Isoflavone Conjugates Are Underestimated in Tissues Using Enzymatic Hydrolysis

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.

Tissue distribution of anthocyanins in rats fed a blackberry anthocyanin‐enriched diet

“…They were largely present as glucurono and/or sulphoconjugate forms [35,36]. A methylated derivative of quercetin (isorhamnetin) was also reported as the main metabolite in testes of quercetin-fed rats [38].…”

“…Flavonoids such as isoflavones and quercetin have been identified in testes from rats fed flavonoid-enriched diets [35,36,38]. They were largely present as glucurono and/or sulphoconjugate forms [35,36].…”

Quantification of Glucuronide Metabolites in Biological Matrices by LC-MS/MS

“…Sometimes, a separate incubation of a commercially obtainable substrate (for example 4-methylumbelliferone glucuronide) in the same matrix is used to monitor the -glucuronidase activity (Blount, Milgram et al 2000). Each incubation should contain 200 -25 000 units of -glucuronidase per 1 mL of incubation medium (Gu, Laly et al 2005;Belanger, Caron et al 2009). Note that vendors define the glucuronidase activity in Roy or in Fishman units, where 1 Fishman unit releases 1 µg phenolphthalein from phenolphthalein-β-glucuronide in 1 h at 38°C.…”

Physiologically based kinetic modelling based prediction of oral systemic bioavailability of flavonoids, their metabolites, and their biological effects

“…Alternatively, tissue:blood partition coefficients can also be obtained using in silico methods based on octanol-water partition coefficient [60]. Kinetic parameters for ADME can be obtained using 1) experimental in vitro models using primary cell cultures, cells from cell lines in culture, subcellular fractions, tissue slices, or relevant recombinants enzymes of organs [61,62], 2) available in silico methods [63][64][65], or 3) by fitting the model to available in vivo data [66].…”

“…In most studies, flavonoid conjugates in plasma were identified and quantified from the amounts of aglycone liberated from conjugated metabolites by using β-glucuronidase and sulfatase to deconjugate the conjugated metabolites to their respective aglycone [60][61][62][63][64][65][66][67][68]. This method has been criticized for running a risk of underestimation and lack of information on the absolute structural conformation of flavonoid conjugates especially with respect to the conjugation position [60,61]. The use of advanced analytical methods in chromatography and mass spectrometry can also not provide the absolute conformation of the chemical structure of individual flavonoid conjugates [69].…”