Kathrin Mandery
email: kathrin.mandery@pharmakologie.med.uni-erlangen.de

Krystyna Bujok
email: krystyna.bujok@t-online.de

Ingrid Schmidt
email: schmidt@pharmakologie.med.uni-erlangen.de

Markus Keiser
email: mkeiser@uni-greifswald.desiegmuw@uni-greifswald.de

Werner Siegmund

Bettina Balk
email: bettina.balk@gmx.de

Jörg König
email: joerg.koenig@pharmakologie.med.uni-erlangen.defromm@pharmakologie.med.uni-erlangen.de

Martin F Fromm

PhD Hartmut Glaeser
email: glaeser@pharmakologie.med.uni-erlangen.de

Influence of the flavonoids apigenin, kaempferol and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K i values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0µM) and OATP2B1 (8.7µM) followed by kaempferol (OATP1A2: 25.2µM, OATP2B1: 15.1µM) and apigenin (OATP1A2: 32.4µM OATP2B1: 20.8µM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC 50 values of 4.3µM, 12.0µM, and 12.6µM, respectively. The OATP1A2-and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC 50 for OATP1A2: 9.3µM, OATP2B1: 13.9µM), kaempferol (IC 50 for OATP1A2: 37.3µM, OATP2B1: 20.7µM) and quercetin (IC 50 for OATP1A2: 13.5µM, OATP2B1: 14.1µM). These data indicate that They are also ingredients of tea, wine, juices, numerous multivitamin preparations and herbal products (e.g. Ginkgo biloba formulations). In vitro and in vivo studies indicate pharmacological effects of flavonoids on prevention of cardiovascular diseases as well as anticancerogenic and antioxidative effects [START_REF] Hertog | Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study[END_REF][START_REF] Hertog | Content of Potentially Anticarcinogenic Flavonoids of Tea Infusions, Wines, and Fruit Juices[END_REF][START_REF] Jagtap | Chemoprotective mechanism of the natural compounds, epigallocatechin-3-Ogallate, quercetin and curcumin against cancer and cardiovascular diseases[END_REF][START_REF] Boots | Health effects of quercetin: from antioxidant to nutraceutical[END_REF].

It has recently been shown that several of these compounds inhibit drug metabolizing enzymes and drug transporters such as cytochrome P450 3A4 and the drug efflux transporter P-glycoprotein (ABCB1) [START_REF] Pal | MDR-and CYP3A4-mediated drug-drug interactions[END_REF], highlighting their potential for food-drug interactions. For example, the flavonoids apigenin, kaempferol, and quercetin which are constituents of Ginkgo biloba formulations, onions, strawberries, and apples [START_REF] Hertog | Content of Potentially Anticarcinogenic Flavonoids of Tea Infusions, Wines, and Fruit Juices[END_REF] were characterized by inhibition and/or induction of CYP3A4, ABCB1, and ABCC2 [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF][START_REF] Pal | MDR-and CYP3A4-mediated drug-herbal interactions[END_REF].

OATP1A2 and OATP2B1 are members of the human OATP family and are localized in the apical membrane of human enterocytes [START_REF] Kobayashi | Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH-dependent A c c e p t e d M a n u s c r i p t 25 transport across intestinal apical membrane[END_REF][START_REF] Glaeser | Intestinal drug transporter expression and the impact of grapefruit juice in humans[END_REF]. OATP1A2 mediates the intracellular uptake of drugs such as fexofenadine [START_REF] Cvetkovic | OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of fexofenadine[END_REF] as well as of endogenous compounds such as taurocholate [START_REF] Kullak-Ublick | Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver[END_REF]. OATP2B1 mediates the cellular uptake of drugs such as fluvastatin [START_REF] Kopplow | Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells[END_REF], atorvastatin [START_REF] Grube | Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart[END_REF] and of endogenous compounds such as estrone-3-sulfate [START_REF] Tamai | Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family[END_REF].

The influence of flavonoids on intestinal drug metabolism and transport is of special interest because flavonoids such as apigenin, kaempferol, and quercetin can reach high concentrations in the gut lumen. The inhibition of an OATP-mediated drug uptake in the intestine may decrease the plasma concentration of a substrate of OATPs, due to a reduced absorption. In vitro studies in HeLa cells and additional in vivo studies showed that the flavonoid naringin, a constituent of grapefruit juice, inhibits the uptake of fexofenadine mediated by OATP1A2 [START_REF] Glaeser | Intestinal drug transporter expression and the impact of grapefruit juice in humans[END_REF][START_REF] Bailey | Naringin is a major and selective clinical inhibitor of organic anion-transporting polypeptide 1A2 (OATP1A2) in grapefruit juice[END_REF][START_REF] Dresser | Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine[END_REF]. This inhibition leads to a reduced area under the plasma concentration time curve (AUC) of the OATP1A2 substrate fexofenadine when grapefruit juice is coadministered with orally taken fexofenadine. Grapefruit juice or orange juice at a concentration of 5% significantly inhibited the OATP2B1-mediated uptake of estrone-3-sulfate by 82% and 53% in HEK293 cells stably expressing OATP2B1 [START_REF] Satoh | Citrus juices inhibit the function of human organic anion-transporting polypeptide OATP-B[END_REF].

Furthermore it was shown in an in vitro study that several herbal extracts, such as Ginkgo biloba extracts, potently inhibited the OATP2B1-mediated uptake of estrone-3sulfate in HEK293 cells. These results suggest that coadministration of some dietary supplements may decrease the absorption of orally administered OATP2B1 substrates [START_REF] Fuchikami | Effects of herbal extracts on the function of human organic anion-transporting polypeptide OATP-B[END_REF].

However, there are no data showing the influence of the flavonoids apigenin, kaempferol, and quercetin, which are chemically related to the grapefruit juice flavonoid naringin, on the function of human intestinal OATPs such as OATP1A2 and OATP2B1.

Therefore, we investigated the potential influence of the flavonoids apigenin, kaempferol, and quercetin on the transport function of OATP1A2 and OATP2B1 in order to gain more insights regarding further possible mechanisms of food-drug or drug-drug interactions. Changes in function of the OATP1A2-and OATP2B1-mediated drug uptake by apigenin, kaempferol, and quercetin may influence the absorption and systemic exposure of OATP substrates. Considering the frequent intake of herbal preparations of Ginkgo biloba, vegetables, and fruits containing these flavonoids, the hypothesized interaction with the intestinal uptake transporters OATP1A2 and

Chemicals

[ 3 H]Sulphobromophthalein (14 Ci/mmol) was obtained from Hartmann Analytic (Braunschweig, Germany). Unlabeled sulphobromophthalein was purchased from Applichem GmbH (Darmstadt, Germany). Sodium butyrate was purchased from Merck KGaA (Darmstadt, Germany). Apigenin (purity ≥ 95.0%, HPLC), kaempferol (purity ≥ 96%, HPLC), quercetin (purity ≥ 98%, HPLC), and fexofenadine hydrochloride were purchased form Sigma-Aldrich Chemie GmbH (Munich, Germany). [ 3 H]Atorvastatin (10 Ci/mmol) and unlabeled atorvastatin were obtained from BioTrend Chemikalien GmbH (Cologne, Germany) and Toronto Research Chemicals Inc. (North York, ON, Canada), respectively. All compounds were dissolved in dimethyl sulfoxide (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). All other chemicals and reagents, unless stated otherwise, were obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany) and were of the highest grade available.

Generation of a HEK293 cell line stably expressing OATP1A2

The SLCO1A2 coding sequence (NM_134431.3) was cloned by reverse transcription reaction and subsequent polymerase chain reaction from human brain cDNA (Human Total RNA Master Panel II, Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) into the pcDNA3.1(+) vector (Invitrogen GmbH, Karlsruhe, Germany) and A c c e p t e d M a n u s c r i p t 10 subcloned into the retroviral vector pQCXIN (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France). Human embryonic kidney cells (HEK293) were transfected with the plasmid pQCXIN-OATP1A2 using a retroviral gene transfer and expression kit (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France). After geneticin (G-418; 500 µg/ml) treatment, single colonies were selected and characterized for OATP1A2 mRNA and protein expression using real-time PCR, immunofluorescence, and immunoblot analysis as previously described [START_REF] Glaeser | Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3[END_REF][START_REF] Mandery | Influence of cyclooxygenase inhibitors on the function of the prostaglandin transporter organic anion-transporting polypeptide 2A1 expressed in human gastroduodenal mucosa[END_REF]. The polyclonal rabbit anti human OATP1A2 antiserum was kindly provided by Professor Richard B. Kim (Division of Clinical Pharmacology, Department of Medicine, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada). The primer for real-time PCR analysis of SLCO1A2 mRNA were forward: 5'-AAGACCAACGCAGGATCCAT -3' and reverse: 5'-GAGTTTCACCCATTCCACGTACA -3' with a resulting amplicon size of 101 base pairs. The primers for the housekeeping gene β-actin were forward: 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and reverse: 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3' with a resulting amplicon size of 661 base pairs. The secondary antibodies for immunofluorescence and immunoblot analyses were used as previously published [START_REF] Glaeser | Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3[END_REF]. HEK293-VC (VC: vector control) cells were established by the same method using the plasmid lacking the insert for transfection.

Cell culture

A c c e p t e d M a n u s c r i p t 11 HEK293-OATP1A2 cells were cultured in minimum essential medium containing 10 % heat-inactivated fetal bovine serum, 2mM non essential amino acids, 500 µg/ml geneticin, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5 % CO 2 .

HEK293-OATP2B1 cells [START_REF] Bachmakov | Interaction of oral antidiabetic drugs with hepatic uptake transporters: focus on organic anion transporting polypeptides and organic cation transporter 1[END_REF][START_REF] Kraft | The prostaglandin transporter OATP2A1 is expressed in human ocular tissues and transports the antiglaucoma prostanoid latanoprost[END_REF] were cultured in minimum essential medium containing 10 % heat-inactivated fetal bovine serum, 800 µg/ml geneticin, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5 % CO 2 .The cells were routinely subcultured by trypsinization using trypsin (0.05 %)-EDTA (0.02 %) solution. All cell culture media supplements were obtained from Invitrogen GmbH (Karlsruhe, Germany).

Transport assays

The uptake experiments were performed as previously described [START_REF] Glaeser | Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3[END_REF][START_REF] Mandery | Influence of cyclooxygenase inhibitors on the function of the prostaglandin transporter organic anion-transporting polypeptide 2A1 expressed in human gastroduodenal mucosa[END_REF]. Briefly, HEK293-OATP1A2, HEK293-OATP2B1, and the respective HEK293-VC cells were seeded in poly-D-lysine coated 12-well plates at an initial density of 7.5x10 5 cells / well. After 24 h, cells were treated with sodium butyrate for 24 h prior to the uptake experiments in order to increase the levels of the recombinant protein [START_REF] Cui | Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells[END_REF]. First, cells were incubated with prewarmed (37 °C) uptake buffer (142 mM NaCl, 5 mM KCl, 1 mM K 2 HPO 4 , 1.2 mM MgSO 4 , 1.5 mM CaCl 2 , 5 mM glucose, and 12.5 mM HEPES, pH 7.3) containing a mix of radiolabeled (0.01 µM) and nonradiolabeled BSP or atorvastatin. HEK293-OATP1A2 cells were incubated with BSP (concentrations see below) and atorvastatin (5 µM) for five minutes and 10 minutes, respectively. OATP2B1 cells were incubated with BSP and atorvastatin for 10 minutes. HEK293-OATP1A2 and HEK293-VC cells were incubated with unlabeled fexofenadine (5 µM) for 5 minutes. Estimation of A c c e p t e d M a n u s c r i p t 12 the inhibitory constants (K i ) of flavonoids for OATP1A2 and OATP2B1 were performed using Dixon-Plot analyses [START_REF] Dixon | The graphical determination of K m and K i[END_REF]. The OATP1A2-mediated BSP net uptake was determined at concentrations of 2.5, 5.0 and 10 µM after an incubation of 5 minutes in absence or presence of increasing concentrations of flavonoids (apigenin: 1, 10, 100 µM; kaempferol: 10, 50, 100 µM for OATP1A2; 1, 10, 100 µM for OATP2B1; quercetin: 25, 50, 100 µM). For OATP2B1, the BSP net uptake was determined using concentrations of 0.5, 1.0 and 2.5 µM at 10 minutes of incubation in absence or presence of increasing concentrations of flavonoids (see above). After incubation cells were lyzed with 0.2% sodium dodecyl sulfate and the intracellular accumulation of radioactivity was determined in 500 µl of cells lysates by liquid scintillation counting (TriCarb 2800, PerkinElmer Life Sciences GmbH). The appropriate protein concentration of each well was determined in 25 µl of cells lysates by bichinchoninic acid assay (BCA Protein Assay Kit; Thermo Scientific). The net uptake was expressed as pmol*mg protein -1 *min -1 . Furthermore, a concentration dependent inhibition of OATP1A2-mediated fexofeandine transport was investigated at concentration of flavonoids ranging from 1 µM to 750 µM. The influence on the OATP1A2-and OATP2B1-mediated uptake of atorvastatin was investigated at a concentration range between 0,1 µM and 250 µM of flavonoids. Measurements were repeated for each concentration three to six times.

Quantitation of fexofenadine by LC-MS/MS

A c c e p t e d M a n u s c r i p t 13The quantitation of fexofenadine by LC-MS/MS was performed as previously described [START_REF] Hofmann | Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry[END_REF]. In brief, 10 µl of samples were prepared by addition of 90 µl of internal standard solution (MDL 026042, kindly provided by Aventis Pharma Deutschland GmbH, Frankfurt a.M., Germany) to 100 µl of the cell lysates. Analysis was performed using the triple quadrupole sytem API 4000™ LC-MS/MS System (Applied Biosystems, Toronto, ON, Canada). The HPLC column used was a Luna 3u CN 100A, New Column 100 x 2.0 mm with SecurityGuard Cartridges, CN 4 x 2.0 mm purchased from Phenomenex (Aschaffenburg, Germany). A mixture of 12 mM ammonium acetate and acetonitril (50:50, v/v) was used as mobile phase. The flow rate was set at 0.25 ml/min. The lower limit of quantitation was 0.5 ng/ml. The peak area ratio of fexofenadine to the internal standard was calculated using Analyst software v1.4.2 (Applied Biosystems, Darmstadt, Germany). The retention time of fexofenadine and of the internal standard was 2.0 min. The calibration curves were linear over the range from 0.5 to 100 ng/ml with a mean correlation coefficient of 0.9992. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH + ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. Coefficient of variation (CV) of the interassay variability (n = 15; quality controls containing 2.5, 25 and 100 ng/ml of fexofenadine) ranged between 4.0 and 6.6 %. The CV of the intraassay variability (n = 5) ranged between 3.4 and 7.4 %. The appropriate protein concentration of each well was determined in 25 µl of cells lysates by bichinchoninic acid assay (BCA Protein Assay Kit; Thermo Scientific). The OATP1A2-and OATP2B1-mediated net uptake was obtained by subtracting the uptake in VC cells from that in OATP1A2-and OATP2B1-expressing cells. The percentage of uptake inhibition was calculated from control experiments in the absence of flavonoids (100% uptake). The corresponding IC 50 values for inhibition of OATP1A2mediated fexofenadine uptake were calculated by fitting the data to a sigmoid doseresponse regression curve (GraphPad Prism 5.00 for Windows; GraphPad Software, San Diego, CA, USA). The IC 50 value is the concentration at which 50% inhibition of substrate uptake is obtained. To determine the type of inhibitory interaction of flavonoids with the OATP1A2-and OATP2B1-mediated BSP uptake, the uptake of 3 [H]BSP was determined in the presence of increasing concentrations of flavonoids. K i values were calculated by the method of Dixon, in which the reciprocal velocity, 1/V, is plotted against the concentration of the inhibitor. The OATP1A2-mediated uptake of BSP and atorvastatin was analyzed using an unpaired t-test with Welch's correction. The calculations were performed using GraphPad Prism 5.00. All data are presented as means ± S.E.M. A value of p≤ 0.05 was considered statistically significant.

Data and statistical Analysis

Characterization of a HEK293 cell line stably expressing OATP1A2

In the present study, we established a HEK293 cell line stably expressing the human uptake transporter OATP1A2. The selected cell clones of the HEK293-OATP1A2 and HEK293-VC cells were investigated regarding their expression, localization, and function of OATP1A2 using real-time PCR, immunoblot and immunofluorescence analysis. The HEK293-OATP1A2 cells were characterized by a significantly higher expression of SLCO1A2 mRNA and OATP1A2 protein compared to the control cells (figure 1a,b). The immunofluorescence analysis revealed that OATP1A2 is localized in the plasma membrane and to a lower extent in the cytosol of HEK293-OATP1A2 cells.

No staining was observed in HEK293-VC cells (figure 1c). The functional characterization showed a 2.4-fold (p < 0.0001, df = 25) and 6.9-fold (p < 0.0001, df = 5) higher uptake of BSP (5 µM) and atorvastatin (5 µM), respectively, into the HEK293-OATP1A2 cells compared to HEK293-VC cells (figure 1d,e) demonstrating that both compounds are substrates of OATP1A2.

Influence of flavonoids on the OATP1A2-and OATP2B1-mediated uptake of BSP

A c c e p t e d M a n u s c r i p t 16

In order to investigate the inhibitory effects of apigenin, kaempferol, and quercetin on the transport function of OATP1A2 and OATP2B1, the K i values of these flavonoids for inhibition of OATP1A2-and OATP2B1-mediated BSP uptake (measured by liquid scintillation counting) were determined. To evaluate the type of interaction of flavonoids with OATP1A2-and OATP2B1-mediated transport of BSP, Dixon plot analyses were performed (figure 2). The lines drawn for each concentration of substrate intersect at a single point above the x-axis indicating that all flavonoids influence both OATP transporters by competitive inhibition. The K i values of all three flavonoids determined from the Dixon plots were lower for OATP2B1 compared to OATP1A2 (figure 2).

Quercetin was the most potent inhibitor of the OATP1A2-mediated BSP uptake followed by kaempferol and apigenin (22.0 ± 5.9 µM, 25.2 ± 13.0 µM, and 32.4 ± 12.2 µM). For OATP2B1 quercetin was also the most potent inhibitor followed by kaempferol and apigenin (8.7 ± 0.1 µM, 15.1 ± 7.8 µM, and 20.8 ± 8.0 µM).

Influence of flavonoids on the OATP1A2-and OATP2B1-mediated uptake of drugs

To determine whether apigenin, kaempferol, and quercetin alter the OATP1A2mediated transport of fexofenadine (measured by LC-MS/MS), inhibition studies were performed (figure 3). Because we previously found that fexofenadine is not a substrate of OATP2B1 [START_REF] Glaeser | Intestinal drug transporter expression and the impact of grapefruit juice in humans[END_REF] only the impact of flavonoids on the OATP1A2-mediated uptake of fexofenadine was investigated. Apigenin was the most potent inhibitor of OATP1A2mediated fexofenadine transport with the lowest IC 50 value followed by kaempferol and The major findings of the present study were that the flavonoids apigenin, kaempferol and quercetin affect the transport function of OATP1A2-and OATP2B1-mediated uptake of BSP and of the drugs fexofenadine and atorvastatin.

Because most of the drugs used in pharmacotherapy are orally administered, the bioavailability of these compounds can already be influenced by intestinal drug transport and metabolism. Drug uptake transporters such as OATP1A2 expressed in the apical membrane of enterocytes [START_REF] Glaeser | Intestinal drug transporter expression and the impact of grapefruit juice in humans[END_REF] seem to influence the intestinal absorption of drugs such as fexofenadine [START_REF] Glaeser | Intestinal drug transporter expression and the impact of grapefruit juice in humans[END_REF][START_REF] Cvetkovic | OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of fexofenadine[END_REF][START_REF] Dresser | Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine[END_REF]. The absorption of fexofenadine, which is not metabolized in humans, is reduced when fexofenadine is coadministered with grapefruit juice due to an inhibition of OATP1A2-mediated fexofenadine uptake by the flavonoid naringin [START_REF] Bailey | Naringin is a major and selective clinical inhibitor of organic anion-transporting polypeptide 1A2 (OATP1A2) in grapefruit juice[END_REF]. Vegetables, fruits, and herbal drugs are rich of flavonoids and therefore, we investigated the influence of the flavonoids apigenin, kaempferol, and quercetin on the transport activity of intestinal transporters OATP1A2 and OATP2B1 which are expressed in the apical membrane of enterocytes [START_REF] Kobayashi | Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH-dependent A c c e p t e d M a n u s c r i p t 25 transport across intestinal apical membrane[END_REF][START_REF] Glaeser | Intestinal drug transporter expression and the impact of grapefruit juice in humans[END_REF].

The amount of apigenin, kaempferol, and quercetin is different within vegetables and herbal drugs. Interestingly, a study indicated that quercetin is more abundant compared to kaempferol in vegetables and fruits such as onions, lettuce, french bean, and apples [START_REF] Hertog | Content of Potentially Anticarcinogenic Flavonoids of 28 Vegetables and 9 Fruits Commonly Consumed in The Netherlands[END_REF]. High amounts of quercetin were detected in onions and apples with mean values of 347 and 36 mg / kg of fresh edible part, respectively [START_REF] Hertog | Content of Potentially Anticarcinogenic Flavonoids of 28 Vegetables and 9 Fruits Commonly Consumed in The Netherlands[END_REF]. Kaempferol showed the highest amounts in leek and endive with values of 30 and 46 mg / kg of fresh edible part, respectively. A further analyses of different beverages revealed that quercetin can A c c e p t e d M a n u s c r i p t 19 be found in red wine (highest concentration measured: 16 mg/l), apple juice, tomato juice, and different tea brands [START_REF] Hertog | Content of Potentially Anticarcinogenic Flavonoids of Tea Infusions, Wines, and Fruit Juices[END_REF]. Taken the high and regular consumption of these vegetables, fruits, and beverages into consideration, it seems comprehensible that the concomitant intake of such foods with drugs such as fexofenadine or atorvastatin may lead to a reduced absorption of these drugs. Therefore, the inhibition of intestinal OATPs by flavonoids could lead, in addition to the modification of cytochrome P450 enzymes and ABC-transporters, to an altered pharmacokinetic and pharmacodynamic of drugs.

The flavonoids apigenin, kaempferol, and quercetin and their glycosides are also the major constituents of herbal drug preparations of Ginkgo biloba. A study investigating different extracts of commercially available Ginkgo biloba preparations showed that in the analyzed extracts approximately 30 % of the active substances were flavone glycosides [START_REF] Kressmann | Pharmaceutical quality of different Ginkgo biloba brands[END_REF]. Most of these glycosides will be deglycosylated by a -glucosidase (lactase-phlorizin hydrolase) localized in the apical membrane of enterocytes [START_REF] Nemeth | Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans[END_REF]. This enzyme may increase the access of the deglycosylated apigenin, kaempferol, and quercetin to OATP1A2 and OATP2B1.

A pure preparation of 500 mg quercetin (Quercetin-500 Plus ® ), available as dietary supplement (http://supplementspot.com), taken with 300 ml of water could lead to a maximal intestinal concentration of 5514 µM. This shows that these supplements could lead to concentrations of quercetin in the intestine high enough to influence the transport activity of OATP1A2 and OATP2B1.

The presented data indicate that the flavonoids apigenin, kaempferol, and quercetin inhibit the OATP1A2-and OATP2B1-mediated uptake of substrates into HEK293 cells by a competitive mechanism. Therefore, these flavonoids could be also substrates of A c c e p t e d M a n u s c r i p t 20 OATP1A2 and OATP2B1. A study with Caco-2 cells provided some in vitro evidence that OATP2B1 may be involved in the translocation of quercetin from the apical to the basal compartment [START_REF] Chabane | Quercetin and naringenin transport across human intestinal Caco-2 cells[END_REF]. Quercetin seems to be a potent inhibitor of the OATP1A2-and OATP2B1-mediated transport represented by the determined K i values in this study.

Interestingly, quercetin is also a potent inhibitor of CYP3A4 and P-glycoprotein [START_REF] Patel | In vitro interaction of the HIV protease inhibitor ritonavir with herbal constituents: changes in P-gp and CYP3A4 activity[END_REF].

Considering that quercetin is also one of the most abundant flavonoids, foods and herbal preparations containing quercetin may be therefore associated with a high risk for food-drug or drug-drug interactions by influencing the intestinal drug uptake, metabolism, and excretion. Furthermore, the potency of inhibition of OATP1A2 and OATP2B1 by apigenin and quercetin was different for the investigated substrates BSP, fexofenadine and atorvastatin. Quercetin was the most potent inhibitor of the OATP1A2and OATP2B1-mediated BSP transport, whereas apigenin was the strongest inhibitor of the OATP1A2-mediated atorvastatin and fexofenadine transport. For the OATP2B1mediated atorvastatin transport no difference in the potency between apigenin and quercetin was observed. Some in vivo studies indicate an interaction between herbal drugs such as Ginkgo biloba preparations and the transporter-mediated absorption of drugs. A study investigated the influence of Ginkgo biloba extract on the pharmacokinetics of fexofenadine [START_REF] Robertson | Effect of Ginkgo biloba extract on lopinavir, midazolam and fexofenadine pharmacokinetics in healthy subjects[END_REF]. After an intake of Ginkgo biloba extract (120 mg bid) for two weeks a non significant reduction by 20 % in fexofenadine AUC was observed. However, it is not stated whether fexofenadine (120 mg) and Ginkgo biloba extract (120 mg) were given simultaneously. Therefore, it could be speculated that the lack of significance is caused by a non simultaneous administration of Ginkgo biloba extract and fexofenadine. In the present study we also established HEK293 cells stably expressing OATP1A2. In previous studies investigating the function of OATP1A2, Xenopus laevis oocytes or Hela cells transiently expressing OATP1A2 were used [START_REF] Cvetkovic | OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of fexofenadine[END_REF][START_REF] Kullak-Ublick | Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver[END_REF]. To the best of our knowledge, this is the first study using HEK293 cells stably expressing OATP1A2. In addition, we were able to show that atorvastatin is a substrate of OATP1A2 which was already published previously [START_REF] Knauer | Human skeletal muscle drug transporters determine local exposure and toxicity of statins[END_REF]. Because atorvastatin is a frequently used drug, the inhibition of OATP1A2-mediated atorvastatin transport by flavonoids should be considered as a possible new mechanism for food-drug or drug-drug interactions. However, atorvastatin is also a substrate of CYP3A4, P-glycoprotein, and OATP2B1. Considering all the possible changes in the pharmacokinetics of atorvastatin due to the modulation of CYP3A4, P-glycoprotein, OATP1A2 and OATP2B1 and also the pharmacogenomics of P-glycoprotein, OATP1A2, and OATP2B1 it becomes obvious that in vivo studies are necessary to clarify the clinical relevance of flavonoid-mediated drug interactions with atorvastatin. An in vivo study investigating the interaction between grapefruit juice and atorvastatin or pravastatin showed an increase in T max of atorvastatin and pravastatin [START_REF] Lilja | Grapefruit juice increases serum concentrations of atorvastatin and has no effect on pravastatin[END_REF]. Grapefruit juice was ingested for two days and on day 3 with the drugs, 0.5 h and 1.5 h after drug intake. It could be speculated that the inhibition of OATP1A2-and OATP2B1-mediated uptake of atorvastatin (OATP1A2 and OATP2B1) and pravastatin (OATP2B1) by grapefruit juice may be responsible for the observed effects. The HEK293-OATP1A2 cells showed a significantly higher uptake (2.4-fold) of BSP (measured by liquid scintillation counting) compared to HEK293-VC cells (*** p< 0.0001, unpaired t-test with Welch's correction). e) Uptake of atorvastatin (5 µM) into HEK293-OATP1A2 and HEK293-VC cells. The HEK293-OATP1A2 cells showed a significantly higher uptake (6.9-fold) of atorvastatin (measured by liquid scintillation counting) compared to HEK293-VC cells (*** p< 0.0001, unpaired t-test with Welch's correction).

All data are presented as means ± S.E.M.

  and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.Flavonoids represent the most abundant polyphenols in vegetables, fruits, and plants.

A c c e p t e d M a n u s c r i p t 8 OATP2B1

 8 might be an important determinant of intraindividual variability of drug disposition.

  3 ± 1.5 µM, 12.0 ± 1.2 µM, and 12.6 ± 1.3 µM). Secondly, the influence of apigenin, kaempferol and quercetin on the OATP1A2-and OATP2B1-mediated atorvastatin transport (measured by liquid scintillation counting) was investigated (figure4). Apigenin and quercetin were the most effective inhibitors for the OATP1A2-and OATP2B1-mediated atorvastatin uptake. For OATP1A2 the IC 50 values of apigenin and quercetin were 9.3 ± 1.5 and 13.5 ± 1.7 µM. The IC 50 values of apigenin and quercetin for OATP2B1 were with 13.9 ± 1.6 µM and 14.1 ± 1.3 µM very similar. Kaempferol showed the lowest inhibitory potency of the OATP1A2-and OATP2B1-mediated atorvastatin transport compared to apigenin and quercetin (IC 50 for OATP1A2: 37.3 ± 1.6 µM, IC 50 for OATP2B1: 20.7 ± 1.8 µM).

A c c e p t e d M a n u s c r i p t 21 Our

 21 data are in line with a previous publication showing that Ginkgo leaf extracts and quercetin-3-glucoside, quercetin-3-rutinoside, kaempferol-3-glucoside, and kaempferol-3-rutinoside inhibited the OATP2B1-mediated transport of estrone-3-sulfate in HEK293cells stably expressing OATP2B1[START_REF] Fuchikami | Effects of herbal extracts on the function of human organic anion-transporting polypeptide OATP-B[END_REF].

A

  c c e p t e d M a n u s c r i p t 22 Taken together, our study shows that the inhibition of OATP1A2-and OATP2B1mediated transport by apigenin, kaempferol, and quercetin should be considered as a potential, new mechanism for food-drug or drug-drug interactions. The clinical relevance of the observed interactions needs to be elucidated in clinical interactions studies.

Figure 1 :

 1 Figure 1: Characterization of the HEK293-OATP1A2 cell line overexpressing OATP1A2.

Figure 2 :

 2 Figure 2: Inhibition of OATP1A2-and OATP2B1-mediated BSP transport (measured by

Figure 3 :

 3 Figure 3: Inhibition of OATP1A2-mediated fexofenadine transport (measured by LC-

Figure 4 :

 4 Figure 4: Inhibition of OATP1A2-and OATP2B1-mediated atorvastatin (5 µM and

Figure 1

 1 Figure 1

Figure 1 grayscale

 1 Figure 1 grayscale

Figure 2

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 Page

 4 Figure 4

7 Acknowledgements This work was funded by a grant of the Deutsche Forschungsgemeinschaft [DFG GL 588/2-1] and the German Federal Ministry of Education and Research [grant, InnoProfile 03IP612].

Abbreviations


Cosima Rughiniş
email: ethnic@surrey.ac.uk

Quantitative tales of ethnic differentiation. Measuring and using Roma / Gypsy ethnicity in statistical analyses

Keywords: Ethnicity, Measurement, Causality, Survey, Roma, Gypsy ethnicity, measurement, causality, survey, Roma, Gypsy

Quantitative tales of ethnic differentiation. Measuring and using Roma / Gypsy ethnicity in statistical analyses

Cosima Rughinis

Conceptual issues

The sociological understanding of ethnicity has gradually moved away from its taken-for-granted understanding as a way of classifying social groups with clear cultural or even biological borders. This shift within the discipline has become more visible after the seminal work of [START_REF] Barth | Little Brown & Co BERGESEN, ALBERT JAMES 1977 'Neo-Ethnicity as Defensive Political Protest[END_REF], 'Ethnic groups and boundaries'. Barth challenges the definition of ethnic groups as 'culturebearing units' based on biological self-perpetuation, focusing on the maintenance of social borders as the key definition of ethnicity (idem, pp. 10-15). Along the same line, the concept of 'emerging ethnicity' proposed by [START_REF] Yancey | Emergent Ethnicity: A Review and Reformulation[END_REF] relies on an understanding of ethnicity as "crystallized under conditions which reinforce the maintenance of kinship and friendship networks" (p. 392), while Bergesen (1977, p.823) adds that it 'ebbs and flows' due to political mobilization. This perspective has been further

developed by scholars such as [START_REF] Ballard | Race, ethnicity and culture[END_REF], Brubaker (2002), Vermeersch (2003), Brubaker, Loveman and Stamatov (2004), Hale (2004), Wacquant (2005), Omi and Winant (2007), and [START_REF] Wimmer | The Making and Unmaking of Ethnic Boundaries: A Multilevel Process Theory[END_REF]. This perspective means, for Brubaker, 'taking as a basic analytical category not the "group" as entity but groupness as a contextually fluctuating conceptual variable ' (2002, pp. 167-68, emphasis in original). The social processes of ethnic differentiation situate individuals in a social landscape that they need to navigate. At the individual level,

"[E]thnic identity" (or ethnicity) is that set of personal points of reference, thick and thin, that involve what we call "ethnic" distinctions between people. An "ethnic group" is thus a set of people who have common points of reference to these ethnic indeed have these things in common and that these similarities are captured by a label, the ethnic group's name" (Hale 2004, p. 473).

This theoretical perspective has important methodological consequences. Firstly, it requires a measurement model for ethnicity that departs from the conventional categorical measurement by ticking the best fitting ethnic label. Secondly, it points to the contextual embeddedness of ethnic identity, and consequently it complicates the relationship between ethnicity and variables usually interpreted as controls, such as age, education, or occupation. Last but not least, it challenges the unidirectionality of causal influences from ethnicity to other social phenomena, which may play a part in the process of ethnic differentiation. If ethnic distinctions are associated with differences in schooling, eating choices, or church attendance, for example, it is plausible that these differences are in turn used to define and re-create ethnic classifications.

Research models

One can differentiate three main uses of ethnicity in quantitative models.

Firstly, 'ethnic disadvantage models' measure a given inequality in access to resources or in risk incidence. Secondly, 'discrimination models' attempt to isolate discrimination from other sources of inequality, and to measure it. The third type, which will be referred to as 'ethnic difference' models, pursues the different relationships between ethnicity and other social phenomena which are theoretically linked to the process of ethnic differentiation. et.al. (2006, pp. 51-52), the use of multivariate analysis to measure discrimination is vulnerable to several sources of bias, including the 'omitted variable bias', when relevant controls are not included, the 'included variable problem', when some controls already capture variation due to discrimination, and the 'diverting variable bias', when controls include variables that should not be controlled, according to the normative concept of discrimination employed.

Ethnic difference models range from simple models that trace differences between ethnic groups in language use or religious denomination, While in ethnic gap models ethnicity is usually measured as a categorical variable, used to chart unequal distributions of resources or risks, in ethnic difference models the focus is on processes of ethnic delineationalso by including more complex measurements of ethnicity, as discussed below.

Measurement issues

Contextual and situational ethnicity

As any other social distinction, ethnicity is shaped in a given social context. This is a serious challenge to cross-cultural harmonization of ethnic terminology (Aspinall 2007, pp. 58-62) and to cross-cultural research using ethnicity. Moreover, contexts that shape ethnic identities vary not only across societies, but also across transient situations in a person's life (Stayman and Deshphande 1989). Public self-identification may be outright strategic, as people may choose their ethnic label to suit their needs in a specific interaction [START_REF] Bovenkerk | The Internal-External Ethnic Identity Measure: Factor-Analytic Structures Based on a Sample of Chinese Americans[END_REF]. This has important measurement consequences, especially if the interview situation is thought to influence ethnic self-identification, as in the case of the Roma.

Measurement models

Aspinall (2007, p. 60) distinguishes two main styles in measuring ethnicity.

On the one hand, there is the analytical approach, measuring the components (1992) builds the 'Multi-Group Ethnic Identity Measure' (MEIM). Subsequent research with the MEIM scale has identified 'ethnic affirmation' and 'ethnic exploration' as two main dimensions of ethnicity (Romero and Roberts 1998, p. 643).

Other multi-dimensional measures of ethnicity are group-specific. For example, Kwan (2000) constructs a scale for measuring the ethnic identity of Chinese-Americans, understood as consisting of internal (cognitive, moral and affective) and external aspects (such as language use, observance of traditions or friendship networks). The Cross Racial Identity Scale (CRIS) for the African American ethnicity [START_REF] Vandiver | Ethnic minority identity and movement politics: The case of the Roma in the Czech Republic and Slovakia[END_REF], Worrel et.al. 2006) are as a rule complemented with dimensional models -such as intensity of belief, religious participation, or private practice. On the contrary, in the studies that use ethnicity as an independent variable, the categorical measure (identification with a specific ethnic label) is usually the sole indicator of ethnicity, while dimensional measures constitute the exception rather than the rule. As a reflection of the same underlying difference in understanding the two phenomena, religiosity is often analyzed as a dependent variable, while ethnicity is most often analyzed as an independent variable.

Model specification issues

Ethnic affiliation and the socio-demographic controls

Control variables in multivariate analysis have the function of isolating the covariation of ethnicity from the covariation of other variables with the dependent variable. Therefore, the choice of control variables plays a decisive role in interpreting the resulting coefficient for ethnic affiliation. As discussed above, some research models impose clear requirements on control variables.

Discrimination models require strict control of relevant competence and preference covariates. In public health studies, ethnicity may be used as an indicator (or proxy) of inherited biological vulnerabilities (Singh 1997, p.307).

In this case, controlling the influence of relevant social confounders and On the contrary, in ethnic difference models with an explanatory focus, ethnic affiliation represents an outcome in a process of social differentiation that builds upon other differences -such as class, gender, or generation distinctions -and in turn shapes these differences. Along similar lines of reasoning, [START_REF] Steinberg | Data Analytic Strategies in Research on Ethnic Minority Youth[END_REF] and [START_REF] Smith | Learning to Live With Complexity: Ethnicity, Socioeconomic Position, and Health in Britain and the United States[END_REF] criticize the overconfident use of socio-economic controls in models that use ethnicity or race variables. Not only are such control indicators often sketchy, allowing for residual confounding; it is also important that 'socioeconomic disadvantages and exclusionary social practices are, in this view, mutually constitutive' [START_REF] Smith | Learning to Live With Complexity: Ethnicity, Socioeconomic Position, and Health in Britain and the United States[END_REF](Smith , p. 1696) -and it is thus impossible to conceptually isolate the influence of ethnicity from the influence of socioeconomic position.

Ethnicity as moderating influence

Ethnicity is likely to be a moderator variable in quantitative modelssignificantly altering the influences of other social attributes [START_REF] Steinberg | Data Analytic Strategies in Research on Ethnic Minority Youth[END_REF]. The moderating influences of ethnicity may be explored by estimating the same model for different ethnic categories of respondents, in order to see whether the configuration of relationships is stable or variable.

Estimates of the size of moderating influences may be obtained by including interaction terms in regression models in pooled samples, together with more detailed tests of interactive hypotheses (Kam and Franzese 2007).

' Direct' or 'residual

Data analysis issues

In ethnic gap models, regression models are the tool of choice, allowing for a measurement of the 'direct effect' of ethnicity after the introduction of proper controls.

In ethnic difference models, given the conceptual linkage of ethnicity to other differentiation processes, analyses that aim to realize theoretically informed evaluations of the causal influence of ethnicity should trace the multiple pathways that connect ethnicity or its dimensions with the dependent variable. Path or structural equation models are better able to conceptualize the mediated associations between ethnicity and the dependent variable than regression models, which focus the attention on the residual associations with the dependent variable. Also, special attention should be paid to the moderating influences of ethnicity -that is, the influence of ethnicity on the effects of other factors included in the model. Kaladydjieva, Gresham andCalafell 2001, Morar et. al. 2004). In what is probably the most controversial field of ethnic gap models, due to its methodological shortcomings [START_REF] Block | How heritability misleads about race[END_REF] and to its relationships with racist

Measurement issues

The literature on Roma ethnicity measurement has been dominated by the debate on the use of hetero-attribution of Roma ethnicity as an indicator of Roma ethnicity [START_REF] Babusik | Legitimacy, Statistics and Research Methodology Who Is Romani in Hungary Today and What Are We (Not) Allowed to Know About Roma[END_REF]). This concern has been prompted by widespread politically-laden debates on the 'true' number of Roma and by the observation that Roma people often prefer not to declare a Roma affiliation in censuses and official contexts (Clark 1998), which led to the assumption that a similar reticence may bias unofficial survey measurements. The use of heteroattribution as an indicator of ethnicity has gradually subsided, partly as a result of the critical approach by [START_REF] Ladányi | The social construction of Roma ethnicity in Bulgaria, Romania and Hungary during market transition[END_REF], [START_REF] Székeli | Ambitious education: the role of family, school and friends in the development of successful Romany life courses[END_REF]Simon (2004), and[START_REF] Ahmed | Approaches to Developing an Improved Cross-National Understanding of Concepts and Terms Relating to Ethnicity and Race[END_REF][START_REF] Ahmed | Approaches to Developing an Improved Cross-National Understanding of Concepts and Terms Relating to Ethnicity and Race[END_REF], who analyzed discrepancies in hetero and auto-attribution, highlighting the socially contingent processes of ethnic labeling. The debate continues to develop, also covering the legitimacy of Roma observers or local experts using hetero-attribution [START_REF] Babusik | Legitimacy, Statistics and Research Methodology Who Is Romani in Hungary Today and What Are We (Not) Allowed to Know About Roma[END_REF][START_REF] Prieto-Flores | Does the canonical theory of assimilation explain the Roma case? Some evidence from Central and Eastern Europe[END_REF], and hetero-attribution for the community as a whole [START_REF] Sandu | Roma Social Mapping. Targeting by a Community Poverty Survey[END_REF].

The dominant measurement model of Roma ethnicity is categorical, using the indicator of self-affiliation with such ethnic labels as Roma, Gypsy,

Roma ethnicity and church attendance in present-day Romania

This section presents a path model using self-declared Roma ethnicity, alongside other variables, to explain church attendance. Given the sketchy specification of the model and its methodological shortcuts, it is useful as a methodological example for interpreting paths and coefficients involving ethnicity, rather than a substantial contribution to understanding religious behaviors.

The analysis relies on two surveys on Roma people from Romania:

The Roma Inclusion Barometer (RIB 2006) and the Work Attitudes Survey (WA 2008) (see Table 2). The two surveys include Roma samples designed with the same methodology [START_REF] Sandu | Multidimensional Model of Racial Identity: A Reconceptualization of African American Racial Identity[END_REF], thus facilitating comparability of results.

(Table 2

Model limitations

Several variables are missing from the available datasets, although they could account for the variation of church attendance: socio-economic status, church accessibility, measured by the time and financial costs involved in reaching the church, and the community norms and personal ideas with respect to church attendance, childhood religious socialization, personal religious worldviews, relevance of religious services, local church policy, or relationships with other churchgoers.

In order to simplify the presentation of model results, the ordinal level variables (education, prayer and church attendance) have been analyzed as as metric. This approximation is justified by the exploratory and illustrative purposes of the model, which does not require precise estimates. Also, for brevity reasons, moderated influences are only explored by means of a comparison of models, without estimating interaction terms. (Figure 1 around here)

Model variables and sampling weights

The comparative path model includes two religiosity predictors and several socio-demographic variables, as detailed in Table 3.

(Table 3 around here)

The distribution of church attendance on ethnic affiliation across the two surveys (Table 4) is quite similar, indicating lower attendance on the part of the Roma people.

(Table 4 around here)

The two religiosity items available in both surveys are the frequency of prayer outside religious service, and affiliation to a Neo-Protestant denomination. The total sample included in the analysis is composed of the weighted non-Roma respondents from the national sample, plus the weighted Roma respondents from the Roma sample 4 . The path model has been estimated using MPlus v.5. Because of the need for sampling weights, the analysis used the MLM estimator (Muthén and Muthén 2007, p. 457).

Model estimates

In this example, in both surveys (see Table 5 andTable 6), the main predictor of church attendance is frequency of prayer (positive), indicative of private religious practice, followed by masculine gender (negative), Neo-Protestant affiliation (positive) and Roma ethnicity (negative).

(Table 6 around here)

The main predictors of frequency of prayer are age (positive), followed by masculine gender (negative), Neo-Protestant affiliation (positive) and Roma ethnicity (negative). Because Roma ethnicity is positively associated with Neo-Protestant affiliation, there is also a positive indirect and non-directional association between Roma ethnicity and church attendance, mediated by the Neo-Protestant affiliation.

(Table 7 around here)

In Table 8 one can see the 'total effect' of ethnicity on church attendance, with its mediated and direct components. Only a small fraction of the difference in church attendance between Roma and non-Roma is explained by the (Table 8 around here)

Moderation effects

A comparison of the models estimated in the non-Roma sample with the Roma sample reveals that ethnicity moderates the influences of gender (in WA 2008) and education on church attendance -although some of these coefficients may not be statistically significant. For Roma and non-Roma alike, men have lower levels of private religious practice compared to women. Still, in WA 2008 the residual gender influence on church attendance is not statistically significant in the Roma sample, unlike the non-Roma sample. This may indicate external reasons that impose a higher uniformity on church attendance across gender categories -such as exclusion from the broader church community.

Education has a weakly positive but statistically significant residual influence on church attendance for the Roma sample, while it has no residual influence in the non-Roma sample. At the same time, education is not directly associated with frequency of prayer for the Roma. If this pattern of associations is indeed more than a statistical artifact, it may indicate an inclusionary influence of education for Roma in mixed communities. Its positive association with Roma church attendance may reflect stronger relevance of religious services for the Roma people with higher levels of There is also an interesting difference concerning the proportion of explained variance for wealth and education across models: while in the non-Roma samples the estimated R square is around 25% for education and 38%

for wealth, in the Roma samples the estimated R square is around 5% for education and 17% for wealth. This difference arises mainly because of age and urban locality, which are powerful predictors in the non-Roma samples but weak predictors in the Roma samples. This indicates that, unlike the non-Roma, the younger generations of Roma people have improved less their educational and material achievements compared to the older generations, when controlling for the other variables. Also, Roma people seem to take less advantage of social and economic opportunities offered by urban localities.

Discussion

The path analysis indicates that ethnicity and church attendance are connected by several circuits, with mixed positive and negative paths. For example, in present-day Romania, Roma ethnicity is associated with an increased probability of Neo-Protestant affiliation, which in turn increases church attendance. On the other hand, the mediated association of ethnicity with attendance through private religious practice (prayer) is negative, and the residual association of Roma ethnicity with attendance is also negative.

The proposed model could be used as an ethnic gap model by a researcher interested in assessing the difference in religious participation between Roma and non-Roma people. Such a difference could be measured

Conclusions

There seems to be a gap between the current theoretical understanding of ethnicity, as a result of successive moves in a process of social differentiation, An in-depth discussion of processes of ethnic differentiation could be impractical, for example, in ethnic disadvantage models for health risks, or in models that are dedicated to measuring ethnic discrimination on the labor market.

Quantitative researchers improve the clarity and relevance of their use of ethnicity variables to the extent that they specify the descriptive or explanatory aims of the analysis, and the choice of control and mediating variables. As previously illustrated for church attendance, the very same direct coefficient for ethnicity in a path model may have a completely different meaning in an 'ethnic disadvantage' model, compared to an 'ethnic difference' model.

In addition to the usual requirements for theoretical relevance and methodological clarity, the use of ethnicity in quantitative models is also subject to political considerations. Ethnic differentiation is interlinked with power relations and political strategies. Therefore, researchers that publish results related to ethnicity are, perforce, players in the wider social process of ethnic border creation and maintenance. A scientific measurement of ethnic differences may be more consequential outside the research community than within it. The most obvious ethical and political risks related to quantitative research using ethnicity refer to the racialization of ethnic groups, by consolidating stereotypical portraits with the cement of scientific authority and

  Quantitative tales of ethnic differentiation. Measuring and using Roma / Gypsy ethnicity in statistical analyses

  social world and who perceive that they

  are not mutually exclusive, because an ethnic disadvantage or a discrimination model may also investigate a phenomenon which is relevant to ethnic differentiation. Also, what counts as a resource or a risk is a matter of normative choice: any feature assumed to be desirable may be analyzed from an 'ethnic gap' perspective -for example, wealth, education, health, but also consumption of a specific product, religious belief or participation, or adherence to a given tradition.The main focus of ethnic disadvantage and discrimination models is precise measurement, and a secondary focus may consist in the identification of relevant predictors. Discrimination models using cross-sectional survey data also face a complicated challenge in model specification. All relevant variables related to the respondents' competence and preference must be controlled for in order to identify unequal treatment. Since discrimination involves unequal treatment for persons who have the same relevant qualifications and claims, the normative issue which arises is what counts as a 'relevant' qualification and what counts as an 'irrelevant', thus discriminatory, criterion. Therefore, as discussed in detail by Hanquinet

  models that aim to study the influences of ethnic affiliation on intergroup attitudes, parenting styles, religious participation, conflict management, and so on.

  sense as a precision tool, aimed to explore the distribution patterns of a given inherited feature.

  views and policies, in particular eugenics -namely, intelligence research,[START_REF] Rushton | General mental ability in South Asians: Data from three Roma (Gypsy) communities in Serbia[END_REF] use an ethnic disadvantage model to discuss differences in 'general mental ability', specifically measured by Raven's matrices, between Roma and non-Roma respondents in Serbia.Sociological analyses also start, most frequently, from an ethnic disadvantage model. There is considerable literature on quality of life (includingZamfir and Zamfir 1993[START_REF] Zamfir | Romii în România, Bucharest, Editura Expert 2 Details about the survey are available on the project website[END_REF], UNDP 2003, Fleck and Rughiniş 2008), and on poverty (such as Emigh and Szelényi 2001, Ladányi and Szelényi 2002). Some pieces of research are also examples of ethnic difference models, insofar as they study the contribution of poverty to the broader process of ethnic differentiation (Emigh and Szelényi 2001, Ladányi and Szelényi 2002, Fleck and Rughiniş 2008). Perceived discrimination is investigated within the framework of an ethnic disadvantage model in the FRA EU-MIDIS research (2009). Discrimination models are relatively rare; examples include Kertesi and Kézdi (s.a.), Mete (2003), and Drydakis (2008).Ethnic difference models of the process of Roma vs. non-Roma ethnic differentiation have addressed the issue of hetero-attribution of Roma ethnicity[START_REF] Ladányi | The social construction of Roma ethnicity in Bulgaria, Romania and Hungary during market transition[END_REF], Csepeli and Simon 2004[START_REF] Ahmed | Approaches to Developing an Improved Cross-National Understanding of Concepts and Terms Relating to Ethnicity and Race[END_REF].[START_REF] Székeli | Ambitious education: the role of family, school and friends in the development of successful Romany life courses[END_REF] and[START_REF] Prieto-Flores | Does the canonical theory of assimilation explain the Roma case? Some evidence from Central and Eastern Europe[END_REF] address the issue of upward Roma mobility and assimilation, while Durst (2002) explores downward mobility and its consequences on fertility. Other ethnic difference models explore the influence of Roma identity on ethnic tolerance(Tufiş 2001) or on political mobilization (Fox 2001).

  More detailed categorical measures introduce multiple affiliations(Fleck and Rughiniş 2008), or an additional classification by tribe (Ro. neam),[START_REF] Zamfir | Romii în România, Bucharest, Editura Expert 2 Details about the survey are available on the project website[END_REF] Zamfir 1993, Zamfir and[START_REF] Zamfir | Romii în România, Bucharest, Editura Expert 2 Details about the survey are available on the project website[END_REF]. Using a formative model,Székeli, Csepeli and Örkény (2003, p. 59) develop a classification of Roma ethnicity by combining the respondents' ethnic affiliation with information about their ethnic background. I did not find any instance of a dimensional model for measuring Roma ethnicity.

  patterns of ethnic differentiation between the Roma and the non-Roma, one should also expect ethnicity to have a moderating influence on other variables -such as gender, religious confession or education.

Figure 1

 1 Figure 1 illustrates the path model that connects Roma ethnicity with church

  in the model, after controlling for Neo-Protestant affiliation. Therefore, the 'direct effect' of ethnicity is likely to include paths mediated by variables which are missing from the model -such as religious beliefs, religious socialization, community relations, and proximity to a church.

  their increased acceptance by the majority of non-Roma churchgoers.

  in statistical models, usually as a categorical indicator, with scarce theoretical justification. Still, if one takes into account the diversity of research models that use ethnicity, a certain amount of variability in the degree of theoretical grounding and interpretation of ethnic differences is to be expected.

around here)

	Ethnic and Racial Studies	Page 14 of 39
	Since this model is built primarily as a tool for methodological exploration and
	not for precise measurement, it is best conceptualized as an 'ethnic difference'
	model, looking for those results that offer valuable information on the process
	of ethnic differentiation, and its consequences on church attendance in
	Romania.
	A considerable body of qualitative research indicates that conversion F to Pentecostalism has had significant consequences for the delineation of o r Roma life trajectories in Romania (Kiss 2009, Fosztó 2009). Therefore,
	P Pentecostal confession is expected to be highly influent on religious
	e r e participation among the Roma.
	R
	e
	v i e
	w
	O n l church frequently. Therefore, although it may not seem intuitive, it does make
	y sense to identify a path that links ethnicity and church attendance and is
	mediated by age.
	While in this example some paths are modeled as unidirectional, such
	as the influence of ethnicity on church attendance via education, wealth or
	Model specification
	15

URL: http://mc.manuscriptcentral.com/rers ethnic@surrey.ac.uk Previous research on church attendance in Romania

[START_REF] Voicu | România religioasă, Iaşi: Institutul European WACQUANT, LOÏC 2005 'Race as civil felony[END_REF]

has shown that its main predictors, besides religiosity, are childhood religious socialization, age and gender. While age and ethnicity seem, prima facie, two unrelated variables, they are connected by processes which are part of ethnic differentiation. Roma people are younger, on average, because of lower life expectancy, higher fertility rates and, maybe, differential migration patterns.

Such life course events are part of an ethnicized life trajectory. One may tentatively say that in Romania the Roma do not get to be old enough to attend prayer, others are modeled as partly non-directional, such as the influences of ethnicity via Neo-Protestant affiliation or age.

attendance Prayer Gender Education Self-id. Roma Age Wealth Urban Neo- Protestant Page 32 of 39 URL: http://mc.manuscriptcentral.com/rers ethnic@surrey.ac.uk Ethnic and Racial Studies

  The model includes all possible paths between variables, and therefore it reproduces the empirical covariance structure.

	Page 31 of 39	Ethnic and Racial Studies Ethnic and Racial Studies	Page 22 of 39
		quantitative precision. This is an obvious risk for quantitative investigations of ZAMFIR, ELENA and ZAMFIR, CĂTĂLIN (Eds.) 1993 łiganii -Figures and Tables
		ethnic differences in 'general mental ability', or deviant behaviors, for Între ignorare şi îngrijorare, Bucharest, Alternative
		example -but also a collateral damage of repeated measurement of ethnic
		gaps in fields like education, employment, or gender equality. As activists in Figure 1. Path model specification
		the civic Roma movement are only too aware, ethnic disadvantage models run
		F o r the risk of portraying members of discriminated ethnic minorities as 'bearers of problems', rather than as resourceful and creative people. Therefore, quantitative research using ethnicity faces several F o r F o r F o r F o r F o r F o r F o r F o r F o r F o r
		P requirements, related both to the theoretical appropriateness of the model, and P P P P P P P P P P
		e e r e e e e e e e e e e to the public understanding of scientific results. In order to de-essentialize ethnicity, heterogeneity within ethnic categories should be systematically e r e r e r e r e r e r e r e r e r e r	Church
		R e investigated. Model estimates in separate ethnic samples are a useful tool for R R R R R R R R R R exploring variability among people that affiliate to a certain ethnic e e e e e e e e e e
		v i e community. Moreover, it is time for ethnicity to become widely understood as a result of choice, not only of ascription. This means, on the one hand, that the v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e
		w directionality of causal relationships between ethnicity and other phenomena w w w w w w w w w w
		O n l should be a matter of theoretical reflection. On the second hand, a clear research priority consists in the development and systematic use of formative and dimensional models for ethnicity measurements, including Roma / Gypsy O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l
		y ethnicity, to complement the current categorical ones. Current standards of y y y y y y y y y y
		measurement and model specification in religiosity research may offer
		valuable reference points for the road ahead.
		23 31 33

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Table 1 .

 1 Uses of ethnicity in three types of quantitative models

	Models	Ethnic gap		Ethnic difference
		Ethnic disadvantage	Discrimination
	Dependent	-Valuable resource or	-Valuable resource	-Variables linked to
	variable	behavior	dependent on others'	the process of ethnic
			decisions	differentiation
	Research focus	-Measurement of	-Measurement of	-Description of
		inequalities	discrimination	ethnic
		-Identification of risk	-Identification of risk	differentiation
		factors	factors	processes
				-Estimates of causal
				influences of
				ethnicity on the
				dependent variables
	Measurement of	-Categorical or	-Categorical or	-Categorical,
	ethnicity	formative	formative	formative or
				dimensional
	Control	-Frequent use of socio-	-Required control of	-Depending on the
	variables	demographic controls	competence and	research question
		-Increasing use of	preference variables	and theoretical
		'reasoned action' models	-Risks: 'omitted	model
			variable bias',
			'included variable
			problem', 'diverting
			variable bias'
	Moderating	-Decreasing returns for	-Are not related to the	-Highly relevant for
	influences	different forms of capital,	research question	the study of ethnic
		or differential influence		differentiation
		of risk factors
	'Direct effects'	-Provides measurements	-Main research focus:	-Causally
		of ethnic disadvantage,	they provide	meaningful for
		without explanatory	measurements of	phenomena which
		force	ethnic discrimination,	are closely linked to
		-Relevant if ethnicity is	if the model is	ethnic
		used as an indicator	correctly specified	differentiation
		(proxy) for biological		-Residual (un-
		heritage, in public health		explained) measure
		studies		of association for
				more distant
				phenomena

Table 4 .

 4 Church attendance recoded on 4 values -dependent variable in the regression model

		RIB 2006		WA 2008
				Non-
		Non-Roma	Roma	Roma	Roma
	1. Less often or never	16	29	16	24
	2. On Holy Days	32	31	33	39
	3. Monthly	27	17	28	18
	4. Weekly	24	20	22	18
	Missing (DK/NR)	1	3	1	1
	Total	100	100	100	100
	Cases	1182	1387	2336	999

Table 5 .

 5 Distribution of religious confessions in Romania. Source: RIB 2006

	Page 35 of 39	Ethnic and Racial Studies
	Religious confession Percent (%)
	Orthodox	88.7
	Romano-Catholic	3.8
	Protestant	3.1
	Neo-Protestant	2.4
	Greek-Catholic	1
	Other	0.6
	No religion	0.3
	Total	100

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