Abstract
Bile acids are the main body of enterohepatic circulation in vivo. They have essential functions such as emulsifying fat, bacteriostasis and regulating multiple metabolic pathways as signal molecules. However, the assignments of NMR signals for some low-concentration bile acids are still needed. This study combined 1D nuclear magnetic resonance (NMR) and 2D NMR techniques including 1H–1H correlation spectroscopy (COSY), 1H–1H total correlation spectroscopy (TOCSY), 1H J-resolved spectroscopy (J-Res), 1H–13C heteronuclear single quantum coherence spectroscopy (HSQC), and 1H–13C heteronuclear multiple bond correlation spectroscopy (HMBC) to assign the 1H and 13C signals of six bile acids in aqueous solution at physiological pH (∼7.4) and nine bile acids in methanol. These data are of importance to the NMR-based studies on lipid digestion, absorption, and metabolism.
Keywords: Bile acids, 1H NMR, 2D NMR, Signal assignments
Graphical abstract
1. Introduction
Bile acids are the main components of bile with numerous essential functions, such as mammalian enterohepatic circulation. These acids are synthesized in the mammalian liver and stored in the gall bladder [1]. Following a meal, the gall bladder contracts and secretes bile, which enters the intestine through the bile duct. Once in the intestine, specific enzymes produced by the gut microbiota dehydroxylate the bile acids to produce some secondary ones, reducing their cytotoxicity [2,3]. Therefore, some bile acids are co-metabolites of the host and gut microbiota.
Bile acids generally have hydrophilic and hydrophobic groups; the former includes hydroxyl, carboxyl, and sulfonic acid groups whereas the latter includes methyl groups and sterol moieties. This unique structural configuration effectively reduces the interfacial tension of oil and water, providing surface activity. As a result, bile acids can promote the emulsification and absorption of hydrophobic chemicals such as lipophilic vitamins, sterol- and fatty acid-containing lipids. In organisms, bile acids dissolve with fat-soluble nutrients in the intestinal tract to form micelles, promoting the absorption of fat-soluble substances, including vitamins, by intestinal epithelial cells [4,5]. Approximately 90%–95% of the bile acids in the intestine are re-absorbed by the intestinal epithelial cells and returned to the liver via the portal vein whilst the other 5%–10% are excreted in feces [6]. Therefore, the levels of bile acids closely reflect the regulation of lipid metabolism, health status of the intestinal tract, and ecological balance of the gut microbiota.
Currently, Liquid Chromatograph-Mass Spectrometer (LC-MS) is the main tool for quantitative analysis of bile acids in biological samples [[7], [8], [9], [10], [11]]. This method allows for the absolute quantitation of bile acid components in biological samples and has good precision and high sensitivity. However, it relies commercially available standards to establish a working curve for each analyte, which can be expensive and time-consuming. In comparison, nuclear magnetic resonance (NMR) analysis requires little preparation and becomes a good tool for metabolomics investigations. For instance, 1H NMR spectra can concurrently provide both structural and quantitative information for all bile acids and their metabolites in complex biological samples. However, the structures of sterol moieties of bile acids have high similarity hence similar chemical shifts resulting in serious signal overlapping for 1H NMR spectra of extracts from complex biological samples hindering their spectral assignments. Huge dynamic range for the concentration of different bile acids poses further analytical challenges. Bile acids with high concentration in biological samples, including cholic acid, deoxycholic acid and their taurine and glycine conjugates have already been characterized in aqueous solutions (pH∼7.4) using 1H and 13C NMR methods [[12], [13], [14]]. Unfortunately, many bile acids with low abundances have not yet been fully assigned in the above complex mixtures using NMR hindering their NMR quantification.
To assist bile acids related metabolomics studies, here, we have systematically assigned the 1H and 13C NMR signals of some important bile acids with incomplete assignments using 1D and 2D NMR spectroscopy including 1H–1H correlation spectroscopy (COSY), 1H–1H total correlation spectroscopy (TOCSY), 1H J-resolved spectroscopy (J-Res), 1H–13C heteronuclear single quantum coherence spectroscopy (HSQC), and 1H–13C heteronuclear multiple bond correlation spectroscopy (HMBC).
2. Materials and methods
2.1. Materials
All standards of bile acids used here were purchased from Steraloids Inc. (Newport, RI, USA). Deuterium oxide (D2O, 99.9 atom% D) was purchased from Sigma-Aldrich Inc. (St. Louis, MO) whereas sodium 3-trimethylsilyl [2,2,3,3-2D4] propionate (TSP) was from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA). NaN3 (analytical grade) was from Tianjin Fuchen Chemical Reagent Company whilst K2HPO4·3H2O and NaH2PO4·2H2O were from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Each of all bile acids (1–2 mg) was dissolved in 600 μL phosphate buffer containing 30% D2O and 0.12 mM TSP or deuterated methanol and centrifugated (16000 g, 4 °C, 10 min) with 550 μL supernatant transferred into a 5-mm NMR tube for NMR analysis.
2.2. NMR spectroscopy
All NMR spectra were recorded at 298 K on a Bruker AVIII 600 MHz NMR spectrometer equipped with a TXI cryoprobe (Bruker Biospin, Germany) with frequencies of 600.13 and 150.90 MHz for 1H and 13C, respectively. The 1H NMR spectra were recorded using the standard noesygppr1d pulse sequence, [RD-90°-t1-90°-tm-90°-ACQ], to achieve effective suppression of solvent signals and flat baslines. Other parameters included the spectral width of 10 ppm, recycle delay (RD) of 2 s, tm of 100 ms, t1 of 4 μs, 90° pulse length of 10 μs, the acquisition time of 1.36 s and 16 scans per spectrum.
All 2D NMR (J-Res, 1H–1H TOCSY, 1H–1H COSY , 1H–13C HSQC and 1H–13C HMBC) spectra were recorded using the standard Bruker pulse sequences [[6], [7], [8]]. In brief, the spectral widths for J-Res spectra were set to 6313.13 Hz in F2 (1H) and 60.013 Hz in F1 (J) with data points set to 2048 for F2 and 128 for F1. For 1H–1H TOCSY, DIPSI was used for spin-locking with a mixing time of 90 ms, and 32 scans per FID were taken. For 1H–1H COSY, 16 scans per FID were taken. For 1H–13C HSQC, the spectral widths were set to 6313.13 Hz in F2 (1H) and 18109.15 Hz in F1 (13C) with data points set to 2048 for F2 and 100 for F1; 120 scans per FID were recorded and Garp was used for decoupling. For 1H–13C HMBC, the spectral widths were set to 6313.13 Hz in F2 (1H) and 33201.94 Hz in F1 (13C) with data points set to 2048 for F2 and 96 for F1; 800 scans per FID were recorded with long-range coupling set to 8 Hz. All these spectra were processed with Topspin (v3.0, Bruker Biospin, Germany) with both dimensions zero-filled to 2048 prior to Fourier transformation.
3. Results and discussion
3.1. Structural dependence of chemical shifts for some bile acids
Fig. 1 shows the structures of bile acids containing a sterol moiety, numerous hydroxyl groups on various carbons and various conjugators at the carboxylic groups. Structural isomers are frequently observed for bile acids with some existing as stereoisomers. For example, CDCA and UDCA are stereoisomers to each other with two hydroxyl groups connected to C3 and C7 on their steroidal rings, respectively. In UDCA, the carbon-oxygen bonds on C3 and C7 are coplanar while in CDCA, these bonds are perpendicular. DCA, UDCA, HDCA, and CDCA all feature steroidal rings with two hydroxyl groups attached while CA, HCA, and other bile acids have steroidal rings linked to three hydroxyl groups. Conjugated bile acids are formed by linking free bile acids with glycine, taurine, and other conjugated groups. These conjugated groups have a variation of protons and carbons in conjugated bile acids with their own unique signals. The structures of GLCA and TLCA show that 18-CH3, 19-CH3, glycine, and taurine are all on the same side of the six-membered ring. The high electron cloud density of these groups resulting from the spatial induction effects of the lone-pair electrons of N and O atoms in the glycine and taurine groups causes repulsion of the electrons on the 18-CH3 and 19-CH3 groups, leading to a deshielding effect on the hydrogen nuclei. Consequently, the signals of protons on the 18-CH3 and 19-CH3 groups of conjugated bile acids shift to a higher field than those of free bile acids. Similar changes in the proton chemical shifts of the 4-CH2, 17-CH3, and 21-CH3 groups of LCA, GLCA, and TLCA are also observed (Fig. 2).
Fig. 1.
Chemical structures of bile acids and their glycine/taurine conjugates. The orientations of some important substitutes are marked relative to the plane of the ring (dashed wedge: α substitution; solid wedge: β substitution). CA: cholic acid; DCA: deoxycholic acid; CDCA: chenodeoxycbolic acid; UDCA: ursodeoxycholic acid; HDCA: hyodeoxycbolic acid; LCA: lithocholic acid; HCA: hyocholic acid.
Fig. 2.
1H NMR chemical shifts for some specific moeties of LCA, GLCA, and TLCA in CD3OD.
3.2. Solvent effects on chemical shift of the bile acids
Solvent selection is a crucial consideration in metabonomics studies as it can significantly affect the chemical shifts of metabolites. In particular, aqueous solution (pH 7.4) and methanol are commonly used solvents for analyzing bile acids though each solvent may result in different chemical shift values. Therefore, we determined the chemical shifts for standards of bile acids in both aqueous and methanol solution. Our results revealed that the hydrogen attached to the hydroxyl-connected carbon of the steroidal ring of bile acids in an aqueous solution exhibited larger chemical shift values than those in methanol (Fig. 3). As hydrogen bonding is a critical factor for the value of chemical shifts, the observed differences may be attributed to the discrepancy of hydrogen bonding strength between the bile acids and two solvents. In particular, the strong polarity of water results in strong hydrogen bonding between the hydroxyl group of the bile acids and water molecules, leading to a deshielding effect on the attached hydrogen and a corresponding shift of the chemical shift to lower fields. Our findings provide important insights into the solvent dependence of the chemical shifts of bile acids which can be used to aid in the identification and characterization of these crucial metabolites in complex biological samples analyzed using different solvents.
Fig. 3.
Some 1H NMR chemical shifts of bile acids in aqueous solution (pH 7.4) and CD3OD.
3.3. Assignment of 1H and 13C NMR chemical shifts for HCA in CD3OD
Using HCA as an example, here, we explain signal assignments for some bile acids with incomplete assignments based on 2D NMR spectra. In proton NMR spectrum of HCA, first, the methyl proton H-21 gave a doublet due to its coupling to neighbor proton H-20 (Fig. 4). Its J-Res spectrum showed a doublet for methyl group at δH 0.95 being readily assignable to H-21 (Fig. 4). Its 1H–1H COSY spectrum, H-21 (δH 0.95) displayed a strong correlation with a signal at δH 1.44 (Fig. 5), which was thus assignable to H-20. The H-20 signal (δH 1.44) was further coupled to signals δH 1.16, δH 1.29 and δH 1.79 assignable to either H17 or two protons on C22, which ought to be confirmed with correlations in its 1H–1H TOCSY and 1H–13C HSQC spectra.
Fig. 4.
The 1H J-Res spectrum for HCA in CD3OD.
Fig. 5.
1H–1H COSY spectrum of HCA in methanol.
The 1H–1H TOCSY spectrum showed corrections for δH 0.95-1.44-1.16-1.29-1.79-2.18-2.32 (Fig. 6), among which the 1H–1H COSY spectrum (Fig. 5) showed obvious correlations for δH 2.19 and δH 2.32, and for δH 1.29 and δH 1.79. Its 1H–13C HSQC spectrum (Fig. 7) showed a correlation between δH 1.16 and δC 59.1 making these signals assignable to H-17 and C-17. Correlations for the carbon at δC 33.8 and two protons at δH 2.18 and δH 2.32 (Fig. 7) indicated that these two protons were from a CH2 moeity with two unequivalent protons. Similarly, correlations between the carbon at δC 34.2 and two protons at δH 1.29 and δH 1.79 in its HSQC spectrum (Fig. 7) suggested the presense of another CH2 moiety with two unequivalent protons. Therefore, δH 1.29 and δH 1.79 were assignale to H-22 (α/β) whilst δH 2.32 and δH 2.18 were assignable to H-23 (α/β). Similarly, NMR signals for H-16, H-15 and H-14 and their attached carbons could be sequentially assigned via the step-by-step analysis of the correlations with H-17 NMR signals in COSY, TOCSY and 1H–13C coupings in HSQC spectrum, which also assisted differentiation for H-14, H-15 and H-16 signals. Proton singlets at δH 0.68 and δH 0.91 coupled to δC 14.0 and δC 25.5, respectively, in its HSQC spectrum (Fig. 7) were assignable to two more methyl groups C-18 and C-19. In its 1H–13C HMBC spectrum (Fig. 8), δH 0.68 showing correlation with C-17 (δC 59.1) was readily assignable to H-18 whilst δH 0.91 was assignable to H-19 similarly.
Fig. 6.
1H–1H TOCSY of HCA in CD3OD.
Fig. 7.
1H–13C HSQC spectrum of HCA in CD3OD.
Fig. 8.
1H–13C HMBC spectrum of HCA in CD3OD.
Links for methyl groups to quaternary carbons could be assigned using the long-range couplings in 1H–13C HMBC spectrum. 1H–13C HMBC spectrum of HCA (Fig. 8) showed such correlations for H-18 (δH 0.68) and a quaternary carbon at δC 45.6 suggested the latter as C-13 whilst the similar coupling for δH 0.91 facilitated assignment of another quaternary carbon (δC 35.8) to C-10. With the same strategy, signals for H-11, H-12, H-14, H-1, H-2 and H-5 together with their attached carbons were assigned (Table 1).
Table 1.
1H and 13C chemical shifts for HCA, TUDCA, GDCA, TLCA, GLCA, LCA, 12-ketodeoxycholic acid (12KDCA), HDCA, and UDCA in CD3OD with carbon atom numbers indicated in Fig. 1.
| Atom NO. | Moieties | HCA |
TUDCA |
GDCA |
TLCA |
GLCA |
LCA |
12KDCA |
HDCA |
UDCA |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | ||
| 1 | CH2-α | 1.82 | 38.6 | 1.81 | 38.3 | 1.78 | 38.3 | 1.80 | 38.5 | 1.80 | 38.4 | 1.79 | 38.4 | 1.67 | 38.3 | 1.79 | 38.6 | 1.02 | 38.3 |
| CH2-β | 1.03 | 38.6 | 1.02 | 38.3 | 0.98 | 38.3 | 0.98 | 38.5 | 0.97 | 38.4 | 0.97 | 38.4 | 1.06 | 38.3 | 1.04 | 38.6 | 1.79 | 38.3 | |
| 2 | CH2-α | 1.31 | 33.2 | 1.26 | 33.2 | 1.42 | 33.0 | 1.31 | 33.2 | 1.31 | 33.1 | 1.30 | 33.2 | 1.27 | 33.0 | 1.29 | 32.9 | 1.25 | 33.1 |
| CH2-β | 1.60 | 33.2 | 1.61 | 33.2 | 1.60 | 33.0 | 1.61 | 33.2 | 1.61 | 33.1 | 1.60 | 33.2 | 1.65 | 33.0 | 1.62 | 32.9 | 1.61 | 33.1 | |
| 3 | CH | 3.32 | 74.6 | 3.48 | 74.1 | 3.51 | 74.4 | 3.52 | 74.5 | 3.52 | 74.4 | 3.52 | 74.4 | 3.52 | 74.2 | 3.49 | 74.2 | 3.48 | 74.2 |
| 4 | CH2-α | 1.97 | 35.1 | 1.61 | 40.2 | 1.80 | 39.2 | 1.76 | 39.2 | 1.76 | 39.1 | 1.75 | 39.1 | 1.64 | 39.0 | 1.87 | 31.8 | 1.79 | 40.6 |
| CH2-β | 1.89 | 35.1 | 1.57 | 40.2 | 1.47 | 39.2 | 1.46 | 39.2 | 1.46 | 39.1 | 1.45 | 39.1 | 1.48 | 39.0 | 1.38 | 31.8 | 1.60 | 40.6 | |
| 5 | CH | 1.55 | 51.3 | 1.48 | 46.3 | 1.39 | 45.5 | 1.37 | 45.6 | 1.37 | 45.5 | 1.37 | 45.5 | 1.47 | 45.0 | 1.56 | 51.6 | 1.45 | 46.4 |
| 6 | CH/CH2-α | 3.76 | 72.6 | 1.54 | 40.7 | 1.27 | 30.4 | 1.26 | 30.4 | 1.89 | 30.3 | 1.88 | 30.3 | 1.32 | 39.1 | 3.99 | 70.4 | 1.54 | 40.5 |
| CH2-β | 1.82 | 40.7 | 1.89 | 30.4 | 1.90 | 30.4 | 1.26 | 30.3 | 1.25 | 30.3 | 1.88 | 39.1 | 1.81 | 40.5 | |||||
| 7 | CH/CH2-α | 3.75 | 74.0 | 3.46 | 74.1 | 1.44 | 29.3 | 1.12 | 29.6 | 1.12 | 29.6 | 1.11 | 29.6 | 1.14 | 29.3 | 1.61 | 37.4 | 3.46 | 74.2 |
| CH2-β | 1.17 | 29.3 | 1.43 | 29.6 | 1.43 | 29.6 | 1.42 | 29.6 | 1.53 | 29.3 | 1.14 | 37.4 | |||||||
| 8 | CH | 1.51 | 41.9 | 1.44 | 46.6 | 1.40 | 38.9 | 1.44 | 43.9 | 1.43 | 43.8 | 1.43 | 43.9 | 1.89 | 38.4 | 1.48 | 38.1 | 1.45 | 46.5 |
| 9 | CH | 1.77 | 35.6 | 1.48 | 42.8 | 1.89 | 36.7 | 1.44 | 43.9 | 1.44 | 43.8 | 1.43 | 43.9 | 1.85 | 47.9 | 1.44 | 38.4 | 1.47 | 42.9 |
| 10 | C | - | 35.8 | - | 42.6 | - | 37.3 | - | 34.8 | - | 34.9 | - | 37.7 | - | 44.8 | - | 43.1 | - | 36.5 |
| 11 | CH2-α | 1.49 | 23.6 | 1.45 | 24.5 | 1.52 | 31.7 | 1.42 | 23.9 | 1.41 | 23.9 | 1.42 | 23.9 | 2.57 | 41.1 | 1.43 | 23.7 | 1.44 | 24.6 |
| CH2-β | 1.27 | 23.6 | 1.31 | 24.5 | 1.52 | 31.7 | 1.31 | 23.9 | 1.28 | 23.9 | 1.27 | 23.9 | 2.00 | 41.1 | 1.22 | 23.7 | 1.31 | 24.6 | |
| 12 | CH2-α | 1.18 | 42.8 | 1.17 | 43.6 | 3.96 | 76.0 | 1.18 | 43.6 | 1.18 | 43.5 | 1.18 | 43.5 | 1.18 | 43.2 | 1.17 | 43.7 | ||
| CH2-β | 1.99 | 42.8 | 2.03 | 43.6 | - | 2.00 | 43.6 | 2.01 | 43.5 | 2.00 | 43.5 | - | 219.9 | 2.01 | 43.2 | 2.02 | 43.7 | ||
| 13 | C | - | 45.6 | - | 46.9 | - | 52.1 | - | 46.0 | - | 45.8 | - | 45.9 | - | 60.9 | - | 45.8 | - | 46.7 |
| 14 | CH | 1.52 | 53.2 | 1.23 | 59.6 | 1.62 | 51.1 | 1.09 | 59.9 | 1.09 | 59.8 | 1.08 | 59.9 | 1.37 | 62.7 | 1.40 | 43.1 | 1.22 | 59.6 |
| 15 | CH2-α | 1.77 | 26.4 | 1.90 | 30.1 | 1.61 | 26.7 | 1.60 | 27.3 | 1.60 | 27.2 | 1.59 | 27.2 | 1.74 | 27.3 | 1.62 | 27.1 | 1.87 | 30.3 |
| CH2-β | 1.11 | 26.4 | 1.46 | 30.1 | 1.10 | 26.7 | 1.08 | 27.3 | 1.07 | 27.2 | 1.08 | 27.2 | 1.37 | 27.3 | 1.14 | 27.1 | 1.45 | 30.3 | |
| 16 | CH2-α | 1.91 | 31.0 | 1.89 | 31.7 | 1.89 | 30.5 | 1.89 | 31.3 | 1.90 | 31.2 | 1.86 | 31.2 | 1.95 | 30.3 | 1.88 | 31.1 | 1.86 | 31.8 |
| CH2-β | 1.34 | 31.0 | 1.29 | 31.7 | 1.30 | 30.5 | 1.30 | 31.3 | 1.31 | 31.2 | 1.30 | 31.2 | 1.37 | 30.3 | 1.31 | 31.1 | 1.30 | 31.8 | |
| 17 | CH | 1.16 | 59.1 | 1.09 | 58.5 | 1.84 | 50.1 | 1.13 | 59.4 | 1.13 | 59.4 | 1.12 | 59.5 | 1.99 | 50.1 | 1.15 | 59.3 | 1.08 | 58.7 |
| 18 | CH3 | 0.68 | 14.0 | 0.71 | 14.7 | 0.71 | 15.0 | 0.68 | 14.5 | 0.68 | 14.4 | 0.67 | 14.4 | 1.06 | 14.0 | 0.68 | 14.2 | 0.70 | 14.6 |
| 19 | CH3 | 0.91 | 25.5 | 0.96 | 26.1 | 0.93 | 25.6 | 0.94 | 26.0 | 0.93 | 25.9 | 0.93 | 25.9 | 1.04 | 25.1 | 0.91 | 25.9 | 0.95 | 26.1 |
| 20 | CH | 1.44 | 38.6 | 1.43 | 39.0 | 1.40 | 38.9 | 1.42 | 39.0 | 1.43 | 38.9 | 1.42 | 38.8 | 1.32 | 30.1 | 1.44 | 38.4 | 1.42 | 38.8 |
| 21 | CH3 | 0.95 | 20.6 | 0.97 | 21.1 | 1.03 | 19.6 | 0.95 | 20.8 | 0.95 | 20.8 | 0.93 | 20.7 | 0.83 | 21.1 | 0.95 | 20.5 | 0.94 | 21.0 |
| 22 | CH2-α | 1.29 | 34.2 | 1.33 | 35.3 | 1.35 | 35.1 | 1.30 | 35.1 | 1.30 | 35.0 | 1.29 | 34.3 | 1.32 | 34.1 | 1.28 | 34.1 | 1.30 | 34.3 |
| CH2-β | 1.79 | 34.2 | 1.79 | 35.3 | 1.80 | 35.1 | 1.77 | 35.1 | 1.78 | 35.0 | 1.78 | 34.3 | 1.83 | 34.1 | 1.78 | 34.1 | 1.79 | 34.3 | |
| 23 | CH2-α | 2.32 | 33.8 | 2.31 | 35.8 | 2.32 | 36.1 | 2.26 | 36.0 | 2.29 | 36.0 | 2.31 | 34.0 | 2.33 | 34.9 | 2.32 | 33.8 | 2.32 | 34.1 |
| CH2-β | 2.18 | 33.8 | 2.14 | 35.8 | 2.16 | 36.1 | 2.10 | 36.0 | 2.13 | 36.0 | 2.18 | 34.0 | 2.19 | 34.9 | 2.19 | 33.8 | 2.19 | 34.1 | |
| 24 | C | - | 184.4 | - | 179.4 | - | 178.2 | - | 178.9 | - | 178.5 | - | 181.8 | - | 180.8 | - | 182.0 | - | 181.9 |
| 25 | CH2/NH | - | 3.61 | 53.2 | 3.47 | 8.06 | 7.62 | ||||||||||||
| 26 | CH2 | - | 2.97 | 51.5 | - | 178.2 | 3.59 | 38.8 | 3.74 | 46.2 | |||||||||
| 27 | CH2/C=O | - | 2.95 | 54.4 | - | 178.3 | |||||||||||||
Additionally, using this NMR-based method allowed us to successfully assign both proton and carbon NMR signals for bile acids in aqueous solution and in methanol (See Tables 1 and 2).
Table 2.
1H and 13C chemical shifts for GUDCA, GHDCA,UDCA, HDCA, Nutriacholic acid and HCA in an aqueous media (D2O) at pH 7.4 with carbon atom numbers indicated in Fig. 1.
| Atom NO. | moieties | GUDCA |
GHDCA |
UDCA |
HDCA |
Nutriacholic acid |
HCA |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | δH | δC | ||
| 1 | CH2-α | 1.82 | 36.8 | 1.81 | 38.1 | 1.83 | 37.3 | 1.82 | 37.3 | 1.86 | 35.4 | 1.86 | 38.6 |
| CH2-β | 1.04 | 36.8 | 1.09 | 38.1 | 1.04 | 37.3 | 1.07 | 37.3 | 1.20 | 35.4 | 1.08 | 38.6 | |
| 2 | CH2-α | 1.30 | 31.5 | 1.37 | 32.0 | 1.29 | 32.1 | 1.37 | 31.6 | 1.25 | 30.8 | 1.35 | 33.7 |
| CH2-β | 1.63 | 31.5 | 1.65 | 32.0 | 1.63 | 32.1 | 1.65 | 31.6 | 1.66 | 30.8 | 1.66 | 33.7 | |
| 3 | CH | 3.63 | 73.7 | 3.65 | 74.3 | 3.62 | 74.1 | 3.64 | 73.5 | 3.66 | 72.3 | 3.49 | 75.8 |
| 4 | CH2-α | 1.63 | 38.5 | 1.75 | 31.2 | 1.63 | 38.5 | 1.74 | 30.5 | 1.65 | 38.0 | 1.92 | 36.3 |
| CH2-β | 1.58 | 38.5 | 1.45 | 31.2 | 1.59 | 38.5 | 1.45 | 30.5 | 1.12 | 38.0 | 1.73 | 36.3 | |
| 5 | CH | 1.55 | 44.7 | 1.64 | 50.9 | 1.52 | 44.9 | 1.63 | 49.9 | 2.03 | 48.6 | 1.64 | 51.6 |
| 6 | CH2-α | 1.57 | 38.7 | 4.12 | 71.3 | 1.57 | 39.1 | 4.12 | 70.5 | 1.90 | 47.4 | 3.90 | 73.6 |
| CH2-β | 1.81 | 38.7 | 1.81 | 39.1 | 3.10 | 47.4 | |||||||
| 7 | CH | 3.63 | 73.7 | 1.64 | 36.8 | 3.62 | 74.1 | 1.65 | 35.9 | - | 226.0 | 3.85 | 75.8 |
| 1.18 | 36.8 | 1.17 | 35.9 | ||||||||||
| 8 | CH | 1.46 | 41.6 | 1.54 | 37.6 | 1.47 | 42.0 | 1.52 | 36.3 | 2.73 | 51.4 | 1.60 | 41.6 |
| 9 | CH | 1.46 | 45.3 | 1.38 | 42.8 | 1.47 | 45.6 | 1.38 | 41.5 | 1.82 | 45.9 | 1.65 | 35.9 |
| 10 | C | - | 45.0 | - | 42.0 | - | 36.9 | - | 36.9 | - | 36.3 | - | 40.6 |
| 11 | CH2-α | 1.46 | 23.4 | 1.44 | 23.6 | 1.46 | 23.7 | 1.43 | 22.8 | 1.56 | 23.7 | 1.51 | 24.3 |
| CH2-β | 1.31 | 23.4 | 1.22 | 23.6 | 1.31 | 23.7 | 1.24 | 22.8 | 1.51 | 23.7 | 1.29 | 24.3 | |
| 12 | CH2-α | 1.23 | 42.2 | 1.21 | 42.7 | 1.21 | 42.6 | 1.19 | 42.0 | 2.02 | 40.5 | 1.19 | 43.3 |
| CH2-β | 2.20 | 42.2 | 2.00 | 42.7 | 2.02 | 42.6 | 1.99 | 42.0 | 1.17 | 40.5 | 2.00 | 43.3 | |
| 13 | C | - | 46.3 | - | 45.5 | - | 46.2 | - | 44.9 | - | 44.3 | - | 47.1 |
| 14 | CH | 1.31 | 57.5 | 1.21 | 59.6 | 1.31 | 57.9 | 1.21 | 57.7 | 1.39 | 50.3 | 1.43 | 53.2 |
| 15 | CH2-α | 1.78 | 28.8 | 1.64 | 26.9 | 1.78 | 29.3 | 1.61 | 26.3 | 1.95 | 27.3 | 1.67 | 26.5 |
| CH2-β | 1.39 | 28.8 | 1.12 | 26.9 | 1.38 | 29.3 | 1.11 | 26.3 | 1.02 | 27.3 | 1.15 | 26.5 | |
| 16 | CH2-α | 1.87 | 30.6 | 1.89 | 30.9 | 1.88 | 31.3 | 1.89 | 30.0 | 1.93 | 30.3 | 1.93 | 36.2 |
| CH2-β | 1.29 | 30.6 | 1.29 | 30.9 | 1.30 | 31.3 | 1.30 | 30.0 | 1.31 | 30.3 | 1.33 | 36.2 | |
| 17 | CH | 1.12 | 56.7 | 1.18 | 58.6 | 1.11 | 57.3 | 1.16 | 57.8 | 1.15 | 56.4 | 1.20 | 59.9 |
| 18 | CH3 | 0.69 | 13.6 | 0.67 | 14.4 | 0.69 | 14.3 | 0.67 | 13.4 | 0.69 | 13.4 | 0.68 | 14.0 |
| 19 | CH3 | 0.95 | 24.9 | 0.93 | 23.6 | 0.95 | 25.5 | 0.93 | 25.0 | 1.23 | 24.3 | 0.94 | 26.0 |
| 20 | CH | 1.44 | 37.4 | 1.46 | 37.9 | 1.43 | 38.1 | 1.44 | 37.5 | 1.43 | 37.2 | 1.44 | 39.0 |
| 21 | CH3 | 0.97 | 20.2 | 0.96 | 20.8 | 0.95 | 20.9 | 0.94 | 20.2 | 0.94 | 20.1 | 0.95 | 22.2 |
| 22 | CH2-α | 1.36 | 34.0 | 1.37 | 34.8 | 1.32 | 35.5 | 1.31 | 34.5 | 1.32 | 34.6 | 1.33 | 36.3 |
| CH2-β | 1.76 | 34.0 | 1.77 | 34.8 | 1.71 | 35.5 | 1.71 | 34.5 | 1.71 | 34.6 | 1.72 | 36.3 | |
| 23 | CH2-α | 2.36 | 35.0 | 2.36 | 35.8 | 2.23 | 37.4 | 2.22 | 36.9 | 2.23 | 36.6 | 2.24 | 38.1 |
| CH2-β | 2.24 | 35.0 | 2.23 | 35.8 | 2.11 | 37.4 | 2.11 | 36.9 | 2.11 | 36.6 | 2.10 | 38.1 | |
| 24 | C=O | - | 179.3 | - | 180.2 | - | 187.5 | - | 187.4 | - | 187.1 | - | 187.5 |
| 25 | NH | 7.90 | - | 7.90 | |||||||||
| 26 | CH2 | 3.75 | 45.6 | 3.74 | 46.4 | ||||||||
| 27 | COOH | - | 181.0 | - | 179.6 | ||||||||
4. Conclusion
This study unambiguously assigned 1H and 13C NMR signals for nine bile acids in methanol-d4 and six bile acids in aqueous solutions at physiological pH (∼7.4) with the combinations of 1D and various 2D NMR (Table 1, Table 2). Our results demonstrated that the 1H chemical shifts of bile acids are structurally dependent and that the solvent influences the 1H chemical shifts of some moieties of bile acids. This provides essential information for these less studied bile acids and is expected to be useful for quantifying bile acids using NMR spectroscopy and studying their metabolism as well as their functions.
CRediT authorship contribution statement
Hong Lin: Data acquisition, Formal analysis, Manuscript preparation and revision. Junbo He: Data check, Validation. Weinong Zhang: Data check, Validation. Huiru Tang: Conceptualization, Supervision, Data interpretation, Manuscript revision.
Declaration of competing interest
The authors declare that they have no competing interests.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grant Nos. 32101967).
Biography
Huiru Tang is a professor at Fudan University. He received Ph. D. degree from the University of London in 1994. From 1992 to 2005, he worked as a Senior Scientist at BBSRC Food Research Institute and Imperial College of Science and Technology, Department of Biomedical Sciences. Then he was a researcher at the Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences. 2014 to date, he joined the School of Life Sciences, Fudan University. He specialized in developing high-throughput and ultrasensitive metabolomics techniques, metabolome/transcriptome/proteome/microbiome data integration techniques, and their application into exploring mechanisms of disease development.
Footnotes
Peer review under responsibility of Innovation Academy for Precision Measurement Science and Technology (APM), CAS.
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