NMR Can be Used to Monitor the Relationship Between Endothelial Lipase and HDL
In atherosclerosis, blood flow becomes restricted due to the deposition of fats, cholesterol, and other substances (collectively known as plaque) on artery walls. A high cholesterol level is a risk factor for atherosclerosis, and it is determined through the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) concentrations. HDL is commonly referred to as “good” cholesterol because it provides anti-oxidative, anti-inflammatory, and cholesterol efflux functions. Therefore, a high level of HDL is beneficial to protect against atherosclerosis.
This protection is partly due to the binding of paraoxonase 1 (PON1), a serum enzyme produced by the liver, which catalyzes anti-oxidative reactions through its arylesterase (AE) activity. Although a serum enzyme, 95% of PON1 is associated with HDL through the formation of an HDL-PON1 complex, which is partially regulated by the endothelial lipase (EL). EL is a phospholipase that participates in the HDL metabolism by hydrolyzing HDL, causing depletion of phospholipids and generating lysophospholipids and free fatty acids. It markedly alters the structure and function of HDL.
Previous studies have demonstrated that EL modification of HDL decreased the HDL-PON1 content in vitro. A recent study has expanded on this data and investigated the effects of EL on the amount of PON1 bound to HDL (HDL-PON1 content) and PON1 AE activity in vitro and in vivo.
Enzymatic Interaction of Endothelial Lipase Displaces PON1 from HDL In Vitro
In vitro studies were performed by incubating purified human HDL with HepG2 cells EL-overexpressing or empty virus (EV)-infected control HepG2 cells in the absence or presence of PON1 co-overexpression. It was found that regardless of PON1 co-overexpression, the HDL-PON1 content and AE activity decreased, indicating that the lipolytic activity of EL plays a crucial role in these changes.
To further understand the mechanism by which EL causes the displacement of PON1 from HDL, an evaluation using fast protein liquid chromatography (FPLC) was performed. In both EL and EV environments, a decrease in HDL size was associated with a decrease in PON1 content and AE activity. The results suggest that EL enzymatic interaction leads to PON1 displacement and not EL-induced alterations in HDL size and composition.
In Vivo Data is Markedly Different from In Vitro Data
In vivo analysis of the EL modification on HDL-PON1 content and AE activity was also performed. Mice were injected with EL-adenovirus (Ad) or control EV-Ad and their serum was analyzed. A marked decrease in HDL levels and AE activity was observed in EL serum, compared to EV serum. However, similar PON1 levels were observed in both EL and EV sera. Due to the decrease in HDL levels, these data suggest either an HDL overload with PON1 or an accumulation of free PON1.
For in vivo studies with humans, the EL serum levels as well as the PON1 content and AE activity of isolated HDL were measured in healthy volunteers using enzyme-linked immunosorbent assay (ELISA). Subjects with a high EL serum level had a positive correlation with a higher PON1 content in HDL, and increased AE activity.
Because the human in vivo data was markedly different from the in vitro data, further investigation was performed using nuclear magnetic resonance (NMR) spectroscopy to assess whether EL serum levels in humans are linked to serum concentrations of HDL parameters.
Using a Bruker Avance IVDr platform* (600 MHz Avance Neo Spectrometer) and the Bruker IVDr lipoprotein subclass analysis module* (B.I.LISA), HDL constituents, such as the cholesterol, triglycerides, and phospholipid content, as well as total HDL were quantified. The results were similar in both high and low EL sera, indicating that HDL is not correlated with EL. Although significantly higher levels of lipolytic products were found in high EL serum in comparison with subjects with low EL serum. These products are known to be generated by EL-mediated phospholipolysis of HDL, indicating some relationship between EL and HDL composition in vivo.
Further Investigation into the Relationship of Endothelial Lipase and HDL In Vivo
This study demonstrated marked differences between EL modulation of HDL in vitro and in vivo. The in vitro data showed a clear link between EL and HDL-PON1 content and AE activity, and the results revealed that it is an enzymatic interaction between EL and HDL that causes PON1 displacement.
Nevertheless, the relationship between EL and HDL in vivo appears to be much more complex. Some correlation between EL and HDL hydrolysis seems to be present, however, additional interactions may be involved in the association between these components. Therefore, further investigation using larger populations is required to elucidate this relationship in humans.
*Bruker NMR Instruments are for Research Use Only. Not for use in clinical diagnostic procedures.
1. Schilcher, I., et al. (2021). Endothelial Lipase Modulated Paraoxonase 1 Content and Arylesterase Activity in HDL. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms22020719.