Journal of Lipid Research, Vol 30, 841-846, Copyright © 1989 by Lipid Research, Inc.
Measurement of apolipoprotein B synthesis in perfused rat liver using stable isotopes: [15N]hippurate as a measure of the intracellular [15N]glycine precursor enrichment
T Matsushima, DR Cryer, KE Winkler, JB Marsh and JA Cortner
Department of Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
Rat livers were perfused by the nonrecirculating technique with medium
containing [15N]glycine and sodium benzoate. At various times, the isotopic
enrichment of hepatic free glycine, hepatic glycyl-tRNA, and perfusate
hippurate was measured by GLC-MS. After 60 min, these parameters had
reached approximately maximal values. At 90 min, the perfusate hippurate
had a 30% greater enrichment of 15N than the intracellular glycine or
glycyl-tRNA. Hippurate enrichment was half that of the medium glycine. The
rat livers secreted apolipoprotein B (B- 100 plus B-48) at a rate of 22
micrograms/g per h. From the 15N enrichment and the secretion rate, an
intrahepatic pool size of 86 micrograms/g of apoB was calculated. From the
minimal intracellular transit time of 30 min, an apoB fractional synthetic
rate (FSR) of 2 pools/h was indicated, whereas the FSR estimated from the
15N- enrichment was 0.26/h. A possible explanation for the discrepancy is
that apoB may recycle within the hepatocyte. On the basis of the present
experiments, when hippurate enrichment is used as a measure of the
enrichment of intrahepatic glycine in in vivo studies with 15N- labeled
glycine, a correction should be applied, under normal metabolic
circumstances, of approximately 20-30%.
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