<Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR characterization and stereochemical assignments of bile acids in aqueous media

The unconjugated bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid; their glycine and taurine conjugated glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid; and a taurine conjugated ursodeoxycholic acid, tauroursodeoxycholic acid, were characterized through ¹H and ¹³C NMR in aqueous media under the physiological pH region (7.4±0.1). Assignments of ¹H and ¹³C signals of all the bile acids were made using a combination of several one- and two-dimensional, homonuclear (¹H−¹H) and heteronuclear (¹H−¹³C) correlations as well as spectral editing NMR methods. Stereochemical assignment of the five-membered ring of the bile acids is reported here for the first time. The complete characterization of various bile acids in aqueous media presented here may have implications in the study of the pathophysiology of biliary diseases through human biliary fluids using NMR spectroscopy.     

One-step analysis of major bile components in human bile using 1H NMR spectroscopy

Human gallbladder bile dissolved in dimethyl-sulfoxide provides sharp and resolved signals for major bile components in ¹H NMR spectra. Characteristic well-resolved marker signals that invariably appear in ¹H NMR spectra of bile were identified for cholesterol (H18 methyl signal at 0.643 ppm), lipids (glycerol CH signal at 5.064 ppm), total bile acids (H18 signals in the range 0.520–0.626 ppm), total glycine conjugated bile acids (NH signal at 6.958 ppm), total taurine conjugated bile acids (NH signal at 7.646 ppm), and urea (NH₂ signal near 5.48 ppm), which enabled their rapid and accurate analysis. Excellent linearity and precision of quantitative analysis was observed for all the identified bile components (R ²>0.99 for all). The method was demonstrated on gallbladder bile from 19 patients with gallbladder diseases. Urea in bile was identified by NMR for the first time and its quantitative analysis, along with several other bile components, is presented. The majority of the bile components could be analyzed in a single step. Accurate and rapid quantification of several bile components noninvasively by using the method presented herein may have far-reaching implications in the study of bile acid metabolism and pathophysiology of various hepatobiliary and gastrointestinal diseases.     

Application of high-resolution, two-dimensional 1H and 13C nuclear magnetic resonance techniques to the characterization of lipid oxidation products in autoxidized linoleoyl/linolenoylglycerols

Subjection of polyunsaturated fatty acid (PUFA)-rich culinary oils to standard frying episodes generates a range of lipid oxidation products (LOP), including saturated and α,β-unsaturated aldehydes which arise from the thermally induced fragmentation of conjugated hydroperoxydiene precursors. Since such LOP are damaging to human health, we have employed high-resolution, two-dimensional ¹H-¹H relayed coherence transfer, ¹H-¹H total correlation, ¹H-¹³C heteronuclear multiple quantum correlation, and ¹H-¹H J-resolved nuclear magnetic resonance (NMR) spectroscopic techniques to further elucidate the molecular structures of these components present in (i) a model linoleoylglycerol compound (1,3-dilinolein) allowed to autoxidize at ambient temperature and (ii) PUFA-rich culinary oils subjected to repeated frying episodes. The above techniques readily facilitate the resolution of selected vinylic and aldehydic resonances of LOP which appear as complex overlapping patterns in conventional one-dimensional spectra, particularly when employed in combination with solvent-induced spectral shift modifications. Hence, much useful multi-component information regarding the identity and/or classification of glycerol-bound conjugated hydroperoxydiene and hydroxydiene adducts, and saturated and α,β-unsaturated aldehydes, present in autoxidized PUFA matrices is provided by these NMR methods. Such molecular information is of much value to researchers investigating the deleterious health effects of LOP available in the diet.     

Analysis of conjugated octadecatrienoic acids inmomordica balsamina seed oil by GLC and13C NMR Spectroscopy

Separation of conjugated octadecatrienoic acids by open tubular gas liquid chromatography (GLC) was performed using glass capillary columns coated with Carbowax 20 M and with OV-1. The equivalent chain length of geometrical isomers of the conjugated octadecatrienoic acids belonging to the two series C_18:3Δ^8.10.12 and C_18:3Δ^9.11.13 were determined. The application of these results to the study of theMomordica balsamina seed oil shows that this oil contains two conjugated octadecatrienoic fatty acids in appreciable amounts, punicic acid (50%) and α-eleostearic acid (13%). The isomerization of conjugated acids inM. balsamina seed oil was followed for one year. Quantitation of octadecatrienoic acids using GLC gave results similar to those obtained with¹³C NMR.     

Synthesis and characterization of poly(aspartic acid) derivatives conjugated with various amino acids

Novel derivatives of poly(aspartic acid) conjugated with various amino acids and their amphiphilic copolymers were synthesized and characterized. Methyl esters of various amino acids (in their hydrochloride form) were synthesized from the reaction of amino acids with methanol in the presence of chlorotrimethylsilane (TMSCl). Aminolysis reaction onto polysuccinimide (PSI) using various amino acid methyl esters in the presence of catalyst and the followed hydrolysis provided the corresponding amino acid-conjugated poly(aspartic acid) derivatives in high reaction yield. Amino acid—conjugated amphiphilic analogs were also prepared by introducing hydrophobic alkylamine along with amino acid using a similar procedure. The chemical structures of copolymers were confirmed by FT-IR and ¹H NMR spectroscopy. The physicochemical properties of amphiphilic copolymers were characterized using dynamic light scattering (DLS), fluorescence spectroscopy and field emission scanning electron microscopy (FE-SEM). In addition, the in vitro cell viability of the copolymers was examined. These polymers have potential applications in the pharmaceutical and cosmetic fields as delivery vehicles for bioactive molecules.     

The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9–10³H₂] or [1¹⁴C] oleate, [1¹⁴C] linoleate or [9–10³H₂] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids. These data suggest that monounsaturated molecular species of biliary phospholipids are more resistant than the diunsaturated ones to in vitro hydrolysis by phospholipase A₂. Ninety min after introduction of the radioactive bile into the upper part of the rat duodenum, high labeling of luminal phospholipids was observed regardless of the bile sample used, although labeling of free fatty acids was always low. The passage of intact biliary phospholipids through the intestinal epithelium is discussed.     

Analysis of the seed oil of Heisteria silvanii (Olacaceae)—A rich source of a novel C18 acetylenic fatty acid

Besides some usual fatty acids (FA), two conjugated ene-yne acetylenic FA [trans-10-heptadecen-8-ynoic acid (pyrulic acid) (7.4%), and trans-11-octadecen-9-ynoic acid (ximenynic acid) (3.5%)], a novel ene-yne-ene acetylenic FA [cis-7, trans-11-octadecadiene-9-ynoic acid (heisteric acid) (22.6%)], and 9,10-epoxystearic acid (0.6%) could be identified in the seed oil of Heisteria silvanii (Olacaceae). Two further conjugated acetylenic FA [9,11-octadecadiynoic acid (0.1%) and 13-octadecene-9,11-diynoic acid (0.4%)] were identified tentatively by their mass spectra. The FA mixture has been analyzed by gas chromatography/mass spectrometry (GC/MS) of their methyl ester and 4,4-dimethyloxazoline derivatives. The structure of heisteric acid was elucidated after isolation via preparative silver ion thin-layer chromatography and by various spectroscopic methods [ultraviolet; infrared; ¹H, ¹³C nuclear magnetic resonance (NMR); ¹H−¹H and ¹H−¹³C correlation spectroscopy]. To determine the position of the conjugated ene-yne-ene system, the NMR spectra were also measured after addition of the lanthanide shift reagent Resolve-Al EuFOD^TM. Furthermore, the triyglyceride mixture was analyzed by high-temperature GC and high-temperature GC coupled with negative chemical ionization MS. A glass capillary column coated with a methoxy-terminated 50%-diphenyl-50%-dimethylpolysiloxane was used for the separation of the triacylglycerol (TAG) species. No evidence of decomposition of the TAG species containing conjugated ene-yne-ene FA was observed. Twenty-six species of the separated TAG were identified by means of their abundant quasi molecular ion [M−H]⁻ and their corresponding carboxylate anions [RCOO]⁻ of the fatty acids, respectively. The major molecular species of the TAG were found to be 16:0/18:1/18:1, 16:0/18:1/18:3 (heisteric acid), 17:2 (pyrulic acid)/18:1/18:1, 18:1/18:1/18:3 (heisteric acid). The TAG containing acetylenic FA showed an unexpected increase of the retention time in comparison to the TAG containing usual FA, thus making the prediction of the elution order of lipid samples containing acetylenic FA difficult.     

Bile Acids Conjugation in Human Bile Is Not Random: New Insights from <Superscript>1</Superscript>H-NMR Spectroscopy at 800 MHz

Bile acids constitute a group of structurally closely related molecules and represent the most abundant constituents of human bile. Investigations of bile acids have garnered increased interest owing to their recently discovered additional biological functions including their role as signaling molecules that govern glucose, fat and energy metabolism. Recent NMR methodological developments have enabled single-step analysis of several highly abundant and common glycine- and taurine- conjugated bile acids, such as glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid. Investigation of these conjugated bile acids in human bile employing high field (800 MHz) ¹H-NMR spectroscopy reveals that the ratios between two glycine-conjugated bile acids and their taurine counterparts correlate positively (R ² = 0.83–0.97; p = 0.001 × 10⁻²–0.006 × 10⁻⁷) as do the ratios between a glycine-conjugated bile acid and its taurine counterpart (R ² = 0.92–0.95; p = 0.004 × 10⁻³–0.002 × 10⁻¹⁰). Using such correlations, concentration of individual bile acids in each sample could be predicted in good agreement with the experimentally determined values. These insights into the pattern of bile acid conjugation 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 disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.     

Detection of [U-13C]eicosapentaenoic acid in rat liver lipids using13C nuclear magnetic resonance spectroscopy

Fatty acid carbons are well-resolved in¹³C nuclear magnetic resonance (NMR) spectra of lipid extracts, but application of this methodology to the metabolism of¹³C-labelled fatty acids has not yet been reported. In the present study,¹³C NMR was used to monitor the presence of 98% [U-¹³C]eicosapentaenoic acid (EPA) in liver and carcass lipids 24 h after it had been injected into the stomach of a rat. Natural abundance¹³C NMR spectra of liver total fatty acid extracts were obtained from four control rats for comparison. At 24 h post-injection, quantitative high resolution¹³C NMR showed¹³C enrichment in liver fatty acid extracts was present mainly at olefinic and at the n−1 to n−4 carbons, but¹³C signal intensities for C−1 to C−4 of [U-¹³C]EPA were markedly reduced or absent. Small¹³C resonances, possibly indicative of some¹³C incorporation into docosahexaenoic acid and saturated or monounsaturated fatty acids, were present in spectra of liver fatty acids. Liver and carcass fatty acid composition was similar in both the controls and the EPA-injected rat, suggesting little accumulation of the injected [U-¹³C]EPA after 24 h. We conclude that the carbon-specific data provided by¹³C NMR of lipid extracts may be useful in monitoring the fate of individual carbons during tracer studies using¹³C-labelled fatty acids.     

Minor components ofLesquerella fendleri seed oil

Routine analysis of fatty ester fractions ofLesquerella fendleri oil suggested the presence of epoxy compounds and other minor components. By a combination of open silica column and high performance liquid chromatography (HPLC) fractionations of the methyl esters prepared from the oil, these constitutents were isolated and then characterized by thin-layer chromatography-mass spectrometry (GC-MS—electron ionization, EI, and chemical ionization, CI) and nuclear magnetic resonance (NMR—¹H and¹³C). Three epoxy acids, 15,16-epoxy-9,12-octadecadienoic, 9,10-epoxy-12-octadecenoic and 9,10-epoxy-octadecanoic, were found. Hydroxy acids present included a C-22 homologue of lesquerolic acid (16-hydroxy-12-docosenoic acid) and 14,15-dihydroxy-tricosanoic acid. Other minor ocmponents included four sterols, brassicasterol, campesterol, β-sitosterol and stigmasterol, and a series of saturated and unsaturated fatty acids up to C30. Visiting postdoctoral scientist sponsored by the government of India.     

Proof of ether-bridged condensation products in UF resins by 2D NMR spectroscopy

The existence of ether-bridged condensation products in urea-formaldehyde (UF) resins is still disputed in the literature as these products have never been isolated or fully characterized. Using ¹H-¹⁵N gradient heteronuclear single quantum correlation (gHSQC) experiment, ¹H-¹³C gHSQC experiment and ¹H-¹³C gradient heteronuclear multiple bond correlation (gHMBC) experiment a, methylolurea hemiformal compound (urea compound with oligomeric chains $-\rm{CH}_{2}\;{\rm O[CH}_{2}{\rm O]}{\mathrm {n}}{\rm H}$ and $n\geq 1$ ) and a symmetrical compound, most likely an ether-bridged condensation product, in a UF resin sample were characterized. The results were confirmed by 2D NMR ¹³C-¹³C gradient correlated spectroscopy (gCOSY) experiment using ¹³C labeled ureaformaldehyde samples. Spectroscopic chemical shifts of the proposed ether-bridged condensation product in ¹⁵N, ¹³C, ¹H NMR spectroscopy were assigned. Furthermore, individual peak assignments are provided for the methylolurea hemiformal moiety.     

Stimulation of bile acid synthesis by dibutyryl cyclic AMP in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to examine the effects of dibutyryl cyclic AMP on the incorporation of¹⁴C-acetate and¹⁴C-cholesterol into bile acids. After an initial lag period, both precursors were incorporated into cholic and chenodeoxycholic acids at a linear rate for the subsequent 60 min. An apparent stimulation of bile acid formation from¹⁴C-acetate by dibutyryl cyclic AMP was complicated by the concomitant inhibition of cholesterol synthesis. In experiments with¹⁴C-cholesterol, dibutyryl cyclic AMP (1 mM) increased the labeled cholic and chenodeoxycholic acids in the medium by 83 and 224%, respectively, but cellular levels of labeled bile acids were unchanged. As a result, the nucleotide stimulated the overall incorporation of¹⁴C-cholesterol into cholic acid by 39% and into chenodeoxycholic acid by 123%. The mean ratio of labeled cholic to chenodeoxycholic acid declined from 55∶45 in control cells to 41∶59 in cells incubated with dibutyryl cyclic AMP. The results demonstrate that label incorporation can be used to study the regulation of bile acid synthesis in isolated hepatocytes. We propose that dibutyryl cyclic AMP enhances bile acid production by phosphorylating, and thus stimulating the activity of, cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis.     

Is there an entero-hepatic circulation of the bile phospholipids?

Bile previously labeled with tritiated oleic acid (the main radioactivity was on bile phospholipids) was mixed with pure isolated phospholipids previously labeled with¹⁴C oleic acid; this mixture was perfused during 6 or 23 hr into the duodenum of test rats bearing a bile fistula. At the time of decapitation, in the small intestine a large hydrolysis of the¹⁴C phospholipids was found. In contrast no bile phospholipid hydrolysis was observed. In the collected bile samples of the test rats, no¹⁴C could be detected (this means a very large decrease of the¹⁴C fatty acids specific activities by the body fatty acids), and the tritiated fatty acids specific activities were only 2.5–12 times lower than in the perfused bile. These results can be explained, assuming that the bile phospholipids enter in an entero-hepatic circulation and are preserved from the dilution in a large pool of lipids.     

Quantitative high-resolution13C and1H nuclear magnetic resonance of ω3 fatty acids from white muscle of atlantic salmon (Salmo salar)

High-resolution¹³C nuclear magnetic resonance (NMR) spectra have been obtained and used to define the ω3 (n-3) fatty acid distribution in lipid extract and white muscle from Atlantic salmon (Salmo salar). The¹³C spectrum of lipid extracted from muscle gives quantitative information about the individual n-3 fatty acids, 18:2n-6, 20:1/22:1 and groups of fatty acids. The quantitative data compare favorably with those obtained by gas-liquid chromatography. The¹H NMR spectrum of the lipid extract gives information about the amount of 22:6n-3 and the total content of n-3 fatty acids. The¹³C NMR technique also revealed the positional distribution (1,3- and 2-acyl) of the important 20:5n-3 and 22:6n-3 acids in the triacylglycerol molecules. In the quantitative¹³C NMR spectrum of white muscle, the methyl region of the acyl chains of triacylglycerols gave rise to sufficiently resolved signals to permit estimation of the total concentration of lipids and the n-3 fatty acid content. The NMR data are in good agreement with corresponding data obtained by traditional methods.     

Simplification and assignment of proton and two-dimensional hetero-correlated NMR spectra of petroleum fractions using gradient selected editing pulse sequences

In this paper, gradient selected (gs) NMR experiments are presented for editing the proton, and two-dimensional (2D) ¹H-¹³C-correlation spectra of different fractions of the petroleum according to the carbon multiplicity. The state of the art experiments allow the much sought after simplification and resolution of the proton, and 2D ¹H-¹³C correlated NMR spectra of such fractions in considerably reduced acquisition times. The proton-edited experiments offer a new and much convenient way of unambiguous estimation of cut-off points between signals from α-methyl and α-methylene protons in ¹H NMR spectra of petroleum fractions. The gs edited 2D ¹H-¹³C correlated spectra of the fractions provide carbon multiplicity information and hetero-nuclear correlation with improved sensitivity in a single experiment leading to complete spectral assignment. These experiments have been applied to complex petroleum fractions for the first time, and show lot of potential to resolve various hitherto unanswered issues concerning the interpretation of the complex NMR spectra of the petroleum products.     

[3-13C] γ-linolenic acid: A new probe for 13C nuclear magnetic resonance studies of arachidonic acid synthesis in the suckling rat

Our objective was to develop a suitable probe to study metabolism of polyunsaturated fatty acids by ¹³C nuclear magnetic resonance (NMR) in the suckling rat pup. [3-¹³C] γ-Linolenic acid was chemically synthesized, and a 20 mg (Experiment 1) or 5 mg (Experiment 2) dose was injected into the stomachs of 6–10-day-old suckling rat pups that were then killed over a 192 h (8 d) time course. ¹³C NMR showed that ¹³C in γ-linolenate peaked in liver total lipids by 12-h post-dosing and that [5-¹³C]-arachidonic acid peaked in both brain and liver total lipids 48–96 h post-dosing. ¹³C enrichment in brain γ-linolenic acid was not detected by NMR, but gas chromatography-combustion-isotope ratio mass spectrometry showed that its mass enrichment in brain phospholipids at 48–96 h post-dosing was 1–2% of that in brain arachidonic acid. ¹³C was present in liver and brain cholesterol and in perchloric acid-extractable water-soluble metabolites in the brain, liver and carcass. We conclude that low but measurable amounts of exogenous γ-linolenic acid do access the suckling rat brain in vivo. The slow time course of [5-¹³C] arachidonic acid appearance in the brain suggests most of it was probably transported there after synthesis elsewhere, probably in the liver. Some carbon from γ-linolenic acid is also incorporated into lipid products other than n−6 long-chain polyunsaturated fatty acids.     

Ion-pair high-performance liquid chromatography of bile salt conjugates: Application to pig bile

The high-performance liquid chromatographic separation and quantitation of conjugated bile salts from pig bile is reported. Synthetic standards and bile samples were chromatographed on a C₁₈ reversed phase column using acetonitrile/water/tetrabutyl ammonium phosphate as an isocratic mobile phase at a flow rate of 1 mL/min. Detection of the ion-pairs was at 214 nm. The method permits efficient separation of all conjugated pig biliary bile salts without prior modification or treatment of the samples. Analysis of 12 pig biles showed that 85% of the bile salts are conjugated to glycine. The three main conjugated bile salts were glyco-3α,6α,7α-trihydroxy-5β-cholanoic acid (GHC), glyco-3α,7α-dihydroxy-5β-cholanoic acid (GCDC), and glyco-3α,6α-dihydroxy-5β-cholanoic acid (GHDC). Glyco-3α-hydroxy-6-oxo-5β-cholanoic acid (G3α6oxo), tauro-3α,7α-dihydroxy-5β-cholanoic acid (TCDC), tauro-3α,6α,7α-trihydroxy-5β-cholanoic acid (THC), and tauro-3α,6α-dihydroxy-5β-cholanoic acid (THDC) were found to contribute each for 4 to 5% ot the total. An excellent correlation was found between the sum of conjugated bile salts quantitated by high-performance liquid chromatography (HPLC) and values obtained by conventional enzymatic assay. Simplicity, efficiency and relative rapidity of the method render it suitable for routine analyses.     

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α‐tocopherol. The existence of hydroperoxide protons was confirmed by D₂O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH₄. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The ¹³C and ¹HNMR spectra of a mixture of 9‐hydroper‐oxy‐10‐trans,12‐cis‐octadecadienoic acid methyl ester (9‐OOH) and 13‐hydroperoxy‐9‐cis, 11‐trans‐octadecadienoic acid methyl ester (13‐OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The ¹³CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The ¹³C and ¹H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α‐tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.