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    • 专利摘要:
    Method for diagnosing Parkinson's disease based on detecting very low serotonin levels in patients' platelets. Initial studies have not found normal serotonin levels in patients, whether or not they were treated with L-DOPA. Similarly, the platelets of patients have impaired their ability to accumulate serotonin. This condition seems to be due to a deficient capacity of the secretory vesicles of the platelets of the patients to accumulate biogenic amines. Given that the biological mechanisms underlying this problem are common in platelets and in dopaminergic neurons, this could explain (given the toxicity of dopamine and its metabolites when they are free in the cytosol) the progressive death of these neurons in Parkinson's disease. Since the affectation of the uptake mechanisms is due to problems in the proteins that mediate their accumulation, this functional failure probably precedes the appearance of the symptoms of the disease by several years. The very early detection of problems in the management of serotonin in platelets can detect possible patients early enough to establish specific measures of neuroprotection. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2752675A1
    申请号:ES201800221
    申请日:2018-10-04
    公开日:2020-04-06
    发明作者:Jurado Ricardo Borges；Escudero Pablo Montenegro；Martinez Maria Dolores Villar
    申请人:Universidad de La Laguna；
    IPC主号:
    专利说明:

    [0001] Platelet functionality for very early diagnosis of Parkinson's disease.
    [0002] TECHNICAL SECTOR
    [0003] Medicine
    [0004] Clinical diagnostic tools.
    [0005] BACKGROUND OF THE INVENTION
    [0006] Parkinson's disease (PD) is a degenerative disorder that involves several neuronal groups in the brain, especially dopaminergic neurons of the nigro-striatal tract. Although numerous diagnostic laboratory tests and imaging techniques have been proposed, they have not demonstrated reliability and diagnosis based on clinical symptoms remains the only recognized method. However, PD is only accompanied by symptoms when neuronal loss reaches 70-80% of nigrostriatal neurons. At that time little can be done by the patient other than symptomatic and palliative treatment. The main drug used is L-DOPA, which is the natural precursor of dopamine, but it will only work during the years when there are still a sufficient number of dopaminergic neurons.
    [0007] Having a diagnostic tool that is capable of detecting PD years, or tens of years, before the onset of clinical symptoms is a primary objective, since it would allow early neuroprotective therapies to be initiated to delay (or prevent) its onset.
    [0008] Dopamine must be stored within the secretory vesicles (VS) in order to be released by a process called exocytosis and also to avoid its degradation in the cytosol (cytoplasm) of neurons. One of the theories about the origin of PD is that part of the dopamine remains in the cytosol of the neurons and is not captured by the VS. The oxidation of dopamine and its main metabolite (DOPAC) generates very toxic oxygen radicals, which end up killing dopaminergic neurons (and to a lesser extent others).
    [0009] The VS of any secretory cell have mechanisms for the concentration of neurotransmitters that are common. Thus the VS of neurons, neuroendocrine cells, and some peripheral cells use similar transporters to concentrate biogenic amines (dopamine, norepinephrine, adrenaline, histamine, or serotonin). Interestingly, platelets and dopaminergic neurons share these mechanisms (acidification: with the V-ATPase; ATP transport: with the vesicular nucleotide transporter -VNUT-; amines: with the vesicular transporter of amines, VMAT-2). It is not easy to carry out human studies with neurons, but it is very easy to work with platelets. PURPOSE OF THE INVENTION
    [0010] Provide a laboratory test that allows a very early diagnosis of Parkinson's disease to be made before the patient presents clinical symptoms of the disease.
    [0011] EXPLANATION OF THE INVENTION
    [0012] Since practically all serotonin (also called 5-hydroxytryptamine or “5-HT”) is found in VS, if the mechanisms are common, platelet levels of 5-HT in Parkinson's patients should be short. Also its ability to capture dopamine, also to retain it, also its release would be reduced. These problems in 5-HT accumulation will be affected long before diagnosis. Maybe even from the moment of birth.
    [0013] We have:
    [0014] 1- Implemented and standardized the technique of isolation, purification and quantification of human platelets (Figure 1)
    [0015] 2- Optimized the conservation in optimal functional state for several days of human platelets.
    [0016] 3- We have implemented analytical methods to measure the 5-HT of platelets seeded in multi-well plates by means of high pressure liquid chromatography in order to create a diagnostic "kit" for general use in the clinical laboratory (Figure 2).
    [0017] 4- Developed the amperometry technique to measure the release of 5-HT from both populations and single platelets (Figure 2).
    [0018] Since practically all of the blood serotonin (also called 5-hydroxytryptamine or "5-HT") is found in the VS of platelets, if the accumulation mechanisms are common, the platelet levels of 5-HT in patients with Parkinson's disease should be low, also their ability to take up dopamine, also to retain it and also its release would be reduced, these problems in the accumulation of 5-HT will be affected long before the diagnosis of PD.
    [0019] Maybe even from the moment of birth.
    [0020] We have:
    [0021] 1- The technique of isolation, purification and quantification of human platelets specifically designed to preserve its secretory function has been implemented and standardized (Figure 1). 2- Optimized the preservation of human platelets in optimal functional state for several days.
    [0022] 3- We have implemented analytical methods to measure the 5-HT of platelets seeded in multi-well plates by means of high pressure liquid chromatography in order to create a diagnostic "kit" for general use in the clinical laboratory (Figure 2).
    [0023] 4- Developed the amperometry technique to measure the release of 5-HT from both populations and single platelets (Figure 2).
    [0024] Results:
    [0025] - 5-HT levels are extremely reduced in patients with PD, both in untreated and in those treated with L-DOPA. The numbers are so different that (in the measured series) there is no overlap between the serotonin values of control patients (presumably healthy subjects) and Parkinson's patients not yet treated with L-DOPA (Figure 3).
    [0026] - Platelets in patients with PD have reduced uptake of 5-HT (Figure 3).
    [0027] - Platelets from PD patients release less 5-HT than control subjects in response to secretagogue stimuli (Figure 4).
    [0028] BRIEF DESCRIPTION OF THE DRAWINGS
    [0029] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is included as an integral part of said description, where, by way of illustration and not limitation, the following:
    [0030] Figure 1. Optimized protocol for the extraction of human platelets and their subsequent maintenance under optimal preservation conditions.
    [0031] The steps for isolation and purification are as follows:
    [0032] 1. Draw 2 9 mL tubes of blood by venipuncture. Vacutainer® tubes with EDTA. Move twice for investment and let rest for about 15-30 min. Room temperature. 2. Centrifuge at 200xg 20 min. Without brake. Temperature 20 ° C.
    [0033] 3. Collect 2/3 of the highest part of the supernatant (to avoid leukocytes) with a plastic Pasteur pipette. Transfer to a 15 mL Falcon.
    [0034] 4. Mix 1: 1 with HEP Buffer. Add prostaglandin E1 (final 1pM concentration).
    [0035] 5. Centrifuge at 100 x g 15 min. Without brake. Temperature 20 ° C.
    [0036] 6. Collect supernatant with a plastic Pasteur pipette without blowing bubbles and transfer it to a 15 mL Falcon
    [0037] 7. Centrifuge at 800 x g 20 min. Without brake. Temperature 20 ° C.
    [0038] 8. Discard the supernatant.
    [0039] 9. Wash twice without resuspending carefully in 1 mL of Buffer citrate.
    [0040] 10. Carefully resuspend in SSP ++ medium. Count cells by Neubauer chamber or optimized turbidimetric analysis.
    [0041] Figure 2. - Optimized general procedure for the quantification of serotonin in human platelets.
    [0042] 1. Arrange the platelet culture in an adapted conical bottom 96-well plate, according to the main figure.
    [0043] 2. Add the corresponding serotonin solutions (5-HT), according to the main figure. Incubate 2h at 20-25 ° C in the dark and gentle shaking.
    [0044] 3. Cool to 0-4 ° C and centrifuge at 3200g 10min. Temperature 4 ° C.
    [0045] 4. Discard the supernatant. Wash without resuspending carefully in washing medium.
    [0046] Temperature 4 ° C.
    [0047] 5. Centrifuge at 3200g 10min. Temperature 4 ° C.
    [0048] 6. Discard the supernatant. Gently resuspend the samples in lysis buffer, avoiding bubbles. Temperature 4 ° C.
    [0049] 7. Centrifuge at 3200g 5min. Temperature 4 ° C.
    [0050] Protein analysis.
    [0051] 8. Take 25uL from the sample wells and bring them to the protein analysis plate, carefully avoiding bubbles. Add BSA solutions to the indicated wells. Temperature 4 ° C.
    [0052] 9. Mix developer solutions A and B and carefully add to each well (sample and BSA). Temperature 4 ° C. Shake lightly and incubate at 37 ° C and in the dark 30-45min. 10. Determine the protein concentration using a plate reader (540-590nm).
    [0053] Serotonin analysis (5-HT analysis).
    [0054] 11. Add the remaining 25uL of sample on the plate to the HPLC-P solution carefully avoiding bubbles. Freeze at -20 ° C 20-30min.
    [0055] 12. Thaw at 0-4 ° C and centrifuge at 3200g 5min Temperature 4 ° C.
    [0056] 13. Determine the serotonin concentration using HPLC.
    [0057] Figure 3.- Serotonin in human platelets.
    [0058] Basal levels and after incubation with serotonin. The data are normalized by protein quantity, in order to correct the possible error derived from having analyzed samples with different numbers of platelets. Starting from the basal levels (B), the platelets were incubated, at the concentrations indicated in abscissa, for two hours. The serotonin concentration reached in control subjects (black line) and PD patients (gray line) is shown for each concentration. The columns show in greater detail the basal serotonin levels and after incubation with 10 pM (10-5 M) of 10 control subjects (black column) and 5 PD patients (gray column). Data show means ± standard error. * p <0.05 t-Student.
    [0059] Figure 4. Serotonin release is reduced in platelets from subjects with Parkinson's disease.
    [0060] Panel A. Schematic of the system used for the quantification of serotonin by populations of «10 million platelets that are packaged in an online perfusion chamber with an electrochemical detector with a potential set at 700 mV. On the right is a typical record of total serotonin release.
    [0061] Panel B. Amperometry system with micro electrodes that allows the detection of the release of a single secretory granule from a human platelet. On the right is a typical 'spike'. A platelet releases its 5-12 granules in very few seconds. The sizes of the spikes (area under the curve) will indicate the total amount of serotonin and, being an all-or-nothing phenomenon, the serotonin content of each one. Both technical approaches show reduced serotonin content and release.
    [0062] PREFERRED EMBODIMENT OF THE INVENTION
    [0063] Figures 2 and 3 show the platelet isolation and maintenance protocol. The compositions of the solutions to be used are set out below:
    [0064] HEP Buffer (140.0mM NaCI, 2.7mM KCI, 5.0mM EGTA, 3.8mM HEPES, pH 7.4 NaOH).
    [0065] Citrate buffer (150.0mM NaCI, 1.0mM EDTA, 50.0mM D-Glucose, Citrate-Na + 10.0mM, pH 7.4 NaOH).
    [0066] SSP ++ (NaCl 69.3mM, KCI 5.0mM, MgCh 1.5mM, Na2HPO4 / NaH2PO4 28.2mM, Citrate-Na + 10.8mM, Acetate-Na + 32.5mM, Penicillin - Streptomycin 100u / mL, pH 7.2 NaOH).
    [0067] Serotonin solutions (NaCI 69.3mM, KCI 5.0mM, MgCI2 1.5mM, Na2HPO4 / NaH2PO4 28.2mM, Citrate-Na + 10.8mM, Acetate-Na + 32.5mM, D-Glucose 5.0mM, Ascorbic 200uM, 5-HT 0-1000uM, pH 7.2 NaOH).
    [0068] Washing medium (NaCl 69.3mM, KCI 5.0mM, MgCI2 1.5mM, Na2HPO4 / NaH2PO4 28.2mM, Citrate-Na + 10.8mM, Acetate-Na + 32.5mM, D-Glucose 5.0mM, pH 7.2 NaOH).
    [0069] Tisis buffer (50mM Tris-HCI, 5mM EDTA, 150mM NaCl, 1% TritonX, pH 7.4 NaOH).
    [0070] BSA solutions (50mM Tris-HCI, 5mM EDTA, 150mM NaCI, 1% TritonX, BSA 0-2mg pH 7.4 NaOH).
    [0071] Solution A (Acidobicinconinico).
    [0072] Solution B (CuSO44% g / L).
    [0073] HPLC-P solution. (PCA 0.45N, IS 225nM).
    权利要求:
    Claims (13)
    [1]
    1. Diagnostic method characterized by the use of basal serotonin concentration in human platelets as a predictive index for the diagnosis of Parkinson's disease before the first symptoms of the disease appear
    2. Diagnostic method according to claim 1, where the quantification of serotonin is determined by HPLC in 96-well plates by means of enzyme-linked immunosorbent assays.
    3. Diagnostic method according to claim 1, where the quantification of serotonin is determined by amperometry in populations or by amperometry with microelectrodes. 4. Diagnostic method according to claim 1, where the platelet extraction and purification protocol comprises the following steps:
    1. Draw 2 9 mL tubes of blood by venipuncture. Vacutainer® tubes with EDTA.
    Move twice for investment and let rest for about 15-30 min. Room temperature.
    2. Centrifuge at 200xg 20 min. Without brake. Temperature 20 ° C.
    3. Collect 2/3 of the highest part of the supernatant (to avoid leukocytes) with a plastic Pasteur pipette. Transfer to a 15 mL Falcon
    4. Mix 1: 1 with HEP Buffer. Add prostaglandin E1 (final 1pM concentration).
    5. Centrifuge at 100 x g 15 min. Without brake. Temperature 20 ° C.
    6. Collect supernatant with a plastic Pasteur pipette without blowing bubbles and transfer it to a 15 mL Falcon.
    7. Centrifuge at 800 x g 20 min. Without brake. Temperature 20 ° C.
    8. Discard the supernatant.
    9. Wash twice without resuspending carefully in 1 mL of Buffer citrate.
    10. Carefully resuspend in SSP ++ medium. Count cells by Neubauer chamber or optimized turbidimetric analysis.
    5. Diagnostic method according to claim 1 where the protocol for the quantification of serotonin comprises the following steps:
    1. Arrange the platelet culture in an adapted conical bottom 96-well plate, according to the main figure.
    [2]
    2. Add the corresponding serotonin solutions (5-HT), according to the main figure. Incubate 2h at 20-25 ° C in the dark and gentle shaking.
    [3]
    3. Cool to 0-4 ° C and centrifuge at 3200g 10min. Temperature 4 ° C.
    [4]
    4. Discard the supernatant. Wash without resuspending carefully in washing medium.
    Temperature 4 ° C.
    [5]
    5. Centrifuge at 3200g 10min. Temperature 4 ° C.
    [6]
    6. Discard the supernatant. Gently resuspend the samples in lysis buffer, avoiding bubbles. Temperature 4 ° C.
    [7]
    7. Centrifuge at 3200g 5min. Temperature 4 ° C.
    [8]
    8. Take 25uL from the sample wells and place them on the protein analysis plate, carefully avoiding bubbles. Add BSA solutions to the indicated wells. Temperature 4 ° C.
    [9]
    9. Mix developer solutions A and B and carefully add to each well (sample and BSA). Temperature 4 ° C. Shake lightly and incubate at 37 ° C and in the dark 30-45min.
    [10]
    10. Determine the protein concentration using a plate reader (540-590nm, Protein analysis).
    [11]
    11. Add HPLC-P solution to the remaining 25uL of sample on the plate.
    carefully avoiding bubbles. Freeze at -20 ° C 20-30min.
    [12]
    12. Thaw at 0-4 ° C and centrifuge at 3200g 5min. Temperature 4 ° C.
    [13]
    13. Determine the serotonin concentration by HPLC (5-HT analysis).
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