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

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

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

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

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

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

Identification of methylated metabolites of oat avenanthramides in human plasma using UHPLC QToF-MS.

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.

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

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

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

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

Aging and alcohol interact to alter hepatic DNA hydroxymethylation.

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

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

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

Retinaldehyde represses adipogenesis and diet-induced obesity.

The metabolism of vitamin A and the diverse effects of its metabolites are tightly controlled by distinct retinoid-generating enzymes, retinoid-binding proteins and retinoid-activated nuclear receptors. Retinoic acid regulates differentiation and metabolism by activating the retinoic acid receptor and retinoid X receptor (RXR), indirectly influencing RXR heterodimeric partners. Retinoic acid is formed solely from retinaldehyde (Rald), which in turn is derived from vitamin A. Rald currently has no defined biologic role outside the eye. Here we show that Rald is present in rodent fat, binds retinol-binding proteins (CRBP1, RBP4), inhibits adipogenesis and suppresses peroxisome proliferator-activated receptor-gamma and RXR responses. In vivo, mice lacking the Rald-catabolizing enzyme retinaldehyde dehydrogenase 1 (Raldh1) resisted diet-induced obesity and insulin resistance and showed increased energy dissipation. In ob/ob mice, administrating Rald or a Raldh inhibitor reduced fat and increased insulin sensitivity. These results identify Rald as a distinct transcriptional regulator of the metabolic responses to a high-fat diet.

A Sensitive Method for Determination 1,25-Dihydroxyvitamin D3 in Human Brain using Ultra-Pressure Liquid Chromatography Tandem Mass Spectrometry.

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 (R 2 = 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.

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

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

Effects of CETP inhibition on triglyceride-rich lipoprotein composition and apoB-48 metabolism.

Cholesteryl ester transfer protein (CETP) facilitates the transfer of HDL cholesteryl ester to triglyceride-rich lipoproteins (TRL). This study aimed to determine the effects of CETP inhibition with torcetrapib on TRL composition and apoB-48 metabolism. Study subjects with low HDL cholesterol (<40 mg/dl), either untreated (n = 9) or receiving atorvastatin 20 mg daily (n = 9), received placebo for 4 weeks, followed by torcetrapib 120 mg once daily for the next 4 weeks. A subset of the subjects not treated with atorvastatin participated in a third phase (n = 6), in which they received torcetrapib 120 mg twice daily for an additional 4 weeks. At the end of each phase, all subjects received a primed-constant infusion of [5,5,5-(2)H(3)]L-leucine, while in the constantly fed state, to determine the kinetics of TRL apoB-48 and TRL composition. Relative to placebo, torcetrapib markedly reduced TRL CE levels in all groups (≥-69%; P < 0.005). ApoB-48 pool size (PS) and production rate (PR) decreased in the nonatorvastatin once daily (PS: -49%, P = 0.007; PR: -49%, P = 0.005) and twice daily (PS: -30%, P = 0.01; PR: -27%, P = 0.13) cohorts. In the atorvastatin cohort, apoB-48 PS and PR, which were already lowered by atorvastatin, did not change with torcetrapib. Our findings indicate that CETP inhibition reduced plasma apoB-48 concentrations by reducing apoB-48 production but did not have this effect in subjects already treated with atorvastatin.

Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and ß-cryptoxanthin by ferret carotene-9',10'-monooxygenase.

Xanthophyll carotenoids, such as lutein, zeaxanthin and ß-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,15'-monooxygenase (CMO1) has been shown to be involved in vitamin A formation, while recent studies suggest that carotene-9',10'-monooxygenase (CMO2) may have a broader substrate specificity than previously recognized. In this in vitro study, we investigated baculovirus-generated recombinant ferret CMO2 cleavage activity towards the carotenoid substrates zeaxanthin, lutein and ß-cryptoxanthin. Utilizing HPLC, LC-MS and GC-MS, we identified both volatile and non-volatile apo-carotenoid products including 3-OH-ß-ionone, 3-OH-α-ionone, ß-ionone, 3-OH-α-apo-10'-carotenal, 3-OH-ß-apo-10'-carotenal, and ß-apo-10'-carotenal, indicating cleavage at both the 9,10 and 9',10' carbon-carbon double bond. Enzyme kinetic analysis indicated the xanthophylls zeaxanthin and lutein are preferentially cleaved over ß-cryptoxanthin, indicating a key role of CMO2 in non-provitamin A carotenoid metabolism. Furthermore, incubation of 3-OH-ß-apo-10'-carotenal with CMO2 lysate resulted in the formation of 3-OH-ß-ionone. In the presence of NAD(+), in vitro incubation of 3-OH-ß-apo-10'-carotenal with ferret hepatic homogenates formed 3-OH-ß-apo-10'-carotenoic acid. Since apo-carotenoids serve as important signaling molecules in a variety of biological processes, enzymatic cleavage of xanthophylls by mammalian CMO2 represents a new avenue of research regarding vertebrate carotenoid metabolism and biological function.

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

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

Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry.

Phylloquinone (vitamin K(1)) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC-APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC-APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC-APCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies.

Gender-specific differences in the kinetics of nonfasting TRL, IDL, and LDL apolipoprotein B-100 in men and premenopausal women.

OBJECTIVE: To investigate mechanisms underlying gender differences in serum lipoprotein concentrations, the kinetic behavior of apoB-100 was assessed. METHODS AND RESULTS: Twenty subjects (<50 years; 12 men and 8 premenopausal women) were provided a Western diet for 4 to 6 weeks, after which the kinetics of apoB-100 in triglyceride-rich, intermediate-density, and low-density lipoprotein (TRL, IDL, and LDL) were determined in the fed state. Nonfasting plasma TC, LDL-C, and triglyceride concentrations were 23%, 34%, and 57% lower, respectively, in the women compared with men. Plasma TRL and LDL apoB 100 pool sizes were lower by 40% and 30%, respectively. These differences were accounted for by higher TRL and LDL apoB 100 fractional catabolic rates (FCR), rather than differences in production rates (PR). Plasma TRL-C and LDL-C were positively correlated with TRL and LDL apoB 100 concentrations and pool size, and negatively correlated with TRL and LDL apoB 100 FCR (women: r=-0.59, P<0.01 and r=-0.54, P<0.04, and men: r=-0.43, P<0.05 and r=-0.44, P<0.05). No significant associations were observed between plasma TRL-C and LDL-C and PR. CONCLUSIONS: These data suggest the mechanism for lower TRL-C and LDL-C concentrations in women was determined predominantly by higher TRL and LDL FCR rather than lower PR. This could explain, in part, the lower CVD risk in premenopausal women relative to men.

Extended-release niacin alters the metabolism of plasma apolipoprotein (Apo) A-I and ApoB-containing lipoproteins.

OBJECTIVE: Extended-release niacin effectively lowers plasma TG levels and raises plasma high-density lipoprotein (HDL) cholesterol levels, but the mechanisms responsible for these effects are unclear. METHODS AND RESULTS: We examined the effects of extended-release niacin (2 g/d) and extended-release niacin (2 g/d) plus lovastatin (40 mg/d), relative to placebo, on the kinetics of apolipoprotein (apo) A-I and apoA-II in HDL, apoB-100 in TG-rich lipoproteins (TRL), intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL), and apoB-48 in TRL in 5 men with combined hyperlipidemia. Niacin significantly increased HDL cholesterol and apoA-I concentrations, associated with a significant increase in apoA-I production rate (PR) and no change in fractional catabolic rate (FCR). Plasma TRL apoB-100 levels were significantly lowered by niacin, accompanied by a trend toward an increase in FCR and no change in PR. Niacin treatment significantly increased TRL apoB-48 FCR but had no effect on apoB-48 PR. No effects of niacin on concentrations or kinetic parameters of IDL and LDL apoB-100 and HDL apoA-II were noted. The addition of lovastatin to niacin promoted a lowering in LDL apoB-100 attributable to increased LDL apoB-100 FCR. CONCLUSIONS: Niacin treatment was associated with significant increases in HDL apoA-I concentrations and production, as well as enhanced clearance of TRL apoB-100 and apoB-48.

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

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

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

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

Asymmetric cleavage of beta-carotene yields a transcriptional repressor of retinoid X receptor and peroxisome proliferator-activated receptor responses.

beta-Carotene and its metabolites exert a broad range of effects, in part by regulating transcriptional responses through specific nuclear receptor activation. Symmetric cleavage of beta-carotene can yield 9-cis retinoic acid (9-cisRA), the natural ligand for the nuclear receptor RXR, the obligate heterodimeric partner for numerous nuclear receptor family members. A significant portion of beta-carotene can also undergo asymmetric cleavage to yield apocarotenals, a series of poorly understood naturally occurring molecules whose biologic role, including their transcriptional effects, remains essentially unknown. We show here that beta-apo-14'-carotenal (apo14), but not other structurally related apocarotenals, represses peroxisome proliferator-activated receptors (PPAR) and RXR activation and biologic responses induced by their respective agonists both in vitro and in vivo. During adipocyte differentiation, apo14 inhibited PPARgamma target gene expression and adipogenesis, even in the presence of the potent PPARgamma agonist BRL49653. Apo14 also suppressed known PPARalpha responses, including target gene expression and its known antiinflammatory effects, but not if PPARalpha agonist stimulation occurred before apo14 exposure and not in PPARalpha-deficient cells or mice. Other apocarotenals tested had none of these effects. These data extend current views of beta-carotene metabolism to include specific apocarotenals as possible biologically active mediators and identify apo14 as a possible template for designing PPAR and RXR modulators and better understanding modulation of nuclear receptor activation. These results also suggest a novel model of molecular endocrinology in which metabolism of a parent compound, beta-carotene, may alternatively activate (9-cisRA) or inhibit (apo14) specific nuclear receptor responses.