The liver plays
a critical role in the metabolism of carbohydrates, alcohol, fatty acids, and lipids.1
Among these functions, the metabolism of cholesterol is critical for maintaining optimal health. Any impairment of this important hepatic function can lead to serious medical complications.2,3
The hydroxylation of cholesterol initiates a cascade of enzymatic reactions that synthesize bile acids (BAs). The hepatic biosynthesis and regulation of BAs is critical to the adsorption and clearance of dietary lipophilic molecules and environmental toxicants from the body.4
Intricate feedback inhibition pathways regulate BA homeostasis, which in turn controls the adsorption of lipophilic nutrients, cholesterol, and fat soluble vitamins by the body.2,5,6
Significant deviations in optimal concentrations and composition of the BA mixture in vivo
can result in serious health complications, including cholestasis, gall stones,3,7–9
and organ damage.4,10
BAs have been shown to participate in their own regulation through nuclear and membrane receptor feedback interactions controlling BA biosynthesis and enterohepatic circulation.4,11
Enterohepatic circulation begins with hepatic BA secretion through the bile canaliculi to the gall bladder for storage (). BAs are reabsorbed into the blood stream along with cholesterol and other lipophilic molecules and transported back to the liver. Up to 85% of BAs are recycled through this process, thereby minimizing daily BA production12
and necessitating its efficient regulation to maintain a tight control over the BA pool size and composition. The role of BAs in cholesterol clearance and catabolism and their emergent role in their own regulation underscore the necessity of monitoring BA biosynthesis and transformation.
A schematic of the enterohepatic circulation through which bile acids are transported into and away from the liver.
There have been some studies on BA pathways in animal models.6,7,13–16
However, such models are expensive and the resulting data can be difficult to interpret. Moreover, as a result of insufficient sensitivity, the composition of BAs has been quantified largely in biological fluids such as blood, urine, bile, and tissue homogenates.5,14,17,18
These approaches have relied on limits of detection ranging from 5 to 50
a range in which small differences in BA biosynthesis are indiscernible.
hepatic models that are capable of mimicking in vivo
liver functions exhibit tremendous potential to elucidate unknown or cryptic BA metabolic pathways.1
Previous work has shown that two-dimensional hepatocyte cultures, specifically the collagen sandwich (CS), are capable of maintaining key liver-specific functions.20–22
Cellular co-cultures and three-dimensional (3D) hepatic constructs have also exhibited enhanced hepatic phenotypic function.23–32
However, the biosynthesis and the resulting homeostasis of BAs, a critical aspect of liver function, have been inadequately studied in these in vitro
Since hepatocyte cultures do not contain high concentrations of BAs, a variety of approaches have been used to obtain precise information on conjugated and unconjugated BAs. Some previous studies have quantified BAs using gas chromatography, which requires extremely laborious sample preparation.9,14,33–36
In other reports, high-pressure liquid chromatography (HPLC) was utilized to simultaneously analyze both unconjugated and conjugated BAs.5,17,18,37,38
However, analytical techniques developed for in vitro
analysis of BAs have inadequate sensitivity (limits of detection were >50
ng/mL) precluding meaningful analysis of the results.33,34,39
Further, the loss of phenotypic function exhibited by hepatocyte monolayers (HMs) because of cellular de-differentiation is likely to result in significant deviations in BA profiles from in vivo
We have recently reported that the assembly of 3D engineered liver tissue mimics comprised of primary rat hepatocytes (parenchymal cells) and liver sinusoidal endothelial cells (LSECs, nonparenchymal cells).40
In these 3D cultures, hepatic parenchymal and nonparenchymal cells were arranged in stratified layers similar to in vivo
structure resulting in stable liver-related functions over a 2-week period. More importantly, these 3D mimics exhibited a 16-fold increase in CYP1A1/2 (cytochrome P450) enzymatic activity, in comparison to just a twofold increase in CS cultures.40
Although CYP1A1/2 does not mediate cholesterol metabolism, other CYP enzymes do so. Therefore, our goal was to test the engineered liver mimetic constructs to determine if they are a suitable platform to conduct detailed studies on BA and cholesterol homeostasis.
We report the development of a HPLC/mass spectrometry (MS) methodology that can simultaneously separate and quantify BAs with a 1
ng/mL limit of detection. To the best of our knowledge, this is the first study that reports the quantification of conjugated and unconjugated primary rat BAs in in vitro
hepatocyte cultures with such a level of sensitivity, which is an order of magnitude better than previous approaches. We achieved increased sensitivity by grouping the BAs based upon their hydrophobicity, before detection. Consequently, not all BAs have to be monitored simultaneously, thereby increasing the sensitivity of the method. A significant advantage of the 1
ng/mL detection limit was that we could identify products of key intermediate steps in the metabolic pathways of primary rat BAs. Mammalian species typically have two primary BA products of cholesterol metabolism, cholic acid (CA) and chenodeoxycholic acid (CDCA). In the rat, beta-muricholic acid (βMCA) is considered an additional primary BA since it is directly synthesized by rat hepatocytes from CDCA.41–44
The final step before functional BA secretion from the liver is conjugation to either glycine or taurine to increase their solubility in vivo.8,12,45
In this study, we exploited the 1
ng/mL detection limit of our HPLC/MS methodology to quantify the following primary rat BAs: CA, glycocholic acid (GCA), taurocholic acid (TCA), CDCA, glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), βMCA, and tauro-beta-muricholic acid (TβMCA).