Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2005
Publication Date: 7/28/2005
Citation: Gu, L., Laly, M., Chang, H.C., Prior, R.L., Fang, N., Ronis, M.J., Badger, T.M. 2005. Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis. Journal of Agriculture and Food Chemistry. 53(17):6858-6863.
Interpretive Summary: Soy protein is used worldwide as an important and excellent protein source. However, soy also has been demonstrated to have other health effects beyond those attributed to usual protein sources. Most of the health effects are thought to be due to isoflavones, phytochemicals that are bound to the soy protein. However, proof of this is indirect. Since isoflavones are converted to other molecules (metabolites), the exact molecules that are responsible for health effects are unknown. We have been developing techniques to identify these molecules and to study them in detail in order to learn more about how soy protein consumption affects health in infants who are fed soy formula. Two methods were applied to hydrolyze the isoflavone conjugates, a traditional enzymatic method and an acid hydrolysis method applying a strong acid, hydrochloric acid. We found acid hydrolysis was much more efficient than enzymes in releasing free isoflavones in rat organs; meanwhile, there was no difference in serum and urine. We concluded that the traditional enzymatic method may have underestimated isoflavones in organ tissues. Our future studies will now use these techniques to study isoflavones in target cells of breast, liver, vessels, etc.
Technical Abstract: Many health effects of soy foods are attributed to isoflavones. Isoflavones upon absorption present as free form, glucuronide, and sulfate conjugates in blood, urine, and bile. Little is known about the molecular forms and the relative concentrations of soy isoflavones in target organs. Acid hydrolysis or enzymatic hydrolysis (glucuronidases and sulfatases) was used to study isoflavone contents in the heart, brain, epididymis, fat, lung, testis, liver, pituitary gland, prostate gland, mammary glands, uterus, and kidney from rats fed diets made with soy protein isolate. The heart had the lowest isoflavone contents (undetectable), and the kidney had the highest (1.8 ± 0.6 nmol/g total genistein; 3.0 ± 1.1 nmol/g total daidzein). Acid hydrolysis released 20-60% more aglycon in tissues than enzymatic digestion (p < 0.05), and both hydrolysis methods gave the same level of isoflavones in serum. Approximately 28-44% of the total isoflavone content within the liver was unconjugated aglycon, and the remainder was conjugated mainly as glucuronide. The subcellular distribution of total isoflavones was 55-60% cytosolic and 13-16% in each of the nuclear, mitochondrial, and microsomal fractions. These results demonstrated that (1) soy isoflavones distribute in a wide variety of tissues as aglycon and conjugates and (2) the concentrations of isoflavone aglycons, which are thought to be the bioactive molecules, are in the 0.2-0.25 nmol/g range, far below the concentrations required for most in vitro effects of genistein or daidzein.