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

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.     

<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.     

Reliability of <Superscript>1</Superscript>H NMR Analysis for Assessment of Lipid Oxidation at Frying Temperatures

The reliability of a method using ¹H NMR analysis for assessment of oil oxidation at frying temperatures was examined. During heating and frying at 180 °C, changes of soybean oil signals in the ¹H NMR spectrum including olefinic (5.16–5.30 ppm), bisallylic (2.70–2.88 ppm), and allylic (1.94–2.15 ppm) proton signals relative to glyceride backbone CH₂ (5.30–5.46 ppm) and aliphatic CH₂ (1.05–1.71 ppm) signals showed strong correlations with conventional analytical methods including total polar compounds, polymerized triacylglycerols, and changes of linoleic acid and linolenic acid peaks in gas chromatography. For oils rich in oleic acid, mid‐oleic sunflower oil (NuSun) and high oleic soybean oil, only the olefinic and allylic proton signals are recommended for analysis due to the relatively low intensity of the bisallylic proton signal. Under these heating and frying conditions, signals indicating intermediate oxidation products, hydroperoxides, were not detected while very small signals corresponding to a variety of aldehydes including alkanal, branched alkenal, 2‐alkenal, and aldehydes of conjugated dienes and epoxides were observed. In this study, it was found that the ¹H NMR method is a fast, convenient, and reliable analytical method to determine the oxidation state of frying oil.     

Spectroscopic Characterization of Ozonated Sunflower Oil

Ozonation reactions are very important in vegetable oil chemistry since their ozonation products are involved in antimicrobial effect in therapeutical uses for several microbiological etiology diseases. Information on the spectroscopic characterization of the products generated by ozonolysis of sunflower oil is limited. In the present study ozonized sunflower oil with 650 mmol-equiv/kg of peroxide index is chemically characterized. Ozonation of sunflower oil produced ozonides, aldehydes and hydroperoxides which were identified by ¹H, ¹³C and two-dimensional ¹H Nuclear Magnetic Resonance (NMR). The virgin sunflower oil and ozonized sunflower oil show very similar ¹H NMR spectra except for the resonances at δ = 9.74 and δ = 9.63 ppm that correspond to both triplet from aldehydic protons, δ = 5.6 ppm (olefinic signal from hydroperoxides), and δ = 5.15 ppm (multiplet from ozonides methylic protons). Other resonance assignments are based on the connectivities provided by the proton scalar coupling constants. These are the following: δ = 3.15 ppm (doublet from methylenic group in α position respect to olefinic proton), δ = 2.45 ppm (multiplet from methylenic group allylic to ozonides methynic protons) and δ = 1.62 ppm (multiplet methylenic protons in β position respect to ozonides methynic protons). From the ¹³C NMR and ¹H-¹³C two- dimensional spectrum of the ozonized sunflower oil, the presence of ozonides was confirmed by the signals δ = 103.43 and δ = 103.49 ppm, respectively. The others new signals found in δ = 42.5 and δ = 42.76 ppm confirm the presence of methylenic carbons from hydroperoxides and ozonides. These results indicate that NMR Spectroscopy can provide valuable information about the amount of reaction compounds of ozonized vegetable oil. From the chemical structural elucidation of ozonated sunflower oils, relevant biochemical and chemical information can be achieved.     

Bile acids LVII. Analysis of bile acids by high pressure liquid chromatography and mass spectrometry

Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associates’ “fatty-acid analysis” or a μBondapak/C₁₈ column eluted with a mixture of 2-propanol/potassium phosphate buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5α- and 5β-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or μPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristic of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.     

NMR characterization of dihydrosterculic acid and its methyl ester

Cyclopropane FA occur in nature in the phosphoplipids of bacterial membranes, in oils containing cyclopropene FA, and in Litchi sinensis oil. Dihydrosterculic acid (2-octyl cyclopropaneoctanoic acid) and its methyl ester were selected for ¹H and ¹³C NMR analysis as compounds representative of cyclopropane FA. The 500 MHz ¹H NMR spectra acquired with CDCl₃ as solvent show two individual peaks at −0.30 and 0.60 ppm for the methylene protons of the cyclopropane ring. Assignments were made with the aid of 2D correlations. In accordance with previous literature, the upfield signal is assigned to the cis proton and the downfield signal to the trans proton. This signal of the trans proton is resolved from the peak of the two methine protons of the cyclopropane ring, which is located at 0.68 ppm. The four protons attached to the two methylene carbons α to the cyclopropane ring also show a split signal. Two of these protons, one from each methylene moiety, display a distinct shift at 1.17 ppm, and the signal of the other two protons is observed at 1.40 ppm, within the broad methylene peak. The characteristic peaks in the ¹³C spectra are also assigned.     

Analysis of the conjugated trienoic acid containing oil fromFevillea trilobata by13C nuclear magnetic resonance spectroscopy

Oil ofFevillea trilobata has the composition palmitic acid 31%, stearic acid 12%, oleic acid 11%, linoleic acid 7%,cis,trans,cis 9,11,13-octadecatrienoic acid 30% andcis,trans,trans-9,11,13-octadecatrienoic acid 9%. The oil was analyzed and components identified by¹³C NMR spectroscopy; the composition was also confirmed by conventional methods. Assignment of¹³C NMR signals of conjugated trienoic acids is discussed and it is shown that mixtures of isomeric conjugated acids can be analyzed by this method. NRCC No. 17797.     

Synthesis and Characterization of Estolide Esters Containing Epoxy and Cyclic Carbonate Groups

The unsaturated sites in oleic 2‐ethylhexyl estolide esters (containing 35 % monoenic fatty acids) were converted into epoxide and five‐membered cyclic carbonate groups and the products characterized by Fourier transform infrared spectra (FTIR), ¹H, and ¹³C nuclear magnetic resonance (NMR) spectroscopies. Epoxidation of the alkene bonds was accomplished using performic acid generated in situ from formic acid and hydrogen peroxide. Greater than 90 % alkenes were converted into their corresponding epoxide groups as determined by oxirane values and the epoxide ring structure was confirmed by ¹H and ¹³C NMR. The estolide ester epoxide material was subsequently reacted with supercritical carbon dioxide in the presence of tetrabutylammonium bromide catalyst to produce the corresponding estolide ester containing the cyclic carbonate group. The signals at 1,807 cm^?1 and δ 82 ppm in the FTIR and ¹³C‐NMR spectra, respectively, confirmed the desired cyclic carbonate was produced. The carbonated estolide ester exhibited a dynamic viscosity, at 25 °C, of 172 mPa·s as compared to 155 mPa·s for the estolide ester starting material. The estolide ester structure of these new derivatives was shown to be consistent throughout their synthesis.     

1H NMR Study of Methyl Linoleate Ozonation.

Unsaturated fatty acids are essential components of vegetable oils and cellular membranes and the involved aspect of unsaturated fatty acids ozonation have been widely studied by different authors. In this paper, in vitro ozonolysis of unsaturated fatty acids with addition of water or ethanol has been studied by Proton Nuclear Magnetic Resonance (¹H NMR) at 250?MHz in order to explore the possibility of this technique for the detection of Criegee ozonides in characterizing ozone reaction with these substrates. The ozonolysis of methyl linoleate showed that signal intensities from formed ozonides were increased with ozone concentration increments. However, the signal intensities with addition of water were higher than those in ethanol addition. Signal intensities from olefinic double bonds were found to decrease with the increment in ozonide signals. Thus, a correspondence of the behavior of these signals is observed with a proportional rate reaction between the number of double bonds in the substrate molecule. Signals from aldehyde formation were poorly detected at lower ozone concentration. It was concluded that the evaluation of ozonide and olefinic double bond signals from 250?MHz ¹H NMR can be a useful tool in assessing ozone reaction with biomolecules. The reaction mechanism for the ozone reaction with unsaturated fatty acids in the presence of water or ethanol is analyzed.     

Crystalline Forms of Cellulose in Softwoods and Hardwoods

Resolution-enhanced ¹³C CP/MAS NMR spectra were obtained for woods from 7 different tree species. Ratios of signal strengths at 90.2 and 88.6 ppm were used to estimate relative proportions of Iα and Iβ crystalline forms of cellulose. These ratios were distinctly higher for softwoods than for hardwoods. The NMR results therefore support published X-ray diffraction evidence for differences between crystalline forms of cellulose in softwoods and hardwoods. Resolution of signals at 84.0 and 84.9 ppm provided evidence for a high degree of molecular order on the surfaces of cellulose crystallites in wood.     

Composition of cecal bile acids in ex-germfree mice inoculated with human intestinal bacteria

Germfree (GF) mice were orally inoculated with human fecal suspension or various components of human fecal microbiota. Three weeks after the inoculation, cecal bile acid composition of these mice was examined. More than 80% of total bile acids was deconjugated in the cecal contents of ex-GF mice associated with human fecal dilutions of 10⁻² or 10⁻⁶, or anaerobic growth from a dilution of 10⁻⁶. In these ex-GF mice, deoxycholic acid accounted for about 20% of total bile acids. In the cecal contents of ex-GF mice associated only with clostridia, unconjugated bile acids made up less than 40% of total bile acids, about half of those in other ex-GF groups. However, the percentage of deoxycholic acid in these mice was the same as that in the other groups. These results indicate that dominant anaerobic bacterial combination is efficient for deconjugation of primary bile acids, and that clostridia in the human feces may play an important role in 7α-dehydroxylation of unconjugated primary bile acids in the intestine.     

Nondestructive quantitative determination of docosahexaenoic acid and n−3 fatty acids in fish oils by high-resolution 1H nuclear magnetic resonance spectroscopy

By using a 500 MHz proton nuclear magnetic resonance (¹H NMR) spectrometer we have developed a quantitative method for determining the contents of docosahexaenoic acid (DHA) in fish oils (mg/g), the molar proportions (mol%) of DHA to all other fatty acids composing the fish oils, and the molar proportions of total n-3 fatty acids to all other non-n-3 fatty acids in the fish oils. After examining the suitability of ethylene glycol dimethyl ether (EGDM), methanol, and 1,4-dioxane as internal standards, experimental conditions were optimized by mainly using EGDM as an internal standard. By setting the pulse repetition time at 30 s, five times longer than the longest T ₁ of the ¹H NMR signals of fish oils, good reproductibility of data and analytical times less than 10 min were achieved. The use of the internal standard also allowed us to quantify DHA on a weight basis (mg/g). Verification of the method was carried out in an interlaboratory study between Japan and Norway on bonito, tuna, and salmon oils. The relative errors in the ¹H NMR data between Japan and Norway were 0.57–5.29% for quantification of DHA, 0.7–2.09% for the molar proportion of DHA, and 0.1–1.41% for the molar proportion of total n-3 fatty acids. Good agreement was observed between the ¹H NMR data and those obtained by gas chromatography (GC). The sample preparation before ¹H NMR measurements required only two steps: sample weighing and preparation of an internal standard solution. Based on the high reproducibility, simplicity of the procedure, and clarity of principle, the proposed ¹H NMR method was judged to be a promising alternative to the GC method in quantification of DHA and n-3 fatty acids in fish oils.     

Preparation and physical properties of some C18 unsaturated fatty esters containingL-amino acid residues and methyl esters ofN-stearoylamino acids

A series of five C₁₈ unsaturated fatty esters (1–5) containing anL-amino acid residue (glycine, alanine, valine, leucine, phenylalanine) was prepared from methyl 12-amino-9-cis-octadecenoate and five methylN-stearoyl-amino acid ester derivatives (6–10) from stearoyl chloride and the sameL-amino acids. The infrared analysis of compounds 1–5 showed characteristic absorption bands at 3300 and 1665 cm⁻¹ for the amino and amido functions, while the amido function in compounds 6–10 gave absorption bands at 3300 and 1680 cm⁻¹. The position of the amido group (peptide linkage) in compounds 1–5 was readily determined by mass spectral analysis.¹H nuclear magnetic resonance (NMR) analysis showed a doublet at 7.15 ppm for the amide proton (NHCO) in compounds 1–5, while in compounds 6–10 the amide proton signal appeared at 6.0 ppm. In¹³C NMR, the amido carbonyl resonance appeared at 171–175 ppm. In compounds 1–5 the effect of the amide function on the ethylene carbon (C-9) gave a signal at 124.1–125.3 ppm, while the remaining ethylenic carbon appeared at 131.9–132.5 ppm. The methine carbon (C-12) of the alkenyl chain was shifted to 48.4–49.8 ppm, and the assignment of the various carbon nuclei in the amino acid residue was readily achieved. In the methylN-stearoylamino acid ester derivatives (6–10), the methine carbon adjacent to the amido system and alpha to the carbomethoxy group appeared between 41.2–57.0 ppm depending on the type of alkyl group present in the amino acid moiety. The phenyl system in compounds 5 and 10 was confirmed by the¹³C signals in the 127–136 ppm range and by the proton signals in the 7.0–7.27 ppm region.     

C NMR study of C-enriched single-wall carbon nanotubes synthesized by catalytic decomposition of methane

¹³C NMR spectra and spin-lattice relaxation times were measured for single-wall carbon nanotubes with 99.9 and 50.0% ¹³C enrichments and natural abundance (1.1% ¹³C) prepared by catalytic decomposition of CH₄. The ¹³C isotropic shift is about 116 ppm from tetramethylsilane, being estimated from magic-angle-spinning (MAS) spectra. The value does not depend on the degree of the ¹³C enrichment. The ¹³C MAS NMR spectra show two additional small peaks at 171 and 152 ppm, which are ascribed to carbon species at defects or edges. The line widths of the main isotropic peak in MAS spectra are about 30 ppm, the origin of which is mostly chemical shift dispersion, reflecting a distribution of diameter and helicity. The line width in the ¹³C static spectra originates from chemical shift dispersion, chemical shift anisotropy and dipole–dipole interactions between ¹³C spins as well as between ¹³C and ¹H spins at defects or edges. ¹H NMR spectra confirm the presence of H-containing species. The ¹³C spin-lattice relaxation is dominated presumably by interaction with magnetic impurities.     

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.     

Rapid and Simple Identification and Quantification of Components in Detergent Formulations by Nuclear Magnetic Resonance Spectroscopy

A rapid, inexpensive, and simple high-resolution NMR spectroscopic method is outlined for determining the content of surfactants and other low-molecular-weight organic compounds in detergent formulations. With simple sample preparation, quantitative results can be obtained from an internal standard and/or the method can be used as a fingerprint analysis of the surfactant composition. The NMR sample is prepared by suspending the detergent sample in deuterated acetic acid and thus dissolving surfactants and other organic compounds. Any content of carbonate will be liberated as CO₂, whereas other inorganic materials are removed by centrifugation. From one-dimensional ¹H and two-dimensional HSQC NMR spectra, the surfactant components and low-molecular-weight organic compounds can be identified from reference spectra. Intensities of signature signals in the one-dimensional ¹H NMR spectrum are used for quantification by comparing with an internal standard. Furthermore, it is demonstrated how ³¹P NMR can be used to identify and quantify phosphonate-type chelators.     

Evidence for distinct precursor pools for biliary cholesterol and primary bile acids in cebus and cynomolgus monkeys

The abnormal metabolism and distribution of plasma lipoproteins have been associated with atherosclerosis and gallstones. To better understand the process of cholesterol excretion, a study was designed to determine whether the contribution of lipoprotein free¹⁴C-cholesterol (as LDL or HDL) to biliary cholesterol or primary bile acids differs in two species of nonhuman primates, cebus and cynomolgus monkeys, having opposite plasma LDL/HDL ratios. Since amino acid conjugation might influence bile acid synthesis or secretion, the taurine and glycine conjugates of newly synthesized primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), were measured in the species capable of conjugating with taurine or glycine (cynomolgus). After total bile acid pool washout, monkeys were infused with human LDL or HDL labeled with free¹⁴C-cholesterol, and the specific activities (SA) of biliary cholesterol and primary bile acid conjugates were determined. In both species, regardless of the lipoprotein infused, the SA of biliary cholesterol and CA were greater than those for total bile acids and CDCA, respectively. In cynomolgus, the SA of glycine conjugates was higher for CA than CDCA, while the SA of taurine conjugates was greater for CDCA than CA. Under these conditions, (i) infused lipoprotein free cholesterol (as either LDL or HDL) contributed more to biliary cholesterol than to bile acids and more to CA than to CDCA; (ii) glycine conjugated preferentially with CA rather than CDCA, while taurine was the preferred conjugate for CDCA. Further, whereas the two primary bile acids had similar rates of synthesis and turnover in cynomolgus, basal bile acid synthesis was much greater in cebus and the CDCA turnover appeared disproportionately large.     

Analysis of conjugated bile acids by high performance liquid chromatography and mass spectrometry

Because of the known advantages of coupling high performance liquid chromatography with mass spectrometry (HPLC-MS) in biological fluids, studies on the reversed-phase HPLC-MS system for direct analysis of conjugated bile acids in human bile samples are described. Ten samples of gallbladder bile of apparently healthy subjects were examined. The amounts of each tauro- and glycoconjugated bile acid as trifluoracetate were determined by mass fragmentography. Quantitation of at least 1 ng of each bile acid was possible.     

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.