CN115078584A - Simple, quick, low-cost and high-flux muscle mass measuring method and detection kit - Google Patents
Simple, quick, low-cost and high-flux muscle mass measuring method and detection kit Download PDFInfo
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Abstract
The application discloses a simple and rapid muscle quality measuring method with low cost and high flux, a calculator and a muscle quality detection kit, which comprise the following steps: (1) the subject takes d 3 An aqueous creatine solution; (2) collecting a urine sample of a tested object; (3) treating a urine sample: adding a diluent into a urine sample of a tested object for dilution; (4) taking the supernatant, injecting and analyzing to obtain a creatinine response value and a D3 creatinine response value: analyzing by adopting a liquid chromatography-tandem mass spectrometry method; (5) and calculating the muscle mass to obtain the muscle mass SMM. This application can be used forThe early diagnosis and screening of the sarcopenia patients effectively solves the problems that the prior analysis method has complex sample pretreatment, long time consumption and high cost, needs to quantify the urine volume to calculate the leakage of the creatine and the like, and improves the detection timeliness and the accuracy.
Description
Technical Field
The application belongs to the field of clinical biomarker detection, relates to muscle mass measurement, and particularly relates to a simple, quick, low-cost and high-flux muscle mass measurement method, a calculator and a muscle mass detection kit.
Background
Sarcopenia, abbreviated as "sarcopenia", refers to a syndrome characterized by progressive and widespread reduction in skeletal muscle strength and mass. Sarcopenia is a relatively less important disease than osteoporosis. Studies have reported that human muscle mass decreases continuously from 20 to 90 years, perhaps by about 50%, particularly from 50 years, at a rate of 15% loss every decade and 30% loss every decade after 70 years. Thus, 5% to 30% of the elderly over the age of 60, 11% to 50% of the elderly over the age of 80 develop sarcopenia to varying degrees, and people with malnutrition and chronic disease may also develop sarcopenia. The occurrence of sarcopenia can affect various physiological functions of patients, cause low physical ability and life quality of the patients, increase risks of falling, fracture, hospitalization, disability and the like, also affect occurrence, development and prognosis of various diseases and bring important burden to the society. As the world population ages, sarcopenia has become a health concern worldwide.
With the aging degree of the population deepening, the early screening and diagnosis of sarcopenia have important significance in the clinical and public health fields. Early diagnosis of sarcopenia is mainly based on measurement of skeletal muscle mass, although there are extensive imaging bases for muscle mass detection, such as CT, MRI, dual energy X-ray absorption (DXA), Bioelectrical Impedance (BIA), etc., and the inherent limitations of these techniques prevent rapid, sensitive, and accurate objective measurement. CT and MRI imaging techniques are used as gold standard methods for evaluating human body components, and are commonly used for measuring muscle mass, but the application of the two techniques in the field of sarcopenia is limited by high cost, long time consumption, low sensitivity, no batch analysis, and the like, measurement errors occur when the water amount in a body changes, and CT also has radiation exposure risk. DXA has the advantages of relatively low cost, low radiation exposure, short scan time, and the like, and is recommended in various guidelines and is the most widely used method in sarcopenia related studies and clinical practice, but DXA detects lean soft tissue of the limbs, including muscle mass, water, and some non-adipose soft tissue, and may overestimate muscle mass, especially for patients with extracellular fluid accumulation. This is also true of BIA, the result of which is not a direct measure of the muscle, but rather is based on individual body resistance parameters, possibly influenced by a number of factors.
Researchers have been looking for biomarkers that can directly reflect muscle mass. 24h urinary creatinine excretion, the most studied biomarker measurement, is based on the biological principles of creatine and creatinine, and the muscle mass can be determined by the size of the muscle pool, considering that the production and excretion of creatinine in urine is directly related to the size of the muscle pool in muscle. The method is researched and applied by students, however, the implementation of completely collecting the total amount of urine discharged within 24 hours is difficult, the compliance of the collected population is poor, the fault tolerance rate in the process is low, and the estimated value of muscle quality is reduced due to the absence of urine sample collection; in addition, subjects must exercise strict diet control, as consumption of creatine-containing foods can lead to an overestimation of total body muscle mass. Based on this, there is a need for a technique that is more practical, ensures accurate measurement, achieves sensitive, simple, fast, nondestructive, low-cost, accurate and standardizable muscle mass measurement, and is used for early diagnosis and screening of sarcopenia.
The researchers turned their eyes to the isotope creatine dilution method based on 24h urinary creatinine excretion, which was proposed by the scholars as early as 1970, but was not further studied and applied due to the technical limitations at that time. The Evans group verified the isotope creatine dilution method, the size of human creatine pool was calculated by measuring steady-state enrichment of D3-creatinine in urine, and the total body muscle mass was obtained, and the D3 creatine dilution method muscle mass was calculated as in formula (I-III). Compared with other methods, the method does not depend on strict and complete urine collection, only needs to collect a single point urine sample in a steady state period after administration, and has the advantages of no radiation risk, good subject compliance, low cost, capability of realizing sensitive, rapid and batch analysis and the like. Therefore, the isotope creatine dilution method is expected to become a conventional measurement method for muscle mass, and can also be used for early diagnosis of sarcopenia and monitoring the change of muscle mass in diseases and treatments.
The MPE calculation formula is:
the size of the creatine pool is calculated as follows:
the SMM calculation formula is:
according to the existing data at home and abroad, the research on measuring the muscle mass by an isotope creatine dilution method is very little, the literature amount is less than 20, and in the existing reports, D3 creatinine, creatinine and D3 creatine need to be accurately quantified, but the quantitative measuring method is very little described and discussed in detail, and the accuracy and comparability are difficult to ensure. At present, high performance liquid chromatography mass spectrometry is adopted for determining D3 creatine and creatinine, and the matrix effect of a urine sample is a serious problem influencing the accuracy and the sensitivity in determination. In the report, isotope internal standard correction is mostly adopted, but for the measurement, the substance to be measured is an isotope labeled substance, so that a suitable isotope internal standard is difficult to obtain, the economic cost is extremely high, and the sensitivity loss caused by the matrix effect cannot be eliminated by the isotope internal standard correction. There are also corrections by means of extractive sample purification or matrix matching, but the complicated procedure is not suitable for clinical practice. These factors are serious obstacles to the spread of this approach. Furthermore, as shown in the above formula, it has been reported that when measuring the size of the creatine pool, it is necessary to specify the amount of D3 creatine actually entering the creatine pool, i.e. to obtain the cumulative leakage amount of D3 creatine, which means that D3 creatine in urine is accurately quantified, and it is necessary to collect a complete urine sample from a single point of urine sample collection from the time point after administration to the steady state and accurately quantify the volume of the urine sample. Obviously, such urine sample collection requirements and analysis requirements will reduce subject compliance to some extent and increase experimental complexity and uncertainty of assay results. Under the background, a quick, sensitive and efficient method is urgently needed to be established, the method is used for the field of clinical detection, the operation is simple and easy, the muscle quality can be accurately measured, the compliance of patients is high, the detection flow can be standardized, so that batch analysis and early diagnosis and screening of sarcopenia patients are realized, and no good solution exists in the existing literature reports.
The foregoing background is provided to facilitate an understanding of the present application and is not admitted to be prior art to the present application by public general knowledge.
Disclosure of Invention
Based on the problems, the muscle quality determination method which is simple, quick, sensitive and standardizable and is suitable for clinical batch sample analysis can be used for early diagnosis and screening of sarcopenia patients. The method effectively solves the problems that the prior analysis method has complex sample pretreatment, long time consumption and high cost, needs to quantify the urine volume to calculate the creatine leakage and the like, and improves the detection timeliness and accuracy.
A simple, quick, low-cost, high-throughput muscle mass measurement method comprises the following steps:
(1) the subject takes d 3 An aqueous creatine solution;
(2) collecting a urine sample of a tested object;
(3) treating a urine sample: adding a diluent into a urine sample of a tested object for dilution;
(4) taking the supernatant, injecting and analyzing to obtain a creatinine response value and a D3 creatinine response value: analyzing by adopting a liquid chromatography-tandem mass spectrometry method;
(5) and (3) carrying out muscle mass calculation, wherein the calculation formula is as follows:
obtaining the muscle mass SMM, wherein the correction coefficient is 3-5.
In one or more specific embodiments of the present application, in the (1), d 3 The creatine water solution is d with the purity of more than or equal to 99 percent 3 Creatine is diluted with sterile ultrapure water to make a 0.5mg/mL or 1.0mg/mL solution.
In one or more specific embodiments of the present application, in said (1), the creatine aqueous solution is administered in a single dose at a dose of 0.5mg/kg.bw or 1.0 mg/kg.bw.
In one or more specific embodiments of the present application, in the step (2), the urine sample of the subject is collected from the random urine of the subject 30 to 72 hours after the subject takes D3-creatine.
In one or more specific embodiments of the present application, in the (3), the diluent is one or more of water, acetonitrile-water, methanol-water, methanol-acetic acid buffer, and acetonitrile-ammonium acetate solution.
In one or more specific embodiments of the present application, in the (3), the dilution ratio of the diluent is 10 to 30 times.
In one or more specific embodiments of the present application, in the (4), the chromatographic conditions are: using ACQUITY
Separating the target compound with BEH HILIC chromatographic column (3.0 × 50mm, 1.7 μm); the mobile phase A is 10mmol/L ammonium acetate water solution containing 5% (v: v) acetonitrile, and the mobile phase B is acetonitrile; performing isocratic elution by using 75% B, wherein the flow rate is 0.2 mL/min; the column temperature is 40 ℃; the temperature of the sample injection plate is 4 ℃; the sample injection volume is 5 mu L;
the mass spectrum conditions are as follows: electrospray ionization (ESI) positive ion mode; the atomization gas is high-purity nitrogen with the flow rate of 3L/min; the heating gas is anhydrous air, and the flow rate is 10L/min; the drying gas is nitrogen, and the flow rate is 10L/min; the collision gas is argon, and the pressure is adjusted to 270 kPa; the interface temperature is 300 ℃; the interface voltage is 3.0 kV; the temperature of the DL pipe is 250 ℃; the temperature of the heating block is 400 ℃; detector voltage 2.24 kV; the multiple reaction monitoring MRM mode was performed for 6min data acquisition, with MRM parameters as shown in table 1, where is the quantitative ion:
TABLE 1 Mass spectrometric detection of MRM parameters
The application also provides a calculator.
A calculator having built in formulas specific to a simple, fast, low cost, high throughput method of measuring muscle mass, said formulas being formulas IV-VI above.
The application also provides a muscle quality detection kit.
A muscle mass detection kit comprises a diluent, a mobile phase for chromatographic mass spectrometry, a chromatographic column for chromatographic mass spectrometry, a calculator and an instruction for use, wherein the instruction for use contains the simple, rapid, low-cost and high-throughput muscle mass measurement method.
In one or more specific embodiments of the present application, the calculator is the calculator described above.
The invention principle and the beneficial effects are as follows:
based on the determination principle of a D3 creatine dilution method, namely, deuterium (D3) labeled creatine is used as a tracer, D3 creatinine enrichment factors (MPE) are calculated by determining the content of D3 creatine and metabolites D3 creatinine and unlabeled creatinine in a urine sample, and the time range of stable D3 creatine in a creatine pool can be reflected by searching the MPE stabilization time period (isotope steady-state time); on the basis, urine samples at any time point within an isotope steady-state time range are collected, MPE values of the urine samples are measured, the size of a creatine pool is calculated by combining D3 creatine conversion absolute quantity (intragastric D3 creatine quantity-urine D3 creatine leakage quantity), and finally the total skeletal muscle quality is evaluated through a creatine pool and a skeletal muscle conversion coefficient. As can be seen from formulas (I-III), the unknown parameters for D3 creatine dilution to measure muscle mass are MPE and urine D3 creatine leak, so accurate quantification of the amount of D3 creatine, D3 creatinine and creatinine in the urine is required, and collection of the integrity and accurate quantification of the volume of the urine sample at all time intervals after administration is required to determine the absolute amount of D3 creatine leak. The HPLC-MS/MS technology is not a second choice for quantifying the content of D3 creatine, D3 creatinine and creatinine at present, but the biggest technical difficulty is to eliminate the influence of the accuracy of the urine sample matrix, and complicated sample pretreatment or costly isotope internal standard correction is required, which is a serious obstacle for popularizing the method for clinical practice.
The inventor finds that the key parameter MPE of the D3 creatine dilution method is only related to the ratio of D3 creatinine to creatinine content in a urine sample through long-term research, so the inventor adopts the HPLC-MS/MS response value ratio of the two to represent MPE, and utilizes D3 creatinine and creatinine to compare with each other to dynamically correct the matrix effect of the urine sample, thereby omitting fussy sample pretreatment and high-cost isotope internal standard correction; the inventor also finds that the leakage amount of D3 creatine is extremely small, and the cumulative leakage amount of D3 creatine of C57 mice and SD rats is less than 5 percent in a proper dosage range. Therefore, the application adopts the ratio of D3 creatinine to creatinine response values to characterize D3 creatinine enrichment factor (MPE), ignores the influence of creatine leakage amount, and improves the measurement and evaluation formulas of muscle quality, and the improved muscle quality calculation formulas are the formulas IV, V and VI.
The formula (IV, V and VI) greatly simplifies the experimental operation, and the form of the kit is adopted to realize the simple, quick, efficient, accurate and low-cost measurement of the muscle mass. The method provided by the application is verified by animal experiments, the obtained steady-state time judgment is consistent with that of a conventional method, and the obtained muscle mass measurement result is consistent with that of a gold standard MRI.
The method can realize rapid, accurate and sensitive detection of the muscle mass, and the accuracy and the practicability of the method are verified by comparing the muscle mass with the gold standard MRI through repeated determination and calculation, and the measurement result is consistent with the determination result of the gold standard MRI. The detection method effectively removes and corrects the matrix effect through single-point urine sample collection, simple sample pretreatment, rapid chromatographic separation and innovative improvement on muscle quality evaluation, greatly simplifies the determination steps and improves the detection efficiency.
This application adopts dedicated muscle mass calculator, according to the operation specification, need not to carry out accurate ration to each determinand, inputs the chromatogram mass spectrum response value of D3 creatinine and creatinine, can obtain the muscle mass value, easy operation measures fast, has also saved loaded down with trivial details computational process, can realize high flux batch sample analysis. Expensive 13C does not need to be purchased as an internal standard to correct matrix effect, the cost for measuring the muscle mass is about 200 yuan/case, and is far lower than the cost of gold standard MRI detection.
The method is optimized in the aspects of chromatographic mass spectrometry conditions and sample treatment, and the sensitivity, accuracy, repeatability and practicability of the method are ensured by adopting the corresponding detection kit through repeated animal experiments, repeated analysis of various labeled samples and actual samples and comparison with the gold standard MRI, so that the clinical rapid diagnosis and screening can be met.
The detection kit realizes batch analysis and standardized operation of multiple samples, and obtains stable results. The urine sample can be processed according to the instruction, the operation is simple, the operation is easy for operators to operate, the flow standardization can be realized, and the work efficiency of the inspection is greatly improved. By adopting the sample treatment process, only 40 mu l of urine sample is needed, the chromatographic mass spectrometry detection can be completed within 8min, and the whole process can be completed within 15 min.
Drawings
FIG. 1 is a D3 creatinine and creatinine chromatogram;
FIG. 2 is a schematic view of a muscle mass detection kit;
FIG. 3 shows the time-varying mean MPE concentration of C57 mice at a 1.0mg/kg. bw gavage dose;
FIG. 4 shows the time-varying trend of MPE signal mean values for C57 mice at a 1.0mg/kg. bw gavage dose;
FIG. 5 is a B-A test of skeletal muscle mass estimation and lean body mass in C57 mice;
FIG. 6 shows the time-varying trend of MPE signals for different gavage dose groups of SD rats, wherein A is a gavage dose of 0.5 mg/kg.bw; b, the stomach dose is 1.0 mg/kg.bw;
FIG. 7 shows the skeletal muscle mass estimation and lean body mass B-A test of SD rats (0.5mg/kg.bw), wherein A is 24h-36 h; FIG. B is a view from 36h to 48 h; c is 48h-54 h; graph D is 54h-60 h; e is 60h-72 h; f is 72h-84 h; g is 84h-96 h;
FIG. 8 shows the skeletal muscle mass estimation and lean body mass B-A test of SD rats (1.0mg/kg. bw), wherein A is 24h-36 h; FIG. B is a view from 36h to 48 h; c is 48h-54 h; graph D is 54h-60 h; e is 60h-72 h; f is 72h-84 h; g is 84h-96 h;
FIG. 9 shows the skeletal muscle mass estimation and lean body mass B-A test in SD rats (dose-pooled), wherein A is 24-36 h; FIG. B is 36h-48 h; c is 48h-54 h; graph D is 54h-60 h; e is 60h-72 h; f is 72h-84 h; and the G picture is 84h-96 h.
Detailed Description
The invention will be further explained with reference to the drawings.
A muscle mass measurement method comprising the steps of:
(1) the subject takes d 3 An aqueous creatine solution;
(2) collecting a urine sample of a tested object;
(3) treating a urine sample: adding a diluent into a urine sample of a tested object for dilution;
(4) taking the supernatant, injecting and analyzing to obtain a creatinine response value and a D3 creatinine response value: analyzing by adopting a liquid chromatography-tandem mass spectrometry method;
(5) and (3) carrying out muscle mass calculation, wherein the calculation formula is as follows:
wherein, the correction coefficient is determined by comparing the experimental determination results of different muscles with the results of gold standard MRI, and the correction coefficient is between 3 and 5.
Figure 1 is a chromatogram of D3 creatinine and creatinine.
Alternatively, (1) wherein d 3 The creatine solution has a purity of more than or equal to 99% 3 Diluting creatine with sterile ultrapure water to obtain solution with concentration of 0.5mg/mL or 1.0 mg/mL; the creatine in aqueous solution is administered in a single dose at a dose of 0.5mg/kg.bw or 1.0 mg/kg.bw.
Optionally, in the step (2), the urine sample of the tested object is collected into random urine of the tested object 30-72 hours after D3-creatine is taken.
Alternatively, in (3), the dilution factor is 10 to 30.
Alternatively, (4), the chromatographic conditions are: using ACQUITY
BEH HILIC column (3.0X 50mm, 1.7 μm) separates the target compound. The mobile phase A is 10mmol/L ammonium acetate water solution containing 5% (v: v) acetonitrile, and the mobile phase B is acetonitrile; isocratically eluted with 75% B at a flow rate of 0.2 mL/min. The column temperature is 40 ℃; the temperature of the sample injection plate is 4 ℃; the injection volume was 5. mu.L.
The mass spectrum conditions are as follows: electrospray ionization (ESI) positive ion mode; the atomization gas is high-purity nitrogen with the flow rate of 3L/min; the heating gas is anhydrous air, and the flow rate is 10L/min; the drying gas is nitrogen, and the flow rate is 10L/min; the collision gas was argon and the pressure was adjusted to 270 kPa. The interface temperature is 300 ℃; the interface voltage is 3.0 kV; the temperature of the DL pipe is 250 ℃; the temperature of the heating block is 400 ℃; the detector voltage was 2.24 kV. Data acquisition was performed for 6min in Multiple Reaction Monitoring (MRM) mode, with MRM parameters as shown in Table 2.
TABLE 2 Mass spectrometric detection of MRM parameters
Note: quantification of ions.
Based on the above formulas IV, V and VI, the present application also provides a calculator.
A calculator has formulas IV, V and VI built in.
Based on the muscle mass measuring method and the calculator, the application also provides a muscle mass detection kit.
Referring to fig. 2, fig. 2 is a muscle mass detection kit of the present application, which includes the above-mentioned diluent for sample processing, the mobile phase for chromatography-mass spectrometry, the chromatographic column for chromatography-mass spectrometry, a calculator dedicated for muscle mass calculation, and an instruction for use, wherein the instruction contains the steps of the above-mentioned muscle mass measurement method.
Example 1
C57 mice: one day before gavage (D) 0 ) Using EchoMRI TM The 500 body composition meter measures sequentially the body composition of each mouse (n-8) including Lean Body Mass (LBM), fat, free water and bound water content, immediately after which the mice are placed in metabolic cages, raised in single cages, normally given a diet, and the collection of urine 24 hours before administration is started as a baseline observation. The next day (D) 1 ) D3 creatine solution was administered to each mouse in a single dose using intragastric administration at a dose of 1.0mg/kgThe mice were returned to the metabolism cage, raised in a single cage, normally given diet, and urine was collected after gavage at intervals of 24 hours for 3 consecutive days (72 hours). After urine collection is finished at each time period, the urine is immediately transferred to a polypropylene centrifugal tube and is stored in a refrigerator at minus 80 ℃ in a sealing way.
The urine sample was taken out and placed at room temperature for thawing, and fully vortexed for 30s after it had thawed and returned to room temperature. Absorbing a proper amount of urine sample, adopting a detection kit, adding a diluent for diluting by 20 times (the diluent is acetonitrile-water), fully and uniformly mixing by vortex, centrifuging for 5min at 10000rpm, taking out supernatant, filtering by a 0.22 mu m filter membrane, and injecting filtrate into UPLC-MS/MS for analysis. UPLC-MS/MS analysis is carried out by adopting the detection kit, and D3 creatinine and creatinine response values are collected and used for determining steady state time and calculating results of creatine pool and muscle mass.
In the embodiment, for simplifying the operation, the MPE calculation is converted into the MPE ratio of the chromatographic mass spectrum signals of D3 creatinine and creatinine under the same dilution multiple Signal And the MPE is calculated and meanwhile the unlabeled creatinine is adopted to realize dynamic matrix effect correction, so that the concentrations of D3 creatinine and creatinine do not need to be accurately quantified, and the feasibility of using the D3 creatinine and the creatinine in isotope steady-state time judgment is explored. The results show that MPE Concentration of (FIG. 3) and MPE Signal (fig. 4) the trends of both over time are consistent. Using formula (IV, V, VI), correction factor is 4.3, MPE Signal Values the size of the C57 mouse creatine pool was calculated for each time of collection and the results are shown in table 3.
TABLE 3C 57 mouse creatine pool size (mg)
| Time of urine sample Collection (h) | Mean value | Standard deviation of | |
| 24 | 144.11 | 48.36 | 84.71-221.66 |
| 48 | 205.22 | 49.58 | 144.22-270.87 |
| 72 | 252.33 | 65.68 | 164.82-375.56 |
In view of the close relationship between creatine pool size and skeletal muscle mass, correlation analysis was performed for creatine pool and MRI-determined lean body mass values at each time interval.
The correlation analysis result of the size of the creatine pool and the lean body mass of the C57 mouse shows that the creatine pool and the lean body mass have good linear trend within the isotope steady-state time range, wherein the creatine pool and the lean body mass have good correlation within 24-48 h and 48-72 h, and the correlation coefficients are all more than 0.7(Pearson's r) 48h ＝0.70567，P＝0.04521；Pearson's r 72h ＝0.72897，P＝0.04021)。
The skeletal muscle quality measured in this example was evaluated for accuracy using MRI as a verification method: the mean values of the MRI and the study measurements were plotted on the abscissa, the absolute difference (lean body mass-measured value) on the ordinate, and the mean ± standard deviation of the difference as the upper and lower limits of 95% agreement, and a Bland-Altman (B-a) test chart (fig. 5) was plotted to analyze the agreement of the two measurement results at each time period in the isotope steady state. The absolute differences all fall within the 95% consistency range.
Example 2
SD rat: subjects (n-20) were randomized into two groups (groups a and B) one day prior to gavage (D) 0 ) Using echo MRI TM The 500 body composition meter measures body composition of each rat in turn, including lean body mass, fat, free water and bound water content, immediately after which the rats are placed in metabolic cages, housed in individual cages, fed a diet normally, and urine collected 24 hours prior to dosing is observed as a baseline. The next day (D1), rats were given a single administration of D3 creatine in water in intragastric doses: group A0.5 mg/kg.bw; bw of 1.0mg/kg in group B, then put back into metabolism cage, raise in single cage, normally give diet, and start collecting urine 12h, 24h, 30h, 36h, 48h, 54h, 60h, 72h, 84h, 96h after gavage. After urine collection is finished at each time period, the urine is immediately transferred to a polypropylene centrifugal tube and is stored in a refrigerator at minus 80 ℃ in a sealing way.
The urine sample was taken out and placed at room temperature for thawing, and after it had melted and returned to room temperature, it was vortexed thoroughly for 30 s. Absorbing a proper amount of urine sample, adopting the detection kit, adding a diluent for diluting by 20 times (the diluent is acetonitrile acetic acid buffer solution), fully and uniformly swirling, centrifuging at 10000rpm for 5min, taking out supernate, filtering by a filter membrane of 0.22 mu m, and injecting the filtrate into UPLC-MS/MS for analysis. And (3) performing UPLC-MS/MS analysis by using a detection kit, collecting D3 creatinine and creatinine response values for determining steady-state time and calculating results of a creatine pool and muscle mass.
Using MPE Signal The values are plotted on the ordinate and the sample times are plotted on the abscissa as a trend graph (fig. 6). The results show that at intragastric doses of 0.5mg/kg.bw and 1.0mg/kg.bw, two groups of MPEs Signal The trend of the change of the value is consistent, the peak value of the D3 creatinine enrichment degree is similar to the time of a mouse and is within 24h after the administration, and the isotope steady-state time is 24h after the administration.
The creatine pool size of SD rats was calculated using the formula (IV, V, VI) with a correction factor of 4.3, and the results are shown in Table 4. The correlation analysis result of the size of the creatine pool and the lean body mass of the SD rat shows that no matter the dosage group is 0.5mg/kg.bw or 1.0mg/kg.bw, the creatine pool and the lean body mass have a good linear trend; in the time periods of 30h-36h, 36h-48h, 48h-60h, 60h-72h and 72h-84h after the administration, the correlation between the size of the creatine pool and the lean body mass is better, and the correlation coefficients are all more than 0.6(P < 0.05).
MRI as a verification method, the skeletal muscle quality measured in this example was evaluated for accuracy: the mean values of the measured values of MRI and this example were plotted on the abscissa, the absolute difference values (lean body mass-measured value) on the ordinate, and the mean ± standard deviation of the difference values as the upper and lower limits of 95% agreement, and a Bland-Altman (B-a) test chart was drawn to analyze the agreement between the two measurement results at each time period in the isotope steady state. For SD rats, both assays showed good agreement between the different dose groups, with the absolute difference falling substantially within the 95% agreement range (fig. 7 and 8).
Considering that the test efficacy is reduced due to the decrease in the amount of the sample after the grouping by dose, the two-sample t-test and the mann-whitney U-test (48-hour creatine pool, non-normal distribution) were used to differentially test the sizes of 0.5mg/kg.bw and 1.0mg/kg.bw of two doses of creatine pools of SD rats by time period, and it was found that there was no significant difference in the sizes of the creatine pools of the two dose groups at the test level of 0.05, and that the relative average deviation of the sizes of the creatine pools of the two dose groups of SD rats at the same time period was small within 24h to 96h after the administration, the value was 2.12% to 8.14%, and it was considered that there was no significant difference in the sizes of the creatine pools at the steady state of the subjects at the two doses. Based on this, all subject assays were performed for concordance with MRI assays, both of which showed good concordance with an absolute difference falling substantially within the 95% concordance range (fig. 9).
TABLE 4 creatine pool size (mg) of SD rat
By adopting the detection kit, the muscle mass of the subject can be calculated only by inputting the chromatographic mass spectrum response values of the samples to be detected, namely creatinine and D3 creatinine.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A simple, quick, low-cost, high-throughput muscle mass measurement method comprises the following steps:
(1) the subject takes d 3 An aqueous creatine solution;
(2) collecting a urine sample of a tested object;
(3) treating a urine sample: adding a diluent into a urine sample of a tested object for dilution;
(4) taking the supernatant, injecting and analyzing to obtain a creatinine response value and a D3 creatinine response value: analyzing by adopting a liquid chromatography-tandem mass spectrometry method;
(5) and (3) carrying out muscle mass calculation, wherein the calculation formula is as follows:
obtaining the muscle mass SMM, wherein the correction coefficient is 3-5.
2. Simple, rapid, low-cost, high-throughput method of measuring muscle mass according to claim 1, wherein (1)In d 3 The creatine solution has a purity of more than or equal to 99% 3 Creatine is diluted with sterile ultrapure water to make a 0.5mg/mL or 1.0mg/mL solution.
3. The simple rapid, low-cost, high-throughput method of measuring muscle mass according to any one of claims 1-2, wherein in (1), the aqueous creatine solution is administered in a single dose of 0.5mg/kg.bw or 1.0 mg/kg.bw.
4. A simple, fast, low-cost, high-throughput method for establishing a muscle mass measurement according to any one of claims 1 to 3, wherein in the step (2), the urine sample of the subject is collected as random urine from the subject 30 to 72 hours after the subject takes D3-creatine.
5. The simple, rapid, low-cost, high-throughput method of measuring muscle mass according to any one of claims 1 to 4, wherein in (3), the diluent is one or more of water, acetonitrile-water, methanol-water, methanol-acetic acid buffer, and acetonitrile-ammonium acetate solution.
6. The simple, rapid, low-cost, high-throughput method of measuring muscle mass according to any one of claims 1 to 5, wherein in (3), the dilution of the diluent is from 10 to 30 times.
7. The simple, rapid, low-cost, high-throughput method of measuring muscle mass according to any one of claims 1 to 6, wherein in (4), the chromatographic conditions are: using ACQUITY
Separating the target compound with BEH HILIC chromatographic column (3.0 × 50mm, 1.7 μm); the mobile phase A is 10mmol/L ammonium acetate water solution containing 5% (v: v) acetonitrile, and the mobile phase B is acetonitrile; performing isocratic elution by using 75% B, wherein the flow rate is 0.2 mL/min; the column temperature is 40 ℃; the temperature of the sample injection plate is 4 ℃; the sample injection volume is 5 mu L;
the mass spectrum conditions are as follows: electrospray ionization (ESI) positive ion mode; the atomization gas is high-purity nitrogen with the flow rate of 3L/min; the heating gas is anhydrous air, and the flow rate is 10L/min; the drying gas is nitrogen, and the flow rate is 10L/min; the collision gas is argon, and the pressure is adjusted to 270 kPa; the interface temperature is 300 ℃; the interface voltage is 3.0 kV; the temperature of the DL pipe is 250 ℃; the temperature of the heating block is 400 ℃; detector voltage 2.24 kV; the multiple reaction monitoring MRM mode was performed for 6min data acquisition, with MRM parameters as shown in table 1, where is the quantitative ion:
TABLE 1 Mass spectrometric detection of MRM parameters
8. A calculator, characterized in that the calculator is provided with a formula which is specially used for a simple, fast, low-cost and high-flux muscle mass measuring method, and the formula is as follows:
9. a muscle mass detection kit comprising a diluent, a mobile phase for chromatographic mass spectrometry, a chromatographic column for chromatographic mass spectrometry, a calculator and instructions for use, wherein the instructions for use comprise a simple, rapid, low cost, high throughput method of measuring muscle mass according to any one of claims 1 to 8.
10. The muscle mass detection kit according to claim 9, wherein the calculator is the calculator of claim 9.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013086070A1 (en) * | 2011-12-07 | 2013-06-13 | Glaxosmithkline Llc | Methods for determining total body skeletal muscle mass |
| WO2014152610A1 (en) * | 2013-03-14 | 2014-09-25 | Abbott Laboratories | Biomarkers, related methods and systems for predicting loss of muscle mass |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013086070A1 (en) * | 2011-12-07 | 2013-06-13 | Glaxosmithkline Llc | Methods for determining total body skeletal muscle mass |
| WO2014152610A1 (en) * | 2013-03-14 | 2014-09-25 | Abbott Laboratories | Biomarkers, related methods and systems for predicting loss of muscle mass |
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| Title |
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| STEPHEN A. STIMPSON 等: "Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyl-d3) dilution in rats", 《JOURNAL OF APPLIED PHYSIOLOGY》 * |
| STIMPSON STEPHEN A 等: "Longitudinal changes in total body creatine pool size and skeletal muscle mass using the D3-creatine dilution method", 《JOURNAL OF CACHEXIA, SARCOPENIA AND MUSCLE》 * |
| WILLIAM J EVANS 等: "D3-Creatine dilution and the importance of accuracy in the assessment of skeletal muscle mass", 《JOURNAL OF CACHEXIA, SARCOPENIA AND MUSCLE》 * |
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