Human Feeding Studies Research Articles

Intake Biomarkers for Nutrition and Health: Review and Discussion of Methodology Issues.

Metabolomics profiles from blood, urine, or other body fluids have the potential to assess intakes of foods and nutrients objectively, thereby strengthening nutritional epidemiology research. Metabolomics platforms may include targeted components that estimate the relative concentrations for individual metabolites in a predetermined set, or global components, typically involving mass spectrometry, that estimate relative concentrations more broadly. While a specific metabolite concentration usually correlates with the intake of a single food or food group, multiple metabolites may be correlated with the intake of certain foods or with specific nutrient intakes, each of which may be expressed in absolute terms or relative to total energy intake. Here, I briefly review the progress over the past 20 years on the development and application intake biomarkers for foods/food groups, nutrients, and dietary patterns, primarily by drawing from several recent reviews. In doing so, I emphasize the criteria and study designs for candidate biomarker identification, biomarker validation, and intake biomarker application. The use of intake biomarkers for diet and chronic disease association studies is still infrequent in nutritional epidemiology research. My comments here will derive primarily from our research group's recent contributions to the Women's Health Initiative cohorts. I will complete the contribution by describing some opportunities to build on the collective 20 years of effort, including opportunities related to the metabolomics profiling of blood and urine specimens from human feeding studies that approximate habitual diets.

Metabolomics-Based Biomarker for Dietary Fat and Associations with Chronic Disease Risk in Postmenopausal Women

BackgroundThe Women’s Health Initiative (WHI) randomized, controlled Dietary Modification (DM) trial of a low-fat dietary pattern suggested intervention benefits related to breast cancer, coronary heart disease (CHD), and diabetes. Here, we use WHI observational data for further insight into the chronic disease implications of adopting this type of low-fat dietary pattern. ObjectivesWe aimed to use our earlier work on metabolomics-based biomarkers of carbohydrate and protein to develop a fat intake biomarker by subtraction, to use the resulting biomarker to develop calibration equations that adjusts self-reported fat intake for measurement error, and to study associations of biomarker-calibrated fat intake with chronic disease risk in WHI cohorts. Corresponding studies for specific fatty acids will follow separately. MethodsProspective disease association results are presented using WHI cohorts of postmenopausal women, aged 50–79 y when enrolled at 40 United States clinical centers. Biomarker equations were developed using an embedded human feeding study (n = 153). Calibration equations were developed using a WHI nutritional biomarker study (n = 436). Calibrated intakes were associated with cancer, cardiovascular diseases, and diabetes incidence in WHI cohorts (n = 81,954) over an approximate 20-y follow-up period. ResultsA biomarker for fat density was developed by subtracting protein, carbohydrate, and alcohol densities from one. A calibration equation was developed for fat density. Hazard ratios (95% confidence intervals) for 20% higher fat density were 1.16 (1.06, 1.27) for breast cancer, 1.13 (1.02, 1.26) for CHD, and 1.19 (1.13, 1.26) for diabetes, in substantial agreement with findings from the DM trial. With control for additional dietary variables, especially fiber, fat density was no longer associated with CHD, with hazard ratio (95% confidence interval) of 1.00 (0.88, 1.13), whereas that for breast cancer was 1.11 (1.00, 1.24). ConclusionsWHI observational data support prior DM trial findings of low-fat dietary pattern benefits in this population of postmenopausal United States women. Trial registration numberThis study is registered with clinicaltrials.gov identifier: NCT00000611.

Metabolomics Biomarkers for Fatty Acid Intake and Biomarker-Calibrated Fatty Acid Associations with Chronic Disease Risk in Postmenopausal Women

BackgroundA substantial observational literature relating specific fatty acid classes to chronic disease risk may be limited by its reliance on self-reported dietary data. ObjectivesWe aimed to develop biomarkers for saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acid densities, and to study their associations with cardiovascular disease (CVD), cancer, and type 2 diabetes (T2D) in Women’s Health Initiative (WHI) cohorts. MethodsBiomarker equations were based primarily on serum and urine metabolomics profiles from an embedded WHI human feeding study (n = 153). Calibration equations were based on biomarker values in a WHI nutritional biomarker study (n = 436). Calibrated intakes were assessed in relation to disease incidence in larger WHI cohorts (n = 81,894). Participants were postmenopausal women, aged 50–79 when enrolled at 40 United States Clinical Centers (1993–1998), with a follow-up period of ∼20 y. ResultsBiomarker equations meeting criteria were developed for SFA, MUFA, and PUFA densities. That for SFA density depended somewhat weakly on metabolite profiles. On the basis of our metabolomics platforms, biomarkers were insensitive to trans fatty acid intake. Calibration equations meeting criteria were developed for SFA and PUFA density, but not for MUFA density. With or without biomarker calibration, SFA density was associated positively with risk of CVD, cancer, and T2D, but with small hazard ratios, and CVD associations were not statistically significant after controlling for other dietary variables, including trans fatty acid and fiber intake. Following this same control, PUFA density was not significantly associated with CVD risk, but there were positive associations for some cancers and T2D, with or without biomarker calibration. ConclusionsHigher SFA and PUFA diets were associated with null or somewhat higher risk for clinical outcomes considered in this population of postmenopausal United States women. Further research is needed to develop even stronger biomarkers for these fatty acid densities and their major components.This study is registered with clinicaltrials.gov identifier: NCT00000611.

Comparative Absorption and Transport of Five Common Carotenoids in Preruminant Calves

Preruminant calves, maintained in a monogastric state by feeding an all-liquid diet, were used to compare the serum appearance and lipoprotein transport of five different carotenoids over 144 h. Thirty newborn calves were fed milk replacer for 1 wk and then randomly assigned to six groups (n = 5), with each group receiving a single 20-mg oral dose of β-carotene in water-soluble beadlets, canthaxanthin in water-soluble beadlets, lutein in oil, lycopene in oil, crystalline α-carotene in oil or crystalline β-carotene in oil as part of a morning meal. Serial blood samples were taken by jugular puncture for up to 1 wk post-dosing. Lipoprotein separation and analysis were completed with selected animals. All carotenoids were absorbed, but in variable amounts. At peak serum carotenoids levels, HDL contained 70–90% of the carotenoids. Canthaxanthin and lutein peaked earlier in serum (8 and 12 h) than did the less polar lycopene, α-carotene, and β-carotene (16, 24 and 24 h). Canthaxanthin and lutein were also cleared more quickly from the serum. Serum concentrations of α-carotene and lycopene displayed slower disappearance rates than did β-carotene. The peak serum level (nmol/L ± SEM) of canthaxanthin (392 ± 136) was lower than that of β-carotene (1245 ± 425), and carotenoids levels of calves receiving these commercial beadlet sources were higher than the serum levels of calves receiving β-carotene (45 ± 17.5), α-carotene (42 ± 18.0), lutein (51 ± 9.5) and lycopene (18 ± 4.6), which were fed in oil. The results show differential uptake and clearance of various carotenoids and enhanced uptake of beadlet forms of two carotenoids. We suggest that the preruminant calf is an excellent model in which to evaluate uptake and transport of carotenoids, especially for those carotenoids that are not approved for human feeding studies.

Biomarker-Calibrated Red and Combined Red and Processed Meat Intakes with Chronic Disease Risk in a Cohort of Postmenopausal Women

The associations of red and processed meat with chronic disease risk remain to be clarified, in part because of measurement error in self-reported diet. We sought to develop metabolomics-based biomarkers for red and processed meat, and to evaluate associations of biomarker-calibrated meat intake with chronic disease risk among postmenopausal women. Study participants were women who were members of the Women's Health Initiative (WHI) study cohorts. These participants were postmenopausal women aged 50-79 y when enrolled during 1993-1998 at 40 US clinical centers with embedded human feeding and nutrition biomarker studies. Literature reports of metabolomics correlates of meat consumption were used to develop meat intake biomarkers from serum and 24-h urine metabolites in a 153-participant feeding study (2010-2014). Resulting biomarkers were used in a 450-participant biomarker study (2007-2009) to develop linear regression calibration equations that adjust FFQ intakes for random and systematic measurement error. Biomarker-calibrated meat intakes were associated with cardiovascular disease, cancer, and diabetes incidence among 81,954 WHI participants (1993-2020). Biomarkers and calibration equations meeting prespecified criteria were developed for consumption of red meat and red plus processed meat combined, but not for processed meat consumption. Following control for nondietary confounding factors, hazard ratios were calculated for a 40% increment above the red meat median intake for coronary artery disease (HR: 1.10; 95% CI: 1.07, 1.14), heart failure (HR: 1.26; 95% CI: 1.20, 1.33), breast cancer (HR: 1.10; 95% CI: 1.07, 1.13) for, total invasive cancer (HR: 1.07; 95% CI: 1.05, 1.09), and diabetes (HR: 1.37; 95% CI: 1.34, 1.39). HRs for red plus processed meat intake were similar. HRs were close to the null, and mostly nonsignificant following additional control for dietary potential confounding factors, including calibrated total energy consumption. A relatively high-meat dietary pattern is associated with somewhat higher chronic disease risks. These elevations appear to be largely attributable to the dietary pattern, rather than to consumption of red or processed meat per se.

Biomarkers for Components of Dietary Protein and Carbohydrate with Application to Chronic Disease Risk in Postmenopausal Women

We recently developed protein and carbohydrate intake biomarkers using metabolomics profiles in serum and urine, and used them to correct self-reported dietary data for measurement error. Biomarker-calibrated carbohydrate density was inversely associated with chronic disease risk, whereas protein density associations were mixed. To elucidate and extend this earlier work through biomarker development for protein and carbohydrate components, including animal protein and fiber. Prospective disease association analyses were undertaken in Women's Health Initiative (WHI) cohorts of postmenopausal US women, aged 50-79 y when enrolled at 40 US clinical centers. Biomarkers were developed using an embedded human feeding study (n=153). Calibration equations for protein and carbohydrate components were developed using a WHI nutritional biomarker study (n=436). Calibrated intakes were associated with chronic disease incidence in WHI cohorts (n=81,954) over a 20-y (median) follow-up period, using HR regression methods. Previously reported elevations in cardiovascular disease (CVD) with higher-protein diets tended to be explained by animal protein density. For example, for coronary heart disease a 20% increment in animal protein density had an HR of 1.20 (95% CI: 1.02, 1.42) relative to the HR for total protein density. In comparison, cancer and diabetes risk showed little association with animal protein density beyond that attributable to total protein density. Inverse carbohydrate density associations with total CVD were mostly attributable to fiber density, with a 20% increment HR factor of 0.89 (95% CI: 0.83, 0.94). Cancer risk showed little association with fiber density, whereas diabetes risk had a 20% increment HR of 0.93 (95% CI: 0.88, 0.98) relative to the HRs for total carbohydrate density. In a population of postmenopausal US women, CVD risk was associated with high-animal-protein and low-fiber diets, cancer risk was associated with low-carbohydrate diets, and diabetes risk was associated with low-fiber/low-carbohydrate diets.

Baseline Gut Metagenomic Functional Gene Signature Associated with Variable Weight Loss Responses following a Healthy Lifestyle Intervention in Humans.

ABSTRACTRecent human feeding studies have shown how the baseline taxonomic composition of the gut microbiome can determine responses to weight loss interventions. However, the functional determinants underlying this phenomenon remain unclear. We report a weight loss response analysis on a cohort of 105 individuals selected from a larger population enrolled in a commercial wellness program, which included healthy lifestyle coaching. Each individual in the cohort had baseline blood metabolomics, blood proteomics, clinical labs, dietary questionnaires, stool 16S rRNA gene sequencing data, and follow-up data on weight change. We generated additional targeted proteomics data on obesity-associated proteins in blood before and after intervention, along with baseline stool metagenomic data, for a subset of 25 individuals who showed the most extreme weight change phenotypes. We built regression models to identify baseline blood, stool, and dietary features associated with weight loss, independent of age, sex, and baseline body mass index (BMI). Many features were independently associated with baseline BMI, but few were independently associated with weight loss. Baseline diet was not associated with weight loss, and only one blood analyte was associated with changes in weight. However, 31 baseline stool metagenomic functional features, including complex polysaccharide and protein degradation genes, stress-response genes, respiration-related genes, and cell wall synthesis genes, along with gut bacterial replication rates, were associated with weight loss responses after controlling for age, sex, and baseline BMI. Together, these results provide a set of compelling hypotheses for how commensal gut microbiota influence weight loss outcomes in humans.IMPORTANCE Recent human feeding studies have shown how the baseline taxonomic composition of the gut microbiome can determine responses to dietary interventions, but the exact functional determinants underlying this phenomenon remain unclear. In this study, we set out to better understand interactions between baseline BMI, metabolic health, diet, gut microbiome functional profiles, and subsequent weight changes in a human cohort that underwent a healthy lifestyle intervention. Overall, our results suggest that the microbiota may influence host weight loss responses through variable bacterial growth rates, dietary energy harvest efficiency, and immunomodulation.

Rare sugars and their health effects in humans: a systematic review and narrative synthesis of the evidence from human trials.

ContextRare sugars are monosaccharides and disaccharides (found in small quantities in nature) that have slight differences in their chemical structure compared with traditional sugars. Little is known about their unique physiological and cardiometabolic effects in humans.ObjectiveThe objective of this study was to conduct a systematic review and synthesis of controlled intervention studies of rare sugars in humans, using PRISMA guidelines.Data SourcesMEDLINE and EMBASE were searched through October 1, 2020. Studies included both post-prandial (acute) and longer-term (≥1 week duration) human feeding studies that examined the effect of rare sugars (including allulose, arabinose, tagatose, trehalose, and isomaltulose) on cardiometabolic and physiological risk factors.Data extractionIn all, 50 studies in humans focusing on the 5 selected rare sugars were found. A narrative synthesis of the selected literature was conducted, without formal quality assessment or quantitative synthesis.Data synthesisThe narrative summary included the food source of each rare sugar, its effect in humans, and the possible mechanism of effect. Overall, these rare sugars were found to offer both short- and long-term benefits for glycemic control and weight loss, with effects differing between healthy individuals, overweight/obese individuals, and those with type 2 diabetes. Most studies were of small size and there was a lack of large randomized controlled trials that could confirm the beneficial effects of these rare sugars.ConclusionRare sugars could offer an opportunity for commercialization as an alternative sweetener, especially for those who are at high cardiometabolic risk.Systematic Review RegistrationOSF registration no. 10.17605/OSF.IO/FW43D.

Effects of Palm Stearin versus Butter in the Context of Low-Carbohydrate/High-Fat and High-Carbohydrate/Low-Fat Diets on Circulating Lipids in a Controlled Feeding Study in Healthy Humans.

Background. Foods rich in saturated fatty acids (SFAs) have been discouraged by virtue of their cholesterol-raising potential, but this effect is modulated by the food source and background level of carbohydrate. Objective. We aimed to compare the consumption of palm stearin (PS) versus butter on circulating cholesterol responses in the setting of both a low-carbohydrate/high-fat (LC/HF) and high-carbohydrate/low-fat (HC/LF) diet in healthy subjects. We also explored effects on plasma lipoprotein particle distribution and fatty acid composition. Methods. We performed a randomized, controlled-feeding, cross-over study that compared a PS- versus a Butter-based diet in a group of normocholesterolemic, non-obese adults. A controlled canola oil-based ‘Run-In’ diet preceded the experimental PS and Butter diets. All diets were eucaloric, provided for 3-weeks, and had the same macronutrient distribution but varied in primary fat source (40% of the total fat). The same Run-In and cross-over experiments were done in two separate groups who self-selected to either a LC/HF (n = 12) or a HC/LF (n = 12) diet track. The primary outcomes were low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein (HDL)-C, triglycerides, and LDL particle distribution. Results. Compared to PS, Butter resulted in higher LDL-C in both the LC/HF (13.4%, p = 0.003) and HC/LF (10.8%, p = 0.002) groups, which was primarily attributed to large LDL I and LDL IIa particles. There were no differences between PS and Butter in HDL-C, triglycerides, or small LDL particles. Oxidized LDL was lower after PS than Butter in LC/HF (p = 0.011), but not the HC/LF group. Conclusions. These results demonstrate that Butter raises LDL-C relative to PS in healthy normocholesterolemic adults regardless of background variations in carbohydrate and fat, an effect primarily attributed to larger cholesterol-rich LDL particles.

The berry health tool chest - an evidence map and interactive resource.

Berry consumption is linked to diverse health benefits, but numerous questions remain regarding mechanism of action, dose efficacy, and optimal duration and frequency of intake. Addressing these outstanding questions requires an organized assessment of current research, to inform future study designs and fill critical knowledge gaps. Tools that organize such information will also facilitate consumer messaging, targeted nutritional health initiatives, and dietary intake guidelines. This review aimed to describe the development and utility of the "Berry Health Tool Chest," an evidence map summarizing trial design features of studies characterizing the impact of berry consumption upon human health biomarkers. A systematic search strategy identified relevant high-quality human feeding studies, whose study design parameters were collected and compiled into an evidence map that is freely available as an interactive online interface enabling tabulated data to be interrogated, filtered, and exported. Of the 231 included studies, approximately 70% were of less than 3 months' duration and/or fewer than 50 participants, illustrating research gaps that could potentially inform the design of future studies.

Avenanthramide Metabotype from Whole-Grain Oat Intake is Influenced by Faecalibacterium prausnitzii in Healthy Adults.

Oat has been widely accepted as a key food for human health. It is becoming increasingly evident that individual differences in metabolism determine how different individuals benefit from diet. Both host genetics and the gut microbiota play important roles on the metabolism and function of dietary compounds. To investigate the mechanism of individual variations in response to whole-grain (WG) oat intake. We used the combination of in vitro incubation assays with human gut microbiota, mouse and human S9 fractions, chemical analyses, germ-free (GF) mice, 16S rRNA sequencing, gnotobiotic techniques, and a human feeding study. Avenanthramides (AVAs), the signature bioactive polyphenols of WG oat, were not metabolized into their dihydro forms, dihydro-AVAs (DH-AVAs), by both human and mouse S9 fractions. DH-AVAs were detected in the colon and the distal regions but not in the proximal and middle regions of the perfused mouse intestine, and were in specific pathogen-free (SPF) mice but not in GF mice. A kinetic study of humans fed oat bran showed that DH-AVAs reached their maximal concentrations at much later time points than their corresponding AVAs (10.0-15.0hours vs. 4.0-4.5hours, respectively). We observed interindividual variations in the metabolism of AVAs to DH-AVAs in humans. Faecalibacterium prausnitzii was identified as the individual bacterium to metabolize AVAs to DH-AVAs by 16S rRNA sequencing analysis. Moreover, as opposed to GF mice, F. prausnitzii-monocolonized mice were able to metabolize AVAs to DH-AVAs. These findings demonstrate that the presence of intestinal F. prausnitzii is indispensable for proper metabolism of AVAs in both humans and mice. We propose that the abundance of F. prausnitzii can be used to subcategorize individuals into AVA metabolizers and nonmetabolizers after WG oat intake. This study was registered at clinicaltrials.gov as NCT04335435.

Anthocyanin Bioactivity in Obesity and Diabetes: The Essential Role of Glucose Transporters in the Gut and Periphery.

Obesity and type-2 diabetes trends continue to worsen in the United States. Dietary anthocyanins (typically provided by berries and other fruits) are reported to have protective effects against both conditions using a variety of experimental research models including animal and human feeding studies. This review highlights studies that explore the biochemical pathways in both tissue and rodent models which could explain clinical improvements noted with anthocyanin consumption. First, the primary mode of intestinal absorption of anthocyanins is through both sGLT1 and GLUT2 glucose transporters. Stronger binding affinities may allow anthocyanins to be more inhibitive to glucose absorption compared to the reverse, where GLUT2 expression may also be affected. Genetic or chemical inhibition of sGLT1 or GLUT2 demonstrate their essential function in anthocyanin absorption across the enterocyte, where the former interacts with a greater variety of anthocyanins but the latter is the major transporter for specific anthocyanin-glycosides. Once absorbed, anthocyanins positively modulate GLUT4 density and function in both skeletal muscle and adipose tissues via the upregulation of AMPK and restoration of insulin sensitivity. Antioxidant properties and phosphodiesterase inhibition by anthocyanins promote both mitochondrial function and density which could be novel targets for dietary management of obesity and its complications.

Separation of postprandial lipoproteins: improved purification of chylomicrons using an ApoB100 immunoaffinity method

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

Dietary methionine influences therapy in mouse cancer models and alters human metabolism.

SummaryNutrition exerts profound effects on health and dietary interventions are commonly used to treat diseases of metabolic etiology. Although cancer has a substantial metabolic component1, the principles that define whether nutrition may be used to influence tumour outcome are unclear2. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. In contrast, whether specific dietary interventions could influence the metabolic pathways that are targeted in standard cancer therapies is not known. We now show that dietary restriction of methionine (MR), an essential amino acid, and the reduction of which has anti-aging and anti-obesogenic properties, influences cancer outcome through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and further is the target of a host of cancer interventions involving chemotherapy and radiation. MR produced therapeutic responses in chemoresistant RAS-driven colorectal cancer patient derived xenografts (PDXs) and autochthonous KRASG12D+/−;TP53−/− -driven soft tissue sarcomas resistant to radiation. Metabolomics revealed the therapeutic mechanisms to occur through tumour cell autonomous effects on the flux through one-carbon metabolism that impacted redox and nucleotide metabolism, thus interacting with the antimetabolite or radiation intervention. Finally, in a controlled and tolerated feeding study in humans, MR resulted in similar effects on systemic metabolism as obtained in responsive mice. These findings provide evidence that a targeted dietary manipulation can affect specific tumour cell metabolism to mediate broad aspects of cancer outcome.

Dietary methionine links nutrition and metabolism to the efficacy of cancer therapies

Nutrition exerts profound effects on health and dietary interventions are commonly used to treat diseases of metabolic etiology. Although cancer has a substantial metabolic component1, the principles that define whether nutrition may be used to influence tumour outcome are unclear2. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. In contrast, whether specific dietary interventions could influence the metabolic pathways that are targeted in standard cancer therapies is not known. We now show that dietary restriction of methionine (MR), an essential amino acid, and the reduction of which has anti-aging and anti-obesogenic properties, influences cancer outcome through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and further is the target of a host of cancer interventions involving chemotherapy and radiation. MR produced therapeutic responses in chemoresistant RAS-driven colorectal cancer patient derived xenografts (PDXs) and autochthonous KRASG12D+/−;TP53−/− -driven soft tissue sarcomas resistant to radiation. Metabolomics revealed the therapeutic mechanisms to occur through tumour cell autonomous effects on the flux through one-carbon metabolism that impacted redox and nucleotide metabolism, thus interacting with the antimetabolite or radiation intervention. Finally, in a controlled and tolerated feeding study in humans, MR resulted in similar effects on systemic metabolism as obtained in responsive mice. These findings provide evidence that a targeted dietary manipulation can affect specific tumour cell metabolism to mediate broad aspects of cancer outcome.

Comparison of Actual and Reported Macronutrient Intake in a Well-controlled Human Feeding Study (P08-003-19)

ObjectivesDietary guidelines are largely based on epidemiological studies that rely on memory-based dietary assessments (MBDAs) such as 24-hour recalls (24 hr). Few reports have compared actual and reported dietary intake in well-controlled feeding studies (WCFS). This study sought to directly compare discrepancies between actual dietary intake from experimental diets and reported 24 hr intake in free-living participants. MethodsFood was provided to 24 subjects (50% female, ages 24.4(3.4); BMI 23.1(2.9)) for 24 days; all diets were eucaloric. Participants were provided a standard diet (15% protein (PRO): 35% fat: 50% carbohydrate (CHO)) for 3 days and then randomly assigned to either a 21-day high fat (60% fat, HF) or a high CHO (75% CHO, HC) diet while PRO was held constant. Diets were ‘typical’ foods with macronutrient compositions adjusted to achieve targeted percentages, either overtly (added sugary drinks) or covertly (added coconut oil). Calories were added to maintain weight (±1 kg). Dietitians administered 4 24-hour recalls (24 hr); one after the standard diet, and once a week during the experimental diets. Actual and reported energy intake (EI) and macronutrient intakes were compared within group via Student’s T-tests (α = 0.05). ResultsNo difference was observed between actual EI and reported EI during the standard or experimental phase for HF or HC groups. %difference was calculated as (g reported/g actual) * 100. PRO intake was over-reported by all subjects during the standard phase – 127.4% (11.3) and during the HC – 115.6% (7.2) and HF – 116.1% (5.6). PRO difference was due to over-reporting of animal PRO. During the experimental diet, HC subjects over-reported CHO – 166.4% (11.4) and under-reported fat –93.7% (4.7) while HF subjects under-reported CHO – 89.2% (6.5) and over-reported fat –119.8 (6.7). (P < 0.05). ConclusionsIn this WCFS in free-living participants, EI is accurately reported. However, subjects over-report animal PRO intake potentially due to their misreporting portion sizes of meat. When given extreme diets, subjects tended to under report the experimental macronutrient (HF or HC). This could potentially be due to covert target macronutrients in the diet. This makes use of the 24hr suspect in WCFS when macronutrients are hidden from participants. Funding SourcesThis study was internally funded through researchers discretionary funds. Supporting Tables, Images and/or Graphs▪▪▪

Abstract P201: Separation of Postprandial Lipoproteins: Purification of Chylomicrons Using an ApoB100 Immunoaffinity Method

Introduction: Elevated fasting and postprandial triglyceride-rich lipoproteins (TRL) are associated with increased risk for atherosclerosis. In the postprandial state circulating lipids consist of dietary fat transported from the intestine, by chylomicrons (containing ApoB48) and fat transported from the liver, in very low-density lipoproteins (VLDL, containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because current methods are inadequate to fully separate these particles. Specifically, chylomicron fractions have considerable contamination from VLDL. Hypothesis: We hypothesized that the generation of resin crosslinked to specific ApoB100 antibodies unique to VLDL would allow purification of chylomicron particles. Proof-of-principle experiments will determine: 1) individual contributions of newly synthesized fatty acids (de novo lipogenesis (DNL)), to triglycerides found in VLDL and chylomicrons; and 2) the proportion of labeled acetyl-CoA (precursor-pool value (PPV)) used to synthesize new palmitate. A difference in DNL and PPV for the purified VLDL and chylomicron fractions will confirm the successful separation of these particles. Methods: Polyclonal antibodies were produced against the C-terminus of the ApoB100 protein and crosslinked to resin generating ApoB100-specific affinity columns. To yield purified chylomicron and VLDL particles we used this technique in conjunction with stable-isotope tracer methodology in a human feeding study. Two healthy volunteers consumed a standardized diet accompanied by oral dosing of 1- 13 C-acetate every 30 minutes for 8 hours. Plasma collected hourly was used for the isolation of TRL, which was applied to the affinity resin and purified chylomicrons and VLDL collected. Triglycerides were isolated and derivatized. The enrichment of methyl-palmitate was measured by GC/MS and Mass Isotopomer Distribution Analysis was used to calculate DNL and PPV. Results: Western blots confirmed the separation of chylomicrons and VLDL by the described method. From the purified TRL fractions, the DNL-AUC (%DNL x 5 hours) values for subject 1 were chylomicron 4% and VLDL 34%, an 88% difference, and for subject 2 were chylomicron 8% and VLDL 83%, a 90% difference. The PPV calculated for each fraction in subject 1 were chylomicron 10% and VLDL 13% and in subject 2 were chylomicron 10%, VLDL 12%. On average, the PPV in the chylomicron fraction were 18% less than the VLDL fraction. Conclusions: In conclusion, results demonstrate that the immunoaffinity method effectively separated postprandial lipoproteins showing fractionated particles carry unique information regarding tissue of origin . Future applications include investigating the respective contribution of fat and sugar to atherosclerosis risk.

Cardiovascular Mechanisms of Action of Anthocyanins May Be Associated with the Impact of Microbial Metabolites on Heme Oxygenase-1 in Vascular Smooth Muscle Cells.

Anthocyanins are reported to have cardio-protective effects, although their mechanisms of action remain elusive. We aimed to explore the effects of microbial metabolites common to anthocyanins and other flavonoids on vascular smooth muscle heme oxygenase-1 (HO-1) expression. Thirteen phenolic metabolites identified by previous anthocyanin human feeding studies, as well as 28 unique mixtures of metabolites and their known precursor structures were explored for their activity on HO-1 protein expression in rat aortic smooth muscle cells (RASMCs). No phenolic metabolites were active when treated in isolation; however, five mixtures of phenolic metabolites significantly increased HO-1 protein expression (127.4–116.6%, p ≤ 0.03). The present study demonstrates that phenolic metabolites of anthocyanins differentially affect HO-1 activity, often having additive, synergistic or nullifying effects.

Dietary long-chain fatty acids and carbohydrate biomarker evaluation in a controlled feeding study in participants from the Women’s Health Initiative cohort ,

Background: Biomarkers of macronutrient intake are lacking. Controlled human feeding studies that preserve the normal variation in nutrient and food consumption are necessary for the development and validation of robust nutritional biomarkers.Objective: We aimed to assess the utility of serum phospholipid fatty acids (PLFAs) as biomarkers of dietary intakes of fatty acids, total fat, and carbohydrate.Design: We used an individualized controlled feeding study in which 153 postmenopausal women from the Women's Health Initiative (WHI) were provided with a 2-wk controlled diet that mimicked each individual's habitual food intake. A total of 41 PLFAs were measured with the use of gas chromatography in end-of-feeding-period fasting serum samples and expressed in both relative and absolute concentrations. R2 values (percentages of variation explained) from linear regressions of (ln-transformed) consumed fatty acids (individual, groups, and broad categories) on (ln-transformed) corresponding measures of serum PLFAs alone and together with selected participant-related variables (age, race/ethnicity, body mass index, season of study participation, education level, and estimated energy intake from doubly labeled water) were used for evaluation against established urinary recovery biomarkers of energy and protein intake as benchmarks. Models to predict intakes of other nutrients were also explored.Results: Intakes of eicosapentaenoic acid and docosahexaenoic acid achieved the benchmark of R2 > 36% with or without covariates. When all 41 serum PLFAs and participant-related covariates were initially included in the model for selection, cross-validated R2 achieved >36% for consumed total carbohydrate (grams per day), total saturated fatty acids (SFAs), percentage of energy from SFAs, and total trans fatty acids with serum PLFAs in both relative and absolute concentrations.Conclusions: Serum PLFA biomarkers perform similarly to established energy and protein urinary recovery biomarkers in describing intake variations for several nutrients and, thus, appear suitable for application in this population of postmenopausal women. This approach represents an important methodologic contribution toward the utilization of nutritional biomarkers to assess macronutrient intake. This trial was registered at clinicaltrials.gov as NCT00000611.

Dietary biomarker evaluation in a controlled feeding study in women from the Women’s Health Initiative cohort ,

Background: Controlled human feeding studies are necessary for robust nutritional biomarker development and validation. Previous feeding studies have typically evaluated single nutrients and tested relatively few diets.Objectives: The objectives were 1) to simultaneously associate dietary intake with a range of potential nutritional biomarkers in postmenopausal women by using a controlled feeding study whereby each participant was provided a diet similar to her usual diet and 2) to evaluate serum concentrations of select nutrients as potential biomarkers with the use of established urinary recovery biomarkers of energy and protein as benchmarks for evaluation.Design: Postmenopausal women from the Women’s Health Initiative (n = 153) were provided with a 2-wk controlled diet in which each individual’s menu approximated her habitual food intake as estimated from her 4-d food record and adjusted for estimated energy requirements. Serum biomarkers, including carotenoids, tocopherols, folate, vitamin B-12, and phospholipid fatty acids, were collected at the beginning and end of the feeding period. Doubly labeled water and urinary nitrogen biomarkers were used to derive estimates of energy and protein consumption, respectively.Results: Linear regression of (ln-transformed) consumed nutrients on (ln-transformed) potential biomarkers and participant characteristics led to the following regression (R2) values for serum concentration biomarkers: folate, 0.49; vitamin B-12, 0.51; α-carotene, 0.53; β-carotene, 0.39; lutein + zeaxanthin, 0.46; lycopene, 0.32; and α-tocopherol, 0.47. R2 values for percentage of energy from polyunsaturated fatty acids and urinary recovery biomarkers of energy and protein intakes were 0.27, 0.53, and 0.43, respectively. Phospholipid saturated fatty acids and monounsaturated fatty acids and serum γ-tocopherol were weakly associated with intake (R2 < 0.25).Conclusions: Serum concentration biomarkers of several vitamins and carotenoids performed similarly to established energy and protein urinary recovery biomarkers in representing nutrient intake variation in a feeding study, and thus are likely suitable for application in this population of postmenopausal women. Further work is needed to identify objective measures of categories of fatty acid intake. This trial was registered at clinicaltrials.gov as NCT00000611.