M. Madalena Caldeira

Sources of hepatic triglyceride accumulation during high-fat feeding in the healthy rat

Hepatic triglyceride (HTG) accumulation from peripheral dietary sources and from endogenous de novo lipogenesis (DNL) was quantified in adult Sprague–Dawley rats by combining in vivo localized 1H MRS measurement of total hepatic lipid with a novel ex vivo 2H NMR analysis of HTG 2H enrichment from 2H‐enriched body water. The methodology for DNL determination needs further validation against standard methodologies. To examine the effect of a high‐fat diet on HTG concentrations and sources, animals (n = 5) were given high‐fat chow for 35 days. HTG accumulation, measured by in vivo 1H MRS, increased significantly after 1 week (3.85 ± 0.60% vs 2.13 ± 0.34% for animals fed on a standard chow diet, P < 0.05) and was maintained until week 5 (3.30 ± 0.60% vs 1.12 ± 0.30%, P < 0.05). Animals fed on a high‐fat diet were glucose intolerant (13.3 ± 1.3 vs 9.4 ± 0.8 mM in animals fed on a standard chow diet, for 60 min glycemia after glucose challenge, P < 0.05). In control animals, DNL accounted for 10.9 ± 1.0% of HTG, whereas in animals given the high‐fat diet, the DNL contribution was significantly reduced to 1.0 ± 0.2% (P < 0.01 relative to controls). In a separate study to determine the response of HTG to weaning from a high‐fat diet, animals with raised HTG (3.33 ± 0.51%) after 7days of a high‐fat diet reverted to basal HTG concentrations (0.76 ± 0.06%) after an additional 7 days of weaning on a standard chow diet. These studies show that, in healthy rats, HTG concentrations are acutely influenced by dietary lipid concentrations. Although the DNL contribution to HTG content is suppressed by a high‐fat diet in adult Sprague–Dawley rats, this effect is insufficient to prevent overall increases in HTG concentrations. Copyright

NMR Derivatives for Quantification of 2H and 13C‐Enrichment of Human Glucuronide from Metabolic Tracers

Quantification of 2H and 13C enrichment distributions in human urinary glucuronide following ingestion of 2H2O and 13C gluconeogenic tracers was achieved by NMR spectroscopy of the 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone and 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivatives. The derivatization process is simple and can be applied to any glucuronide species. The derivatives are highly soluble in acetonitrile and generate well‐resolved and narrow 2H and 13C NMR signals. The 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided resolution of the six glucuronide 13C signals and numerous 13C isotopomer populations through one‐ and two‐bond 13C‐13C‐coupling, while the 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided complete resolution of the 2H NMR signals for the five glucuronide hydrogens. The isopropylidene methyl signals were also resolved and provided an internal 2H enrichment standard following the acetonation of glucuronolactone with deuterated acetone.

NMR Study of uronic acids and their complexation with molybdenum(VI) and tungsten(VI) oxoions

Abstract A multinuclear 1D and 2D NMR study of d -galacturonic and d -glucuronic acids in aqueous solution and their complexation with tungstate and molybdate ions for variable concentration and pH conditions has been undertaken. The acids exist mainly in the pyranose forms, but complexes were detected involving the less stable α- and β-furanose anomers as well as the α-pyranose form. Thus, NMR evidence was gathered for the formation of two 1:2 (metal-ligand) complexes of W(VI) with the furanose forms. These are stronger with d -galacturonic acid and when the β forms are involved. The same was found with Mo(VI), but, in addition, 2:1 complexes also form. In the case of d -galacturonic acid, three such complexes were detected, two involving the α-pyranose form, in an approximately 4 C 1 , and a 1 C 4 conformation, respectively, and the other presumably involving the β-furanose isomer. With d -glucuronic acid, only one such complex could be characterized, involving the α-pyranose isomer in a distorted 1 C 4 conformation. More detailed information on the structure of the various complexes was obtained from 1 H, 13 C, 17 O, 95 Mo, and 183 W NMR data. The 2:1 complexes with the α-pyranose forms, insofar as they involve metal binding to the ring oxygen atom, are considered to play an important role in the oxidation of the acids especially by Mo(VI).

Noninvasive Analysis of Hepatic Glycogen Kinetics Before and After Breakfast with Deuterated Water and Acetaminophen

The contributions of hepatic glycogenolysis to fasting glucose production and direct pathway to hepatic glycogen synthesis were quantified in eight type 1 diabetic patients and nine healthy control subjects by ingestion of 2H2O and acetaminophen before breakfast followed by analysis of urinary water and acetaminophen glucuronide. After overnight fasting, enrichment of glucuronide position 5 relative to body water (G5/body water) was significantly higher in type 1 diabetic patients compared with control subjects, indicating a reduced contribution of glycogenolysis to glucose production (38 ± 3 vs. 46 ± 2%). Following breakfast, G5/body water was significantly higher in type 1 diabetic patients, indicating a smaller direct pathway contribution to glycogen synthesis (47 ± 2 vs. 59 ± 2%). Glucuronide hydrogen 2 enrichment (G2) was equivalent to body water during fasting (G2/body water 0.94 ± 0.03 and 1.02 ± 0.06 for control and type 1 diabetic subjects, respectively) but was significantly lower after breakfast (G2/body water 0.78 ± 0.03 and 0.82 ± 0.05 for control and type 1 diabetic subjects, respectively). The reduced postprandial G2 levels reflect incomplete glucose-6-phosphate–fructose-6-phosphate exchange or glycogen synthesis from dietary galactose. Unlike current measurements of human hepatic glycogen metabolism, the 2H2O/acetaminophen assay does not require specialized on-site clinical equipment or personnel.

Complexes of Mo(VI) with lactic, thiolactic and thiomalic acids studied by NMR spectroscopy

Proton and 13C evidence is presented on the number, stoichiometry, geometry and relative stability of the complexes formed in the systems Mo(VI) + L-lactic acid, Mo(VI) + D,L-thiolactic acid and Mo(VI) + D,L-thiomalic acid in aqueous solutions at pH values in the range 3–8. In particular, 1:2 complexes are always formed and, in addition, complexes of higher metal content are detected in the case of thiolactic and thiomalic acids.

Peroxovanadium(V) complexes of glycolic acid as studied by NMR spectroscopy

Multinuclear ( 1 H, 13 C, 17 O, 51 V) 1D and 2D NMR spectroscopy has been used to characterize the peroxovanadium(V) complexes of glycolic acid in aqueous solution. One 2:2:2 (metal:ligand:peroxo) complex, together with a 1:1:1 and a 2:2:1 species, are found in the pH range 1‐7. The 2:2:2 complex is a monoperoxo (one peroxide unit per vanadium atom) dinuclear species having a V2O3 4 seven-coordinated metal centre. In this structure, the two vanadium atoms are triple bridged, two of those bridges being formed by oxygen atoms of the hydroxyl group of the acid. The 1:1:1 species has a seven-coordinated VO 3 metal centre. Glycolic acid bonds to the vanadium atoms in a bidentate way, through both the carboxylic and the hydroxyl groups. The peroxo groups are bound in the equatorial plane relative to the apical VO and the geometry around each vanadium atom is close to pentagonal bipyramidal. The 2:2:1 complex is similar to the 2:2:2 species, except for one of the vanadium centres, which is now a five-coordinated oxovanadium centre. Three additional complexes are found in very small amounts for some pH and concentration conditions. Further support for the proposal of monoperoxovanadium species is given by UV‐visible spectroscopy results.

MULTINUCLEAR NMR STUDY OF THE COMPLEXATION OF D-GLUCARIC ACID WITH MOLYBDENUM(VI) AND TUNGSTEN(VI)

Abstract Proton and 13 C NMR spectra of aqueous solutions of sodium molybdate and D -glucaric acid for variable molar ratios and pH values (range 1–9) clearly show the existence of five complexes dominating for specific concentration and pH conditions: a 2:1 metal to ligand complex, a , dominant at low pH (pH ≈ 2) for dilute solutions; two other complexes, b and c , which seem to have 2:2 and 1:1 composition, respectively, and which dominate at low pH for higher concentrations; another 2:1 complex, d , formed at intermediate pH (pH ≈ 4.5–6); still another 2:1 complex, e , formed at higher pH (pH ≈ 6–8.5). From the 1 H and 13 C chemical shifts observed on complexation, the binding sites of D -glucaric acid to the metal are established. From the proton-proton coupling constants the approximate conformation of the bound ligand is determined. The structures proposed on this basis are partially supported by 95 Mo chemical shifts, and the structure changes for the 2:1 complexation with pH are rationalized. Similar results are expected with W(VI) but due to close chemical shifts and exchange phenomena, only a 2:1 complex at low pH is adequately characterized.

MULTINUCLEAR NMR STUDY OF COMPLEXATION OF D-GALACTARIC AND D-MANNARIC ACIDS WITH TUNGSTEN(VI) OXOIONS

Abstract The coordination compounds formed between W(VI) and D-galactaric and D-mannaric acids, in aqueous solution, have been studied by 1H, 13C, 17O and 183W NMR spectroscopy. In the pH range 3–8 for D-galactaric acid and 2–10 for D-mannaric acid, the acids are found to form n:n species (mainly 2:2) with tungstate, in which the ligands are bound to the metal by the two carboxylate groups and their adjacent OH groups. Above pH 6.5, a 2:1 species is also formed, in which all the OH functions are coordinated to the metal, the two carboxylate groups remaining free. The formation of symmetrical or asymmetrical species is discussed, taking into account the configuration of the ligands. Structures for the various complexes are formulated.

NMR spectroscopy study of the peroxovanadium(V) complexes of L-malic acid

Abstract Continuing the solution speciation studies of peroxovanadium(V) complexes with α-hydroxycarboxylic acids, we now report on the complexes formed with l -malic acid, studied by multinuclear (1H, 13C, 51V) 1D and 2D NMR spectroscopy. The system V(V)– l -malic acid–H2O2 in aqueous solution was found to involve a large number of species: nine major peroxovanadium(V) complexes in the pH range 2–7, the structures of which have been deduced, and eight weaker complexes in very low concentrations. The former are monoperoxo species with the stoichiometries (metal:acid:peroxide) 2:2:2 (two complexes), 2:2:1 (two complexes), 2:1:1 (two complexes) and 1:1:1 (three complexes). The 2:2:2 complexes have seven-coordinated metal centres which are doubly bridged by the hydroxyl oxygen atom of malic acid. In one case, all three functional groups of malic acid are bound to each of the two VO3+ metal centres, whereas in the other the C4 carboxylic group of the ligand is dangling, two possibilities being advanced for the vanadium centres: either one V2O34+ centre or two VO3+ centres. The two complexes of stoichiometry 2:2:1 are the result of degradation (loss of one peroxide unit) of the 2:2:2 species in which l -malic acid is a bidentate ligand; they have seven- and five-coordinated metal centres, respectively, the peroxo and the oxo centres, and the malate groups are equally bidentate. The two complexes of stoichiometry 2:1:1 have presumably V2O42+ metal centres and all three functional groups of l -malic acid are involved in chelation; isomerism is interpreted as due to which of the two non-equivalent V(V) atoms is the peroxo centre. The three 1:1:1 complexes have either seven- or six-coordinated VO3+ metal centres; in one of the complexes, l -malic acid acts as a tridentate ligand, whereas in the other two, the ligand is bidentate.

Multinuclear NMR study of the complexation of D-galactaric and D-mannaric acids with molybdenum(VI)

The coordination compounds formed between MoVI and d-galactaric and d-mannaric acids, in aqueous solution, have been studied by 1H, 13C, 17O and 95Mo NMR spectroscopy, and compared taking into account the configuration of the ligands. In the pH range ca. 2–9, four major complexes are detected (in slow exchange at room temperature). The results point to the following structures: a 4 : 2 complex, in acidic solution, two ligand molecules bridging two cis Mo2O2+5 moieties via all hydroxyl and carboxylate groups; a 2 : 1 complex, in basic solution, having a tetradentate ligand molecule bound to an Mo2O2+5 centre via the four hydroxyl groups; 2 : 2 and 1 : 1 species, all along the pH range, in which the ligands are bound to MoO2+2 centres via the carboxylate and their adjacent OH groups.