A new picture of HDL metabolism is unveiled by state-of-the-art mass spectrometry

The metabolism of apoA-I/HDL may lead to an improved understanding of HDL function in humans. Endogenous labeling with stable isotopes can be used to study the metabolism of apolipoproteins during a clinical trial/study.... [ view full abstract ]

The metabolism of apoA-I/HDL may lead to an improved understanding of HDL function in humans. Endogenous labeling with stable isotopes can be used to study the metabolism of apolipoproteins during a clinical trial/study. However, traditional detection methods such as multiple reaction monitoring mass spectrometry cannot measure precisely the low tracer enrichment in slowly turning over proteins as in HDL. Using the high resolution/accurate mass Q Exactive mass spectrometer, we have established a novel targeted mass spectrometry approach to measure low-abundant D3-Leu tracer enrichment (<<1.0%), which is necessary to determine the kinetic parameters of HDL apolipoproteins.

Subjects were administered a bolus of D3-Leu. Plasma was collected up to 70 hours for HDL isolation via anti-apoA-I immunopurification. Five HDL size fractions were isolated using non-denaturing gel electrophoresis: alpha0, alpha1, alpha2, alpha3 and prebeta. D3-Leu enrichment was measured with the QExactive for subsequent compartmental modeling. Contrary to the canonical view that posits prebeta is released first and then converted to alpha3, alpha2 and alpha1, our multicompartmental analysis revealed that the alpha apoA-I/HDL sizes are simultaneously released from liver. Our analysis also revealed that the major source for prebeta is alpha3. We analyzed additional apolipoproteins, including apoA-II, apoC-III, apoE, apoM and apoA-IV; their compartmental modeling also revealed that their appearance on a given HDL size is due to direct secretion, with minor contributions of exchange among the HDL sizes.

Our findings provide a novel picture of HDL metabolism that may lead to the development of more reliable clinical markers of coronary heart disease.