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Acute consumption of Black walnuts increases fullness and decreases lipid peroxidation in humans
Journal Pre-proof Acute consumption of Black walnuts increases fullness and decreases lipid peroxidation in humans
Liana L. Rodrigues, Jamie A. Cooper, Chad M. Paton PII:
S0271-5317(19)30585-8
DOI:
https://doi.org/10.1016/j.nutres.2019.09.002
Reference:
NTR 8047
To appear in:
Nutrition Research
Received date:
17 June 2019
Revised date:
22 August 2019
Accepted date:
4 September 2019
Please cite this article as: L.L. Rodrigues, J.A. Cooper and C.M. Paton, Acute consumption of Black walnuts increases fullness and decreases lipid peroxidation in humans, Nutrition Research(2019), https://doi.org/10.1016/j.nutres.2019.09.002
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© 2019 Published by Elsevier.
Journal Pre-proof Acute Consumption of Black Walnuts Increases Fullness and Decreases Lipid Peroxidation in Humans Authors: Liana L. Rodrigues1 Jamie A. Cooper1
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Chad M. Paton1, 2
Author Affiliations: 1
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Department of Foods and Nutrition, University of Georgia, Athens, GA, USA
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Department of Food Science and Technology, University of Georgia, Athens, GA, USA
Corresponding Author:
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Chad M. Paton, Department of Food Science and Technology, University of Georgia, 100 Cedar Street,
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Athens, GA 30602, Phone: 706-542-3750, Email: [email protected]
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Abbreviations ANOVA: Analysis of Variance BMI: body mass index BW: black walnut CVD: cardiovascular disease EW: English walnut HDL: high-density lipoprotein HNL: Human Nutrition Laboratory LDL: low-density lipoprotein MUFA: monounsaturated fatty acid ORAC: Oxygen Radical Absorbance Capacity PUFA: polyunsaturated fatty acid SEM: Standard Error of Mean SFA: saturated fatty acid TAC: Total Antioxidant Capacity TBARS: Thiobarbituric Acid Reactive Substances TC: total cholesterol TG: triglycerides VAS: Visual Analog Scale WHR: Waist-to-Hip Ratio
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Journal Pre-proof
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Journal Pre-proof Abstract
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Walnuts are a nutrient dense food, but most health research is on English walnuts (EW). Black walnuts (BW) contain a different antioxidant and fatty acid profile, and more protein, compared to EW. The purpose of the study was to compare postprandial responses following the consumption of 3 breakfast meals containing either butter (control), BW, or EW. We hypothesized that walnut-containing meals would mitigate post-meal increases in glucose, insulin, triglycerides, and lipid peroxidation while increasing TAC compared to the traditional meal without nuts. Furthermore, we hypothesized that the BW meal would exhibit greater TAC and subjective fullness while mitigating postprandial increases in lipid peroxidation better than the EW. This was a randomized, double-blind control crossover study in 30 healthy adults with three testing visits. At each visit, subjects consumed either the control, BW, or EW meal. Blood draws and visual analog scale appetite ratings were obtained at fasting, 30, 60, 120, and 180min postprandially. The BW and EW meals resulted in greater suppression of appetite vs. control (p<0.01 and p=0.03, respectively), and the BW meal also increased fullness more than EW and control (p<0.01 and p<0.001, respectively). Finally, the BW meal also had a greater suppression of lipid peroxidation vs. control (p=0.01). There were no other treatment differences in the other measures of appetite or for glycemia, triglycerides, or total antioxidant capacity. Substituting butter in a breakfast meal with BW or EW increased fullness; however, the BW meal was superior for suppressing overall appetite while also lowering postprandial lipid peroxidation.
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Key Words: Antioxidant; Lipid Peroxidation; Appetite; Breakfast; Nut
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Journal Pre-proof 1. Introduction Traditional breakfast in developed nations is often rich in energy, sugar, and saturated fatty acids (SFA) and low in fiber and other essential nutrients [1]. This type of poor diet quality may increase one’s risk for developing obesity [2] and chronic diseases such as cardiovascular disease (CVD) and diabetes [3,4]. Conversely, foods that are rich in fiber and unsaturated fatty acids, and are low in sodium, added sugars and SFA, improve diet quality and may decrease
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chronic disease risk [4,5]. In addition, consuming foods rich in antioxidants can mitigate the
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further preventing the onset of chronic diseases [6,7].
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oxidative stress that is induced by regular metabolic processes and environmental stresses,
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Walnuts are rich in unsaturated fatty acids, antioxidants, fiber and protein [8,9] and have
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been associated with weight maintenance [10,11] and a reduced risk for CVD, diabetes, and certain cancers [8]. Therefore, substituting sources of SFA with walnuts in a breakfast meal may
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promote weight stability and overall health. Long-term intervention studies have demonstrated
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that walnut consumption reduces disease risk factors by improving blood lipids [12-20],
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endothelial function [21], and satiety [22]. However, the effects of walnut consumption on measures of glycemia and antioxidant status are less clear. Some studies report decreases in hemoglobin A1c, glucose and insulin [23,24], while other studies report no changes [25]. Similarly, one study reported improved total antioxidant capacity following long-term walnut supplementation [26], yet other studies reported no changes [27,28]. There is also limited evidence on the health benefits associated with walnut consumption in an acute setting, and the studies that do exist have conflicting findings. Some studies report increased subjective satiety following a meal containing walnuts [29,30], while others report no differences [31-33]. Likewise, postprandial attenuation of insulin and glucose has been shown in 4
Journal Pre-proof one study [30], while two others found no difference in these measures or blood lipids [29,34]. Finally, numerous studies report acute increases in plasma polyphenol concentration and total antioxidant capacity (TAC) with decreases in lipid peroxidation [35-37]; yet these results were also not consistent among all studies [36-38]. Therefore, more research is needed to elucidate the potential health benefits of walnuts during an acute meal challenge. There are two main types of walnuts commonly used as food ingredients; English
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walnuts (EW) (Juglans regia) and black walnuts (BW) (Juglans nigra). The aforementioned
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positive health effects associated with walnut consumption have been demonstrated in EW while
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BW have not been studied as thoroughly. BW contain a different nutrient and phytochemical
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profile than EW, so their effects on health may differ. To date, there is only one study
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investigating the impact of BW on health, which showed that daily consumption of BW for 4 weeks changed blood lipids in a gender dependent manner [39]. However, there were no changes
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in in antioxidant status or low-density lipoprotein (LDL) oxidation. Therefore, more research is
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needed on the potential health effects of BW consumption as well as how it will compare to EW.
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We hypothesized that the walnut-containing meals would mitigate post-meal increases in glucose, insulin, triglycerides (TG), and lipid peroxidation while improving all measures of subjective appetite and TAC compared to the traditional meal without nuts. Furthermore, we hypothesized that the BW meal would exhibit greater TAC and subjective fullness while mitigating postprandial increases in lipid peroxidation better than the EW due to the BW’s higher phytonutrient and protein content. Finally, we hypothesized that the sensory responses for taste and overall preference would be similar between test meals. To test these hypotheses, the objective of the current study was to evaluate the health effects of consuming isocaloric breakfast meals containing either BW, EW, or no nuts (control). To associate the findings to the type of 5
Journal Pre-proof breakfast meal consumed, we employed a randomized, double-blind control trial in which all participants completed all 3 acute meal challenges. 2. Methods and materials 2.1 Study Design This study was a randomized, double-blind control trial consisting of 3 study visits for 3
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different treatments. The treatments were high-fat breakfast muffins containing either butter
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(control), BW, or EW. Study visits were completed in a random order with at least 72 hours
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between each visit. This study was approved by the Institutional Review Board for human
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subjects, and informed written consent was obtained from each participant prior to testing.
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2.2 Participants
Thirty normal weight (body mass index (BMI) = 18-24.9kg/m2) adults (n = 16 women, n
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= 14 men) between the ages of 18 and 45y were recruited for the study. Exclusion criteria
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included allergies to test meal ingredients, medication use, chronic disease, supplement use, pregnancy or plans to become pregnant, special diets (i.e. ketogenic diet, intermittent fasting), or
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tobacco use. Subjects were recruited through flyers, campus emails, and word of mouth. Monetary compensation was provided for participation in the study. 2.3 Protocol 2.3.1 Lead-In Diet For 24 hours prior to each visit, participants were prescribed a diet consisting of 55-60% carbohydrates, 15-20% protein, and 20-25% fat. They were encouraged to choose meals from a pre-designed menu in which all items followed the prescribed macronutrient distribution. 6
Journal Pre-proof Participants then ate the exact same meals for the 24h period prior to each subsequent testing visit and kept a food diary. The menu and food diaries were designed to promote compliance to the lead-in diet protocol. In addition, subjects were instructed to avoid consuming fruits or vegetables for 12h prior to the visit and to avoid all nuts, nut butters, and food or drinks that were high in antioxidants or omega-3 fatty acids (a list of these foods was provided) for at least 24h prior to each visit.
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2.3.2 Visit 1
The morning following the lead-in diet, participants reported to the Human Nutrition
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Laboratory (HNL) at 0700h following an overnight fast (8-12h) and no exercise or alcohol for at
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least 16h. Research personnel first confirmed that these instructions were followed before
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anthropometric measurements were collected. Weight was measured to the nearest 0.05 kg using a Health O Meter Professional clinical scale (Sunbeam Products, Inc.; McCook, IL, USA), and
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height was measured to the nearest millimeter (mm) using an Invicta Plastics Limited
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stadiometer (London, England). Next, subjects rested quietly for 5 minutes before blood pressure
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was measured in triplicate using an Intelli Sense digital blood pressure monitor (Omron Healthcare, Inc.; Lake Forest, IL, USA). There were 30 seconds between each measurement, and the average across all three measurements were used in analyses. Finally, waist and hip circumference were measured in triplicate to the nearest mm using a Seca ergonomic circumference measuring tape (Chino, CA, USA), and the average measurement was used in analyses. Measurements of the waist were made at the approximate midpoint between the last palpable rip and the top of the iliac crest. Hip circumference measurements were measured at the widest portion of the buttocks. Wasit-to-hip ratio was calculated by dividing waist measurement (cm) by the hip measurement (cm). 7
Journal Pre-proof Following anthropometric measurements, participants answered standardized questions on appetite by marking on a continuous 100 mm visual analog scale (VAS) [40]. The questions were: 1) How hungry are you, 2) How full are you, 3) How much do you think you could eat right now (prospective consumption), and 4) How strong is your desire to eat. The anchor phrases of the 100 mm were appropriate for each question. For example, the question about hunger was anchored by “not at all hungry” at the 0 mm point and “extremely hungry” at the 100
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mm point. The distance from the lower end of the scale to the participant’s mark was measured
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and recorded in mm.
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Next, an intravenous catheter was placed in the antecubital vein and a fasting blood
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sample (t=0) was drawn. The line was kept patent with saline. Participants then consumed the
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test meal in the form of a breakfast muffin along with 200mL of water within a 5-minute period. Research personnel supervised the breakfast to ensure the entire muffin was consumed.
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Following consumption, participants completed a questionnaire to evaluate sensory appeal of the
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muffin [41,42]. The muffin was rated on several sensory modalities such as appearance, taste/flavor, texture/consistency and aroma/smell as well as overall acceptance. A modified 9-
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point hedonic scale was used with ‘1’ indicating ‘dislike extremely’ and ‘9’ indicating ‘like extremely’. Thus, the sensory evaluation was a forced categorical variable because subjects were instructed to circle a number from 1-9 to indicate their ratings for each sensory modality. Postprandial blood draws then occurred at 30min, 1h, 2h, and 3h, along with the same hunger/fullness VAS questionnaire. Overall appetite score was calculated at each time point using the following equation: [desire to eat + hunger + (100 – fullness) + prospective consumption]/4 [43,44]. For blood sample collection, all samples were immediately placed on ice and then spun at 3,000rpm for 15 minutes at 4°C. A small portion of the sample from t=0 was
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Journal Pre-proof used to measure blood lipids (Piedmont Athens Regional, Athens, Georgia, USA). The remaining plasma from all time points was aliquoted and stored at -80°C until assayed. 2.3.3 Breakfast muffins There were three different high-fat breakfast muffins. The control muffin was a traditional muffin that contained butter as the predominate source of fat. For the other two
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muffins, part of the butter was substituted for either one serving (28 grams) of BW or EW. All
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three muffins were matched for total energy (kcal), sugar, and total fat content. By design, the fatty acid composition differed slightly between all three muffins. The muffin recipes were
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developed using a nutrient analysis software program (The Food Processor, ESHA Research,
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Salem, OR). Table 1 shows the nutrient profile of each breakfast muffin.
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2.3.4 Visits 2 and 3
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At least 72h after visit 1, participants reported to the HNL for visit 2. The exact same procedures from visit 1 were repeated, except for the type of breakfast muffin consumed. Finally,
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at least 72h after visit 2, subjects returned to the lab for their final visit. Again, the exact same
consumed.
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procedures from visits 1 and 2 were repeated. The only difference was the type of muffin
2.4 Biochemical Assays The fasting blood sample from visit 1 was used to assess a standard lipid panel of total cholesterol (TC), LDL cholesterol, high-density lipoprotein (HDL) cholesterol, and TGs (Piedmont Athens Regional, Athens, Georgia, USA). In addition, fasting and postprandial TGs were measured by enzyme-based calorimetric assays (Wako Chemicals USA, Inc., Richmond, Virginia), glucose was measured using a colorimetric glucose oxidase method [45], and insulin 9
Journal Pre-proof was measured by radioimmunoassay (MilliporeSigma, Damstadt, Germany). Finally, lipid peroxidation levels were measured by the Thiobarbituric Acid Reactive Substances (TBARS) assay (Cayman Chemical Inc., Ann Arbor, MI, USA) and TAC was measured by an Oxygen Radical Absorbance Capacity (ORAC) assay (Zen Bio, Inc., Morrisville, NC) at fasting, and 60, 120, and 180min postprandially.
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2.5 Statistical analyses
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A sample size of 28 subjects was estimated using G*Power program 3.1.9.2., assuming at least 80% power (beta) and an alpha of 0.05. The SAS version 9.4 statistical package (SAS
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Institute Inc, Cary, NC) was used for all other statistical analyses, and statistical significance was
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set at α≤0.05. All results are reported as mean ± SEM unless otherwise noted. To compare
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sensory evaluation responses for each muffin and differences in baseline anthropometric measurements from the three visits, a one-way analysis of variance (ANOVA) was performed. A
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repeated measures ANOVA was conducted to determine main effects and interaction effects of
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the muffin treatment and time on TGs, insulin, glucose, lipid peroxidation, TAC and VAS
analyses. 3. Results
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responses. When significance was found, a least squares means test was used to perform post hoc
3.1 Participants Thirty-four participants consented to the study, however, four subjects dropped out during the study (Figure 1). Therefore, thirty normal weight adults (16=female, 14=male), with the average age of 21.6 ± 2.8y (range was 18 to 31y) completed all three study visits and were included in the final analyses. Their baseline characteristics are displayed in Table 2. There were 10
Journal Pre-proof no significant differences between males and females for BMI, diastolic blood pressure, TC, LDL, TG, or HDL. Height, weight, waist-to-hip ratio (WHR), systolic blood pressure, and age were significantly greater in males vs. females (p<0.01, p=0.01, p<0.001, and p=0.02, respectively). The nutrient analysis of the self-selected 24h lead-in diet the day prior to the 3 study visits showed no significant differences in total energy intake (1,978.502±102.1, 1,735.1±110.8, and 1,830.2±97.1 kcals/d) or the distribution of carbohydrate (51.1±1.5,
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50.4±2.0, and 53.0±1.6%), fat (32.1±1.2, 31.6±1.6, and 30.0±1.4%) or protein (16.8±0.7,
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18.0±0.9, and 17.0±0.7%) for visits 1, 2, and 3, respectively.
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3.2. Subjective Ratings
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Subjective VAS responses for the overall appetite score, fullness, hunger, prospective
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consumption and desire to eat are displayed in Figure 2. For overall appetite, there was a significant effect of treatment (p=0.03) and time (p<0.001), but no treatment by time interaction
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(ns) (Figure 2A). The significant treatment effect was for a greater suppression of appetite in
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both the BW and EW muffins vs. control (p<0.01 and p=0.03, respectively). There was no
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difference in appetite suppression between the BW vs. EW muffins (ns). For fullness, there was also a significant effect of treatment (p<0.001) and time (p<0.001), but no treatment by time interaction (ns) (Figure 2B). The significant treatment effect was for greater fullness in the BW muffin vs. both the EW and control muffins (p<0.01 and p<0.001, respectively). There was no difference between the EW and control (ns). Furthermore, for hunger, prospective consumption, and desire to eat, there was a significant effect of time (p<0.001 for all), but no main effect of treatment or treatment by time interactions (ns) (Figures 2C, 2D, and 2E). However, there was a trend for greater suppression of prospective consumption (p=0.06) in the EW vs. BW treatment. Finally, sensory evaluation data for each muffin is displayed in Table 3. Taste/flavor was rated 11
Journal Pre-proof significantly lower for BW muffin compared to EW (p<0.01) and control (p=0.01), and there was no difference in taste/flavor preference between the EW vs. control muffins (ns). There were also no other significant differences between any of the three muffins for appearance, texture/consistence, aroma/smell or overall acceptance. 3.3. Physiological Responses
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The postprandial changes in TBARS and ORAC are shown in Figure 3. For TBARS,
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there was a significant main effect of treatment (p=0.01), but no effect of time or treatment by time interaction (ns) (Figure 3A). The significant treatment effect was for a greater suppression
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of lipid peroxidation in the BW vs. control (p<0.01), but no difference between BW vs. EW or
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EW vs. control (ns). For ORAC, there was a significant main effect of time (p=0.03), but no
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treatment effect or treatment by time interactions (ns) (Figure 3C).
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The postprandial changes in glucose, insulin, and TGs are presented in Figure 4. For all three variables, there was a significant effect of time (p<0.001 for all), but no treatment effects or
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4. Discussion
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treatment by time interactions (ns) (Figures 4A, 4B, and 4C).
While numerous health studies have been conducted on EW, only one previous study has explored the potential health benefits of BW consumption. This is the first study to show the acute health benefits of BW consumption in healthy adults. Our results indicated that a breakfast meal containing either BW or EW induced a greater suppression of appetite compared to a butter-containing meal following an acute meal challenge. Additionally, the BW meal increased fullness more than the EW or control meals and led to reduced postprandial lipid peroxidation. These results support some of our hypotheses for appetite and lipid peroxidation, but we did not 12
Journal Pre-proof see beneficial changes in all measures of subjective appetite as expected. In addition, there was a slightly lower rating for taste, which was contrary to our sensory hypothesis, while overall preference ratings for the meals was not different. Both walnut-containing meals resulted in a greater suppression of overall appetite, but the BW meal also induced greater fullness than the EW meal. Although these results were statistically significant, it is important to consider if they are also clinically meaningful. It is
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accepted that a difference of 8-10% in the relative response between a control and treatment is
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considered to be clinically relevant [46]. On average, the difference between BW vs both EW
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and control muffins for fullness was 7.6-12.4%. In addition, the difference between the BW and
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EW vs control muffin for overall appetite was 4.8% and 4.3%, respectively. Therefore, the
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magnitude of difference for fullness for the BW vs EW and control muffins is clinically relevant, and the overall appetite outcomes, although important, should be interpreted with caution.
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Furthermore, similar beneficial effects on subjective measures of fullness have been
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previously reported following an EW-containing breakfast, (high in polyunsaturated fatty acids (PUFA), compared to the placebo which was rich in monounsaturated fatty acids (MUFA) [29].
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Conversely, others have shown no differences in subjective appetite ratings when comparing EW meals to those rich in MUFA and/or SFA [32,33]. Therefore, it is possible that other nutrients or phytochemicals, aside from the fatty acid profile, led to the differences in some measures of appetite between our three treatments. Protein is well-established as being more satiating than carbohydrate or fat during isocaloric conditions [47-49], so the slightly higher protein content of the walnut-containing meals may be one reason for our observed appetite differences. This may also explain why we found greater fullness for the BW vs. EW as the protein content is higher for BW vs. EW (14.0 vs. 11.5g). 13
Journal Pre-proof Additionally, the higher fiber content of the walnut-containing meals may have led to the better overall appetite score compared to the control meal since dietary fiber has also been shown to improve satiety [50]. Higher fiber intake is associated with lower body weights and enhanced satiety possibly due to an increase in gastric distention, fermentation, and satiety hormones [5153]. Although previous acute meal challenge studies have been unable to show greater reductions in appetite among EW meals, the amount or “dose” of fiber may be important. In those studies,
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walnut meals contained 26-32% more fiber compared to the control meals [29,32], whereas our
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walnut-enriched meals contained approximately 46% more fiber than the control. This larger
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difference between test meals may have contributed to our improved postprandial appetite responses. Regardless of the exact nutrient, or combination of nutrients, that are responsible for
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the improved appetite scores and fullness, our study shows that substituting a common fat source
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with walnuts, especially BW, improves markers of appetite.
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Due to the higher fiber content of the walnut-containing meals, we expected to see an attenuation of postprandial glucose, insulin or triglyceride compared to the control meal. We did
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not observe such differences, which is contrary to one of our hypotheses. The lack of glycemic
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differences observed may be explained by the carbohydrate-matching of our test meals. In another acute study, postprandial glycemic responses were compared among high-fat shakes with varying FA compositions (MUFA vs. PUFA), but similar carbohydrate contents, to a low-fat, high carbohydrate control [30]. The two high-fat shakes attenuated insulin and glucose more than control, but did not differ from one another. Since our carbohydrate contents were similar, the differences in fiber content or fatty acid composition likely were not strong enough to override the similar total carbohydrate content between our test meals.
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Journal Pre-proof In support of our first hypothesis, the BW meal also suppressed lipid peroxidation more than the control meal, while there was no difference between EW vs. control. Both BW and EW are high in polyphenols [54], which have been shown to protect plasma from lipid peroxidation [35,55]. However, BW have a slightly greater total phenolic content compared to the EW [54], which may explain the superior lipid peroxidation outcomes observed from the BW meal. It is also important to note that lipid peroxides, such as malondialdehyde (MDA) which was
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measured by the TBARS assay, are derived from PUFAs [56]. The EW muffin contained slightly
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more PUFA than the BW muffin (14.6 g vs. 11.6 g), thus the EW muffin was likely more
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susceptible to lipid peroxidation.
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Despite the enhanced suppression of lipid peroxidation with the BW muffin, there were no
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differences in TAC among the three test meals, which was contrary to our hypothesis. The lack of agreement between these two antioxidant measures may be explained by the ORAC assay
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mechanism. The ORAC assay works through the Hydrogen Atom Transfer (HAT) mechanism in which the antioxidant donates a hydrogen atom to the reactive oxygen species (ROS) [57].
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However, the specific antioxidants within the BW that resulted in less lipid peroxidation may not
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undergo the HAT mechanism, limiting the ability of the ORAC assay to comprehensively determine TAC. It is also possible that lipid peroxidation occurs in the gastrointestinal tract prior to absorption; however, previous research has shown that lipid peroxidation byproducts are absorbed [58-60] and should therefore still appear in plasma. Unfortunately, TAC has not been previously measured in human plasma following BW ingestion limiting our ability to make comparisons with other studies. Previous studies in EW have found increases in TAC when measured by hydrophilic ORAC when incorporating 80-90g of walnuts into test meals [35,37].
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Journal Pre-proof Since our test meals only contained 28g of walnuts, our dose of walnuts may not have been substantial enough to observe differences in plasma TAC. There were some limitations to the current study. First, we did not provide a 24h lead-in diet, so slight deviations to our prescribed macronutrient distribution existed based on the results from the nutrient analysis. Importantly, however, the total energy and macronutrient distribution did not differ between any of the 3 visits. We also relied on self-report intake data for the lead-in
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diet, which is another limitation as omissions or mistakes by the participants could have
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occurred. In addition, we did not introduce a buffet meal to determine if increased subjective
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fullness measures translated into reduced intake at a subsequent meal. We also did not control for
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weight loss histories, which could have affected subjective appetite outcomes. Finally, we had a
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relatively young, healthy population, so extrapolating the current results into older age populations may not be appropriate. Notably, there were significant pre-treatment between males
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and females, but these differences were unlikely to affect our outcomes due to the crossover
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design and within subjects analysis. All subjects of both genders received all treatments. In conclusion, we found that substituting BW for butter in a breakfast meal improved
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markers of health through decreased appetite, increased fullness and suppressed lipid peroxidation. We also observed improved appetite scores for the EW, but these scores did not differ from BW. These findings for both walnuts, but especially the BW, are clinically relevant because consuming a satiating breakfast may reduce overall intake throughout the day, which will promote weight stability. In addition, reducing lipid peroxidation may reduce the risk for chronic diseases that are associated with oxidative stress [61]. Future studies are needed in other populations at risk for chronic disease, as well as measured appetite hormones, subsequent energy intake, and TAC following meals containing different doses of BW and EW. 16
Journal Pre-proof Acknowledgment The authors have no conflicts of interest to declare. This work was supported by Hammons
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Product Company [grant number AWD00006706].
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[16] Olmedilla-Alonso B, Granado-Lorencio F, Herrero-Barbudo C, Blanco-Navarro I, Blázquez-García S, Pérez-Sacristán B. Consumption of restructured meat products with added walnuts has a cholesterol-lowering effect in subjects at high cardiovascular risk: a randomised, crossover, placebo-controlled study. J Am Coll Nutr. 2008;27:342-348. [17] Torabian S, Haddad E, Cordero-MacIntyre Z, Tanzman J, Fernandez ML, Sabate J. Longterm walnut supplementation without dietary advice induces favorable serum lipid changes in free-living individuals. Eur J Clin Nutr. 2010;64:274-279. [18] Njike VY, Ayettey R, Petraro P, Treu JA, Katz DL. Walnut ingestion in adults at risk for diabetes: effects on body composition, diet quality, and cardiac risk measures. BMJ Open Diabetes Res Care. 2015;3:e000115. [19] Bamberger C, Rossmeier A, Lechner K, Wu L, Waldmann E, Stark RG, Altenhofer J, Henze K, Parhofer KG. A walnut-enriched diet reduces lipids in healthy caucasian subjects, independent of recommended macronutrient replacement and time point of consumption: A prospective, randomized, controlled trial. Nutrients. 2017;9. [20] Rock CL, Flatt SW, Barkai HS, Pakiz B, Heath DD. Walnut consumption in a weight reduction intervention: effects on body weight, biological measures, blood pressure and satiety. Nutr J. 2017;16:76. [21] Katz DL, Davidhi A, Ma Y, Kavak Y, Bifulco L, Njike VY. Effects of walnuts on endothelial function in overweight adults with visceral obesity: a randomized, controlled, crossover trial. J Am Coll Nutr. 2012;31:415-423. [22] Stevenson JL, Paton CM, Cooper JA. Hunger and satiety responses to high-fat meals after a high-polyunsaturated fat diet: A randomized trial. Nutrition. 2017;41:14-23. [23] Tapsell LC, Batterham MJ, Teuss G, Tan SY, Dalton S, Quick CJ, Gillen LJ, Charlton KE. Long-term effects of increased dietary polyunsaturated fat from walnuts on metabolic parameters in type II diabetes. Eur J Clin Nutr. 2009;63:1008-1015. [24] Kalgaonkar S, Almario RU, Gurusinghe D, Garamendi EM, Buchan W, Kim K, Karakas SE. Differential effects of walnuts vs almonds on improving metabolic and endocrine parameters in PCOS. Eur J Clin Nutr. 2011;65:386-393. [25] Tapsell LC, Gillen LJ, Patch CS, Batterham M, Owen A, Baré M, Kennedy M. Including walnuts in a low-fat/modified-fat diet improves HDL cholesterol-to-total cholesterol ratios in patients with type 2 diabetes. Diabetes Care. 2004;27:2777-2783. [26] Hudthagosol C, Haddad E, Jongsuwat R. Antioxidant activity comparison of walnuts and fatty fish. J Med Assoc Thai. 2012;95:179-188. [27] Davis L, Stonehouse W, du Loots T, Mukkuddem-Petersen J, van der Westhuizen F, Hanekom S, Jerling J. The effects of high walnut and cashew nut diets on the antioxidant status of subjects with metabolic syndrome. Eur J Nutr. 2007;46:155-164. [28] Rorabaugh JM, Singh AP, Sherrell IM, Freeman MR, Vorsa N, Fitschen P, Malone C, Maher MA, Wilson T. English and black walnut phenolic antioxidant activity in vitro and following human nut consumption. Food Nutr Sci. 2011;2:193-200. [29] Brennan AM, Sweeney LL, Liu X, Mantzoros CS. Walnut consumption increases satiation but has no effect on insulin resistance or the metabolic profile over a 4-day period. Obesity (Silver Spring). 2010;18:1176-1182. [30] Burton-Freeman B. Sex and cognitive dietary restraint influence cholecystokinin release and satiety in response to preloads varying in fatty acid composition and content. J Nutr. 2005;135:1407-1414.
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[31] Eastep A, Kern S, Schmitz S, Sebranek P, Hoppe M, Scott H, Horwath I, Wilson T. Effect of snack-choice on pre-meal hunger and post-meal satiety in college freshmen. FASEB. 2017;31. [32] Rock CL, Flatt SW, Barkai HS, Pakiz B, Heath DD. A walnut-containing meal had similar effects on early satiety, CCK, and PYY, but attenuated the postprandial GLP-1 and insulin response compared to a nut-free control meal. Appetite. 2017;117:51-57. [33] Casas-Agustench P, López-Uriarte P, Bulló M, Ros E, Gómez-Flores A, Salas-Salvadó J. Acute effects of three high-fat meals with different fat saturations on energy expenditure, substrate oxidation and satiety. Clin Nutr. 2009;28:39-45. [34] Cortés B, Núñez I, Cofán M, Gilabert R, Pérez-Heras A, Casals E, Deulofeu R, Ros E. Acute effects of high-fat meals enriched with walnuts or olive oil on postprandial endothelial function. J Am Coll Cardiol. 2006;48:1666-1671. [35] Torabian S, Haddad E, Rajaram S, Banta J, Sabaté J. Acute effect of nut consumption on plasma total polyphenols, antioxidant capacity and lipid peroxidation. J Hum Nutr Diet. 2009;22:64-71. [36] Berryman C, Grieger J, West S, Chen C, Blumberg J, Rothblat G, Sankaranarayanan S, Kris-Etherton P. Acute consumption of walnut and walnut components differentially affect postprandial lipemia, endothelial function, oxidative stress, and cholesterol efflux in humans with mild hypercholesterolemia. J Nutr. 2013;146:788-794. [37] Haddad EH, Gaban-Chong N, Oda K, Sabaté J. Effect of a walnut meal on postprandial oxidative stress and antioxidants in healthy individuals. Nutr J. 2014;13:4. [38] McKay DL, Chen CY, Yeum KJ, Matthan NR, Lichtenstein AH, Blumberg JB. Chronic and acute effects of walnuts on antioxidant capacity and nutritional status in humans: a randomized, cross-over pilot study. Nutr J. 2010;9:21. [39] Fitschen PJ, Rolfhus KR, Winfrey MR, Allen BK, Manzy M, Maher MA. Cardiovascular effects of consumption of black versus English walnuts. J Med Food. 2011;14:890-898. [40] Kral TV, Roe LS, Rolls BJ. Combined effects of energy density and portion size on energy intake in women. Am J Clin Nutr. 2004;79:962-968. [41] Peryam D, Pilgrim F. Hedonic scale method of measuring food preferences. Food Technology. 1957;11:9-14. [42] Buhaly M, Bordi PL. Development and sensory evaluation of a high-protein, vitaminfortified fruit roll-up for children with Cystic Fibrosis. Food Res Int. 2004;14:243-256. [43] Stewart L, Black R, Wolever T, Anderson G. The relationship between the glycemic response to breakfast cereals and subjective appetite and food intake. Nutr Res. 1997;17:12491260. [44] Flint A, Raben A, Blundell JE, Astrup A. Reproducibility, power and validity of visual analogue scales in assessment of appetite sensations in single test meal studies. Int J Obes Relat Metab Disord. 2000;24:38-48. [45] Blake DA, McLean NV. A colorimetric assay for the measurement of D-glucose consumption by cultured cells. Anal Biochem. 1989;177:156-160. [46] Blundell J, de Graaf C, Hulshof T, Jebb S, Livingstone B, Lluch A, Mela D, Salah S, Schuring E, van der Knaap H, Westerterp M. Appetite control: methodological aspects of the evaluation of foods. Obes Rev. 2010;11:251-270. [47] Astrup A. The satiating power of protein--a key to obesity prevention? Am J Clin Nutr. 2005;82:1-2.
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[48] Westerterp-Plantenga MS, Rolland V, Wilson SA, Westerterp KR. Satiety related to 24 h diet-induced thermogenesis during high protein/carbohydrate vs high fat diets measured in a respiration chamber. Eur J Clin Nutr. 1999;53:495-502. [49] Paddon-Jones D, Westman E, Mattes RD, Wolfe RR, Astrup A, Westerterp-Plantenga M. Protein, weight management, and satiety. Am J Clin Nutr. 2008;87:1558S-1561S. [50] Slavin J, Green H. Dietary fibre and satiety. Nutrition Bulletin. 2007;32:32-42. [51] Clark M, Slavin J. The effect of fiber on satiety and food intake: a systematic review. J Am Coll Nutr. 2013;32. [52] Ye Z, Arumugam V, Haugabrooks E, Williamson P, Hendrich S. Soluble dietary fiber (Fibersol-2) decreased hunger and increased sateity hormones in humans when ingested with a meal. Nutr Res. 2015;35. [53] Adam CL, Williams PA, Garden KE, Thomson LM, Ross AW. Dose-dependent effects of a soluble dietary fibre (pectin) on food intake, adiposity, gut hypertrophy and gut satiety hormone secretion in rats. PLoS One. 2015;10:e0115438. [54] Rodrigues C, Camara S, Shlegel V. A review on the potential human health benefits of the black walnut: A comparison with the other English walnuts and other tree nuts. Int J Food Prop. 2016;19. [55] Nigdikar SV, Williams NR, Griffin BA, Howard AN. Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivo. Am J Clin Nutr. 1998;68:258-265. [56] Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev. 2014;2014:360438. [57] Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem. 2005;53:42904302. [58] Gorelik S, Ligumsky M, Kohen R, Kanner J. A novel function of red wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation products. The FASEB Journal. 2007;22:41-46. [59] Girón-Calle J, Alaiz M, Millán F, Ruiz-Gutiérrez V, Vioque E. Bound Malondialdehyde in Foods: Bioavailability of the N-2-Propenals of Lysine. Journal of Agricultural and Food Chemistry. 2002;50:6194-6198. [60] Suomela J-P, Ahotupa M, Kallio H. Triacylglycerol oxidation in pig lipoproteins after a diet rich in oxidized sunflower seed oil. Lipids. 2005;40:437-444. [61] Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Biomed Sci. 2008;4:89-96.
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Journal Pre-proof Figure Captions Figure 1. Consolidating Standards of Reporting (CONSORT) flow diagram of selection of study participants.
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Figure 2. Time course for change in subjective appetite score, fullness, hunger, prospective consumption and desire to eat for fasting (t=0) and after consuming a control, black walnut, or English walnut-containing muffin, measured on a 100-mm scale (Figure 2A, B, C, D, and E, respectively). Repeated Measures Analysis of Variance (ANOVA) was used to determine main and interaction effects of treatment and time. There was a significant treatment effect for greater suppression of appetite for the black walnut (BW) and English walnut (EW) meals vs. control (p<0.01 and p=0.03, respectively) (Figure 2A). There was a significant treatment effect for greater fullness for the BW meal vs. both the EW and control (p<0.01 and p<0.0001, respectively) (Figure 2B). Data are presented as means ± SEM (n=30). Significance was defined as P < .05.
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Figure 3. Time course of meal responses for Thiobarbituric Acid Reactive Substances (TBARS) and Oxygen Radical Absorbance Capacity (ORAC) assays at fasting (t=0) and after consuming a control, black walnut, or English-walnut containing muffin (Figure 3A and B, respectively). Repeated Measures Analysis of Variance (ANOVA) was used to determine main and interaction effects of treatment and time. There was a significant treatment effect for lower postprandial TBARS for the black walnut meal vs. control (p<0.01) (Figure 3A). Data are presented as means ± SEM (n=30). Significance was defined as P < .05.
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Figure 4. Time course of meal responses for glucose, insulin, and triglycerides (TG) at fasting (t=0) and after consuming a control, black walnut, or English-walnut containing muffin (Figure 3A, B, and C, respectively). Repeated Measures Analysis of Variance (ANOVA) was used to determine main and interaction effects of treatment and time. There were no significant differences between meal treatments. Data are presented as means ± SEM (n=30).
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English Walnut Muffin 696.3 56.9 9.1 34.1 72.2 4.1 11.5 43.3 16.6 9.8 14.6 2.9 11.6 59.9
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Table 1. Nutrient Breakdown of each Test Meal Control Black Walnut Composition Muffin Muffin Energy (kcal) 692.1 686.4 % Carbohydrate 56.4 60.1 % Protein 6.1 11.9 % Fat 37.4 35.2 Carbohydrate (g) 68.3 71.0 Fiber (g) 2.2 4.2 Protein (g) 7.4 14.0 Fat (g) 45.3 41.6 Saturated Fat (g) 27.7 15.9 Monounsaturated Fat (g) 13.2 11.7 Polyunsaturated Fat (g) 2.0 11.6 Omega-3 FA (g) 0.7 1.13 Omega-6 FA (g) 1.3 10.4 Total Nut Phenolics (mg)* n/a 68.6
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Abbreviations: FA = Fatty Acid; g = grams; kcal = kilocalories; n/a = not applicable *Total phenolic data for the 28g serving of black and English walnuts [54].
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Female (n=16) 20.7 ± 1.4 165.6 ± 4.4 63.6 ± 6.8 23.2 ± 2.0 0.7 ± 0.0 112.6 ±7.3 70.6 ±7.3 150.4 ± 22.7 81.6 ± 17.3 53.5 ± 13.2 76.5 ± 30.6
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Table 2. Baseline Demographics Male (n=14) Age (y) 22.7 ± 3.6* Height (cm) 176.8 ± 8.9* Weight (kg) 74.1 ± 9.3* BMI (kg/m2) 23.6 ± 1.7 WHR (cm) 0.8 ± 0.0* SBP (mm Hg) 122.3 ± 13.8* DBP (mm Hg) 75.6 ± 9.0 Total Cholesterol (mg/dL) 157.6 ± 34.7 LDL (mg/dL) 88.4 ± 31.2 HDL (mg/dL) 49.4 ± 10.0 Triglycerides (mg/dL) 98.9 ± 45.4
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Values are presented as mean ± SD, and two-sample t-tests were used to compare the baseline demographics of males vs females at baseline. Abbreviations: BMI= Body mass index; WHR= Waist to hip ratio; SBP= Systolic blood pressure; DBP= Diastolic blood pressure; LDL= Low Density Lipoprotein; HDL= High Density Lipoprotein. *Indicates significant difference between males and females at P<0.05.
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Journal Pre-proof Table 3. Sensory Evaluation of Test Meals Control Muffin Black Walnut Muffin Appearance 7.4 ± 0.3 7.5 ± 0.2 Taste/Flavor 7.8 ± 0.2 7.0 ± 0.3* Texture/Consistency 7.7 ± 0.2 7.2 ± 0.3 Aroma/Smell 7.3 ± 0.3 7.4 ± 0.2 Overall Acceptance 7.6 ± 1.0 7.4 ± 0.3
English Walnut Muffin 7.8 ± 0.2 8.0 ± 0.1 8.0 ± 0.2 7.8 ± 0.2 8.0 ± 0.1
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Values are presented as mean ± SEM, and a one-way Analysis of Variance (ANOVA) was used to compare sensory ratings between test meal. * Indicates significantly lower score for Taste/Flavor for Black Walnut vs. both control and English Walnut meals.
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Journal Pre-proof Highlights:
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Black walnuts are an adequate substitute for butter fat in breakfast meals based on sensory and physiological responses. Incorporating black walnuts into a breakfast meal increased feelings of fullness more than the addition of English walnuts. Black walnuts reduced lipid peroxidation more than a traditional butter muffin.
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Figure 1
Figure 2
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Liana L. Rodrigues, Jamie A. Cooper, Chad M. Paton PII:
S0271-5317(19)30585-8
DOI:
https://doi.org/10.1016/j.nutres.2019.09.002
Reference:
NTR 8047
To appear in:
Nutrition Research
Received date:
17 June 2019
Revised date:
22 August 2019
Accepted date:
4 September 2019
Please cite this article as: L.L. Rodrigues, J.A. Cooper and C.M. Paton, Acute consumption of Black walnuts increases fullness and decreases lipid peroxidation in humans, Nutrition Research(2019), https://doi.org/10.1016/j.nutres.2019.09.002
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof Acute Consumption of Black Walnuts Increases Fullness and Decreases Lipid Peroxidation in Humans Authors: Liana L. Rodrigues1 Jamie A. Cooper1
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Chad M. Paton1, 2
Author Affiliations: 1
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Department of Foods and Nutrition, University of Georgia, Athens, GA, USA
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Department of Food Science and Technology, University of Georgia, Athens, GA, USA
Corresponding Author:
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Chad M. Paton, Department of Food Science and Technology, University of Georgia, 100 Cedar Street,
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Athens, GA 30602, Phone: 706-542-3750, Email: [email protected]
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Abbreviations ANOVA: Analysis of Variance BMI: body mass index BW: black walnut CVD: cardiovascular disease EW: English walnut HDL: high-density lipoprotein HNL: Human Nutrition Laboratory LDL: low-density lipoprotein MUFA: monounsaturated fatty acid ORAC: Oxygen Radical Absorbance Capacity PUFA: polyunsaturated fatty acid SEM: Standard Error of Mean SFA: saturated fatty acid TAC: Total Antioxidant Capacity TBARS: Thiobarbituric Acid Reactive Substances TC: total cholesterol TG: triglycerides VAS: Visual Analog Scale WHR: Waist-to-Hip Ratio
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Journal Pre-proof Abstract
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Walnuts are a nutrient dense food, but most health research is on English walnuts (EW). Black walnuts (BW) contain a different antioxidant and fatty acid profile, and more protein, compared to EW. The purpose of the study was to compare postprandial responses following the consumption of 3 breakfast meals containing either butter (control), BW, or EW. We hypothesized that walnut-containing meals would mitigate post-meal increases in glucose, insulin, triglycerides, and lipid peroxidation while increasing TAC compared to the traditional meal without nuts. Furthermore, we hypothesized that the BW meal would exhibit greater TAC and subjective fullness while mitigating postprandial increases in lipid peroxidation better than the EW. This was a randomized, double-blind control crossover study in 30 healthy adults with three testing visits. At each visit, subjects consumed either the control, BW, or EW meal. Blood draws and visual analog scale appetite ratings were obtained at fasting, 30, 60, 120, and 180min postprandially. The BW and EW meals resulted in greater suppression of appetite vs. control (p<0.01 and p=0.03, respectively), and the BW meal also increased fullness more than EW and control (p<0.01 and p<0.001, respectively). Finally, the BW meal also had a greater suppression of lipid peroxidation vs. control (p=0.01). There were no other treatment differences in the other measures of appetite or for glycemia, triglycerides, or total antioxidant capacity. Substituting butter in a breakfast meal with BW or EW increased fullness; however, the BW meal was superior for suppressing overall appetite while also lowering postprandial lipid peroxidation.
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Key Words: Antioxidant; Lipid Peroxidation; Appetite; Breakfast; Nut
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Journal Pre-proof 1. Introduction Traditional breakfast in developed nations is often rich in energy, sugar, and saturated fatty acids (SFA) and low in fiber and other essential nutrients [1]. This type of poor diet quality may increase one’s risk for developing obesity [2] and chronic diseases such as cardiovascular disease (CVD) and diabetes [3,4]. Conversely, foods that are rich in fiber and unsaturated fatty acids, and are low in sodium, added sugars and SFA, improve diet quality and may decrease
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chronic disease risk [4,5]. In addition, consuming foods rich in antioxidants can mitigate the
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further preventing the onset of chronic diseases [6,7].
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oxidative stress that is induced by regular metabolic processes and environmental stresses,
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Walnuts are rich in unsaturated fatty acids, antioxidants, fiber and protein [8,9] and have
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been associated with weight maintenance [10,11] and a reduced risk for CVD, diabetes, and certain cancers [8]. Therefore, substituting sources of SFA with walnuts in a breakfast meal may
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promote weight stability and overall health. Long-term intervention studies have demonstrated
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that walnut consumption reduces disease risk factors by improving blood lipids [12-20],
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endothelial function [21], and satiety [22]. However, the effects of walnut consumption on measures of glycemia and antioxidant status are less clear. Some studies report decreases in hemoglobin A1c, glucose and insulin [23,24], while other studies report no changes [25]. Similarly, one study reported improved total antioxidant capacity following long-term walnut supplementation [26], yet other studies reported no changes [27,28]. There is also limited evidence on the health benefits associated with walnut consumption in an acute setting, and the studies that do exist have conflicting findings. Some studies report increased subjective satiety following a meal containing walnuts [29,30], while others report no differences [31-33]. Likewise, postprandial attenuation of insulin and glucose has been shown in 4
Journal Pre-proof one study [30], while two others found no difference in these measures or blood lipids [29,34]. Finally, numerous studies report acute increases in plasma polyphenol concentration and total antioxidant capacity (TAC) with decreases in lipid peroxidation [35-37]; yet these results were also not consistent among all studies [36-38]. Therefore, more research is needed to elucidate the potential health benefits of walnuts during an acute meal challenge. There are two main types of walnuts commonly used as food ingredients; English
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walnuts (EW) (Juglans regia) and black walnuts (BW) (Juglans nigra). The aforementioned
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positive health effects associated with walnut consumption have been demonstrated in EW while
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BW have not been studied as thoroughly. BW contain a different nutrient and phytochemical
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profile than EW, so their effects on health may differ. To date, there is only one study
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investigating the impact of BW on health, which showed that daily consumption of BW for 4 weeks changed blood lipids in a gender dependent manner [39]. However, there were no changes
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in in antioxidant status or low-density lipoprotein (LDL) oxidation. Therefore, more research is
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needed on the potential health effects of BW consumption as well as how it will compare to EW.
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We hypothesized that the walnut-containing meals would mitigate post-meal increases in glucose, insulin, triglycerides (TG), and lipid peroxidation while improving all measures of subjective appetite and TAC compared to the traditional meal without nuts. Furthermore, we hypothesized that the BW meal would exhibit greater TAC and subjective fullness while mitigating postprandial increases in lipid peroxidation better than the EW due to the BW’s higher phytonutrient and protein content. Finally, we hypothesized that the sensory responses for taste and overall preference would be similar between test meals. To test these hypotheses, the objective of the current study was to evaluate the health effects of consuming isocaloric breakfast meals containing either BW, EW, or no nuts (control). To associate the findings to the type of 5
Journal Pre-proof breakfast meal consumed, we employed a randomized, double-blind control trial in which all participants completed all 3 acute meal challenges. 2. Methods and materials 2.1 Study Design This study was a randomized, double-blind control trial consisting of 3 study visits for 3
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different treatments. The treatments were high-fat breakfast muffins containing either butter
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(control), BW, or EW. Study visits were completed in a random order with at least 72 hours
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between each visit. This study was approved by the Institutional Review Board for human
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subjects, and informed written consent was obtained from each participant prior to testing.
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2.2 Participants
Thirty normal weight (body mass index (BMI) = 18-24.9kg/m2) adults (n = 16 women, n
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= 14 men) between the ages of 18 and 45y were recruited for the study. Exclusion criteria
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included allergies to test meal ingredients, medication use, chronic disease, supplement use, pregnancy or plans to become pregnant, special diets (i.e. ketogenic diet, intermittent fasting), or
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tobacco use. Subjects were recruited through flyers, campus emails, and word of mouth. Monetary compensation was provided for participation in the study. 2.3 Protocol 2.3.1 Lead-In Diet For 24 hours prior to each visit, participants were prescribed a diet consisting of 55-60% carbohydrates, 15-20% protein, and 20-25% fat. They were encouraged to choose meals from a pre-designed menu in which all items followed the prescribed macronutrient distribution. 6
Journal Pre-proof Participants then ate the exact same meals for the 24h period prior to each subsequent testing visit and kept a food diary. The menu and food diaries were designed to promote compliance to the lead-in diet protocol. In addition, subjects were instructed to avoid consuming fruits or vegetables for 12h prior to the visit and to avoid all nuts, nut butters, and food or drinks that were high in antioxidants or omega-3 fatty acids (a list of these foods was provided) for at least 24h prior to each visit.
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2.3.2 Visit 1
The morning following the lead-in diet, participants reported to the Human Nutrition
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Laboratory (HNL) at 0700h following an overnight fast (8-12h) and no exercise or alcohol for at
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least 16h. Research personnel first confirmed that these instructions were followed before
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anthropometric measurements were collected. Weight was measured to the nearest 0.05 kg using a Health O Meter Professional clinical scale (Sunbeam Products, Inc.; McCook, IL, USA), and
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height was measured to the nearest millimeter (mm) using an Invicta Plastics Limited
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stadiometer (London, England). Next, subjects rested quietly for 5 minutes before blood pressure
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was measured in triplicate using an Intelli Sense digital blood pressure monitor (Omron Healthcare, Inc.; Lake Forest, IL, USA). There were 30 seconds between each measurement, and the average across all three measurements were used in analyses. Finally, waist and hip circumference were measured in triplicate to the nearest mm using a Seca ergonomic circumference measuring tape (Chino, CA, USA), and the average measurement was used in analyses. Measurements of the waist were made at the approximate midpoint between the last palpable rip and the top of the iliac crest. Hip circumference measurements were measured at the widest portion of the buttocks. Wasit-to-hip ratio was calculated by dividing waist measurement (cm) by the hip measurement (cm). 7
Journal Pre-proof Following anthropometric measurements, participants answered standardized questions on appetite by marking on a continuous 100 mm visual analog scale (VAS) [40]. The questions were: 1) How hungry are you, 2) How full are you, 3) How much do you think you could eat right now (prospective consumption), and 4) How strong is your desire to eat. The anchor phrases of the 100 mm were appropriate for each question. For example, the question about hunger was anchored by “not at all hungry” at the 0 mm point and “extremely hungry” at the 100
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mm point. The distance from the lower end of the scale to the participant’s mark was measured
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and recorded in mm.
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Next, an intravenous catheter was placed in the antecubital vein and a fasting blood
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sample (t=0) was drawn. The line was kept patent with saline. Participants then consumed the
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test meal in the form of a breakfast muffin along with 200mL of water within a 5-minute period. Research personnel supervised the breakfast to ensure the entire muffin was consumed.
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Following consumption, participants completed a questionnaire to evaluate sensory appeal of the
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muffin [41,42]. The muffin was rated on several sensory modalities such as appearance, taste/flavor, texture/consistency and aroma/smell as well as overall acceptance. A modified 9-
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point hedonic scale was used with ‘1’ indicating ‘dislike extremely’ and ‘9’ indicating ‘like extremely’. Thus, the sensory evaluation was a forced categorical variable because subjects were instructed to circle a number from 1-9 to indicate their ratings for each sensory modality. Postprandial blood draws then occurred at 30min, 1h, 2h, and 3h, along with the same hunger/fullness VAS questionnaire. Overall appetite score was calculated at each time point using the following equation: [desire to eat + hunger + (100 – fullness) + prospective consumption]/4 [43,44]. For blood sample collection, all samples were immediately placed on ice and then spun at 3,000rpm for 15 minutes at 4°C. A small portion of the sample from t=0 was
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Journal Pre-proof used to measure blood lipids (Piedmont Athens Regional, Athens, Georgia, USA). The remaining plasma from all time points was aliquoted and stored at -80°C until assayed. 2.3.3 Breakfast muffins There were three different high-fat breakfast muffins. The control muffin was a traditional muffin that contained butter as the predominate source of fat. For the other two
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muffins, part of the butter was substituted for either one serving (28 grams) of BW or EW. All
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three muffins were matched for total energy (kcal), sugar, and total fat content. By design, the fatty acid composition differed slightly between all three muffins. The muffin recipes were
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developed using a nutrient analysis software program (The Food Processor, ESHA Research,
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Salem, OR). Table 1 shows the nutrient profile of each breakfast muffin.
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2.3.4 Visits 2 and 3
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At least 72h after visit 1, participants reported to the HNL for visit 2. The exact same procedures from visit 1 were repeated, except for the type of breakfast muffin consumed. Finally,
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at least 72h after visit 2, subjects returned to the lab for their final visit. Again, the exact same
consumed.
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procedures from visits 1 and 2 were repeated. The only difference was the type of muffin
2.4 Biochemical Assays The fasting blood sample from visit 1 was used to assess a standard lipid panel of total cholesterol (TC), LDL cholesterol, high-density lipoprotein (HDL) cholesterol, and TGs (Piedmont Athens Regional, Athens, Georgia, USA). In addition, fasting and postprandial TGs were measured by enzyme-based calorimetric assays (Wako Chemicals USA, Inc., Richmond, Virginia), glucose was measured using a colorimetric glucose oxidase method [45], and insulin 9
Journal Pre-proof was measured by radioimmunoassay (MilliporeSigma, Damstadt, Germany). Finally, lipid peroxidation levels were measured by the Thiobarbituric Acid Reactive Substances (TBARS) assay (Cayman Chemical Inc., Ann Arbor, MI, USA) and TAC was measured by an Oxygen Radical Absorbance Capacity (ORAC) assay (Zen Bio, Inc., Morrisville, NC) at fasting, and 60, 120, and 180min postprandially.
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2.5 Statistical analyses
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A sample size of 28 subjects was estimated using G*Power program 3.1.9.2., assuming at least 80% power (beta) and an alpha of 0.05. The SAS version 9.4 statistical package (SAS
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Institute Inc, Cary, NC) was used for all other statistical analyses, and statistical significance was
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set at α≤0.05. All results are reported as mean ± SEM unless otherwise noted. To compare
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sensory evaluation responses for each muffin and differences in baseline anthropometric measurements from the three visits, a one-way analysis of variance (ANOVA) was performed. A
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repeated measures ANOVA was conducted to determine main effects and interaction effects of
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the muffin treatment and time on TGs, insulin, glucose, lipid peroxidation, TAC and VAS
analyses. 3. Results
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responses. When significance was found, a least squares means test was used to perform post hoc
3.1 Participants Thirty-four participants consented to the study, however, four subjects dropped out during the study (Figure 1). Therefore, thirty normal weight adults (16=female, 14=male), with the average age of 21.6 ± 2.8y (range was 18 to 31y) completed all three study visits and were included in the final analyses. Their baseline characteristics are displayed in Table 2. There were 10
Journal Pre-proof no significant differences between males and females for BMI, diastolic blood pressure, TC, LDL, TG, or HDL. Height, weight, waist-to-hip ratio (WHR), systolic blood pressure, and age were significantly greater in males vs. females (p<0.01, p=0.01, p<0.001, and p=0.02, respectively). The nutrient analysis of the self-selected 24h lead-in diet the day prior to the 3 study visits showed no significant differences in total energy intake (1,978.502±102.1, 1,735.1±110.8, and 1,830.2±97.1 kcals/d) or the distribution of carbohydrate (51.1±1.5,
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50.4±2.0, and 53.0±1.6%), fat (32.1±1.2, 31.6±1.6, and 30.0±1.4%) or protein (16.8±0.7,
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18.0±0.9, and 17.0±0.7%) for visits 1, 2, and 3, respectively.
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3.2. Subjective Ratings
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Subjective VAS responses for the overall appetite score, fullness, hunger, prospective
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consumption and desire to eat are displayed in Figure 2. For overall appetite, there was a significant effect of treatment (p=0.03) and time (p<0.001), but no treatment by time interaction
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(ns) (Figure 2A). The significant treatment effect was for a greater suppression of appetite in
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both the BW and EW muffins vs. control (p<0.01 and p=0.03, respectively). There was no
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difference in appetite suppression between the BW vs. EW muffins (ns). For fullness, there was also a significant effect of treatment (p<0.001) and time (p<0.001), but no treatment by time interaction (ns) (Figure 2B). The significant treatment effect was for greater fullness in the BW muffin vs. both the EW and control muffins (p<0.01 and p<0.001, respectively). There was no difference between the EW and control (ns). Furthermore, for hunger, prospective consumption, and desire to eat, there was a significant effect of time (p<0.001 for all), but no main effect of treatment or treatment by time interactions (ns) (Figures 2C, 2D, and 2E). However, there was a trend for greater suppression of prospective consumption (p=0.06) in the EW vs. BW treatment. Finally, sensory evaluation data for each muffin is displayed in Table 3. Taste/flavor was rated 11
Journal Pre-proof significantly lower for BW muffin compared to EW (p<0.01) and control (p=0.01), and there was no difference in taste/flavor preference between the EW vs. control muffins (ns). There were also no other significant differences between any of the three muffins for appearance, texture/consistence, aroma/smell or overall acceptance. 3.3. Physiological Responses
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The postprandial changes in TBARS and ORAC are shown in Figure 3. For TBARS,
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there was a significant main effect of treatment (p=0.01), but no effect of time or treatment by time interaction (ns) (Figure 3A). The significant treatment effect was for a greater suppression
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of lipid peroxidation in the BW vs. control (p<0.01), but no difference between BW vs. EW or
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EW vs. control (ns). For ORAC, there was a significant main effect of time (p=0.03), but no
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treatment effect or treatment by time interactions (ns) (Figure 3C).
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The postprandial changes in glucose, insulin, and TGs are presented in Figure 4. For all three variables, there was a significant effect of time (p<0.001 for all), but no treatment effects or
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4. Discussion
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treatment by time interactions (ns) (Figures 4A, 4B, and 4C).
While numerous health studies have been conducted on EW, only one previous study has explored the potential health benefits of BW consumption. This is the first study to show the acute health benefits of BW consumption in healthy adults. Our results indicated that a breakfast meal containing either BW or EW induced a greater suppression of appetite compared to a butter-containing meal following an acute meal challenge. Additionally, the BW meal increased fullness more than the EW or control meals and led to reduced postprandial lipid peroxidation. These results support some of our hypotheses for appetite and lipid peroxidation, but we did not 12
Journal Pre-proof see beneficial changes in all measures of subjective appetite as expected. In addition, there was a slightly lower rating for taste, which was contrary to our sensory hypothesis, while overall preference ratings for the meals was not different. Both walnut-containing meals resulted in a greater suppression of overall appetite, but the BW meal also induced greater fullness than the EW meal. Although these results were statistically significant, it is important to consider if they are also clinically meaningful. It is
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accepted that a difference of 8-10% in the relative response between a control and treatment is
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considered to be clinically relevant [46]. On average, the difference between BW vs both EW
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and control muffins for fullness was 7.6-12.4%. In addition, the difference between the BW and
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EW vs control muffin for overall appetite was 4.8% and 4.3%, respectively. Therefore, the
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magnitude of difference for fullness for the BW vs EW and control muffins is clinically relevant, and the overall appetite outcomes, although important, should be interpreted with caution.
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Furthermore, similar beneficial effects on subjective measures of fullness have been
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previously reported following an EW-containing breakfast, (high in polyunsaturated fatty acids (PUFA), compared to the placebo which was rich in monounsaturated fatty acids (MUFA) [29].
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Conversely, others have shown no differences in subjective appetite ratings when comparing EW meals to those rich in MUFA and/or SFA [32,33]. Therefore, it is possible that other nutrients or phytochemicals, aside from the fatty acid profile, led to the differences in some measures of appetite between our three treatments. Protein is well-established as being more satiating than carbohydrate or fat during isocaloric conditions [47-49], so the slightly higher protein content of the walnut-containing meals may be one reason for our observed appetite differences. This may also explain why we found greater fullness for the BW vs. EW as the protein content is higher for BW vs. EW (14.0 vs. 11.5g). 13
Journal Pre-proof Additionally, the higher fiber content of the walnut-containing meals may have led to the better overall appetite score compared to the control meal since dietary fiber has also been shown to improve satiety [50]. Higher fiber intake is associated with lower body weights and enhanced satiety possibly due to an increase in gastric distention, fermentation, and satiety hormones [5153]. Although previous acute meal challenge studies have been unable to show greater reductions in appetite among EW meals, the amount or “dose” of fiber may be important. In those studies,
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walnut meals contained 26-32% more fiber compared to the control meals [29,32], whereas our
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walnut-enriched meals contained approximately 46% more fiber than the control. This larger
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difference between test meals may have contributed to our improved postprandial appetite responses. Regardless of the exact nutrient, or combination of nutrients, that are responsible for
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the improved appetite scores and fullness, our study shows that substituting a common fat source
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with walnuts, especially BW, improves markers of appetite.
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Due to the higher fiber content of the walnut-containing meals, we expected to see an attenuation of postprandial glucose, insulin or triglyceride compared to the control meal. We did
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not observe such differences, which is contrary to one of our hypotheses. The lack of glycemic
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differences observed may be explained by the carbohydrate-matching of our test meals. In another acute study, postprandial glycemic responses were compared among high-fat shakes with varying FA compositions (MUFA vs. PUFA), but similar carbohydrate contents, to a low-fat, high carbohydrate control [30]. The two high-fat shakes attenuated insulin and glucose more than control, but did not differ from one another. Since our carbohydrate contents were similar, the differences in fiber content or fatty acid composition likely were not strong enough to override the similar total carbohydrate content between our test meals.
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Journal Pre-proof In support of our first hypothesis, the BW meal also suppressed lipid peroxidation more than the control meal, while there was no difference between EW vs. control. Both BW and EW are high in polyphenols [54], which have been shown to protect plasma from lipid peroxidation [35,55]. However, BW have a slightly greater total phenolic content compared to the EW [54], which may explain the superior lipid peroxidation outcomes observed from the BW meal. It is also important to note that lipid peroxides, such as malondialdehyde (MDA) which was
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measured by the TBARS assay, are derived from PUFAs [56]. The EW muffin contained slightly
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more PUFA than the BW muffin (14.6 g vs. 11.6 g), thus the EW muffin was likely more
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susceptible to lipid peroxidation.
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Despite the enhanced suppression of lipid peroxidation with the BW muffin, there were no
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differences in TAC among the three test meals, which was contrary to our hypothesis. The lack of agreement between these two antioxidant measures may be explained by the ORAC assay
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mechanism. The ORAC assay works through the Hydrogen Atom Transfer (HAT) mechanism in which the antioxidant donates a hydrogen atom to the reactive oxygen species (ROS) [57].
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However, the specific antioxidants within the BW that resulted in less lipid peroxidation may not
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undergo the HAT mechanism, limiting the ability of the ORAC assay to comprehensively determine TAC. It is also possible that lipid peroxidation occurs in the gastrointestinal tract prior to absorption; however, previous research has shown that lipid peroxidation byproducts are absorbed [58-60] and should therefore still appear in plasma. Unfortunately, TAC has not been previously measured in human plasma following BW ingestion limiting our ability to make comparisons with other studies. Previous studies in EW have found increases in TAC when measured by hydrophilic ORAC when incorporating 80-90g of walnuts into test meals [35,37].
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Journal Pre-proof Since our test meals only contained 28g of walnuts, our dose of walnuts may not have been substantial enough to observe differences in plasma TAC. There were some limitations to the current study. First, we did not provide a 24h lead-in diet, so slight deviations to our prescribed macronutrient distribution existed based on the results from the nutrient analysis. Importantly, however, the total energy and macronutrient distribution did not differ between any of the 3 visits. We also relied on self-report intake data for the lead-in
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diet, which is another limitation as omissions or mistakes by the participants could have
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occurred. In addition, we did not introduce a buffet meal to determine if increased subjective
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fullness measures translated into reduced intake at a subsequent meal. We also did not control for
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weight loss histories, which could have affected subjective appetite outcomes. Finally, we had a
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relatively young, healthy population, so extrapolating the current results into older age populations may not be appropriate. Notably, there were significant pre-treatment between males
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and females, but these differences were unlikely to affect our outcomes due to the crossover
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design and within subjects analysis. All subjects of both genders received all treatments. In conclusion, we found that substituting BW for butter in a breakfast meal improved
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markers of health through decreased appetite, increased fullness and suppressed lipid peroxidation. We also observed improved appetite scores for the EW, but these scores did not differ from BW. These findings for both walnuts, but especially the BW, are clinically relevant because consuming a satiating breakfast may reduce overall intake throughout the day, which will promote weight stability. In addition, reducing lipid peroxidation may reduce the risk for chronic diseases that are associated with oxidative stress [61]. Future studies are needed in other populations at risk for chronic disease, as well as measured appetite hormones, subsequent energy intake, and TAC following meals containing different doses of BW and EW. 16
Journal Pre-proof Acknowledgment The authors have no conflicts of interest to declare. This work was supported by Hammons
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Product Company [grant number AWD00006706].
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Journal Pre-proof Figure Captions Figure 1. Consolidating Standards of Reporting (CONSORT) flow diagram of selection of study participants.
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Figure 2. Time course for change in subjective appetite score, fullness, hunger, prospective consumption and desire to eat for fasting (t=0) and after consuming a control, black walnut, or English walnut-containing muffin, measured on a 100-mm scale (Figure 2A, B, C, D, and E, respectively). Repeated Measures Analysis of Variance (ANOVA) was used to determine main and interaction effects of treatment and time. There was a significant treatment effect for greater suppression of appetite for the black walnut (BW) and English walnut (EW) meals vs. control (p<0.01 and p=0.03, respectively) (Figure 2A). There was a significant treatment effect for greater fullness for the BW meal vs. both the EW and control (p<0.01 and p<0.0001, respectively) (Figure 2B). Data are presented as means ± SEM (n=30). Significance was defined as P < .05.
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Figure 3. Time course of meal responses for Thiobarbituric Acid Reactive Substances (TBARS) and Oxygen Radical Absorbance Capacity (ORAC) assays at fasting (t=0) and after consuming a control, black walnut, or English-walnut containing muffin (Figure 3A and B, respectively). Repeated Measures Analysis of Variance (ANOVA) was used to determine main and interaction effects of treatment and time. There was a significant treatment effect for lower postprandial TBARS for the black walnut meal vs. control (p<0.01) (Figure 3A). Data are presented as means ± SEM (n=30). Significance was defined as P < .05.
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Figure 4. Time course of meal responses for glucose, insulin, and triglycerides (TG) at fasting (t=0) and after consuming a control, black walnut, or English-walnut containing muffin (Figure 3A, B, and C, respectively). Repeated Measures Analysis of Variance (ANOVA) was used to determine main and interaction effects of treatment and time. There were no significant differences between meal treatments. Data are presented as means ± SEM (n=30).
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English Walnut Muffin 696.3 56.9 9.1 34.1 72.2 4.1 11.5 43.3 16.6 9.8 14.6 2.9 11.6 59.9
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Table 1. Nutrient Breakdown of each Test Meal Control Black Walnut Composition Muffin Muffin Energy (kcal) 692.1 686.4 % Carbohydrate 56.4 60.1 % Protein 6.1 11.9 % Fat 37.4 35.2 Carbohydrate (g) 68.3 71.0 Fiber (g) 2.2 4.2 Protein (g) 7.4 14.0 Fat (g) 45.3 41.6 Saturated Fat (g) 27.7 15.9 Monounsaturated Fat (g) 13.2 11.7 Polyunsaturated Fat (g) 2.0 11.6 Omega-3 FA (g) 0.7 1.13 Omega-6 FA (g) 1.3 10.4 Total Nut Phenolics (mg)* n/a 68.6
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Abbreviations: FA = Fatty Acid; g = grams; kcal = kilocalories; n/a = not applicable *Total phenolic data for the 28g serving of black and English walnuts [54].
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Journal Pre-proof
Female (n=16) 20.7 ± 1.4 165.6 ± 4.4 63.6 ± 6.8 23.2 ± 2.0 0.7 ± 0.0 112.6 ±7.3 70.6 ±7.3 150.4 ± 22.7 81.6 ± 17.3 53.5 ± 13.2 76.5 ± 30.6
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Table 2. Baseline Demographics Male (n=14) Age (y) 22.7 ± 3.6* Height (cm) 176.8 ± 8.9* Weight (kg) 74.1 ± 9.3* BMI (kg/m2) 23.6 ± 1.7 WHR (cm) 0.8 ± 0.0* SBP (mm Hg) 122.3 ± 13.8* DBP (mm Hg) 75.6 ± 9.0 Total Cholesterol (mg/dL) 157.6 ± 34.7 LDL (mg/dL) 88.4 ± 31.2 HDL (mg/dL) 49.4 ± 10.0 Triglycerides (mg/dL) 98.9 ± 45.4
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Values are presented as mean ± SD, and two-sample t-tests were used to compare the baseline demographics of males vs females at baseline. Abbreviations: BMI= Body mass index; WHR= Waist to hip ratio; SBP= Systolic blood pressure; DBP= Diastolic blood pressure; LDL= Low Density Lipoprotein; HDL= High Density Lipoprotein. *Indicates significant difference between males and females at P<0.05.
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Journal Pre-proof Table 3. Sensory Evaluation of Test Meals Control Muffin Black Walnut Muffin Appearance 7.4 ± 0.3 7.5 ± 0.2 Taste/Flavor 7.8 ± 0.2 7.0 ± 0.3* Texture/Consistency 7.7 ± 0.2 7.2 ± 0.3 Aroma/Smell 7.3 ± 0.3 7.4 ± 0.2 Overall Acceptance 7.6 ± 1.0 7.4 ± 0.3
English Walnut Muffin 7.8 ± 0.2 8.0 ± 0.1 8.0 ± 0.2 7.8 ± 0.2 8.0 ± 0.1
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Values are presented as mean ± SEM, and a one-way Analysis of Variance (ANOVA) was used to compare sensory ratings between test meal. * Indicates significantly lower score for Taste/Flavor for Black Walnut vs. both control and English Walnut meals.
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Journal Pre-proof Highlights:
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Black walnuts are an adequate substitute for butter fat in breakfast meals based on sensory and physiological responses. Incorporating black walnuts into a breakfast meal increased feelings of fullness more than the addition of English walnuts. Black walnuts reduced lipid peroxidation more than a traditional butter muffin.
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