[Monitoring of adipocyte responsiveness by in situ microdialysis in lipodystrophy tissue: adjustment of a glycerol quantification method in small samples].
In situ microdialysis allows monitoring of metabolic cellular processes at the tissue level in vivo. In the assessment of physiopathologic alterations seen in lipodystrophy, monitoring of glycerol release is pivotal. Indeed, it allows to quantify the pharmacological responsiveness of subcutaneous adipose tissue in humans. Until now, the small volume of microdialysate collected (5-15 microL/sample) restricted the assessment of glycerol level to the use of the radio-enzymatic method or the reference spectrophotometric microanalysis technique. The aim of this study was to adapt the method of glycerol measurement by iminequinone spectrophotometry colorimetric assay (520 nm) using the following reagent: 0.5 IU Glycerokinase, 1.23 IU glycerophosphate oxidase, 0.98 IU peroxidase, 4.6 mM Mg, 5.4 mM 4-chlorophenol, 0.25 mM 4-aminoantipyrine and 1.4 mM ATP. The assay was setup to run on Olympus AU 2700 automate (15 pL sample volume). The sensitivity of the method was improved by adding a 0.2 mmol triglyceride (TG) solution and 1.5 IU lipase to samples, reducing the limit of free glycerol quantification to 0.020 mmol/L. The analytical repeatability was 2.0% and the reproducibility was 7.9%. The present method thus demonstrated the feasibility of pharmacodynamic exploration of local cutaneous responsiveness in vivo in clinical trials.
Belhadj-Tahar
H
H
Groupe Santé Recherche, Département de Pharmacologie Clinique, 31300 Toulouse, France. hafid.belhadj@wanadoo.fr
Nassar
B B
BB
Sadeg
N
N
Coulais
Y
Y
D017667:Adipocytes / Q000378:metabolism* / Q000473:pathology; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D005990:Glycerol / Q000378:metabolism*; D006801:Humans; D008060:Lipodystrophy / Q000378:metabolism* / Q000473:pathology; D017551:Microdialysis / Q000379:methods*; D008875:Middle Aged; D013053:Spectrophotometry
D015415:Biomarkers; D005990:Glycerol