Magnetic resonance spectroscopy of bile in the detection of cholangiocarcinoma
Authors: Omkar B. Ijare, Tedros Bezabeh, Nils Albiin, Ian C.P. Smith
Journal: Journal of Hepatology (2010)

A new NMR-based metabolomics approach for the diagnosis of biliary tract cancerJournal of HepatologyVol. 52Issue 2PreviewBiliary tract cancer is highly lethal at presentation, with increasing mortality worldwide. Current diagnostic measures employing multiple criteria such as imaging, cytology, and serum tumor markers are not satisfactory, and a new diagnostic tool is needed. Because bile is a cognate metabolite-rich bio-fluid in the biliary ductal system, we tested a new metabolomic approach to develop an effective diagnostic tool. Full-Text PDF A new NMR metabolomics-based diagnosis for hepatobiliary tract cancerJournal of HepatologyVol. 54Issue 2PreviewBezabeh and co-workers wrote a letter for their concerns about our recent article [1], and we would like to provide our reply as below. Full-Text PDF Open Access To the Editor:We would like to draw your attention to some of the drawbacks in a recent article published by Wen et al. in your journal [[1]Wen H. Yoo S.S. Kang J. Kim H.G. Park J.S. Jeong S. et al.A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer.J Hepatol. 2010; 52: 228-233Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar], on NMR-based metabolomics of bile samples in distinguishing cholangiocarcinoma from benign biliary diseases. The authors performed orthogonal partial least square discriminant analysis (OPLS-DA) of 1H NMR spectra of bile samples obtained from patients with cholangiocarcinoma and benign biliary diseases. They classified both groups with a sensitivity of 88% and a specificity of 81%. We are currently working on the utility of magnetic resonance spectroscopy in the study of hepatopancreatobiliary diseases [2Albiin N. Smith I.C.P. Arnelo U. Lindberg B. Bergquist A. Dolenko B. et al.Detection of cholangiocarcinoma with magnetic resonance spectroscopy of bile in patients with and without primary sclerosing cholangitis.Acta Radiol. 2008; 49: 855-862Crossref PubMed Scopus (40) Google Scholar, 3Ijare O.B. Bezabeh T. Albiin N. Arnelo U. Bergquist A. Lindberg B. et al.Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: a 1H MR study.NMR Biomed. 2009; 22: 471-479Crossref PubMed Scopus (30) Google Scholar, 4Bezabeh T. Ijare O.B. Albiin N. Arnelo U. Lindberg B. Smith I.C.P. Detection and quantification of d-glucuronic acid in human bile using 1H NMR spectroscopy: relevance to the diagnosis of pancreatic cancer.MAGMA. 2009; 22: 267-275Crossref PubMed Scopus (31) Google Scholar, 5Ijare OB, Smith ICP, Mohajeri S, Bezabeh T. Magnetic resonance spectroscopy of human bile in the detection of hepatopancreaticobiliary disease: past, present and future. In: Khetrapal CL, Kumar A, Ramanathan KV, editors. Future directions of NMR. 1st ed. New Delhi: Springer (India) Private Limited; 2010 [chapter 4].Google Scholar, 6Ijare O.B. Bezabeh T. Albiin N. Bergquist A. Arnelo U. Smith I.C.P. Simultaneous quantification of glycine- and taurine-conjugated bile acids, total bile acids, and choline-containing compounds in human bile using 1H NMR spectroscopy.J Pharm Biomed Anal. 2010; 53: 667-673Crossref PubMed Scopus (23) Google Scholar] and have noticed some shortcomings in the above study. The authors have mentioned that their work was the first metabolomics approach reported in the diagnosis of human hepatobiliary diseases [[1]Wen H. Yoo S.S. Kang J. Kim H.G. Park J.S. Jeong S. et al.A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer.J Hepatol. 2010; 52: 228-233Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. However, a similar metabolomic study using bile samples for the detection of cholangiocarcinoma was published by our group about two years ago [[2]Albiin N. Smith I.C.P. Arnelo U. Lindberg B. Bergquist A. Dolenko B. et al.Detection of cholangiocarcinoma with magnetic resonance spectroscopy of bile in patients with and without primary sclerosing cholangitis.Acta Radiol. 2008; 49: 855-862Crossref PubMed Scopus (40) Google Scholar]. In our study, we performed multivariate analysis of 1H NMR spectra of bile samples obtained from patients with cholangiocarcinoma and other benign biliary diseases (primary sclerosing cholangitis/choledocholithiasis) with a comparable sample size as that of Wen et al. [[1]Wen H. Yoo S.S. Kang J. Kim H.G. Park J.S. Jeong S. et al.A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer.J Hepatol. 2010; 52: 228-233Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. We reported a sensitivity of 88.9% and a specificity of 87.1% in classifying cancer and control groups. Khan et al. [[7]Khan S.A. Cox I.J. Thillainayagam A.V. Bansi D.S. Thomas H.C. Taylor-Robinson S.D. Proton and phosphorus-31 nuclear magnetic resonance spectroscopy of human bile in hepatopancreaticobiliary cancer.Eur J Gastroenterol Hepatol. 2005; 17: 733-738Crossref PubMed Scopus (43) Google Scholar] had also previously reported a study in which they analyzed bile samples from cholangiocarcinoma, pancreatic cancer, and other hepatobiliary diseases using 1H NMR spectroscopy. Although our study [[2]Albiin N. Smith I.C.P. Arnelo U. Lindberg B. Bergquist A. Dolenko B. et al.Detection of cholangiocarcinoma with magnetic resonance spectroscopy of bile in patients with and without primary sclerosing cholangitis.Acta Radiol. 2008; 49: 855-862Crossref PubMed Scopus (40) Google Scholar] and the one by Khan et al. [[7]Khan S.A. Cox I.J. Thillainayagam A.V. Bansi D.S. Thomas H.C. Taylor-Robinson S.D. Proton and phosphorus-31 nuclear magnetic resonance spectroscopy of human bile in hepatopancreaticobiliary cancer.Eur J Gastroenterol Hepatol. 2005; 17: 733-738Crossref PubMed Scopus (43) Google Scholar] were undertaken with similar objectives as that of Wen et al., they were not cited. We also have concerns regarding some of the metabolites quantified in the targeted metabolic profiling. Wen et al. [[1]Wen H. Yoo S.S. Kang J. Kim H.G. Park J.S. Jeong S. et al.A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer.J Hepatol. 2010; 52: 228-233Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar] compared the levels of choline in both groups and the difference was not found to be statistically significant (p= 0.85). However, in our study, we quantified the predominant choline-containing phospholipid, phosphatidylcholine (PC) which was decreased in cancer patients compared to the benign group with the difference being statistically significant (p= 0.02). Khan et al. had also reported a reduced phosphatidylcholine signal in cancer patients (cholangiocarcinoma/pancreatic cancer) compared to the non-cancer patients (p= 0.007) [[7]Khan S.A. Cox I.J. Thillainayagam A.V. Bansi D.S. Thomas H.C. Taylor-Robinson S.D. Proton and phosphorus-31 nuclear magnetic resonance spectroscopy of human bile in hepatopancreaticobiliary cancer.Eur J Gastroenterol Hepatol. 2005; 17: 733-738Crossref PubMed Scopus (43) Google Scholar]. In a similar study, Nagana Gowda et al. observed decreased levels of phospholipids in cholangiocarcinoma patients compared to the non-liver disease control group (p= 0.001) [[8]Nagana Gowda G.A. Shanaiah N. Cooper A. Maluccio M. Raftery D. Visualization of bile homeostasis using 1H-NMR spectroscopy as a route for assessing liver cancer.Lipids. 2009; 44: 27-35Crossref PubMed Scopus (34) Google Scholar]. Moreover, in a recent study, Sharif et al. made similar observations in comparing patients with cholangiocarcinoma and gallstone disease (p= 0.01) [[9]Sharif A.W. Williams H.R.T. Lampejo T. Khan S.A. Bansi D.S. Westaby D. Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy.HPB. 2010; 12: 396-402Abstract Full Text Full Text PDF Scopus (38) Google Scholar]. The above observations are consistent with the fact that phosphatidylcholine is an important component of bile protecting bile ducts from the harmful effects of bile acids and its depletion can lead to bile duct injury [[3]Ijare O.B. Bezabeh T. Albiin N. Arnelo U. Bergquist A. Lindberg B. et al.Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: a 1H MR study.NMR Biomed. 2009; 22: 471-479Crossref PubMed Scopus (30) Google Scholar]. Second, the authors reported that citrate levels were elevated in cancer patients compared to the benign subjects with the difference being statistically significant [[1]Wen H. Yoo S.S. Kang J. Kim H.G. Park J.S. Jeong S. et al.A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer.J Hepatol. 2010; 52: 228-233Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. Although citrate is a component of bile acting as a calcium chelator in gallbladder disease [[10]Vitetta L. Sali A. Citrate: a component of bile and calcium chelator in gallbladder disease.J Nutr Environ Med. 1999; 9: 199-207Crossref Scopus (3) Google Scholar], its levels are considerably low (0–406 μM). As a result, detection of citrate in bile using 1H NMR spectroscopy would be difficult. Moreover, they have associated citrate with a signal resonating at 1.5 ppm (one of the signals found to be discriminatory in the OPLS-DA). This assignment is not correct as the –CH2– protons in citrate resonate at totally different chemical shift (in the region 2.55–2.75 ppm). Furthermore, the authors have misassigned a signal resonating at 3.70 ppm to a –CHn–OR moiety. We believe that this signal arises from the –CH2– protons of glycine conjugated to bile acids [[6]Ijare O.B. Bezabeh T. Albiin N. Bergquist A. Arnelo U. Smith I.C.P. Simultaneous quantification of glycine- and taurine-conjugated bile acids, total bile acids, and choline-containing compounds in human bile using 1H NMR spectroscopy.J Pharm Biomed Anal. 2010; 53: 667-673Crossref PubMed Scopus (23) Google Scholar]. Finally, the bile samples in our study were analyzed without any sample pretreatment which reduced the experimental time, whereas in the study by Wen et al., the samples had undergone pretreatment. More specifically, the samples were freeze-dried and later dissolved in the solvent system, D2O–CD3OD (buffered with 10 mM sodium phosphate, pH 6). Their approach increased the procedure time considerably, which makes it less appealing to be used in the clinic. In a clinical setting, it is highly recommended that the minimum number of steps be used between sample collection and the generation of diagnostic output. Moreover, their methodology is expected to be more expensive as they used deuterated solvents to prepare the samples. Although bile is a complex biofluid, it is still possible to use the neat samples for the quantification of major biliary biochemicals, such as glycine-conjugated bile acids, taurine-conjugated bile acids, and choline-containing phospholipids [[6]Ijare O.B. Bezabeh T. Albiin N. Bergquist A. Arnelo U. Smith I.C.P. Simultaneous quantification of glycine- and taurine-conjugated bile acids, total bile acids, and choline-containing compounds in human bile using 1H NMR spectroscopy.J Pharm Biomed Anal. 2010; 53: 667-673Crossref PubMed Scopus (23) Google Scholar]. Although Wen et al. have presented some important NMR-based results in the diagnosis of cholangiocarcinoma and have done a thorough job in comparing their NMR data with other diagnostic markers (CEA, CA 19-9, and bile cytology), the errors we have noticed may mislead readers if left uncorrected. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.