Study On Lipid Metabolism And Metabolomics In Stable Coronary Heart Disease With Phlegm-blood Stasis Syndrome

ObjectiveTo explore the difference of lipid metabolism level and to find the diagnostic markers and differential metabolites of patients with phlegm-stasis syndrome of stable coronary heart disease（CHD）.MethodsPart I:Study on the level of lipid metabolism in stable CHD with phlegm-stasis syndrome.121 patients with stable CHD were included.According to the clinical diagnostic criteria of phlegm dampness syndrome of CHD and the study on the diagnostic criteria of blood stasis syndrome of CHD,the patients were divided into four groups:phlegm-dampness syndrome,blood stasis syndrome,intermingled phlegm-stasis syndrome,and non-phlegm and non-stasis syndrome.First,Spearman rank correlation analysis was used to analyze the relationship between age,gender,body mass index（BMI）,total cholesterol（TC）,triglyceride（TG）,highdensity lipoprotein cholesterol（HDL-C）,low-density lipoprotein cholesterol（LDL-C）,apolipoprotein A（ApoA1）,apoB,oxidized low density lipoprotein（ox LDL）,oxidized high density lipoprotein（ox-HDL）,glycosylated low density lipoprotein（GlyLDL）),lecithin cholesterol acyltransferase（LCAT）,cholesteryl ester transfer protein（CETP）,paraoxonase（PON）and phlegm-stasis syndrome of CHD;Second,ANOVA（or rank sum test）was used to analyze the distribution differences of blood lipid level,modified lipoprotein and lipoprotein metabolic enzyme in phlegm-stasis syndrome of CHD;Frequency statistics was used to analyze the difference of ApoE genotype distribution in patients with phlegm-stasis syndrome of CHD;Third,univariate and multivariate logistic regression were used to analyze the risk factors of phlegm-stasis syndrome of CHD;Fourth,the specific indexes were screened according to the results of logistic regression,and the receiver operating characteristic curve（ROC）was used to diagnose the phlegm-stasis syndrome of CHD;Fifth,the classification and expression trees（CART）was used to predict the critical value of diagnosing phlegm-stasis syndrome of CHD.Part II:The metabolic difference of phlegm-stasis syndrome of CHD was analyzed based on metabolomics.People were divided into five groups:control,phlegm-dampness syndrome group,blood stasis syndrome group,intermingled phlegm-stasis syndrome group and non-phlegm and non-stasis syndrome group.Liquid chromatography tandem mass spectrometry（LC-MS/MS）was used to detect the metabolites.According to the variable weight in the project（VIP）>1.5 and P<0.05,the differential metabolites among the groups were screened,the types of differential metabolites were annotated according to the human metabolome database（HMDB）,the signal pathway of differential metabolite enrichment was analyzed with Metabo analyst platform,and the biomarkers at the metabolomics level were screened.The Wayne diagram was used to further screen the common differential metabolites between the control and CHD group（phlegmdampness syndrome,blood stasis syndrome,intermingled phlegm-stasis syndrome and nonphlegm and non-stasis syndrome group）,and the common differential metabolites between the CHD phlegm-stasis syndrome group（phlegm-dampness syndrome,blood stasis syndrome,intermingled phlegm-stasis syndrome）and non-phlegm and non-stasis syndrome group.The differential metabolites were screened again with the fold change（FC）>2,and the ROC curve was drawn to judge the diagnostic ability of differential metabolites to diseases and syndrome types.ResultsPart Ⅰ:The relationship between phlegm-stasis syndrome and lipid metabolism of 121 patients with stable CHD were analyzed.The results are as follows:（1）Spearman rank correlation analysis showed that phlegm-dampness syndrome of CHD was negatively correlated with age,HDL-C,ApoAl and positively correlated with BMI,blood stasis syndrome of CHD was positively correlated with TC,TG,ApoB and ApoA1,and intermingled phlegm-stasis syndrome of CHD was negatively correlated with age.（2）ANOVA（or rank sum test）showed that the levels of TC,LDL-C and ApoB in patients with blood stasis syndrome and intermingled phlegm-stasis syndrome were significantly higher than those in patients with non-phlegm and non-stasis syndrome,and the levels of LDL-C in patients with phlegm-dampness syndrome were also higher than those in patients with non-phlegm and non-stasis syndrome（p<0.05）.HDL-C and ApoAl levels in blood stasis syndrome were significantly higher than those in phlegm-dampness syndrome and intermingled phlegm-stasis syndrome（p<0.05）.The expression of GlyLDL in phlegm-dampness syndrome and intermingled phlegm-stasis syndrome was significantly higher than that in non-phlegm and non-stasis syndrome（p<0.05）.The results of lipoprotein metabolic enzymes showed that the expression of LCAT was the lowest and the expression of LPL was the highest in intermingled phlegm-stasis syndrome.The expression of CETP was the highest in blood stasis syndrome while PON was the highest in non-phlegm and non-stasis syndrome and the lowest in blood stasis syndrome.But all of them were not statistically significant.（3）The results of ApoE genotype detection showed that the proportion of risk genotypes（E3/E4 or E4/E4）was the highest in intermingled phlegm-stasis syndrome group（18.75%）,followed by phlegm-dampness syndrome group（10.81%）,non-phlegm and non-stasis syndrome group（10.71%）and blood stasis syndrome group（8.33%）.The frequency of E4 allele in intermingled phlegm-stasis syndrome group was the highest（10.93%）while E2 allele in blood stasis syndrome was the highest（12.5%）,followed by non-phlegm and non-stasis syndrome（7.14%）.（4）Univariate logistic regression showed that compared with non-phlegm and non-stasis syndrome,LDL-C（OR=2.06,95%CI:1.35-2.77）was positively correlated with phlegm-dampness syndrome.ApoB（OR=19.56,95%CI:15.57-23.67）,ox-HDL（OR=3.98,95%CI:2.89-5.06）and TC（OR=2.10,95%CI:1.44-2.75）were positively correlated with blood stasis syndrome.The age（OR=0.94,95%CI:0.89-1.00）was negatively correlated with intermingled phlegm-stasis syndrome,while TC（OR=1.78,95%CI:1.20-2.35）and ApoB（OR=12.53,95%CI:9.04-16.02）were positively correlated with intermingled phlegm-stasis syndrome.Multivariate logistic regression showed that HDL-C was negatively correlated with blood stasis syndrome（OR=0.01,95%CI:0-13.95）.ApoB was positively correlated with blood stasis syndrome（OR=16.38,95%CI:13.38-19.38）and intermingled phlegm-stasis syndrome（OR=15.36,95%CI:12.16-18.55）.（5）Combined with univariate and multifactorial logistic results,the specific indexes of phlegm-stasis syndrome of CHD were age,LDL-C,ox-HDL and GlyLDL,which were used as combined indexes to draw ROC curve to diagnose phlegm-stasis syndrome of CHD.The results showed that the area under the curve（AUC）was 0.772,sensitivity（79.3%）,specificity（70%）,p<0.001,indicating that young age,high LDL-C,high ox-HDL and high GlyLDL has moderate efficacy in diagnosing CHD with phlegm-stasis syndrome.（6）Ox-HDL and GlyLDL were introduced into CART to predict phlegm-stasis syndrome of CHD and the results are as follows:①phlegm-dampness syndrome:8 ug/L<ox-HDL<14 ug/L or ox-HDL<8 ug/L and GlyLDL≥1.7 g/mL or 7 ug/L≤ox-HDL<8 ug/L and GlyLDL≥1.3 g/mL;②Blood stasis syndrome:8 ug/L≤ox-HDL≤8.6 ug/L.③Intermingled phlegm-stasis syndrome:ox-HDL>14ug/L or ox-HDL<8 ug/L and 0.93 g/mL≤GlyLDL<1.3 g/mL;④Non-phlegm and non-stasis syndrome:ox-HDL<8ug/L and GlyLDL<0.93 g/mL or ox-HDL<7ug/L and 0.93 g/mL≤GlyLDL<1.3 g/mL.Part Ⅱ:The differential metabolites between groups were screened by LC-MS/MS technique,it was found that:（1）Compared with the control group,the CHD group（phlegm-dampness,blood stasis,intermingled phlegm-stasis and non-phlegm-non-stasis syndrome）had a total of 71 differential metabolites in the positive ion mode and 70 in the negative ion mode.The differential metabolites are mainly benzenoids（benzene and substituted derivatives,phenolic compounds）,organic acids and derivatives（organic sulfonic acids and derivatives）,lipids and lipid-like molecules（fatty acids）,organoheterocyclic compounds（indole and derivatives）.The pathways involved mainly include biotin metabolism,phenylpropane biosynthesis,arachidonic acid metabolism and so on.（2）There were 86 differential metabolites between phlegm-dampness syndrome group and non-phlegm and non-stasis syndrome group,52 differential metabolites were screened in positive ion mode and 34 in negative ion mode,FC>2 and the metabolic differential substance that could be enriched to the pathway was cis-4-Hydroxy-D-proline,its expression was down-regulated in phlegm-dampness syndrome,and the enrichment pathway was arginine and proline metabolism.Other differential metabolites were mainly organic acids and derivatives（carboxylic acids and derivatives）,organoheterocyclic compounds（piperazines）and lipids（fatty acids）.The main enrichment pathways of differential metabolites were involved in metabolic pathway,porphyrin and chlorophyll metabolism,cyano amino acid metabolism and so on.（3）There were 69 differential metabolites between blood stasis syndrome group and nonphlegm and non-stasis syndrome group,41 differential metabolites were screened in positive ion mode while 28 in negative ion mode.The metabolic differential substance whose FC>2 was Paracetamol,which was down-regulated in blood stasis syndrome,and its metabolic pathway was related to bile secretion.The other differential metabolites were mainly organoheterocyclic compounds,organic acids and derivatives,lipids（fatty acids）,benzenoids（benzene and substituted derivatives）.The main enrichment pathway of differential metabolites involves folic acid synthesis and so on.（4）There were 52 differential metabolites between intermingled phlegm-stasis syndrome group and non-phlegm and non-stasis syndrome group,and there were 26 in positive ion mode and 26 in negative ion mode.The metabolic differences with FC>2 were 3-（4-hydroxyphenyl）propanoate and 4-（beta-Acetylaminoethyl）imidazole,which were up-regulated in intermingled phlegm-stasis syndrome.The metabolic pathway was mainly related to tyrosine metabolism and histidine metabolism.Other differential metabolites were mainly lipids（fatty acids）,organic acids and derivatives（carboxylic acids and derivatives）.The main enrichment pathways of differential metabolites were related to lysine degradation,cyano amino acid metabolism,toluene degradation and so on.（5）There were 120 differential metabolites in phlegm-dampness syndrome group and intermingled phlegm-stasis syndrome,and 60 differential metabolites in positive and negative ion mode,respectively.Daidzein and（S）-1-Pyrroline-5-carboxylate were up-regulated whose FC>2 and can be enriched into the pathway.The main enrichment pathways were isoflavone biosynthesis and biosynthesis of secondary metabolites.The other differential metabolites are mainly organic oxygen compounds and organic acids and derivatives（carboxylic acids and derivatives）.The other enrichment pathways were involved in the regulation of actin cytoskeleton,insulin secretion,pancreatic secretion,phenylpropane biosynthesis,sesquiterpene and triterpene biosynthesis.（6）There were 76 different metabolites between the blood stasis syndrome group and intermingled phlegm-stasis syndrome,and there were 38 metabolites in the positive and negative ion mode,respectively.The metabolic differences with FC>2 and enriched to the pathway were 2-Oxindole and 3Miminomel 3-（4-hydroxyphenyl）propanoate.The expression of 2-Oxindole was down-regulated while 3Miminomel 3-（4-hydroxyphenyl）propanoate was up-regulated in intermingled phlegm-stasis syndrome.The metabolic pathways were the biosynthesis of benzoxazazinones and tyrosine metabolism.Other differential metabolites were mainly organic acids and derivatives（carboxylic acids and derivatives）and organic oxygen compounds.The main enrichment pathways of differential metabolites were involved in ABC transporter,monoterpene biosynthesis,oxidative phosphorylation.（7）There were 44 differential metabolites between phlegm-dampness syndrome group and blood stasis syndrome group,23 in positive ion model and 21 in negative ion model.The metabolic difference with FC>2 and enriched into the pathway was（S）-1-Pyrroline-5-carboxylate,which was down-regulated in phlegm-dampness syndrome.It was organic acid and derivatives（carboxylic acid and derivatives）,and the metabolic pathway was related to carbapenem biosynthesis.The other differential metabolites were mainly organoheterocyclic compounds（piperazines）.The main enrichment pathways of differential metabolites were involved in sesquiterpene and triterpene biosynthesis.（8）The Venn diagram was used to further screen the common differential metabolites of the 4 syndrome types in the CHD group and the healthy control group,and found that there were 11 common differential metabolites in the positive ion mode and 8 in the negative ion mode.According to the screening results of differential metabolites,the differential metabolites with FC>2 were selected to draw the ROC curve,and methyl（2e₆e）-（10r₁1s）-10₁1-epoxy-3₇₁1trimethyltrideca-2₆-dienoate down-regulated（AUC was 0.969,sensitivity was 100%,specificity was 93.8%,95%CI:0.919-1.0,P<0.001）;D-Iditol up-egulated（AUC was 0.867,sensitivity was 78.1%,specificity was 87.5%,95%CI:0.721-1.0,P=0.0015）;（2S,4e,6e,8s,9R）-9-hydroxy2,8-dimethyl-3-oxo-4,6-undecadienoic acid up-egulated（AUC was 0.852,sensitivity was 71.9%,specificity was 87.5%,95%CI:0.723-0.980,P=0.0023）could be the diagnostic indexes of CHD.It shows that the above three indexes have good diagnostic value for CHD.（9）The common difference of metabolites between phlegm-stasis syndrome（phlegmdampness,blood stasis,intermingled phlegm-stasis syndrome）and non-phlegm and non-stasis syndrome were（2e）-octenoylcarnitine and N-Nitrosoproline,and their expression were downregulated in phlegm-stasis syndrome group.Selecting the differential metabolites with FC>2 to draw the ROC curve and（2e）-octenoylcarnitine down-regulation（AUC was 0.839,sensitivity was 0.701-0.976,specificity was 70.8%,95%CI:0.701-0.976,p=0.005）was used as the diagnostic index of CHD with phlegm-stasis syndrome.Conclusions（1）The blood lipid levels in blood stasis syndrome group and intermingled phlegm-stasis syndrome group with CHD were significantly higher than that in phlegm-dampness syndrome group and non-phlegm and non-stasis syndrome group,which may be related to the expression of lipoprotein metabolic enzyme and ApoE gene polymorphism.（2）High GlyLDL was the independent risk factor of phlegm-dampness syndrome;high ApoB was the independent risk factor of blood stasis syndrome and intermingled phlegm-stasis syndrome.The use of CART can better distinguish phlegm-blood stasis syndrome in CHD.（3）The differential metabolites between the control and CHD group were mostly lipids.Methyl（2E₆E）-（10R₁1S）-10₁1-epoxy-3₇₁1-trimethyltrideca-2₆-dienoate,D-Iditol,（2SGravity 4e,6e,8s,9R）-9kw,hydroxylily,2e-octenoylcarnitine can be used as metabolic diagnostic markers of CHD.The differential metabolites of CHD with phlegm-stasis syndrome and non-phlegm and non-stasis syndrome are mostly lipids,organic acids and their derivatives.（2e）-octenoylcamitine can be used as metabolic diagnostic markers of CHD with phlegm-stasis syndrome.There are differential expression profiles in phlegm-stasis syndrome of CHD.