2.4.2. Plasma determinations
At the end of the treatment, mice were sacrificed under isoflurane
anesthesia. Blood samples were collected in ice-cold tubes containing
heparin and centrifuged at 5000 g at 4°C for 20 min, and the plasma was
frozen at −80°C. Plasma glucose, low-density lipoprotein
(LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol and
triglycerides concentrations were measured by colorimetric methods using
Spinreact kits (Spinreact, S.A., Girona, Spain). Plasma insulin
concentrations were quantified using a mouse insulin ELISA kit (Alpco
Diagnosis, Salem, NH, USA). Homeostatic model assessment of insulin
resistance (HOMA-IR) was calculated using the formula: fasting glucose
(mM)×fasting insulin (μU/mL)/22.5. Plasma levels of LPS were measured
using a PierceTM Chromogenic Endotoxin Quant Kit
(Thermo Scientific, Inc., Waltham, MA, USA) following manufacturer’s
instructions. The plasma samples were pre-treated with a 10 mM
MgCl2 solution and PyrosperseTMDispersing Agent (Lonza, Walkersville, MD, USA) in order to remove
residual heparin used during blood extraction and fat, respectively.
Sterile and pyrogen-free material was always used to guarantee sample
and test integrity.