Jim Fang

Bioavailability of anthocyanins

Abstract Anthocyanins are a subgroup of flavonoids responsible for the blue, purple, and red color of many fruits, flowers, and leaves. Consumption of foods rich in anthocyanins has been associated with a reduced risk of cardiovascular disease and cancer. The fate of anthocyanins after oral administration follows a unique pattern rather different from those of other flavonoids. Anthocyanins could be absorbed from the stomach as well as intestines. Active transporters may play a role in the absorption of anthocyanins from the stomach as well as in their transfer within the kidney or liver. Anthocyanins such as cyanidin-3-glucoside and pelargonidin-3-glucoside could be absorbed in their intact form into the gastrointestinal wall; undergo extensive first-pass metabolism; and enter the systemic circulation as metabolites. Phenolic acid metabolites were found in the blood stream in much higher concentrations than their parent compounds. These metabolites could be responsible for the health benefits associated with anthocyanins. Some anthocyanins can reach the large intestine in significant amounts and undergo decomposition catalyzed by microbiota. In turn, these decomposition products may contribute to the health effects associated with anthocyanins in the large intestine. This review comprehensively summarizes the existing knowledge about absorption, distribution, metabolism, and elimination of anthocyanins as well as their decomposition within the gastrointestinal lumen.

Some anthocyanins could be efficiently absorbed across the gastrointestinal mucosa: extensive presystemic metabolism reduces apparent bioavailability.

Despite the accumulating evidence supporting the health effects of anthocyanins, their plasma concentrations were found to be very low. However, 30 and 56% of cyanidin 3-glucoside (Cy-3-glc) and pelargonidin 3-glucoside (Pg-3-glc) were found as protocatechuic acid (PCA) and 4-hydroxybenzoic acid, respectively, in plasma following oral administration in humans. Second, 12.4% of (13)C was recovered from urine and breath following oral ingestion of [(13)C]-Cy-3-glc in humans. The actual percentage of [(13)C]-Cy-3-glc absorbed across the gastrointestinal wall could be higher because of the involvement of enterohepatic recycling in the disposition of anthocyanins. In animal studies, high total urinary recoveries were found following oral ingestion of (14)C-labeled anthocyanins. Third, anthocyanins seem to be efficiently absorbed following in situ gastric and intestinal perfusions in rats. Therefore, some anthocyanins could be efficiently absorbed from the gastrointestinal lumen, undergo extensive first-pass metabolism, and enter the systemic circulation as metabolites.

Determination of clozapine, and its metabolites, N-desmethylclozapine and clozapine N-oxide in dog plasma using high-performance liquid chromatography.

Clozapine and its two major metabolites, N-desmethylclozapine and clozapine N-oxide were quantified using a high-performance liquid chromatographic method with UV detection in dog plasma following a single dose of clozapine. The analysis was performed on a 5-micrometer Hypersil CN (CPS-1; 250x4.6 mm) column. The mobile phase consisted of acetonitrile-water-1 M ammonium acetate (50:49:1, v/v/v), which was adjusted to pH 5.0 with acetic acid. The detection wavelength was 254 nm. A liquid-liquid extraction technique was used to extract clozapine and its metabolites from dog plasma. The recovery rates for clozapine, N-desmethylclozapine, and the internal standard (I.S.) were close to 100% using this method. The recovery rate for clozapine N-oxide (62-66%) was lower as expected because it is more polar. The quantitation limits for clozapine, clozapine N-oxide, and N-desmethylclozapine were 0.11, 0.05 and 0.05 microM, respectively. Intra-day reproducibility for concentrations of 0.1, 1.0 and 5.0 microM were 10.0, 4.4 and 4.2%, respectively, for N-oxide; 11.2, 4.3 and 4.9%, respectively, for N-desmethylclozapine; and 10.8, 2.2 and 4.9%, respectively, for clozapine. Inter-day reproducibility was <15% for clozapine N-oxide, <8% for N-desmethylclozapine and <19% for clozapine. This simple method was applied to determine the plasma concentration profiles of clozapine, N-desmethylclozapine and clozapine N-oxide in dog following administration of a 10 mg/kg oral dose of clozapine.

Enantioselective analysis of ritalinic acids in biological samples by using a protein-based chiral stationary phase.

AbstractPurpose. This study was to develop and validate a new chiral HPLC-UV method for the quantitative analysis of enantiomeric ritalinic acid (RA) in human plasma. Methods. An α1-acid glycoprotein column was used with the mobile phase containing 0.4% acetic acid and 0.1% dimethyloctylamine, pH 3.4. The detection of enantiomeric RAs was at 220 nm. Results. A baseline separation for d- and l-RA was achieved by a separation factor of 2.08. Methylphenidate, the precursor of RA, was eluted with the front solvent, and thus does not interfere in the analysis of RA in our method. The assay was successfully applied for the in vitro analysis of enantiomeric ritalinic acids produced by human and dog plasma and dog liver. Conclusions. Data demonstrated that the esterase(s) in human plasma metabolize d-methylphenidate faster than its l-isomer. The yielded intrinsic clearances (Clint) are 1.02 and 2.17 μl/min/mg protein, respectively, for d and l-methylphenidate.

In vitro identification of the human cytochrome p450 enzymes involved in the oxidative metabolism of loxapine.

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) enzymes responsible for the oxidative metabolism of loxapine to 8‐hydroxyloxapine, 7‐hydroxyloxapine, N‐desmethylloxapine (amoxapine) and loxapine N‐oxide. These studies included use of cDNA‐expressed enzymes, correlation analysis with 12 phenotyped human liver microsomal samples, and use of selective inhibitors of cytochrome P450s. The resultant data indicated that loxapine was mainly metabolized by human liver microsomes to (i) 8‐hydroxyloxapine by CYP1A2, (ii) 7‐hydroxyloxapine by CYP2D6, (iii) N‐desmethyloxapine by CYP3A4 and (iv) loxapine N‐oxide by CYP3A4. The involvement of flavin‐containing monooxygenase (FMO) in the formation of loxapine N‐oxide was also observed. Copyright

HPLC-MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute-and-shoot sample preparation methods, respectively

A high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP-Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water-1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute-and-shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, cyanidin-3-xyloside and quercetin-3-galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.