Detection of advanced glycation endproducts in a cerebrospinal fluid sample

FIELD OF THE INVENTION

[0001] The present invention relates to the measurement of advanced glycation endproducts in cerebrospinal fluid and especially to the assessment and monitoring of neurodegenerative disorders based on such measurement.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive dementia that ultimately leads to death. It is also one of the most devastating brain diseases and a severe medical, psychological, and economic problem in modern societies. For example, there are about 600,000 patients suffering from Alzheimer's disease in Japan.

[0003] Despite the huge number of cases and the tremendous costs to healthcare systems, the appropriate diagnosis of Alzheimer's disease is a major challenge to the clinician in the field. At least three different approaches, all of some, despite limited, utility, are available.

[0004] First, there are so-called mental or cognitive examinations. Such examinations are very difficult to standardize and to transfer from one institution to an other. One of the most widely used examinations follows the instructions given by the National Institute of Neurological and Communicative Disorders and Strokes-Alzheimer's disease and Related Disorders Association, the so-called NINCDS-ADRDA criteria (McKhann, G., et al., Neurology 34:939-944, 1984).

[0005] Second, there are physical approaches to diagnose Alzheimer's disease which are largely based on metabolic activities of the brain investigated. Such physical methods include, for example, near-infrared spectroscopy (NIRS) and positron emission tomography (PET). These methods may, for example, be used to investigate the level of oxygenation of hemoglobin in the brain as, for example, described by C. Hock et al., Brain Research 755:293-303, 1997.

[0006] Third to be mentioned is the approach to diagnose Alzheimer's disease by biological markers. The major problem and obstacle to the use of biological markers is the fact that Alzheimer's disease is rather slowly progressing. It is assumed that there is quite a long preclinicaf phase, i.e., a phase in which the disease starts and progresses without leading to the manifestation of clearly visible or diagnostically accessible symptoms or markers. Even after clinical onset, the progress of the disease is slow, and it takes five to ten years until a patient dies due to the disease (C. Hock, Neurobiology of Aging 19:149-155, 1998). Biological markers, nonetheless, represent extremely attractive tools to better detect, monitor, and diagnose AD. They can be collected repetitively, their measurement is comparatively cheap and easy to perform, and the result of such measurement is not as difficult to standardize as, for example, mental examinations.

[0007] Several protein markers have attracted major attention during the past ten years, for example, the protein known as amyloid precursor protein (APP) and especially the breakdown product of this APP that is known as amyloid β-peptide (Aβ, Weidemann, A., et al., Cell 57:115-126, 1989), the tau protein, and the apolipoprotein E4. Only apolipoprotein E4 is diagnosed from blood. The tau protein or its variants containing different degrees of phosphorylation, as well as the various forms of Aβ, usually are tested in cerebrospinal fluid (CSF).

[0008] Advanced glycation endproducts (AGE's), in contrast, are a hallmark of diabetic disease and have been studied there extensively. In the formation of AGE's in a first step, glucose and other reducing sugars attach non-enzymatically to the amino groups of proteins in a concentration-dependent manner. The non-enzymatic reaction between glucose and the free amino groups on proteins to form a stable amino, 1-deoxy ketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein a rearrangement of the amino terminus of the B-chain of hemoglobin by reaction with glucose forms an adduct and gives rise to a product known as hemoglobin A1c. Similar reactions have also been found to occur with a variety of other body proteins such as lens crystallin, collagen, and nerve proteins (see Bunn et al., Biochem. Biophys. Res. Commun. 67:103-109, 1975; Koenig et al., J. Biol. Chem. 252:2992-2997, 1975; Monnier and Cerami, Maillard Reaction in Food and Nutrition, Ed. Waller, G. A., American Chemical Society, 431-448, 1983; and Monnier and Cerami, Clinics in Endocrinology and Metabolism 11:431-452, 1982.

[0009] Over time, these initial Amadori adducts can undergo secondary reactions such as further oxidation, rearrangements, dehydrations, and cross-linking with other protein groups, and finally accumulate as a family of complex structures referred to as AGE's. Substantial progress has been made towards the elucidation of the biological roles and clinical significance of advanced glycation endproducts so that it is now acknowledged that many of the conditions heretofore attributed to the aging process or to the pathological effects of diseases such as diabetes are attributable at least in part to the formation, accumulation, and/or activity of AGE's in vivo.

[0010] Because the above mentioned secondary reactions occur slowly, proteins may accumulate significant amounts of Amadori products before accumulating a measurable amount of AGE's in vivo. AGE's may modify important biological molecules or structures such as receptors, membranes, and enzymes. They can cause protein cross-linking, which in turn may reduce the structural and/or functional integrity of organs and organ parts, thus ultimately reducing or impairing organ function.

[0011] The advanced glycation process is particularly noteworthy in that it predominately affects proteins with long half-lives, e.g., collagen under conditions of relatively high sugar concentration, such as in diabetes mellitus. Numerous studies have suggested that AGE's play an important role in the structural and functional alteration which occurs in proteins during aging and in chronic disease.

[0012] A second physiological condition giving rise to AGE-modified biomolecules is summarized as oxidative stress. It is known that oxidative stress leads, among other events, to the formation of advanced glycation endproducts and may be one of the patho-mechanisms in neurodegenerative disorders, e.g., in AD (Behl, Chr., Progress in Neurobiology 57:301-323, 1999. It is also known that striking similarities are found when, e.g., comparing in vitro properties of Aβ and the so-called prion proteins known as hallmarks or pathogenic agents from diseases summarized as transmissible spongiform encephalopathies (TSE's). Oxidative stress is of major importance in the pathogenesis of TSE's, e.g., of bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jacob disease (CJD) in humans (Kretzschmar, et al., Nature 380:345-347, 1996. Oxidative stress phenomena thus trigger the formation of AGE in such neurodegenerative diseases as AD, BSE, and CJD.

[0013] Recently, antibody reagents have become available, and methods have been described which may be used in the reproducible detection of well-defined AGE structures. U.S. Pat. No. 5,610,076 discloses the specific detection of hemoglobin-carrying AGE structures (Hb-AGE). The improvement described herein resides in the pretreatment of samples with detergents and/or chaotropic reagents. The positive effects of such pretreatment are explained by exposure of Hb-AGE epitopes that otherwise would not be accessible to anti-AGE antibodies.

[0014] U.S. Pat. No. 5,698,197 describes a monoclonal antibody (4G9) reactive with in vivo formed AGE's. In one embodiment this antibody is used in a sandwich type ELISA to detect apo-B AGE, IgG AGE, collagen AGE, serum AGE peptides and proteins as well as urinary AGE peptides and proteins. In order to perform these AGE sandwich assays, monoclonal antibody 4G9 or an immunoreactive fragment thereof is directly coated to the solid phase. Competitive immunoassay parameters are also described using 4G9. BSA-AGE is used as antigen and coated to the solid phase of such competition type assays.

[0015] Whereas all the approaches in the field of Alzheimer's disease as discussed above, such as PET scans, mental examinations, or certain biochemical markers may be used to some extent in the assessment of Alzheimer's disease, there still is a tremendous need especially for improved and easy to use biochemical or biological markers to further improve the tools available in order to detect, diagnose, or monitor neurodegerative diseases such as Alzheimer's disease, BSE, and CJD.

[0016] It has now surprisingly been found that the level of advanced glycation endproducts in CSF samples can be used with great advantage in the assessment and diagnosis of neurodegenerative diseases such as AD, BSE, and CJD, especially when measures are taken to expose AGE epitopes.

SUMMARY OF THE INVENTION

[0017] The present invention discloses a method for assessment of neurodegenerative diseases, especially of Alzheimer's disease and Creutzfeldt-Jacob disease in humans and of bovine spongiform encephalopathy in cattle, based on measurement of at least one advanced glycation endproduct in a cerebrospinal fluid sample, characterized in that said CSF sample is pretreated to expose AGE epitopes.

[0018] It has been found that pretreatment of cerebrospinal fluid is an effective means of exposing AGE epitopes. It has also been found that the level of AGE epitopes which can be exposed is much higher in CSF obtained from patients diagnosed with Alzheimer's disease, as compared to CSF samples collected from controls.

[0019] In one embodiment, a method according to the present invention is used to assess Alzheimer's disease by comparing AGE-CSF levels measured in a sample collected from a patient known to have, or suspected of having, Alzheimer's disease and to compare such levels to levels measured in parallel or known from control samples.

[0020] The method according to the present invention is used as well to monitor Alzheimer's disease.

[0021] A further embodiment of the present invention is a method to expose AGE epitopes as present in a CSF sample, said method comprising use of proteolytic enzymes such as esperase or proteinase K.

[0022] The use of methods according to the present invention in screening efforts for drugs affecting Alzheimer's disease which influence the level of AGE in CSF is, of course, a major area of potential application representing another preferred embodiment of the present invention which is of major relevance to the field of AD.

DESCRIPTION OF THE DRAWING

[0023]
FIG. 1 is a graph showing AGE-CML levels in pretreated and untreated CSF samples. Cerebrospinal fluid samples from healthy controls and from patients with Alzheimer's disease have been investigated with and without sample pretreatment. As can be seen, the levels of the AGE structure carboxymethyl lysine (CML) as detected by monoclonal antibody 4G9 are approximately the same for both groups before treatment. However, after exposure of CML epitopes by pretreatment, an increased AGE-CML level is found with CSF samples from AD patients as compared to pretreated CSF samples from control individuals. The vertical bars indicate the standard deviation as calculated.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In a preferred embodiment, the present invention relates to a method of measurement of at least one advanced glycation endproduct in a cerebrospinal fluid sample, characterized in that said CSF sample is pretreated. Such pretreatment leads to the exposure of AGE epitopes which are not recognized by a specific binding partner in another aliquot of the same sample without such pretreatment.

[0025] Advanced glycation endproducts are a hallmark of diabetic disease and have been studied there extensively. As mentioned further above, AGE's are known and used as markers of diabetes. It is therefore surprising that AGE structures contained in cerebrospinal fluid have now been found and demonstrated to be of major importance in the assessment of neurodegenerative diseases such as AD, CJD, and BSE. Even more surprising is the fact that a pretreatment of CSF fluid is required to expose additional AGE epitopes, and especially that such exposure has different effects in CSF samples collected from patients suspected or diagnosed with Alzheimer's disease as compared to samples taken from healthy controls. Obviously, the CSF of patients with Alzheimer's disease must contain a significant proportion of AGE epitopes which can be exposed by appropriate treatments, whereas comparatively low levels of such exposable AGE epitopes exist in CSF from healthy people.

[0026] As mentioned above, several quite different disease entities in the field of neurodegenerative diseases are characterized by loss of neurons, congnitive impairments, and clinical manifestations of different type and nature. These neurodegenerative diseases comprise, among others, Alzheimer's disease, Parkinson's disease, and diseases grouped under TSE's. These TSE's include such different diseases of animals and humans such as scrapie, BSE, kuru-kuru, Gerstmann-Sträussler-Scheinker syndrome, and CJD. Oxidative stress is known as one of the factors also contributing to diseases such as CJD and BSE (Kretzschmar, supra). These diseases now also have been found to exhibit changes in AGE-CSF levels.

[0027] A preferred embodiment according to the present invention is, therefore, a method for assessment of a neurodegenerative disease based on measurement of at least one advanced glycation endproduct in cerebrospinal fluid characterized in that said CSF sample is pretreated to expose AGE epitopes.

[0028] The term “AGE epitope” is used to describe a structure which is the result of a non-enzymatic glycation plus oxidation process or is a result of reactions accompanied with oxidative stress which is bound or recognized by a specific binding partner, especially by an antibody specifically reacting with this structure. One well-known example of such an AGE epitope-specific binding partner is monoclonal antibody 4G9, as described in U.S. Pat. No. 5,698,197.

[0029] There are many ways of pretreatment, some of which will be discussed in more detail below. Such pretreatment may be performed completely separate from the test mixture which is finally used to assess AGE levels. It is, however, also possible to use rather short preincubations with a denaturing reagent. At its extremes, pretreatment may be performed simultaneously with the analysis. It is, however, preferred that the CSF sample is first pretreated with an appropriate agent and/or method, and thereafter the level of AGE is measured using an appropriate assay, e.g., an immunoassay.

[0030] The term “exposed” is used to indicate that epitopes which are not accessible to a specific binding partner without pretreatment are accessible (exposed) after appropriate treatment.

[0031] Out of the many methods possible to be used in the pretreatment, it has been found that methods resulting in protein denaturation are most appropriate. Such denaturation results, for example, in unfolding of globular protein structures and in dissociation of protein and/or peptide complexes. It is therefore preferred that a method according to the present invention is performed by including a pretreatment step which results in protein denaturation.

[0032] Out of the various methods and agents known in the art to perform such pretreatment, it is preferred to use reagents selected from the group of detergents, chaotropic reagents, and proteolytic enzymes. It is also possible to apply a method of heat treatment or a method of acid treatment to expose additional AGE epitopes in a CSF sample. All these reagents or methods are known to result in protein unfolding and dissociation of protein and peptide aggregates.

[0033] AGE epitopes usually represent rather small structures. Such epitopes may be masked or hidden within a protein or peptide molecule. One of the preferred ways to expose these epitopes is the use of proteolytic enzymes. A preferred embodiment according to the present invention is a method to expose AGE epitopes as present in a CSF sample, said method comprising use of a proteolytic enzyme.

[0034] The use of the proteolytic enzyme may preferably be combined with the use of other pretreatment methods. E.g., it is possible to first use a chaotropic denaturing agent and thereafter to apply a proteolytic enzyme. It is also a preferred embodiment of the present invention to use several proteolytic enzymes in combination.

[0035] It is possible to measure the level of various AGE structures or AGE epitopes which are present in CSF by different methods such as HPLC (high performance liquid chromatography), MS (mass spectroscopy), and specific binding assays. Immunological detection by specific antibodies is preferred. By standard immunoassay procedures, e.g., as described in relevant textbooks such as “Practice and Theory of Enzyme Immunoassays” by Tijssen, Elsevier, Amsterdam, 1990, and many others, or as illustrated by the examples below, the level of the AGE structure under investigation is measured.

[0036] In a preferred method according to the present invention, at least one proteolytic enzyme is used in the pretreatment of a CSF sample. Theoretically, many different proteolytic enzymes may be used. However, it is preferred to use highly active and cheap enzymes that are easy to handle. Examples of such enzymes are esperase and proteinase K. Use of proteinase K is most preferred.

[0037] Enzymatic pretreatment with proteolytic enzymes may be performed under various conditions with respect to, e.g., enzyme concentration, time, or temperature used. It is preferred to standardize this step in order to not introduce additional variation into the overall measurement. Preferably conditions are chosen which lead to maximum exposure of AGE epitopes. E.g., in the detection of AGE-CML (an AGE epitope comprising a structure derived from carboxymethyl lysine, CML), proteinase K is preferably used in a concentration from 0.3 to 5 mg/mL, or also preferred from 0.5 to 2 mg/mL, for preferably 0.5 to 24 hours, more preferred from 1 to 6 hours. It is also preferred to control for the temperature during enzymatic digestion. Temperature may, e.g., be set to 25° C. or 37° C. An elevated temperature such as 37° C. is preferred.

[0038] In order to better and more reliably assess the clinical meaning of an AGE value measured as described above, it is advantageous to compare the levels measured to levels known or obtained in parallel from one or more CSF control samples. It is therefore preferred to use the above measurements in a method to assess a neurodegenerative disease comprising comparing at least one AGE-CSF level in a sample to be assessed to an AGE-CSF level known or obtained from one or more CSF control samples. The term “AGE-CSF” is used to indicate that an AGE from a CSF sample is investigated. Of course, it is also possible to compare the AGE level measured in a clinical CSF sample to the average value or cutoff value as obtained for said AGE in a large group of CSF samples obtained from control individuals.

[0039] It is especially preferred to use a method of the present invention to assess a neurodegenerative disease selected from the group consisting of AD, CJD, and BSE. Most preferred, the inventive methods are used to assess AD in humans or CJD in cattle.

[0040] It is further preferred to use a method according to the present invention for the purpose of diagnosing AD. For a diagnostic application, usually the level of a marker of interest is carefully investigated in healthy individuals or for differential diagnosis, in clinical samples which are obtained from a group of patients with a clinically related disease but a different diagnosis. From the values measured in these control samples, the mean value as well as the standard deviation from this mean value are calculated. Based on these values, a cutoff value, which usually represents the mean plus or minus 2, or in some cases plus or minus 3, standard deviations is defined. Values measured in clinical samples which are above or below this cutoff value may be indicative or diagnostic of a certain disease. With such cutoff values established, the method according to the present invention is used to diagnose Alzheimer's disease. The use of the present invention to diagnose Alzheimer's disease or to differentiate AD from other related neurological disorders therefore represents a further preferred embodiment of the present invention.

[0041] As mentioned above, Alzheimer's disease is a rather slow progressing disorder. There are few, if any, means to assess progressing or amelioration of Alzheimer's disease, and biochemical markers so far are of limited use in that respect.

[0042] AGE levels in CSF may change during the course of the disease as well as may be dependent on usage of drugs. Such changes now can be followed and measured over time, i.e., monitored. It represents a further preferred embodiment to use a method according to the present invention to monitor AD.

[0043] Treatment measures for Alzheimer's disease are scarce, and only very few drugs are available to treat this important disease. Screening for new drugs is hampered by the fact that not many reliable biochemical markers are available. It is known that many AGE structures are similar, if not identical, in animal species as well as in humans.

[0044] Based on the improved methods of measurement of CSF AGE levels according to this invention, it is now possible to analyze and investigate CSF samples of laboratory animals or of patients in order to assess drug efficacy via changes in AGE-CSF levels. It is now feasible to screen for drugs which are affecting AGE-CSF levels, and it is further preferred to use a method according to the present invention to screen for drugs in the field of mental disorders, especially AD, which influence the level or affect the level of an AGE in CSF.

[0045] The examples, references, and figure are provided herein to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the procedures set forth without departing from the spirit of the invention.

EXAMPLES

Example 1

Pretreatment of Cerebrospinal Fluid

[0046] Buffer and Reagents

[0047] a) Buffer

[0048] 10 mM TRIS

[0049] 150 mM NaCl

[0050] 0,001% (w/v) N-methylisothiazolone

[0051] 0,01% (w/v) 2-chloroacetamide

[0052] 0,05% (v/v) TWEEN 20 (ICI America, Inc.)

[0053] pH 7.4

[0054] b) Proteinase K

[0055] Roche Molecular Biochemicals, Cat. No. 236 608. A solution of 13 mg/mL in the above buffer solution was used.

[0056] c) PMSF (phenylmethylsulfonyl Fluoride)

[0057] Roche Molecular Biochemicals, Cat. No. 236 608. PMSF, which was dissolved in ethanol to yield 100 mM/L, was diluted shortly before use 1+50 into the above buffer.

[0058] Digestion of CSF

[0059] To 60 μL of CSF, 5 μL of proteinase K solution (final concentration 1 mg/mL) was added. Reagents were mixed and incubated for 3 hours at 37° C. To inhibit the proteinase K activity, 65 μL of PMSF were added (final concentration, 1 mM), and the reagent mixture was further incubated for 30 to 60 minutes. Thereafter, the pretreated sample was ready for AGE-CML determination.

Example 2

Measurement of AGE-CML

[0060] Reagents and Solutions

[0061] a) Incubation Buffer

[0062] A commercially available incubation buffer (Fibrin Monomer Assay from Roche Diagnostics GmbH, Product No. 565 440) was used.

[0063] b) Washing Solution

[0064] 10 mM TRIS

[0065] 150 mM NaCl

[0066] 0.001% (w/v) N-methylisothiazolone

[0067] 0.01% (w/v) 2-chloroacetamide

[0068] 0.05% (v/v) TWEEN 20

[0069] pH 7.4

[0070] c) Substrate

[0071] The commercially available ENZYMUN assay substrate (Roche Diagnostics GmbH, Product No. 1 295 250) was used.

[0072] Reagents Used to Perform the Assay

[0073] a) Bi-BSA-AGE

[0074] Bovine serum albumin (BSA, Calbiochem, Order no. 12657) was subjected to advanced glycation in vitro. For this purpose, BSA-AGE was obtained after incubation of BSA (50 mg/mL in 50 mM potassium phosphate, 150 mM sodium chloride, 20 μM copper sulfate, pH 7.4) with D-glucose (0.5 M) for 3 weeks at 35° C. and then dialysed against 100 mM potassium phosphate buffer, 100 mM sodium chloride, pH 8.0.

[0075] For biotinylation, 50 mg BSA-AGE was incubated with 3.4 mg D-biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester for 90 minutes, and the reaction was stopped by addition of lysine monochloride to yield a final concentration of 10 mM. Finally the solution was dialysed overnight against phosphate buffered saline, 50 mM potassium phophate, 150 mM sodium chloride, pH 7.5. The biotinylated product was called “bi-BSA-AGE”. Bi-BSA-AGE was used as a 1 μg/mL solution in the above incubation buffer.

[0076] b) Standards

[0077] 6-(N-carboxymethylamino)caproic acid, disodium salt (a CML-type synthetic molecule with MW 233) was used as standard material. Solutions containing 0, 6.25, 12.5, 25, 50, and 100 ng/ml of standard were prepared by appropriate dilution of this material in incubation buffer.

[0078] c) Detection Reagent

[0079] Monoclonal antibody 4G9, which is known to react with carboxymethyl lysine structures, e.g, such as the above mentioned 6-(N-carboxymethylamino) caproic acid, was purified and conjugated with horseradish peroxidase (POD) according to standard procedures. The activity of such conjugate can be expressed in terms of peroxidase units. In the assay as described below, the monoclonal antibody-POD conjugate was diluted in incubation buffer to obtain roughly 90 mU/mL of POD activity.

[0080] Streptavidin-Coated Solid Phase

[0081] BSA-biotin was prepared according to EP 0 331 127, example 1, dissolved, and diluted to 10 μg/mL in 50 mM potassium phosphate, pH 7.4 and 300 μL, each, incubated in the wells of a microtiter plate (NUNC MAXISORP) for 5 hours at room temperature. The plate was emptied and refilled with 300 μL/well homogeneously crosslinked streptavidin according to example 2 of EP 0 331 127, 10 μg/mL in 50 mM potassium phosphate, pH 7.4. After 18 hours incubation at room temperature, the wells were emptied and refilled with a solution of 9 g/L sodium chloride, 10 g/L Dextran T40, and 3 g/L BSA (300 μL/well). After 30 minutes, the plates were completely emptied, dried during 3 hours at 25° C., 2% relative humidity, and sealed in an airtight bag.

[0082] Immunoassay for AGE-CML

[0083] The wells of a streptavidin-coated micro titer plate were incubated with bi-BSA-AGE (1 μg/mL). 100 μL/well were incubated at room temperature for 1 hour. Any unbound bi-BSA-AGE was removed by washing all wells with 3×300 μL washing solution.

[0084] Sample and peroxidase-conjugated monoclonal antibody were co-incubated. 50 μL of sample (either proteinase K pretreated CSF, untreated CSF, or standard material) was added to the wells immediately followed by 50 μL of conjugate solution as prepared above. This mixture was incubated for 1 hour at room temperature.

[0085] Non-bound reagents were removed by washing as described above.

[0086] Bound peroxidase was detected by standard substrate reaction. For this purpose, 100 μL of substrate solution were added per well and incubated for roughly 30 minutes at room temperature. Peroxidase activity was measured via the change in substrate at a wavelength of 405 nm.

[0087] During all incubation steps, the reaction mixture in the wells of the microtiter plate was gently moved using a plate shaker device.

[0088] Concentration of AGE-CML in CSF was extrapolated from the standard curve according to standard procedures.

[0089] Representative results as obtained with untreated CSF samples and samples pretreated as described above are summarized in Tables 1 and 2. Table 1 gives the individual data measured, and Table 2 is a statistical evaluation thereof.

1TABLE 1AGE-CML in CSF samplesCML without pretreatmentCML with pretreatmentDiagnosis[ng/ml][ng/ml]Control16.612.0Control7.211.9Control10.08.5Control33.819.1Control14.120.5Control8.86.0Control11.49.8Control11.03.7Control0.03.1Control12.09.6Control9.511.0Control11.113.5Control7.412.3AD3.622.9AD4.816.4AD10.319.1AD25.629.5AD30.454.5AD26.147.8AD13.814.2AD0.317.4AD10.238.1AD13.634.2AD9.222.6AD10.932.0

[0090]

2

TABLE 2

Mean values (MW) and standard deviation (SD)

Without pretreatment
With pretreatment

Controls, n = 13

MW
11.8
10.8

SD
7.7
5.1

AD, n = 12

MW
13.2
29.1

SD
9.5
12.8

Number	Date	Country	Kind
01102826.3	Feb 2001	EP
00127948.8	Dec 2000	EP

Detection of advanced glycation endproducts in a cerebrospinal fluid sample

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Priority Claims (2)

PCT Information