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Am J Physiol Endocrinol Metab 270: E1022-E1036, 1996;
0193-1849/96 $5.00
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AJP - Endocrinology and Metabolism, Vol 270, Issue 6 E1022-E1036, Copyright © 1996 by American Physiological Society

ARTICLES

Sensitive methods to study human apolipoprotein B metabolism using stable isotope-labeled amino acids

T. Demant, C. J. Packard, H. Demmelmair, P. Stewart, A. Bedynek, D. Bedford, D. Seidel and J. Shepherd
Institut fur Klinische Chemie, Ludwig-Maximilians Universitat, Munich, Germany.

The objective of the study was to develop a sensitive method using stable isotope-labeled tracers that would permit the determination of apolipoprotein B (apoB) metabolism in very low-density lipoprotein subfractions (VLDL1, Sf 60-400; VLDL2, Sf 20-60), intermediate-density lipoprotein (IDL, Sf 12-20), and low-density lipoprotein (LDL, Sf 0-12). Six normolipidemic subjects were given trideuterated leucine, and its clearance from plasma and appearance in the four apoB-containing lipoprotein fractions were followed by use of a highly sensitive gas chromatography-mass spectrometry technique in which the m + 3-to-m + 2 ion ratio was selectively monitored. This analytic approach permitted the precise measurement of low enrichments in IDL and LDL and extension of the turnover out to 250-300 h. A compartmental model was developed to derive rate constants from the plasma and apoB enrichment curves. The model was uniquely identifiable once parameter dependencies had been introduced to reduce the number of unknowns. Values were obtained for apoB input into all lipoprotein density intervals, together with rates of interconversion and catabolism; these agreed well with results from radioiodinated tracer experiments. An alternative model structure was also explored in which input occurred only into VLDL1. Altering the protocol of tracer administration (bolus vs. primed constant infusion) and dose (over a 10-fold range) had no influence on the results obtained. The analytic and modeling approach described will permit stable isotopes to be used to elucidate key features of apoB metabolism in normal and pathological states.


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