TWI769956B - Methods for predicting survival rate of sarcopenia patients - Google Patents

Abstract

Disclosure herein is a method for predicting survival rate of a subject suffering from sarcopenia. According to some embodiments of the present disclosure, the method comprises determining the level of PCaa C38:6 in the biological sample isolated from a subject so as to make a prognosis of the survival rate of the subject.

Description

Methods for predicting survival in patients with sarcopenia

The present invention relates to a detection method. Specifically, the present invention is a method for predicting the 5-year survival rate of a sarcopenia patient by analyzing the content of a target phospholipid in a biological sample derived from a sarcopenia patient.

According to the definition of the World Health Organization, 14% of the total population over the age of 65 is an advanced society, and 20% is a super-aged society. According to statistics, as of the end of July 2021, Taiwan's population over the age of 65 has reached 16.5%, indicating that Taiwan's population structure has officially entered an aging society. Due to demographic changes, many issues related to the health status and care of the elderly population have arisen. In addition to the well-known three highs (high cholesterol, blood sugar and blood lipids), heart-related diseases, diabetes and cancer, other aging-related diseases cannot be ignored.

Sarcopenia is a disease of loss of muscle mass and function due to aging. Patients with sarcopenia have many adverse functional and clinical effects, including disability, poor lower extremity function, metabolic syndrome and cardiovascular risk, and even death in severe cases.

Most sarcopenia is caused by multiple diseases or risk factors, not explained by a single etiology or pathology. Factors known to influence the progression of sarcopenia in addition to age and gender, lifestyle Diet, mobility, nutritional status, and chronic diseases (eg, osteoporosis or cardiovascular disease) are all associated with it, with malnutrition being the most common cause of decreased muscle mass and reduced mobility in older adults. There is currently no specific drug available for the treatment of sarcopenia, and general medical advice is to improve muscle loss through diet and exercise. The clinical judgment of the severity of sarcopenia is mainly based on the patient's mobility. However, these assessment methods are easily affected by other factors that affect the individual's performance during testing, and it is difficult to clearly determine the severity of sarcopenia.

In view of this, a method for clearly judging the severity of sarcopenia is urgently needed in the related art of the present invention.

SUMMARY The purpose of this summary is to provide a simplified summary of the disclosure to give the reader a basic understanding of the disclosure. This summary is not an exhaustive overview of the disclosure, and it is not intended to identify key/critical elements of embodiments of the invention or to delineate the scope of the invention.

；以及(b)基於步驟(a)的結果來預斷該個體的5年存活率，其中 當PCaa C38：6的含量等於或高於71微莫耳濃度且等於或低於80微莫耳濃度時，則該個體的5年存活率為60%；當PCaa C38：6的含量高於80微莫耳濃度時，則該個體的5年存活率大於60%；或當PCaa C38：6的含量低於71微莫耳濃度時，則該個體的5年存活率小於60%。 One aspect of the present invention relates to a method for predicting the survival rate of an individual with sarcopenia from a biological sample of the individual; the method comprises: (a) determining phospholipid choline diacyl group in the biological sample Content of (phosphatidylcholine diacyl, PCaa) C38:6 (PCaa C38:6), wherein PCaa C38:6 has the structure of formula (I):

and (b) predicting a 5-year survival rate for the individual based on the results of step (a), wherein the PCaa C38:6 level is equal to or higher than 71 micromolar and equal to or lower than 80 micromolar , the 5-year survival rate of the individual is 60%; when the content of PCaa C38:6 is higher than 80 micromolar concentration, the 5-year survival rate of the individual is greater than 60%; or when the content of PCaa C38:6 is low At 71 micromolar, the 5-year survival rate for this individual is less than 60%.

According to a further embodiment of the present disclosure, when the content of PCaa C38:6 is equal to or higher than 85 micromolar concentration, the 5-year survival rate of the individual is greater than 90%; or when the content of PCaa C38:6 is equal to or higher than 90% Below 62 micromolar concentrations, the 5-year survival rate for the individual is less than 31%.

According to some embodiments of the present disclosure, the biological sample may be blood, plasma, serum, or urine. According to certain embodiments of the present disclosure, the biological specimen is plasma.

According to certain embodiments of the present disclosure, the individual is a human.

After referring to the following embodiments, those with ordinary knowledge in the technical field of the present invention can easily understand the basic spirit and other purposes of the present invention, as well as the technical means and implementation aspects of the present invention.

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows: Fig. 1 is a graph showing the cumulative death occurrence curve according to a specific embodiment of the present invention. , which describes the cumulative death rate of subjects in different groups at different times; (A) is the cumulative death curve of subjects grouped according to the content of PCaa C38:6 in plasma; (B) is The cumulative mortality curve of subjects grouped according to the content of PCaa C38:6 in plasma and gender; (C) is based on the content of PCaa C38:6 in plasma, and the subjects grouped according to the presence or absence of hypertension. cumulative death Incidence curve; (D) is the cumulative death curve of subjects grouped according to the content of PCaa C38:6 in plasma and walking speed. L: low content; M: medium content; H: high content; L-F: low content-female; M-F: medium content-female; H-F: high content-female; L-M: low content-male; M-M: medium content-male; H-M: high content - male; L-NonHTN: low content - no hypertension; M-NonHTN: medium content - no hypertension; H-NonHTN: high content - no hypertension; L-HTN: low content - hypertension; M-HTN: moderate content-hypertension; H-HTN: high content-hypertension; L-NGS: low content-normal walking speed; M-NGS: moderate content-normal walking speed; H-NGS: high content-normal Walking speed; L-LGS: low content - low walking speed; M-LGS: medium content - low walking speed; H-LGS: high content - low walking speed.

Fig. 2 is a survival function diagram according to another embodiment of the present invention, which illustrates the cumulative survival of subjects divided into 3 groups according to the content of PCaa C38:6 in plasma at different times during the test period rate analysis results. L: low content; M: medium content; H: high content.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description for the embodiments and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The features of various specific embodiments as well as method steps and sequences for constructing and operating these specific embodiments are encompassed in the detailed description. However, other embodiments may also be utilized to achieve the same or equivalent function and sequence of steps.

Unless otherwise defined in this specification, scientific and technical terms used herein have the same meanings as understood and commonly used by those of ordinary skill in the art to which this invention belongs.

Unless contradicting the context, the singular nouns used in this specification cover the plural form of the noun; and the plural nouns used also cover the singular form of the noun. In addition, in this manual and In the scope of the patent application, expressions such as "at least one" and "one or more" have the same meaning, and both mean that one, two, three or more are included. What's more, in this specification and the scope of the patent application, "at least one of A, B and C", "at least one of A, B or C" and "at least one of A, B and/or C" ” means A only, B only, C only, both A and B, B and C, both A and C, and A, B and C.

Notwithstanding that the numerical ranges and parameters setting forth the broader scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains the standard deviation resulting from individual testing methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value lies within an acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. Except for the experimental examples, or unless expressly stated otherwise, all ranges, quantities, values and percentages used herein (for example, to describe material amounts, time durations, temperatures, operating conditions, quantity ratios and the like) should be understood ) are modified by "covenant". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying claims are approximate numerical values and may be changed as required. At a minimum, these numerical parameters should be construed to mean the number of significant digits indicated and the numerical values obtained by applying ordinary rounding. Numerical ranges are expressed herein as being from one endpoint to the other or between the endpoints; unless otherwise indicated, the numerical ranges recited herein are inclusive of the endpoints.

In this disclosure, the terms "subject" or "patient" are used interchangeably and refer to animals, including humans, that can be diagnosed and/or predicted using the methods of the present invention. Thus, "individual" or "patient" includes any mammal that may benefit from the treatment of the present disclosure. The mammals encompass all members of the class Mammalia, including humans, primates, domestic and farm animals (such as rabbits, pigs, sheep, and cattle), zoo or competition animals, pets, and rodents (such as, Small rat and rat). The term "non-human mammals" covers all members of the class Mammalia except humans. In one embodiment, the patient is a human.

In this disclosure, the term "biological sample" is meant to encompass any phospholipid (ie, PC) obtained from an individual (which may be a healthy or diseased individual) that can be used to analyze the present disclosure. aa C38:6) of the sample or specimen. According to embodiments of the present disclosure, specimens obtained from an individual include, but are not limited to, samples of bodily fluids (eg, blood, serum, plasma, or urine). According to some embodiments, the biological sample is a body fluid sample, especially a plasma sample. A biological sample obtained from an individual can be processed using techniques well known to those skilled in the art, and then the content of phospholipids in the sample can be determined.

In the present disclosure, the term "sarcopenia" refers to the imbalance between muscle cell growth, synthesis and catabolism, resulting in loss of skeletal muscle and reduced muscle strength, which gradually results in many activities in daily life. reduce. It usually occurs in the elderly and belongs to an aging disease. The diagnostic criteria for sarcopenia is a decrease in muscle mass (low muscle mass) plus a decrease in muscle strength (low muscle strength) or a decrease in mobility (low physical performance). Sarcopenia can be divided into primary and secondary. If sarcopenia is only caused by aging, it is called primary sarcopenia; : Severe organ failure, cancer, endocrine disease), malnutrition (including insufficient intake, malabsorption or anorexia caused by drugs) caused by sarcopenia is secondary sarcopenia. Most sarcopenia are caused by multiple diseases or risk factors that are not explained by a single etiology or pathology.

In this disclosure, the term "5-year survival rate" refers to the percentage of all diagnosed individuals who are still alive 5 years after a patient is diagnosed with a particular disease. In general, the 5-year survival rate can be used to confirm the prognosis of a patient's disease.

In this disclosure, the term "prognosis" or prediction refers to predicting the likelihood that a patient with sarcopenia will still be alive after 5 years, for example, a 5-year survival rate higher or lower than 60% . When understood, the term "prediction" does not necessarily refer to the likelihood that a certain course or outcome can be predicted with 100% accuracy. Relatively, those of ordinary skill in the art to which the present invention pertains can understand that the term "prediction" refers to increasing the probability of a certain course or result occurring; that is, compared to those without specific conditions (eg PCaa C38: A sarcopenic patient with a level of 6 below 71 micromolar), a sarcopenic patient with a specific condition (eg PCaa C38:6 level above 80 micromolar) is more likely to be alive after 5 years the result of.

Detailed ways

The calculated survival rate is one of the indicators commonly used clinically to judge the prognosis of an individual after suffering from a specific disease, and is often expressed as a 1-year survival rate or a 5-year survival rate. The current method for diagnosing sarcopenia still uses the individual's mobility (eg, walking speed or grip strength) as the main judgment criterion, and is easily affected by other external factors that affect the performance of the test and affect the accuracy of the test. As mentioned above, motor performance capabilities are currently used in related fields to assess the severity of sarcopenia. In order to improve the accuracy of detection, the present disclosure provides an alternative assessment method. Specifically, the present disclosure is based in part on the inventor's discovery that the content of phospholipids (PC aa C38:6) in the plasma of patients with sarcopenia correlates with the survival rate of patients, and thus can be used as a determinant of 5-year survival in patients with sarcopenia rate indicator.

The present disclosure thus provides a method for predicting 5-year survival in individuals with sarcopenia. According to an embodiment of the present disclosure, the method is a method for determining the 5-year survival rate of a sarcopenic individual from the content of specific phospholipids in a biological specimen isolated from the individual. The method comprises (a) determining the content of PCaa C38:6 in the biological specimen; and (b) predetermining the 5-year survival rate of the individual based on the results of step (a).

According to embodiments of the present disclosure, the biological specimen is derived from an individual suffering from sarcopenia. According to some embodiments of the present disclosure, the biological specimen is a blood specimen, a plasma specimen, a serum specimen, or a urine specimen. According to certain embodiments of the present disclosure, the biological specimen is a plasma specimen.

The biological specimen can be separated from the individual with sarcopenia by using the methods commonly used by those with ordinary knowledge in the technical field to which the present invention pertains; After centrifugation, the supernatant is taken as a serum sample; if an anticoagulant is added to the test tube, after centrifugation, the supernatant is taken to obtain a plasma sample.

In step (a), after the biological sample is isolated from the individual with sarcopenia, the content of PCaa C38:6 in the biological sample can be determined by analytical methods well known to those skilled in the art. According to certain embodiments of the present disclosure, PCaa C38:6 has the structure of formula (I):

According to certain embodiments of the present disclosure, methods for analyzing the content of PCaa C38:6 include, but are not limited to, gas chromatography (Gas Chromatography, GC), high performance liquid chromatography (High Performance Liquid Chromatography, HPLC), Ultra-high Performance Liquid Chromatography (UPLC), mass spectrometry (MS), nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR), etc. According to a specific embodiment of the present disclosure, using ultra-high performance liquid chromatography-mass spectrometry (Ultra-high Performance Liquid Chromatography-tandem mass, UPLC-MSMS) or flow injection analysis (flow injection analysis, FIA)-MSMS To analyze the content of PCaa C38:6 in biological samples.

Step (b) is to determine the 5-year survival rate of the individual with sarcopenia based on the analysis result of step (a). Specifically, when the content of PCaa C38:6 is equal to or higher than 71 micromolar and equal to or lower than 80 micromolar (eg 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 micromolar concentration), the 5-year survival rate of the individual is 60%; 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or higher micromolar concentrations), the 5-year survival rate of the individual was greater than 60%; and when the content of PCaa C38:6 was low at 71 micromolar (e.g. 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46 or lower micromolar concentrations), the 5-year survival rate for that individual is less than 60%. According to a specific embodiment of the present disclosure, in individuals with a 5-year survival rate greater than 60%, PCaa C38:6 levels are greater than 80 micromolar and less than or equal to 122 micromolar; and at 5 years survival In less than 60% of individuals, the PCaa C38:6 level was greater than or equal to 46 micromolar and less than 71 micromolar.

According to certain embodiments of the present disclosure, when the PCaa C38:6 level is equal to or higher than 85 micromolar concentration, the 5-year survival rate of the individual is greater than 90%; and when the PCaa C38:6 level Below or equal to 62 micromolar, the 5-year survival rate of the individual is less than 31%. According to a particular embodiment of the present disclosure, PCaa C38:6 is present at or above 85 micromolar and less than or equal to 122 micromolar in individuals with a 5-year survival rate greater than 90%; Individuals with an annual survival rate of less than 31% have PCaa C38:6 levels at or below 62 micromolar and above or equal to 46 micromolar.

According to certain embodiments of the present disclosure, an individual whose 5-year survival rate can be predicted by the methods of the present disclosure is a mammal. In certain embodiments of the present disclosure, the individual is a human.

Several experimental examples are provided below to illustrate certain aspects of the present invention, so as to facilitate the practice of the present invention by those skilled in the art to which the present invention pertains, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art, after reading the description presented herein, can fully utilize and practice the present invention without undue interpretation. All publications cited herein are considered part of this specification in their entirety.

Example

Materials and Methods

1. Research groups

The study guidelines were approved by the Human Trials Ethics Committee of Chang Gung Memorial Hospital. The experimental data described in the present disclosure were obtained from the subjects of this experiment. The trial included a total of 234 individuals aged 65 years or older and divided these subjects into 3 cohorts (44 without sarcopenia; 81 with early-stage sarcopenia; and 98 patients with sarcopenia). The trial period was 5 years (2014 to 2019). During the trial, subjects were physically examined once a year to obtain follow-up data.

2. Analysis of Metabolites in Plasma

Metabolites in plasma were analyzed using a commercially available metabolite assay kit (AbsoluteIDQ p180). Briefly, 10 microliters of each plasma sample were taken and the samples were prepared according to the manufacturer's instructions. Plasma biogenic amine levels were then measured by UPLC-MSMS, and plasma lipid metabolite levels were analyzed by FIA-MSMS. The concentrations of metabolites are all expressed in micromolar concentrations (μM).

3. Statistical analysis

All statistical analyses were two-tailed, and statistical analyses were performed using statistical software (SPSS version 19.0). Differences between the two groups were compared with the independent student's t-test. Judgment criteria and metabolite concentrations were expressed as mean ± standard deviation (Standard Deviation, SD).

Example 1 Demographics and clinical characteristics of subjects

A total of 223 subjects participated in this trial and were divided into 3 groups based on no sarcopenia, early sarcopenia, and sarcopenia. After 5 years of continuous follow-up, a total of 4 subjects without sarcopenia, 6 subjects with early sarcopenia, and 35 subjects with sarcopenia during the trial (2014 died within 2019). The results of the study indicated that subjects with sarcopenia had a higher 5-year mortality rate compared to the other two groups. In addition, among all surviving subjects, 2 subjects without sarcopenia, 7 subjects with early sarcopenia, and 9 subjects with sarcopenia in the trial Physiological function decreased significantly during the period (Table 1). In order to analyze the key factors most associated with patient survival, subsequent experiments will exclude sarcopenic subjects with reduced physiology, and only analyze samples from 89 sarcopenic subjects who survived and died.

Example 2 Metabolome analysis of sarcopenia patients

Identify the metabolites most associated with sarcopenia severity by analyzing differences in plasma metabolites in surviving and deceased sarcopenia subjects to understand factors affecting 5-year survival in sarcopenia patients . 89 subjects (54 surviving and 35 dead sarcopenia) were treated with a commercially available metabolome assay kit Subjects) plasma samples, followed by UPLC-MSMS or FIA-MSMS to characterize and quantify biogenic amines and lipid metabolites in plasma. The results indicated that the contents of PCaa C38:6, PCaa C40:6, PCaa C40:8 and PCaa C42:8 were significantly different in surviving and dead subjects, respectively, compared with sarcopenia patients who were still alive after 5 years , in the plasma of sarcopenia patients who died within 5 years, the content of the above four phospholipids was lower (results not shown), and the content of PCaa C38:6 in the plasma of surviving and dead subjects was relatively low (results not shown). significant difference. This result suggests that less PCaa C38:6 in plasma may be a key factor in increasing mortality in patients with sarcopenia.

Example 3 Determining 5-year survival in sarcopenia patients

3.1 Correlation between the content of phospholipids in plasma and the survival rate of patients with sarcopenia

According to the content of phospholipids in the subjects' plasma, 89 patients with sarcopenia were divided into 3 groups, namely high content group (H), medium content group (M) and low content group (L), respectively. Expressed as the mean ± SD of its concentration (micromolar concentration) (as shown in Table 2), there were significant differences in the phospholipid concentration among the groups.

In order to identify other factors that jointly affect the survival rate of sarcopenia, the cumulative incidence of death within 5 years of subjects who were divided into 3 groups based on the content of plasma PCaa C38:6 during the trial period was as shown in the first As shown in panel A, the results indicated that the mortality rates of the three groups were L>M>H in sequence, indicating that the The higher the phospholipid content, the lower the mortality rate; in other words, the higher the phospholipid content in the plasma, the higher the survival rate; the H, M and L groups were further divided according to gender, the presence or absence of hypertension or the speed of walking. Divided into two groups to determine whether gender, high blood pressure and walking speed affect the mortality of the subjects. Mortality was higher when subjects were male, had high blood pressure, or walked slowly (Panel 1, panels B to D); subjects with high blood pressure or slow walking If the plasma of the subjects contained more PCaa C38:6, the mortality rate was significantly lower than that of the subjects in the L and M groups. The results indicate that the content of PCaa C38:6 in plasma may be a key factor affecting the severity of sarcopenia, and the mortality rate of patients can be inferred based on its content, in other words, it can be used as a judgment basis for determining the 5-year survival rate of patients candidate factor.

3.2 The 5-year survival rate of sarcopenia patients is determined by the content of PCaa C38:6 in plasma

Plasma levels of phospholipids (eg PCaa C38:6) are known to affect 5-year survival in sarcopenia patients. According to this correlation, the content of phospholipids is used as a criterion for determining the 5-year survival rate of patients, and is used to confirm the prognosis of sarcopenia patients and/or the severity of sarcopenia. First, the cumulative survival rate of 89 subjects was divided into 3 groups according to the content of PCaa C38:6 in the plasma, and the survival function graph as shown in Figure 2 was drawn. The results indicated that during the test period, 20 subjects in the low-level group and 12 subjects in the medium-level group died, respectively, while among the subjects in the high-level group, only 3 subjects died during the test period. Divide the number of subjects who died in each group by the total number of subjects in each group to obtain the mortality rate of subjects in each group, and then convert the mortality rate into the survival rate, and then obtain the 5-year mortality rate of subjects in the high-content group. The survival rate was 90%; the 5-year survival rate in the medium-content group was 60%; and the 5-year survival rate in the low-content group was 31%.

Further, according to the average concentration of phospholipids in the plasma of the subjects in each group (Table 2), the upper and lower limits of the concentration of phospholipids (PCaa C38:6) in the plasma of each group were calculated to determine the 5-year survival rate of the patients. the threshold. As described in Table 2, in the medium level group (group M) where the 5-year survival rate of the subjects was 60%, the level of PCaa C38:6 in the plasma of the subjects was between about 71 micromolar to about 80 between micromolar concentrations; In the high-content group (group H) where the 5-year survival rate of the subjects was higher than 90%, the PCaa C38:6 in the subjects' plasma was between 85 micromolar and 122 micromolar; In the low-level group (group L) with a 5-year survival rate of less than 31%, the plasma PCaa C38:6 level of the subjects was between about 46 micromolar and 62 micromolar. From the results, it can be seen that the content of PCaa C38:6 in plasma is proportional to the survival rate of patients.

Summarizing the above, the present disclosure provides a method for predicting 5-year survival in sarcopenia patients using PC aa C38:6 as a biomarker. Clinical medical personnel can judge the severity of the disease based on the prediction results, and then give appropriate treatment recommendations.

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains, without departing from the principle and spirit of the present invention, should Various changes and modifications can be made to it, so the protection scope of the present invention should be defined by the appended claims.

Claims (3)

A method for predicting survival of an individual with sarcopenia from a biological sample of the individual, wherein the biological sample is plasma, comprising: (a) determining the phospholipid choline diacyl C38 in the biological sample: The content of 6 (PCaa C38:6), wherein the PCaa C38:6 has the structure of formula (I):

and (b) predicting a 5-year survival rate for the individual based on the results of step (a), wherein when the PCaa C38:6 level is equal to or higher than 71 micromolar and equal to or lower than 80 micromolar When the concentration of PCaa C38:6 is higher than 80 micromolar concentration, the 5-year survival rate of the individual is greater than 60%; or when the PCaa C38:6: When the concentration of 6 is lower than 71 micromolar, the 5-year survival rate of the individual is less than 60%. The method of claim 1, wherein the 5-year survival rate of the individual is greater than 90% when the PCaa C38:6 content is equal to or higher than 85 micromolar; or when the PCaa C38:6 content At levels equal to or less than 62 micromolar, the 5-year survival rate for the individual is less than 31%. The method of claim 1, wherein the individual is a human.

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