Increased Bioavailability of Ubiquinol Compared to That of

Jun 30, 2014 - Solubilized ubiquinol and ubiquinone were added to yogurt and ... significantly greater during digestion of yogurt enriched with ubiqui...
2 downloads 0 Views 1016KB Size
Download PDF
Article pubs.acs.org/JAFC

Increased Bioavailability of Ubiquinol Compared to That of Ubiquinone Is Due to More Efficient Micellarization during Digestion and Greater GSH-Dependent Uptake and Basolateral Secretion by Caco‑2 Cells Mark L. Failla,*,† Chureeporn Chitchumroonchokchai,† and Fumiki Aoki§ †

Human Nutrition Program, The Ohio State University, Columbus, Ohio 43210, United States Kaneka North America LLC, 6161 Underwood Road, Pasadena, Texas 77507, United States

§

ABSTRACT: The oral bioavailability of ubiquinol recently has been reported to be greater than that of ubiquinone in healthy adults. The basis for this influence of redox state of coenzyme Q (CoQ) on bioavailability has been investigated using the coupled in vitro digestion/Caco-2 cell model. Solubilized ubiquinol and ubiquinone were added to yogurt and subjected to simulated gastric and small intestinal digestion. Partitioning of CoQ in mixed micelles during small intestinal digestion was significantly greater during digestion of yogurt enriched with ubiquinol. Similarly, apical uptake from mixed micelles and transepithelial transport of CoQ by Caco-2 cells were significantly greater after digestion of the ubiquinol-rich yogurt compared to digested ubiquinone-rich yogurt. Reduction of cellular GSH significantly decreased cell uptake and basolateral secretion of both ubiquinol and ubiquinone, although the adverse impact was much greater for ubiquinol. These data suggest that the enhanced bioaccessibility and bioavailability of ubiquinol compared to ubiquinone results from reduced coenzyme being more efficiently incorporated into mixed micelles during digestion and its greater uptake and basolateral secretion in a glutathionedependent mechanism. KEYWORDS: ubiquinol, ubiquinone, coenzyme Q, bioaccessibility, bioavailability, Caco-2 cells

■

INTRODUCTION Coenzyme Q10 (CoQ10) is a lipophilic compound composed of a quinoid moiety and a hydrophobic tail of 10 isoprenoid units in humans. The compound exists in both the reduced (CoQH) and oxidized (CoQ10) states, which are referred to as ubiquinol and ubiquinone, respectively. CoQ10 is predominantly localized within the inner mitochondrial membrane, where it has an essential role in the electron transport chain, accepting two electrons from complexes I and II. CoQ also is present in other cellular membranes, where the reduced moiety functions as an antioxidant. In this role, CoQ prevents peroxidative damage to membrane phospholipids and free radical induced oxidative damage of proteins and mitochondrial DNA and mediates the regeneration of α-tocopherol.1 This antioxidant activity has promoted the use of CoQ as a supplement to prevent and attenuate the pathology of cardiovascular1−4 and neurodegenerative diseases,5,6 improve exercise performance,7 and reduce exercise-induced muscular injury.8 To mediate its health-promoting effects, orally administered CoQ must be absorbed and delivered to target tissues; that is, it must be bioavailable. The bioavailability of ingested compounds is markedly influenced by their physicochemical characteristics and the nature of the delivery system.9 As for other lipophilic dietary compounds and drugs, the absorption of CoQ requires solubilization, partitioning in mixed micelles during small intestinal digestion, uptake by absorptive epithelial cells, and incorporation into lipoproteins for secretion into the lymph.10 Ubiquinone, the most common form in CoQ supplements, has very low bioavailability because of its considerable lipophilicity, crystalline state, and high molecular weight (MW 863).11−13 © 2014 American Chemical Society

The activity of P-glycoprotein (Pgp) located in the brush border membrane of absorptive epithelial cells also decreases bioavailability as it effluxes newly acquired CoQ10 into the small intestinal lumen.14,15 Various formulations of CoQ have been developed during the past decade to enhance its solubility and absorption. Improved bioavailability has been demonstrated for formulations containing CoQ in water-soluble inclusion complexes with cyclodextrins, oily mixtures, phospholipids, self-emulsifying delivery systems, and nanoparticles.8,16 It has been shown that the oxidation state of CoQ also influences bioavailability as the absorption of ubiquinol exceeded that of ubiquinone.11−13 The enhanced bioavailability of ubiquinol has been associated with greater anti-inflammatory activity in human monocyte-like cells,17 attenuated UVAassociated mitochondrial damage in human dermal fibroblasts,18 decelerated senescence of SAMP1 mice,19,20 protection against renal ischemia and reperfusion injury in rats,21 improved ventricular function in patients with advanced congestive heart failure,22 and greater glycemic control in type 2 diabetics.23 Insights regarding the basis for the greater absorption of ubiquinol than of ubiquinone are limited. The first objective of the present study was to compare the stability and partitioning of ubiquinone and ubiquinol in mixed micelles during simulated digestion of yogurt supplemented with the coenzyme solubilized in polyethylene-60 hydrogenated castor Received: Revised: Accepted: Published: 7174

April 15, 2014 June 29, 2014 June 30, 2014 June 30, 2014 dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

containing 2 mL of 5% potassium hexacyanoferrate(III) in PBS and mixed. Sealed tubes were incubated in a shaking water bath for 10 min at 37 °C at 85 rpm before extraction as described below. Replicate aliquots of starting material, chyme, and its aqueous fraction were transferred to empty glass tubes and immediately extracted. Extracts were analyzed by HPLC with a diode array detector. Ubiquinol content was calculated by the difference in the amount of ubiquinone in tubes lacking potassium hexacyanoferrate and total CoQ in tubes containing potassium hexacyanoferrate. Recovery of CoQ after digestion was calculated as nanomoles of total CoQ in chyme divided by total CoQ in the starting material multiplied by 100%. The efficiency of micellarization was calculated as total CoQ in the aqueous fraction of chyme divided by nanomoles of total CoQ in yogurt matrix multiplied by 100%. CoQ Uptake and Transport by Caco-2 Cells. Caco-2 HTB37 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) at passage 19 and maintained as previously described.28 Cells (2.5 × 105) were seeded on membrane insets (3.0 μm pore diameter; EMD Millipore, Billerica, MA, USA) in six-well dishes. Washed monolayers (passages 20−26) were used for experiments 21−24 days after monolayers became confluent. Filtered aqueous fraction of chyme generated during in vitro digestion of yogurt containing ubiquinone or ubiquinol solubilized in HCO60 was diluted 1:3 with DMEM (pH 6.5) containing 500 μmol/ L phenol red along with oleate (1 mmol/L): taurocholate (0.5 mmol/ L) micelles to induce chylomicron assembly and secretion.29,30 Phenol red-free DMEM (3 mL) containing 1% FBS and 1% NEAA was added to the basolateral compartment. Cultures were incubated for 4 h before apical and basolateral media were collected. Monolayers were washed once with cold PBS containing 2 g/L albumin followed by two additional washes with cold PBS. Cell pellets were collected in cold PBS and centrifuged at 400 × g for 5 min at 4 °C. Cell pellets and basolateral medium were stored frozen under nitrogen gas at −80 °C overnight. CoQ uptake was calculated as follows: [(ubiquinone + ubiquinol) in cells of CoQ supplemented cultures − (endogenous ubiquinone + ubiquinol)] + [CoQ] in basolateral compartment. Endogenous CoQ content in monolayers of Caco-2 cells grown on membrane inserts to 21 days postconfluency was 220 ± 12 pmol/mg protein (n = 10) with ubiquinol representing 65 ± 2.0% of total CoQ (n = 10). To determine possible differences in retrotransport of newly acquired CoQ from medium prepared after digestion of yogurt with ubiquinone or ubiquinol, 100 μmol/L verapamil was added to both apical and basolateral medium to block Pgp-mediated efflux into the apical compartment. The cell monolayer was not adversely affected during the 4 h of incubation with this concentration of verapamil as assessed by phenol red flux from the apical to basolateral compartment and cell protein content per membrane insert. The effects of altered GSH status and inhibition of Trx reductase activity in Caco-2 cells on uptake and transport of CoQ across the monolayer of Caco-2 cells also were examined to determine the impact of redox imbalance on CoQ flux. For these experiments, cells were treated with inhibitors as described below. Control and treated cells were incubated in DMEM with 500 μmol/L phenol red and Tween-40 micelles containing ubiquinone (80 ± 5 μmol/L) or ubiquinol (65 ± 3 μmol/L) and 10 μmol/L α-tocopherol were prepared as described elsewhere.28 Apical medium also contained oleate (1.0 mmol/L), sodium taurocholate (0.5 mmol/L), and glycerol (50 μmol/L) to induce chylomicron assembly and secretion into the basolateral compartment.29 Phenol red-free DMEM containing 1% FBS and 1% NEAA was added to the basolateral compartment. After 4 h, basolateral medium and washed monolayers were collected and extracted immediately for CoQ analysis. Alteration of Glutathione Status of Caco-2 Cells. The protocol described by Noda et al.31 was used to decrease the ratio of GSH to GSSG in cells to induce cell redox imbalance without oxidative stress. This involves pretreatment of monolayers with apical medium containing 5 mmol/L DL-buthioninesulfoximine (BSO) for 12.5 h and 30 μmol/L 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) for 30 min. Then fresh DMEM containing BSO, BCNU, diamide (30 μmol/ L), phenol red (500 μmol/L), Tween-40 micelles containing either

oil (HCO60). Uptake of CoQ in mixed micelles generated during digestion across the brush border membrane (i.e., apical uptake) by Caco-2 human intestinal cells and the extent of transport of CoQ across the monolayer into the basolateral compartment (i.e., basolateral secretion) also were determined. This coupled model has been used previously to compare the micellarization and cellular uptake of ubiquinone from various formulations.24 The bioaccessibility of ubiquinone was greater for supplements containing solubilized CoQ. CoQ exists primarily as ubiquinol in extra-mitochondrial cellular compartments and in plasma. Reduction of ubiquinone to ubiquinol has been proposed to be dependent on the activities of a number of enzymes located in various cellular compartments. These include glutathione (GSH) reductase,25 thioredoxin (Trx) reductase,26 and NAD(P)H quinone oxidoreductase 1 (NQO1, also commonly referred to as DTdiaphorase)27 among others. As these enzymes are components of antioxidant systems, it is likely that a balanced redox status is essential for maintaining the elevated ratio of ubiquinol to ubiquinone. The second objective of this study was to examine the effect of disruption of the redox imbalance on the apical uptake and basolateral secretion of CoQ by Caco-2 cells. Cellular redox balance was altered by either decreasing the cellular concentration of GSH or inhibiting the activity of thioredoxin (Trx) reductase in Caco-2 cells.

■

MATERIALS AND METHODS

Reagents. Ubiquinone, ubiquinol, and polyethylene glycol 60linked hydrogenated castor oil (HCO60) were provided by Kaneka Corp., Japan. Fetal bovine serum, fungizone, penicillin−streptomycin, L-glutamine, and nonessential amino acids were purchased from Life Technologies Corp. (Grand Island, NY, USA). Bile extract, pepsin, lipase, and pancreatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were purchased from ThermoFisher Scientific (Pittsburgh, PA, USA) unless otherwise indicated. Preparation of HCO60 Enriched with CoQ. Briefly, 1 g of HCO60 in a 50 mL glass tube was melted at 60 °C in a water bath before the addition of 0.2 g of ubiquinone or ubiquinol. After mixing well and incubating for 10 min at 60 °C, glycerol (0.6 g) was added, and tubes were mixed and incubated for an additional 10 min. Then 1.0 g of the stock solution was transferred to a glass tube (20 mL) containing 7 mL of DI water at 60 °C, mixed well, and incubated for 10 min. Finally, aqueous CoQ solution (1 g) was diluted with hot water to 10 mL to yield yellow and colorless clear solutions of ubiquinone and ubiquinol, respectively. Stock solutions were prepared fresh for each experiment. Simulated Digestion of CoQ-Enriched HCO60. Details for simulated gastric and small intestinal digestion have been described previously.28 Initial experiments showed that partitioning of ubiquinol in the filtered aqueous fraction of chyme (i.e., samples at termination of small intestinal phase of simulated digestion) markedly exceeded that of ubiquinone. Thus, the concentration of ubiquinol in 2 mL of HCO60 added to 14.2 g of fat-free yogurt was decreased to generate aqueous fractions of chyme containing similar concentrations of CoQ (sum of ubiquinone + ubiquinol) as that in the aqueous fraction after digestion of HCO60 containing ubiquinone. The total amounts of yogurt, HCO60, ubiquinone, and ubiquinol for in vitro digestion were 2.0 ± 0.1 g, 25 mg, 7.1 μmol, and 2.5 μmol, respectively. α-Tocopherol (50 μmol/L) and ascorbic acid (500 μmol/L) also were added to all tubes containing CoQ-rich yogurt to limit oxidation of ubiquinol during digestion. The efficiencies (%) of micellarization of CoQ during digestion of yogurt supplemented with 7.1 or 2.3 μmol of ubiquinol were similar (62.1 ± 1.8 vs 64.7 ± 3.1%, respectively, p = 0.197). Aliquots of the predigestion mixture of yogurt supplemented with CoQ-rich HCO60 (2 mL), after completion of the simulated small intestinal phase of digestion, and filtered (0.2 μm pores) aqueous fraction (2 mL) of chyme were transferred immediately to glass tubes 7175

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

■

ubiquinone or ubiquinol, and taurocholate-oleate micelles was added to the apical compartment of cultures. Basolateral medium and washed monolayers were collected after 4 h. To quantify intracellular reduced (GSH) and oxidized (GSSG) glutathione, cell pellets were immediately suspended in ice-cold PBS (0.25 mL) containing an equal volume of 10% PCA and 1 mmol/L DTPA and disrupted with a hand-held homogenizer. Homogenate was centrifuged at 16,000 × g at 4 °C for 5 min. Acidified supernatant was collected and stored at −80 °C for glutathione analysis by HPLC-EC using a boron-doped diamond electrode.32 GSH and GSSG were separated on a Shiseido C18 column (5 μm, 250 × 3.0 mm; Phenomenex, Torrance, CA, USA) with a C18 guard column containing the identical solid phase. Mobile phase was 6% acetonitrile in 25 mM sodium dihydrogen phosphate containing 1.4 mM 1-octanesulfonic acid, pH 2.5, at a flow rate of 1.0 mL/min. Areas under the curve for cell GSH and GSSG were compared with six-point standard curves using pure GSH and GSSG. GSH reductase activity was measured in cell homogenates by monitoring reduction of GSSG by NADPH coupled to the reaction of GSH with 5,5′-dithiobis(2-nitrobenzoic acid).25 Inhibition of Trx Reductase Activity in Caco-2 Cells. Auranofin (10 μmol/L) was added to DMEM containing Tween-40 micelles with CoQ and taurocholate-oleate micelles. This medium (2 mL) was added to the apical compartment of Caco-2 cells grown on membrane inserts. After 4 h of incubation, basolateral medium was collected to quantify CoQ transport across the monolayer. Trx reductase activity and redox status of CoQ in washed monolayers were analyzed to compare with cells identically treated in the absence of auranofin. Trx reductase activity was determined by monitoring the reduction of DTNB substrate in the absence versus in the presence of inhibitor at 412 nm (Thioredoxin Reductase Kit, Abnova, Walnut, CA, USA). Analysis of Coenzyme Q by HPLC-DAD. CoQ was analyzed by HPLC-DAD as described by Okamoto et al.33 Briefly, aliquots (1 mL) of either medium or cell pellets resuspended in PBS were extracted in glass tubes with 2 mL of ethanol followed by 6 mL of hexane. The mixture was vortexed for 60 s and centrifuged at 800 × g for 10 min at 4 °C. Supernatant was transferred to clean glass vial, and samples were re-extracted. Combined supernatant was dried under a stream of nitrogen gas, the film was resolubilized with 0.4 mL of MeOH/hexane (75:25), and 20−60 μL was injected onto the HPLC column. Because this method lacked sufficient sensitivity for accurate quantification of ubiquinol in cells and basolateral medium, ubiquinol was oxidized by the addition of an equal volume of 5% potassium hexacyanoferrate(III). After incubation at 37 °C with shaking (85 rpm) for 10 min, ubiquinone was extracted into ethanol and hexane. Ubiquinone was separated on a Sunfire C18 column (5 μm, 4.6 × 250 mm; Waters) following elution from a Sunfire C18 guard column. Mobile phase was a mixture of methanol and hexane (75:25) flowing at a rate of 1.0 mL/ min. Peak area was monitored at 275 nm. A standard curve was prepared for quantification with the limit of detection (signal-to-noise ratio of 5:1) being 58 nmol/L. Ubiquinol was calculated as the difference between total CoQ in samples incubated with potassium hexacyanoferrate and the quantity of ubiquinone in the absence of the oxidant. Miscellaneous. Cell protein was determined by the bicinchoninic acid assay (Pierce Chemical Co.) using bovine serum albumin as standard. Mean cell protein per insert was 1.2 ± 0.05 mg and independent of the presence or absence of ubiquinone and ubiquinol in the apical compartment. Flux of phenol red from the apical to the basolateral compartment was determined by measuring absorbance at 546 nm at 15 s after the addition of 20 μL of 1 mol/L NaOH to 150 μL of basolateral medium at room temperature.34 Analysis of Data. Results are expressed as means ± SD for the number (n) of observations indicated. Statistical analyses were performed using Student’s paired t test and one-way analysis of variance and Tukey’s HSD (honestly significant difference). A minimum p value of 0.05 was used to determine statistically significant differences among means for all tests.

Article

RESULTS AND DISCUSSION

Bioaccessibility of Ubiquinol Exceeds That of Ubiquinone. We previously reported that the efficiency of micellarization of ubiquinone during simulated digestion was greater for commercial formulations in which the coenzyme was solubilized by incorporation into either γ-cyclodextrin or oils containing emulsifiers.24 Here ubiquinone and ubiquinol were solubilized in HCO60 to directly compare their micellarization during simulated digestion. HCO60 is polyethylene glycol 60 linked to hydrogenated castor oil and is used as an emulsifier for cosmetic and pharmaceutical formulations.35,36 Hydroxystearic acid, the most abundant fatty acid in this preparation, appears to have a relatively high affinity for CoQ and the PEG60 moiety facilitates emulsification in the water matrix. Also, PEGylated oils have low toxicity, teratogenicity, and mutagenicity. Preliminary studies showed that the efficiency of micellarization of ubiquinol during the small intestinal phase of digestion of HCO60 added to yogurt was 2−3 times greater than that of ubiquinone. Thus, the concentration of ubiquinone in HCO60 was increased to generate mixed micelles containing similar amounts of ubiquinone and ubiquinol for investigating uptake and transport of CoQ by monolayers of differentiated Caco-2 cells (Table 1). Table 1. Effect of Redox State of CoQ on Stability and Efficiency of Micellarization during Simulated Digestion of Yogurt Supplemented with Ubiquinone or Ubiquinol in PEG60 Hydrogenated Castor Oil Vehiclea

starting material (CoQ-enriched yogurt) μmol of CoQ total CoQb ubiquinone ubiquinol chyme generated during digestion μmol of CoQ ubiquinone ubiquinol recovery of CoQ (%)c filtered aqueous fraction of chyme μmol of CoQ ubiquinone ubiquinol % ubiquinol % micellarization of CoQd

ubiquinonerich yogurt

ubiquinol-rich yogurt

7.1 ± 0.1a 7.1 ± 0.1 nd

2.34 ± 0.05b 0.58 ± 0.02 1.78 ± 0.02

5.84 ± 0.26 nd 82.0 ± 3.7a

0.81 ± 0.02 1.17 ± 0.03 84.5 ± 1.6a

2.00 ± 0.05 ND 0 28.1 ± 0.7b

0.66 0.86 56.4 65.0

± ± ± ±

0.04 0.04 2.5 2.5a

a Data are the mean ± SD for four independent digestions. ND, not detected. Different letters in the same row indicate that means differ significantly (p < 0.01). bTotal CoQ = ubiquinone + ubiquinol. c Percentage of total CoQ in chyme versus starting material. d Percentage of total CoQ in aqueous fraction of chyme versus starting material.

Recovery of ubiquinone after in vitro digestion of the yogurt containing the ubiquinone supplement was 82 ± 3.7% with 28% of the initial content of the coenzyme transferred to the aqueous fraction of chyme during the small intestinal phase of digestion (Table 1). All CoQ in the aqueous fraction, that is, the bioaccessibility, was ubiquinone. After digestion of the yogurt supplemented with ubiquinol, 84% of CoQ (ubiquinone + ubiquinol) in the food matrix was retained and 65% of initial content of CoQ partitioned in the bioaccessible fraction. Partial oxidation of ubiquinol occurred during both preparation of the ubiquinol-enriched yogurt and simulated digestion with 7176

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

Figure 1. Apparent uptake of CoQ and secretion of the coenzyme into the basolateral compartment by Caco-2 cells incubated with aqueous fraction of chyme generated during digestion of yogurt containing either ubiquinone (20.0 nmol/well) or ubiquinol (15.2 nmol/well) supplement. Apparent uptake (A) represents the percentage of CoQ in the apical compartment present in cells (corrected for endogenous CoQ) and the basolateral compartment (B) of cultures of Caco-2 cells after 4 h of incubation. The percentage of CoQ accumulated by cells that was secreted into the basolateral medium was calculated by dividing the quantity of the coenzyme in the basolateral compartment by the corrected amount in cells plus the quantity in the basolateral compartment. Data are the mean ± SD, n = 6. Different letters above bars indicate that means differ significantly (p < 0.01).

Figure 2. Inhibition of Pgp increases apparent uptake (A) and basolateral secretion (B) of ubiquinone and ubiquinol in apical medium. Caco-2 cells were treated with medium containing verapamil (100 μmol/L) in the apical and basolateral compartments for 30 min before addition of apical medium containing diluted aqueous fractions generated during digestion of yogurt supplemented with either ubiquinone. Apical medium contents of ubiquinone and ubiquinone were 12.8 and 12.3 nmol/well, respectively. Cultures were incubated for 4 h before collection of basolateral medium and washed cells. Data are the mean ± SD, n = 3. Different letters above bars indicate significantly (p < 0.05) different means.

ubiquinol, accounting for 59 and 56% of total CoQ in chyme and the filtered aqueous fraction, respectively. Nevertheless, the efficiency with which total CoQ partitioned in the aqueous fraction of chyme during digestion of the ubiquinolsupplemented yogurt was more than twice as great as that of CoQ after digestion of yogurt enriched with ubiquinone (p < 0.001). The similar recoveries of CoQ after digestion and its more efficient partitioning in the aqueous fraction of chyme demonstrated that solubilized ubiquinol has greater bioaccessibility than solubilized ubiquinone. The extent of oxidation of ubiquinol during manual manipulations for solubilization in HCO, mixing in the yogurt food matrix, and simulated digestion likely exceeded that in standard commercial preparations of supplements and in situ digestion. The commercial form of ubiquinol is generally a softgel capsule. The content of the capsule is prepared in a deoxygenated environment under a blanket of inert gas before the soft-gel capsule shell is filled in the absence of air. These processes result in ≤2% oxidation of ubiquinol. Moreover, the shell of the soft-gel capsule has low oxygen permeability for ubiquinol to stabilize the reduced state until the capsule disintegrates in the stomach. Some oxidation of ubiquinol in the stomach and small intestine is possible as foods contain air and swallowing also introduces air into the stomach. However, oxygen diffuses from the gastrointestinal lumen into gut tissues, resulting in a decreasing oxygen gradient as chyme progresses

from the stomach to the anaerobic environment in the colon.37,38 Therefore, ubiquinol oxidation is expected to be lower in the commercial preparation of the delivery system and in situ environment than we observed in the laboratory. Apical Uptake and Transepithelial Transport of CoQ by Caco-2 Cells from Micelles Generated during Digestion. Apical uptake of CoQ incorporated into mixed micelles during digestion of yogurt containing either ubiquinone or ubiquinol by Caco-2 cells and subsequent transfer of acquired CoQ to the basolateral compartment were determined. The quantities of CoQ in medium prepared with aqueous fraction of chyme after digestion of yogurt containing ubiquinone and ubiquinol were 20.0 ± 0.4 and 15.2 ± 0.4 nmol/well, respectively. Apparent uptake of CoQ from the apical compartment (%) was calculated as the corrected intracellular CoQ content (i.e., total CoQ minus endogenous CoQ) plus the quantity of CoQ in the basolateral compartment after 4 h of incubation. Apparent uptake of CoQ was twice as great for monolayers exposed to filtered aqueous fraction generated during digestion of yogurt enriched with ubiquinol as for cells incubated with aqueous fraction after digestion of yogurt supplemented with ubiquinone (Figure 1A). The corrected intracellular quantity of CoQ was 17% greater (p < 0.001) in cultures incubated with the filtered aqueous fraction of chyme after digestion of the ubiquinol supplement compared to the ubiquinone supplement (1.17 ± 0.02 vs 1.00 ± 0.04 7177

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

Figure 3. Altered cellular GSH status is associated with decreased apical uptake and basolateral secretion of CoQ. Caco-2 cells were incubated in the absence or presence of a cocktail of inhibitors of GSH synthesis and reduction as described under Materials and Methods. Apical medium containing ubiquinone (5.0 nmol/well) or ubiquinol (4.85 nmol/well) was added and cultures were incubated for 4 h. Data are the mean ± SD, n = 6. Different letters above bars indicate that means differ significantly (p < 0.01).

nmol/well, respectively). Ubiquinol accounted for 55% of intracellular pool of accumulated CoQ regardless of origin of the mixed micelles delivering CoQ. The quantity of CoQ secreted into basolateral medium was 5 times greater (p < 0.0001) in cultures incubated with micellar CoQ generated during digestion of ubiquinol-rich yogurt compared to CoQ incorporated into mixed micelles during digestion of ubiquinone-rich yogurt (0.42 ± 0.04 vs 0.08 ± 0.01 nmol CoQ/well). This represents 2.4-fold greater secretion of the quantity of the coenzyme apparently taken up by cells incubated with micelles generated during digestion of yogurt with ubiquinol compared to ubiquinone (Figure 1B). This difference was not associated with changes in barrier integrity of the monolayers as apical to basolateral flux of the paracellular marker phenol red was not significantly different for monolayers incubated with medium containing filtered aqueous fraction of chyme generated during digestion of yogurt enriched with either ubiquinone (86 ± 5 pmol/cm2/h, p = 0.4656) or ubiquinol (87 ± 4 pmol/cm2/h, p = 0.3173). These rates for paracellular transport of the dye were similar to that across monolayers of control cells, that is, 83.9 ± 8.1 pmol/ cm2/h. Verapamil-mediated inhibition of the Pgp effluxer increased apparent uptake and basolateral secretion of CoQ from micelles generated during digestion of yogurt supplemented with either ubiquinone or ubiquinol (Figure 2). Apparent apical uptake of CoQ increased 38 and 140% when verapamil-treated Caco-2 cells were incubated with diluted aqueous fraction of chyme after digestion of ubiquinone and ubiquinol supplemented yogurt, respectively (Figure 2A). This change was associated with 19% (p < 0.01) and 31% (p < 0.0001) increases in intracellular CoQ in verapamil-treated cells incubated in medium containing diluted aqueous fraction from digested yogurt supplemented with ubiquinone and ubiquinol, respectively. Similarly, secretion of CoQ into the basolateral compartment increased 2.4- and 5.3-fold in Pgp-treated cells incubated with micelles generated during digestion of yogurt containing ubiquinone and ubiquinol, respectively (Figure 2B). These data suggest that apical uptake and secretion of CoQ into the basolateral compartment are more efficient for supplemented ubiquinol than for ubiquinone. It is unknown if the redox state of CoQ affects its transfer from mixed micelles to the apical surface of Caco-2 cells, transfer across or incorporation into the plasma membrane, or distribution to other subcellular compartments including the Golgi during chylomicron assembly. CoQ content and redox status in medium containing the mixed micelles generated during digestion of yogurt with the

supplements were stable during 4 h of incubation in cell-free wells. Recovery of total CoQ and ubiquinol exceeded 95 and 90%, respectively. Only ubiquinone was detected in the basolateral medium after collected samples were stored under nitrogen at −80 °C overnight, whereas both ubiquinone and ubiquinol were present in basolateral medium that was analyzed immediately upon termination of the incubation after 4 h. These data suggest that the presence of 19% oxygen (95% air, 5% CO2) in the cell culture environment, the relative absence of other antioxidants in the basolateral compartment, and overnight storage of medium likely contributed to oxidation of ubiquinol after its transport across the basolateral membrane. Effect of Altered Cellular Redox Status on Uptake and Basolateral Secretion of CoQ. Because uptake and basolateral secretion of CoQ from micelles generated during digestion of yogurt with ubiquinol exceeded that for micelles containing only ubiquinone, we hypothesized that efficient secretion of CoQ across the basolateral membrane required a reduced intracellular environment. Interactive redox systems are responsible for maintaining a reduced intracellular environment.39 Reduced glutathione is the most abundant intracellular antioxidant in aerobic cells. Alterations in glutathione status previously have been shown to affect glucose uptake,40 calcium absorption,41 and the activity of organic solute transporters and ion channels.42 The concentration of total glutathione and the high ratio of glutathione to glutathione disulfide are maintained by the activities of γ-glutamyl cysteine synthetase and NADPHdependent GSH reductase, respectively. GSH reductase also has been reported to directly reduce ubiquinone to ubiquinol in tissue homogenates and cytosol.25 The protocol of Noda et al.31 was used to decrease GSH content of Caco-2 cells without inducing oxidative stress. GSH reductase activity and cellular GSH decreased 41 and 99%, respectively, and cell GSSG increased 23% in monolayers incubated with a cocktail. Despite this marked decrease in cellular GSH, there was no overt cytotoxicity as cellular morphology, mitochondrial activity as assessed by reduction of MTT, and cell protein content per well were not significantly different in control cultures and cultures exposed to the cocktail of inhibitors (data not shown). Similarly, there was no significant change in barrier integrity as apical to basolateral flux of phenol red was not significantly different in control in cultures with control and low glutathione status. Finally, the amounts of ubiquinone or ubiquinol in medium were not altered during co-incubation with the inhibitors in the absence of cells. Apparent uptake of ubiquinone and ubiquinol from micelles in apical medium was decreased 48% (p < 0.0001) and 65% (p < 0.0001), respectively, in cells with low glutathione status 7178

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

Figure 4. Intracellular GSH (A) and GSSG (B) are altered by exposure of Caco-2 cells to micellar CoQ and inhibitors of GSH status. Cultures were incubated in the absence (control) or presence of inhibitors of GSH status as described under Materials and Methods. Data are the mean ± SD, n = 6. Different letters above error bars indicate that means differ significantly (p < 0.01).

Figure 5. Inhibition of Trx reductase activity decreases apical uptake (A) of ubiquinol and basolateral secretion of CoQ (B) in cultures containing micellar ubiquinol or ubiquinone. Monolayers of Caco-2 grown were co-incubated in apical medium with auranofin (10 μmol/L) and either ubiquinone (6.3 nmol/well) or ubiquinol (6.1 nmol/well) for 4 h. Data are the mean ± SD, n = 6. Different letters above bars indicate significantly (p < 0.001) different means.

ubiquinone and ubiquinol attenuated the extent of the reduction in cell GSH and increase in GSSG when cells were treated with BSO, BCNU, and diamide (Figure 4). We next assessed whether the decreased uptake and transport of micellar CoQ in cells with low glutathione status was specific or reflected a more general effect of cell redox status on the greater bioavailability of ubiquinol than of ubiquinone. The thioredoxin/thioredoxin disulfide couple represents the other major intracellular redox system.41 Trx reductase activity was 7.3−10.0 nmol/min/mg protein in cultures ± apical CoQ, but not detected in cells incubated with 10 μmol/L auranofin for 4 h. Loss of Trx reductase activity was associated with 50 and 40% decreases in the apical uptake (Figure 5A) and basolateral secretion (Figure 5B) of CoQ, respectively, in cultures incubated with apical medium containing ubiquinol. In contrast, cell uptake and basolateral secretion were not altered in auranofin-treated cultures incubated with apical medium containing ubiquinone. Similarly, the intracellular ratio of ubiquinol to ubiquinone also significantly decreased in auranofin-treated cells incubated with micelles containing ubiquinol, but not ubiquinone (Figure 6A). The discrepancy in the response of monolayers to auranofin led us to question whether the presence of the inhibitor in medium was associated with extracellular oxidation of ubiquinol. This interaction was confirmed by comparison of the stability of ubiquinol in Tween-40 micelles in the absence and presence of auranofin in cell-free medium. Ubiquinol decreased 31% during the 4 h of incubation in cell-free medium containing auranofin (Figure 6B). Oxidation of ubiquinol to ubiquinone accounted for only one-third of the total loss. The concentration of ubiquinone in medium ± auranofin incubated for 4 h in the absence of cells was not significantly altered after 4 h (Figure 6B). These results suggest the decreased uptake

(Figure 3). Similarly, secretion of CoQ into the basolateral compartment decreased 30 and 65% in cultures incubated in medium with ubiquinone and ubiquinol, respectively. The quantities of CoQ in cells and in basolateral medium were not significantly different in cultures with low GSH status regardless of whether ubiquinone or ubiquinol was present in apical medium. Elucidation of the mechanism by which depletion of cell GSH adversely affects transport of both reduced and oxidized CoQ across the brush border and basolateral membranes of differentiated Caco-2 cells requires investigation. There were additional interactions between CoQ and glutathione status. The endogenous ratio of ubiquinol to ubiquinone decreased by 20% (p < 0.05) in cells treated with the inhibitors of glutathione status in the absence of apical CoQ. The presence of 5.0 nmol of ubiquinone in apical medium was associated with a doubling of cellular GSH (Figure 4A) and a 14% reduction in GSSG (Figure 4B). Similarly, Wagner et al.43 reported that treatment of C2C12 myotubes with 100 μmol/L ubiquinone plus 250 μmol/L lipoic acid increased expression of γ-glutamyl cysteine synthase and the activities of GSH reductase and glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase, the latter two being key enzymes in the synthesis of NADPH. These changes resulted in a 4-fold increase in intracellular GSH. Hwang et al.44 also reported that addition of 10 μmol/L ubiquinone to medium increased intracellular GSH concentration in UV-A irradiated keratinocytes. In contrast with the stimulatory effect of ubiquinone on cellular GSH content, incubation of cells in apical medium containing 4.85 nmol of ubiquinol per well decreased GSH and GSSG by 16 and 46%, respectively. The basis for the differential effects of ubiquinone and ubiquinol on glutathione status of Caco-2 cells in the present study is unknown. It is also noteworthy that apical medium containing 7179

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

without alteration of the ratio of ubiquinol to ubiquinone when cultures were incubated in medium containing ubiquinol and dicoumarol. There was no extracellular interaction between dicoumarol and ubiquinol as recovery of ubiquinol during incubation in cell-free medium with dicoumarol for 6 h was 98%, with 95% of the coenzyme remaining in the reduced state. Together, these data suggest that partial loss of NQO1 activity minimally affected accumulation of ubiquinol by HT-29 cells. Clarity regarding the possible influence of NQO1 activity on the intestinal absorption of ubiquinol will require investigation in an animal model. In summary, the present in vitro studies suggest that the greater bioavailability of ubiquinol compared to that of ubiquinone is the result of increased efficiency of several processes associated with the absorption of orally administered lipophilic compounds. These included partitioning into mixed micelles during simulated small intestinal digestion, apical uptake by Caco-2 cells, and subsequent secretion of the coenzyme across the basolateral membrane. The extent of the observed differences in these processes between ubiquinol and ubiquinone likely underestimate the situation in situ because oxygen tension in the laboratory exceeded that for commercial preparation of ubiquinol in soft gel capsules, exposure to compounds in the meal prior to ingestion, and during gastric and small intestinal phases of digestion. This led to the partial oxidation of ubiquinol to ubiquinone, resulting in the presence of both ubiquinol and ubiquinone in mixed micelles added to the apical compartment of Caco-2 cells. The present results also show that a reduced intracellular environment is necessary to maintain the elevated ratio of ubiquinol to ubiquinone within cells, which in turn facilitates both the apical uptake and basolateral secretion of CoQ. This was demonstrated by markedly decreasing cellular GSH and the activity of TrxR. Because the actual compartmentalization of the bolus of CoQ taken up by the cell is not known, it is unclear which flavoenzyme or combination of such enzymes shown to reduce extramitochondrial ubiquinone in cell homogenates and once purified are responsible for doing so in absorptive cells lining the gut.

Figure 6. Decreased intracellular CoQ in monolayers co-incubated in apical medium containing 10 μmol/L auranofin and ubiquinol (A) is due to extracellular oxidation and degradation of the reduced coenzyme (B). The amounts of unbiquinone and ubiquinol in apical medium of Caco-2 cell cultures were 6.3 and 5.4 nmol/well, respectively. TrxR, thioredoxin reductase; CoQ10, ubiquinone; CoQH, ubiquinol. Data are the mean ± SD for n = 6 and 4 in panels A and B, respectively. Different letters above error bars indicate that means differ significantly different (p < 0.001 for panel A and p < 0.01 for panel B).

■

AUTHOR INFORMATION

Corresponding Author

*(M.L.F.) E-mail: [email protected]. Phone: (614) 560-9401. Fax: (614) 292-4339.

and transport of ubiquinol in medium containing auranofin resulted from both oxidation and degradation of micellar ubiquinol in the apical compartment by an unknown chemical reaction. NQO1 catalyzes the two-electron reduction of quinones in human tissues and has been suggested to participate in the reduction of ubiquinone.27 Unlike normal intestinal epithelial cells, Caco-2 cells lack NQO1 activity because they express a mutant form of the enzyme that is rapidly eliminated by the proteosomal degradation pathway.45−47 Therefore, we were not able to determine whether NQO1 activity affected CoQ uptake, transport, and the redox status of intracellular CoQ using this cell line. We did test the possibility that the enzyme might affect CoQ uptake and redox status using the HT-29 human colonic cell line that expresses wild-type protein. NQO1 activity was decreased by 25% in cells exposed to 10 μmol/L dicoumarol, a specific inhibitor of NQO1.27 This change did not alter the intracellular content of CoQ or the ratio of ubiquinol to ubiquinone during incubation in medium supplemented with ubiquinone (80 μmol/L) (data not shown). In contrast, there was a 19% decline in intracellular cell CoQ content (p < 0.05)

Notes

The authors declare the following competing financial interest(s): F.A. is an employee of Kaneka North America LLC, the sponsor for this project.

■

ACKNOWLEDGMENTS The authors thank Drs. Richard S. Bruno and Eunice Mah for guidance with analysis of GSH and GSSG using the Ultimate HPLC-BDD detector (Thermo Fisher Scientific, Waltham, MA, USA).

■

ABBREVIATIONS USED BCNU, 1,3-bis(2 chloroethyl)-1-nitrosourea; BSO, DL-buthioninesulfoximine; CoQ, coenzyme Q; CoQ10, ubiquinone; CoQH, ubiquinol; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5′-dithiobis(2-nitrobenzoic) acid; DTPA, diethylenetriaminepentaacetic acid; FBS, fetal bovine serum; GSH, glutathione; GSSG, glutathione disulfide; HCO, hydro7180

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

(19) Yan, J.; Fujii, K.; Yao, J.; Kishida, H.; Horose, K.; Sawashita, J.; Takeda, T.; Mori, M.; Higuchi, K. Reduced coenzyme Q10 decelerates senescence in SAMP1 mice. Exp. Gerontol. 2006, 41, 130−140. (20) Schmelzer, C.; Kubo, M.; Sawashita, J.; Kitano, M.; Hosoe, K.; Boomgaarrden, I.; Doring, F.; Higuchi, K. Supplementation with the reduced form of coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferator-activated receptor-α gene expression signature in SAMP1 mice. Mol. Nutr. Food Res. 2010, 54, 805−815. (21) Peerapanyasut, W.; Thamprasert, K.; Wongmekiat, O. Ubiquinol supplementation protects against renal ischemia and reperfusion injury in rats. Free Radical Res. 2013, 48, 180−189. (22) Langsjoen, P. H.; Langsjoen, A. M. Supplemental ubiquinol in patients with advanced congestive heart failure. Biofactors 2008, 32, 119−128. (23) Mezawa, M.; Takemoto, M.; Onishi, S.; Ishibashi, R.; Ishikawa, T.; Yamaga, M.; Fujimoto, M.; Okabe, E.; He, P.; Kobayashi, K.; Yokote, K. The reduced form of coenzyme Q10 improves glycemic control in patients with type 2 diabetes. Biofactors 2012, 38, 416−421. (24) Bhagavan, H.; Chopra, B. K.; Craft, N. E.; Chitchumroonchokchai, C.; Failla, M. L. Assessment of coenzyme Q10 absorption using an in vitro digestion-Caco-2 cell model. Int. J. Pharm. 2007, 333, 112−117. (25) Bjornstedt, M.; Nordman, T.; Olsson, J. M. Extramitochondrial reduction of ubiquinone by flavoenzymes. Methods Enzymol. 2004, 378, 131−138. (26) Xia, L.; Nordman, T.; Olsson, J. M.; Damdimoupolos, A.; Bjorkhem-Bergman, L.; Nalvarte, I.; Eriksson, L. C.; Arner, E. S. J.; Spyrou, G.; Bjornstedt, M. The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone: a novel mechanism for defense against oxidative stress. J. Biol. Chem. 2003, 278, 2141−2146. (27) Landi, L.; Fiorentinin, D.; Galli, M.; Segura-Aguilar, J.; Beyer, R. E. DT-diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function. Free Radical Biol. Med. 1997, 22, 329−335. (28) Chitchumroonchokchai, C.; Schwartz, S. J.; Failla, M. L. Assessment of lutein bioavailability from meals and supplements using simulated digestion and Caco-2 human intestinal cells. J. Nutr. 2004, 134, 2280−2286. (29) Luchoomun, J.; Hussain, M. M. Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly. J. Biol. Chem. 1999, 274, 19565−19572. (30) During, A.; Hussain, M. M.; Morel, A. W.; Harrison, E. H. Carotenoid uptake and secretion by Caco-2 cells: β-carotene isomer selectivity and carotenoid interactions. J. Lipid Res. 2002, 43, 1086− 1095. (31) Noda, T.; Iwakiri, R.; Fujimoto, K.; Aw, T. Y. Induction of mild intracellular redox imbalance inhibits proliferation of Caco-2 cells. FASEB J. 2001, 15, 2131−2139. (32) Park, J. H.; Mah, E.; Bruno, R. S. Validation of high performance liquid chromatography-boron-doped diamond detector for assessing hepatic glutathione redox status. Anal. Biochem. 2010, 407, 151−159. (33) Okamoto, T.; Fukui, K.; Nakamoto, M.; Kishi, T.; Okishio, T.; Yamagami, T.; Kanamori, N.; Kishi, H.; Hiraoka, E. High-performance liquid chromatography of coenzyme Q-related compounds and it application to biological materials. J. Chromatogr. 1985, 342, 35−46. (34) Lewis, S. A. Assessing epithelial cell confluence by spectroscopy. In Methods in Molecular Biology: Epithelial Cell Culture Protocols; Wise, C., Ed.; Humana Press: Totowa, NJ, USA, 2002; pp 329−336. (35) Burnett, C.; Heldreth, B. Amended safety assessment of PEGylated oils as used in cosmetics. Amended Report, Cosmetic Ingredient Review 2013, pp 1−29; http://www.cir-safety.org/sites/ default/files/pegoil122012final_faa-final%20for%20posting.pdf (accessed Jan 12, 2014). (36) Committee for Veterinary Medicinal Products. Summary Report of Polyoxyl Castor Oil, Polyoxylhydrogenated Castor Oil; The European Agency for the Evaluation of Medicinal Products, Veterinary Medicines Evaluation Unit: London, UK, 1999; pp 1−3.

genated castor oil; NQO1, NAD(P)H:quinone oxidoreductase family 1; PBS, phosphate buffer saline; PCA, perchloric acid; Pgp, Pgp-glycoprotein; Trx, thioredoxin

■

REFERENCES

(1) Ernster, L.; Dallner, G. Biochemical, physiological and medical aspects of ubiquinone function. Biochim. Biophys. Acta 1995, 1271, 195−204. (2) Littarru, G. P.; Tiano, L. Clinical aspects of coenzyme Q10: an update. Nutrition 2010, 26, 250−254. (3) Celik, T.; Iyisoy, A. Coenzyme Q10 and coronary artery bypass surgery: what we have learned from clinical trials. J. Cardiothorac. Vasc. Anesth. 2009, 23, 935−936. (4) Makhija, N.; Sendasgupta, C.; Kiran, U.; Lakshmy, R.; Hote, M. P.; Choudhary, S. K.; Airan, B.; Abraham, R. The role of oral coenzyme Q10 in patients undergoing coronary artery bypass graft surgery. J. Cardiothorac. Vasc. Anesth. 2008, 22, 832−839. (5) Henchcliffe, C.; Beal, M. F. Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nat. Clin. Pract. Neurol. 2008, 4, 600−609. (6) Naia, L.; Ribeiro, M. J.; Rego, A. C. Mitochondrial and metabolicbased protective strategies in Huntington’s disease: the case of creatine and coenzyme Q. Rev. Neurosci. 2011, 23, 13−28. (7) Cooke, M.; Losia, M.; Buford, T.; Shelmadine, B.; Hudson, G.; Kerksick, C.; Rasmussen, C.; Greenwood, M.; Leutholtz, B.; Willoughby, D.; Kreider, R. Effect of acute and 14-day coenzyme Q10 supplementation on exercise performance in both trained and untrained individuals. J. Int. Soc. Sports Nutr. 2008, 5, 1−14. (8) Kon, M.; Tanabe, K.; Akimoto, T.; Kimura, F.; Tanimura, Y.; Shimizu, K.; Okamoto, T.; Kono, I. Reducing exercise-induced muscular injury in kendo athletes with supplementation of coenzyme Q10. Br. J. Nutr. 2008, 100, 903−909. (9) Bhagavan, H. N.; Chopra, R. K. Coenzyme Q10: absorption, tissue uptake, metabolism and pharmacokinetics. Free Radical Res. 2006, 40, 445−453. (10) Borel, P. Factors affecting intestinal absorption of highly lipophilic food microconstituents (fat-soluble vitamins, carotenoids and phytosterols). Clin. Chem. Lab. Med. 2003, 41, 979−994. (11) Miles, M. V.; Horn, P.; Miles, L.; Tang, P.; Steele, P.; DeGrauw, T. Bioequivalence of coenzyme Q10 from over the counter supplements. Nutr. Res. (N.Y.) 2002, 22, 919−929. (12) Hosoe, K.; Kitano, M.; Kishida, H.; Kubo, H.; Fuji, K.; Kitahara, M. Study on safety and bioavailability of ubiquinol (Kaneka QHTM) after single and 4-week multiple oral administration to healthy volunteers. Regul. Toxicol. Pharmacol. 2007, 47, 19−28. (13) Evans, M.; Baisley, J.; Barss, S.; Guthrie, N. A randomized, double-blind trial on the bioavailability of two CoQ10 formulations. J. Funct. Food 2009, 1, 65−73. (14) Itagaki, S.; Ochiai, A.; Kobayashi, M.; Sugawara, M.; Hirano, T.; Iseki, K. Interaction of coenzyme Q10 with the intestinal drug transporter P-glycoprotein. J. Agric. Food Chem. 2008, 56, 6923−6927. (15) Xia, S.; Xu, S.; Zhang, X.; Zhong, F.; Wang, Z. Nanoliposomes mediate coenzyme Q10 transport and accumulation across the human intestinal Caco-2 cell monolayer. J. Agric. Food Chem. 2009, 57, 7989− 7996. (16) Beg, S.; Javed, S.; Kohli, K. Bioavailability enhancement of coenzyme Q10: an extensive review of patents. Recent Pat. Drug Delivery Formulation 2010, 4, 45−257. (17) Schmelzer, C.; Kohl, C.; Rimbach, G.; Doring, F. The reduced form of coenzyme Q decreases the expression of lipopolysaccharidesensitive genes in human THP-1 cells. J. Med. Food 2010, 14, 391− 397. (18) Bruge, F.; Damiani, E.; Puglia, C.; Offerta, A.; Armeni, T.; Littaru, G. P.; Tiano, L. Nanostructured lipid carriers loaded with CoQ10: effect on human dermal fibroblasts under normal and UVAmediated oxidative conditions. Int. J. Pharm. 2013, 455, 348−356. 7181

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182

Journal of Agricultural and Food Chemistry

Article

(37) Forster, R. E. Physiological basis of gas exchange in the gut. Ann. N.Y. Acad. Sci. 1968, 150, 4−12. (38) He, G.; Shankar, R. A.; Chzhan, M.; Samouilov, A.; Kuppusamy, P.; Zweier, J. L. Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4586−4591. (39) Circu, M. L.; Aw, T. K. Intestinal redox biology and oxidative stress. Stem Cell Dev. Biol. 2012, 23, 729−737. (40) Iantomasi, T.; Favilli, F.; Marracccini, P.; Vincenzini, M. T. Glutathione involvement on the intestinal Na+-dependent D-glucose active transporter. Mol. Cell. Biochem. 1998, 178, 387−392. (41) Xiao, Y.; Cui, J.; Shi, Y. H.; Sun, J.; Wang, Z. P.; Le, G. W. Effects of duodenal redox status on calcium absorption and related genes expression in high-fat diet-fed mice. Nutrition 2010, 26, 1188− 1194. (42) Hammond, C. L.; Lee, T. K.; Ballatori, N. Novel roles for glutathione in gene expression, cell death, and membrane transport of organic solutes. J. Hepatol. 2001, 34, 946−954. (43) Wagner, A.; Ernst, I. M. A.; Birringer, M.; Sancak, O.; Barella, L.; Rimbach, G. A combination of lipoic acid plus coenzyme Q10 induces PGC1α, a master switch of energy metabolism, improves stress response, and increases cellular glutathione levels in cultured C2C12 skeletal muscle cells. Oxidative Med. Cell. Longevity 2012, DOI: 10.1155/2012/835970. (44) Hwang, T. L.; Tsai, C. K.; Chen, J. L.; Changchien, T. T.; Wang, C. C.; Wu, C. M. Magnesium ascorbyl phosphate and coenzyme Q10 protect keratinocytes against UVA irradiation by suppressing glutathione depletion. Mol. Med. Rep. 2012, 6, 375−383. (45) Traver, R.; Horikoshi, T.; Danenberg, K. D.; Stadlbauer, H. W.; Danenberg, P. V.; Ross, D.; Gibson, N. W. NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity. Cancer Res. 1992, 52, 797−802. (46) Karczewski, J. M.; Peters, J. G. P.; Noordhoek, J. Quinone toxicity in DT-diaphorase-efficient and -deficient colon carcinoma cell lines. Biochem. Pharmacol. 1999, 57, 27−37. (47) Siegel, D.; Anwar, A.; Winski, S. L.; Kepa, J. K.; Zolman, K. L.; Ross, D. Rapid polyubiquitination and proteosomal degradation of a mutant form of NAD(P)H:quinone oxidoreductase 1. Mol. Pharmacol. 2001, 59, 263−268.

7182

dx.doi.org/10.1021/jf5017829 | J. Agric. Food Chem. 2014, 62, 7174−7182