Binding of Plant Polyphenols to Serum Albumin and LDL: Healthy

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Bioactive Constituents, Metabolites, and Functions

Binding of Plant Polyphenols to Serum Albumin and LDL: Healthy Implications for Heart Disease Dana M. Poloni, Olivier Dangles, and Joe A. Vinson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06674 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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Journal of Agricultural and Food Chemistry

Binding of Plant Polyphenols to Serum Albumin and LDL: Healthy Implications for Heart Disease

Dana M. Poloni†, Olivier Dangles‡ and Joe A. Vinson*†

†Department

of Chemistry, Loyola Science Center, University of Scranton, 925 Ridge Row, Scranton, PA 18510 USA ‡ Avignon University, INRA, UMR408 SQPOV, 84000, Avignon, France

______________________________________________________________________________ *Author for correspondence (email: [email protected]; fax (+)-1-570-941-7572 telephone: (+)-1-941-7551.

ACS Paragon Plus Environment


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ABSTRACT

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Cardiovascular disease (CVD) is the leading cause of death in industrialized nations. The

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initiating event in atherosclerosis or hardening of the arteries, is oxidation of low density

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lipoprotein (LDL). Binding with serum albumin and LDL of 41 polyphenols (major antioxidants

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in plant foods) constituting four classes of flavonoids, three types of phenolic acids and seven

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polyphenol conjugate metabolites was investigated indirectly by fluorescence quenching and

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directly by affinity separation/HPLC (four of the polyphenols). Stern -Volmer plots yielded K

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values for the two proteins. Albumin binding of the polyphenols was significantly stronger than

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LDL. K values were highly correlated with the lipophilicity of the polyphenols. The number of

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polyphenol molecules determined by quenching was ~1 for both proteins. Direct analysis

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yielded 2 to 13 molecules of polyphenols/LDL particle. Multiple substituent effects on binding

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were analyzed. Evidence was put forward that binding of polyphenols to these proteins is

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protective for CVD by multiple mechanisms.

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______________________________________________________________________________

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KEY WORDS: albumin; binding constant; cardiovascular disease; LDL; oxidation;

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polyphenols


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INTRODUCTION

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Cardiovascular disease (CVD) is the leading global cause of death, responsible for 17.3 million

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lives in 2013.1 Diet, which contributed to an estimated one in two heart disease deaths in the US

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in 2010, may also play an important beneficial role in decreasing oxidative stress and

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inflammation.2-4 The evidence for the effects of diet on CV outcomes mainly relies on

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observational data from epidemiology studies. The first large scale intervention studies studying

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plant foods' ingredients focused on the antioxidant vitamins C, E and β-carotene given as

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supplements.5.6 The results were disappointing in that these vitamins had no effect on risk

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factors in long-term trials. The first long-term intervention of a whole diet was the multicenter

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"Primary Prevention of Cardiovascular Disease with a Mediterranean Diet" PREDIMED trial.

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The study tested the hypothesis that the Mediterranean diet (MedDiet) is more effective in

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preventing CVD than a relatively low-fat diet using a very rigorous randomized-control

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methodology. They enrolled 7447 participants free of CVD at baseline but at a high risk for

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CVD. Subjects were divided into one of three groups received either 1 liter per week of extra-

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virgin olive oil or 30 g of mixed nuts or counseled to follow a low-fat diet.7 After 5 years the

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study was halted due to a there large and significant CVD risk reduction of 30% for both

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MedDiet groups compared to the low-fat control diet group. What in the MedDiet is responsible

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of the beneficial CVD effect? The major non-nutritive components of the diet are plant-derived

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phenolic compounds which are collectively known as polyphenols (PP). Their contribution in the

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average diet is about 1 g/day.8 This intake is much larger than the other dietary antioxidant

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vitamins. The PREDIMED study showed that the risk of CVD as well as diabetes and total

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mortality were significantly reduced with a diet rich in PP.9,10 In the very large Nurses' Health

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Study in the USA there was an association of flavonoid-rich foods and flavonoids (a large sub-



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class of polyphenols) with the risk of all-cause mortality.11 The MedDiet intervention study with

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olive and nuts was also applied to a female cohort (n=110). Urine lipid and DNA oxidation

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markers were significantly decreased relative to the low fat control diet.12 Total urinary

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polyphenols were inversely associated with obesity (CVD risk factor) in the PREDIMED

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study.13 Urine polyphenols were also inversely associated with decreased mortality in older

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adults in the 12 year follow-up of the Chianti study in Italy.14

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PP are an abundant and structurally diverse group of secondary plant metabolites. They

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can be classified as flavonoids and non-flavonoids. Among the flavonoids, various groups can be

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distinguished: flavonols, flavan-3-ols (monomeric and polymeric structures), flavones,

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isoflavones, flavanones, and anthocyanidins. Non-flavonoids include stilbenes, hydrolyzable

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tannins, lignans and phenolic acids. The latter is the major type of PP in fruits, vegetables and

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most beverages. The number one source of PP in the US diet is coffee15 and the major PP in

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coffee are phenolic acids; namely hydroxycinnamic acids. Grains and beer also contain

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primarily phenolic acids. Very little of the dietary PP are absorbed in the small intestine and a

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large proportion of the PP are partially metabolized in the colon by normal flora by esterification,

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hydrolysis of ester and glycosidic bonds, demethylation, dehydroxylation, and decarboxylation.16

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However for PP that are absorbed by the GI tract, extensive conjugation, primarily in the liver

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results in a variety of O-methylated, O-sulfated and O-glucuronated compounds. Thus, except

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for a few anomalies, PP exist primarily as metabolites within the body's circulatory system, cells,

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tissues and organs.

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Besides metabolism, interactions of PP with plasma proteins must be explored when examining transport, biological activity, delivery to the tissues and organs, and ultimate


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clearance from the body. Serum albumin (HSA) with a molecular mass of 66,500 is known as a

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transport protein and is the major protein in human blood (660 µM). LDL, although a minor

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plasma protein (~2 µM), plays a major role in atherogenesis. When LDL is oxidized it is taken

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up by with no feedback control by macrophages and converted into foam cells that are the

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precursors of atherosclerotic plaques. This process is the basis for the "oxidative theory of

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atherosclerosis" and initiated the intensive research of PP as antioxidants.17 However, the

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antioxidant activity cannot be the sole property of PP which determines their effects in vivo due

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to, a) the low concentration of PP (generally nM to µM) in the blood, tissues and organs, and b)

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the extensive metabolism which typically weakens their electron-donating (antioxidant)

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properties. Compared to the abundant literature on the binding of PP and both bovine and

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human serum albumin (BSA and HSA, respectively), the nature and binding of PP to LDL has

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not been studied previously. Thus we have investigated the PP-LDL binding using fluorescence

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quenching methods for a large number and variety of PP classes and compared LDL and BSA

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for their affinity for PP.

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MATERIALS AND METHODS

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Materials. All PP (except those generously gifted), chemicals, and affinity column

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resins were purchased from Sigma-Aldrich, St. Louis, MO. All water was ultrapure water from a

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Waters Reverse Osmosis Ultrapurifier. All aqueous solutions were run through a Chelex-100

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column to remove trace metal ions from the solutes that catalyze the oxidation of PP and

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proteins.



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Biologicals. Plasma was freshly prepared the same day as received from porcine blood

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stabilized with EDTA (Lampire Biological Laboratories, Coopersburg, PA) which was air

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shipped at 00C. Plasma was prepared by centrifugation for 7 minutes at 3300 RPM and 4°C.

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Plasma was aliquoted into 1.5 ml portions in 1.5 microcentrifuge tubes, bubbled with nitrogen,

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flash frozen with liquid nitrogen, and stored at -800C until use.

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LDL was prepared by a standard technique using 2-3 cm of 4% heparin agarose affinity

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columns.18 Multiple columns were prepared and upon collection of LDL eluate; it was pooled

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and N2 was bubbled through the solution. An aliquot was removed for protein measurement by

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Coomasie Blue reagent and the remaining LDL frozen at -80oC. Fractions containing LDL were

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only thawed once. Any unused material that day was discarded as refreezing and thawing

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resulted in oxidation of the LDL. In order to regenerate the columns, previously used columns

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were uncapped, and allowed to drain. The column was then washed with 3.0 mL of nanopure

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H2O, followed by 4 mL of urea (1M) in nanopure H2O, 1 mL of 1% (v/v) Triton X-100 in H2O,

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10 mL of H2O, and then 5 mL of phosphate buffered saline (PBS), followed by capping of the

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column at both ends, and storage at 40C until future use.

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Fluorescence Quenching. A PerkinElmer LS-45 Luminescence Spectrometer along with a Fisher Scientific temperature controlled water bath set at 25°C ± 1°C were used to

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determine fluorescence quenching. Excitation was set at 280 nm at and emission spectra were

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acquired in the wavelength range between 300 and 500 nm for tryptophan residues in BSA and

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LDL. A solution of BSA was prepared in PBS at 515 µM and refrigerated at 40C until used.

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LDL was thawed and diluted with PBS to give similar fluorescence as BSA, and thus

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presumably similar tryptophan concentration. The protein solution (2.7 ml) was put in a cuvette


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and 3 µL of 245 µM polyphenol in methanol was added (less than 1% methanol in the final

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solution), mixed by inversion and fluorescence measured after 5 minutes to allow for

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equilibration of binding. The addition of the PP standard to the protein was repeated 4 times with

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duplicate samples. For such low PP concentrations and low extinction coefficients, PP

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absorption at the protein's excitation and emission wavelength was negligible and no inner filter

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corrections were necessary.

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Calculations. We determined of protein-PP binding constants by 5 different methods;

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Stern-Volmer, Double-Logarithm, Scatchard, Quadratic, and Benesi-Hildebrand. Each method

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uses different assumptions to calculate the K values. The plots were compared with regards to

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correlation coefficient and precision of duplicate experiments. The double-logarithm method

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gave the best plots (as expected from a log-log plot) but the Stern-Volmer method gave the best

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precision. The most commonly used method in the literature, Stern-Volmer was used.

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Fluorescence quenching by PP can occur via two different mechanisms, static, which

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results from the formation of a ground state complex between the PP and the protein, or dynamic

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as a result of the collisional encounter between the PP-protein complex. These pathways are

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described in the Stern-Volmer equation: F0/F = 1 + KSV[free PP]. For dynamic quenching, KSV

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can be written as kqτ0 where kq is the quenching rate constant of the flurophore-quencher pair

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and τ0 is the lifetime of the fluorophore in the absence of the quencher PP, which is ~10-8s for a

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tryptophan residue.19 Statistics were performed with SigmaStat Version 3.01 software and

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significance was defined as p < 0.05. The octanol/water partition coefficient (P) is the

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equilibrium concentration ratio of non-dissociated compound partitioning between 2 immiscible

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phases; octanol and water. It is referred to as a measure of lipophilicity or hydrophobicity. For



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phenolic acids and sulfates which exist as ions, and glucuronides which are completely ionized

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to carboxyl anions at pH 7.4 in aqueous solutions, the distribution coefficient (D) was used,

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which is corrected for the presence of the anion(s) in water. Values for log P and log D and

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other theoretical properties such as calculated Hill affinity binding sites (n), polar surface area

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and number of hydrogen bond acceptors/donors for the PP were obtained from ChemSpider and

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secondarily from The Human Metabolome Database via the WorldWide Web.

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Determination of actual number of PP molecules bound to LDL. Under saturation

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conditions (6 µM) quercetin, chrysin, pelargonidin, or ferulic acid in methanol (maximum 1%)

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were added to a heparin agarose isolated LDL (70 µg/ml protein solution). LDL was previously

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diluted to 0.9% NaCl with PBS (pH 7.4) and nanopure water, and allowed to equilibrate for 30

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min at room temperature. The solution was then run through the heparin agarose column.

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Unbound PP came off immediately. LDL-bound PP were eluted with 2.5 ml of 2.7% NaCl and

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the PP concentration measured with HPLC.

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RESULTS AND DISCUSSION

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Lipoproteins. Pigs, due to their grain-based diet, have plasma lipids and lipoproteins

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that are lower in concentration than in human plasma. Pigs have an average cholesterol of 80

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mg/dL and an LDL of 47.5 mg/dL20 However the molecular weights and the composition of the

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lipoproteins are both very similar to those of humans.21 Pig plasma was used rather than human

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due to biological safety concerns. We used our standard published affinity column method18 to

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isolate lower density lipoproteins as it provides an inexpensive and quick protocol. The affinity

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column fraction contained LDL and VLDL. By weight pig plasma contains 93% LDL and 7%


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VLDL.22 The calculated LDL mole % was 98.9%. So for discussion purposes the affinity

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column isolated protein will be called LDL. By comparison human LDL is 95 mole % of LDL.23

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Polyphenols. In Figure 1 are shown the chemical structures of the PP arranged

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alphabetically. Most are phenolic acids and one compound flavone, displays a flavonoid

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skeleton without phenol groups. The Stern-Volmer binding constants K and the number (n) of

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PP bound to the tryptophan residue(s) of the two proteins are shown in Table 1 which is arranged

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according to PP classification and sub-classification.

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Nature of the Interaction. Assuming dynamic quenching, the kqs (quenching constants)

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of BSA with PP ranged from 2x1013 to 4x1014 M-1s-1 and for LDL 6x1013 to 3x1014 M-1s-1. For

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all PP the values of Kq are much greater than the 2x1010 M-1s-1 characteristic of a diffusion-

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controlled collisional rate constant.24 Thus the assumption of dynamic quenching does not hold

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and the interaction of PP with both BSA and LDL appears to be static in nature.

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Binding Constants (Table 1). The average % standard deviation was 4.0% for KBSA and

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4.7% for KLDL. It is apparent that the KBSA is larger than the KLDL. Indeed for the 41 PP

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compounds the average KBSA was 2.49 ± 1.56 x105 and for KLDL was 1.75 ± 0.74 x105,

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respectively, p < 0.001. There was also a highly significant Pearson correlation between the two

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K values, r2 = 0.1018 with the large outlier ellagic acid, and r2 = 0.6545 without it, p

Binding of Plant Polyphenols to Serum Albumin and LDL: Healthy

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