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Journal of Hepatology, 54, 2, pp. 398-399, 2010-11-10
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Magnetic resonance spectroscopy of bile in the detection of cholangiocarcinoma
Ijare, Omkar B.; Bezabeh, Tedros; Albiin, Nils; Smith, Ian C. P.
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Letter to the Editor
Magnetic resonance spectroscopy of bile in the detection of
cholangiocarcinoma
Omkar B. Ijare1, Tedros Bezabeh1, , Nils Albiin2,
Ian C.P. Smith1
1 National Research Council Institute for Biodiagnostics, Winnipeg, Manitoba,
Canada
2 Karolinska University Hospital, Karolinska Institutet, Huddinge, Stockholm, Sweden
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], 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 [2], [3], [4], [5] and [6] 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]. However, a similar metabolomic study using bile samples for the detection of cholangiocarcinoma was published by our group about two years ago [2]. 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]. We reported a sensitivity of 88.9% and a specificity of 87.1% in classifying cancer and control groups. Khan et al. [7] 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] and the one by Khan et al. [7] 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] 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]. 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]. Moreover, in a recent study, Sharif et al. made similar observations in comparing patients with
cholangiocarcinoma and gallstone disease (p = 0.01) [9]. 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]. Second, the authors reported that citrate levels were elevated in cancer patients compared to the benign subjects with the difference being statistically significant [1]. Although citrate is a component of bile acting as a calcium chelator in gallbladder disease [10], 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].
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].
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.
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Conflict of interest
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.
References
1. H. Wen, S.S. Yoo, J. Kang, H.G. Kim, J.S. Park, S. Jeong et al. A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer J Hepatol, 52 (2010), pp. 228–233
2. N. Albiin, I.C.P. Smith, U. Arnelo, B. Lindberg, A. Bergquist, B. Dolenko et al. Detection of cholangiocarcinoma with magnetic resonance spectroscopy of bile in patients with and without primary sclerosing cholangitis Acta Radiol, 49 (2008), pp. 855–862
3. O.B. Ijare, T. Bezabeh, N. Albiin, U. Arnelo, A. Bergquist, B. Lindberg et al. Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of
cholestasis: a 1H MR study NMR Biomed, 22 (2009), pp. 471–479
4. T. Bezabeh, O.B. Ijare, N. Albiin, U. Arnelo, B. Lindberg, I.C.P. Smith Detection and quantification of d-glucuronic acid in human bile using 1H NMR spectroscopy:
relevance to the diagnosis of pancreatic cancer MAGMA, 22 (2009), pp. 267–275 5. Ijare 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].
6. O.B. Ijare, T. Bezabeh, N. Albiin, A. Bergquist, U. Arnelo, I.C.P. Smith 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, 53 (2010), pp. 667–673
7. S.A. Khan, I.J. Cox, A.V. Thillainayagam, D.S. Bansi, H.C. Thomas, S.D. Taylor-Robinson Proton and phosphorus-31 nuclear magnetic resonance spectroscopy of human bile in hepatopancreaticobiliary cancer Eur J Gastroenterol Hepatol, 17 (2005), pp. 733–738
8. G.A. Nagana Gowda, N. Shanaiah, A. Cooper, M. Maluccio, D. Raftery Visualization of bile homeostasis using 1H-NMR spectroscopy as a route for assessing liver cancer Lipids, 44 (2009), pp. 27–35
9. A.W. Sharif, H.R.T. Williams, T. Lampejo, S.A. Khan, D.S. Bansi, D. Westaby Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy HPB, 12 (2010), pp. 396–402
10. L. Vitetta, A. Sali Citrate: a component of bile and calcium chelator in gallbladder disease J Nutr Environ Med, 9 (1999), pp. 199–207
