The hormonally active form of vitamin D, 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], has been associated with neuroprotective effects in the brain, but has been difficult to measure in human brain tissue because of its low concentration. The aim of this study was to develop and validate a sensitive method to quantify 1,25(OH)2D3 in the human brain. Prior to analysis by the LC-MS/MS, the samples were derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione. The method showed good linearity of 1,25(OH)2D3 over the physiological range (R2 = 0.9998). The limit of detection was 2.5 pg/g, >10 times lower than the previously reported limit of detection. The average 1,25(OH)2D3 concentrations in 3 regions of human brain tissue samples were: anterior watershed 30.7 pg/g; mid-temporal cortex 19.2 pg/g; and cerebellum 18.5 pg/g. This validated method to quantify 1,25(OH)2D3 in human brain tissue can be applied to obtain information about its presence in various regions of the human brain associated with neurodegenerative diseases.

Background: Dietary stearic acid (18:0), a saturated fatty acid (SFA) commonly present in Western diets, has an LDL-C lowering effect compared to shorter chain SFAs such as palmitic acid (16:0), and a similar effect compared to oleic acid (18:1). However, the underlying mechanisms remain unclear. Hypothesis: We tested the hypothesis that the hypocholesterolemic effect of dietary 18:0 and 18:1 relative to 16:0 is modulated by alterations in cholesterol and bile acid (BA) metabolism. Methods: This secondary analysis used archived plasma and fecal samples from a randomized crossover feeding study (N=20 mildly hypercholesteremic postmenopausal women, 64±7 years, BMI 26.4±3.4kg/m 2 ). Participants consumed each of 3 isocaloric diets enriched in either 18:0, 16:0 or 18:1 for five weeks with a 2-week washout. Primary (P) and secondary (S) BAs, and their conjugates were measured in fecal, fasting and non-fasting (NF) plasma samples using the Biocrates MxP Quant 500 kit and Quadrupole Time-of-Flight mass spectrometry. Fasting and NF plasma cholesterol synthesis (lathosterol) and absorption (-sitosterol) markerswere quantified using gas chromatography. Mixed-effect and generalized linear mixed models were used to test the difference in outcome measures among diets, with Tukey-Kramer post hoc comparison. Spearman correlation coefficients with FDR adjustment was calculated between BA, cholesterol synthesis/absorption markers, and CVD risk factors. Results: Compared to the 16:0 diet, consumption of the 18:0 diet resulted in significantly lower fasting and NF plasma lathosterol (-22%); higher -sitosterol (19%); higher fecal PBAs (31%) and lower fecal SBAs (-17%) concentrations. Plasma PBAs were significantly lower in the fasted state (-34%), but higher in the NF state (21%; 18:0 vs. 16:0). Interestingly, conjugated PBA and SBA concentrations in the NF state were significantly higher after participants consumed the 18:0 compared to the 18:1 diet (all p < 0.05). Plasma NF PBAs were positively associated with -sitosterol (r=0.56, p < 0.05), while plasma fasting PBAs were negatively associated with lathosterol (r=-0.58, p < 0.05). Conjugated PBA and SBA concentrations were negatively associated with LDL-C, hsCRP, E-selectin and insulin concentrations in the fasted but not NF state. Conclusion: The favorable effects of 18:0 on CVD risk factors may be modulated, in part, by changes in BA and cholesterol metabolism, with distinct effects in the fasted and NF states.

When subject to damage or stress, cells develop responses in order to maintain tissue homeostasis. Two such decisions are ferroptosis and cellular senescence, but how cells decide between these outcomes remains unclear. Here we show that senescent cells increase levels of multiple membrane bound polyunsaturated fatty acids (PUFAs), but a specific PUFA, dihomo−gamma−linolenic acid (DGLA, 20:3−ω3) is reduced. Exogenous repletion of DGLA or inhibition of the enzyme that metabolizes DGLA, delta−5−desaturase, instead results in cell death by ferroptosis. Senescent cells also show elevated levels of other ferroptosis sensitizers, including labile iron and expression of lipoxygenases − but also increase Gpx4 levels. Oral administration of exogenous DGLA lowers senescent cell burden in aged mice and improves age−related functional outcomes. Finally, obese humans with lowered DGLA desaturation rates showed lower markers of adipose tissue senescence. Together, our data implicate DGLA and its desaturation as a major driver of decisions between senescence and ferroptosis. ### Competing Interest Statement CW is an inventor on patents related to the elimination of senescent cells using DGLA, D5D inhibitors, and other ferroptosis inducers. The content is the sole responsibility of the authors and does not necessarily represent the official views of the USDA.

Nonalcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is expected to increase globally in the upcoming years. Diets high in refined carbohydrates and added sugars (HRCD) have been linked to NAFLD progression. Previous studies have demonstrated that retinoids possess anti-tumorigenic properties, yet less is known about whether upstream provitamin A carotenoids exhibit similar characteristics. We recently demonstrated that dietary beta-cryptoxanthin (BCX), a provitamin A carotenoid, decreased NAFLD severity and HCC progression in diethylnitrosamine (DEN)-initiated, high refined carbohydrate diet (HRCD)-fed mice. In this study, we evaluated the effects of dietary BCX supplementation on modulating plasma biomarkers, hepatic vitamin A status, and plasma metabolomic profiles in our DEN-initiated, HRCD-promoted model of HCC. Male, six-week-old C57BL/6J mice were injected with DEN (25 mg/kg BW) and a fed a HRCD (66.5% carbs including sucrose) with or without BCX supplementation (10 mg/kg diet) for 24 weeks. Using plasma samples, we performed metabolomics using our UHPLC-QTOF-MS platform, a targeted Biocrates MxP Quant 500 kit, and a complimentary untargeted analysis of phospholipids from the METLIN database to identify metabolites which correlate with primary outcomes. Hepatic vitamin A levels were determined via HPLC. Compared to HRCD-fed, DEN-injected mice, mice fed BCX displayed significantly lower tumor burden and steatosis, higher hepatic vitamin A status, and lower plasma levels of alanine transaminase (ALT) and lactate dehydrogenase (LDH). Moderate negative correlations were also identified amongst hepatic vitamin A levels and primary outcomes, such as HCC tumor number, steatosis score, and relevant biomarkers. Our metabolomics data showed significant modulations in metabolites with BCX treatment from phosphatidylcholine, sphingomyelin, ceramide, bile acid, and other phospholipid classes. BCX supplementation significantly reduced HCC severity, increased hepatic vitamin A status, reduced plasma levels of ALT and LDH, and altered several lipid-related classes of metabolites, thus supporting a chemopreventive potential for BCX against HRCD-promoted HCC development. USDA/ARS and NIFA/AFRI.

β-Carotene-15,15′-oxygenase (BCO1) and β-carotene-9′,10′-oxygenase (BCO2) cleave carotenoids to form vitamin A or apo-carotenoid species. Beyond these roles, BCO1 and BCO2 have been implicated in modulating lipid, cholesterol, and oxidative stress-related processes, yet less is known about their combined influence on shaping gut microbial and plasma metabolic profiles. Current intake of refined carbohydrates and added sugars exceeds the recommendations within the Dietary Guidelines for Americans, which may contribute to the development of nonalcoholic fatty liver disease (NAFLD). In the present study, we aimed to uncover whether mice with systemic ablation of BCO1-/-/BCO2-/- (DKO) display distinctive phenotypes in response to a high refined carbohydrate diet (HRCD) model of NAFLD. Male, six-week-old C57BL/6J wild type mice (WT) and DKO mice were fed a chow diet or a HRCD (66.5% carbs including sucrose) for 24 weeks. We used fecal and plasma samples to respectively perform 16S rDNA sequencing, and both targeted and untargeted metabolomics using our UHPLC-QTOF-MS platform, a targeted Biocrates MxP Quant 500 kit, and a complimentary untargeted analysis of phospholipids from the METLIN database. Data were analyzed using QIIME2-DEseq2, MetaboAnalyst 5.0, MassHunter Qualitative Analysis, and MicrobiomeAnalyst. HRCD-fed WT and DKO mice developed similar levels of hepatic steatosis although DKO mice had significantly higher hepatic levels of malondialdehyde (MDA). Interestingly, in both chow-fed and HRCD-fed DKO mice, plasma adiponectin and hepatic bile acids were significantly lower compared to respective WT groups. Our metabolomics data revealed that DKO mice displayed significant alterations in ceramide, cholesterol ester, triglyceride, and phospholipid classes under both chow and HRCD conditions. Additionally, when compared to HRCD-fed WT mice, HRCD-fed DKO mice displayed significantly reduced alpha diversity and possessed significant compositional differences based on Bray Curtis dissimilarity measures. BCO1/BCO2 DKO mice display altered metabolomic and gut microbial profiles. Carotenoid cleavage enzymes affect diverse physiological processes, which involve lipid metabolism and the gut microbiome. USDA/ARS and NIFA/AFRI.

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.

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.

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. 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 −80o 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. 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). 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. Supported by the National Institute of Aging.

RationaleConsumption 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.MethodsWe 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.ResultsSeparation 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).ConclusionsThis 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.

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.

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<0.05), hsCRP (r=0.31 to 0.37, p<0.02), TG (r=0.46 to 0.60; p<0.001), VLDL-C (r=0.47 to 0.63; p<0.001), TC/LDL-C (r=0.42 to 0.63; p<0.001) and LDL-C/HDL-C (0.37 to 0.57; p<0.001) ratios, and negatively with HDL-C (r= -0.51 to -0.44; p<0.001) levels. LA levels were also positively correlated with LDL-C levels (r=0.33; p=0.011). 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<0.05) after subjects consumed the unrefined (124.8±55.8 pmol/mL) compared to the simple (29.5±10.3 pmol/mL) and refined (26.8±9.0 pmol/mL) diets. C21:0 and C19:0 were the major ARs present in plasma. 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.

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.

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.

Background: Low serum total 25-hydroxyvitamin D-3 [25(OH)D-3] concentrations have been associated with cognitive impairment. However, it is unclear if serum 25(OH)D-3 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 D-3, 25(OH)D-3, and 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] in human brain. Methods: The assay developments were performed using porcine brains. Liquid extraction was used in homogenized samples (similar to 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 D-3, 25(OH)D-3, and 1,25(OH)(2)D-3 over the physiological range (R-2 = 0.9995, 0.9968, and 0.9970, respectively). The lowest detection limit for vitamin D-3, 25(OH)D-3, and 1,25(OH)(2)D-3 in porcine brain was 25, 50 and 25 pg/g, respectively. The method was successfully applied to the determination of vitamin D-3 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)D-3, with corpus callosum containing 334 pg/g compared with 158 pg/g in cerebellum. 1,25(OH)(2)D-3 was only detected in prefrontal and middle frontal cortices at a very low level. No vitamin D-3 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.

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. 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 < 0.001. The average concentrations of vitamin D3, 25(OH)D and MK4 were 604 pg/g, 535 pg/g, and 3.4 pmol/g, respectively. 25(OH)D and MK4 were detected in >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 < 0.0001). The 1,25(OH)2D was significantly correlated between the MF and CR regions only (Spearman r = 0.30, P < 0.001, all other P ≥ 0.002). MK4 and PK were positively correlated across the four regions studied (MK4 all Spearman r ≥ 0.78, phylloquinone r ≥ 0.49, all P < 0.001). To the best of our knowledge, this study is the first evaluation of the concentrations of vitamin D and vitamin K forms in multiple regions of the human brain. Overall, the vitamin D and vitamin K forms were each positively correlated across the four brain regions studied. Future studies are needed to clarify the roles of these nutrients in AD and dementia. National Institute of Aging.

Supported by the National Institute of Aging.

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.

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.