Microcomputed Tomography Technique for In Vivo Three-Dimensional Fat Tissue Volume Evaluation After Polymer Injection

Tissue engineering technologies are new and promising techniques in fat tissue reconstruction. However, to assess their efficacy before any clinical application, in vivo experiments are mandatory. This study assesses whether microcomputed tomography (CT) scan imaging is suitable to analyze in vivo the behavior of injected engineered polymer and changes in fat tissue. The volume of mice inguinal fat pads and the resorption rate of different polymers were analyzed by CT scan for up to 3 months. Different biomaterials were used, including our innovative microspheres loaded with oleic acid. We were able to follow in vivo the polymer and the fat volume of the same animals during a long-term follow-up of 90 days. Semiautomatic three-dimensional quantification allowed to determine the fat volume enhancement after injection, as well as the resorption rate of our product compared to other biomaterials (i.e., polylactic and hyaluronic acid) until 90 days. Our results demonstrate the encouraging proof-of-principle evidence for the application of micro-CT scan technology to follow in vivo biodegradable polymers in a fat tissue engineering approach. This noninvasive technique offers the advantages of the long-term follow-up of fat tissue and synthetic materials in the same animals, which allows both a scientific evaluation of the measurements and the reduction of the number of animals used in in vivo protocols in accordance with the 3 R principles governing the use of animals in science

Absolute quantification of human serum transferrin by species-specific isotope dilution laser ablation ICP-MS

We report for the first time the absolute quantification of a metalloprotein separated by nondenaturing gel electrophoresis (GE) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in combination with species-specific isotope dilution mass spectrometry (IDMS). The proposed method is based on the use of an isotopically enriched 57Fe-transferrin complex to quantify natural transferrin (Tf) in human serum samples. First, the saturation process of Tf with natural abundance or isotopically enriched 57Fe was accomplished by using freshly synthesized Fe-citrate solutions. The stability of the metal-protein complex as well as its stoichiometry was investigated by spectrophotometry and ICP-MS, demonstrating a satisfactory stability over a period of at least one month and a molar ratio Fe:Tf of 1.94 ± 0.09, which is close to the expected value of 2. The species-specific IDMS method was compared with external calibration using the Fe-Tf (absolute Tf amount between 2 and 10 μg) and different sample preparation procedures (stained and nonstained gels) as well as two laser ablation strategies (single line ablation in the direction perpendicular or horizontal to the electrophoretic migration) were evaluated. The proposed species-specific GE-LA-ICP-IDMS method was tested for the analysis of a serum certified reference material (ERM-DA470k/IFCC). The results were in good agreement with the certified value with relative standard deviation values in the range of 0.9–2.7% depending on the data treatment procedure used. Furthermore, the analysis time has been drastically reduced in comparison with previous approaches to less than 15 min. The quantification by species-specific GE-LA-ICP-IDMS allowed us to obtain accurate and precise results not only by analyzing the protein spot in the middle position but also in the adjacent ablation line to the center

A global HILIC-MS approach to measure polar human cerebrospinal fluid metabolome: Exploring gender-associated variation in a cohort of elderly cognitively healthy subjects.

Cerebrospinal fluid (CSF) is a key body fluid that maintains the homeostasis in central nervous system (CNS). As a biofluid whose content reflects the brain metabolic activity, the CSF has been profiled in the context of neurological diseases to provide novel insights into the disease mechanisms. However, a global high-throughput approach to measure a broad diversity of polar metabolites present in CSF is lacking. Although still perceived as challenging and less reproducible, hydrophilic interaction liquid chromatography (HILIC) has recently evolved to offer the unprecedented coverage capacity of water-soluble metabolome. Here, we present a global HILIC high-resolution mass spectrometry-based (HRMS) approach that combines the profiling in acidic pH ESI (+) and basic pH ESI (-) mode to extend the coverage of CSF polar metabolome. This approach allowed us to annotate and measure a broad range of central carbon metabolites (implicated in glycolysis, TCA cycle, nucleotide, amino acid and fatty acid metabolism) in CSF collected from cognitively healthy elderly volunteers (n = 32), using a single extraction method. Metabolite annotation was achieved using the accurate mass, RT and MS/MS criteria, allowing for the characterization of 146 measurable metabolites. Exploration of characterized individual CSF profiles allowed for a discovery of intriguing gender-associated differences, with significantly higher acylcarnitine levels in men and higher taurine levels women. With this case study, we demonstrate the value of combined HILIC ESI ± HRMS profiling to assess CSF metabolome in clinical research studies