Nutrient Metabolism—Research Communication

More documents

Recommendations

Info

3228 lected for 24 h after the beginning of the meal. All of the biological samples were stored at 20°C until analysis. HPLC analysis. Plasma samples were acidified (to pH 4.9) with 0.1 volume of 0.58 mol/L acetic acid and incubated at 37°C for2h (plasma) or for 30 min (urine) with or without -glucuronidase/ sulfatase (100 U/L). Plasma proteins were precipitated by the addition of 500 L of methanol/200 mmol/L HCl and the extract was centrifuged for 50 min at 14000 g. After this extraction step, 20 L of supernatant was injected and analyzed by HPLC. The concentrations of conjugated derivatives were estimated as the difference between the concentrations of phloretin measured before and after the enzymatic treatment. For the analysis of phloretin in plasma, plasma standards containing 0, 0.25, 0.5, 1, 5 and 10 mol/L added phloretin were prepared. The standards were treated exactly as the samples (hydrolysis and extraction). Day-to day-variation and withinday variation for phloretin from all matrices were 10%. The recovery of phloretin from all matrices reached 97%. The limit of detection for phloretin was 25 nmol/L. The HPLC analysis was performed using isocratic conditions (1.5 mL/min) with a 150 4.6 mm Hypersil BDS C18–5 m (Life Sciences International, Cergy, France). The mobile phase consisted of 30 mmol/L NaH 2PO 4 buffer, pH 3, containing 25% acetonitrile. The detection was performed using a multielectrode coulometric detection (4-electrodes CoulArray, Eurosep, France) with potentials set at 375, 500, 600 and 700 mV. To visualize the conjugated forms of phloretin and to detect the presence of phloridzin, the chromatographic conditions were as follows (flow rate 1 mL/min): 0–2 min, solvent A 85%/solvent B15%; 2–22 min, solvent A 85%/solvent B15% 3 solvent A 63%/solvent B 37%; 22–28 min, solvent A 63%/solvent B 37%; 28–32 min, solvent A 63%/solvent B 37% 3 solvent A 85%/solvent B15%; solvent A contained water and 30mmol/L NaH 2PO 4 buffer, pH 3, and solvent B, acetonitrile. Glucose measurements. The glucose concentration in urine, sampled from bladder, was determined by an enzymatic procedure as described by Bergmeyer et al. (15). Data analysis. Values are means SEM. Significance of differences between means was determined by ANOVA and the Student- Newman-Keuls multiple comparison test (Instat; GraphPad, San Diego, CA). Differences were considered significant at P 0.05. RESULTS When rats were fed a meal containing phloridzin, this compound was not recovered in plasma that was not subjected to enzymatic hydrolysis. However, unconjugated phloretin was detected in the plasma of rats fed phloridzin as in those fed phloretin (9.0 3.0 and 6.9 0.9 mol/L, respectively) (Fig. 1A). After hydrolysis by -glucuronidase/sulfatase, the phloretin peak markedly increased (Fig. 1B), suggesting that the major circulating forms were glucuronidated and/or sulfated derivatives of phloretin. We did not detect any methoxylated forms of phloretin in plasma. These data indicate that the nature of the circulating metabolites was independent of the administrated form of phloretin (aglycone vs. glucoside). At 4 h after the beginning of the meal containing phloretin, 22.8 2.8 mol/L of phloretin was measured in hydrolyzed plasmas (Fig. 2A). Of this total amount, expressed as phloretin equivalents, 5% was represented by unconjugated phloretin and 95% by conjugated forms. When measured 4 h after the meal, the plasma concentration of phloretin in rats fed phloridzin was markedly lower (50%; P 0.05) (Fig. 2B) than that found in plasma of rats fed phloretin. Thus, phloretin appeared more rapidly in plasma when it was administered to rats in the aglycone rather than in the glucosidic form (Fig. 2). Whatever the supplementation (phloretin or phloridzin), the ratio between unconjugated aglycone and total forms was of the same magnitude (5%). When rats were sampled 10 h after the meal, the plasma concentrations of total phloretin were not significantly differ- CRESPY ET AL. FIGURE 1 Representative chromatograms of plasma from rats fed phloretin or phloridzin before ( panel A) or after ( panel B) enzymatic hydrolysis by -glucuronidase/sulfatase. The detection was performed using multielectrode coulometric detection (4-electrodes CoulArray, Eurosep, France) with potentials set at 375, 500, 600 and 700 mV. ent between rats fed the two diets, i.e., they were 66.9 19.4 mol/L for those fed the phloridzin meal and 54.2 8.0 mol/L for those fed the phloretin meal. Whatever compound was administered (phloretin or phloridzin), the level of unconjugated aglycone recovered in plasma represented 10% of the total. At 24 h after food intake, the total plasma concentrations in phloretin dramatically decreased to 4.8 2.1 and 7.7 4.0 mol/L after phloridzin and phloretin intake, respectively (Fig. 2). In both cases, 14% of the total was constituted by the aglycone forms. The urinary excretion of phloretin was measured over a 24-h period after the ingestion of each experimental meal. Excretion rates did not differ between rats fed the phloretin meal (8.5 0.9 mol/24 h) and those fed the phloridzin meal (8.2 1.7 mol/24 h). These urinary excretions corresponded to 10.4% of the ingested dose. Because phloridzin increases glucosuria in diabetic rats (6), we checked whether the consumption of phloretin (22 mg) affected glucosuria. The measurements were made in urine sampled from the bladder 10 h after food intake when the total plasma concentration of phloretin was high. Glucosuria was 73.4 13 mol/L in the phloretin group and 74.0 14 mol/L in the phloridzin group, not different from that of control rats (38.8 4 mol/L; P 0.05). DISCUSSION The present study clearly demonstrates that whatever form was administered (phloretin or phloridzin), their bioavailability was similar, as reflected by the absence of significant dif- Downloaded from jn.nutrition.org by guest on August 3, 2013
FIGURE 2 Plasma concentrations of total (unconjugated and conjugated forms) phloretin over 24 h in rats fed 0.157% phloretin (panel A) or 0.25% phloridzin (panel B). Values are means SEM, n 6. *P 0.05; (A) vs. (B). ferences in the 24-h urinary excretions. However, the plasma kinetics of phloretin and phloridzin differed because phloretin appeared more rapidly in plasma when rats were fed phloretin vs. phloridzin. The high level of phloretin measured in plasma 4 h after the beginning of the meal supplemented with phloretin suggests that its absorption occurred chiefly in the small intestine. In a previous study, using an in situ intestinal perfusion model, we showed that these two compounds were absorbed in the small intestine and that phloretin was absorbed more efficiently than its glucoside (16). In the plasma of rats fed a phloridzin meal, no trace of this glucoside was detected, indicating that it must be hydrolyzed, probably by LPH, before its absorption and metabolism. The analysis of plasma from rats fed a meal supplemented with phloridzin or phloretin showed that 85–95% of the circulating forms are conjugated metabolites of phloretin (glucuronides and/or sulfates) and that the remainder was present as the unconjugated form. This last result is quite surprising because when flavonoid aglycones are administered in a meal, all of the circulating forms are generally conjugated derivatives (17–20). The few studies reporting the presence of unconjugated aglycone in rat plasma used gavage as the mode of administration (21,22). Such a procedure delivers a large amount of compound in short time, leading to a direct diffusion of the compound administered through the intestinal wall; thus, this does not reflect a phenomenon that could occur under physiologic conditions. Our present data suggest that when dihydrochalcones were administered in a meal, a part of the compound added to the diet could be metabolized by conjugative enzymes, and thus could be recovered intact in plasma. Moreover we did not detect any methoxylated forms of phloretin in plasma, confirming the importance of the presence of the BIOAVAILABILITY OF PHLORETIN AND PHLORIDZIN IN RATS 3229 catechol group in the methoxylation process, as previously reported (23,24). It has been shown in vivo that some flavonoid glucosides, especially quercetin-3-O-glucose, are absorbed more rapidly than their corresponding aglycones (25,26). Different hypotheses have been proposed to explain the rapid absorption of the glucosides. Hollman et al. (25) suggested that the active SGLT1 could be involved in the transport of flavonol glucosides. Phloridzin blocks SGLT1 (4) but is not absorbed into enterocytes by this transporter (27). Similarly, Day et al. (28) proposed that if in vivo LPH is responsible for the hydrolysis of flavonoid glucosides, the proximity of the released aglycone to the membrane may facilitate the passive diffusion of the flavonoid into the enterocytes. The present study showed that when rats were fed control diets containing phloridzin or phloretin, phloretin was absorbed more rapidly than its glucoside. This is not consistent with the hypothesis of Day et al. (28). During the first hours after ingestion, this hydrolysis step by LPH seems to represent a bottleneck for absorption. Nevertheless, the hydrolysis did not seem to constitute a limiting step because 10 h after the beginning of the experimental meal, the plasma concentrations of phloretin did not differ in rats fed the phloridzin or phloretin meals. At 24 h after the beginning of food intake, the plasma concentration of phloretin metabolites returned to a low level. By contrast, it has been reported that flavonoids such as quercetin or naringenin are still present at high concentrations in rat plasma 24 h after administration (19,29). This phenomenon could be due to the fact that the elimination of quercetin may be balanced by some digestive absorption, which still occurred during the postabsorptive period, and by some from enterohepatic cycling. This phenomenon allows an increase in the half-lives of these compounds. Because the conjugated forms of phloretin were quickly eliminated via the urinary route and enterohepatic cycling activity was insufficient to maintain the plasma concentration during the postabsorptive period, its half-life was decreased. We noted an enhancement in the proportion of unconjugated phloretin measured in the plasma between 4 (5%) and 24 h (14%). This rise could be due to the easier elimination of the conjugated metabolites of phloretin than of the aglycone itself. When phloretin was administered as the aglycone or as the glucoside, 10.4% of the ingested dose was recovered in urine. Both ingested compounds were excreted to the same extent in urine. Nevertheless, phloretin was excreted more efficiently in this biological fluid, as in a previous study (4% of the ingested dose) (10). This excretion difference could be explained by the dose and the mode of administration (200 vs. 88 mg/kg in our study and gavage vs. meal). The biological properties of phloridzin, which have been investigated extensively, include its ability to block the absorption of glucose by SGLT1. Moreover, phloretin inhibits the facilitated glucose transporter protein GLUT2, located on the basolateral side of the enterocytes (30). By this mechanism, phloretin could also limit the intestinal absorption of glucose. These properties have been demonstrated in in vivo studies using diabetic rats. Their plasma glucose concentration was normalized by treatment with phloridzin (7,8), notably by an increase of glucosuria, which limited hyperglycemia (6). The identification of unconjugated phloretin in plasma could be of physiologic interest. Because phloretin interacts with GLUT2, which is also present in kidney (31), it is conceivable that this aglycone could increase glucose urinary excretion by limiting its reabsorption. In conclusion, the present study shows that phloretin, administered as the aglycone or as the glucoside, is absorbed Downloaded from jn.nutrition.org by guest on August 3, 2013
Page 1: Nutrient Metabolism—Research Comm

Nutrient Metabolism—Research Communication

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?