Controlled Feeding Study Research Articles

Gut Microbial Protein Expression in Response to Dietary Patterns in a Controlled Feeding Study: A Metaproteomic Approach.

Although the gut microbiome has been associated with dietary patterns linked to health, microbial metabolism is not well characterized. This ancillary study was a proof of principle analysis for a novel application of metaproteomics to study microbial protein expression in a controlled dietary intervention. We measured the response of the microbiome to diet in a randomized crossover dietary intervention of a whole-grain, low glycemic load diet (WG) and a refined-grain, high glycemic load diet (RG). Total proteins in stools from 9 participants at the end of each diet period (n = 18) were analyzed by LC MS/MS and proteins were identified using the Human Microbiome Project (HMP) human gut microbiome database and UniProt human protein databases. T-tests, controlling for false discovery rate (FDR) <10%, were used to compare the Gene Ontology (GO) biological processes and bacterial enzymes between the two interventions. Using shotgun proteomics, more than 53,000 unique peptides were identified including microbial (89%) and human peptides (11%). Forty-eight bacterial enzymes were statistically different between the diets, including those implicated in SCFA production and degradation of fatty acids. Enzymes associated with degradation of human mucin were significantly enriched in the RG diet. These results illustrate that the metaproteomic approach is a valuable tool to study the microbial metabolism of diets that may influence host health.

Abstract MP43: Urine And Plasma Metabolome of Healthy Adults Consuming the Dietary Approaches to Stop Hypertension Diet: A Pilot Study

The mechanisms of the Dietary Approaches to Stop Hypertension (DASH) diet on various health markers remain unclear. The objective of this study was to identify plasma and urine metabolites altered by DASH that may elucidate mechanisms of benefit and suggest biomarkers to facilitate research and monitoring. We tested metabolomics differences in a controlled feeding study of 20 people consuming a Control diet for one wk followed by 2 wks of random assignment to Control or DASH. Fasting plasma and 24-hour urine samples collected at the end of each wk were profiled for metabolites using non-targeted GC/MS and amino acids and acylcarnitines were measured in plasma using a targeted panel. Linear models compared metabolite levels between DASH and Control during the final two randomized feeding wks [nominal p&lt;0.05 and false discovery rate (FDR) adjusted p&lt;0.2]. Compared to Control, DASH is designed to be lower in total fat and saturated fat, but higher in protein, fiber, potassium, magnesium and calcium. Overall, we found 161 identifiable urine metabolites. In FDR adjusted tests, urine excretion of a variety of polyphenolic acids were higher on DASH. In plasma, gamma tocopherol, acylcarnitine C5 from branched chain amino acid (BCAAs) metabolism, and amino acids including BCAAs leucine/isoleucine and valine were lower on DASH (Table 1). Several small and medium chain dicarboxylic acylcarnitines were higher on DASH. Consumption of DASH increases urine levels of a set of polyphenolic compounds many of which have been linked to lower blood pressure in animal and small human studies. In plasma, BCAAs are lower on DASH despite higher protein intake. Further research is needed to investigate these metabolites as a panel for dietary monitoring in hypertension. Table 1. Nontargeted and targeted metabolites (Selected metabolites are shown here) in DASH and Control.

Proof-of-concept study of the efficacy of a microbiota-directed complementary food formulation (MDCF) for treating moderate acute malnutrition

BackgroundChildhood undernutrition remains a significant global health challenge accounting for over half of all under 5 child mortality. Moderate acute malnutrition (MAM), which leads to wasting [weight-for-length z-scores (WLZ) between − 2 and − 3], affects 33 million children under 5 globally and more than 2 million in Bangladesh alone. We have previously reported that acute malnutrition in this population is associated with gut microbiota immaturity, and in a small, 1-month pre-proof-of-concept (POC) study demonstrated that a microbiota-directed complementary food formulation (MDCF-2) was able to repair this immaturity, promote weight gain and increase plasma biomarkers and mediators of healthy growth. Here we describe the design controlled feeding study that tests whether MDCF-2 exhibits superior efficacy (ponderal growth, host biomarkers of a biological state) than a conventional Ready-to-use Supplementary Food (RUSF) in children with MAM over intervention period of 3 months.MethodsTwo separate cohorts of 12–18-month-old children will be enrolled: 124 with primary MAM, and 124 with MAM after having been treated for severe acute malnutrition (post-SAM MAM). We have established several field sites in an urban slum located in the Mirpur district of Dhaka, Bangladesh and at a rural site, Kurigram in the north of Bangladesh. The two groups of children receiving MDCF-2 and RUSF will be compared at baseline (pre-intervention), after 1 month, at the end of intervention (3 months), 1 month after cessation of intervention, and every 6 months thereafter for 4 years.DiscussionThis study will determine whether daily, controlled administration of MDCF-2 for 3 months provides superior improvements in weight gain, microbiota repair, and elevated levels of key plasma biomarkers/mediators of healthy growth compared to the control RUSF formulation. The pathogenesis of MAM is poorly defined and there are currently no WHO-approved treatments; results from the current study of children with primary MAM and post-SAM MAM will shed light on the effects of the gut microbiota on childhood growth/development and will provide a knowledge base that may help improve complementary feeding practices.Trial registrationThe primary MAM and post-SAM MAM trials are registered in Clintrials.gov (NCT04015999 and NCT04015986, registered on July 11, 2019, retrospectively registered).

PCDD/Fs, DL-PCBs, and NDL-PCBs in Dairy Cows: Carryover in Milk from a Controlled Feeding Study.

A feeding study was carried out to investigate the kinetics in cow milk of the 17 polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), the 12 dioxin-like polychlorinated biphenyls (DL-PCBs), and the 6 non-dioxin-like PCBs (NDL-PCBs) regulated by the European (EU) legislation. A fortified ration (ΣPCDD/Fs and DL-PCBs: 24.68 ng TEQ/day/cow; ΣNDL-PCBs: 163.99 μg/day/cow) was given to the animals for 49 days, followed by 42 days on clean feed. EU maximum limit for TEQPCDD/F+DL-PCB was exceeded in milk after 1 week of exposure, while for ΣNDL-PCBs, after 5 weeks. Milk compliance was restored after 1 week on clean feed, but to return to the basal TEQPCDD/F+DL-PCB it took 42 days. At the end of the study, ΣNDL-PCBs had not yet reached the basal level. The carryover rate of ΣNDL-PCBs was 25.4%, while the carryover rate of TEQPCDD/F+DL-PCB was 36.9%. The latter was mainly affected by the 12 congeners contributing most to the toxic equivalent (TEQ) level, explaining the fast overcome of the maximum limit in milk.

Effects of regular-fat and low-fat dairy consumption on daytime ambulatory blood pressure and other cardiometabolic risk factors: a randomized controlled feeding trial

The extent to which dairy products and their fat content influence cardiovascular health remains uncertain. This study aimed to assess how consumption of low-fat milk and regular-fat cheese enriched in γ-aminobutyric acid (GABA) influences daytime ambulatory blood pressure (BP) and other cardiometabolic risk factors. In this crossover controlled feeding study, 55 healthy men and women with high-normal daytime BP were randomly assigned to sequences of three 6-wk isoenergetic diets, each comprising 1) no dairy (control diet), 2) 3 daily servings of 1% fat milk, and 3) 1 daily serving of 31% fat cheddar cheese naturally enriched in GABA. Total proteins, carbohydrates, and fats were matched across all 3 diets. The additional 2% of energy from SFAs in the cheese diet was replaced by n-6 PUFAs in the other diets. Comparison of postdiet ambulatory systolic BP revealed no difference (P=0.34), which was also the case for ambulatory diastolic BP (P=0.45). The cheese diet increased serum LDL-cholesterol concentrations compared with the control and milk diets (+5.8%, P=0.006 and +7.0%, P=0.0008, respectively) and increased LDL particle size compared with the milk diet (P=0.02). HDL-cholesterol concentrations after the milk diet were lower than after the control diet (-4.1%; P=0.009). The milk and cheese diets increased triglycerides compared with the control diet (+9.9%, P=0.01 and +10.5%, P=0.007, respectively). There was no significant difference between all diets for C-reactive protein concentrations and markers of glucose/insulin homeostasis. These results suggest that short-term consumption of dairy products, whether low or regular in fat, has no overall effect on daytime ambulatory BP compared with a dairy-free diet. Other cardiometabolic risk factors may be differently modified according to the fat content of the dairy product. This trial was registered at clinicaltrials.gov as NCT02763930.

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children.

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.INTRODUCTIONThere is a dimension to post-natal human development that involves assembly of microbial communities in different body habitats, including the gut. Children with acute malnutrition have impaired development of their gut microbiota, leaving them with communities that appear younger (more immature) than those of chronologically age-matched healthy individuals. Current therapeutic foods given to children with acute malnutrition have not been formulated based on knowledge of how they affect the developmental biology of the gut microbiota. Moreover, they are largely ineffective in ameliorating the long-term sequelae of malnutrition that include persistent stunting, neurodevelopmental abnormalities, and immune dysfunction.RATIONALERepairing microbiota immaturity and determining the degree to which such repair restores healthy growth requires identification of microbial targets that are not only biomarkers of community assembly but also mediators of various aspects of growth. Identifying ingredients in complementary foods, consumed during the transition from exclusive milk feeding to a fully weaned state, that increase the representation and expressed beneficial functions of growth-promoting bacterial taxa in the developing microbiota could provide an effective, affordable, culturally acceptable, and sustainable approach to treatment.RESULTSMetabolomic and proteomic analyses of serially collected plasma samples were combined with metagenomic analyses of serially collected fecal samples from Bangladeshi children with severe acute malnutrition (SAM) treated with standard therapy. The results provided a readout of their biological features as they transitioned from SAM to a state of persistent moderate acute malnutrition (MAM) with accompanying persistent microbiota immaturity. Significant correlations were identified between levels of plasma proteins, anthropometry, plasma metabolites, and the representation of bacteria in their microbiota. Gnotobiotic mice were subsequently colonized with a defined consortium of bacterial strains that represent various phases of microbiota development in healthy Bangladeshi children. Administration of different combinations of Bangladeshi complementary food ingredients to colonized mice and germ-free controls revealed diet-dependent increases in the abundance and changes in the metabolic activities of targeted weaning-phase strains as well as diet- and colonization-dependent augmentation of growth-promoting host signaling pathways. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes were subsequently examined in gnotobiotic mice colonized with immature microbiota from children with post-SAM MAM and in gnotobiotic piglets colonized with a defined consortium of targeted age- and growth-discriminatory taxa. A randomized, double-blind study of standard therapy versus various MDCF prototypes emerging from these preclinical models, conducted in Bangladeshi children with MAM, identified a lead MDCF that increased levels of biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function toward a state resembling healthy children. Using an approach inspired by statistical methods applied to financial markets, we show in the accompanying paper by Raman et al. that this lead MDCF was most effective in repairing the microbiota.CONCLUSIONThese findings demonstrate the translatability of results obtained from pre-clinical gnotobiotic animal models to humans, directly support the hypothesis that healthy microbiota development is causally linked to healthy growth, illustrate an approach for treating childhood undernutrition, and with the capacity to deliberately reconfigure immature microbiota, suggest a means to decipher how elements of the gut microbial community operate to regulate various host systems involved in healthy growth.Overview of therapeutic food discovery and testing. The approach used for integrating preclinical gnotobiotic animal models with human studies to understand the contributions of perturbed gut microbiota development to childhood malnutrition and to identify MDCFs.

Compound‐specific isotope analysis of amino acids as a new tool to uncover trophic chains in soil food webs

AbstractFood webs in soil differ fundamentally from those aboveground; they are based on inputs from both living plants via root exudates, and from detritus, which is a complex mixture of fungi, bacteria, and dead plant remains. Trophic relationships are difficult to disentangle due to the cryptic lifestyle of soil animals and inevitable microbial contributions to their diet. Compound‐specific isotope analysis of amino acids (AAs) is increasingly used to explore complex food webs. The combined use ofAAδ13C and δ15N values is a promising new approach to disentangle trophic relationships since it provides independent but complementary information on basal resources, as well as the trophic position of consumers. We conducted a controlled feeding study in which we reconstructed trophic chains from main basal resources (bacteria, fungi, plants) to primary consumers (springtails, oribatid mites) and predators (gamasid mites, spiders). We analyzed dual compound‐specific isotopeAAvalues of both resources and consumers. By applying an approach termed “stable isotope (13C) fingerprinting” we identified basal resources, and concomitantly calculated trophic positions using15N values of trophic and sourceAAs in consumers. In the13C fingerprinting analysis, consumers in general grouped close to their basal resources. However, higher than usual offsets inAAδ13C between diet and consumers suggest either gut microbial supplementation or the utilization of specific resource fractions. Identification of trophic position crucially depends on correct estimates of the trophic discrimination factor (TDFGlu‐Phe), which was close to the commonly applied value of 7.6‰ in primary consumers feeding on microbial resources, but considerably lower in arachnid predators (~2.4‰), presumably due to higher diet quality, excretion of guanine, and fluid feeding. While our feeding study demonstrates that dual compound‐specificAAanalyses hold great promise in delineating trophic linkages among soil‐dwelling consumers and their resources, it also highlights that a “one‐size‐fits‐all” approach to TDFGlu‐Phedoes not apply to soil food webs.

Maternal choline supplementation alters vitamin B-12 status in human and murine pregnancy

Despite participation in overlapping metabolic pathways, the relationship between choline and vitamin B-12 has not been well characterized especially during pregnancy. We sought to determine the effects of maternal choline supplementation on vitamin B-12 status biomarkers in human and mouse pregnancy, hypothesizing that increased choline intake would improve vitamin B-12 status. Associations between common genetic variants in choline-metabolizing genes and vitamin B-12 status biomarkers were also explored in humans. Healthy third-trimester pregnant women (n=26) consumed either 480 or 930 mg choline/day as part of a 12-week controlled feeding study. Wild-type NSA and Dlx3 heterozygous (Dlx3+/−) mice, which display placental insufficiency, consumed a 1×, 2× or 4× choline diet and were sacrificed at gestational days 15.5 and 18.5. Serum vitamin B-12, methylmalonic acid (MMA) and homocysteine were measured in all samples; holotranscobalamin (in humans) and hepatic vitamin B-12 (in mice) were also measured. The 2× choline supplementation for 12 weeks in pregnant women yielded higher serum concentrations of holotranscobalamin, the bioactive form of vitamin B-12 (~24%, P=.01). Women with genetic variants in choline dehydrogenase (CHDH) and betaine-homocysteine S-methyltransferase (BHMT) had higher serum MMA concentrations (~31%, P=.03) and lower serum holotranscobalamin concentrations (~34%, P=.03), respectively. The 4× choline dose decreased serum homocysteine concentrations in both NSA and Dlx3+/− mice (~36% and~43% respectively, P≤.015). In conclusion, differences in choline supply due to supplementation or genetic variation modulate vitamin B-12 status during pregnancy, supporting a functional relationship between these nutrients.

Circulating 25-Hydroxyvitamin D Concentrations in Overweight and Obese Adults Are Explained by Sun Exposure, Skin Reflectance, and Body Composition

ABSTRACTBackgroundObese individuals are known to be at higher risk for vitamin D deficiency than normal-weight individuals. Cutaneous synthesis is a major source of vitamin D; however, objective measurements of sun exposure are lacking in this population.ObjectiveTo assess the validity of a regression model using sun exposure in lean individuals to estimate serum 25-hydroxyvitamin D [25(OH)D] in overweight and obese individuals, and to develop a prediction equation for serum 25(OH)D in overweight and obese adults.MethodsThis study was a secondary analysis of a 15-wk controlled feeding study investigating the effects of dairy consumption on body composition. Information regarding sun exposure, including day, hour, time outside, and clothing, were self-assessed in sun exposure diaries. Personal sun exposure energy (joules) was assessed by downloading time-specific ultraviolet B energy data from climate stations. Skin reflectance was measured using a Minolta 2500d spectrophotometer. Dietary intake of vitamin D was known. Serum 25(OH)D concentration was measured by radioimmunoassay. Body composition was determined from whole-body dual energy x-ray absorptiometry and computed tomography scans.ResultsSun exposure was positively related to serum 25(OH)D (r = 0.26; P ≤ 0.05) and inversely related to total fat mass, android fat, and BMI (r = −0.25, −0.30, and −0.32, respectively). The modified Hall model significantly overestimated serum 25(OH)D in overweight and obese adults by 27.33–80.98 nmol/L, depending on the sun exposure calculation. A new regression model was developed for overweight and obese persons that explained 29.1% of the variance in postintervention 25(OH)D concentrations and included sun exposure, skin reflectance, total fat mass, total lean mass, and intra-abdominal adipose tissue as predictors.ConclusionMajor determinants of serum 25(OH)D concentration in healthy overweight and obese individuals include sun exposure, skin reflectance, and adiposity. Addition of adiposity terms to the prior model significantly improved predictive ability in overweight and obese men and women. (clinicaltrials.gov: NCT00858312)

Plasma Lipidomics Profiles After a Diet Characterized by Whole Grains Compared to a Diet High in Refined Grains and Added Sugars (FS03-07-19)

ObjectivesDietary patterns high in fiber from sources including whole grains, legumes, fruits, vegetables, nuts and seeds, are associated with lower risk of chronic disease, such as cardiovascular disease and cancer. We investigated how plasma lipidomics profiles differed between a diet high in whole grains (WG) versus a diet high in refined grains and added sugars (RG). MethodsUsing a randomized, crossover, controlled feeding study, 80 healthy participants (n = 40 men, n = 40 women, 40 normal weight, 40 overweight/obese), 18–45 y, were randomized to receive either a WG or RG diet for 28 days. After a 28-day washout period where participants resumed their habitual diet, they crossed over to the other diet. Targeted, differential mobility mass spectrometry was performed on fasting plasma samples collected at the baseline and end of each diet period and quantified the concentrations of 863 lipids from 13 classes. Paired t-tests and pairwise partial least squares-discriminant analysis (PLS-DA) were used to evaluate differences in lipid profiles between the two diets. ResultsAt a class level, only ceramides were significantly different when comparing the two diets. After removing lipid species with > 20% missing values or CVs < 25%, 606 were retained for species analysis. Sixty-seven lipid species were significantly different between diets at day 28 (FDR < 0.05): 38 of 414 detected triglycerides, 9 of 59 phosphatidylethanolamines, 9 of 63 phosphatidylcholines, 4 of 22 cholesterol esters, 3 of 11 sphingomyelins, 2 of 13 lysophosphatidylcholines, and 1 of 5 ceramides. The majority of significant lipids were higher in plasma after the WG diet. PLSDA analysis showed the first and second components explaining 49% and 8.4%, respectively. Based on the selected components, lipidomic profiles showed fair separation for the two groups of diet. R2 values were 0.07 and 0.43, and Q2 values were -0.03 and 0.04 for components 1 and 2, respectively. ConclusionsHigher concentrations of some lipid species such as cholesterol ester 12:0, a carrier of high-density lipoprotein, could indicate a favorable shift in lipid profiles. Further investigation using more complex models are being conducted. Funding SourcesNational Cancer Institute - National Institutes of Health.

331-OR: Glycemic Index and Postprandial Blood Glucose Excursions, Insulin Requirements, and Hunger in T1D

High compared to low glycemic index (GI) foods elicit larger glycemic and insulinemic excursions, and are associated with lower satiety. However, it is unclear whether the satiety effect is attributable to differences in glycemia or insulinemia, and whether insulin delivery in individuals with T1D could be optimized for satiety per se. Fifteen men with T1D, BMI 25 (SD 3) kg/m2, aged 23 (5) years, HbA1c 7.4 (0.7)% underwent three experimental conditions in a randomized cross-over design: High GI, high insulin (HGI/HI): meal GI of 84 with intravenous insulin (IVI) to maintain euglycemia; Low GI (LGI): meal GI of 37 with IVI to maintain euglycemia (comparison condition); High GI, low insulin (HGI/LI): high GI meal as above with IVI matched to LGI. Test meals provided 25% of daily calories (60% carbohydrate, 15% protein, 25% fat). IVI was adjusted using a PID algorithm with blood glucose measurements every 5 minutes for 4.5 hr. When using matched insulin doses, blood glucose area under the curve (AUC) was 789 (145) vs. 493 (48) mg/dl*h (p&amp;lt;0.001) for HGI/LI vs. LGI. To maintain euglycemia (HGI/HI vs. LGI), 25 (9) vs. 17 (5) units of insulin were needed (p=0.005). Late postprandial (3.5-4.5 hr) free fatty acid (FFA) concentrations were higher for HGI/HI (0.62 [0.31] mmol/L) vs. LGI (0.26 [0.09] mmol/L p=0.008). Hunger was lower at peak glycemia (1 hr) for HGI/HI vs. LGI (2.3 vs. 3.2 on a 0-10 scale, p=0.01), but was greater in the late postprandial phase after either HGI/HI or HGI/LI vs. LGI (6.3 [p=0.05] or 6.3 [p=0.03] vs. 5.3, respectively). In a controlled feeding study, a high GI meal was associated with a 1.5-fold higher insulin requirement to obtain similar glycemia, or a 1.6-fold higher glucose AUC when using the same insulin dose. The high GI meal also increased late postprandial hunger regardless of insulin dose, and resulted in a greater rise in FFA (high insulin condition). These meal effects, with or without aggressive insulin management, may predispose to cardiovascular risk, metabolic dysfunction or weight gain. Disclosure B.S. Lennerz: None. D.C. Simonson: Stock/Shareholder; Self; GI Windows, Inc. Stock/Shareholder; Spouse/Partner; Phase V Technologies, Inc. G.M. Steil: Consultant; Self; Eli Lilly and Company, Profusa, Inc. D.S. Ludwig: Other Relationship; Self; Various publishers. Funding National Institute of Diabetes and Digestive and Kidney Diseases (K12DK094721, K24DK082730)

Walnuts and Vegetable Oils Differentially Affect the Gut Microbiome and Associations with Cardiovascular Risk Factors (OR29-06-19)

ObjectivesTo examine gut bacterial composition following a higher-saturated fatty acids (SFA) run-in, standard Western diet (SWD) and 3 study diets where SFA was replaced with: whole walnuts (WD); vegetable oils in a fatty acid matched diet devoid of walnuts (WFMD); vegetable oils in an oleic acid replaces α-linolenic acid (ALA) diet (ORAD) and devoid of walnuts. Correlations between the bacterial composition and cardiovascular disease (CVD) risk factors were also explored. MethodsAdults at CVD risk (n = 42) were included in this secondary analysis of a randomized, crossover, controlled-feeding study that reported improvements in CVD risk factors. Only significantly changed risk factors were correlated with significantly enriched bacteria. Fecal collections followed a 2-week run-in diet and three 6-week diet periods. Linear discriminant analysis Effect Size plots were used to determine enriched taxa. ResultsFollowing the WD, Roseburia (mean relative abundance = 4.2%, LDA = 4, P = 0.0008), Eubacterium eligensgroup (1.4%, 4, 0.05), Lachnospiraceae UCG001 (1.2%, 3, 0.03), Lachnospiraceae UCG004 (1.0%, 3, 0.04) and Leuconostocaceae (0.03%, 3, 0.05) were abundant relative to the SWD. Relative to the SWD, Roseburia (mean relative abundance = 3.6%, LDA = 4, P = 0.02) and Eubacterium eligensgroup (1.5%, 3, 0.02) were enriched following the WFMD, and following the ORAD Clostridialesvadin BB60 (0.3%, 2, 0.04) was most abundant. Significant associations existed between enriched bacteria following the WD and CVD risk factors. Eubacterium eligens correlated with brachial (R = –0.5, P = 0.0009) and central mean arterial pressure (MAP; –0.5, 0.002), and central diastolic blood pressure (cDBP; –0.5, 0.0006). Lachnospiraceae correlated with brachial (R = –0.4, P = 0.02) and central MAP (–0.4, 0.02), cDBP (–0.3, 0.04), total cholesterol (TC; –0.4, 0.03), and non-HDL-C (–0.4, 0.02). Leuconostocaceae correlated with brachial (R = 0.3, P = 0.03) and central MAP (0.3, 0.03). ConclusionsSimilar eubiotic bacteria were enriched by the WD and WFMD suggesting ALA and linoleic acid affect the gut microbiome. Enrichment of Lachnospiraceae and associations with improved CVD risk factors suggests the microbiome contributes to the beneficial health effects of walnuts. Funding SourcesThe California Walnut Commission and National Center for Advancing Translational Sciences, NIH. Supporting Tables, Images and/or Graphs▪▪

Effects of Physical Activity on the Performance of 24-h Urinary Sucrose and Fructose as a Biomarker of Total Sugars Intake (P18-128-19)

ObjectivesUrinary sucrose and fructose has been suggested as a predictive biomarker of total sugars intake based on research involving UK adults. The purpose of this study was to determine the association between sugars consumption and 24-hour urinary sucrose and fructose (24uSF) in US adult population and to investigate the effect of physical activity on this association. MethodsFifty seven free-living healthy subjects 20 to 68 years old, participated in a 15-day highly controlled feeding study, consuming their habitual diet, provided by the research metabolic kitchen. Dietary sugars were estimated using Nutrition Data System for Research (NDSR). Subjects collected eight 24-hour urine samples measured for urinary sucrose and fructose. Physical activity was assessed daily using a validated 15-day log that inquired about 38 physical activities across six domains; home activities, transportation, occupation, conditioning, sports and leisure. ResultsThe 15-d mean total sugars and added sugars intake (SD) of the study population was 112.2 (33.1) g/day and 65.8 (29.0) g/day (9.7%EI), respectively. Significant moderate positive correlation was found between 15-d mean total sugars intake and 8-day mean 24uSF (r = 0.56, P < 0.001). Similarly, added sugars were moderately correlated with 24uSF (r = 0.56, P < 0.001), while no correlation was found between naturally-occurring sugars and 24uSF (r = 0.070, P < 0.001). In a linear multiple regression, total and added sugars each explained 30% of variability in 24uSF (Adjusted R2, p value; total sugars: 0.297, 0.001; added sugars: 0.301, P < 0.001). We found no effect of physical activity on the association between dietary and urinary sugars in neither the correlation nor the linear regression analysis. Conclusions24uSF showed moderate association with both total and added sugars consumption in US adults. No evidence was found showing that physical activity levels affect the association between 24uSF and sugars intake in US adults. Funding SourcesNational Cancer Institute.

Estimated 24-hour Sodium Excretion from Available Equations Is a Poor Predictor of Intake (OR15-06-19)

ObjectivesAccurate estimates of sodium (Na) intake are critical for advancing our understanding of Na and cardiovascular disease. The aim of this study was to utilize data from a controlled feeding study to assess the agreement between estimated 24hUNa, calculated from several timed spot urine collections, and known Na intake. MethodsThis secondary analysis used data from the control phase of a potassium intervention study in normotensive men and women (n = 39) to avoid the potential confounding factor of dietary potassium manipulation. Participants followed the controlled diet containing on average 3463 mg Na/d for 3 days. On day 4, participants ate 3 meals in a clinic setting and collected urine every 2 hours starting with a fasting second void. The 24-hour urine collection was completed with the first morning void the following day. Spot urine samples were analyzed for Na and estimated 24hUNa was calculated from all samples using the INTERSALT and Tanaka equations, and from only the fasting second collection using the Kawasaki equation. The relationship between estimated 24hUNa and Na intake was assessed using Spearman correlations, and agreement between estimated 24hUNa and intake was assessed using Wilcoxon Signed Rank Tests and Bland Altman plots. ResultsFor all timed spot urine samples, estimated 24hUNa using INTERSALT and Kawasaki were significantly correlated with Na intake (all P < 0.05), whereas estimates using Tanaka were not significantly correlated with intake (all P > 0.05). For all timed spot urine samples, estimated 24hUNa using INTERSALT was significantly different from Na intake (all P < 0.05). All estimates of 24hUNa using Tanaka were significantly different from Na intake (all P < 0.05) except from estimates from the 2-hour sample (P = 0.172) and the first void at 24 hours (P = 0.102). Estimate of 24hUNa using Kawasaki was not significantly different from intake (P = 0.635). Bland Altman plots showed high levels of bias, especially at higher and lower levels of intake. ConclusionsThese results suggest that, regardless of timing of collection and choice of equation, estimated 24hUNa is not a valid indicator of actual Na intake. New equations that directly estimate Na intake are needed. Funding SourcesThe parent study was funded by the Alliance for Potato Research and Education (APRE).

Prenatal Choline Supplementation Improves Child Color-location Memory Task Performance at 7 Y of Age (FS05-01-19)

ObjectivesTo assess the effect of 3rd trimester maternal choline supplementation on child memory at 7 y of age. Animal studies have provided strong evidence that maternal choline supplementation improves offspring cognition, including memory, but few experimental studies have evaluated this intervention in humans. MethodsPregnant women (n = 26) were randomized to consume 480 mg (approx. the Adequate Intake [AI]) or 930 mg choline/d from gestational wk 27 until delivery as part of a controlled-feeding study. All food (providing 380 mg choline/d), choline supplements (100 mg or 550 mg/d), and prenatal vitamins were provided by the study. An ancillary follow-up was conducted to assess offspring cognitive functioning, including memory, at age 7 y (n = 20). Children performed a computer-based color-location memory task in which the location of dots on a cartoon figure were recalled after a retention interval (RI) of 1 or 8 s. The task increased in difficulty level (number of dots to be recalled; 1 to 5 dots) every 4 trials. Data were analyzed using mixed models; pairwise comparisons were corrected for multiplicity. ResultsChildren in the 930 mg/d group passed more levels than those in the 480 mg/d group (P = .048; see Figure), indicative of superior memory span. While the choline group by RI interaction was non-significant (P = .31), contrasts revealed that the 930 (v. 480) mg/d group tended to pass more levels at the 1 s RI (P = .052), but not at the 8 s RI (P = .58). Similarly, the analysis of total dots correct revealed a choline group by RI interaction (P = .08) such that children in the 930 (v. 480) mg/d group recalled more dots correctly at the 1 s RI (P = .02), but not at the 8 s RI (P > .99). ConclusionsChildren whose mothers consumed 930 (v. 480) mg choline/d performed significantly better on a task of color-location memory at age 7 y, suggesting a long-term beneficial effect of prenatal choline supplementation on offspring cognition in humans. These findings indicate that the choline AI for pregnant women may not be sufficient for optimal offspring cognitive functioning and raise concerns about choline intake during pregnancy in North America, which on average is only approx. 70% of the AI. Funding SourcesFunded by NIFA/USDA and the Balchem Corp. JEHN and CLB were supported by NIH Traineeships. CLB was supported by an Egg Nutrition Center Young Investigator Research Award for Early Exploration. Supporting Tables, Images and/or Graphs▪

Prenatal Choline Supplementation Improves Child Color-location Memory Task Performance at 7 Y of Age (FS05-01-19)

Abstract Objectives To assess the effect of 3rd trimester maternal choline supplementation on child memory at 7 y of age. Animal studies have provided strong evidence that maternal choline supplementation improves offspring cognition, including memory, but few experimental studies have evaluated this intervention in humans. Methods Pregnant women (n = 26) were randomized to consume 480 mg (approx. the Adequate Intake [AI]) or 930 mg choline/d from gestational wk 27 until delivery as part of a controlled-feeding study. All food (providing 380 mg choline/d), choline supplements (100 mg or 550 mg/d), and prenatal vitamins were provided by the study. An ancillary follow-up was conducted to assess offspring cognitive functioning, including memory, at age 7 y (n = 20). Children performed a computer-based color-location memory task in which the location of dots on a cartoon figure were recalled after a retention interval (RI) of 1 or 8 s. The task increased in difficulty level (number of dots to be recalled; 1 to 5 dots) every 4 trials. Data were analyzed using mixed models; pairwise comparisons were corrected for multiplicity. Results Children in the 930 mg/d group passed more levels than those in the 480 mg/d group (P = .048; see Figure), indicative of superior memory span. While the choline group by RI interaction was non-significant (P = .31), contrasts revealed that the 930 (v. 480) mg/d group tended to pass more levels at the 1 s RI (P = .052), but not at the 8 s RI (P = .58). Similarly, the analysis of total dots correct revealed a choline group by RI interaction (P = .08) such that children in the 930 (v. 480) mg/d group recalled more dots correctly at the 1 s RI (P = .02), but not at the 8 s RI (P &gt; .99). Conclusions Children whose mothers consumed 930 (v. 480) mg choline/d performed significantly better on a task of color-location memory at age 7 y, suggesting a long-term beneficial effect of prenatal choline supplementation on offspring cognition in humans. These findings indicate that the choline AI for pregnant women may not be sufficient for optimal offspring cognitive functioning and raise concerns about choline intake during pregnancy in North America, which on average is only approx. 70% of the AI. Funding Sources Funded by NIFA/USDA and the Balchem Corp. JEHN and CLB were supported by NIH Traineeships. CLB was supported by an Egg Nutrition Center Young Investigator Research Award for Early Exploration. Supporting Tables, Images and/or Graphs

Abstract MP37: Estimating Sodium Intake Using Timed Urine Collections From a Controlled Feeding Study

Introduction: Hypertension is a major risk factor for CVD affecting 34% of American adults. High sodium (Na) intake is identified as a modifiable risk factor for high blood pressure, but our ability to assess Na intake is poor. The gold standard is to measure Na excretion in a 24h urine collection, but this is burdensome. An alternative method is to predict 24h Na excretion from a spot urine sample. However, we do not know how well predicted 24h Na excretion reflects intake, the true outcome of interest, or the ideal time to collect a spot urine sample. These gaps can only be filled using a controlled feeding study in which intake is known and timing of urine collections is controlled. Hypothesis: We hypothesized that, with currently available equations, 24h Na excretion predicted from spot urine samples will not accurately reflect intake. Methods: This is a secondary analysis of a randomized, multi-phase, crossover, full-feeding trial in healthy adults ( n = 39, aged 29.7 ± 11.2 y). Data from the two control phases were used in which dietary intake and testing conditions were identical. Participants ate three meals in a clinic setting and spot urine samples were collected every 2 hours as part of a 24h collection. Urine samples were analyzed for sodium, potassium, and creatinine. The INTERSALT equations were used to predict 24h Na excretion from each of the 9 spot urine samples collected. Predicted 24h Na excretion values were compared with observed 24h excretion and known dietary intake using repeated measures ANOVA. Intra-class correlations for each timed spot urine sample were calculated to determine the variability in average spot urine Na excretion between the two control phases. Results: Predicted 24h Na excretion using the spot urines at hour 10, 12, overnight, and early morning collection were not significantly different from observed 24h Na excretion (all p ≥ 0.21). However, predicted 24h Na excretion underestimates observed 24h Na excretion by about &gt;600 mg. Importantly, all predicted 24h excretions as well as observed 24h excretion were significantly lower than intake by &gt;1000 mg (all p &lt; 0.001). Intraclass correlations were significant at hour 0, 2, 8, 12, and evening/overnight (all p &lt; 0.05), suggesting low day-to-day variability in Na excretion at these times. Conclusions: Regardless of time of spot urine collection, predicted 24h Na excretion is lower than Na intake, suggesting currently-available equations do not provide adequate estimates of Na intake. The next step is to develop equations to accurately predict Na intake from a controlled feeding study model using spot urine samples collected at a time when Na excretion is consistent from day to day. These improved equations could be used to implement effective dietary interventions to reduce blood pressure and CVD risk.

Abstract P091: Short-term Increased Dietary Potassium From Potato and Potassium Gluconate Has No Effect on Blood Pressure and Microcirculation in Pre-hypertensive-to-hypertensive Adults

Potassium is an essential nutrient of concern according to recent Dietary Guidelines for Americans Advisory Committees, with less than 3% of the population meeting the current adequate intake (AI) level of 4700mg/d. Increases in potassium intake have been linked to improvements in cardiovascular and other metabolic health outcomes. Blood pressure (BP) is currently the primary criterion for determining potassium requirements. In this clinical trial, we assessed the effects of increasing potassium intake on BP and microcirculation (endothelial function). Thirty pre-hypertensive-to-hypertensive (Systolic BP ≥ 120mmHg) men (N=15) and Women (N=15) with a mean ± SD age, BMI, and BP (systolic(SBP)/ diastolic(DBP)) of 48.2 ± 15 y, 31.4 ± 6.1, and 136.3 ± 11.9/ 86.1 ± 7.1 mmHg, respectively, were enrolled in a cross-over randomized control diet trial. Participants were assigned to a random order of four 16-day dietary potassium interventions including a basal diet (control) of 2300mg/d(~60mmol/d), and three periods of an additional 1000mg/d(3300mg/d(~85mmol/d) total) of potassium in the form of potatoes (baked, boiled, or pan-heated with no additional fat), French fries, or a potassium gluconate supplement. Each intervention period was separated by two or more weeks of wash out. Blood pressure was measured in triplicate using manual auscultation on days 1, 4, 6, 8, 11, 13, 15, 16, and 17 of each intervention. Microvascular and endothelial function were assessed via thermal hyperemia, utilizing laser Doppler flowmetry (LDF), at baseline and at the end of each intervention. Results for differences in both SBP and DBP ± SE were assessed at the end treatment (average of day 15, 16, and 17 measurements) using a mixed model with repeated measures and Dunnett adjustment (P &lt; 0.05), with no significant differences among groups (control: 128.7 ± 2.1/ 83.6 ± 1.6, French fries: 127.7 ± 2.2/ 83.7 ± 1.6, potatoes: 127.6 ± 2.3/ 85.1 ± 1.6, supplement: 128.0 ± 2.2/ 83.7 ± 1.7). Utilizing the same statistical analysis, there were also no significant differences in endothelial function (measured as percent of cutaneous vascular conductance max (%CVCmax) ± SE) among groups (control: 87.4 ± 1.5%, French Fries: 87.7 ± 1.4%, potatoes: 88.3 ± 1.1%, supplement: 88.9 ± 1.0%). In this small, controlled feeding study of short duration, increased potassium from potatoes or supplement achieved 70% of recommended intakes, but had no significant benefit to blood pressure or microvascular function in men and women with higher cardiometabolic risk.

Reducing Dietary Sodium to 1000 mg per Day Reduces Neurovascular Transduction Without Stimulating Sympathetic Outflow.

The American Heart Association recommends no more than 1500 mg of sodium/day as ideal. Some cohort studies suggest low-sodium intake is associated with increased cardiovascular mortality. Extremely low-sodium diets (≤500 mg/d) elicit activation of the renin-angiotensin-aldosterone system and stimulate sympathetic outflow. The effects of an American Heart Association-recommended diet on sympathetic regulation of the vasculature are unclear. Therefore, we assessed whether a 1000 mg/d diet alters sympathetic outflow and sympathetic vascular transduction compared with the more commonly recommended 2300 mg/d. We hypothesized that sodium reduction from 2300 to 1000 mg/d would not affect resting sympathetic outflow but would reduce sympathetic transduction in healthy young adults. Seventeen participants (age: 26±2 years, 9F/8M) completed 10-day 2300 and 1000 mg/d sodium diets in this randomized controlled feeding study (crossover). We measured resting renin activity, angiotensin II, aldosterone, blood pressure, muscle sympathetic nerve activity, and norepinephrine. We quantified beat-by-beat changes in mean arterial pressure and leg vascular conductance (femoral artery ultrasound) following spontaneous sympathetic bursts to assess sympathetic vascular transduction. Reducing sodium to 1000 mg/d increased renin activity, angiotensin II, and aldosterone ( P<0.01 for all) but did not alter mean arterial pressure (78±2 versus 77±2 mm Hg, P=0.56), muscle sympathetic nerve activity (13.9±1.3 versus 13.9±0.8 bursts/min, P=0.98), or plasma/urine norepinephrine. Sympathetic vascular transduction decreased ( P<0.01). These data suggest that reducing sodium from 2300 to 1000 mg/d stimulates the renin-angiotensin-aldosterone system, does not increase resting basal sympathetic outflow, and reduces sympathetic vascular transduction in normotensive adults.

3318 Phosphorus Absorption in Healthy Adults and in Patients with Moderate Chronic Kidney Disease

OBJECTIVES/SPECIFIC AIMS: The aim of this study is to compare intestinal phosphorus absorption in healthy adults and moderate stage chronic kidney disease patients in the context of a controlled feeding study METHODS/STUDY POPULATION: Participants are 30-75 years old and include 10 healthy subjects and 10 moderate-staged CKD patients. Each subject pool will be enrolled in a 9-day study period including 7 days of controlled feeding of a 1500 mg phosphorus diet. Following the controlled feeding, two days of absorption tests will take place (oral and IV tests) utilizing radioisotopic phosphorus to calculate fractional absorption efficiency. RESULTS/ANTICIPATED RESULTS: Current enrollment has produced 7 total matched subject (current n = 14/20). Four of the 7 pairs of completed subjects are female and 3 of 7 are black. Preliminary kinetic modeling data from the first enrolled subject show a moderate CKD patient with fractional absorption of 0.375. With forthcoming analyses, we expect that this fractional absorption result will not be statistically different from this subject’s matched pair, nor will each groups average absorption be different from the other. Additionally, we expect absorption to be maintained even with changes in secondary outcomes measures in serum (FGF23, 1,25-dihydroxyvitamin D, parathyroid hormone, and total phosphorus) in CKD patients. DISCUSSION/SIGNIFICANCE OF IMPACT: Lack of statistical difference in fractional phosphorus absorption between gropus would support that intestinal phosphorus absorption is inappropriately normal in CKD patients compared to healthy adults, despite evidence of abnormal phosphorus homeostatic mechanisms. Future studies will consider the effect of dietary P restriction, the most common nutrition intervention in moderate stage CKD, on fractional absorption efficiency in CKD.