases caused by the accumulation of glycosphingolipids according to the present invention is carried out using subjects such as kidney, liver and other organ tissues, blood, cerebrospinal fluid and other body fluids, urine, skin, muscle, and interstitial tissues as specimens. Collected and quantified glycosphingolipid contained in these. In particular, blood and urine are collected as specimens for easy collection. When collecting organ tissue as a specimen, needle biopsy is generally used to puncture the organ and collect a part of it, but not limited to this, other methods such as incisional biopsy and excisional biopsy should also be used. Can do. As a result of quantification, when the obtained quantitative value shows an abnormally high value, the subject can be estimated or diagnosed as a patient or carrier of these diseases.

In the present invention, when a specimen or material containing glycosphingolipid is treated with SCDase, the specimen or material is dissolved or dispersed or suspended in an aqueous solution in advance to obtain a glycosphingolipid-containing liquid. SCDase treatment may be performed after thawing what has been frozen by ultrasonic treatment. At this time, the aqueous solution in which the specimen or the material is dissolved or dispersed or suspended is preferably a buffer, and the pH is preferably pH 5.5 to 6.5, more preferably pH 5.8 to 6.2. Particularly preferred is about pH 6.0. The buffer used at this time is not particularly limited, but is preferably an acetate buffer or a phosphate buffer. In addition, the “sphingoglycolipid-containing liquid” herein refers to not only a state in which the glycosphingolipid is completely dissolved in the buffer solution, but also a liquid in which the glycosphingolipid is dispersed or suspended in the buffer solution. Including.
The concentration of the glycosphingolipid in the glycosphingolipid-containing solution is not particularly limited as long as the glycosphingolipid or the glycosphingolipid-containing solution can be prepared by dispersing or suspending, but is preferably a concentration of 4 to 2000 ng / mL. It is.

In addition, the ultrasonic treatment of the glycosphingolipid-containing solution is performed for the purpose of sufficiently dissolving the glycosphingolipid in the buffer solution (that is, dissolving, dispersing or suspending, etc.). As long as the glycosphingolipid is not subject to physical degradation, it may be performed under any conditions. For example, an ultrasonic treatment device (sonicator), an ultrasonic cleaner (for example, USK-3R (As One Corporation)), etc. Can be used. The temperature of the glycosphingolipid-containing liquid at the time of sonication is not particularly limited as long as the liquid phase state of the liquid is maintained and the glycosphingolipid is not denatured. C.). The sonication time is not particularly limited as long as the purpose of the sonication is achieved, but the preferred time when using an ultrasonic cleaner is, for example, 15 minutes to 60 minutes, more preferably. Examples include 20 to 40 minutes.
Freezing of the glycosphingolipid-containing solution after ultrasonic treatment is not particularly limited as long as the solution is completely frozen and the glycosphingolipid as a content is not denatured. A preferable freezing temperature is, for example, −20 ° C. to −180 ° C., more preferably −60 ° C. to −100 ° C. Regarding the freezing rate, for example, without any particular management, a room temperature sphingoglycolipid-containing liquid is placed in a freezer set at a predetermined freezing temperature and frozen as it is.
The melting of the frozen glycosphingolipid-containing solution is not particularly limited as long as the content of the glycosphingolipid is not affected by denaturation or the like, but can be carried out, for example, by leaving it at room temperature.

The sphingolipid ceramide deacylase (SCDase) used in the present invention has an activity capable of specifically hydrolyzing an amide bond in a glycosphingolipid to liberate a fatty acid, thereby converting the glycosphingolipid into a lysosphingoglycolipid. As long as it has, there is no particular limitation, but SCDase derived from Pseudomonas (for example, Pseudomonas sp. SCDase derived from SP H-37 (FERM P-13822), SCDase derived from non-fermentative Gram-negative bacilli AI-2 (FERM P-16124), etc. can be suitably used, of which SCDase derived from Pseudomonas is preferred. Good.
Hydrolysis with SCDase can be carried out by a conventional method. For example, the reaction temperature is preferably in the range of 36 to 40 ° C., and the reaction time is preferably in the range of 10 to 20 hours. The enzyme reaction can be stopped by heating the lysosphingoglycolipid-containing solution after the reaction to deactivate the enzyme. Heating can be carried out, for example, by maintaining a temperature of 90 ° C. or higher for 3 to 10 minutes.

In the present invention, the lysosphingoglycolipid-containing solution obtained by SCDase treatment may be subjected to ultrasonic treatment before analysis by mass spectrometry. The sonication is performed for the purpose of sufficiently dissolving the lysosphingoglycolipid, and so may be performed under any conditions as long as this purpose can be achieved and the lysosphingoglycolipid is not subject to physical degradation. , And can be carried out in the same manner as described above.

In the present invention, there is no particular limitation on the mass spectrometry used for the analysis of glycosphingolipid, but tandem mass spectrometry (MS / MS) is preferably used. The high performance liquid chromatograph tandem mass spectrometry (LC / MS / MS) described in the examples is an example.
When performing a high performance liquid chromatograph, selection of a column and other various settings can be appropriately performed according to a conventional method.
Tandem mass spectrometry (MS / MS) can also be carried out according to conventional methods. For example, lysosphingoglycolipids are ionized (parent ions) and cleaved by colliding them with a collision gas. This can be carried out by generating only one daughter ion and selectively detecting only specific daughter ions. Examples of the ionization method include soft ionization methods such as electroion spray ionization, atmospheric pressure chemical ionization, and thermospray ionization. Among these, the electro ion spray ionization method can be preferably used.

Glycosphingolipid quantification using mass spectrometry in the present invention is carried out by subjecting a standard product containing a known concentration of glycosphingolipid to SCDase treatment and analyzing it by mass spectrometry to determine the ion chromatogram of lysosphingoglycolipid. Obtain the peak area, then draw a calibration curve that shows the correlation between the glycosphingolipid concentration and the peak area, and then analyze the sample or material by mass spectrometry on the ion chromatogram of lysosphingoglycolipid. The amount of glycosphingolipid contained in the specimen or material is calculated by interpolating the peak area. However, the present invention is not limited to this, and the maximum value of the ion intensity of the ion chromatogram can be used as a parameter instead of the peak area.

In the present invention, the term “lysosphingoglycolipid” or “lyso form” refers to a deacylated form obtained by hydrolyzing the amide bond of the glycosphingolipid and removing the fatty acid from the glycosphingolipid.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not intended to be limited to the examples.

[Preparation of globotriaosylceramide standard solution]
1.6022 g of taurodeoxycholic acid (Nacalai Tesque) is dissolved in 200 mL of 50 mM acetate buffer (pH 6.0), filtered through a 0.22 μm membrane filter, and 0.8% (w / v) taurodeoxycholic acid solution (TDC solution) Adjusted. 100 μg of porcine erythrocyte-derived globotriaosylceramide (Nacalai Tesque) was dissolved in 1 mL of TDC solution to prepare a 100 μg / mL globotriaosylceramide solution (GL-3 standard solution). This GL-3 standard solution was serially diluted with a TDC solution to prepare 2000, 1000, 400, 200, 40, 20, and 4 ng / mL GL-3 standard solutions. 50 μL of each GL-3 standard solution is dispensed into a 500 μL PP test tube (assist) and ultrasonic cleaner [USK-3R, ASONE CORPORATION, main specifications (tank volume: 5.9 L, oscillation frequency] : 40kHz, Oscillator circuit: Separately excited oscillation method, Oscillator: BLT (Bolt-tightened Langevin), Output: 120W], ultrasonic treatment was performed at room temperature (25 ° C) for 30 minutes, then about 15 at -80 ° C Freeze for hours.

[SCDase treatment of globotriaosylceramide standard solution]
5 μL (25 mU) of Pseudomonas
SCDase derived from sp. (Sphingolipid ceramide N-deacylase (Takara Bio) was mixed with 995 μL of TDC solution to make 2XSCDase solution. After thawing each GL-3 standard solution (50 μL) at room temperature, 50 μL of 2XSCDase solution was added thereto and mixed, and allowed to stand at 37 ° C. for about 15 hours to hydrolyze globotriaosylceramide to obtain a lyso form (globotriaosyl sphingosine). SCDase was inactivated by heating at 5 ° C. for 5 minutes.

[Extraction of total lipid from mouse kidney]
Tissue (kidney) was collected from 4 mice (C57Black 6N, 24 weeks old, male), chloroform: methanol = 2: 1 solution was added to about 1 mg of tissue wet weight 10 mg, homogenized, and centrifuged. Separation (2000 rpm, 5 minutes, room temperature). The supernatant was transferred to a centrifuge tube, 4 mL of water for injection was added, and the mixture was vigorously mixed for 30 seconds, followed by centrifugation (2000 rpm, 5 minutes, room temperature). The lower layer (organic layer) was collected, dispensed in 500 μL aliquots into glass test tubes, and dried with a centrifugal concentrator. The dried sample was stored at −20 ° C. or lower until measurement. This was taken as the total lipid fraction.

[SCDase treatment of total lipid fraction]
1 mL of 0.8% TDC solution is added to the total lipid fraction and dissolved, transferred to a 1.5 mL tube (Eppendorf), and subjected to ultrasonic treatment using an ultrasonic cleaner (USK-3R, ASONE CORPORATION). ) At room temperature (25 ° C.) for 30 minutes. This is diluted 4-fold with 0.8% TDC solution, then 50 μL is dispensed into a 500 μL PP test tube (assist), transferred to a -80 ° C. freezer and stored overnight (about 15 hours). And frozen. The frozen sample was allowed to stand at room temperature and thawed. After adding 50 μL of 2XSCDase solution and mixing, the mixture was reacted at 37 ° C. overnight (about 15 hours). The solution after the reaction was heated at 90 ° C. for 5 minutes to inactivate the enzyme. This was defined as Condition 1 group. In addition, at this time, those subjected to SCDase treatment without performing the above ultrasonic treatment and freezing and thawing were classified as Condition 2 group. The SCDase treatment was performed on 6 samples independently for each group.

[Tandem mass spectrometry]
As a tandem mass spectrometer, API2000 (ABI) comprising an ion spray part, three quadrupole parts (Q1, Q2, Q3) and a detection part was used. The analysis conditions were Q1 (m / z = 786.3), Q3 (m / z = 282.3), and positive ion mode. The measurement sample introduced into this apparatus is continuously ionized by the ion spray unit. The ionized sample is first introduced into Q1. As the introduced measurement sample ions, only specific ions (m / z = 786.3) are selectively introduced into Q2 in Q1. The ions introduced into Q2 collide with collision gas (nitrogen) and cleave, producing various daughter ions with globotriaosylsphingosine as the parent ion. The resulting daughter ions are then introduced into Q3. In Q3, ions derived from globotriaosylsphingosine are specifically detected by selectively introducing only a specific daughter ion (m / z = 282.3) into the detector. Q1 m / z = 786.3 is the monovalent cationized parent ion of globotriaosylsphingosine (molecular weight: 785.9), and Q3 m / z = 282.3 is globotriaosylsphingosine cleaved by Q2. This is a condition for selectively detecting a daughter ion in which a sphingosine skeleton part lacking the sugar chain part produced by ionization is ionized.

[Preparation of calibration curve by high performance liquid chromatography tandem mass spectrometry (LC / MS / MS method)]
After adding 100 μL of acetonitrile to the SCDase-treated standard solution and mixing, sonication was performed at room temperature (25 ° C.) for 30 minutes using an ultrasonic cleaner (USK-3R, ASONE CORPORATION). . The standard solution after sonication was dispensed into a 300 μL vial for HPLC (Shimadzu Corporation) and analyzed by the LC / MS / MS method. The LC / MS / MS method was performed using API2000 (ABI) as a tandem mass spectrometer and LC20 (Shimadzu Corporation) as a high performance liquid chromatography apparatus. For high performance liquid chromatography, ODS (Cadenza CD-C18, 2.0
mm × 75.0 mm, Immtak), the column temperature was previously set to 40 ° C. in a column oven, and the column was equilibrated with a mixed solution of methanol: 0.1% (v / v) formic acid aqueous solution (75:25). 20 μL of the above standard solution sonicated was added to the column. The flow rate was 0.2 mL / min, and after addition of the sample, methanol: 0.1% (v / v) aqueous formic acid (75:25) was allowed to flow for 2 minutes, and then methanol: 0.1% (v / v) aqueous formic acid (85: Then, the methanol concentration ratio was linearly increased to 95% over 4 minutes, and finally 100% methanol was allowed to flow for 2 minutes. The solution that passed through the column under the above conditions was continuously introduced into the tandem mass spectrometer. The measurement conditions of the tandem mass spectrometer are Q1 (m / z = 786.3) and Q3 (m / z = 282.3), and the ionic strength derived from globotriaosylsphingosine (lyso form) is performed in positive ion mode. Was measured and an ion chromatogram was prepared. The measurement was performed twice for each standard solution. From the obtained ion chromatogram, the peak area of the globotriaosylsphingosine-derived ion with respect to the globotriaosylceramide (GL-3) concentration was calculated, and a calibration curve was prepared.

[Sample analysis by high-performance liquid chromatograph tandem mass spectrometry (LC / MS / MS method)]
For condition 1 group, 100 μL of acetonitrile was added to the SCDase-treated sample, mixed, and then sonicated using an ultrasonic cleaner (USK-3R, ASONE CORPORATION) at room temperature (25 ° C). For 30 minutes. The sample after sonication was dispensed into a 300 μL vial for HPLC (Shimadzu Corporation) and analyzed by the LC / MS / MS method. The analysis by LC / MS / MS method was performed in the same manner as the preparation of the above calibration curve, and the ion intensity derived from globotriaosylsphingosine (lyso form) was measured to prepare an ion chromatogram. Using this and the calibration curve created above, the concentration of globotriaosylceramide contained in the sample was calculated. On the other hand, for the condition 2 group, the above analysis was performed without ultrasonic treatment. The measurement was performed twice for each sample in each group, and the average value was taken as the measurement value.

[Results of analysis by high performance liquid chromatograph tandem mass spectrometry (LC / MS / MS method)]
FIG. 4 shows an ion chromatogram of globotriaosylsphingosine (lyso form) obtained using a globotriaosylceramide standard solution (2000 ng / mL). The horizontal axis represents the retention time (minutes), and the vertical axis represents the ionic strength (cps: count / second). The peak derived from globotriaosylsphingosine reached its maximum intensity with a retention time of 3.68 minutes. The area surrounded by the horizontal axis and the peak was calculated as the peak area.

Fig. 5 shows a calibration curve prepared using a standard solution prepared by serial dilution of globotriaosylceramide. At this time, when a regression equation by the quadratic method was obtained in the range of 4 to 2000 ng / mL which is the quantification range of the calibration curve, the correlation coefficient of the measured value with respect to this regression equation was calculated as 0.999, and the peak area and globotriaosyl A very strong correlation was found with the concentration of ceramide. Using this calibration curve, globotriaosylceramide contained in mouse kidney-derived total lipid was quantified.

In the group (condition 1 group) that was sonicated and freeze-thawed at the time of SCDase treatment and sonicated at the time of analysis by mass spectrometry (at the time of mass spectrometry), the six quantitative values of globotriaosylceramide were The standard deviation was 32.36, and the relative standard deviation was 1.15% (the upper part of Table 1). On the other hand, the quantitative values of 6 times in the condition 2 group were in the range of 2540-3072 ng, the standard deviation was 197.5, and the relative standard deviation was 7.14% (lower part of Table 1). In both condition group 1 and condition group 2, globotriaosylceramide contained in the specimen could be quantified. Moreover, in the condition 1 group, a quantitative value having less variation than the condition 2 group could be obtained (the upper part of Table 1).

The above results are obtained by hydrolyzing globotriaosylceramide contained in the sample with sphingolipid ceramide deacylase to obtain the lyso form, and then analyzing the lyso form by mass spectrometry. It shows that ceramide can be quantified. In particular, by sonicating and freezing and thawing at the time of SCDase treatment, and by subjecting the sample to sonication in advance at the time of analysis by mass spectrometry (at the time of mass spectrometry), mass spectrometry is used to obtain 4 to 2000 ng / This shows that the amount of globotriaosylceramide contained in a sample in the range of mL can be quantified very accurately.

According to the present invention, it is possible to diagnose a patient having a disease caused by glycosphingolipid accumulation or a carrier thereof, particularly a patient with Gaucher's disease and Fabry disease.

Claims

A method for quantifying glycosphingolipid,
(A) A specimen containing glycosphingolipid is dissolved or dispersed in an aqueous solution, and a sphingolipid ceramide deacylase is added to the glycosphingolipid-containing liquid thus obtained to deacylate the glycosphingolipid contained therein by hydrolysis. Releasing lysosphingoglycolipid, and
(B) A quantification method comprising a step of quantifying the glycosphingolipid by measuring the lysosphingoglycolipid by mass spectrometry.
The specimen is obtained from any one selected from the group consisting of kidney, liver and other organ tissues collected from the subject, blood, spinal fluid and other body fluids, urine, skin, muscle, and interstitial tissue. The quantification method according to claim 1.
The method according to claim 1 or 2, wherein the glycosphingolipid is selected from the group consisting of glucocerebroside, globotriaosylceramide, galactocerebroside, sulfatide, ganglioside GM1 and ganglioside GM2.
3. The method according to claim 1 or 2, wherein the glycosphingolipid is glucocerebrosid or globotriaosylceramide.
The sphingolipid ceramide deacylase is selected from the group consisting of SCDase from Pseudomonas, SCDase from Shewanella, SCDase from Streptomyces, and SCDase from non-fermentative Gram-negative bacilli AI-2 (FERM P-16124) The quantification method according to any one of claims 1 to 4, wherein
The quantification method according to claim 1, wherein the mass spectrometry method is tandem mass spectrometry (MS / MS).
7. The quantification method according to claim 1, wherein the mass spectrometry method is high performance liquid chromatograph tandem mass spectrometry (LC / MS / MS).
A method for diagnosing a disease caused by glycosphingolipid accumulation by quantifying the glycosphingolipid using the quantification method according to any one of claims 1 to 7.
The disease caused by glycosphingolipid accumulation is selected from the group consisting of Gaucher disease, Fabry disease, Krabbe disease, metachromatic leucodystrophy, GM1-gangliosidosis and GM2-gangliosidosis, The diagnostic method according to Item 8.
The diagnostic method according to claim 8, wherein the disease caused by glycosphingolipid accumulation is Gaucher's disease or Fabry disease.
A method for preparing a lysosphingoglycolipid-containing liquid,
(A) dissolving or dispersing a specimen containing glycosphingolipid in an aqueous solution, and sonicating the glycosphingolipid-containing liquid thus obtained;
(B) freezing the sonicated liquid;
(C) thawing the frozen solution; and
(D) a step of adding a sphingolipid ceramide deacylase to the melted liquid, deacylating the glycosphingolipid contained in the liquid by hydrolysis, and releasing lysosphingoglycolipid. , The preparation method.
The specimen is obtained from any one selected from the group consisting of kidney, liver and other organ tissues collected from the subject, blood, spinal fluid and other body fluids, urine, skin, muscle, and interstitial tissue. The preparation method of claim 11, wherein
The preparation method according to claim 11 or 12, wherein the glycosphingolipid is selected from the group consisting of glucocerebroside, globotriaosylceramide, galactocerebroside, sulfatide, ganglioside GM1 and ganglioside GM2.
The preparation method according to claim 11 or 12, wherein the glycosphingolipid is glucocerebrosid or globotriaosylceramide.
The sphingolipid ceramide deacylase is selected from the group consisting of SCDase from Pseudomonas, SCDase from Shewanella, SCDase from Streptomyces, and SCDase from non-fermentative Gram-negative bacilli AI-2 (FERM P-16124) The preparation method according to any one of claims 11 to 14, wherein
A method for quantifying glycosphingolipid, comprising:
(A) sonicating the glycosphingolipid-containing solution prepared in any one of claims 11 to 15,
(B) A quantification method comprising a step of quantifying a glycosphingolipid by measuring lysosphingoglycolipid contained in the ultrasonically treated liquid by mass spectrometry.
The quantification method according to claim 16, wherein the mass spectrometry method is tandem mass spectrometry (MS / MS).
The quantitative method according to claim 16, wherein the mass spectrometry is high-performance liquid chromatograph tandem mass spectrometry (LC / MS / MS).
A method for diagnosing a disease caused by glycosphingolipid accumulation by quantifying the glycosphingolipid using the quantification method according to any one of claims 16 to 18.
The disease caused by glycosphingolipid accumulation is selected from the group consisting of Gaucher's disease, Fabry disease, Krappe disease, metachromatic leucodystrophy, GM1-gangliosidosis and GM2-gangliosidosis, Item 20. The diagnosis method according to Item 19.
The diagnostic method according to claim 19, wherein the disease caused by accumulation of glycosphingolipid is Gaucher's disease or Fabry disease.
A method for producing a lysosphingoglycolipid-containing liquid,
(A) dissolving or dispersing a material containing glycosphingolipid in an aqueous solution, and sonicating the glycosphingolipid-containing liquid thus obtained;
(B) freezing the sonicated liquid;
(C) thawing the frozen solution; and
(D) a step of adding a sphingolipid ceramide deacylase to the melted liquid, deacylating the glycosphingolipid contained in the liquid by hydrolysis, and releasing lysosphingoglycolipid. ,Production method.