DIAGNOSTIC METHOD OF MUCOPOLYSACCHARIDOSES

Abstract

Provision of a method for accurate diagnosis of mucopolysaccharidoses, including determining the level of glycosaminoglycan in a biological sample with high sensitivity and with ease. A diagnostic method of mucopolysaccharidoses including the following steps (1) and (2): (1) a step including (a) filtering a biological sample with an ultrafiltration filter, digesting the sample on the filter with a glycosaminoglycan-specific enzyme, centrifuging the digested sample to obtain a filtrate, or (b) digesting a biological sample with a glycosaminoglycan-specific enzym, filtering the sample with an ultrafiltration filter to obtain a filtrate, applying the filtrate obtained by (a) or (b) to a liquid chromatograph/mass spectrometer, and analyzing glycosaminoglycan-derived disaccharides, and (2) a step of diagnosing a subject as having mucopolysaccharidosis, chemically diagnosing effect of treatment of mucopolysaccharidoses, or determining types of mucopolysaccharidoses, on the basis of quantitative concentration data and disaccharide composition obtained in step (1).

Description

FIELD OF THE INVENTION

The present invention relates to a diagnostic method of mucopolysaccharidoses.

BACKGROUND ART

Mucopolysaccharidoses are a group of lysosomal storage diseases caused by deficiency of the lysosomal enzymes needed to degrade glycosaminoglycans (GAGs). In patients suffering mucopolysaccharidosis, degradation products of mucopolysaccharides systemically accumulate, gradually impairing the functions of tissue and organs. Mucopolysaccharidoses are primarily classified into 7 types depending on the identity of the lacking enzyme. Most mucopolysaccharidosis cases are progressive and accompanied by mental retardation, and in some types of the disease, the clinical outcome is often death in early adult life. Clinical abnormalities primarily include significantly deformed bones, a short neck, joint stiffness and coarse facial features. In addition, diffuse cornea opacification, hearing disorder, liver enlargement, heart diseases, and abnormally low height are observed.

In diagnosis of mucopolysaccharidoses, glycosaminoglycans (hereinafter reffered to as GAG) content of a biological sample, such as blood, is determined. Conventionally known assays of GAGs include the following methods.

JP-A-4-135496 discloses a method of analyzing GAG, which method includes transforming GAG into disaccharides by use of an enzyme that specifically degrades GAG, and analyzing the composition of the resultant disaccharides by means of high performance liquid chromatography (hereinafter referred to as HPLC). Chem. Pharm. Bull. 46 (1), 97 to 101 (1998) discloses a method of analyzing KS, which method includes transforming keratan sulfate (hereinafter referred to as KS) in urine into disaccharides by use of keratanase, which is an enzyme that specifically degrades KS, and analyzing the resultant disaccharides by means of HPLC. Journal of Chromatography B, 765, 151 to 160 (2001) discloses an analysis method of GAG, including hydrolysis of plasma GAG or serum GAG, and formed galactose and aminosugar are analyzed by means of HPLC. Analytical Biochemistry 302, 169 to 174 (2002) discloses an analysis method of chondroitin sulfate (hereinafter referred to as CS), which method include filtration of plasma CS or urine CS through an ultrafiltration filter, followed by degradation of CS into disaccharides with chondroitinase ABC on the filter, and analyzing the disaccharides contained in the filtrate by means of HPLC. Analytical Biochemistry 290, 68 to 73 (2001) discloses a method of analyzing the composition of KS-derived disaccharides, which method includes pretreatment of tissue KS through ethanol precipitation, degrading the pretreated product with keratanase II into disaccharides, followed by liquid chromatography/tandem mass spectrometry of the resultant disaccharides (hereinafter referred to as LC/MS/MS), whereby the KS-derived disaccharide composition is investigated. Journal of Chromatography B, 754, 153 to 159 (2001) discloses an analysis method of the heparan sulfate (HS) derived disaccharide composition, which method includes pretreatment of tissue through ethanol precipitation, degradation into disaccharides by use of an enzyme specifically directed to HS, and injecting the disaccharides by means of LC/MS/MS. JP-A-2003-265196 and Clinica Chimica Acta, 264, 245 to 250 (1997) respectively describe a method of diagnosing mucopolysaccharidoses through measurement of urine GAG using 1,9-dimethylmethylene blue.

Also, JP-A-10-153600 discloses an assay method using a polypeptide that is capable of specifically binding to KS and Hyaluronic acid (hereinafter referred to as HA)-containg molecule.

DISCLOSURE OF THE INVENTION

Problem to be Solved by the Invention

However, conventional methods have various problems, including a scatter of measured concentrations, low measurement sensitivity, and intricate pretreatment procedure. Moreover, only one type of GAG can be measured in a single test. Thus, no conventional diagnostic method has been satisfactory for the diagnosis of mucopolysaccharidoses.

Accordingly, the present invention provides a method for accurate diagnosis of mucopolysaccharidoses, including determining the level of glycosaminoglycan in a biological sample with high sensitivity and with ease.

Means to Solve the Problem

The present inventors have carried out extensive studies with an aim to develop a method for simultaneous measurement of a plurality of glycosaminoglycans in a biological sample with high sensitivity, and have found that accurate diagnosis of mucopolysaccharidoses can be rendered from highly sensitive simultaneous quantification of a plurality of glycosaminoglycans contained in a biological sample, which is realized when use of an ultrafiltration filter and enzymatic digestion performed on the filter is further combined with LC/MS/MS. The present invention has been accomplished on the basis of this finding.

The present invention provides (A) to (E) below.

(A) A diagnostic method of mucopolysaccharidoses including the following steps (1) and (2):

• (1) a step including (a) filtering a biological sample with an ultrafiltration filter, digesting the biological sample on the filter with a GAG-specific enzyme, and centrifuging the digested sample to obtain a filtrate, or (b) digesting a sample with with a GAG-specific enzyme, filtering the digested sample with an ultrafiltration filter to obtain a filtrate, applying the filtrate obtained by (a) or (b) to LC/MS/MS, and analyzing GAG-derived disaccharides, and
• (2) a step of diagnosing a subject as having mucopolysaccharidosis or determining types of mucopolysaccharidoses, on the basis of quantitative concentration data and disaccharide composition obtained in step (1).

(B) A method as described in (A), wherein, in step (1), the HPLC is performed under such conditions that the analytical column is a carbon graphite column and an alkaline solution is employed as a mobile phase, to thereby elute GAG-derived disaccharides at optimal elution positions that facilitate the MS analysis.

(C) A method as described in (A) or (B), wherein, in step (1), the disaccharides are produced through use of a solution containing, as the GAG-specific degrading enzyme, keratanase II, heparitinase, and chondroitinase B; and KS, HS, and DS are analyzed simultaneously.

(D) A method as described in (A) or (B), wherein, in step (1), the disaccharides are produced using, as the GAG-specific degrading enzyme, any one of keratanase II, heparitinase, and chondroitinase B; and one or two of KS, HS, and DS are analyzed.

(E) A method as described in any one of (A) to (D), wherein, in step (1), the biological sample is selected from among plasma, serum, blood, urine, and body fluid.

Advantageous Effect of the Invention

Hence, the method of the present invention in its broadest scope provides an accurate, highly sensitive, and convenient diagnosis of mucopolysaccharidoses. Thus, if the diagnostic method of the present invention is performed on newborns, mucopolysaccharidoses can be detected in an early stage after birth, and appropriate enzyme replacement therapy or gene therapy performed in an early stage would restrain development of the pathological conditions of the patient.

In addition to the use in diagnosis of mucopolysaccharidoses, the method of the present invention can also be used to comprehend the therapeutic effect of the aforementioned therapy, to decide on therapeutic options, and to evaluate drug efficacy in the development of pharmaceuticals.

Moreover, the method of the present invention finds utility in biomarker assays performed for identifying GAG-related pathological conditions, such as inflammations associated with arthrosis deformans, chronic articular rheumatism, or diseases accompanied by abnormalities in corneal tissue; carcinomas; and liver diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relation between mobile phase pH and elution position.

FIG. 2 is a graph showing the relation between salt concentration of the mobile phase and elution position.

FIGS. 3A and 3B provide chromatograms showing peak profiles of mobile phase pH, which affect the separation.

BEST MODES FOR CARRYING OUT THE INVENTION

No particular limitation is imposed on the biological sample employed in step (1) of the method of the present invention, so long as the sample contains mucopolysaccharides. Examples of the biological sample include plasma, serum, blood, urine, and body fluid. Of these, plasma and serum are particularly preferred.

No particular limitation is imposed on the ultrafiltration filter employed in the present invention, so long as the filter does not allow mucopolysaccharides to pass therethrough, but allow passage of molecules smaller than mucopolysaccharides in molecular weight. Preferably, the filter can isolate molecules having a molecular weight of about 5000. Examples of commercially available ultrafiltration filters which may be employed in the present invention include ULTRAFREET™-MC (BIOMAX-5) (product of MILLIPORE). When an AcroPrep 96 filter plate (10K) (product of PALL Life Sciences) is employed, simultaneous processing can be performed on multiple samples.

No particular limitation is imposed on the GAG-specific enzymes employed in the present invention, so long as the enzymes degrade glycosaminoglycans. Exemplary enzymes are those which act specifically on KS, HS or DS and degrade the same. These enzymes may be employed singly or in combination of two or more species. When the three enzymes; i.e., keratan sulfate degrading enzyme, heparan sulfate degrading enzyme, and dermatan sulfate degrading enzyme, are employed in combination, keratan sulfate, heparan sulfate, and dermatan sulfate are all degraded simultaneously, whereas when one of these enzymes is employed, one or two species of these glycosaminoglycans can be analyzed. Preferred examples of the GAG-degrading enzymes include keratanase, heparitinase, and chondroitinase B. Examples of commercially available GAG-specific enzymes include keratanase, keratanase II, heparitinase, heparitinase I, heparitinase II, heparinase, and chondroitinase B (produced and sold by SEIKAGAKU CORPORATION). As for the HS degrading enzyme, an enzyme having a similar effect, which is commercially available from Sigma Co., may be employed. Of the above-mentioned enzymes, most preferably, the three enzymes of keratanase II, heparitinase, and chondroitinase B are employed in combination, or alternatively, one of these three enzymes is employed.

Enzymatic digestion by the GAG-specific enzyme(s) performed according to the present invention is complete after, for example, 1- to 30-hour digestion at 30 to 40° C. Preferably, enzymatic digestion is performed in a 37° C. incubator for 15 hours.

In one application of the present invention, when CS or HA is a target substance which is desired to be measured, chondroitinase ABC, chondroitinase ACII, or hyaluronidase SD may be used to specifically degrade CS or HA, followed by LC/MS/MS for analysis.

Glycosaminoglycans are degraded to disaccharides through enzymatic digestion using the above-mentioned GAG-specific enzymes. Some abbreviations of disaccharides are provided below.

ΔDiHS-0S: ΔHexA α1→4GlcNAc: 2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-enopyranosyluronic acid)-D-glucose, ΔDiHS-NS: ΔHexA α1→4GlcNS: 2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-D-glucose, ΔDiHS-6S: ΔHexA α1→4GlcNAc(6S): 2-acetamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-O-D-glucose, MSD: Galβ1→3GlcNAc(6S), DSD: Gal(6S)β1→3GlcNAc(6S).

The step (1) of the present invention includes (a) a means which comprises filtering a biological sample with an ultrafiltration filter, and digesting the biological sample on the filter with a GAG-specific enzyme, and (b) a means which comprises digesting a biological sample with a GAG-specific enzym, and filtering the digested biological sample with an ultrafiltration filter. The means (b) may be performed, for instance, by drawing a small amount of blood from the ear lobe of a subject, digesting a blood-impregnated filter paper with a GAG-specific enzyme, and filtering the digested substance with an ultrafiltration filter.

The disaccharides which are measurement targets in the present invention are MSD and DSD (degradation products of KS by keratanase II); ΔDiHS-0S, ΔDiHS-NS, and ΔDiHS-6S (degradation products of HS by heparitinase); and ΔDi-4S (degradation products of DS by chondroitinase B).

A digestion product obtained from the above process is centrifuged and the filtrate is injected to LC/MS/MS for analysis of disaccharides. Preferably, centrifugation is performed, for example, at 5000 to 8000×g for 10 to 15 minutes.

No particular limitation is imposed on the analytical column of LC/MS/MS, so long as the column can separate the above-mentioned disaccharides. Examples of the column include a carbon graphite column and a reverse phase HPLC column in which ODS (octadecylsilane) is employed as a stationary phase. For obtaining good resolution, a carbon graphite column is preferred. Examples of commercially available carbon graphite columns include Hypercarb (2.0 mm i.d.×150 mm, 5 μm) (product of Thermo Electron Corp). When a column having a shorter length is employed, retention time of disaccharides can be shortened.

In the present invention, in order to optimize the elution positions of disaccharides, preferably, the mobile phase is an alkaline solution. The alkaline solution is preferably of pH 7 to 11, more preferably pH 8 to 10, still more preferably pH 9 to 10, particularly preferably pH 10, and gradient conditions are preferably established together with an organic solvent. A preferred salt for adjusting pH to fall within an alkaline range is aqueous ammonia or an ammonium salt. Exemplary aqueous ammonium salt solutions include aqueous ammonium bicarbonate solution, aqueous ammonium formate solution, and aqueous ammonium acetate solution, with aqueous ammonium bicarbonate solution being preferred. For attaining good elution positions, the salt concentration of any of the above solutions is preferably 3 to 100 mmol/L, more preferably 3 to 50 mmol/L, even more preferably 10 mmol/L. Examples of the organic solvent include acetonitrile, methanol, ethanol, and 2-propanol. Most preferably, gradient conditions are conducted using a solution of pH 10 prepared through addition of 28% aqueous ammonia to 10 mmol/L ammonium bicarbonate solution (10 mmol/L ammonium bicarbonate buffer (pH 10)) and acetonitrile.

As shown in FIGS. 1 and 2, when the pH and the salt concentration of the mobile phase are regulated, GAG-derived disaccharides can be eluted at elution positions (i.e., optimal retention times) that are optimal for the MS analysis. In addition, as shown in FIGS. 3A and 3B, through maneuvering the pH of the mobile phase, the peak shape was improved significantly. Thus, this approach enables retention time regulation of saccharides, which has otherwise been very difficult according to conventional methods.

Through the above-described sub-steps in step (1), the GAG level and the disaccharide composition of a biological sample can be obtained. In step (2), on the basis of the data obtained in step (1), diagnosis of mucopolysaccharidosis can be rendered, and moreover, the type of mucopolysaccharidosis can be determined. Furthermore, effect of a therapy of mucopolysaccharidosis can be assessed. Table 1 shows a classification of mucopolysaccharidoses.

	TABLE 1
	Class name	Lacking enzyme
IH	Hurler syndrome	α-L-iduronidase
IS	Scheie syndrome	α-L-iduronidase
IH/S	Hurler-Scheie syndrome	α-L-iduronidase
IIA	Hunter syndrome, severe type	sulfoiduronate sulfatase
IIB	Hunter syndrome, mild type	sulfoiduronate sulfatase
IIIA	Sanfilippo syndrome A	heparan sulfate N-sulfatase
IIIB	Sanfilippo syndrome B	N-acetyl-α-D-
		glucosaminidase
IIIC	Sanfilippo syndrome C	acetyl-CoA-α-glucosaminide
		N-acetyltransferase
IIID	Sanfilippo syndrome D	N-acetylgiucosamine-6-
		sulfatase
IVA	Morquio syndrome A	N-acetylgalactosamine-6-
		sulfatase
IVB	Morquio syndrome B	β-galactosidase
VIA	Maroteaux-Lamy syndrome,	N-acetylgalactosamine-4-
	severe type	sulfatase
VIB	Maroteaux-Lamy syndrome,	N-acetylgalactosamine-4-
	mild type	sulfatase
VII	β-glucuronidase deficiency	β-glucuronidase

EXAMPLES

The present invention will next be described in detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1

In order to check whether the assay method of the present invention provides a successful screening on plasma or serum samples, the following experiment was performed using plasma samples from mucopolysaccharidosis patients and control plasma samples (human).

Pretreatment of a plasma or serum sample:

• 1) Add a plasma or serum sample (0.01 mL) to ULTRAFREE™-MC (BIOMAX-5);
• 2) Centrifuge at 4,000×g for 15 minutes;
• 3) Replace the collection tube in ULTRAFREE™-MC (BIOMAX-5) by a new tube;
• 4) Add a 50-μg/mL aqueous chondrosine solution (0.02 mL) (produced and sold by SEIKAGAKU CORPORATION) as an internal standard substance onto the filter (note: throughout the procedures, water should be purified water);
• 5) Add 50-mmol/L Tris-HCl buffer (0.02 mL, pH 7) onto the filter;
• 6) Add an enzyme mixture solution (0.02 mL) containing keratanase II, heparitinase, and chondroitinase B (2 mU each) onto the filter;
• 7) Mix the resultant mixture using a vortex mixer for about ten seconds;
• 8) Incubate the mixture at 37° C. for 15 hours;
• 9) Centrifuge the resultant mixture at 8,000×g for 15 minutes;
• 10) Add water (0.02 mL) to the filtrate;
• 11) Mix the resultant mixture using a vortex mixer for about 10 seconds; and
• 12) Transfer the-thus obtained liquid sample into an injection vial for an autosampler.

Pretreatment of a sample for producing a calibration curve:

• 1) KS standard solutions: Bovine-cornea-derived KS (produced and sold by SEIKAGAKU CORPORATION) is employed.
• Concentrations are shown in Table 2.
• 2) HS standard solutions: An unsaturated heparan/heparin-disaccharide kit (H kit) (produced and sold by SEIKAGAKU CORPORATION) is employed. Aqueous solutions each containing ΔDiHS-0S, ΔDiHS-6S, and ΔDiHS-NS are prepared.
• Concentrations are shown in Table 3.
• 3) Add an aliquot (0.01 mL) of each of the above-prepared KS standard solutions and an aliquot (0.01 mL) of each of the above-prepared HS standard solutions to ULTRAFREE™-MC (BIOMAX-5).
• 4) Add an 50-pg/mL aqueous solution (0.02 mL) of chondrosine (produced and sold by SEIKAGAKU CORPORATION) as an internal standard substance onto the filter.
• 5) Adding 50-mmol/L Tris-HCl buffer (0.02 mL, pH 7) on the filter.
• 6) Add an enzyme-mixed aqueous solution (0.02 mL) containing keratanase II, heparitinase, and chondroitinase B (2 mU each) onto the filter.
• 7) Mix the resultant mixture by use of a vortex mixer for about ten seconds.
• 8) Incubate the mixture at 37° C. for 15 hours.
• 9) Centrifuge the resultant mixture at 8,000×g for 15 minutes. 10) Add blank plasma or blank serum to ULTRAFREE™-MC (BIOMAX-5) then centrifuge at 8,000×g for 15 minutes, to thereby prepare a blank filtrate.
• 11) Add the thus-prepared blank filtrate (0.01 mL) to the filtrate obtained in step 9).
• 12) Mix the resultant mixture using a vortex mixer for about 10 seconds.

13) Transfer the-thus obtained liquid sample into an injection vial for an autosampler.

TABLE 2
Concentration of standard solution (KS)
	(Unit: μg/mL)
	S7	S6	S5	S4	S3	S2	S1
MSD	7.1	3.6	2.8	1.4	0.71	0.36	0.14
DSD	2.9	1.5	1.2	0.58	0.29	0.15	0.058
Total	10	5	4	2	1	0.5	0.2

TABLE 3
Concentration of standard solution (HS)
	(Unit: ng/mL)
	S7	S6	S5	S4	S3	S2	S1
ΔDiHS-0S	1000	500	200	100	50	20	10
ΔDiHS-NS	500	250	100	50	25	10	5
ΔDiHS-6S	1000	500	200	100	50	20	10

The LS/MS/MS apparatus employed are as follows:

HPLC apparatus: HP1100 system (Agilent Technology Inc.) (Palo Alto, Calif., USA), autosampler: HTC PAL (CTC Analytics Inc.) (Zwingen, Switzerland), mass spectrometer: API 4000 (Applied Biosystems Inc.) (Lincoln Centre Drive Foster City, Calif., USA).

The HPLC conditions employed are as follows.

Analytical column: Hypercarb (2.0 mm i.d.×150 mm, 5 μm) (Thermo Electron Corp.) (Waltham, Mass., USA), mobile phase: (A) 10 mmol/L Ammonium bicarbonate buffer (pH 10), (B) Acetonitrile, gradient conditions: [Time(min)/B(%)]; [0/0]→[0.9/0]→[1.0/30]→[6.0/30]→[6.1/0]→[8.0/0], rate flow: 0.2 mL/min, column temperature 45° C., the volume of injection into an autosampler: 0.01 mL.

The MS/MS conditions employed are as follows.

Ionization method: turbo ionspray, detection mode: multiple reaction monitoring (MRM)-negative mode, turbospray temperature: 650° C., monitoring ion (CID energy): Galβ^1-3GlcNAc(6S)m/z 462.1-m/z 97.0 (CID: −80 eV); Gal(6S)β^1-3GlcNAc(6S)m/z 462.1-m/z 97.0 (CID: −80 eV); ΔDiHS-0S m/z 378.1-m/z 174.9 (CID: −22 eV); ΔDiHS-NS m/z 416.0-m/z 137.9 (CID: −34 eV); ΔDiHS-6S m/z 458.2-m/z 97.1 (CID: −52 eV); I.S.m/z 354.0-m/z 113.0 (CID: −22 eV).

For calculation of concentrations, a linear first-order regression equation was established using concentrations on the calibration curve, peak area ratio (“peak area of the standard substance of each analyte”/“peak area of an internal standard substance”), and the method of least squares. A weighting of 1/“calibration curve concentration” was used for curve fit.

Three different control serum samples were measured for three days (N=5). The results are shown in Tables 4 and 5.

	TABLE 4
	MSD	DSD
	Replicates	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3
Batch 1	n1	0.97	0.52	0.60	0.34	0.18	0.21
	n2	0.96	0.51	0.64	0.36	0.19	0.22
	n3	1.1	0.54	0.58	0.37	0.18	0.19
	n4	0.97	0.54	0.62	0.35	0.18	0.21
	n5	1.1	0.55	0.61	0.36	0.19	0.21
	Mean	1.0	0.53	0.61	0.36	0.18	0.21
	SB	0.073	0.016	0.022	0.0114	0.0055	0.0110
	CV %	7.2	3.1	3.7	3.2	3.0	5.3
Batch 2	n1	0.94	0.59	0.70	0.35	0.19	0.22
	n2	0.93	0.59	0.67	0.37	0.18	0.21
	n3	1.1	0.54	0.65	0.34	0.18	0.21
	n4	1.0	0.58	0.65	0.35	0.19	0.20
	n5	1.1	0.58	0.63	0.35	0.18	0.20
	Mean	1.01	0.58	0.66	0.35	0.18	0.21
	SD	0.083	0.021	0.026	0.0110	0.0055	0.0084
	CV %	8.2	3.6	4.0	3.1	3.0	4.0
Batch 3	n1	1.0	0.52	0.62	0.35	0.18	0.19
	n2	1.0	0.52	0.63	0.36	0.17	0.19
	n3	0.96	0.56	0.60	0.35	0.17	0.19
	n4	1.1	0.52	0.61	0.37	0.17	0.19
	n5	0.96	0.53	0.62	0.35	0.17	0.19
	Mean	1.00	0.53	0.62	0.36	0.17	0.19
	SD	0.057	0.017	0.011	0.0089	0.0045	0.0000
	CV %	5.7	3.3	1.9	2.5	2.6	0.0
Overall	Mean	1.01	0.55	0.63	0.35	0.18	0.20
(N = 15)	SD	0.067	0.028	0.030	0.010	0.0076	0.0115
	CV %	6.6	5.1	4.8	2.8	4.2	5.7

	TABLE 5
	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, 6S*
	Replicates	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3
Batch 1	n1	59	53	72	19	15	19	54	17	18
	n2	58	51	70	19	16	18	53	17	24
	n3	61	55	73	22	16	21	53	19	23
	n4	60	52	77	19	15	20	52	19	28
	n5	66	53	74	20	17	18	53	17	26
	Mean	61	53	73	20	16	19	53	18	24
	SD	3.1	1.5	2.6	1.3	0.84	1.3	0.71	1.1	3.8
	CV %	5.1	2.8	3.5	6.6	5.3	6.8	1.3	6.2	15.8
Batch 2	n1	54	49	74	20	16	19	64	25	25
	n2	57	49	74	22	16	19	60	18	23
	n3	61	48	70	21	14	20	66	19	24
	n4	55	49	70	22	14	19	51	22	22
	n5	63	50	71	19	14	19	57	19	26
	Mean	58	49	72	21	15	19	60	21	24
	SD	3.9	0.71	2.0	1.3	1.1	0.45	5.9	2.9	1.6
	CV %	6.7	1.4	2.9	6.3	7.4	2.3	10.0	14.0	6.6
Batch 3	n1	58	48	62	20	14	18	60	15	19
	n2	59	48	71	22	15	19	67	18	21
	n3	59	49	68	21	14	16	75	18	20
	n4	61	52	67	19	15	18	81	18	21
	n5	57	49	67	18	15	19	66	15	20
	Mean	59	49	67	20	15	18	70	17	20
	SD	1.5	1.6	3.2	1.6	0.55	1.2	8.2	1.6	0.84
	CV %	2.5	3.3	4.8	7.9	3.8	6.8	11.8	9.8	4.1
Overall	Mean	59	50	71	20	15	19	61	18	23
(N = 15)	SD	3.0	2.2	3.7	1.4	0.96	1.1	9.0	2.5	2.9
	CV %	5.1	4.4	5.2	6.8	6.4	6.1	14.8	13.6	12.7
*ΔDi-4S, 6S: These disaccharides were produced from DS and HS(6S).

Three different control plasma samples were measured for one day (N=5). The results are shown in Tables 6 and 7.

	TABLE 6
	MSD	DSD
	Replicates	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3
Concen-	n1	0.40	0.32	0.34	0.12	0.13	0.11
tration	n2	0.38	0.34	0.38	0.12	0.12	0.11
(μg/mL)	n3	0.35	0.32	0.33	0.12	0.12	0.10
	n4	0.37	0.33	0.30	0.12	0.12	0.095
	n5	0.37	0.33	0.33	0.11	0.12	0.11
Mean		0.37	0.33	0.34	0.12	0.12	0.11
SD		0.018	0.008	0.029	0.0045	0.0045	0.0071
CV %		4.9	2.6	8.6	3.8	3.7	6.7

	TABLE 7
	Repli-	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, -6S*
	cates	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3	No. 1	No. 2	No.3
Concen-	n1	91	64	53	13	14	12	54	140	90
tration	n2	110	67	52	13	14	12	55	180	130
(μg/mL)	n3	100	58	50	15	11	12	55	170	91
	n4	96	54	47	15	13	13	55	170	97
	n5	83	53	53	15	14	12	68	180	130
Mean		96	59	51	14	13	12	57	168	108
SD		10.1	6.1	2.5	1.1	1.3	0.4	5.9	16.4	20.6
CV %		10.5	10.4	5.0	7.7	9.9	3.7	10.4	9.8	19.2
*ΔDi-4S, 6S: These disaccharides were produced from DS and HS(6S).

In Tables 5 and 7, the concentration data of ΔDi-4S, 6S represent a total concentration of DS-derived ΔDi-4S and HS-derived ΔDiHS-6S.

As is clear from Tables 4 to 7, the method of the present invention is an accurate, precise analytical method.

The results of measurement on plasma samples from mucopolysaccharidosis patients and control plasma samples are shown in Tables 8 and 9.

TABLE 8
			Concentarations	Composition
Sample		Age	(μg/mL)	(%)
No.	Categoly	(years)	MSD	DSD	Total	MSD	DSD
1	MPS I	1.2	3.1	0.50	3.6	86	14
2	MPS I	0.1	4.6	1.0	5.6	82	18
3	MPS II	15	4.0	0.76	4.8	84	16
4	MPS II	19	4.5	0.86	5.4	84	16
5	MPS II	19	5.0	1.3	6.3	79	21
6	MPS IIIA	4.5	2.4	0.72	3.1	77	23
7	MPS IIIA	0.7	2.6	0.51	3.1	84	16
8	MPS IIIB	4.5	2.2	0.40	2.6	85	15
9	MPS IIIB	6.5	2.4	0.80	3.2	75	25
10	MPS IIIC	6	3.0	0.83	3.8	78	22
11	MPS IV	3.3	7.0	2.4	9.4	74	26
12	MPS IV	3.5	3.7	1.1	4.8	77	23
13	MPS VI	NA	1.9	0.32	2.2	86	14
14	MPS VI	6.7	4.0	1.3	5.3	75	25
15	MPS VII	7	1.3	0.28	1.6	82	18
16	MPS VII	0.5	2.6	0.63	3.2	80	20
17	Control	43	0.76	0.16	0.92	83	17
18	Control	14	0.96	0.22	1.2	81	19
19	Control	51	0.89	0.29	1.2	75	25
20	Control	30	0.60	0.18	0.78	77	23
21	Control	34	0.76	0.26	1.0	75	25
22	Control	12	2.2	0.45	2.7	83	17
23	Control	4	1.1	0.36	1.5	75	25
24	Control	1	1.8	0.36	2.2	83	17
25	Control	14	2.2	0.71	2.9	76	24
26	Control	23	0.46	0.13	0.59	78	22
27	Control	26	0.73	0.21	0.94	78	22
28	Control	31	0.43	0.13	0.56	77	23
29	Control	36	1.6	0.38	2.0	81	19
NA: Not available.

TABLE 9
			Concentarations	Composition
Sample		Age	(ng/mL)	(%)
No.	Categoly	(years)	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, -6S*	Total	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, -6S*
1	MPS I	1.2	1200	250	590	2040	59	12	29
2	MPS I	0.1	8500	3300	12000	23800	36	14	50
3	MPS II	15	850	190	230	1270	67	15	18
4	MPS II	19	670	160	320	1150	58	14	28
5	MPS II	19	1100	270	1800	3170	35	9	57
6	MPS IIIA	4.5	1400	320	68	1788	78	18	4
7	MPS IIIA	0.7	2900	590	640	4130	70	14	15
8	MPS IIIB	4.5	1200	270	61	1531	78	18	4
9	MPS IIIB	6.5	2600	770	530	3900	67	20	14
10	MPS IIIC	6	1200	280	470	1950	62	14	24
11	MPS IV	3.3	520	90	700	1310	40	7	53
12	MPS IV	3.5	360	59	780	1199	30	5	65
13	MPS VI	NA	340	73	590	1003	34	7	59
14	MPS VI	6.7	340	62	1400	1802	19	3	78
15	MPS VII	7	210	19	33	262	80	7	13
16	MPS VII	0.5	980	180	700	1860	53	10	38
17	Control	43	120	20	88	228	53	9	39
18	Control	14	130	23	240	393	33	6	61
19	Control	51	120	24	260	404	30	6	64
20	Control	30	130	26	260	416	31	6	63
21	Control	34	130	24	260	414	31	6	63
22	Control	12	150	25	170	345	43	7	49
23	Control	1	290	46	320	656	44	7	49
24	Control	14	350	55	350	755	46	7	46
25	Control	31	220	22	69	311	71	7	22
26	Control	36	470	78	340	888	53	9	38
*ΔDi-4S, 6S: These disaccharides were produced from DS and HS(6S).
NA: Not available.

As is clear from Tables 8 and 9, the method of the present invention has been found to be useful in an assay of a clinical sample and also in screening. A mucopolysaccharidosis type IV case (No. 11 in Table 8) showed a high KS concentration. Also, mucopolysaccharidosis type I, II, and III cases (Nos. 1 to 10 in Table 9) showed high values of HS-derived ΔDiHS-0S concentration and HS-derived ΔDiHS-NS concentration. Moreover, a mucopolysaccharidosis type VI case (No. 14 in Table 9) showed a high value of DS-derived ΔDi-4S,6S concentration.

In cases where ΔDi-4S,6S level was high, DS or HS was also found to be high. However, when ΔDi-4S,6S has a high compositional proportion of disaccharides, a high value of ΔDi-4S,6S reflects a high DS value. In other words, the method of the present invention, which can provide analyses of concentration data of respective disaccharides and compositional proportions, is very useful for attaining a detailed analysis.

As described above, with the present method, KS, HS, and DS levels can be analyzed simultaneously. If some correlation is identified in future research between age, pathological conditions, etc. of a patient and KS, HS, and DS levels, it is believed that a single assay provides separate, simultaneous diagnosis of different types of mucopolysaccharidoses.

Example 2

In order to check whether the assay method of the present invention provides a successful screening on urine samples, the following experiment was performed using urine samples from mucopolysaccharidosis patients and control urine samples (human).

Pretreatment of a urine sample:

• 1) Add a urine sample (0.01 mL) to ULTRAFREE™-MC (BIOMAX-5);
• 2) Centrifuge at 4,000×g for 15 minutes;
• 3) Replace the collection tube in ULTRAFREE™-MC (BIOMAX-5) by a new tube;
• 4) Add a 50μg/mL aqueous chondrosine solution (0.02 mL) (produced and sold by SEIKAGAKU CORPORATION) as an internal standard substance onto the filter;
• 5) Add 50-mmol/L Tris-HCl buffer (0.02 mL, pH 7) onto the filter;
• 6) Add an enzyme mixture solution (0.02 mL) containing keratanase II, heparitinase, and chondroitinase B (2 mU each) onto the filter;
• 7) Mix the resultant mixture using a vortex mixer for about ten seconds;
• 8) Incubate the mixture at 37° C. for 15 hours;
• 9) Centrifuge the resultant mixture at 8,000×g for 15 minutes;
• 10) Add water (0.02 mL) to the filtrate;
• 11) Mix the resultant mixture using a vortex mixer for about 10 seconds; and
• 12) Transfer the-thus obtained liquid sample into an injection vial for an autosampler.

Pretreatment of a sample for producing a calibration curve: 1) KS standard solutions: Bovine-cornea-derived KS (produced and sold by SEIKAGAKU CORPORATION) is employed.

Concentrations are shown in Table 10.

• 2) HS standard solutions: An unsaturated heparan/heparin-disaccharide kit (H kit) (produced and sold by SEIKAGAKU CORPORATION) is employed. Aqueous solutions each containing ΔDiHS-0S, ΔDiHS-6S, and ΔDiHS-NS are prepared. Concentrations are shown in Table 11.
• 3) Add an aliquot (0.01 mL) of each of the above-prepared KS standard solutions and an aliquot (0.02 mL) of each of the above-prepared HS standard solutions to ULTRAFREE™-MC (BIOMAX-5).
• 4) Add an 50-μg/mL aqueous solution (0.02 mL) of chondrosine (produced and sold by SEIKAGAKU CORPORATION) as an internal standard substance onto the filter.
• 5) Adding 50-mmol/L Tris-HCl buffer (0.02 mL, pH 7) on the filter.
• 6) Add an enzyme-mixed aqueous solution (0.02 mL) containing keratanase II, heparitinase, and chondroitinase B (2 mU each) onto the filter.
• 7) Mix the resultant mixture by use of a vortex mixer for about ten seconds.
• 8) Incubate the mixture at 37° C. for 15 hours.
• 9) Centrifuge the resultant mixture at 8,000×g for 15 minutes.
• 10) Allow a blank urine sample to pass through a stationery column, Bond Elute SAX column (500 mg/3 mL), to thereby prepare a blank solution.
• 11) Add the thus-prepared blank solution (0.01 mL) to the filtrate obtained in step 9).
• 12) Mix the resultant mixture using a vortex mixer for about 10 seconds.

13) Transfer the-thus obtained liquid sample into an injection vial for an autosampler.

TABLE 10
Concentration of standard solution (KS)
	(Unit: μg/mL)
	S7	S6	S5	S4	S3	S2	S1
MSD	7.1	3.6	2.8	1.4	0.71	0.36	0.14
DSD	2.9	1.5	1.2	0.58	0.29	0.15	0.058
Total	10	5	4	2	1	0.5	0.2

TABLE 11
Concentration of standard solution (HS)
	(Unit: ng/mL)
	S6	S5	S4	S3	S2	S1
ΔDiHS-0S	2500	1250	500	250	100	50
ΔDiHS-NS	1250	625	250	125	50	25
ΔDIHS-6S	625	313	125	63	25	13

In the analysis of urine samples, LC/MS/MS conditions employed and concentration calculation method are the same as those used for the analyses of plasma and serum samples.

Three different control urine samples were measured for three days (N-5). The results are shown in Tables 12 and 13.

	TABLE 12
	MSD	DSD
	Replicates	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3
Batch 1	n1	1.3	0.97	1.5	0.65	0.47	0.98
	n2	1.1	1.1	1.5	0.54	0.52	0.98
	n3	1.2	1.1	1.6	0.62	0.49	0.96
	n4	1.2	1.0	1.5	0.58	0.50	0.94
	n5	1.2	1.1	1.6	0.62	0.50	1.1
	Mean	1.2	1.1	1.5	0.60	0.50	1.0
	SD	0.071	0.064	0.055	0.043	0.018	0.063
	CV %	5.9	6.1	3.6	7.1	3.7	6.3
Batch 2	n1	1.3	1.1	1.6	0.63	0.50	1.0
	n2	1.3	1.1	1.6	0.72	0.50	1.1
	n3	1.2	1.2	1.6	0.55	0.52	1.1
	n4	1.3	1.1	1.6	0.66	0.49	1.0
	n5	1.2	1.2	1.6	0.54	0.53	1.1
	Mean	1.3	1.1	1.6	0.62	0.51	1.1
	SD	0.055	0.055	0.000	0.076	0.016	0.055
	CV %	4.3	4.8	0.0	12.2	3.2	5.2
Batch 3	n1	1.4	1.2	1.8	0.65	0.47	1.0
	n2	1.5	1.2	1.8	0.71	0.47	1.1
	n3	1.4	1.3	1.8	0.51	0.54	1.1
	n4	1.5	1.2	1.8	0.66	0.49	1.0
	n5	1.3	1.2	1.9	0.52	0.52	1.1
	Mean	1.4	1.2	1.8	0.61	0.50	1.1
	SD	0.084	0.045	0.045	0.090	0.031	0.055
	CV %	5.9	3.7	2.5	14.7	6.3	5.2
Overall	Mean	1.3	1.1	1.7	0.61	0.50	1.0
(N = 15)	SD	0.12	0.087	0.13	0.067	0.022	0.063
	CV %	9.0	7.6	7.9	11.0	4.4	6.0

	TABLE 13
	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, 6S*
	Replicates	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3	No. 1	No. 2	No. 3
Batch 1	n1	1100	880	1800	430	340	1100	3100	2000	6100
	n2	900	920	1900	350	370	1100	2400	2200	6700
	n3	980	890	1800	450	370	1100	2900	2200	7000
	n4	1000	890	1800	410	350	1100	3100	2100	6300
	n5	1000	880	1800	450	360	1100	3100	2200	6900
	Mean	996	892	1820	418	358	1100	2920	2140	6600
	SD	71	16	45	41	13	0	303	89	387
	CV %	7.2	1.8	2.5	9.9	3.6	0.0	10.4	4.2	5.9
Batch 2	n1	990	810	1800	450	330	980	3200	2300	6400
	n2	1100	820	1800	490	330	1000	2900	2200	6600
	n3	890	930	1900	410	360	1100	3000	2400	6300
	n4	1000	790	1800	460	310	1000	3100	2100	6500
	n5	900	910	1800	420	330	1000	2900	2300	6300
	Mean	976	852	1820	446	332	1016	3020	2260	6420
	SD	86	63	45	32	18	48	130	114	130
	CV %	8.8	7.4	2.5	7.2	5.4	4.7	4.3	5.0	2.0
Batch 3	n1	1100	990	2000	460	350	1100	3100	2300	6500
	n2	1200	830	2000	500	360	1100	3200	2000	6900
	n3	890	810	2000	390	390	1200	2600	2600	6500
	n4	1100	940	1900	480	340	1100	3300	2000	7000
	n5	990	790	2000	420	330	1100	2800	2300	6400
	Mean	1056	872	1980	450	354	1120	3000	2240	6660
	SD	119	88	45	45	23	45	292	251	270
	CV %	11.3	10.1	2.3	9.9	6.5	4.0	9.7	11.2	4.1
Overall	Mean	1009	872	1873	438	348	1079	2980	2213	6560
(N = 15)	SD	94	61	88	40	21	58	240	164	282
	CV %	9.3	7.0	4.7	9.1	6.0	5.4	8.0	7.4	4.3
*ΔDi-4S, 6S: These disaccharides were produced from DS and HS(6S).

As is apparent from Tables 12 and 13, the present method has been shown to be an accurate, precise analytical method.

The results of measurement on urine samples from mucopolysaccharidosis patients are shown in Tables 14 and 15.

TABLE 14
		Concentarations	Composition
Sample		(μg/mg creatinine)	(%)	Creatinine
No.	Data	MSD	DSD	Total	MSD	DSD	(mg/mL)
1	MPS I	21	5.1	26	80	20	0.1324
2	MPS I	14	3.5	17	79	21	0.244
3	MPS I	5.1	1.1	6.3	82	18	0.107
4	MPS II	11	3.5	14	75	25	0.111
5	MPS II	12	3.9	16	76	24	0.633
6	MPS II	2.3	0.91	3.2	71	29	0.836
7	MPS IIIA	38	10	48	80	20	0.0288
8	MPS IIIA	8.7	2.8	12	75	25	0.172
9	MPS IIIA	22	6.5	29	77	23	0.054
10	MPS IIIB	14	4.7	19	75	25	0.188
11	MPS IIIB	7.2	2.6	9.8	74	26	0.47
12	MPS IIIB	79	61	140	56	44	0.105
13	MPS IIIC	5.2	2.4	7.6	69	31	0.463
14	MPS IIIC	2.1	1.0	3.1	67	33	0.765
15	MPS IIIC	30	6.1	37	83	17	0.493
16	MPS IVA	19	18	37	50	50	0.468
17	MPS IVA	4.2	3.2	7.4	57	43	0.688
18	MPS IVA	13	12	25	51	49	1.38
19	MPS IVB	37	13	50	74	26	0.105
20	MPS IVB	6.1	2.6	8.8	70	30	0.797
21	MPS IVB	15	4.8	20	76	24	0.2711
22	MPS VI	4.5	3.3	7.8	58	42	0.799
23	MPS VI	4.3	1.9	6.2	69	31	0.304
24	MPS VI	3.9	2.3	6.1	63	37	0.618
25	MPS VII	2.8	0.88	3.7	76	24	0.193
26	MPS VII	22	8.8	30	71	29	0.694
27	MPS VII	1.9	0.74	2.7	72	28	0.43
28	Adult control 1	0.63	0.27	0.90	70	30	1.0319
29	Adult control 2	0.53	0.37	0.89	59	41	2.2735
30	Adult control 3	0.46	0.19	0.65	71	29	1.9874
31	Adult control 4	0.52	0.24	0.76	69	31	2.1103
32	Adult control 5	1.0	0.34	1.3	74	26	0.7045
33	Adult control 6	0.28	0.15	0.43	64	36	3.1815
34	Adult control 7	0.44	0.25	0.69	64	36	2.0811
35	Adult control 8	0.49	0.27	0.76	65	35	2.0401
36	Adult control 9	0.49	0.22	0.71	69	31	1.9045
37	Adult control 10	0.72	0.23	1.0	76	24	1.3672
38	Adult control 11	0.53	0.35	0.88	60	40	2.6606
39	Adult control 12	0.47	0.30	0.77	61	39	1.7903

TABLE 15
		Concentarations	Composition
Sample		(ng/mg creatinine)	(%)	Creatinine
No.	Data	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, -6S*	Total	ΔDiHS-0S	ΔDiHS-NS	ΔDi-4S, -6S*	(mg/mL)
1	MPS I	110000	23000	580000	713000	15	3	81	0.1324
2	MPS I	98000	30000	980000	1108000	9	3	88	0.244
3	MPS I	15000	3100	41000	59100	25	5	69	0.107
4	MPS II	70000	13000	200000	283000	25	5	71	0.111
5	MPS II	63000	25000	440000	528000	12	5	83	0.633
6	MPS II	950	400	2600	3950	24	10	66	0.836
7	MPS IIIA	330000	66000	32000	428000	77	15	7	0.0288
8	MPS IIIA	110000	21000	18000	149000	74	14	12	0.172
9	MPS IIIA	240000	48000	35000	323000	74	15	11	0.054
10	MPS IIIB	170000	53000	32000	255000	67	21	13	0.188
11	MPS IIIB	110000	36000	26000	172000	64	21	15	0.47
12	MPS IIIB	260000	130000	2800000	3190000	8	4	88	0.105
13	MPS IIIC	63000	20000	19000	102000	62	20	19	0.463
14	MPS IIIC	30000	8400	5500	43900	68	19	13	0.765
15	MPS IIIC	3200	1300	5300	9800	33	13	54	0.493
16	MPS IVA	1400	750	19000	21150	7	4	90	0.468
17	MPS IVA	550	200	2900	3650	15	5	79	0.688
18	MPS IVA	1600	1000	15000	17600	9	6	85	1.38
19	MPS IVB	2200	760	4200	7160	31	11	59	0.105
20	MPS IVB	650	340	1300	2290	28	15	57	0.797
21	MPS IVB	1800	700	5500	8000	23	9	69	0.2711
22	MPS VI	2100	1000	160000	163100	1	1	98	0.799
23	MPS VI	1700	630	110000	112330	2	1	98	0.304
24	MPS VI	1900	940	140000	142840	1	1	98	0.618
25	MPS VII	470	140	980	1590	30	9	62	0.193
26	MPS VII	45000	20000	190000	255000	18	8	75	0.694
27	MPS VII	7000	1800	8100	16900	41	11	48	0.43
28	Adult control 1	510	190	610	1310	39	15	47	1.0319
29	Adult control 2	700	290	1100	2090	33	14	53	2.2735
30	Adult control 3	440	170	650	1260	35	13	52	1.9874
31	Adult control 4	440	150	900	1490	30	10	60	2.1103
32	Adult control 5	540	170	1200	1910	28	9	63	0.7045
33	Adult control 6	310	140	690	1140	27	12	61	3.1815
34	Adult control 7	430	160	720	1310	33	12	55	2.0811
35	Adult control 8	540	230	1200	1970	27	12	61	2.0401
36	Adult control 9	450	170	840	1460	31	12	58	1.9045
37	Adult control 10	370	130	700	1200	31	11	58	1.3672
38	Adult control 11	750	380	1900	3030	25	13	63	2.6606
39	Adult control 12	530	200	610	1340	40	15	46	1.7903
*ΔDi-4S, 6S: These disaccharides were produced from DS and HS(6S).

As is clear from Tables 14 and 15, the method of the present invention has been found to be useful in an assay of a clinical sample and also in screening. Mucopolysaccharidosis types I, II, III cases showed high HS concentrations, and a mucopolysaccharidosis type VI case showed a high value of ΔDi-4S,6S concentration.

In particular, KS-derived DSD ratio differs between mucopolysaccharidosis type IV A (No. 16 to 18 in Table 14) and mucopolysaccharidosis type IV B (No. 19 to 21 in Table 14). That is, type IV A showed a high DSD ratio. Therefore, analysis of compositional ratio can distinguish between type IV A and type IV B.

As described above, with the present method, KS, HS, and DS levels can be analyzed simultaneously. If some correlation is identified in future research between age, pathological conditions, etc. of a patient and KS, HS, and DS levels, it is believed that a single assay provides simultaneous diagnosis of different types of mucopolysaccharidoses.

Claims

1. A diagnostic method of mucopolysaccharidoses including the following steps (1) and (2): (1) a step including (a) filtering a biological sample with an ultrafiltration filter, digesting the sample on the filter with a glycosaminoglycan-specific enzyme, centrifuging the digested sample to obtain a filtrate, or (b) digesting a biological sample with a glycosaminoglycan-specific enzyme, filtering the sample with an ultrafiltration filter, applying the filtrate obtained by (a) or (b) to a liquid chromatograph/mass spectrometer, and analyzing glycosaminoglycan-derived disaccharides, and (2) a step of diagnosing a subject as having mucopolysaccharidosis, chemically diagnosing effect of treatment of mucopolysaccharidoses, or determining types of mucopolysaccharidoses, on the basis of quantitative concentration data and disaccharide composition obtained by step (1).

2. The method according to claim 1, wherein, in step (1), liquid chromatography is performed under such conditions that the analytical column is a carbon graphite column and an alkaline solution is employed as a mobile phase, to thereby elute glycosaminoglycan-derived disaccharides at optimal elution positions that facilitate the MS analysis.

3. The method according to claim 1, wherein, in step (1), the disaccharides are produced through use of a solution containing, as the glycosaminoglycan-specific degrading enzyme, keratanase II, heparitinase, and chondroitinase B; and keratan sulfate, heparan sulfate, dermatan sulfate are analyzed simultaneously.

4. The method according to claim 1, wherein, in step (1), the disaccharides are produced through use of a solution containing, as the glycosaminoglycan-specific degrading enzyme, keratanase II, heparitinase, and chondroitinase B; and keratan sulfate, heparan sulfate, dermatan sulfate are analyzed simultaneously.

5. The method according to claim 1, wherein, in step (1), the biological sample is selected from among plasma, serum, blood, urine, and body fluid.