The author outlines his philosophy and practice of management of gall bladder cancer based on his more than three-decade experience at a large tertiary level super-specialty referral hospital attached to a university-status teaching institution at Lucknow in northern India where GBC is very common.

High concentrations of taurine are present in the developing human brain and maternal breast milk. Taurine is thought to influence fetal growth and brain development based on experimental rodent studies. As fish is an important dietary source of taurine, we investigated associations between taurine concentrations and child outcomes in a high fish consuming population.

To examine associations between maternal and cord serum taurine concentrations and birth anthropometric measures and cognitive development in children at 20 months of age.

Pregnant women were recruited between 2008 and 2011 as part of Nutrition Cohort 2 (NC2) of the Seychelles Child Development Study (SCDS). Maternal taurine serum concentrations were measured at 28 week's gestation and in cord serum. Child weight, length and head circumference were measured at birth and neurodevelopment was assessed using Bayley Scales of Infant Development II (BSID-II) at 20 months of age. Associations between taurine status, birth measures and neurodevelopmental outcomes were examined (n = 300) using regression models and adjusted for relevant covariates.

Mean (SD) maternal and cord taurine concentrations were 124.9 (39.2) µmol/L (range 28.2-253.9 µmol/L) and 187.6 (60.0) µmol/L (range 55.0-417.4 µmol/L) respectively. We found no associations between maternal taurine concentrations and child anthropometric and neurodevelopmental measures (weight β = -0.001, SE=0.001; length β = -0.006, SE=0.006; head circumference β = -0.002, SE=0.002; MDI β = -0.005, SE=0.015; PDI β = -0.004, SE=0.016; all P > 0.05), or between cord taurine concentrations and outcomes (weight β = -0.001, SE<0.000; length β = -0.001, SE=0.004; head circumference β < 0.000, SE=0.002; MDI β = 0.004, SE=0.010; PDI β = -0.015, SE=0.012; all P > 0.05).

The Seychellois population have high maternal and cord taurine concentrations owing to their high fish intake and may be considered taurine replete compared to individuals who consume a Westernised diet. This high taurine status may explain why there were no significant associations between maternal and cord taurine concentrations and outcomes after adjusting for covariates.

Bile acids are acidic steroids which help in lipid absorption, act as signaling molecules, and are key intermediate molecules between host and gut microbial metabolism. Perturbations in the circulating bile acid pool can lead to dysregulated metabolic and immunological function which may be associated with liver and intestinal disease. Bile acids have chemically diverse structures and are present in a broad range of concentrations in a wide variety of samples with complex biological matrices. Advanced analytical methods are therefore required to identify and accurately quantify individual bile acids. Though enzymatic determination of total bile acid is most popular in clinical laboratories, these methods provide limited information about individual bile acids. Advanced analytical methods such as gas chromatography- and liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy are highly informative techniques which help in identification and quantification of individual bile acids in complex biological matrices. Here, we review the detection technologies currently used for bile acid identification and quantification. We further discuss the advantages and disadvantages of these analytical techniques with respect to sensitivity, specificity, robustness, and ease of use. Graphical abstract.

Primary sclerosing cholangitis (PSC) has been considered to be either an "autoimmune disease" or a "bile acid-induced injury." In vitro MRS studies on PSC patients have limitations due to the contamination of bile with contrast agent (commonly administered during endoscopic retrograde cholangiopancreatography) and/or the use of patient cohorts with other diseases as controls. The objective of this study was to quantify biliary metabolites using in vivo ¹ H MRS and gain insight into the pathogenesis of PSC. Biliary metabolites in 10 PSC patients and 14 healthy controls were quantified in vivo using ¹ H MRS on a 3 T MR scanner. The concentrations of total bile acids plus cholesterol, glycine-conjugated bile acids, taurine-conjugated bile acids, and choline-containing phospholipids (chol-PLs) were compared between the two groups. There were statistically significant decreases in the levels of the above mentioned biliary metabolites in the PSC patients compared with controls. The reduction in bile acid secretion in bile of PSC patients indicates accumulation of bile acids in hepatocytes. Moreover, reduction in the levels of chol-PLs in bile may increase the toxic effects of bile acids. Our findings suggest that the bile duct injury in PSC patients is most likely due to "bile acid-induced injury."

Increased sugar intake is implicated in Type-2 diabetes and fatty liver disease; however, the mechanisms through which glucose and fructose promote these conditions are unclear. We hypothesize that alterations in intestinal metabolite and microbiota profiles specific to each monosaccharide are involved. Two groups of six adult C57BL/6 mice were fed for 10-weeks with diets with glucose (G) or fructose (F) as sole carbohydrates, and a third group was fed with a normal chow carbohydrate mixture (N). Fecal metabolites were profiled by nuclear magnetic resonance (NMR) and microbial composition by real-time polymerase chain reaction (qPCR). Although N, G and F mice exhibited similar weight gains (with slight slower gains for F) and glucose tolerance, multivariate analysis of NMR data indicated that F mice were separated from N and G, with decreased butyrate and glutamate and increased fructose, succinate, taurine, tyrosine, and xylose. The different sugar diets also resulted in distinct intestinal microbiota profiles. That associated with fructose seemed to hold more potential to induce host metabolic disturbances compared to glucose, mainly by promoting bile acid deconjugation and taurine release and compromising intestinal barrier integrity. This may reflect the noted nonquantitative intestinal fructose absorption hence increasing its availability for microbial metabolism, a subject for further investigation.

Convenient multiple dosing makes oral administration an ideal route for delivery of therapeutic siRNA. However, hostile GI environments and nonspecific biological trafficking prevent achieving appropriate bioavailability of siRNA. Here, an orally administered AuNP-siRNA-glycol chitosan-taurocholic acid nanoparticle (AR-GT NPs) was developed to selectively deliver Akt2 siRNA and treat colorectal liver metastases (CLM). AR-GT NPs are dual padlocked nonviral vectors in which the initially formed AuNP-siRNA (AR) conjugates are further encompassed by bifunctional glycol chitosan-taurocholic acid (GT) conjugates. Covering the surface of AR with GT protected the Akt2 siRNA from GI degradation, facilitated active transport through enterocytes, and enhanced selective accumulation in CLM. Our studies in CLM animal models resulted in the reduction in Akt2 production, followed by initiation of apoptosis in cancer cells after oral administration of Akt2 siRNA-loaded AR-GT. This therapeutic siRNA delivery system may be a promising approach in treating liver-associated diseases.

Improved models of the gastrointestinal environment have great potential to assist the complex process of drug formulation. Molecular dynamics (MD) is a powerful method for investigating phase behavior at a molecular level. In this study we use multiple MD simulations to calculate phase diagrams for bile before and after digestion. In these computational models, undigested bile is represented by mixtures of palmitoyl-oleoylphosphatidylcholine (POPC), sodium glycodeoxycholate (GDX), and water. Digested bile is modeled using a 1:1 mixture of oleic acid and palmitoylphosphatidylcholine (lysophosphatidylcholine, LPC), GDX, and water. The computational phase diagrams of undigested and digested bile are compared, and we describe the typical intermolecular interactions that occur between phospholipids and bile salts. The diffusion coefficients measured from MD simulation are compared to experimental diffusion data measured by DOSY-NMR, where we observe good qualitative agreement. In an additional set of simulations, the effect of different ionization states of oleic acid on micelle formation is investigated.

30-99% of administered nanoparticles will accumulate and sequester in the liver after administration into the body. This results in reduced delivery to the targeted diseased tissue and potentially leads to increased toxicity at the hepatic cellular level. This review article focuses on the inter- and intra-cellular interaction between nanoparticles and hepatic cells, the elimination mechanism of nanoparticles through the hepatobiliary system, and current strategies to manipulate liver sequestration. The ability to solve the "nanoparticle-liver" interaction is critical to the clinical translation of nanotechnology for diagnosing and treating cancer, diabetes, cardiovascular disorders, and other diseases.

In vitro (1)H MRS of human bile has shown potential in the diagnosis of various hepatopancreatobiliary (HPB) diseases. Previously, in vivo (1)H MRS of human bile in gallbladder using a 1.5 T scanner demonstrated the possibility of quantification of choline-containing phospholipids (chol-PLs). However, other lipid components such as bile acids play an important role in the pathophysiology of the HPB system. We have employed a higher magnetic field strength (3 T), and a custom-built receive array coil, to improve the quality of in vivo (1)H MRS of human bile in the gallbladder. We obtained significant improvement in the quality of 1D spectra (17 healthy volunteers) using a respiratory-gated PRESS sequence with well distinguished signals for total bile acids (TBAs) plus cholesterol resonating at 0.66 ppm, taurine-conjugated bile acids (TCBAs) at 3.08 ppm, chol-PLs at 3.22 ppm, glycine-conjugated bile acids (GCBAs) at 3.74 ppm, and the amide proton (-NH) arising from GCBAs and TCBAs in the region 7.76-8.05 ppm. The peak areas of these signals were measured by deconvolution, and subsequently the molar concentrations of metabolites were estimated with good accuracy, except for that of TBAs plus cholesterol. The concentration of TBAs plus cholesterol was overestimated in some cases, which could be due to lipid contamination. In addition, we report the first 2D L-COSY spectra of human gallbladder bile in vivo (obtained in 15 healthy volunteers). 2D L-COSY spectra will be helpful in differentiating various biliary chol-PLs in pathological conditions of the HPB system.

There is large usage of polybrominated diphenyl ethers (PBDEs) especially for decabromodiphenyl ether (BDE-209, Deca-BDE) in controlling the risks of fire. The toxicological effects of PBDEs are worth being concerned about. Female SD rats were daily gavaged with BDE-209 ether at the dose of 100 mg/kg for 20 days. Histological observation was performed for the screening of the target organs for BDE-209 exposure. The distribution and metabolism of PBDEs in the exposed main organs were evidenced by HRGC-HRMS. Alterations of the endogenous metabolite concentrations in urine were investigated using metabonomic approaches based on (1)H NMR spectrum. Histopathological changes including serious edema in kidney, hepatocellular spotty necrosis and perivasculitis in liver indicated that BDE-209 caused potential influences on endogenous metabolism in the exposed liver and the kidney. BDE-209 was found to be highly accumulated in lipid, ovary, kidney and liver after 20 days' exposure. Occurrence of other lower brominated PBDEs in the rats demonstrated that reductive debromination process happened in vivo. Hydroxylated and methoxylated-BDEs, as metabolism products, were also detected in the rat tissues. A total of 12 different endogenous metabolites showed obvious alterations in urine from the exposed rats, indicating the disturbance of the corresponding internal biochemical processes induced by BDE-209 exposure. These findings in vivo suggested the potential health risk might be of concern due to the toxicological effects of BDE-209 as a ubiquitous compound in the environment.

Human liver synthesizes bile; bile, containing a large number of metabolites, is transported through the canaliculi and bile ducts, and stored in the gallbladder before entering into the intestine. In the intestine, a large number of bile metabolites are reabsorbed and sent back to the liver for recirculation. Owing to close association of the bile with the gastrointestinal system, the bile metabolic profile is highly sensitive to the onset of numerous gastrointestinal disease processes. A growing number of studies suggest that hepatobiliary disease biomarkers are richly populated in human bile. These studies stress the potential of profiling the human bile metabolome for early diagnostics as well as deeper insights into gastrointestinal disease processes. Once the biomarkers are established reliably using human bile, they can be targeted in easily accessible fluids such as blood and urine or targeted in bile itself using noninvasive methods such as in vivo magnetic resonance spectroscopy. NMR spectroscopy is one of the most powerful bioanalytical tools, which promises profiling of human bile metabolome and exploring early biomarkers for hepatobiliary diseases. Comprehensive analysis of human bile using NMR spectroscopy has lead to identification and quantification of major bile metabolites. This review describes the discovery and quantitation of biomarkers of hepatobiliary diseases in human bile using NMR spectroscopy.

Bile acids, phospholipids, and cholesterol are the major lipid components in human bile. The composition of bile is altered in various cholestatic diseases, and determining such alterations will be of great clinical importance in understanding the pathophysiology of these diseases. A robust method for the simultaneous quantification of major biliary lipids--glycine-conjugated bile acids (GCBAs), taurine-conjugated bile acids (TCBAs), total bile acids (TBAs) and choline-containing phospholipids (choline-PLs) has been devised using (1)H NMR spectroscopy. Bile samples were obtained from patients with various hepatopancreatobiliary diseases (n=10) during an endoscopic retrograde cholangiopancreatography (ERCP) examination. Peak areas of metabolite-signals of interest were obtained simultaneously by deconvoluting the experimental spectrum, making the present method robust. GCBAs and TCBAs have been quantified using the peak areas of their characteristic methylene (CH(2)) signals resonating at 3.73 and 3.07 ppm, whereas TBA and choline-PLs were quantified using their methyl (CH(3)) and trimethylammonium (-N(+)(CH(3))(3)) signals resonating at 0.65 and 3.22 ppm respectively. The present method was compared with an NMR-based literature method (which involves dissolving bile in DMSO), and a good correlation was observed between the two methods with regression coefficients - 0.97, 0.99, 0.98 and 0.93 for GCBAs, TCBAs, TBAs, and choline-PLs respectively. This method has the potential to be extended to in vivo applications for the simultaneous quantification of various biliary lipids non-invasively.

Metabolic profiling of biofluids is emerging as an important area with a promising number of applications in clinical medicine, including early diagnosis of numerous diseases that normally remain silent until late in the progress of disease. While blood and urine are more often used to explore biomarkers that distinguish he healthy from disease conditions, human bile is emerging as a rich source of biomarkers specifically for the cancers of the liver (hepatocellular carcinoma), bile ducts (cholangiocarcinoma), gallbladder and pancreas. This is owing to the fact that metabolites linked to the pathways of tumor cell metabolism are rich in bile by virtue of its association or proximity to the pathological source. Recent methodological developments have enabled the identification of a number of bile metabolites that have links with hepatopancreatobiliary diseases. Investigations of human bile are also considered to help the biomarker discovery process in vitro and provide avenues for translational research in detecting and following dynamic variations of biomarkers in clinical settings using noninvasive approaches, such as in vivo magnetic resonance spectroscopy. This article reviews the current status and potential applications of human bile as a source of biomarkers, with emphasis on metabolites, for early detection of cancers associated with the hepatopancreatobiliary system.

In vitro, high-resolution (1)H and (31)P NMR based qualitative and quantitative analyses of the lipid components of the tissue, serum, and CSF of patients with primary brain tumors were performed. Proton NMR spectra of the lipid extract of serum (blood specimen collected before the surgical procedure) and surgically discarded tissue showed that the total cholesterol (T.CHOL) and choline containing phospholipids (PL) were significantly higher in quantity in medulloblastoma and glioblastoma multiforme as compared to normal subjects. Serum lipid extracts of grade II/ III gliomas showed a higher quantity of PL than normal subjects. Cholesterol esters (CHOLest) were detectable in the tissue lipid extract of the patients with tumors and absent in normal tissue. There was a reduction in the quantity of CHOLest in the serum lipid extract of the tumor patients as compared to normal subjects. Ratio of PL to T.CHOL in serum lipid extract showed a significant difference between different grades of tumors versus normal subjects, while, a significant difference was observed only in medulloblastoma versus normal subjects in tissue lipid extract. Ratio of CHOL to CHOLest distinguishes the different grades of tumors versus normal subjects as well as between different grades of tumors (except medulloblastoma versus glioblastoma). The ratio of the Ph (total phospholipids except phosphatidylcholine) to PC (phosphatidylcholine) in (31)P NMR based study showed a significant difference in all grades of tumors (except medulloblastoma) in normal subjects in tissue lipid extract as well as between different grades of tumors. Medulloblastoma could be differentiated from glioblastoma as well as from normal subjects in serum lipid extract by the ratio of the Ph to PC. Proton NMR spectra of the lipid extract of CSF showed that the CHOL, CHOLest, and PL were present in the patients with tumors, although these were absent in the patients with meningitis, motor neuron disease, and mitochondrial myopathies as well as in normal subjects. PL and T.CHOL provided discrimination between different grades of tumors (except glioblastoma versus medulloblastoma) in the lipid extract of the CSF. This study suggests the role of lipid estimation in CSF and serum as a complementary diagnostic tool for the evaluation of brain tumors preoperatively. NMR-based lipid estimation of post-surgical tumor tissue may also contribute to differentiating the tumor types.

The utility of (1)H MR spectroscopy in detecting chronic cholestasis has been investigated. The amide proton region of the (1)H MR spectrum of human bile plays a major role in differentiating cholestatic (Ch) patterns from the normal ones. Bile obtained from normal bile ducts contains both taurine and glycine conjugates of bile acids--cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA). Absence of a glycine-conjugated bile acid glycochenodeoxycholic acid (GCDCA) has been observed in bile samples obtained from primary sclerosing cholangitis (PSC) patients. A total of 32 patients with various hepatobiliary diseases were included in the study. Twenty-one patients had PSC and 11 had normal cholangiograms. One PSC patient was excluded from the study because of a bad spectrum. Seventeen out of the 20 PSC patients showed an absence of GCDCA in their (1)H MR spectrum of bile. Six of the 11 reference patients with normal cholangiogram also showed spectra similar to those of PSC, indicating the possibility of cholestasis. DQF-COSY and TOCSY experiments performed on bile samples from PSC patients also revealed absence of phosphatidylcholine (PC) in some of the bile samples, suggesting possible damage to the cholangiocytes by the toxic bile. These observations suggest that analysis of human bile by (1)H MRS could be of value in the diagnosis of chronic Ch liver disorders.

Bile acids constitute a group of structurally closely related molecules and represent the most abundant constituents of human bile. Investigations of bile acids have garnered increased interest owing to their recently discovered additional biological functions including their role as signaling molecules that govern glucose, fat and energy metabolism. Recent NMR methodological developments have enabled single-step analysis of several highly abundant and common glycine- and taurine- conjugated bile acids, such as glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid. Investigation of these conjugated bile acids in human bile employing high field (800 MHz) (1)H-NMR spectroscopy reveals that the ratios between two glycine-conjugated bile acids and their taurine counterparts correlate positively (R2 = 0.83-0.97; p = 0.001 x 10(-2)-0.006 x 10(-7)) as do the ratios between a glycine-conjugated bile acid and its taurine counterpart (R2 = 0.92-0.95; p = 0.004 x 10(-3)-0.002 x 10(-10)). Using such correlations, concentration of individual bile acids in each sample could be predicted in good agreement with the experimentally determined values. These insights into the pattern of bile acid conjugation in human bile between glycine and taurine promise useful clues to the mechanism of bile acids' biosynthesis, conjugation and enterohepatic circulation, and may improve our understanding of the role of individual conjugated bile acids in health and disease.

This (1)H nuclear magnetic resonance metabonomics study was aimed to determine urinary biomarkers of cholestasis resulting from inhibition of biliary secretion of bile or obstruction of bile flow. To inhibit biliary secretion of bile, cyclosporine A was administered to male Sprague-Dawley rats. Obstruction of bile flow was induced by administration of 4,4'-methylene dianiline, alpha-naphthylisothiocyanate or bile duct ligation. Clinical pathological and histopathological examinations were performed to confirm cholestatic injury and (1)H nuclear magnetic resonance spectral data for urine samples were analysed to determine similarities and differences in profiles of metabolites using the Spotfire. In cyclosporine A-treated groups, serum total bilirubin and bile acid were significantly increased but no remarkable hepatic histopathological-changes were observed. In 4,4'-methylene dianiline-, alpha-naphthylisothiocyanate- and bile duct ligation-treated groups, serum alkaline phosphatase, gamma-glutamyltranspeptidase and total bilirubin levels increased significantly, and hepatic histopathological-changes were observed. On urinary (1)H nuclear magnetic resonance spectral analysis, area intensities derived from 0.66 to 1.90 ppm were decreased by cyclosporine A, whereas they were increased by other treatments. These metabolites were identified using the NMR suite as bile acids, branched-chain amino acids, n-butyrate, propionate, methyl malonate and valerate. These metabolites were further investigated by K-means clustering analysis. The cluster of these metabolites is considered to be altered by cholestasis. We conclude that bile acids, valine and methyl malonate have a possibility to be urinary cholestatic biomarkers, which distinguish a difference in mechanism of toxicity. (1)H nuclear magnetic resonance metabonomics thus appears to be useful for determining the mechanisms of toxicity and can be front-loaded in drug safety evaluation and biomarker discovery.

Changes in bile synthesis by the liver or alterations in the enterohepatic circulation due to a variety of etiological conditions may represent a novel source of liver disease-specific biomarkers. Bile from patients with liver diseases exhibited significant changes in the levels of glycine- and taurine-conjugated bile acids, phospholipids, cholesterol and urea relative to non-liver disease controls. Cholangiocarcinoma and non-malignant liver diseases (NMLD) showed the most significant alterations. Further, hepatocellular carcinoma (HCC) could be differentiated from NMLD (p = 0.02), as well as non-liver disease controls (p = 0.02) based on the amounts of bile acids, phospholipids and/or cholesterol. HCC also differed with cholangiocarcinoma although not significantly. Urea increases somewhat in non-malignant liver disease relative to non-liver disease controls, while the bile acids, phospholipids and cholesterol all decrease significantly. The ratio between some major bile metabolites also distinguished NMLD (p = 0.004-0.01) from non-liver disease controls. This snapshot view of bile homeostasis, is obtainable from a simple nuclear magnetic resonance (NMR) approach and demonstrates the enormous opportunity to assess liver status, explore biomarkers for high risk diseases such as cancers and improve the understanding of normal and abnormal cellular functions.

Gallbladder cancer (GBC) is frequently associated with gallstones (GS). At the same time, however, a very small number of patients with GS develop GBC. Cholesterol and metal salts are the common constituents of all GS. To understand their role in the etiopathogenesis of GBC, cholesterol, calcium, and magnesium composition in GS is compared in cancerous and benign gallbladders.

GS from patients with GBC (n = 11), chronic cholecystitis (CC; n = 23), and xanthogranulomatous cholecystitis (XGC; n = 11) undergoing cholecystectomy were analyzed using proton nuclear magnetic resonance spectroscopy. The diagnosis of the gallbladder disease was based on histopathological examinations. Cholesterol, calcium, and magnesium in the GS of GBC, XGC, and CC were analyzed, compared, and correlated using statistical methods.

The quantity of cholesterol was significantly less in the GS of GBC than in benign gallbladder diseases (CC or XGC, P < 0.0001 for both). Both calcium and magnesium were significantly higher in GBC than in benign disease (calcium: P < 0.0005 and magnesium: P < 0.0001 for GBC vs CC; calcium: P < 0.02 and magnesium: P < 0.04 for GBC vs XGC). In all the GS, calcium was higher than magnesium. Calcium and magnesium were positively correlated in GBC (R = 0.69) and XGC (R = 0.75), and cholesterol and calcium were negatively correlated in CC (R =-0.61).

Differences in the GS composition between malignant and benign gallbladder patients may provide useful clues to the etiopathogenesis of GBC. These clues could lead to the identification of patients with GS in vivo who are at high risk of developing GBC, and advocate prophylactic cholecystectomy to prevent GBC.

Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid components often have to be determined in the same tissue sample. Perchloric acid (PCA) used for the extraction of aqueous metabolites has a deleterious effect on lipid components; the severity of the damage is not known. In this study, human muscle tissue was first treated with PCA to extract aqueous metabolites, and the residue was then used for lipid extraction by conventional methods, i.e. the methods of Folch and Bligh & Dyer and a standardised one using methanol/chloroform (1:3, v/v) used in our laboratory. A (1)H-NMR spectrum was obtained for each lipid extract. Lipid was quantified by measuring the integral area of N(+)-(CH(3))(3) signals of phospholipids (PLs). Triacylglycerol (TG) and cholesterol (CHOL) were quantified using the -CH(2)- signals of glycerol and the C18 methyl signal, respectively. This study shows that prior use of PCA caused marked attenuation of TG, PL, and CHOL. This was confirmed by recovery experiments and observation of the direct effect of PCA on the standard lipid components. On the basis of the quantity of lipid lost in each case, three novel equations (with respect to TG, PL, and CHOL) were derived. Application of these equations to lipid quantities estimated in different pathological tissues after PCA pre-treatment produced values equivalent to those estimated without PCA use. This study conclusively shows that PCA pre-treatment damages all three lipid moieties, TG, PL, and CHOL. When PCA is used in a fixed ratio to the tissue, the lipid damage is also proportional and correctable by statistically derived equations. These equations will be useful in human biopsy specimens where aqueous and lipid components have to be studied using the same tissue sample because of the small quantity available.

Gallbladder cancer (GBC) is common in northern India. The western world has a pessimistic attitude towards GBC resulting in inadequate management of even early GBC. At the other extreme is the Japanese aggressivism with high mortality but very few actual long-term survivors. The Indian surgeons have adopted a Buddhist "middle path"--aggressive surgical approach for "less advanced" GBC and non-surgical palliative approach for "more advanced" GBC. We rely heavily on staging laparoscopy to detect metastatic deposits on liver, peritoneum and omentum, and upper gastrointestinal endoscopy (UGIE) to detect duodenal infiltration which indicates unresectability as we do not perform pancreatico-duodenectomy for GBC. Our favoured procedure is extended cholecystectomy (EC) which includes a 2 cm nonanatomical wedge of liver in the GB bed and the lymph nodes in hepatoduodenal ligament, behind the duodenum and head of pancreas and along the hepatic artery to the right of celiac axis. EC can achieve R0 resection in patients with T1-T2 and T3 (fundus/body--hepatic bed type) disease. For T3 (neck--hepatic hilum type) and T4 disease major hepatic resection is required. In selected patients with nodally advanced GBC, a non-curative simple cholecystectomy with post-operative chemoradiotherapy may improve survival. GBC is an "Indian disease" and Indian surgeons have to be prepared to accept the "challenge" of GBC.

Symbiotic gut microorganisms (microbiome) interact closely with the mammalian host's metabolism and are important determinants of human health. Here, we decipher the complex metabolic effects of microbial manipulation, by comparing germfree mice colonized by a human baby flora (HBF) or a normal flora to conventional mice. We perform parallel microbiological profiling, metabolic profiling by (1)H nuclear magnetic resonance of liver, plasma, urine and ileal flushes, and targeted profiling of bile acids by ultra performance liquid chromatography-mass spectrometry and short-chain fatty acids in cecum by GC-FID. Top-down multivariate analysis of metabolic profiles reveals a significant association of specific metabotypes with the resident microbiome. We derive a transgenomic graph model showing that HBF flora has a remarkably simple microbiome/metabolome correlation network, impacting directly on the host's ability to metabolize lipids: HBF mice present higher ileal concentrations of tauro-conjugated bile acids, reduced plasma levels of lipoproteins but higher hepatic triglyceride content associated with depletion of glutathione. These data indicate that the microbiome modulates absorption, storage and the energy harvest from the diet at the systems level.

Biochemicals in the upper-gut aspirate in 31 patients with malabsorption syndrome (MAS) with and without small intestinal bacterial overgrowth (SIBO), and 10 disease-free controls were analyzed using high-resolution (1)H-NMR spectroscopy, and were correlated with the degree of SIBO and severity of MAS. Compared to controls, the patients had higher quantities (micromol/L: median [range]) of total bile acids/cholesterol (2000 [0-12000] vs. 300 [0-600]), lactate (700 [0-5200] vs. nil [0-30]), acetate (200 [0-6500] vs. 20 [0-200]), and formate (80 [0-900] vs. nil [0-50]) (P < 0.01, Mann-Whitney U-test). However, amino acids and glucose were comparable in both. Quantities (micromol/L: median [range]) of acetate (1330 [220-6500] vs. 100 [0-1430]), lactate (1430 [670-3300] vs. 300 [0-5200]), formate (360 [0-600] vs. 25 [0-800]), and unconjugated bile acids (500 [40-600] vs. 10 [0-300]) were higher in MAS patients with SIBO than those without SIBO (P < 0.01, Mann-Whitney U-test, for all). In patients with MAS the quantity of acetate positively correlated with the degree of SIBO, and unconjugated bile acids correlated with the degree of steatorrhoea (Spearman's rank correlation coefficient, two-tailed, P < 0.05: 0.46 and 0.52, respectively). This study demonstrates the bacterial production of metabolites and deconjugation of bile acids in patients with MAS.

Human gallbladder bile dissolved in dimethylsulfoxide provides sharp and resolved signals for major bile components in 1H NMR spectra. Characteristic well-resolved marker signals that invariably appear in 1H NMR spectra of bile were identified for cholesterol (H18 methyl signal at 0.643 ppm), lipids (glycerol CH signal at 5.064 ppm), total bile acids (H18 signals in the range 0.520-0.626 ppm), total glycine conjugated bile acids (NH signal at 6.958 ppm), total taurine conjugated bile acids (NH signal at 7.646 ppm), and urea (NH2 signal near 5.48 ppm), which enabled their rapid and accurate analysis. Excellent linearity and precision of quantitative analysis was observed for all the identified bile components (R2 > 0.99 for all). The method was demonstrated on gallbladder bile from 19 patients with gallbladder diseases. Urea in bile was identified by NMR for the first time and its quantitative analysis, along with several other bile components, is presented. The majority of the bile components could be analyzed in a single step. Accurate and rapid quantification of several bile components noninvasively by using the method presented herein may have far-reaching implications in the study of bile acid metabolism and pathophysiology of various hepatobiliary and gastrointestinal diseases.

1H and 13C NMR spectra of intact human bile were assigned using one-dimensional (1H and 13C) and two-dimensional (1H-1H and 1H-13C) experiments. Individual conjugated bile acids--glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid--were identified. The bile acids were quantified accurately and individually in a single step by using distinct and characteristic amide signals. Making use of 13C NMR, the study also suggests a way to analyze unconjugated bile acids separately, if present. Chemical shift assignments and rapid single-step analysis of individual conjugated bile acids from intact bile presented herein may have immense utility in the study of bile acid metabolism and deeper understanding of hepatobiliary diseases.

Gallbladder cancer, the commonest malignancy of the biliary tract worldwide, is common in northern India. It can be clinically obvious, an unexpected finding at laparotomy, detected incidentally on histological examination or may be missed only to present with recurrence during follow up. US, CECT, uppeer gastro-intestinal endoscopy, and laparoscopy are useful for diagnosis and staging. We have adopted a 'middle path'--between pessimistic nihilism of the West and aggressive radicalism of Japan--of management, i.e., extended cholecystectomy for early disease confined to the gallbladder and hepato-dudodenal ligament, and non-surgical palliation for advanced disease. The aetiological role of gallstones in the causation of gallbladder cancer needs to be investigated to decide the place of prophylactic cholecystectomy, if any.

The unconjugated bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid; their glycine and taurine conjugates glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid; and a taurine conjugated ursodeoxycholic acid, tauroursodeoxycholic acid, were characterized through 1H and 13C NMR in aqueous media under the physiological pH region (7.4 +/- 0.1). Assignments of 1H and 13C signals of all the bile acids were made using a combination of several one- and two-dimensional, homonuclear (1H-1H) and heteronuclear (1H-13C) correlations as well as spectral editing NMR methods. Stereochemical assignment of the five-membered ring of the bile acids is reported here for the first time. The complete characterization of various bile acids in aqueous media presented here may have implications in the study of the pathophysiology of biliary diseases through human biliary fluids using NMR spectroscopy.