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.     

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.     

Linseed oil and mixture with maleic anhydride: <Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR

It was shown that ¹H NMR allowed a rapid determination of the ratio of the linolenic residues over all the others (linoleic + oleic + saturated) and ¹³C NMR allowed a rapid determination of the ratio of linolenic over (linoleic + oleic) residues as well as the linoleic/oleic ratio in a few minutes on less than 20 mg of crude LO. After thermal treatment (220°C for 2 h, followed by cooling to room temperature), a 1∶1 mixture of linseed oil (LO) and maleic anhydride (MA), which was a suspension, became limpid and remained limpid. Moreover, the viscosity of the mixture was higher than that of pure LO treated in the same way. It was shown by ¹³C NMR and quantitative recovery of the constituents (LO and MA) through solvent separation that no reaction occurred between LO and MA during this thermal treatment. This result is in accord with DSC analysis of such a 1∶1 LO/MA mixture that exhibited an exothermic effect too small (about 34 kcal/mol) to correspond to formation of a C−C bond.     

<Superscript>1</Superscript>H- and <Superscript>13</Superscript>C-NMR Characterization of the Molecular Components of the Lipid Fraction of Pecorino Sardo Cheese

In this work the molecular fatty components of Pecorino Sardo Protected Designation of Origin (PS PDO) cheese were characterized through an exhaustive investigation of the ¹H- and ¹³C-NMR spectra of the extracted lipids. Several fatty acids (FA), such as long chain saturated, oleic, linoleic, linolenic, butyric, capric, caprylic, caproic, trans vaccenic, conjugated linoleic acid (cis9, trans11–18:2), and caproleic (9–10:1) were unambiguously detected. The positional isomery of some acyl groups in the glycerol backbone of triacylglycerols (TAG) was assessed. Furthermore, the NMR signals belonging to sn-1,2/2,3, sn-1,3 diacylglycerols (DAG), and free fatty acids (FFA) were analysed as a measure of lipolytic processes on cheese. Lastly, ¹H-NMR resonances of saturated aldehydes and hydroperoxides were detected, their very low intensity indicating that the lipid oxidation process can be considered to be of minor relevance in Pecorino Sardo cheese.     

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.     

Novel ceramides and a new glucoceramide from the roots of <Emphasis Type="Italic">Incarvillea arguta</Emphasis>

Novel ceramides, rel-(3S,4S,5S)-3-[(2R)-2-hydroxycosanoyl-hexacosanoylamino]-4-hydroxy-5-[(4Z)-tetradecane-4-ene]-2,3,4,5-tetrahydrofuran (1a-g), and a new glucoceramide, 1-O-β-d-glucopyranosyl-(2S,3S,4R,8E)-2-[(2R)-2-hydroxytetracosanoylamino]-1,3,4-octodecanetriol-8-ene (2) were isolated from the aqueous ethanolic extract of the roots of Incarvillea arguta, together with eight known compounds: β-sitosterol (3), oleanolic acid (4), ursolic acid (5), piperin (6), maslinic acid (7), β-sitosterol 6′-O-acyl-β-d-glucopyranoside (8), 8-epideoxyloganic acid (9), and plantarenaloside (10). Their structures were elucidated on the basis of spectral data including IR, MS, NMR [¹H NMR, ¹³C NMR (distortionless enhancement by polarization transfer), ¹H−¹H COSY, heteronuclear multiplequantum coherence, and heteronuclear multiple-bond coherence correlations]. The relative configurations were established by nuclear Overhauser effect spectroscopy experiments and by comparison of the NMR spectral data and coupling constants with those already reported in the literature.     

Acyl Migration Kinetics of 2-Monoacylglycerols from Soybean Oil via <Superscript>1</Superscript>H NMR

The acyl migration kinetics of neat 2-monoacylglycerol (2-MAG) to form 1-MAG was determined using ¹H NMR spectroscopy to monitor the β-proton integration ratios of the two species over time. 2-MAG was synthesized by the Novozym 435-catalyzed alcoholysis of soybean oil and isolated by solvent extraction or molecular distillation at a mole fraction (X _2-MAG) of 0.94 relative to total MAG. The kinetics parameters of the neat 2-MAG acyl migration were investigated over the temperature range of 23–80 °C. The 2-MAG mol fraction remained unchanged at 23 °C over the course of 168 h and reached an equilibrium of X _2-MAG = 0.09 at only 80 °C. Modeling of the kinetics data revealed a 2-MAG half life (t _1/2) of 3,500 and 22.8 h at 23 and 80 °C, respectively, with an activation energy of 79.0 ± 6.5 kJ mol⁻¹. The use of ¹H NMR spectroscopy proved an expedient method for monitoring the acyl migration in 2-MAG compared to other reported methods (e.g. GC, HPLC, and ¹³C-NMR spectroscopy), requiring no sample manipulation and minimizing the deleterious effects of high temperatures and solvent exposure. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

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.     

Characterization of sacha inchi (<Emphasis Type="Italic">Plukenetia volubilis</Emphasis> L.) oil by FTIR spectroscopy and <Superscript>1</Superscript>H NMR. Comparison with linseed oil

Three oil samples obtained from sacha inchi (Plukenetia volubilis L.) seeds were studied by means of FTIR and ¹H NMR. Frequency data of the most significant bands of the IR spectrum of this oil are given. These data show that sacha inchi oil has a high degree of unsaturation. The same fact is deduced from the ratio between the absorbance of the bands due to the stretching vibrations of the cis olefinic CH double bonds at 3010.5 cm⁻¹ and to the methylene symmetrical stretching vibrations at 2855.1 cm⁻¹. The proportions of monounsaturated, polyunsaturated, and saturated acyl groups were predicted from the frequency of some IR bands, and these were in satisfactory agreement with the values obtained through FAME generation and their quantification by GC. Likewise, simple observation of the ¹H NMR spectra provided a great deal of information about sacha inchi oil, with regard not only to the relative proportions of the different acyl groups but also to their nature. Thus, the presence of γ-linolenic acyl groups was discounted. Furthermore, the area of some ¹H NMR signals was used to determine the proportion of saturated and mono-, di-, and triunsaturated acyl groups, which also were in satisfactory agreement with the values obtained by classical methods. IR and ¹H NMR determinations take very little time in comparison with classical methods and do not require chemical manipulation or transformation of the sample. A comparison was also made between the compositions of sacha inchi and linseed oil. Both oils are important sources of the healthful n−3 linolenic acyl groups, and sacha inchi also contains high proportions of the n−6 linoleic acyl groups.     

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.     

Conversion of linoleic acid into novel oxylipins by the mushroom <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Oxylipins are associated with important processes of the fungal life cycle, such as spore formation. Here, we report the formation of FA metabolites in Agaricus bisporus. Incubation of a crude extract of lamellae with linoleic acid (18∶2) led to the extensive formation of two oxylipins. They were identified as 8(R)-hydroxy-9Z,12Z-octadecadienoic acid (8-HOD) and 8(R),11(S)-dihydroxy-9Z,12Z-octadecadienoic acid (8,11-diHOD) by using RP-HPLC, GC-MS, IR, GC-MS analysis of diastereomeric derivatives, and ¹H NMR and ¹³C NMR spectroscopy. Neither compound has been reported before in A. bisporus. Oleic (18∶1), α-linolenic (18∶3n−3), and γ-linolenic (18∶3n−6) acids were converted into their 8-hydroxy derivatives as well, and 18∶3n−3 was further metabolized to its 8,11-diol derivative. Reactions with [U-¹³C]18∶2 demonstrated that the compounds 8-HOD and 8,11-diHOD were formed from exogenously supplied 18∶2. When [U-¹³C]8-HOD was supplied, it was not converted into 8,11-diHOD, indicating that it was not an intermediate in the formation of 8,11-diHOD. When a crude extract of A. bisporus was incubated under an atmosphere of ¹⁶O₂/¹⁸O₂, the two hydroxyl groups of 8,11-diHOD contained either two ¹⁸O atoms or two ¹⁶O atoms. Species that contained one of each isotope could not be detected. We propose that the formation of the 8,11-dihydroxy compounds occurs through either an 8,11-endoperoxy, an 8-peroxo free radical, or an 8-hydroperoxy intermediate. In the latter case, the reaction should be catalyzed by dioxygenase with novel specificity.     

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.     

<Superscript>29</Superscript>Si NMR Chemical Shift Tensor and Electronic Structure of 7-Silanorbornadienes

The structure and bonding of 7-silanorbornadienes was investigated using X-ray Diffraction (XRD), solid-state NMR spectroscopy and density functional calculations. The solid state structures of four benzo-7-silanorbornadienes (4a, c, d, e) and of one dibenzo-7-silabenzonorbornadiene (5a) are reported and compared with the results of previous structural investigations. The most prominent features of the molecular structures of all 7-silanorbornadienes are very long Si-C(bridgehead) bonds (d(SiC) = 190.6–196.8 pm) and very acute CSiC bond angles α (α(CSiC) = 78.7–83.9°). All newly investigated 7-silanorborndienes show for tetracoordinated silicon nuclei extremely deshielded ²⁹Si NMR resonances (δ²⁹Si = 65.6–31.6). Solid State NMR investigations for 7-silanorbornadienes anti-4a, b reveal highly anisotropic chemical shift tensors of axial or nearly axial symmetry (4a: δ₁₁ = 161, δ₂₂ = δ₃₃ = −11; 4b: δ₁₁ = 113, δ₂₂ = 14, δ₃₃ = −15). The dominating, strongly deshielding δ₁₁ component is oriented almost perpendicular to the plane spanned by the two bridgehead carbon atoms and the bridging silicon atom. The DFT calculations suggest that the origin of the strong deshielding is a small energy difference between the frontier orbitals, which are strongly localized at the silicon atom. In addition the computations reveal that both the long SiC bonds and the strongly deshielded ²⁹Si NMR chemical shift are direct consequences of the bonding situation in 7-silanorbornadienes which are characterized by through space interaction of the C=C double bonds and by hyperconjugation between the SiC σ-bonds and the unoccupied orbitals of the C=C double bonds.     

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.     

Use of a <Superscript>13</Superscript>C tracer to investigate lutein as a ligand for plasma transthyretin in humans

The selective accumulation of lutein in the macula of the human retina is likely to be mediated by specific transport and/or binding proteins. Our objective was to determine whether transthyretin (TTR) is a plasma transport protein for lutein. We used a biosynthetic ¹³C-lutein tracer and GC-combustion interfaced-isotope ratio MS to gain the requisite sensitivity to detect the minute amounts of lutein expected as a physiological ligand for TTR. Subjects (n=4) each ingested 1 mg of ¹³C-lutein daily for 3 d and donated blood 24 h after the final dose. For three subjects, the plasma TTR-retinol-binding protein (RBP) complex was partially purified by anion-exchange (diethylaminoethyl, DEAE) chromatography and then dissociated by hydrophobic-interaction chromatography to yield the TTR component. For subject 4, the initial DEAE purification step was omitted and total plsma TTR (RBP-bound and free) was isolated by hydrophobic-interaction chromatography. In each case, the crude TTR fractions were then purified to homogeneity by RBP-sepharose affinity chromatography. Pure TTR was extracted with chloroform, and unlabeled lutein was added to the extract as a carrier. The mean ¹³C/¹²C ratio (expressed in delta notation, δ¹³C) of the lutein fraction isolated from the plasma TTR extracts of the four subjects was −30.53±3.29‰. The δ¹³C value of the unlabeled lutein carrier was −30.97±0.27‰. Thus, no ¹³C enrichment was detected in association with TTR. We conclude that lutein is not associated with TTR in human plasma after being ingested in physiological amounts.     

Synthesis of poly(propylene-<Emphasis Type="Italic">co</Emphasis>-lactide carbonate) and hydrolysis of the terpolymer

A new aliphatic polycarbonate, terpolymer of carbon dioxide, propylene oxide, and dl-lactide, was synthesized by using a polymer-supported bimetallic complex as a catalyst. The terpolymer prepared was characterized by FT-IR, ¹H NMR, ¹³C NMR, ¹H–¹H COSY, DSC, and WAXD measurements. The influence of molar ratio on the terpolymerization progress was investigated. The results showed that lactide unit was inserted into the backbone of CO₂–PO successfully. Because of the existence of the lactide ester unit, the terpolymers had stronger degradability than poly(propylene carbonate).     

Subsoil <Superscript>15</Superscript>N-N<Subscript>2</Subscript>O Concentrations in a Sandy Soil Profile After Application of <Superscript>15</Superscript>N-fertilizer

Studies on emissions of nitrous oxide (N₂O) from agricultural soils mostly focus on fluxes between the soil and the atmosphere or are limited to the atmosphere in the topsoil. However, in soils with shallow water tables, significant N₂O formation may occur closer to the groundwater. The aims of this study were (i) to determine the importance of subsoil N₂O formation in a sandy soil; and (ii) to obtain a quantitative insight in the contribution of subsoil N₂O to the overall losses of N₂O to the environment. We applied ¹⁵N labeled fertilizer at a rate of 5.22 kg ¹⁵N ha⁻¹; 50% as Ca(NO₃)₂ and 50% as NH₄Cl, on a mesic typic Haplaquod seeded with potatoes (Solanum tuberosum L.), and traced soil N₂O concentrations and fluxes over a one-year period. Throughout the year, total N₂O and the amount of ¹⁵N recovered in soil N₂O were highest in the subsoil, with a maximum concentration at 48 cm depth in mid-February of 19900 μl m⁻³ and 24 μg ¹⁵N m⁻³, respectively. The maximum concentration coincided with the highest water-filled pore space of 71%. The cumulative flux of N₂O was 446 g N₂O-N ha⁻¹, the recovery of ¹⁵N in this flux was 0.06%. During the summer, maximum fluxes followed high soil N₂O concentrations. During winter, no such relation was found. We concluded that the formation of N₂O was the highest in the subsoil, largely controlled by water-filled pore space rather than NO₃⁻ concentration or temperature. Although high subsoil N₂O concentrations did not lead to high surface fluxes of N₂O in the winter, artificial draining may lead to high indirect N₂O emissions through supersaturated drainage water.     

Structural and Activity Investigation into Al<Superscript>3+</Superscript>, La<Superscript>3+</Superscript> and Ce<Superscript>3+</Superscript> Addition to the Phosphomolybdate Heteropolyanion for Isobutane Selective Oxidation

Abstract  

Twelve phosphomolybdate compounds were synthesized via cationic exchange and were of the form: M _x H_3–3x [PMo₁₂O₄₀] (M = Al, La or Ce; 0 ≤ x ≤ 1). These compounds were analyzed by XRD and adsorption isotherm. Aluminum addition causes a primitive cubic phase, while lanthanum and cerium yield body-centered structures. La and Ce addition reduces surface area of phosphomolybdate structure. Temperature-programmed experiments for the selective oxidation of isobutane yielded methacrolein, 3-methyl-2-oxetanone (lactone), acetic acid (not with aluminous compounds), propene (only with aluminous compounds), carbon dioxide and water. The preference for propene rather than acetic acid formation with Al³⁺ may be due to the smaller cation size, or primitive cubic structure. These products form via two distinct reaction processes, labeled categories 1 and 2. Category 1 formation is associated with isobutane forming products on the surface, but reaction rate determined by bulk migration of charged particles. Category 2 formation is concerned with isobutane penetrating deep within the bulk of the substrate and forming products which subsequently desorb in a series of bell-shaped humps. Methacrolein forms via both category 1 and 2, whilst all other products form via category 2 exclusively. Kinetic analysis showed apparent activation barriers for category 1 methacrolein formation range from 67 ± 2 kJ mol⁻¹ to >350 kJ mol⁻¹, and occur in groups with small, medium and large activation barriers. The addition of +3 metal cations to the phosphomolybdate anion increase thermal stability, significantly decreasing deactivation; IR spectroscopy shows that the Keggin structure remains intact during temperature-programmed experiments with the Al, La and Ce salts.     

A 2<Superscript>7-3</Superscript> fractional factorial optimization of polybenzimidazole based membrane electrode assemblies for H<Subscript>2</Subscript>/O<Subscript>2</Subscript> fuel cells

We describe the usefulness of a statistical fractional factorial design to obtain consistent and reproducible behavior of a membrane-electrode-assembly (MEA) based on a phosphoric acid (PA) doped polybenzimidazole (PBI) membrane, which allows a H₂/O₂ fuel cell to operate above 150 °C. Different parameters involved during the MEA fabrication including the catalyst loading, amount of binder, processing conditions like temperature and compaction load and also the amount of carbon in the gas diffusion layers (GDL) have been systematically varied according to a 2^7-3 fractional factorial design and the data thus obtained have been analyzed using Yates’s algorithm. The mean effects estimated in this way suggest the crucial role played by carbon loading in the gas diffusion layer, hot compaction temperature and the binder to catalyst ratio in the catalyst layer for enabling continuous performance. These statistically designed electrodes provide a maximum current density and power density of 1,800 mA cm⁻² and 280 mW cm⁻², respectively, at 160 °C using hydrogen and oxygen under ambient pressure.