Protein Synthesis Determined from Non-Radioactive Phenylalanine Incorporated by Antarctic Fish

Direct measurements of temperature-dependent weight gains are experimentally challenging and time-consuming in long-lived/slow-growing organisms such as Antarctic fish. Here, we reassess methodology to quantify the in vivo protein synthesis rate from amino acids, as a key component of growth. We tested whether it is possible to avoid hazardous radioactive materials and whether the analytical pathway chosen is robust against analytical errors. In the eelpout, Pachycara brachycephalum, (C9H11N1O2)-C-13-N-15 phenylalanine was injected intraperitoneally and muscle tissue was sampled before injection and at 1.5 h time intervals up to 6 h thereafter. The incorporation of (CN)-C-13-N-15-labeled-phenylalanine into muscle was monitored by quantification of bound and free phenylalanine through liquid chromatography-mass spectrometry. We found an increase in the pool of labeled, free phenylalanine in the cytosolic fraction that leveled off after 4.5 h. The labeled phenylalanine bound in the proteins increased linearly over time. The resulting protein synthesis rate (Ks) for P. brachycephalum was as low as 0.049 +/- 0.021% day(-1). This value and its variability were in good agreement with literature data obtained from studies using radioactive labels, indicating that this methodology is well suited for characterizing growth in polar fish under in situ conditions in remote areas or on research vessels.

Automated summary

We assessed a methodology to measure the in vivo protein synthesis rate in Antarctic fish. By injecting labeled phenylalanine into the eelpout species Pachycara brachycephalum and monitoring its incorporation into muscle tissue, we found a low protein synthesis rate for this species. The results were in agreement with previous studies that used radioactive labels, indicating the reliability of this non-radioactive method for studying growth in polar fish.