Le dosage de vitamines du groupe B (acide pantothénique et cobalamines) dans les aliments après isolement chromatographique et détection fluorimétrique

La méthode mise au point pour le dosage de l'acide pantothénique libre consiste en une purification des échantillons par passages successifs sur des cartouches échangeuses d'anions et de cations, puis en un isolement chromatographique en phase inverse suivi d'une dérivation post-colonne de l'acide pantothénique en isoindole fluorescent (réaction de la -alanine, obtenue par hydrolyse alcaline à chaud de l'acide pantothénique, avec l'ortho-phthaldialdéhyde en présence d'acide mercaptopropionique). Une hydrolyse enzymatique (pepsine, puis pantéthéinase et phosphatase alcaline), effectuée avant l'étape de purification, permet de libérer l'acide pantothénique lié et ainsi d'obtenir la teneur en vitamine B5 totale dans les aliments.En ce qui concerne la vitamine B12 libre, le protocole proposé comporte une purification très spécifique par passage sur colonne d'immunoaffinité, avec concentration de l'échantillon, puis une transformation de la vitamine en ribazole (hydrolyse alcaline à chaud, puis traitement par une phosphatase alcaline). Le ribazole obtenu est par la suite dosé par fluorimétrie après un isolement chromatographique en phase inverse. Un traitement par la pepsine permet de libérer les différentes formes de la vitamine B12 liées à des protéines et ainsi de pouvoir estimer la teneur en vitamine B12 totale dans les aliments.Ces méthodes, sensibles et répétables, donnent de bons taux de recouvrement. Etant données leur faible limite de détection (0,65 g.g-1 pour la vitamine B5 et 1 ng.g-1 pour la vitamine B12) et la bonne résolution des pics chromatographiques (respectivement d'acide pantothénique et de ribazole), elles devraient pouvoir très vraisemblablement être utilisées pour la détermination de ces vitamines dans n'importe quel échantillon alimentaire.The developped method for the free pantothenic acid consists of a purification of the samples by successive passages through anion and cation exchange cartridges, then an isolation by reversed-phase chromatography followed by a post-column derivatization of pantothenic acid as a fluorescent isoindole (reaction of -alanin, formed by hot alkaline hydrolysis of pantothenic acid, with ortho-phthaldialdehyde in the presence of mercaptopropionic acid). An extraction procedure in two steps, first treatment by pepsin (hydrolysis of forms bound to proteins), then treatment by alkaline phosphatase and pantetheinase (hydrolysis of coenzyme A), allows to release the total vitamin B5 in the pantothenic acid form.As for free vitamin B12, the proposed protocol contains a very specific purification by passage through an immunoaffinity column, with concentration of the extract, then a transformation of the vitamin into ribazole (obtained by hot alkaline hydrolysis followed by a treatment by alkaline phosphatase). The fluorescent compound is then analysed by fluorimetry after a separation by reversed-phase chromatography. An enzymatic hydrolysis by pepsin allows to release the different forms of vitamin B12 bound to proteins and thus to obtain an estimation of the total vitamin B12 amount.These sensitive and repetable methods give good recovery rates. Owing to their low detection limit (0,65 g.g-1 for the vitamin B5 and 1 ng.g-1 for the vitamine B12) and the good resolution of the peaks (respectively of pantothenic acid and of ribazole), they could most probably be applied to the determination of these vitamins in any foodstuff.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Re-emergence of papulonodular napkin dermatitis with use of reusable diapers: report of 5 cases

International audienceThe use of reusable diapers, often made of cotton and bamboo material, is becoming more widespread in France with the "eco" fashion, as they are considered more natural, ecologic and economic. We report 5 cases of papulonodular lesions in convex skin areas associated with the use of these diapers in infants. One case was typical Sevestre and Jacquet erosive dermatitis. The 4 others presented skin-coloured umbilicated papules or nodules with slight or no erythema and could be considered early-stage Sevestre and Jacquet erosive dermatitis, granuloma gluteale (1 case) or pseudo-verrucous papules. These 3 diagnoses probably belong to the same disease spectrum, proposed as "irritant napkin papulonodules". Napkin lesions occurred subsequent to 1) in all cases, use of reusable diapers, which are probably less absorbent than disposable diapers; 2) in 2 cases, insufficient food, which was responsible for lack of weight gain and delayed healing; and 3) in 1 case, diarrhea, which worsened the moisture. We alert physicians to possible papulonodular napkin dermatitis in infants wearing reusable diapers

Arginine reduces bacterial invasion in rats with head injury

International audienceOBJECTIVES:The benefit of arginine in intensive care unit patients with severe sepsis is still controversial. An excessive supply of arginine could lead to an overproduction of nitric oxide and could be responsible for septic shock and multiorgan failure. However, this claim is not supported by any experimental or clinical data. We set out to determine whether an enteral supply of arginine would modulate bacterial invasion in rats with head injury.METHODS:Male Sprague-Dawley rats with head injury were randomized into two groups. Group 1 included rats with head injury fed a standard enteral nutrition (Sondalis HP, n = 10) and group 2 included rats with head injury fed the standard enteral nutrition plus arginine (4 g/kg/d, n = 11). Two days after head injury, the rats received a single enteral bolus of luminescent Escherichia coli Xen 14. Bacterial proliferation was evaluated in vivo at time + 2 hrs and time + 6 hrs after E. coli challenge. Four days after head injury, blood was sampled for arginine and fibrinogen assay. Muscles, intestine, spleen, and thymus were removed and weighed.RESULTS:There was no mortality in either group. The luminescence signal was similar in the two groups at time +2 hrs (group 1: 414 [5-823] vs. group 2: 496 [0.1-993] (median value[min-max]; not significant) and was significantly lower at time +6 hrs in group 2 (group 1: 71 [0-142] vs. group 2: 8.5 [0-17]; p = .026). Arginine treatment did not improve any nutritional parameters.CONCLUSIONS:Arginine was not responsible for mortality in rats with head injury with infectious complications and reduced the intensity of bacterial invasion

Mutations in NFKB2 and potential genetic heterogeneity in patients with DAVID syndrome, having variable endocrine and immune deficiencies.

International audienceDAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated. We ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies. Mutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC). We confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function