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BACKGROUND - Fatty acids, including n-6 series, modulate immune function, but their effect on CD4 cell counts, death, or hospitalization in HIV-infected patients on antiretroviral therapy is unknown.
METHODS - In a randomized trial for effects of multivitamins in HIV-infected patients in Uganda, we used gas chromatography to measure plasma n-6 fatty acids at baseline; determined CD4 counts at baseline, 3, 6, 12, and 18 months; and recorded hospitalization or death events. The associations of fatty acids with CD4 counts and events were analyzed using repeated-measures analysis of variance and Cox regression, respectively.
RESULTS - Among 297 patients with fatty acids measurements, 16 patients died and 69 were hospitalized within 18 months. Except for linoleic acid, n-6 fatty acids levels were positively associated with CD4 counts at baseline but not during follow-up. In models that included all 5 major n-6 fatty acids, age; sex; body mass index; anemia status; use of antiretroviral therapy, multivitamin supplements, and alcohol; and the risk of death or hospitalization decreased significantly with an increase in linoleic acid and gamma-linolenic acid levels, whereas associations for dihomo-gamma-linolenic acid, arachidonic acid, and aolrenic acid were null. The hazard ratios (95% confidence intervals) per 1 SD increase in linoleic acid and gamma-linolenic acid were 0.73 (0.56-0.94) and 0.51 (0.36-0.72), respectively. Gamma-linolenic acid remained significant (hazard ratio = 0.51; 95% confidence interval: 0.35 to 0.68) after further adjustment for other plasma fatty acids.
CONCLUSIONS - Lower levels of gamma-linolenic acid are associated with lower CD4 counts and an increased risk of death or hospitalization. These results suggest a potential for using n-6 fatty acids to improve outcomes from antiretroviral therapy.
Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.
OBJECTIVE - To evaluate the association of uric acid (UA) levels with a panel of markers of oxidative stress and inflammation.
METHODS - Plasma UA levels, along with a panel of oxidative stress and inflammatory markers, were measured in 755 Chinese women.
RESULTS - Plasma UA levels were inversely associated with urinary levels of the oxidative stress marker F2-isoprostanes and positively correlated to levels of inflammatory markers, such as C-reactive protein and some proinflammatory cytokines (tumor necrosis factor-α and interleukin-6) in blood as well as prostaglandin E2 metabolites in urine.
CONCLUSIONS - Plasma UA levels correlate to oxidation and inflammation biomarkers in opposite directions in women.
Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.
Copyright © 2015 the American Physiological Society.
A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat-fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (SNi) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
Population-based investigations suggest that red blood cells (RBCs) are therapeutically effective when collected, processed, and stored for up to 42 days under validated conditions before transfusion. However, some retrospective clinical studies have shown worse patient outcomes when transfused RBCs have been stored for the longest times. Furthermore, studies of RBC persistence in the circulation after transfusion have suggested that considerable donor-to-donor variability exists and may affect transfusion efficacy. To understand the limitations of current blood storage technologies and to develop approaches to improve RBC storage and transfusion efficacy, we investigated the global metabolic alterations that occur when RBCs are stored in AS-1 (AS1-RBC). Leukoreduced AS1-RBC units prepared from 9 volunteer research donors (12 total donated units) were serially sampled for metabolomics analysis over 42 days of refrigerated storage. Samples were tested by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, and specific biochemical compounds were identified by comparison to a library of purified standards. Over 3 experiments, 185 to 264 defined metabolites were quantified in stored RBC samples. Kinetic changes in these biochemicals confirmed known alterations in glycolysis and other pathways previously identified in RBCs stored in saline, adenine, glucose and mannitol solution (SAGM-RBC). Furthermore, we identified additional alterations not previously seen in SAGM-RBCs (eg, stable pentose phosphate pathway flux, progressive decreases in oxidized glutathione), and we delineated changes occurring in other metabolic pathways not previously studied (eg, S-adenosyl methionine cycle). These data are presented in the context of a detailed comparison with previous studies of SAGM-RBCs from human donors and murine AS1-RBCs. Global metabolic profiling of AS1-RBCs revealed a number of biochemical alterations in stored blood that may affect RBC viability during storage as well as therapeutic effectiveness of stored RBCs in transfusion recipients. These results provide future opportunities to more clearly pinpoint the metabolic defects during RBC storage, to identify biomarkers for donor screening and prerelease RBC testing, and to develop improved RBC storage solutions and methodologies.
Copyright © 2014 Elsevier Inc. All rights reserved.
The anti-atherogenic effects of omega 3 fatty acids, namely eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) are well recognized but the impact of dietary intake on bioactive lipid mediator profiles remains unclear. Such a profiling effort may offer novel targets for future studies into the mechanism of action of omega 3 fatty acids. The present study aimed to determine the impact of DHA supplementation on the profiles of polyunsaturated fatty acids (PUFA) oxygenated metabolites and to investigate their contribution to atherosclerosis prevention. A special emphasis was given to the non-enzymatic metabolites knowing the high susceptibility of DHA to free radical-mediated peroxidation and the increased oxidative stress associated with plaque formation. Atherosclerosis prone mice (LDLR(-/-)) received increasing doses of DHA (0, 0.1, 1 or 2% of energy) during 20 weeks leading to a dose-dependent reduction of atherosclerosis (R(2) = 0.97, p = 0.02), triglyceridemia (R(2) = 0.97, p = 0.01) and cholesterolemia (R(2) = 0.96, p<0.01). Targeted lipidomic analyses revealed that both the profiles of EPA and DHA and their corresponding oxygenated metabolites were substantially modulated in plasma and liver. Notably, the hepatic level of F4-neuroprostanes, a specific class of DHA peroxidized metabolites, was strongly correlated with the hepatic DHA level. Moreover, unbiased statistical analysis including correlation analyses, hierarchical cluster and projection to latent structure discriminate analysis revealed that the hepatic level of F4-neuroprostanes was the variable most negatively correlated with the plaque extent (p<0.001) and along with plasma EPA-derived diols was an important mathematical positive predictor of atherosclerosis prevention. Thus, oxygenated n-3 PUFAs, and F4-neuroprostanes in particular, are potential biomarkers of DHA-associated atherosclerosis prevention. While these may contribute to the anti-atherogenic effects of DHA, further in vitro investigations are needed to confirm such a contention and to decipher the molecular mechanisms of action.
Mass spectrometry (MS) offers a sensitive, reliable, and highly accurate method for measurement of isotopic labeling, which is required for generating comprehensive flux maps using metabolic flux analysis (MFA). We present protocols for assessing isotope labeling in a wide range of biochemical species, including proteinogenic amino acids, free organic and amino acids, sugar phosphates, lipids, starch-glucose, and RNA-ribose. We describe the steps of sample preparation, MS analysis, and data handling required to obtain high-quality isotope labeling measurements that are applicable to MFA. By selecting target analytes that maximize identifiability of the key fluxes of interest, MS measurements of isotope labeling can provide a powerful platform for assessing metabolic fluxes in complex biochemical networks.
Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [(1-13)C]glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.
Copyright © 2013 Elsevier Inc. All rights reserved.