Nutritional Sciences

Background Intake of an edible blue-green alga Nostoc commune var. sphaeroides Ku¨tzing (N. Commune) has been shown to lower plasma total cholesterol concentration, but the mechanisms behind the hypocholesterolemic effect have not been elucidated. Aim of the study To elucidate the mechanisms underlying the cholesterol-lowering effect of N. commune in mice. Methods Male C57BL/6J mice were fed the AIN-93 M diet supplemented with 0 or 5% (wt/wt) dried N. Commune for 4 weeks. Lipid levels in the plasma and liver, intestinal cholesterol absorption and fecal sterol excretion were measured. Expression of hepatic and intestinal genes involved in cholesterol metabolism was evaluated by quantitative realtime PCR. Results N. commune supplementation significantly reduced total plasma cholesterol and triglyceride concentrations by * 20% compared to controls. Intestinal cholesterol absorption was significantly decreased, while fecal neutral sterol output was significantly increased in N. communefed mice. mRNA levels of the cholesterol transporters such as Niemann Pick C1 Like 1, scavenger receptor class B type 1, ATP-binding cassette transporters G5 and A1 in small intestine were not significantly different between two groups. Hepatic lipid contents including total cholesterol, triglyceride and free cholesterol in N. commune-fed mice were not significantly altered. However, the expression of cholesterol modulating genes including sterol regulatory element binding protein-2 and 3-hydroxy-3-methylglutaryl coenzyme A reductase were significantly increased in mice fed N. commune. Conclusions N. commune supplementation exerted a hypocholesterolemic effect in mice, largely in part, by reducing intestinal cholesterol absorption and promoting fecal neutral sterol excretion.

Background Adipocyte fatty acid binding protein (A-FABP) present in macrophages has been implicated in the integration of lipid metabolism and inflammatory response, contributing to development of insulin resistance and atherosclerosis. Aim of the study This study was conducted to test the hypothesis that the role of fatty acids in the inflammatory pathways is mediated through the modulation of A-FABP expression in macrophages. Methods Murine RAW 264.7 macrophages were treated with inflammatory insults and fatty acids for quantitative real-time PCR and Western blot analysis. The cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor, for elucidating mechanisms for the regulation of A-FABP expression by fatty acids. RNA interference (RNAi) to knock down A-FABP was utilized to assess its role in inflammatory gene expression. Results When RAW 264.7 were incubated with lipopolysaccharides (LPS; 100 ng/ml) or 2.5 ng/ml of tumor necrosis factor a for 18 h, A-FABP mRNA and protein levels were drastically increased. Unsaturated fatty acids (100 lmol/l in complexed with BSA) such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid, significantly repressed the basal as well as LPS-induced A-FABP expression, whereas palmitic acid did not elicit the same effect. TSA increased A-FABP mRNA levels and abolished the repressive effect of linoleic acid on A-FABP expression in unstimulated and LPS-stimulated macrophages. Depletion of A-FABP expression by 70-80% using RNAi markedly decreased cyclooxygenase 2 mRNA abundance and potentiated the repression by linoleic acid. Conclusion Unsaturated fatty acids inhibited the basal as well as LPS-induced A-FABP expression. The mechanism may involve histone deacetylation and anti-inflammatory effect of unsaturated fatty acids may be at least in part attributed to their repression of A-FABP expression in RAW 264.7 macrophages.

High density lipoprotein (HDL) is an atheroprotective element which is critical in the reverse cholesterol transport (RCT) pathway to eliminate excess cholesterol from the periphery of the body. A low plasma HDL cholesterol levels indicate a higher risk of getting cardiovascular diseases (CVD). Formation and metabolism of HDL largely rely on ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1) that are transporters to facilitate the efflux of cellular cholesterol to outside of cells. ABCA1 in the liver and small intestine is the major contributing factor for HDL formation. On the other hand, ABCA1 and ABCG1 present in macrophages contribute to cellular cholesterol homeostasis and protection against foam cell formation in artery. The objective of the study was to elucidate the regulatory mechanism for the expression of ABCA1 and ABCG1by fatty acids in HepG2 (a human hepatoma cell line), FHs 74 Int (a human small intestine epithelial cell line) and RAW264.7 macrophages. In all these cell lines, unsaturated fatty acids, but not saturated fatty acids, repressed the expression of ABCA1 and ABCG1 at the transcriptional and/or post-transcription levels in a cell type specific manner. The results of this study suggest the following mechanisms for the repressive effect of unsaturated fatty acids on the ABCA1 and ABCG1 expression: 1) ABCA1 in iii iii HepG2 and FHs 74 Int cells is inhibited by unsaturated fatty acids at the posttranscriptional level via calpain-independent mechanism; 2) ABCA1 and ABCG1 in RAW264.7 macrophages are repressed by unsaturated fatty acids at the transcriptional level by a liver X receptor-dependent mechanism and at the post-transcriptional level by increasing protein degradation. iv iv ACKNOWLEDGEMENTS The journey to this thesis contains hard work, excitement, expectation and frustration. It would never happen without generous help from different people. It is my pleasure to take this golden opportunity to thank every individual who makes this journey possible. First and foremost, I would like to express my sincere gratitude and appreciation to my respectable and honorable advisor, Dr. Ji-Young Lee for her continuous guidance and effort throughout my master program. Her constructive suggestions and invaluable insights have greatly impacted on my research works. Without her help and support, both morally ad financially, this thesis would not be possible. Besides that, I also would like to thank Drs. Timothy Carr and Janos Zempleni for their patience and efforts of being my thesis committee. Apart from that, I also would like to address special thanks to my lab members, and students from Dr. Carr's lab, especially my lab manager Young-Ki Park for his generous assistance in solving my daily experiment problems. Their advice and suggestion have greatly improved the speed and quality of my research. It has been crucial for me to complete the research works in time with their enthusiastic help and inspiration. Last but not least, I would like to thank my family (my parents and sisters) and my significant other, Chia-Yiaw Chong, for their love and consistent support, both mentally and financially. Without their encouragement and kind words, the thesis works would have been much more difficult. v v

Background Adipocyte fatty acid binding protein (A-FABP) present in macrophages has been implicated in the integration of lipid metabolism and inflammatory response, contributing to development of insulin resistance and atherosclerosis. Aim of the study This study was conducted to test the hypothesis that the role of fatty acids in the inflammatory pathways is mediated through the modulation of A-FABP expression in macrophages. Methods Murine RAW 264.7 macrophages were treated with inflammatory insults and fatty acids for quantitative real-time PCR and Western blot analysis. The cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor, for elucidating mechanisms for the regulation of A-FABP expression by fatty acids. RNA interference (RNAi) to knock down A-FABP was utilized to assess its role in inflammatory gene expression. Results When RAW 264.7 were incubated with lipopolysaccharides (LPS; 100 ng/ml) or 2.5 ng/ml of tumor necrosis factor a for 18 h, A-FABP mRNA and protein levels were drastically increased. Unsaturated fatty acids (100 lmol/l in complexed with BSA) such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid, significantly repressed the basal as well as LPS-induced A-FABP expression, whereas palmitic acid did not elicit the same effect. TSA increased A-FABP mRNA levels and abolished the repressive effect of linoleic acid on A-FABP expression in unstimulated and LPS-stimulated macrophages. Depletion of A-FABP expression by 70-80% using RNAi markedly decreased cyclooxygenase 2 mRNA abundance and potentiated the repression by linoleic acid. Conclusion Unsaturated fatty acids inhibited the basal as well as LPS-induced A-FABP expression. The mechanism may involve histone deacetylation and anti-inflammatory effect of unsaturated fatty acids may be at least in part attributed to their repression of A-FABP expression in RAW 264.7 macrophages.

Objectives We determined if multivitamin supplementation (MV) affected serum folate levels in women during a 12-week intervention, and if genotypes affected these outcomes. Methods Women (21–40 years old) were recruited from the local community. PRE testing occurred between 0600–0900 AM 5 ± 2 days following each participants’ last menstrual cycle, and involved a buccal cell collection and a fasted blood draw. Participants were then randomly assigned in a double-blinded fashion to the MV group (n = 43) or placebo group (n = 51). MV (per 2 beadlets-in-capsule serving) contained 50 mcg vitamin D3, 6.7 mg alpha tocopherol of vitamin E as mixed tocopherols, 90 μg vitamin K2 MK7, 600 μg 5-methyltetrahydrofolate, 8 μg B12, 8 mg iron, 50 mg magnesium, 1 mg boron, and 320 mg omega-3 fatty acids. The placebo capsules contained safflower oil and cellulose. Participants consumed two capsules per day with breakfast for 12 weeks. Following the intervention, participants reported to the laboratory...

Dietary protein and fiber have been shown to independently improve subjective measures of appetite control. The aim of this study was to determine the acute effects of a high-protein, high-fiber (HP/HFb) beverage taken as a preload compared with an isocaloric lower-protein, lower-fiber (LP/LFb) placebo beverage on subjective appetite ratings and subsequent energy intake at an ad libitum meal in healthy adults. A total of 50 overweight/obese men and women [ = 25 men, 25 women; age 30 ± 2 y; body mass index (BMI) 29.6 ± 0.3 kg/m] received a 160 kcal HP/HFb beverage containing 17 g protein and 6 g fiber on one occasion and an isocaloric LP/LFb placebo beverage containing 1 g protein and 3 g fiber on another occasion in a randomized, double-blind, crossover design. Thirty min after consumption of the beverage preload, an ad libitum pizza meal was provided to be consumed over a 30-min period. Visual analog scales (VAS) were used to assess subjective appetite ratings throughout the testin...

The purpose of this study was to validate the efficacy of a customized vitamin-mineral supplement on blood biomarkers in pre-menopausal females. Women (21–40 years old) who were apparently healthy were recruited from the local community (ClinicalTrials.gov trial registration NCT03828097). Pretesting (PRE) occurred in the morning 5 ± 2 days following each participant's menses and involved a fasted blood draw, body mass assessment, and blood pressure assessment. Participants were then randomly assigned in a double-blinded fashion to either the multivitamins (MV) (n = 43) or placebo group (n = 51). Participants consumed two capsules per day with breakfast for 12 weeks. Following the trial, participants reported to the laboratory for POST assessments, which replicated PRE procedures. Red blood cell fatty acid and serum micronutrient analyses were performed in a blinded fashion at hematology laboratories. A group × time interaction was observed for serum vitamin D levels (p < 0.00...

Being born prematurely associates with greater cardiovascular disease (CVD) risk in adulthood. Less understood are the unique and joint associations of dietary patterns and behaviors to this elevated risk among adults who are born prematurely. We aimed to model the associations between term status, dietary and lifestyle behaviors with CVD risk factors while accounting for the longitudinal effects of family protection, and medical or environmental risks. In wave-VIII of a longitudinal study, 23-year olds born prematurely (PT-adults, n=129) and full term (FT-adults, n=38) survey-reported liking for foods/beverages and activities, constructed into indexes of dietary quality and sensation-seeking, dietary restraint and physical activity. Measured CVD risk factors included fasting serum lipids and glucose, blood pressure and adiposity. In bivariate relationships, PT-adults reported lower dietary quality (including less affinity for protein-rich foods and higher affinity for sweets), less liking for sensation-seeking foods/activities, and less restrained eating did FT-adults. In comparison to nationally-representative values and the FTadults, PT-adults showed greater level of CVD factors for blood pressure and serum lipids. In structural equation modeling, dietary quality completely mediated the association between term status and HDL-cholesterol (higher quality, lower HDL-cholesterol) yet joined term status to explain variability in systolic blood pressure (PT-adults with lowest dietary quality had highest

Interest in nutrient-rich berry juices is growing, but their high polyphenol levels render them sensorily unappealing. Fifty adults, who were assessed for sensory phenotype and dietary behaviors, provided sensory and palatability ratings of juices from 'Viking' aronia berries for each of seven harvest weeks. By peak harvest, juice preference increased twofold , averaging neither like/dislike. This hedonic shift was associated with: increases in juice sugars paralleling increases in perceived sweetness (maximum=weak); reductions in percent acidity paralleling reductions in sourness (minimum= moderate), astringency (minimum=to just above weak) and bitterness (minimum=just below weak). About 25% of adults liked the aronia juice, including adults who also liked an aqueous citric acid solution (average rating=moderately sour) or those who reported adventurous eating behaviors. Bitter taste phenotype, measured by propylthiouracil or quinine bitterness, failed to explain significant variation in juice sensation or preference. We also collected sensory and preference ratings from juice collected at peak harvest blended with sugar and/or sweet olfactory flavoring (10ppm ethyl butyrate). Increasing juice sweetness by adding 5% sucrose decreased sourness and improved preference from weak dislike to weak like. Adding sweet olfactory flavoring decreased juice sourness without changing preference. Adding sweet flavoring and 3% sucrose resulted in reduction of sourness and

Objectives The ability of a novel beadlet-in-oil, gastric-resistant, vegetarian capsule containing a multiple vitamin-mineral (MVM) composition to deliver capsule contents past the stomach was tested by standard in vitro tablet disintegration procedures using magnesium as the marker of capsule contents dissolution. Methods A novel capsule design using a gastric-resistant vegetarian hypromellose/gellan gum capsule (DRcaps®, Capsugel®) was tested for disintegration in a standard tablet disintegration apparatus according to compendial United States Pharmacopeia methods, as per Good Manufacturing Practices for dietary supplements. The MVM ingredients were encapsulated into size 0 Vcaps® (hypromellose with no gastric acid resistance) and DRcaps®. Individual capsules were placed into chambers containing simulated gastric fluid (0.1 M HCl) for 120 minutes then changed to simulated intestinal fluid (buffered 2% sodium lauryl sulfate, pH 6.0) for an additional 300 minutes. Aliquots were test...

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Objectives In vitro dissolution tests are valuable first-step tools in the development of bioavailable delivery systems, making it possible to assess the performance of novel technologies in releasing active ingredients through the amount dissolved in a dissolution medium. The objective of this study was to evaluate whether phosphatidyl choline-enriched lipid encapsulation releases the majority of vitamin C as ascorbic acid past the stomach in a standard in vitro dissolution procedure and to assess the release profile. Methods A novel phosphatidyl choline-enriched lipid encapsulation that is solid at room temperature was tested for dissolution in a standard dissolution apparatus according to compendial United States Pharmacopeia methods, as per Good Manufacturing Practices for dietary supplements. One serving (included 1000 mg ascorbic acid) was placed into vessels containing simulated gastric fluid (0.1 M HCl) for 120 minutes then changed to simulated intestinal fluid (buffered 2% ...

In a secondary analysis, we assessed the ability of dietary and physical activity surveys to explain variability in weight loss within a worksite-adapted Diabetes Prevention Program. The program involved 58 overweight/obese female employees (average age = 46 ± 11 years SD; average body mass index = 34.7 ± 7.0 kg/m2 SD) of four long-term care facilities who survey-reported liking and frequency of dietary and physical activity behaviors. Data were analyzed using a latent variable approach, analysis of covariance, and nested regression analysis to predict percent weight change from baseline to intervention end at week 16 (average loss = 3.0%; range—6% gain to 17% loss), and follow-up at week 28 (average loss = 2.0%; range—8% gain to 16% loss). Using baseline responses, restrained eaters (reporting liking but low intakes of high fat/sweets) achieved greater weight loss at 28 weeks than those reporting high liking/high intake (average loss = 3.5 ± 0.9% versus 1.0 ± 0.8% S.E., respectivel...

Consumption of dark green vegetables falls short of recommendations, in part, because of unpleasant bitterness. A laboratory-based study of 37 adults was used to determine bitter and hedonic responses to vegetables (asparagus, Brussels sprouts, kale) with bitter masking agents (1.33 M sodium acetate, 10 and 32 mM sodium chloride, and 3.2 mM aspartame) and then characterized by taste phenotype and vegetable liking. In repeated-measures ANOVA, aspartame was most effective at suppressing bitterness and improving hedonic responses for all sampled vegetables. Among the sodium salts, 32 mM sodium chloride decreased bitterness for kale and sodium acetate reduced bitterness across all vegetables with a tendency to increase liking for Brussels sprouts, as release from mixture suppression increased perceived sweetness. Participants were nearly equally divided into three 6-n-propylthiouracil (PROP) phenotype groups. Those tasting the least PROP bitterness (non-tasters) reported least vegetable bitterness, and the additives produced little change in vegetable liking. Aspartame persisted as the most effective bitter blocker for the PROP tasters (medium, supertasters), improving vegetable liking for the medium tasters but too much sweetness for supertasters. The sodium salts showed some bitter blocking for PROP tasters, particularly sodium acetate, without significant gains in vegetable liking. Via a survey, adults characterized as low vegetable likers reported greater increase in vegetable liking with the maskers than did vegetable likers. These results suggest that bitter masking agents (mainly sweeteners) can suppress bitterness to increase acceptance if they are matched to perceived vegetable bitterness or to self-reported vegetable disliking.

Consumption of dark green vegetables falls short of recommendations, in part, because of unpleasant bitterness. A laboratory-based study of 37 adults was used to determine bitter and hedonic responses to vegetables (asparagus, Brussels sprouts, kale) with bitter masking agents (1.33 M sodium acetate, 10 and 32 mM sodium chloride, and 3.2 mM aspartame) and then characterized by taste phenotype and vegetable liking. In repeated-measures ANOVA, aspartame was most effective at suppressing bitterness and improving hedonic responses for all sampled vegetables. Among the sodium salts, 32 mM sodium chloride decreased bitterness for kale and sodium acetate reduced bitterness across all vegetables with a tendency to increase liking for Brussels sprouts, as release from mixture suppression increased perceived sweetness. Participants were nearly equally divided into three 6-n-propylthiouracil (PROP) phenotype groups. Those tasting the least PROP bitterness (non-tasters) reported least vegetable bitterness, and the additives produced little change in vegetable liking. Aspartame persisted as the most effective bitter blocker for the PROP tasters (medium, supertasters), improving vegetable liking for the medium tasters but too much sweetness for supertasters. The sodium salts showed some bitter blocking for PROP tasters, particularly sodium acetate, without significant gains in vegetable liking. Via a survey, adults characterized as low vegetable likers reported greater increase in vegetable liking with the maskers than did vegetable likers. These results suggest that bitter masking agents (mainly sweeteners) can suppress bitterness to increase acceptance if they are matched to perceived vegetable bitterness or to self-reported vegetable disliking.

Food liking-disliking patterns may strongly influence food choices and health. Here we assess: (1) whether food preference patterns are genetic/environmentally driven; and (2) the relationship between metabolomics profiles and food preference patterns in a large population of twins. 2,107 individuals from TwinsUK completed an online food and lifestyle preference questionnaire. Principle components analysis was undertaken to identify patterns of food liking-disliking. Heritability estimates for each liking pattern were obtained by structural equation modeling. The correlation between blood metabolomics profiles (280 metabolites) and each food liking pattern was assessed in a subset of 1,491 individuals and replicated in an independent subset of monozygotic twin pairs discordant for the liking pattern (65 to 88 pairs). Results from both analyses were meta-analyzed. Four major food-liking patterns were identified (Fruit and Vegetable, Distinctive Tastes, Sweet and High Carbohydrate, an...

Feasible, valid, and reliable tools are needed to assess dietary quality among preschoolers. We aimed to test construct-/criterion-related validity and reliability of a dietary quality index generated from a liking survey and novel dietary patterns for characterizing food hedonism and parental feeding practices. Participants included 416 economically disadvantaged, diverse preschoolers (41% overweight/obese) from educational centers and their parents. Parents completed liking and frequency surveys; the liking survey took half as long to complete. Preschoolers' skin carotenoid status (measured by Resonance Raman spectroscopy) and BMI percentile (measured weight/height) were assessed. The Healthy Eating Preference Index (HEPI) was constructed from weighted averages of liking scores for food groups and healthy variety score and Healthy Eating Index 2010 (HEI) from nutrient analysis of the frequency survey. The HEPI was normally distributed and showed construct validity and adequate...

Objectives To test the effect of probiotic strains (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12; PS), a prebiotic (bacteriophage blend; BB), a postbiotic (tributyrin; TB), or a combination of all three (full mix; FM) on microbial fermentation and metabolite production in vitro. Methods Short term colonic incubation was performed under conditions representative of the proximal large intestine, using the fecal microbiota of a single healthy human donor. At the start of the experiment, treatments and a blank control (BC), were added to the reactors. Each reactor also received a carbohydrate-supplemented nutritional medium and freshly prepared fecal inoculum. Reactors were closed with a rubber septum, made anaerobic through flushing with nitrogen, and incubated for 48 hours. Each condition was run in triplicate. Overall fermentative activity (pH and gas production), changes in microbial metabolite production (short chain fatty acid, lactate, and ammonium ...

Objectives To test the effect of probiotic strains (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12), a prebiotic (bacteriophage blend), and a postbiotic (tributyrin) on the human intestinal microbiota in vitro. Methods Short term colonic incubation was performed under conditions representative of the proximal large intestine, using the fecal microbiota of a single healthy human donor. At the start of the experiment, treatments and a blank control were added to the reactors. Each reactor also received a carbohydrate-supplemented nutritional medium and freshly prepared fecal inoculum. Reactors were closed with a rubber septum, made anaerobic through flushing with nitrogen, and incubated for 48 hours. Each condition was run in triplicate. Changes in microbial community composition were determined via 16S-targeted Illumina sequencing and flow cytometry. Enriched bacterial genera were identified using LEfSe (LDA > 2, p < 0.05). Results A significant inc...