High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma

We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.     

The therapeutic potential of chemical chaperones in protein folding diseases

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.     

The histidin-loop is essential for transport activity of human MDR3. A novel mutation of MDR3 in a patient with progressive familial intrahepatic cholestasis type 3

Experimental evidence has been provided that a histidine-loop within the nucleotide binding domain of ABC transporter is essential for efficient function of this class of transporter proteins. Here we report the first patient with a mutation of the putative histidine-loop of a human ABC transporter, the multi drug resistance protein 3 (MDR3). The patient presented at the age of 4years with a history of severe pruritus, elevated serum gamma-glutamyltransferase and bile acid levels since several years suggesting the diagnosis of progressive familial intrahepatic cholestasis type 3 (PFIC-3) due to defects in MDR3. Liver biopsy demonstrated an apparently normal MDR3 expression, however, genetic analysis revealed a novel homozygous mutation in the ABCB4 gene (c.3691C>T) in the patient. This mutation was associated with a change of histidine to tyrosine at amino acid position 1231 of MDR3 (p.H1231Y). As shown by sequence alignment, this amino acid corresponds to the highly conserved histidine of the "H-loop", which is critical for ATP-hydrolysis, suggesting an essential role of histidine 1231 of human MDR3.     

Drug-induced bile duct injury

Drug-induced liver injury includes a spectrum of pathologies, some related to the mode of injury, some to the cell type primarily damaged. Among these, drug-induced bile duct injury is characterized by the destruction of the biliary epithelium following exposure to a drug. Most of the drugs associated with bile duct injury cause immune-mediated lesions to the epithelium of interlobular ducts. These share common histopathological features with primary biliary cholangitis, such as inflammation and necrosis at the expense of cholangiocytes and, if the insult persists, bile duct loss and biliary cirrhosis. Some drugs selectively target larger ducts. Such injury is often dose-dependent and thought to be the result of intrinsic drug toxicity. The histological changes resemble those seen in primary sclerosing cholangitis. This overview focuses on the clinical and pathological features of bile duct injury associated with drug treatment and on the immunological and biochemical effects that drugs exert on the biliary epithelium. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Sphingolipids in colon cancer

Colorectal cancer is one of the major causes of death in the western world. Despite increasing knowledge of the molecular signaling pathways implicated in colon cancer, therapeutic outcomes are still only moderately successful. Sphingolipids, a family of N-acyl linked lipids, have not only structural functions but are also implicated in important biological functions. Ceramide, sphingosine and sphingosine-1-phosphate are the most important bioactive lipids, and they regulate several key cellular functions. Accumulating evidence suggests that many cancers present alterations in sphingolipids and their metabolizing enzymes. The aim of this review is to discuss the emerging roles of sphingolipids, both endogenous and dietary, in colon cancer and the interaction of sphingolipids with WNT/β-catenin pathway, one of the most important signaling cascades that regulate development and homeostasis in intestine. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Genetic determinants of cholangiopathies: Molecular and systems genetics

Familial cholangiopathies are rare but potentially severe diseases. Their spectrum ranges from fairly benign conditions as, for example, benign recurrent intrahepatic cholestasis to low-phospholipid associated cholelithiasis and progressive familial intrahepatic cholestasis (PFIC). Many cholangiopathies such as primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC) affect first the bile ducts (“ascending pathophysiology”) but others, such as PFIC, start upstream in hepatocytes and cause progressive damage “descending” down the biliary tree and leading to end-stage liver disease. In recent years our understanding of cholestatic diseases has improved, since we have been able to pinpoint numerous disease-causing mutations that cause familial cholangiopathies. Accordingly, six PFIC subtypes (PFIC type 1–6) have now been defined. Given the availability of genotyping resources, these findings can be introduced in the diagnostic work-up of patients with peculiar cholestasis. In addition, functional studies have defined the pathophysiological consequences of some of the detected variants. Furthermore, ABCB4 variants do not only cause PFIC type 3 but confer an increased risk for chronic liver disease in general. In the near future these findings will serve to develop new therapeutic strategies for patients with liver diseases. Here we present the latest data on the genetic background of familial cholangiopathies and discuss their application in clinical practice for the differential diagnosis of cholestasis of unknown aetiology. As look in the future we present “system genetics” as a novel experimental tool for the study of cholangiopathies and disease-modifying genes. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.