Quantitative determination of Hex4, lyso-GM1, Fuc-GlcNAc-Asn, and lyso-sulfatide in cerebrospinal fluid

Abstract

To provide a method of quantifying Hex4, lyso-GM1, Fuc-GlcNAc-Asn, or lyso-sulfatide contained in a brain.SOLUTION: A method of quantifying Hex4, lyso-GM1, Fuc-GlcNAc-Asn, or lyso-sulfatide contained in cerebrospinal fluid is provided, the method comprising: adding an internal standard substance to a solution containing cerebrospinal fluid; subjecting the solution containing the cerebrospinal fluid with the added internal standard substance to liquid chromatography to obtain an eluate; and subjecting the eluate to mass spectrometry.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a method for quantifying Hex4, lyso-GM1, Fuc-GlcNAc-Asn, or lyso-sulfatide contained in cerebrospinal fluid, and more specifically, to a method for quantifying the amount of Hex4, lyso-GM1, Fuc-GlcNAc-Asn, or lyso-sulfatide contained in cerebrospinal fluid, the method comprising the steps of subjecting a solution containing the cerebrospinal fluid to liquid chromatography to obtain an eluate, and subjecting the eluate to mass spectrometry. [Background technology]

[0002] Pompe disease is a type of glycogen storage disease (type II glycogen storage disease) caused by a deficiency of acid α-glucosidase. Acid α-glucosidase is a type of lysosomal enzyme that has the activity of breaking down glycogen into glucose in lysosomes. When this enzyme is deficient, glycogen accumulates in the lysosomes. This enzyme is pancellular, but in Pompe disease, the main symptoms are due to invasion of the myocardium, skeletal muscle, and diaphragm. Clinically, Pompe disease is classified into three types: classical infantile type, infantile type, and adult type, and in all cases, muscle weakness and respiratory disorders due to glycogen accumulation in the myocardium, skeletal muscle, and diaphragm are observed. It has been reported that respiratory disorders are not only caused by impaired muscle contractility of the diaphragm, but also by impairment of the phrenic nerve, which controls the respiratory system (Non-Patent Document 1).

[0003] As a treatment for Pompe disease, enzyme replacement therapy is performed by injecting acid α-glucosidase into the patient via intravenous drip infusion. The efficacy of enzyme replacement therapy is generally evaluated by quantifying glycogen concentration in muscle tissue.

[0004] Hexose tetrasaccharide (Hex4) has been reported as a biomarker for Pompe disease other than glycogen (Patent Document 1, Non-Patent Documents 2-7), and it has been reported that its level is elevated in the urine and blood of patients with Pompe disease (Non-Patent Document 5). Hex4 is thought to be a metabolic product of glycogen, and its main components include Glc4 (Glc α1-6 Glc α1-4 Glc α1-4 Glc) and M4 (Glc α1-4 Glc α1-4 Glc α1-4 Glc) (Non-Patent Document 3).

[0005] GM1 gangliosidosis is a genetic disease caused by the deficiency of β-galactosidase. β-galactosidase is a type of lysosomal enzyme that breaks down monosialoganglioside GM1, a type of sphingolipid, into monosaccharides in lysosomes. When this enzyme is deficient, monosialoganglioside GM1 accumulates in the brain and internal organs. Clinically, GM1 gangliosidosis is classified into four types: infantile type (type 1), which develops from early infancy and shows widespread central nervous system disorders including spastic paraplegia, cherry red spots on the fundus, hepatosplenomegaly, and bone abnormalities, juvenile type (type 2), which develops from around 1 year of age and shows progressive central nervous system disorders, adult type (type 3), which shows symptoms such as speech disorder from school age and is characterized by extrapyramidal symptoms such as gait disturbance and dystonia, and Morquio disease type B, which shows severe bone deformation without central nervous system symptoms.

[0006] Treatment of GM1 gangliosidosis is limited to symptomatic treatment, and no enzyme replacement therapy is currently in clinical use.

[0007] In lysosomal diseases in which sphingolipids accumulate, there have been reported cases in which not only the substrate substance itself but also the lyso form (deacylated form) of the accumulated substance has been evaluated as a biomarker (Non-Patent Document 8), and it has been reported that lyso-monosialoganglioside GM1 (lyso-GM1), the lyso form of monosialoganglioside GM1, is elevated in the blood of patients with GM1 gangliosidosis (Non-Patent Document 9).

[0008] Fucosidosis is a type of genetic disease caused by the deficiency of α-L-fucosidase (FUCA1). FUCA1 is a type of lysosomal enzyme that has the activity of hydrolyzing α-L-fucoside to L-fucose and alcohol in lysosomes. When this enzyme is deficient, glycolipids and glycoproteins containing fucose (α-L-fucoside, etc.) accumulate in the brain and internal organs. Clinically, fucosidosis is classified into two types: a severe type (type 1) that develops in infancy and is characterized by facial abnormalities, retarded psychomotor development, and an elevated concentration of NaCl in sweat, and a mild type (type 2) that develops from 1 to 2 years of age, is mainly characterized by angiokeratoma, has a normal concentration of NaCl in sweat, and progresses slowly.

[0009] Treatment of fucosidosis is limited to supportive care for neurological symptoms, and no enzyme replacement therapy is currently in clinical use.

[0010] In fucosidosis, in which α-L-fucoside and the like accumulate, a method for evaluating Fuc1-α-6GlcNAc1-β-Asn (hereinafter referred to as Fuc-GlcNAc-Asn) as a biomarker has been reported. It has been reported that Fuc1-α-6GlcNAc1-β-Asn accumulates in the urine of patients with fucosidosis and in the brains of fucosidosis model dogs (Non-Patent Documents 10 to 14).

[0011] Metachromatic leukodystrophy is a genetic disease caused by a deficiency of arylsulfatase A (ARSA). ARSA is a type of lysosomal enzyme that has the activity of decomposing sulfatide, a type of sphingolipid, into galactosylceramide in lysosomes. When this enzyme is deficient, sulfatides and other substances accumulate in the brain white matter, peripheral nerves, kidneys, etc. Clinically, metachromatic leukodystrophy is classified into three types: infantile type, which develops by the age of 2 and presents with hypotonia, loss of deep tendon reflexes, and gait disturbance; juvenile type, which develops around the age of 4 to 6 and presents with optic atrophy, intellectual disability, spastic paralysis, etc.; and adult type, which develops from the late teens onwards and presents with emotional disorders, speech disorders, dementia, and psychiatric symptoms, and progresses over a period of 5 to 10 years.

[0012] Treatment of metachromatic leukodystrophy is limited to symptomatic treatment, and no enzyme replacement therapy is currently in clinical use.

[0013] It has been reported that not only sulfatide but also lyso-sulfatide, the lyso form of sulfatide, accumulates in the brain, kidneys, and liver of patients with metachromatic leukodystrophy (Patent Document 2, Non-Patent Documents 15-19). [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Special Publication No. 2004-501365 [Patent Document 2] Special Publication No. 2016-506501 [Non-patent literature]

[0015] [Non-Patent Document 1] DeRuisseau LR. et al., Proc Natl Acad Sci US A. 106(23), 9419-24 (2009) [Non-Patent Document 2] Chien YH. et al., JIMD Rep. 19, 67-73 (2015) [Non-Patent Document 3] Sluiter W. et al., Clin Chem. 58(7), 1139-47 (2012) [Non-Patent Document 4] Young SP. et al., Genet Med. 11(7), 536-41 (2009) [Non-Patent Document 5] An Y. et al., Mol Genet Metab. 85(4), 247-54 (2005) [Non-Patent Document 6] Young SP. et al., Anal Biochem. 316(2), 175-80 (2003)

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[0016] An object of the present invention is to provide a method for quantifying Hex4, lyso-GM1, Fuc-GlcNAc-Asn, or lyso-sulfatide contained in cerebrospinal fluid. [Means for solving the problem]

[0017] In the course of research aimed at the above-mentioned objective, the present inventors discovered that Hex4, lyso-GM1, Fuc-GlcNAc-Asn, or lyso-sulfatide contained in cerebrospinal fluid collected from experimental animals can be quantitatively determined with good sensitivity by mass spectrometry, and thus completed the present invention. That is, the present invention includes the following. 1. A method for quantifying substances contained in cerebrospinal fluid, comprising: adding an internal standard to a solution containing the cerebrospinal fluid to which the internal standard has been added; subjecting the solution containing the cerebrospinal fluid to liquid chromatography to obtain an eluate; subjecting the eluate to mass spectrometry; The method comprises: 2. The method according to 1 above, wherein the substance contained in the cerebrospinal fluid is Hex4. 3. The method according to claim 2, wherein the liquid chromatography is hydrophilic interaction liquid chromatography. 4. The method according to claim 2 or 3 above, wherein said Hex4 is measured by comparison with an internal standard substance. 5. The method according to any one of 2 to 4 above, wherein the internal standard substance is represented by the following formula [VII] or formula [XXV]. [ka] [In the formula, at least one of the carbon atoms indicated by an asterisk is carbon 13.] [ka] [In the formula, at least one of the carbon atoms indicated by an asterisk is carbon 13.] 6. The method according to any one of 2 to 5 above, wherein the cerebrospinal fluid is obtained from a patient suffering from a disease in which Hex4 and / or glycogen accumulates in the body. 7. The method according to claim 6, wherein the disease is Pompe's disease. 8. The method according to 6 or 7 above, wherein the patient has undergone treatment to reduce Hex4 and / or glycogen present in the body. 9. The method according to 1 above, wherein the substance contained in the cerebrospinal fluid is lyso-monosialganglioside GM1. 10. The method according to claim 9, wherein the liquid chromatography is reverse phase chromatography. 11. The method according to claim 9 or 10, wherein the lyso-monosialganglioside GM1 is measured by comparison with an internal standard substance. 12. The method according to any one of 9 to 11 above, wherein the internal standard substance is represented by the following formula [VIII]: [ka] 13. The method according to any one of 9 to 12 above, wherein the cerebrospinal fluid is obtained from a patient suffering from a disease in which lyso-monosialganglioside GM1 and / or monosialoganglioside GM1 accumulates in the body. 14. The method according to claim 13, wherein the disease is GM1 gangliosidosis. 15. The method according to claim 13 or 14, wherein the patient has undergone treatment to reduce lyso-monosialganglioside GM1 and / or monosialoganglioside GM1 present in the body. 16. The method according to 1 above, wherein the substance contained in the cerebrospinal fluid is Fuc-GlcNAc-Asn. 17. The method according to claim 16, wherein the liquid chromatography is normal phase chromatography or anion exchange chromatography. 18. The method according to claim 16 or 17, wherein the Fuc-GlcNAc-Asn is measured by comparison with an internal standard substance. 19. The method according to any one of 16 to 18 above, wherein the internal standard substance is represented by the following formula [IX]. [ka] 20. The method according to any one of 16 to 19 above, wherein the cerebrospinal fluid is obtained from a patient suffering from a disease in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulates in the body. 21. The method according to claim 20, wherein the disease is fucosidosis. 22. The method according to claim 20 or 21 above, wherein the patient has undergone treatment to reduce Fuc-GlcNAc-Asn and / or α-L-fucoside present in the body. 23. The method according to 1 above, wherein the substance contained in the cerebrospinal fluid is lysosulfatide. 24. The method according to claim 23, wherein the liquid chromatography is reverse phase chromatography. 25. The method according to claim 23 or 24, wherein the lyso-sulfatide is measured by comparison with an internal standard substance. 26. The method according to any one of 23 to 25 above, wherein the internal standard substance is represented by the following formula [X]: [ka] 27. The method according to any one of 23 to 26 above, wherein the cerebrospinal fluid is obtained from a patient suffering from a disease in which lyso-sulfatide and / or sulfatide accumulates in the body. 28. The method according to claim 27, wherein the disease is metachromatic leukodystrophy. 29. The method according to claim 27 or 28, wherein the patient has undergone treatment to reduce lyso-sulfatide and / or sulfatide present in the body. Effect of the Invention

[0018] According to the present invention, it is possible to diagnose diseases that develop due to accumulation of Hex4 and / or glycogen in the central nervous system, and to examine the effectiveness of treatments for such diseases that aim to reduce Hex4 and / or glycogen accumulated in the central nervous system.

[0019] Furthermore, according to the present invention, it is possible to diagnose diseases that develop due to the accumulation of lyso-monosialoganglioside GM1 and / or monosialoganglioside GM1 in the central nervous system, and to examine the effectiveness of treatments performed for such diseases in order to reduce lyso-monosialoganglioside GM1 and / or monosialoganglioside GM1 accumulated in the central nervous system.

[0020] Furthermore, the present invention makes it possible to diagnose diseases that develop due to the accumulation of Fuc-GlcNAc-Asn and / or α-L-fucoside in the central nervous system, and to examine the effectiveness of treatments for such diseases that are intended to reduce the amount of Fuc-GlcNAc-Asn and / or α-L-fucoside that has accumulated in the central nervous system.

[0021] Furthermore, according to the present invention, it is possible to diagnose diseases that develop due to accumulation of lyso-sulfatide and / or sulfatide in the central nervous system, and to examine the effectiveness of treatments for such diseases that are intended to reduce lyso-sulfatide and / or sulfatide accumulated in the central nervous system. [Brief description of the drawings]

[0022] [Figure 1]A diagram showing the calibration curve for Hex4. The vertical axis shows the area ratio (Hex4 detection peak area / H-IS detection peak area), and the horizontal axis shows the Hex4 concentration (ng / mL). [Diagram 2] Figure showing the results of measuring brain Hex4 concentration in wild-type mice and Pompe disease model mice. The vertical axis shows brain Hex4 concentration (μg / wet tissue weight (g)). The white bars show the measured values ​​of brain Hex4 concentration in wild-type mice (WT), and the black bars show the measured values ​​of brain Hex4 concentration in Pompe disease model mice (KO). Error bars show standard deviation (WT: n=5, KO: n=4). [Diagram 3] Figure showing the results of measuring the Hex4 concentration in cerebrospinal fluid (CSF) in wild-type mice and Pompe disease model mice. The vertical axis shows the CSF Hex4 concentration (ng / mL). The white bars show the measured values ​​of the Hex4 concentration in CSF of wild-type mice (WT), and the black bars show the measured values ​​of the Hex4 concentration in CSF of Pompe disease model mice (KO). The error bars show the standard deviation (WT: n = 5, KO: n = 3). [Figure 4] Figure showing the results of measuring brain glycogen concentration in wild-type mice and Pompe disease model mice. The vertical axis shows glycogen concentration (mg / wet tissue weight (g)). The white bars show the measured values ​​of brain glycogen concentration in wild-type mice (WT), and the black bars show the measured values ​​of brain glycogen concentration in Pompe disease model mice (KO). Error bars show standard deviation (WT: n=5, KO: n=4). [Diagram 5] A graph showing the relationship between brain glycogen concentration and brain Hex4 concentration in the same mouse. The vertical axis shows brain glycogen concentration (mg / wet tissue weight (g)), and the horizontal axis shows brain Hex4 concentration (μg / wet tissue weight (g)). White diamonds represent wild-type mice (WT), and black diamonds represent Pompe disease model mice (KO). [Figure 6] A graph showing the relationship between brain glycogen concentration and CSF Hex4 concentration in the same mouse. The vertical axis shows brain glycogen concentration (mg / wet tissue weight (g)), and the horizontal axis shows CSF Hex4 concentration (ng / mL). White diamonds represent wild-type mice (WT), and black diamonds represent Pompe disease model mice (KO). [Figure 7]The relationship between brain and CSF Hex4 concentrations in the same mouse. The vertical axis indicates brain Hex4 concentration (μg / wet tissue weight (g)), and the horizontal axis indicates CSF Hex4 concentration (ng / mL). White diamonds indicate wild-type mice (WT), and black diamonds indicate Pompe disease model mice (KO). [Figure 8] A diagram showing the calibration curve for lyso-GM1. The vertical axis shows the area ratio (lyso-GM1 detection peak area / G-IS detection peak area), and the horizontal axis shows the lyso-GM1 concentration (ng / mL). [Figure 9] This figure shows the results of measuring lyso-GM1 concentration in the brain of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of lyso-GM1 in the brain (μg / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 10] This figure shows the results of measuring lyso-GM1 concentration in the spinal cord of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of lyso-GM1 in the spinal cord (μg / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 11] This figure shows the results of measuring lyso-GM1 concentration in the lungs of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of lyso-GM1 in the lungs (ng / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 12]This figure shows the results of measuring lyso-GM1 concentration in the liver of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis shows the concentration of lyso-GM1 in the liver (ng / wet tissue weight (g)). 12wks and 38wks indicate 12 weeks of age and 38 weeks of age, respectively. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 13] This figure shows the results of measuring kidney lyso-GM1 concentration in wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates kidney lyso-GM1 concentration (ng / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 14] This figure shows the results of measuring lyso-GM1 concentration in the quadriceps muscle of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis shows the concentration of lyso-GM1 in the quadriceps muscle (ng / wet tissue weight (g)). 12wks and 38wks indicate 12 weeks of age and 38 weeks of age, respectively. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 15] This figure shows the results of measuring lyso-GM1 concentration in the heart of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of lyso-GM1 in the heart (ng / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 16]This figure shows the results of measuring lyso-GM1 concentration in the spleen of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of lyso-GM1 in the spleen (ng / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 17] This figure shows the results of plasma lyso-GM1 concentration measurement in wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the plasma lyso-GM1 concentration (ng / mL). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 18] This figure shows the results of measuring lyso-GM1 concentrations in CSF in wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of lyso-GM1 in the CSF (ng / mL). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks, KO12wks: n=1, WT38wks, KO38wks: n=2). [Figure 19] This figure shows the results of measuring GM1 concentration in the brain of wild-type mice (WT), β-Gal KO homozygous mice (KO), and β-Gal KO heterozygous mice (Hetero). The vertical axis indicates the concentration of GM1 in the brain (mg / wet tissue weight (g)). 12wks indicates 12 weeks of age, and 38wks indicates 38 weeks of age. Error bars indicate standard deviation (WT12wks, Hetero12wks: n=1, WT38wks, KO12wks, KO38wks: n=2). [Figure 20]A graph showing the relationship between lyso-GM1 concentrations in the brain and CSF in the same mouse. The vertical axis shows the lyso-GM1 concentration in the brain (μg / wet tissue weight (g)), and the horizontal axis shows the lyso-GM1 concentration in the CSF (ng / mL). White diamonds represent wild-type mice (WT), white circles represent β-Gal KO heterozygous mice (Hetero), and black diamonds represent β-Gal KO homozygous mice (KO). [Figure 21] A graph showing the relationship between brain GM1 concentration and CSF lyso-GM1 concentration in the same mouse. The vertical axis shows brain GM1 concentration (mg / wet tissue weight (g)), and the horizontal axis shows CSF lyso-GM1 concentration (ng / mL). White diamonds represent wild-type mice (WT), white circles represent β-Gal KO heterozygous mice (Hetero), and black diamonds represent β-Gal KO homozygous mice (KO). [Figure 22] A graph showing the relationship between brain lyso-GM1 concentration and brain GM1 concentration in the same mouse. The vertical axis shows brain GM1 concentration (mg / wet tissue weight (g)), and the horizontal axis shows brain lyso-GM1 concentration (μg / wet tissue weight (g)). White diamonds represent wild-type mice (WT), white circles represent β-Gal KO heterozygous mice (Hetero), and black diamonds represent β-Gal KO homozygous mice (KO). [Diagram 23] A diagram showing the calibration curve for Fuc-GlcNAc-Asn. The vertical axis indicates the area ratio (Fuc-GlcNAc-Asn detection peak area / F-IS detection peak area), and the horizontal axis indicates the concentration of Fuc-GlcNAc-Asn (ng / mL). [Figure 24] Figure showing the results of measuring Fuc-GlcNAc-Asn concentrations in the brains of wild-type mice and fucosidosis model mice. The vertical axis shows the concentration of Fuc-GlcNAc-Asn in the brain (mg / g dry tissue weight). The white bars show the measured values ​​of Fuc-GlcNAc-Asn concentrations in the brains of wild-type mice (WT), and the black bars show the measured values ​​of Fuc-GlcNAc-Asn concentrations in the brains of fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=3). [Diagram 25]Figure showing the results of measuring Fuc-GlcNAc-Asn concentrations in the livers of wild-type mice and fucosidosis model mice. The vertical axis shows the Fuc-GlcNAc-Asn concentration in the liver (mg / g dry tissue weight). The white bars show the measured values ​​of Fuc-GlcNAc-Asn concentrations in the livers of wild-type mice (WT), and the black bars show the measured values ​​of Fuc-GlcNAc-Asn concentrations in the livers of fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=3). [Figure 26] Figure showing the results of measuring kidney Fuc-GlcNAc-Asn concentrations in wild-type mice and fucosidosis model mice. The vertical axis shows kidney Fuc-GlcNAc-Asn concentration (mg / dry tissue weight (g)). The white bars show the measured kidney Fuc-GlcNAc-Asn concentrations in wild-type mice (WT), and the black bars show the measured kidney Fuc-GlcNAc-Asn concentrations in fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=3). [Figure 27] Figure showing the results of measuring Fuc-GlcNAc-Asn concentrations in the spleen of wild-type mice and fucosidosis model mice. The vertical axis shows the concentration of Fuc-GlcNAc-Asn in the spleen (mg / g dry tissue weight). The white bars show the measured values ​​of Fuc-GlcNAc-Asn concentrations in the spleen of wild-type mice (WT), and the black bars show the measured values ​​of Fuc-GlcNAc-Asn concentrations in the spleen of fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=3). [Figure 28] Figure showing the results of plasma Fuc-GlcNAc-Asn concentration measurements in wild-type mice and fucosidosis model mice. The vertical axis shows the plasma Fuc-GlcNAc-Asn concentration (μg / mL). The white bars show the measured plasma Fuc-GlcNAc-Asn concentration in wild-type mice (WT), and the black bars show the measured plasma Fuc-GlcNAc-Asn concentration in fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=3). [Figure 29]Figure showing the results of urinary Fuc-GlcNAc-Asn concentration measurements in wild-type mice and fucosidosis model mice. The vertical axis shows the concentration of urinary Fuc-GlcNAc-Asn (μg / mg). The white bars show the measured values ​​of urinary Fuc-GlcNAc-Asn concentration in wild-type mice (WT), and the black bars show the measured values ​​of urinary Fuc-GlcNAc-Asn concentration in fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=2, no error bar for KO only). [Diagram 30] Figure showing the results of measuring the CSF Fuc-GlcNAc-Asn concentration in wild-type mice and fucosidosis model mice. The vertical axis shows the CSF Fuc-GlcNAc-Asn concentration (μg / mL). The white bars show the measured values ​​of CSF Fuc-GlcNAc-Asn concentration in wild-type mice (WT), and the black bars show the measured values ​​of CSF Fuc-GlcNAc-Asn concentration in fucosidosis model mice (KO). Error bars show standard deviation (WT: n=3, KO: n=3). [Diagram 31] A graph showing the relationship between Fuc-GlcNAc-Asn concentrations in the brain and CSF of the same mouse. The vertical axis shows the Fuc-GlcNAc-Asn concentration in the brain (mg / g dry tissue weight), and the horizontal axis shows the Fuc-GlcNAc-Asn concentration in the CSF (μg / mL). White diamonds represent wild-type mice (WT), and black diamonds represent fucosidosis model mice (KO). [Diagram 32] A diagram showing the calibration curve for lyso-sulfatide. The vertical axis shows the area ratio (lyso-sulfatide detection peak area / S-IS detection peak area), and the horizontal axis shows the lyso-sulfatide concentration (ng / mL). [Diagram 33]Fig. 1 shows the results of measuring lyso-sulfatide concentration in the brain of wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice. The vertical axis shows the concentration of lyso-sulfatide in the brain (μg / g dry tissue weight). WT (young) indicates 20-23 week old wild-type mice, WT indicates 93-100 week old wild-type mice, KO (young) indicates 20-23 week old ARSA KO homozygous mice, KO indicates 93-100 week old ARSA KO homozygous mice, and Hetero indicates 93-100 week old ARSA KO heterozygous mice. Error bars indicate standard deviation (WT, WT (young): n = 2, KO, KO (young): n = 3, Hetero: n = 4). [Diagram 34] This figure shows the results of measuring lyso-sulfatide concentration in the brain of wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice. The vertical axis shows the concentration of lyso-sulfatide in the spinal cord (μg / g dry tissue weight). WT (young) indicates 20-23 week old wild-type mice, WT indicates 93-100 week old wild-type mice, KO (young) indicates 20-23 week old ARSA KO homozygous mice, KO indicates 93-100 week old ARSA KO homozygous mice, and Hetero indicates 93-100 week old ARSA KO heterozygous mice. Error bars indicate standard deviation (WT, WT (young): n = 2, KO, KO (young): n = 3, Hetero: n = 4). [Diagram 35] This figure shows the results of measuring lyso-sulfatide concentration in the brain of wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice. The vertical axis shows the concentration of lyso-sulfatide in the sciatic nerve (μg / dry tissue weight (g)). WT (young) indicates 20-23 week old wild-type mice, WT indicates 93-100 week old wild-type mice, KO (young) indicates 20-23 week old ARSA KO homozygous mice, KO indicates 93-100 week old ARSA KO homozygous mice, and Hetero indicates 93-100 week old ARSA KO heterozygous mice. Error bars indicate standard deviation (WT, WT (young): n = 2, KO, KO (young): n = 3, Hetero: n = 4). [Diagram 36]Figure showing the results of measuring brain lyso-sulfatide concentration in wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice. The vertical axis shows kidney lyso-sulfatide concentration (μg / dry tissue weight (g)). WT (young) indicates 20-23 week old wild-type mice, WT indicates 93-100 week old wild-type mice, KO (young) indicates 20-23 week old ARSA KO homozygous mice, KO indicates 93-100 week old ARSA KO homozygous mice, and Hetero indicates 93-100 week old ARSA KO heterozygous mice. Error bars indicate standard deviation (WT, WT (young): n = 2, KO, KO (young): n = 3, Hetero: n = 4). BLOQ indicates that the measured value is the lower limit of quantification. [Figure 37] Figure showing the results of measuring lyso-sulfatide concentrations in the brain of wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice. The vertical axis shows the plasma lyso-sulfatide concentration (ng / mL). WT (young) indicates 20-23 week old wild-type mice, WT indicates 93-100 week old wild-type mice, KO (young) indicates 20-23 week old ARSA KO homozygous mice, KO indicates 93-100 week old ARSA KO homozygous mice, and Hetero indicates 93-100 week old ARSA KO heterozygous mice. Error bars indicate standard deviation (WT, WT (young): n = 2, KO, KO (young): n = 3, Hetero: n = 4). BLOQ indicates that the measured value is the lower limit of quantification. [Figure 38]Figure showing the results of measuring lyso-sulfatide concentration in the brain of wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice. The vertical axis shows the CSF lyso-sulfatide concentration (ng / mL). WT (young) indicates 20-23 week old wild-type mice, WT indicates 93-100 week old wild-type mice, KO (young) indicates 20-23 week old ARSA KO homozygous mice, KO indicates 93-100 week old ARSA KO homozygous mice, and Hetero indicates 93-100 week old ARSA KO heterozygous mice. Error bars indicate standard deviation (WT, WT (young): n = 2, KO, KO (young): n = 3, Hetero: n = 4). BLOQ indicates that the measured value is the lower limit of quantification. [Figure 39] A graph showing the relationship between lyso-sulfatide concentrations in the brain and CSF in the same mouse. The vertical axis shows brain lyso-sulfatide concentration (μg / g dry tissue weight), and the horizontal axis shows CSF lyso-sulfatide concentration (ng / mL). White diamonds represent wild-type mice (WT), white circles represent ARSA KO heterozygous mice (Hetero), and black diamonds represent ARSA KO homozygous mice (KO). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] In the present invention, the term "glucose tetrasaccharide" refers to four molecules of D-glucose linked in order via α1-6 bonds, α1-4 bonds, and α1-4 bonds, and is a compound represented by the following formula (I). Salts of the compound represented by formula (I) and their equivalents are also included in glucose tetrasaccharide. Glucose tetrasaccharide can be abbreviated as Glc4. The term 6-α-D-Glucopyranosyl Maltotriose used in Example 2 is synonymous with glucose tetrasaccharide.

[0024] [ka]

[0025] In the present invention, the term "lyso-monosialo-ganglioside GM1" refers to a compound represented by the following formula (III) in which monosialoganglioside GM1, a sphingolipid represented by the following formula (II), is a deacylated lyso form, and salts of the compound represented by the following formula (III) and their equivalents are also included in lyso-monosialo-ganglioside GM1. Monosialoganglioside GM1 can be abbreviated as GM1, and lyso-monosialo-ganglioside GM1 can be abbreviated as lyso-GM1.

[0026] [ka]

[0027] [ka]

[0028] In the present invention, "Fuc1-α-6GlcNAc1-β-Asn" refers to the compound represented by the following formula (IV), and the salt of the compound represented by the following formula (IV) and its equivalents are also included in Fuc1-α-6GlcNAc1-β-Asn. Fuc1-α-6GlcNAc1-β-Asn can be abbreviated as Fuc-GlcNAc-Asn.

[0029] [ka]

[0030] In the present invention, the term "lyso-sulfatide" refers to a compound represented by the following formula (VI) in which sulfatide, a glycolipid represented by the following formula (V), is deacylated lyso form, and salts of the compound represented by the following formula (VI) and their equivalents are also included in lyso-sulfatide. Lyso-sulfatide can be written as lyso-sulfatide.

[0031] [ka]

[0032] [ka]

[0033] The method of the present invention for quantifying Hex4 contained in cerebrospinal fluid preferably comprises the steps of adding an internal standard substance to a solution containing cerebrospinal fluid, subjecting the solution containing cerebrospinal fluid to liquid chromatography to obtain an eluate, and subjecting the eluate to mass spectrometry.

[0034] The method of the present invention for quantifying lyso-GM1 contained in cerebrospinal fluid preferably comprises the steps of adding an internal standard substance to a solution containing cerebrospinal fluid, subjecting the solution containing cerebrospinal fluid to liquid chromatography to obtain an eluate, and subjecting the eluate to mass spectrometry.

[0035] The method of the present invention for quantifying Fuc-GlcNAc-Asn contained in cerebrospinal fluid preferably comprises the steps of adding an internal standard substance to a solution containing cerebrospinal fluid, subjecting the solution containing cerebrospinal fluid to liquid chromatography to obtain an eluate, and subjecting the eluate to mass spectrometry.

[0036] The method of the present invention for quantifying lyso-sulfatide contained in cerebrospinal fluid preferably comprises the steps of adding an internal standard substance to a solution containing cerebrospinal fluid, subjecting the solution containing cerebrospinal fluid to liquid chromatography to obtain an eluate, and subjecting the eluate to mass spectrometry.

[0037] In this invention, the term "internal standard substance" refers to a standard sample for preparing a calibration curve, and a substance added in a fixed amount to a measurement sample for use in a method for calculating the relative amount of an analyte by determining the ratio between the measurement value derived from the measured internal standard substance and the measurement value derived from the analyte.

[0038] There is no particular restriction on the species from which the cerebrospinal fluid is derived, but it may be an experimental animal, including mouse, rat, or monkey, or a human.

[0039] The quantitative value of Hex4 contained in the cerebrospinal fluid has a positive correlation with the quantitative value of glycogen contained in the brain. Therefore, by quantifying Hex4 contained in the cerebrospinal fluid, the glycogen contained in the brain can be indirectly quantified. In addition, by measuring the change in the concentration of Hex4 contained in the cerebrospinal fluid, the change in the concentration of glycogen contained in the brain can be indirectly quantified. Therefore, for example, by quantifying Hex4 contained in the cerebrospinal fluid of an experimental animal, it is possible to screen a drug having a medicinal effect of reducing Hex4 and / or glycogen accumulated in the cerebrospinal fluid. In addition, by quantifying Hex4 contained in human cerebrospinal fluid, it is possible to diagnose patients suffering from diseases in which Hex4 and / or glycogen accumulate in the central nervous system. Furthermore, it is possible to investigate the effect of treatments performed for such diseases to reduce Hex4 and / or glycogen accumulated in the central nervous system. An example of a disease in which glycogen accumulates in the central nervous system is Pompe disease, which develops due to a genetic deficiency of acid α-glucosidase (GAA). Treatments for patients with Pompe disease to reduce the amount of glycogen stored in the body include enzyme replacement therapy, which supplements GAA, but this enzyme replacement therapy is not performed to reduce the amount of glycogen stored in the central nervous system.

[0040] The quantitative value of lyso-GM1 contained in cerebrospinal fluid has a positive correlation with the quantitative value of lyso-GM1 contained in the brain. Therefore, by quantifying lyso-GM1 contained in cerebrospinal fluid, the lyso-GM1 contained in the brain can be indirectly quantified. Furthermore, by measuring the change in the concentration of lyso-GM1 contained in cerebrospinal fluid, the change in the concentration of lyso-GM1 contained in the brain can be indirectly quantified. Therefore, for example, by quantifying lyso-GM1 contained in the cerebrospinal fluid of an experimental animal, it is possible to screen for a drug having a medicinal effect of reducing lyso-GM1 and / or monosialoganglioside GM1 accumulated in the cerebrospinal fluid. Furthermore, by quantifying lyso-GM1 contained in human cerebrospinal fluid, it is possible to diagnose patients suffering from diseases in which lyso-GM1 and / or monosialoganglioside GM1 accumulate in the central nervous system. Furthermore, it is possible to investigate the effect of a treatment performed for such a disease to reduce lyso-GM1 and / or monosialoganglioside GM1 accumulated in the central nervous system. An example of a disease in which monosialoganglioside GM1 accumulates in the central nervous system is GM1 gangliosidosis, which develops due to a genetic deficiency of β-galactosidase (β-Gal). For example, enzyme replacement therapy to replenish β-Gal is expected as a treatment for patients with GM1 gangliosidosis to reduce monosialoganglioside GM1 accumulated in the body, but this enzyme replacement therapy is not performed to reduce monosialoganglioside GM1 accumulated in the central nervous system.

[0041] The quantitative value of Fuc-GlcNAc-Asn contained in cerebrospinal fluid has a positive correlation with the quantitative value of Fuc-GlcNAc-Asn contained in the brain. Therefore, by quantifying Fuc-GlcNAc-Asn contained in cerebrospinal fluid, the Fuc-GlcNAc-Asn contained in the brain can be indirectly quantified. Furthermore, by measuring the change in the concentration of Fuc-GlcNAc-Asn contained in cerebrospinal fluid, the change in the concentration of Fuc-GlcNAc-Asn contained in the brain can be indirectly quantified. Therefore, for example, by quantifying Fuc-GlcNAc-Asn contained in the cerebrospinal fluid of an experimental animal, it is possible to screen for a drug having a medicinal effect of reducing Fuc-GlcNAc-Asn and / or α-L-fucoside accumulated in the cerebrospinal fluid. Furthermore, by quantifying Fuc-GlcNAc-Asn contained in human cerebrospinal fluid, it is possible to diagnose patients suffering from diseases in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulate in the central nervous system. Furthermore, it is possible to examine the effects of treatments for such diseases performed to reduce Fuc-GlcNAc-Asn and / or α-L-fucosides accumulated in the central nervous system. An example of a disease in which α-L-fucosides accumulate in the central nervous system is fucosidosis, which develops due to a genetic deficiency of α-L-fucosidase (FUCA1). For example, enzyme replacement therapy to replenish FUCA1 is expected as a treatment performed to reduce α-L-fucosides accumulated in the body of patients with fucosidosis, but this enzyme replacement therapy is not performed to reduce α-L-fucosides accumulated in the central nervous system.

[0042] The quantitative value of lyso-sulfatide contained in cerebrospinal fluid has a positive correlation with the quantitative value of lyso-sulfatide contained in the brain. Therefore, by quantifying the amount of lyso-sulfatide contained in cerebrospinal fluid, the amount of lyso-sulfatide contained in the brain can be indirectly quantified. Furthermore, by measuring the change in the concentration of lyso-sulfatide contained in cerebrospinal fluid, the change in the concentration of lyso-sulfatide contained in the brain can be indirectly quantified. Therefore, for example, by quantifying the amount of lyso-sulfatide contained in the cerebrospinal fluid of an experimental animal, it is possible to screen for a drug having a medicinal effect of reducing lyso-sulfatide and / or sulfatide accumulated in the cerebrospinal fluid. Furthermore, by quantifying the amount of lyso-sulfatide contained in human cerebrospinal fluid, it is possible to diagnose patients suffering from diseases in which lyso-sulfatide and / or sulfatide accumulate in the central nervous system. Furthermore, it is possible to investigate the effect of a treatment performed for such a disease to reduce lyso-sulfatide and / or sulfatide accumulated in the central nervous system. An example of a disease in which sulfatides accumulate in the central nervous system is metachromatic leukodystrophy, which develops due to a genetic deficiency of arylsulfatase A (ARSA). Enzyme replacement therapy to replenish ARSA is expected as a treatment for reducing sulfatides accumulated in the body of patients with metachromatic leukodystrophy, but this enzyme replacement therapy is not performed to reduce sulfatides accumulated in the central nervous system.

[0043] In the present invention, cerebrospinal fluid may be collected from a subject such as an experimental animal and then analyzed immediately, or may be frozen and stored before being thawed and analyzed. When analyzing multiple cerebrospinal fluid samples, the efficiency of the work can be improved by storing the cerebrospinal fluid frozen and analyzing them all at once.

[0044] In one embodiment of the present invention, the cerebrospinal fluid is subjected to liquid chromatography, and an eluate containing Hex4 is fractionated. The stationary phase material used in this liquid chromatography is not particularly limited as long as it can fractionate an eluate containing Hex4 from the cerebrospinal fluid, but preferably, it is a material that can adsorb Hex4 once and then elute it. The stationary phase material is preferably one that can adsorb Hex4 by ionic interaction, hydrophobic interaction, hydrophilic interaction, etc. Hex4 has high polarity. Therefore, as a material that can adsorb Hex4 once and then elute it, a material that can retain Hex4 by hydrophilic interaction can be preferably used. An example of a preferred stationary phase material for such high performance liquid chromatography is one that contains a primary amide group. The ACQUITY UPLC BEH Amide Column (Nihon Waters) used in Example 6 below is a preferred example of a stationary phase material that contains a primary amide group.

[0045] In one embodiment of the present invention, cerebrospinal fluid is subjected to liquid chromatography, and an eluate containing lyso-GM1 is fractionated. There is no particular limitation on the stationary phase material used in this liquid chromatography, as long as it can fractionate an eluate containing lyso-GM1 from cerebrospinal fluid, but it is preferably one that can adsorb lyso-GM1 once and then elute it. The stationary phase material is preferably one that can adsorb lyso-GM1 by ionic interaction, hydrophobic interaction, hydrophilic interaction, etc. Lyso-GM1 has a long carbon chain. Therefore, as a material that can adsorb it once and then elute it, one that can retain lyso-GM1 by hydrophobic interaction can be suitably used. One example of a preferred stationary phase material for such high-performance liquid chromatography is porous silica gel whose surface is modified with ODS (octadecylsilyl group). Cadenza CW-C18 (Intact Co., Ltd.) used in the following Example 16 is a suitable example of an ODS column.

[0046] In one embodiment of the present invention, cerebrospinal fluid is subjected to liquid chromatography, and an eluate containing Fuc-GlcNAc-Asn is fractionated. There is no particular limitation on the stationary phase material used in this liquid chromatography, as long as it can fractionate an eluate containing Fuc-GlcNAc-Asn from cerebrospinal fluid, but it is preferably one that can adsorb Fuc-GlcNAc-Asn once and then elute it. The stationary phase material is preferably one that can adsorb Fuc-GlcNAc-Asn by ionic interaction, hydrophobic interaction, hydrophilic interaction, etc. Fuc-GlcNAc-Asn has high polarity. Therefore, as a material that can adsorb it once and then elute it, one that can retain Fuc-GlcNAc-Asn by hydrophilic interaction can be preferably used. An example of a preferred stationary phase material for such high performance liquid chromatography is porous silica gel whose surface is modified with aminopropyl groups. Unison UK-Amino (Intact) used in the following Example 26 is a preferred example of an aminopropyl type column.

[0047] In one embodiment of the present invention, cerebrospinal fluid is subjected to liquid chromatography, and an eluate containing lyso-sulfatide is fractionated. The stationary phase material used in this liquid chromatography is not particularly limited as long as it can fractionate an eluate containing lyso-sulfatide from cerebrospinal fluid, but is preferably one that can adsorb lyso-sulfatide once and then elute it. The stationary phase material is preferably one that can adsorb lyso-sulfatide by ionic interaction, hydrophobic interaction, hydrophilic interaction, etc. Lyso-sulfatide has a long carbon chain. Therefore, as a material that can adsorb it once and then elute it, one that can retain lyso-sulfatide by hydrophobic interaction can be preferably used. One example of a preferred stationary phase material for such high-performance liquid chromatography is porous silica gel whose surface is modified with ODS (octadecylsilyl group). Cadenza CW-C18 (Intact Co., Ltd.) used in the following Example 35 is a preferred example of an ODS column.

[0048] A certain amount of an internal standard substance is added to the cerebrospinal fluid before subjecting it to liquid chromatography. In one embodiment of the present invention, the internal standard substance is, in particular, a Glc4 molecule represented by the following formula (VII), in which one or more of the six carbon atoms marked with an asterisk are 13 C, preferably all six of these carbon atoms are 13 The internal standard is Glc4 (6-aD-Glucopyranosyl maltotriose-13C6), which is C. This internal standard is added especially when quantifying Hex4 contained in cerebrospinal fluid.

[0049] [ka]

[0050] As an internal standard substance added when quantifying Hex4 in cerebrospinal fluid, one or more of the six carbon atoms marked with an asterisk in the M4 molecule represented by the following formula (XXV) is 13 C. All six of these carbon atoms can be used. 13 M4, which is C, is suitable as an internal standard.

[0051] [ka]

[0052] A certain amount of an internal standard substance is added to the cerebrospinal fluid before subjecting it to liquid chromatography. In one embodiment of the present invention, the internal standard substance is, in particular, the compound N-Glycinated lyso-ceramide trihexoside represented by the following formula (VIII). This internal standard substance is added particularly when quantifying lyso-monosialoganglioside GM1 contained in the cerebrospinal fluid.

[0053] [ka]

[0054] A certain amount of an internal standard substance is added to the cerebrospinal fluid before subjecting it to liquid chromatography. In one embodiment of the present invention, the internal standard substance is, in particular, a compound Fuc1-α-3GlcNAc1-β-OMe (Fuc-GlcNAc-OMe) represented by the following formula (IX). This internal standard substance is added particularly when quantifying Fuc-GlcNAc-Asn contained in the cerebrospinal fluid.

[0055] [ka]

[0056] A certain amount of an internal standard substance is added to the cerebrospinal fluid before subjecting it to liquid chromatography. In one embodiment of the present invention, the internal standard substance is, in particular, N-glycinated lyso-sulfatide, a compound represented by the following formula (X). This internal standard substance is added particularly when quantifying lyso-sulfatide contained in the cerebrospinal fluid.

[0057] [ka]

[0058] The flow path from the outlet of the liquid chromatography is connected to a mass spectrometer, and the eluate from the liquid chromatography is sequentially sent to the mass spectrometer.

[0059] There is no particular limitation on the mass spectrometer that can be used in this case. For example, the mass spectrometer may be one that employs any ionization method, including photoionization (APPI), electron ionization (EI), chemical ionization, electric desorption, fast atom bombardment (FAB), matrix assisted laser desorption ionization (MALDI), and electrospray ionization (ESI), as an ion source for ionizing the molecules to be analyzed. In addition, the mass spectrometer may be one in which the analysis section for separating the ionized molecules is of any type, including a magnetic deflection type, a quadrupole type, an ion trap type, and a tandem quadrupole type.

[0060] A mass spectrometer having an ion source operated in electrospray ionization (ESI) in a positive ion mode and a tandem quadrupole type analysis section can be suitably used in the method of the present invention. A tandem quadrupole type mass spectrometer is a mass spectrometer in which a quadrupole (Q1) functioning as a mass filter, a quadrupole (Q2) functioning as a collision cell, and a quadrupole (Q3) functioning as a mass filter are arranged in series. In the quadrupole (Q1), a precursor ion of interest is separated from a plurality of ions generated by ionization based on the mass-to-charge ratio (m / z) of the ion. Next, in the collision cell (Q2), the precursor ion is collided with an inert gas or the like (e.g., argon) to generate product ions (fragment ions). Next, in the quadrupole (Q3), the obtained product ions can be selectively detected based on the mass-to-charge ratio (m / z), and thus the substance that is the source of the product ions can be quantified.

[0061] Below, we take Glc4, which is one of the Hex4 species, as an example to illustrate a specific example of a method for generating precursor ions and product ions from Glc4 using a tandem quadrupole mass spectrometer. First, Glc4 is ionized to obtain a precursor ion with a mass-to-charge ratio (m / z) of 665.1. One of the theoretically conceivable chemical formulas for the precursor ion is shown in formula (XI) below.

[0062] [ka]

[0063] The above precursor ion was separated and fragmented in the collision cell (Q2) to give a product ion ([MH] - ) is obtained. The product ion ([MH] - A theoretically conceivable chemical formula for this is shown in formula (XII) below.

[0064] [ka]

[0065] The amount of Glc4 contained in a sample can be measured by measuring the product ions generated from Glc4 using the above-mentioned method using liquid chromatography and a tandem quadrupole mass spectrometer. Note that Glc4 and Hex4 other than Glc4 exist in cerebrospinal fluid, and some Hex4 other than Glc4 is not completely separated from Glc4 by liquid chromatography and generates the same product ions as those generated by the tandem quadrupole mass spectrometer measurement. Therefore, the measurement value of cerebrospinal fluid also includes values ​​derived from Hex4 other than Glc4. However, since most of the Hex4 present in cerebrospinal fluid is considered to generate the product ion shown by formula (XII), the measurement value of Glc4 can also be considered as the measurement value of Hex4. For example, M4 shown by formula (XXV) is also considered to generate the product ion shown by formula (XII) by the above-mentioned treatment. The eluate obtained by subjecting cerebrospinal fluid to liquid chromatography is analyzed using a tandem quadrupole mass spectrometer, and the area of ​​the peak (detected peak) detected on the chromatographic chart corresponding to the product ion (XII) with a mass-to-charge ratio (m / z) of 179.0 is calculated. This area is divided by the area of ​​the peak (internal standard peak) corresponding to the product ion derived from the internal standard contained in the same eluate. Note that the internal standard is Glc4 molecule represented by the above formula (VII), in which all six carbon atoms marked with an asterisk are 13 C, the mass-to-charge ratio (m / z) of the product ion derived from the internal standard is 185.0.

[0066] The standard samples for the calibration curve are analyzed in the same manner by liquid chromatography and mass spectrometry, the area of ​​the detection peak for each standard sample for the calibration curve is divided by the area of ​​the internal standard peak, and a calibration curve is created based on the obtained values. Then, by interpolating the values ​​obtained by analyzing the eluate obtained by subjecting cerebrospinal fluid to liquid chromatography onto this calibration curve, it is possible to measure a value that can be regarded as the concentration of Hex4 contained in the sample. In one embodiment of the present invention, the concentration of Hex4 refers to the measured value obtained in this manner. M4 can also be used as the internal standard instead of Glc4.

[0067] Below is a specific example of a method for generating precursor ions and product ions from lyso-GM1 using a tandem quadrupole mass spectrometer. First, lyso-GM1 is ionized to obtain a precursor ion with a mass-to-charge ratio (m / z) of 640.8. One of the theoretically conceivable chemical formulas of the precursor ion is shown in the following formula (XIII).

[0068] [ka]

[0069] The above precursor ion was separated and fragmented in the collision cell (Q2) to give a product ion with a mass-to-charge ratio (m / z) of 282.3 ([M+2H] 2+ ) is obtained. Product ion ([M+2H] 2+ A theoretically conceivable chemical formula for this compound is shown in formula (XIV) below.

[0070] [ka]

[0071] Next, a specific example of a method for generating precursor ions and product ions from the internal standard N-glycinated lyso-ceramide trihexoside using a tandem quadrupole mass spectrometer will be given. First, a precursor ion with a mass-to-charge ratio (m / z) of 843.5 is obtained by ionizing N-glycinated lyso-ceramide trihexoside. One of the theoretically possible chemical formulas of the precursor ion is shown in the following formula (XV).

[0072] [ka]

[0073] The above precursor ions were separated and fragmented in the collision cell (Q2) to give product ions with a mass-to-charge ratio (m / z) of 264.3 ([M+H] + ) is obtained. Product ions ([M+H] + A theoretically conceivable chemical formula for this compound is shown in formula (XVI) below.

[0074] [ka]

[0075] The amount of lyso-GM1 contained in a sample can be measured by measuring the product ions generated from lyso-GM1 using the above-mentioned method using liquid chromatography and a tandem quadrupole mass spectrometer. The eluate obtained by subjecting cerebrospinal fluid to liquid chromatography is analyzed using a tandem quadrupole mass spectrometer to calculate the area of ​​the peak (detection peak) detected on the chromatographic chart corresponding to the product ion (XIV) with a mass-to-charge ratio (m / z) of 282.3. This area is divided by the area of ​​the peak (internal standard peak) corresponding to the product ion derived from the internal standard contained in the same eluate. The mass-to-charge ratio (m / z) of the product ion derived from the internal standard is 264.3.

[0076] The standard samples for the calibration curve are analyzed in the same manner by liquid chromatography and mass spectrometry, and the area of ​​the detection peak for each standard sample for the calibration curve is divided by the area of ​​the internal standard peak, and a calibration curve is created based on the obtained values. The concentration of lyso-GM1 contained in the sample can be measured by interpolating the values ​​obtained by analyzing the eluate obtained by subjecting the cerebrospinal fluid to liquid chromatography onto this calibration curve.

[0077] Below is a specific example of a method for generating precursor ions and product ions from Fuc-GlcNAc-Asn using a tandem quadrupole mass spectrometer. First, Fuc-GlcNAc-Asn is ionized to obtain a precursor ion with a mass-to-charge ratio (m / z) of 482.2. One of the theoretically conceivable chemical formulas of the precursor ion is shown in formula (XVII) below.

[0078] [ka]

[0079] The above precursor ion was separated and fragmented in the collision cell (Q2) to give a product ion with a mass-to-charge ratio (m / z) of 336.1 ([M+H] + ) is obtained. Product ions ([M+H] + A theoretically conceivable chemical formula for this compound is shown in formula (XVIII) below.

[0080] [ka]

[0081] Next, a specific example of a method for generating precursor ions and product ions from the internal standard Fuc-GlcNAc-OMe using a tandem quadrupole mass spectrometer will be given. First, Fuc-GlcNAc-OMe is ionized to obtain a precursor ion with a mass-to-charge ratio (m / z) of 382.2. One of the theoretically conceivable chemical formulas of the precursor ion is shown in the following formula (XIX).

[0082] [ka]

[0083] The above precursor ions were separated and fragmented in the collision cell (Q2) to give product ions with a mass-to-charge ratio (m / z) of 204.1 ([M+H] + ) is obtained. Product ions ([M+H] + A theoretically conceivable chemical formula for this compound is shown in formula (XX) below.

[0084] [ka]

[0085] The amount of Fuc-GlcNAc-Asn contained in a sample can be measured by measuring the product ions generated from Fuc-GlcNAc-Asn using the above-mentioned method using liquid chromatography and a tandem quadrupole mass spectrometer. The eluate obtained by subjecting cerebrospinal fluid to liquid chromatography is analyzed using a tandem quadrupole mass spectrometer to calculate the area of ​​the peak (detection peak) detected on the chromatographic chart corresponding to the product ion (XVIII) with a mass-to-charge ratio (m / z) of 336.1. This area is divided by the area of ​​the peak (internal standard peak) corresponding to the product ion derived from the internal standard contained in the same eluate. The mass-to-charge ratio (m / z) of the product ion derived from the internal standard is 204.1.

[0086] The standard samples for the calibration curve are analyzed in the same manner by liquid chromatography and mass spectrometry, and the area of ​​the detection peak for each standard sample for the calibration curve is divided by the area of ​​the internal standard peak to create a calibration curve based on the values ​​obtained. The concentration of Fuc-GlcNAc-Asn contained in the sample can be measured by interpolating the values ​​obtained by analyzing the eluate obtained by subjecting cerebrospinal fluid to liquid chromatography onto this calibration curve.

[0087] Below is a specific example of a method for generating precursor ions and product ions from lyso-sulfatide using a tandem quadrupole mass spectrometer. First, lyso-sulfatide is ionized to obtain a precursor ion with a mass-to-charge ratio (m / z) of 542.3. One of the theoretically conceivable chemical formulas of the precursor ion is shown in formula (XXI) below.

[0088] [ka]

[0089] The above precursor ion was separated and fragmented in the collision cell (Q2) to give a product ion with a mass-to-charge ratio (m / z) of 282.3 ([M+H] + ) is obtained. Product ions ([M+H] + A theoretically conceivable chemical formula for this compound is shown in formula (XXII) below.

[0090] [ka]

[0091] Next, a specific example of a method for generating precursor ions and product ions from the internal standard N-glycinated lyso-sulfatide using a tandem quadrupole mass spectrometer will be given. First, a precursor ion with a mass-to-charge ratio (m / z) of 599.3 is obtained by ionizing N-glycinated lyso-sulfatide. One of the theoretically conceivable chemical formulas of the precursor ion is shown in the following formula (XXIII).

[0092] [ka]

[0093] The above precursor ion was separated and fragmented in the collision cell (Q2) to give a product ion with a mass-to-charge ratio (m / z) of 339.3 ([M+H] + ) is obtained. Product ions ([M+H] + A theoretically conceivable chemical formula for this compound is shown in formula (XXIV) below.

[0094] [ka]

[0095] The amount of lyso-sulfatide contained in a sample can be measured by measuring the product ions generated from lyso-sulfatide using the above-mentioned method using liquid chromatography and a tandem quadrupole mass spectrometer. The eluate obtained by subjecting cerebrospinal fluid to liquid chromatography is analyzed using a tandem quadrupole mass spectrometer to calculate the area of ​​the peak (detection peak) detected on the chromatographic chart corresponding to the product ion (XXII) with a mass-to-charge ratio (m / z) of 282.3. This area is divided by the area of ​​the peak (internal standard peak) corresponding to the product ion derived from the internal standard contained in the same eluate. The mass-to-charge ratio (m / z) of the product ion derived from the internal standard is 339.3.

[0096] The standard samples for the calibration curve are analyzed in the same manner by liquid chromatography and mass spectrometry, and the area of ​​the detection peak for each standard sample for the calibration curve is divided by the area of ​​the internal standard peak, and a calibration curve is created based on the obtained values. The concentration of lyso-sulfatide contained in the sample can be measured by interpolating the values ​​obtained by analyzing the eluate obtained by subjecting cerebrospinal fluid to liquid chromatography onto this calibration curve.

[0097] Pompe disease is one of the diseases in which glycogen accumulates in the body. Enzyme replacement therapy is used to treat Pompe disease, in which acid α-glucosidase is administered intravenously to replenish the patient. The most common method for evaluating the effectiveness of enzyme replacement therapy is to quantify the glycogen concentration in muscle tissue. However, this is not effective for central nervous system (CNS) abnormalities because the enzyme cannot pass through the blood-brain barrier (BBB). Therefore, the glycogen concentration in cerebrospinal fluid is not measured for the purpose of evaluating the effectiveness of regular enzyme replacement therapy.

[0098] Pompe disease is a genetic disease in which there is a partial or complete deficiency in GAA activity. Due to the deficiency or absence of this enzyme, glycogen accumulates in the patient's body. The symptoms of Pompe disease are mainly those affecting the heart muscle, skeletal muscle, and diaphragm. Clinically, Pompe disease is classified into three types: classical infantile type, infantile type, and adult type. In all cases, muscle weakness and respiratory disorders due to the accumulation of glycogen in the heart muscle, skeletal muscle, and diaphragm are observed. In recent years, it has been thought that respiratory disorders associated with Pompe disease are not only caused by impaired muscle contractility of the diaphragm, but also by disorders of the phrenic nerve that controls the respiratory system. In this light, it is considered important to remove glycogen accumulated in the central nervous system, even in Pompe disease, which was thought not to be accompanied by abnormalities in the central nervous system. However, measuring glycogen accumulated in the tissues of the central nervous system is not practical because it requires biopsy of cells from the central nervous system.

[0099] As shown in Example 10, the quantitative value of Hex4 contained in cerebrospinal fluid measured by the method of one embodiment of the present invention correlates with the concentration of glycogen contained in the brain tissue of the same individual. That is, according to the method of the present invention, the amount of glycogen accumulated in the central nervous system can be evaluated by measuring Hex4 contained in cerebrospinal fluid.

[0100] As mentioned above, in the case of Pompe disease, it is considered important to remove glycogen accumulated in the central nervous system. In order for a drug administered by intravenous injection or the like to reach the central nervous system, it is necessary to pass through the blood-brain barrier. When developing a therapeutic drug for Pompe disease that can pass through the blood-brain barrier and be effective in the central nervous system, it becomes necessary to evaluate the efficacy of the drug in the central nervous system. The method of the present invention meets such a demand. The same can be said for the development of a type of drug that can be administered directly to the central nervous system without passing through the blood-brain barrier.

[0101] Patients with diseases in which Hex4 and / or glycogen accumulate in the central nervous system, including Pompe disease, can be screened based on the measured value obtained by measuring the concentration of Hex4 in the cerebrospinal fluid using the method of one embodiment of the present invention. If the measured value is abnormally higher than that of normal humans, the subject can be determined to be a patient with the disease. The method of the present invention can be carried out for the purpose of making such a determination.

[0102] When treating a patient with a disease in which Hex4 and / or glycogen accumulate in the central nervous system, the effectiveness of the treatment can be confirmed by measuring the concentration of Hex4 contained in the patient's cerebrospinal fluid before and after the treatment, and measuring the amount of decrease in these concentrations after the treatment. The greater the amount of decrease, the higher the therapeutic effect can be judged to be. The method of the present invention can be carried out for the purpose of confirming such therapeutic effect. For example, when the concentration of Hex4 contained in the patient's blood decreases, preferably by 10% or more, before and after the treatment, for example, by 20% or more, 30% or more, or 50% or more, it can be judged that the treatment is effective.

[0103] GM1 gangliosidosis is one of the diseases in which monosialoganglioside GM1 accumulates in the body. Enzyme replacement therapy, in which β-galactosidase is administered intravenously to patients, is expected to be a treatment for GM1 gangliosidosis, but no such treatment is currently being used clinically. If enzyme replacement therapy were performed, the effectiveness of the therapy would be evaluated by quantifying the concentration of monosialoganglioside GM1 in muscle tissue. However, this is not effective for central nervous system (CNS) abnormalities because the enzyme cannot pass through the blood-brain barrier (BBB). Therefore, the concentration of monosialoganglioside GM1 in cerebrospinal fluid is not measured for the purpose of evaluating the effectiveness of conventional enzyme replacement therapy.

[0104] GM1 gangliosidosis is a disease in which there is a genetic partial or complete deficiency of β-galactosidase activity. Due to the deficiency or absence of this enzyme, monosialoganglioside GM1 accumulates in the patient's body. The main symptom of GM1 gangliosidosis is central nervous system damage. Clinically, GM1 gangliosidosis is classified into four types: infantile type, juvenile type, adult type, and Morquio disease type B. Central nervous system damage is observed in all types except Morquio disease type B. Therefore, it is considered important to remove monosialoganglioside GM1 accumulated in the central nervous system for GM1 gangliosidosis. However, measuring monosialoganglioside GM1 accumulated in the tissues of the central nervous system is not realistic because it requires biopsy of cells of the central nervous system.

[0105] As shown in Example 20, the quantitative value of lyso-GM1 contained in cerebrospinal fluid measured by the method of one embodiment of the present invention correlates with the concentration of monosialoganglioside GM1 contained in the brain tissue of the same individual. That is, according to the method of the present invention, the amount of monosialoganglioside GM1 accumulated in the central nervous system can be evaluated by measuring lyso-GM1 contained in cerebrospinal fluid.

[0106] As described above, in the case of GM1 gangliosidosis, it is considered important to remove monosialoganglioside GM1 accumulated in the central nervous system. In order for a drug administered by intravenous injection or the like to reach the central nervous system, it is necessary to pass through the blood-brain barrier. When developing a therapeutic drug for GM1 gangliosidosis that can pass through the blood-brain barrier and be effective in the central nervous system, it becomes necessary to evaluate the efficacy of the drug in the central nervous system. The method of the present invention meets such a demand. The same can be said for the development of a type of drug that can be administered directly to the central nervous system without passing through the blood-brain barrier.

[0107] Patients with diseases in which lyso-GM1 and / or monosialoganglioside GM1 accumulate in the central nervous system, including but not limited to GM1 gangliosidosis, can be screened based on the measured value obtained by measuring the concentration of lyso-GM1 in the cerebrospinal fluid using the method of one embodiment of the present invention. If the measured value is abnormally higher than that of normal humans, the subject can be determined to be a patient with the disease. The method of the present invention can be carried out for the purpose of making such a determination.

[0108] When treating a patient with a disease in which lyso-GM1 and / or monosialoganglioside GM1 accumulates in the central nervous system, the effectiveness of the treatment can be confirmed by measuring the concentration of lyso-GM1 contained in the patient's cerebrospinal fluid before and after the treatment, and measuring the amount of decrease in these concentrations after the treatment. The greater the amount of decrease, the higher the therapeutic effect can be judged to be. The method of the present invention can be carried out for the purpose of confirming such therapeutic effect. For example, when the concentration of lyso-GM1 contained in the patient's blood decreases, preferably by 10% or more, for example, by 20% or more, 30% or more, or 50% or more, before and after the treatment, the therapeutic effect can be judged to be present.

[0109] Fucosidosis is one of the diseases in which α-L-fucoside accumulates in the body. Enzyme replacement therapy, in which α-L-fucosidase is administered intravenously to patients, is expected to be a treatment for fucosidosis, but no such treatment is currently being used clinically. If enzyme replacement therapy were performed, a method for evaluating its effectiveness would be to quantify the concentration of α-L-fucoside in muscle tissue. However, this is not effective for abnormalities in the central nervous system (CNS) because the enzyme cannot pass through the blood-brain barrier (BBB). Therefore, the concentration of α-L-fucoside in cerebrospinal fluid is not measured for the purpose of evaluating the effectiveness of conventional enzyme replacement therapy.

[0110] Fucosidosis is a disease in which there is a genetic partial or complete deficiency of α-L-fucosidase activity. Due to the deficiency or absence of this enzyme, α-L-fucoside accumulates in the patient's body. The main symptom of fucosidosis is central nervous system damage. Clinically, fucosidosis is classified into two types, severe and mild, but central nervous system damage is observed in both cases. Therefore, it is considered important to remove α-L-fucoside that has accumulated in the central nervous system in fucosidosis. However, measuring α-L-fucoside that has accumulated in central nervous system tissues requires biopsy of central nervous system cells, which is not realistic.

[0111] As shown in Example 29, the quantitative value of Fuc-GlcNAc-Asn contained in cerebrospinal fluid measured by the method of one embodiment of the present invention correlates with the concentration of Fuc-GlcNAc-Asn contained in brain tissue of the same individual. In other words, according to the method of the present invention, the amount of Fuc-GlcNAc-Asn accumulated in the central nervous system can be evaluated by measuring Fuc-GlcNAc-Asn contained in cerebrospinal fluid.

[0112] As mentioned above, in the case of fucosidosis, it is considered important to remove α-L-fucoside accumulated in the central nervous system. In order for a drug administered by intravenous injection or the like to reach the central nervous system, it is necessary to pass through the blood-brain barrier. When developing a therapeutic drug for fucosidosis that can pass through the blood-brain barrier and be effective in the central nervous system, it becomes necessary to evaluate the efficacy of the drug in the central nervous system. The method of the present invention meets such a demand. The same can be said for the development of a type of drug that can be administered directly to the central nervous system without passing through the blood-brain barrier.

[0113] Patients with diseases in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulate in the central nervous system, including fucosidosis, can be screened based on the measured value obtained by measuring the concentration of Fuc-GlcNAc-Asn in cerebrospinal fluid using the method of one embodiment of the present invention. If the measured value is abnormally higher than that of normal humans, the subject can be determined to be a patient with the disease. The method of the present invention can be carried out for the purpose of making such a determination.

[0114] When treating a patient with a disease in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulates in the central nervous system, the effectiveness of the treatment can be confirmed by measuring the concentration of Fuc-GlcNAc-Asn contained in the patient's cerebrospinal fluid before and after the treatment, and measuring the amount of decrease in these concentrations after the treatment. The greater the amount of decrease, the higher the therapeutic effect can be judged to be. The method of the present invention can be carried out for the purpose of confirming such therapeutic effect. For example, when the concentration of Fuc-GlcNAc-Asn contained in the patient's blood decreases, preferably by 10% or more, for example, by 20% or more, 30% or more, or 50% or more, before and after the treatment, the therapeutic effect can be judged to be present.

[0115] Metachromatic leukodystrophy is one of the diseases in which sulfatides accumulate in the body. Enzyme replacement therapy, in which arylsulfatase A (ARSA) is administered intravenously to patients, is expected to be a treatment for metachromatic leukodystrophy, but no such treatment is currently being used clinically. If enzyme replacement therapy were performed, the effectiveness of the therapy would be evaluated by quantifying the sulfatide concentration in muscle tissue. However, this is not effective for central nervous system (CNS) abnormalities because the enzyme cannot pass through the blood-brain barrier (BBB). Therefore, the sulfatide concentration in cerebrospinal fluid has not been measured to evaluate the effectiveness of conventional enzyme replacement therapy.

[0116] Metachromatic leukodystrophy is a disease caused by a genetic deficiency of partial or complete arylsulfatase A (ARSA) activity. Deficiency or absence of this enzyme leads to accumulation of sulfatides in the patient's body. The main symptom of metachromatic leukodystrophy is central nervous system damage. Clinically, metachromatic leukodystrophy is classified into three types: infantile, juvenile, and adult, and central nervous system damage is observed in all types. Therefore, it is considered important to remove sulfatides accumulated in the central nervous system in metachromatic leukodystrophy. However, measuring sulfatides accumulated in central nervous system tissue requires biopsy of central nervous system cells, which is not practical.

[0117] As shown in Example 38, the quantitative value of lyso-sulfatide contained in cerebrospinal fluid measured by the method of one embodiment of the present invention correlates with the concentration of lyso-sulfatide contained in the brain tissue of the same individual. That is, according to the method of the present invention, the amount of lyso-sulfatide accumulated in the central nervous system can be evaluated by measuring the lyso-sulfatide contained in cerebrospinal fluid.

[0118] As mentioned above, in metachromatic leukodystrophy, it is also considered important to remove sulfatides accumulated in the central nervous system. In order for a drug administered by intravenous injection or the like to reach the central nervous system, it is necessary to pass through the blood-brain barrier. When developing a therapeutic drug for metachromatic leukodystrophy that can pass through the blood-brain barrier and be effective in the central nervous system, it becomes necessary to evaluate the efficacy of the drug in the central nervous system. The method of the present invention meets such a demand. The same can be said for developing a type of drug that can be administered directly to the central nervous system without passing through the blood-brain barrier.

[0119] Patients with diseases in which lyso-sulfatide and / or sulfatide accumulate in the central nervous system, including metachromatic leukodystrophy, can be screened based on the measured value obtained by measuring the concentration of lyso-sulfatide in the cerebrospinal fluid using the method of one embodiment of the present invention. If the measured value is abnormally higher than that of normal humans, the subject can be determined to be a patient with the disease. The method of the present invention can be carried out for the purpose of making such a determination.

[0120] When treating a patient with a disease in which lyso-sulfatide and / or sulfatide accumulate in the central nervous system, the effectiveness of the treatment can be confirmed by measuring the concentration of lyso-sulfatide contained in the patient's cerebrospinal fluid before and after the treatment, and measuring the amount of decrease in these concentrations after the treatment. The greater the amount of decrease, the higher the therapeutic effect can be judged to be. The method of the present invention can be carried out for the purpose of confirming such therapeutic effect. For example, when the concentration of lyso-sulfatide contained in the patient's blood decreases, preferably by 10% or more, for example, by 20% or more, 30% or more, or 50% or more, before and after the treatment, the therapeutic effect can be judged to be present.

[0121] The method of one embodiment of the present invention may also be used to search for a therapeutic agent for a disease in which Hex4 and / or glycogen accumulate in the central nervous system, using a model animal. The therapeutic effect of administering a drug to a model animal for a disease in which Hex4 and / or glycogen accumulate in the central nervous system can be confirmed by measuring the concentration of Hex4 contained in the cerebrospinal fluid of the model animal before and after administration, and measuring the amount of reduction after administration. The greater the amount of reduction, the higher the therapeutic effect of the drug can be determined. The method of the present invention can be carried out for the purpose of confirming such therapeutic effect. For example, when the concentration of Hex4 contained in the blood of the model animal decreases, preferably by 10% or more, for example, by 20% or more, 30% or more, or 50% or more before and after treatment, it can be determined that there is a therapeutic effect. Here, the animal species of the model animal is not particularly limited, but examples include monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, and preferably monkeys, mice, and rats.

[0122] In other words, there is no particular limitation on the species from which the cerebrospinal fluid to be analyzed is obtained, but examples include humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, and in particular humans, monkeys, mice, and rats.

[0123] The method of one embodiment of the present invention can also be used to search for a therapeutic agent for a disease in which lyso-GM1 and / or monosialoganglioside GM1 accumulates in the central nervous system, using a model animal. The therapeutic effect of administering a drug to a model animal for a disease in which lyso-GM1 and / or monosialoganglioside GM1 accumulates in the central nervous system can be confirmed by measuring the concentration of lyso-GM1 contained in the cerebrospinal fluid of the model animal before and after administration, and measuring the amount of decrease after administration. The greater the amount of decrease, the higher the therapeutic effect of the drug can be determined. The method of the present invention can be carried out for the purpose of confirming such a therapeutic effect. For example, when the concentration of lyso-GM1 contained in the blood of the model animal decreases, preferably by 10% or more, for example by 20% or more, 30% or more, or 50% or more, before and after the treatment, it can be determined that the therapeutic effect is present. Here, the species of the model animal is not particularly limited, but examples include monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, with monkeys, mice, and rats being preferred.

[0124] In other words, there is no particular limitation on the species from which the cerebrospinal fluid to be analyzed is obtained, but examples include humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, and in particular humans, monkeys, mice, and rats.

[0125] The method of one embodiment of the present invention can also be used to search for a therapeutic agent for a disease in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulates in the central nervous system, using a model animal. The therapeutic effect of administering a drug to a model animal for a disease in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulates in the central nervous system can be confirmed by measuring the concentration of Fuc-GlcNAc-Asn contained in the cerebrospinal fluid of the model animal before and after administration, and measuring the amount of decrease after administration. The greater the amount of decrease, the higher the therapeutic effect of the drug can be determined. The method of the present invention can be carried out for the purpose of confirming such a therapeutic effect. For example, it can be determined that the therapeutic effect has been achieved when the concentration of Fuc-GlcNAc-Asn contained in the blood of the model animal decreases, preferably by 10% or more, for example, by 20% or more, 30% or more, or 50% or more, before and after the treatment. Here, the species of the model animal is not particularly limited, but examples include monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, with monkeys, mice, and rats being preferred.

[0126] In other words, there is no particular limitation on the species from which the cerebrospinal fluid to be analyzed is obtained, but examples include humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, and in particular humans, monkeys, mice, and rats.

[0127] The method of one embodiment of the present invention can also be used to search for a therapeutic agent for a disease in which lyso-sulfatide and / or sulfatide accumulates in the central nervous system, using a model animal. The therapeutic effect of administering a drug to a model animal for a disease in which lyso-sulfatide and / or sulfatide accumulates in the central nervous system can be confirmed by measuring the concentration of lyso-sulfatide contained in the cerebrospinal fluid of the model animal before and after administration, and measuring the amount of reduction after administration. The greater the amount of reduction, the higher the therapeutic effect of the drug can be determined. The method of the present invention can be carried out for the purpose of confirming such a therapeutic effect. For example, when the concentration of lyso-sulfatide contained in the blood of the model animal decreases, preferably by 10% or more, for example, by 20% or more, 30% or more, or 50% or more, before and after each treatment, it can be determined that the therapeutic effect is present. Here, the species of the model animal is not particularly limited, but examples include monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, with monkeys, mice, and rats being preferred.

[0128] In other words, there is no particular limitation on the species from which the cerebrospinal fluid to be analyzed is obtained, but examples include humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, horses, cows, pigs, dogs, and cats, and in particular humans, monkeys, mice, and rats. EXAMPLES

[0129] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to the examples.

[0130] The following Examples 1 to 10 relate to the measurement of Hex4.

[0131] [Example 1] Preparation of reagent solution Mobile phase A: 4 mL of 25% ammonium hydroxide solution (Merck) and 996 mL of water were mixed to prepare mobile phase A.

[0132] Mobile phase B: 4 mL of 25% ammonium hydroxide solution (Merck) and 996 mL of acetonitrile (for LC / MS, Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and used as mobile phase B.

[0133] [Example 2] Preparation of standard solution for Hex4 measurement Standard stock solution for Hex4 measurement: 1 mg of 6-aD-glucocopyranosyl maltotriose (Glc4, Carbosynth) was dissolved in 0.5 mL of pure water to prepare a 2 mg / mL solution, which was used as the standard stock solution for Hex4 measurement.

[0134] Standard solutions for the calibration curve for Hex4 measurement (H-SS-1 to H-SS-8): Using methanol, the standard stock solution for Hex4 measurement was serially diluted according to Table 1 below to prepare standard solutions for the calibration curve for Hex4 measurement (H-SS-1 to H-SS-8).

[0135] [Table 1]

[0136] QC standard solutions for Hex4 measurement (H-QS-1 to H-QS-4): Using methanol, the standard solution for the calibration curve for Hex4 measurement, H-SS-8, was serially diluted according to Table 2 below to prepare QC standard solutions for Hex4 measurement (H-QS-1 to H-QS-4).

[0137] [Table 2]

[0138] [Example 3] Preparation of internal standard solution for Hex4 measurement Internal standard stock solution for Hex4 measurement: 1 mg of 6-aD-Glucopyranosyl maltotriose-13C6 (Toronto Research Chemicals) (H-IS) was dissolved in 0.5 mL of pure water to prepare a 2 mg / mL solution, which was used as the internal standard stock solution for Hex4 measurement.

[0139] Internal standard solutions for Hex4 measurement (H-IS-1, H-IS-2): Using methanol, the internal standard stock solution for Hex4 measurement was serially diluted according to Table 3 below to prepare internal standard solutions for Hex4 measurement (H-IS-1, H-IS-2).

[0140] [Table 3]

[0141] [Example 4] Preparation of brain tissue extract and separation of CSF The brains of wild-type mice (C57BL / 6, 53 weeks old, Oriental Bioservices) and GAA KO mice (C57BL / 6, hTfR KI, GAA KO, 50 weeks old, Oriental Bioservices), a model mouse for Pompe disease, were removed and their wet weights were measured. Brain tissue, pure water in an amount 20 times the wet weight of the brain tissue, and 15 SUS beads Φ5 mm (Taitec) were added to a 5 mL self-supporting Mailing tube (Watson), and the tissue was crushed (2500 rpm, 120 seconds) using a bead crusher (μT-12, Taitec). The crushed tissue fluid was transferred to a 1.5 mL sampling tube (Sarstedt), heated at 100°C for 5 minutes, and then placed on ice. The samples were then centrifuged at 13,000 rpm and 4°C for 5 minutes using a microcentrifuge (MX-307, Tommy Seiko Co., Ltd.), and the supernatant was collected as a brain tissue extract. In addition, cerebrospinal fluid (CSF) was separated from each individual from which the brain was extracted, at approximately 15 μL.

[0142] [Example 5] Preparation of various samples Standard samples for calibration curve for Hex4 measurement (H-S0 to H-S7): 100 μL each of the standard solutions for calibration curve for Hex4 measurement (H-SS-1 to H-SS-8) prepared in Example 2 and 100 μL of the internal standard solution for Hex4 measurement (H-IS-1) prepared in Example 3 were mixed and stirred according to Table 4 below, and then centrifuged at 16,000 rpm and 20°C for 5 minutes using a high-speed micro centrifuge (MX-307, Tommy Seiko Co., Ltd.). The resulting supernatant was filled into vials to prepare standard samples for calibration curve for Hex4 measurement (H-S0 to H-S7).

[0143] [Table 4]

[0144] QC standard samples for Hex4 measurement (H-Q1 to H-Q4): 100 μL each of QC standard solutions for Hex4 measurement (H-QS-1 to H-QS-4) prepared in Example 2 and 100 μL of internal standard solution for Hex4 measurement (H-IS-1) prepared in Example 3 were mixed according to Table 5 below, stirred, and then centrifuged at 16,000 rpm and 20°C for 5 minutes. The resulting supernatant was filled into vials to prepare QC standard samples for Hex4 measurement (H-Q1 to H-Q4).

[0145] [Table 5]

[0146] Measurement sample (brain): 630 μL of methanol was added to 70 μL of the brain tissue extract obtained in Example 4, and after stirring, the mixture was centrifuged at 16,000 rpm and 20°C for 5 minutes to obtain a supernatant. 500 μL of the obtained supernatant was collected and the solvent was removed using a centrifugal concentrator (CC-105, Tommy Seiko Co., Ltd.), and then 25 μL of the internal standard solution for Hex4 measurement H-IS-1 prepared in Example 3 and 25 μL of methanol were added to redissolve the mixture. After stirring, the mixture was centrifuged at 16,000 rpm and 20°C for 5 minutes, and the obtained supernatant was filled into a vial to be used as a measurement sample.

[0147] Measurement sample (CSF): 10 μL of the internal standard solution for Hex4 measurement H-IS-1 prepared in Example 3 and 8 μL of methanol were added to 2 μL of CSF obtained in Example 4, and the mixture was stirred and then centrifuged at 16,000 rpm and 20°C for 5 minutes. The resulting supernatant was filled into a vial and used as the measurement sample.

[0148] Example 6 LC / MS / MS analysis The LC / MS / MS analysis was carried out using a combination of hydrophilic interaction ultra-high performance liquid chromatography and a tandem quadrupole mass spectrometer. The mass spectrometer (MS / MS device) used was a QTRAP5500 (AB Sciex), and the HPLC device was a NexeraX2 (Shimadzu). The analytical column was an ACQUITY UPLC BEH Amide Column, 2.1 mm x 5 mm (Nihon Waters), and the guard column was an ACQUITY UPLC BEH Amide VanGuard Pre-column, 2.1 mm x 50 mm (Nihon Waters). The mobile phases used were mobile phase A and mobile phase B prepared in Example 1. The column temperature was set to 40°C.

[0149] After equilibrating the column with a mixture of 45% (v / v) mobile phase A and 55% (v / v) mobile phase B, 10 μL of sample was injected and chromatography was performed under the mobile phase conditions shown in Table 6. The flow rate of the mobile phase was 0.4 mL / min.

[0150] [Table 6]

[0151] The ion source parameters, MS internal parameters, and valve switching program of the MS / MS instrument were set according to the instruction manual for QTRAP5500 (AB Sciex), as shown in Tables 7 to 9, respectively.

[0152] [Table 7]

[0153] [Table 8]

[0154] [Table 9]

[0155] The measured values ​​(ng / mL) and accuracy (%) were each expressed to three significant digits. Accuracy (%) was calculated based on the following formula: Accuracy (%) = measured value / theoretical concentration x 100 In addition, the theoretical concentration in the above calculation formula refers to the concentration of Hex4 added to the standard sample for the calibration curve for Hex4 measurement or the standard sample for QC for Hex4 measurement.

[0156] [Example 7] Evaluation of calibration curve and quantitative range for Hex4 measurement The standard sample for the calibration curve for Hex4 measurement and the standard sample for QC for Hex4 measurement prepared in Example 5 were measured, and the area of ​​the peak (detection peak) detected on the chromatogram of the product ion derived from Hex4 contained in each standard sample for the calibration curve for Hex4 measurement and the standard sample for QC for Hex4 measurement was calculated. The area of ​​the detection peak of the product ion derived from the internal standard solution was also calculated. The accuracy at each preparation concentration (theoretical concentration) of the standard sample for the calibration curve for Hex4 measurement is shown in Table 10, and the accuracy at each preparation concentration (theoretical concentration) of the standard sample for the QC for Hex4 measurement is shown in Table 11. In the range of 1 to 1000 ng / mL, the standard sample for the calibration curve for Hex4 measurement could be measured with an accuracy of 96.8 to 109%, and the standard sample for the QC for Hex4 measurement could be measured with an accuracy of 93.8 to 103%.

[0157] [Table 10]

[0158] [Table 11]

[0159] In addition, the area ratio (Hex4 detection peak area / H-IS detection peak area) of the detection peak area derived from Hex4 contained in each standard sample for the calibration curve for Hex4 measurement (Hex4 detection peak area) to the detection peak area derived from the internal standard solution for Hex4 measurement (H-IS detection peak area) was calculated. This value was plotted on the vertical axis, and the Hex4 concentration (Hex4 concentration) of each standard sample for the calibration curve for Hex4 measurement was plotted on the horizontal axis. A regression equation was calculated using quadratic programming to create a calibration curve for Hex4 measurement. The obtained calibration curve for Hex4 measurement showed good linearity in the range of 1 to 1000 ng / mL (Figure 1). The correlation coefficient (r) was 1.0000.

[0160] [Example 8] Measurement of Hex4 concentration in brain and CSF Using the calibration curve for Hex4 measurement obtained in Example 7 and the measurement sample prepared in Example 5, the Hex4 concentration in the brain and CSF of each of the wild-type mice and the Pompe disease model mice was calculated. The measurement results are shown in Figures 2 and 3, respectively. The results showed that the Hex4 concentration was higher in both the brain and CSF of the Pompe disease model mice compared to the wild-type mice. This result indicates that the Hex4 concentration in the brain and CSF is increased in the Pompe disease model mice compared to the wild-type mice.

[0161] [Example 9] Measurement of brain glycogen concentration In order to evaluate the correlation between the measured values ​​of Hex4 concentration in the brain and CSF obtained in Example 8 and the brain glycogen concentration, the brain glycogen concentration was measured for each of wild-type mice and Pompe disease model mice. The brain glycogen concentration was measured using a Glycogen Assay Kit (Biovision). The Glycogen Assay Kit breaks down glycogen contained in a sample into glucose and quantifies the amount of this glucose to quantify glycogen.

[0162] The brain tissue extract and CSF prepared in Example 4, which were used to measure the Hex4 concentrations in the brain and CSF, were used as glycogen concentration measurement samples. Note that the glycogen concentration measurement samples that were predicted to exceed the upper limit concentration of the calibration curve were appropriately diluted.

[0163] Preparation of standard solutions for glycogen calibration curve: 10 μL of standard solution in the Glycogen Assay Kit was mixed with 990 μL of water to prepare a 20 μg / mL glycogen solution. Next, the 20 μg / mL glycogen solution was diluted with the Hydrolysis Buffer in the Glycogen Assay Kit according to Table 12 below to prepare standard solutions for glycogen calibration curve (STD. 1 to STD. 7, Blank).

[0164] [Table 12]

[0165] Measurement of brain glycogen concentration: 50 μL of the standard solution for the glycogen calibration curve and the sample for measuring glycogen concentration were added to each well of an F96 Black plate (ThermoFisher Scientific). Measurements were performed using two wells for all the standard solutions for the glycogen calibration curve and the tissue extract for measuring glycogen concentration. 1 μL of Hydrolysis Enzyme Mix from the Glycogen Assay Kit was added to two wells of the standard solution for the glycogen calibration curve and one well of the tissue extract for measuring glycogen concentration, mixed, and reacted at room temperature for 30 minutes. To measure the endogenous glucose concentration contained in the sample, a well to which Hydrolysis Enzyme Mix had not been added was provided. Development Buffer, Development Enzyme Mix, and OxiRed Probe from the Glycogen Assay Kit were mixed in a ratio of 48.7:1:0.3 to prepare the reaction solution. 50 μL of reaction solution was added to each well, and the reaction was allowed to proceed for 30 minutes at room temperature in the dark. Fluorescence was measured using a fluorescence plate reader (Gemini XPS, Molecular Devices Japan). (Ex / Em = 535 / 587 nm)

[0166] Calculation of brain glycogen concentration: A standard curve was created from the theoretical concentration of the glycogen standard solution and the average signal value using a linear fit curve, and each glucose concentration was calculated. For diluted samples, correction was made using the dilution rate. The glycogen concentration was calculated by subtracting the glucose concentration of the measurement sample to which Hydrolysis Enzyme Mix was not added (endogenous glucose concentration value) from the value of the measurement sample to which Hydrolysis Enzyme Mix was added (total glucose concentration value).

[0167] Results: The measurement results of brain glycogen concentration are shown in Figure 4. As with the brain Hex4 concentration (Figure 2) and CSF Hex4 concentration (Figure 3) obtained in Example 8, the brain glycogen concentration was shown to be increased in Pompe disease model mice compared to wild-type mice.

[0168] [Example 10] Comparison of Hex4 concentration in brain and CSF with brain glycogen concentration The relationship between the glycogen concentration in the brain and the Hex4 concentration in the brain, the relationship between the glycogen concentration in the brain and the Hex4 concentration in the CSF, and the relationship between the Hex4 concentration in the brain and the Hex4 concentration in the CSF in the same mouse are plotted respectively in Figures 5 to 7. The coefficient of determination (R 2 ) were 0.9227, 0.9345, and 0.9467, respectively, and a high correlation was observed in all cases.

[0169] These results indicate that the Hex4 concentration in CSF reflects the Hex4 and glycogen concentrations in the brain, and that the Hex4 concentration in CSF can be used as a central nervous system biomarker in diseases in which Hex4 and / or glycogen accumulate in the brain. In particular, the Hex4 concentration in CSF can be used as a central nervous system biomarker in Pompe disease.

[0170] The following Examples 11 to 20 relate to the measurement of lyso-GM1.

[0171] [Example 11] Preparation of reagent solution Mobile phase C: 1 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and 499 mL of water were mixed to form mobile phase C.

[0172] Mobile phase D: 1 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and 499 mL of acetonitrile (for LC / MS, Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and used as mobile phase D.

[0173] 60% methanol solution: Methanol and water were mixed in a ratio of 60:40 (v / v) to prepare a 60% methanol solution.

[0174] 65% methanol solution: Methanol and water were mixed in a ratio of 65:35 (v / v) to prepare a 65% methanol solution.

[0175] 90% methanol solution: Methanol and water were mixed in a ratio of 90:10 (v / v) to prepare a 90% methanol solution.

[0176] [Example 12] Preparation of standard solution for lyso-GM1 measurement Standard stock solution for lyso-GM1 measurement: lyso-Monosialoganglioside GM1(NH4 + A 1 mg / mL solution was prepared by dissolving 0.5 mg of lyso-GM1 (lyso-GM1, Matreya LLC) in 0.5 mL of methanol, and this was used as the standard stock solution for lyso-GM1 measurement.

[0177] Standard solutions for the calibration curve for lyso-GM1 measurement (G-SS-1 to G-SS-8): Using methanol, the standard stock solution for lyso-GM1 measurement was serially diluted according to Table 13 below to prepare standard solutions for the calibration curve for lyso-GM1 measurement (G-SS-1 to G-SS-8).

[0178] [Table 13]

[0179] QC standard solutions for lyso-GM1 measurement (G-QS-1 to G-QS-4): Using methanol, the standard solution G-SS-8 for the calibration curve for lyso-GM1 measurement was serially diluted according to Table 14 below to prepare QC standard solutions for lyso-GM1 measurement (G-QS-1 to G-QS-4).

[0180] [Table 14]

[0181] [Example 13] Preparation of internal standard solution for lyso-GM1 measurement Internal standard stock solution for lyso-GM1 measurement: 1 mg of N-glycinated lyso-ceramide trihexoside (Matreya LLC) (G-IS) was dissolved in 1 mL of pure water to prepare a 1 mg / mL solution, which was used as the internal standard stock solution for lyso-GM1 measurement.

[0182] Internal standard solutions for lyso-GM1 measurement (G-IS-1 to G-IS-3): Using methanol, the internal standard stock solution for lyso-GM1 measurement was serially diluted according to Table 15 below to prepare internal standard solutions for lyso-GM1 measurement (G-IS-1 to G-IS-3).

[0183] [Table 15]

[0184] [Example 14] Preparation of tissue extracts, plasma and CSF Tissues (brain, spinal cord, lung, liver, spleen, kidney, heart, and quadriceps) were removed from β-Gal KO homozygous mice (C57BL / 6, 12 weeks and 38 weeks old, Oriental Bioservices), β-Gal KO heterozygous mice (C57BL / 6, 12 weeks old, Oriental Bioservices), and wild-type mice (C57BL / 6, 12 weeks old and 38 weeks old, Oriental Bioservices). The wet weight of each tissue was measured, and the tissue, pure water in an amount 9 times the wet weight of the tissue, and 15 SUS beads Φ5 mm (Taitec) were added to a 5 mL self-supporting mailing tube (Watson), and the tissue was disrupted (2500 rpm, 300 seconds) using a bead disrupter (μT-12, Taitec). Approximately 1 mL of tissue fluid after disruption was collected and used as tissue extract. For the brain and spinal cord, tissue fluid was also prepared by diluting it 10-fold with pure water and used appropriately for the measurements. In addition, approximately 300 μL of plasma was taken from each individual. In addition, approximately 15 μL of cerebrospinal fluid (CSF) was taken from each individual.

[0185] [Example 15] Preparation of various samples Standard samples for calibration curve for lyso-GM1 measurement (G-S0 to G-S7): 150 μL each of the standard solutions for calibration curve for lyso-GM1 measurement (G-SS-1 to G-SS-7) prepared in Example 12 and 150 μL of the internal standard solution for lyso-GM1 measurement (G-IS-1) prepared in Example 13 were mixed and stirred according to Table 16 below, and then centrifuged at 16,000 × g and 20°C for 5 minutes using a high-speed micro centrifuge (MX-307, Tommy Seiko Co., Ltd.). The resulting supernatant was filled into vials to prepare standard samples for calibration curve (G-S0 to G-S7).

[0186] [Table 16]

[0187] QC standard samples for lyso-GM1 measurement (G-Q1 to G-Q4): 150 μL each of QC standard solutions for lyso-GM1 measurement (G-QS-1 to G-QS-4) prepared in Example 12 and 150 μL of internal standard solution for lyso-GM1 measurement (G-IS-1) prepared in Example 13 were mixed according to Table 17 below, stirred, and then centrifuged at 16,000 × g and 20°C for 5 minutes. The resulting supernatant was filled into vials to prepare QC standard samples (G-Q1 to G-Q4).

[0188] [Table 17]

[0189] Measurement samples (brain, spinal cord, lung, liver, kidney, quadriceps, plasma): 210 μL of pure water and 408 μL of methanol were added to each of 70 μL of tissue extracts and plasma from the brain, spinal cord, lung, liver, kidney, and quadriceps obtained in Example 14, and after stirring, the mixture was centrifuged at 16,000×g and 20°C for 5 minutes to obtain a supernatant. 590 μL of the obtained supernatant was collected, and 10 μL of the internal standard solution G-IS-2 for lyso-GM1 measurement prepared in Example 13 was added. Of the 600 μL of the obtained solution, 500 μL was loaded onto an Oasis HLB 1 cc Vac Cartridge (30 mg, 30 μm, Nihon Waters). After washing with 1 mL of 60% methanol solution, it was eluted with 1 mL of 90% methanol solution. After removing the solvent from 800 μL of the eluted fraction using a centrifugal concentrator (CC-105, Tommy Seiko Co., Ltd.), 40 μL of methanol was added to redissolve the fraction. After stirring, the fraction was centrifuged at 16,000×g and 20°C for 5 minutes, and the resulting supernatant was filled into a vial and used as the measurement sample.

[0190] Measurement sample (heart): 210 μL of pure water and 408 μL of methanol were added to 70 μL of the heart tissue extract obtained in Example 14, and the mixture was stirred and then centrifuged at 16,000×g and 20°C for 5 minutes to obtain a supernatant. 590 μL of the obtained supernatant was collected, and 10 μL of the internal standard solution G-IS-2 for lyso-GM1 measurement prepared in Example 13 was added. Of the 600 μL of the obtained solution, 500 μL was loaded onto an Oasis HLB 1 cc Vac Cartridge (30 mg, 30 μm, Nihon Waters). After washing with 1 mL of 60% methanol solution and then 250 μL of 65% methanol solution, elution was performed with 1 mL of 90% methanol solution. After distilling off the solvent from the eluted fraction of 800 μL using a centrifugal concentrator (CC-105, Tommy Seiko Co., Ltd.), 40 μL of methanol was added and the fraction was redissolved. After stirring, the mixture was centrifuged at 16,000×g and 20°C for 5 minutes, and the resulting supernatant was filled into a vial to prepare a measurement sample.

[0191] Measurement sample (spleen): 210 μL of pure water and 408 μL of methanol were added to 70 μL of the spleen tissue extract obtained in Example 14, and the mixture was stirred and then centrifuged at 16,000×g and 20°C for 5 minutes to obtain a supernatant. 590 μL of the obtained supernatant was collected, and 10 μL of the internal standard solution G-IS-2 for lyso-GM1 measurement prepared in Example 13 was added. Of the 600 μL of the obtained solution, 500 μL was loaded onto an Oasis HLB 1 cc Vac Cartridge (30 mg, 30 μm, Nihon Waters). After washing with 1 mL of 60% methanol solution, elution was performed with 400 μL of 90% methanol solution. After distilling off the solvent from 320 μL of the eluted fraction using a centrifugal concentrator (CC-105, Tommy Seiko Co., Ltd.), 40 μL of methanol was added and the fraction was redissolved. After stirring, the mixture was centrifuged at 16,000×g and 20°C for 5 minutes, and the resulting supernatant was filled into a vial to prepare a measurement sample.

[0192] Measurement sample (CSF): 10 μL of the internal standard solution G-IS-1 for lyso-GM1 measurement prepared in Example 13 and 8 μL of methanol were added to 2 μL of CSF obtained in Example 14, and the mixture was stirred and then centrifuged at 16,000 × g and 20°C for 5 minutes. The resulting supernatant was filled into a vial and used as the measurement sample.

[0193] Example 16 LC / MS / MS analysis LC / MS / MS analysis was performed using a combination of reversed-phase chromatography and a tandem quadrupole mass spectrometer. A QTRAP5500 or TripleQuad6500+ (AB Sciex) was used as the mass spectrometer (MS / MS device), and a NexeraX2 (Shimadzu) was set as the HPLC device. Cadenza CW-C18 2 mm x 150 mm (Intact) was used as the analytical column, and CW-C18 5 x 2 mm (Intact) was used as the guard column. Mobile phases C and D prepared in Example 11 were used as the mobile phases. The column temperature was set to 40°C.

[0194] After equilibrating the column with a mixture of 62.5% (v / v) mobile phase C and 37.5% (v / v) mobile phase D, 10 μL of sample was injected and chromatography was performed under the mobile phase conditions shown in Table 18. The flow rate of the mobile phase was 0.4 mL / min.

[0195] [Table 18]

[0196] The ion source parameters, MS internal parameters, and valve switching program of the MS / MS instrument were set according to the instruction manual of QTRAP5500 or TripleQuad6500+ (AB Sciex), as shown in Tables 19 to 21, respectively.

[0197] [Table 19]

[0198] [Table 20]

[0199] [Table 21]

[0200] The measured values ​​(ng / mL) and accuracy (%) were each expressed to three significant digits. Accuracy (%) was calculated based on the following formula: Accuracy (%) = measured value / theoretical concentration x 100 In addition, the theoretical concentration in the above calculation formula refers to the concentration of lyso-GM1 added to the standard sample for the calibration curve for lyso-GM1 measurement or the standard sample for QC for lyso-GM1 measurement.

[0201] [Example 17] Evaluation of calibration curve and quantitative range for lyso-GM1 measurement The standard sample for the calibration curve for lyso-GM1 measurement and the standard sample for QC for lyso-GM1 measurement prepared in Example 15 were measured, and the area of ​​the peak (detection peak) detected on the chromatographic chart of the product ion derived from lyso-GM1 contained in each standard sample for the calibration curve for lyso-GM1 measurement and the standard sample for QC for lyso-GM1 measurement was calculated. In addition, the area of ​​the detection peak of the product ion derived from the internal standard solution for lyso-GM1 measurement was calculated. The accuracy at each preparation concentration (theoretical concentration) of the standard sample for the calibration curve for lyso-GM1 measurement is shown in Table 22, and the accuracy at each preparation concentration (theoretical concentration) of the standard sample for the QC for lyso-GM1 measurement is shown in Table 23. In the range of 1 to 1000 ng / mL, the standard sample for the calibration curve for lyso-GM1 measurement could be measured with an accuracy of 91.1 to 105%, and the standard sample for the QC for lyso-GM1 measurement could be measured with an accuracy of 98.2 to 107%.

[0202] [Table 22]

[0203] [Table 23]

[0204] In addition, the area ratio (lyso-GM1 detection peak area / G-IS detection peak area) of the detection peak derived from lyso-GM1 contained in each standard sample for the calibration curve for lyso-GM1 measurement (lyso-GM1 detection peak area) to the detection peak derived from the internal standard solution for lyso-GM1 measurement (G-IS detection peak area) was calculated. This value was plotted on the vertical axis, and the concentration of lyso-GM1 in each standard sample for the calibration curve for lyso-GM1 measurement (lyso-GM1 concentration) was plotted on the horizontal axis. A regression equation was calculated using quadratic programming to create a calibration curve for lyso-GM1 measurement. The resulting calibration curve for lyso-GM1 measurement showed good linearity in the range of 1 to 1000 ng / mL (Figure 8). The correlation coefficient (r) was 0.9999.

[0205] [Example 18] Measurement of lyso-GM1 concentrations in each tissue, CSF, and plasma of wild-type and model mice Using the calibration curve for lyso-GM1 measurement obtained in Example 17 and the measurement sample prepared in Example 15, the lyso-GM1 concentration in each tissue, plasma, and CSF of wild-type mice, β-Gal KO homozygous mice, and β-Gal KO heterozygous mice was calculated. The measurement results are shown in Figures 9 to 18, respectively. In all tissues, plasma, and CSF, the lyso-GM1 concentration was higher in β-Gal KO homozygous mice compared to wild-type mice. In addition, the lyso-GM1 concentration was lower in β-Gal KO heterozygous mice, similar to that of wild-type mice. These results indicate that the lyso-GM1 concentration in each tissue, plasma, and CSF is increased in β-Gal KO homozygous mice compared to wild-type mice. In addition, an age-dependent increase in lyso-GM1 concentration can be confirmed in the brain, CSF, and spinal cord, i.e., the central nervous system, of β-Gal KO homozygous mice.

[0206] [Example 19] Measurement of GM1 concentration in brain tissue of wild-type mice and model mice In order to evaluate the correlation between the measured lyso-GM1 concentrations in the brain and CSF obtained in Example 18 and the GM1 concentration in brain tissue, the GM1 concentrations in the brain tissue of wild-type mice, β-Gal KO homozygous mice, and β-Gal KO heterozygous mice were measured.

[0207] The measurement sample (brain) prepared in Example 15 and used for measuring the lyso-GM1 concentration in the brain and CSF was used as the sample for measuring the GM1 concentration in brain tissue, and the measurement was carried out as follows.

[0208] [Preparation of GM1 measurement reagent solution] 0.5 M ammonium acetate solution: 19.27 g of ammonium acetate (Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with water to make a total volume of 500 mL to prepare a 0.5 M ammonium acetate solution.

[0209] 25 mM ammonium acetate solution: 25 mL of 0.5 M ammonium acetate solution and 475 mL of water were mixed to prepare a 25 mM ammonium acetate solution.

[0210] 50 mM ammonium acetate solution: 1 mL of 0.5 M ammonium acetate solution and 9 mL of water were mixed to prepare a 50 mM ammonium acetate solution.

[0211] Mobile phase C': 25 mL of 0.5 M ammonium acetate solution and 400 mL of acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed, and then water was added to make the total volume 500 mL. This was used as mobile phase C'.

[0212] Mobile phase D': 25 mL of 0.5 M ammonium acetate solution and 350 mL of acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed, and then water was added to make the total volume 500 mL. This was used as mobile phase D'.

[0213] [Preparation of standard solution for GM1 measurement] Standard stock solution for GM1 measurement: 5 mg of monosialoganglioside GM1 (NH4+ salt) (GM1, Matreya LLC) was dissolved in 1 mL of methanol to prepare a 5 mg / mL solution, which was used as the standard stock solution for GM1 measurement.

[0214] Standard solutions for GM1 measurement calibration curve (GM1-SS-1 to GM1-SS-8): The standard stock solution for GM1 measurement was serially diluted using dimethyl sulfoxide (DMSO) according to Table 24 below to prepare standard solutions for GM1 measurement calibration curve (GM1-SS-1 to GM1-SS-8).

[0215] [Table 24]

[0216] [Preparation of internal standard solution for GM1 measurement] Internal standard stock solution for GM1 measurement: 0.5 mg of N-omega-CD3-Octadecanoyl monosialoganglioside GM1 (NH4+ salt) (Matreya LLC) (hereinafter referred to as GM1-IS) was dissolved in 0.5 mL of methanol to prepare a 1 mg / mL solution, which was used as the internal standard stock solution for GM1 measurement.

[0217] Internal standard solutions for GM1 measurement (GM1-IS-1, GM1-IS-2): The internal standard stock solution for GM1 measurement was serially diluted with DMSO according to Table 25 below to prepare internal standard solutions for GM1 measurement (GM1-IS-1, GM1-IS-2).

[0218] [Table 25]

[0219] [Preparation of various samples] Standard samples for GM1 measurement calibration curve (GM1-S0 to GM1-S8): 50 μL each of standard solutions for GM1 measurement calibration curve (GM1-SS-1 to GM1-SS-8), 50 μL of internal standard solution for GM1 measurement (GM1-IS-1), 100 μL of 25 mM ammonium acetate solution, and 800 μL of acetonitrile were mixed according to Table 26 below to prepare standard samples for GM1 measurement calibration curve (GM1-S0 to GM1-S8).

[0220] [Table 26]

[0221] [LC / MS / MS analysis] LC / MS / MS analysis was performed using a combination of reversed-phase chromatography and a tandem quadrupole mass spectrometer. A QTRAP5500 (AB Sciex) was used as the mass spectrometer (MS / MS device), and a Nexera LC-30A (Shimadzu) was set as the HPLC device. A UK amino column 2.1 x 150 mm (Intact) was used as the primary column, and a Kinetex HILIC 2.1 x 150 mm (Phenomenex) was used as the secondary column. The mobile phases used were mobile phase C' and mobile phase D' prepared above. The column temperature was set to 60°C.

[0222] After the primary and secondary columns were equilibrated with mobile phase C' and mobile phase D', respectively, 10 μL of sample was injected and chromatography was performed under the mobile phase conditions shown in Table 27. The flow rate of mobile phase C' was 0.2 mL / min, and the flow rate of mobile phase D' was 0.25 mL / min.

[0223] [Table 27]

[0224] The MS / MS settings, GM1 detection ion species, detection conditions for each GM1 ion species, and ESI parameters were set according to the QTRAP5500 (AB Sciex) instruction manual as shown in Tables 28 to 31. For the detection ions, the peaks on the ion chromatogram detected for each isoform (GM1_d18:1_C18:0, GM1_d20:1_C18:0, GM1_d18:1_C16:0, GM1_d16:1_C18:0) were integrated and used as one GM1 peak for analysis.

[0225] [Table 28]

[0226] [Table 29]

[0227] [Table 30]

[0228] [Table 31]

[0229] The measured values ​​(ng / mL) were reported to three significant figures.

[0230] The area ratio (GM1 detection peak area / GM1-IS detection peak area) of the detection peak derived from GM1 contained in each standard sample for the GM1 measurement calibration curve to the detection peak derived from the GM1 internal standard solution (GM1-IS detection peak area) was calculated. This value was plotted on the vertical axis, and the concentration of GM1 in each standard sample for the GM1 measurement calibration curve (GM1 concentration) was plotted on the horizontal axis. A regression equation was calculated using quadratic programming to create a calibration curve for GM1 measurement.

[0231] [Measurement of GM1 concentration in brain tissue of wild-type and model mice] Using the calibration curve for GM1 measurement obtained above and the measurement sample (brain) prepared in Example 15, the GM1 concentration in the brain tissue of each of wild-type mice, β-Gal KO homozygous mice, and β-Gal KO heterozygous mice was calculated. The measurement results are shown in Figure 19. Compared to wild-type mice, the GM1 concentration was higher in β-Gal KO homozygous mice. In addition, the GM1 concentration was lower in β-Gal KO heterozygous mice, similar to that of wild-type mice. This result indicates that the GM1 concentration in brain tissue is increased in β-Gal KO homozygous mice compared to wild-type mice. An age-dependent increase in GM1 concentration can be confirmed in β-Gal KO homozygous mice.

[0232] [Example 20] Comparison of lyso-GM1 concentrations in the brain and CSF with GM1 concentrations in the brain The relationship between the lyso-GM1 concentration in the brain and the lyso-GM1 concentration in the CSF, the relationship between the GM1 concentration in the brain and the lyso-GM1 concentration in the CSF, and the relationship between the lyso-GM1 concentration in the brain and the GM1 concentration in the brain in the same mouse are plotted respectively in Figures 20 to 22. The coefficient of determination (R 2 ) were 0.9909, 0.9051, and 0.9375, respectively, and a high correlation was observed in all cases.

[0233] These results indicate that the lyso-GM1 concentration in CSF reflects the lyso-GM1 and GM1 concentrations in the brain, and that the lyso-GM1 concentration in CSF can be used as a biomarker of the central nervous system in diseases in which lyso-GM1 and / or GM1 accumulate in the brain. In particular, we show that the lyso-GM1 concentration in CSF can be used as a biomarker of the central nervous system in GM1 gangliosidosis.

[0234] The following Examples 21 to 29 relate to the measurement of Fuc-GlcNAc-Asn.

[0235] [Example 21] Preparation of reagent solution Mobile phase E: 2 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and 998 mL of water were mixed to form mobile phase E.

[0236] Mobile phase F: 2 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and 998 mL of acetonitrile (for LC / MS, Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and used as mobile phase F. [Example 22] Preparation of standard solution for measuring Fuc-GlcNAc-Asn Standard stock solution for Fuc-GlcNAc-Asn measurement: 3.3 mg of Fuc1-α-6GlcNAc1-β-Asn (Fuc-GlcNAc-Asn, Peptide Institute) was dissolved in 16.5 mL of pure water to prepare a 0.2 mg / mL solution, which was used as the standard stock solution for Fuc-GlcNAc-Asn measurement.

[0237] Standard solutions for the calibration curve for Fuc-GlcNAc-Asn measurement (F-SS-1 to F-SS-7): The standard stock solution for Fuc-GlcNAc-Asn measurement was serially diluted with pure water according to Table 32 below to prepare standard solutions for the calibration curve for Fuc-GlcNAc-Asn measurement (F-SS-1 to F-SS-7).

[0238] [Table 32]

[0239] QC standard solutions for Fuc-GlcNAc-Asn measurement (F-QS-1 to F-QS-4): Using pure water, the standard solution for the calibration curve for Fuc-GlcNAc-Asn measurement, F-SS-7, was serially diluted according to Table 33 below to prepare QC standard solutions for Fuc-GlcNAc-Asn measurement (F-QS-1 to F-QS-4).

[0240] [Table 33]

[0241] [Example 23] Preparation of internal standard solution for measuring Fuc-GlcNAc-Asn Internal standard stock solution for Fuc-GlcNAc-Asn measurement: 2 mg of Fuc1-α-3GlcNAc1-β-OMe (Fuc-GlcNAc-OMe, Toronto Research Chemicals) (F-IS) was dissolved in 1 mL of methanol to prepare a 2 mg / mL solution, which was used as the internal standard stock solution for Fuc-GlcNAc-Asn measurement.

[0242] Internal standard solutions for Fuc-GlcNAc-Asn measurement (F-IS-1 to F-IS-3): The internal standard stock solution for Fuc-GlcNAc-Asn measurement was serially diluted with acetonitrile according to Table 34 below to prepare internal standard solutions for Fuc-GlcNAc-Asn measurement (F-IS-1 to F-IS-3).

[0243] [Table 34]

[0244] [Example 24] Preparation of tissue extracts, separation of plasma, CSF and urine Tissues (brain, liver, kidney, and spleen) were extracted from wild-type mice (C57BL / 6, 13-15 weeks old, Oriental Bioservices) and FUCA1 KO mice (C57BL / 6, 13-15 weeks old, Oriental Bioservices), a fucosidosis model mouse. Each tissue was freeze-dried and then weighed. The tissue, 39 times the freeze-dried weight of the tissue, pure water, and 15 SUS beads Φ5 mm (Taitec) were added to a 5 mL self-supporting mailing tube (Watson), and the tissue was crushed (2500 rpm, 120 seconds) using a bead crusher (μT-12, Taitec). Approximately 1 mL of tissue fluid was collected after crushing and used as tissue extract. In addition, approximately 300 μL of plasma, approximately 15 μL of cerebrospinal fluid (CSF), and approximately 1 mL of urine were collected from each individual.

[0245] [Example 25] Preparation of various samples Standard samples for calibration curve for Fuc-GlcNAc-Asn measurement (F-S0 to F-S7): 20 μL each of the standard solutions for calibration curve for Fuc-GlcNAc-Asn measurement (F-SS-1 to F-SS-7) prepared in Example 22 and 80 μL of the internal standard solution for Fuc-GlcNAc-Asn measurement (F-IS-1) prepared in Example 23 were mixed according to Table 35 below, stirred, and then centrifuged at 16,000 rpm and 20°C for 5 minutes using a high-speed micro centrifuge (MX-307, Tommy Seiko Co., Ltd.). The resulting supernatant was filled into vials to prepare standard samples for calibration curve for Fuc-GlcNAc-Asn measurement (F-S0 to F-S7).

[0246] [Table 35]

[0247] QC standard samples for Fuc-GlcNAc-Asn measurement (F-Q1 to F-Q4): 20 μL each of the QC standard solutions for Fuc-GlcNAc-Asn measurement (F-QS-1 to F-QS-4) prepared in Example 22 and 80 μL of the internal standard solution for Fuc-GlcNAc-Asn measurement (F-IS-1) prepared in Example 23 were mixed according to Table 36 below, stirred, and then centrifuged at 16,000 × g and 20°C for 5 minutes. The resulting supernatant was filled into vials to prepare QC standard samples for Fuc-GlcNAc-Asn measurement (F-Q1 to F-Q4).

[0248] [Table 36]

[0249] Measurement samples (brain, liver, kidney, spleen): The tissue extracts of the brain, liver, kidney, and spleen obtained in Example 24 were diluted 100-fold with pure water as necessary. 4 μL of pure water and 32 μL of the internal standard solution F-IS-1 for Fuc-GlcNAc-Asn measurement prepared in Example 23 were added to 4 μL of each tissue extract (or each tissue extract diluted 100-fold), and after stirring, the mixture was centrifuged at 16,000×g and 20° C. for 5 minutes. The obtained supernatant was filled into a vial and used as a measurement sample.

[0250] Measurement samples (CSF, plasma): The CSF and plasma obtained in Example 24 were diluted 10-fold with pure water as necessary. 4 μL of pure water and 32 μL of the internal standard solution for Fuc-GlcNAc-Asn measurement F-IS-1 prepared in Example 23 were added to 4 μL of CSF or plasma (or 10-fold diluted CSF or plasma), stirred, and then centrifuged at 16,000×g and 20° C. for 5 minutes. The resulting supernatant was filled into a vial and used as the measurement sample.

[0251] Measurement sample (urine): 18 μL of pure water was added to 2 μL of urine obtained in Example 24 to prepare a 10-fold diluted urine. If necessary, the 10-fold diluted urine was further diluted 100-fold with pure water to prepare a 1000-fold diluted urine. 4 μL of pure water and 32 μL of the internal standard solution F-IS-1 for measuring Fuc-GlcNAc-Asn prepared in Example 24 were added to 4 μL of the 10-fold diluted urine (or 1000-fold diluted urine), stirred, and then centrifuged at 16,000×g and 20° C. for 5 minutes. The resulting supernatant was filled into a vial and used as the measurement sample.

[0252] [Example 26] LC / MS / MS analysis LC / MS / MS analysis was performed using a combination of normal phase chromatography and a tandem quadrupole mass spectrometer. A QTRAP5500 (AB Sciex) was used as the mass spectrometer (MS / MS device), and a NexeraX2 (Shimadzu Corporation) was set as the HPLC device. A Unison UK-Amino 2 mm x 150 mm (Intact) was used as the analytical column, and a guard cartridge UK-Amino 5 x 2 mm (Intact) was used as the guard column. Mobile phase E and mobile phase F prepared in Example 21 were used as the mobile phase. The column temperature was set to 40°C.

[0253] After equilibrating the column with a mixture of 48% (v / v) mobile phase E and 52% (v / v) mobile phase F, 10 μL of sample was injected and chromatography was performed under the mobile phase conditions shown in Table 37. The flow rate of the mobile phase was 0.4 mL / min.

[0254] [Table 37]

[0255] The ion source parameters, MS internal parameters, and valve switching program of the MS / MS instrument were set according to the QTRAP5500 (AB Sciex) instruction manual as shown in Tables 38 to 40, respectively.

[0256] [Table 38]

[0257] [Table 39]

[0258] [Table 40]

[0259] The measured values ​​(ng / mL) and accuracy (%) were each expressed to three significant digits. Accuracy (%) was calculated based on the following formula: Accuracy (%) = measured value / theoretical concentration x 100 The theoretical concentration in the above formula refers to the concentration of Fuc-GlcNAc-Asn added to the standard sample for the calibration curve for Fuc-GlcNAc-Asn measurement or the QC standard sample for Fuc-GlcNAc-Asn measurement.

[0260] [Example 27] Evaluation of calibration curve and quantitative range for Fuc-GlcNAc-Asn measurement The standard sample for the calibration curve for Fuc-GlcNAc-Asn measurement and the standard sample for QC for Fuc-GlcNAc-Asn measurement prepared in Example 25 were measured, and the area of ​​the peak (detection peak) detected on the chromatogram of the product ion derived from Fuc-GlcNAc-Asn contained in each standard sample for the calibration curve for Fuc-GlcNAc-Asn measurement and the standard sample for QC for Fuc-GlcNAc-Asn measurement was calculated. In addition, the area of ​​the detection peak of the product ion derived from the internal standard solution for Fuc-GlcNAc-Asn measurement was calculated. The accuracy at each preparation concentration (theoretical concentration) of the standard sample for the calibration curve for Fuc-GlcNAc-Asn measurement is shown in Table 41, and the accuracy at each preparation concentration (theoretical concentration) of the standard sample for QC for Fuc-GlcNAc-Asn measurement is shown in Table 42. In the range of 0.3-300 ng / mL, the calibration standards for Fuc-GlcNAc-Asn measurement were measurable with an accuracy of 95.7-107%, and the QC standards for Fuc-GlcNAc-Asn measurement were measurable with an accuracy of 93.1-106%.

[0261] [Table 41]

[0262] [Table 42]

[0263] In addition, the area ratio (Fuc-GlcNAc-Asn detection peak area / F-IS detection peak area) of the detection peak area derived from Fuc-GlcNAc-Asn contained in each standard sample for the calibration curve for Fuc-GlcNAc-Asn measurement to the detection peak area derived from the internal standard solution for Fuc-GlcNAc-Asn measurement (Fuc-GlcNAc-Asn detection peak area) was calculated. This value was plotted on the vertical axis, and the concentration of Fuc-GlcNAc-Asn (Fuc-GlcNAc-Asn concentration) of each standard sample for the calibration curve for Fuc-GlcNAc-Asn measurement was plotted on the horizontal axis, and a regression equation was calculated using quadratic programming to create a calibration curve for Fuc-GlcNAc-Asn measurement. The obtained calibration curve for Fuc-GlcNAc-Asn measurement showed good quadratic curve properties in the range of 0.3 to 300 ng / mL (Figure 23). The correlation coefficient (r) was 0.9999.

[0264] [Example 28] Measurement of Fuc-GlcNAc-Asn concentrations in each tissue, CSF, plasma, and urine of wild-type and model mice Using the calibration curve obtained in Example 27 and the measurement samples prepared in Example 25, the concentrations of Fuc-GlcNAc-Asn in each tissue, plasma, urine, and CSF of wild-type mice and FUCA1 KO mice were calculated. The measurement results are shown in Figures 24 to 30, respectively. The results showed that the concentrations of Fuc-GlcNAc-Asn were higher in each tissue, plasma, urine, and CSF of FUCA1 KO mice compared to wild-type mice. This result indicates that the concentrations of Fuc-GlcNAc-Asn in each tissue, plasma, urine, and CSF of FUCA1 KO mice are increased compared to wild-type mice.

[0265] [Example 29] Comparison of Fuc-GlcNAc-Asn concentrations in brain and CSF The relationship between the Fuc-GlcNAc-Asn concentration in the brain and the Fuc-GlcNAc-Asn concentration in the CSF of the same mouse is plotted in Figure 31. The coefficient of determination (R 2 ) was 0.981, indicating a high correlation.

[0266] These results indicate that the Fuc-GlcNAc-Asn concentration in CSF reflects that in the brain and suggest that the Fuc-GlcNAc-Asn concentration in CSF can be used as a central nervous system biomarker for diseases in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulate in the brain.

[0267] The following Examples 30 to 38 relate to the measurement of lyso-sulfatide.

[0268] [Example 30] Preparation of reagent solution Mobile phase G: Mobile phase G was prepared by mixing 1 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and 499 mL of pure water.

[0269] Mobile phase H: 1 mL of formic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and 499 mL of acetonitrile (for LC / MS, Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to prepare mobile phase H.

[0270] 60% methanol solution: Methanol and water were mixed in a ratio of 60:40 (v / v) to prepare a 60% methanol solution.

[0271] 90% methanol solution: Methanol and water were mixed in a ratio of 90:10 (v / v) to prepare a 90% methanol solution.

[0272] [Example 31] Preparation of standard solution for measuring lyso-sulfatide Standard stock solution for lyso-sulfatide measurement: lyso-sulfatide (NH4 + 1 mg of lyso-sulfatide (lyso-sulfatide salt, Matreya LLC) was dissolved in 1 mL of methanol to prepare a 1 mg / mL solution, which was used as the standard stock solution for measuring lyso-sulfatide.

[0273] Standard solutions for the calibration curve for lyso-sulfatide measurement (S-SS-1 to S-SS-8): Using methanol, the standard stock solution for lyso-sulfatide measurement was serially diluted according to Table 43 below to prepare standard solutions for the calibration curve for lyso-sulfatide measurement (S-SS-1 to S-SS-8).

[0274] [Table 43]

[0275] Lyso-sulfatide QC standard solutions (S-QS-1 to S-QS-4): Lyso-sulfatide QC standard solutions (S-QS-1 to S-QS-4) were prepared by serially diluting lyso-sulfatide calibration standard solution S-SS-8 with methanol according to Table 44 below.

[0276] [Table 44]

[0277] [Example 32] Preparation of internal standard solution for measuring lyso-sulfatide Internal standard stock solution for lyso-sulfatide measurement: 1 mg of N-glycinated lyso-sulfatide (S-IS, Matreya LLC) was dissolved in 1 mL of methanol to prepare a 1 mg / mL solution, which was used as the internal standard stock solution for lyso-sulfatide measurement.

[0278] Internal standard solutions for lyso-sulfatide measurement (S-IS-1 to S-IS-3): Using methanol, the internal standard stock solution for lyso-sulfatide measurement was serially diluted according to Table 45 below to prepare internal standard solutions for lyso-sulfatide measurement (S-IS-1 to S-IS-3).

[0279] [Table 45]

[0280] [Example 33] Preparation of tissue extracts, plasma and CSF Tissues (brain, spinal cord, sciatic nerve, and kidney) from ARSA KO homozygous mice (C57BL / 6, 20-23 weeks old and 93-100 weeks old, Oriental Bioservices), ARSA KO heterozygous mice (C57BL / 6, 93-100 weeks old, Oriental Bioservices), and wild-type mice (C57BL / 6, 20-23 weeks old and 93-100 weeks old, Oriental Bioservices) were excised and lyophilized, and the dry weights were measured. The tissues, 39 times the dry weight of the tissue, and 3 or 15 SUS beads Φ5 mm (Taitec) were added to a 2 mL Shatter-Resistant tube (Scientific Specialties) or a 5 mL self-supporting Mailing tube (Watson), and the tissues were disrupted using a bead disrupter (μT-12, Taitec) (2500 rpm, 300 s). After crushing, the tissue fluid was left on ice for 30 minutes or more while stirring occasionally, and approximately 1 mL of tissue extract was taken. In addition, approximately 300 μL of plasma was taken from each individual. Furthermore, approximately 15 μL of cerebrospinal fluid (CSF) was taken from each individual.

[0281] [Example 34] Preparation of various samples Standard samples for lyso-sulfatide measurement calibration curve (S-S0 to S-S7): 50 μL each of the standard solutions for lyso-sulfatide measurement calibration curve (S-SS-1 to S-SS-7) prepared in Example 31 and 50 μL of the internal standard solution for lyso-sulfatide measurement (S-IS-1) prepared in Example 32 were mixed and stirred according to Table 46 below, and then centrifuged at 16,000 × g and 20 ° C for 5 minutes using a high-speed micro centrifuge (MX-307, Tommy Seiko Co., Ltd.). The resulting supernatant was filled into a vial to prepare standard samples for lyso-sulfatide measurement calibration curve (S-S0 to S-S7).

[0282] [Table 46]

[0283] QC standard samples for lyso-sulfatide measurement (S-Q1 to S-Q4): 50 μL each of the QC standard solutions for lyso-sulfatide measurement (S-QS-1 to S-QS-4) prepared in Example 31 and 50 μL of the internal standard solution for lyso-sulfatide measurement (S-IS-1) prepared in Example 31 were mixed according to Table 47 below, stirred, and then centrifuged at 16,000 × g and 20°C for 5 minutes. The resulting supernatant was filled into vials to prepare QC standard samples for lyso-sulfatide measurement (S-Q1 to S-Q4).

[0284] [Table 47]

[0285] Measurement samples (brain, spinal cord, sciatic nerve, and kidney): 240 μL of pure water and 467.2 μL of methanol were added to 80 μL of each of the tissue extracts of the brain, spinal cord, sciatic nerve, and kidney obtained in Example 33, respectively, and the mixture was stirred and then centrifuged at 16,000×g and 20°C for 5 minutes to obtain the supernatant. 590.4 μL of the obtained supernatant was collected, and 9.6 μL of the internal standard solution S-IS-2 for lyso-sulfatide measurement was added. Of the 600 μL of the obtained solution, 500 μL was loaded onto an Oasis HLB 1 cc Vac Cartridge (30 mg, 30 μm, Japan Waters). After washing with 1 mL of 60% methanol solution, it was eluted with 1 mL of 90% methanol solution. After stirring, the mixture was centrifuged at 16,000×g and 20° C. for 5 minutes, and 20 μL of the resulting supernatant was filled into a Sample Cup IA (Shinwa Kakosha) to prepare a measurement sample.

[0286] Measurement sample (plasma): 240 μL of pure water and 467.2 μL of methanol were added to 80 μL of plasma obtained in Example 33, and the mixture was stirred and then centrifuged at 16,000×g and 20°C for 5 minutes to obtain a supernatant. 590.4 μL of the obtained supernatant was collected, and 9.6 μL of internal standard solution S-IS-2 for lyso-sulfatide measurement was added. 500 μL of the obtained solution (600 μL) was loaded onto an Oasis HLB 1 cc Vac Cartridge (30 mg, 30 μm, Nihon Waters). After washing with 1 mL of 60% methanol solution, elution was performed with 1 mL of 90% methanol solution. The solvent of 400 μL of the eluted fraction was distilled using a centrifugal concentrator (CC-105, Tommy Seiko Co., Ltd.), and then 40 μL of methanol was added to redissolve the fraction. After stirring, the mixture was centrifuged at 16,000×g and 20° C. for 5 minutes, and 20 μL of the resulting supernatant was filled into a Sample Cup IA (Shinwa Kakosha) to prepare a measurement sample.

[0287] Measurement sample (CSF): 10 μL of the internal standard solution S-IS-1 for lyso-sulfatide measurement prepared in Example 32 and 6 μL of methanol were added to 4 μL of CSF obtained in Example 33, and the mixture was stirred and then centrifuged at 16,000×g and 20°C for 5 minutes. The resulting supernatant was filled into a Sample Cup IA (Shinwa Chemical Industry Co., Ltd.) to prepare the measurement sample.

[0288] [Example 35] LC / MS / MS analysis The LC / MS / MS analysis was carried out using a combination of reversed-phase chromatography and a tandem quadrupole mass spectrometer. A QTRAP5500 (AB Sciex) was used as the mass spectrometer (MS / MS device), and a NexeraX2 (Shimadzu Corporation) was set as the HPLC device. Cadenza CW-C18 2 mm x 150 mm (Intact) was used as the analytical column, and CW-C18 5 x 2 mm (Intact) was used as the guard column. Mobile phase G and mobile phase H prepared in Example 30 were used as the mobile phase. The column temperature was set to 40°C.

[0289] After equilibrating the column with a mixture of 62.5% (v / v) mobile phase G and 37.5% (v / v) mobile phase H, 10 μL of sample was injected and chromatography was performed under the mobile phase conditions shown in Table 48. The flow rate of the mobile phase was 0.4 mL / min.

[0290] [Table 48]

[0291] The ion source parameters, MS internal parameters, and valve switching program of the MS / MS instrument were set according to the instruction manual for QTRAP5500 (AB Sciex), as shown in Tables 49 to 51, respectively.

[0292] [Table 49]

[0293] [Table 50]

[0294] [Table 51]

[0295] The measured values ​​(ng / mL) and accuracy (%) were each expressed to three significant digits. Accuracy (%) was calculated based on the following formula: Accuracy (%) = measured value / theoretical concentration x 100 In addition, the theoretical concentration in the above calculation formula refers to the concentration of lyso-sulfatide added to the standard sample for the calibration curve for lyso-sulfatide measurement or the standard sample for QC for lyso-sulfatide measurement.

[0296] [Example 36] Evaluation of calibration curve and quantitative range for lyso-sulfatide measurement The standard sample for the calibration curve for lyso-sulfatide measurement and the standard sample for QC for lyso-sulfatide measurement prepared in Example 34 were measured, and the area of ​​the peak (detection peak) detected on the chromatographic chart of the product ion derived from lyso-sulfatide contained in each standard sample for the calibration curve for lyso-sulfatide measurement and the standard sample for QC for lyso-sulfatide measurement was calculated. In addition, the area of ​​the detection peak of the product ion derived from the internal standard solution for lyso-sulfatide measurement was calculated. Table 52 shows the accuracy at each preparation concentration (theoretical concentration) of the standard sample for the calibration curve for lyso-sulfatide measurement, and Table 53 shows the accuracy at each preparation concentration (theoretical concentration) of the standard sample for the QC for lyso-sulfatide measurement. In the range of 0.01-10 ng / mL, the calibration standards for lyso-sulfatide measurement were measurable with an accuracy of 95.3-105%, and the QC standards for lyso-sulfatide measurement were measurable with an accuracy of 98.3-103%.

[0297] [Table 52]

[0298] [Table 53]

[0299] In addition, the area ratio (lyso-sulfatide detection peak area / S-IS detection peak area) of the detection peak area derived from lyso-sulfatide contained in each standard sample for the calibration curve for lyso-sulfatide measurement (lyso-sulfatide detection peak area) to the detection peak area derived from the internal standard solution for lyso-sulfatide measurement (S-IS detection peak area) was calculated. This value was plotted on the vertical axis, and the concentration of lyso-sulfatide in each standard sample for the calibration curve for lyso-sulfatide measurement (lyso-sulfatide concentration) was plotted on the horizontal axis. A regression equation was calculated using quadratic programming to create a calibration curve for lyso-sulfatide measurement. A good approximation curve was obtained in the range of 0.01 to 10 ng / mL (Figure 32). The correlation coefficient (r) was 0.9999.

[0300] [Example 37] Measurement of lyso-sulfatide concentrations in each tissue, plasma, and CSF of wild-type and model mice Using the calibration curve for lyso-sulfatide measurement obtained in Example 36 and the measurement sample prepared in Example 34, the lyso-sulfatide concentration in each tissue, plasma, and CSF of wild-type mice, ARSA KO homozygous mice, and ARSA KO heterozygous mice was calculated. The measurement results are shown in Figures 33 to 38, respectively. The results showed that the lyso-sulfatide concentration was higher in ARSA KO homozygous mice than in wild-type mice in all tissues, plasma, and CSF. In addition, the lyso-sulfatide concentration was lower in ARSA KO heterozygous mice, similar to that of wild-type mice. This result shows that the lyso-sulfatide concentration in each tissue, plasma, and CSF is increased in ARSA KO homozygous mice compared to wild-type mice. An age-dependent increase in lyso-sulfatide concentration can be confirmed in each tissue, CSF, and plasma of ARSA KO homozygous mice.

[0301] [Example 38] Comparison of lyso-sulfatide concentrations in the brain and CSF The relationship between the lyso-sulfatide concentration in the brain and the lyso-sulfatide concentration in the CSF of the same mouse is plotted in Figure 39. The coefficient of determination (R 2 ) was 0.9187, indicating a high correlation.

[0302] These results indicate that lyso-sulfatide concentrations in CSF reflect those in the brain and suggest that CSF lyso-sulfatide concentrations can be used as a central nervous system biomarker for diseases in which lyso-sulfatide and / or sulfatides accumulate in the brain. [Industrial Applicability]

[0303] According to the present invention, it is possible to provide a method and a diagnostic kit for diagnosing diseases that develop due to the accumulation of glucose tetrasaccharide and / or glycogen in the central nervous system, and for examining the effectiveness of treatments for such diseases that aim to reduce glucose tetrasaccharide and / or glycogen accumulated in the central nervous system.

[0304] Furthermore, according to the present invention, it is possible to provide a method and a diagnostic kit for diagnosing diseases that develop due to the accumulation of lyso-monosialoganglioside GM1 and / or monosialoganglioside GM1 in the central nervous system, and for examining the effectiveness of treatments for such diseases that aim to reduce lyso-monosialoganglioside GM1 and / or monosialoganglioside GM1 accumulated in the central nervous system.

[0305] Furthermore, the present invention can provide a method and a diagnostic kit for diagnosing diseases that develop due to the accumulation of Fuc-GlcNAc-Asn and / or α-L-fucoside in the central nervous system, and for examining the effectiveness of treatments for such diseases that aim to reduce Fuc-GlcNAc-Asn and / or α-L-fucoside accumulated in the central nervous system.

[0306] Furthermore, according to the present invention, it is possible to provide a method and a diagnostic kit for diagnosing diseases that develop due to accumulation of lyso-sulfatide and / or sulfatide in the central nervous system, and for examining the effectiveness of treatments for such diseases that are intended to reduce lyso-sulfatide and / or sulfatide accumulated in the central nervous system.

Claims

1. A method for quantifying Fuc-GlcNAc-Asn contained in cerebrospinal fluid, comprising: adding an internal standard to the solution containing cerebrospinal fluid; subjecting a solution containing the cerebrospinal fluid to which the internal standard has been added to liquid chromatography to obtain an eluate; subjecting the eluate to mass spectrometry; wherein The method, wherein the internal standard substance is represented by the following formula [IX]. 【Chemistry 9】

2. 2. The method of claim 1, wherein the liquid chromatography is normal phase chromatography or anion exchange chromatography.

3. 3. The method of claim 1 or 2, wherein the Fuc-GlcNAc-Asn is measured by comparison with an internal standard.

4. The method according to any one of claims 1 to 3, wherein the cerebrospinal fluid is obtained from a patient suffering from a disease in which Fuc-GlcNAc-Asn and / or α-L-fucoside accumulates in the body.

5. The method of claim 4, wherein the disease is fucosidosis.

6. The method according to claim 4 or 5, wherein the patient has undergone treatment to reduce Fuc-GlcNAc-Asn and / or α-L-fucoside present in the body.

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