IN VITRO METHOD FOR THE DETERMINATION OF NEURODEGENERATIVE DISEASES

Abstract

The present invention concerns an in vitro method for the determination of a neurodegenerative disease wherein separately from each other the content of kynurenine and kynurenic acid in a body fluid is determined and the quotient of the content of kynurenine to the content of kynurenic acid is calculated.

Claims

1. An in vitro method for the determination of a neurodegenerative disease wherein separately from each other the content of kynurenine and kynurenic acid in a body fluid is determined and the quotient of the content of kynurenine to the content of kynurenic acid is calculated.

2.-14. (canceled)

Description

[0073] FIG. 1 shows a schematic overview of the kynurenine pathway, the major route of tryptophan degradation in higher eukaryotes. Enzymes are indicated in italics. The neurotoxic metabolites QUIN and 3-HK are shown as well as the neuroprotective metabolite KYNA (13).

[0074] FIG. 2 shows L-kynurenine and kynurenic acid concentrations in M for normal controls and differences between <60 and >60 m years of age. Measurements were made in serum and saliva as well. FIG. 2 shows that the mean values of kynurenine and kynurenic acid in cohorts of healthy volunteers are very similar regardless whether the volunteers have an age above or below 60 years.

[0075] FIG. 3 shows a comparison of kynurenine (measured in serum) in 2 groups: controls (n=194); patients with neurodegenerative disorders (n=42). There was a significant difference for kynurenine and kynurenine acid (p<0.001).

[0076] FIG. 4 shows values given for the correlation between serum and saliva in kynurenine and kynurenic acid measurement.

[0077] FIG. 5 shows the difference of the quotient Kyn/KynA between normal controls (n=194) and patients (n=42) with neurodegenerative (ND) disorders (p<0.0000071) when measured in saliva.

[0078] FIG. 6 shows a comparison of the concentration of kynurenine vs. kynurenic acid for normal controls (n=181). The ratio of both is similar in serum and saliva.

[0079] FIG. 7 shows the results for kynurenine values in serum and saliva in the normal control group. The results are shown for the whole control group and for female (n=143) and male (n=159) members of the group, respectively.

[0080] FIG. 8 shows a comparison of the kynurenine content in serum and saliva for normal controls and for patients (n=49) with long-standing neurodegenerative disease. The difference for serum and for saliva is significant (p<0.001). Kynurenine is significantly increased in the patient group.

[0081] FIG. 9 shows a comparison of the kynurenic acid content in serum and saliva for normal controls and patients as in FIG. 8. Higher values of kynurenic acid can be observed for the normal control group while the concentration is decreased in patients with ND. The difference between the groups is significant for the value in serum as well as for the value in saliva.

[0082] FIG. 10 shows the values of kynurenine for male and female members of the normal control group. The median concentration in serum is 2.630.64 M (female group) and 2.790.64 M (male group); the corresponding concentrations in saliva are 0.790.37 M and 0.880.34 M. No statistically significant difference was observed in these values for female or male persons.

[0083] FIG. 11 A shows the values for kynurenine in serum and saliva for the normal control group (n=302). The median concentration in serum was 2.690.6 M, in saliva 0.820.28 M.

[0084] FIG. 11 B shows the correlation of the kynurenine serum values vs. the kynurenine saliva values. A correlation of r.sup.2=0.90 was determined which remained the same also for higher values. This is a clear indication that the saliva values are generally useful for diagnostic purposes.

[0085] FIG. 12 shows serum and saliva concentrations for patients and normal control persons (NP). The results show that in the samples of patients the ratio of kynurenine to kynurenic acid has changed to the disadvantage of the neuroprotective substance kynurenic acid.

[0086] FIG. 13 shows a comparison of the determination of kynurenic acid via a fluorescence based method compared to a commercially available ELISA test.

[0087] The present invention is described in more detail in the Figures and the following Examples.

Example 1

[0088] Forty-two patients with cerebral dementia (mean age 71+5.3 years, mean MMS-score 22) were enrolled in a comparative study with normal controls (n=194; mean age 48.8 years, range 16-88 years). Aim of our study was to detect changes in the tryptophan metabolism in patients with cerebral dementia, by estimating either kynurenine, kynurenic acid and ratio of kyn/kynA in plasma and in saliva. There was no age related difference between a group I of normal controls (n=93, age>60, mean age 71.3 years, range 60-88) and group II (n=101, age<60, mean age 38.8, range 16-60; FIG. 2). This is demonstrating that the disease must not necessarily be age related in general. The neurodegenerative diseases may be caused by different reasons whereby, however, the frequency of neurodegenerative diseases increases statistically with increasing age.

Example 2

[0089] Kynurenine was significantly higher and kynurenic acid lower and ratio was different in patients with neurodegenerative disorders. This could be demonstrated in serum as well as in saliva. The measured values are shown in FIG. 3.

[0090] There was a correlation between the values of kynurenine and kynurenic acid in serum compared to saliva (saliva 1:3.5 in serum for kynurenine and 1:3.2 in saliva for kynurenic acid in normal controls, FIG. 4).

Example 3

[0091] Patients with neurodegenerative disease showed a total different pattern: mean values for kynurenine in serum as well as in saliva were significant higher (4.800.6 M for serum and 1.34+0.3 M for saliva) whereas values for kynurenic acid were significant lower (1.580.3 in serum and 1.30.2 in saliva, FIG. 3). This is in correspondence with the theory of the pathophysiology of the disease: the neuro-protective part (kynurenic acid) is downregulated and the inflammatory part (kynurenine) is upregulated.

Example 4

[0092] Concerning the measurement of kynurenine and kynurenic acid in serum and saliva we could demonstrate the correlation for serum and saliva. The small numeric difference between both values is related to the different method of measurement.

[0093] We could demonstrate that measurement of kynurenine and kynurenic acid is possible in serum as well as in saliva. There is a relationship between the values in serum compared to the values in saliva.

[0094] Compared to the data evaluated in normal controls, data in patients showed significant different pattern and could be easy identified.

[0095] In total, already in this small group of patients it could be demonstrated, that kynurenine/kynurenic acid measurement is a tool to identify cerebral disorders as well as to monitor them. The measured kynurenine/kynurenic acid quotient is a clear indicator for neurodegenerative diseases if the quotient is 1.0 or higher.

Example 5

[0096] The collective of normal control persons is comprised of blood donors. These were healthy persons who consented to the use of serum and saliva samples for the purposes of the present research and examples. Serum was obtained from 326 persons, 302 of which were included in the determination of kynurenine. For 12 persons, no corresponding saliva samples were available or could not be used for the test. For 12 persons, test values in a pathological range were determined, probably based on inflammations (kynurenine above 4.2 in serum). Such persons could not be included in the group of healthy controls.

[0097] The obtained values corresponded well with values published in the prior art. Saliva samples generally had lower levels of kynurenine and kynurenic acid.

[0098] From the 302 persons of the normal control group, 143 were female and 159 male. The average age was 47.6 (18 to 75 years old) for the female group and 47.1 (18 to 75 years old) for the male group. The age was determined to not be statistically significant. Results are shown in FIGS. 7 and 10.

[0099] In a second series of tests, kynurenine and kynurenic acid were determined in serum and saliva. For 181 samples a determination for both kynurenine and kynurenic acid could be performed in saliva as well as in serum. The results are shown in FIG. 6.

[0100] Further test results are shown in the remaining Figures attached herewith.

Example 6

[0101] Kynurenic acid concentration was determined by a fluorescence based test as described in the following. The test result was compared to the results obtained using a commercially available ELISA test kit (KYNA ELISA human, Cloud-Clone Corporation, 11271 Richmond Avenue Suite H104, Houston Tex. 77082, USA, Lot: L 150525449. The assay employs the competitive inhibition enzyme immunoassay technique and was performed in the manner as described in the instruction manual).

[0102] The fluorescence based test was performed using FluoStar BMG and the following conditions:

Reagents:

[0103] Perchloric acid (HClO.sub.4), 60%=10 M, and 6M (1:1.67 dilution of 10 M acid; e.g. for 10 ml: 4 ml H.sub.2O+6 ml HClO.sub.4 10M)

Standards:

[0104] Kynurenic acid (MW: 189.17, Sigma K3375) standard curve from 10 M to 0.156 M: KynA (1.89 mg) dissolved in 500 l DMSO, then addition of 5 ml H.sub.2O+4.5 ml HClO.sub.4 6 M: 1000 M

[0105] Dilution 1:50 with HClO.sub.4 6M (.fwdarw.20 M) and further dilution 1:2 using 6M HClO.sub.4 up to 0.16 M

Sample Preparation:

[0106] To 30 l HClO.sub.4 in Eppendorf Tubes 300 l serum/saliva are added, centrifuged (10/15000 g/10 C.) and 200 l of the clear supernatant are used as sample

Test Batches (in White MT-Plate):

[0107] 200 l of standard/sample are filled into corresponding wells. Fluorescence is measured at Ex=365 nm and Em=460 nm, Gain is adjusted to 65-85 (as assessed from the graphic plot)

Evaluation:

[0108] The concentration of KynA is determined based on the calibration line taking into account a dilution factor of 1.1 resulting from the sample preparation

[0109] The result of this comparison is shown in FIG. 13. The fluorescence based test provided more accurate results than the ELISA test kit.

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