Method for estimating muscle mass

Abstract

To provide a method for estimating a muscle mass of a subject.SOLUTION: A method for estimating a muscle mass of a subject, the method includes the steps of: (1) measuring an amount of a specific modified nucleoside contained in a biological sample collected from the subject; and (2) estimating a muscle mass of the subject from the measured amount of the specific modified nucleoside. The modified nucleoside is at least one modified nucleoside selected from the group consisting of 5-methoxycarbonyl methoxyuridine (mcmo5U), 1-methylguanosine (m1G), 2'-O-methyladenosine (Am), N4-acetylcytidine (ac4C), and N6-threonylcarbamoyladenosine (t6A).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for estimating muscle mass, and more specifically, to a method for estimating muscle mass of a subject by measuring modified nucleosides in urine or blood collected from the subject. [Background technology]

[0002] Japan is currently entering a super-aging society, and social security costs, primarily for medical care and nursing care, are steadily increasing. Muscle weakness is particularly common among the elderly, which can lead to falls and fractures. Falls and fractures can lead to a decline in activities of daily living (ADL). A decline in ADLs can lead to the need for long-term nursing care. Therefore, it is important to maintain and improve muscle strength to reduce the risk of falls and fractures and maintain ADLs.

[0003] Muscle mass estimation is commonly used to assess muscle weakness in subjects. Bioelectrical impedance analysis (BIA) is a convenient method for estimating muscle mass. This method involves passing a weak current through the subject's body, and body composition information (e.g., muscle mass, total body water percentage, total body fat percentage) is calculated based on the bioelectrical impedance measured during the process (see, for example, Patent Documents 1 and 2). While BIA has the advantages of being simple, rapid, and noninvasive, accurate results require appropriate measurement conditions and expert interpretation because results are affected by fluctuations in water balance and measurement conditions. For example, diurnal variations can lead to inaccuracies, and measurement results can change depending on skin conditions such as sweating and dermatitis. Furthermore, the correction formulas used to calculate muscle mass from measurement results differ depending on the device, resulting in different results depending on the model of the measurement device.

[0004] In addition, reliable measurement of muscle mass using the bioelectrical impedance method requires the use of expensive equipment (mainly medical equipment), and measurements are limited to medical institutions and rehabilitation facilities equipped with such equipment.Furthermore, because bioelectrical impedance measurement involves passing an electric current through the body, there is a problem that it cannot be performed on subjects with implanted medical devices such as pacemakers. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2004-344348 [Patent Document 2] WO2019 / 012897 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for estimating muscle mass that is simple, inexpensive, minimally invasive, and has few limitations on the subjects to be tested. [Means for solving the problem]

[0007] The present inventors have intensively studied the physiological significance of chemical modifications in ribonucleic acid (RNA) in mammals, and as a result have found that specific modified nucleosides, such as 5-methoxycarbonylmethoxyuridine (MCM), are present in the urine of test subjects. 5 The present inventors have found that the amount of α-glucan (U) significantly correlates with the muscle mass of the subject, and have completed the present invention. The present invention includes the following aspects. [1] A method for estimating muscle mass of a subject, comprising measuring the amount of a modified nucleoside contained in a biological sample collected from the subject, The modified nucleoside is 5-methoxycarbonylmethoxyuridine (mcmo 5 U), 1-methylguanosine (m1G), 2'-O-methyladenosine (Am), N4 -acetylcytidine (ac4C), and N 6 -threonylcarbamoyl adenosine (t6A), and at least one modified nucleoside selected from the group consisting of: [2] The following steps: (1) measuring the amount of modified nucleosides contained in a biological sample collected from a subject; and (2) estimating the subject's muscle mass from the measured amount of modified nucleoside; The method for estimating muscle mass of a subject according to [1] above, comprising: [3] The method according to [2] above, wherein the amount of the modified nucleoside is measured using a mass spectrometer in the step (1). [4] The method according to [2] above, wherein the step (1) involves measuring the amount of the modified nucleoside using an antibody specific to the modified nucleoside. [5] The method according to any one of [1] to [4] above, wherein the biological sample is urine or blood collected from a subject. [6] The biological sample is urine collected from a subject, and the amount of the modified nucleoside is measured using an internal standard (e.g., creatinine, urea nitrogen, uric acid, adenosine, uridine, or 3-amino-3-carboxypropyluridine (ACP)). 3 The method according to any one of [1] to [4] above, wherein the method is carried out by internal standard correction using the chromatogram of the present invention. [7] The method according to any one of [1] to [6] above, wherein the subject is a subject at risk of muscle mass loss (e.g., a person at risk of or diagnosed with sarcopenia, an elderly person, a patient who has been bedridden for a long period of time, a patient with a neuromuscular disease, a patient with an endocrine disease, a patient with a chronic disease, or a person who is malnourished). [8] A method for providing information useful for diagnosing whether a subject has sarcopenia, wherein the useful information is muscle mass estimated by a method according to any one of [1] to [6] above. [Effects of the Invention]

[0008] According to the present invention, muscle mass of a subject can be estimated simply, inexpensively, with little invasiveness, and with few restrictions on the subject. [Brief explanation of the drawings]

[0009] [Figure 1] The figure shows the correlation between muscle mass (% of body weight) measured by bioelectrical impedance analysis and the amount of urinary 5-methoxycarbonylmethoxyuridine (mcmo5U) in healthy individuals. [Figure 2] The graph shows the results of measuring the amount of mcmo5U in each mouse organ. The bars in the graph represent the mean ± SEM, and the points represent individual measurement results. [Figure 3] This shows the correlation between skeletal muscle index (SMI) measured by bioelectrical impedance analysis and the amount of urinary 1-methylguanosine (m1G) and 2'-O-methyladenosine (Am). [Figure 4] The figures show the results of measuring urinary 1-methylguanosine (m1G), N4-acetylcytidine (ac4C), and N6-threonylcarbamoyladenosine (t6A) in healthy individuals and patients with sarcopenia. The upper figure shows the values ​​of each modified nucleoside after correction with 3-amino-3-carboxypropyluridine (acp3U), and the lower figure shows the receiver operating characteristic curves (ROC) for the sensitivity and specificity at each cutoff value. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described below by way of illustrative embodiments, along with preferred methods and materials that can be used in carrying out the present invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Furthermore, any materials and methods equivalent or similar to those described herein can also be used in carrying out the present invention. Furthermore, all publications and patents cited in this specification in connection with the present invention are incorporated herein by reference and constitute a part of this specification, for example, to describe methods, materials, and the like that can be used in the present invention.

[0011] In this specification, the expression "A to B" indicating a range of values ​​means a range of values ​​including the endpoints A and B.

[0012] As used herein, the term "subject" or "test subject" refers to any mammal, including, but not limited to, humans; non-human primates such as chimpanzees, other apes, and monkey species; domesticated mammals such as cows, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; and small or laboratory animals, including rodents such as mice, rats, and guinea pigs, with humans being preferred. The term "subject" also includes adults, the elderly, infants, and newborns, as well as patients with any disease and healthy individuals who have not been diagnosed with a specific disease.

[0013] The subject or test subject for whom muscle mass is estimated by the method of the present invention is preferably a subject suspected of muscle mass loss.For example, but not limited to, those suspected of or diagnosed with sarcopenia, elderly people, and those suffering from certain diseases that may cause muscle mass loss, such as long-term bedridden patients, neuromuscular disease patients, endocrine disease patients, chronic disease patients, or malnourished people.Particularly preferred subjects are those suspected of or diagnosed with sarcopenia, or elderly people.

[0014] The method of the present invention estimates the muscle mass of a subject. The muscle mass of a subject can be expressed by an index used in the art. Examples include, but are not limited to, muscle mass (relative to body weight), skeletal muscle index (SMI), and muscle mass index (MMI). Muscle mass (relative to body weight) can be expressed as muscle mass (%) = (muscle mass ÷ body weight) × 100. Skeletal muscle index (SMI) is expressed as SMI = appendicular skeletal muscle mass (kg) ÷ height. 2 (m 2 ) The muscle mass index is MMI = total body muscle mass (kg) ÷ height 2 (m 2 ) can be expressed as

[0015] SMI is used as a diagnostic criterion for age-related muscle loss (sarcopenia). SMI values ​​for men <7.0 kg / m 2 , Female<5.5kg / m 2 If so, it is considered sarcopenia.

[0016] In the present invention, the term "biological sample" refers to any sample derived from a subject that may contain modified nucleosides. Although not particularly limited, a body fluid sample derived from a subject is preferably used as the biological sample. The term "body fluid sample" refers to any liquid sample that can be isolated from an individual's body, including, but not limited to, blood, plasma, serum, saliva, urine, tears, sweat, etc. Preferably, the biological sample is plasma, serum, or urine. In one embodiment, the biological sample used in the present invention is plasma, serum, or urine derived from a human.

[0017] In the method of the present invention, the modified nucleoside to be measured is 5-methoxycarbonylmethoxyuridine (mcmo 5 U), 1-methylguanosine (m1G), 2'-O-methyladenosine (Am), N 4 -Acetylcytidine (ac4C) or N 6-threonylcarbamoyl adenosine (t6A). Each modified nucleoside has the following structural formula:

[0018] [ka]

[0019] Modified nucleosides (e.g., mcmo 5 The detection and measurement of the modified nucleoside (e.g., mcmo) is performed using the 5 There are no particular limitations on the method as long as it can detect U), but it is preferably detected by mass spectrometry or ELISA.

[0020] "Mass spectrometry" (also referred to as "MS") is an analytical technique for identifying compounds by their mass. It involves applying energy such as high voltage to a sample to be analyzed to ionize it, and then filtering, detecting, and / or measuring the ions based on their mass-to-charge ratio (m / z). There are many types of mass spectrometers, depending on the method of ionizing the sample and the detection method. The modified nucleosides (e.g., mcmo) targeted by the present invention are well known. 5 Any mass analyzer can be used without particular limitation as long as it can be used to detect U. Examples of mass analyzers that can be used in the present invention include, but are not limited to, mass spectrometers (MS), and their improved versions such as TOF-MS and MALDI-TOF-MS (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometer), quadrupole mass analyzers, ion trap mass analyzers, and Fourier transform mass analyzers. Various mass analyzers are commercially available and can be used as appropriate in the present invention.

[0021] When using mass spectrometry, detection can be achieved with a single mass spectrometer, but a tandem mass spectrometer (tandem MS / MS) with two mass spectrometers connected in tandem is preferred. A tandem MS / MS is an instrument in which two mass spectrometers (MS) are connected in series with a collision activation chamber between them. First, the sample is ionized in the first MS, and then only ions with a specific mass number are selected and introduced into the collision activation chamber, where they are collided with an inert gas such as Xe (xenon). Then, secondary ions (product ions) generated from the ions selected in the first MS are detected by the second MS.

[0022] Ionization methods include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electron ionization (EI), fast electron impact (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Those skilled in the art can select an appropriate ionization method based on the analyte to be measured, the type of sample, the type of detector, the selection of positive ion mode or negative ion mode, and other factors. The modified nucleosides (e.g., mcmo) targeted by the present invention can be used to detect nucleosides. 5 There are no particular limitations as long as U) can be detected, and the above-mentioned methods can be used appropriately.

[0023] Tandem MS / MS is typically performed using selected reaction monitoring (SRM). Selective reaction monitoring refers to the operation of a mass spectrometer to continuously detect only the signal levels of specific product ions generated from the analyte, instead of acquiring product ion spectra in multistage mass analysis with two or more stages. In SRM, tandem mass analysis can be spatial or temporal.

[0024] When mass spectrometry is used, it is preferable to couple liquid chromatography (LC) or gas chromatography (GC) before a mass spectrometer (MS or MS / MS), more preferably LC, and the sample is separated by LC or GC before being introduced into the mass spectrometer for analysis. By coupling LC or GC before the mass spectrometer, for example, even when blood samples or urine samples are used, analysis can be performed satisfactorily.

[0025] Types of chromatography that can be used for LC include partition chromatography, normal phase liquid chromatography (NPLC), displacement chromatography, reverse phase liquid chromatography (RPLC), size exclusion chromatography, ion exchange chromatography, and affinity chromatography.

[0026] Generally, a mass spectrometer consists of a sample introduction section, an ionization section (ion source), a mass separation section (analyzer), a detection section (detector), a vacuum pump (vacuum pump), an instrument control section, and a data processing section (data system).

[0027] Examples of analyzers used in mass spectrometers include triple quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers, with triple quadrupole analyzers or quadrupole time-of-flight (QTOF) analyzers being preferred. Given that many commercially available instrument platforms advantageous for SRM assays use triple quadrupole analyzers, it is more preferable to perform tandem MS / MS using a triple quadrupole analyzer in the detection method of the present invention. As commonly understood by those skilled in the art, the term "triple quadrupole" as used herein encompasses not only quadrupoles but also multipole or stacked electrodes instead of quadrupoles. Furthermore, in the detection of the present invention, mass spectrometry may be performed in either negative ion mode or positive ion mode, or, in the case of tandem MS / MS, both ion modes may be combined.

[0028] In the detection method of the present invention, a triple quadrupole LC / MS / MS in which an LC is coupled before a mass spectrometer is preferably used.

[0029] In the methods of the present invention, modified nucleosides (e.g., mcmo) can be isolated using mass spectrometry. 5 When detecting U), it is preferable to deproteinize and / or desalt the sample beforehand. These pretreatments allow for accurate and sensitive detection of the target substance.

[0030] Deproteinization methods generally include insolubilization by denaturation of proteins (addition of acids such as perchloric acid, trichloroacetic acid, metaphosphoric acid, etc., addition of water-miscible organic solvents such as acetone, acetonitrile, methanol, ethanol, heating, cooling), and physical removal (ultrafiltration using membrane filters (centrifugal filtration devices, etc.), dialysis using dialysis tubing, ultracentrifugation). Deproteinization can also be performed using permeation-limiting packing materials such as internal reversed-phase packing materials, hybrid packing materials, and hydrophilic polymer packing materials. Modified nucleosides (e.g., mcmo 5 As long as the detection of β-lactamase (U) is not impaired, the preferred deproteinization method is not particularly limited, but an example is deproteinization using an insolubilization method involving protein denaturation using a water-miscible organic solvent, such as methanol. Deproteinization methods are well known and can be performed according to standard procedures. For example, deproteinization may be performed by adding 0.2 to 20 volumes, preferably 1 to 5 volumes, of ethanol or methanol to a sample (preferably a body fluid sample), allowing the reaction to proceed for a sufficient time (e.g., 15 minutes) for protein denaturation, followed by centrifugation under conditions sufficient to precipitate the denatured protein (e.g., 12,000 × g for 15 minutes), and recovering the supernatant (organic solvent layer), thereby obtaining a deproteinized sample. The deproteinized sample can be used directly or after drying using a centrifugal evaporator or the like and dissolving it in an appropriate solvent such as distilled water for LC.

[0031] As the desalting method, any known desalting method used in analysis can be used as appropriate. In addition, by using the above-mentioned protein removal treatment method, it is possible to also use the method for desalting.

[0032] In one embodiment of the method of the present invention, the amount of modified nucleosides (e.g., mcmo) in a sample is determined based on the results measured using a mass spectrometer. 5 Calculate or measure the amount of modified nucleosides (e.g., mcmo 5 The amount of urinary tract ATP (U) may be measured with or without correction using an internal standard, but the use of an internal standard allows for more accurate measurement. The internal standard is a substance in a sample (e.g., urine or blood) for correction. Examples of internal standards include, but are not limited to, adenosine and uridine in plasma or serum when the sample is a blood sample, such as plasma or serum, and creatinine, urea nitrogen, uric acid, adenosine, uridine, and 3-amino-3-carboxypropyluridine (ACP) in urine when the sample is urine. 3 Examples of internal standards include 1-methylinosine (mI), 1-methylinosine (mI), and 1-methylinosine (mI). Using the detection values ​​of these internal standards, the detected modified nucleosides (e.g., mcmo 5 By correcting the amount of U), the target substance can be measured more accurately. Only one type of internal standard may be used, or two or more types may be used in combination.

[0033] In one embodiment of the method of the present invention, modified nucleosides (e.g., mcmo) in a sample are 5 The amount of modified nucleosides (e.g., mcmo) can be measured using ELISA. ELISA is a method in which a specific antibody is bound to a target antigen contained in a sample, and detection and quantification are performed using an enzyme reaction. In the method of the present invention, modified nucleosides (e.g., mcmo) are used. 5Measurement can be performed using an antibody against modified nucleosides (U). The antibody against the modified nucleoside can be prepared according to a conventional method, or an antibody prepared by a contract manufacturer or a commercially available antibody can also be used. The antibody can be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred.

[0034] In the method of the present invention, modified nucleosides (e.g., mcmo) are detected using an ELISA method. 5 The amount of U) can be measured by a conventional method, for example, a direct method, an indirect method, a sandwich method, or a competitive method, but the sandwich method is preferred.

[0035] The estimation of muscle mass in the method of the present invention is based on the measured modified nucleosides (e.g., mcmo 5 The muscle mass can be calculated from the amount of modified nucleosides (e.g., mcmo) that are the target of the present invention. 5 Using a calibration curve showing the correlation between the amount of modified nucleosides (e.g., mcmo) and muscle mass, 5 The muscle mass of a subject can be estimated from the amount of U. Since muscle mass is expected to vary depending on the subject's age, sex, and other factors, it is preferable to use calibration curves prepared for various factors, for example, for sex and age.

[0036] One embodiment of the method of the present invention comprises the steps of: (1) measuring the amount of a specific modified nucleoside contained in a biological sample collected from a subject, for example, by mass spectrometry or ELISA; wherein the modified nucleoside is 5-methoxycarbonylmethoxyuridine (mcmo 5 U), 1-methylguanosine (m1G), 2'-O-methyladenosine (Am), N 4 -acetylcytidine (ac4C), and N 6-threonylcarbamoyl adenosine (t6A), and at least one modified nucleoside selected from the group consisting of (2) estimating the subject's muscle mass from the measured amount of modified nucleoside; It is also a method for estimating muscle mass in a subject, including: [Example]

[0037] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. Example 1: Muscle mass and urinary modified nucleoside levels in healthy individuals Muscle mass (% of body weight) was measured for 11 healthy subjects using a medical body composition analyzer (InBody S10). Urine samples were also collected from 11 healthy volunteers. 100 μL of the sample was then deproteinized and desalted using a column (Nanosep with 3K Omega) to prepare the analytical sample. 2 μL of the sample was analyzed comprehensively for modified nucleosides using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8060NX). Measurement results for each modified nucleoside were normalized using urinary 3-amino-3-carboxypropyluridine (acp3U). As a result, muscle mass (% of body weight) measured using InBody S10 and urinary 5-methoxycarbonylmethoxyuridine (mcmo 5 The results are shown in Figure 1. Equation: Y=2.867 × 10 -5 × X - 0.001008 R-squared: 0.3815 p-value:0.0429 It was.

[0038] (Example 2) mcmo in RNA extracted from mouse organs 5 Measurement of U amount Organs (heart, lung, liver, kidney, skeletal muscle, and testis) were removed from three mice, homogenized, and RNA from each organ was extracted with Trizol. The RNA concentration was adjusted to 1000 ng / μL using a spectrophotometer (NanoDrop ND-1000). RNA was then degraded to single nucleosides using RNase. Two μL of each sample was analyzed using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8060NX) to determine the mCMO content of the RNA. 5 The results were corrected using 3-amino-3-carboxypropyluridine (acp3U). The results are shown in Figure 2. 5 The highest levels of U were detected in skeletal muscle, with levels two to three times higher than in other organs.

[0039] From the above results, mcmo 5 It was found that U was detected at the highest level in skeletal muscle. 5 It was found that the amount of U can estimate the muscle mass of the subject.

[0040] (Example 3) Muscle mass (SMI value) and urinary modified nucleoside amount in subjects Skeletal muscle index (SMI) was measured for 50 subjects, including healthy individuals, elderly healthy individuals, and sarcopenia patients, using a medical body composition analyzer (Seca mBCA525). Urine samples were collected from each subject and deproteinized and desalted as in Example 1 to prepare analytical samples. The modified nucleosides 1-methylguanosine (m1G) and 2'-O-methyladenosine (Am) were measured for each sample using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8060NX). Measurement results for each modified nucleoside were corrected for urinary 3-amino-3-carboxypropyluridine (acp3U). As a result, a significant correlation was confirmed between the SMI value measured using the seca mBCA525 and the amount of 1-methylguanosine (m1G) and 2'-O-methyladenosine (Am) in urine. The results are shown in Figure 3.

[0041] (Example 4) Urinary modified nucleoside levels in sarcopenic patients The subjects of the measurement were healthy individuals (31 individuals) and patients diagnosed with sarcopenia (19 individuals). Urine was collected from each subject and deproteinized and desalted in the same manner as in Example 1 to prepare analytical samples. Each sample was analyzed using an ultrafast triple quadrupole mass spectrometer (Shimadzu Corporation LCMS-8060NX) to determine the amount of modified nucleosides, 1-methylguanosine (m1G), N 4 -acetylcytidine (ac4C) and N 6 The results are shown in Figure 4. The modified nucleoside was significantly decreased in patients with sarcopenia.

[0042] The above detailed description is merely illustrative of the objects and scope of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein, without departing from the scope of the appended claims. [Industrial Applicability]

[0043] The present invention provides a new method for estimating muscle mass. The method of the present invention can be performed at any location using a biological sample such as urine collected from a subject, eliminating the need for measurement at a medical institution or other facility equipped with expensive measuring equipment, making it simple, convenient, and virtually non-invasive. In addition, the method of the present invention requires only the collection of a biological sample such as urine from the subject, reducing the limitations on the subjects.

Claims

1. A method for estimating muscle mass of a subject, comprising measuring the amount of a modified nucleoside contained in a biological sample collected from the subject, The method, wherein the modified nucleoside is at least one modified nucleoside selected from the group consisting of 5-methoxycarbonylmethoxyuridine (mcmo5U), 1-methylguanosine (m1G), 2'-O-methyladenosine (Am), N4-acetylcytidine (ac4C), and N6-threonylcarbamoyladenosine (t6A).

2. The following steps: (1) measuring the amount of modified nucleosides contained in a biological sample collected from a subject; and (2) estimating the subject's muscle mass from the measured amount of modified nucleoside; The method of claim 1 , comprising:

3. The method according to claim 2, wherein the step (1) measures the amount of modified nucleosides using a mass spectrometer.

4. The method according to claim 2, wherein the step (1) measures the amount of the modified nucleoside using an antibody specific to the modified nucleoside.

5. The method according to any one of claims 1 to 4, wherein the biological sample is urine or blood collected from a subject.

6. The method according to any one of claims 1 to 4, wherein the biological sample is urine collected from a subject, and the amount of the modified nucleoside is measured by internal standard correction using an internal standard.

7. The method according to any one of claims 1 to 4, wherein the subject is a subject at risk of losing muscle mass.

8. The method of claim 7, wherein the subject is selected from the group consisting of those at risk of or diagnosed with sarcopenia, elderly people, patients who have been bedridden for a long time, patients with neuromuscular diseases, patients with endocrine diseases, patients with chronic diseases, and malnourished people, and subjects at risk of losing muscle mass.

9. A method for providing information useful for diagnosing whether a subject has sarcopenia, wherein the useful information is muscle mass estimated by a method according to any one of claims 1 to 4.

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