Direct capillary electrophoretic detection of carbohydrate-deficient transferrin in neat serum

Abstract

Transferrin, an iron transport protein found in serum and cerebrospinal fluid, is known to be microheterogeneous with respect to its carbohydrate and sialic acid content. The forms of transferrin deficient in sialic acid and/or carbohydrate, termed carbohydratedeficient transferrin (CDT), have been of clinical interest for almost two decades as a result of the initial finding that elevated CDT concentrations are associated with chronic, excessive alcohol abuse. We demonstrate the utility of capillary electrophoresis for examining the CDT sialoform profile via the direct electrophoresis of serum. The need for negligible preelectrophoresis sample preparation and absence of postelectrophoresis processing dramatically decreases analysis time compared to slab gel-based separations. Using a fluorocarbon-coated capillary containing a hydroxyethyl cellulose/ borate buffer, the high resolution separation of serum components is effected in less than 30 min. Under these conditions, the beta region proteins (including transferrin) are well resolved from the alpha-2 and gamma zone proteins in a window where the individual transferrin sialoforms can be detected. The usefulness of this method is demonstrated with the electrophoresis of serum from subjects known to be either nonalcoholic and alcoholic.Abbreviations: CDT, carbohydrate deficient transferrin; HEC, hydroxyethyl cellulose; If, transferrin 2376

Capillary electrophoresis of proteins 1999-2001

“…CE can detect chronic or repeated alcohol abuse by analyzing carbohydrate-deficient transferrin in human serum [43,46,148]. The main transferrin isoform in blood of healthy subjects contains four sialic acids, while in subjects who abuse alcohol the sialic acid content of transferrin is reduced.…”

“…It is less stable at low ionic strength (50.003), in the presence of SDS and after an application of a hydrophobic analyte such as polystyrene carbonate [40]. EOF and adsorption of proteins to the capillary wall can be manipulated by other dynamic wall coatings as published during the last two years: fluorosurfactants [42,43], polyvinyl alcohol (PVA) [44], tentacle-like oligourethanes [45], putrescine [46], Pluronic liquid crystals [47,48], sulfonated polymers [49], Polybrene [50], linear hydrocarbons and ethylbenzene [51], glycerylpropyltrimethoxysilane [52].…”

On-line capillary isoelectric focusing–mass spectrometry for quantitative analysis of peptides and proteins

“…Therefore, in order to cancel the charge distribution caused by the iron load, saturation with ferric ion is necessary for cIEF analysis of transferrin. Ferric citrate (500 M) was added to human apo transferrin (2 g/L) and left for 15 min to saturate its iron-binding site [13]. Then, the iron-saturated tetrasialo-transferrin was subjected to anion exchange chromatography according to a previous report using a Mono-Q HR 5/5 column (Amersham, Uppsala, Sweden) [14].…”

La transferrine hyposialylée et le % CDT. Mesure par électrophorèse capillaire avec le coffret CEofix CDT (ANALIS) chez des sujets consommateurs modérés

“…Dans leur revue, Petersen et al reprennent le cheminement des développements de l'EC pour ce dosage [17]. Sans oublier la phase préanalytique (l'échantillon), peu à peu les modifications ont porté sur les longueurs, les diamètres et revêtements des capillaires, sur les tampons d'électrophorèse, et sur les temps de voltage [8,13,14,17,21,24]. Cependant l'asialotransferrine n'était pas détectée par exemple pour un taux de CDT à 61 U/I alors que l'IEF la mettait en évidence pour des échantillons plus faibles en CDT [cité dans 17].…”