CN110824172A - Method for measuring LEP/APN ratio in venous blood - Google Patents
Method for measuring LEP/APN ratio in venous blood Download PDFInfo
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Abstract
The invention provides a method for measuring LEP/APN ratio in venous blood, which comprises the following steps: (1) preparation of reagents: LEP and APN detection ELISA kit; (2) preparing an instrument: (2-1) an enzyme-labeled analyzer; (2-2) an ultra-low temperature refrigerator; (2-3) a desk centrifuge; (3) and (3) experimental operation: (3-1) specimen collection: collecting early morning fasting venous blood of a selected subject by using a vacuum negative pressure tube, standing for 30min, centrifuging for 10min at 3000rpm by using a desktop centrifuge, separating serum, storing in an ultra-low temperature refrigerator at-80 ℃, and detecting LEP and APN; (3-2) measuring the APN and LEP levels of the serum by using an ELISA method; and (3-3) calculating the LEP/APN ratio according to the result. The invention adopts blood sample detection, sample collection is simple and convenient, and the occurrence of patient uncooperative condition can be reduced to the maximum extent.
Description
Technical Field
The invention belongs to the technical field of medical clinical examination, and particularly relates to a method for measuring a ratio of LEP/APN in venous blood. The method provided by the invention is only used for obtaining an intermediate result LEP/APN ratio and is not used for diagnosing diseases.
Background
Non-alcoholic fatty liver disease (NAFLD) has been identified as one of the risk factors for this primary liver cancer. Due to the lack of a uniform monitoring management mechanism, some NAFLD patients are often diagnosed with hepatocellular carcinoma at an advanced stage. Therefore, early prevention and early treatment, and reduction of incidence of NAFLD are new challenges facing the basic medicine and clinical medicine of various countries in the world. Comprehensive analysis of the non-alcoholic fatty liver disease in China shows that the prevalence rates of NAFLD in urban areas such as Beijing, Shanghai and hong Kong are respectively 39.5%, 43.5% and 42.0%, and the prevalence rate of NALFD in the elderly in Taiwan (the average age is 70.3 years) is as high as 54.4%. Most NAFLD is hidden from disease and often lacks specific clinical manifestations and typical symptoms at an early stage.
Current studies indicate that there is a correlation between NAFLD pathogenesis and insulin resistance. Insulin resistance and obesity cause newly increased lipid influx into the liver, leading to accumulation of TG in the liver, causing fatty liver. In the state of disturbed lipid metabolism, the dynamic balance of intrahepatic fat is disrupted. High-density lipoprotein cholesterol (HDL-C) in common biochemical indexes has a protective effect on reducing blood fat, and the content of HDL-C is obviously lower than the normal level when the BMI is more than 25. With the increase of BMI and body fat, blood Free Fatty Acid (FFA) and liver synthesized triglyceride of NAFLD patients increase, synthesized TG of liver increases, and TG clearing capacity is reduced; when FFA entering the liver and FFA synthesized by the liver itself exceed the oxidative capacity of the liver, endogenous TG synthesized by the liver cannot be metabolized, resulting in accumulation of lipids in hepatocytes. The blood FFA increase reversely inhibits insulin signal transmission and promotes generation and development of NAFLD.
The fat factors Leptin (LEP) and Adiponectin (APN) play an important role in the occurrence and development processes of the nonalcoholic fatty liver disease (NAFLD), and are novel molecular markers of the NAFLD at present. LEP is a proteinaceous hormone secreted by adipose tissue. The precursor consists of 167 amino acid residues, the N-terminal has 21 amino acid residues of signal peptide, the signal peptide of the precursor is cut off in blood to be 146 amino acids, and the molecular weight is 16 kDa. LEP has a wide range of biological effects, and more importantly, it binds to LEP receptors in the hypothalamus to suppress appetite, increase energy consumption, and suppress fat synthesis, thereby maintaining metabolic balance in the body. APN is an endogenous biologically active polypeptide or protein secreted by adipocytes, consisting of 244 amino acids, and is also called Acrp30 because of its relative molecular weight of 30 kDa. APN acts as an insulin hypersensitizing hormone, the level of which decreases with increasing fat content. And the APN has certain anti-inflammatory effect, so that the protective effect on liver cell damage is realized.
LEP is a secreted protein synthesized by adipocytes, and is of great significance in the occurrence and development of NAFLD because TG synthesis is increased by inhibiting gene expression of phosphoenolpyruvate carboxylase (PEPCK) of hepatocytes in the liver, resulting in increased deposition of lipids in the liver, so LEP is used as an independent prediction index of the severity of hepatic steatosis. Studies have shown that plasma LEP levels are positively correlated with the severity of SP suffering from NALFD.
It has been shown that a decrease in APN levels decreases fatty acid oxidation, glucose uptake and insulin gluconeogenesis inhibition in skeletal muscle cells, and also decreases the ability to inhibit hepatic glycogen production due to reduced specific binding to G protein-coupled receptors on the liver cell membrane. Meanwhile, with the reduction of the level of the APN, the original anti-inflammatory effect is correspondingly reduced, the liver cell injury cannot be protected to a certain extent, and the possibility that a patient suffers from NAFLD is increased.
LEP and APN are adipokines which have opposite effects in the development of insulin resistance and MetS. Studies have shown that LEP/APN values are good biomarkers of MetS in the general population, and in studies with SP APN levels, in addition to glucose levels, were found to be negatively correlated with most metabolic parameters. In addition to GLU and HDL levels, LEP/APN values are positively correlated with most metabolic parameters, and there are significant gender differences in LEP, APN levels and LEP/APN values, the discrimination of the LEP/APN values for MetS is better in men than in women.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the LEP/APN ratio in venous blood, adopting blood specimen detection, being simple and convenient for sample collection, and being capable of reducing the occurrence of patient uncooperative condition to the greatest extent.
In order to solve the above technical problem, an embodiment of the present invention provides a method for measuring an LEP/APN ratio in venous blood, including the following steps:
(1) preparation of reagents: LEP and APN detection ELISA kit;
(2) instrument preparation
(2-1) an enzyme-labeled analyzer;
(2-2) an ultra-low temperature refrigerator;
(2-3) a desk centrifuge;
(3) experimental procedures
(3-1) specimen Collection
Collecting early morning fasting venous blood of a selected subject by using a vacuum negative pressure tube, standing for 30min, centrifuging for 10min at 3000rpm by using a desktop centrifuge, separating serum, storing in an ultra-low temperature refrigerator at-80 ℃, and detecting LEP and APN;
(3-2) determination of serum APN and LEP levels by ELISA
Preparing: taking out the reagent from the ultra-low temperature refrigerator 20 minutes in advance, and balancing the room temperature;
preparing liquid: diluting the concentrated washing liquid with distilled water at a ratio of 1:20 for later use;
dissolution of the standard:
LEP standard curve: adding 1.0 ml of specimen universal diluent into each standard substance, standing at room temperature for 15 minutes, and slightly and uniformly mixing after the specimen universal diluent is fully dissolved; at a concentration of 2000 pg/ml, and then diluted to 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml and 0 pg/ml in this order;
APN standard curve: adding 1.0 ml of specimen universal diluent into each standard substance, standing at room temperature for 15 minutes, after the specimen universal diluent is fully dissolved, gently mixing uniformly to obtain a mixture with the concentration of 10 ng/ml, and then sequentially diluting into 5ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625ng/ml, 0.31 ng/ml, 0.15 ng/ml and 0 ng/ml;
numbering: numbering the micropores corresponding to the sample in sequence;
sample adding: when detecting APN, diluting serum with a 1:2500 reagent matched universal diluent for later use, directly adding LEP, respectively adding blank, standard substance and 100 mu l of sample to be detected into corresponding holes, lightly shaking and uniformly mixing, sealing a plate by a sealing plate film, and then incubating for 90 minutes at 37 ℃;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
adding a biotin antibody: diluting the concentrated biotin antibody with a biotin antibody diluent 1:30 20 minutes in advance to prepare a biotinylated antibody working solution, adding 100 mu l of biotin antibody into each hole, sealing the holes with a sealing plate film, and then incubating for 60 minutes at 37 ℃;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
adding an enzyme: diluting the concentrated enzyme conjugate by using an enzyme conjugate diluent 1:30 20 minutes in advance to prepare an enzyme conjugate working solution; adding 100 mu l of enzyme conjugate into each hole, slightly shaking and uniformly mixing, sealing the plate by a sealing plate membrane, and then placing at 37 ℃ for incubation for 30 minutes;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
color development: adding 100 mul of color development liquid A, B into each hole, gently oscillating and mixing uniformly, and developing for 15 minutes in a dark place at 37 ℃;
and (3) determination: adding 100 mu l of stop solution into each hole, patting and uniformly mixing, setting the wavelength of an enzyme-labeled analyzer to be 450nm, zeroing by using a blank hole, and then measuring the OD value of each hole;
data processing: performing data processing by using a polynomial linear fitting mode, drawing by taking the background-subtracted OD value of each calibrator as a vertical coordinate and the concentration of each calibrator as a horizontal coordinate to obtain a standard curve, and calculating the concentrations of LEP and APN in a sample according to the standard curve;
and (3-3) calculating the LEP/APN ratio according to the result.
The technical scheme of the invention has the following beneficial effects:
the invention adopts blood sample detection, sample collection is simple and convenient, and the occurrence of patient uncooperative condition can be reduced to the maximum extent.
Drawings
FIG. 1 is a graph of a LEP standard plotted in the present invention;
FIG. 2 is a APN standard curve plotted in the present invention;
FIG. 3 is a table comparing the incidence of NAFLD in SP groups versus control combinations of the present invention;
FIG. 4 is a graph comparing the incidence of NAFLD in the SP group and control combinations of the present invention;
FIG. 5 is a table comparing the biochemical index (%) of the SP group and the control group in accordance with the present invention;
FIG. 6 is a table of quantitative analysis of biochemical indicators (mean. + -. SD) of SP group and control group according to the present invention;
FIG. 7 is a table of quantitative analysis (mean. + -. SD) of the indexes in the SP group and the control group according to the present invention;
FIG. 8 is a graph showing the quantitative analysis of biochemical markers in the SP group and the control group according to the present invention;
FIG. 9 is a comparative table of the PANSS scale of psychiatric symptoms in the SP group of the present invention;
FIG. 10 is a table comparing the administration of NAFLD group and fatty liver free group in SP group according to the present invention;
FIG. 11 is a graph showing the comparison between the administration of NAFLD group and fatty liver free group in SP group according to the present invention;
FIG. 12 is a table showing the effect of SP group internal drug administration on NAFLD concomitant in the present invention;
FIG. 13 is a table of the effect of different modes of administration of AAS drugs on SP with NAFLD in accordance with the present invention;
FIG. 14 shows NAFLD population T of the present invention2DM, hypertension incidence and BMI typing difference rate comparison table;
FIG. 15 is a table of quantitative analysis of biochemical indicators (mean. + -. SD) among groups of NAFLD population according to the present invention;
FIG. 16 is a table of risk factors affecting SP combined NAFLD by Logistic regression analysis in the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The invention provides a method for measuring LEP/APN ratio in venous blood, which comprises the following steps:
(1) preparation of reagents: LEP, APN detection ELISA kit (euphb bosch biotechnology limited, china);
(2) instrument preparation
(2-1) enzyme-labeled analyzer (Reynolds Co.);
(2-2) an ultra-low temperature refrigerator (Mitsubishi of Zhongke);
(2-3) a table centrifuge (Shanghai medical instruments Co., Ltd.);
(3) experimental procedures
(3-1) specimen Collection
Collecting early morning fasting venous blood of a selected subject by using a vacuum negative pressure tube, standing for 30min, centrifuging for 10min at 3000rpm by using a desktop centrifuge, separating serum, storing in an ultra-low temperature refrigerator at-80 ℃, and detecting LEP and APN;
(3-2) determination of serum APN and LEP levels by ELISA
Preparing: taking out the reagent from the ultra-low temperature refrigerator 20 minutes in advance, and balancing the room temperature;
preparing liquid: diluting the concentrated washing liquid with distilled water at a ratio of 1:20 for later use;
dissolution of the standard:
LEP standard curve: adding 1.0 ml of specimen universal diluent into each standard substance, standing at room temperature for 15 minutes, and slightly and uniformly mixing after the specimen universal diluent is fully dissolved; at a concentration of 2000 pg/ml, and then diluted sequentially to 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml and 0 pg/ml, an LEP standard curve was plotted, as shown in fig. 1;
APN standard curve: adding 1.0 ml of specimen universal diluent into each standard, standing for 15 minutes at room temperature, after the specimen universal diluent is fully dissolved, gently mixing the mixture to obtain a mixture with the concentration of 10 ng/ml, then diluting the mixture to 5ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625ng/ml, 0.31 ng/ml, 0.15 ng/ml and 0 ng/ml in sequence, and drawing an APN standard curve, wherein the APN standard curve is shown in figure 2.
Numbering: numbering the micropores corresponding to the sample in sequence;
sample adding: when detecting APN, diluting serum with a 1:2500 reagent matched universal diluent for later use, directly adding LEP, respectively adding blank, standard substance and 100 mu l of sample to be detected into corresponding holes, lightly shaking and uniformly mixing, sealing a plate by a sealing plate film, and then incubating for 90 minutes at 37 ℃;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
adding a biotin antibody: diluting the concentrated biotin antibody with a biotin antibody diluent 1:30 20 minutes in advance to prepare a biotinylated antibody working solution, adding 100 mu l of biotin antibody into each hole, sealing the holes with a sealing plate film, and then incubating for 60 minutes at 37 ℃;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
adding an enzyme: diluting the concentrated enzyme conjugate by using an enzyme conjugate diluent 1:30 20 minutes in advance to prepare an enzyme conjugate working solution; adding 100 mu l of enzyme conjugate into each hole, slightly shaking and uniformly mixing, sealing the plate by a sealing plate membrane, and then placing at 37 ℃ for incubation for 30 minutes;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
color development: adding 100 mul of color development liquid A, B into each hole, gently oscillating and mixing uniformly, and developing for 15 minutes in a dark place at 37 ℃;
and (3) determination: adding 100 mu l of stop solution into each hole, patting and uniformly mixing, setting the wavelength of an enzyme-labeled analyzer to be 450nm, zeroing by using a blank hole, and then measuring the OD value of each hole;
data processing: performing data processing by using a polynomial linear fitting mode, drawing by taking the background-subtracted OD value of each calibrator as a vertical coordinate and the concentration of each calibrator as a horizontal coordinate to obtain a standard curve, and calculating the concentrations of LEP and APN in a sample according to the standard curve;
and (3-3) calculating the LEP/APN ratio according to the result.
Schizophrenia (SZ) is a disease that seriously affects the physical and mental health and the quality of life of humans, often accompanied by special disorders in thinking, perception, emotion and behavior. Patients have unstable conditions and are easy to repeatedly attack, and the long-term hospitalization brings huge economic burden to families and society. In 2013, research and investigation of electronic database big data show that the lifetime prevalence rate of a continental region Schizophrenia Patient (SP) is estimated to be 4.62%, wherein the prevalence rate of a male is 4.63%, the prevalence rate of a female is 4.95%, and the prevalence rates of the male and the female are not statistically different; but the incidence rate of urban population is 5.15 per mill, and the incidence rate of rural population is 4.44; the prevalence rate of urban residents is higher than that of rural residents. 2016 research data show that the sign of overweight and obesity combined with chronic SP in China has obvious sex difference. Female patients are about 2 times as obese as male patients, and women have more obvious lipid metabolism disorder.
Once SP has been found to be unable to accurately express liver discomfort due to its disease specificity, NAFLD has often been complicated by SP, and in addition to SP disease specificity, it is clinically difficult to alleviate and cure fatty liver symptoms by physical exercise enhancement, reduction or replacement of antipsychotic drugs. In addition, considering economic reasons, practicability and operability of patients, ultrasonic diagnosis technology is mostly adopted for diagnosing NAFLD in China at present. Often, the SP attacks and treatment periods have unstable mental symptoms, the ultrasonic examination is carried out for a long time, and the patients often have uncooperative conditions.
The gold standard for SP diagnosis of NAFLD is a pathology report. However, the pathological detection has large damage, so that the pathological detection is mostly not adopted in China in clinic. In the imaging diagnosis of SP combined NAFLD, (1) ultrasonic diagnosis: the SP liver in vivo abnormal image is characterized by diffuse fatty liver expressed by 'fatty zone', 'bright liver', obviously reduced intrahepatic blood vessels, unclear texture and the like in the liver area, and diffuse non-uniform fatty liver expressed by high or slightly high echo area on gray scale ultrasound, clear but irregular edge, similar hemangioma and synchronously enhanced or synchronously weakened high echo area and liver parenchyma. The positive rate of the B ultrasonic can be increased by the factors of age increase, medication time extension, combined medication and the like. (2) X-ray: in fatty liver, the abnormality cannot be found by X-ray examination, and the clinical value is limited. CT: is a valuable imaging examination technique. The flat scan showed a decrease in liver density, and diffuse fat infiltration was manifested as a decrease in total liver density. Focal infiltrates produce a local decrease in liver density in the lobe, segment or sub-segment of the liver. MRI: because the resonance frequencies of the hydrogen protons in the fat and the water are different, the same phase and the opposite phase of the chemical shift imaging are not imaged, and liver fat infiltration can be shown. In the anti-phase image, the fat-infiltrated signal is significantly reduced in intensity compared to the signal in the in-phase image, which is characteristic. In the literature, the diagnosis value is inferior to that of CT probably because MRI is more expensive than CT and B-ultrasonic examination. Therefore, the related literature of CT detection related to SP combined NAFLD is less in China. NAFLD is still in the scientific research stage in the aspect of gene diagnosis, and the clinical routine examination is difficult to popularize.
The prevalence of NAFLD (55.65%) was found to be much higher in middle aged and elderly women SP than in the normal population control group without SZ (26.32%) in women of the same age group (fig. 3, fig. 4), and higher than the overall prevalence of NAFLD in adults in china. It can be seen that SP is a high-incidence population of NAFLD in middle and old aged women. The prevalence of metabolic syndrome (MetS) in SP is increasing and receiving more and more attention. Previous studies have shown that Uric Acid (UA) is associated with MetS in the general population and has gender differences. As a selective antioxidant, UA reduction was also found in SP and more pronounced in men. Increased UA concentrations in SP are associated with hypertriglyceridemia (TG), Low Density Lipoprotein (LDL) levels, male hypertension and female hypertriglyceridemia. The results of UA and MetS risk correlation analysis show significance in male patients, i.e. lower UA concentrations are associated with lower MetS risk, and none in females.
Previous studies on prevalence and risk factors for SP-incorporated NAFLD in young men showed that prevalence of SP-incorporated NAFLD in young men was higher than in normal young men. And the ALT and AST in the indexes reflecting liver functions have obvious difference among groups.
BMI is an international important standard for measuring the obesity degree and health of a human body, and the weight/height is determined by two relatively objective parameters of the weight and the height of the human body2And calculating a result. Refer to Chinese patent medicineHuman overweight/obesity prevention control guidelines standard: BMI < 18.5 kg/m2Has low bulk mass and a BMI of 18.5 to 23.9 kg/m2The BMI is not less than 24 kg/m for the quality of a normal body2Is overweight, and has a BMI of more than or equal to 28kg/m2For obesity, BMI was typed. The data from the 2009 chinese SP large sample study showed that SP overweight and obese patients were higher compared to the normal population. Female obese patients were almost twice as obese as men compared to men SP.
The presence of NAFLD, on the one hand, leads to an increased risk of endothelial dysfunction, further increasing the risk of cardiovascular and cerebrovascular diseases, and, on the other hand, NAFLD primary pathological liver changes cause structural and functional changes in the liver, increasing the incidence and mortality of cirrhosis, liver failure and hepatocellular carcinoma. Metabolic syndromes such as weight gain, obesity and disorders of carbohydrate and lipid metabolism may occur after treatment with antipsychotics. With the prolongation of the course of the disease, the long-term use of Atypical Antipsychotics (AAS) is associated to varying degrees with weight gain and, due to this, mild elevation of transaminases by Mets and NAFLD. There are scholars who believe that SP is receiving antipsychotic medication as a risk factor for its complications with NAFLD.
During clinical rehabilitation diagnosis and treatment of psychiatric department, doctors regularly monitor the levels of conventional biochemical project examination, LEP, APN and other fat factors of patients and general clinical information data of the patients such as height, weight, BMI, blood pressure, heart rate, mental symptoms and the like in the early hospitalization period and the anti-SP treatment process, and make reasonable risk assessment, so that the incidence rate of SP combined with NAFLD can be effectively reduced, and the survival quality of the people is improved.
On the basis of the technical scheme provided by the invention, the collected samples are subjected to biochemical detection at the same time, intermediate data can be provided for whether SP combines NAFLD, and the method comprises the following specific operation steps:
first, reagent preparation
Typical biochemical reagents include Total Bilirubin (TBIL), Direct Bilirubin (DBIL), Total Protein (TP), albumin (albumin, ALB), alanine aminotransferase (ALNANE aminotransferase, ALT), aspartate transferase (AST), Gamma Glutamyltransferase (GGT), alkaline phosphatase (ALKALINEPHARATASE, ALP), uric acid (uric acid, UA), urea (blood urea, BUN), creatinine (Creatine, CRE), β -microglobulin (β -microlobulin, β MG), fasting GLU, total Cholesterol (CHOL), triglyceride (apolipoprotein, TG), high density lipoprotein (HDL-protein), low density lipoprotein (APB, LDL-36), and so on, and low cholesterol protein (LDL-12A), LDL-2 protein (lipoprotein A, LDL-12), and so on.
Secondly, preparing an instrument: au1000 full-automatic biochemical analyzer (beckmann), enzyme labeling analyzer (reynolds corporation), ultra-low temperature refrigerator (zhongke meiling), desktop centrifuge (shanghai medical instruments ltd), ultrasonic instrument (siemens oxana 1).
Third, statistics of medical records
3.1, 115 cases of schizophrenia in middle-aged and elderly women, which are divided by B-ultrasonic diagnosis: NAFLD group 64 cases, non-NAFLD group 51 cases; middle-aged and elderly women healthy control 95 cases, NAFLD group 25 cases, non-NAFLD group 70 cases; counting the age, disease course, height, weight, blood pressure, heart rate, taking medicine, hypertension and T2A DM disease condition;
3.2, BMI calculation formula: BMI = weight ÷ height2（kg/m2）；
3.3, psychiatric symptoms scale: PANSS scale (positive symptom scale, negative symptom scale, general spirit).
Fourth, experiment operation
4.1, collecting 5ml of early morning fasting venous blood of 2 selected subjects by using a vacuum negative pressure tube, standing for 30 minutes, centrifuging at 3000rpm for 10 minutes, separating serum, directly carrying out biochemical detection by using one tube, and carrying out LEP and APN detection by using the technical scheme of the invention by using the other tube.
4.2 general Biochemical project detection
The serum biochemical detection project is quantitatively detected on an Au1000 full-automatic biochemical instrument, and the operation steps are strictly carried out according to instrument standard procedures. The clinical samples and the quality control products of the two batches are simultaneously tested on the computer, and the indoor quality is strictly controlled.
Liver function test items: TBIL, DBIL, TP, ALB, ALT, AST, GGT, and ALP.
Renal function test items UA, BUN, CRE and β2MG。
Fasting glucose: GLU.
And (3) blood fat detection items: CHOL, TG, HDL, LDL, APOA1 and APOB.
Wherein, fig. 8A is age and disease course, fig. 8B is fasting blood glucose, fig. 8C is liver function index, fig. 8D is kidney function index, fig. 8E is blood lipid index, fig. 8F is fat factor. P < 0.05; p < 0.01; p < 0.001.
In the research, the biochemical index detection result of the kidney function of the middle-aged and old women shows that the items with positive increase of the SP group result are BUN, UA and β2MG, wherein UA was significantly higher in the SP group than the normal control group (fig. 5, fig. 6), and NAFLD was also significantly higher in the SP group than the fatty liver free group (fig. 8D). UA was not significantly associated in risk analysis of SP incorporation NAFLD in middle and old aged women, possibly associated with gender differences.
In this study, the biochemical test results of liver function showed that DBIL was significantly increased in middle and old aged women SP group compared to the same age group (fig. 8C), suggesting abnormal bilirubin metabolism; TP and ALB were significantly lower than the control group (FIG. 8C), indicating that SP liver synthesized protein function was reduced. In SP, GGT and ALP were elevated in NAFLD group compared to fatty liver-free group (fig. 8C), suggesting bilirubin metabolism disorder; ALT was significantly elevated in the NAFLD group (fig. 8C), suggesting that SP-incorporated NAFLD patients had severe liver parenchymal damage, and ALT was an important relevant risk factor for SP-incorporated NAFLD in middle-aged and elderly women (fig. 16). Therefore, liver function detection has important prompting significance in SP combined NAFLD auxiliary diagnosis and treatment of middle-aged and old women, and particularly has significant ALT significance.
In this study, BMI data showed that the SP group was higher in middle-aged and elderly women than the control group without SP (fig. 6), NAFLD group was higher in both the SP group and the control group than the fatty liver free group (fig. 7), SP group was also higher in NAFLD group than the control group (fig. 15), and the SP group was both overweight and obese with abnormal rate higher than the control group (fig. 14); the results of the risk correlation analysis showed that BMI typing is a high risk factor for SP incorporation NAFLD in middle and old aged women (figure 16). SP was more prone to clinical signs of overweight, obesity, consistent with the results of Subramaniam M et al. Thus, supporting the notion that obesity, being overweight, may be one of the risk factors leading to an increased prevalence of SP-incorporated NAFLD.
In this study, it was found that the total score of NAFLD group positive scale, the score of anxiety term in general psychopathology scale and the total score of PANSS in SP group were significantly lower than those of fatty liver free group, while the negative scale showed emotional withdrawal, passive/apathy social withdrawal, and three items of bradykinesia in general psychopathology scale were significantly higher than those of fatty liver free group (fig. 9). Indicating the difference of mental symptoms between the NAFLD group and the fatty liver free group in the SP group. The positive symptoms of the patients without fatty liver are more obvious than those of the patients with NAFLD, and the patients are easy to show the mental symptoms of anxiety and restlessness. Patients in the NAFLD group are socially passive, have indifferent mental symptoms to social activities, and have a lifestyle with bradykinesia and sedentary movements that are more likely to increase the risk of SP suffering from NAFLD.
In the research, the discreteness of samples of each group of LEP is large, so that the statistical significance among the groups is reduced finally. However, in the correlation analysis, LEP was found to be a risk factor for SP incorporation NAFLD in elderly women (figure 16).
In this study, the level of APN was not different between the SP group and the control group in elderly women, but NAFLD was significantly lower in the SP group than in the fatty liver free group (fig. 8F), indicating that APN levels were negatively correlated with SP incorporation of NAFLD in elderly women. The results of independent risk factor analysis were consistent with SP-incorporated NAFLD (figure 16), suggesting that APN is a protective factor for SP-incorporated NAFLD in middle and old age women.
The results of this study showed that the LEP/APN values were not significantly different in the SP group of middle and old aged women compared to the fatty liver free group, although the NAFLD group was higher than the fatty liver free group, and were associated with a large degree of dispersion in LEP values (fig. 8F). LEP/APN values were not statistically different between the SP group and the control group (figure 4), and between the SP group and the control group of the NAFLD population (figure 15).
And selecting possible risk factors as independent variables according to the result of the t test of each index, and finally influencing the risk factors of SP and NAFLD combination by using whether NAFLD is concurrent or not as dependent variable through Logistic regression analysis. The results show that age, ALT, TG, LDL, LEP, APN and BMI typing has a clear correlation with middle aged and elderly women SP combined NAFLD, wherein age, ALT, TG, LDL, LEP and BMI typing is a risk factor, and wherein TG, LDL and BMI typing is a high risk factor. The APN is a protective factor (figure 16) consistent with the physiological role of the APN.
In clinical treatment of SP, AAS and typical Antipsychotics (AS) are commonly used, and in this study, the middle-aged and elderly women mostly used AAS, and AAS combination is frequently used (fig. 11). However, whether the drugs are administered or not (fig. 10), or analyzed by classification of AAS and AS or by specific drug grouping (fig. 11), or even if the drugs are administered in combination (fig. 12), the results suggest that the administration factors do not affect the SP of middle-aged and elderly women who accompany NAFLD, and there is no significant difference between the different administration modes of the common AAS (fig. 12). In addition, because the treatment of SP is often the particularity of combined use of a plurality of medicines with different dosages, and some patients have poor compliance of taking medicines before admission to the hospital for standard treatment and have irregular dosage, the study does not take the medicine condition into the risk factors influencing SP and NAFLD for analysis.
The serological detection method has the advantages that the sample collection only needs 3-5 minutes, and the SP is easy to cooperate. Liver function, renal function, blood sugar, blood fat are SP conventional serological test items, can detect in hospitals of various cities and counties and even in primary health hospitals, and have low price and high regular monitoring feasibility. LEP, APN project experiment operation is comparatively simple, can do benefit to clinical popularization.
In conclusion, the incidence of SP combined NAFLD in the middle-aged and the elderly is high, and NAFLD seriously affects the life quality of SP in women of the middle-aged and the elderly. There are many different indexes of SP in middle and old age compared with normal control group. The age, ALT, TG, LDL, LEP, APN and BMI typing are risk factors of SP concurrent NAFLD of middle-aged and old women, wherein the age, ALT, TG, LDL, LEP and BMI typing are risk factors of the middle-aged and old women, and the TG, LDL and BMI typing is a high-risk factor; APN is its protective factor. Therefore, the method for measuring the LEP/APN ratio in venous blood provided by the invention can only provide an intermediate result for judging whether SP is concurrent with NAFLD, cannot be used for directly obtaining a disease diagnosis result, and does not belong to a disease diagnosis method.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (1)
1. A method for measuring LEP/APN ratio in venous blood is characterized by comprising the following steps:
(1) preparation of reagents: LEP and APN detection ELISA kit;
(2) instrument preparation
(2-1) an enzyme-labeled analyzer;
(2-2) an ultra-low temperature refrigerator;
(2-3) a desk centrifuge;
(3) experimental procedures
(3-1) specimen Collection
Collecting early morning fasting venous blood of a selected subject by using a vacuum negative pressure tube, standing for 30min, centrifuging for 10min at 3000rpm by using a desktop centrifuge, separating serum, storing in an ultra-low temperature refrigerator at-80 ℃, and detecting LEP and APN;
(3-2) determination of serum APN and LEP levels by ELISA
Preparing: taking out the reagent from the ultra-low temperature refrigerator 20 minutes in advance, and balancing the room temperature;
preparing liquid: diluting the concentrated washing liquid with distilled water at a ratio of 1:20 for later use;
dissolution of the standard:
LEP standard curve: adding 1.0 ml of specimen universal diluent into each standard substance, standing at room temperature for 15 minutes, and slightly and uniformly mixing after the specimen universal diluent is fully dissolved; at a concentration of 2000 pg/ml, and then diluted to 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml and 0 pg/ml in this order;
APN standard curve: adding 1.0 ml of specimen universal diluent into each standard substance, standing at room temperature for 15 minutes, after the specimen universal diluent is fully dissolved, gently mixing uniformly to obtain a mixture with the concentration of 10 ng/ml, and then sequentially diluting into 5ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625ng/ml, 0.31 ng/ml, 0.15 ng/ml and 0 ng/ml;
numbering: numbering the micropores corresponding to the sample in sequence;
sample adding: when detecting APN, diluting serum with a 1:2500 reagent matched universal diluent for later use, directly adding LEP, respectively adding blank, standard substance and 100 mu l of sample to be detected into corresponding holes, lightly shaking and uniformly mixing, sealing a plate by a sealing plate film, and then incubating for 90 minutes at 37 ℃;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
adding a biotin antibody: diluting the concentrated biotin antibody with a biotin antibody diluent 1:30 20 minutes in advance to prepare a biotinylated antibody working solution, adding 100 mu l of biotin antibody into each hole, sealing the holes with a sealing plate film, and then incubating for 60 minutes at 37 ℃;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
adding an enzyme: diluting the concentrated enzyme conjugate by using an enzyme conjugate diluent 1:30 20 minutes in advance to prepare an enzyme conjugate working solution; adding 100 mu l of enzyme conjugate into each hole, slightly shaking and uniformly mixing, sealing the plate by a sealing plate membrane, and then placing at 37 ℃ for incubation for 30 minutes;
washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
color development: adding 100 mul of color development liquid A, B into each hole, gently oscillating and mixing uniformly, and developing for 15 minutes in a dark place at 37 ℃;
and (3) determination: adding 100 mu l of stop solution into each hole, patting and uniformly mixing, setting the wavelength of an enzyme-labeled analyzer to be 450nm, zeroing by using a blank hole, and then measuring the OD value of each hole;
data processing: performing data processing by using a polynomial linear fitting mode, drawing by taking the background-subtracted OD value of each calibrator as a vertical coordinate and the concentration of each calibrator as a horizontal coordinate to obtain a standard curve, and calculating the concentrations of LEP and APN in a sample according to the standard curve;
and (3-3) calculating the LEP/APN ratio according to the result.
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