Vitamin A relative dose response test: validation by intravenous injection in children with liver disease.

The relative dose response (RDR) test has been proposed as a tool for estimation of total body stores of vitamin A. In this study we have validated a variation of this test in children with or without liver disease. Administration of vitamin A was done by intravenous injection of 1000 micrograms of retinyl palmitate. We conclude that the RDR test by intravenous injection performed after 5 h is a reliable and sensitive indicator of vitamin A status. Values found were greater than 20% when liver vitamin A concentration is less than 20 micrograms/g liver and less than 10% when liver vitamin A concentration is greater than 20 micrograms/g liver. We also conclude that the RDR test can be applied to evaluate the efficiency of vitamin A therapy and we confirm that plasma retinol levels should not be used to screen vitamin A status.

Short- and long-term vitamin A treatment in children with cholestasis.

Newborns have limited reserve supplies of vitamin A. Infants with chronic cholestasis are in a precarious nutritional state because of their limited ability to build these stores even though the vitamin is present in their diet. In this study, we investigated liver concentrations of vitamin A in 30 children with extrahepatic biliary atresia. We demonstrate that correction of the deficiency occurs after intramuscular administration of a water-miscible solution of retinyl palmitate (100,000 IU, or 30 mg retinol equivalent). Furthermore, we evaluated the effect of vitamin A injections on liver and blood concentrations in nine children with chronic cholestasis over a 1-y period. We conclude this treatment is efficient and is well tolerated.

Detection of staphylococcal enterotoxin B. A comparative study of ELISA and ELIFA systems.

The enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunofiltration assay (ELIFA) sandwich methods have been compared for their ability to detect staphylococcal enterotoxin B (SEB) in biological samples. Using the avidin-biotin amplification system in the ELISA, high sensitivity was observed and staphylococcal protein A interference was completely prevented. In contrast, ELIFA was less sensitive than ELISA, but very easy to perform, fast and simple to read routinely.

Validation of antibody-based recognition by piezoelectric transducers through electroacoustic admittance analysis.

The development of immunosensors based on piezoelectric transducers is widely investigated due to their attractive potentialities. The quartz crystal microbalance (QCM) may give a direct response signal which characterizes the binding event between a sensitive layer, immobilized onto the surface transducer, and the analysis to be detected. However, for small biomolecules, such as some antigens, it is quite difficult to obtain an observable signal. This is mainly due to the lack of sensitivity of the commonly used QCM (5 to 10 MHz quartz crystal). Moreover, the mass estimated with the QCM response through the Sauerbrey equation and the mass which can be measured thanks to other analytical techniques, in our case an enzymatic assay, are different: the deposited mass is generally overestimated by the QCM. To validate QCM mass measurements and, therefore antigens recognition, the interactions of acoustic shear waves with a biolayer were investigated during enzyme adsorption onto the microbalance gold electrode or during the antibody/antigen binding. Electroacoustic admittance was measured around the resonance frequency of a 27 MHz quartz resonator in parallel with microbalance measurements. The parameters which characterize the quartz microbalance equivalent circuit were compared with the classical microbalance frequency. The mass overestimation, given by the microbalance, could be explained either by modification of the rheological properties of the sensitive layers and/or by an inadequacy of the assay performed.

Study of immunoglobulin G thin layers obtained by the Langmuir-Blodgett method: application to immunosensors.

Nowadays, immunosensors play a leading part in the field of bioanalytical chemistry research. As with any biosensor, they need appropriate transducers and a suitable technique to immobilize the active biocomponents. In this study, two transduction modes were chosen: mass effects (quartz microbalance measurements) and geometric and dielectric effects (capacitance measurements). The Langmuir-Blodgett (LB) method appears to be quite suitable for generating biospecific surfaces. This work has focused on the detection of staphylococcal enterotoxin B, the corresponding antibody being immobilized at the surface of fatty acids by a variant of the LB method. The composition of the film and the nature of antibody-fatty acid interactions were studied by means of the two transducers mentioned above. FTIR (Fourier transform infra-red) spectroscopy and protein diagnostic assay. Influence of several parameters (pH, ionic strength, transfer pressure, antibody concentration in the subphase) was investigated. The immobilization rate reached its maximum when experimental conditions allowed optimal electrostatic interactions. In this case, the quartz crystal microbalance response, in air, reached 55 Hz per monolayer of immobilized immunoglobulin G and the equivalent capacitance variation, measured in liquid media, was around 300 pF cm-2. Activity of the biospecific LB films, when binding enterotoxin, was checked by the classical ELISA (enzyme immuno-linked assay) technique.

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis.

A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis.

Quantitation of polymerase chain reaction-amplified DNA fragments by capillary electrophoresis and laser-induced fluorescence detection.

Quantitation of DNA fragments amplified by polymerase chain reaction (PCR) is needed for the determination of target DNA in molecular biology. Capillary electrophoresis in entangled polymer solution coupled to laser-induced fluorescence detection was assessed as an alternative technique to conventional slab gel methods to monitor competitive PCR, which consists of amplifying an internal standard fragment under the same conditions as the target fragment. The fluorescence signal was generated either through end-labeling of the fragments using 5'-fluorescein-labeled primers or through intercalation of ethidium bromide along the double strand. It is shown that the more accurate and reliable results were obtained using this latter pathway.

[Vitamin A status in liver diseases in children. Study based on 79 hepatic biopsies]. / Réserves vitaminiques A dans les hépatopathies de l'enfant. Etude portant sur 79 biopsies hépatiques.

The authors studied the vitamin A status of 79 children, 71 of whom had liver disease. Plasma levels indicate low vitamin A concentration only when they are below 10 micrograms/dl. Study of liver concentration is currently the reference to appreciate vitamin A status. These concentrations are low in children with cholestasis. The lower the liver concentration, the higher the percentage of retinol in the liver. The percentage of the various retinyl esters is however independent of liver concentrations. The authors propose a model for the regulation of vitamin A metabolism within 4 pools.