Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-04-21
2001-11-20
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S195000, C435S200000, C435S232000
Reexamination Certificate
active
06319680
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for the quantitative determination of a monosaccharide, such as sialic acid, constituting a sugar composition.
2. Description of Related Art
Sugar compositions such as glycoproteins and glycolipids are important substances that constitute a mechanism of function-regulation for human body, particularly immunity related portions and, recently, investigations have been being made intensively in the top front of medicine, such as therapy of AIDS or tumors. It has already been considered that the sugar chain of glycoprotein present on the surface of AIDS virus bonds to the CD4 molecule on the surface of host cells to cause infection and there have been many reports that the sugar moiety of the sugar composition varies due to tumors.
Among such methods, particularly quantitative determination of various monosaccharides constituting the sugar composition composed mainly of sialic acid is the key of the investigation and various methods have been proposed for achieving more rapid and higher sensitivity analysis.
Various conventional methods and their advantages and disadvantages will be explained below.
For example,
(1) A method for determining sialic acid, neutral sugars and amino sugars by gas-liquid chromatography (GLC) after converting the sample into a volatile sugar derivative by methanolysis and trimethylsilylation:
This method has defects that although it can determine sialic acid, neutral sugars and amino sugars simultaneously, it requires a large amount of sample (10 nmol or more as monosaccharide), that the analysis by GLC is not suitable for treating multiple samples, and that N-acetylglucosamine bonded to an asparagine residue cannot be determined (Anal. Biochem., 119, 17-24 (1982)).
(2) A method for the determination of sialic acid in which sialic acid liberated by sialidase or an acid is converted to 1,2-diamino-4,5-methylenedioxybenzene acid (DMB) derivative, followed by high performance liquid chromatography (HPLC):
This method is simple and has high sensitivity. However, it can analyze only sialic acid and the derivative is unstable and has to be analyzed within 12 hours (“Method for Studying Glycoprotein Sugar Chain (Biochemistry Experimental Methods 23)”, Gakkai Shuppan Center, p.20 (1989)) and is limited in its application.
(3) A method for the determination of amino sugars by amino acid analysis:
This method is simple and has high sensitivity but it can analyze only amino sugars.
(4) A method for the determination of neutral sugars and amino sugars that involves labeling the sugars with pyridylamino, converting them into ethyl 4-aminobenzoate derivatives, followed by HPLC:
This method cannot analyze sialic acid.
(5) A method for the determination of sialic acid, neutral sugars and amino sugars by converting the sialic acid and sugars into 1-phenyl-3-methyl-5-pyrazolone (PMP) derivative, followed by HPLC:
This method can be determined with high sensitivity, but has such an inconvenience that sialic acid can not be simultaneously determined together with neutral sugars and amino sugars.
(6) A method for the determination of sialic acid, neutral sugars and amino sugars that involves converting the sialic acid and sugars into cyanoacetamide derivatives after isolation by HPLC:
This method is a post column labeling method and is simple but takes a long time for analysis and cannot afford simultaneous determination of sialic acid together with neutral sugars and amino sugars so that it is inconvenient.
(7) A method for the determination of sialic acid, neutral sugars and amino sugars using anion exchange chromatography and an electrochemical detector:
This method can save the trouble of labeling but it is inconvenient in that it requires a special apparatus and that it cannot determine sialic acid together with neutral sugars and amino sugars simultaneously (Methods in Enzymology, 230, 208-225 (1994)).
As stated above, conventional methods for the determination of monosaccharides constituting sugar compositions are advantageous in a way and disadvantageous in another. Whereas the method for the determination of sugar compositions that enables diagnosis of affected sites without surgical invasion gives less stress to patients and hence are being increasingly used. Thus, a method that can treat many samples in a simple manner is desired. Accordingly, there has been a keen desire for urgently developing a method for simultaneous determination of monosaccharides that constitute sugar compositions including sialic acid that has recently attracted much attention as an indices for monitoring changes in pathological state affected by, e.g., tumors, with high rate, high sensitivity and accuracy.
BRIEF SUMMARY OF THE INVENTION
Therefore, it is an object of the present invention to provide a method for the simultaneous determination of monosaccharides constituting a sugar composition.
With view to solving the above object, the present inventors have made intensive research and as a result they have found that in the analysis of monosaccharides in a sugar composition such as glycoprotein, glycolipid, etc. which are constituted by bonding of respective monosaccharides, etc., a sialic acid and respective monosaccharides can be determined simultaneously by converting liberated sialic acid into N-acylmannosamine followed by acid hydrolysis, thus achieving the present invention.
That is, the present invention provides a method for analyzing monosaccharides in a sugar composition, comprising the steps of:
(1) liberating sialic acid from the sugar composition with sialidase or an acid;
(2) converting the liberated sialic acid into N-acylmannosamine with sialic acid aldolase; and
(3) acid hydrolyzing N-acylmannosamine and a sugar residue.
Furthermore, in the present invention, upon determination of the respective monosaccharides, the de-N-acylated monosaccharides during hydrolyss may be N-acetylated, and further the monosaccharides that are N-acetylated again and N-acylmannosamine may be labeled with ethyl 4-aminobenzoate (ABEE).
In the above description, the sugar residue means a remaining sugar composition after only sialic acid is liberated from a sugar composition to be determined, i.e., a bonded substance of remaining monosaccharides.
The sialic acid (N-acylmannosamine) and the respective monosaccharides may be analyzed by MPLC, capillai electrophoresis, GLC or the like method. It is particularly preferred that they are analyzed by HPLC, which is suitable for treating many samples.
In the present invention, the determination of respective monosaccharides by HPLC may be performed using a reverse phase column and it is desirable to use a borate buffer as an eluent.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, sialic acid and respective monosaccharides constituting a sugar composition can be determined simultaneously. In the conventional methods, sialic acid was completely decomposed and could not be detected under the conditions under which neutral sugars and amino sugars are hydrolyzed. Therefore, when sialic acid, neutral sugars and amino sugars are determined, treatment and analysis must have been performed twice or more on a single sample. In the present invention, sialic acid is preliminarily converted into a compound which is not decomposed under the condition that neutral sugars and amino sugars are hydrolyzed, so that the determination of sialic acid, neutral sugars and amino sugars at a time. This provides excellent operability and enables rapid determination of respective monosaccharides constituting a sugar composition.
The sugar composition, which is the target of analysis in the present invention, includes glycoproteins, glycolipids, oligosaccharides and polysaccharides.
The reagent used in the present invention includes sialidase, sialic acid aldolase and acids. Sialidase may be originated from
Arthrobacter ureafaciens, Clostridium perfringens, Streptococcus s., Vibrio cholerae, Salmonella typhimurium
, New castle disease virus, etc. The reagent o
Arai Junko
Kamei Masugu
Saito Keiko
Yasuno Shoichi
Kabushiki Kaisha Honen Corporation
Oliff & Berridg,e PLC
Prats Francisco
LandOfFree
Method for analyzing monosaccharide in a sugar composition does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for analyzing monosaccharide in a sugar composition, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for analyzing monosaccharide in a sugar composition will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2603333