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.

Figure 1

Enzymatic method used for quantification of cholesterol based on cholesterol oxidase-peroxidase coupled reaction. Amplex Red^®: 10-acetyl-3, 7-dihidroxyphenoxazine.

Figure 2

Vegetable extracts interfere with cholesterol analyses in phosphate buffered saline based systems using cholesterol oxidase peroxidase-coupled reactions. Both cocoa (a) and tea-derived (b) extracts showed, in a dose dependent fashion, reactivity in systems used for cholesterol analyses using cholesterol oxidase-peroxidase coupled reactions. Cholesterol independent fluorescence was defined as fluorescence arising from the complete system without the enzymes cholesterol oxidase and cholesterol esterase. Cholesterol content was obtained by subtracting cholesterol independent fluorescence from the complete system. c. Interference was not sensible to heat-denaturation (96°C, 3 min) or metal chelation (EDTA and DTPAC 1 mM), but to catalase (7 mg/ml). Linearity of a cholesterol standard curve was sensible to the presence of either cocoa (d) and tea-derived (e) extracts diluted in phosphate buffered saline in different concentrations (from 0 to 20 mg/ml). Values are means ± SEM. Statistical analysis was done by ANOVA followed by Tukey HSD post hoc test (* p < 0.05).

Figure 3

Vegetable extracts interfere with cholesterol analyses in in the presence of porcine bile based systems using cholesterol oxidase peroxidase-coupled reactions. Both cocoa (a) and tea-derived (b) extracts inhibited, in a dose dependent fashion, bile-derived cholesterol reactivity in systems used for cholesterol analyses using cholesterol oxidase-peroxidase coupled reactions. Cholesterol-dependent and independent fluorescences were as defined in figure 2. Linearity of a cholesterol standard curve was sensible to the presence of either cocoa (c) and tea-derived (d) extracts diluted in bile in different concentrations (from 0 to 20 mg/ml). (e) Vegetable-extracts (10 mg/mL) inhibited cholesterol oxidase activity in the presence of bile, based on the cholesterol chromatographic assay. Values are means ± SEM. Statistical analysis was done by ANOVA followed by Tukey HSD post hoc test (* p < 0.05).

Figure 4

Vegetable extract impairs cholesterol bioavailability in a model of duodenal content, but this impairment could be overestimated (tea) or underestimated (cocoa) by enzyme-based cholesterol assays. Values are means ± SEM. Statistical analysis was done by ANOVA followed by Tukey HSD post hoc test (* p < 0.05).