Single‐Step analysis of individual conjugated bile acids in human bile using ¹H NMR spectroscopy

Abstract

1H and 13C NMR spectra of intact human bile were assigned using one-dimensional (1H and 13C) and two-dimensional (1H-1H and 1H-13C) experiments. Individual conjugated bile acids--glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid--were identified. The bile acids were quantified accurately and individually in a single step by using distinct and characteristic amide signals. Making use of 13C NMR, the study also suggests a way to analyze unconjugated bile acids separately, if present. Chemical shift assignments and rapid single-step analysis of individual conjugated bile acids from intact bile presented herein may have immense utility in the study of bile acid metabolism and deeper understanding of hepatobiliary diseases.

Detection and quantification of d-glucuronic acid in human bile using 1H NMR spectroscopy: relevance to the diagnosis of pancreatic cancer

“…NMR spectroscopy has been previously utilized for the analysis of major biliary biochemicals such as bile salts, phospholipids, and cholesterol [15]. Khan et al [16] have explored the possibility of using 31 P NMR of bile for the detection of pancreaticobiliary malignancies, focusing on the analysis of PC levels.…”

Bile Acids Conjugation in Human Bile Is Not Random: New Insights from 1H‐NMR Spectroscopy at 800 MHz

“…While D 2 O served as a field-frequency locking solvent, TSP served as chemical shift as well as a quantitative reference. Recently, from the comprehensive analysis of bile 1 H-NMR spectra, we have shown that the characteristic amide signals constituting three glycine-conjugated bile acids, glycocholic acid (GCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), and their taurine counterparts, taurocholic acid (TCA), taurodeoxycholic acid (TDCA) and taurochenodeoxycholic acid (TCDCA), invariably appear in the region 7.8–8.1 ppm [20]. We have also shown that these bile acids signals are better distinguished when the coupling between amide protons and the attached methylene protons is removed by decoupling.…”

“…It is shown from the experiments performed at different pH that the amide signals of individual conjugated bile acids represent more quantitatively over the pH range 6 ± 0.5 [18, 20]. Hence, the pH of each bile solution was adjusted to this range by the addition of 1–2 μL of 1 N hydrochloric acid.…”

“…Hence, it is an attractive technique for analyzing a large number of bile acids in a single-step, particularly when the concentration is not a limiting factor. Recently, from a series of comprehensive NMR studies of human bile and authentic bile acids, we reported simple NMR methodologies for detecting and quantifying major bile metabolites including a number of conjugated bile acids in human bile [17–20]. Subsequently, using these methods we have shown significant differences in the concentrations of bile metabolites between liver disease patients and controls [21].…”

Highly Sensitive Luminescence Assessment of Bile Acid Using a Balofloxacin-Europium(III) Probe in Micellar Medium

“…So, the estimation of bile acid in biological fluids has always presented technical difficulty owing to their low concentration and complex structure. Because of the growing interest on bile acid, a number of analytical methods have been developed in the literature for the estimation of bile acid in biological matrices, based on high performance liquid chromatograph (HPLC) with ultraviolet (UV), 9,10 fluorescence, 11 evaporative light scattering 12,13 or electrospray tandem mass spectrometer (MS) detection, 14,15 LC-MS/MS, 16 gas chromatography-mass spectrometry (GC-MS), 17 thin-layer chromatography, 18 nuclear magnetic resonance (NMR), 19 electrochemical sensor, 20-22 chemiluminescence, 23 spectrofluorimetry, 24 ultraviolet-visible spectrophotometry (UV-vis), 25 radioimmunoassay detection and enzymatic-colorimetric method. 26 However, these assay methodologies often suffer from disadvantages of low sensitivity, time consuming, costly reagents and instruments, and complicated pretreatment.…”