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Journal of Lipid Research, Vol 32, 1063-1072, Copyright © 1991 by Lipid Research, Inc.
BW Patterson, DL Hachey, GL Cook, JM Amann and PD Klein
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
micrograms 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 less than 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.
ARTICLES
Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins
USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030.
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