Julia Dittrich (1), Richard Wagner (1), Joachim Thiery (1), Susen Becker (1), Ralph Burkhardt (1), Uta Ceglarek (1) (Presenter)
(1) University Hospital Leipzig
Apoproteins are key components of lipoproteins and mediate diverse functions in lipid transport and metabolism. A LC-MS/MS methods to analyze apoproteins using species-unique peptide sequences for Apo A-I, Apo A-II, Apo A-IV, Apo B48, Apo B100, Apo C-I, Apo E and Apo J in 3 µl human or murine plasma. In human plasma Apo A-I and Apo A-II showed the highest concentrations. The simultaneous analysis of apoproteins in only 3 µl murine and human plasma allows us a comprehensive characterization of apolipoprotein levels using normo- and hyperlipidemic patients and different mice strains under different nutritional conditions.
Introduction: Apolipoproteins are key components of lipoproteins and mediate diverse functions in lipid transport and metabolism. Plasma concentrations of apolipoproteins ApoB100 and ApoA1 are used for risk assessment of cardiovascular disease (CVD). Validated immunological assays are only available for analysis of Apo B which is associated with LDL particles and Apo A-I reflecting the number of HDL particles. For other apolipoproteins which may improve the risk assessment of cardiovascular disease validated methods are missing.
For the investigation of genetic factors for the development of CVD knock-out mice models are used. The analysis of apolipoproteins in murine plasma lacks due to the limited sample volume. Our laboratory has previously described a LC-MS/MS method to analyze a panel of apolipoproteins using proteotypic peptides in 3 µl human plasma. The aim of the present study was to establish an adapted method for a simultaneous quantification of eight different apolipoproteins in murine plasma samples.
Methods: Murine and human plasma samples (3 µl) and isolated lipoprotein nfractions were denaturized, reduced, alkylated and digested by trypsin prior LC-MS/MS analysis. Different digestion times were compared and within-day and in between days (n=10) precision were determined.
Results: We identified species-unique peptide sequences for Apo A-I, Apo A-II, Apo A-IV, Apo B48, Apo B100, Apo C-I, Apo E and Apo J using Blast and NCBI database. Tryptic digestion time ranged from 30 min (Apo A-I, Apo A-II, Apo A-IV, Apo B100, Apo J) to 16 h (Apo E, Apo B 48) until complete apolipoprotein digestion. The lower limits of quantification was found between 0, 04 µmol/L and 240 µmol/L. Coefficients of variation <13% were determined. Recovery rates ranged between 92 and 155 %. Comparison with immunoassays showed a good agreement for Apo A-I and Apo B in human plasma samples. In human plasma Apo A-I and Apo A-II showed the highest concentrations (50 µmol/L). Apolipoprotein plasma concentrations in C57-Bl6 wild type mice and hyperlipidemic Apo E knockout mice ranged between 0.29 µmol/L (Apo B100) and 60.6 µmol/L (Apo A-II). Characteristic apolipoprotein distributions could be identified in isolated human and murine lipoprotein fractions.
Differences in fasting and non-fasting status were identified for human normo-lipemic and hyperlipidemic plasma. Also for the wild-type and knock-out mice models differences in lipid metabolism between fasting and non-fasting conditions could be identified.
Conclusion: We developed a novel LC-MS/MS approach for the simultaneous measurement of eight different apolipoproteins in only 3 µl murine or human plasma. This allows us a comprehensive characterization of murine apolipoprotein levels under normo- and hyperlipidemic conditions in mice and man.