title: When cholesterol is not cholesterol: a note on the enzymatic determination of its concentration in model systems containing vegetable extracts.
abstract: BACKGROUND
  Experimental evidences demonstrate that vegetable derived extracts inhibit cholesterol absorption in the gastrointestinal tract. To further explore the mechanisms behind, we modeled duodenal contents with several vegetable extracts.
  
  
  RESULTS
  By employing a widely used cholesterol quantification method based on a cholesterol oxidase-peroxidase coupled reaction we analyzed the effects on cholesterol partition. Evidenced interferences were analyzed by studying specific and unspecific inhibitors of cholesterol oxidase-peroxidase coupled reaction. Cholesterol was also quantified by LC/MS. We found a significant interference of diverse (cocoa and tea-derived) extracts over this method. The interference was strongly dependent on model matrix: while as in phosphate buffered saline, the development of unspecific fluorescence was inhibitable by catalase (but not by heat denaturation), suggesting vegetable extract derived H(2)O(2) production, in bile-containing model systems, this interference also comprised cholesterol-oxidase inhibition. Several strategies, such as cholesterol standard addition and use of suitable blanks containing vegetable extracts were tested. When those failed, the use of a mass-spectrometry based chromatographic assay allowed quantification of cholesterol in models of duodenal contents in the presence of vegetable extracts.
  
  
  CONCLUSIONS
  We propose that the use of cholesterol-oxidase and/or peroxidase based systems for cholesterol analyses in foodstuffs should be accurately monitored, as important interferences in all the components of the enzymatic chain were evident. The use of adequate controls, standard addition and finally, chromatographic analyses solve these issues.
authors.affiliation: Institut de Recerca Biomèdica de Lleida-Universitat de Lleida-Parc Científic i Agroalimentari Tecnològic de Lleida, Spain.
authors.forename: Mariona
authors.initials: M
authors.lastname: Jové
authors.forename: José C E
authors.initials: JC
authors.lastname: Serrano
authors.forename: Maria Josep
authors.initials: MJ
authors.lastname: Bellmunt
authors.forename: Anna
authors.initials: A
authors.lastname: Cassanyé
authors.forename: Neus
authors.initials: N
authors.lastname: Anglès
authors.forename: Jordi
authors.initials: J
authors.lastname: Reguant
authors.forename: José R
authors.initials: JR
authors.lastname: Morelló
authors.forename: Reinald
authors.initials: R
authors.lastname: Pamplona
authors.forename: Manuel
authors.initials: M
authors.lastname: Portero-Otín
mesh_terms: D002784:Cholesterol / Q000032:analysis*; D002789:Cholesterol Oxidase / Q000378:metabolism; D005504:Food Analysis / Q000379:methods* / Q000592:standards; D007408:Intestinal Absorption; D013058:Mass Spectrometry; D010936:Plant Extracts / Q000737:chemistry; D014675:Vegetables / Q000737:chemistry*
chemical_list: D010936:Plant Extracts; D002784:Cholesterol; D002789:Cholesterol Oxidase