Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins

Procedures are presented for the separation and determination of the isotopic enrichment of multiple human apolipoproteins labeled in vivo with a stable isotope amino acid. The isotopic enrichments of plasma lysine and plasma apolipoproteins were monitored for 16 days after a single intravenous dose of [4,4,5,5-²H₄]lysine (5 mg/kg body weight). The use of a multiply deuterated amino acid enabled the measurement of isotopic enrichments above background over the entire 16-day time course in all proteins. Individual apolipoproteins were separated on a specially designed gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis system cast in a conventional slab gel apparatus which resolved apoB-100, apoE, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III-1, and apoC-III-2 on a single gel. After staining with Coomassie blue, proteins bands (containing 5 to 30 μg of individual apolipoprotein) were excised from the gel. Amino acids were recovered from hydrolyzed gel slices, derivatized, and analyzed by gas chromatography-mass spectrometry for determination of lysine isotopic enrichments. The utility of the method is demonstrated using examples of apolipoproteins B-100, A-I, A-II, C-I, C-II, and C-III from either total plasma d < 1.21 g/ml lipoproteins or selected lipoprotein subfractions. Lysine isotopic enrichments of proteins were generally determined with a precision of better than 5%. The isotopic enrichment profiles were consistent with literature reports of apolipoprotein metabolic kinetics based on the use of radioiodinated apolipoproteins. The procedures outlined can be used to separate and measure the isotopic enrichment of virtually any apolipoprotein from any chosen lipoprotein fraction. Thus, these procedures should find wide application in the study of apolipoprotein metabolic kinetics.