Final Report Summary - CARDIOBIOLIP (Novel mass spectrometry-based integrated analytical platforms for lipidomics studies on blood lipoproteins for cardiovascular disease research)
Cardiovascular disease (CVD) is the most common cause of death in developed countries but its symptoms only become evident late in the course of the disease. Therefore, there is an urgent need to discover novel biomarkers which may allow risk prediction at an individual’s level and it is necessary to gain a deeper understanding on the mechanisms involved in order to design improved and personalized treatments.
Lipoproteins are a wide range of heterogeneous blood particles comprising different proteins and lipid classes. Their main role is the delivery or uptake of specific lipids to/from different sites in the body. Low levels of high density lipoproteins and high levels of low density lipoproteins are connected with the development of atherosclerosis. There is also evidence that inflammation and lipids may be linked, as inflammation as well as cholesterol levels are the main driver of atherosclerosis, and lipoproteins can have important signaling functions. Therefore, in-depth lipid profiling of lipoprotein fractions would increase the understanding of the biochemistry of the processes involved behind CVD.
The CardioBioLip project was envisioned with the main aim to enable the integrated analysis of lipids in blood lipoprotein particles in order to better understand the connectivity of lipid composition, lipid metabolism and lipoprotein levels with the development of CVD.
Different separation techniques were compared for serum/plasma lipoprotein species and capillary isotachophoresis (cITP) resulted to be the most appropriate one for analytical purposes. However, cITP of lipoproteins had first shortcomings like limited resolving of lipoprotein fractions and limited robustness. For repeatable and reproducible results, we therefore explored and developed a number of highly stable capillary coatings for ITP protein separations at high pH.
An improved method for the separation of lipoproteins in plasma/serum samples was developed, basing on the optimization of the spacer compounds added to the serum/plasma sample, resulting in the separation of 18 lipoprotein fractions. These have been classified according to the main lipoprotein types (HDL, chylomicrons, VLDL, LDL). The method has been validated with human plasma samples and compared with a validated NMR method of lipoprotein analysis. The method developed is superior in its performance to previous published approaches, with just a very small amount of sample required (in the low microliter range) to relatively quantify lipoproteins. In addition, this method offers fractionation of serum into lipoprotein fractions next to a detailed profiling of them For this, a fraction collection method for the ITP-separated lipoprotein species has been developed, allowing subsequent analysis of the lipoprotein fractions. This allows to determine apolipoproteins, mRNA and other compounds of interest in the fractions; for this analysis collaborations with other groups have been established.
The developed lipoprotein profiling method and several mass-spectrometry based analytical lipid profiling platforms have been applied to the study of blood serum samples from both atherosclerosis animal models and patients. Clearly different lipoprotein and lipid profiles and levels can be observed depending on the lipid content of the diet. LDLr-/- mice under a high cholesterol diet tend to quickly develop atherosclerosis and present high levels of certain pro-inflammatory oxidized lipids, which indicate the role of the inflammatory response in the triggering of atherosclerosis. On the other hand, their lipoprotein levels result appeared to be altered, with clear changes in the particle types present in their blood. Interpretation of the results obtained with respect to lipid metabolism pathways will provide extremely useful information for the understanding of the biochemical processes involved in CVD.
In another collaboration project human serum samples from patients with different degrees of atherosclerosis and presence or absence of major cardiac events have been analyzed with the lipoprotein and lipidomics methods. The first results of the data analysis are very promising, and data analysis is on-going and will extend the time of the project due to the complexity of the available clinical metadata. The data and studies will reveal important information that might help to understand the onset of atherosclerosis and understanding of the occurrence of major cardiac events.
The developed methods and the results obtained in preclinical and clinical studies will contribute to an earlier diagnosis and prediction of the prognosis of CVD; a better diagnosis and prediction of CVD complications are crucial to develop and apply new treatments to prevent upcoming CVD. The novel methodology to separate lipoproteins was developed in such a way to allow coupling and integration with lipid profiling and other omics profiling in an high throughput set-up. This will be realized within the next 1-2 years by the host group based on the results obtained, and will allow the cost-efficient analysis of larger number of samples. This will allow to obtain more in-detail information on the patient status with regards to lipid metabolism, and at the same underpins the economic potential of the platform and relevance of this project to one of the society´s most important concerns, cardiovascular disease.
Lipoproteins are a wide range of heterogeneous blood particles comprising different proteins and lipid classes. Their main role is the delivery or uptake of specific lipids to/from different sites in the body. Low levels of high density lipoproteins and high levels of low density lipoproteins are connected with the development of atherosclerosis. There is also evidence that inflammation and lipids may be linked, as inflammation as well as cholesterol levels are the main driver of atherosclerosis, and lipoproteins can have important signaling functions. Therefore, in-depth lipid profiling of lipoprotein fractions would increase the understanding of the biochemistry of the processes involved behind CVD.
The CardioBioLip project was envisioned with the main aim to enable the integrated analysis of lipids in blood lipoprotein particles in order to better understand the connectivity of lipid composition, lipid metabolism and lipoprotein levels with the development of CVD.
Different separation techniques were compared for serum/plasma lipoprotein species and capillary isotachophoresis (cITP) resulted to be the most appropriate one for analytical purposes. However, cITP of lipoproteins had first shortcomings like limited resolving of lipoprotein fractions and limited robustness. For repeatable and reproducible results, we therefore explored and developed a number of highly stable capillary coatings for ITP protein separations at high pH.
An improved method for the separation of lipoproteins in plasma/serum samples was developed, basing on the optimization of the spacer compounds added to the serum/plasma sample, resulting in the separation of 18 lipoprotein fractions. These have been classified according to the main lipoprotein types (HDL, chylomicrons, VLDL, LDL). The method has been validated with human plasma samples and compared with a validated NMR method of lipoprotein analysis. The method developed is superior in its performance to previous published approaches, with just a very small amount of sample required (in the low microliter range) to relatively quantify lipoproteins. In addition, this method offers fractionation of serum into lipoprotein fractions next to a detailed profiling of them For this, a fraction collection method for the ITP-separated lipoprotein species has been developed, allowing subsequent analysis of the lipoprotein fractions. This allows to determine apolipoproteins, mRNA and other compounds of interest in the fractions; for this analysis collaborations with other groups have been established.
The developed lipoprotein profiling method and several mass-spectrometry based analytical lipid profiling platforms have been applied to the study of blood serum samples from both atherosclerosis animal models and patients. Clearly different lipoprotein and lipid profiles and levels can be observed depending on the lipid content of the diet. LDLr-/- mice under a high cholesterol diet tend to quickly develop atherosclerosis and present high levels of certain pro-inflammatory oxidized lipids, which indicate the role of the inflammatory response in the triggering of atherosclerosis. On the other hand, their lipoprotein levels result appeared to be altered, with clear changes in the particle types present in their blood. Interpretation of the results obtained with respect to lipid metabolism pathways will provide extremely useful information for the understanding of the biochemical processes involved in CVD.
In another collaboration project human serum samples from patients with different degrees of atherosclerosis and presence or absence of major cardiac events have been analyzed with the lipoprotein and lipidomics methods. The first results of the data analysis are very promising, and data analysis is on-going and will extend the time of the project due to the complexity of the available clinical metadata. The data and studies will reveal important information that might help to understand the onset of atherosclerosis and understanding of the occurrence of major cardiac events.
The developed methods and the results obtained in preclinical and clinical studies will contribute to an earlier diagnosis and prediction of the prognosis of CVD; a better diagnosis and prediction of CVD complications are crucial to develop and apply new treatments to prevent upcoming CVD. The novel methodology to separate lipoproteins was developed in such a way to allow coupling and integration with lipid profiling and other omics profiling in an high throughput set-up. This will be realized within the next 1-2 years by the host group based on the results obtained, and will allow the cost-efficient analysis of larger number of samples. This will allow to obtain more in-detail information on the patient status with regards to lipid metabolism, and at the same underpins the economic potential of the platform and relevance of this project to one of the society´s most important concerns, cardiovascular disease.