Resumen
[0001] The present invention relates to a Raman spectroscopy method for determining protein structure associated with global cognitive deterioration in Alzheimer's disease from the Raman spectroscopic analysis of a human blood sample, preferably of a human blood plasma sample, comprising obtaining at least one spectral value of at least one of the Raman spectrum regions comprised between 1600-1700 cm<−1>, between 910-980 cm<−1>, between 730-760 cm<−1 >and/or between 390-450 cm<−1>, and where said spectral value allows obtaining an indication of the presence or absence of protein structure associated with a condition of Alzheimer's disease. The present invention also relates to a method for the infrared spectroscopic analysis of a blood sample, by means of additionally obtaining at least one spectral value of at least one of the infrared spectrum regions comprised between 1600 and 1700 cm<−1>, and/or between 1000 and 1150 cm<−1 >and/or between 1140 and 1190 cm<−1>. Optionally, the Raman spectroscopy method further comprises the infrared spectroscopic analysis of the same blood sample. The invention also relates to a diagnostic method for diagnosing Alzheimer's disease comprising measuring a Raman spectrum and/or an infrared spectrum of a plasma sample.
Reivindicaciones
1. A vibrational spectroscopy method for determining protein structure associated with global cognitive deterioration in Alzheimer's disease in a blood sample comprising the following steps: a) recording a Raman spectrum of a previously obtained human blood sample; b) obtaining a Raman spectral value of at least one of the following Raman spectrum regions: R.1. region comprised between 1600 and 1700 cm<-1> ; R.2. region comprised between 910 and 980 cm<-1> ; R.3. region comprised between 730 and 760 cm<-1> ; R.4. region comprised between 390 and 450 cm<-1> ; where said spectral value is selected from a value of an interband Raman intensity ratio, a value of the area of said spectral region and/or a frequency value. c) classifying the blood sample in one of the following classes: I. blood sample containing the protein structure associated with non-cognitive deterioration in Alzheimer's disease; II. blood sample containing the protein structure associated with cognitive deterioration in Alzheimer's disease; by comparison of the Raman spectral value obtained in step b with a reference spectral value which allows distinguishing between class I or II, or by multivariate analysis comparison of the Raman spectral value obtained in step b with class I and class II reference Raman spectral values. 2. A vibrational spectroscopy method for determining protein structure associated with global cognitive deterioration in Alzheimer's disease in a blood sample comprising the following steps: a) recording an infrared spectrum of a previously obtained human blood sample; b) obtaining an infrared spectral value of at least one of the following infrared spectrum regions: IR.1. region comprised between 1600 and 1700 cm<-1> ; IR.2. region comprised between 1000 and 1150 cm<-1> ; where said infrared spectral value is selected from a value of the area of said spectral region and/or a percentage value of the interband areas of said spectral region; c) classifying the blood sample in one of the following classes: I. blood sample containing the protein structure associated with non-cognitive deterioration in Alzheimer's disease; II. blood sample containing the protein structure associated with cognitive deterioration in Alzheimer's disease; by comparison of the infrared spectral value obtained in step b with a reference infrared value which allows distinguishing between class I or II, or by multivariate analysis comparison of the infrared value obtained in step b with class I or II reference infrared spectral values. 3. The method according to claim 1, additionally comprising the steps of a method according to claim 2 applied to a sample from the same subject. 4. The method according to claim 3, wherein the sample is classified by multivariate analysis comparison of the spectral values obtained in step b with class I and class II reference Raman values and with class I and class II reference infrared spectral values. 5. The method according to any one of claims 1 to 4, where the blood sample is a blood plasma fraction sample. 6. The method according to any one of claims 1 and 3-5, where the spectral value of the Raman spectrum region defined in R.1 comprises the ratio of Raman intensities obtained around 1671 cm<-1> and 1658 cm<-1> , the spectral value of the Raman spectrum region defined in R.4 comprises the ratio of Raman intensities obtained around 409 cm<-1> and 423 cm<-1> , the spectral value of the Raman spectrum region defined in R.2 comprises the area of the Raman spectral profile comprised between 910 and 980 cm<-1> , and/or the spectral value of the Raman spectrum region defined in R.3 is selected from the group consisting of the ratio of Raman intensities obtained at the maximum of the band around 758 cm<-1> and at the maximum of the band located in the 740-750 cm<-1> region or of the frequency of the band comprised in said Raman spectrum region between 740 and 750 cm<-1> . 7. The method according to any one of claims 2-5, where the spectral value of the infrared spectrum region defined in IR.1 comprises the percentage of the area of the spectral profile between 1640 and 1623 cm<-1> with respect to the area of the amide I region between 1670-1623 cm<-1> expressed in second derivatives, and/or the spectral value of the infrared spectrum region defined in IR.2 comprises the percentage of the area of the spectral profile between 1150 and 1000 cm<-1> with respect to the area in the 3010-2800 cm<-1> region. 8. The method according to any one of claims 1, 3-6, where step b comprises obtaining the spectral values of the Raman spectrum regions defined in R.1, R.2, R.3 and R.4; and where the classification of step c comprises multivariate analysis comparison of said spectral values with a class I reference Raman spectral value and with a class II reference Raman spectral value, where said reference spectral values are comparable to those obtained in step b. 9. The method according to any one of claims 3-4 or 5-8, where step b comprises obtaining the spectral values of the Raman spectrum regions defined in R.1, R.2, R.3 and R.4; obtaining the spectral values of the infrared spectrum regions defined in IR.1 and IR.2; and step c comprises classifying said spectral values by means of multivariate analysis comparison with a class I reference Raman spectral value, with a class II reference Raman spectral value, with a class I reference infrared spectral value and with a class II reference infrared spectral value, where said reference spectral values are comparable to those obtained in steps b. 10. The method according to any one of claims 8 or 9, where the class II reference value comprises at least one reference spectral value of a blood sample of the following sub-classes: II-a. blood sample associated with mild cognitive deterioration in Alzheimer's disease, with a value of GDS-3 on Reisberg's global cognitive deterioration scale. II-b. blood sample associated with mild cognitive deterioration in Alzheimer's disease, with a value of GDS-4 on Reisberg's global cognitive deterioration scale. II-c. blood sample associated with mild cognitive deterioration in Alzheimer's disease, with a value of GDS-5 on Reisberg's global cognitive deterioration scale. II-d. blood sample associated with mild cognitive deterioration in Alzheimer's disease, with a value of GDS-6 on Reisberg's global cognitive deterioration scale. II-e. blood sample associated with mild cognitive deterioration in Alzheimer's disease, with a value of GDS-7 on Reisberg's global cognitive deterioration scale. 11. The method according to any one of claims 8 or 9, where the reference spectral value is obtained under the same conditions described in steps a-b according to claims 1 or 2, from reference blood samples as they are defined in class I and in class II, or from reference blood samples as they are defined in class I, in sub-class II-a, in sub-class II-b, in sub-class II-c, in sub-class II-d and in sub-class II-e. 12. The method according to any one of claims 8 to 11, where the multivariate analysis is a discriminant analysis using the following as independent variables: - the spectral value or values obtained in step b, and the reference spectral values; and the following as a grouping variable: - a variable indicative of classes I and II of the reference spectral values. 13. A diagnostic method for diagnosing Alzheimer's disease in a subject comprising the steps of: a) measuring a Raman spectrum of a plasma sample from said subject obtaining at least one Raman band selected from the group consisting of a Raman band comprising vibrations specific to β-protein structure, a Raman band of amyloid peptides comprising vibrations specific to angular deformation of the peptide bond, a Raman band comprising vibrations specific to α-helix protein structure and a Raman band comprising vibrations specific to tertiary protein structure with tryptophan residues and b) comparing the Raman spectrum obtained in step a) with the spectrum of a reference sample wherein a Raman spectrum variation indicative of an increase in intensities specific to β-protein structure with respect to the reference spectrum, a Raman spectrum variation indicative of an increase in intensities specific to angular deformation of the peptide bond with respect to the reference spectrum, a Raman spectrum variation indicative of a reduction in intensities specific to α-helix protein structure with respect to the reference spectrum and/or a Raman spectrum variation indicative of an increase in vibrations specific to tryptophan residues in a tertiary protein structure with respect to the reference spectrum, is indicative of the patient having Alzheimer's disease. 14. The method according to claim 13, wherein the Raman band comprising vibrations specific to beta sheet protein is the band approximating 1671 cm<-1> , the Raman band comprising vibrations specific to angular deformation of the peptide bond is the band approximating 409 cm<-1> , the Raman band comprising vibrations specific to α-helix protein structure is defined by the spectral profile between 980 and 910 cm<-1> and/or the Raman band comprising vibrations specific to tryptophan residues in a tertiary protein structure is the band approximating the 740-750 cm<-1> region. 15. The method according to any one of claims 13 to 14, wherein - the Raman spectrum variation indicative of an increase in intensities specific to β-protein structure with respect to the reference spectrum is an increase in the ratio between the intensities located around 1671 cm<-1> and 1658 cm<-1> , - the Raman spectrum variation indicative of an increase in intensities specific to tryptophan residues in a tertiary protein structure with respect to the reference spectrum is an increase in the ratio between the intensity of the maximum of the band located around 758 cm<-1> and the intensity of the band located around 743 cm<-1> or an increase in the frequency of the band in the 740-750 cm<-1> range, - the Raman spectrum variation indicative of a reduction in intensities specific to α-helix protein structure with respect to the reference spectrum is a reduction in the area of the spectral profile between 980-910 cm<-1> and/or - the Raman spectrum variation indicative of an increase in vibrations specific to angular deformation of the peptide bond with respect to the reference spectrum is an increase in the ratio between the intensities of the bands located around 409 cm<-1> and 423 cm<-1> . 16. A diagnostic method for diagnosing Alzheimer's disease in a subject comprising the steps of: a) measuring an IR spectrum of a plasma sample from said subject obtaining at least one band specific to the presence of β-protein structure and/or in at least one specific band indicative of the presence of compounds generated during oxidative stress and b) comparing the IR spectrum obtained in step (a) with the spectrum of a reference sample wherein an IR spectrum variation indicative of an increase in β-protein structure with respect to the reference spectrum and/or an IR spectrum variation indicative of an increase in the concentration of compounds generated in the sample during oxidative stress with respect to the reference spectrum is indicative of the patient having Alzheimer's disease. 17. The method according to claim 16, wherein the band specific to the presence of β-protein structure is the band approximating 1640-1623 cm<-1> and/or the band specific to the presence of compounds generated during oxidative stress is the band between 1150 cm<-1> and 1000 cm<-1> . 18. The method according to any of claims 16 or 17, wherein the IR spectrum variation indicative of an increase in β-protein structure with respect to the reference spectrum is an increase in the spectral profile area in the region between 1640-1623 cm<-1> and/or the IR spectrum variation indicative of an increase in the concentration of compounds generated in the sample during oxidative stress with respect to the reference spectrum is an increase in the spectral profile area in the region between 1150 cm<-1> and 1000 cm<-1> . 19. The method according to claim 18, wherein said increase in the spectral profile area in the region between 1640-1623 cm<-1> is determined as a percentage of the area in the region between 1640-1620 cm<-1> with respect to the area of the amide I region between 1670-1623 cm<-1> expressed in second derivatives and/or wherein said increase in the concentration of compounds generated during oxidative stress is determined as a percentage of the area in the region between 1150 cm<-1> and 1000 cm<-1> of the spectrum with respect to the area in the region between 3010 cm<-1> and 2800 cm<-1> . 20. The method according to any one of claims 13 to 15, additionally comprising the steps of a method according to any of claims 16 to 19 applied to a sample from the same subject. 21. The method according to claim 20, wherein the method according to any one of claims 13 to 15 comprises measuring the Raman spectrum obtaining a Raman band comprising vibrations specific to β-protein structure, a Raman band comprising vibrations specific to angular deformation of the peptide bond, a Raman band comprising vibrations specific to α-helix protein structure and a Raman band comprising vibrations specific to tertiary protein structure with tryptophan residues. and wherein the method according to any one of claims 16 to 19 comprises measuring an IR spectrum obtaining a band specific to the presence of β-protein structure and a specific band indicative of the presence of compounds generated during oxidative stress wherein a Raman spectrum variation indicative of an increase in intensities specific to β-protein structure with respect to the reference spectrum, a Raman spectrum variation indicative of an increase in intensities specific to angular deformation of the peptide bond with respect to the reference spectrum, a Raman spectrum variation indicative of a reduction in intensities specific to α-helix protein structure with respect to the reference spectrum, a Raman spectrum variation indicative of a reduction in intensities specific to tryptophan residues in a protein structure with respect to the reference spectrum, an IR spectrum variation indicative of an increase in beta sheet protein structure with respect to the reference spectrum, and an IR spectrum variation indicative of an increase in the concentration of compounds generated in the sample during oxidative stress with respect to the reference spectrum is indicative of the patient having Alzheimer's disease.
