Comparison of progesterone concentration determination by 12 non-isotopic immunoassays and gas chromatography/mass spectrometry in 99 human serum samples.

A single serum progesterone determination may be highly predictive for early pregnancy and in vitro fertilisation and embryo-transfer outcomes. We therefore compared 12 direct non-isotopic progesterone immunoassays with gas-chromatography/mass spectrometry (GC/MS). For each assay, data from the analysis of 99 individual sera were compared with data obtained by GC/MS, using regression and bias plot analyses and the ratio method. We observed a larger difference in concentration between high and low values and a broader distribution of results for immunoassays than for GC/MS. All immunoassays displayed bias in the calibration process and a lack of specificity and/or sensitivity, to various degrees. We tried to identify the parameters of the assay procedure that might contribute to these discrepancies. None of the criteria investigated (antibodies, control and preparation of calibrators, blocking agents and choice of tracer) had a significant effect when studied alone.

[Validity of immunochemical methods for bloody progesterone: a study performed in 1998]. / Validité des dosages immunochimiques de la progestérone dans le sang: étude réalisée en 1998.

The aim of this study was to compare, with manufacturer's agreement, twelve direct and non-isotopic commercial assays of progesterone. We have evaluated the analytical performances: low limit detection, imprecision, accuracy (recovery and dilution tests) and we have tested some patient samples. Results were compared to a reference method using isotope dilution Gas Chromatography and Mass Spectrometry combination (GC-MS). For each assay, analytical qualities and defaults are established. Large differences are found between progesterone concentration measured on the same sample with the different methods essentially for the low concentrations. Comparison with GC-MS raised questions about the accuracy of the different assays. This work will be aid laboratories to their choice and/or validation.

Purification and properties of a monoacylglycerol lipase in human erythrocytes.

A membrane-bound monoacylglycerol lipase (MAGL) activity, previously demonstrated in intact human erythrocytes [Boyer, Somma, Vérine, L'Hôte, Finidori, Merger and Arnaud (1981) J. Clin. Endocrinol. Metab. 53, 143-148], has now been purified to apparent homogeneity by a five-step procedure involving solubilization in CHAPS and sequential chromatographies on Sephacryl S-400, DEAE-Trisacryl, Zn(2+)-chelating Sepharose and Superose 12 columns. The purified protein has a molecular mass of 68 +/- 2 kDa, as determined by SDS/PAGE and gel filtration, suggesting that the enzyme behaves as a monomer. The concentration-dependence of MAGL activity with monooleoylglycerol, the preferred substrate showed kinetics typical of an interfacial lipolytic enzyme displaying optimal activity on emulsified substrate particles; apparent Km values were 0.27 mM and 0.49 mM for the sn-1(3)- and sn-2-isomers respectively. MAGL had no, or negligible, activity towards tri-oleoylglycerol, di-oleoylglycerol, oleoylcholesterol, oleoyl-CoA and phosphatidylcholine; it was inhibited by di-isopropylfluorophosphate, PMSF and diethyl p-nitrophenyl phosphate, suggesting that MAGL is a serine hydrolase. MAGL activity was not modified by bile salt or apolipoprotein C-II, whereas a dose-dependent inhibition was observed with apolipoprotein A-I.

Reversible inhibition by ethanol of Mg(2+)-dependent phosphatidate phosphohydrolase: an in vitro study in the rat reticulocyte.

By using a tracer method, we demonstrate that short-term in vitro exposure of intact rat reticulocytes to ethanol elicits a biphasic response of cell-bound Mg(2+)-dependent phosphatidate phosphohydrolase (PAP). An initial concentration-dependent (200-750 mM) activity decrease is rapidly (< 10 min) followed by reversal of the inhibition in the presence of ethanol, suggesting the development of a cell resistance to the inhibitory agent. Addition to the cell suspension of propranolol (100 microM), a known PAP inhibitor, does elicit PAP inhibition but unlike ethanol, inhibition is not followed by a return with time to control value. Ethanol-induced inhibition of cell-bound PAP was also demonstrated in cell-free extracts, where the Mg(2+)-dependent activity was decreased both in the particulate and soluble fractions. In the intact cells, the transient PAP inhibition occurs in concomitance with an overall increase in total glycerolipid biosynthesis, which is constant over 60-min incubation. We suggest that the biphasic mode of response to ethanol of Mg(2+)-dependent PAP activity may play a role in the mechanism of membrane adaptation to ethanol, and thereby to the pathogenesis of alcoholism.

Increased monoester lipase activity in red blood cells during hyperthyroidism.

Human red blood cells (RBC) contain a monoester lipase (MEL) activity which is tightly associated with the cell membrane and has its active site externally oriented, as inferred from the ability of the intact cell to hydrolyse an exogenously added lipid substrate. Membrane-bound MEL activity was measured by a radiochemical assay in intact RBC from 29 untreated hyperthyroid patients. The mean (+/- S.D.) MEL level was higher (P less than 0.01) in these patients (1220 +/- 212 mu./10(12) RBC) than in the control group (1010 +/- 120 mu./10(12) RBC). There was no difference between men and women. The increase in MEL values was associated with significantly (P less than 0.001) decreased values of mean cellular volume and mean cellular haemoglobin content. A continued study of 13 patients, who became euthyroid with treatment, showed a normalization of the MEL values in RBC. The increased lipolytic potency of RBC represents a new characteristic of hyperthyroid patients. Further exploration of the possible diagnostic or prognostic implications of this enzymatic change seems warranted.