Application of elemental analysis-coupled isotope ratio mass spectrometry for protein turnover analysis using deuterium labeling: Purification and analysis of hydrogen isotope ratio of non-derivatized protein-bound alanine.

RATIONALE: Heavy water can be used as a tracer for the evaluation of protein turnover. By adding heavy water (D2 O) to the precursor pool, nonessential amino acids, including alanine, can be isotopically labeled in vivo. Protein turnover can then be quantified by measuring the hydrogen isotope ratio of protein-bound alanine. METHODS: In this study, we constructed a novel method to apply deuterium labeling of alanine to the evaluation of protein turnover using elemental analysis-coupled isotope ratio mass spectrometry (EA-IRMS). We established a preparative high-performance liquid chromatography method to isolate alanine from protein hydrolysates. EA-IRMS was then used to determine the hydrogen isotope ratio of alanine isolated from hydrolysates of protein from mouse myoblast C2C12 cells that had been treated with D2 O over the course of 72 h. RESULTS: In cells treated with 4% D2 O, the deuterium enrichment of alanine increased to approximately 0.9% over time, while that of cells treated with 0.017% D2 O increased to approximately 0.006%. The rate of protein synthesis calculated by fitting the increase of deuterium excess to rise-to-plateau kinetics was similar regardless of the concentration of D2 O. When C2C12 cells treated with insulin and rapamycin were analyzed 24 h after the addition of 0.017% D2 O, protein turnover was found to be accelerated by insulin, but this effect was offset by co-treatment with rapamycin. CONCLUSION: The derivative-free measurement of the hydrogen isotope ratio of protein-bound alanine using EA-IRMS can be applied to the evaluation of protein turnover. The proposed method is an accessible option for many laboratories to perform highly sensitive IRMS-based evaluations of protein metabolic turnover.

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube.

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

Inorganic Iron Supplementation Rescues Hematological Insufficiency Even Under Intense Exercise Training in a Mouse Model of Iron Deficiency with Anemia.

Iron deficiency anemia (IDA) due to malnutrition and/or blood loss is a common condition, especially in women of reproductive age. Intense exercise can induce anemia via an inflammatory response, but whether intense exercise affects the efficacy of iron supplementation to treat IDA is unclear. Here, we show in a mouse model of IDA that acute intense swimming increased IL-6 levels in the blood, but did not affect the maximum elevation of plasma iron following oral administration of 0.5 mg/kg Bw iron. However, compared with the control group without intense exercise, acute intense swimming was associated with a significant decrease in plasma iron 2 and 4 h after iron loading that could be attributed to rapid iron absorption in peripheral tissues. In the chronic experiment, IDA mice administered 0.36, 1.06, or 3.2 mg/kg Bw iron per day that were subjected to 11 intense swimming sessions over 3 weeks showed significantly decreased recovery levels for hemoglobin and red blood cell count during the early phase of the experimental period. At the end of the experimental period, significant, dose-dependent effects of iron, but not the main effect of intense exercise, were seen for recovery of hemoglobin and red blood cell counts, consistent with the acute exercise study. These results suggested that intense exercise in the presence of IDA does not inhibit iron absorption from the gastrointestinal tract and that iron supplementation can enhance the recovery process even after intense exercise.