Recently, the team led by Professor Zhang Mei of the Tianjin Armed Police Characteristic Medical Center published an article on the Research Square preprint server, titled "NMR Metabolomics Risk Factors for Coronary Heart Disease in Postmenopausal Women Under Moderate Intensity Statins". Based on nuclear magnetic resonance (NMR) spectroscopy, the article examines lipoprotein particle subclasses as risk factors for the occurrence of coronary heart disease in postmenopausal women taking moderate-intensity statins. The publication makes a valuable contribution to more precise prediction of coronary heart disease, and the authors give special thanks to ProteinT for its support in NMR testing and analysis. Building on its powerful NMR platform, ProteinT continues to explore early warning, medication guidance and long-term monitoring for cardiovascular and cerebrovascular disease, driving screening and medication guidance — for cardiovascular and cerebrovascular conditions as well as common cancer types — towards a more precise approach.
Background
Dyslipidaemia, and in particular an excessively high level of low-density lipoprotein cholesterol (LDL-C), is an important risk factor for the occurrence of coronary heart disease (CHD). Postmenopausal women are a distinct group, characterised by elevated LDL-C and an increased risk of CHD. Patients with diabetes mellitus (DM) and metabolic syndrome (MS), owing to abnormal lipid metabolism, are also more likely to develop CHD.
After proprotein convertase subtilisin/kexin type 9 (PCSK9) is synthesised and secreted by hepatocytes, it binds to the LDL receptor (LDL-R), leading to a decline in LDL-C metabolism. PCSK9 has in recent years become a new target for intervention in lipid metabolism. However, although the PCSK9 monoclonal antibodies evolocumab and alirocumab have achieved reductions in LDL-C of approximately 50%-60% and a 15% relative reduction in major adverse cardiac events (MACE), statins remain the most widely used first-line lipid-lowering therapy.
Nuclear magnetic resonance (NMR) spectroscopy is an advanced measurement method that can provide a large volume of metabolomics data, including lipid indicators, metabolites and amino acids. With regard to lipids, NMR can not only group different lipoproteins according to particle size (very low-density lipoprotein (VLDL-1~5), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL-1~6) and high-density lipoprotein (HDL-1~4)), it can also, on the basis of the different responses of substances in a magnetic field, quantitatively determine the content of lipoprotein components (cholesteryl ester (CE), free cholesterol (FC), triglyceride (TG) and phospholipid (PL)). Several studies have reported correlations between lipid indicators and the risk of CHD. In this study, we combined NMR spectroscopy with laboratory testing to explore risk factors for the occurrence of CHD among 139 NMR results, and sought to identify the relationship between NMR metabolomics, PCSK9 and coronary atherosclerosis in a cohort of postmenopausal women treated with moderate-intensity statins.
Methods
1. Study population
Patients who met the inclusion and exclusion criteria were enrolled in this study, giving a total of 300 samples. The inclusion criteria were: female, aged over 46 years, more than one year since natural menopause, and continuous once-daily use of atorvastatin 10 mg QD or rosuvastatin 20 mg. Coronary angiography (CAG) was performed between June 2019 and June 2020 after at least 6 months of treatment. Exclusion criteria included previous percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), oophorectomy and other conditions.
2. Grouping
All enrolled patients were grouped according to three criteria. Patients with coronary artery stenosis ≥50% were assigned to the CHD group (242 cases) on the basis of CAG results, while those with coronary artery stenosis < 50% were assigned to the non-CHD group (58 cases) on the basis of CAG results. According to the diagnosis of DM and MS, patients were further divided into a DM group and a non-DM group, and an MS group and a non-MS group.
3. Laboratory testing and NMR spectroscopic lipid measurement
Patients' clinical characteristics were collected from the medical record system. Fasting blood samples were collected on the day before CAG, then centrifuged and stored at -70°C. The Gensini score was calculated on the basis of CAG, and NMR spectroscopy, lipoprotein(a) (Lp(a)), PCSK9 and C-reactive protein (C-RP) testing were performed.
4. Statistical analysis
Categorical variables were expressed as n (%), and differences between groups were assessed using the chi-square test. The normality of the distribution of continuous variables was examined using the Kolmogorov-Smirnov test. Student's t-test was used to assess differences in continuous variables between groups. Variables that were clinically relevant and showed a univariate relationship with the diagnosis of CHD were entered into a multivariate logistic regression model. Spearman correlation analysis of variables with the Gensini score or PCSK9 was plotted as a heat map.
Study approach
In all patients, as well as in patients with coronary heart disease, diabetes mellitus (DM) and metabolic syndrome (MS), multivariate logistic regression and Spearman correlation analysis were used to explore the risk factors for CHD and the relationship between the Gensini score, PCSK9 and the NMR results.
Results
1. Clinical demographic characteristics, and routine and advanced lipid indicators
This study enrolled 300 patients, of whom the 242 with coronary heart disease were assigned to the CHD group. The 123 with diabetes mellitus were assigned to the DM group, and the 75 with metabolic syndrome to the MS group. As shown in Supplementary Tables 1–3, nine items of clinical information and 139 NMR results were listed and compared between the CHD and non-CHD groups, the DM and non-DM groups, and the MS and non-MS groups. Among these, age, fasting plasma glucose (FPG) and LDL particle/concentration (P/C) were discordant, while apolipoprotein A2 (ApoA2), apolipoprotein B (ApoB), total particles, LDL-6 particles, HDL-PL, LDL-6-TG, LDL-6-CE, LDL-6-FC, LDL-6-PL, LDL-6-ApoB, HDL-3-PL, HDL-2-ApoA1 and HDL-3-ApoA1 all differed across the three comparisons; notably, ApoA2, HDL-PL, HDL-3-PL, HDL-2-ApoA1 and HDL-3-ApoA1 were all significantly reduced in the CHD, DM and MS groups.
As shown in Supplementary Table 1, the LDL particle count was markedly higher in the CHD group, rather than the concentration. Further comparison revealed that the difference was caused mainly by LDL-1 and LDL-6 particles. In the comparison between patients with and without DM, as shown in Supplementary Table 2, IDL-PL and VLDL-PL were considerably higher in DM patients, while HDL-FC was considerably lower. The comparison between patients with and without MS is shown in Supplementary Table 3: VLDL-PL, IDL-PL, LDL-PL and HDL-TG were considerably higher in MS patients, whereas HDL-CE and HDL-FC were considerably lower. Compared with the undiagnosed population, PCSK9 was lower in both CHD patients and MS patients.
2. Risk factors for coronary heart disease
Multivariate logistic regression analysis was used to further assess the relationship between lipoprotein particles and CHD; the results are shown in Figure 2. Age, LDL-6 particles, LDL-TG and LDL-FC were risk factors for CHD, whereas ethanol and glycerol were protective factors. Even where statistical significance was not reached, the effect of PCSK9 on CHD was neutral.
3. Relationship between the Gensini score and NMR results
Spearman correlation analysis was used to assess the relationship between the Gensini score and the NMR results; the results are shown in Figure 3. In CHD patients, DM patients and all patients, the indicators positively correlated with the Gensini score included ApoB/A1, VLDL and IDL particles, TG in VLDL-3~5, LDL-1~2 and LDL-5~6, CE in VLDL-2~5 and IDL, FC in VLDL-2~5, LDL-1 and IDL, and PL content in VLDL-3~5. Indicators negatively correlated with the Gensini score included ApoA1, ApoA2, CE and PL in HDL-2~4, FC in ApoA1 and HDL-3, and ApoA2 content in HDL-4. In MS patients, the indicators positively correlated with the Gensini score were limited to TG, CE and PL in VLDL-3~5, while those negatively correlated were FC and PL content in LDL-2, and ApoA1, CE and PL content in HDL-1. PCSK9 was negatively correlated with the Gensini score in all patients and in DM patients.
4. Relationship between PCSK9 and NMR results
Spearman correlation analysis was used to assess the correlation between plasma PCSK9 and the NMR results; the results are shown in Figure 4. In all patients and in CHD and DM patients, PCSK9 was negatively correlated with total cholesterol (TC), ApoB/A1, VLDL, IDL and LDL particles, and with almost all lipid contents in VLDL-3~5, IDL and LDL-1. In the MS group, there was little correlation between PCSK9 and the NMR results.
Discussion
Cardiovascular disease (CVD) is the leading cause of death among women. Conventional lipid measurement focuses mainly on the pathogenic role of lipoprotein content in CHD, yet the number and structure of lipoproteins are also risk factors for CVD.
TG in VLDL is converted into CE via cholesteryl ester transfer protein acting on LDL and HDL, producing CE-depleted LDL and HDL. The TG at the core of VLDL, LDL and HDL is subsequently hydrolysed by hepatic lipase, giving rise to IDL and to smaller, denser LDL and HDL. NMR determined the lipoprotein subclasses VLDL-1 to VLDL-5, IDL, LDL-1 to LDL-6 and HDL-1 to HDL-4, arranged progressively smaller and denser, with less TG and more PL. Some researchers used NMR to measure the specific effects of pravastatin 40 mg daily on lipoprotein particle concentration and content, and found a "V"-shaped relationship between the lipid-lowering effect and lipoprotein particle size. The downward trends in lipoprotein content (CE, FC and TG) were similar, but the overall decline in TG and FC within LDL was markedly smaller. In our study, even under moderate-intensity statin treatment, lipoprotein TG and FC content remained risk factors for CHD. In the course of the correlation analysis in this study, we noted that the positive correlations between the Gensini score and the NMR results were concentrated mainly in the lipoprotein content of VLDL-2 to VLDL-5, IDL and LDL-1. These results also showed a trend related to lipoprotein size, consistent with previous studies.
In contrast, the results of the correlation analysis between PCSK9 and the NMR findings were the opposite of those reported in earlier studies. Some studies have shown that circulating PCSK9 levels are positively correlated with VLDL, large IDL particles, small and very small LDL particles, small HDL particles and VLDL particle size, and negatively correlated with LDL size and HDL size. It has also been reported that serum PCSK9 levels may be influenced by sex, HDL-C, TG and physical activity. However, apart from the general heterogeneity of the patients enrolled in the two studies, the biggest difference is that the patients in our study were receiving statin therapy. Statins have been reported to abolish the correlation between PCSK9 and LDL-C, and a number of studies have confirmed that statins can increase PCSK9. Our hypothesis is that statins lower lipid levels while raising circulating PCSK9, giving rise to the negative correlation between them.
This study also demonstrated that ethanol and glycerol are protective factors for CHD. Glycerol is released mainly from the hydrolysis of TG. It has been reported that inflammation and homocysteine inhibit TG metabolism. We found no evidence, however, of a direct protective effect of glycerol against CHD. We are inclined to regard glycerol merely as a marker of reduced TG hydrolysis. Few studies have confirmed the clinical effect of ethanol on CHD in non-drinkers. The protective effect of endogenously produced ethanol remains to be investigated further.
Summary
In this study we explored the risk factors for the occurrence of coronary heart disease among the NMR results of a cohort of postmenopausal women receiving statin therapy. The results showed that age, LDL-6 particles, LDL-TG and LDL-FC are risk factors for CHD, while ethanol and glycerol are protective factors. The NMR results showed that the lipoprotein content of VLDL-2~5, IDL, LDL-1 and LDL-2 was positively correlated with the Gensini score, whereas the lipoprotein content of ApoA1, ApoA2 and HDL-1~4 was negatively correlated with it. The PCSK9 and NMR results showed negative correlations with VLDL, IDL and LDL particles, TC and ApoB/A1, as well as with almost all lipid contents in VLDL-3~5, IDL and LDL-1. The clinical significance and value of these indicators await further study.
