A Sample Preparation Method for the Simultaneous Profiling of Signaling Lipids and Polar Metabolites in Small Quantities of Muscle Tissues from a Mouse Model for Sarcopenia

Abstract

The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1∆/− mouse mutant exhibiting multiple–morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia.

Metabolomic analysis of dietary‐restriction‐induced attenuation of sarcopenia in prematurely aging DNA repair‐deficient mice

“…The significantly decreased grip strength, motor coordination and learning performance in AL progeroid mice compared to AL‐WT mice (Figure 1 ) may be related to altered muscle function and quality. Hence, muscle specimens of the quadriceps femoris muscle (Quad) were profiled using recently developed metabolomics methods for the detection of signalling lipids and polar metabolites 34 and the identified molecules related to muscle function and quality, that is, oxidative stress, pro‐ and anti‐inflammatory markers, and energy status, analysed by Partial Least Squares Discriminant Analysis (PLS‐DA). Separation between WT and Ercc1 ∆/− or Xpg −/− mice was observed, especially for the anti‐inflammatory metabolites (Figure 2A ), indicating large alterations of accelerated aging in these metabolites.…”

“…The chemicals, solvents, internal standards (ISTDs) used for muscle tissue extraction as well as the muscle tissue extraction method were described in He et al 34 and detailed in the supporting information .…”

Advances in Metabolic Profiling of Biological Samples

“…Some authors have explored the ability to extract meaningful information from small sample volumes, which is especially important in clinical settings where sample availability can be limited. He et al [6] developed a sample preparation method for simultaneously extracting non-polar and polar metabolites from limited amounts of mouse muscle tissues (5-50 mg dry weight). Overall, 109 lipids (e.g., oxylipins, fatty acids, and lysophospholipids) and 62 polar targeted metabolites (e.g., amino acids, sugars, and nucleotides) were successfully detected via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and capillary electrophoresis-mass spectrometry (CE-MS).…”