Solid phase 125i human specific radioimmunoassay

Whole blood for determination of serum analytes (triglycerides and insulin) was collected into microtubes with coagulation activator (Sarstedt, Nümbrecht, Germany) and left to clot before being centrifuged (15 000 g for 2 min), and the serum was frozen at −80°C. Whole blood was added to lithium heparin microtubes (Sarstedt, Nümbrecht, Germany) containing ethylene glycol tetraacetic acid–glutathione and tetrahydrolipostatin, gently mixed, and the plasma was removed and frozen at −80°C until analysis of NEFA. Triglycerides were analysed using enzymic photometry (ABX Pentra 400, HORIBA Medical, Montpellier, France) and insulin concentration using a solid‐phase ¹²⁵I human‐specific radioimmunoassay (Merck Millipore, Billerica, MA, USA). NEFA was analysed by the ACS‐ACOD Method (Wako Diagnostics, Richmond, VA, USA).

Whole blood collected during the insulin clamp was immediately analysed for glucose concentration (YSI2300; Yellow Springs Inc., OH, USA). Serum insulin concentration was measured using a solid‐phase ¹²⁵I human‐specific radioimmunoassay (Merck Millipore, Billerica, MA, USA).