Patent Application
20190056413
The present invention relates to a method for detecting specific diseases or detecting onset risk of these diseases and a detection kit therefor, more specifically to a method for detecting specific diseases or detecting onset risk of these diseases, comprising a step of measuring a ganglioside GM3 with a specific structure, and a detection kit therefor.
Diabetes and cardiovascular disease (CVD) are related diseases that share a number of overlapping risk factors including obesity, hypertension and low levels of physical activity. Atherosclerosis which is one of the CVDs is a pathological process characterized by the accumulation of cholesterol and fatty material in the lumen of blood vessels resulting in plaque formation. Due to plaque rupture and clot formation, stroke, heart attack and the like may be caused (Non-Patent Document 1).
There is scientific evidence that metabolic and behavioral risk factors heavily influence the aetiology of atherosclerosis. Behavioral risk factors include a lack of physical activity, unhealthy diet such as excessive calories, salt and fat intake, and tobacco and alcohol abuse. Traditional metabolic risk factors include hypertension, heightened blood lipids such as cholesterol, hyperglycemia. Additionally, increased carotid intima-media thickness (CIMT) has been reported to be associated with CVD risk factors.
The most well-known forms of diabetes are type 1 and type 2. Type 2 diabetes is generally regarded as a lifestyle-related disease associated with a chronic caloric surplus, lack of exercise, obesity and high serum levels of low-density lipoprotein (LDL). Type 2 diabetes has insulin resistance and/or insulin deficiency, and hyperglycemia (Non-Patent Document 2). It has been made clear that dysfunction of adipocytes associated with an over-accumulation of visceral fat in obesity, namely, abnormality of adipocytokine secretion induces insulin resistance, which plays an important role as various causes of pathological conditions of type 2 diabetes and arteriosclerotic diseases.
Macrophages being myeloid cells are always in visceral adipose tissue. On the other hand, adipocytokine (MCP-1) is secreted from visceral adipocytes having insulin resistance with enlargement, macrophages are mobilized from surrounding blood vessels. Further, a large number of free fatty acids secreted from enlarged adipose tissue activate macrophages through Toll-like receptors, and secrete inflammatory cytokines such as TNFα and IL-6. These inflammatory cytokines act on adipocytes, resulting in the negative effect of adipocyte insulin resistance. Thus, the abnormality of secreting adipocytokine is enhanced. In recent years, it has been found that chronic inflammation of these adipocytes evokes systemic insulin resistance, and the pathophysiology of adipose tissue has been noted (Non-Patent Document 3).
In recent years, type 2 diabetes has been typically diagnosed by means of hematology. In hematology, fasting blood glucose, HbA1c, glycoalbumin and the like are measured as an index. However, to fully grasp the condition of type 2 diabetes, a plurality of indices must be measured and evaluated, not only one index.
The inventors found that diseases having insulin-resistant conditions selected from the group consisting of type 2 diabetes, hyperlipidemia, hypertension and obesity can be detected by quantifying a ganglioside GM3 (α-Neu5Ac-(2-3)β-Gal-(1-4)-β-Glc-(1-1)-ceramide) which is one kind of ganglioside in the blood (Patent Document 1, Non-Patent Document 4). Gangliosides collectively refer to sphingoglycolipids containing sialic acid, and the sphingoglycolipids are glycolipids which are biosynthesized by enzymatic stepwise addition of sugars, or sulfuric acid, such as glucose, galactose, N-acetylgalactosamine, N-acetylglucosamine and sialic acid, to ceramide and are expressed outside the lipid bilayer of the cell membrane.
Further, it has been found that the total amount of GM3 is correlated with lipoprotein cholesterol (LDL-c) (Non-Patent Document 4).
There are a large number of species of ganglioside GM3, the diversity of which depends on the number of carbons or double bonds, as well as the type and number of substituents that comprise the ceramide moiety. Depending on the species, the biophysical properties and physiological functions of GM3 are different so that it has been thought that measuring a ganglioside GM3 with a specific structure is useful for detecting lifestyle-related diseases such as metabolic syndrome. In fact, to date, ganglioside GM3 with a specific structure useful for the detection of lifestyle-related diseases has not been reported.
The present invention has been accomplished in view of the above-mentioned background art, and an object thereof is to provide a method and a kit for detecting diseases or detecting onset risk of these diseases, comprising a step of measuring a ganglioside GM3 with a specific structure which is correlated with a large number of molecules regarding lifestyle-related diseases (specific diseases) such as metabolic syndrome.
The present inventors have earnestly investigated to solve the above-mentioned problems, and as a result, they have found that one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, or onset risk of these diseases can be detected by measuring a ganglioside GM3 in which the ceramide moiety is hydroxylated.
They have further found that a ganglioside GM3 with the specific structure is correlated with a maker regarding at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, whereby they have accomplished the present invention.
That is, the present invention is directed to [1] A method for detecting at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined or detecting onset risk of these diseases, which comprises
(a) a step of measuring a ganglioside GM3 in a blood sample derived from a subject,
wherein the ganglioside GM3 has a structure represented by the following formula (1).
[In the formula (1), R1 represents a glycan constituting the ganglioside GM3, and R2—C(═O)— represents a fatty acid residue having 16 or more and 24 or less carbon atoms which may have a double bond, and having an OH group as a substituent.]
[2] The detection method according to [1], wherein the disease is metabolic syndrome.
[3] The detection method according to [1] or [2], wherein the structure represented by the formula (1) is a hydrocarbon group having 22 or 24 carbon atoms which may have a double bond, and R2—C(═O)— has an OH group as a substituent.
[4] The detection method according to [1] or [2], wherein the structure represented by the formula (1) is a hydrocarbon group having 24 carbon atoms which has a double bond, and R2—C(═O)— has an OH group as a substituent.
[5] The detection method according to [1] or [2], wherein the structure represented by the formula (1) is a structure represented by the following formula (2).
[In the formula (2), R1 represents a glycan constituting the ganglioside GM3.]
[6] The detection method according to [5], wherein the method which further comprises,
(b) a step of comparing an amount of the ganglioside GM3 with the structure represented by the formula (2) measured in the Step (a), with an amount of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from a normal subject,
(c) a step of determining the value of the marker with regard to at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined of the subject, to be abnormal as compared with those of the normal subject, when there is a significant difference in the step (b).
[7] The detection method according to [6], wherein the marker with regard to the visceral fat accumulation, hyperglycemia, dyslipidemia and hypertension is at least one marker selected from the group consisting of autotaxin, HOMA-IR, HbA1c, aspartic acid aminotransferase, alanine-aminotransferase, low-density lipoprotein cholesterol, fasting blood sugar and blood pressure.
[8] The detection method according to [6] or [7], wherein the marker with regard to the atherosclerosis is at least one selected from the group consisting of sphingomyelin, lysophosphatidylcholine, C-reactive protein and mean intima-media thickness.
[9] The detection method according to any of [1] to [8], wherein the measurement of the ganglioside GM3 is carried out by a method of reacting an antibody to the GM3, a high performance liquid chromatography method, mass spectrometry or liquid chromatography/mass spectrometry.
[10] A detection kit, for detecting at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, or onset risk of these diseases, which contains a ganglioside GM3 as a standard substance,
wherein the ganglioside GM3 has a structure represented by the following formula (1).
[In the formula (1), R1 represents a glycan constituting the ganglioside GM3, and R2—C(═O)— represents a fatty acid residue having 16 or more and 24 or less carbon atoms which may have a double bond, and having an OH group as a substituent.]
According to the detection method of the present invention, the above-mentioned problems can be solved, the above-mentioned tasks can be settled, and by measuring the ganglioside GM3 with a specific structure in a blood sample, the conditions of the subject can be grasped more accurately and more simply and easily from a more fundamental point of view, as compared with the conventional detection methods.
By measuring the specific ganglioside GM3 represented by the formula (1), a specific disease or the onset risk of the specific diseases can be detected more accurately as compared with the measurement of the general ganglioside GM3 (measurement of the entire ganglioside GM3).
Further, the conditions relating to a lifestyle-related disease of the subject can be detected more accurately as compared with the conventional detection method of the lifestyle-related diseases.
More specifically, according to the detection method of the present invention, “at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined” can be detected by measuring the ganglioside GM3 with a specific structure more accurately and more simply and easily from a more fundamental point of view, as compared with the conventional methods.
In the present specification, the above-mentioned matter in the double quotation marks (including the combined diseases) is sometimes abbreviated as “specific disease(s)”.
It is also possible to predict onset risk of the specific diseases more accurately, more simply and easily at an earlier stage from a more fundamental point of view, as compared with the conventional methods, and is possible to take measures early so as not to suffer from the specific diseases, etc.
Further, according to the detection method of the present invention, even when the specific diseases cannot be detected by measuring the total GM3 amount, it is possible to detect the specific diseases, or to detect onset risk of the specific diseases.
In particular, according to the detection method of the present invention, it is possible to detect whether the subject is suffered from the metabolic syndrome or not, or to determine the onset risk.
In addition, the ganglioside GM3 with a specific structure has correlation with markers of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis, and it is possible to detect whether it is suffered from the specific diseases or to predict whether there is a risk of developing the specific disease, by the simple and easy operation that the ganglioside GM3 with a specific structure is measured.
According to the present invention, by detecting the specific diseases or the diseases such as metabolic syndrome, lifestyle-related disease, etc., or to detect onset risk of these diseases, detection and prevention of circulatory diseases, caused by them, such as diabetes; cardiovascular disorders such as cardiac infarction and angina; cerebrovascular disorders such as cerebral infarction; renal dysfunction; vascular disorders such as extremital artery obstruction; become easy.
Hereinafter, the present invention will be described, but the present invention is not limited by the following specific embodiments, and can be practiced by optionally modifying these.
The detection method of the present invention is a method for detecting at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, or detecting onset risk of these diseases which comprises
(a) a step of measuring a ganglioside GM3 in a blood sample derived from a subject, wherein the ganglioside GM3 has a structure represented by the following formula (1).
[In the formula (1), R1 represents a glycan constituting the ganglioside GM3, and R2—C(═O)— represents a fatty acid residue having 16 or more and 24 or less carbon atoms which may have a double bond, and having an OH group as a substituent.]
In the present specification, each disease is defined as follows.
<1> When a waist size at the position of the navel of a subject is 85 cm or more in the case of male, and 90 cm or more in the case of female, it is determined to be visceral fat accumulation (hereinafter sometimes abbreviated as “VFA”).
<2> When a fasting blood sugar level of the subject is 126 mg/dL or more, it is determined to be hyperglycemia.
<3> When one satisfies at least one condition among the following conditions (1) to (3), it is determined to be dyslipidemia.
(1) A triglyceride (neutral fat) value of the subject is 150 mg/dL or more.
(2) A low-density lipoprotein cholesterol (LDL-c) value of the subject is 140 mg/dL or more.
(3) A high-density lipoprotein cholesterol (HDL-c) value of the subject is less than 40 mg/dL.
<4> When a systolic blood pressure (the maximum blood pressure) of the subject is 140 mmHg or higher and/or a diastolic blood pressure (the minimal blood pressure) thereof is 90 mmHg or higher, it is determined to be hypertension.
<5> Atherosclerosis is also referred to as atheromatous arteriosclerosis, and refers to the state in which plaque (porridge species) is formed at an inner membrane of a blood vessel by accumulating fat or cholesterol at the inner membrane of the blood vessel.
<6> Nephropathy is a disease in which kidney function is lowered. There is nephropathy which is generated by causing disorder at the capillaries of the kidney by continuing the state of a high blood glucose level due to hyperglycemia, etc.
<7> In addition, the diseases considered to be caused by lifestyle habit such as the VFA, hyperglycemia, dyslipidemia, hypertension, atherosclerosis, etc., are sometimes abbreviated as “lifestyle-related disease”.
The detection method of the present invention is a method for detecting at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, or for detecting onset risk of these diseases.
“To detect the disease two or more of which are combined” means “to detect whether or not the subject is developed at least two or more diseases selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis”.
Further, “to detect onset risk of the disease two or more of which are combined” means “to detect whether or not the subject has onset risk of at least two or more diseases selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis”.
The detection method of the present invention is a detection method of a specific disease or a detection method of onset risk of the specific disease, and the detection method of the present invention is also a method for detecting a lifestyle-related disease, or for detecting onset risk of the lifestyle-related disease.
Moreover, the detection method of the present invention is also a method for detecting metabolic syndrome, or for detecting onset risk of the metabolic syndrome.
In the present specification, the “metabolic syndrome” refers to the state in which the subject is in visceral fat accumulation, and is developed at least two diseases among “hyperglycemia, dyslipidemia and hypertension”. Therefore, the state of the “metabolic syndrome” is included in the state that the subject is developed specific diseases.
<Step (a)>
In the step (a), the specific ganglioside GM3 with a structure represented by the following formula (1) in the blood sample derived from the subject is measured. The “measurement” is a concept that includes to confirm the presence of the specific ganglioside GM3 in the blood, or to quantitate the specific ganglioside GM3 in the blood. The “measurement” may further comprise other steps, if necessary.
In general, the term “Ganglioside” refers to a glycosphingolipid in which one or more sialic acid is bound onto the glycan.
The terms “ganglioside GM3” means that the glycan of the glycosphingolipid is “α-Neu5Ac-(2-3) β-Gal-(1-4) -β-Glc-(1-1)-ceramide”.
Here, “α-Neu5Ac” means N-acetyl-α-neuraminic acid, “β-Gal” means β-galactose, and “β-Glc” means β-glucose.
The ganglioside GM3 (hereinafter, it is sometimes simply abbreviated as “GM3”) to be measured in the present invention is a GM3 with the structure particularly represented by the following formula (1).
[In the formula (1), R1 represents a glycan constituting the ganglioside GM3, and R2—C(═O)— represents a fatty acid residue having 16 or more and 24 or less carbon atoms which may have a double bond, and having an OH group as a substituent.]
Here, from the definition of the “ganglioside GM3”, “R1 which is a glycan constituting the ganglioside GM3” in the formula (1) means that “α-Neu5Ac”, “β-Gal” and “β-Glc” are bonded as in the definition of the “ganglioside GM3”.
Hereinafter, the “GM3 with the structure represented by the formula (1)” is sometimes abbreviated simply as “hydroxylated GM3”.
With regard to the structure represented by the formula (1), “R2—C(═O) —” in the formula (1) is preferably a hydrocarbon group having 22 or 24 carbon atoms which may have a double bond and having an OH group as a substituent in the points that detection of the specific disease can be carried out more accurately, and detection of onset risk of the specific disease can be carried out more accurately as compared with the measurement of the entire GM3 such as quantification of the total amount of GM3.
Also, it is more preferable that R2 is a hydrocarbon group having 23 carbon atoms which has a double bond, and having an OH group as a substituent from the above-mentioned points.
Further, it is particularly preferable that the structure represented by the formula (1) is a structure represented by the following formula (2) from the above-mentioned points.
[In the formula (2), R1 represents a glycan constituting the ganglioside GM3.]
Hereinafter, “GM3 with a structure represented by the formula (2)” is sometimes abbreviated as “[hC24:1]GM3”. The “[hC24:1]GM3” is a fatty acid having 24 carbon atoms (the carbon to which the oxygen of the carboxyl group of the fatty acid is bonded by a double bond is counted as one.) in which the fatty acid of GM3 has an OH group as a substituent, and it means that it has a double bond in the hydrocarbon group.
The “subject” in the present invention represents mammals such as humans, dogs, cats, horses, cows, pigs, sheep and monkeys, and the subject is preferably humans.
For example, “blood sample derived from the subject” to be used in the present invention is whole blood, serum, and plasma. It is preferable to use “serum or plasma derived from the subject” in the point that highly accurate measurement can be carried out.
For example, the whole blood collected from the subject is charged in a blood-collecting tube to which an anticoagulant is added, and after mixing with inversion, it is centrifuged whereby plasma can be recovered. Also, after coagulating the whole blood collected from the subject, it is centrifuged whereby serum can be recovered.
The extraction method of the “GM3 with the specific structure represented by the formula (1)” in the blood sample can use conventionally known methods. More specifically, the GM3 can be extracted from the blood sample by the method described in Examples described below, etc.
In addition, the detection method of the present invention is preferably a detection method which further comprises,
(b) a step of comparing an amount of the ganglioside GM3 with the structure represented by the formula (2) measured in the step (a), with an amount of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from a normal subject,
(c) a step of determining the value of the marker with regard to at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined of the subject, to be abnormal as compared with those of the normal subject, when there is a significant difference in the step (b).
<Step (b)>
In the step (b), the amount of the ganglioside GM3 with the structure represented by the formula (2) measured in the step (a), and the amount of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from a normal subject are compared with each other.
<Step (c)>
In the step (c), when there is a significant difference in the step (b), the value of the marker with regard to at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined of the subject is determined to be abnormal as compared with those the normal subject.
The “significant difference” means when the amount of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from the subject, and the amount of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from the normal subject are subjected to statistical analysis by using Mann-Whitney U test, two-sample t-test or the like, and as a result, the P value is 0.05 or less.
When the lower limit of the value (fold) dividing the value of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from the subject, by the value of the ganglioside GM3 with the structure represented by the formula (2) in the blood sample derived from the normal subject is within the range of 1.2-fold or more to 2.5-fold or more, it is preferable to determine the marker value of the specific disease to be abnormal, when it is within the range of 1.4-fold or more to 2.3-fold or more, it is more preferable to determine the same to be abnormal, and when it is within the range of 1.6-fold or more to 2-fold or more, it is particularly preferable to determine the same to be abnormal. If the lower limit of lower limit value of the divided value is too low, there is sometimes a case where the normal subject is determined to be abnormal, while if the upper limit of the lower limit of the divided value is too high, there is sometimes a case where it does not determine to be abnormal even when it is the abnormal case.
The “marker with regard to the disease in which two or more are combined” means the “marker with regard to at least two or more diseases selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis”.
The step (c) is based on the fact that when the GM3 with the structure represented by the formula (1) is in particular the ganglioside GM3 with the structure represented by the formula (2) ([hC24:1]GM3), the inventors have found that it had a correlation with the marker (biomarker) regarding the specific disease.
That is, for example, a particularly preferable method is that, by measuring one molecule as [hC24:1]GM3, “the value of the marker of the specific disease” which has a correlation with the [hC24:1]GM3 can be predicted, whether the value of the marker is abnormal or not as compared with the normal subject can be estimated or determined, and based on such a determination, the specific disease is detected, or onset risk of the specific disease is detected.
The marker with regard to the visceral fat accumulation, hyperglycemia, dyslipidemia and hypertension is preferably at least one marker selected from the group consisting of autotaxin, HOMA-IR (Homeostasis model assessment-Insulin Resistance), HbA1c (Hemoglobin A1c), aspartic acid aminotransferase (AST), alanine-aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-c), fasting blood sugar and blood pressure.
The marker with regard to the atherosclerosis is preferably at least one marker selected from the group consisting of sphingomyelin, lysophosphatidylcholine, C-reactive protein and mean intima-media thickness (IMT).
The measurement method of the specific ganglioside GM3 is not particularly limited, and it is preferable to use a method of reacting an antibody to the GM3, a high performance liquid chromatography method, mass spectrometry or liquid chromatography/mass spectrometry. These methods may be used in combination.
The method of acting an antibody to the GM3 is not particularly limited, and is ELISA method, for example.
The high performance liquid chromatography method (hereinafter sometimes abbreviated as “HPLC”) is not particularly limited and may be used, for example, normal phase chromatography, reverse phase chromatography, ion exchange chromatography or the like.
The mass spectrometry is not particularly limited and may be used, for example, as an ionization method, MALDI method, ESI method, APCI method or the like. Also, as a separating portion, a time-of-flight type (TOF type), a double focusing type, a quadrupole focusing type, an ion trap type or the like may be used. In the measurement (detection, quantification) of the GM3 with the specific structure, it is preferable to use the ESI method or the APCI method as the ionization method, and it is preferable to use the quadrupole focusing type as the separating portion.
The “liquid chromatography/mass spectrometry (LC/MS)” is an analytical method in which the high performance liquid chromatography method and the mass spectrometry are combined.
The present invention further provides a detection kit, for detecting at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, or onset risk of these diseases, which contains a ganglioside GM3 as a standard substance, wherein the ganglioside GM3 has a specific structure represented by the following formula (1).
[In the formula (1), R1 represents a glycan constituting the ganglioside GM3, and R2—C(═O)— represents a fatty acid residue having 16 or more and 24 or less carbon atoms which may have a double bond, and having an OH group as a substituent.]
When the detection kit of the present invention is used, the above-mentioned effects can be more suitably exerted. That is, when the detection kit of the present invention is used, by using the detection method of the present invention, it is easily possible to detect at least one disease selected from the group consisting of hyperglycemia, dyslipidemia, hypertension and atherosclerosis or a disease two or more of which are combined, or to detect onset risk of these diseases.
Further, when the detection kit of the present invention is used, it is possible to detect whether or not the subject is suffering from a specific disease such as lifestyle-related diseases, etc., or to detect whether or not the subject has a risk of developing a specific disease such as lifestyle-related disease, etc., whereby the state of the subject can be more accurately grasped.
Hereinafter, the present invention will be explained more specifically by showing Examples; but the present invention is not limited to them unless beyond its scope.
Subjects were 125 Japanese people. Among the 125 people, normal subjects were 26, patients with only visceral fat accumulation (VFA) were 39, patients with VFA and hyperglycemia were 15, and patients with VFA and dyslipidemia were 28.
Patients with VFA, hyperglycemia and dyslipidemia, namely patients with metabolic syndrome were 17.
All subjects gave their written informed consent prior to their inclusion in the study.
100 ng of 13C-labeled GM3 (hereinafter abbreviated as “d18:1-[13C]16:0”) was added for an internal standard to 50 μL of serum, the solution was lyophilized, and then dissolved in 5 mL of a mixture of chloroform and methanol (chloroform:methanol=1:1). The mixture was sonicated, incubated at 40° C. for 1 hour, and then centrifuged at 4° C. for 30 minutes (15,000×g). The supernatant was collected and the pellet was subjected to the same extraction procedure. Obtained supernatants were combined and evaporated, thereby extracting lipids from the serum.
Each GM3 species was measured using LC-MS/MS with electrospray ionization, in multiple reaction monitoring negative ionization mode. Accela 1250 Pump (Thermo Fisher Scientific Inc.) as LC and Vantage AM (quadrupole type, Thermo Fisher Scientific Inc.) as a mass spectrometry device were used. Calibration of the mass spectrometry device was performed using a mixture of GM3 species extracted from milk.
Lipids extracted from 50 μL of serum were dissolved in 50 μL of methanol, and 6 μL of the mixture was injected into LC-MS/MS. In the 6 μL of the mixture, 12 ng of d18:1-[13C]16:0 being the internal standard was contained.
The settings of LC are as follows.
(2) Flow rate: 50 μL/min
(3) Mobile phase: (i) A mixture of 20% of H2O, 50% of 2-propanol and 30% of methanol, containing 0.1% acetic acid and 0.1% ammonia (hereinafter referred to as “solvent A”). (ii) A mixture of 2% of H2O, 50% of 2-propanol and 48% of methanol, containing 0.1% acetic acid and 0.1% ammonia (hereinafter referred to as “solvent B”).
(4) Gradient program: The gradient program employed started with 100% solvent A for 5 min then ramped to 100% solvent B over 30 min. 100% solvent B was maintained for 4 min, then the solvent was returned to 100% solvent A over 1 min and held there for 10 min.
The settings of mass spectrometry device are as follows.
(1) Spray voltage: −2500 V
(2) Capillary temperature: 204° C.
(3) Vaporizer temperature: 373° C.
(4) Sheath gas pressure (nitrogen gas): 50 (arbitrary units)
(5) Ion sweep gas pressure: 0 (arbitrary units)
(6) Auxiliary gas pressure (nitrogen gas): 15 (arbitrary units)
(7) S-lens RF amplitude: 276
(8) Ionic dissociation (CID) collision energy: 0
(9) Collision pressure: 1.0 mTorr
(10) Collision energy: 53 eV
(11) Scan time: 0.01 second
The amount of each GM3 species was quantified based on the internal standard (d18:1-[13C]16:0). The total amount of GM3 was calculated based on the total amount of 23 kinds of GM3 detected.
The results of quantitative analysis were shown as mean±SD. For all statistical analysis, P≤0.05 was deemed statistically significant. In statistical analysis, Mann-Whitney U test or two-sample t-test was used to obtain Spearman's rank correlation coefficients.
Further, in Example 5, all GM3 species were subjected to multivariate analysis. Body mass index (BMI), waist, calcaneus stiffness, diastolic blood pressure (DBP), systolic blood pressure (SBP), Brinkman index, ejection fraction, HOMA-IR, hemoglobin (HbA1c), platelets, AST (glutamic oxaloacetic transaminase (GOT)), ALT (glutamate-pyruvate transaminase (GPT)), γ-glutamyl transpeptidase (γ-GTP), triglyceride, creatinine, uric acid, high-density lipoprotein cholesterol (HDL-c), C-reactive protein, LDL-c, free fatty acids, total adiponectin, autotaxin, sphingomyelin, phosphotidylcholine, lysophosphatidylcholine, and IMT were investigated as explanatory variables. All statistical analysis were conducted using GraphPad Prism 6.04 (GraphPad Software Inc.) except multivariate analysis when XLSTAT (version 2014.4.02, Addinsoft Corporation) was utilized.
Markers (indices) with regard to specific diseases such as metabolic syndrome in (1) normal subjects (n=26), (2) patients with only VFA (n=39), (3) patients with VFA and hyperglycemia (n=15), (4) patients with VFA and dyslipidemia (n=28), and (5) patients with VFA, hyperglycemia and dyslipidemia (namely, patients with metabolic syndrome) (n=17) were measured, respectively. The results are shown in Table 1. In Table 1, (1) shows normal subjects, (2) shows patients with only visceral fat accumulation, (3) shows patients with visceral fat accumulation and hyperglycemia, (4) shows patients with visceral fat accumulation and dyslipidemia, and (5) shows patients with visceral fat accumulation, hyperglycemia and dyslipidemia.
Further, in the table, “ns” shows P>0.05, “*” shows P≤20.05, “**” shows P≤0.01, “***” shows P≤0.001, “****” shows P≤0.0001. All pieces of data in the table are reported as mean±SD.
59.9 ± 2.1 **
168.2 ± 1.5 ***
78.0 ±1.7 ****
7.2 ± 0.7 *
1.7 ± 0.2 *
The total amount of GM3 of (1) normal subjects (n=26), (2) patients with only VFA (n=39), (3) patients with VFA and hyperglycemia (n=15), (4) patients with VFA and dyslipidemia (n=28), and (5) patients with VFA, hyperglycemia and dyslipidemia (namely, patients with metabolic syndrome) (n=17) was measured, respectively. The results are shown in
The results of
Correlation between total GM3 and each disease marker (total cholesterol, LDL-c, autotaxin, mean IMT, fasting blood glucose, insulin, HOMA-IR, HbA1c) was analyzed using Spearman's rank correlation. The results are shown in
As shown in
The results of Examples 2 and 3 suggested that, the possibility to be or become afflicted by specific diseases such as metabolic syndrome was not precisely determined by measuring or comparing the total amount of GM3.
Regarding GM3, there are a large number of molecular species depending on carbon numbers, the number of double bonds, presence or absence of substituents, or kinds of substituents, in a hydrocarbon group of the ceramide moiety. On the basis of results of Examples 2 and 3, it was thought that measuring specific GM3 molecular species can provide more precise information on subjects relating to lifestyle-related diseases or specific diseases, not measuring the total amount of GM3, so that correlation between each GM3 species and disease was analyzed.
Similar to Example 1, the amount of each GM3 species of (1) normal subjects (n=26), (2) patients with only VFA (n=39), (3) patients with VFA and hyperglycemia (n=15), (4) patients with VFA and dyslipidemia (n=28), and (5) patients with VFA, hyperglycemia and dyslipidemia (namely, patients with metabolic syndrome) (n=17) was measured. The results are shown in
In the horizontal axis of
Regarding the structure of each GM3 species, for example, in a case of “d18:1-24:0,” “d18:1” means that the sphingomyelin moiety of GM3 is a hydrocarbon group having 18 carbon atoms and the hydrocarbon group has a double bond. “24:0” means that the fatty acid of GM3 is a hydrocarbon group having 24 carbon atoms and the hydrocarbon group has no double bond. For example, when “d18:1-24:0” has an OH group as a substituent in the fatty acid, this is described as “d18:1-h24:0”.
The vertical axis of
The results of
Particularly, “d18:1-h24:0”, “d18:1-h24:1”, “d18:1-h22:0” of (5) patients with VFA, hyperglycemia and dyslipidemia was statistically significant, with levels 1.7 times or more higher than that of normal subjects.
These results suggested that, GM3 having an OH group as a substituent in the fatty acid of GM3 (hydroxylated GM3) was useful for detecting specific diseases or lifestyle-related diseases such as metabolic syndrome.
Correlation between each disease marker, and GM3 species which comprised a large proportion of GM3 in the serum (12 kinds described in the horizontal axis of
The disease markers are as follows.
(1) Marker for obesity: BMI
(2) Marker for metabolic syndrome: LDL-c, autotaxin (in Table 2, abbreviated as “ATX”), HOMA-IR, AST, ALT, HbA1c
(3) Marker for atherosclerosis: lysophosphatidylcholine (in Table 2, abbreviated as “LPC”), sphingomyelin (in Table 2, abbreviated as “SM”), ejection fraction (in Table 2, abbreviated as “EF”), C-reactive protein (in Table 2, abbreviated as “CRP”), platelets, triglyceride (TG)
(4) Markers for hypertension: blood pressure (BP)
(5) Markers for nephropathy: creatinine (CRE)
(6) Other markers: age
“C24:0” in Table 2 corresponds to “d18:1-24:0” in
The results of Table 2 showed that hydroxylated GM3 (hC24:0, hC24:1, hC22:0) was correlated with makers with regard to specific disease more than non-hydroxylated GM3. Particularly, “hC24:1” with a structure represented by the following formula (2) ([hC24:1]GM3) was correlated with a large number of markers such as age, autotaxin, sphingomyelin, blood pressure, HOMA-IR, lysophosphatidylcholine, HbA1c, AST, ALT, C-reactive protein, and thus it was suggested that “hC24:1” was useful for detecting or diagnosing specific diseases.
Further, the results of Table 2 showed that all GM3 species were not correlated with molecules regarding specific disease. Since it is thought that the total amount of GM3 is increased with increase of molecular species not regarding specific disease, it is suggested that using the total amount of GM3 as a maker for detecting or diagnosing specific disease may induce false positive or erroneous detection or diagnosis.
[In the formula (2), R1 represents a glycan constituting the ganglioside GM3.]
<Correlation Between [hC24:1]GM3 and Each Disease Marker>
The results of Example 5 suggested that [hC24:1]GM3 was useful as a marker for specific diseases such as metabolic syndrome, or a maker for lifestyle-related diseases or specific diseases. Next, correlation between [hC24:1]GM3 and each disease marker (total cholesterol, LDL-c, autotaxin, IMT, fasting blood glucose, insulin, HOMA-IR, HbA1c) was analyzed using Spearman's rank correlation. The results are shown in
The results of
<Measurement of Content of [hC24:1]>
The amount of [hC24:1] relative to [C24:1] contained in each of the whole GM3, the whole sphingomyelin and the whole ceramide in the serum was measured. The results are shown in
The vertical axis of
The results of
On the other hand, 0.67% of [C24:1] contained in sphingomyelin was [hC24:1], and 0.45% of [C24:1] contained in ceramide was [hC24:1].
These results demonstrated that, on the basis of the fact that patients with specific disease have a large amount of GM3, since “a fatty acid having 24 carbon atoms, having an OH group as a substituent, and having a double bond” was bonded to (existed in) GM3 particularly, [hC24:1]GM3 was particularly suitable as a marker with regard to specific diseases.
The results of
Each amount of hydroxylated GM3 (“d18:1-h24:0”, “d18:1-h24:1”, “d18:1-h22:0”) and non-hydroxylated GM3 (“d18:1-24:0”, “d18:1-24:1”, “d18:1-22:0”) of (1) normal subjects (n=26), (2) patients with only VFA (n=39), (3) patients with VFA and hyperglycemia (n=15), (4) patients with VFA and dyslipidemia (n=28), and (5) patients with VFA, hyperglycemia and dyslipidemia (namely, patients with metabolic syndrome) (n=17) was measured. The results are shown in
The vertical axis of
The results of
On the other hand, the results of
Correlation between hydroxylated GM3 or non-hydroxylated GM3, and each disease marker (total cholesterol, LDL-c, autotaxin, IMT, fasting blood glucose, insulin, HOMA-IR, HbA1c) was analyzed using Spearman's rank correlation. The results are shown in
The results of
On the other hand, the results of
The results of Examples 8 and 9 showed that GM3 having an OH group as a substituent in the fatty acid of GM3 (hydroxylated GM3) was useful for detecting specific diseases or lifestyle-related diseases such as metabolic syndrome.
Detection methods of the present invention can detect at least one disease selected from the group consisting of visceral fat accumulation, hyperglycemia, dyslipidemia, hypertension and atherosclerosis, or a disease two or more of which are combined or detect onset risk of these diseases, can grasp the condition of subjects precisely. Thus, the present invention can be used in fields such as diagnostic agents, reagents for research and clinical examinations.
The present application is based on the Japanese Patent Application No. 2014-224811 filed on Nov. 5, 2011; and entire contents of the application are cited and incorporated herein as the disclosure of the specifications of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-224811 | Nov 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/080801 | 10/30/2015 | WO | 00 |
