Method for qualitative and quantitative detection of monounsaturated fatty acids (MUFAS) in a human or animal lipid biological sample

Abstract

The present invention concerns a method for qualitative and quantitative detection of monounsaturated fatty acids (MUFAs) in a human or animal lipid biological sample, preferably in a sebum sample. In particular, the method of the present invention can be advantageously used for the qualitative and quantitative detection of MUFAs in a human lipid biological sample, such as, human sebum, for the diagnosis of excessive secretion of MUFAs occurring in Parkinson's disease (PD), acne or seborrheic dermatitis.

Description

[0001] METHOD FOR QUALITATIVE AND QUANTITATIVE DETECTION OF MONOUNSATURATED FATTY ACIDS (MUFAS) IN A HUMAN OR ANIMAL LIPID BIOLOGICAL SAMPLE

[0002] The present invention concerns a method for qualitative and quantitative detection of monounsaturated fatty acids (MUFAs) in a human or animal lipid biological sample, preferably in a sebum sample. In particular, the method of the present invention can be advantageously used for the qualitative and quantitative detection of monounsaturated fatty acids (MUFAs) in a human lipid biological sample, such as, human sebum, for the diagnosis of Parkinson's disease (PD), or a cutaneous disorder like acne, rosacea, psoriasis, atopic or seborrheic dermatitis.

[0003] Sebum is a complex biofluid composed of neutral lipids, wherein the MUFAs, e.g., sapienate, palmitoleate, oleate, are present in both the free and the bound form. Recent studies describe abnormal abundance of MUFAs in sebum in presence of a cutaneous disorder, like acne, and a neurodegenerative disease, i.e. PD. Both dermatological and neurological diseases, associated with abnormal activity of the sebaceous gland and dysregulated sebum composition, with it, have the potential to be screened for the levels of MUFAs in sebum (Okoro E, et al. 2021 ; Briganti S, et al. 2021 ).

[0004] Therefore, the determination of the overall unsaturation degree of sebum is particularly important in the above conditions and other disorders that may involve deregulated fatty acids (FAs) desaturation.

[0005] Known methods for quantifying MUFAs in the sebum are chromatographic methods, either gas chromatography (GC) or liquid chromatography (LC), coupled with different detectors. GC coupled to mass spectrometry (MS) is widely used in the determination of MUFAs. GCMS and LCMS methods are costly due to the requirement of sophisticated equipment and trained personnel. Moreover, to be applied, both GCMS and LCMS methods require lengthy sample preparation and long chromatographic runtime. Moreover, such analytical methods are performed in laboratory settings and are not immediately transferrable to the direct application in the clinical setting. To adapt the requirements of immediate readings of indexes of the unsaturation degree of sebum in the same context of sebum sampling, a colorimetric assay is preferable.

[0006] In the light of the above, it is therefore apparent the need to provide new approaches for quantifying MUFAs in a human or animal lipid biological sample, for example in the human sebum, that overcome the disadvantages of known methods for quantifying MUFAs. In this context, the aim of the present invention is for example overcoming the disadvantages of sophisticated methods and make measurements directly applicable.

[0007] It is known that Zhang F, et al. , 2018, have described a method applied to the measurement of MUFAs in vegetable oils. This assay exploits the reactivity of the carbon-carbon double bond in the MUFAs with silver ions (Ag+). To perform the assay, the lipid matrix containing MUFAs is mixed with a solution of Ag+ ion in excess. The Ag+ ions bind the double bond in a stoichiometric proportion, while the excess of Ag+ ions is available to bind with reagents, i.e. the chromophore 3, 3', 5,5'- Tetramethylbenzidine (TMB), that develops a complex absorbing the light in the visible range. The intensity of the developed color is inversely proportional to the concentration of double bonds, ranging from dark blue (MUFAs at low concentration) to colorless (MUFAs at high concentration). However, vegetable oils have a composition of fatty molecules different from the composition of fatty molecules of a human or animal lipid biological sample. Specifically, the composition of fatty molecules in sebum is characterized by a prevalent fatty acid chain length of 16 carbon atoms, whereas vegetable oils are characterized by oleic and linoleic acid, which have a C18 chain length. In addition, sebum contains also more complex unsaturated fatty molecules such as for example squalene and wax esters that can interfere with the measurement of MUFAs. More specifically, squalene is a triterpene that has as many as six double bonds and is present in sebum in a high percentage of 15-20% w / w %, whereas it is present in a percentage of only 0.1 -1 % in olive oil. Therefore, squalene can potentially interfere with the measurement of MUFAs in sebum.

[0008] The Applicant has surprisingly found that the principle underlying the aforementioned assay can also be exploited for the quantification of MUFAs in a human or animal lipid biological sample, for example in a sebum sample, despite the fact that it also contains other, much more complex, unsaturated fatty molecules (for example squalene and wax esters). In addition, the method of the invention is also reliable despite the difficulties in collecting comparable amounts of sebum, a problem that does not exist for vegetable oil samples. To this end, the applicant modified substantially the known method for the detection of MUFAs in vegetable oils and was able to apply it to mammalian biological samples in order to provide an effective method in supporting the diagnosis of pathological conditions. In particular, the applicant selected suitable systems to obtain comparable amount of biological sample and suitable solvents, which at the same time solubilize the biological sample for the analysis and avoid the possible interference of unsaturated fatty acids different from MLIFA. The applicant has therefore developed a method for the quantification of MUFAs in a human or animal lipid biological samples that is simple, fast, cost effective, yet reliable than the known methods available to date.

[0009] In particular, according to the present invention, a preliminary set-up has been carried out wherein the linearity and the repeatability have been tested in a given concentration range of the reference MLIFA, sapienic acid (C16:1 ), which is accounted among the most abundant MUFAs in sebum. Furthermore, according to the present invention, the method has been optimized for the application to human sebum collected with adhesive tapes, then extracted and dissolved in alcohol. In particular, the assay was applied to sebum extracts of PD patients and Alzheimer's disease (AD) patients and healthy controls (HC), whose content of MUFAs had been previously analysed (Briganti S, et al., 2021 ) with GCMS. The Applicant investigated the application of the assay also on sebum sampled directly from the skin with a miniaturized cotton swab: the preliminary evidence supports the transferability of the assay to the development of a device for the assessment of overall unsaturation degree of sebum in a clinical setting (lab-on-chip, point-of-care test) (Lovecchio N, et al. 2022). This test has never been applied to human sebum and is not available as a ready to use kit or a lab-on-chip. Adhesive tapes and cotton swab are sampling means or devices providing measurable quantity of biological samples so that comparable conditions for the sampling of the biological matrix to be analysed with the method of the present invention are assured. In fact, defined areas of the adhesive tapes or volumes of cotton swab allow the collection of comparable quantities of biological samples. The quantities of collected biological sample can be measured by calculating the difference between the weight of the sampling means before and after collecting the biological sample. In addition, the solvents used in the method of the present invention are surprisingly able to solubilise MUFA from the sampling means, without solubilising relevant quantities of highly hydrophobic compounds, such as waxes, triglycerides and compounds of the sampling supports that can interfere with the measurement of MUFAs, and to provide a reliable measurement of MUFAs.

[0010] It is therefore a specific objective of the present invention a method for qualitative and / or quantitative detection of MUFAs in a human or animal lipid biological sample (or lipid matrix) comprising the following steps: a) dissolving a known (or measured) quantity of said sample in a known volume of an organic solvent to obtain a lipid solution, b) mixing the lipid solution a) with Ag+ions so that said Ag+ions react with the MUFAs of the lipid biological sample, c) adding a chromophore able to bind unreacted Ag+ ions, obtaining a complex chromophore-Ag+ absorbing the light in the visible range, d) observing the colour of the solution of step c),

[0011] Ag+ ions moles and chromophore moles of step b) and c) being chosen so that Ag+ ions moles are higher than a chosen threshold value of MUFAs moles contained in the same quantity (amount) of biological sample (the chosen threshold is a likely threshold value; for example it can be the mean normal quantity of MUFAs in the same type of biological sample or the mean quantity of MUFAs in the same type of biological sample of a subject with excessive MUFAs in sebum in comparison to normal quantity, such as in a subject suffering from PD and less than the chromophore moles, the molar concentration of Ag+ ions and of chromophore being chosen so that a calibration curve of the absorbance against known MUFAs molar concentration is linear; wherein, the colour intensity of the solution c) provides a qualitative indication of the MUFAs amount in the biological sample, for example in comparison to the colour intensity of the chosen threshold value of MUFAs.

[0012] According to the present invention, the intensity of the colour of the solution of step d) is related to the moles of Ag+ ions that have not reacted with MUFAs and, therefore, have reacted with the chromophore. Thus, darker colour means low amount of MUFAs and lighter colour means high amount of MUFAs.

[0013] According to the present invention, the method can further comprise the step e) that is obtaining a measure of MUFAs amount in the biological sample by obtaining a measurement of the absorbance of the solution of step c) and comparing said measurement with the calibration curve of the absorbance against known molar concentrations of MUFAs.

[0014] A person skilled in the art is able to prepare a calibration curve wherein the same method described above is carried out with known molar concentrations of MUFAs and with a fixed molar concentration of Ag+ ions and a fixed molar concentration of chromophore. Therefore, different concentrations of unreacted Ag+ ions correspond to different absorbance values, which, in turn, correspond to different MUFAs molar concentrations. The person skilled in the art is able to choose the concentration of Ag+ ions and the concentration of chromophore so that the calibration curve of the absorbance against known MUFAs molar concentration is linear.

[0015] The term “known volume of organic solvent” in step a) means that the volume is a given volume in order to quantify the concentration of MUFAs in the collected biological sample (i.e. the biological sample without organic solvent. In other words, the quantity of biological sample and the volume of solvent in which the biological sample is diluted have to be known.

[0016] In addition, according to the present invention, the method can further comprise a step cO) of adding a buffer solution, such as a sodium acetate -acetic acid (NaAc) buffer solution, before step c) (i.e. the buffer solution can be added before step a) or before step b) or before step c)).

[0017] According to the method of the present invention, said lipid biological sample can be chosen from the group consisting of sebum, lipid extract from cells, tissues, plasma and serum, preferably sebum, more preferably human sebum.

[0018] A person skilled in the art is able to choose a suitable organic solvent in order to solubilize said lipid sample, however, it is important also that the organic solvent does not solubilize relevant amounts of compounds of the biological sample, such as waxes and triglycerides, or of the sampling means, such as plastic material, used for collecting the biological sample, that can interfere with the measurement of MUFAs. For these reasons, according to the present invention, said organic solvent can be preferably a moderately polar organic solvent. For example, the organic solvent can have a relative polarity value chosen in the range from 0.1 and 0.8 in comparison to the polarity of water considered as 1 . Preferably, the organic solvent can be chosen in the group consisting of methanol, ethanol, 2-propanol, 1 -propanol, 1 -butanol, 2-butanol, acetone, ethyl acetate, acetonitrile, methyl-ethyl ether, diethyl ether, methyl-tert-buthyl ether (MTBE), glycerol and t-butyl alcohol, or a mixture thereof which resulted surprisingly suitable in the method of the present invention. Organic solvents having high toxicity such as aromatics (benzene, tetrahydrofuran) and chlorinated solvents (chloroform, dichloromethane, dichloroethane) are excluded so as not to expose operators to chemical hazards.

[0019] According to the method of the present invention, said Ag+ ions can be in a form of an Ag salt, for example a salt chosen from the group consisting of AgNO2, AgNOs, Ag2COs and mixture thereof, preferably AgNOs, or a solution thereof.

[0020] According to an embodiment of the present invention, said chromophore can be TMB.

[0021] According to the method of the present invention, the concentration of the lipid biological sample in the lipid solution of step a) can range from 0,2 mg / ml to 1 mg / ml. For example, when the biological sample is sebum, the range 0.2 mg / ml - 1 mg / ml indicates the amounts of sebum consistent with the range of the calibration curve to ensure linearity of the response: a sample with amounts of sebum outside this range might not respond linearly to the assay.

[0022] In addition, according to the present invention, the method can comprise a step a1 ) after step a) wherein the lipid solution is diluted in an organic solvent at a volume ratio of from 1 :10 to 1 :100. According to an embodiment of the present invention, said Ag+ ions of step b) can be at a molar concentration ranging from 1 .75 mM to 2.5 mM when said Ag+ ions are in the form of a solution of a silver (Ag+) salt.

[0023] According to a further embodiment of the present invention, the molar ratio between the Ag+ ions and the chosen threshold value of MUFAs can be from 38:1 to 175:1. For example, the molar ratio between Ag+ (1 ,75 mM, 0,04375 pmol) and C16:1 (10 pM, 0,00025 pmol) is 175 / 1 ; molar ratio between Ag+ (1 ,75 mM 0,04375 pmol) and C16:1 (45 pM, 0,001125 pmol) is 38,9 / 1.

[0024] According to the method of the present invention, the chromophore of step c) can be used in form of a solution having a molar concentration ranging from 1 .5 mM to 2 mM.

[0025] According to the present invention, the molar ratio between the Ag+ ions and the chromophore can be from 0.55:1 to 0.85:1 , preferably 0.58:1 to 0.83:1.

[0026] According to the method of the present invention, the calibration curve of the absorbance can be linear for a concentration of MUFAs, such as C16:1 , in an organic solvent, such as isopropyl-alcohol, ranging from 10 pM to 45 pM, by using a volume of the solution of MUFAs of 25 pL, in a volume of buffer solution, such as sodium acetate-acetic acid buffer solution 0.2 M, pH=5, of 150 pL, a volume of 25 pL of a solution of Ag+ ions at a concentration of 1.75 mM, for example in sodium acetate-acetic acid buffer solution 0.2 M, pH=5, and a volume of 50 pL of a solution of TMB at a concentration of 1.5 mM for example in ethanol.

[0027] The colour of the solution of MUFAs used for the calibration curve ranges from colourless to dark blue, whereas the colour of a solution without MUFAs (blank solution) is deep blue.

[0028] In particular, the present invention concerns the above mentioned method, wherein in step a) the sample is dissolved in the organic solvent, such as isopropylalcohol, in the 1 :100 ratio to obtain the lipid solution and a volume of 25 pL of the lipid solution is used in the method; in step b), the lipid solution is mixed with a volume of 25 pL of a solution of Ag+ ions at the concentration of 1 .75 mM; in step c) a volume of 50 pL of a solution of TMB at the concentration of 1 .5 mM is added; wherein before step c), the step cO) is carried out by adding a volume of 150 pL of a buffer solution, such as a sodium acetate-acetic acid (NaAc) buffer solution, (i.e. the buffer solution can be added before step a) or before step b) or before step c)) and in step d) if the intensity of colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve (i.e. the colour of the solution is from colourless to dark blue), it means that the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs of a subject presenting non physiological levels of MUFAs, i.e. excessive levels of MUFAs as in the case of PD; whereas, if the intensity of colour of the solution of step c) does not correspond to a concentration of MUFAs in the range of linearity of the calibration curve (i.e. the colour of the solution is deep blue colour), steps a) to c) are repeated wherein the sample is dissolved in the organic solvent, such as isopropyl-alcohol, in a ratio of 1 :10 instead of 1 :100, keeping unchanged the other parameters of concentration, volume and compounds the same, in order the concentration of MUFAs of the sample to be comprised in the range of linearity of the calibration curve; and in step d) if the intensity of colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve, the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs of a subject with physiological levels of MUFAs.

[0029] According to an embodiment of the present invention, the samples are Sebutape patches extracted in 500 pL isopropyl-alcohol.

[0030] In addition, according to a further embodiment, the present invention concerns the above-mentioned method wherein in step a) the sample is dissolved in a volume of organic solvent, such as isopropyl-alcohol, of 250 pL to obtain the lipid solution; in step b), the lipid solution is mixed with a volume of 250 pL of a solution of Ag+ ions at a concentration of 1 .75 mM; in step c) a volume of 500 pL of a solution of TMB at a concentration of 1.5 mM is added; wherein before step c), the step cO) is carried out by adding a volume of 1500 pL of a buffer solution, such as a sodium acetate-acetic acid (NaAc) buffer solution (i.e. the buffer solution can be added before step a) or before step b) or before step c)), and in step d) if the intensity of colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve (i.e. the colour of the solution is from colourless to dark blue), the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs above the physiological levels, as in the case of a subject suffering from PD; whereas, if the intensity of colour of the solution of step c) does not correspond to a concentration of MUFAs in the range of linearity of the calibration curve (i.e. the colour of the solution is deep blue colour), steps a) to c) are repeated wherein in step a) the sample is dissolved in a volume of organic solvent, such as isopropyl-alcohol, of 25 pL to obtain the lipid solution; in step b), the lipid solution is mixed with a volume of 25 pL of a solution of Ag+ ions at a concentration of 1 .75 mM; in step c) a volume of 50 pL of a solution of TMB at a concentration of 1.5 mM is added; wherein before step c), the step cO) is carried out by adding a volume of 150 pL of a buffer solution, such as a sodium acetate-acetic acid (NaAc) buffer solution, (i.e. the buffer solution can be added before step a) or before step b) or before step c)) and in step d) if the intensity of colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve (i.e. the colour of the solution is from colourless to dark blue), the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs of a subject with physiological levels of MUFAs in sebum.

[0031] According to the method of the present invention, when the intensity of colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve, the method can comprise also step e).

[0032] For example, in this method the sample can be taken by a swab.

[0033] According to the present invention, the steps a), b) and cO) can be carried out in any order before step c) (i.e. a)+ b)+ cO); a) + cO) + b); b) + a)+ cO); b) + cO) + a); c0)+ a) + b); cO) + b)+ a). According to an embodiment of the present invention, the sample can be added in a mixture of the organic solvent and Ag+ ions and optionally the buffer solution; or in the organic solvent optionally mixed with the buffer solution and then adding Ag+ ions before step c); or to Ag+ ions optionally mixed with the buffer solution and then adding the organic solvent before step c). According to an embodiment of the present invention, step c) is carried out after at least 10 minutes from the previous step (i.e. the last step carried out before step c)).

[0034] According to a preferred embodiment, the biological sample is human sebum.

[0035] The present invention concerns also a method for in vitro diagnosis of excessive MUFAs in sebum as in the case of PD, said method comprising the steps a) to e) according to any one of the previous claims, wherein the excessive MUFAs secretion as in the PD is diagnosed when the concentration of MUFAs in the sample is greater than that of a sample from a healthy control, i.e. subjects that do not suffer from PD.

[0036] According to an embodiment of the present invention, excessive MUFAs levels as in Parkinson's disease is diagnosed when the molar concentration of MUFAs in the human sebum sample is higher than 400 pM, preferably comprised in the range from 400 pM to 5000 pM.

[0037] According to the method of the present invention, the biological sample can be collected by a swab and followed by no sample processing. Alternatively, the biological sample can be collected by adhesive patch to be used in the method of the present invention. The patch advantageously provides the possibility of multiple testing, sample back-up, use for multiple analytical modes.

[0038] On the basis of the above, table 6 shows the concentration range of Ag+ ions and TMB that can be used according to the present invention.

[0039] Table 6

[0040] However, the volumes indicated in table 6 can be multiplied by 10 when the sample comprises high amount of MUFAs so that Ag+ ions and chromophore moles are not sufficient for the reaction with MUFAs and detection. Volumes can be multiplied as long as the ratios of components and of concentrations of them remain the same.

[0041] The present invention concerns also a kit for qualitative and / or quantitative detection MUFAs in a human or animal lipid biological sample (or lipid matrix) comprising Ag+ ions and a chromophore or solutions comprising them.

[0042] Said Ag+ ions can be in a form of a Ag salt, for example a salt chosen from AgNO2, AgNOs, Ag2COs and mixture thereof, preferably AgNOs.

[0043] According to the invention, the chromophore used in the kit can be TMB.

[0044] Regarding the Ag+ ions, they can be in a form of a solution having a concentration of 1 .75 mM.

[0045] According to the invention, the chromophore used in the kit can be in the form of a solution having a concentration of 1 .5 mM.

[0046] The kit according to the present invention can further comprise an organic solvent having a relative polarity in the range from 0.1 to 0.8 in comparison to the polarity of water considered as 1 , for example it can be chosen from the group consisting of methanol, ethanol, 2-propanol, 1 -propanol, 1 -butanol, 2-butanol, acetone, ethyl acetate, acetonitrile, methyl-ethyl ether, diethyl ether, methyl-tert- buthyl ether (MTBE), glycerol and t-butyl alcohol, or a mixture thereof.

[0047] In addition, according to the present invention, the kit can further comprise means of sampling the lipid biological sample.

[0048] According to specific embodiment, said means can be chosen between adhesive tape or cotton swab.

[0049] According to a further embodiment, the kit can comprise a standard such as for example palmitoleic acid or sapienic acid. The standard can be provided in an ampule such as a vial or a test tube and can be used for preparing a calibration curve by diluting the standard in different concentrations. Alternatively, it can be used in only one concentration in order to develop a colour intensity of reference. In addition or alternatively to the standard, the kit of the present invention can comprise a scale of intensity of colour, for example on cardboard, as a reference tool for an approximate measurement of MUFAs concentration.

[0050] The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the examples and the enclosed drawings, wherein Figure 1 shows a calibration curve of palmitoleate (C16:1 , which has the same reactivity of sapienate) ranging from 10 to 45 pM, wherein the mean absorbance was plotted against the concentration expressed in pM (mean abs ± SD).

[0051] EXAMPLE 1 : Study for the development of a method for quantifying MUFAs in a sebum sample according to the invention

[0052] Evaluation of the linearity and the reproducibility of the assay

[0053] Silver nitrate (AgNOs) and 3,3',5,5'-Tetramethylbenzidine (TMB) were used to conduct the assay. A 0.2 M acetate buffer solution was prepared to conduct the reaction at pH 5. The reaction was performed in a 96-well plate: 25 pL of different concentrations of a isopropyl-alcohol solution of sapienate (from 10 pM to 45 pM) and 25 pL of a acetate buffer solution of AgNO3 1 ,75 mM were added to 150 pL of an acetate buffer solution into the well (the same results are obtained by the use of palmitoleate that is the isomer 16:1 n-7). After 10 minutes, the reaction was complete, and 50 pL of solution of TMB 1 ,5 mM in ethanol were added to start the colorimetric reaction. After 10 minutes of stabilization in the dark at room temperature (RT), the absorbance was measured with a spectrophotometer at the wavelength of 652 nm: the corresponding absorbance of Ag+-TMB complex decreased as the C16:1 concentration increased. A calibration curve was plotted using average values from 6 replicates (Table 1 ) to evaluate the linearity and reproducibility, as shown in Figure 1 . As shown by standard deviation (SD) (values reported in Table 1 ) and the correlation coefficient (R2) in the insert, both linearity and reproducibility were satisfactory.

[0054] Table 1

[0055] (RSD (%)= relative standard deviation (%))

[0056] The assay was tested on other authentic standards of lipids present in the sebum composition, to investigate the reactivity of double bonds in their chemical structure: squalene (SQ), triglycerides (TGs) and diglycerides (DGs) (the last two lipid classes were chosen among those binding at least one MLIFA). The calibration curves obtained showed unsatisfactory linearity (R2 = 0.4250 for SQ, R2 = 0.5829 for TG 56:1 , R2 = 0.0321 for DG 34:1 ) suggesting that the assay responds linearly only to double bonds in MUFAs.

[0057] Other silver (Ag+) salts were tested to extend the applicability of the assay. Silver nitrite (AgNO2) and silver carbonate (Ag2COs) showed a similar reactivity as AgNOs (R2=0.9539 for AgNO2, R2=0.9924 for Ag2CO3), at same concentration and experimental conditions, suggesting an interchangeable use of different Ag salts.

[0058] APPLICATION TO SEBUM EXTRACTS The developed test was applied to sebum extracts from patients with neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer's disease (AD) and from healthy controls (HC). Sebum extracts belonging to the three groups (HC, AD and PD) of 20 samples each, previously analysed by GCMS to quantify fatty acids and other components such as squalene, were analysed with the optimized assay. The assay showed a slightly higher concentration of double bonds in AD and PD sebum extracts compared to HC. Difference were not statistically significant. This result was expected for the AD samples, since it was previously demonstrated by GCMS that the concentration of MUFAs in AD is comparable to that of HC (Briganti S, et al. 2021 ). In contrast, the results of the assay applied to the PD sebum was inconsistent with the GCMS results that demonstrated a significantly higher concentration of MUFAs in PD’s sebum (Table 2, correlation value -0.3). This raised the question whether the assay reached a saturation level (all the Ag+ ions have reacted with MUFAs) when the concentration of MUFAs was outside the concentration range demonstrated to have linear correlation with the assay response. To verify the hypothesis, the assay was repeated on samples diluted 1 :100. As shown in the Table 2, the correlation between the assay performed on PD samples diluted 1 :100 and the GCMS results reached a satisfactory value (R=0.8).

[0059] In contrast, when the assay was applied to samples from HC and AD after 1 :100 dilutions, the correlation with GCMS results was deteriorated (data not shown). This suggests that, keeping the amount of Ag+ ions and TMB used constant, the assay needs to be performed in two-steps to best address quantification of double bonds in MUFA-rich sebum samples typical of PD. Moreover, while the GCMS quantitative data showed that PD sebum contained sapienate at a concentration 3 fold higher than sebum from HC, according to the colorimetric assay the level of MUFAs in PD patients is more than 3-fold higher than HC. This observation indicates that beyond sapienate, other MUFA species detected with the colorimetric assay present high abundance in PD sebum.

[0060] Table 2

[0061] Direct application to sebum

[0062] The Applicant investigated the applicability of the assay to sebum collected directly from the skin with a cotton swab, in order to develop in the future a simple device that could extend the use of this diagnostic tool. The cotton swab was rubbed gently on the skin for 5 seconds and then dipped for 10 seconds into the reaction well containing 150 pL acetate buffer solution and 25 pL isopropyl alcohol. Then, 25 pL of an acetate buffer solution of AgNO3 1 ,75 mM were added, and after 10 minutes the assay was completed with the addition of 50 pL of an ethanol solution of TMB 1 ,5 mM. After 10 minutes in the dark at RT, the absorbance values proved that the sebum samples taken directly from the skin of healthy volunteers were reactive similarly to sebum extracted from tapes. To confirm that the reactivity was due to the sebum collected with the swab, 100 pL of the reaction mixture were extracted with ethyl acetate and analysed with GCMS after the assay was concluded. MUFAs C16:1 were detectable in the swab extracts at the end of the double bond assay and its amounts were correlated with the absorbance of the complex TMB-Ag+ ions determined by the assay. This confirmed that the test was applicable to detect rapidly the degree of sebum unsaturation in swabs. In Table 3, the results of the assay applied to three samples taken from three different sites on the face of two healthy volunteers accounting for total six samples are reported.

[0063] The three samples from the same subject (samples 1 , 2 and 3) assayed for double bonds provided results distinguishable from those obtained on the three samples from the second volunteer (samples 4, 5, and 6), indicating consistency within the same donor and donor-to-donor variability. The reaction mixture after the assay was processed to be analysed by GCMS to determine the abundance of sapienate relative to the internal standard (iStd). As shown in Table 3, the assay of double bonds was consistent with the abundance of sapienate in the same sample (correlation coefficient > 0.9).

[0064] Table 3

[0065] CONCLUSIONS

[0066] The assay was successfully applied to the quantification of double bonds in sebum extracts from patches and from sebum sampled with swabs. In the first mode of sampling, the assay was successfully applied to the discrimination of sebum from PD patients. The second sampling approach provided to be directly and rapidly applicable to the assay of double bonds, which proved to be reproducible and consistent with quantitative assessments of MUFAs.

[0067] EXAMPLE 2: Application of the method of the present invention by using Sebutape patches extracted in 500 pL of isopropyl alcohol

[0068] Table 4 shows the steps and optimal conditions of the method of the present invention when the biological sample is the Sebutape patch extracted in 500 pL of IPA.

[0069] Table 4

[0070] EXAMPLE 3: Application of the method of the present invention by using sebum taken directly from the skin with a cotton swab

[0071] Table 5 shows the steps and optimal conditions of the method of the present invention when the biological sample is sebum taken directly from the skin with a cotton swab.

[0072] Table 5

[0073] According to the above experimental results, the AgN03-TMB acceptable concentration range in the assay of the present invention is reported in the following Table 6. Table 6

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Claims

CLAIMS1 ) Method for qualitative and / or quantitative detection of monounsaturated fatty acids in a human or animal lipid biological sample comprising the following steps: a) dissolving said sample in an organic solvent obtaining a lipid solution, b) mixing the lipid solution a) with Ag+ions so that said Ag+ions react with the MUFAs of the lipid biological sample, c) adding a chromophore able to bind unreacted Ag+ ions, obtaining a complex chromophore-Ag+ absorbing the light in the visible range, d) observing the colour of the solution of step c),Ag+ ions moles and chromophore moles of step b) and c) being chosen so that Ag+ ions moles are higher than a chosen threshold value of MUFAs moles contained in the same quantity of biological sample and less than the chromophore moles, the molar concentration of Ag+ ions and of chromophore being chosen so that a calibration curve of the absorbance against known MUFAs molar concentration is linear; wherein, the colour of the solution c) provides a qualitative indication of the MUFAs amount in the biological sample.2) Method according to claim 1 , wherein said method further comprises step e) of obtaining a measure of MUFAs amount in the biological sample by obtaining a measurement of the absorbance of the solution of step c) and comparing said measurement with the calibration curve of the absorbance against known MUFAs molar concentrations.3) Method according to any one of claims 1 -2, wherein said method further comprises a step cO) of adding a buffer solution, such as a sodium acetate-acetic acid (NaAc) buffer solution, before step c).4) Method according to any one of claims 1 -3, wherein said lipid biological sample is chosen from the group consisting of sebum, lipid extract from cells, tissues, plasma, and serum preferably sebum, more preferably human sebum.5) Method according to any one of claims 1 -4, wherein said organic solvent has a relative polarity in the range from 0.1 to 0.8 in comparison to the polarity of water considered as 1 , for example it is chosen from the group consisting of methanol, ethanol, 2-propanol, 1 -propanol, 1 -butanol, 2-butanol, acetone, ethyl acetate, acetonitrile, methyl-ethyl ether, diethyl ether, methyl-tert-buthyl ether(MTBE), glycerol and t-butyl alcohol, or a mixture thereof.6) Method according to any one of claims 1 -5, wherein said Ag+ ions are in form of a Ag salt, for example a salt chosen from AgNO2, AgNOs, Ag2COs and mixture thereof, preferably AgNOs, or a solution thereof.7) Method according to any one of claims 1 -6, wherein said chromophore is TMB.8) Method according to any one of claims 1 -7, wherein the concentration of lipid biological sample in the lipid solution of step a) ranges from 0,2 mg / ml to 1 mg / ml.9) Method according to any one of claims 1 -8, wherein said method comprises a step a1 ) after step a) wherein the lipid solution is diluted in an organic solvent at a volume ratio of from 1 : 10 to 1 :

100. 10) Method according to any one of claims 6-9, wherein said Ag+ ions of step b) are at a molar concentration ranging from 1 .75 mM to 2.5 mM when said Ag+ ions are in form of a solution of a Ag salt.11 ) Method according to any one of claims 1 -10, wherein the molar ratio between the Ag+ ions and the chosen threshold value of MUFAs is from 38:1 to 175:1.12) Method according to claim 10 or 11 , wherein the chromophore of step c) is used in form of a solution having a molar concentration ranging from 1 .5 mM to 2 mM.13) Method according to claim 9 or 11 or 12, wherein the molar ratio between the Ag+ ions and the chromophore is from 0.55:1 to 0.85:1 , preferably 0.58:1 to 0.83:1.14) Method according to any one of claims 1 -13, wherein the calibration curve of the absorbance is linear for a concentration of MUFAs, such as C16:1 , in an organic solvent, such as isopropyl-alcohol, ranging from 10 pM to 45 pM, by using a volume of the solution of MUFAs of 25 pL, in a volume of buffer solution of 150 pL, a volume of 25 pL of a solution of Ag+ ions at a concentration of 1 .75 mM, and a volume of 50 pL of a solution of TMB at a concentration of 1 .5 mM.15) Method according to any one of claims 1 -14, wherein in step a) the sample is dissolved in the organic solvent, such as isopropyl-alcohol, in a ratio of 1 :100 to obtain the lipid solution and a volume of 25 pL of the lipid solution is used in the method; in step b), the lipid solution is mixed with a volume of 25 pL of a solution of Ag+ ions at a concentration of 1 .75 mM; in step c) a volume of 50 pL of a solution of TMB at a concentration of 1.5 mM is added; wherein before step c), the step cO) is carried out by adding a volume of 150 pL of a buffer solution, such as a sodium acetate-acetic acid buffer solution, and in step d) if the colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve, it means that the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs of a subject presenting non physiological levels of MUFAs, such as a subject suffering from Parkinson’s disease; whereas, if the colour of the solution of step c) does not correspond to a concentration of MUFAs in the range of linearity of the calibration curve, steps a) to c) are repeated wherein the sample is dissolved in the organic solvent, such as isopropyl-alcohol, in a ratio of 1 : 10 instead of 1 : 100; and in step d) if the colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve, the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs of a subject with physiological levels of MUFAs.16) Method according to any one of claims 1 -14, wherein in step a) the sample is dissolved in a volume of organic solvent, such as isopropyl-alcohol, of 250 pL to obtain the lipid solution; in step b), the lipid solution is mixed with a volume of 250 pL of a solution of Ag+ ions at a concentration of 1 .75 mM; in step c) a volume of 500 pL of a solution of TMB at a concentration of 1.5 mM is added; wherein before step c), the step cO) is carried out by adding a volume of 1500 pL of a buffer solution, such as a sodium acetate-acetic acid (NaAc) buffer solution, and in step d) if the colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve, the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs abovethe physiological levels, such as for example that of a subject suffering from Parkinson’s disease; whereas, if the colour of the solution of step c) does not correspond to a concentration of MUFAs in the range of linearity of the calibration curve, steps a) to c) are repeated wherein in step a) the sample is dissolved in a volume of organic solvent, such as isopropyl-alcohol, of 25 pL to obtain the lipid solution; in step b), the lipid solution is mixed with a volume of 25 pL of a solution of Ag+ ions at a concentration of 1 .75 mM; in step c) a volume of 50 pL of a solution of TMB at a concentration of 1.5 mM is added; wherein before step c), the step cO) is carried out by adding a volume of 150 pL of a buffer solution, such as a sodium acetate-acetic acid (NaAc) buffer solution, and in step d) if the colour of the solution of step c) corresponds to a concentration of MUFAs in the range of linearity of the calibration curve, the concentration of MUFAs in the biological sample corresponds to a concentration of MUFAs of a subject with physiological levels of MUFAs.17) Method according to any one of the previous claims wherein the biological sample is human sebum.18) Method for in vitro diagnosis of Parkinson's disease, said method comprising the steps a) to e) according to any one of the previous claims, wherein the Parkinson's disease is diagnosed when the concentration of MUFAs in the sample is greater than that of a sample from a healthy control.19) Method according to claim 18, wherein Parkinson's disease is diagnosed when the molar concentration of MUFAs in the human sebum sample is higher than 400 pM, preferably comprised in the range from 400 pM to 5000 pM.20) Kit for qualitative and / or quantitative detection of monounsaturated fatty acids in a human or animal lipid biological sample, said kit comprising Ag+ ions and a chromophore or solutions comprising them.21 ) Kit according to claim 20, wherein said Ag+ ions are in form of a Ag salt, for example a salt chosen from AgNO2, AgNOs, Ag2COs and mixture thereof, preferably AgNOs.22) Kit according to any one of claims 20-21 , wherein the chromophore isTMB.23) Kit according to any one of claims 20-22, wherein the Ag+ ions are in form of a solution having a concentration of 1.75 mM.24) Kit according to any one of claims 20-23, wherein the chromophore is in the form of a solution having a concentration of 1 .5 mM.25) Kit according to any one of claims 20-24, said kit further comprising an organic solvent having a relative polarity in the range from 0.1 to 0.8 in comparison to the polarity of water considered as 1 , for example it is chosen from the group consisting of methanol, ethanol, 2-propanol, 1 -propanol, 1 -butanol, 2-butanol, acetone, ethyl acetate, acetonitrile, methyl-ethyl ether, diethyl ether, methyl-tert- buthyl ether (MTBE), glycerol and t-butyl alcohol, or a mixture thereof.26) Kit according to any one of claims 20-25, said kit further comprising means of sampling the lipid biological sample.27) Kit according to claim 26 wherein said means are chosen between adhesive tape or cotton swab.28) Kit according to any one of claims 20-27, wherein said kit further comprises a standard such as palmitoleic acid or sapienic acid, and or a scale of intensity of colour, for example on cardboard, as a reference tool for an approximate measurement of MUFAs concentration.

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