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©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
Differential diagnosis of gallstones by using hypericin as a fluorescent optical imaging agent
Marlein Miranda Cona, Ye-Wei Liu, Antoine Hubert, Ting Yin, Yuan-Bo Feng, Peter de Witte, Etienne Waelkens, Yan-Sheng Jiang, Jian Zhang, Stefaan Mulier, Qian Xia, Gang Huang, Raymond Oyen, Yi-Cheng Ni
Marlein Miranda Cona, Ye-Wei Liu, Ting Yin, Yuan-Bo Feng, Yan-Sheng Jiang, Stefaan Mulier, Raymond Oyen, Yi-Cheng Ni, Department of Imaging and Pathology, Faculty of Medicine, Biomedical Sciences Group, University Hospitals, KU Leuven, B-3000 Leuven, Belgium
Antoine Hubert, Etienne Waelkens, Department of Cellular and Molecular Medicine, Biomedical Sciences Group, KU Leuven, B-3000 Leuven, Belgium
Peter de Witte, Molecular Biodiscovery Laboratory, Faculty of Pharmaceutical Sciences, Biomedical Sciences Group, KU Leuven, B-3000 Leuven, Belgium
Jian Zhang, Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, Jiangsu Province, China
Qian Xia, Gang Huang, Department of Nuclear Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
Author contributions: Miranda Cona M, Liu YW, Hubert A, Yin T, Feng YB, Jiang YS, Zhang J, Xia Q and Ni YC carried out all the experiments, performed data analysis and wrote the manuscript; Zhang J, Mulier S and Huang G contributed to collecting gallstones from patients, concept designing and manuscript writing; de Witte P, Waelkens E and Huang G contributed new reagents/analytic tools; Miranda Cona M, Oyen R and Ni YC contributed to concept designing and all authors approved the final version for submission.
Supported by Research Foundation - Flanders (FWO), the KU Leuven Molecular Small Animal Imaging Center MoSAIC, No. KUL EF/05/08; and the center of excellence in vivo molecular imaging research (IMIR); KU Leuven projects, No. IOF-HB/08/009, and No. IOF-HB/12/018; the European Union, Asia-Link CfP 2006-EuropeAid/123738/C/ACT/Multi-Proposal, No. 128-498/111; National Natural Science Foundation of China, No. 81071828; and Jiangsu Province Natural Science Foundation, No. BK2010594.
Institutional review board statement: This experimental research was approved by the Ethical Committee of Medical School, KU Leuven, Belgium.
Conflict-of-interest statement: None of the authors have any conflict of interest.
Data sharing statement: No additional data available are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Yi-Cheng Ni, MD, PhD, Professor, Department of Imaging and Pathology, Faculty of Medicine, Biomedical Sciences Group, University Hospitals, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
yicheng.ni@med.kuleuven.be
Telephone: +32-16-322752 Fax: +32-16-343765
Received: May 11, 2015
Peer-review started: May 12, 2015
First decision: September 9, 2015
Revised: April 1, 2016
Accepted: May 4, 2016
Article in press: May 4, 2016
Published online: August 7, 2016
Processing time: 445 Days and 12.6 Hours
AIM: To explore the feasibility of using hypericin as an optical imaging probe with affinity for cholesterol for differential fluorescent detection of human gallstones.
METHODS: Cholesterol, mixed and pigment stones from cholecystectomy patients were incubated with hypericin or solvent. After 72 h, the stones were analysed for fluorescence (365 nm) and treated with 2-propanol/dimethyl sulfoxide for high performance liquid chromatography (HPLC) analysis. Rats with virtual gallbladder containing human cholesterol, mixed or pigment gallstones (VGHG) received 5 mg/kg hypericin or solvent and VGHG rats with cholesterol stones were given different hypericin doses (5-15 mg/kg). Twelve hours later, the stones were analysed at 365 nm. Biliary excretion and metabolites of hypericin were assessed in common bile duct (CBD) cannulated rats for 9 h using fluorospectrometry, HPLC and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).
RESULTS: Homogeneous high fluorescence was seen on cholesterol stones either pre-incubated with hypericin or extracted from VGHG rats receiving hypericin. Mixed stones showed a dotted fluorescent pattern, whereas pigment and solvent-treated ones lacked fluorescence. HPLC showed 7.68, 6.65 and 0.08 × 10-3 M of cholesterol in extracts from cholesterol, mixed, and pigment gallstones, respectively. Hypericin accounted for 2.0, 0.5 and 0.2 × 10-6 M in that order. On cholesterol stones from VGHG rats receiving different hypericin doses, a positive correlation was observed between dose and fluorescence. In the bile from CBD-cannulated rats, fluorescence represented 20% of the injected dose with two peaks in 9 h. HPLC analysis revealed that hypericin conjugates reached 60% of the peak area. By MALDI-TOF MS, hypericin-glucuronide was detected.
CONCLUSION: This study proves the potential use of hypericin for differential fluorescent detection of human gallstones regarding their chemical composition.
Core tip: Cholelithiasis refers to cholesterol, mixed and pigment gallstones in the gallbladder. Although the current diagnostic methods may detect their presence in the biliary system, none of them may offer an in situ differential diagnosis of gallstones regarding their chemical composition. Hypericin is a fluorophore plant pigment tending to bind membrane lipids and is excreted via bile. Because cholesterol is likely the target that hypericin binds, hypericin could become a potential optical imaging agent for differential fluorescent detection of gallstones. Such imaging procedures would allow identifying the origin, composition and formation of gallstones in patients and permit adopting strategies to minimize cholelithiasis recurrence.
