Measurement of apolipoprotein B synthesis in perfused rat liver using stable isotopes: [15N]hippurate as a measure of the intracellular [15N]glycine precursor enrichment.
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%.
Matsushima
T
T
Department of Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
Cryer
D R
DR
Winkler
K E
KE
Marsh
J B
JB
Cortner
J A
JA
D000818:Animals; D001055:Apolipoproteins B / Q000096:biosynthesis*; D002852:Chromatography, Ion Exchange; D008401:Gas Chromatography-Mass Spectrometry; D005998:Glycine / Q000378:metabolism*; D006626:Hippurates / Q000032:analysis*; D008099:Liver / Q000378:metabolism*; D008297:Male; D009587:Nitrogen Isotopes; D010477:Perfusion; D012353:RNA, Transfer, Gly / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D001055:Apolipoproteins B; D006626:Hippurates; D009587:Nitrogen Isotopes; D012353:RNA, Transfer, Gly; D005998:Glycine