Raw Bile Research Articles

Rapid diagnosis and recurrence prediction of choledocholithiasis disease using raw bile with machine learning assisted SERS

Surface-enhanced Raman spectroscopy (SERS) analysis based on body fluids has been widely applied in disease diagnose. Choledocholithiasis is a widespread and often recurrent digestive system disease, with limited data on factors predicting its formation and reappearance. Bile contains many components that could provide valuable diagnostic information; however, the current diagnosis of biliary disease by SERS focuses on detecting specific component in the bile, overlooking the complex interplay and correlations among multiple factors that could be crucial for accurate diagnosis. This work directly obtained multi-component SERS spectral information of raw bile from 46 patients. Characteristic information was extracted from bile SERS spectra using Principal Component Analysis (PCA), revealing variations in the content of characteristic components associated with different choledocholithiasis types and their recurrence frequency. Pearson correlation analysis was also introduced to reveal the interactions of primary active substances pertinent to choledocholithiasis diagnosis. The efficacy of PCA and Support Vector Machine (SVM) models in classifying stone types, presented an accuracy of 99.2 %. Furthermore, the interaction patterns among SERS characteristic components in choledocholithiasis recurrence frequency were revealed, and with the support of SVM, the prediction for different recurrence rates reached an accuracy of 95.2 %. Overall, this work demonstrates that integrating SERS with machine learning can support disease diagnosis and the interpretation of correlations among multiple components, facilitating elucidating the disease mechanisms.

Identification of gallbladder cancer by direct near-infrared measurement of raw bile combined with two-trace two-dimensional correlation analysis.

We demonstrated the utility of direct near-infrared (NIR) bile analysis for the identification of gallbladder (GB) cancer by employing two-trace two-dimensional (2T2D) correlation analysis to recognize dissimilar spectral features among diverse bile samples for potential improvement of discrimination accuracy. To represent more diverse clinical cases for reliable assessment, bile samples obtained from five normal, 44 gallstone, 25 GB polyp, six hepatocellular cancer (HCC), and eight GB cancer subjects were analyzed. Due to the altered metabolic pathways by carcinogenesis, the NIR spectral features of GB cancer samples, including intensity ratios of main peaks, were different from those of other sample groups. The differentiation of GB cancer in the principal component (PC) score domain was mediocre and subsequent discrimination accuracy based on linear discriminant analysis (LDA) was 88.5%. When 2T2D slice spectra were obtained using a reference spectrum constructed by the linear combination of the spectra of five pure representative bile metabolites and employed, the accuracy was improved to 95.6%. The sensitive recognition of dissimilar spectral features in GB cancer by 2T2D correlation analysis was responsible for the enhanced discrimination.

Feasibility for SERS-based discrimination of gallbladder cancer from polyp by indirect recognition of components in bile

The aqueous solution extracted from raw bile juice is composed primarily of bile salts, with lower levels of bilirubin and its derivatives. Among them, the bilirubin and bilirubin-derived metabolites are the only surface-enhanced Raman scattering (SERS)-active components. An analytical scheme indirectly responsive and able to utilize all bile components, including SERS-inactive bile salts, was explored for SERS-based discrimination of gallbladder (GB) polyp and GB cancer. Initially, the surface of a SERS substrate (Au nanodendrite on Ni foil (AuND@NiF)) was covered with an alkanethiol molecule to generate a SERS signal and attract bile components by mutual interaction. For more effective recognition of bile components, 4 independent substrates covered with 4 different alkanethiols with various functional groups (SH(CH2)2CH3, SH(CH2)2NH2, SH(CH2)2COOH, and SH(CH2)2OH) were prepared. The SERS peaks of each substrate clearly varied on interaction with pure bile components as well as aqueous bile samples, and the SERS peaks in each substrate were individually characteristic. When the principal component (PC) scores of spectra obtained using the SH(CH2)2CH3- and SH(CH2)2OH-covered substrates were combined, the k-Nearest Neighbor-based discrimination accuracy was 100%, superior to those (90.6–96.9%) using individual substrates. The use of complementary bile component-induced spectral information provided by the two substrates was responsible for accurate discrimination. On the other hand, when bare AuND@NiF recognizing only SERS-active bilirubin derivatives was used, discrimination was unsatisfactory (accuracy: 75.0%).

Feasibility of laser-induced breakdown spectroscopy as a direct raw bile analysis tool for screening of gallbladder cancer

Laser-induced breakdown spectroscopy (LIBS) was explored for the first time for discrimination of gallbladder (GB) polyp and GB cancer (GBC) by direct measurement of raw bile juice without pretreatment.

Combination of LIBS-based elemental analysis and near-infrared molecular fingerprinting of bile juice to enhance identification of gallbladder cancer.

Laser-induced breakdown spectroscopy (LIBS) and near-infrared (NIR) spectroscopy, enabling the measurement of raw bile directly without sample pretreatment, were cooperatively combined to enhance the discrimination of gallbladder cancer (GBC) from other diseases of gallstone and gallbladder (GB) polyp. Since elemental contents and metabolite compositions of bile vary according to the pathological conditions of pancreaticobiliary patients, the use of complementary information could be synergetic to improve disease identification accuracy. The ratios of Mg and Na peak areas (AMg/ANa) and Na and K peak areas (ANa/AK) in the LIBS spectra of GBC samples were different from those of the remaining samples. Also, the intensity ratios of main NIR peaks differed in GBC. Nonetheless, the use of only element peak area ratio or NIR peak intensity ratio was not sufficient to clearly discriminate GBC. On the other hand, when the ANa/AK values and second NIR principal component scores were combined, the discrimination of GBC from normal/gallstone/GB polyp was substantially enhanced owing to incorporation of both complementary GBC-discriminant spectroscopic signatures.

Pretreatment-free SERS analysis of raw bile juice for identification of gall bladder disease using paper-coupled Au nanodendrite-encaged nickel foam

For pretreatment-free SERS measurement of raw bile juice for the identification of gall bladder (GB) disease, a paper strip bridging Au nanodendrite-encaged nickel foam (PS-AuND@NF) was demonstrated. To build a medium for facile sample diffusion as well as SERS generation, Au nanodendrite (AuND) with a high SERS efficiency was constructed on the porous frame of the NF (AuND@NF). Then, the PS-AuND@NF was built by simply positioning AuND@NF on a paper strip, which functioned as a channel for sample introduction to AuND@NF and a subsequent sample drain. With the continuous elution of raw bile juice, non-specific adsorption of SERS-inactive constituents such as lipids on the Au surface could be minimized. Using the PS-AuND@NF, raw bile juice samples obtained from 6 GB polyp and 21 GB stone patients were measured. Since only a few components in bile juice are expected to be SERS-active such as bilirubin-containing metabolites, the information obtained from the SERS spectra is not descriptive enough for the discrimination of both GB diseases. Therefore, two-trace two-dimensional (2T2D) correlation analysis, which is able to effectively feature out asynchronous (dissimilar) spectral behaviors in the comparison of two spectra, was carried out in parallel. When the principal component (PC) scores obtained from the 2T2D slice spectra were used for the k-nearest neighbor (k-NN), the discrimination accuracy improved to 88.9 % from 81.5 % obtained when using the PC scores of the raw spectra. Overall, the PS-AuND@NF enabled simple pretreatment-free SERS analysis of complex and viscous bile juice without interference from other existing components.

Feasibility study for rapid near-infrared spectroscopic identification of different gallbladder diseases by direct analysis of bile juice.

A whole-sample-covering near-infrared (NIR) spectroscopy scheme has been adopted for the simple drop-and-dry measurement of raw bile juice for the identification of gallbladder (GB) diseases of stone, polyp, and cancer. For reproducible measurement, a non-NIR absorbing polytetrafluoroethylene (PTFE) providing a hydrophobic surface was chosen as a substrate to form bile juice droplets of a consistent shape. To ensure representative spectroscopic sampling, NIR radiation illuminated the whole area of the dried sample for spectral acquisition. The NIR band shapes and relative band intensities of GB cancer differed moderately from those of GB stone and GB polyp. The composition of GB cancer samples was presumed to be dissimilar from other sample compositions. Differentiation between GB polyp and GB stone, however, was less facile; nevertheless, in the case of GB polyp samples, the obtained NIR features were informative in the identification of various pathological conditions such as adenomyomatosis (abnormal growth of epidermal tissue) and hepatitis B. To elucidate the NIR features of bile juice samples, separate NIR spectra of major bile constituents such as conjugated bile salts, lecithin, cholesterol, and albumin were analyzed. The demonstrated NIR spectroscopy scheme requiring no sample pretreatment or separation of bile juice could be useful for fast bile juice-based screening of GB diseases, especially the identification of early GB cancer.

Current Status of Hepatitis E Virus Infection in Korea

Hepatitis E virus (HEV) is an emerging pathogen associated with acute viral hepatitis, and HEV is becoming increasingly recognized. Approximately 2% of acute viral hepatitis is caused by HEV, and 18 cases of hepatitis E have been reported in Korea. Of these cases, only two have involved a history of travel from India, which suggests that they were imported cases. The remaining reported cases include a sporadic case of acute hepatitis E with genotype 4 HEV isolates and identification of the full genome sequence, as well as another case of genotype 4 HEV hepatitis that developed after ingestion of the raw bile juice of a wild bear living on a mountain in southern Korea. Moreover, genotype 3 HEV, which shows close genetic homology with swine HEV in Korea, has been detected in collected human serum samples. Therefore, genotypes 3 and 4 HEV are currently circulating in the Korean community and may be related to zoonotic transmission and food-borne infection. The reported anti-HEV seroprevalence of 17% to 27% in the Korean population suggests that HEV infection has been autochthonously circulating, thereby resulting in subclinical infection in Korea. Given the discrepancies among anti-HEV assays, the diagnosis of hepatitis E should be made with caution using adequate antibody assays, and HEV RNA should be preferably detected from the stool. Further virological characterization and epidemiological study of the virus are warranted.

31P High Resolution NMR Spectroscopy in Analysis of Phosphate-containing Compounds of Bile

31 P high resolution nuclear magnetic resonance (NMR) spectroscopy was used to examine phospholipid metabolism and to analyze the phosphate-containing compounds in the bile in the transplanted liver recipients, the cholelithiasis patients’ and the living donors’ groups. Three signals of NMR spectrum of raw bile were determined: inorganic phosphate (P i ), lysophosphatidylcholine (LPtdC), and phosphatidylcholine (PtdC) in all investigated groups. Pi concentration was significantly higher in the recipients’ group than in the living donors’ group (Mann-Whitney test, p p 31 P NMR spectral data set provided good classifications between the living donors’ and recipients’ groups and the poor one among all groups. Results of our study suggest that 31 P NMR spectroscopy in vitro may be used for assessment of graft function, for the early signs of rejection and for the predisposition to gallstone formation.

The first case of genotype 4 hepatitis E related to wild boar in South Korea

Several cases of serologically diagnosed hepatitis E have been reported in South Korea; however, there have been no documented cases of zoonotic transmission with virologic characterization of hepatitis E virus (HEV). We report on a sporadic case of autochthonous acute hepatitis E after ingestion of raw bile juice obtained from a wild boar living on a domestic mountain in a southern part of Korea. It is the first case of hepatitis E acquired through presumably zoonotic transmission, which was verified as genotype 4 HEV.

Trichinella spiralis: Differential effect of host bile on the in vitro invasion of infective larvae into epithelial cells

The differential effect of fox and pig bile and its corresponding low molecular weight fraction (LMW) was investigated on the in vitro invasion of MDCK-AA7 epithelial cell monolayers by Trichinella spiralis muscle larvae. Seven invasion experiments were performed and a total of 274 cell monolayers were examined. Fox and pig raw bile at 1:10 and 1:20 dilution and their LMW fractions at 1:10 dilution activated T. spiralis larvae to invade the cell monolayers. In addition, fox raw bile caused significantly larger cell damage than pig raw bile at both dilutions. The area of cell damage was larger at 1:10 than at 1:20 dilution for both fox and pig raw bile ( p < 0.05). On the other hand, there was no significant difference between the areas of cell damage caused by the LMW fractions of fox and pig bile. It is concluded that differences between host bile actions may account for differences in host susceptibility to T. spiralis.

Effects of raw biles and their non-protein fractions from fox, pig, sheep and chicken on the survival of Trichinella spp. in vitro

The in vitro differential effect of fox, pig, sheep and chicken bile and corresponding non-protein fractions at various concentrations on the motility of released muscle larvae of Trichinella spiralis, Trichinella nativa and Trichinella nelsoni was examined. In many cases, the percentages of motile (live) larvae of the three Trichinella species cultured in the presence of the non-protein fractions of bile from the study animals were significantly higher ( p < 0.001) compared to their respective control cultures. In addition, the percentages of motile (live) larvae of all Trichinella species cultured in the presence of the non-protein fraction of bile at every concentration from all study animals were significantly higher ( p < 0.001) compared to their respective cultures in the presence of raw bile. Not only did results with non-protein fractions of bile differ from those with raw bile, but also the non-protein fraction, with increased dilution, showed a decrease in the percentages of motile larvae while the opposite was true with the raw bile ( p < 0.001). These observations indicate that the non-protein fraction of bile prolongs the in vitro survival of larvae.

Acute Hepatitis and Renal Failure Following Ingestion of Raw Carp Gallbladders-Maryland and Pennsylvania, 1991 and 1994

In some cultures, eating gallbladders from certain species of snakes, birds, or fish is believed to improve health. A syndrome of acute hepatitis and renal failure following the ingestion of raw carp gallbladders has been described previously among persons living in Asia (1-4). This report summarizes two cases of this syndrome that occurred in residents of Pennsylvania who had eaten the raw bile and gallbladders of carp caught in Maryland.

コイの水溶性中毒成分としてのシプリノールについて

Two types of intoxication, caused by intake of carp gallbladder or mainly its muscle (Sasimi), have been reported. In order to identify the substances responsible for these carp intoxications, we initially examined the distribution of toxin in normal carp viscera and muscle by means of an acute toxicity test in ddY male mice (19-20g). It was found that the toxic substances were in gallbladder and liver-pancreas, especially bile, but not muscle. Thus, intoxication by carp gallbladder could be attributable to components normally present in carp bile. Based on the LD50 values, it was concluded that essentially all of the bile toxicity can be explained by the toxicity of cyprinol sulfate, a major component of carp bile. Furthermore, the acute toxic symptoms induced by raw bile and cyprinol sulfate were similar. On the other hand, from the result of a 50% hemolysis test, it was found that some bile acids contained in raw bile were more hemolytic than cyprinol sulfate. Taking these results into consideration, it was suggested that carp intoxication resulting from ingestion of the gallbladder could be caused by not only cyprinol sulfate but also other components, especially bile acid.

Toxic Acute Renal Failure and Hepatitis after Ingestion of Raw Carp Bile

The raw carp bile has both nephrotoxic and hepatotoxic effects which are not well known. Recently, we studied 13 patients who had toxic acute renal failure and toxic hepatitis after ingestion of raw bile of carp in 3, grass carp in 8 and silver carp in 2 cases. The purpose of this report is to alert physicians to this very rare cause of toxic acute renal failure and hepatitis. All patients presented initially with gastrointestinal upset after eating. These symptoms were followed by oliguria in 7 patients (54%), hematuria was noted in 10 (77%) and jaundice in 8 patients (62%). Elevation of blood urea nitrogen, creatinine and transaminases lasted for about 3 weeks. The severity of the symptoms depended on the amount of bile ingested. All the patients recovered with conservative therapy and hemodialysis. Biopsy of the kidney revealed findings compatible with acute tubular necrosis similar to that produced by other nephrotoxins. Biopsy of the liver revealed findings consistent with acute toxic hepatitis. Both suggest toxic effects of carp bile as a cause of toxic acute renal failure and hepatitis.

The effects of bile salt and raw bile on the intestinal absorption of micellar fatty acid in the rat in vitro.

1. The uptake, esterification and transport of [(14)C]oleic acid were studied using sacs of rat everted small intestine incubated in 25 ml. of a buffered mixture of sodium taurocholate, glyceryl mono-oleate and (14)C-labelled oleic acid in micellar form.2. Intestine obtained from bile fistula rats (bile duct cannulated 48 hr previously) showed elevated rates of (14)C uptake into the tissue total lipid compared with sham-operated controls.3. Nearly all of the excess (14)C uptake in the bile fistula group was in the form of free fatty acid. Both groups showed similar rates of [(14)C]oleic acid incorporation into tissue triglyceride and also similar, though small, amounts transported into the serosal fluid.4. In further experiments using intestine from bile fistula rats the addition of 1 ml. of fresh rat bile to the incubation mixture reduced the (14)C uptake to approximately control levels. The addition of 2-3 ml. of fresh bile similarly reduced the uptake and increased (14)C incorporation into the triglycerides of mucosal tissue and serosal fluid.5. These responses were not entirely the result of the bile salts contained in fresh bile since increasing the taurocholate concentration per se caused uptake, esterification and transport all to increase. In the presence of the higher taurocholate concentration the addition of fresh bile still caused a decrease in (14)C uptake.6. There was no significant effect of either fresh bile or taurocholate on the transport of the 3-O-methyl analogue of D-glucose under comparable conditions.7. It is concluded that raw bile contains one or more components other than bile salts which may be important in determining fatty acid absorption.