Separation of postprandial lipoproteins: improved purification of chylomicrons using an ApoB100 immunoaffinity method.

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

Effects of Dietary Fructose Restriction on Liver Fat, De Novo Lipogenesis, and Insulin Kinetics in Children With Obesity.

BACKGROUND & AIMS: Consumption of sugar is associated with obesity, type 2 diabetes mellitus, nonalcoholic fatty liver disease, and cardiovascular disease. The conversion of fructose to fat in liver (de novo lipogenesis [DNL]) may be a modifiable pathogenetic pathway. We determined the effect of 9 days of isocaloric fructose restriction on DNL, liver fat, visceral fat (VAT), subcutaneous fat, and insulin kinetics in obese Latino and African American children with habitual high sugar consumption (fructose intake >50 g/d). METHODS: Children (9-18 years old; n = 41) had all meals provided for 9 days with the same energy and macronutrient composition as their standard diet, but with starch substituted for sugar, yielding a final fructose content of 4% of total kilocalories. Metabolic assessments were performed before and after fructose restriction. Liver fat, VAT, and subcutaneous fat were determined by magnetic resonance spectroscopy and imaging. The fractional DNL area under the curve value was measured using stable isotope tracers and gas chromatography/mass spectrometry. Insulin kinetics were calculated from oral glucose tolerance tests. Paired analyses compared change from day 0 to day 10 within each child. RESULTS: Compared with baseline, on day 10, liver fat decreased from a median of 7.2% (interquartile range [IQR], 2.5%-14.8%) to 3.8% (IQR, 1.7%-15.5%) (P < .001) and VAT decreased from 123 cm3 (IQR, 85-145 cm3) to 110 cm3 (IQR, 84-134 cm3) (P < .001). The DNL area under the curve decreased from 68% (IQR, 46%-83%) to 26% (IQR, 16%-37%) (P < .001). Insulin kinetics improved (P < .001). These changes occurred irrespective of baseline liver fat. CONCLUSIONS: Short-term (9 days) isocaloric fructose restriction decreased liver fat, VAT, and DNL, and improved insulin kinetics in children with obesity. These findings support efforts to reduce sugar consumption. ClinicalTrials.gov Number: NCT01200043.

Celecoxib affects estrogen sulfonation catalyzed by several human hepatic sulfotransferases, but does not stimulate 17-sulfonation in rat liver.

Celecoxib is known to alter the preferred position of SULT2A1-catalyzed sulfonation of 17ß-estradiol (17ß-E2) and other estrogens from the 3- to the 17-position. Understanding the effects of celecoxib on estrogen sulfonation is of interest in the context of the investigational use of celecoxib to treat breast cancer. This study examined the effects on celecoxib on cytosolic sulfotransferases in human and rat liver and on SULT enzymes known to be expressed in liver. Celecoxib's effects on the sulfonation of several steroids catalyzed by human liver cytosol were similar but not identical to those observed previously for SULT2A1. Celecoxib was shown to inhibit recombinant SULT1A1-catalyzed sulfonation of 10nM estrone and 4µM p-nitrophenol with IC50 values of 2.6 and 2.1µM, respectively, but did not inhibit SULT1E1-catalyzed estrone sulfonation. In human liver cytosol, the combined effect of celecoxib and known SULT1A1 and 1E1 inhibitors, quercetin and triclosan, resulted in inhibition of 17ß-E2-3-sulfonation such that the 17-sulfate became the major metabolite: this is of interest because the 17-sulfate is not readily hydrolyzed by steroid sulfatase to 17ß-E2. Investigation of hepatic cytosolic steroid sulfonation in rat revealed that celecoxib did not stimulate 17ß-E2 17-sulfonation in male or female rat liver as it does with human SULT2A1 and human liver cytosol, demonstrating that rat is not a useful model of this effect. In silico studies suggested that the presence of the bulky tryptophan residue in the substrate-binding site of the rat SULT2A homolog instead of glycine as in human SULT2A1 may explain this species difference.

Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1.

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17ß-estradiol (17ß-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3ß, 17ß-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low µM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17ß-dihydroequilenin (17ß-Eqn), 17ß-dihydroequilin (17ß-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17ß-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17ß-Eq was stimulated at celecoxib concentrations below 40 µM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.

Fiber supplementation lowers plasma p-cresol in chronic kidney disease patients.

OBJECTIVE: To determine the effects of supplemental fiber on plasma p-cresol, stool frequency, and quality of life (QoL) in chronic kidney disease (CKD) patients. DESIGN AND SETTING: In a 12-week single-blind study, participants were provided with control muffins and supplements (5.5 g sucrose/day) for 2 weeks, muffins containing 10 g/day pea hull fiber and control supplements for 4 weeks, and muffins with 10 g/day pea hull fiber and 15 g/day inulin as a supplement for 6 weeks. SUBJECTS: Individuals with CKD (n = 13; 6 males, 7 females; aged 65 ± 3 years; estimated glomerular filtration rate <50 mL/minute/1.73(2)) completed the study. MAIN OUTCOME MEASURES: Plasma p-cresol was determined by gas chromatography-mass spectrometry, stool frequency by 5-day journals, and QoL by the KDQOL-36™. RESULTS: Plasma p-cresol decreased from 7.25 ± 1.74 mg/L during week 1 to 5.82 ± 1.72 mg/L during week 12 (P < .05), and in participants with high compliance (>70% inulin intake), from 6.71 ± 1.98 mg/L to 4.22 ± 1.16 mg/L (P < .05). Total fiber intake increased from 16.6 ± 1.7 g/day during control to 26.5 ± 2.4 g/day (P < .0001) with the added pea hull and to 34.5 ± 2.2 g/day with pea hull and inulin (P < .0001). Stool frequency increased from 1.4 ± 0.2 stools/day during control to 1.9 ± 0.3 stools/day during both fiber periods (P < .05). No change in overall QoL was observed. CONCLUSIONS: Supplementing the diet of CKD patients with fiber may be a dietary therapy to reduce p-cresol and improve stool frequency.

Triuret as a potential hypokalemic agent: Structure characterization of triuret and triuret-alkali metal adducts by mass spectrometric techniques.

Triuret (also known as carbonyldiurea, dicarbamylurea, or 2,4-diimidotricarbonic diamide) is a byproduct of purine degradation in living organisms. An abundant triuret precursor is uric acid, whose level is altered in multiple metabolic pathologies. Triuret can be generated via urate oxidation by peroxynitrite, the latter being produced by the reaction of nitric oxide radical with superoxide radical anion. From this standpoint, an excess production of superoxide radical anions could indirectly favor triuret formation; however very little is known about the potential in vivo roles of this metabolite. Triuret's structure is suggestive of its ability to adopt various conformations and act as a flexible ligand for metal ions. In the current study, HPLC-MS/MS, energy-resolved mass spectrometry, selected ion monitoring, collision-induced dissociation, IRMPD spectroscopy, Fourier transform-ion cyclotron resonance mass spectrometry and computational methods were employed to characterize the structure of triuret and its metal complexes, to determine the triuret-alkali metal binding motif, and to evaluate triuret affinity toward alkali metal ions, as well as its affinity for Na(+) and K(+) relative to other organic ligands. The most favored binding motif was determined to be a bidentate chelation of triuret with the alkali metal cation involving two carbonyl oxygens. Using the complexation selectivity method, it was observed that in solution triuret has an increased affinity for potassium ions, compared to sodium and other alkali metal ions. We propose that triuret may act as a potential hypokalemic agent under pathophysiological conditions conducive to its excessive formation and thus contribute to electrolyte disorders. The collision- or photo-induced fragmentation channels of deprotonated and protonated triuret, as well as its alkali metal adducts, are likely to mimic the triuret degradation pathways in vivo.

Reactions of peroxynitrite with uric acid: formation of reactive intermediates, alkylated products and triuret, and in vivo production of triuret under conditions of oxidative stress.

Hyperuricemia is associated with hypertension, metabolic syndrome, preeclampsia, cardio-vascular disease and renal disease, all conditions associated with oxidative stress. We hypothesized that uric acid, a known antioxidant, might become prooxidative following its reaction with oxidants; and, thereby contribute to the pathogenesis of these diseases. Uric acid and 1,3-(15)N(2)-uric acid were reacted with peroxynitrite in different buffers and in the presence of alcohols, antioxidants and in human plasma. The reaction products were identified using liquid chromatography-mass spectrometry (LC-MS) analyses. The reactions generate reactive intermediates that yielded triuret as their final product. We also found that the antioxidant, ascorbate, could partially prevent this reaction. Whereas triuret was preferentially generated by the reactions in aqueous buffers, when uric acid or 1,3-(15)N(2)-uric acid was reacted with peroxynitrite in the presence of alcohols, it yielded alkylated alcohols as the final product. By extension, this reaction can alkylate other biomolecules containing OH groups and others containing labile hydrogens. Triuret was also found to be elevated in the urine of subjects with preeclampsia, a pregnancy-specific hypertensive syndrome that is associated with oxidative stress, whereas very little triuret is produced in normal healthy volunteers. We conclude that under conditions of oxidative stress, uric acid can form reactive intermediates, including potential alkylating species, by reacting with peroxynitrite. These reactive intermediates could possibly explain how uric acid contributes to the pathogenesis of diseases such as the metabolic syndrome and hypertension.

Inactivation of nitric oxide by uric acid.

The 1980 identification of nitric oxide (NO) as an endothelial cell-derived relaxing factor resulted in an unprecedented biomedical research of NO and established NO as one of the most important cardiovascular, nervous and immune system regulatory molecule. A reduction in endothelial cell NO levels leading to "endothelial dysfunction" has been identified as a key pathogenic event preceding the development of hypertension, metabolic syndrome, and cardiovascular disease. The reduction in endothelial NO in cardiovascular disease has been attributed to the action of oxidants that either directly react with NO or uncouple its substrate enzyme. In this report, we demonstrate that uric acid (UA), the most abundant antioxidant in plasma, reacts directly with NO in a rapid irreversible reaction resulting in the formation of 6-aminouracil and depletion of NO. We further show that this reaction occurs preferentially with NO even in the presence of oxidants peroxynitrite and hydrogen peroxide and that the reaction is at least partially blocked by glutathione. This study shows a potential mechanism by which UA may deplete NO and cause endothelial dysfunction, particularly under conditions of oxidative stress in which UA is elevated and intracellular glutathione is depleted.

Host-guest chemistry of the chromium-wheel complex [Cr8F8(tBuCO2)16]: prediction of inclusion capabilities by using an electrostatic potential distribution determined by modeling synchrotron X-ray structure factors at 16 K.

The structure and detailed electron density distribution (EDD) of the large octanuclear chromium-wheel host complex [Cr8F8(tBuCO2)16] (1) has been determined from synchrotron X-ray structure factors collected at 16(5) K. The complex has a central cavity with a minimum entry distance between carbon atoms of the pivalate methyl groups (pivalic acid = tBuCO2H) of 4.027(4) A on one side of the molecule and 7.273(4) A on the other. The screened side of the molecule can be "opened" by rotation of methyl groups to create a strained host structure, which is compensated for by improved host-guest and host-solvent interaction. The EDD of the 272-atom complex (1144 e-) was determined by multipole modeling based on the experimental structure factors. 3d orbital populations on the Cr atoms and topological analysis of the EDD show that the covalent part of the metal-ligand interactions consists mainly of sigma donation from the ligands, but that overall the interactions are predominantly electrostatic. The electrostatic potential (EP) has been calculated from the experimental EDD. Knowledge of the geometry of the naked complex 1 as well as the EP in the central cavity of this molecule allows us to deduce which characteristic properties guest molecules must have to be accepted into the void. To probe these predictions, a series of complexes of 1 with different guest inclusions were synthesized (2 = 1 + N,N'-dimethylformamide (DMF), 3 = 1 + N,N'-dimethylacetamide (DMA), 4 = 1 + DMA + DMF, 5 = 1 + 2CH3CN), and their structures were examined by using X-ray diffraction data measured at 120(1) K. Results of these studies indicate that in the crystalline state, the optimal guest molecule should be linear and possess a permanent dipole. Attempts to crystallize the host complex with cations incorporated into the cavity were fruitless, although electrospray ionization mass spectrometry showed that a [1 + potassium]+ entity pre-exists in solution and can be transferred intact into the gas phase.

Enhancement of ionization efficiency by electrochemical reaction products in on-line electrochemistry/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

A miniaturized two-electrode electrochemical (EC) cell was developed and was coupled on-line with an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-FTICR MS). Electrochemistry on-line with mass spectrometry, EC/ESI-FTICR MS, of triphenylamine (TPA), which undergoes one-electron oxidation to form a radical cation (TPA*+), demonstrates a significant sensitivity enhancement compared to ESI-FTICR MS. The on-line EC cell configuration with a stainless steel ES needle as the working electrode produces the highest sensitivity in EC/ESI-MS. The results provide evidence that, during the ES ionization, electrolytic reactions occur mainly in the ES tip region, as previously predicted. The results demonstrate that ESI-MS signal suppression by tetrabutylammonium perchlorate electrolyte, which can be a problem, is minimized in EC/ESI-MS. TPA*+ dimer tetraphenylbenzidine (TPB) can be detected by EC/ESI-MS, together with TPA*+, as TPB*+ and TPB2+. The high mass resolving power of FTICR MS was exploited to identify TPB2+ dication in the presence of [TPA*+ - H*]+ ions of the same m/z, from their respective isotopic distributions. The dimer dication TPB2+ can be detected only in EC/ESI-MS.