Measurement of hepatic protein synthesis in unrestrained mice‐evaluation of the ‘flooding technique’

Abstract

Controversy exists regarding the validity of various techniques for estimating rates of protein synthesis in vivo. In the present report, we have compared estimates of hepatic protein synthesis in normal mice with a pulse labelling of [1-14C]leucine and calculated hepatic protein synthetic rates in a conventional two-pool model and in a five-pool compartment analysis. Results obtained with pulse labelling were also compared to those obtained in animals receiving a flooding dose of 1.5 mumol L-phenylalanine and 0.4 microCi [U-14C]phenylalanine per gram of body weight or 1.0 mumol L-leucine and 0.4 microCi [l-14C]leucine per gram of body weight. Estimates of protein synthesis were calculated with plasma free amino acid, liver acid-soluble fraction and acylated tRNA specific radioactivities as being representative of the precursor pool for protein synthesis. Rates of hepatic protein synthesis obtained with pulse labelling and either leu-tRNA or acid-soluble fractions of liver leucine as the precursor for protein synthesis gave similar results (37 +/- 5 vs 42 +/- 5% per day) in a two-pool model, but disagreed in a five-pool model (37 +/- 5 vs 6 +/- 2% per day). Estimates based on plasma enrichment in leucine were only one fifth of values obtained with tRNA in labelling experiments. When the plasma pool with tracer amino acids was used to indicate the precursor labelling of protein synthesis, values obtained with the flooding dose of either phenylalanine or leucine agreed with those obtained with pulse labelling and enrichment in tRNA (30 +/- 3 nmol min-1 vs 28 +/- 4 nmol min-1); with however no agreement when the enrichment in the liver mixed tissue pool was used (76 +/- 5 nmol min-1). Complete equilibration of the amino acid pools did not occur despite flooding. Therefore, the flooding technique may only represent an approximate method to measure protein synthesis in vivo, although it gives absolute values that agree well with results from labelling techniques based on tRNA enrichment provided the plasma pool is used as the precursor enrichment.