Oncogenic Integration of Nucleotide Metabolism via Fatty Acid Synthase in Non-Hodgkin Lymphoma.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP-NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin's lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN-PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

Vitamin D and Vitamin K Concentrations in Human Brain Tissue Are Influenced by Freezer Storage Time: The Memory and Aging Project.

BACKGROUND: Vitamins D and K, which are present in human brain, may have a role in neurodegenerative disease. OBJECTIVES: Given the interest in measuring nutrient concentrations in archived brain samples, it is important to evaluate whether freezer storage time affects these concentrations. Therefore, we evaluated differences in vitamin D and vitamin K concentrations in human brain samples stored for various lengths of time. METHODS: Postmortem brain samples were obtained from 499 participants in the Rush Memory and Aging Project (mean age 92 y, 72% female). Concentrations of vitamins D and K and their metabolites were measured in 4 regions (midtemporal cortex, midfrontal cortex, cerebellum, anterior watershed white matter) using LC-MS/MS and HPLC, respectively. The predominant forms were 25-hydroxycholecalciferol [25(OH)D3] and menaquinone-4 (MK4). ANOVA was used to determine if concentrations differed according to storage time. RESULTS: The geometric mean of the mean 25(OH)D3 concentration (across 4 regions) in brains stored for 1.1 to 6.0 y did not differ from that in brains stored ≤1.0 y (all P ≥ 0.37), whereas 25(OH)D3 in brains stored >6.0 y was 31-40% lower (P ≤ 0.003). MK4 had similar results, with the geometric mean MK4 concentration in the brains stored ≥9.0 y being 48-52% lower than those in brains stored ≤1.0 y (P ≤ 0.012). The 25(OH)D3 and MK4 concentrations were positively correlated across all 4 regions (all Spearman ρ ≥ 0.79, P < 0.001). CONCLUSIONS: 25(OH)D3 and MK4 appear to be stable in brain tissue from older adults stored at -80°C for up to 6 and 9 y, respectively, but not longer. Freezer storage time should be considered in the design and interpretation of studies using archived brain tissue.

Comparison of the Postprandial Metabolic Fate of U-13C Stearic Acid and U-13C Oleic Acid in Postmenopausal Women.

OBJECTIVE: Compare the postprandial fatty acid metabolism of isotopically labeled stearate (U-13C18:0) and oleate (U-13C18:1). Approach and Results: In conjunction with a randomized-controlled crossover trial, 6 hypercholesterolemic postmenopausal women (≥50 years; body mass index: 25.6±3.0 kg/m2; LDL [low-density lipoprotein]-cholesterol ≥110 mg/dL) consumed isocaloric diets enriched in 18:0 or 18:1 (10%-15% E) for 5 weeks each. On day 1 of week 5, following a 12-hour fast, participants receive their experimental diet divided into 13 hourly meals beginning at 8 am. U-13C18:0 or U-13C18:1 was incorporated into the 1:00 pm meal (1.0 mg/kg body weight). Serial blood and breath samples were collected over 12 hours and fasting samples at 24 and 48 hours. Plasma and lipid subfraction fatty acid profiles were assessed by gas chromatography-flame ionization detector, isotope-enrichment by liquid chromatography time-of-flight mass spectrometry, and fatty acid oxidation rate (expired 13CO2) by isotope ratio mass spectrometry. Both diets resulted in similar plasma LDL-cholesterol concentrations. Kinetic curves showed that U-13C18:0 had a higher plasma area under the curve (66%), lower plasma clearance rate (-46%), and a lower cumulative oxidation rate (-34%) than U-13C18:1. Three labeled plasma metabolites of U-13C18:0 were detected: 13C16:0, 13C16:1, and 13C18:1. No plasma metabolites of U-13C18:1 were detected within the study time-frame. Higher incorporation of 18:0 in cholesteryl ester and triglyceride fractions was observed on the 18:0 compared with the 18:1 diet. CONCLUSIONS: The neutrality of 18:0 on plasma LDL-cholesterol concentrations is not attributable to a single factor. Compared with 18:1, 18:0 had higher plasma area under the curve because of lower clearance and oxidation rates, underwent both a direct and a multistage conversion to 18:1, and was preferentially incorporated into cholesteryl esters and triglycerides.

Simplified method for the measurement of plasma alkylresorcinols: Biomarkers of whole-grain intake.

RATIONALE: Consumption of whole grains is negatively associated with cardiovascular disease (CVD) risk but quantification of whole-grain intake is challenging. Alkylresorcinols (ARs) are biomarkers of whole-grain intake. Current methods for AR quantification involve a time-consuming multi-step separation process that hampers applicability in large-scale studies. METHODS: We developed a streamlined method to quantify ARs in human plasma based on protein precipitation and direct injection into an ultra-high-performance liquid chromatograph coupled to a quadrupole time-of-flight mass spectrometer operating in atmospheric pressure chemical ionization negative ion mode. RESULTS: Separation of five major ARs was achieved, with linearity in the 5 to 550 nmol/L range and a lower limit of detection (LOD) of 0.5 nmol/L and quantification (LOQ) of 5 nmol/L. The within-run and between-run precision and accuracy were below 15%, and recoveries above 90%. Once validated, the method was applied to measure concentrations of plasma ARs in subjects who participated in a randomized, crossover trial evaluating the effect of carbohydrate type on CVD risk factors. The unrefined carbohydrate diet with the highest fiber content resulted in the highest plasma AR concentration (93 ± 78 nmol/L), and was significantly different (p <0.01) from lower fiber diets (18 ± 26 nmol/L and 19 ± 26 nmol/L, simple and unrefined carbohydrate, respectively). CONCLUSIONS: This method offers a simplified approach to measure concentrations of plasma ARs as an objective biomarker of whole-grain intake that can be applied to large-scale cohort studies.

Determination of Vitamin D and Its Metabolites in Human Brain Using an Ultra-Pressure LC-Tandem Mass Spectra Method.

BACKGROUND: Low serum total 25-hydroxyvitamin D3 [25(OH)D3] concentrations have been associated with cognitive impairment. However, it is unclear if serum 25(OH)D3 concentrations are a valid indicator of the concentrations of vitamin D and its metabolites in human brain. OBJECTIVES: The aim of this study was to develop and validate a method to quantify vitamin D3, 25(OH)D3, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in human brain. METHODS: The assay developments were performed using porcine brains. Liquid extraction was used in homogenized samples (∼0.1 g each) prior to analysis by LC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. This method was then applied to the determination of vitamin D and its metabolites in a whole human brain obtained from the National Development and Research Institutes. RESULTS: The method showed good linearity of vitamin D3, 25(OH)D3, and 1,25(OH)2D3 over the physiological range (R 2 = 0.9995, 0.9968, and 0.9970, respectively). The lowest detection limit for vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in porcine brain was 25, 50 and 25 pg/g, respectively. The method was successfully applied to the determination of vitamin D3 and its metabolites in the prefrontal cortex, middle frontal cortex, middle temporal cortex, cerebellum, corpus callosum, medulla, and pons of a human brain. All analyzed human brain regions contained 25(OH)D3, with corpus callosum containing 334 pg/g compared with 158 pg/g in cerebellum. 1,25(OH)2D3 was only detected in prefrontal and middle frontal cortices at a very low level. No vitamin D3 was detected in any examined areas of this single human brain. CONCLUSIONS: To the best of our knowledge, this study is the first report of the measurement of concentrations of vitamin D metabolites in human brain. This validated method can be applied to postmortem studies to obtain accurate information about the presence and role of vitamin D and its metabolites in human brain and neurodegenerative diseases.

Comparison of diets enriched in stearic, oleic, and palmitic acids on inflammation, immune response, cardiometabolic risk factors, and fecal bile acid concentrations in mildly hypercholesterolemic postmenopausal women-randomized crossover trial.

BACKGROUND: Direct comparisons between SFAs varying in chain length, specifically palmitic acid (16:0) and stearic acid (18:0), relative to the latter's metabolic product, oleic acid (18:1), on cardiometabolic risk factors are limited. OBJECTIVE: The aim of this study was to determine the relative comparability of diets enriched in palmitic acid, stearic acid, and oleic acid on inflammation and coagulation markers, T lymphocyte proliferation/ex-vivo cytokine secretion, plasma cardiometabolic risk factors, and fecal bile acid concentrations. METHODS: Hypercholesterolemic postmenopausal women (n = 20, mean ± SD age 64 ± 7 y, BMI 26.4 ± 3.4 kg/m2, LDL cholesterol ≥ 2.8 mmol/L) were provided with each of 3 diets [55% energy (%E) carbohydrate, 15%E protein, 30%E fat, with ∼50% fat contributed by palmitic acid, stearic acid, or oleic acid in each diet; 5 wk/diet phase] using a randomized crossover design with 2-wk washouts between phases. Outcome measures were assessed at the end of each phase. RESULTS: Fasting LDL-cholesterol and non-HDL-cholesterol concentrations were lower after the stearic acid and oleic acid diets than the palmitic acid diet (all P < 0.01). Fasting HDL-cholesterol concentrations were lower after the stearic acid diet than the palmitic acid and oleic acid diets (P < 0.01). The stearic acid diet resulted in lower lithocholic acid (P = 0.01) and total secondary bile acid (SBA) concentrations (P = 0.04) than the oleic acid diet. All other outcome measures were similar between diets. Lithocholic acid concentrations were positively correlated with fasting LDL-cholesterol concentrations (r = 0.33; P = 0.011). Total SBA, lithocholic acid, and deoxycholic acid concentrations were negatively correlated with fasting HDL cholesterol (r = -0.51 to -0.44; P < 0.01) concentrations and positively correlated with LDL cholesterol:HDL cholesterol (r = 0.37-0.54; P < 0.01) ratios. CONCLUSIONS: Dietary stearic acid and oleic acid had similar effects on fasting LDL-cholesterol and non-HDL-cholesterol concentrations and more favorable ones than palmitic acid. Unlike oleic acid, the hypocholesterolemic effect of stearic acid may be mediated by inhibition of intestinal hydrophobic SBA synthesis. These findings add to the data suggesting there should be a reassessment of current SFA dietary guidance and Nutrient Facts panel labeling.This trial was registered at clinicaltrials.gov as NCT02145936.

Differential Effects of Estrogen and Progestin on Apolipoprotein B100 and B48 Kinetics in Postmenopausal Women.

The distinct effects of the estrogen and progestin components of hormonal therapy on the metabolism of apolipoprotein (apo) B-containing lipoproteins have not been studied. We enrolled eight healthy postmenopausal women in a placebo-controlled, randomized, double-blind crossover study. Each subject received placebo, conjugated equine estrogen (CEE, 0.625 mg/day) and CEE plus medroxyprogesterone acetate (MPA, 2.5 mg/day) for 8 weeks in a randomized order, with a 4-week washout between phases. Main outcomes were the fractional catabolic rate (FCR) and production rate (PR) of apo B100 in triglyceride-rich lipoproteins (TRL), intermediate-density lipoproteins (IDL) and low -density lipoprotein (LDL) and of apo B48 in TRL. Compared to placebo, CEE increased TRL apo B100 PR (p = 0.04). CEE also increased LDL apo B100 FCR (p = 0.02), but this effect was offset by a significant increase in LDL apo B100 PR (p = 0.04). Adding MPA to CEE negated the CEE effects resulting in no significant changes in TRL apo B100 PR and LDL apo B100 FCR and PR relative to placebo. Relative to placebo, during CEE there was a trend toward a reduction in plasma apo B48 concentrations and PR (p = 0.07 and p = 0.12, respectively). Compared with CEE, CEE + MPA significantly increased TRL apo B48 FCR (p = 0.02) as well as apo B48 PR (p = 0.01), resulting in no significant changes in apo B48 concentration. Estrogen and progestin have independent and opposing effects on the metabolism of the atherogenic apo B100- and apo B48-containing lipoproteins.

Curcumin and piperine supplementation of obese mice under caloric restriction modulates body fat and interleukin-1ß.

BACKGROUND: Dietary bioactive compounds capable of improving metabolic profiles would be of great value, especially for overweight individuals undergoing a caloric restriction (CR) regimen. Curcumin (Cur), a possible anti-obesity compound, and piperine (Pip), a plausible enhancer of Cur's bioavailability and efficacy, may be candidate agents for controlling body fat, metabolism and low grade inflammation. METHODS: 47 eight-week-old male C57BL/6 mice were fed a high fat diet (HFD) for 23 weeks to induce obesity. Then, mice were divided into 5 groups. Group 1 continued on HFD ad libitum. The other 4 groups underwent CR (reduced 10% HFD intake for 10 weeks, 20% for 20 weeks) with Cur, Pip, Cur + Pip or none of these. Percent body fat, plasma inflammatory markers associated with obesity (interferon (IFN)-γ, interleukin (IL)-10, IL-12 p70, IL-1ß, IL-6 and KC/GRO), plasma Cur metabolites and liver telomere length were measured. RESULTS: Compared to the other groups, obese mice who underwent CR and received Cur + Pip in their diet lost more fat and had significantly lower IL-1ß and KC/GRO. Tandem mass spectrometry analysis of plasma from obese mice under CR showed no difference in Cur metabolite levels between groups supplemented with Cur alone or combined with Pip. However, plasma IL-1ß levels were inversely correlated with curcumin glucuronide. Minor modulation of telomere length were observed. CONCLUSIONS: It is plausible that supplementing the high fat diet of CR mice with Cur + Pip may increase loss of body fat and suppresses HFD induced inflammation. Combination of Cur and Pip has potential to enhance CR effects for the prevention of metabolic syndrome.

Hepatic DNA hydroxymethylation is site-specifically altered by chronic alcohol consumption and aging.

PURPOSE: Global DNA hydroxymethylation is markedly decreased in human cancers, including hepatocellular carcinoma, which is associated with chronic alcohol consumption and aging. Because gene-specific changes in hydroxymethylcytosine may affect gene transcription, giving rise to a carcinogenic environment, we determined genome-wide site-specific changes in hepatic hydroxymethylcytosine that are associated with chronic alcohol consumption and aging. METHODS: Young (4 months) and old (18 months) male C57Bl/6 mice were fed either an ethanol-containing Lieber-DeCarli liquid diet or an isocaloric control diet for 5 weeks. Genomic and gene-specific hydroxymethylcytosine patterns were determined through hydroxymethyl DNA immunoprecipitation array in hepatic DNA. RESULTS: Hydroxymethylcytosine patterns were more perturbed by alcohol consumption in young mice than in old mice (431 differentially hydroxymethylated regions, DhMRs, in young vs 189 DhMRs in old). A CpG island ~2.5 kb upstream of the glucocorticoid receptor gene, Nr3c1, had increased hydroxymethylation as well as increased mRNA expression (p = 0.015) in young mice fed alcohol relative to the control group. Aging alone also altered hydroxymethylcytosine patterns, with 331 DhMRs, but alcohol attenuated this effect. Aging was associated with a decrease in hydroxymethylcytosine ~1 kb upstream of the leptin receptor gene, Lepr, and decreased transcription of this gene (p = 0.029). Nr3c1 and Lepr are both involved in hepatic lipid homeostasis and hepatosteatosis, which may create a carcinogenic environment. CONCLUSIONS: These results suggest that the location of hydroxymethylcytosine in the genome is site specific and not random, and that changes in hydroxymethylation may play a role in the liver's response to aging and alcohol.

Plasma 12- and 15-hydroxyeicosanoids are predictors of survival in pulmonary arterial hypertension.

This study aimed to characterize alterations in select eicosanoids in experimental and human pulmonary arterial hypertension (PAH) and to assess their potential utility as predictors of outcome. Using liquid chromatography-mass spectrometry, we performed targeted lipidomic analyses of the lungs and right ventricles (RVs) of chronically hypoxic rats and plasma of consecutive PAH patients and healthy controls. In rat lungs, chronic hypoxia was associated with significantly decreased lung prostacyclin (PGI2)/thromboxane B2 (TXB2) ratio and elevated lung 8-hydroxyeicosanoid (HETE) acid concentrations. RV eicosanoids did not exhibit any changes with chronic hypoxia. PAH treatment-naïve patients had significantly increased plasma concentrations of TXB2 and 5-, 8-, 12-, and 15-HETE. The PGI2/TXB2 ratio was lower in PAH patients than in controls, especially in the treatment-naïve cohort (median: 2.1, 0.3, and 1.3 in controls, treatment-naïve, and treated patients, respectively, P = 0.001). Survival was significantly worse in PAH patients with 12-HETEhigh (≥57 pg/mL) and 15-HETEhigh (≥256 pg/mL) in unadjusted and adjusted analyses (hazard ratio [HR]: 2.8 [95% confidence interval (CI): 1.1-7.3], P = 0.04 and HR: 4.3 [95% CI: 1.6-11.8], P = 0.004, respectively; adjustment was performed with the REVEAL [Registry to Evaluate Early and Long-Term PAH Disease Management] risk score). We demonstrate significant alterations in eicosanoid pathways in experimental and human PAH. We found that 12- and 15-HETE were independent predictors of survival in human PAH, even after adjusting for the REVEAL score, suggesting their potential role as novel biomarkers.

Distinct metabolism of apolipoproteins (a) and B-100 within plasma lipoprotein(a).

OBJECTIVES: Lipoprotein(a) [Lp(a)] is mainly similar in composition to LDL, but differs in having apolipoprotein (apo) (a) covalently linked to apoB-100. Our purpose was to examine the individual metabolism of apo(a) and apoB-100 within plasma Lp(a). MATERIALS AND METHODS: The kinetics of apo(a) and apoB-100 in plasma Lp(a) were assessed in four men with dyslipidemia [Lp(a) concentration: 8.9-124.7nmol/L]. All subjects received a primed constant infusion of [5,5,5-(2)H3] L-leucine while in the constantly fed state. Lp(a) was immunoprecipitated directly from whole plasma; apo(a) and apoB-100 were separated by gel electrophoresis; and isotopic enrichment was determined by gas chromatography/mass spectrometry. RESULTS: Multicompartmental modeling analysis indicated that the median fractional catabolic rates of apo(a) and apoB-100 within Lp(a) were significantly different at 0.104 and 0.263 pools/day, respectively (P=0.04). The median Lp(a) apo(a) production rate at 0.248nmol/kg·day(-1) was significantly lower than that of Lp(a) apoB-100 at 0.514nmol/kg·day(-1) (P=0.03). CONCLUSION: Our data indicate that apo(a) has a plasma residence time (11days) that is more than twice as long as that of apoB-100 (4days) within Lp(a), supporting the concept that apo(a) and apoB-100 within plasma Lp(a) are not catabolized from the bloodstream as a unit in humans in the fed state.

Diminished anabolic signaling response to insulin induced by intramuscular lipid accumulation is associated with inflammation in aging but not obesity.

The loss of skeletal muscle mass is observed in many pathophysiological conditions, including aging and obesity. The loss of muscle mass and function with aging is defined as sarcopenia and is characterized by a mismatch between skeletal muscle protein synthesis and breakdown. Characteristic metabolic features of both aging and obesity are increases in intramyocellular lipid (IMCL) content in muscle. IMCL accumulation may play a mechanistic role in the development of anabolic resistance and the progression of muscle atrophy in aging and obesity. In the present study, aged and high-fat fed mice were used to determine mechanisms leading to muscle loss. We hypothesized the accumulation of bioactive lipids in skeletal muscle, such as ceramide or diacylglycerols, leads to insulin resistance with aging and obesity and the inability to activate protein synthesis, contributing to skeletal muscle loss. We report a positive association between bioactive lipid accumulation and the loss of lean mass and muscle strength. Obese and aged animals had significantly higher storage of ceramide and diacylglycerol compared with young. Furthermore, there was an attenuated insulin response in components of the mTOR anabolic signaling pathway. We also observed differential increases in the expression of inflammatory cytokines and the phosphorylation of IκBα with aging and obesity. These data challenge the accepted role of increased inflammation in obesity-induced insulin resistance in skeletal muscle. Furthermore, we have now established IκBα with a novel function in aging-associated muscle loss that may be independent of its previously understood role as an NF-κB inhibitor.

Rosuvastatin Enhances the Catabolism of LDL apoB-100 in Subjects with Combined Hyperlipidemia in a Dose Dependent Manner.

Dose-associated effects of rosuvastatin on the metabolism of apolipoprotein (apo) B-100 in triacylglycerol rich lipoprotein (TRL, d < 1.019 g/ml) and low density lipoprotein (LDL) and of apoA-I in high density lipoprotein (HDL) were assessed in subjects with combined hyperlipidemia. Our primary hypothesis was that maximal dose rosuvastatin would decrease the apoB-100 production rate (PR), as well as increase apoB-100 fractional catabolic rate (FCR). Eight subjects received placebo, rosuvastatin 5 mg/day, and rosuvastatin 40 mg/day for 8 weeks each in sequential order. The kinetics of apoB-100 in TRL and LDL and apoA-I in HDL were determined at the end of each phase using stable isotope methodology, gas chromatography-mass spectrometry, and multicompartmental modeling. Rosuvastatin at 5 and 40 mg/day decreased LDL cholesterol by 44 and 54% (both P < 0.0001), triacylglycerol by 14% (ns) and 35% (P < 0.01), apoB by 30 and 36% (both P < 0.0001), respectively, and had no significant effects on HDL cholesterol or apoA-I levels. Significant decreases in plasma markers of cholesterol synthesis and increases in cholesterol absorption markers were observed. Rosuvastatin 5 and 40 mg/day increased TRL apoB-100 FCR by 36 and 46% (both ns) and LDL apoB-100 by 63 and 102% (both P < 0.05), respectively. HDL apoA-I PR increased with low dose rosuvastatin (12%, P < 0.05) but not with maximal dose rosuvastatin. Neither rosuvastatin dose altered apoB-100 PR or HDL apoA-I FCR. Our data indicate that maximal dose rosuvastatin treatment in subjects with combined hyperlipidemia resulted in significant increases in the catabolism of LDL apoB-100, with no significant effects on apoB-100 production or HDL apoA-I kinetics.

Aging and alcohol interact to alter hepatic DNA hydroxymethylation.

BACKGROUND: Aging and chronic alcohol consumption are both modifiers of DNA methylation, but it is not yet known whether chronic alcohol consumption also alters DNA hydroxymethylation, a newly discovered epigenetic mark produced by oxidation of methylcytosine. Furthermore, it has not been tested whether aging and alcohol interact to modify this epigenetic phenomenon, thereby having an independent effect on gene expression. METHODS: Old (18 months) and young (4 months) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18% of energy) or an isocaloric Lieber-DeCarli control diet for 5 weeks. Global DNA hydroxymethylation and DNA methylation were analyzed from hepatic DNA using a new liquid chromatography-tandem mass spectrometry method. Hepatic mRNA expression of the Tet enzymes were measured via quantitative real-time polymerase chain reaction. RESULTS: In young mice, mild chronic alcohol exposure significantly reduced global DNA hydroxymethylation compared with control mice (0.22 ± 0.01 vs. 0.29 ± 0.06%, p = 0.004). Alcohol did not significantly alter hydroxymethylcytosine levels in old mice. Old mice fed the control diet showed decreased global DNA hydroxymethylation compared with young mice fed the control diet (0.24 ± 0.02 vs. 0.29 ± 0.06%, p = 0.04). This model suggests an interaction between aging and alcohol in determining DNA hydroxymethylation (pinteraction  = 0.009). Expression of Tet2 and Tet3 was decreased in the old mice relative to the young (p < 0.005). CONCLUSIONS: The observation that alcohol alters DNA hydroxymethylation indicates a new epigenetic effect of alcohol. This is the first study demonstrating the interactive effects of chronic alcohol consumption and aging on DNA hydroxymethylation.

Quantification of phylloquinone and menaquinones in feces, serum, and food by high-performance liquid chromatography-mass spectrometry.

Vitamin K, comprising phylloquinone (PK) and menaquinones (MKn), is a family of vitamers found in multiple biological and environmental matrices. Advancing emerging evidence for novel and distinct physiologic roles of these vitamers in human health and disease necessitates sensitive and selective methods for quantifying PK and MKn in these matrices. We developed a novel method employing high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS) for simultaneous quantification of 11 vitamin K vitamers that can be applied in feces, serum and food. Minimal detectable concentrations of vitamin K vitamers ranged from 1 pmol/g to 30 pmol/g. Limits of quantification ranged from 5 pmol/g to 90 pmol/g. Inter-assay and intra-assay variations were <17% and <8%, respectively, in food, and <12% and <8%, respectively, in feces. Recovery exceeded 80% for all vitamers in both food and feces. The method successfully quantified PK and MKn concentrations in rat chow, feces and serum. In summary, this LC-APCI-MS method provides a sensitive and selective tool for quantifying vitamin K vitamers in feces, serum and food. This method can be applied in human and animal studies examining the role of vitamin K vitamers derived from the diet and gut bacteria synthesis in health and disease.

Differential cellular uptake and metabolism of curcuminoids in monocytes/macrophages: regulatory effects on lipid accumulation.

We have previously shown that curcumin (CUR) may increase lipid accumulation in cultured human acute monocytic leukaemia cell line THP-1 monocytes/macrophages, but that tetrahydrocurcumin (THC), an in vivo metabolite of CUR, has no such effect. In the present study, we hypothesised that the different cellular uptake and/or metabolism of CUR and THC might be a possible explanation for the previously observed differences in their effects on lipid accumulation in THP-1 monocytes/macrophages. Chromatography with tandem MS revealed that CUR was readily taken up by THP-1 monocytes/macrophages and slowly metabolised to hexahydrocurcumin sulphate. By contrast, the uptake of THC was low. In parallel with CUR uptake, increased lipid uptake was observed in THP-1 macrophages but not with the uptake of THC or another CUR metabolite and structurally related compounds. From these results, it is possible to deduce that CUR and THC are taken up and metabolised differently in THP-1 cells, which determine their biological activity. The remarkable differential cellular uptake of CUR, relative to THC and other similar molecules, may imply that the CUR uptake into cells may occur via a transporter.

Aging alters hepatic DNA hydroxymethylation, as measured by liquid chromatography/mass spectrometry.

BACKGROUND: Aging is one of the most important risk factors for cancer. It appears that aberrant epigenetic changes might be a common driver of aging and cancer. Among them are changes in DNA methylation and DNA hydroxymethylation. The 5' carbon of cytosines in CpG dinucleotides of DNA can be either methylated or hydroxymethylated. Like 5'-methylcytosine, changes in 5'-hydroxymethylcytosine may occur due to aging, potentially leading to downstream changes in transcription and cancer development. METHODS: We set up a method to measure 5'-methyl-2'-deoxycytidine and 5'-hydroxymethyl-2'-deoxycytidine in DNA using liquid chromatography/mass spectrometry (LC/MS-MS) and used this method to measure the percentage of total cytosine that was either methylated or hydroxymethylated in the liver tissues of young and old C57Bl/6 male mice. The DNA was enzymatically hydrolyzed by sequential digestion with nuclease P1, phosphodiesterase I and alkaline phosphatase. The isotopomers [(15)N3]-2'-deoxycytidine and (methyl-d 3, ring-6-d 1)-5-methyl-2'-deoxycytidine were added to the DNA hydrolysates as internal standards. DNA methylation and hydroxymethylation were calculated as a percentage of total deoxycytidine in genomic DNA. RESULTS: Within day variations for DNA methylation and hydroxymethylation were 3.45% and 8.40%, while day to day variations were 6.14% and 17.68%, respectively. Using this method it was determined that hepatic DNA of old mice had increased levels of hydroxymethylation relative to young (0.32 ± 0.02% vs. 0.24 ± 0.01%, P = 0.02), with no significant changes in 5'-methylcytosine. CONCLUSIONS: DNA hydroxymethylation measured by LC/MS-MS method can be a novel biomarker of aging. It will be useful to investigate the potential role of DNA hydroxymethylation in the development and prevention of age-associated cancer.

Differences between basal lung levels of select eicosanoids in rat and mouse.

Metabolites of arachidonic acid play an important role in mediating inflammation, cell proliferation, and oxidative stress that contribute to many pulmonary diseases. We hypothesized that the substantial differences between rats and mice in their responses to experimental pulmonary hypertensive stimuli would be associated with parallel differences in their basal eicosanoid profile. Rat and mouse lung extracts were subjected to liquid chromatography tandem mass spectrometry that was optimized for simultaneous separation and rapid quantification of the major hydroxyeicosatetraenoic acids (HETEs) and prostaglandins (PGs). Basal levels (pg/µg protein) of arachidonic acid metabolites differed significantly between rat and mouse lungs. Median values of the following major eicosanoids were significantly higher in mouse than in rat lungs: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGE2, and PGI2, as well as isoprostane-E2 and -F2α. In addition, the PGI2/TXB2 ratio was increased in mouse relative to rat lungs. On the basis of the important roles that these compounds play in determining pulmonary vascular tone, the differences in select eicosanoid profiles, especially the PGI2/TXB2 ratio, between rat and mouse lungs may underlie the interspecies differences in susceptibility to the development of pulmonary hypertension.

Effect of blueberry juice on clearance of buspirone and flurbiprofen in human volunteers.

AIM: The present study evaluated the possibility of drug interactions involving blueberry juice (BBJ) and substrate drugs whose clearance is dependent on cytochromes P4503A (CYP3A) and P4502C9 (CYP2C9). METHODS: A 50:50 mixture of lowbush and highbush BBJ was evaluated in vitro as an inhibitor of CYP3A activity (hydroxylation of triazolam and dealkylation of buspirone) and of CYP2C9 activity (flurbiprofen hydroxylation) using human liver microsomes. In clinical studies, clearance of oral buspirone and oral flurbiprofen was studied in healthy volunteers with and without co-treatment with BBJ. RESULTS: BBJ inhibited CYP3A and CYP2C9 activity in vitro, with 50% inhibitory concentrations (IC50 ) of less than 2%, but without evidence of mechanism-based (irreversible) inhibition. Grapefruit juice (GFJ) also inhibited CYP3A activity, but inhibitory potency was increased by pre-incubation, consistent with mechanism-based inhibition. In clinical studies, GFJ significantly increased area under the plasma concentration-time curve (AUC) for the CYP3A substrate buspirone. The geometric mean ratio (GMR = AUC with GFJ divided by AUC with water) was 2.12. In contrast, the effect of BBJ (GMR = 1.39) was not significant. In the study of flurbiprofen (CYP2C9 substrate), the positive control inhibitor fluconazole significantly increased flurbiprofen AUC (GMR = 1.71), but BBJ had no significant effect (GMR = 1.03). CONCLUSION: The increased buspirone AUC associated with BBJ is quantitatively small and could have occurred by chance. BBJ has no effect on flurbiprofen AUC. The studies provide no evidence for concern about clinically important pharmacokinetic drug interactions of BBJ with substrate drugs metabolized by CYP3A or CYP2C9.

Linkage between C-reactive protein and triglyceride-rich lipoprotein metabolism.

OBJECTIVE: Inflammation plays an important role in atherosclerosis. Elevated C-reactive protein (CRP) levels are associated with a greater risk of cardiovascular disease. Our goal was to study CRP metabolism, and to determine its relationship with lipoprotein metabolism using stable isotope methodology. MATERIAL/METHODS: Eight subjects with combined hyperlipidemia underwent a 15-h primed-constant infusion with deuterated leucine. CRP was purified from the plasma density fraction greater than 1.21g/ml by affinity chromatography. Lipoprotein fractions were separated by sequential ultracentrifugation. Isotope enrichment was determined by gas chromatography/mass spectrometry. RESULTS: The subjects had mean LDL-C levels of 147.5mg/dl and mean CRP levels of 3.4mg/l. The mean CRP production rate (PR) was 0.050±0.012mg/kg/day and the mean CRP fractional catabolic rate (FCR) was 0.343±0.056 pools/day (residence time 2.92days). CRP pool size (PS) was significantly related to production (r=0.93; p<0.001), but not FCR. CRP PS was also related to body mass index (r=0.79; p=0.02). There was a significant association between CRP FCR and TRL apoB-100 FCR (r=0.74, p=0.04), as well as between CRP PS and TRL apoB-48 FCR (r=-0.90, p=0.002), indicating linkage between CRP and TRL metabolism. CONCLUSION: The main determinant of plasma CRP levels was CRP production rate. Moreover a significant linkage between CRP metabolism and both TRL apoB-100 and apoB-48 catabolism was noted.