**2.3 Analytical methods**

The gasometric and acid-base analyses (pH, pO2, pCO2, bicarbonate, SaO2) were carried out using the ABL 300 analyser (Radiometer, Copenhagen, Denmark).

The glucose and lactate concentrations were determined with the Ektachem 500 automated analyser (Kodak, New York, United States) by an enzymatic method using glucose oxidase (EC 1.1.3.4) and lactate oxidase (EC 1.13.12.4) and a measurement by reflectometry (reference interval in adult blood: 3.6 – 5.8 mmol/l for glucose and 0.7 – 2.1 mmol/l for lactate).

The total free fatty acid concentration was measured by a manual colorimetric enzymatic assay (Okabe et al., 1980), using an acyl-coenzyme A synthetase (EC 6.2.1.3), an acylcoenzyme A oxidase (EC 1.3.3.6) and a peroxidase (EC 1.11.1.7) (Biomérieux, Marcy l'Etoile, France).

The ketone bodies content was determined by a fluorimetric enzymatic micromethod based on the measurement of NADH fluorescence (Olsen, 1971). Fluorescence was quantified on a spectrofluorimeter, the Kontron SFM (Kontron, Zurich, Switzerland), the excitation and emission wavelengths being 350 and 460 nm respectively. The reagents (lactate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADH, NAD) were supplied by Boehringer (Mannheim, Germany).

The adult blood reference values with the techniques used are from 0.018 to 0.078 mmol/l for aceto-acetate and 0.050 to 0.100 mmol/l for beta-hydroxybutyrate.

The cholesterol concentrations were measured by an enzymatic assay using a cholesterol esterase (EC 3.1.1.13) and a cholesterol oxidase (EC 1.1.3.6.) (Biomérieux, Marcy l'Etoile, France); the reference interval in adult blood is 3.6 – 7 mmol/l.

The hCG content was determined using the IMx automated analyser (Abbott Diagnostics, Abbott Park, United States), by a microparticle enzyme immunoassay.
