Direct infrared analysis of post-translational modifications of proteins

Abstract

The present invention relates to a method for measuring post-translational modification of proteins in a subject. The method comprises recording infrared radiation in a predetermined wave number range attenuated by an integument of the subject, wherein the integument is still attached to the subject; and comparing the attenuation of the infrared radiation to a predetermined value in order to obtain information about post-translational modification of proteins in the integument. The present invention also relates to a corresponding system and post-translationally modified integument proteins as markers for in vivo diagnosis of diseases, such as diabetes or renal insufficiency.

Description

[0001] This application is a divisional application of Chinese application filed on March 6, 2017, with application number 201780015122.X (PCT / EP2017 / 055229) and entitled "Direct Infrared Analysis of Post-Translational Modifications of Proteins". Technical Field

[0002] This invention relates to measuring post-translational modifications of proteins in subjects, and more specifically to measuring post-translational modifications of proteins using infrared spectroscopy, whereby post-translational modifications of proteins can represent diseases such as diabetes or renal insufficiency. Background Technology

[0003] Although the prevalence of diabetes is rising dramatically in sub-Saharan Africa, accurate diagnosis and monitoring of this metabolic disease remain problematic for local health professionals. According to revised criteria from the American Diabetes Association, the European Association for the Study of Diabetes, and the International Diabetes Federation, the diagnosis of diabetes is based on plasma glucose or hemoglobin A1c (HbA1c) concentrations. For plasma glucose concentrations, diagnosis is based on a fasting plasma glucose concentration ≥126 mg / dL (7.0 mmol / L), a random plasma glucose concentration ≥200 mg / dL (11.1 mmol / L), or a plasma glucose value ≥200 mg / dL (11.1 mmol / L) in a 2-hour 75 g oral glucose tolerance test (OGTT). For hemoglobin A1c (HbA1c), diagnosis is based on a concentration ≥48 mmol / mol.

[0004] However, the use of these gold standards in sub-Saharan Africa is hampered for several reasons. Venous blood glucose, a widely used tool for diagnosing and monitoring diabetes, is prone to pre-analytical changes. Reported HbA1c results are affected by hemoglobinopathies, iron deficiency, factors affecting red blood cell age and survival, uremia, and the presence of hyperbilirubinemia. Furthermore, a blood sample usually needs to be drawn by a doctor or nurse. Additionally, African patients frequently refuse blood analyses due to cultural or religious objections.

[0005] Increased carbamylation is also known to be observed in patients with renal insufficiency. It should be noted that many adverse health effects observed in end-stage renal failure (e.g., atherosclerosis, anemia) are associated with increased carbamylation. However, to date, there are no clinically useful biomarkers for assessing carbamylation that are readily available in routine clinical laboratories.

[0006] Therefore, there is still a need in the art for inexpensive, rapid, portable and accurate methods to measure post-translational modifications of proteins in subjects, such as glycation or carbamylation, without relying on blood sampling. Summary of the Invention

[0007] One object of the present invention is to provide good methods, systems, and biomarkers for the post-translational modifications of proteins in subjects. One advantage of protein post-translational modifications is that they can provide information for the diagnosis of diseases such as diabetes or renal insufficiency.

[0008] One advantage of embodiments of the present invention is that it allows for the non-invasive measurement of post-translational modifications of proteins, such as glycation or carbamylation. In the case of glycation, it can be an indirect measure of average blood glucose levels.

[0009] One advantage of the embodiments of the present invention is that it can measure protein post-translational modifications in a cheap, fast and portable manner, while still being sufficiently accurate.

[0010] One advantage of the embodiments of the present invention is that, in the case of protein post-translational modification, which is protein glycosylation, glycosylated protein markers can reflect the glycosylation of most proteins associated with target organ damage.

[0011] One advantage of the embodiments of the present invention is that it can obtain the average post-translational modifications of proteins over a longer period of time in a single measurement.

[0012] One advantage of the embodiments of the present invention is that it allows for the observation of the evolution of translational modifications of proteins over a longer period of time.

[0013] One advantage of the embodiments of the present invention is that, in the case of protein post-translational modification, which is protein glycosylation, the measurement of glycosylation can be used to diagnose diabetes.

[0014] One advantage of the embodiments of the present invention is that, in the case of protein carbamylation as a post-translational modification of protein, the measurement of carbamylation can be used to diagnose renal insufficiency.

[0015] The above objectives are achieved by the methods, systems, markers, and uses according to the present invention.

[0016] In a first aspect, the present invention relates to a method for measuring post-translational modifications of proteins in a subject, the method comprising recording infrared radiation attenuated by the subject’s epidermis within a predetermined wavenumber range, wherein the epidermis is still attached to the subject, and comparing the attenuation of the infrared radiation with a predetermined value to obtain information about post-translational modifications of proteins in the epidermis.

[0017] Surprisingly, the attenuation of infrared radiation within a specific wavenumber range allows for the identification of post-translational modifications of proteins, such as glycation or carbamylation, in the cuticle, such as nails, that have not been removed from the subject (e.g., human). In other words, a non-invasive technique for detecting post-translational modifications of nail keratin, such as glycation or carbamylation, has been obtained. Since the measurement technique is based on infrared spectroscopy, an advantage of embodiments of the present invention is that it provides an inexpensive, rapid, and portable, yet sufficiently accurate, method for measuring post-translational modifications. Because no blood sample is required, no doctor or nurse is needed for the test. Another advantage of embodiments of the present invention is that the method does not require nail trimming.

[0018] One advantage of some embodiments of the present invention that measure glycation is that information about fructosamines formed in the nail matrix can be obtained. Another advantage of such embodiments of the present invention is that information about diabetes in subjects can be obtained.

[0019] The method may include irradiating the subject's fingernails with the infrared radiation.

[0020] The subject's outer skin may be the subject's fingernail.

[0021] When the post-translational modification of the protein consists of glycosylation of nail keratin, the method includes recording from 400 to 5500 cm -1 Within the wavenumber range, preferably from 4000 to 5500 cm⁻¹ -1 Within, the optimal selection is from 4200 to 4500 cm. -1 The infrared radiation within the range is compared, and the attenuation is compared with a predetermined value within the said wavenumber range. In some embodiments, the infrared radiation can be between 400 and 5500 cm⁻¹. -1 Within the wavenumber range, preferably between 4000 and 5500 cm⁻¹ -1 Within this range, the optimal selection is between 4200 and 4500 cm. -1 Recording within a range. In some embodiments, comparing the attenuation of the infrared radiation with a predetermined value includes comparing values ​​from a range of 4000 cm. -1 Up to 4500 cm -1 Within, for example, a range of 4000 cm -1 Up to 4500 cm -1 The attenuation of infrared radiation in the region. Recording can also be limited to this wavenumber range. One advantage of the embodiments of the present invention is that it is available from a range of 4000 cm⁻¹. -1 Up to 4500 cm -1 Within, for example, a range of 4000 cm -1 Up to 4500 cm -1The information on the attenuation within allows for 100% accurate detection. The attenuation of the infrared radiation then relates to the attenuation caused by glycation of epidermal proteins in the epidermis. An advantage of embodiments of the invention is that nail protein glycation can be used as a biomarker for detecting diabetes. An advantage of embodiments of the invention is that glycation of diabetic target organs such as the lens of the eye and the kidneys can be studied. An advantage of embodiments of the invention is that the novel biomarker, glycated nail proteins, reflects the glycation of most proteins associated with target organ damage, such as the lens of the eye and the kidneys, via the enzyme fructosamine 3 instead of the currently used biomarker HbA1c, which typically undergoes deglycation.

[0022] When the post-translational modification of the protein consists of carbamylation of nail keratin, the method includes recording from 4650 to 7700 cm. -1 Infrared radiation in the wavenumber range, for example, 4650 to 7700 cm⁻¹. -1 The infrared radiation is measured within a certain wavenumber range, and the attenuation comparison includes comparing it with a predetermined value within said wavenumber range. In some embodiments, comparing the attenuation of the infrared radiation with the predetermined value includes a comparison range of 4650 cm⁻¹. -1 Up to 7700 cm -1 The attenuation of infrared radiation within this range. Recording can also be limited to this wavenumber range. One advantage of the embodiments of the present invention is that it comes from a range of 4650 cm⁻¹. -1 Up to 7700cm -1 Information on attenuation within the range allows for 100% accurate detection. Recording and / or comparing infrared radiation may include recording and / or comparing wavelengths from 1300 nm to 2150 nm, for example, from 1300 nm to 1500 nm and / or from 1525 nm to 1575 nm, and / or from 1625 nm to 1700 nm and / or from 1725 nm to 1775 nm and / or from 1825 nm to 2100 nm, for example, from 1825 nm to 1950 nm and / or from 1925 nm to 2050 nm and / or from 2050 nm to 2100 nm. The attenuation of the infrared radiation then relates to attenuation caused by carbamylation of exoskeleton proteins in the exoskeleton. An advantage of embodiments of the invention is that carbamylation of nail proteins can be used as a biomarker for detecting renal insufficiency. One advantage of embodiments of the present invention is that, based on carbamylation, it allows for the study of adverse health effects observed in end-stage renal failure, such as atherosclerosis and anemia. Another advantage of embodiments of the present invention is that the novel biomarker, carbamylated nail proteins, reflects the carbamylation of most proteins associated with renal failure.

[0023] The protein may be keratin. One advantage of embodiments of the present invention is that post-translational modifications of nail keratin can be used as a marker, since keratin is the most abundant nail protein.

[0024] The measured post-translational modifications of the protein, such as glycosylation or carbamylation, can reflect the average post-translational modifications of the protein over a period of 0.5 to 9 months (preferably 1 to 6 months) prior to the recording. An advantage of embodiments of the invention is that the measurements provide information on long-term post-translational modifications of the protein, i.e., post-translational modifications that have occurred over the past few months, thus yielding averaged and reliable values.

[0025] The method may include performing Fourier transform infrared spectroscopy. One advantage of embodiments of the invention is that an efficient measurement method is obtained, allowing for rapid detection of desired results. The method may include infrared reflectance spectroscopy. Another advantage of embodiments of the invention is that measurements can be performed with little or no sample preparation, as in vivo measurements are possible. Another advantage is that the technique is optical rather than invasive.

[0026] The method may include comparing the contributions of different spectral bands to determine the degree of post-translational modifications of proteins in the subject, such as glycation or carbamylation. One advantage of embodiments of the invention is that accuracy can be further improved by considering different spectral bands.

[0027] The method may include first removing contaminants from the outer skin. One advantage of embodiments of the invention is that contaminants such as nail polish are removed first to avoid affecting the measurement signal and to obtain more accurate results.

[0028] Predetermined values ​​can be selected based on the subject's race and / or gender. An advantage of embodiments of the invention is that it takes into account inter-racial differences.

[0029] The method may include recording the attenuation of infrared radiation at different locations on the nail bed, comparing the attenuation of the infrared radiation at different locations with predetermined values, and based on this, obtaining the time dependence of post-translational modifications of proteins, such as glycation or carbamylation. An advantage of embodiments of the invention is that by measuring at different locations on a subject's nail along the nail's growth direction, the temporal evolution of diseases such as diabetes or renal insufficiency can be obtained.

[0030] In a second aspect, the present invention relates to a system for measuring post-translational modifications of proteins in a subject, the system comprising an infrared radiation source, an infrared radiation detector, and a data analyzer, the data analyzer being used to analyze the attenuation of infrared radiation within a predetermined wavenumber range by which the attenuation is still attached to the subject's skin by comparing the attenuation with a predetermined value, in order to obtain information about post-translational modifications of proteins in the skin.

[0031] When the post-translational modifications of the protein consist of glycosylation of nail keratin, the data analyzer is adapted to analyze samples from 400 to 5500 cm⁻¹. -1 Within the wavenumber range, preferably from 4000 to 5500 cm⁻¹ -1 Within, the optimal selection is from 4200 to 4500 cm. -1 The attenuation of infrared radiation within the range. In a specific implementation, the range is 4000 cm. -1 Up to 4500 cm -1 .

[0032] When the post-translational modification of the protein consists of carbamylation of nail keratin, the data analyzer is tuned to operate at 4650 to 7700 cm⁻¹. -1 The data analyzer is adapted to analyze the attenuation of infrared radiation within the wavenumber range of 1300 nm to 2150 nm, for example, from the wavelength range of 1300 nm to 1500 nm and / or from the range of 1525 nm to 1575 nm, and / or from the range of 1625 nm to 1700 nm and / or from the range of 1725 nm to 1775 nm and / or from the range of 1825 nm to 2100 nm, for example, from the range of 1825 nm to 1950 nm and / or from the range of 1925 nm to 2050 nm and / or from the range of 2050 nm to 2100 nm.

[0033] The system may include a retainer or positioning device for positioning fingers or toes, such that the subject's fingernails are positioned relative to the infrared radiation source and the infrared radiation detector.

[0034] The system may include a Fourier transform infrared spectrometer.

[0035] The system can be configured to measure reflected infrared radiation.

[0036] The analyzer can be adapted to compare the contributions of different spectral bands in subjects in order to obtain the degree of post-translational modifications of proteins, such as glycosylation or carbamylation.

[0037] The system can be adapted to take measurements at different locations on the subject's skin.

[0038] The system may include a scanner for scanning the skin of a subject with an irradiation beam.

[0039] In a third aspect, the present invention relates to a post-translational modified epithelial protein for in vivo diagnosis of aberrant post-translational modifications. An advantage of embodiments of the invention is the availability of biomarkers for in vivo diagnosis of aberrant post-translational modifications representing disease. The post-translational modified epithelial protein can be a biomarker for the in vivo diagnosis of the aberrant post-translational modifications. Aberrant post-translational modifications are typically caused by disease.

[0040] In some embodiments, the present invention relates to a glycated epithelial protein for in vivo diagnosis of abnormal blood glucose. One advantage of embodiments of the invention is that it provides a biomarker for in vivo diagnosis of abnormal blood glucose levels representative of diabetes.

[0041] Glycated epithelial proteins can be used as a biomarker for diagnosing abnormal blood glucose levels in vivo, a condition caused by diabetes.

[0042] In some embodiments, the present invention relates to a carbamylated epithelial protein for in vivo diagnosis of abnormal carbamylation. One advantage of the embodiments of the present invention is that it provides a biomarker for in vivo diagnosis of abnormal carbamylation representing renal insufficiency.

[0043] Carbamylated epithelial proteins can serve as biomarkers for the in vivo diagnosis of abnormal carbamylation, which is caused by renal insufficiency.

[0044] Post-translational modifications of the outer skin protein can be keratin.

[0045] In a fourth aspect, the present invention relates to the use of post-translational modified epithelial proteins for in vivo diagnosis of abnormal post-translational modifications. Post-translational modifications may be, for example, glycosylation or carbamylation.

[0046] In vivo diagnoses with aberrant post-translational modifications can include diseases such as diabetes or renal insufficiency.

[0047] Specific and preferred aspects of the invention are set forth in the appended independent and dependent claims. Features from dependent claims may be combined with features of the independent claims and other appropriate features of dependent claims, rather than being explicitly set forth in the claims.

[0048] Despite the continuous improvement, change, and development of equipment in this field, existing concepts are considered to represent substantial new and novel improvements, including deviations from existing practices, thereby providing more efficient, stable, and reliable equipment of this nature.

[0049] The above and other features, characteristics, and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings, which illustrate the principles of the invention by way of example. This description is given for illustrative purposes only and does not limit the scope of the invention. Reference numerals cited below are taken with reference to the accompanying drawings. Attached Figure Description

[0050] Figure 1 A system for measuring post-translational modifications of proteins according to an embodiment of the present invention is shown.

[0051] Figures 2a to 4b Near-infrared spectra (a) and spectral analysis (b) according to examples of the present invention for different wavenumber ranges are shown.

[0052] Figure 5 An embodiment according to the present invention is shown, showing the change of glycation over time during treatment.

[0053] Figure 6a and 6b The control group was shown. Figure 6a ) and diabetes group ( Figure 6b The difference in glycation between the proximal and distal regions of the fingernail illustrates a feature of an embodiment according to the invention.

[0054] Figure 7a and 7b An embodiment according to the present invention is shown, for the infrared spectrum of carbamate ( Figure 7a ) and spectral analysis ( Figure 7b ).

[0055] In different figures, the same reference numerals refer to the same or similar elements. Detailed Implementation

[0056] The invention will be described with reference to specific embodiments and certain accompanying drawings, but the invention is not limited thereto, but is limited only by the claims. The described drawings are illustrative only and not restrictive. In the drawings, the sizes of some elements may be exaggerated and not drawn to scale for illustrative purposes. Dimensions and relative dimensions do not correspond to actual reductions in practice.

[0057] Furthermore, the terms first, second, third, etc., used in the specification and claims are used to distinguish similar elements and are not necessarily used to describe an order in time, space, sequence, or any other manner. It should be understood that the terms thus used are interchangeable where appropriate, and embodiments of the invention described herein can operate in orders other than those described or shown herein.

[0058] Furthermore, the terms top, bottom, above, below, etc., used in the specification and claims are for descriptive purposes and are not necessarily used to describe relative positions. It should be understood that such terms are interchangeable where appropriate, and embodiments of the invention described herein can operate in orientations other than those described or shown herein.

[0059] It should be noted that the term "comprising" as used in the claims should not be construed as limiting itself to the devices listed thereafter; it does not exclude other elements or steps. Therefore, it should be interpreted as specifying the presence of the features, integers, steps, or components mentioned, but not excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the expression "device comprising devices A and B" should not be limited to devices consisting solely of components A and B. This means that for the purposes of this invention, the only relevant components of the device are A and B.

[0060] Throughout this specification, references to "an embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the phrase "in an embodiment" or "in an embodiment" in various places throughout this specification does not necessarily refer to all but may refer to the same embodiment. Furthermore, as will be appreciated by those skilled in the art from this disclosure, a particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0061] Similarly, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description therein to facilitate improvement of this disclosure and to aid in understanding one or more of the various inventive aspects. However, this approach of the disclosure should not be construed as reflecting an intention that the claimed invention requires more features than are expressly stated in each claim. Rather, as reflected in the appended claims, inventive aspects lie in fewer than all features of a single foregoing disclosed embodiment. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, wherein each claim is independently a separate embodiment of the invention.

[0062] Furthermore, while some embodiments described herein include features not included in other embodiments, it will be understood by those skilled in the art that combinations of features from different embodiments are intended to fall within the scope of the invention and form different embodiments. For example, any of the claimed embodiments in the appended claims may be used in any combination.

[0063] Furthermore, some of the embodiments described herein are methods or combinations of method elements that can be implemented by a processor of a computer system or by other means of implementing the functions described herein. Therefore, a processor having the necessary instructions for executing the elements of such a method or method forms a means for implementing the elements of such a method or method. Moreover, the elements of the apparatus embodiments described herein are examples of means for implementing the functions performed by said elements for the purposes of this invention.

[0064] Numerous specific details are set forth in the description provided herein. However, it should be understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this specification.

[0065] Embodiments of the present invention relate to the detection of protein modifications in the human exoskeleton. In some embodiments, detecting protein glycation in the exoskeleton, such as nails, allows for the measurement of glycation in a subject. Glycation can be an indirect measure of blood glucose levels, which will be discussed in detail below. In other embodiments, protein carbamylation in the exoskeleton is detected as a biomarker of carbamylation in renal insufficiency. Carbamylation is a chemical reaction that occurs when the isocyanate urea isomer reacts with lysine residues of a protein. Since human nails are composed of a specific protein (keratin), carbamylation of keratin can be considered an excellent model for assessing carbamylation in a living individual. However, it should be noted that the present invention is not limited to glycation and carbamylation. In a first aspect, the present invention relates to a method for measuring post-translational modifications of proteins in a subject. The method according to embodiments of the present invention is particularly applicable to measuring glycation or carbamylation in the human body, although embodiments of the present invention are not limited thereto, and the method is applicable in principle to any organism.

[0066] According to an embodiment of the invention, the method includes recording infrared radiation attenuated by the subject's skin within a predetermined wavenumber range, wherein the skin is still attached to the subject. The method can typically be performed after irradiating the subject's skin with an infrared radiation beam containing radiation within the predetermined wavenumber range; however, the irradiation step can be performed before applying the method, making it not necessarily a step of the method itself. Alternatively, the method may include irradiating the subject's skin with the infrared radiation. In an embodiment of the invention, the method further includes comparing the attenuation of the infrared radiation with a predetermined value and obtaining information about post-translational modifications of proteins based thereon. Embodiments of the invention are particularly advantageous for obtaining information about a subject's disease, such as diabetes or renal insufficiency. According to an embodiment of the invention, in the case of glycation, wavenumbers from 400 to 5500 cm⁻¹ can be used. -1 Within, advantageously derived from the wavenumber range of 4000 cm⁻¹-1 Up to 4500 cm -1 Radiation within the range of 4650 to 7700 cm⁻¹ can be used in the case of carbamylation, according to some other embodiments. -1 Radiation within.

[0067] As an illustration, the features and advantages of additional optional and standard steps of the method according to embodiments of the present invention will be further discussed below. Reference will thus be made to glycosylation of proteins, such as those used in diabetes. Clearly, similar details are applicable to other post-translational modifications of proteins, such as carbamylation, but the corresponding wavenumber ranges, optical components, etc., require appropriate adaptation with necessary modifications. Such optical components are readily available.

[0068] For irradiating the skin, an infrared laser beam or a focused irradiation beam can be used. To generate infrared radiation, an infrared laser, such as a near-infrared diode laser, can be used, but other infrared radiation sources, such as halogen NIR sources, can also be used. The infrared radiation beam spot advantageously has a size such that the irradiation does not extend beyond the surface of the skin, such as the surface of a fingernail. Typical beam spot sizes that can be used correspond to a diameter of up to 3 mm, although embodiments are not limited to this.

[0069] According to an embodiment of the invention, the infrared radiation can originate from the range of 4000 to 4500 cm. -1 Inside, the attenuation of infrared radiation in this range is very significant due to the glycated epidermal proteins.

[0070] For irradiating the skin, the subject is typically required to position the body part containing the skin in a specific location so that irradiation can be performed in a controlled manner. For example, the subject may be asked to position a finger or toe in a holder so that the nail can be irradiated in a controlled manner. The holder or positioning device may be such that the skin is positioned below the infrared radiation source and below a detector that is adjusted to detect reflected radiation.

[0071] Detection can be performed by detecting the spectrum of radiation attenuated by the outer skin. Alternatively, detection at several specific wavelengths or wavelength ranges can also be performed, allowing for the identification of glycosylation of outer skin proteins.

[0072] In embodiments, the detection techniques that can be used may include performing near-infrared (NIR) spectroscopy, or may include performing Fourier transform infrared spectroscopy, such as ATR-FTIR. Both techniques allow for relatively short scan times for a given resolution. Therefore, one advantage of embodiments of the present invention is that an efficient measurement method is obtained that allows for rapid detection of desired results.

[0073] In some embodiments, the method may include infrared reflectance spectroscopy. In a preferred embodiment, the infrared reflectance spectroscopy may include attenuated total reflectance spectroscopy.

[0074] Attenuated total internal reflection (ATR) is a sampling technique that can be used in conjunction with infrared spectroscopy. It advantageously allows for the measurement of solid or liquid samples without further preparation, requiring only contact between the sample and the ATR crystal. Therefore, one advantage of embodiments of the invention is that measurements can be performed with little or no sample preparation, enabling in vivo measurements. Another advantage is that the technique is optical rather than invasive.

[0075] As noted above, the subject's cuticle can typically be the subject's fingernail. In a preferred embodiment, the nail area probed during measurement can be the fingernail plate. Fingicles typically contain a large proportion, such as about 85%, of keratin, which is one of the markers that can be used to measure the glycation of proteins in the cuticle or indirectly measure blood glucose levels. Therefore, one advantage of embodiments of the present invention is that the recorded infrared radiation may have been attenuated by the nail, and thus typically contains a relatively strong marker signal. Furthermore, the nail plate typically does not contain blood vessels and is metabolically inactive, which reduces the possible factors that could affect or interfere with the measurement results. However, embodiments are not limited to using fingernails as the cuticle. In some embodiments, the cuticle can also be the subject's skin.

[0076] In some implementations, comparing the attenuation of infrared radiation with a predetermined value may include comparing radiation from a range of 4000 cm. -1 Up to 4500 cm -1 The attenuation of infrared radiation within the body. It has been found that using this specific infrared radiation allows for very accurate measurements of glycated blood sugar, and therefore very accurate detection of diabetes. Comparisons of attenuation can be performed, for example, using a predetermined algorithm or a lookup table. This can be performed, for example, using a processor programmed to perform such comparisons on input and / or automatically. The comparison provides results that can be provided as output, such as a displayed comparison result. The comparison result can provide an indication of whether the measured level is above or below a specific threshold. However, based on the comparison result, an indication of whether a subject has diabetes can be given; this diagnosis is not part of the method in some embodiments and can be performed independently by a medically trained person, i.e., outside of the method. Therefore, the diagnosis may not be part of the method.

[0077] In some implementations, predetermined values ​​for comparison can be determined based on additional subject characteristics such as race and / or sex. Therefore, such information can be used as input and prompted by the processor. This information is then combined with the obtained measurements to arrive at the comparison results.

[0078] One advantage of the embodiments of the present invention is that it allows for the study of glycation in diabetic target organs such as the lens of the eye and the kidneys.

[0079] One advantage of the embodiments of the present invention is that the new glycosylated epidermal protein markers reflect the glycosylation of most proteins associated with target organ damage, such as the lens of the eye and the kidneys, rather than the currently used marker HbA1c, through the enzyme fructosamine 3, which typically undergoes deglycosylation.

[0080] In this implementation, the protein may be keratin.

[0081] One advantage of the embodiments of the present invention is that cutaneous keratin glycation can be used as a marker, because keratin is an abundant cutaneous protein, especially in nails.

[0082] According to some embodiments, the method provides results representing average blood glucose levels, for example, blood glucose levels occurring over a period of 0.5 to 9 months, or over a period of 1 to 6 months. An advantage of embodiments of the invention is that the measurements provide long-term blood glucose information, i.e., blood glucose levels occurring over the past few months, thus yielding average, reliable values.

[0083] According to some embodiments, the measurement allows for the measurement and tracking of the temporal evolution of cuticle proteins. The method may, for example, include recording the attenuation of infrared radiation at different locations on the cuticle, comparing the attenuation of said infrared radiation at different locations to predetermined values, and obtaining a time dependence of blood glucose based on this. If the measurement can be performed at different locations, for example, along the direction of nail growth, this allows for obtaining results representing the temporal evolution of glycation. For example, the tip of the nail plate typically reflects earlier glycation, such as glycation 6 to 9 months prior to the measurement, while areas of the nail plate closer to the nail matrix typically reflect more recent glycation, such as glycation 0.5 to 1 month prior to the measurement. Therefore, measurements at different locations on the nail can be used to establish, for example, the temporal evolution of glycation over the most recent 6 to 9 months. Furthermore, by sampling larger or smaller portions of the nail, average glycation values ​​over larger or smaller time windows can be obtained; for example, by sampling the entire nail plate, average glycation values ​​over the most recent 6 to 9 months can be obtained.

[0084] In some embodiments, the method may include first removing contaminants from the outer skin. This can be done, for example, using conventional cleaning products. An advantage of embodiments of the invention is that contaminants such as nail polish are removed first to avoid affecting the measurement signal and to obtain more accurate results.

[0085] In a second aspect, the present invention relates to a system for measuring post-translational modifications of proteins in a subject, such as glycation or formamide, the system comprising an infrared radiation source, an infrared radiation detector, and a processor, such as a data analyzer, for analyzing the attenuation of infrared radiation within a predetermined wavenumber range still attached to the subject's skin by comparing the attenuation with predetermined values, in order to obtain information about the protein's post-translational modifications. According to an embodiment of the invention, in the case of glycation, a data analyzer can be used to analyze radiation from a wavenumber range of 400 to 5500 cm⁻¹. -1 Within, advantageously derived from the wavenumber range of 4000 cm⁻¹ -1 Up to 4500 cm -1 Radiation within the range. According to some other embodiments, in the case of carbamylation, a data analyzer can be used to analyze radiation from the wavenumber range of 4650 to 7700 cm⁻¹. -1 Radiation within the body. The system is particularly suitable for performing methods as described in the first aspect for measuring protein post-translational modifications.

[0086] As an explanation, Figure 1 The system according to an embodiment of the invention is schematically illustrated. Similarly, the described system is particularly suitable for measuring glycosylation, but it should be understood that corresponding systems for measuring other post-translational modifications of proteins are described by adjusting the wavenumber range and corresponding optical components, and thus such systems are also described. Figure 1 A system 100 for measuring blood glucose is shown. System 100 includes a radiation source 110; a detector 120 configured to detect infrared radiation from the radiation source 110 after it has been attenuated by the subject's skin; and a holder or positioning device 130 for positioning the subject's skin relative to the radiation source 110 and the detector 120. The holder or positioning device 130 may, for example, be adapted to hold a hand or finger or foot or toe in a position such that the fingernail or toe is accurately positioned for measurement. Alternatively, the positioning device may correspond to a positioning device of the system such that it can be mounted to a hand, finger, foot, or toe, and such that the fingernail or toe is accurately positioned relative to the system, making measurement possible. The system also includes a processing device or processor 140 for processing the detected attenuated radiation received at the detector 120. The processor is thus adapted to compare the obtained measurement result with a predetermined value. This comparison or result based thereon may be output via an output device 150. As noted in the first aspect, the radiation source and detector may be adapted to generate radiation from a range of 4000 cm⁻¹. -1 Up to 4500 cm -1The detector can be an infrared radiation within this frequency range, and / or the processor can be adapted to process infrared radiation within this frequency range. The detector can be a Fourier transform infrared spectrometer, a detector suitable for measuring reflected IR radiation, or a detector configured to perform attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR).

[0087] In the implementation, the processor can be adapted to compare the contributions of different spectral bands in order to obtain the blood glucose level in the subject.

[0088] In some embodiments, the system can be adapted to perform measurements at different locations on the subject's skin, for example, by providing scanning motion of an irradiation beam across the skin. In some embodiments, the system may therefore include a scanner for scanning the subject's skin with the irradiation beam.

[0089] Other features and advantages of the system may correspond to those described for the corresponding method in the first aspect.

[0090] In a third aspect, the present invention relates to a post-translational modified epidermal protein, such as a glycosylated epidermal protein or a carbamylated epidermal protein, for in vivo diagnosis of aberrant post-translational modifications of proteins. An advantage of embodiments of the invention is the availability of biomarkers for in vivo diagnosis of aberrant post-translational modifications of proteins. Aberrant post-translational modifications of proteins typically represent, for example, a hyperglycemic form, a hypoglycemic form, or a hypercarbamylated form, as caused by diabetes. In other cases, aberrant post-translational modifications of proteins may be time-varying or irregular post-translational modification forms. In embodiments, the post-translational modified epidermal protein can be a biomarker for in vivo diagnosis of aberrant post-translational modifications of proteins. In embodiments, the aberrant post-translational modification of the protein may be caused by diabetes. In embodiments, the post-translational modified epidermal protein may be keratin. The epidermis may be a nail.

[0091] In a fourth aspect, the present invention relates to the use of post-translational modified epithelial proteins for in vivo diagnosis of aberrant post-translational modifications of proteins. In embodiments, the in vivo diagnosis of said aberrant post-translational modifications of proteins may include the diagnosis of diseases such as diabetes or renal insufficiency.

[0092] The invention will now be described in detail through several embodiments thereof. It will be apparent that other embodiments of the invention may be configured based on the knowledge of those skilled in the art without departing from the true technical teachings of the invention, which is limited only by the terms of the appended claims.

[0093] Example: Infrared spectra of fingernails in diabetic patients and control groups

[0094] Near-infrared spectra were taken from the nail plates of 5 patients diagnosed with diabetes who belonged to the same ethnic group and 25 people in the control group.

[0095] NIR spectra at 4200 cm⁻¹ -1 -5400 cm -1 Shot and plotted within the wavenumber range Figure 2a As can be seen, the spectra of the two groups are clearly distinguishable. To better quantify this spectral difference, for each measurement, by... Figure 2b The standard normal variables (SNVs) were listed to analyze the spectrum. A fairly good separation was observed between the data points corresponding to the diabetes group on one hand and the control group on the other.

[0096] To further improve separation, the spectral range was reduced to 4200-4500 cm⁻¹. -1 And the obtained spectrum is as follows Figure 3a As shown. Similarly, for each measurement, by... Figure 3b The SNVs are listed for spectral analysis. Further improvement in separation between the two groups can be observed. Furthermore, after training the evaluation model using a set of training data, it can predict with 100% accuracy which group a given sample belongs to.

[0097] Similarly, the spectral range is limited to 5060–5400 cm⁻¹. -1 And the corresponding spectrum is as follows Figure 4a As shown. Similarly, for each measurement, in Figure 4b The spectrum was analyzed to determine the second derivative. However, in this case, the separation was not improved, and a 100% conclusive prediction could not be performed, similar to the evaluation of the first derivative (not shown).

[0098] Example: Monitoring glycation during treatment

[0099] Near-infrared spectra of the patient's nail plate were captured and analyzed to monitor changes during treatment. As glucose diffuses from the capillary network into the fingernail and the nail grows over time, fluctuations in the NIR spectrum due to changes in glucose diffusion from the blood to the nail can be observed. Therefore, this allows for the observation of data improvements as diabetic patients undergo treatment over time. Figure 5In this study, patients with type 2 diabetes were monitored during treatment with metformin (3 x 850 mg daily). Standardized normal values ​​for the first derivative of the spectra are also given. Results for specific patients (empty squares) are discussed in detail, with reference values ​​for the control group (C) and the diabetes group (DM) also shown. Measurement number 26 is the first measurement at the start of treatment and falls within the diabetes (blue) group. Measurement number 27 is the second measurement, several weeks after the patient received treatment. It can be seen that the second measurement is progressing towards the control group. This means the patient's condition is improving (mean blood glucose decreased from 279 mg / dL to 105 mg / dL). Measurement number 30 is the third measurement, also several weeks after the second measurement. This graph (data collected over 5-week intervals) nicely illustrates the ability to assess glycemic control using non-invasive NIR monitoring.

[0100] Example: Evaluating time evolution based on NIR measurements of different regions of the fingernail.

[0101] For both the control and diabetic groups, NIR spectroscopy in the spectral range of 1460 nm to 1630 nm was used to measure different regions of the fingernails. A difference between the proximal and distal regions of the fingernail indicated glucose accumulation in the nail during nail growth. Results for the control group showed... Figure 6a In the middle, while the results of the diabetes group showed Figure 6b In both cases, roughly two sets are visible on the score chart. The distal measurements are more to the left, and the proximal measurements are more to the right. However, some overlap still exists between the two areas due to variations in glucose concentration in the fingernails of different test subjects.

[0102] Example: Infrared spectra of fingernails in patients with renal insufficiency and control groups

[0103] Near-infrared spectra were taken from the nail plates of many patients with end-stage renal insufficiency and many control group members who belonged to the same ethnic group.

[0104] NIR spectra were captured in the wavelength range of 1300 nm to 2150 nm. Figure 7a As can be seen, a spectral difference can be observed between the spectra of people in the control group (c) and those with renal insufficiency (N). To better quantify this spectral difference, principal component analysis of the spectra was performed, and by... Figure 7b The standard normal variable (SNV) was listed to analyze the spectrum. It can be seen that there is a significant difference between the control group (C) and those with renal insufficiency (N). It can be seen that near-infrared spectroscopy on human fingernails provides an excellent tool for the non-invasive assessment of carbamylation.

[0105] It should be understood that although preferred embodiments, specific constructions and configurations, and materials have been discussed herein with respect to the device according to the invention, various changes or modifications in form and detail may be made without departing from the scope of the invention and the technical teachings. For example, any formulas given above merely represent usable procedures. Functions may be added or removed from the block diagrams, and operations may be exchanged between function blocks. Steps may be added or removed from the methods described within the scope of the invention.

Claims

1. A system for measuring post-translational modifications of proteins in a subject, the system comprising an infrared radiation source and an infrared radiation detector configured to record infrared radiation within a predetermined wavenumber range on the skin attached to the subject, the skin referring to a plate, the infrared radiation being attenuated by the skin; and A data analyzer, programmed to analyze the attenuation of recorded infrared radiation by comparing the attenuation with predetermined values, in order to obtain information about post-translational modifications of proteins in the outer skin.

2. The system of claim 1, wherein the system includes a retainer for positioning a finger or toe such that the subject's fingernail is positioned relative to the infrared radiation source and the infrared radiation detector while the fingernail remains attached to the subject.

3. The system according to any one of claims 1 or 2, wherein the system is adapted to perform measurements at different locations on the subject's skin.

4. The system according to any one of claims 1 to 3, wherein the data analyzer is configured to detect the attenuation of post-translational modifications of epidermal proteins in the epidermis.

5. The system of claim 4, wherein the data analyzer is configured to detect attenuation caused by post-translational modifications of keratin.

6. The system according to any one of claims 1 to 5, wherein the post-translational modification of the protein consists of glycosylation of nail keratin, and the data analyzer is adapted to analyze samples from 400 to 5500 cm⁻¹ -1 Within the wavenumber range, preferably from 4000 to 5500 cm⁻¹ -1 The attenuation of infrared radiation within.

7. The system according to any one of claims 1 to 5, wherein the post-translational modification of the protein comprises carbamylation of nail keratin, and the data analyzer is adapted to analyze data from 4650 cm⁻¹. -1 Up to 7700 cm -1 The attenuation of infrared radiation within the wavenumber range.

8. The system according to any one of claims 1 to 7, wherein the system is configured to record the attenuation of infrared radiation at different locations on a nail plate, compare the attenuation of infrared radiation with a predetermined value, and obtain the time dependence of protein post-translational modifications based on the attenuation recorded at different locations on the nail plate.

9. The system according to any one of claims 1 to 8, wherein the system is configured to perform any one of near-infrared spectroscopy, Fourier transform infrared spectroscopy, or infrared reflectance spectroscopy.

10. The system according to any one of claims 1 to 9, wherein the data analyzer is configured to compare the contributions of different spectral bands to obtain the blood glucose level in the subject.

11. The system according to any one of claims 1 to 10, wherein the system includes input to obtain information about the race or sex of the subject, and wherein the data analyzer is configured to select a predetermined value based on the obtained information about the race or sex of the subject.

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