To identify the mechanisms underlying muscle aging, we have undertaken a high-resolution differential proteomic analysis of gastrocnemius muscle in young adults, mature adults, and old LOU/c/jall rats. Two-dimensional gel electrophoresis and subsequent MALDI-ToF mass spectrometry analyses led to the identification of 40 differentially expressed proteins. Strikingly, most differences characterized old (30-month) animals, whereas young (7-month) and mature (18-month) adults exhibited similar patterns of expression. Important modifications in contractile (actin, myosin light-chains, troponins-T) and cytoskeletal (desmin, tubulin) proteins, and in essential regulatory proteins (gelsolin, myosin binding proteins, CapZ-beta, P23), likely account for dysfunctions in old muscle force generation and speed of contraction. Other features support decreases in cytosolic (triose-phosphate isomerase, enolase, glycerol-3-P dehydrogenase, creatine kinase) and mitochondrial (isocitrate dehydrogenase, cytochrome-c oxidase) energy metabolisms. Muscle aging is often associated with increased oxidative stress. Accordingly, we observed differential regulation of molecular chaperones (hsp20, hsp27, reticuloplasmin ER60) and of proteins implicated in reactive aldehyde detoxification (aldehyde dehydrogenase, glutathione transferase, glyoxalase). We further noticed up-regulation of proteins involved in transcriptional elongation (RNA capping protein) and RNA-editing (Apobec2). Most of these proteins were previously unrecognized as differentially expressed in old muscles, and they represent novel starting points for elucidating the mechanisms of muscle aging.
Hierarchical clustering methodology is a powerful data mining approach for a first exploration of proteomic data. It enables samples or proteins to be grouped blindly according to their expression profiles. Nevertheless, the clustering results depend on parameters such as data preprocessing, between-profile similarity measurement, and the dendrogram construction procedure. We assessed several clustering strategies by calculating the F-measure, a widely used quality metric. The combination, on logged matrix, of Pearson correlation and Ward's methods for data aggregation is among the best clustering strategies, at least with the data sets we studied. This study was carried out using PermutMatrix, a freely available software derived from transcriptomics.
24,25(OH)D is the product of 25(OH)D catabolism by CYP24A1. The measurement of serum 24,25(OH)D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC-MS/MS method to determine: (1) the relationships between serum 24,25(OH)D and 25(OH)D; (2) serum reference intervals in healthy individuals; (3) the diagnostic accuracy of 24,25(OH)D measurement as an indicator for vitamin D status; 4) 24,25(OH)D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC-MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)D and 25(OH)D concentrations. The 25(OH)D:24,25(OH)D ratio was significantly higher (P<.001) at 25(OH)D<50 nmol/L. The reference interval for 25(OH)D:24,25(OH)D ratio in healthy subjects was 7-23. Measurement of serum 24,25(OH)D can be used as predictor of vitamin D status, a concentration of>4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities.
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