title: Development of an UPLC mass spectrometry method for measurement of myofibrillar protein synthesis: application to analysis of murine muscles during cancer cachexia.
abstract: Cachexia, characterized by skeletal muscle mass loss, is a major contributory factor to patient morbidity and mortality during cancer. However, there are no reports on the rate of myofibrillar protein synthesis (MPS) in skeletal muscles that vary in primary metabolic phenotype during cachexia, in large part because of the small-size muscles and regional differences in larger muscles in the mouse. Here, we describe a sensitive method for measurement of MPS and its application to analysis of MPS in specific muscles of mice with (Apc(Min/+)) and without (C57BL/6) cancer cachexia. Mice were injected with a loading dose of deuterated phenylalanine (D5F), and myofibrillar proteins were extracted from skeletal muscles at 30 min. The relative concentrations of D5F and naturally occurring phenylalanine (F) in the myofibrillar proteins and the amino acid pool were quantified by ultra-performance liquid chromatograph (UPLC) mass spectrometry (MS). The rate of MPS was determined from D5F-to-F ratio in the protein fraction compared with the amino acid pool. The rate of MPS, measured in 2-5 mg of muscle protein, was reduced by up to 65% with cachexia in the soleus, plantaris, diaphragm, and oxidative and glycolytic regions of the gastrocnemius. The rate of MPS was significantly higher in the oxidative vs. glycolytic gastrocnemius muscle. A sufficiently sensitive UPLC MS method requiring a very small amount of muscle has been developed to measure the rate of MPS in various mouse muscles. This method should be useful for studies in other animal models for quantifying effects of cancer and anti-cancer therapies on protein synthesis in cachexia, and particularly for analysis of sequential muscle biopsies in a wide range of animal and human studies.
authors: Research and Innovation Centre, Fondaizone Edmund Mach, San Michele all' Adige Trento, Italy.
authors: Maria
authors: M
authors: Lima
authors: Shuichi
authors: S
authors: Sato
authors: Reilly T
authors: RT
authors: Enos
authors: John W
authors: JW
authors: Baynes
authors: James A
authors: JA
authors: Carson
mesh_terms: D011125:Adenomatous Polyposis Coli / Q000150:complications* / Q000235:genetics; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001706:Biopsy; D002100:Cachexia / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D002853:Chromatography, Liquid; D003964:Diaphragm / Q000378:metabolism; D015536:Down-Regulation; D017491:Genes, APC; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000096:biosynthesis*; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology; D009210:Myofibrils / Q000378:metabolism* / Q000473:pathology; D010649:Phenylalanine / Q000008:administration & dosage / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D015415:Biomarkers; D009124:Muscle Proteins; D010649:Phenylalanine