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Technical Briefs |
1
Departament de Bromatologia i Nutrició, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain;
2
Unitat de Lipids i Epidemiologia Cardiovascular, Institut Municipal d'Investigació Médica (IMIM), Carrer Doctor Aiguader, 80, 08003 Barcelona, Spain.;
3
Laboratori de Referència de Catalunya, 8027 Barcelona, Spain;
a author for
correspondence: fax 34-93-2213237, e-mail mcovas{at}imim.es
There is growing interest in the role of phenolic compounds in the diet as antioxidants. Epidemiological studies support a relationship between the consumption of phenolic rich food products (1)(2) and a low incidence of coronary heart disease. Strong evidence exists that oxidation of LDL lipids is a risk factor for atherosclerosis and coronary heart disease (3). Oxidation of LDL appears to occur predominantly in arterial intima in microdomains sequestered from antioxidants of plasma (4). In this situation, phenolics that bind LDL are good candidates for preventing lipid peroxidation and atherosclerotic processes. The reduction of LDL oxidation observed after consumption of foods rich in phenolic compounds (5)(6) provides indirect evidence of a binding of phenols to LDL. However and to our knowledge, the binding of individual phenolic compounds to LDL has not yet been demonstrated.
Using a solid-phase extraction with HPLC-diode array detection (DAD), we have identified and quantified some phenolic compounds in LDL isolated after long-term spins to ensure the purity of the lipoprotein. Blood samples from 10 male healthy volunteers on nonsupplemented diets were obtained by venipuncture and collected in EDTA-containing tubes after an overnight fast. The study was done in accordance with the Helsinki Declaration of 1975, as revised in 1989. The protocol was approved by a local ethics committee. Plasma was pooled, and LDL was isolated by a two-step sequential flotation ultracentrifugation. Isotonic saline (1 mL; d = 1.006 kg/L) containing 1.091 mmol/L EDTA was layered on top of plasma (2 mL) in centrifuge tubes (Beckman polycarbonate tubes, total volume, 5 mL; Beckman). The tubes were centrifuged in a Beckman XL-70 ultracentrifuge with a Beckman 50.4 rotor at 227 000g for 18 h at 4 °C. The VLDL fraction was separated by aspiration. The plasma (3 mL) was deposited in a centrifuge tube (Kontron polyallomer tubes, total volume, of 13 mL; Kontron) containing 0.1155 g of KBr (Sigma). Isotonic saline (3 mL; d = 1.21 kg/L) containing 1.091 mmol/L EDTA and 2.704 mol/L KBr was layered on top of the plasma. Tubes were centrifuged in a Kontron 41.14 rotor at 215 000g for 20 h at 4 °C. The LDL-containing middle layer was aspirated. Protein content was determined by a red pyrogallol method (Sigma).
Acidulated LDL (1 mL of LDL plus 20 µL of concentrated phosphoric acid) was applied to a Waters OasisTM HLB extraction cartridge and washed with water (1 mL) and 50 mL/L methanol in water (1 mL). The phenols were eluted with methanol, and the methanolic extract was evaporated under a nitrogen stream. The residue was dissolved in mobile phase and analyzed by HPLC-DAD. Briefly, the eluted fraction (100 µL) was injected in a 1050 Hewlett-Packard gradient liquid chromatograph (Hewlett Packard) equipped with an automatic injector. A Nucleosil (Tracer) C18 120 (25 x 0.4 cm, 5 µm) reversed-phase column was used with a precolumn of the same material, both columns set at 40 °C. The flow rate was 1.5 mL/min. The HPLC conditions were similar to those described previously (7); however the elution profile was slightly modified as follows: 0 min, 100% A, 0% B; 5 min, 98% A, 2% B; 10 min, 96% A, 4% B; 15 min, 90% A, 10% B; 26 min, 81% A, 19% B; 26.5 min, 79.5% A, 20.5% B; 35 min, 70% A, 30% B; 40 min, 60% A, 40% B; 45 min, 0% A, 100% B, where solvent A was acidulated water containing 27 mL/L glacial acetic acid, and solvent B was 200 mL/L phase A and 800 mL/L acetonitrile. Detection was performed using a Hewlett Packard 1050M diode array ultraviolet-visible detector coupled to a Hewlett Packard Chem Station. The chromatogram was monitored at 280, 320, and 365 nm. To study the recovery, known amounts of rutin (270 ng) were added to the LDL, and then the LDL samples were acidulated and extracted with the same procedure described above.
The results obtained at 280 nm are shown in Fig. 1
. Two compounds were identified by retention times and spectral
properties as rutin (peak 2) and quercetin-3-glucuronide (peak 3).
Three major peaks had spectra consistent with those of flavonoids
(peaks 1, 4, and 5), but all remain unidentified. Phenolic compounds
with flavonoid-like spectra were present in the highest concentrations
in LDL (16.27, 0.92, and 5.37 nmol catequin equivalents/mg LDL protein
for peaks 1, 4 and 5, respectively). The concentrations of rutin and
quercetin-3-glucuronide were 0.093 nmol/mg LDL protein and 0.073 nmol
rutin equivalents/mg LDL protein, respectively. The analytical
within-run imprecision (n = 10), expressed as the CV, ranged from
4.2% for peak 1 to 6.3% for rutin. The analytical between-run
imprecision (n = 10), expressed as the CV, ranged from 4.6% for
peak 1 to 13% for rutin. The recovery obtained (n = 6) was 80.6%
± 6.7% (mean ± SD).
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Rutin, quercetin, and flavonoids are phenolics present in fruits, vegetables, spices, olive oil, and red wine (5)(6)(7)(8)(9). Quercetin-rich foods such as onions or red wine have been shown to delay the susceptibility of LDL to oxidation after their supplementation in in vivo human studies (5)(6). Rutin and quercetin have been shown to delay the susceptibility of VLDL plus LDL to oxidation in in vitro studies after their addition to the isolated lipoproteins (10). Increased total phenol content of LDL after ingestion of red wine, as measured by the Folin-Ciocalteau method, has been reported (6). This method, however, is sensitive to nearly all oxidizable compounds, and the development of a reliable HPLC method to detect and identify phenolic compounds in LDL is needed (6).
Our results provide evidence that several dietary phenolic compounds
are bound to LDL lipoproteins. The binding of these phenolic compounds
to LDL supports the role of phenolics in the protection of the
autocatalytic chain reaction of fatty acid peroxidation in LDL directly
and/or by preservation of other chain-breaking antioxidants such as
-tocopherol (9). Phenolics bound to LDL were found in
nonsupplemented individuals, and thus routine diets already appear to
achieve this binding. HPLC-DAD with previous solid-phase extraction can
potentially be used to evaluate the relationship between the binding of
phenolic compounds to LDL and their biological activity as antioxidants
in supplementation studies with dietary or nutraceutical foods. The
fact that these phenolic compounds remain associated to LDL after
long-term spins could suggest a strong binding with LDL. However,
further studies are required to measure and compare the in vivo and in
vitro kinetics of association and release, to see whether there is any
process that actively partitions these substances into LDL. Our results
provide further evidence that dietary phenolics are likely to protect
LDL lipoproteins from oxidation in the subendothelial spaces of the
arterial intima and thereby delay the onset or progression of
atherosclerotic processes.
In conclusion, through the integration of a solid-phase extraction system with HPLC-DAD, we have demonstrated the presence of individual dietary phenolic compounds bound to human LDL particles in nonsupplemented individuals. This method provides a simple, rapid, and accurate identification and quantification of phenolic compounds present in LDL.
Acknowledgments
This work was supported by Grant ALI97-1607-CO2-01 from Comisión Interministerial de Ciencia y Tecnología and by FPI Grant 98/9562 from Fondo de Investigación Sanitaria. We thank A. Waterhouse from the University of California at Davis for reviewing the manuscript.
References
The following articles in journals at HighWire Press have cited this article:
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  F. Natella, M. Nardini, F. Belelli, and C. Scaccini Coffee drinking induces incorporation of phenolic acids into LDL and increases the resistance of LDL to ex vivo oxidation in humans Am. J. Clinical Nutrition, September 1, 2007; 86(3): 604 - 609. [Abstract] [Full Text] [PDF]
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 S. Benito, S. Buxaderas, and M. T. Mitjavila Flavonoid metabolites and susceptibility of rat lipoproteins to oxidation Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2819 - H2824. [Abstract] [Full Text] [PDF]
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 R. M. Lamuela-Raventos, C. Andres-Lacueva, J. Permanyer, and M. Izquierdo-Pulido More Antioxidants in Cocoa J. Nutr., March 1, 2001; 131(3): 834 - 834. [Full Text]
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