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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)D(3)] 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)D(3) 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)D(3) 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)D(3) and MK4 concentrations were positively correlated across all 4 regions (all Spearman ρ ≥ 0.79, P

Compare the postprandial fatty acid metabolism of isotopically labeled stearate (U-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.

OBJECTIVES: Vitamins D and K are present in human brain tissue, and evidence is emerging that these nutrients may have a role in neurodegenerative disease. Given the increasing interest in conducting analyses of archived brain samples, it is important to evaluate the stability of these nutrients in brain tissue over long-term storage. Therefore, we evaluated the influence of freezer storage time on vitamin D and vitamin K concentrations in human brain samples. METHODS: Post-mortem brain samples were obtained from 500 participants in the Rush Memory and Aging Project (mean age 91 yrs, 71% female). At autopsy and brain dissection, the tissues were immediately frozen on brass plates atop dry ice then stored at −80(o) C until analysis. Storage time was categorized in 1-year increments. Vitamin D [25(OH)D] and vitamin K [menaquinone-4 (MK4)] concentrations were measured in four regions (mid-temporal cortex, mid-frontal cortex, cerebellum, anterior watershed white matter) using LC/MS/MS and HPLC, respectively. We calculated the mean 25(OH)D and MK4 concentrations across the four regions and then applied a natural-log transformation to the means to improve normality. Analysis of variance was used to determine if the geometric mean ± SEM 25(OH)D and MK4 concentrations differed according to storage time. The samples stored ≤1.0 year served as the reference group. RESULTS: The mean 25(OH)D concentration in brains stored >6.0 years (n = 203; 0.8 ± 0.05 pmol/g), was significantly lower than the concentration in brains stored ≤1.0 year (n = 79; 1.2 ± 0.09 pmol/g, P ≤ 0.004). The 25(OH)D in the brains stored ≤1.0 year did not differ from the concentration in brains stored for 1.1–6.0 years (n = 212; 1.2 ± 0.05 pmol/g, P ≥ 0.37). The mean MK4 concentration in the brains stored ≥9.0 years (n = 81; 0.7 ± 0.6 pmol/g) was significantly lower than that in the brains stored ≤1.0 year (1.2 ± 1.3 pmol/g; P ≤ 0.01). The MK4 in brains stored ≤1.0 year did not differ from the concentration in brains stored for 1.1–9.0 years (n = 339; 1.3 ± 0.8 pmol/g; P ≥ 0.11). CONCLUSIONS: 25(OH)D and MK4 appear to be stable in brain tissue stored at −80 °C for up to 6 and 9 years, respectively, but not longer. This should be considered in the design and interpretation of studies linking brain concentrations of these nutrients to neurodegenerative diseases. FUNDING SOURCES: Supported by the National Institute of Aging.

Objective: Compare the postprandial fatty acid metabolism of isotopically labeled stearate (U- 13 C18:0) and oleate (U- 13 C18:1). 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/m 2 ; 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- 13 C18:0 or U- 13 C18:1 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 13 CO 2 ) by isotope ratio mass spectrometry. Both diets resulted in similar plasma LDL-cholesterol concentrations. Kinetic curves showed that U- 13 C18:0 had a higher plasma area under the curve (66%), lower plasma clearance rate (−46%), and a lower cumulative oxidation rate (−34%) than U- 13 C18:1. Three labeled plasma metabolites of U- 13 C18:0 were detected: 13 C16:0, 13 C16:1, and 13 C18:1. No plasma metabolites of U- 13 C18:1 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.

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.

Background: Observational studies suggest an inverse association between whole-grain (WG) consumption and inflammation. However, evidence from interventional studies is limited, and few studies have included measurements of cell-mediated immunity.Objective: We assessed the effects of diets rich in WGs compared with refined grains (RGs) on immune and inflammatory responses, gut microbiota, and microbial products in healthy adults while maintaining subject body weights.Design: After a 2-wk provided-food run-in period of consuming a Western-style diet, 49 men and 32 postmenopausal women [age range: 40-65 y, body mass index (in kg/m2)

OBJECTIVES: Very little is known about the forms of vitamin D and vitamin K in the human brain. The objective of this study is to evaluate concentrations of vitamin D and vitamin K forms in human brain and their correlations across four human brain regions. METHODS: Vitamin D [D3, 25(OH)D and 1,25(OH)2D] and vitamin K [phylloquinone and menaquinone-4 (MK4)] concentrations were measured by LC/MS/MS and HPLC, respectively, in four brain regions from post-mortem samples obtained from participants in the Rush Memory and Aging Project (n = 130, mean age 82 yrs, 81% female). The brain regions analyzed were the mid-frontal cortex (MF) and mid-temporal cortex (MT) [two regions important for memory in Alzheimer's Disease (AD)], the cerebellum (CR, a region not affected by AD), and the anterior watershed white matter (AWS, a region associated with vascular disease). The correlations among the vitamin forms across brain regions were calculated using Spearman rank order correlation coefficients. Significance was set at P 95% of the brain samples. Nearly 92% of 1,25(OH)2D and 80% of phylloquinone samples had concentrations below the limit of assay detection (LOD) 1,25(OH)2D = 20 ng/g, phylloquinone = 0.1 pmol/g). Vitamin D3 and 25(OH)D concentrations were positively correlated across all four regions (all Spearman r ≥ 0.78, P

OBJECTIVES: Vitamins D and K are detected in the human brain, but the associations among the forms of vitamin D and vitamin K within brain region is unclear. The objective of this study is to evaluate the correlations of vitamin D forms and vitamin K forms within four regions of the human brain. METHODS: Post-mortem brain samples were obtained from 130 participants in the Rush Memory and Aging Project, a prospective cohort of community-dwelling adults from Chicago, IL (age range 78–86 y; 81% Female). Vitamin D [D3, 25(OH)D and 1,25(OH)2D] and vitamin K (phylloquinone and MK4) concentrations were measured by LC/MS/MS and HPLC, respectively, in the following regions: middle frontal cortex (MF) and middle temporal cortex (MT) [two key memory regions affected by Alzheimer's Disease (AD)], cerebellum, (CR, which is unaffected by AD), and the anterior watershed white matter (AWS, associated with vascular disease). The correlations among nutrient forms within each region were calculated using Spearman rank order correlation coefficients. Statistical significance was set at P

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.

Introduction: Dietary fat quality has a significant impact on CVD risk. Among the saturated fatty acids (FA), we and others have documented that stearic (18:0) is unique because, unlike shorter chain FA (12:0, 14:0, 16:0), it does not raise plasma LDL-C levels, relative to monounsaturated FA, such as oleic acid (18:1). The mechanism(s) responsible for this effect are not fully elucidated. Hypothesis: We tested the hypothesis that the hypocholesterolemic effect of dietary 18:0 and 18:1 relative to 16:0 is mediated by alterations in fecal bile acid metabolism. Methods: Primary (cholic, chenodeoxycholic) and secondary bile acids (SBA: lithocholic [LA], deoxycholic [DCA]) were quantified in stool samples from a randomized controlled cross-over trial examining the effect of dietary FA on CVD risk factors. Subjects (N=20 postmenopausal women, 50-85 years, BMI 25-35kg/m 2 , LDL-C >100mg/dL) consumed each diet for 35 days separated by a 2 week washout period. Diets provided 55%E carbohydrate, 15%E protein and 30%E fat with half of the fat provided by 16:0, 18:0 or 18:1, respectively. CVD risk factors (lipids, glucose, insulin, inflammatory markers) were measured using standard methodology. For fecal bile acids analysis, freeze dried samples were spiked with the corresponding deuterated internal standards, followed by an overnight extraction, purification and analysis using reversed-phase liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry. Quantification was by isotopic dilution. Spearman correlation coefficients were calculated between bile acids and CVD risk factors. Results: Fecal total SBA levels were significantly lower after subjects consumed the 18:0 (4.5±3.9 umol/g) compared to the 18:1 (6.8±5.7 umol/g) diet, with intermediate levels after the 16:0 diet (5.2±3.6 umol/g). This was predominantly due to significantly lower LA, and to a lesser extent DCA levels. No detectable differences were observed in primary bile acids levels. Total, LA and DCA levels were positively correlated with insulin (r=0.38 to 0.45; p Conclusion: These data suggest that the hypocholesterolemic effect of dietary 18:0 was not mediated by increased bile acid excretion as was observed with dietary 18:1. Instead, dietary 18:0 appears to have an inhibitory effect on hydrophobic SBA synthesis in the intestine, which in turn could reduce the efficiency of cholesterol solubilization and thus cholesterol absorption. Further studies on the effects of dietary 18:0 on gut microbiome populations involved in SBA synthesis are warranted.

Introduction: Consumption of whole grains is associated with improvements in cardiometabolic risk factors and decreased CVD risk. Quantification of whole grain intake is challenging due to the limitations of self-reported intake; diversity among and differences in the interpretation of the term “whole grain”. Alkylresorcinols (ARs) are phenolic lipids present in the outer layer of wheat and rye grains that are considered objective biomarkers of whole grain intake. Current methods for ARs quantification involve a multi-step separation, extraction and purification processes that hampers applicability in large-scale studies. Hypothesis: Our aim was to develop a single-step method to measure 5 predominant ARs (C17:0, C19:0, C21:0, C23:0 and C25:0) in human plasma, and to validate this method by measuring plasma ARs levels in subjects who participated in a randomized, cross-over trial evaluating the effect of carbohydrate quality on CVD risk factors. We hypothesized that the direct method would distinguish between low- and high-whole grain intake and be more rapid and cost effective than prior methods. Methods: A dilute-and-shoot strategy based on plasma (20 μL) dilution with methanol for protein precipitation, followed by centrifugation and direct injection into an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UHPLC/Q-TOF-MS; Agilent Technologies), using negative electrospray ionization. C19:0-D 4 was used as internal standard. Separation was performed using a C18 column. Run time was 11 minutes/sample. Method validation was based on the following criteria: linearity, working range, lowest limit of quantification (LOQ), accuracy and precision. This method was then used to quantify ARs in fasting plasma samples from postmenopausal women and men (N=11, 65±8 years, BMI 29.8±3.2 kg/m 2 , LDL-C ≥2.6 mmol/L) who consumed each of 3 isocaloric diets (60%E carbohydrate, 15%E protein, 25%E fat) enriched in either simple, refined, or unrefined carbohydrate-containing foods for 4.5 weeks in a randomized crossover design, with 2-week washout periods. Results: (1) Analytical: The method showed linearity (>0.999) in the range 2 to 100 ng/mL, and had acceptable values for accuracy and precision with a LOQ of 2 ng/mL. (2) Applicability: Total fiber content of the simple, refined and unrefined carbohydrate diets was 8.6, 9.6 and 19.5g/1000kcal, respectively. This was reflected in plasma AR levels, being significantly higher (p Conclusion: This simplified method offers a direct and rapid strategy to accurately measure AR in human plasma that can be scaled up for large studies, and provides an objective assessment of whole grain intake.

ObjectivesLipoprotein(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 methodsThe 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.ResultsMulticompartmental 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).ConclusionOur 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.

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-1β, 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-1β 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-1β 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.

Background The plasma 25-hydroxyvitamin D response to supplementation with vitamin D varies widely, but vitamin D absorption differences based on diet composition is poorly understood. Objectives We tested the hypotheses that absorption of vitamin D-3 is greater when the supplement is taken with a meal containing fat than with a fat-free meal and that absorption is greater when the fat in the meal has a higher monounsaturated-to-polyunsaturated fatty acid ratio (MUFA:PUFA). Design Open, three-group, single-dose vitamin D-3 comparative absorption experiment. Participants/setting Our 1-day study was conducted in 50 healthy older men and women who were randomly assigned to one of three meal groups: fat-free meal, and a meal with 30% of calories as fat with a low (1:4) and one with a high (4:1) MUFA:PUFA. After a 12-hour fast, all subjects took a single 50,000 IU vitamin D-3 supplement with their test breakfast meal. Main outcome measures Plasma vitamin D-3 was measured by liquid chromatography–mass spectrometry before and 10, 12 (the expected peak), and 14 hours after the dose. Statistical analyses performed Means were compared with two-tailed t tests for independent samples. Group differences in vitamin D-3 absorption across the measurement time points were examined by analysis of variance with the repeated measures subcommand of the general linear models procedure. Results The mean peak (12-hour) plasma vitamin D-3 level after the dose was 32% (95% CI 11% to 52%) greater in subjects consuming fat-containing compared with fat-free meals ( P =0.003). Absorption did not differ significantly at any time point in the high and low MUFA and PUFA groups. Conclusions The presence of fat in a meal with which a vitamin D-3 supplement is taken significantly enhances absorption of the supplement, but the MUFA:PUFA of the fat in that meal does not influence its absorption.

Oat avenanthramides (AVAs) are a group of phenolic alkaloids, consisting of an anthranilic acid and a hydroxycinnamic acid linked by a pseudo-peptide bond. Bioavailability of AVA is poor in humans, suggesting transformations for rapid excretion. Thus, we aim to identify metabolites of AVA isomers in plasma of humans after consuming AVA-enriched oats. After lipid removal, AVA and their metabolites in plasma were extracted with ethyl acetate and analysed using an Agilent UHPLC-QToF-MS. Pharmacokinetics of AVA-O showed a bimodal distribution with Cmax1 and 2 for AVA-O at 5.9 ± 5.2 and 7.9 ± 7.0 ng/mL and Tmax1 and 2 at 1.7 ± 0.7 and 3.1 ± 1.2 h, respectively. Only the methyl-AVA-O showed a single Cmax at 14 ± 9.9 ng/mL AVA-O equivalents and a Tmax of 2.4 ± 2.7 h. This analysis is the first to identify methylated metabolites of AVAs and AVA aglycones in human blood after acute AVA consumption.

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.

Introduction: FASN catalyzes de novo fatty acid (FA) biosynthesis, which is an oncogenic function observed in many cancers, including NHL. HIF1a inducible FASN activity is responsible for overcoming negative hypoxic influence and upregulation of glucose metabolism as observed during premalignant transformation. FASN catalytic activity is also dependent on precursors derived from glucose metabolism. However, implications of FASN targeted therapies on these interdependent metabolic interactions and the overall impact on cancer cell proliferation remains unknown. Methods: FASN small molecule inhibitors cerulenin, orlistat, TVB3657 and TVB3166 were evaluated using a diverse panel of B cell NHL lines and primary NHL cells for the impact on FASN inhibition signaling & induction of cell death (MTT, caspases & AnnexinV/PI). Global transcriptomics were done with Affymetrix Human 2.0 ST Genechip with Gene Set Enrichment and Ingenuity Pathway Analysis (GSEA & IPA). Metabolomic profiling was performed using mass spectrometry. TXNRD1, GSR, NQO1 expression and activity were evaluated by western blot and using enzyme assay kits. Global DNA & RNA synthesis were evaluated using ClickIt Edu and EU assay kits. RNAseq data available from 775 NHL patients were utilized for metabolic transcriptome mapping. Results: Treatment with all FASN inhibitors resulted in dose- and time-dependent reduction (>90%) in cell viability and cell death in all NHL cell lines. GSEA and IPA identified cell cycle & RNA metabolism as downregulated biological processes with carbohydrate and oxidative stress as upregulated biological processes observed. "Key gene" analysis predicted TNF signaling as a prominent response to FASN inhibition, validated as increased TNF secretion by ELISA with cell fractionation and western blot analysis revealing activation of TNF-PI3K signaling. Activation of PI3K with FASN inhibition also corresponded with increased expression of PI3K-dependent metabolic genes associated with glucose and lipid metabolism. Furthermore, metabolomic profiling in SUDHL10 cells revealed accumulation of the FASN precursor acetyl-CoA with FASN inhibition that was accompanied by increased ketogenic glycolytic and citric acid cycle activity. In addition, NADPH accumulation (FASN substrate) occurred with FASN inhibition, which was accompanied with reduction in ribose-phosphate and nucleotide pools as these processes are relevant to NADPH-generating PPP function. G6PD, TXNRD1, GSR & NQO1 were identified as "key genes" responsive to FASN inhibition. Interestingly, this cluster of "key genes" represented the entire enzymatic activity related to the first rate-limiting step in PPP. Subsequently, we determined that increased NADP(H) pools were responsible for impaired NADPH regenerating functions of TXNRD1 and GSR antioxidant enzymes, with PI3K-dependent activation of NQO1 and uptake of glucose facilitating de novo glutathione synthesis, which substituted for the loss of antioxidant functions in SUDHL10, SUDHL4 and Raji cells. All responses were sensitive to inhibition by PI3K inhibition (e.g., BKM120) with co-targeting via FASN & PI3K inhibition resulting in markedly increased oxidative stress, loss of mitochondrial membrane potential & synergistic cell death in NHL cell lines and primary NHL cells. Finally, FASN inhibition was associated with reduction in ribo/deoxy-ribonucleotide pools that decreased global transcriptional activity (de novo RNA synthesis, by EU labeling) and replication (by EdU incorporation in DNA) (Fig 1). Analysis of RNAseq data and mapping of metabolic transcriptome from 772 NHL patients showed consistently elevated expression of genes related to glycolysis, citric acid cycle, fatty acid and nucleotide metabolism in patients with mutations in p53, MYC, BCL2, mTOR, MYD88, PIM2 & CREBP genes, suggesting that these onco-metabolic interactions may be important for lymphomagenesis. Conclusions: Taken together, FASN oncogenic activity appears to extend beyond de novo fatty acid biosynthesis, serving as a central onco-metabolic regulator of malignant cell proliferation vis-à-vis integrating glucose, nucleotides, and antioxidant metabolic functions in NHL. In addition, co-targeting FASN and PI3K induced synergistic cell death. Altogether, blocking FASN and the dependent onco-metabolic functions represent highly novel targets for therapeutic strategies in NHL. Disclosures Chen: Oncomics, LLC: Consultancy, Patents & Royalties. Dave:Data Driven Bioscience: Equity Ownership. Evens:Seattle Genetics: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Tesaro: Research Funding; Verastem: Consultancy, Honoraria.

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.

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.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.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; p0.001), 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.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.

Objective Statins are known to reduce plasma C-reactive protein (CRP) concentrations. Our goal was to define the mechanisms by which CRP was reduced by maximal dose atorvastatin. Methods Eight subjects with combined hyperlipidemia (5 men and 3 postmenopausal women) were enrolled in a randomized, placebo-controlled double-blind, cross over study. Subjects underwent a 15-h primed-constant infusion with deuterated leucine after 8 weeks of placebo and 80 mg/day of atorvastatin. CRP was isolated from lipoprotein deficient plasma, (density > 1.21 g/ml) by affinity chromatography. Isotopic enrichment was determined by gas chromatography/mass spectrometry. Kinetic parameters were determined using compartmental modeling. Paired t test and Wilcoxon signed ranks test were used to compare differences between placebo and atorvastatin. Results Compared with placebo, atorvastatin decreased median CRP pool size by 28.4% (13.31 ± 3.78 vs 10.26 ± 3.93 mg; p = 0.16), associated with a median CRP fractional catabolic rate increase of 39.9% (0.34 ± 0.06 vs 0.50 ± 0.11 pools/day; p = 0.09), with no significant effect on median CRP production rate (0.050 ± 0.01 vs 0.049 ± 0.01 mg/kg/day; p = 0.78). Conclusion Our data indicate that maximal doses of atorvastatin lower plasma CRP levels by substantially decreasing the median CRP plasma residence time from 2.94 days to 2.0 days, with no significant effect on the median CRP production rate.

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.

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-naive 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-naive cohort (median: 2.1, 0.3, and 1.3 in controls, treatment-naive, 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.

Reverse phase HPLC coupled to negative mode electrospray ionization (ESI) mass spectrometry (MS) was used to quantify 16 flavonoids and 2 phenolic acids from almond skin extracts. Calibration curves of standard compounds were run daily and daidzein was used as an internal standard. The inter-day relative standard deviation (RSD) of standard curve slopes ranged from 13% to 25% of the mean. On column (OC) limits of detection (LOD) for polyphenols ranged from 0.013 to 1.4 pmol, and flavonoid glycosides had a 7-fold greater sensitivity than aglycones. Limits of quantification were 0.043 to 2.7 pmol OC, with a mean of 0.58 pmol flavonoid OC. Mean inter-day RSD of polyphenols in almond skin extract was 6.8% with a range of 4% to 11%, and intra-day RSD was 2.4%. Liquid nitrogen (LN(2)) or hot water (HW) blanching was used to facilitate removal of the almond skins prior to extraction using assisted solvent extraction (ASE) or steeping with acidified aqueous methanol. Recovery of polyphenols was greatest in HW blanched almond extracts with a mean value of 2.1 mg/g skin. ASE and steeping extracted equivalent polyphenols, although ASE of LN(2) blanched skins yielded 52% more aglycones and 23% less flavonoid glycosides. However, the extraction methods did not alter flavonoid profile of HW blanched almond skins. The recovery of polyphenolic components that were spiked into almond skins before the steeping extraction was 97% on a mass basis. This LC-MS method presents a reliable means of quantifying almond polyphenols.

The literature describing vitamin D content of fat tissue is extremely limited. We conducted a pilot study that measured the concentrations of vitamin D(3) in the fat tissue and serum of obese adults. These measurements were performed using a new liquid chromatography mass spectrometry (LC/MS) method. The objectives of this study were: to measure and report the vitamin D(3) concentration in serum and subcutaneous fat samples from obese individuals and to examine the association of vitamin D(3) in fat with vitamin D(3) in serum. This cross-sectional study was conducted in 17 obese men and women who were scheduled to undergo gastric bypass surgery. The mean vitamin D(3) concentration in subjects' subcutaneous fat tissue samples was 102.8 +/- 42.0 nmol/kg. The mean vitamin D(3) concentration in serum was 7.78 +/- 3.99 nmol/l. Vitamin D(3) concentrations of fat tissue and serum were positively correlated (r = 0.68, P = 0.003). Consistent with previous findings in obese subjects, subjects in this study had suboptimal vitamin D status as demonstrated by a mean 25-hydroxyvitamin D concentration of 43.3 +/- 15.4 nmol/l. In conclusion, fat tissue vitamin D(3) can be measured by LC/MS and is detectable in obese subjects with suboptimal vitamin D status. Compatible with the long-standing concept that fat tissue is a storage site for vitamin D, fat tissue and serum vitamin D(3) concentrations were positively correlated.

Cholesteryl ester transfer protein (CETP) inhibition leads to changes in lipoprotein metabolism. We studied the effect of the CETP inhibitor torcetrapib on VLDL apolipoprotein E (apoE) metabolism. Subjects, pretreated with atorvastatin (n = 9) or untreated (n = 10), received placebo followed by torcetrapib (4 weeks each). After each treatment, subjects underwent a primed-constant infusion of D(3)-leucine to determine the VLDL apoE production rate (PR) and fractional catabolic rate (FCR). Torcetrapib alone reduced the VLDL apoE pool size (PS) (-28%) by increasing the VLDL apoE FCR (77%) and leaving the VLDL apoE PR unchanged. In subjects pretreated with atorvastatin, torcetrapib increased the VLDL apoE FCR (25%) and PR (21%). This left the VLDL apoE PS unchanged but increased the VLDL apoE content, likely enhancing VLDL clearance and reducing LDL production in this group. Used alone, torcetrapib reduces the VLDL apoE PS by increasing the apoE FCR while leaving the VLDL apoE content unchanged. In contrast, torcetrapib added to atorvastatin treatment increases both the VLDL apoE FCR and PR, leaving the VLDL apoE PS unchanged. Adding torcetrapib to atorvastatin treatment increases the VLDL apoE content, likely leading to decreased conversion of VLDL to LDL, reduced LDL production, and lower levels of circulating VLDL and LDL.

Nine hypercholesterolemic and hypertriglyceridemic subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study to test the effect of atorvastatin 20 mg/day and 80 mg/day on the kinetics of apolipoprotein B-100 (apoB-100) in triglyceride-rich lipoprotein (TRL), intermediate density lipoprotein (IDL), and LDL, of apoB-48 in TRL, and of apoA-I in HDL. Compared with placebo, atorvastatin 20 mg/day was associated with significant reductions in TRL, IDL, and LDL apoB-100 pool size as a result of significant increases in fractional catabolic rate (FCR) without changes in production rate (PR). Compared with the 20 mg/day dose, atorvastatin 80 mg/day caused a further significant reduction in the LDL apoB-100 pool size as a result of a further increase in FCR. ApoB-48 pool size was reduced significantly by both atorvastatin doses, and this reduction was associated with nonsignificant increases in FCR. The lathosterol-campesterol ratio was decreased by atorvastatin treatment, and changes in this ratio were inversely correlated with changes in TRL apoB-100 and apoB-48 PR. No significant effect on apoA-I kinetics was observed at either dose of atorvastatin. Our data indicate that atorvastatin reduces apoB-100- and apoB-48-containing lipoproteins by increasing their catabolism and has a dose-dependent effect on LDL apoB-100 kinetics. Atorvastatin-mediated changes in cholesterol homeostasis may contribute to apoB PR regulation.

BACKGROUND Strategies for improving the vitamin A status of vulnerable populations are needed. OBJECTIVE We studied the influence of the amounts of dietary fat on the effectiveness of carotene-rich plant foods in improving vitamin A status. DESIGN Schoolchildren aged 9-12 y were fed standardized meals 3 times/d, 5 d/wk, for 9 wk. The meals provided 4.2 mg provitamin A carotenoids/d (mainly beta-carotene) from yellow and green leafy vegetables [carrots, pechay (bok choy), squash, and kangkong (swamp cabbage)] and 7, 15, or 29 g fat/d (2.4, 5, or 10 g fat/meal) in groups A, B, and C (n = 39, 39, and 38, respectively). Other self-selected foods eaten were recorded daily. Before and after the intervention, total-body vitamin A pool sizes and liver vitamin A concentrations were measured with the deuterated-retinol-dilution method; serum retinol and carotenoid concentrations were measured by HPLC. RESULTS Similar increases in mean serum beta-carotene (5-fold), alpha-carotene (19-fold), and beta-cryptoxanthin (2-fold) concentrations; total-body vitamin A pool size (2-fold); and liver vitamin A (2-fold) concentrations were observed after 9 wk in the 3 study groups; mean serum retinol concentrations did not change significantly. The total daily beta-carotene intake from study meals plus self-selected foods was similar between the 3 groups and was 14 times the usual intake; total fat intake was 0.9, 1.4, or 2.0 times the usual intake in groups A, B, and C, respectively. The overall prevalence of low liver vitamin A (

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.

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. 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. 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. 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.

Limited information is available concerning the qualitative and quantitative composition of polyphenolic compounds, especially flavonoids, in almonds. We determined total phenols, flavonoids, and phenolic acids in California almond (Prunus dulcis) skins and kernels among the principal almond varieties (Butte, Carmel, Fritz, Mission, Monterey, Nonpareil, Padre, and Price) with high-performance liquid chromatography (HPLC)/electrochemical detection and UV detection. Liquid chromatography/tandem mass spectrometry under identical HPLC conditions was utilized to verify identities of the predominant flavonoids and phenolic acids. Total phenols ranged from 127 (Fritz) to 241 (Padre) mg gallic acid equivalents/100 g of fresh weight. The analyses were compiled to produce a data set of 18 flavonoids and three phenolic acids. The predominant flavonoids were isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-glucoside (in combination), catechin, kaempferol-3-O-rutinoside, epicatechin, quercetin-3-O-galactoside, and isorhamnetin-3-O-galactoside at 16.81, 1.93, 1.17, 0.85, 0.83, and 0.50 mg/100 g of fresh weight almonds, respectively. Using the existing approach of calculating only the aglycone form of flavonoids for use in the U.S. Department of Agriculture nutrient database, whole almonds would provide the most prevalent aglycones of isorhamnetin at 11.70 (3.32), kaempferol at 0.60 (0.17), catechin at 1.93 (0.55), quercetin at 0.72 (0.20), and epicatechin at 0.85 (0.24) mg/100 g of fresh weight (mg/oz serving), respectively. These data can lead to a better understanding of the mechanisms of action underlying the relationship between almond consumption and health-related outcomes and provide values for whole and blanched almonds suitable for inclusion in nutrient databases.

Objective— Plasma high-density lipoprotein (HDL) cholesterol levels are inversely correlated with the risk of developing coronary heart disease. Hormonal replacement therapy (HRT) affects plasma HDL cholesterol levels, with estrogen increasing HDL cholesterol levels and progestins blunting this effect. This study was designed to assess the mechanism responsible for these effects. Materials and Methods— HDL apolipoprotein A-I (apoA-I) kinetics were studied in 8 healthy postmenopausal women participating in a double-blind, randomized, crossover study comprising 3 phases: placebo, conjugated equine estrogen (CEE) (0.625 mg/d), and CEE plus medroxyprogesterone acetate (MPA) (2.5 mg/d). Compared with placebo, treatment with CEE resulted in an increase in apoA-I pool size (+20%, P P P P Conclusion— Postmenopausal estrogen replacement increases apoA-I levels and production rate. When progestin is added to estrogen, it opposes these effects by reducing the production of apoA-I.

Objective— To determine mechanisms contributing to the altered lipoprotein profile associated with aging and menopause, apolipoprotein B-100 (apoB-100) and apoA-I kinetic behavior was assessed. Methods and Results— Eight premenopausal (25±3 years) and 16 postmenopausal (65±6 years) women consumed for 6 weeks a standardized Western diet, at the end of which a primed-constant infusion of deuterated leucine was administered in the fed state to determine the kinetic behavior of triglyceride-rich lipoprotein (TRL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB-100, and high-density lipoprotein (HDL) apoA-I. Data were fit to a multicompartmental model using SAAM II to calculate fractional catabolic rate (FCR) and production rate (PR). Total cholesterol, LDL cholesterol (LDL-C), TRL-C, and triglyceride levels were higher (50%, 55%, 130%, and 232%, respectively) in the postmenopausal compared with the premenopausal women, whereas HDL-C levels were similar. Plasma TRL, IDL, and LDL-apoB-100 levels and pool sizes (PS) were significantly higher in the postmenopausal than premenopausal women. These differences were accounted for by lower TRL, IDL, and LDL apoB-100 FCR ( P r =−0.46; P r =0.62; P r =−0.70; P Conclusions— The mechanism for the increase in TRL and LDL apoB-100 PS observed in the postmenopausal women was determined predominantly by decreased TRL and LDL catabolism rather than increased production. No differences were observed in HDL apoA-I kinetics between groups.

Current knowledge of the bioavailability of lycopene in humans is limited due to the inability to distinguish newly administered lycopene from the body reserves of lycopene. A quantitative method to assess the absorption and relative bioavailability of newly absorbed synthetic or natural lycopene was developed using two deuterated lycopene sources, in conjunction with an advanced LC/APCI-MS (liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry) to analyze newly absorbed lycopene in blood samples of study subjects. Two subjects (1 male and 1 female) consumed hydroponically grown tomatoes containing deuterium-enriched lycopene (80-84 g wet weight tomato containing 16.3 and 17.4 micromol lycopene, respectively) and two subjects (1 male, and 1 female) consumed 11 micromol synthetic (2)H(10) lycopene in 6 g of corn oil. Tomatoes were steamed and pureed. The doses were given together with a liquid formulated drink with 25% energy from fat. Our results showed that up to 34 days after taking an oral (2)H(10) lycopene dose (synthetic or from tomato) with a liquid formula drink, the area under the curve of the average serum percent enrichment response of synthetic lycopene reached 33.9 (+/-1.7) nmol-day/micromol lycopene in the dose, whereas that of lycopene from the tomato dose was 11.8 (+/-0.3) nmol-day/mumol lycopene in the dose. Our study provides evidence that the absorption of physiological levels of lycopene in intrinsically labeled tomatoes can be studied in humans. From these preliminary investigations, we find that the bioavailability of synthetic lycopene in oil appears to be about three times higher than that of lycopene from steamed and pureed tomatoes.

The metabolism of apolipoproteins (apo) (a) and B-100 within plasma lipoprotein (a) [Lp(a)] was examined in the fed state in 23 subjects aged 41 to 79 years who received a primed-constant infusion of [5,5,5-2H3] leucine over 15 hours. Lipoprotein (a) was isolated from the whole plasma using a lectin affinity-based method. Apolipoprotein (a) and apoB-100 were separated by gel electrophoresis, and tracer enrichment of each apolipoprotein was measured using gas chromatography/mass spectrometry. Data were fit to a multicompartmental model to determine fractional catabolic rates (FCRs) and secretion rates (SRs). The FCRs of apo(a) and apoB-100 (mean +/- SEM) within plasma Lp(a) were significantly different (0.220 +/- 0.030 pool/d and 0.416 +/- 0.040 pool/d, respectively; P < .001). Apolipoprotein (a) SR (0.50 +/- 0.08 mg/[kg per d]) was significantly lower than that of apoB-100 SR (1.53 +/- 0.22 mg/[kg per d]; P < .001) of Lp(a). Plasma concentrations of Lp(a) were correlated significantly with both apo(a) SR and apoB-100 SR (r = 0.837 and r = 0.789, respectively; P < .001) and negatively with apo(a) FCR and Lp(a) apoB-100 FCR (r = -0.547 and r = -0.717, respectively; P < .01). These data implicate different metabolic fates for apo(a) and apoB-100 within Lp(a) in the fed state. We therefore hypothesize that apo(a) does not remain covalently linked to a single apoB-100 lipoprotein but that it rather reassociates at least once with another apoB-100 particle, probably newly synthesized, during its plasma metabolism.

Obesity is recognized as a major public health problem. Obesity is a multifactorial disease and is often associated with a wide range of comorbidities including hypertension, non-insulin dependent (Type II) diabetes mellitus, and cardiovascular disease, all of which contribute to morbidity and mortality. This review deals with stable isotope mass spectrometric methods and the application of stable isotopes to metabolic studies of obesity. Body composition and total energy expenditure (TEE) can be measured by mass spectrometry using stable isotope labeled water, and the metabolism of protein, lipid, and carbohydrate can be measured using appropriate labeled tracer molecules.

Objective— Our purpose was to determine the relationship between apolipoprotein (apo) A-I and apoB-48 and apoB-100 metabolism in moderately hypercholesterolemic humans. Methods and Results— The kinetics of apoA-I within high-density lipoprotein (HDL), apoB-48 and apoB-100 within triglyceride-rich lipoproteins, and apoB-100 within intermediate-density lipoprotein and low density-lipoprotein (LDL) were examined with a primed constant infusion of [5,5,5- 2 H 3 ] leucine in the fed state (hourly feeding) in 23 subjects after consumption of a 36% total fat diet. Lipoproteins were isolated by ultracentrifugation; apolipoproteins by SDS-PAGE gels; and isotope enrichment assessed by gas chromatograph/mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II. ApoA-I production rate (PR) was correlated with LDL apoB-100 pool size (PS; r =0.49; P =0.017) and LDL cholesterol ( r =0.61; P =0.002), whereas apoA-I fractional catabolic rate (FCR) was inversely correlated with apoB-48 FCR ( r =−0.40; P =0.05) but not with very low-density lipoprotein apoB-100 FCR. Conclusions— Two links exist between apoA-I and apoB kinetics: 1) when LDL apoB-100 PS is high, there is increased apoA-I PR; and 2) delayed chylomicron remnant clearance (represented by apoB-48 FCR) is associated with enhanced apoA-I FCR, a finding indicating that alterations in intestinal lipoproteins may be more important in determining HDL cholesterol levels than changes in liver lipoproteins.

Objective— To determine mechanisms contributing to decreased high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein cholesterol (LDL-C) concentrations associated with hydrogenated fat intake, kinetic studies of apoA-I, apoB-100, and apoB-48 were conducted using stable isotopes. Methods and Results— Eight postmenopausal hypercholesterolemic women were provided in random order with 3 diets for 5-week periods. Two-thirds of the fat was soybean oil (unsaturated fat), stick margarine (hydrogenated fat), or butter (saturated fat). Total and LDL-C levels were highest after the saturated diet ( P P P r =−0.56, P =0.03) and LDL-C concentrations were negatively correlated with LDL apoB-100 FCR ( r =−0.48, P =0.05). Conclusions— The mechanism for the adverse lipoprotein profile observed with hydrogenated fat intake is determined in part by increased apoA-I and decreased LDL apoB-100 catabolism.

The plasma transport of stable isotope-labeled phylloquinone at physiologic doses from food was studied. A single bolus of 100 g (396 +/- 28 microg phylloquinone) deuterium-labeled collard greens was fed with a breakfast containing 24 g fat to 5 men (26 to 71 years). Eleven blood samples were obtained over 216 hours. Phylloquinone concentrations in plasma and lipoprotein subfractions were measured using high-performance liquid chromatography (HPLC), and the ion abundances of deuterated and endogenous phylloquinone were determined using gas chromatography/mass spectrometry (GC/MS). Plasma total phylloquinone concentrations peaked at 6 to 9 hours (10.51 +/- 4.38 to 8.30 +/- 4.64 nmol/L) and returned to baseline by 24 hours (1.26 +/- 0.38 nmol/L). The triglyceride-rich lipoprotein (TRL) fraction was the major carrier of phylloquinone; low-density lipoprotein (LDL) and high-density lipoprotein (HDL) fractions contained smaller amounts. Maximum enrichment of plasma and TRL phylloquinone with deuterium (88% and 89%, respectively) was reached at 6 hours, respectively; t(1/2) was 22.8 hours (n = 3). Deuterated-phylloquinone was not detectable in plasma or TRL fraction at 72 hours. These results suggest rapid uptake and transport of physiologic doses of phylloquinone.

The aim of the investigation was to assess a stable isotope method for determining the relative bioavailability of food-derived lutein in humans. Subjects were administered a single dose of deuterium-labeled carotenoids from intrinsically labeled spinach or collard green; 10 mL blood samples were drawn at various time points over a 34 days period. The vegetables had been hydroponically grown using 25 atom-% deuterated water. Lutein molecules in the vegetables were partially deuterated with a highest abundance isotopomer at M 0 + 8 (unlabeled molecular mass, M 0, plus 8 additional mass units from 8 deuterium atoms in the molecules). This allowed labeled lutein to be distinguished from endogenous lutein in serum samples after consuming the labeled meal. The presence of labeled lutein in the circulation was determined by liquid chromatography-mass spectrometry (LC/MS) equipped with an atmospheric pressure chemical ionization (APCI) interface. The quantification of the labeled lutein in serum samples enabled the calculation of the enrichment for each time point after the dose; these values were plotted vs. time to generate absorption-clearance curves for each of the subjects. Area under the curve analyses of four different subjects (integrated over 29 days) yielded serum lutein responses of 128, 145, 149, and 262 μg-day/mg dietary lutein, following an acute dose of spinach containing 15.4, 18.8, 18.8 and 9.8 mg labeled lutein, respectively. This technique will facilitate the study of lutein bioavailability from different foods of diverse carotenoid composition and/or following various food preparation procedures.

Elder adulthood and diminished folate status are each associated with an enhanced risk of colorectal carcinogenesis. We therefore examined whether these two factors are mechanistically related. Weanling male Sprague-Dawley rats (n = 44) and 1-y-old rats (n = 44) were each divided into three groups and fed diets containing 0, 4.5 or 18 micro mol folic acid/kg (deplete, replete and supplemented groups, respectively). Rats were killed at 0, 8 and 20 wk. The folate concentrations, the distribution of the different coenzymatic forms of folate, uracil incorporation into DNA and genomic DNA methylation were measured in the colonic mucosa. Folate-deplete and folate-replete elder rats had 30-45% lower colonic folate concentrations than young rats. Furthermore, 5-methyltetrahydrofolate was uniformly depleted in colons of the elder, folate-deplete rats, whereas this depletion occurred in only a minority of the younger rats. By the end of the experiment, the folate-deplete and folate-replete elder rats had approximately 50% more uracil incorporated into their colonic DNA than the corresponding young groups (P < 0.05). In elder rats, this uracil misincorporation was incremental across the three diet groups (P-test for trend < 0.05), whereas no excess uracil incorporation was observed in young rats. Neither age nor dietary folate affected genomic DNA methylation in the colon. In conclusion, the colon of elder rats is more susceptible to biochemical and molecular consequences of folate depletion than that of young rats. However, folate supplementation is as effective at sustaining adequate colonic folate status in elder rats as it is in the young.

Eukaryotic DNA is methylated at some cytosine residues, and this epigenetic feature performs critical functions. We developed a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The DNA was enzymatically hydrolyzed by sequential digestion with three enzymes. DNA hydrolyzates were subsequently separated by reversed-phase high-performance liquid chromatography in isocratic mode. The four major DNA bases and 5-methyl-2'-deoxycytidine were resolved and eluted in 13 min. Identification of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine was obtained by combined diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomers [15N3]-2'-deoxycytidine and (methyl-d3,ring-6-d1)-5-methyl-2'-deoxycytidine were used as internal standards. Ions of m/z 126 and 130 were used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 were used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status was calculated on the basis of the amount of 5-methyl-2'-deoxycytidine per microgram of DNA with percent relative standard deviations (%RSD) for a method precision of 7.1 (within-day) and 5.7 (day-to-day). This method also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with %RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). This LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective, and precise.

Objective: To examine total energy expenditure (TEE) in relation to occupation and reported leisure time activities in free-living Chinese adults, and to determine whether measured TEE values differ from current international dietary energy recommendations. Setting and subjects: Seventy three weight-maintaining adults aged 35–49 y, leading unrestricted lives in urban Beijing, with a wide variety of occupations. Design and methods: A cross-sectional study in which TEE was determined by doubly labeled water, body composition by deuterium oxide (2H2O) dilution, resting energy expenditure (pREE) by prediction equations, and occupational and leisure time activities by questionnaire. Results: For men and women respectively, TEE averaged 12.10±0.32 and 9.53±0.23 MJ/day (P

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S -adenosyl- l -methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S -adenosyl- l -methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation ( T / T ) and 187 homozygous for the wild-type ( C / C ) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels ( P < 0.01). T / T genotypes had a diminished level of DNA methylation compared with those with the C / C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T / T subjects with low levels of folate accounted for the diminished DNA methylation ( P < 0.0001). Moreover, in T / T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates ( P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status.