Faculty Bibliography

The purpose of this minireview is to show that a new paradigm is developing regarding hepatic bile flow. The focus thus far has been on carrier-mediated transport of bile acids and other solutes, such as glutathione, which create an osmotic gradient for the transcellular and paracellular flow of water into canaliculi. In addition to the physicochemical properties of bile acids, which govern the osmotic gradient, data now exist showing that the tight junctions governing paracellular water flow and Aquaporin-8 water channels governing transcellular water flow are regulated independently. Thus, the rate of water flow into the canaliculus in response to bile acid transport is variable and determines canalicular bile acid concentration, which affects the production and solubilization of cholesterol-lecithin vesicles. These new considerations modify thinking regarding the occurrence of cholestasis and its progression and reorient the design of experimental studies that can distinguish the different determinants of bile flow. SIGNIFICANCE STATEMENT: The paradigm that water flow into the canaliculus is determined only by the rate of carrier-mediated transport has been challenged recently by the changes that occur in hepatic bile composition in the Claudin-2 knockout mouse and with the cholestatic effect of estradiol 17β-d-glucuronide. Thus, a respective reduction in paracellular or transcellular canalicular water flow, probably via Aquaporin 8, has no significant effect on bile acid excretion.

INTRODUCTION: Fecal microbiota transplant (FMT) is an effective treatment for recurrent Clostridioides difficile infection (rCDI), but its precise mechanism remains unknown (1-2). Recent studies suggest that the restoration of gut bacteria, which carry necessary enzymes to convert primary bile acids (PBAs) to secondary bile acids (SBAs), may play a key role in rCDI risk (3, Figure 1). For example, in vitro studies have demonstrated that PBAs stimulate C. difficile spore germination and that deoxycholic acid, a SBA, inhibits toxin-producing C. difficile (4). As normal fecal bile acid pools consist predominantly of SBAs, we hypothesize that a contributor of rCDI risk is the decrease in SBAs from enteric bacteria loss following antibiotic use (5-8).We present the first case enrolled in our clinical trial using ursodeoxycholic acid (UDCA), a SBA, as an adjunct treatment to prevent future rCDI. CASE DESCRIPTION/METHODS: An otherwise healthy 46-year-old man presented with S. aureusbacteremia secondary to septic knee arthritis. While on an extended course of cefazolin, he developed profuse, watery diarrhea and stool C. difficile toxin PCR was positive. He was then treated with intravenous metronidazole for one week and oral vancomycin for two weeks, with resolution of symptoms. Six weeks later, CDI recurred and was successfully treated with oral vancomycin for two weeks, in conjunction with concurrent UDCA 300 mg twice daily for eight weeks. Stool samples were collected at two-week intervals and fecal bile acid compositions were assessed via liquid chromatography - mass spectrometry. Fecal bile acid analyses demonstrated a shift towards protective SBAs by the end of the 8-week course of ursodiol (Figure 2). This profile of increased SBAs remained consistent despite subsequent retreatment with cefazolin at 5 months for recurrent septic arthritis. The patient has been without rCDI now for almost 2 years. DISCUSSION: The findings in this case are consistent with our hypothesis that increasing SBAs through adjunct treatment with UDCA can be effective in breaking the rCDI cycle. While some CDI patients have adequate enteric bacteria to produce SBAs after antibiotic treatment, others may have a delayed normalization of their gut microbiome, and may therefore require ?bridge therapy? with supplementary SBAs (e.g., UDCA) until their endogenous microbial community is restored. Treatment and analysis of additional patients with rCDI in this clinical trial are in progress. (Figure Presented)

Background: Canalicular water flow which initiates hepatic bile formation is accepted as being osmotically determined and thus dependent on the physical properties of the solutes transported across the canalicular membrane. Although many methods have been applied to estimate the colligative properties of bile acids different value are obtained depending, in part,on the sensitivity and technique utilized (1 ). Vapor pressure osmometry provides the direct determination of the osmotic effects of bile acids and other solutes transported into the canaliculus. Methods: Using a Vapro 360 instrument which permits determination of osmolality in the physiologic range we have determined the osmolality of the naturally occurring conjugated bile acids which are actively transported and the effect of lecithin and cholesterol on aggregation. Results: Sodium taurousodeoxycholate has approximately twice the osmotic activity of other primary conjugated bile acids (Figure 1) and thus will provide greater rates of canalicular flow. Solubilized lecithin and cholesterol do not significantly affect the osmotic activity of conjugated bile acids. Taurolithocholate solubilized in taurochlate does not decrease osmotic activity and thus is not the mechanism for its cholestatic effect. Conclusion: Tauroursodeoxycholate, in addition to enhancing canalicular water flow, is reported to stimulate water flow from cholangiocytes and to protect against the deleterious effects of increased hepatocellular concentration of other bile acids. Because of active ileal transport it may prove more beneficial than ursodeoxycholic acid by achieving a greater therapeutic effect at lower doses which will also diminish fecal lithocholic acid. (1) Natalini B. et al 2014. Determination of bile salt critical micellization concentration on the road to drug discovery. (Figure Presented)

(25R)-26-Hydroxycholesterol (27-hydroxycholesterol) has been found to accumulate in breast tissue and to stimulate tumor growth via the mu estrogen receptor. Although most tissues express CYP27A1, the highest levels are in macrophages and most attention had been given to the production of 27-hydroxycholesterol in sub-endothelial macrophages as part of reverse cholesterol transport. In view of the newly identified biologic activity, it is important to consider the determinants of the levels of 27-hydroxycholesterol in macrophages that infiltrate breast tissue. Among these determinants are the oxysterol binding proteins expressed in macrophages, the level of expression of CYP7B1, the oxysterol 7 alpha hydroxylase that generates an inactive triol, and further oxidation of 27-hydroxycholestrol to the C27 acid by multifunctional CYP27A1. Transport of 27-hydroxycholesterol from macrophages to plasma is HDL-associated. In many tissues the ratio of 27-hydroxycholesterol to cholesterol (ng/mug) is higher than that in plasma. Tamoxifen, an effective estrogen receptor antagonist that prevents breast cancer, also has the biologic property of blocking several steps in the lanosterol to cholesterol metabolic pathway. In genetically disposed women, tamoxifen may increase the amount of 27-hydroxycholesterol in breast tissue.

Studies of hepatic bile formation reported in 1958 established that it was an osmotically generated water flow. Intravenous infusion of sodium taurocholate established a high correlation between hepatic bile flow and bile acid excretion. Secretin, a hormone that stimulates bicarbonate secretion, was also found to increase hepatic bile flow. The sources of the water entering the biliary system with these two stimuli were differentiated by the use of mannitol. An increase in its excretion parallels the increase in bile flow in response to bile acids but not secretin, which led to a quantitative distinction between canalicular and ductular water flow. The finding of aquaglyceroporin-9 in the basolateral surface of the hepatocyte accounted for the rapid entry of mannitol into hepatocytes and its exclusion from water movement in the ductules where aquaporin-1 is present. Electron microscopy demonstrated that bile acids generate the formation of vesicles that contain lecithin and cholesterol after their receptor-mediated canalicular transport. Biophysical studies established that the osmotic effect of bile acids varies with their concentration and also with the proportion of mono-, di-, and trihydroxy bile acids and provides a basis for understanding their physiological effects. Because of the varying osmotic effect of bile acids, it is difficult to quantify bile acid independent flow generated by other solutes, such as glutathione, which enters the biliary system. Monohydroxy bile acids, by markedly increasing aggregation number, severely reduce water flow. Developing biomarkers for the noninvasive assessment of normal hepatic bile flow remains an elusive goal that merits further study.

Desmosterol is a C27 sterol intermediate in cholesterol synthesis generated during the metabolic pathway that transforms lanosterol into cholesterol. It has become of particular interest in the pathogenesis of Alzheimer's disease (AD) because of the report that the activity of the gene coding for the enzyme DHCR24, which metabolizes desmosterol to cholesterol, is selectively reduced in the affected areas of the brain. Any change in the pattern of C27 sterol intermediates in cholesterol synthesis merits investigation with respect to the pathogenesis of AD, since neurosteroids such as progesterone can modulate the tissue levels. We therefore analyzed the C27 sterol composition using a metabolomics approach that preserves the proportion of the different sterol intermediates. In AD, the proportion of desmosterol was found to be less than that of age-matched controls. The findings do not directly support the focus on Seladin-1, although they could reflect different stages of a slowly progressive disease.

Evidence is emerging that during the development of Alzheimer's disease (AD), changes in the synthesis and metabolism of cholesterol and progesterone are occurring that may or may not affect the progression of the disease. The concept arose from the recognition that dehydrocholesterol 24-reductase (DHCR24/Seladin-1), one of the nine enzymes in the endoplasmic reticulum that determines the transformation of lanosterol to cholesterol, is selectively reduced in late AD. As a consequence, the tissue level of desmosterol increases, affecting the expression of ABC transporters and the structure of lipid rafts, both determinants of amyloid-beta processing. However, the former effect is considered beneficial and the latter detrimental to processing. Other determinants of desmosterol tissue levels are 24,25 epoxycholesterol and the ABCG1 and ABCG4 transporters. Progesterone and its metabolites are determinants of tissue levels of desmosterol and several other sterol intermediates in cholesterol synthesis. Animal models indicate marked elevations in the tissue levels of these sterols at early time frames in the progression of neurodegenerative diseases. The low level of neuroprogesterone and metabolites in AD are consonant with the low level of desmosterol and may have a role in amyloid-beta processing. The sparse data that has accumulated appears to be a sufficient basis for proposing a systematic evaluation of the biologic roles of sterol intermediates in the slowly progressive neurodegeneration characteristic of AD.