Quantitation of phlorizin and phloretin using an ultra high performance liquid chromatography-electrospray ionization tandem mass spectrometric method.

A sensitive and selective ultra high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for the determination of phlorizin and phloretin in human plasma has been firstly developed. Samples were prepared after protein precipitation and analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. Negative electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.02% formic acid), using a gradient procedure. The analytes and internal standard dihydroquercetin were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 2.5-1000.0 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 9.2%. The accuracy determined at three concentrations was within ± 7.3% in terms of relative error. The total run time was 12.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of phlorizin and phloretin in various biological fluids.

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport.

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.

Polyphenols from alcoholic apple cider are absorbed, metabolized and excreted by humans.

We determined the uptake and excretion of low doses of polyphenols in six subjects who each consumed 1.1 L of an alcoholic cider beverage. Over a 24-h period, no phloretin was detected in plasma (detection limit = 0.036 micromol/L), but 21 +/- 5% of the dose (4.8 mg) was excreted in the urine. In contrast, from a low dose of 1.6-mg quercetin equivalents, no quercetin was found in urine or plasma, but 3'-methyl quercetin was detected in plasma [C(max) (maximum concentration) = 0.14 +/- 0.19 micromol/L; range: 0 to 0.44 micromol/L]. No flavanol monomers (dose of free (+)-catechin and (-)-epicatechin = 3.5 mg) were detected in urine or plasma (detection limit: 0.01 micromol/L). Caffeic acid (total dose including esters = 11 mg) was detected only in plasma within 2 h, with C(max) = 0.43 +/- 0.3 micromol/L (range: 0.18 to 0.84 micromol/L). An almost 3-fold increase in hippuric acid was detected in 24-h urine (74 +/- 29 micromol/L; range: 38-116 micromol/L), compared with a prestudy value of 19 +/- 9 micromol/L. These data show that polyphenols are taken up from cider, that phloretin is excreted in the urine and suggest that low doses of quercetin are extensively methylated in humans.

Bioavailability of phloretin and phloridzin in rats.

Phloretin is a flavonoid found exclusively in apples and in apple-derived products where it is present as the glucosidic form, namely, phloridzin (phloretin 2'-O-glucose). In the present study, we compared the changes in plasma and urine concentrations of these two compounds in rats fed a single meal containing 0.25% phloridzin or 0.157% phloretin (corresponding to the ingestion of 22 mg of phloretin equivalents). In plasma, phloretin was recovered mainly as the conjugated forms (glucuronided and/or sulfated) but some unconjugated phloretin was also detected. By contrast, no trace of intact phloridzin was detected in plasma of rats fed a phloridzin meal. These compounds presented different kinetics of absorption; phloretin appeared more rapidly in plasma when rats were fed the aglycone than when fed the glucoside. However, whatever compound was administered, no significant difference in the plasma concentrations of total phloretin were observed 10 h after food intake. At 24 h after the beginning of the meal, the plasma concentrations of phloretin were almost back to the baseline, indicating that this compound was excreted rapidly in urine. The total urinary excretion rate of phloretin was not affected by the forms administered, and was estimated to be 8.5 micromol/24 h in rats fed phloretin or phloridzin. Thus, 10.4% of the ingested dose was recovered in urine after 24 h.

Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes.

The sidedness of phloretin binding to the glucose carrier has been determined by comparing the type of inhibition produced in zero trans entry and zero trans exit experiments. Initial rates of zero trans entry were measured by the method of R.D. Taverna and R.G. Langdon (Biochim. Biophys. Acta 298:412-421, 1973), which involves pink ghosts loaded with glucose oxidase; this obviates the problem of rapid substrate accumulation inside the cells. With phloretin equilibrated across the membrane, the inhibition of entry was competitive, and the inhibition of exit noncompetitive. The experimental procedures were validated by showing that the inhibition by cytochalasin B, known to bind inside but not outside, was noncompetitive in entry and competitive in exit, as predicted. It was also demonstrated that even after pre-incubation of the cells with a relatively high concentration of phloretin, the phloretin adsorbed in the membrane did not significantly alter the rate of carrier reorientation. The results show that the outward-facing form of the glucose carrier, but not the inward-facing form, bears a phloretin binding site; thus phloretin, as well as cytochalasin B, is bound asymmetrically, phloretin outside and cytochalasin B inside.

The placental transfer of hexoses and polyols in the guinea-pig, as shown by umbilical perfusion of the placenta.

1. Experiments involving polyol and sugar transfer across the guineapig placenta have been described.2. Regarding the former, sorbitol, dulcitol and mannitol are all transferred at approximately the same rate and more slowly than meso-inositol. Erythritol is not transferred.3. Concerning the hexose transfers, glucose and galactose are transferred at approximately the same rate and more rapidly than fructose.4. With regard to glucose transfer, there appears to be a mechanism with the following properties: (a) It tends towards saturation under certain conditions. (b) It favours saturation rather than diffusion kinetics for forward flows. (c) It exhibits competition with fructose and with galactose. Glucose/galactose competition has been used to demonstrate uphill transport by counterflow. (d) Competitive inhibition by phlorrhizin or phloretin could not be demonstrated.