High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy of Paired Clinical Liver Tissue Samples from Hepatocellular Cancer and Surrounding Region.

The global burden of liver cancer is increasing. Timely diagnosis is important for optimising the limited available treatment options. Understanding the metabolic consequences of hepatocellular carcinoma (HCC) may lead to more effective treatment options. We aimed to document metabolite differences between HCC and matched surrounding tissues of varying aetiology, obtained at the time of liver resection, and to interpret metabolite changes with clinical findings. High-resolution magic angle spinning nuclear magnetic resonance (HRMAS-NMR) spectroscopy analyses of N = 10 paired HCC and surrounding non-tumour liver tissue samples were undertaken. There were marked HRMAS-NMR differences in lipid levels in HCC tissue compared to matched surrounding tissue and more subtle changes in low-molecular-weight metabolites, particularly when adjusting for patient-specific variability. Differences in lipid-CH3, lipid-CH2, formate, and acetate levels were of particular interest. The obvious differences in lipid content highlight the intricate interplay between metabolic adaptations and cancer cell survival in the complex microenvironment of liver cancer. Differences in formate and acetate might relate to bacterial metabolites. Therefore, documentation of metabolites in HCC tissue according to histology findings in patients is of interest for personalised medicine approaches and for tailoring targeted treatment strategies.

Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells.

Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells' maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

The Role of Mass Spectrometry in Hepatocellular Carcinoma Biomarker Discovery.

Hepatocellular carcinoma (HCC) is the main liver malignancy and has a high mortality rate. The discovery of novel biomarkers for early diagnosis, prognosis, and stratification purposes has the potential to alleviate its disease burden. Mass spectrometry (MS) is one of the principal technologies used in metabolomics, with different experimental methods and machine types for different phases of the biomarker discovery process. Here, we review why MS applications are useful for liver cancer, explain the MS technique, and briefly summarise recent findings from metabolomic MS studies on HCC. We also discuss the current challenges and the direction for future research.

Mapping of population disparities in the cholangiocarcinoma urinary metabolome.

Phenotypic diversity in urinary metabolomes of different geographical populations has been recognized recently. In this study, urinary metabolic signatures from Western (United Kingdom) and South-East Asian (Thai) cholangiocarcinoma patients were characterized to understand spectral variability due to host carcinogenic processes and/or exogenous differences (nutritional, environmental and pharmaceutical). Urinary liquid chromatography mass spectroscopy (LC-MS) spectral profiles from Thai (healthy = 20 and cholangiocarcinoma = 14) and UK cohorts (healthy = 22 and cholangiocarcinoma = 10) were obtained and modelled using chemometric data analysis. Healthy metabolome disparities between the two distinct populations were primarily related to differences in dietary practices and body composition. Metabolites excreted due to drug treatment were dominant in urine specimens from cholangiocarcinoma patients, particularly in Western individuals. Urine from participants with sporadic (UK) cholangiocarcinoma contained greater levels of a nucleotide metabolite (uridine/pseudouridine). Higher relative concentrations of 7-methylguanine were observed in urine specimens from Thai cholangiocarcinoma patients. The urinary excretion of hippurate and methyladenine (gut microbial-host co-metabolites) showed a similar pattern of lower levels in patients with malignant biliary tumours from both countries. Intrinsic (body weight and body composition) and extrinsic (xenobiotic metabolism) factors were the main causes of disparities between the two populations. Regardless of the underlying aetiology, biological perturbations associated with cholangiocarcinoma urine metabolome signatures appeared to be influenced by gut microbial community metabolism. Dysregulation in nucleotide metabolism was associated with sporadic cholangiocarcinoma, possibly indicating differences in mitochondrial energy production pathways between cholangiocarcinoma tumour subtypes. Mapping population-specific metabolic disparities may aid in interpretation of disease processes and identification of candidate biomarkers.

Characterisation of the Serum Metabolic Signature of Cholangiocarcinoma in a United Kingdom Cohort.

BACKGROUND: A distinct serum metabonomic pattern has been previously revealed to be associated with various forms of liver disease. Here, we aimed to apply mass spectrometry to obtain serum metabolomic profiles from individuals with cholangiocarcinoma and benign hepatobiliary diseases to gain an insight into pathogenesis and search for potential early-disease biomarkers. METHODS: Serum samples were profiled using a hydrophilic interaction liquid chromatography platform, coupled to a mass spectrometer. A total of 47 serum specimens from 8 cholangiocarcinoma cases, 20 healthy controls, 8 benign disease controls (bile duct strictures) and 11 patients with hepatocellular carcinoma (as malignant disease controls) were included. Data analysis was performed using univariate and multivariate statistics. RESULTS: The serum metabolome disparities between the metabolite profiles from healthy controls and patients with hepatobiliary disease were predominantly related to changes in lipid and lipid-derived compounds (phospholipids, bile acids and steroids) and amino acid metabolites (phenylalanine). A metabolic pattern indicative of inflammatory response due to cirrhosis and cholestasis was associated with the disease groups. The abundance of phospholipid metabolites was altered in individuals with liver disease, particularly cholangiocarcinoma, but no significant difference was seen between profiles from patients with benign biliary strictures and cholangiocarcinoma. CONCLUSION: The serum metabolome in cholangiocarcinoma exhibited changes in metabolites related to inflammation, altered energy production and phospholipid metabolism. This study serves to highlight future avenues for biomarker research in large-scale studies.

Cholangiocarcinoma: a guide for the nonspecialist.

Cholangiocarcinoma (CCA) is a tumor with increasing prevalence around the world. The prevalence of CCA is highest in East Asia and most significantly in the countries through which the Mekong River flows, owing to the presence of liver flukes, which are consumed in raw fish dishes. Outside Asia, the causes of bile duct cancers for the most part are unknown. In this review, we assess the current state of knowledge in both fluke-associated and sporadic CCA, from etiological, diagnostic, and treatment perspectives.

The Plasma and Serum Metabotyping of Hepatocellular Carcinoma in a Nigerian and Egyptian Cohort using Proton Nuclear Magnetic Resonance Spectroscopy.

BACKGROUND/AIMS: Previous studies have observed disturbances in the 1H nuclear magnetic resonance (NMR) blood spectral profiles in malignancy. No study has metabotyped serum or plasma of hepatocellular carcinoma (HCC) patients from two diverse populations. We aimed to delineate the HCC patient metabotype from Nigeria (mostly hepatitis B virus infected) and Egypt (mostly hepatitis C virus infected) to explore lipid and energy metabolite alterations that may be independent of disease aetiology, diet and environment. METHODS: Patients with HCC (53) and cirrhosis (26) and healthy volunteers (19) were recruited from Nigeria and Egypt. Participants provided serum or plasma samples, which were analysed using 600 MHz 1H NMR spectroscopy with nuclear Overhauser enhancement spectroscopy pulse sequences. Median group spectra comparison and multivariate analysis were performed to identify regions of difference. RESULTS: Significant differences between HCC patients and healthy volunteers were detected in levels of low density lipoprotein (P = 0.002), very low density lipoprotein (P < 0.001) and lactate (P = 0.03). N-acetylglycoproteins levels in HCC patients were significantly different from both healthy controls and cirrhosis patients (P < 0.001 and 0.001). CONCLUSION: Metabotype differences were present, pointing to disturbed lipid metabolism and a switch from glycolysis to alternative energy metabolites with malignancy, which supports the Warburg hypothesis of tumour metabolism.

Urinary metabolic profiling by 1H NMR spectroscopy in patients with cirrhosis may discriminate overt but not covert hepatic encephalopathy.

To date urinary metabolic profiling has been applied to define a specific metabolic fingerprint of hepatocellular carcinoma on a background of cirrhosis. Its utility for the stratification of other complications of cirrhosis, such as hepatic encephalopathy (HE), remains to be established. Urinary proton nuclear magnetic resonance (1H-NMR) spectra were acquired and NMR data from 52 patients with cirrhosis (35 male; 17 female, median (range) age [60 (18-81) years]) and 17 controls were compared. A sub-set of 45 patients (33 male; 12 female, [60 (18-90) years, median model for end stage liver disease (MELD) score 11 (7-27)]) were fully characterised by West-Haven criteria, Psychometric Hepatic Encephalopathy Score (PHES) and electroencephalogram (EEG), and defined as overt HE (OHE, n = 21), covert HE (cHE, n = 7) or no HE (n = 17). Urinary proton nuclear magnetic resonance (1H-NMR) spectra were analysed by partial-least-squares discriminant analysis (PLS-DA). The results showed good discrimination between patients with cirrhosis (n = 52) and healthy controls (n = 17) (R2X = 0.66, R2Y = 0.47, Q2Y = 0.31, sensitivity-60 %, specificity-100 %) as the cirrhosis group had higher 1-methylnicotinamide with lower hippurate, acetate, phenylacetylglycine and N-methyl nicotinic acid levels. While patients with OHE could be discriminated from those with no HE, with higher histidine, citrate and creatinine levels, the best models lack robust validity (R2X = 0.65, R2Y = 0.48, Q2Y = 0.12, sensitivity-100 %, specificity-64 %) with the sample size used. Urinary 1H-NMR metabolic profiling did not discriminate patients with cHE from those without HE, nor discriminate subjects on the basis of PHES/EEG result or MELD score. In conclusion, patients with cirrhosis showed different urinary 1H-NMR metabolic profiles compared to healthy controls and those with OHE may be distinguished from those with no HE although larger studies are required. However, urinary 1H-NMR metabolic profiling did not discriminate patients with differing grades of HE or according to severity of underlying liver disease.

Hepatic steatosis and fibrosis: Non-invasive assessment.

Chronic liver disease is a major cause of morbidity and mortality worldwide and usually develops over many years, as a result of chronic inflammation and scarring, resulting in end-stage liver disease and its complications. The progression of disease is characterised by ongoing inflammation and consequent fibrosis, although hepatic steatosis is increasingly being recognised as an important pathological feature of disease, rather than being simply an innocent bystander. However, the current gold standard method of quantifying and staging liver disease, histological analysis by liver biopsy, has several limitations and can have associated morbidity and even mortality. Therefore, there is a clear need for safe and non-invasive assessment modalities to determine hepatic steatosis, inflammation and fibrosis. This review covers key mechanisms and the importance of fibrosis and steatosis in the progression of liver disease. We address non-invasive imaging and blood biomarker assessments that can be used as an alternative to information gained on liver biopsy.

Urinary Metabotyping of Hepatocellular Carcinoma in a UK Cohort Using Proton Nuclear Magnetic Resonance Spectroscopy.

BACKGROUND: Discriminatory metabolic profiles have been described in urinary 1H nuclear magnetic resonance (NMR) spectroscopy studies of African patients with hepatocellular carcinoma (HCC). This study aimed to assess similarities in a UK cohort, where there is a greater etiological diversity. METHODS: Urine from cirrhosis and HCC patients was analyzed using a 600 MHz 1H NMR system. Multivariate analysis and median group MR spectra comparison identified metabolite alterations between groups. Metabolite identification was achieved through literature reference and statistical total correlation spectroscopy. Diagnostic accuracy was compared to serum alpha-fetoprotein (AFP). RESULTS: Of the 52 patients recruited, 13 samples from HCC and 25 from cirrhosis patients were selected. At 200 IU mL-1, diagnostic sensitivity of AFP was 27%. Multivariate analysis of urinary spectra generated diagnostic models with a sensitivity/specificity of 53.6%/96%. p-Cresol sulfate (P = 0.04), creatinine (P = 0.03), citrate (P = 0.21) and hippurate (P = 0.52) were reduced in the HCC patients. Carnitine (P = 0.31) and formate (P = 0.44) were elevated. CONCLUSION: Diagnostic sensitivity was lower than previous African studies, but still outperformed serum AFP. Reduced creatinine, citrate and hippurate and elevated carnitine are comparable with the African studies. p-Cresol sulfate alteration is a novel finding and may indicate an altered sulfonation capacity of the liver in patients with HCC.

Urinary nuclear magnetic resonance spectroscopy of a Bangladeshi cohort with hepatitis-B hepatocellular carcinoma: A biomarker corroboration study.

AIM: To establish if a distinct urinary metabolic profile could be identified in Bangladeshi hepatitis-B hepatocellular carcinoma (HCC) patients compared to cirrhosis patients and controls. METHODS: Urine samples from 42 Bangladeshi patients with HCC (39 patients with hepatitis-B HCC), 47 with cirrhosis on a background of hepatitis B, 46 with chronic hepatitis B, and seven ethnically-matched healthy controls were analyzed using nuclear magnetic resonance (NMR) spectroscopy. A full dietary and medication history was recorded for each subject. The urinary NMR data were analyzed using principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) techniques. Differences in relative signal levels of the most discriminatory metabolites identified by PCA and OPLS-DA were compared between subject groups using an independent samples Kruskal-Wallis one-way analysis of variance (ANOVA) test with all pairwise multiple comparisons. Within the patient subgroups, the Mann-Whitney U test was used to compare metabolite levels depending on hepatitis B e-antigen (HBeAg) status and treatment with anti-viral therapy. A Benjamini-Hochberg adjustment was applied to acquire the level of significance for multiple testing, with a declared level of statistical significance of P < 0.05. RESULTS: There were significant differences in age (P < 0.001), weight (P < 0.001), and body mass index (P < 0.001) across the four clinical subgroups. Serum alanine aminotransferase (ALT) was significantly higher in the HCC group compared to controls (P < 0.001); serum α-fetoprotein was generally markedly elevated in HCC compared to controls; and serum creatinine levels were significantly reduced in the HCC group compared to the cirrhosis group (P = 0.004). A three-factor PCA scores plot showed clustering of the urinary NMR spectra from the four subgroups. Metabolites that contributed to the discrimination between the subgroups included acetate, creatine, creatinine, dimethyamine (DMA), formate, glycine, hippurate, and trimethylamine-N-oxide (TMAO). A comparison of relative metabolite levels confirmed that carnitine was significantly increased in HCC; and creatinine, hippurate, and TMAO were significantly reduced in HCC compared to the other subgroups. HBeAg negative patients showed a significant increase in creatinine (P = 0.001) compared to HBeAg positive patients in the chronic hepatitis B subgroup, whilst HBeAg negative patients showed a significant decrease in DMA (P = 0.004) in the cirrhosis subgroup compared to HBeAg positive patients. There were no differences in metabolite levels in HCC patients who did or did not receive antiviral treatment. CONCLUSION: Urinary NMR changes in Bangladeshi HCC were identified, corroborating previous findings from Egypt and West Africa. These findings could form the basis for the development of a cost-effective HCC dipstick screening test.

Hepatocellular carcinoma: Review of disease and tumor biomarkers.

Hepatocellular carcinoma (HCC) is a common malignancy and now the second commonest global cause of cancer death. HCC tumorigenesis is relatively silent and patients experience late symptomatic presentation. As the option for curative treatments is limited to early stage cancers, diagnosis in non-symptomatic individuals is crucial. International guidelines advise regular surveillance of high-risk populations but the current tools lack sufficient sensitivity for early stage tumors on the background of a cirrhotic nodular liver. A number of novel biomarkers have now been suggested in the literature, which may reinforce the current surveillance methods. In addition, recent metabonomic and proteomic discoveries have established specific metabolite expressions in HCC, according to Warburg's phenomenon of altered energy metabolism. With clinical validation, a simple and non-invasive test from the serum or urine may be performed to diagnose HCC, particularly benefiting low resource regions where the burden of HCC is highest.

The role of intestinal microbiota in murine models of acetaminophen-induced hepatotoxicity.

BACKGROUND & AIMS: Variations in intestinal microbiota may influence acetaminophen metabolism. This study aimed to determine whether intestinal microbiota are a source of differential susceptibility to acetaminophen-induced hepatotoxicity. METHODS: Conventionally housed C3H/HeH (CH) and C3H/HeH germ-free (GF) mice were administered a 200 mg/kg IP dose of acetaminophen. The severity of hepatotoxicity at 8 h was assessed by histology and biochemical indices. A urinary metabolic profile was obtained using (1) H-NMR. Baseline hepatic glutathione content and CYP2E1 expression were quantified. An additional group of C3H/HeJ (LPS-r) mice were assessed to determine the contribution of LPS/TLR4 signalling. RESULTS: Baseline glutathione levels were significantly reduced (P = 0.03) in GF mice. CYP2E1 mRNA expression and protein levels were not altered. Interindividual variability did not differ between GF and CH groups. No significant differences in the extent of hepatocellular injury (ALT or percentage necrosis) were demonstrated. However, a milder acute liver failure (ALF) phenotype was shown in GF compared with CH mice, with reduced plasma bilirubin and creatinine and increased blood glucose. Differential acetaminophen metabolism was demonstrated. GF mice displayed a higher urinary acetaminophen-sulphate:glucuronide ratio compared with CH (P = 0.01). Urinary analysis showed metabolic differentiation of GF and CH groups at baseline and 8 h (cross-validated anova P = 1 × 10(-22) ). Interruption of TLR4 signalling in LPS-r mice had additional protective effects. CONCLUSION: Variations in intestinal microbiota do not fully explain differential susceptibility to acetaminophen-induced hepatotoxicity. GF mice experienced some protection from secondary complications following acetaminophen overdose and this may be mediated through reduced TLR4/LPS signalling.

Lipid profiling of pre-treatment liver biopsy tissue predicts sustained virological response in patients with chronic hepatitis C.

AIM: Hepatic lipid is important in the pathogenesis and progression of hepatitis C-related liver disease. Polyunsaturated fatty acids have been shown to reduce viral replication in cell culture. Proton magic angle spinning magnetic resonance spectroscopy ((1) H MAS MRS) enables metabolic analysis of intact tissue. The aim was to examine the relationship between hepatic lipid composition by metabolic profiling of liver tissue at baseline and treatment response to pegylated-Interferon alfa2 and Ribavirin. METHODS: Baseline liver biopsy samples from 31 patients with chronic hepatitis C were analyzed histologically and by (1) H MAS MRS. Indices of lipid composition were derived and partial least squares discriminant analysis with cross-validation was used to predict treatment outcome. RESULTS: Of 31 patients, 14 achieved sustained virological response (SVR). Lipid polyunsaturation (median (IQR)) was higher in SVR (3.41% (2.31)) than in treatment failure (TF) (2.15% (1.51)), P = 0.02. Lipid saturation was lower in SVR (85.9% (3.39)) than TF (86.7% (2.17)), P = 0.04. The total lipid content was lower in SVR (1.54% (0.81)) than TF (2.72% (3.47)), P = 0.004. Total choline to lipid ratio was higher in SVR (11.51% (9.99)) than TF (7.5% (6.82)), P = 0.007. Cross-validation correctly predicted the SVR group in 13 of 14 samples with 1 sample misclassified, and the TF group in all 17 samples. CONCLUSIONS: Lipid polyunsaturation was greater and total lipid lower in those with SVR, compared with TF. Metabolic profiling of intact liver biopsy samples predicted SVR with high accuracy. Hepatic lipid composition may impact on treatment success.

Serum metabolic profiling in inflammatory bowel disease.

BACKGROUND: The inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis (UC), are chronic inflammatory conditions of the gastrointestinal tract whose pathogenesis is not completely understood. (1)H nuclear magnetic resonance (NMR) spectroscopy of serum generates comprehensive metabolic profiles, reflecting systemic metabolism, which may be altered in disease states. AIM: The aim of this study was to use (1)H NMR-based serum metabolic profiling in the investigation of CD patients, UC patients, and controls, potentially to provide insights into disordered metabolism in IBD, and into underlying mechanisms of disease. METHODS: Serum metabolic profiles were acquired from 67 individuals (24 CD patients, 20 UC patients, and 23 healthy controls). The multivariate pattern-recognition techniques of principal components analysis (PCA) and partial least squares discriminant analysis with orthogonal signal correction (OSC-PLS-DA) were used to investigate differences between cohorts. RESULTS: OSC-PLS-DA distinguished CD and UC cohorts with significant predictive accuracy, highlighting differences in lipid and choline metabolism. Metabolic profiles of both CD and UC cohorts, and the combined IBD cohort, differed significantly from controls: metabolites of importance in the OSC-PLS-DA models included lipoproteins (especially HDL cholesterol), choline, N-acetylglycoprotein, and amino acids. CONCLUSIONS: (1)H NMR-based metabolic profiling has identified distinct differences in serum metabolic phenotype between CD and UC patients, as well as between IBD patients and controls.

Urinary metabolic biomarkers of hepatocellular carcinoma in an Egyptian population: a validation study.

The advent of metabonomics has seen a proliferation of biofluid profiling studies of patients with hepatocellular carcinoma. The majority of these studies have been conducted in single indigenous populations making the widespread applicability of candidate metabolite biomarkers difficult. Presented here is a urinary proton nuclear magnetic resonance spectroscopy study of mainly hepatitis C virus infected Egyptian patients with hepatocellular carcinoma, which corroborates findings of a previous study from our group of mainly hepatitis B-infected Nigerian patients with hepatocellular carcinoma. Using multivariate statistical analysis, in the form of orthogonal signal-corrected partial least squared discriminant analysis, the sensitivity and specificity of the technique for distinguishing patients with tumors from healthy controls and patients with cirrhosis was 100%/94% and 81%/71%, respectively. Discriminatory metabolites included glycine, trimethylamine-N-oxide, hippurate, citrate, creatinine, creatine, and carnitine. This metabolic profile bears similarity to profiles identified in the Nigerian cohort of subjects indicative of tumor effects on physiology, energy production, and aberrant chromosomal methylation. This is the first study to identify similarly altered urine metabolic profiles of hepatocellular carcinoma in two etiologically and ethnically distinct populations, suggesting that altered metabolism as a result of tumorogenesis is independent of these two factors.

Differences in gut microbial metabolism are responsible for reduced hippurate synthesis in Crohn's disease.

BACKGROUND: Certain urinary metabolites are the product of gut microbial or mammalian metabolism; others, such as hippurate, are mammalian-microbial 'co-metabolites'. It has previously been observed that Crohn's disease (CD) patients excrete significantly less hippurate than controls. There are two stages in the biosynthesis of this metabolite: 1) gut microbial metabolism of dietary aromatic compounds to benzoate, and 2) subsequent hepatorenal conjugation of benzoate with glycine, forming hippurate. Differences in such urinary co-metabolites may therefore reflect systemic consequences of altered gut microbial metabolism, though altered host metabolic pathways may also be involved. METHODS: It was hypothesised that reduced hippurate excretion in CD patients was due to alterations in the gut microbiota, and not differences in dietary benzoate, nor defective host enzymatic conjugation of benzoate. 5 mg/kg sodium benzoate were administered orally to 16 CD patients and 16 healthy controls on a low-benzoate diet. Baseline and peak urinary hippurate excretion were measured. RESULTS: Baseline hippurate levels were significantly lower in the CD patients (p = 0.0009). After benzoate ingestion, peak urinary levels of hippurate did not differ significantly between the cohorts. Consequently the relative increase in excretion was significantly greater in CD (p = 0.0007). CONCLUSIONS: Lower urinary hippurate levels in CD are not due to differences in dietary benzoate. A defect in the enzymatic conjugation of benzoate in CD has been excluded, strongly implicating altered gut microbial metabolism as the cause of decreased hippurate levels in CD.

Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy.

OBJECTIVES: Cholangiocarcinoma (CCA) has a poor prognosis and its aetiology is inadequately understood. Magnetic resonance spectroscopy (MRS) of bile may provide insights into the pathogenesis of CCA and help identify novel diagnostic biomarkers. The aim of this study was to compare the chemical composition of bile from patients with CCA with that of bile from patients with benign biliary disease. METHODS: Magnetic resonance spectra were acquired from the bile of five CCA patients and compared with MRS of control bile from patients with benign biliary disease (seven with gallstones, eight with sphincter of Oddi dysfunction [SOD], five with primary sclerosing cholangitis [PSC]). Metabolic profiles were compared using both univariate and multivariate pattern-recognition analysis. RESULTS: Univariate analysis showed that levels of glycine-conjugated bile acids were significantly increased in patients with CCA, compared with the benign disease groups (P= 0.002). 7 beta primary bile acids were significantly increased (P= 0.030) and biliary phosphatidylcholine (PtC) levels were reduced (P= 0.010) in bile from patients with CCA compared with bile from gallstone patients. These compounds were also of primary importance in the multivariate analysis: the cohorts were differentiated by partial least squares discriminant analysis (PLS-DA). CONCLUSIONS: These preliminary data suggest that altered bile acid and PtC metabolism play an important role in CCA aetiopathogenesis and that specific metabolites may have potential as future biomarkers.

Hepatic lipid profiling in chronic hepatitis C: an in vitro and in vivo proton magnetic resonance spectroscopy study.

BACKGROUND & AIMS: Hepatic steatosis is an important factor in pathogenesis, progression and response to treatment in hepatitis C. We aimed to investigate differences in hepatic lipid composition in liver biopsies from patients with chronic hepatitis C using proton magnetic resonance spectroscopy ((1)H MRS) and to translate these findings to the in vivo clinical setting. METHODS: Two cohorts of patients with histologically defined chronic hepatitis C were studied. High-resolution MR spectra were obtained from 47 liver biopsy samples. These data were used to derive biologically relevant prior knowledge for the assignment and interpretation of lower-resolution in vivo hepatic MRS data acquired at 1.5T from a second cohort of 59 patients. MRS data were obtained both in vitro and in vivo from a subset of 11 patients. RESULTS: Multivariate factor analysis demonstrated characteristic MR spectral differences by fibrosis stage and genotype. Total lipid increased with fibrosis stage (r=0.43, p=0.003) and was higher in genotype 3 compared to genotype 1 (p=0.03), while lipid polyunsaturation decreased with increasing fibrosis stage (r=-0.55, p<0.0005) and, independently, with increasing steatosis. Non-invasive assessment using in vivo hepatic (1)H MRS corroborated in vitro findings, but the signal-to-noise ratio was insufficient for reliable assessment of lipid polyunsaturation in vivo. CONCLUSIONS: Hepatic lipid composition was analysed using MRS in patients with chronic hepatitis C in vitro and in vivo, demonstrating significant differences in indices by disease severity. High-resolution data informed the analysis and interpretation of in vivo spectra, but further improvements in spectral quality in vivo are required.