Permeability and Conjugative Metabolism of Flaxseed Lignans by Caco 2 Human Intestinal Cells

Reports in the literature associate the dietary intake of flaxseed lignans with a number of health benefits. The major lignan found in flaxseed, secoisolariciresinol diglucoside, undergoes metabolism principally to secoisolariciresinol (2), enterodiol (3), and enterolactone (4) in the human gastrointestinal tract. Systemically, lignans are present largely as phase II enzyme conjugates. To improve understanding of the oral absorption characteristics, a systematic evaluation of the intestinal permeation was conducted and the conjugative metabolism potential of these lignans using the polarized Caco 2 cell system was analyzed. For permeation studies, lignans (100 microMole ) were added to acceptor or donor compartments and samples were taken at 2 h. For metabolism studies, lignans (100 microMole ) were incubated in Caco-2 for a maximum of 48 h. Cell lysates and media were treated with βglucuronidase/sulfatase, and lignin concentrations were determined using HPLC. Apical-to-basal permeability coefficients for 2 to 4 were 8.0 plus and minus  0.4, 7.7 plus and minus  0.2, and 13.7 plus and minus  0.2 (×10−6) cm/s, respectively, whereas efflux ratios were 0.8 to 1.2, consistent with passive diffusion. The permeation of compound 1 was not detected. The extent of conjugation after 48 h was less than  3 percent, 95 percent, 90 percent, and  greater than 99 percent for 1 to 4, respectively. These data suggest 2 to 4, but not 1 undergo passive permeation and conjugative metabolism by Caco-2 cells. (Authors abstract)

Plant polyphenolic compounds continue to receive prominent attention for their health benefits in a number of chronic disease states.  Lignans, a class of phenylpropanoid compounds widely distributed in the plant kingdom, have generated particular interest due to their ability to favorably modulate biological risk factors of cardiovascular disease and cancer.  Flaxseed is a rich source of the lignan secoisolariciresinol diglucoside (1), and human clinical trials and animal model studies have reported important beneficial cardiovascular effects of flaxseed lignan supplementation such as improvement in blood lipid profiles,  reduction in the development of aortic atherosclerosis, and delayed onset of type II diabetes.  Furthermore, several studies suggest a protective role for flaxseed lignans against cancers of the breast, prostate, and colon.  In the flaxseed, 1 exists as an oligomer with ester linkages to 3-hydroxy-3-methylglutaric acid, cinnamic acid, and other phenolic glucosides.  It is assumed, but not yet demonstrated, that when the ester-linked 1 containing oligomer is ingested, the first metabolic step is the cleavage of the ester linkage and the release of free 1. Subsequently, the glucosidic groups are cleaved from 1 to yield the aglycone form, secoisolariciresinol (2), in the upper gastrointestinal tract. Compound 2 either is absorbed or will undergo subsequent metabolism by the colonic microflora to the mammalian lignans enterodiol (3) and enterolactone (4).  Although attempts have been made in the literature to draw an epidemiological association between 4 and the positive health effects of flaxseed consumption, it still remains controversial whether plant-derived lignans (i.e., 1 and 2) and/or the mammalian lignans (i.e., 4 and 3) mediate the putative health benefits associated with flaxseed lignin consumption. Hence, the purpose of this study was to evaluate the permeation characteristics of the plant-derived lignans, 1 and 2, and the mammalian lignans, 3 and 4, and to characterize the extent of phase II conjugative metabolism by the Caco-2 model. This information should provide insight into the oral bioavailability of each lignan and the possible bioactive lignan form associated with the health benefits of flaxseed lignan consumption.

As an important step to an enhanced understanding of the oral absorption properties of 1 and its metabolites, 2 to 4, a systematic evaluation of lignan permeation across intestinal cells and the contribution of intestinal cells to phase II metabolism of 1 and its metabolites, 2 to 4, was conducted. This evaluation involved the use of the Caco 2 cell monolayer, as its use is widely accepted as an intestinal permeability screening assay and has been used to gain preliminary understandings on the propensity for intestinal phase II enzyme metabolism in an intact cellular system.  The following key observations were reported: Secoisolariciresinol Diglucoside (1) Does Not Permeate across the Caco-2 Polarized Epithelium; Metabolites (2 to 4) of Secoisolariciresinol Diglucoside (1), But Not 1, Undergo Extensive Phase II Enzyme Metabolism in Caco-2 Monolayers. Compound 4 exhibited the highest apparent permeability and predicted absorbed fraction, which may explain in part its greater extent of conjugation in the Caco-2 monolayer. Conversely, the poor permeability characteristics of 1 across the Caco-2 cell monolayer are consistent with its poor conjugative metabolism.  This study found that the principal lignan of flaxseed, 1, undergoes very limited intestinal cell permeation and metabolism and is not likely to be systemically available following oral administration. The metabolites, 2 to 4, though, passively permeate across the intestinal epithelium and exhibit appreciable conjugative metabolism. The ability of the Caco-2 cell monolayer to extensively metabolize 2 to 4 indicates a role for the intestine in the presystemic metabolism of these lignans. Further studies are required to determine the oral bioavailability and the exact contributions of the intestine and liver to presystemic metabolism. Furthermore, glucuronide conjugates are pharmacologically inactive with a few exceptions, but the compellin evidence of health benefits associated with flaxseed lignin consumption coupled with the present knowledge of their extensive phase II metabolism may warrant an investigation into the possible pharmacological activity of these conjugated metabolites. (Editors comments)