NMR Spectroscopy-Based Metabolic Profiling for Detecting

Chapter 23

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NMR Spectroscopy-Based Metabolic Profiling for Detecting Hepatobiliary Diseases G. A. Nagana Gowda* and Daniel Raftery Department of Chemistry, Purdue University, West Lafayette, IN 47907 *E-mail: [email protected]

The emerging area of metabolomics deals with the detection of a large number of small molecules (metabolites) from human biofluids or tissue in a single step and promises immense utility for early diagnosis, therapy monitoring and understanding pathogenesis of many diseases. Metabolomics methods are focused on the information rich analytical techniques of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). While MS is highly sensitive, NMR spectroscopy provides more reproducible and quantifiable data. Focused on the development of simple NMR based tools for metabolomics applications, new NMR methodologies have been explored. The methods were applied to detect hepatobiliary disease biomarkers using human bile, which is closely associated with the pathological source. Causes or consequences of numerous diseases including hepatocellular carcinoma, cholangiocarcinoma and gallbladder cancer have been attributed to the altered human bile metabolic profile.

Profiling of small molecules (metabolites), in biological samples such as blood, urine, bile, saliva, cerebrospinal fluid and tissue specimens, which are the downstream products of genes, mRNAs and proteins, promises numerous clinical applications including early disease diagnostics (1–4). The significant interest in metabolite profiling arises from the high sensitivity of metabolites to subtle changes in the biological state. Such high sensitivity of metabolites is valuable for early detection of onset of many diseases that are normally asymptomatic until late in the disease process. An important advantage of metabolite profiling © 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


over other approaches is that a large number of metabolites can be analyzed quantitatively and reproducibly using advanced analytical techniques. Further, this approach is either non- or minimally-invasve and requires minimal or no sample preparation steps. These characteristics are very important and attractive for translating the findings to clinical applications. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are the most powerful analytical techniques used for metabolic profiling (2). NMR spectrosocpy offers numerous advantages because of its ability to provide accurate and reproducible information on a large number of metabolites, in a single-step and using intact samples. However, it is challenging to obtain NMR spectra for biological samples with well resolved peaks from a single experiment, and the knowledge of identity of a large number of metabolites in complex NMR spectra is lacking. The high complexity to the NMR spectra arises due to the presence of thousands of metabolites in the biological samples. Such vast chemical diversity and the concentration range of over eight orders of magnitude (5) render detection of all the metabolites from a single analytical technique impractical Hence novel analytical methods are required to analyze complex biological mixtures.

Advanced NMR Methods for Metabolite Profiling To circumvent the challenges associated with high complexity of the biological samples, we focused on the development of new NMR methods targeting detection of individual classes of metabolites based on their chemical functional groups. Specifically, metabolites containing amino, carboxyl and hydroxyl functional groups were targeted individually by tagging with isotopes such as 13C, 15N or 31P. One-dimensional (1D) or two-dimensional (2D) NMR experiments involving these heteronuclei selectively detect isotope labeled metabolites with greatly improved resolution and sensitivity, free of background signals from the rest of the tagged molecule as well as the untagged molecules (6–9). Tagging of the amine class of metabolites involves a simple chemical reaction on intact biological samples using 13C labeled acetic anhydride. A subsequent 1H13C 2D HSQC (heteronuclear single quantum coherence) NMR experiment detects amine class of metabolites with improved sensitivity and resolution (6). More recently, we introduced an alternative method, which involves isotope tagging using 13C labeled formic acid to further improve the performance of 13C isotope tagging experiments (7). This method is more robust than the earlier method since the short one-bond distance between the labeled 13C and its closest 1H produces a large J-coupling constant and enables efficient transfer of polarization between 13C and 1H using shorter interpulse delays in the 2D experiments. Furthermore, the close distance between the isotope tag and metabolite leads to somewhat wider dispersion of the tagged metabolites’ signals in the resultant 2D spectrum. Tagging carboxyl group containing metabolites in biological samples involves derivatization with 15N labeled ethanolamine. The 1H-15N 2D HSQC NMR experiment then selectively detects the 15N tagged carboxylic acids in a 408 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


single measurement with enhanced resolution and sensitivity (8). This method exploits the broad chemical shift dispersion of 15N nuclei and imparts a large dispersion to the individual metabolites signals. This approach, enables detection of nearly two hundred carboxyl group containing metabolites in biological samples, both quantitatively and reproducibly. Detection of such a large number of metabolites is unprecedented from the NMR spectroscopy point of view (Fig. 1).

Figure 1. 1H-15N 2D HSQC NMR spectrum showing the detection of carboxylic acid class of metabolites using 15N isotope tagging (8).

Isotope tagging of hydroxyl group containing metabolites involves derivatization of metabolites using an agent containing 31P, 2-chloro-4,4,5,5tetramethyldioxaphospholane (9). 31P tagging approach also detects metabolites containing aldehyde and carboxyl groups. 31P tagged metabolites are detected with enhanced resolution using one-dimensional 31P NMR by exploiting the 100% abundance and wide chemical shift range of 31P NMR spectrum. All the isotope labeling approaches that we have developed are simple, highly reproducible and quantitative. Metabolites containing amino, carboxyl and hydroxyl functional groups constitute major classes of metabolites in biological processes. Hence, our ability to detect and quantify them using the new isotope tagging approaches represents a significant step forward in the area of metabolites based biomarkers detection for a number of diseases. 409 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Human Blood and Urine For a number of reasons human blood plasma/serum and urine have been extensively used in the area of metabolomics. Urine can be acquired non-invasively and it provides several hundreds of detectable signals even by relatively less sensitive analytical methods such as NMR spectroscopy. Blood, on the other hand, maintains normal metabolic homeostasis and any changes to this status represent a pathological state. However, the complexity due to numerous confounding factors such as age, gender, diet, body mass, lifestyle, drug, environment and diurnal variation, and pathological conditions involving multiple organs poses a significant challenge to detect biomarkers for disease of interest using human urine and blood plasma/serum samples. To overcome such challenges, metabolic profiling efforts were focused on other biological specimens such as human bile and tissue. A growing number of studies suggest that disease biomarkers are richly populated in such specimens due to their close association with the pathological source (10–13).

Human Bile as a Source of Hepatobiliary Disease Biomarkers Human bile is a complex fluid synthesized and secreted by the liver (14), and transported through bile canaliculi and bile ducts, and stored in the gallbladder. The bile gets concentrated in the gallbladder and emptied into the small intestine (duodenum) upon ingestion of food. Bile constitutes a mixture of organic molecules such as cholesterol, phospholipids, bile acids, urea, glucose, steroid hormones and bilirubin, and a variety of proteins and peptides. Under normal conditions, the constituents of bile are tightly controlled through homeostasis. Alterations in normal metabolism due to etiological conditions of hepatobiliary system represent a novel source of liver disease-specific biomarkers (12). Since the bile constitutes a pool of metabolites closely associated with the gastrointestinal system, metabolic profile of bile closely represents normal or abnormal cellular processes associated with the liver, bile ducts and gallbladder. Hence analysis of bile provides valuable clues to potential biomarkers associated with numerous hepatobiliary diseases. Among a large number of bile metabolites, cholesterol, glycerophospholipids and bile acids represent the most abundant constituents of human bile. Bile acids constitute a group of a large number of structurally closely related molecules, which are synthesized starting from cholesterol and conjugated mainly with glycine or taurine before secretion from the hepatocytes (15, 16).

Metabolite Profiling of Human Bile Using NMR Spectroscopy Severe overlap of signals from a large number of bile metabolites, which exist in aggregated form because of amphipathic nature of the molecules, is a major challenge in the analysis of human bile using NMR spectroscopy. To circumvent this problem, numerous advanced NMR methodologies have recently been applied and unraveled the complexity of human bile spectrum (17–22). This work involved extensive analysis of synthetic compounds and establishment of a 410 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


library of 1H and 13C NMR chemical shifts for numerous bile metabolites under physiological conditions (22). Subsequently, using a combination of the chemical shift library, and homonuclear and heteronuclear NMR experiments at 400, 700 and 800 MHz, comprehensive analysis of bile NMR spectra was made. An important outcome of this study is the identification of all major bile metabolites including bile acids, cholesterol, phospholipids, unsaturated lipids and urea, all of which are known to be commonly found in healthy human bile (18, 19). In human bile, six conjugated bile acids are commonly present at high concentration. These constitute two primary bile acids, cholic acid and chenodeoxycholic acid, and a secondary bile acid, deoxycholic acid. Both primary and secondary bile acids are conjugated to glycine, resulting in glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, and to taurine, resulting in taurocholic acid, taurodeoxycholic acid and taurochenodeoxycholic acid. We identified all these six bile acids individually in bile for the first time from the point of view of NMR. Subsequently, simple NMR methods were developed for high-throughput analysis of all major bile metabolites in a single step for routine applications.

Visualization of Human Bile Homeostasis for Assessing Hepatobiliary Diseases Concentrations of biliary metabolites are regulated through homeostatic control. Any alterations in metabolic conditions arising from etiological conditions of hepatobiliary diseases, including alterations in the bile synthesis by the liver or enterohepatic circulation, represent a novel source of liver disease-specific biomarkers. Since the bile constitutes a pool of both endogenous and exogenous compounds excreted from the cells of the liver, gallbladder and bile ducts, the metabolic profile of bile closely represents normal as well as abnormal cellular metabolic processes associated with these organs, and hence investigations of bile composition potentially provide valuable clues to the biomarkers of hepatobiliary diseases for diagnostic and therapeutic applications. Recent investigations of bile using single-step NMR methods showed significant alterations in bile synthesis and enterohepatic circulation for a number of hepatobiliary diseases, including malignancies. Cholangiocarcinoma and non-malignant liver diseases showed most significant alterations. Further, hepatocellular carcinoma could be differentiated from cholangiocarcinoma based on the amounts of bile acids, phospholipids and cholesterol (Fig. 2) (23). Such a snapshot view of alerted bile homeostasis, is obtainable from a simple NMR approach and demonstrates the enormous opportunity to assess liver status, explore biomarkers for high risk diseases such as cancer, and improve the understanding of normal and abnormal cellular functions. Investigation of hepatobiliary diseases using human bile and NMR methods has the added advantage that results of such in vitro studies can be utilized for clinical applications using non-invasive in vivo magnetic resonance spectroscopy (24).

411 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Bile Acids Conjugation in Human Bile In healthy individuals, bile acids perform a number of important functions including promoting cholesterol elimination by converting cholesterol to bile acids and transporting cholesterol from hepatocytes to intestine through micelles formation, transporting phospholipids through mixed micelles formation, fat absorption, and signaling bile acids synthesis through negative feedback regulation (25). In the small intestine, bile acids are thought to inhibit the growth of bacteria (26). Numerous studies link the bile acids to a number of hepatobiliary and intestinal diseases. As mentioned earlier, methodological developments in NMR have enabled single-step analysis of abundant and common glycineand taurine- conjugated bile acids. Investigation of these conjugated bile acids in human bile employing high field NMR has shown that ratios between two glycine-conjugated bile acids and their taurine counterparts correlate positively as do ratios between a glycine-conjugated bile acid and its taurine counterpart (27). These insights into bile acids conjugation pattern 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 diseases.

Figure 2. Distinguishing hepatocellular carcinoma (HCC) and cholangio-carcinoma (CC) from one another and from controls based on bile metabolite concentrations. 412 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Altered Bile Composition, Gallstones, and Gallbladder Cancer Understanding the risk factors of gallbladder carcinogens in terms of bile composition and gallstones is an important step in the development of biomarkers for gallbladder cancer (GBC). Altered bile composition causes formation of gallstones (GS) in the gallbladder. GS disease is a common medical problem all over the world (28) and GBC is the most common malignancy (cancer) of the biliary tract. GS are suspected to be a contributing factor to the aetio-pathogenesis of GBC (29). GBC is frequently associated with GS (30) and the risk of GBC is 4 to 5 times higher in patients with GS than those without GS (31). At the same time, a very small number of patients with GS develop GBC. Several studies report the link between GS with GBC (32–36). While many studies report that patients with GBC are associated more frequently with large GS (32–34), a subsequent study contradicted such findings (35). Considering the importance of determining biochemical composition of GS under varied pathological conditions for understanding the aetio-pathogenesis of GBC, we recently analyzed bile metabolites in GS from patients with cancerous and non-cancerous gallbladder using NMR spectroscopy (37). Unlike gallstones from benign disease, those from GBC were cholesterol non-predominant mixed stones. Moreover, both calcium and magnesium were higher in GBC than in benign disease. Such differences in GS composition between malignant and benign gallbladder patients may provide useful clues to the aetio-pathogenesis of GBC and lead to identification of patients with GS, in vivo, who are at high risk of developing GBC and advocate prophylactic cholecystectomy to prevent GBC.

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NMR Spectroscopy-Based Metabolic Profiling for Detecting

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