Hervé Galons

The Application of a New Highly-Sensitive Radioimmunoassay for Plasma 21-Deoxycortisol to the Detection of Steroid-21-Hydroxylase Deficiency

21-deoxycortisol (21-DF) is a steroid of strictly adrenal origin formed by the 11-hydroxylation of 17-hydroxyprogesterone. This metabolic pathway is minor in normal subjects, in whom basal plasma concentrations range from 0·03 to 0·63 nmol/L and from 0·865 to 1·50 nmol/L after adrenocorticotropic hormone (ACTH; Synacthène Immédiat, Ciba/Geigy, France). However, this metabolic pathway becomes major in 21-hydroxylase-deficient patients: in those who have the classical form of congenital adrenal hyperplasia (CAH) basal plasma 21-DF levels can attain more than 144 nmol/L. The synthesis of two isomers, E and Z, of the 21-deoxycortisol-3-carboxymethyloxime (CMO) hapten enabled us to prepare the corresponding E and Z immunogens by coupling them to bovine serum albumin (BSA), as well as the corresponding iodinated E and Z 21-DF-3-CMO-histamine tracers. We developed a very sensitive radioimmunoassay for 21-DF in plasma by associating an anti-21-DF-3-CMO-BSA-E isomer antibody to an iodinated 21-DF histamine-Z isomer (standard curve IC 50 = 8 pg/tube). This plasma 21-DF radioimmunoassay allowed diagnosis of the classical form of CAH in untreated newborn (basal 21-DF levels greater than 144 nmol/L), as well as the late-onset form (post-ACTH 21-DF levels greater than 11 · 54 nmol/L), and also permitted detection of 21-hydroxylase-deficient heterozygotes of both forms of CAH among the general population (post-ACTH 21-DF levels between 2·02 and 9·52 nmol/L).

Easy alkylation of purine bases by solid-liquid phase transfer catalysis without solvent. : structural analysis by 2D heteronuclear 1H 13C correlated nmr spectroscopy.

Abstract Solid-liquid PTC without added organic solvent promotes alkylation of purine derivatives leading in particular to an efficient synthesis of the antiviral DHPA. The location of the substituent on the ring was determined by analysis of coupling interactions through 2D δ-δ heteronuclear 1H 13C correlated NMR spectroscopy.

Catalyse par transfert de phase solide-liquide sans solvant : application a la reaction de michael

Abstract Solid-liquid phase transfer catalysis without added organic solvent efficiently promotes MICHAEL reactions. The method is applied here to the addition of acetylacetone, methylacetoacetate and fluorene anions on hindered acceptors.

Plasma 21-deoxycortisol: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunossay using 125iodine

Plasma 21-deoxycortisol (21DF) is an excellent marker of 21-hydroxylase deficiency. Currently, it is the only marker able to detect heterozygous carriers with 21-hydroxylase deficiency after ACTH stimulation. We have already developed radioimmunoassays for 21DF using first tritiated, then 125I-21DF which had a ten-fold higher sensitivity. However, because the lifespan of 125I-21DF is short, the tracer needs to be reprepared every two months and this multiplies the risk of contamination by radioactive 125I vapours. We therefore developed a non-isotopic 21DF assay that uses a 21DF-biotin conjugate with a original bridge, a diaminopropyl arm, linking the steroid to biotin. The 21DF-biotin conjugate was measured by time-resolved fluorescence after adding streptavidin-europium to the microtitration wells. The analytical qualities of this assay were very similar to those of the radioimmunoassay using 125I-21DF as tracer. The results obtained by the two methods, in either normal subjects or patients with 21-hydroxylase deficiency, were virtually the same.

Solid-Liquid Phase Transfer Catalysis Without Solvent: Further Improvement in SNAr Reactions

Abstract Solid-liquid PTC without added solvent efficiently promotes SNAr reactions of a variety of anionic nucleophiles generated in situ. This methodology is applied with success to some examples concerning non-activated aromatic systems. TDA-1 is the most effective catalyst.

A new specific and sensitive time resolved-fluoroimmunoassay of 11-deoxycortisol in serum

A biotinylated 11-deoxycortisol tracer was synthesized from 11-deoxycortisol-3-carboxymethyloxime and the conjugate obtained by acylation of biotinylaminopropylammonium trifluoroacetate. This biotinylated tracer was used to develop an 11-deoxycortisol time-resolved-fluoroimmunoassay (TR-FIA). The tracer was quantified after adding streptavidine-Europium. A TR-FIA sensitive standard curve, with displacement of 20, 50, and 80% of tracer was obtained with 12.4, 70.7, and 512.8 pg of 11-deoxycortisol, respectively. After extraction followed by Celite chromatography, purified serum samples were simultaneously assayed by RIA and TR-FIA. The results obtained by the two methods were practically identical, however, this new specific, non-isotopic 11-deoxycortisol assay has the advantage of being more sensitive than RIA, thus well-suited to accurate measurement in endocrinological studies, particularly when serum 11-deoxycortisol levels in patients are just above the highest normal values. Moreover, this non-isotopic assay is cheaper than RIA.

Small-molecule inducers of Aβ-42 peptide production share a common mechanism of action

The pathways leading specifically to the toxic Aβ42 peptide production, a key event in Alzheimers disease (AD), are unknown. While searching for pathways that mediate pathological increases of Aβ42, we identified Aftin‐4, a new compound that selectively and potently increases Aβ42 compared to DMSO (N2a cells: 7‐fold; primary neurons: 4‐fold; brain lysates: 2‐fold) with an EC50 of 30 μM. These results were confirmed by ELISA and IP‐WB. Using affinity chromatography and mass spectrometry, we identified 3 proteins (VDAC1, prohibitin, and mitofilin) relevant to AD that interact with Aftin‐4, but not with a structurally similar but inactive molecule. Electron microscopy studies demonstrated that Aftin‐4 induces a reversible mitochondrial phenotype reminiscent of the one observed in AD brains. Sucrose gradient fractionation showed that Aftin‐4 perturbs the subcellular localization of γ‐secretase components and could, therefore, modify γ‐secretase specificity by locally altering its membrane environment. Remarkably, Aftin‐4 shares all these properties with two other “AD accelerator” compounds. In summary, treatment with three Aβ42 raising agents induced similar biochemical alterations that lead to comparable cellular phenotypes in vitro, suggesting a common mechanism of action involving three structural cellular targets.—Bettayeb, K., Oumata, N., Zhang, Y., Luo, W., Bustos, V., Galons, H., Greengard, P., Meijer, L., Flajolet, M. Small‐molecule inducers of Aβ‐42 peptide production share a common mechanism of action. FASEB J. 26, 5115–5123 (2012). www.fasebj.org

Easy Michael Addition by Solid-Liquid Phase Transfer Catalysis Abnormal Reaction of N-Acetylaminomalonate

Abstract Solid-liquid phase transfer catalysis (PTC) without added solvent efficiently promotes Michael addition of substituted malonates on hindered acceptors. A rearrangement of adducts formed by addition of diethyl N-acetylaminomalonate is described.

Organic reactions without solvent: Michael additions on an unsaturated sulfone and sulfoxide

Abstract Solid-liquid phase transfer catalysis in the absence of any solvent efficiently promotes Michael additions of nitro alkanes and of diethyl N-acetylaminomalonate to phenyl vinyl sulfone and to phenyl vinyl sulfoxide. The adduct of the Michael addition to diethyl N-acetylaminomalonate to phenyl vinyl sulfoxide was converted by distillation under partial vacuum to diethyl N-acetylamino vinyl malonate which gave (±)-vinylglycine by hydrochloric acid catalysis.

The measurement of 11β-hydroxy-4-pregnene-3,20-dione (21-Deoxycorticosterone) by radioimmunoassay in human plasma

A specific radioimmunoassay (RIA) method is described for the determination of 21-deoxycorticosterone (21 DB) in human plasma. 21-Deoxycorticosterone-3-(O-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate antisera in rabbits. Steroids which reacted significantly with the antisera were found to be progesterone, pregnenolone, corticosterone and 11-oxo progesterone. However, after extraction of plasma and column chromatography on Celite, all these steroids were separated from 21-deoxycorticosterone and consequently did not interfere with the radioimmunoassay. The intra- and interassays coefficients of variation were 8% and 11% respectively. Mean plasma 21-deoxycorticosterone level for healthy subjects was very low: 17.8 +/- 14.8 pmol/l (mean +/- SD) with no statistical difference between males and females. During the ACTH stimulation test, the 21-deoxycorticosterone levels of healthy subjects increased to 84.7 +/- 26.3 pmol/l (mean +/- SD) for males and 79.3 +/- 31.6 pmol/l (mean +/- SD) for females. Consequently high levels of plasma 21-deoxycorticosterone were found in treated patients suffering from congenital adrenal hyperplasia (CAH) with 21-hydroxylase deficiency, particularly in CAH salt-losers with high plasma renin activity (PRA), where the plasma level reached 40,545 pmol/l. Thus, 21-deoxycorticosterone may be a new marker for adrenal 21-hydroxylase deficiency.