Salivary biomarker for use in the diagnosis, differentiation and neuropsychological evaluation of stroke patients

Abstract

The invention relates to a salivary biomarker for non-invasive differentiation of stroke patients. More particularly, the invention relates to xanthine oxidase for use in diagnosing stroke patients and differentiating patients with a haemorrhagic stroke from patients with an ischemic stroke. The invention also relates to xanthine oxidase for use in the neuropsychological evaluation of patients, i.e. for distinguishing patients with mild cognitive impairment from patients with moderate cognitive impairment.

Description

[0001] Salivary biomarker for use in the diagnosis, differentiation and neuropsychological evaluation of stroke patients

[0002] The invention relates to a salivary biomarker for non-invasive differentiation of stroke patients. More particularly, the invention relates to xanthine oxidase for use in diagnosing stroke patients and differentiating patients with a haemorrhagic stroke from patients with an ischemic stroke. The invention also relates to xanthine oxidase for use in the neuropsychological evaluation of patients, i.e. for distinguishing patients with mild cognitive impairment from patients with moderate cognitive impairment.

[0003] Cerebrovascular diseases are one of the most common causes of adult deaths and disabilities in the world. Despite considerable scientific advances in research on the pathogenesis of the disease, the number of stroke patients continues to increase significantly. Therefore, laboratory stroke biomarkers which would allow for a quick and non-invasive diagnosis, are being intensely sought. Although the diagnosis of stroke is based on clinical examination and computed tomography, in this way it is not always possible to exclude other stroke mimics, such as subdural or epidural haematoma, brain tumours, craniocerebral or cervical spine trauma, infections (meningitis, encephalitis, cerebral abscess), seizures, migraine complications and metabolic disorders. In addition, CT sensitivity in newly diagnosed ischemic stroke does not exceed 30-35%, which, in the absence of widespread availability of perfusion MRI and CT, indicates that new diagnostic strategies need to be sought. Also, not all patients are treated in specialist stroke / neurological medical facilities. Therefore, it is not surprising that fast and non-invasive stroke biomarkers are constantly sought.

[0004] In the pathogenesis of stroke and its vascular complications, a key role is played by a local increase in the activity of an oxidoreductase class brain enzyme, i.e. xanthine oxidase (XO; EC 1.17.3.2). XO catalyses the reaction of conversion of hypoxanthine to xanthine and xanthine to uric acid, which is used to quantify the activity of the enzyme. In the pathogenesis of stroke, especially during ischaemia, the production of both XO and its substrates in the brain is increased. When oxygen is delivered to the brain during reperfusion, XO causes rapid conversion of hypoxanthine to xanthine. Therefore, it has been proposed that XO should be used in the diagnosis of stroke patients. Although brain tissues showed a statistically significant increase in XO activity in patients with stroke (compared to healthy individuals), changes in enzyme activity were no longer observed in routinely collected diagnostic fluids such as plasma, serum or urine. In the publication by Aygul R., 2006, it was confirmed that the assessment of XO activity has no diagnostic yield for the diagnosis of stroke.

[0005] In patients with stroke, the blood-brain barrier is damaged, and thus various brain biomolecules are released into the blood. From the circulation, due to very good vascularisation of the salivary glands and close proximity to the tissues of the brain, various biomolecules can pass into the saliva by passive, facilitated or active diffusion, which indicates that the use of saliva in medical diagnostics would be justified. The results of recent studies have confirmed that the concentration of various brain biomarkers is characterized by a much higher correlation coefficient for saliva than for blood or urine, which makes saliva very attractive in laboratory diagnostics. The advantages of saliva include also its easy availability, non-invasive and painless collection and relatively high durability compared to blood and cerebrospinal fluid. In the prior art, methods for diagnosing diseases by testing the level of markers in biological samples collected from the patient are known.

[0006] Maciejczyk M. et al., 2021 describes a potential use of tumour necrosis factor (TNF-a) in saliva, as assessed by ELISA, to differentiate healthy individuals from stroke patients.

[0007] Gerreth P. et al., 2020 describes a potential use of oxidative stress biomarkers (e.g. reduced glutathione, advanced oxidation protein products (AOPP), lipid hydroperoxides (LOOH)) in unstimulated and stimulated saliva as assessed by ELISA, colourimetric and fluorimetric methods to differentiate healthy individuals from stroke patients.

[0008] Jabs C.M. et al., 1990 describes a method for evaluating tissue and plasma adenosine, inosine, and hypoxanthine / xanthine levels based on luminescence. At the same time, it was emphasized that the method can be adapted to assay markers in other body fluids.

[0009] Patent US7608406 discloses a method for diagnosing a stroke in a subject, comprising contacting a blood, serum, or plasma sample from a patient with an antibody which binds myeloperoxidase and reading a signal indicating the presence or amount of the peptide bound by the antibody. Correlating the test result with the occurrence or absence of stroke in the subject, the stroke including an ischemic stroke and haemorrhagic stroke. The method may comprise one or more additional assays for one or more markers selected from a large group.

[0010] This correlation step involves correlating the results of performed test(s) and the results of computed tomography performed in the subject to assess the occurrence of stroke in the subject. Basic and additional assays are performed using the same sample obtained from the subject. The method and composition described in the patent constitute rapid, sensitive and specific assaying means for use in the diagnosis and differentiation of various forms of stroke and transient ischemic attacks.

[0011] EP 2185926 discloses a method for diagnosing acute myocardial ischaemia or stroke in a patient for point-of-care testing based on measuring the concentration of metabolic by-products of xanthine oxidase (XO) activity on xanthine and hypoxanthine, such as inosine and hypoxanthine, in a biological sample by luminescence; and determining whether the patient experiences acute ischaemia or stroke on the basis of the measurement, wherein in the first step XO is added to a biological sample obtained from the patient, and the measurement step comprises a step of using a chemiluminescent agent and signal enhancers in the biological sample during the measurement of intermediate products, and no clean-up of the sample before the analysis is required.

[0012] The steps of collecting, measuring and reading are carried out in less than 10 minutes, preferably in 1 minute or less. The tested intermediate products of the reaction are hydrogen peroxide, superoxide anionic radicals (SARs) and oxygen free radicals. If the measurement in relative luminescence units corresponds to the measured value of 4.6 μM or more of inosine, hypoxanthine or xanthine in the biological sample, then the result indicates that the patient has experienced acute cardiac hypoxia or stroke. When the level of by-product in the patient's sample exceeds 95% or more of the confidence interval threshold compared to normal controls, this represents a positive indicator of acute ischaemia.

[0013] In the method disclosed in EP 2185926, a measurement step comprises the use of a substrate to which the XO enzyme is bound, preferably the substrate is selected from a strip test or a bed. The method further includes a step of reducing an antioxidant effect of uric acid (UA) in a biological sample, which increases sensitivity during measurement. Further, the method comprises a step of diluting the biological sample prior to the measurement step, preferably with a diluent buffering the pH of the sample to pH 7.2-7.8. Further, the method includes adding purine nucleoside phosphorylase (PNP) prior to the XO addition step. According to the method, the biological sample may be a biological fluid selected from blood, plasma, saliva, cerebrospinal fluid, urine.

[0014] The object of the present invention is the use of assessment of salivary xanthine oxidase (XO) activity for diagnosing a stroke, while differentiating healthy individuals from individuals experiencing a stroke, as well as for determining the type of stroke (a haemorrhagic or ischemic one). It has been shown that the assessment of specific salivary XO activity (XO activity in saliva / protein content in saliva) allows to distinguish patients with an ischemic stroke from patients with a haemorrhagic stroke and healthy individuals with a very high specificity and accuracy, because of which this biomarker can be used in non-invasive differential stroke diagnosis. Salivary XO also distinguishes patients with mild cognitive impairment from patients with moderate cognitive impairment and, as a result, it can be a potential screening tool used for comprehensive neuropsychological evaluation in stroke patients.

[0015] In the prior art, there is no data as regards the possibility of using XO in stroke diagnosis, including the use of saliva to assess XO activity. This is the first report pointing that salivary XO may be used in the differential diagnosis of stroke patients. So far, a method for diagnosing acute myocardial ischaemia or a stroke by measuring plasma concentrations of inosine and hypoxanthine, i.e. metabolic by- products of xanthine oxidase (XO) activity, after adding a certain amount of XO enzyme to a biological sample collected from a patient, has only been disclosed (EP 2185926).

[0016] Said solution indicates the use of assessment of specific XO activity in saliva (enzyme activity expressed as the content of salivary protein) using the fluorimetric method to diagnose the type of stroke (a haemorrhagic and ischemic one) and assess cognitive functions in patients. Following the collection of saliva from the patient at a medical facility or at home, the sample is centrifuged, and thus obtained supernatant is used to assess XO activity. The XO activity is evaluated using a fast and reproducible fluorimetric method, which requires a very small volume of test material (50 μl sample). In the first step of the assay, xanthine / hypoxanthine is added to the sample. The XO present in the saliva catalyses the oxidation of xanthine / hypoxanthine to superoxide radical and uric acid (UA). The superoxide radical is then reduced to hydrogen peroxide (H2O2), which reacts 1:1 with the Amplex Red reagent. Then a reaction with horseradish peroxidase takes place to form the red fluorescent compound resorufin, whose fluorescence is proportional to the XO activity in the assayed sample. This method is characterized by the ease and non-invasiveness of test material collection, as well as the simplicity and speed of analysis by the fluorimetric method, which could be performed in a routine diagnostic laboratory.

[0017] The biomarker described in the invention meets all the criteria for a stroke biomarker: it is measured by standardized, validated and generally available methods, is easy to interpret and exhibits appropriate sensitivity and specificity. It can be used in non-invasive stroke diagnosis, by differentiating both healthy individuals from stroke patients, as well as patients with an ischemic stroke from patients with a haemorrhagic stroke. It should be noted that the collection of saliva as diagnostic material involves low costs, is non-invasive and painless.

[0018] Saliva can be collected at home, without assistance from specialist medical personnel, and the transport of such material does not require a special procedure. Saliva is also non-infectious material, unlike blood commonly used in medical diagnostics. In addition, compounds assayed in saliva tend to be more stable than those present in blood or cerebrospinal fluid. The proposed invention is therefore distinguished not only by the non-invasiveness of collecting the material for testing, but also by the simplicity of assays and a low price of the analysis. Moreover, salivary XO also differentiates patients with mild cognitive impairment from patients with moderate cognitive impairment, thus it can be also a potential screening tool used for comprehensive neuropsychological evaluation in stroke patients.

[0019] An object of the invention is xanthine oxidase for use as a biomarker in diagnosing stroke patients.

[0020] Preferably, the activity of xanthine oxidase is measured in a body fluid, which is preferably saliva.

[0021] Preferably, the activity of xanthine oxidase is measured by the fluorimetric method.

[0022] Preferably, the volume of test material (saliva) is at least 50 μl.

[0023] Another object of the invention is xanthine oxidase for use as a biomarker in differentiating stroke patients.

[0024] Preferably, the activity of xanthine oxidase is measured in a body fluid, which is preferably saliva.

[0025] Preferably, differentiation applies to patients with a haemorrhagic stroke and patients with an ischemic stroke.

[0026] Still another object of the invention is xanthine oxidase for use as a biomarker in neuropsychological evaluation of stroke patients.

[0027] Preferably, the activity of xanthine oxidase is measured in a body fluid, which is preferably saliva.

[0028] Preferably, patients with mild cognitive impairment are distinguished from patients with moderate cognitive impairment.

[0029] The object of the present invention is presented in the embodiments in the drawing, in which:

[0030] Figure 1 shows specific activity of xanthine oxidase (XO; nU / mg total protein) in saliva in healthy subjects (control group, n = 30), patients with a haemorrhagic stroke (n = 15) and patients with an ischemic stroke (n = 15). **— p < 0.01; ***— p < 0.001; ****— p < 0.0001.

[0031] Figure 2 shows diagnostic yield ROC curves for specific activity of xanthine oxidase (XO; nU / mg total protein) in saliva differentiating patients with a haemorrhagic stroke versus the control group, patients with an ischemic stroke versus the control group and patients with a haemorrhagic stroke versus patients with an ischemic stroke.

[0032] Figure 3 shows the correlation between specific activity of xanthine oxidase (XO; nU / mg total protein) in saliva and cognitive function in ACE III.

[0033] Figure 4 shows specific activity of xanthine oxidase (XO; nU / mg total protein) in the saliva of patients with a stroke and normal cognitive function, patients with a stroke and mild cognitive impairment and patients with a stroke and moderate cognitive impairment, ns— statistically not significant; **** — p < 0.0001.

[0034] Figure 5 shows diagnostic yield ROC curves for specific activity of xanthine oxidase (XO; nU / mg total protein) in saliva differentiating patients with a stroke and normal cognitive function, patients with a stroke and mild cognitive impairment, and patients with a stroke and moderate cognitive impairment. The invention is presented in the following embodiments, which are not limiting in any way.

[0035] Embodiment 1

[0036] The study was approved by the Bioethics Committee of the Poznan University of Medical Sciences (Resolutions 59 / 19 and 890 / 19). 30 stroke patients were enrolled in the study and divided into 2 subgroups depending on the type of the stroke experienced: 15 patients with an ischemic stroke and 15 patients with a haemorrhagic stroke. A control group consisted of 30 generally healthy individuals matching the study group in terms of age, sex, periodontal status, dentition and oral hygiene.

[0037] The patients' cognitive and functional status was measured by the following scales:

[0038] • Addenbrooke's Cognitive Examination III (ACE III) to differentiate patients with and without cognitive impairment. ACE III assesses attention and orientation, memory, verbal fluency, language and visuospatial skills;

[0039] • Barthel Index (Bl) for measuring an individual's daily life functioning, especially as regards activities of daily living (ADL);

[0040] • Berg Balance Scale (BBS) to determine a patient's ability or inability to safely maintain balance while performing a series of specific tasks;

[0041] • Functional Independence Measure (FIM) to study a person's social, psychological, and physical functioning.

[0042] An oral examination and saliva collection were performed between 30 and 35 days after the onset of stroke.

[0043] Following the saliva collection, a dental examination was carried out. The examination was performed in artificial headlamp lighting with the use of a plane mouth mirror and periodontal probe, in accordance with the World Health Organization criteria (WHO 5th edition). A dentition status was assessed using the CFE index according to Szpringer-Nodzak, 1999, which is the sum of teeth with primary or secondary active caries (C) and following caries, the teeth extracted due to caries (E) or filled (F). The condition of gingiva was determined with the use of the Gingival Index (Gl) according to Loe and Silness, 1963, at teeth No. 16, 12, 24, 36, 32, 44, according to the following scale: 0 - normal gingiva (pale pink colour of the gingiva); 1 - mild inflammation - slight discolouration of the gingiva, slight oedema, no bleeding on probing; 2 - moderate inflammation - redness, oedema and glazing of the gingiva, bleeding on probing; 3 -severe inflammation - marked redness and oedema of the gingiva, ulceration, tendency to spontaneous bleeding. The Plaque Index according to Silness and Loe, 1964, was used to assess oral hygiene. The index was determined at teeth No. 16, 12, 24, 36, 32, 44, according to the following criteria: 0 - no plaque; 1 - thin plaque layer at the gingival margin invisible to the naked eye; 2 - moderate accumulation of soft deposits in the periodontal pocket, at the gingival margin - visible to the naked eye; 3 - abundant accumulation of soft deposits inside the periodontal pocket and / or at the gingival margin and tooth surface.

[0044] The test material was mixed unstimulated saliva. Saliva samples were collected between 7:00 and 9:00 a.m. by the spitting method developed by Maciejczyk et al., 2019.

[0045] Prior to the saliva collection, the patients were advised not to consume any solid or liquid food other than pure water for at least two hours prior to the saliva collection procedure. The patients were also instructed to refrain from strenuous physical exertion and hygiene procedures (mouth rinsing, tooth brushing, gum chewing, etc.) for 12 hours before the saliva collection procedure.

[0046] As all the patients were in the subacute phase of stroke and took medication regularly, the time from the last dose of the drug was set at not less than 2 hours. Subjects from the control group were not allowed to take any medication for 8 hours prior to the saliva collection procedure.

[0047] Before the collection, the mouth was rinsed twice with distilled water at room temperature to avoid contamination from other sources. Saliva was collected in a separate room after 5 minutes of adaptation to the environment. A patient sat in a comfortable seat position with their head slightly tilted downwards in a comfortable position during the procedure of saliva collection into a sterile Falcon tube. The saliva collected during 1 minute was discarded. The saliva was collected for 10 minutes. The volume of saliva was measured with a calibrated pipette. A saliva flow rate was measured by dividing the volume by the time required for collection and expressed in mL / min.

[0048] After the collection, the saliva was centrifuged (+4°C, 20 min., 3000 x g in a MPW 351 centrifuge, MPW Med. Instruments) and, to equalize supernatants, butylated hydroxytoluene (BHT, Sigma-Adrich, USA) was added in the amount of 10 μL of 0.5 M BHT in acetonitrile (ACN) / 1 ml saliva, to protect the samples from oxidative processes. The saliva samples were placed in - 80°C and stored frozen for further assays.

[0049] There were no statistically significant differences between the study and control groups in terms of sex, age, concomitant diseases, medications taken, and oral hygiene and periodontal status. Only functional and cognitive performance as measured by Bl, FIM, BBS, and ACE III scores and total salivary protein concentrations were significantly lower in stroke patients than in the control group. The results are shown in Table 1.

[0050] * results were expressed as mean ± standard deviation (SD) (95% confidence interval (95% Cl))

[0051] XO activity was examined in saliva by the fluorimetric method using a commercial Amplex® Red Xanthine / Xanthine Oxidase Assay kit (Invitrogen, USA). In this kit, XO catalyses the oxidation of xanthine / hypoxanthine to superoxide radical and uric acid (UA). The superoxide radical is then reduced to hydrogen peroxide (H2O2), which reacts 1:1 with the Amplex Red reagent. Then a reaction with horseradish peroxidase takes place and the red fluorescent compound resorufin is formed. Resorufin fluorescence was measured at a wavelength of 540 / 590 nm. It is assumed that one unit (U) of XO activity is 1 pmol UA obtained from hypoxanthine at 25°C. To measure the absorbance and fluorescence of the sample a BioTek Synergy Hl 96-well plate reader (Winooski, VT, USA) was used. All the analyses were performed in triplicate. The results were normalized to total protein (TPC) content and presented as specific activity (nU / mg protein).

[0052] Total protein content (TPC) was assayed colourimetrically with the use of bicinchoninic acid (BCA) with bovine serum albumin (BSA) as a standard (Thermo Scientific PIERCE BCA Protein Assay Kit, Rockford, IL, USA). The assays were performed using a commercially available diagnostic kit. The obtained results were normalized to the content of the protein in saliva as assayed by the BCA colourimetric method.

[0053] A statistical analysis of the data obtained was performed. The data analysis was carried out using GraphPad Prism 8 for macOS (GraphPad software, La Jolla, CA, USA). The distribution of variables was evaluated by the Kolmogorov-Smirnov test, while the equality of variances was assessed by Levene's test. To compare two experimental groups, the Student's t-test was used, while for three groups an analysis of variance (ANOVA) with Tukey's post hoc test was applied. The statistical significance defined was p < 0.05. The p-values were calculated with adjustment for multiple comparisons. The results were expressed as mean ± standard deviation (SD) and 95% confidence interval (95% Cl)) Correlations between clinical data and saliva biomarkers were established with the use of the Pearson correlation coefficient. The impact of multiple independent variables on one quantitative dependent variable was also assessed by a linear regression analysis. Stroke types, ACE III, Bl, FIM, BBS, and saliva flow in minutes were used as independent variables, and the results were reported as 95% Cl with regression parameters. The diagnostic yield of saliva biomarkers was additionally assessed with the use of receiver operating characteristic curve (ROC). The area under the curve (AUC) and the cut-off point for the biomarker characterized by the highest sensitivity and specificity were calculated.

[0054] The specific activity of xanthine oxidase (XO; nU / mg total protein) was shown to be significantly higher in both the saliva of patients with a haemorrhagic and ischemic stroke compared to healthy individuals (the control group). Salivary XO activity is also significantly higher in the saliva of patients with an ischemic stroke compared to patients with a haemorrhagic stroke (Figure 1).

[0055] A multivariate regression analysis showed that the specific activity of xanthine oxidase (XO; nU / mg total protein) depended on the type of stroke as well as cognitive function (ACE III), but did not depend on the patient's functional status according to Bl, FIM and BBS scales and the saliva flow (Table 2).

[0056] The ROC (Receiver Operating Characteristic) curve is a graphical representation of the relationship between the sensitivity and specificity of a diagnostic marker. It helps determine the optimal cut-off point dividing the set of results into two groups: a group where a specific phenomenon (e.g. a disease) is present and a group where it is absent. The area under the ROC curve evaluates the ability of the test to separate normal and abnormal results. The AUC assumes values in the range of 0 to 1. The larger the area, the more accurately, based on the result of the analysed diagnostic variable, we may classify patients into the group with a positive test result or into the group with a negative test result, and the AUC should be significantly greater than 0.5. With the use of the diagnostic yield ROC analysis, it was shown that the XO biomarker as assessed in saliva significantly statistically differentiates patients with an ischemic stroke from patients with a haemorrhagic stroke (AUC: 0.764) and healthy individuals (AUC: 0.973) (Figure 2). This biomarker is characterized by both very high sensitivity (an ischemic versus haemorrhagic stroke: 66.67%; an ischemic stroke versus the control group: 93.33%) and specificity (an ischemic versus haemorrhagic stroke: 66.67%; an ischemic stroke versus the control group: 90%), which confirms its diagnostic yield.

[0057] With the use of the Pearson correlation coefficient it was shown that XO specific activity (nU / mg total protein) in saliva correlates negatively with postural balance on the BBS scale (r = -0.61, p < 0.001) and performance in daily activities on the Bl scale (r = -0.58, p = 0.001). Salivary XO was also negatively correlated with cognitive function on the ACE III scale (r = -0.71, p < 0.001), including specifically with attention and orientation (r = -0.57, p = 0.001), memory (r = -0.63, p = 0.0002), visual perception (r = - 0.54, p = 0.002), language (r = -0.56, p = 0.001), and visuospatial skills (r = -0.53, p = 0.003) (Figure 3).

[0058] Embodiment 2

[0059] In the next stage of the study, patients with a haemorrhagic and ischemic stroke (the study group) were divided into three subgroups based on the severity of cognitive impairment as assessed on the ACE III scale: no cognitive impairment (score 100-89 in ACE III), mild cognitive impairment (score 88-61 in ACE III) and moderate cognitive impairment (score < 61 in ACE III).

[0060] XO specific activity (nU / mg total protein) in saliva was shown to be significantly higher in patients with moderate cognitive impairment compared to patients with stroke and mild cognitive impairment and individuals with a stroke without cognitive impairment as assessed in ACE III (Figure 4).

[0061] The ROC analysis showed that specific XO activity differentiated patients with stroke and mild cognitive impairment compared to moderate cognitive impairment with high accuracy and specificity (Figure 5).

[0062] XO specific activity in saliva also distinguishes patients with mild cognitive impairment from patients with moderate cognitive impairment, which can be a potential screening tool used for comprehensive neuropsychological evaluation in stroke patients. References:

[0063] 1. Jabs C.M., Neglen P., Eklof B., Thomas E J., Adenosine, inosine, and hypoxanthine / xanthine measured in tissue and plasma by a luminescence method; Clin. Chem. 1990: 36 (1) p.81- 87; https: / / doi.Org / 10.1093 / clinchem / 36.l.81

[0064] 2. Aygul R, Kotan D, Demirbas F, Ulvi H, Deniz O. Plasma Oxidants and Antioxidants in Acute Ischaemic Stroke. Journal of International Medical Research.; 2006: 413-418. doi:10.1177 / 147323000603400411

[0065] 3. Maciejczyk M, Zalewska A, tadny JR. Salivary Antioxidant Barrier, Redox Status, and Oxidative Damage to Proteins and Lipids in Healthy Children, Adults, and the Elderly. Oxid Med Cell Longev.;2019:4393460. doi: 10.1155 / 2019 / 4393460.

[0066] 4. Maciejczyk M, Pawlukianiec C, Zendzian-Piotrowska M, tadny JR, Zalewska A. Salivary Redox Biomarkers in Insulin Resistance: Preclinical Studies in an Animal Model. Oxid Med Cell Longev. 2021: 3734252. doi: 10.1155 / 2021 / 3734252.

[0067] 5. Gerreth P., Maciejczyk M., Zalewska A., Gerreth K., Hojan K. Comprehensive Evaluation of the Oral Health Status, Salivary Gland Function, and Oxidative Stress in the Saliva of Patients with Subacute Phase of Stroke: A Case-Control Study. Journal of Clinical Medicine, 2020. 9. 2252. 10.3390 / jcm9072252.

Claims

AMENDED CLAIMS received by the International Bureau on 23 August 2023 (23.08.2023)1. Use of salivary xanthine oxidase as a biomarker for diagnosing stroke patients in vitro.

2. Use of salivary xanthine oxidase according to claim 1, characterised in that the activity of xanthine oxidase is measured by the fluorimetric method.

3. Use of salivary xanthine oxidase according to claim 2, characterised in that the saliva volume is at least 50 μl.

4. Use of salivary xanthine oxidase as a biomarker for differentiating stroke patients, characterised in that the salivary xanthine oxidase activity from 46 nU / mg to 60 nU / mg means haemorrhagic stroke, and the salivary xanthine oxidase activity higher than 60nU / mg means ischemic stroke.

5. Use of salivary xanthine oxidase as a biomarker for neuropsychological evaluation of stroke patients, characterised in that the salivary xanthine oxidase activity from 51nU / mg to 62 nU / mg means mild cognitive impairment, and the salivary xanthine oxidase activity higher than 62 nU / mg means moderate cognitive impairment.STATEMENT UNDER ARTICLE 19(1)Amendments to the submitted claims are intended to clarify the scope of the invention and to precisely define that the invention concerns the use of salivary xanthine oxidase as a biomarker of stroke. Moreover based on the salivary enzyme activity levels, Applicants are able to distinguish between hemorrhagic and ischemic stroke, and to distinguish between mild and moderate cognitive impairment, what is not obvious in a light of documents from the art cited in ISR.The category of independent Claim 1 was changed by adding the word "use" of salivary xanthine oxidase and precisely define that the invention concerns only to use of salivary xanthine oxidase as a biomarker for diagnosis stroke patients in vitro. That changes indicate the inventive step of said invention.In view of this change, claims 2 and 3 were canceled.Claims 4 and 5, which after the amendments are claim 2 and 3, respectively, were also redrafted as"Use of salivary xanthine oxidase..."Claim 6, which in the present version of the claims is numbered 4, in addition to the editorial adjustment as all the preceding claims, further specifies the salivary xanthine oxidase activity level ranges allowing for distinguishing between hemorrhagic and ischemic stroke, in such a way that activity from 46 nU / mg to 60 nU / mg indicates hemorrhagic stroke, and an activity result above60nU / mg indicates ischemic stroke. The confirmation of such a range of values can be found in the graph in Fig.l. So taking into account of these ranges to the content of the claim, it does not add any new matter.Claims from 7 to 9 are cancelled.Claim 10, which in the current version of the claims is numbered 5, except for redaction in accordance with the formula of all the preceding claims, in the characterizing part, it clarifies the activity level ranges of salivary xanthine oxidase allowing to distinguish cognitive disorders in such a way that activity from 51-62 nU / mg indicates mild cognitive impairment and activity above 62 nU / mg means moderate cognitive impairment. Confirmation of such a range of values is found in example 2 in the table on page 10 of the description.Claims 11 to 13 are cancelled.In response, the Applicant attaches redrafted claims.In addition, as obvious result od cancellation of some claims, numbering of claims is amended accordingly.No new matter was added.

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