Quantification of bioactive lipids

Bruxelles Woluwe

The biological activities of most bioactive lipids are controlled by the balance between their production and degradation. However, because numerous bioactive lipids are produced and degraded by multiple pathways, measuring the expression or activities of the enzymes involved is often not enough. Thus it is essential to directly quantify their endogenous levels, as these represent the final result of the synthetic and degradation pathways. We therefore develop analytical methods that will help us understand the role and implication of these bioactive lipids in pathophysiology.

These three families of bioactive lipids are suggested to be involved in obesity and metabolic syndrome. To facilitate the quantification of these potentially interconnected lipids, we have developed and validated a HPLC-MS method allowing for their simultaneous quantification from tissues (Mutemberezi et al. Anal Bioanal Chem, 2016). A critical step of the method is the extraction and purification of as many oxysterols as possible. Indeed, oxysterolquantification methods are affected by analytical artifacts due to the oxidation of cholesterol during the analytical procedure. The sensitivity and specificity of the method allow us to quantify the 23 analytes of interest in a number of different tissues (including the liver, adipose tissue and plasma). We found that 16 weeks of high-fat diet strongly impacted the hepatic levels of several oxysterols, ceramides, and endocannabinoids and that a partial leastsquaresdiscriminant analysis (PLS-DA) based on the variations of the hepatic levels of these 23 bioactive lipids allowed for differentiating the lean mice from the obese mice (Figure 1).

Figure 1: Partial least-squares (PLS) discriminant analysis based on the hepatic levels of oxysterols, ceramides and endocannabinoids found in control and obese mice. Each dot on the graph represents a mouse. (adapted from Mutemberezi  et al. Anal Bioanal Chem, 2016).

Next to the oxysterols, bile acids are the second family of important bioactive lipids issued from cholesterol metabolism. As for oxysterols, a better understanding of their roles can only be reached through their quantification in physiopathological situations.

Thus this year, we developed an HPLC-MS method for the quantification of bile acids from several matrices, including mice liver and gallbladder content. Furthermore, to favor translational studies to humans, we adapted the method to quantify bile acids from serum/plasma samples (using either 30 μL of mouse serum or 50 μL of human serum) (Figure 2). Currently our method allows us to quantify 19 bile acids.

Figure 2: Representative EIC for the main bile acids present in mouse and human plasma. Analysis carried out by HPLCMS using an LTQ-orbitrap operated in negative mode.