Valérie Lamothe

ELISA as a new method to measure genistein and daidzein in food and human fluids

Widely distributed in the plant kingdom, phytoestrogens, like soy isoflavones are found in plant protein extracts at varying levels depending on culture conditions and cultivars. Their increasingly used in human, as natural estrogens and their varying levels in raw materials raise the question concerning the measurement of isoflavones both in food or food supplements as well as in human fluid. To validate our new ELISA technique, isoflavones measurements in food-supplements, in soy food and in human fluid from volunteers participating to a kinetic study or a survey, were done. Our method was also compared with other physico-chemical techniques in an inter-laboratory assay (23 laboratories). In conclusion, our new ELISA technique is reliable, sensitive, cheap, rapid and can be used either for a great number of human fluid analysis, for crude matter or food analysis as long as the appropriate extraction technique is performed prior to ELISA procedure. # 2003 Elsevier Ltd. All rights reserved.

Syntheses of Novel Hapten–Protein Conjugates for Production of Highly Specific Antibodies to Formononetin, Daidzein and Genistein

Abstract The syntheses of novel hapten–carrier protein conjugates are described. Isoflavones functionalised in C2 were coupled to bovine serum albumin or swine thyroglobulin. The antibodies obtained were used for the development of ELISAs (enzyme-linked immunosorbent assays) to measure the three isoflavones with IC50 ranging from 15.6 to 100xa0ng/mL.

Higher bioavailability of isoflavones after a single ingestion of a soya-based supplement than a soya-based food in young healthy males

Soya isoflavones, genistein and daidzein, are the focus of numerous studies investigating their potential effects on health and results remain controversial. Bioavailability is clearly a crucial factor influencing their bioefficacy and could explain these discrepancies. This study aimed at assessing: (1) the isoflavone content of sixty-nine European soya-derivative products sold on the French market; (2) the bioavailability of isoflavones comparing supplement with food. Twelve healthy volunteers were recruited in a randomized two-way crossover trial and received 35 mg isoflavones equivalent aglycone either through supplements or through cheese, both containing different patterns of isoflavone conjugates and different daidzein:genistein ratios. A specific ELISA method was used to assess the plasma and urinary concentrations of isoflavones and thus the pharmacokinetic parameters, which were then normalized to mg of each isoflavone ingested. Results showed that the normalized Cmax of daidzein (P = 0.002) and similarly the normalized AUC0 --> infinity and Cmax of genistein (P = 0.002) from soya-based capsules were higher than that from soya-based cheese. In conclusion, this work completes studies on isoflavone bioavailability and presents new data regarding isoflavone concentrations in soya-derivative products. Assuming that isoflavone conjugation patterns do not influence isoflavone bioavailability, this study shows that isoflavones contained in capsules are more bioavailable than those contained in soya-based cheese. Although the supplement is more bioavailable, the relative importance of this is difficult to interpret as there is little evidence that supplements are biologically active in human subjects to date and further studies will be necessary for this specific supplement to prove its efficacy.

Temporal Memory and Its Enhancement by Estradiol Requires Surface Dynamics of Hippocampal CA1 N-Methyl-D-Aspartate Receptors

BACKGROUNDnIdentifying the underlying cellular mechanisms of episodic memory is an important challenge, since this memory, based on temporal and contextual associations among events, undergoes preferential degradation in aging and various neuropsychiatric disorders. Memory storage of temporal and contextual associations is known to rely on hippocampal N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity, which depends ex vivo on dynamic organization of surface NMDARs. Whether NMDAR surface trafficking sustains the formation of associative memory, however, remains unknown.nnnMETHODSnWe tested this hypothesis, using single nanoparticle imaging, electrophysiology, and behavioral approaches, in hippocampal networks challenged with a potent modulator of NMDAR-dependent synaptic plasticity and memory, 17β-estradiol (E2).nnnRESULTSnWe demonstrate that E2 modulates NMDAR surface trafficking, a necessary condition for E2-induced potentiation at hippocampal cornu ammonis 1 synapses. Strikingly, cornu ammonis 1 NMDAR surface trafficking controls basal and E2-enhanced mnemonic retention of temporal, but not contextual, associations.nnnCONCLUSIONSnNMDAR surface trafficking and its modulation by the sex hormone E2 is a cellular mechanism critical for a major component of episodic memory, opening a new and noncanonical research avenue in the physiopathology of cognition.

Influence of ethnic origin (Asian v . Caucasian) and background diet on the bioavailability of dietary isoflavones

Soya isoflavones: genistein and daidzein are increasingly consumed in Western countries. Their beneficial effects are discussed considering nutrition and health in Asia. The present study aimed to check whether chronic ingestions, ethnic origin and dietary context can influence soya phyto-oestrogen bioavailability. Two prospective trials were carried out to blindly assess the pharmacokinetics after acute and chronic intake of soya-based cheese (45.97 (sd1.57) mg isoflavones) taken once a day for 10 d. Twelve healthy young Asians immersed for 2 months in France were randomised in a cross-over design to compare the influence of a Western v. Asian dietary context. The second trial partly nested in the first one, compared Asians under the Western diet to twelve healthy young male Caucasians under the same diet. All volunteers were non-equol producers. After an acute intake of soya in Western diet, Asians exhibited higher maximum concentration measured in plasma (Cmax) and area under the plasma concentration-time curve (AUC) for genistein and daidzein than Caucasians (P = 0.005, 0.006, 0.032 and 0.008, respectively). In Caucasians under Western diet, AUC and Cmax values significantly increased after chronic intake. This was not the case for daidzein in Asians whatever the dietary context. For the first time, it is evidenced that on acute intake of soya cheese, Asians absorb soya phyto-oestrogens better than Caucasians, regardless of whether the background diet is Western or Asian. On chronic ingestions, AUC and Cmax values were increased for daidzein and genistein in Caucasians but not in Asians. There are ethnic differences in isoflavone pharmacokinetic and bioavailability. This may influence health outcomes.

Synthesis of haptens and conjugates for ELISA of glycitein: development and validation of an immunological test.

Two carboxylic acid haptens of glycitein were synthesized, with a spacer arm at the C2 position. They differed in the length of the spacer arm, with the length of the spacer arms being three or four carbon atoms, and were named Delta3-glycitein and Delta4-glycitein haptens, respectively. The different haptens were coupled to bovine serum albumin (BSA), and the coupling efficiency was assessed by MALDI mass spectrometry. Polyclonal antibodies were generated against the BSA conjugates. An additional conjugate of Delta4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta3-glycitein and 62.5 ng mL(-1) with anti-Delta4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta3-glycitein antibody, interassay and intra-assay variations were 12.2 and 11.5%, respectively. Specificity tests did not show any significant cross-reaction with any other soy isoflavone. This specificity is not influenced by the length of the spacer arm. The assay was validated by measurements performed on plasma samples as well as on soy-based foodstuffs and on soy-based food supplements.

Removing isoflavones from modern soyfood: Why and how?

Estrogenic isoflavones were found, in the 1940s, to disrupt ewe reproduction and were identified in soy-consumers urine in 1982. This led to controversy about their safety, often supported by current Asian diet measurements, but not by historical data. Traditional Asian recipes of soy were tested while assaying soy glycosilated isoflavones. As these compounds are water-soluble, their concentration is reduced by soaking. Pre-cooking or simmering time-dependently reduces the isoflavone:protein ratio in Tofu. Cooking soy-juice for 15 or 60min decreases the isoflavone:protein ratios in Tofu from 6.90 to 3.57 and 1.80, respectively (p<0.001). Traditional Tempeh contains only 18.07% of the original soybean isoflavones (p<0.001). Soy-juice isoflavones were reduced by ultra-filtration (6.54 vs 1.24 isoflavone:protein; p<0.001). Soy-protein and isoflavones are dissociated by water rinsing and prolonged cooking, but these have no equivalent in modern processes. As regards human health, a precise definition of the safety level of isoflavone intake requires additional studies.

Original preparation of conjugates for antibody production against Amicoumacin-related anti-microbial agents

Amicoumacins are natural products with potent anti-ulcerogenic and anti-bacterial activities, and have been isolated from different Bacillus genera. They belong to a family of 3,4-dihydroisocoumarin derivatives bearing hydroxylated amino acid side chains. The 3,4-dihydroisocoumarin moiety of Amicoumacins has been obtained in two steps from 2-methoxybenzoic acid by combining directed and benzylic metalation strategies. The use of s-BuLi in both steps gave satisfactory and reproducible yields. For the development of an immunoassay (ELISA) of Amicoumacin-related compounds in biological media, the deprotected 3,4-dihydroisocoumarin moiety has been coupled to the BSA carrier protein via a homobifunctional linker deriving from d-tartaric acid. This approach enabled to introduce the hydroxylated portion of Amicoumacin directly during the preparation of hapten-protein conjugates. The coupling ratio was evaluated by mass spectrometry. The hapten-protein conjugate showing the best coupling ratio was used to generate polyclonal immunosera in rabbits. After immunoserum titration, ELISA conditions were set up and specificity tests were performed on solutions of pure parent compounds, semi-purified Amicoumacin B as well as on culture supernatants of strains known for their Amicoumacin production. This immunoassay is suitable for a rapid and simple screening test for the production of Amicoumacins and its related compounds by bacterial strains.

Estradiol enhances retention but not organization of hippocampus-dependent memory in intact male mice

Because estrogens have mostly been studied in gonadectomized females, effects of chronic exposure to environmental estrogens in the general population are underestimated. Estrogens can enhance hippocampus-dependent memory through the modulation of information storage. However, declarative memory, the hippocampus-dependent memory of facts and events, demands more than abilities to retain information. Specifically, memory of repetitive events of everyday life such as where I parked requires abilities to organize/update memories to prevent proactive interference from similar memories of previous parking events. Whether such organizational processes are estrogen-sensitive is unknown. We here studied, in intact young and aged adult mice, drinking-water (1μM) estradiol effects on both retention and organizational components of hippocampus-dependent memory, using a radial-maze task of everyday-like memory. Demand on retention vs organization was manipulated by varying the time-interval separating repetitions of similar events. Estradiol increased performance in young and aged mice under minimized organizational demand, but failed to improve the age-associated memory impairment and diminished performance in young mice under high organizational demand. In fact, estradiol prolonged mnemonic retention of successive events without improving organization abilities, hence resulted in more proactive interference from irrelevant memories. c-Fos imaging of testing-induced brain activations showed that the deterioration of young memory was associated with dentate gyrus dysconnectivity, reminiscent of that seen in aged mice. Our findings support the view that estradiol is promnesic but also reveal that such property can paradoxically impair memory. These findings have important outcomes regarding health issues relative to the impact of environmental estrogens in the general population.

Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration

Abstract17β-Estradiol (17β-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17β-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17β-E2 effects in the brain. However, 17β-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17β-E2, its isomer17α-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17β-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5xa0pmolxa0mL−1, respectively, were reached for both 17α-E2 and 17β-E2. LOD values for E1 and E3 were 0.01 and 0.025xa0pmolxa0mL−1, respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17β-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17α-E2 was not detected in biological samples, 17β-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water.n Graphical abstractMethod workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ß-E2, its isomer17α-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates