[001 ] The invention concerns application of hydrogen peroxide and 17f3-estradiol and its metabolites as biomarkers in a method of diagnosing neurodegenerative diseases in vitro, the Parkinson’s disease in particular. The invention also concerns a method of diagnosing neurodegenerative diseases, the Parkinson’s disease in particular.

[002] Parkinson’s disease (PD) is, after Alzheimer’s disease (AD), the second most frequently diagnosed neurodegenerative disease with the occurrence rate of approximately 0.3% of the entire population and approximately 1 % in the group of persons aged above 60.

[003] In the recent years, genetic mutations related to the PD have been identified though in vast majority cases the disease occurs sporadically. Irrespective of the cause, PD is characterised by a loss of dopaminergic neurons (DA) in the pars compacta of substantia nigra in the cerebellum which ends in the striatum. Any disorders in the flow of the dopaminergic stimuli from the pars compacta of substantia nigra to the striatum causes movement disorders such as psychomotor sluggishness, resting tremor, and rigidity of the muscles.

[004] The telltale symptom of the PD are intraneuronal inclusions composed mainly of a-synuclein (a-syn) aggregates known as the Lewy bodies. The bodies, however, cannot be used as a PD biomarker as this would require taking a sample of the cerebellum. The presence of the Lewy bodies is discovered post mortem. The PD symptoms such as bradykinesia, rigidity, and tremor depend on the advancing loss of dopaminergic neurons of the substantia nigra which project to the striatum.

[005] In the tracer-based dopamine transporter test named Datscan SPECT the density of dopaminergic innervation of the striatum is identified, with changes in the parameter used to objectivise the PD diagnosis [1 -3], The test, however, requires expensive equipment, which limits accessibility of this type of diagnosis substantially because of the costs of running the test.

[006] Despite extensive literature discussing the clinical and pathological aspects of PD, the specific molecular basis of the DA neuron degeneration remain insufficiently studied.

[007] Diagnosis and treatment of PD should take into account both kinetic and non-kinetic symptoms. Diagnosing PD is based primarily on identification of typical kinetic symptoms. Non-kinetic symptoms, however, such as behaviour and mood disorders, cognitive function disorders, disaesthesia, autonomic disorders, or sleep-wake disorders occur in most patients suffering from PD and may affect the quality of their lives substantially. Moreover, the neurodegeneration process begins in the non-dopaminergic structures of the cerebellum or in the peripheral nervous system many years before the occurrence of the first kinetic symptoms.

[008] The prodromal phase of PD is dominated by non-kinetic symptoms, such as REM sleep behaviour disorders, dysomia, depressive disorders, and constipation.

[009] Therefore, patients in the pre-clinical and prodromal phase are the best candidates for modem therapies which modify the course of the disease [1-3], but the Data Scan test used to diagnose the disease is a very expensive method of limited accessibility.

[010] Known in the prior art are methods of identifying PD and other neurodegenerative diseases, for example the method disclosed in international patent application No. WO2016130793A1 where PD is diagnosed in its early stage using the early stage PD autoantibodies as blood biomarkers which specifically combine with an antigen selected from the group composed of target antigens. In one embodiment of the invention, the method of diagnosing PD consists in identifying the presence or absence of four or a larger number of PD antibody biomarkers in a biological sample, where the biomarkers combine specifically with the serine/threonine-protein kinase MARK1 , pseudoridine synthase 1 tRNA (PLISL 1 ), interleukin 20 (IL20), and C-C motif chemokine 19 (CCL19). According to another international application (WO2015042326A1 ), on the other hand, in order to identify a higher probability or risk of neurodegenerative disorders, the collected blood samples should be tested for the level of lysozyme protein and/or cathepsin S using an enzyme assay.

[011 ] A similar solution is known from patent EP3022560 B1 , which describes a method of identifying neurodegenerative disorders in basic health care, which involves measurements of at least four biomarkers: A2M, FVII, TNFa, and PPY in a blood sample collected from the patient and comparing the biomarker levels against the biomarker levels in a regular blood sample, where an increased level of the biomarkers in the tested blood sample versus a regular blood sample indicates that the patient probably suffers from one of the neurodegenerative diseases: AD, PD, the Down syndrome, frontotemporal dementia (FTD), or dementia with the Lewy bodies (DLB).

[012] Known from international application No. WO2014018650 A1 , on the other hand, is a PD diagnosing method which consists in assessing the expression level of at least one microRNA (miRNA) of the SEQ ID NO: 1- 283 in a biological sample collected from the patient, and comparing the expression level in the sample with the reference expression level. An increased or decreased level of expression in the sample compared to the reference expression level may identify the patient as a person either suffering from PD or at risk of PD development.

[013] Moreover, known from patent US9746482 B2 is a method of diagnosing PD in the patient, which consists in testing a biological sample so as to identify the presence or absence of one or more PD diagnostic biomarkers, and in particular to detect any bindings between at least two antibodies and at least two antigens selected from the group consisting of an intracellular adhesion molecule (ICAM4), the elongation factor 1 -alpha 1 , the tripartite motif containing protein 21 (TRIM21 ), and the haematopoietic domain containing SH2 (HSH2D). [014] In view of the fact that the solutions presented in the prior art do not provide a quick, cheap, and commonly accessible PD diagnostics, the purpose of the invention is to develop a diagnostic tool to identify the biomarkers of neurodegenerations caused by PD, especially for diagnosing patients in the early stages of PD development, which will then enable application of effective treatment with modern drugs.

[015] In the course of the research, it was found that the solution according to the invention provides an entirely new tool for diagnosing neurogenerative diseases, PD in particular. The invention provides an effective solution to the problem of diagnosing the onset of PD too late, where the diagnosis has been to date conducted in the phase of clinical symptoms. This will enable beginning the treatment in very early stages of the disease, before any kinetic symptoms of advancing neurodegeneration occur.

[016] Estradiol may be metabolised in the organism in a number of processes with hydroxylation in the -2, -4, and 16 positions, and methylation to methoxyestrogens being the most common ones. In their publication, Behl et al. [4] demonstrated that 17f3-estradiol and some estradiol derivatives may prevent intramolecular accumulation of hydrogen peroxide, and ultimately the degeneration of the ©prime neurons, clonal cells of the hippocampus, and cells in the organotypic hippocampus. Interestingly, the neuroprotective antioxidant action of oestrogens depends on the presence of the hydroxyl group in the C3 position on the steroid A ring, but is independent of oestrogen receptor activation [4],

[017] It turns out that not all estradiol metabolites have protective properties with respect to neurons. Based on the earlier research conducted by the Creators of this invention on the HT22 hippocampal cells, a hypothesis was formulated that 2-methoxyestradiol may demonstrate neurotoxic action [5], However, it was only the in vivo tests performed in the course of the works on this invention that made it possible to identify the relation between concentration of oestrogen and its selected derivatives: hydroxy and methoxyoestrogens in correlation to induction of hydrogen peroxide, and their role in diagnosing the Parkinson’s disease. [018] The invention concerns application of hydrogen peroxide and 17f3-estradiol and its metabolites in a method of diagnosing neurodegenerative diseases in vitro, Parkinson’s disease in particular, characterised in that the method of diagnosing neurodegenerative diseases includes an assay of the level of hydrogen peroxide and 17f3-estradiol and its metabolites in the patient’s blood serum and that of the control group (reference sample), where in order to determine the levels of 17f3-estradiol and its metabolites samples of blood serum are subject to derivatisation using dansyl chloride, and a neurodegenerative disease is diagnosed when the concentration of hydrogen peroxide in the tested sample is higher than that determined for the healthy control group, and when the concentration of hydroxylated derivatives of 17f3-estradiol (hydroxy derivatives) in the tested sample is lower while the concentration of methoxylated derivatives of 17f3-estradiol (methoxy-derivatives) therein is higher than those assayed for the healthy control. A neurodegenerative disease involves changes in non- dopaminergic structures of the cerebellum and/or peripheral nervous system, especially in the prodomal phase of the Parkinson’s disease.

[019] According to the invention, determined in the blood serum (plasma) samples are levels of 17f3-estradiol (known as estradiol E2) and its metabolites selected from the group including: a-estradiol (a-E2 or ALPHA E2); 2-hydroxyestrone (2-OH-E1 ); 2-hydroxy-estradiol (2-OH-E2);) 2- methoxyestradiol (2-ME-O-E2); 4-hydroxyestrone (4-OH-E1 ); 4- m ethoxyestrone (4-ME-O-E1 ), and 16-hydroxyestrone (16-OH-E1 ), following which the concentrations of the above-listed compounds in the two samples (tested and control) are compared. Preferably, the differences between the concentrations of the tested analytes in samples collected from patients and the healthy control are analysed using the Games-Howell post-hoc test.

[020] In the conducted experimental tests, it was found that the occurring differences in the concentrations of hydrogen peroxide and 17 |3- estradiol and its metabolites in the tested patient samples versus the reference samples collected from healthy individuals can be used as a predictive indicator of the occurrence of neurodegenerative diseases or the risk of their development.

[021 ] In addition, the invention concerns a method of diagnosing of neurodegenerative diseases, the Parkinson’s disease in particular, in vitro, characterised in that collected from a patient or control group is a blood sample from which the plasma is isolated, following which determined in the sample are the levels of hydrogen peroxide, 17|3-estradiol and its metabolites, and the obtained results are compared against the results for the healthy control.

[022] According to the invention, the level of hydrogen peroxide in the sample is determined by the stopped-flow method using a biosensor in the form of a coordination compound of chromium (III) and pyridoxamine {cis- [Cr(C2O4)(pm)(OH2)2]+}, while the level of 17f3-estradiol and its metabolites selected from the group including a-estradiol, 2- hydroxyestrone, 2-hydroxy-estradiol, 2-methoxyestradiol, 4- hydroxyestrone, 4-methoxyestrone, and 16-hydroxyestrone in the sample is determined by liquid chromatography coupled with tandem mass detection - LC-MS/MS upon prior extraction of the analytes by the liquidliquid technique using an organic solvent and derivatisation of the sample using dansyl chloride.

[023] Based on the conducted analyses, substantial differences in the level of hydrogen peroxide and oestrogen derivatives were observed. In the case of hydrogen peroxide levels, its concentration in the tested sample is 275 times higher on average than the concentration determined for the healthy control. Meanwhile, as concerns hydroxylated derivatives of oestrogen (hydroxyderivatives: 2-OH-E1 , 2-OH-E2, 4-OH-E1 , or 16-OH-E1 ), their concentration in the samples collected from PD patients was at least 3 times lower than in the healthy control, whereas as concerns methoxylated derivatives of oestrogen (methoxy-derivatives: 2-MEO-E2, 4-ME-OE1 ), their concentration in the samples from PD patients was at least 2 times higher than in the healthy control. [024] Pursuant to the method according to the invention, to improve detection, the tested oestrogens and their derivatives were subject to derivatisation which resulted in increased molecular mass of the monitored ions and changed properties of the hydroxyl derivatives from hydrophilic to lipophilic. Derivatisation was performed using dansyl chloride as the derivatising agent. Due to short derivatisation time and friendly conditions (30 s, room temperature) it stands out among other reagents earlier used in the literature. The use of dansyl chloride in the invention is further advantageous in that the agent is easily and cheaply available in the market. Before it is applied, the derivatised analytes may be concentrated using SPME extraction. Oestrogens and their derivatives subject to assay were isolated from the biological matrix by the liquid-liquid extraction technique using an organic solvent (dichloromethane).

[025] It was observed that the procedure of derivatising the tested analytes enabled enhancement of their identification by the mass detector, as the signal from the analyte was amplified at the expense of noise signals, which improved the sensitivity of the method. As the result, a low pg LOD value was achieved. In addition, thanks to the changed properties of the hydroxyl derivatives it was possible to use the C18 column and perfectly separate the assayed analytes in the reversed-phase system.

[026] Analysis of the results enabled making the observation that statistically significant differences between the PD patients and healthy subjects occur in the case of most metabolites, except for 2-ME-O-E1 , and E1 (Tables Nos. 1 -5). In the PD patient group a higher median is observed for the following parameters: 4-ME-O-E1 , 2-ME-O-E2, and E2. The opposite relation is characteristic for the other variables highlighted in bold print. The effect size, measured with the eta-squared coefficient, is highest for 5 variables, i.e. 4-OH-E1 , 2-OH-E2, 16-OH-E1 , as well as for 4-ME-O-E1 , and 2-OH-E1 (Table 4).

[027] To recapitulate, in view of the statistical data, estradiol (E2), a-estradiol (ALFA E2), 2-4-hydroxyestrone (2-0H-E1 ), 2-hydroxy-estradiol (2- OH-E2), 2-methoxy-estradiol (2-ME-O-E2), 4-hydroxyestrone (4-OH-E1 ), 4-m ethoxyestrone (4-ME-0-E1 ), and 16-4 hydroxyestrone (16-OH-E1 ) are the most essential PD biomarkers.

[028] In addition, according to the performed tests (Tables Nos. 1 -4), the 4-OH- E1 level is five times lower in PD patients than in the control group, although due to massive accumulation of one of its metabolites, the compound is constantly metabolised to 4-ME-O-E1 and, alternatively, to an extremely potent electrophile of estrone-3,4-quinones (E1 -3,4-Q) [12] most likely responsible for the oxidative stress (leading to neurodegeneration) and DNA damage (resulting in carcinogenesis in a longer perspective). Because of high reactivity of E1 -3,4-Q and its tendency to form DNA and glutathione complexes, its level in the cell is hard to capture employing direct methods [12] and could not be effectively measured by the Inventors.

[029] The obtained results of the research can be explained considering the electrophilic potential of the tested compounds. Therefore, at the subsequent stage of their research, the Inventors performed a series of experiments aimed at explaining the mechanisms of neuron protection or induction of their death by individual estradiol metabolites.

[030] In the tests of the electrophilic potential of the analysed compounds (Table 5), it was demonstrated that the key 4-OH-E1 metabolites, i.e. 4-ME-O-E1 and E1 -3,4-Q, are relatively lipophilic (high logP and relatively small polar surface area, PSA, of the molecule), which enables their free penetration through the lipid bilayers of the neural cells and exacerbation of the oxidative stress in various cell compartments. The calculations were performed based on the density functional theory, in accordance with Koopman’s theorem assumptions. Electrophilicity range acc. to [13],

Estrogens substantially regulate the oxidative stress level. In their publication, Felty et al. [6] proved that E2 induces the mitochondrial level of the reactive oxygen species (ROS) as signal transmitters. Estradiol per se, on the other hand, acts as an antioxidant. Moreover, it has been proved that 2-ME-O-E2 induces reactive oxygen and nitrogen species which leads to the death of cancerous cells [5,7-10], Now, He et al. [11] proved that the biomarkers of the Parkinson’s disease related to the oxidattive stress are: uric acid, DJ-1 protein, coenzyme Q, homocysteine, and 8- hydroxydeoxyguanosine.

[031 ] Hence, based on the research conducted by the Inventors, it has been proved that the level of hydrogen peroxide in blood serum can also serve as a PD biomarker. This is because it has turned out that patients suffering from clinically diagnosed Parkinson’s disease were observed to have an elevated level of hydrogen peroxide in their blood serum versus the control group where the hydrogen peroxide level was below the detection limit. This high hydrogen peroxide level comes most likely from non- enzymatic conversion of 4-OHE1 to E1 -3,4-Q where large amounts of peroxide radical, 02*-, are generated, then converted to H2O2 through intra- and extracellular form of superoxide dismutase, SOD (see fig.5).

[032] Therefore, in the method according to the invention one preferably assays the levels of E2, ALFA E2, 2-0H-E1 , 2-OH-E2, 2-ME-O-E2, 4-OH-E1 , 4- ME-O-E1 , 16-OH-E1 , and hydrogen peroxide in blood serum, which gives the complete picture of the PD patient’s clinical condition

[033] The advantage of the invention lies in the fact that in terms of concentration, the analysed 17f3-estradiol metabolites are characteristic for both PD patients and the healthy, and as such they can be employed as reliable biomarkers differentiating between the existence or non-existence of a neurodegenerative disease. Therefore, according to the invention, hydrogen peroxide, and 17 [3-estradiol and its metabolites are the biomarkers used to diagnose degenerative diseases which involve changes in the non-dopaminergic structures of the cerebellum and/or the peripheral nervous system, Parkinson’s disease in its prodomal phase in particular. The solution according to the invention provides a swift and uncomplicated method of assessing the patient’s condition and the risk of the development of a neurodegenerative disease even before somatic symptoms develop.

[034] The gist of the invention is shown on drawing figures where:

[035] Fig. 1 presents the relation between the age of patients with Parkinson’s disease and the concentration of 2OHE2 in the serum.

[036] Fig. 2 presents the relation between the duration of the disease in the Parkinson’s disease patient group and the E1 concentration.

[037] Fig. 3 presents graphically the results showing the concentrations of the 17|3-estradiol derivatives in the blood serum of patients with Parkinson’s disease compared to the healthy control group.

[038] Fig.4 presents a comparison of the hydrogen peroxide levels in the blood serum of patients with Parkinson’s disease versus the healthy control group by the stopped flow analysis. T-Student test **** p<0.0001 .

[039] Fig.5 (which does not represent the gist of the invention) presents the metabolism and DNA adducts of estrogens, quoted after Cavalieri et al. [12]

[040] Fig. 6 a)- j) presents the levels of 17|3-estradiol and its derivatives (E1 , E2, 2-E2, 2-OH-E1 , 2-OH-E2, 2-MEO-E1 , 2-MEO-E2, 4-OH-E1 , 4-MEO-E1 , 16-OH-E1 ) in the PD patients’ blood serum compared to that of the healthy subjects. The data were analysed using the GraphPad Prism Software, rev. 8.0.1 , and the analysis was run with the t-test * p <0,1 , ** p <0,01 , *** p <0,001 , **** p <0,0001 versus the control.

[041] An embodiment of the invention is presented below.

Embodiment 1

THE TESTED GROUP

Samples of blood serum were collected from healthy volunteers participating in the research project pursued in cooperation between the Neurology and Stroke Department of St. Adalbert’s Hospital in Gdansk and the Chair and Department of Medical Chemistry of the Medical University of Gdansk (MUG) on approval from the Bioethics Committee No. 195/2020. All participants in the research project gave their informed written consent.

The number of the tested subjects: 9 healthy volunteers forming the control group, and 22 patients suffering from Parkinson’s disease.

The criteria of including the PD patients and the healthy control in the study:

• confirmed diagnosis of the earlier non-treated Parkinson’s disease, stadium H-Y l-ll, age range: 45-70. The clinical diagnosis of the Parkinson’s disease formulated based on the disgnostic criteria of the United Kingdom Parkinson’s Disease Society Brain Bank (UKPDS BB) (slowness of movement and at least one of the following symptoms: extrapyramidal stiffness, resting tremor, or posture stimuli disorder, plus the UKPDS BB criteria supporting the diagnosis),

• no interview data or symptoms in clinical examination and in the imaging scan of the brain which would suggest diagnosing the Parkinson syndrome (parkinsonism with symptoms, or another neurodegenerative disease) in accordance with the UKPDS BB criteria excluding the diagnosis of Parkinson’s disease,

The criteria of excluding patients and the healthy control from the study:

• suspected or diagnosed Parkinson syndrome (patients suffering from secondary parkinsonism, G21 acc. to ICD-10) or the Parkinson syndrome accompanying other neurodegenerative diseases (G23 acc. to ICD-10), atypical parkinsonism.

• Blood samples from both groups (patients and healthy control) were collected by qualified medical staff under the supervision of clinicians. The obtained material @ was frozen and stored in deep frozen at the temperature of -80°C until the time of the LC-MS/MS analysis [liquid chromatography coupled with tandem mass detection] and the stopped-flow analysis [stoppedflow method],

A. Test of the estrogen and estrogen metabolite levels by LC-MS/MS.

Before the levels of estrogens and their hydroxy- and methoxy-derivatives are determined, the samples are subject to the derivatisation process. a) Sample derivatisation

500 pL were collected from defrozen serum samples (of each: the patients and healthy control) and transferred to 10 mL threaded test tubes of glass with an automatic pipette. Then, 4 mL of dichloromethane were added to each, the samples were closed with screwed on caps and placed on a rotary vortexer; their content was mixed for 30 min at 40 r/min. After the time, the tubes were removed and the bottom organic layer was collected with a pipette to be transferred to new glass test tubes. Further on, 10 pL of radiolabelled solutions of estrogen derivatives at the concentration of 1 pg/mL were added to each, and mixed on a single-tube vortexer for 30 s. The so processed samples were placed in a void condenser in the temperature of 30°C and evaporated to dryness over 40 min. The subsequent step consisted in analyte derivatisation process. To that aim, 100 pL of sodium bicarbonate 01 M buffer solution, pH 9.0, and 100 pL of dansyl chloride in acetone at the concentration of 1 mg/ml were added to each as the derivatising reagent. The tube content was mixed for 30 s on a single-tube vortexer, then transferred to 250 pL tubes of the Eppendorf type, and centrifuged for 5 min. at 8000 r/min. b) Test of the estrogen and estrogen metabolite levels by LC-MS/MS. Following the derivatisation process, 100 pL of each of the so-processed samples were collected and transferred to 200 pL glass inserts, which were then placed in an automatic sample changer. The were analysed by liquid chromatography coupled with tandem mass detection - LC-MS/MS method using a Shimadzu (Japan) LCMS-8050 set to assay the levels of estrogens (E1 ), (E2), (ALFA-E2), (2-OH-E2), (2-0H-E1 ), (2-ME-O-E2), (2-ME-0-E1 ), (4- 0H-E1 ), (4-ME-0-E1 ), and (16-0H-E1 )) in each.

The mass spectrometer was fitted with a source of ions of the electrospray, ESI, type and an analyser of the MS/MS triple quadrupole type. The estrogens were separated on a C18 column of the ‘core-shell’ type called Poroshell 120, sized 100 x 3.0 mm; 2.7 pm, in the reversed phase arrangement (RP). Gradient elution was employed where the mobile phase A was composed of ultrapure water with 0.1 % of formic acid added, while methanol was used as phase B.

The optimised conditions of analytical separation of estrogens and their derivatives by the LC-MS/MS technique were as follows:

• sample volume dosed into the column: 3 pL

• aggregate duration of a single test: 55 min

• volumetric flow speed of the mobile phase in time: 0.3 mL/min

• column thermostating temperature: 40°C

• temperature in the chamber of the automatic sample changer: 4°C

• MS work mode: positive

• temperature of the ESI ion source: 300°C

• temperature of the heatblock: 400°C

• volumetric flow speed of the heating gas (nitrogen) over time unit: 10 L/min

• volumetric flow speed of the drying gas (nitrogen) over time unit: 10 L/min

• volumetric flow speed of the nebulising (dispersing) gas over time unit:3 L/min

• temperature of the desolvation line (DL): 250°C

• voltage applied to the capillary tube: 3 kV

By employing the optimised and validated LC-MS/MS method one can assay 13 analytes: estrogens and their hydroxy- and methoxy-derivatives over the time of 55 min. Obtaining satisfying separation of 13, structurally very similar compounds required application of a highly sensitive apparatus - a tandem mass spectrometer with an analyser of the triple quadrupole type, and the signals were recorded in the MRM follow-up reaction mode.

The first quadrupole, Q1 , selects the m/z of the parent ion (quasi-particle) relevant for each analysed compound, and thereon, the ion enters the collision chamber Q2. Once collision has been induced, the ions undergo fragmentation and are filtered by the third quadrupole. Each analyte was identified and assayed by monitoring the respective ion passages in the MRM follow-up reaction mode.

The samples collected from patients were analysed by the proposed LC- MS/MS method at random so as to avoid a systematic error. c) Statistical analysis of the results

Statistical analysis was performed using the IBM SPSS Statistics 25 package.

The two groups of subjects were compared using the II Mann-Whitney test.

A Spearman correlation analysis enabled determination whether a statistically significant correlation existed between the analysed variables. The effect size was measured with the Eta-squared coefficient.

The following descriptive statistics were used in the statistical analysis: mean, median, standard deviation, minimum, maximum, the first and third quartiles.

Analysis by the Kruskal-Wallis test enabled verification whether there were any statistically significant differences between individual subject groups. Wherever statistically significant differences were detected, the Games- Howell post-hoc test was applied

The selection was based on the fact that variances in the compared subject groups were not homogenous, and because the number of subjects was low. This enabled checking between which groups statistically significant differences did occur.

The value adopted as the statistically significant level was p < 0.05.

The developed analytical method was statistically appraised for specificity, linearity, LOD, and LOQ. The specificity was tested by comparing the chromatogram obtained from the sample devoid of any reference (blank) with the chromatogram of a sample enhanced with solutions of reference estrogens and their derivatives. No interference between peaks of endogen substances of the biological matrix and peaks of the analysed estrogens confirmed specificity of the method.

Linearity of the methods was confirmed for concentrations ranging from 0.1 ng/mL to 50 ng/mL based on the plotting of the directly proportional correlation between the nominal concentrations of the analytes and the relation of the surface area under the peak of the analysed substance to the surface area of the peak of the internal reference. Then, the regression equations were calculated by the least squares method, and the numerical values of the R2 determination coefficient close to 1 are the measure of the variable correlation degree. The detection limit was calculated based on the signal to noise ratio (S/N = 3). The detection limit was calculated in accordance with the principle that it represents three times the LOD level.

[042] The results of the analyses are shown in tables.

[043] Table No. 1 presents the average level of selected 17|3-estradiol derivatives in the blood serum of patients suffering from Parkinson’s disease compared to the healthy control

[044] Table No. 2 presents the concentrations of estrogens and their metabolites above or below which predisposition to develop Parkinson’s disease may occur.

[045] Tables Nos. 3, 4, 5 present the results of the statistical analysis.

[046] Table 6 presents the electrophilicity index of estradiol derivatives.

Table 1 Average level of estrogens and their metabolites in the serum of patients compared to the healthy control. The data were analysed using GraphPad Prism Software, rev. 8.0.1 , with the analysis performed by the t- test * p <0.1 , ** p <0.01 , *** p <0.001 , **** p <0.0001 , ns - no statistical significance versus the control.

Table 2 Concentrations of estrogens and their metabolites above or below which predisposition to develop Parkinson’s disease may occur Table 3. Descriptive statistics of the analysed metabolites in the group of those suffering from Parkinson’s disease and in the control group. * Mann-Whitney

II Test-; 1 - Parkinson’s disease 2 - control group; concentration [ng/ml].

Table 4. Correlation between the analysed variables in the control group matching the group of Parkinson patients- 1 - age; 2 - 2ME0E1 concentration; 3 - 4ME0E1 concentration; 4 - E1 concentration; 5 - 2MeOE2 concentration; 6 - 16OHE1 concentration; 7 - E2 concentration; 8 - ALFA E2 concentration; 9 - 2OHE2 concentration; 10 - 4OHE1 concentration; 11 - 2OHE1 concentration. Concentration expressed in [ng/ml].

Table 5. Correlation between the analysed variables in the Parkinson patient group-. 1 - age-; 2 - Duration of the disease (in years); 3 - Hoehn-Yah scale-; 4 -2ME0E1 concentration; 5 -4ME0E1 concentration; 6 -E1 concentration; 7 -2MEOE2 concentration; 8 -16OHE1 concentration; 9 -E2 concentration; 10 -ALFA-E2 concentration; 11 -2OHE2 concentration; 12 - 4OHE1 concentration. Concentration expressed in [ng/ml].

Table 6. Electrophilicity index of estradiol derivatives. Lipophilicity expressed as the logP coefficient and the molecule polar surface area. Strong electrophiles co> 1.5 eV, week electrophiles co < 0.8 eV.

A. Determining hydrogen peroxide in blood serum by the stoppedflow method.

[047] 500 pL of serum was collected from the defrozen serum samples (of each: the patients and the healthy control) and transferred to 10 mL threaded test tubes of glass with an automatic pipette.

[048] The samples of both groups were analysed to assay the concentration of hydrogen peroxide. To that aim, the stopped-flow method was applied using the micro-volumetric set for the stopped-flow method tests, i.e. the SX.18MV-R1 , which is a piece of equipment on stock of the Chemistry Department of the University of Gdansk.

[049] The stopped-flow method consists in assaying hydrogen peroxide by measuring the speed of the reaction of uptake of the released carbon oxide (IV) and at the same time determining concentration of hydrogen peroxide in the biological material.

[050] Used in the carbon oxide assay method (IV) was sodium pyruvate which is a direct source of CO2, the fact which can be illustrated with the following reaction equation: pyruvate + H2O2 acetate + CO2 + H2O [051 ] As apparent from the above reaction, the stoichiometric ratio of carbon oxide (IV) to hydrogen peroxide is 1 :1 , i.e. the calculated volume of CO2 is the same as the volume of hydrogen peroxide in the analysed system.

[052] Used in the tests was a biosensor previously synthesized by the Inventors - the coordination compound of chromium (III) and pyridoxine {cis- [Cr(C2O4)(pm)(OH2)2]+}. Assumed was selective reaction of a-ketoacid- pyruvate with hydrogen peroxide followed by decarboxylation of the intermediate - pyruvic peracid, and uptake of the released carbon oxide (IV) by cis-[Cr(C2O4)(pm)(OH2)2]+ (hereinafter: the biosensor).

[053] Reagent solutions were placed separate in two working syringes A and B, with the biological material (sample) and 5 mM solution of potassium pyruvate mixed in the molar ratio of 1 :1 placed in syringe A, and 1 mM solution of the biosensor in phosphate buffer solution of pH of 7.4 in syringe B. Then, the solutions were passed through a mixer (the mixing performed very fast - 10-3 s), following which through a measuring cell, and up to the return syringe B. The filling of the return syringe B with the solution pushed out the piston which hit a microswitch. At that moment, the flow was stopped and measuring began. The progress of the reaction in the portion of the solution stopped in the measuring cell was controlled spectrophotometrically. At the measuring stage, measured was the change in substrate concentration over time.

[054] The concentrations of hydrogen peroxide were calculated by global analysis which is based on the collection of results in the form of a set of absorption spectra measured for the entire wavelength range (characteristic for the specific compound; 330-700 nm), over the defined period of time.

[055] The obtained results were subject to statistical analysis in the GraphPad Prism 20 v. 8.0.1 software using the t-test. The significance level was determined at p<0.0001 .

Conclusions [056] The average concentration of 2-OH-E1 in healthy subjects was 0.62 ng/ml, whereas in PD patients it fell below the detection limit, and that is why it was concluded that no 2-OH-E1 in the blood serum may predispose to PD development. In addition, the average concentration of 2-OH-E2 in the control group was 1.48 ng/ml, and 9 times lower in patients (0.16 ng/ml); the 4-OH-E1 level, on the other hand, was 4.5 times lower in PD patients (0.62 ng/ml) compared to the healthy control (2.79 ng/ml). Moreover, the average concentration of 16-OH-E1 in the patients was 3 times lower (0.05 ng/ml) than in the healthy subjects (0.15 ng/ml). On the other hand, the tests revealed a substantial increase of methoxyestrogens in the PD patients. The average concentration of 2-MEO-E2 in the PD patients was more than 2 times higher (3.07 ng/ml) than the concentration in the healthy subjects (1.48 ng/ml), and the average level of 4-MEO-E1 in the patients was over 11 times higher (0.94 ng/ml) compared to the control (0.08 ng/ml).

[057] The sizes of the differences in concentrations of the tested compounds are also related to the age of the tested subjects. The older the patient from whom a serum sample is collected, the lower is the 2OHE2 value, and the higher it falls in the

[058] Hoehn-Yahr scale. Moreover, the E1 concentration went down over the duration of the disease. Hence, the 2OHE2 level in the serum seems of significance for diagnosing PD.

[059] In addition, the concentration of H2O2 found in the blood of the PD patients was 275.56 times higher on average than in healthy subjects.

[060] The tested patients were found to demonstrate a reduced level of hydroxylated derivatives of estradiols, which was compensated for by methylated derivatives, plus a high level of H2O2. The above data indicate that a reduced level of the above-specified hydroxy-estradiols and an increased level of the above-mentioned methoxyestradiols, correlated with the H2O2 level serve as credible biomarkers in diagnosing neurodegenerative diseases, Parkinson’s disease (PD) in particular. BIBLIOGRAPHY

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