The human colonic microflora influences the alterations of xenobiotic-metabolizing enzymes by catechins in male F344 rats.

As other xenobiotics, polyphenols are metabolized both by the endogenous detoxication system and the gut microflora. We hypothesized that the presence of a gut microflora may account for the effect of catechins on phase I and II xenobiotic-metabolizing enzymes and that the human bacterial metabolites may be different from those of a rodent gut microflora. Therefore, the effects of 2% (+)-catechin or 2% (-)-epicatechin were studied in germ free (GF) rats and rats inoculated with the flora of a human volunteer (HFA). In addition, the catechins were administered in ethanol as a vehicle. In the liver, (+)-catechin or (-)-epicatechin decreased the total amount of CYP450 in both GF and HFA rats while the isoenzyme CYP2E1 decreased. In GF rats only, CYP2C11 increased when compared to the rats treated with the vehicle alone. (+)-catechin increased the specific activity of UGT-chloramphenicol in GF rats only and that of cytosolic glutathion-S-transferase (GST) in HFA rats only. In the intestine, (+)-catechin and (-)-epicatechin increased the specific activity of UGT-4-methylumbelliferone in both GF and HFA rats and that of UGT- chloramphenicol in HFA rats only. In conclusion, the presence of a human flora in rats is able to modify the inducing effect of catechins on the UGT and GST activities suggesting the involvement of bacterial metabolites. The alterations on CYP 450 are independent of the presence of a human gut flora.

Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids.

Polymeric proanthocyanidins are common constituents of many foods and beverages. Their fate in the human body remains largely unknown. Their metabolism by human colonic microflora incubated in vitro in anoxic conditions has been investigated using nonlabeled and (14)C-labeled purified proanthocyanidin polymers. Polymers were almost totally degraded after 48 h of incubation. Phenylacetic, phenylpropionic and phenylvaleric acids, monohydroxylated mainly in the meta or para position, were identified as metabolites by gas chromatography coupled to mass spectrometry (GC-MS). Yields were similar to those previously reported for flavonoid monomers. These results provide the first evidence of degradation of dietary phenolic polymers into low-molecular-weight aromatic compounds. To understand the nutritional properties of proanthocyanidins, it is therefore essential to consider the biological properties of these metabolites.

Methyl phenylalkanoates as substrates to probe the active sites of esterases.

We have used methyl esters of phenylalkanoic acids to probe the active site of two esterases (FAE-III and CinnAE) from Aspergillus niger. Only methyl 4-hydroxy-3-methoxycinnamate and 4-hydroxy-3-methoxyphenylpropionate out of 19 substrates tested were significant substrates for both enzymes (k(cat) values about 10(2) s(-1) and 10(3) s(-1), respectively). Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Differences in Km values for FAE-III were small (0.45-2.08 mM) but there were two orders of magnitude difference in k(cat) values (12.1-1063 s(-1)), whereas for CinnAE, there were large differences in values for both Km (0.014-1.32 mM) and k(cat) (41.3-1410 s(-1)). Lability of the ester bonds, as estimated from second-order rate constants (k2) for chemical reaction with sodium hydroxide, did not correlate to k(cat) for CinnAE (r = 0.33) or for FAE-III (r = 0.43). Maintaining the phenylpropenoate structure but altering the substitutions on the aromatic ring demonstrated the following: a 3-methoxy group is essential for FAE-III activity, whereas a 3-methoxy group precluded activity of CinnAE, with the exception of methyl 4-hydroxy-3-methoxycinnamate which was a relatively poor substrate for CinnAE; (b) increasing the number of methoxy substitutions increased the activity of FAE-III, and decreased the activity of CinnAE; (c) 4-hydroxy substituents, and additional hydroxy substituents, increased the activity of CinnAE, but decreased that of FAE-III; (d) the rate of hydrolysis with sodium hydroxide of the methyl esters in general is decreased by hydroxy substitutions on the aromatic ring but increased by methoxy substitutions. Analysis of kinetic data obtained in the presence of inhibitors indicated that substrate analogs were able to bind to both free CinnAE and to a CinnAE-substrate complex, but conversely, were only able to bind to free FAE-III. The results show that the specificities of the two A. niger esterases are complementary. The rate of hydrolysis by this class of carboxylic ester hydrolase does not depend on the intrinsic lability of the ester bond, but depends on both the distance between the aromatic ring and the ester bond, and the substitutions on the aromatic ring.